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https://openalex.org/W4206527580	https://www.researchsquare.com/article/rs-95577/latest.pdf	English	null	Age at menarche, age at natural menopause and risk of rheumatoid arthritis – a Mendelian randomization study	Research Square (Research Square)	2,021	cc-by	6,803	Age at menarche, age at natural menopause and risk of rheumatoid arthritis – a Mendelian randomization study g g p risk of rheumatoid arthritis – a Mendelian randomization study Jingjing Zhu The First Affiliated Hospital of Zhejiang Chinese Medical University Zheng Niu Affiliated Hangzhou First People's Hospital Zhejiang University School of Medicine: Hangzhou First People's Hospital Lars Alfredsson Karolinska Institutet Department of Clinical Neuroscience Lars Klareskog karolinska institute Leonid Padyukov Karolinska Institutet Xia Jiang (  xia.jiang@ki.se ) Karolinska Institute, Harvard T.H. Chan School of Public Health https://orcid.org/0000-0001-5878-8986 Research article Keywords: Mendelian randomization, Rheumatoid arthritis, Age at menarche, Age at natural menopause, Age at first birth Posted Date: February 3rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-95577/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 at Arthritis Research & Therapy on April 9th, 2021. See the published version at https://doi.org/10.1186/s13075-021-02495-x. Jingjing Zhu The First Affiliated Hospital of Zhejiang Chinese Medical University Zheng Niu Affiliated Hangzhou First People's Hospital Zhejiang University School of Medicine: Hangzhou First People's Hospital Lars Alfredsson Karolinska Institutet Department of Clinical Neuroscience Lars Klareskog karolinska institute Leonid Padyukov Karolinska Institutet Xia Jiang (  xia.jiang@ki.se ) Karolinska Institute, Harvard T.H. Chan School of Public Health https://orcid.org/0000-0001-5878-8986 g Affiliated Hangzhou First People's Hospital Zhejiang University School of Medicine: Hangzhou First eople's Hospital lf d Research article Posted Date: February 3rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-95577/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 at Arthritis Research & Therapy on April 9th, 2021. See the published version at https://doi.org/10.1186/s13075-021-02495-x. Page 1/15 Page 1/15 Abstract Background Hormonal reproductive factors have been suggested to play an important role in the etiology of rheumatoid arthritis (RA), an autoimmune inflammatory disorder affecting primarily women. We conducted a two-sample Mendelian randomization (MR) study examining three relevant exposures, age at menarche (AAM), age at natural menopause (ANM) and age at first birth (AFB) with the risk of RA. Methods We collected summary statistics from the hitherto largest GWAS conducted in AAM (N=329,345), ANM (N=69,360), AFB (N=251,151) and RA (Ncase=14,361, Ncontrol=43,923), all of European ancestry. We constructed strong instruments using hundreds of exposure-associated genetic variants and estimated causal relationship through different MR approaches including an inverse-variance weighted method, an MR-Egger regression and a weighted-median method. We conducted a multivariable MR to control for pleiotropic effect acting in particular through obesity and socioeconomic status. We also performed important sensitivity analyses to verify model assumptions. Results We did not find any evidence in support for a causal association between genetically predicted reproductive factors and risk of RA (ORper-SD increment in AAM=1.06 [0.98-1.15]; ORper-SD increment in ANM=1.05 [0.98-1.11], OR per-SD increment in AFB=0.85 [0.65-1.10]). Results remained consistent after removing palindromic SNPs (ORper-SD increment in AAM=1.06 [0.97-1.15], ORper-SD increment in ANM=1.05 [0.98-1.13], ORper- SD increment in AFB=0.81 [0.61-1.07]) or excluding SNPs associated with potential confounding traits (ORper- SD increment in AAM=1.03 [0.94-1.12], ORper-SD increment in ANM=1.04 [0.95-1.14]). No outlying instrument was identified through the leave-one-out analysis. Conclusions Our MR study does not convincingly support a casual effect of reproductive factors, as reflected by age at menarche, age at menopause and age at first birth, on the development of RA. Despite the largely augmented set of instruments we used, these instruments only explained a modest proportion of phenotypic variance of exposures. Our knowledge regarding this topic is still insufficient and future studies with larger sample size should be designed to replicate or dispute our findings. Background Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder affecting mainly women of reproductive age and often leads to disabling outcomes as well as shortened life expectancy if left untreated or not properly controlled. The sex difference in the prevalence of RA has been well documented where the disease strikes women more frequently and severe. For example, the incidence of RA has been estimated to be 4-5 times higher in women than in men below age 50 and twice higher during age 60-70. [1] In addition, observational studies have suggested that in general female RA patients do worse than male patients. [2] The reasons for this overrepresentation of women remain unclear but X-linked genetic factors and hormonal components are likely to be involved. Some women develop RA at transitional periods when Page 2/15 sex hormones are shifting, for example after pregnancy and/or before menopause. [3] Medications that modulate hormone levels including long-term oral contraceptive use [4] and/or postmenopausal hormone therapy [5] have been found to be associated with a reduced risk of RA. These observations highlight a role of hormonal and reproductive factors in the disease etiology. Several large-scale epidemiological studies have investigated the relationship between female reproductive factors and RA using three most readily available measures – age at menarche (AAM), age at natural menopause (ANM) and age at first birth (AFB) – yet results remain controversial. For example, the longitudinal Nurses’ Health Study enrolling 121,700 women during 1976-2002 identified an association of age at menarche ≤ 10 years with an increased risk of seropositive RA (RR 1.6, 95%CI 1.1- 2.4) but not a significant association with risk of overall RA. [6] The study (NHS, 1976-2010; NHSII 1989- 2011) also revealed that early age at natural menopause (≤ 44 years) was associated with an increased risk of seronegative RA (pooled HR 2.4, 95%CI 1.5-4.0). [7] Data from the Swedish EIRA study, a population-based case-control study of female incident RA cases (2035 cases and 2911 controls, aged 18-70 years) showed an increased risk of ACPA (antibodies to citrullinated peptide antigens)-negative RA in those who were at a young age at first birth (< 23 years) (OR 2.5, 95%CI 1.5-4.1) compared to nulliparous women. [8] An analysis using cross-sectional data from 1,892 participants in the Third National Health and Nutrition Examination Survey did not find any association between age at menarche or pregnancy history with RA after menopause. (N=251,151). [11-13] IV-outcome associations were extracted from the largest GWAS conducted in RA (NRA=14,361, Ncontrol=43,923). [14] (N=251,151). [11-13] IV-outcome associations were extracted from the largest GWAS conducted in RA (NRA=14,361, Ncontrol=43,923). [14] Methods We conducted current study applying a standard two-sample framework where IV-exposure associations and IV-outcome associations come from two sets of independent non-overlapping individuals. To reduce population stratification, we included only individuals of European ancestry. Exposure Three reproductive exposures demonstrated by previous GWAS(s) as having polygenic components were involved. Age at menarche, a milestone in female pubertal development, varies markedly among females. The genetic regulation in AAM has been highlighted by a recent meta-GWAS incorporating 329,345 women collected by the ReproGen consortium, 23andMe and UK Biobank and identified 389 independent AAM-associated signals spreading over 10 biological pathways. [11] Age at natural menopause poses a substantial impact on infertility and disease risk including breast cancer and cardiovascular events. The genetic architecture of ANM has been examined by a recent GWAS of 69,360 women identifying 54 independent signals located in 44 genomic regions, most of which were found to contain one or more DNA damage response pathway genes. [12] Reproductive behavior such as age at first birth is known to be partly driven by biological processes. A recent GWAS has examined the genetic architecture of reproductive tempo defined by AFB in 251,151 women and identified 10 AFB-associated loci. [13] In all three GWAS(s), independent signals (our IVs) were defined as following. A list of index variants was first defined using a distance-based metric, by which any SNPs passing the two-tailed threshold of significance (P<5×10−8) within 1 Mb of another significant SNP were considered to be located in the same locus. This list of signals was then further augmented using approximate conditional analysis in GCTA, using an LD reference panel from the UK Biobank study. Only secondary signals that were uncorrelated (r2<0.05) were included in the final list. All these IVs passed quality control procedures under minor allele frequency > 0.001 and Hardy-Weinberg Equilibrium > 1´10-6. IV-exposure associations were extracted from each GWAS. [11-13] Outcome IV-outcome associations were obtained from a meta-GWAS involving 18 participating cohorts totaling 14,361 RA cases and 43,923 controls of European ancestry. To the best of our knowledge, none of the participants in these 18 studies overlapped with participants in the exposure GWAS(s). [14] Background [9] These discrepancies are perhaps not surprising since conventional epidemiological studies generally rely on environmental information and results are likely to be influenced by measurement error, confounding, and reverse causality. Hormonal reproductive factors including puberty and fertility are influenced by genetic, nutritional, socioeconomic and environmental factors, and can be highly heterogeneous among women. Nevertheless, the genetic regulations in AAM, ANM and AFB have been recently highlighted by discoveries from large-scale genome-wide association studies (GWAS) leveraging millions of women of European ancestry. These results provide a valuable opportunity to utilize a novel statistical approach Mendelian randomization (MR) to make causal inference – an approach that uses genetic variants (single nucleotide polymorphisms, SNPs) as instrumental variables (IVs) to assess a causal effect of a risk factor on an outcome from observational data. Since SNPs are randomly assigned at conception and always precede disease onset, results from MR are less susceptible to confounding and reverse causation, which are the major limitations of conventional observational studies. [10] To the best of our knowledge, no MR analysis has been performed to examine a potential causal association between hormonal reproductive factors and development of RA, of which findings may help address patient concerns in topics of puberty, fertility, motherhood and RA as well as improve our knowledge on the biological mechanisms underlying RA. Therefore, we aim to conduct the first and also the largest two-sample MR on three female reproductive factors (AAM, ANM and AFB) with the risk of RA. Genetic variants associated with each reproductive event were used as instrumental variable (IVs). IV-exposure associations were extracted from the recently published and also the largest GWAS(s) conducted in AAM (N=329,345), ANM (N=69,360) and AFB Page 3/15 Page 3/15 Statistical analysis Mendelian randomization analysis We applied several MR methods including an inverse variance weighted approach (IVW) [15], a maximum likelihood-based method [16], an MR-Egger regression [17] and a weighted median approach [18]. Page 4/15 Briefly, IVW represents the main conventional approach which only gives consistent estimates if all genetic variants in the analysis are valid instrumental variables. When the IVs are weak, IVW tends to underestimate the true variation of the estimate, while the likelihood method gives appropriately estimated confidence intervals. MR-Egger evaluates the directional pleiotropic effect of instrumental variables, of which the intercept term can be interpreted as an estimate of the average pleiotropy of genetic variations. The weighted median method is robust to outliers and provides consistent estimates even when 50% of the genetic variants are invalid IVs; and is considered as relatively more robust to horizontal pleiotropy. Briefly, IVW represents the main conventional approach which only gives consistent estimates if all genetic variants in the analysis are valid instrumental variables. When the IVs are weak, IVW tends to underestimate the true variation of the estimate, while the likelihood method gives appropriately estimated confidence intervals. MR-Egger evaluates the directional pleiotropic effect of instrumental variables, of which the intercept term can be interpreted as an estimate of the average pleiotropy of genetic variations. The weighted median method is robust to outliers and provides consistent estimates even when 50% of the genetic variants are invalid IVs; and is considered as relatively more robust to horizontal pleiotropy. Sensitivity analysis A valid MR analysis is defined by three key model assumptions – the IVs are strongly associated with the risk factor of interest (relevance), share no common cause with the outcome (independence), and affect outcome solely through the exposure (exclusion restriction). [10] Upon the satisfaction of all three assumptions, causal inferences between exposure(s) and outcome(s) can be made based on observational data. We performed several important sensitivity analyses to verify MR model assumptions. For each index SNP, we searched for its potential association with confounding traits in GWAS catalog and conducted analysis excluding pleiotropic SNPs. Moreover, we used a robust adjusted profile score (MR-RAPS) approach which is robust to both systemic and idiosyncratic pleiotropy and performed excellently in all the numerical examples. [19] Educational attainment and obesity are two important confounders affecting both reproductive traits and risk of RA. Statistical analysis [20] We further integrated GWAS summary statistics and additional IVs on education and BMI, and conducted an IVW-based multivariable MR (MVMR) to estimate the direct effect of reproductive factors controlling for the effect of BMI and education. [21, 22] Finally, we excluded one SNP at-a-time and performed IVW on the remaining SNPs to identify outlying IVs. We calculated statistical power using the non-centrality parameter of the test statistic as suggested by Brion et al. (http://cnsgenomics.com/shiny/mRnd/). All analyses were conducted with packages “TwoSampleMR”, “MRInstruments” and “Mendelian Randomization” in R v3.6.3. Results Overall, we did not find convincing evidence in support for a causal relationship between the three hormonal related exposures and risk of RA. Specifically, genetically predicted AAM did not significantly influence the risk of RA using IVW approach (ORper-SD increment in AAM [95%CI]: 1.06 [0.98-1.15]). Estimates remained consistent across different methods (ORper-SD increment in AAM [95%CI] for maximum likelihood 1.07 [1.00-1.14]; for MR-Egger regression 1.11 [0.90-1.36]; for weighted-median 1.08 [0.97-1.21]). We did not observe apparent sign of pleiotropy (P for MR-Egger intercept = 0.69). Similarly, we did not find any compelling evidence supporting a casual association of genetically instrumented ANM with RA either using IVW (ORper-SD increment in ANM [95%CI]: 1.05 [0.98-1.11]) or MR-Egger regression (ORper-SD increment in ANM [95%CI]: 1.04 [0.90-1.20]). Null finding was identified using the weighted-median approach (ORper-SD increment in ANM [95%CI]: 1.03 [0.98-1.08]). Consistently, for genetically predicted AFB, we did not observe Page 5/15 any significant association with RA using IVW (ORper-SD increment in AFB [95%CI]: 0.85 [0.65-1.10]), MR-Egger regression (ORper-SD increment in AFB [95%CI]: 3.32 [0.36-30.81]) or weighted-median approach (ORper-SD increment in AFB [95%CI]: 0.90 [0.73-1.10]). Indeed, for both ANM and AFB, significant results appeared using the maximum likelihood approach, yet this method is known to provide better power by neglecting horizontal pleiotropy and results were not supported by other methods. We did not observe apparent signs of horizontal pleiotropy (P for MR-Egger intercept = 0.92 for ANM and = 0.26 for AFB) (Table 1). Results remained consistent after removing palindromic SNPs (OR [95%CI]: 1.06 [0.97-1.14] for AAM; 1.05 [0.98-1.13] for ANM; 0.81 [0.61-1.07] for AFB). As shown in Supplementary Table 1 and Supplementary Table 2, the AAM and ANM IVs were also found to be associated with potential confounders while none of the 10 AFB-associated IVs was cited by the NHGRI-EBI Catalog (Supplementary Table 3). Using 283 AAM-associated IVs and 42 ANM-associated IVs excluding pleiotropic SNPs, we did not detect a causal effect of AAM or ANM on RA risk (OR [95%CI]: 1.03 [0.94-1.12] for AAM; 1.04 [0.95-1.14] for ANM), corroborating our primary findings (Table 1). To effectively control for pleiotropy, we next looked into the Robust Adjusted Profile Score (RAPS) approach which is robust to both systemic and idiosyncratic pleiotropy. [19] We performed MR-RAPS estimator and found that results remain largely consistent with our primary findings (Table 2). Discussion In this study, we examined a putative causal relationship between three hormonal reproductive traits (AAM, ANM and AFB) and an autoimmune inflammatory disease RA which affects mainly women. We capitalized on the summary statistics of the largest GWAS(s) conducted for these traits in European ancestry populations and constructed strong instruments using hundreds of SNPs associated with the exposures (F-statistic for AAM 11.89, for ANM 65.18, for AFB 11.68). We did not find convincing evidence in support for a causal effect of reproductive factors on RA using univariable MR analyses. Consistent null associations were identified by sensitivity analysis and multivariable MR analysis, demonstrating the robustness of our findings. Current results from conventional epidemiological studies on this topic remain controversial, yet many studies point towards a positive association. For example, a study enrolling 121,700 female nurses found that age at menarche ≤ 10 years was associated with an increased risk of seropositive RA (RR 1.60, 95%CI 1.10-2.40). [6] A community-based health survey including 30,447 subjects (18,326 women) between 1991 and 1996 found an association between early age at menopause (≤ 45 years) and subsequent development of RA (OR 2.42, 95%CI 1.32-4.45), which remained significant after adjusting for smoking, level of education and length of breastfeeding (OR 1.92, 95%CI 1.02-3.64). [23] A prospective cohort study of 31,336 North America women reported similar findings (RRmenopause >51 vs. menopause <45 0.64, 95%CI 0.41-1.00). [24] Our large-scale MR, however, did not identify a putative causal link between the three well-defined hormonal exposures and risk of RA. Several reasons underlie such a discrepancy. First of all, reproductive factors are highly complicated and heterogenous traits shaped by both genetic and environmental factors and genetics alone does not fully capture the phenotypic variance of these traits. For example, age at first birth is a human behavioral trait influenced largely by psychosocial, cultural and financial factors rather than the genetics. Secondly, results from previous epidemiological studies are likely to be impaired by confounding factors. For example, obesity is an important confounder affecting both the exposure and the outcome. An MR study demonstrated that a one-year delay in age at menarche reduced adult BMI by 0.38kg/m2 (95%CI 0.25-0.51kg/m2). [25] Global adiposity is a robust causal risk factor for RA as demonstrated by our recently published MR. [26] It is likely that traditional epidemiological investigations did not adequately control for the confounding effects from obesity. Results Education and BMI are two modifiable risk factors, both of which play an important role in the etiology of RA and shape the reproductive exposures. We next conducted an IVW-based MVMR to estimate a direct effect of reproductive factors on RA accounting for the confounding effect from obesity and socioeconomic status. The results of MVMR remained consistent with our primary findings. The effect of AAM with RA did not alter substantially after adjusting for BMI (OR [95%CI]: 0.97 [0.83-1.13]) or education (OR [95%CI]: 1.07 [0.98-1.16]). Similarly, for ANM, we did not observe any significant effect with RA after adjusting for BMI (OR [95%CI]: 1.06 [0.99-1.27]) or education (OR [95%CI]: 1.04 [0.98-1.11]). For AFB, similar null effect was found after adjusting for BMI (OR [95%CI]: 0.85 [0.57-1.24]) or education (OR [95%CI]: 1.10 [0.58-2.11]) (Table 3). In the leave-one-out analysis where we iteratively removed one SNP at a time and performed IVW using the remaining SNPs, we did not observe apparent outlying SNPs and the odds ratios were in accordance with our primary findings, aggregating closely around the expected value of estimation (Figure 1). Finally, we calculated the power of our analysis. As shown in Table 4, the sample size of the RA GWAS was 58,284 with 24.64% cases. According to the three exposure GWAS(s), 7.4% of phenotypic variance of AAM could be explained by the 389 index SNPs, 5.7% of ANM phenotypic variance could be explained by the 54 index SNPs and 0.2% of AFB phenotypic variance could be explained by the 10 SNPs. Under current situation, for AAM, our study had 80% power to detect a causal effect of a 10.4% (i.e., ORs of 1.104) increase in RA risk. For ANM, the minimal detectable effect was 12% increase (i.e., ORs of 1.12). For AFB, the minimal detectable effect was 70% increase (i.e., ORs of 1.70). We presented a range of power estimations in Table 4. Page 6/15 Discussion The protective effect of education on RA has been reported by observational studies. [27, 28] An MR study identified that a one- year later in age at menarche increased 0.14 years (53 days) of time spent in education. [29] We performed a MVMR to control for the effect of adiposity and education, and the negative results corroborating our main findings on a null association. Finally, it is also likely that the true causal effect of reproductive factors on RA is modest, which our study is underpowered to identify. The biological mechanisms underlying hormonal factors and the development of RA remain unclear. The effect of sex hormones on the immune system and their interaction with environmental and genetic factors may partly explain the higher prevalence of RA observed among women. Estrogen is a complex Page 7/15 Page 7/15 modulator to the immune system exerting both a stimulatory and an inhibitory effect. [30] For example, estrogens at periovulatory to pregnancy levels stimulate B cells and the Th2 response and support the survival of auto-reactive T and B cell clones. On other hand, estrogens could inhibit cell-mediated responses such as the differentiation to Th17 cells. [30-32] A reduced risk of RA onset during pregnancy compared to an increased risk postpartum, suggests a role the hormonal changes or the exposure to fetus paternal HLA in RA onset. [33] Our study has several strengths. To the best of our knowledge, no MR has been performed to assess the relationship between reproductive factors and RA. We incorporated three different reproductive traits (age at menarche, age at natural menopause and age at first birth) reflecting the length of reproductive period and complementing each other well. Moreover, we conducted important sensitivity analyses to verify MR model assumptions. We selected the most significant independent SNPs identified by the largest GWAS, so all were robustly associated with exposure of interest, guaranteeing “relevance” assumption. We excluded SNPs associated with potential confounders on the exposure-outcome relationship to satisfy “exclusion restriction” assumption. The consistent results observed across different approaches, further lend support to our findings. We have to acknowledge several limitations. Firstly, our analysis was performed using the European populations which restricted its generalizability. Secondly, the genetic instruments of three exposures (AAM, ANM and AFB) we used as proxies for hormonal reproductive characteristics captured only a modest proportion of phenotypic variance. Discussion Reproductive factors are complex traits influenced by different components such as genetic, environmental and socioeconomic factors as well as their complex interactions. The design of our study disables us to take into account environmental impacts. Thirdly, the association between genetically predicted age at each of the reproductive events and risk of RA was evaluated fitting the exposure as a continuous variable – we can still not exclude a non-linear effect which was not captured by our study with the current availability of data. Future work on such topics may be focused on categorized age of reproductive events. Fourthly, our study was conducted using overall RA (a majority of which are seropositive RA, >85%) without specifying disease subsets characterized by the presence/absence of antibodies to citrullinated peptides or rheumatic factors. It is possible that hormonal factors influence different RA subsets via a distinct way. It is also likely that other factors such as hormone use and health conditions confound our results, in addition to the only two confounders (obesity and education) considered in the current study. However, it is difficult to control for the effect of hormone therapy due to limited availability of genetic data underlying this trait. Finally, power calculations showed that potential weak effects were difficult to be detected in our analysis. Conclusions In summary, using both univariable and multivariable Mendelian randomization approaches, we could not provide evidence supporting a casual effect of reproductive factors as reflected by age at menarche, age at menopause or age at first birth in the development of RA. Our result is not so surprising considering the relatively weak genetic instruments and power. The findings represent a preliminary but Page 8/15 Page 8/15 Page 8/15 important step towards the identification of causal associations between female hormonal reproductive factors and a female disease RA. As some hormonal factors are potentially modifiable, understanding their precise role is essential for future preventive interventions focusing on women at high risk. Our knowledge regarding this topic is still insufficient and future studies with larger sample size and better power should be designed to increase our knowledge in this field. important step towards the identification of causal associations between female hormonal reproductive factors and a female disease RA. As some hormonal factors are potentially modifiable, understanding their precise role is essential for future preventive interventions focusing on women at high risk. Our knowledge regarding this topic is still insufficient and future studies with larger sample size and better power should be designed to increase our knowledge in this field. Abbreviations RA rheumatoid arthritis AAM age at menarche ANM age at natural menopause AFB age at first birth ACPA antibodies to citrullinated peptide antigens MR Mendelian randomization GWAS genome-wide association studies SNPs single nucleotide polymorphisms IV instrumental variable IVW inverse-variance weighted method BMI body mass index MVMR multivariable Mendelian randomization Ethics approval and consent to participate The summary statistics used in the present study are aggregated level of data which do not contain any personal information. The original GWAS have obtained ethical approval from relevant ethics review committees. Authors’ contributions JJZ and XJ designed the study, analyzed the data, interpreted the results and wrote the manuscript. ZN, LA, LK, and LP interpreted the results and revised the manuscript. All authors provided the corresponding author with permission to be named in the manuscript. XJ is the guarantor of this study. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or nonprofit sectors. Consent for publication Not applicable Availability of data and materials Page 9/15 Page 9/15 The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgments Summary statistics for the genetic associations with AAM, ANM, AFB, BMI, education and RA were obtained from GWAS respectively by Day FR et al., Day FR et al., Barban N et al., Yengo L et al., Lee JJ et al. and the RA meta-GWAS conducted by Okada Y et al. We thank all investigators for sharing the genome-wide summary statistics. Competing interests Nothing declared References 1. Kvien TK, Uhlig T, Ødegård S, Heiberg MS: Epidemiological aspects of rheumatoid arthritis: the sex ratio. Annals of the New York Academy of Sciences 2006, 1069:212-222. 1. Kvien TK, Uhlig T, Ødegård S, Heiberg MS: Epidemiological aspects of rheumatoid arthritis: the sex ratio. Annals of the New York Academy of Sciences 2006, 1069:212-222. 2. van Vollenhoven RF: Sex differences in rheumatoid arthritis: more than meets the eye. BMC medicine 2009, 7:12. 2. van Vollenhoven RF: Sex differences in rheumatoid arthritis: more than meets the eye. BMC medicine 2009, 7:12. 3. Desai MK, Brinton RD: Autoimmune Disease in Women: Endocrine Transition and Risk Across the Lifespan. Frontiers in endocrinology 2019, 10:265. 3. Desai MK, Brinton RD: Autoimmune Disease in Women: Endocrine Transition and Risk Across the Lifespan. Frontiers in endocrinology 2019, 10:265. 4. Orellana C, Saevarsdottir S, Klareskog L, Karlson EW, Alfredsson L, Bengtsson C: Oral contraceptives, breastfeeding and the risk of developing rheumatoid arthritis: results from the Swedish EIRA study. Annals of the rheumatic diseases 2017, 76(11):1845-1852. 4. Orellana C, Saevarsdottir S, Klareskog L, Karlson EW, Alfredsson L, Bengtsson C: Oral contraceptives, breastfeeding and the risk of developing rheumatoid arthritis: results from the Swedish EIRA study. Annals of the rheumatic diseases 2017, 76(11):1845-1852. 5. Orellana C, Saevarsdottir S, Klareskog L, Karlson EW, Alfredsson L, Bengtsson C: Postmenopausal hormone therapy and the risk of rheumatoid arthritis: results from the Swedish EIRA population- based case-control study. Eur J Epidemiol 2015, 30(5):449-457. 5. Orellana C, Saevarsdottir S, Klareskog L, Karlson EW, Alfredsson L, Bengtsson C: Postmenopausal hormone therapy and the risk of rheumatoid arthritis: results from the Swedish EIRA population- based case-control study. Eur J Epidemiol 2015, 30(5):449-457. 6. Karlson EW, Mandl LA, Hankinson SE, Grodstein F: Do breast-feeding and other reproductive factors influence future risk of rheumatoid arthritis? Results from the Nurses' Health Study. Arthritis and rheumatism 2004, 50(11):3458-3467. Page 10/15 Page 10/15 7. Bengtsson C, Malspeis S, Orellana C, Sparks JA, Costenbader KH, Karlson EW: Association Between Menopausal Factors and the Risk of Seronegative and Seropositive Rheumatoid Arthritis: Results From the Nurses' Health Studies. Arthritis Care Res (Hoboken) 2017, 69(11):1676-1684. 8. Orellana C, Wedren S, Kallberg H, Holmqvist M, Karlson EW, Alfredsson L, Bengtsson C, Group ES: Parity and the risk of developing rheumatoid arthritis: results from the Swedish Epidemiological Investigation of Rheumatoid Arthritis study. Annals of the rheumatic diseases 2014, 73(4):752-755. 9. References Beydoun HA, el-Amin R, McNeal M, Perry C, Archer DF: Reproductive history and postmenopausal rheumatoid arthritis among women 60 years or older: Third National Health and Nutrition Examination Survey. Menopause 2013, 20(9):930-935. 9. Beydoun HA, el-Amin R, McNeal M, Perry C, Archer DF: Reproductive history and postmenopausal rheumatoid arthritis among women 60 years or older: Third National Health and Nutrition Examination Survey. Menopause 2013, 20(9):930-935. 10. Davies NM, Holmes MV, Davey Smith G: Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians. BMJ 2018, 362:k601. 10. Davies NM, Holmes MV, Davey Smith G: Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians. BMJ 2018, 362:k601. 11. Day FR, Thompson DJ, Helgason H, Chasman DI, Finucane H, Sulem P, Ruth KS, Whalen S, Sarkar AK, Albrecht E et al: Genomic analyses identify hundreds of variants associated with age at menarche and support a role for puberty timing in cancer risk. Nat Genet 2017, 49(6):834-841. 12. Day FR, Ruth KS, Thompson DJ, Lunetta KL, Pervjakova N, Chasman DI, Stolk L, Finucane HK, Sulem P, Bulik-Sullivan B et al: Large-scale genomic analyses link reproductive aging to hypothalamic signaling, breast cancer susceptibility and BRCA1-mediated DNA repair. Nat Genet 2015, 47(11):1294-1303. 13. Barban N, Jansen R, de Vlaming R, Vaez A, Mandemakers JJ, Tropf FC, Shen X, Wilson JF, Chasman DI, Nolte IM et al: Genome-wide analysis identifies 12 loci influencing human reproductive behavior. Nat Genet 2016, 48(12):1462-1472. 14. Okada Y, Wu D, Trynka G, Raj T, Terao C, Ikari K, Kochi Y, Ohmura K, Suzuki A, Yoshida S et al: Genetics of rheumatoid arthritis contributes to biology and drug discovery. Nature 2014, 506(7488):376-381. 15. Burgess S, Scott RA, Timpson NJ, Davey Smith G, Thompson SG, Consortium E-I: Using published data in Mendelian randomization: a blueprint for efficient identification of causal risk factors. Eur J Epidemiol 2015, 30(7):543-552. 16. Burgess S, Butterworth A, Thompson SG: Mendelian randomization analysis with multiple genetic variants using summarized data. Genet Epidemiol 2013, 37(7):658-665. 16. Burgess S, Butterworth A, Thompson SG: Mendelian randomization analysis with multiple genetic variants using summarized data. Genet Epidemiol 2013, 37(7):658-665. 17. Bowden J, Davey Smith G, Burgess S: Mendelian randomization with invalid instruments: effect estimation and bias detection through Egger regression. Int J Epidemiol 2015, 44(2):512-525. 17. Bowden J, Davey Smith G, Burgess S: Mendelian randomization with invalid instruments: effect estimation and bias detection through Egger regression. References Tang B, Shi H, Alfredsson L, Klareskog L, Padyukov L, Jiang X: Obesity-related traits and the development of rheumatoid arthritis - evidence from genetic data. Arthritis & rheumatology (Hoboken, NJ) 2020. 26. Tang B, Shi H, Alfredsson L, Klareskog L, Padyukov L, Jiang X: Obesity-related traits and the development of rheumatoid arthritis - evidence from genetic data. Arthritis & rheumatology (Hoboken, NJ) 2020. 27. Kwon JM, Rhee J, Ku H, Lee EK: Socioeconomic and employment status of patients with rheumatoid arthritis in Korea. Epidemiol Health 2012, 34:e2012003. 27. Kwon JM, Rhee J, Ku H, Lee EK: Socioeconomic and employment status of patients with rheumatoid arthritis in Korea. Epidemiol Health 2012, 34:e2012003. 28. Bergstrom U, Jacobsson LT, Nilsson JA, Wirfalt E, Turesson C: Smoking, low formal level of education, alcohol consumption, and the risk of rheumatoid arthritis. Scand J Rheumatol 2013, 42(2):123-130. 28. Bergstrom U, Jacobsson LT, Nilsson JA, Wirfalt E, Turesson C: Smoking, low formal level of education, alcohol consumption, and the risk of rheumatoid arthritis. Scand J Rheumatol 2013, 42(2):123-130. 29. Gill D, Del Greco MF, Rawson TM, Sivakumaran P, Brown A, Sheehan NA, Minelli C: Age at Menarche and Time Spent in Education: A Mendelian Randomization Study. Behav Genet 2017, 47(5):480-485. 30. Straub RH: The complex role of estrogens in inflammation. Endocrine reviews 2007, 28(5):521-574. 29. Gill D, Del Greco MF, Rawson TM, Sivakumaran P, Brown A, Sheehan NA, Minelli C: Age at Menarche and Time Spent in Education: A Mendelian Randomization Study. Behav Genet 2017, 47(5):480-485. 30. Straub RH: The complex role of estrogens in inflammation. Endocrine rev 30. Straub RH: The complex role of estrogens in inflammation. Endocrine reviews 2007, 28(5):521-574. 31. Sthoeger ZM, Chiorazzi N, Lahita RG: Regulation of the immune response by sex hormones. I. In vitro effects of estradiol and testosterone on pokeweed mitogen-induced human B cell differentiation. Journal of immunology (Baltimore, Md : 1950) 1988, 141(1):91-98. 31. Sthoeger ZM, Chiorazzi N, Lahita RG: Regulation of the immune response by sex hormones. I. In vitro effects of estradiol and testosterone on pokeweed mitogen-induced human B cell differentiation. Journal of immunology (Baltimore, Md : 1950) 1988, 141(1):91-98. 32. Hughes GC, Choubey D: Modulation of autoimmune rheumatic diseases by oestrogen and progesterone. Nature reviews Rheumatology 2014, 10(12):740-751. 32. Hughes GC, Choubey D: Modulation of autoimmune rheumatic diseases by oestrogen and progesterone. Nature reviews Rheumatology 2014, 10(12):740-751. 33. References Silman A, Kay A, Brennan P: Timing of pregnancy in relation to the onset of rheumatoid arthritis. Arthritis and rheumatism 1992, 35(2):152-155. 33. Silman A, Kay A, Brennan P: Timing of pregnancy in relation to the onset of rheumatoid arthritis. Arthritis and rheumatism 1992, 35(2):152-155. Tables References Int J Epidemiol 2015, 44(2):512-525. 18. Bowden J, Davey Smith G, Haycock PC, Burgess S: Consistent Estimation in Mendelian Randomization with Some Invalid Instruments Using a Weighted Median Estimator. Genet Epidemio 2016, 40(4):304-314. 18. Bowden J, Davey Smith G, Haycock PC, Burgess S: Consistent Estimation in Mendelian Randomization with Some Invalid Instruments Using a Weighted Median Estimator. Genet Epidemiol 2016, 40(4):304-314. 19. Zhao Q, Wang J, Hemani G, Bowden J, Small D: Statistical inference in two-sample summary-data Mendelian randomization using robust adjusted profile score. The Annals of Statistics 2018, 48(3). 19. Zhao Q, Wang J, Hemani G, Bowden J, Small D: Statistical inference in two-sample summary-data Mendelian randomization using robust adjusted profile score. The Annals of Statistics 2018, 48(3). 20. Sanderson E: Multivariable Mendelian Randomization and Mediation. Cold Spring Harbor Perspectives in Medicine 2020. 20. Sanderson E: Multivariable Mendelian Randomization and Mediation. Cold Spring Harbor Perspectives in Medicine 2020. Page 11/15 Page 11/15 21. Lee JJ, Wedow R, Okbay A, Kong E, Maghzian O, Zacher M, Nguyen-Viet TA, Bowers P, Sidorenko J, Karlsson Linnér R et al: Gene discovery and polygenic prediction from a genome-wide association study of educational attainment in 1.1 million individuals. Nat Genet 2018, 50(8):1112-1121. 22. Yengo L, Sidorenko J, Kemper KE, Zheng Z, Wood AR, Weedon MN, Frayling TM, Hirschhorn J, Yang J, Visscher PM: Meta-analysis of genome-wide association studies for height and body mass index in ∼700000 individuals of European ancestry. Hum Mol Genet 2018, 27(20):3641-3649. 23. Pikwer M, Bergstrom U, Nilsson JA, Jacobsson L, Turesson C: Early menopause is an independent predictor of rheumatoid arthritis. Annals of the rheumatic diseases 2012, 71(3):378-381. 24. Merlino LA, Cerhan JR, Criswell LA, Mikuls TR, Saag KG: Estrogen and other female reproductive risk factors are not strongly associated with the development of rheumatoid arthritis in elderly women. Seminars in Arthritis and Rheumatism 2003, 33(2):72-82. 25. Gill D, Brewer CF, Del Greco MF, Sivakumaran P, Bowden J, Sheehan NA, Minelli C: Age at menarche and adult body mass index: a Mendelian randomization study. Int J Obes (Lond) 2018, 42(9):1574- 1581. 25. Gill D, Brewer CF, Del Greco MF, Sivakumaran P, Bowden J, Sheehan NA, Minelli C: Age at menarche and adult body mass index: a Mendelian randomization study. Int J Obes (Lond) 2018, 42(9):1574- 1581. 26. Tables Page 12/15 Page 12/15 Page 12/15 Genetically predicted age at menarche, age at menopause and age at first birth with the risk of toid arthritis. Results from primary mendelian randomization analysis as well as sensitivity based on a subset of instruments. Genetically predicted age at menarche, age at menopause and age at first birth with the risk of toid arthritis. Results from primary mendelian randomization analysis as well as sensitivity based on a subset of instruments. y p g , g p g toid arthritis. Results from primary mendelian randomization analysis as well as sensitivity based on a subset of instruments. ods #SNP OR (95%CI) P- value #SNP OR (95%CI) P- value #SNP OR (95%CI) P- value Full set Remove palindromic SNPs Remove confounding SNPs enarche W 340 1.06 (0.98- 1.15) 0.11 284 1.06 (0.97- 1.15) 0.19 283 1.03 (0.94- 1.12) 0.57 mum hood 340 1.07 (1.00- 1.14) 0.06 284 1.06 (0.99- 1.14) 0.12 283 1.03 (0.95- 1.11) 0.50 gger 340 1.11 (0.90- 1.36) 0.34 284 1.08 (0.85- 1.37) 0.51 283 0.97 (0.72- 1.30) 0.83 gger cept 0.69 0.85 0.68 hted ian 340 1.08 (0.97- 1.21) 0.15 284 1.10 (0.97- 1.25) 0.12 283 1.01 (0.90- 1.14) 0.86 tural menopause W 54 1.05 (0.98- 1.11) 0.15 47 1.05 (0.98- 1.13) 0.13 42 1.04 (0.95- 1.14) 0.40 mum hood 54 1.05 (1.02- 1.08) 4´10-3 47 1.06 (1.02- 1.10) 9´10-4 42 1.05 (1.00- 1.10) 0.05 gger 54 1.04 (0.90- 1.20) 0.61 47 1.05 (0.90- 1.23) 0.51 42 1.05 (0.74- 1.48) 0.79 gger cept 0.92 0.99 0.96 hted ian 54 1.03 (0.98- 1.08) 0.30 47 1.05 (1.00- 1.11) 0.07 42 0.94 (0.88- 1.01) 0.09 st birth W 10 0.85 (0.65- 1.10) 0.22 9 0.81 (0.61- 1.07) 0.14 NA NA NA mum hood 10 0.84 (0.73- 0.97) 0.02 9 0.81 (0.70- 0.94) 5´10-3 NA NA NA gger 10 3.32 (0.36- 30.81) 0.32 9 3.05 (0.33- 28.49) 0.36 NA NA NA gger cept 0.26 0.28 NA NA NA hted ian 10 0.90 (0.73- 1.10) 0.30 9 0.85 (0.69- 1.05) 0.13 NA NA NA ne of the 10 age at first birth associated SNPs was found to be associated with other traits ng to GWAS catalog. IVW: inverse-variance weighted method. Table 2. Genetically predicted age at menarche, age at menopause and age at first birth with the risk of rheumatoid arthritis. A sensitivity analysis using MR-RAPS assuming over-dispersion. Figure 1 Sensitivity analysis leaving one SNP out at a time for the association between reproductive factors and RA risk. a. The distribution of odds ratios from 389 leave-one-out analysis conducted for age at menarche and RA risk. b. The distribution of odds ratios from 54 leave-one-out analysis conducted for age at menopause and RA risk. c. The distribution of odds ratios from 10 leave-one-out analysis conducted for age at first birth and RA risk. Tables Methods of robust loss OR (95%CI) P-value Age at menarche Huber method 1.07 (0.98-1.16) 0.13 Tukey method 1.07 (0.99-1.16) 0.08 Age at natural menopause Huber method 1.02 (0.97-1.07) 0.52 Tukey method 1.01 (0.97-1.05) 0.51 Age at first birth Huber method 0.90 (0.70-1.15) 0.41 Tukey method 0.89 (0.70-1.14) 0.37 Page 13/15 Table 3. Genetically predicted age at menarche, age at menopause and age at first birth with the risk of rheumatoid arthritis. Multivariable analysis adjusting for the effect of body mass index and year of education. Table 3. Genetically predicted age at menarche, age at menopause and age at first birth with the risk of rheumatoid arthritis. Multivariable analysis adjusting for the effect of body mass index and year of education. Methods #SNP OR (95%CI) P-value Age at menarche Body mass index 140 0.97 (0.83-1.13) 0.12 Year of education 316 1.07 (0.98-1.16) 0.11 Age at natural menopause Body mass index 51 1.06 (0.99-1.27) 0.08 Year of education 54 1.04 (0.98-1.11) 0.18 Age at first birth Body mass index 10 0.85 (0.57-1.24) 0.40 Year of education 10 1.10 (0.58-2.11) 0.76 Table 4. Power calculation of the current analysis. Exposure Variance explained by index SNPs Sample size (% cases) Power to detect OR 1.05/0.95 1.10/0.91 1.15/0.87 1.20/0.83 Age at menarche 0.074 58284 (24.64%) 28% 78% 98% 100% Age at natural menopause 0.057 23% 67% 95% 100% Age at first birth 0.002 6% 7% 10% 14% Table 4. Power calculation of the current analysis. E V i S l Figures Page 14/15 1 ivity analysis leaving one SNP out at a time for the association between reprod k. a. The distribution of odds ratios from 389 leave-one-out analysis conducted A risk. b. The distribution of odds ratios from 54 leave-one-out analysis conduct pause and RA risk. c. The distribution of odds ratios from 10 leave-one-out analy first birth and RA risk. l t Fil Figure 1 Sensitivity analysis leaving one SNP out at a time for the association between reproductive factors and RA risk. a. The distribution of odds ratios from 389 leave-one-out analysis conducted for age at menarche and RA risk. b. The distribution of odds ratios from 54 leave-one-out analysis conducted for age at menopause and RA risk. c. The distribution of odds ratios from 10 leave-one-out analysis conducted for age at first birth and RA risk. This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. menarcheRASupplementaryTables13.docx Page 15/15 menarcheRASupplementaryTables13.docx Page 15/15
https://openalex.org/W2972549055	https://www.nature.com/articles/s41467-019-12138-0.pdf	English	null	Synchronous 500-year oscillations of monsoon climate and human activity in Northeast Asia	Nature communications	2,019	cc-by	10,237	1 Key Laboratory of Cenozoic Geology and Environment, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, China. 2 CAS Center for Excellence in Tibetan Plateau Earth Sciences, Beijing 100101, China. 3 University of Chinese Academy of Sciences, Beijing 100049, China. 4 CAS Center for Excellence in Life and Paleoenvironment, Beijing 100044, China. 5 Department of East Asian Languages and Cultures, Stanford University, Stanford, CA 94305, USA. 6 Yunnan Key Laboratory of Plateau Geographical Processes and Environmental Changes, College of Tourism and Geographical Sciences, Yunnan Normal University, Kunming 650500, China. 7 Institutions of Earth Science, Chinese Academy of Sciences, Beijing 100029, China. 8 Department of Archaeology, School of History and Culture, Shandong University, Jinan 250100, China. 9These authors contributed equally: Deke Xu, Houyuan Lu. Correspondence and requests for materials should be addressed to D.X. (email: ccxudeke@mail.iggcas.ac.cn) or to H.L. (email: houyuanlu@mail.iggcas.ac.cn) ARTICLE ARTICLE Synchronous 500-year oscillations of monsoon climate and human activity in Northeast Asia The Neolithic Hon- gshan culture, especially, has attracted much attention in the study of Chinese civilization, because it developed prior to the emergence of dynastic civilization in the Central Plain. The Hongshan culture is characterized by large-scale public archi- tecture and elite burials associated with the abundant use of jade at the ritual centers of Niuheliang and Dongshanzui sites. The disappearance of the Hongshan culture, often described as a rapid and total collapse due to climatic change, has also generated considerable speculation14,15. Despite the cultural importance of NE China, there has been no systematic evaluation using accu- rately dated quantitative records of human activity which are correlated with settlement patterns and cultural change in the region. In the last decade, an integrated dataset of archeological radiocarbon dates has been developed as a proxy for prehistoric human population, activity, and cultural change. It is based on the proposition that a larger population would result in the increased production and deposition of cultural carbon, thus providing more material evidence for age determinations1,16–18. This approach has been used to reconstruct early human activity and population history in different regions, including Europe, North America and China1,16–18, and therefore we used this proven approach to analyze the data from NE China. M onsoonal climate change at the decadal-centennial scale played a crucial role in inﬂuencing past and modern ecosystem evolution and human activities in East Asia1,2. The Holocene is one of the key time intervals for understanding the relationship between changes in the East Asian Summer Monsoon (EASM) and human activity. Holocene cli- mate change can affect human society in two principal ways: (1) abrupt catastrophic climatic events, such as ﬂoods and droughts3,4, have major impacts, and it has been suggested that El Niño-Southern Oscillation (ENSO)5, and volcanic6 and solar activity7 may have played a key role in driving abrupt monsoon changes and human activity7; (2) cyclic climate changes, such as quasi-centennial oscillations, were likely associated with human developments such as nomadic migration and dynastic alterna- tions in China4,8. However, studies4,8 of the latter have focused on the historical period, and the few that have focused on the early-middle Holocene are controversial9, mainly due to the lack of annual- to decadal-resolution climate records and quantitative proxies of human activity and cultural evolution. Here we report the results of a study of annually laminated Maar Lake Xiao- longwan (Fig. Synchronous 500-year oscillations of monsoon climate and human activity in Northeast Asia Deke Xu1,2,9, Houyuan Lu 1,2,3,9, Guoqiang Chu1,4, Li Liu5, Caiming Shen6, Fengjiang Li 1,7, Can Wang8 & Naiqin Wu1,3,7 Deke Xu1,2,9, Houyuan Lu 1,2,3,9, Guoqiang Chu1,4, Li Liu5, Caiming Shen6, Fengjiang Li 1,7, Can Wang8 & Naiqin Wu1,3,7 Prehistoric human activities were likely inﬂuenced by cyclic monsoon climate changes in East Asia. Here we report a decadal-resolution Holocene pollen record from an annually-laminated Maar Lake in Northeast China, a proxy of monsoon climate, together with a compilation of 627 radiocarbon dates from archeological sites in Northeast China which is a proxy of human activity. The results reveal synchronous ~500-year quasi-periodic changes over the last 8000 years. The warm-humid/cold-dry phases of monsoon cycles correspond closely to the intensiﬁcation/weakening of human activity and the ﬂourishing/decline of prehistoric cul- tures. Six prosperous phases of prehistoric cultures, with one exception, correspond approximately to warm-humid phases caused by a strengthened monsoon. This ~500-year cyclicity in the monsoon and thus environmental change triggered the development of pre- historic cultures in Northeast China. The cyclicity is apparently linked to the El Niño-Southern Oscillation, against the background of long-term Holocene climatic evolution. These ﬁndings reveal a pronounced relationship between prehistoric human activity and cyclical climate change. 1 Key Laboratory of Cenozoic Geology and Environment, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, China. 2 CAS Center for Excellence in Tibetan Plateau Earth Sciences, Beijing 100101, China. 3 University of Chinese Academy of Sciences, Beijing 100049, China. 4 CAS Center for Excellence in Life and Paleoenvironment, Beijing 100044, China. 5 Department of East Asian Languages and Cultures, Stanford University, Stanford, CA 94305, USA. 6 Yunnan Key Laboratory of Plateau Geographical Processes and Environmental Changes, College of Tourism and Geographical Sciences, Yunnan Normal University, Kunming 650500, China. 7 Institutions of Earth Science, Chinese Academy of Sciences, Beijing 100029, China. 8 Department of Archaeology, School of History and Culture, Shandong University, Jinan 250100, China. 9These authors contributed equally: Deke Xu, Houyuan Lu. Correspondence and requests for materials should be addressed to D.X. (email: ccxudeke@mail.iggcas.ac.cn) or to H.L. (email: houyuanlu@mail.iggcas.ac.cn) 1 NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12138-0 M Holocene: Xinglongwa, Zhaobaogou, Hongshan, Xiaoheyan, Lower Xiajiadian, and Upper Xiajiadian. Archaeological investi- gations have demonstrated ﬂuctuations in population density and settlement distribution through time12,13. Synchronous 500-year oscillations of monsoon climate and human activity in Northeast Asia 1) (42°18.0′N, 126°21.5′E) in Northeast (NE) China, which has recently yielded a decadal-resolution pollen record spanning the past 5350 years (yr)10,11. This site provides an opportunity to establish a decadal-resolution climate record for the entire Holocene. NE China is one of the regions where millet-based agriculture and early complex societies developed. The region has experi- enced six major Neolithic and Bronze Age cultures during the Here, we present two sets of completely independent proxies: one is a decadal-resolution Holocene pollen record derived from 130°E 120°E 110°E 130°E 120°E 110°E Winter monsoon Summer Monsoon Inner Mongolia Plateau Greater Khingan Mts. Changbai Mts. 50°N 40°N –6000 Chinese Loess Plateau North China Plain Maar Lake Xiaolongwan 0 105 210 420 630 840 km 4000 0 Elevation Value (m.a.s.l) 170°0'0"E 170°0'0"E 120°0'0"E 120°0'0"E 70°0'0"E 14C dated archeological sites Study Area 70°0'0"E 60°0'0"N 10°0'0"N g. 1 Locations of Maar Lake Xiaolongwan (red star) and radiocarbon dates from Neolithic archeological sites in NE China (open circles). Trajectories of e East Asian Summer Monsoon and Winter Monsoon are shown by red and blue arrows, respectively. This ﬁgure was generated using DIVA-GIS 7.5 50°N 40°N 120°E 130°E 110°E Fig. 1 Locations of Maar Lake Xiaolongwan (red star) and radiocarbon dates from Neolithic archeological sites in NE China (open circles). Trajectories of the East Asian Summer Monsoon and Winter Monsoon are shown by red and blue arrows, respectively. This ﬁgure was generated using DIVA-GIS 7.5 (http://www.diva-gis.org/) Fig. 1 Locations of Maar Lake Xiaolongwan (red star) and radiocarbon dates from Neolithic archeological sites in NE China (open circles). Trajectories of the East Asian Summer Monsoon and Winter Monsoon are shown by red and blue arrows, respectively. This ﬁgure was generated using DIVA-GIS 7.5 (http://www.diva-gis.org/) NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12138-0 most likely annual10,11,19 (Supplementary Fig. 4). The pollen record from the upper 271 cm (~−50–5350 cal yr B.P., cal yr B.P.: before 1950 AD) of this core was published by Xu et al.11. In this study, we extend the study interval from ~−50 cal yr B.P. to ~9210 cal yr B.P.10. an annually laminated Maar Lake Xiaolongwan, which is a proxy of monsoon climate; and the other is a dataset of integrated archaeological radiocarbon dates from NE China1, which is a proxy of human activity and prehistoric cultures. Results d Site description, sediment sampling and chronologies. Maar Lake Xiaolongwan is located in the Changbai Mountains, Jilin Province, NE China (Fig. 1 and Supplementary Fig. 1). The lake has a surface area of 0.079 km2, a maximum depth of 15 m, and a catchment area of 0.16 km2; it is a closed basin with no inﬂows or outﬂows19. The lake is dystrophic and humic, with brown-colored water which contains a high concentration of organic matter and has a low pH20. The lake has seasonal dinoﬂagellate blooms and is annually laminated21 (Supplementary Note 1). Broad-leaved deciduous forest (<720 m a.s.l), mixed deciduous and coniferous forest (720–1100 m a.s.l.) and coniferous forest (1100–1800 m a.s. l.) are present, with increasing altitude, in the mountainous area around the lake11. The climate of the region is controlled mainly by the EAM system10,22. High rainfall is usually coupled with high temperatures during the summer season10,22. 14C dates for the above-mentioned six prehistoric cultures in NE China were assembled from the datasets (Methods). We calibrated these averaged dates (95.4% conﬁdence level) and generated summed probability values for each culture using the Sum function in the CALIB 7.04 program23 and the IntCal13 calibration curve24. The frequency distribution values for each culture were plotted using colorized ﬁlled contours. ~500-yr cycle of vegetation and monsoon oscillations. The pollen record (Supplementary Data 1) from Maar Lake Xiaolongwan is illustrated in Fig. 2. The percentages of the dominant pollen types, including the elements of temperate mixed deciduous broadleaved and coniferous forest (e.g. Pinus, Betula, Carpinus, Quercus and Ulmus) and herb taxa (e.g. Arte- misia), show signiﬁcant changes on centennial and millennial timescales during the last 9260 yr. According to the pollen assemblages and the results of cluster analysis (CONISS)26, we ~500-yr cycle of vegetation and monsoon oscillations. The pollen record (Supplementary Data 1) from Maar Lake Xiaolongwan is illustrated in Fig. 2. The percentages of the dominant pollen types, including the elements of temperate mixed deciduous broadleaved and coniferous forest (e.g. Pinus, Betula, Carpinus, Quercus and Ulmus) and herb taxa (e.g. Arte- misia), show signiﬁcant changes on centennial and millennial timescales during the last 9260 yr. According to the pollen assemblages and the results of cluster analysis (CONISS)26, we A 387-cm-long piston core was raised from a water depth of 14.5 m near the center of the lake in early spring 2006. Synchronous 500-year oscillations of monsoon climate and human activity in Northeast Asia The results provide robust evidence for synchronous ~500-yr cyclical changes in monsoon climate, human activity and prehistoric cultural development in the East Asian Monsoon (EAM) region during the Holocene. Six prosperous phases of Neolithic and Bronze Age cultures correspond approximately to warm-humid phases caused by a strengthened EASM, except for the ﬁrst expansion of the Hongshan culture, which corresponds to the phase of strongest EASM in the middle Holocene. We suggest that humans responded to climatic ﬂuctuations with different social strategies, leading to the rise and fall of early complex societies in the region. y We also compiled a dataset of 627 14C dates from 98 archeological sites in NE China, bounded by the Chinese Loess Plateau-North China Plain in the south and the Inner Mongolian Plateau-Greater Khingan Mountains in the west. 584 of the dates were extracted from the database for China (N = 4656)1 and the others were from recent publications (Methods) (Fig. 1). 550 averaged 14C dates were calibrated using standard methods and software, following rigorous screening and elimination of dates from unsuitable materials such as shells, soils, and materials from temples, pagodas and canoes1. The resulting summed probability values were then plotted along the abscissa in decadal intervals using the Sum function in the CALIB 7.0.4 program23 and the IntCal13 calibration curve24. The major peaks and troughs in these summed probability distributions are regarded as evidence of increased and decreased human activity, respectively, with the steepness of the gradient of an increase/decrease indicating the rapidity of development and the intensity of human activity25. 14 ARTICLE As Quercus decreases to a minimum, Pinus increases to a maximum and the PCA F1 sample scores increase. The interval between two Quercus minima (and two peaks in PCA F1 sample scores and in Pinus) is ~500-yr. The thermophilous and hygrophilous taxa, Quercus, Ulmus, Juglans and Fraxinus, have negative loadings on axis 1, whereas the cold-tolerant taxa, Pinus, Abies, Betula, and drought-tolerant taxa such as Artemisia and Chenopodiaceae, have positive loadings. Quercus is a major component of deciduous broad- leaved forest, while Pinus is the main component of temperate mixed deciduous broadleaved and coniferous forest27. The anti- phased ﬂuctuations between Pinus and Quercus thus represent changes in the relative proportions of coniferous and deciduous broadleaved trees in the forest communities28,29 and thus the dynamics of regional vegetation change in the Changbai Mountains30. Hence, PCA F1 reﬂects major changes in regional vegetation in the pollen source area of the lake. We performed Hilbert–Huang Transform (HHT) Ensemble empirical mode decomposition (EEMD)35,36 on the Quercus percentages and PCA F1 sample scores to remove the long-term –20 0 20 –20 0 20 –20 0 20 –20 0 20 –20 0 20 –20 0 20 –20 0 20 –20 0 20 20 40 20 40 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 –10 0 10 c1 c2 c3 c4 c5 c6 c7 c8 c9 c0 Age (cal yr B.P.) c2-5 EEMD decomposition of Quercus percentages (An=0.1; Nesb=10) a b c ~500 yr 0.000 0.002 0.004 0.006 0.008 0.010 0 32 64 32 16 8 4 2 1 1/2 1/4 1/8 1/16 1/32 1/64 64 128 256 512 1024 2048 0 2000 4000 Age (cal yr B.P.) 6000 8000 1 × 104 8 × 103 6 × 103 4 × 103 2 × 103 Period (yr) Power Frequency (yr–1) Quercus percentage Significant >99% Fig. 3 Results of time-series analysis of the Quercus percentages for NE China. a EEMD35,36: white noise (An) of 0.1 and the component number (Nesb) of 10 Log922 2 þ 1 are used for the ﬁrst EEMD component. Component c0 represents the original Quercus percentages. To remove high-frequency ﬂuctuations and orbital and millennial trends, a new detrended time series (c2-5) was generated by summing components c2-c5 from the ﬁrst decomposition (c0). b Results of univariate spectral analysis37 of the Quercus percentages time-series over the past 9260 yr. c Wavelet power spectrum38 of Quercus percentages. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12138-0 divided the sequence into two major stages: (1) from ~9210 to 5700 cal yr B.P., broadleaved trees (e.g. Juglans, Fraxinus, Ulmus, and Quercus) are dominant, and the percentages of Quercus exhibit regular oscillations; and (2) after ~5700 cal yr B.P., Pinus, Betula, and Artemisia exhibit an increasing trend, and Quercus, Carpinus and Ulmus a decreasing trend. Note that both Pinus and Quercus exhibit anti-phased oscillations with the large amplitude of ~10–20%. divided the sequence into two major stages: (1) from ~9210 to 5700 cal yr B.P., broadleaved trees (e.g. Juglans, Fraxinus, Ulmus, and Quercus) are dominant, and the percentages of Quercus exhibit regular oscillations; and (2) after ~5700 cal yr B.P., Pinus, Betula, and Artemisia exhibit an increasing trend, and Quercus, Carpinus and Ulmus a decreasing trend. Note that both Pinus and Quercus exhibit anti-phased oscillations with the large amplitude of ~10–20%. The Changbai Mountains region is a typical temperate monsoon climate zone27. Studies of the factors controlling the distribution of montane vegetation zones indicate that the vegetation dynamics in the study region are controlled by temperature and precipitation31–34. Furthermore, deciduous broadleaved trees favor a warm and moist environment, while temperate mixed deciduous broadleaved and coniferous forest can tolerate cooler and drier conditions than deciduous broadleaved forest in the montane region of NE China. Therefore, PCA F1 not only reﬂects regional vegetation changes, but it also indicates monsoon climate changes: i.e. high Pinus frequencies and positive PCA F1 sample scores represent a predominance of coniferous forest and cold-dry climatic conditions, whereas high Quercus percentages and negative PCA F1 sample scores imply the expansion of temperate deciduous forest and warm-humid conditions. Principal components analysis (PCA) was conducted on the terrestrial pollen percentages to extract the main gradients in the vegetation. All pollen taxa with a relative abundance >2% in at least two samples were used. The ﬁrst and second principal components (PCA F1 and PCA F2) have eigenvalues of 0.83 and 0.08, explaining 83% and 8% of the total variance, respectively (Supplementary Fig. 5). Although our pollen record reveals a gradual, long-term, insolation-forced pattern (Supplementary Fig. 6 and Supplemen- tary Note 2) of vegetation succession and climate change (Fig. 2), the most striking feature is that the frequencies of Pinus and Quercus, and PCA F1 sample scores, exhibit a series of multi- centennial oscillations superimposed on this general pattern (Fig. 2). Results d Initial studies using scanning electron and optical microscopy, sediment trap observations, and an independent chronology (Methods) [lamination counting (Supplementary Fig. 2), and 137Cs, 210Pb (Supplementary Fig. 3) and AMS 14C dating (Supplementary Table 1)] indicated that the laminated sediments of the core are Age (cal yr B.P.) Warm-humid Cold-dry 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 20 40 Pinus Abies Picea 20 Betula 20 Carpinus Corylus 20 % Juglans 20 Ulmus 20 Fraxinus Tilia 20 40 Quercus Chenopodiaceae 20 Artemisia 0 2 PCA F1 400 800 N Total Pollen Sum Zone I II Conifer Broad-leaved tree Herb 10 20 30 40 Total sum of squares CONISS Fig. 2 Pollen diagram of major pollen types and PCA F1 sample scores for Lake Xiaolongwan. The green, red, and blue silhouettes represent conifers, deciduous broad-leaved trees, and herbs, respectively. Positive PCA F1 sample scores represent cold-dry climatic conditions, whereas negative PCA F1 sample scores represent warm-humid conditions Fig. 2 Pollen diagram of major pollen types and PCA F1 sample scores for Lake Xiaolongwan. The green, red, and blue silhouettes represent conifers, deciduous broad-leaved trees, and herbs, respectively. Positive PCA F1 sample scores represent cold-dry climatic conditions, whereas negative PCA F1 sample scores represent warm-humid conditions NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunicatio ONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications 3 ARTICLE ARTICLE The spectra of the detrended (c2-5) Quercus percen- tages and PCA F1 sample scores reveal peaks at an ~500-yr period, signiﬁcant at the 99% conﬁdence level (Fig. 3b and Supplementary Fig. 7). The results of the wavelet analysis show that the ~500-yr quasi-periodic oscillations in Quercus percen- tages and PCA F1 sample scores are near-stable and dominant throughout the last 9260 yr, except for a period of weak representation from ~7500 to 6000 cal yr B.P. (Fig. 3c and Supplementary Fig. 7c). The band-pass ﬁltering results of Quercus percentages reveals a total of 13 ~500-yr cold-dry and warm- humid oscillations from ~9210 to 2500 cal yr B.P. (Supplementary Fig. 8). The warm-humid climatic optima occur at ~8970, ~8470, ~7970, ~7420, ~6830, ~6350, ~5940, ~5450, ~4980, ~4390, ~3900, ~3400, ~2810 cal yr B.P. (Supplementary Fig. 8). Pollen records from Maar Lake Sihailongwan34 (20 km from Maar Lake Xiaolongwan) and Jeju Island in the Yellow Sea5, and the speleothems record from Heshang cave39 in South China, also show ~500-yr summer monsoon oscillations during the mid-late Holocene (Supplementary Fig. 9 and Supplementary Note 3). Our record of long-chain n-alkanes δ13C27–31, an effective humidity proxy from the same core, shows a strong ~500-yr periodicity10 (Supplementary Fig. 10 and Supplementary Note 3). In addition, a 200-yr precipitation record from an adjacent weather station in Changchun City40 maps convincingly on the ﬁnal stage of the pollen-based ~500-yr monsoon cycles (Supplementary Fig. 11), Moreover, the temporal framework demonstrates that a northward advance of the EASM rain belt could have led to warm and humid conditions favorable for Quercus, whereas a southward retreat of the EASM rain belt could have caused a cold and arid environment unfavorable for Quercus in NE Asia5,41. Thus, these studies all support the conclusion that our pollen record clearly shows that temporal vegetation and environmental changes were driven by ~ 500-yr periodic EASM oscillations. However, the EASM was the strongest during the period of 7500–5500 cal yr B.P., and such climatic conditions may have suppressed the signal of ~ 500-yr monsoon cyclicity in the study area. ~ 500-yr cyclical change in human activity. The summed 14C probability (SCP) record exhibits lower values ( < 0.286) and weak variability ( < 0.184) before 8000 cal yr B.P. and after 2000 cal yr B.P. (Fig. 4a: c0, Supplementary Fig. 12 & Supplementary Note 4). ARTICLE This may be due to the relative paucity of archaeological sites and dates before 8000 cal yr B.P. and the increased availability of historical documentary evidence after 2500 cal yr B.P.. The SCP remains high (from 0.042 to 0.998), with notable ﬂuctuations (from 0.167 to 0.648), from 8000 to 2000 cal yr B.P. (Fig. 4a: c0); the peak values are at ~ 7570, ~ 6770, ~ 6240, ~ 5440, ~ 4930, ~ 4420, ~ 3820, ~ 3300 and ~ 2800 cal yr B.P. (Supplementary Fig. 13b). g The SCP curve for NE China was detrended using HHT EEMD35,36 (Fig. 4a). The c2-c5 summed components (Fig. 4a: –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 0.2 0.4 0.0 0.5 1.0 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 0.0 0.5 c1 c2 c3 c4 c5 c6 c7 c8 c9 c0 c2-5 Age (cal yr B.P. ) EEMD decomposition of summed 14C probability (An=0.1; Nesb=10) ~500 yr a b c 0.000 0.002 0.004 0.006 0.008 0.010 0 1 2 3 4 5 Power Summed 14C probability Frequency (yr–1) Significant >99% 32 64 128 256 512 1024 2048 Period (yr) 0 2000 4000 Age (cal yr B.P.) 6000 8000 64 32 16 8 4 2 1 1/2 1/4 1/8 1/16 1/32 1/64 Fig. 4 Results of time-series analysis of the SCP record for NE China. (a) EEMD35,36: white noise (An) of 0.1 and the component number (Nesb) of 10 Log922 2 þ 1 are used for the ﬁrst EEMD component. Component c0 represents the original SCP data. To remove high-frequency ﬂuctuations and orbital and millennial trends, a new detrended time series (c2-5) was formed by summing components c2-c5 from the ﬁrst decomposition (c0). (b) Results of univariate spectral analysis37 of the SCP time-series over the past 8000 yr. (c) Wavelet power spectrum38 of SCP. The 95% conﬁdence level is outlined in black. ARTICLE The 95% conﬁdence level is outlined in black. The blue dotted box indicates weak oscillations of ~500-yr cyclicity during the period of ~7500 to 5700 cal yr B.P 4 NATURE COMMUNICATIONS \| (2019)10:4105 \| https://doi org/10 1038/s41467 019 12138 0 \| www nature com/naturecommunications –20 0 20 –20 0 20 –20 0 20 –20 0 20 –20 0 20 –20 0 20 –20 0 20 –20 0 20 20 40 20 40 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 –10 0 10 c1 c2 c3 c4 c5 c6 c7 c8 c9 c0 Age (cal yr B.P.) c2-5 EEMD decomposition of Quercus percentages (An=0.1; Nesb=10) a b a ~500 yr 0.000 0.002 0.004 0.006 0.008 0.010 0 1 × 104 8 × 103 6 × 103 4 × 103 2 × 103 Power Frequency (yr–1) Quercus percentage Significant >99% c 32 64 32 16 8 4 2 1 1/2 1/4 1/8 1/16 1/32 1/64 64 128 256 512 1024 2048 0 2000 4000 Age (cal yr B.P.) 6000 8000 Period (yr) c Period (yr) Age (cal yr B.P.) Fig. 3 Results of time-series analysis of the Quercus percentages for NE China. a EEMD35,36: white noise (An) of 0.1 and the component number (Nesb) of 10 Log922 2 þ 1 are used for the ﬁrst EEMD component. Component c0 represents the original Quercus percentages. To remove high-frequency ﬂuctuations and orbital and millennial trends, a new detrended time series (c2-5) was generated by summing components c2-c5 from the ﬁrst decomposition (c0). b Results of univariate spectral analysis37 of the Quercus percentages time-series over the past 9260 yr. c Wavelet power spectrum38 of Quercus percentages. The 95% conﬁdence level is outlined in black. The blue dotted box indicates weak oscillations of ~500-yr cyclicity during the period of ~7500 to 5700 cal yr B.P NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12138-0 supporting the conclusion that our pollen record is a monsoonal climate proxy. trend and decadal-scale noise (Fig. 3a and Supplementary Fig. 7a). The c2-c5 summed components (Fig. 3a: c2-5 and Supplementary Fig. 7a: c2-5) of Quercus percentages and PCA F1 sample scores show clear oscillations. Redﬁt spectral37, wavelet38 and ﬁlter analysis were applied to determine the periodicities and periodic stabilities. ARTICLE The red dotted box indicates the strong oscillations of ~ 500-yr cycles during the period of ~ 8000 to 2000 cal yr B.P NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications 5 b b –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 –0.5 0.0 0.5 0.2 0.4 0.0 0.5 1.0 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 0.0 0.5 c1 c2 c3 c4 c5 c6 c7 c8 c9 c0 c2-5 Age (cal yr B.P. ) EEMD decomposition of summed 14C probability (An=0.1; Nesb=10) a b c Period (yr) a EEMD decomposition of summed 14C probability (An=0.1; Nesb=10) EEMD decomposition of summed 14C probability (An=0.1; Nesb=10) ~500 yr 0.000 0.002 0.004 0.006 0.008 0.010 0 1 2 3 4 5 Power Summed 14C probability Frequency (yr–1) Significant >99% c 32 64 128 256 512 1024 2048 Period (yr) 0 2000 4000 Age (cal yr B.P.) 6000 8000 64 32 16 8 4 2 1 1/2 1/4 1/8 1/16 1/32 1/64 Period (yr) Age (cal yr B.P. ) Fig. 4 Results of time-series analysis of the SCP record for NE China. (a) EEMD35,36: white noise (An) of 0.1 and the component number (Nesb) of 10 Log922 2 þ 1 are used for the ﬁrst EEMD component. Component c0 represents the original SCP data. To remove high-frequency ﬂuctuations and orbital and millennial trends, a new detrended time series (c2-5) was formed by summing components c2-c5 from the ﬁrst decomposition (c0). (b) Results of univariate spectral analysis37 of the SCP time-series over the past 8000 yr. (c) Wavelet power spectrum38 of SCP. The 95% conﬁdence level is outlined in black. The red dotted box indicates the strong oscillations of ~ 500-yr cycles during the period of ~ 8000 to 2000 cal yr B.P 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12138-0 c2-5) of EEMD and wavelet analysis results indicate a strong ~500-yr oscillation from 8000 to 2500 cal yr B.P. (Fig. 4a, c). Redﬁt spectral analysis also shows a dominant ~500-yr cyclicity during this interval (Fig. 4b). Thus, the consistency of the characteristics of the time-series analysis results of the SCP indicates a dominant ~500-yr cycle of the intensity of human activity in NE China (Fig. 4). lags by several decades (cycle 3) the ~500-yr cycle of warm- humid/cold-dry climatic oscillations (Fig. 5a, Supplementary Fig. 13). ARTICLE In addition, the timing of the ~500-yr cycles of the intensity of human activity (SCP) and the ﬂourishing/decline of prehistoric cultures (Fig. 5b, c) show similar temporal patterns. Thus, the cyclicity of human activity in NE China during the Holocene was almost synchronous and highly coherent with the EASM ﬂuctuations. Based on the 14C probability record from the archeological sites in NE China, the intervals during when prehistoric cultures ﬂourished are: ~7700–7200 cal yr B.P. (Xinglongwa); ~7000–6700 cal yr B.P. (Zhaobaogou); ~6500–5900, ~5700–5300 and ~5100–4800 cal yr B.P. (Hongshan); ~4600–4300 and ~4100–3700 cal yr B.P. (Xiaoheyan) and ~3600–3200 cal yr B.P. (Lower Xiajiadian) and ~3000–2600 cal yr B.P. (Upper Xiajia- dian) (Fig. 5b). In general, the phases of prosperous prehistoric Synchronous ~500-yr cyclic changes in EASM and human activity. Figure 5 displays the ~500-yr band pass ﬁlter results of the detrended Quercus percentages (summed components c2-c5), and the detrended SCP (summed components c2-c5), together with the frequency distribution of prehistorical cultural 14C probability in NE China. The ~500-yr cycle of changing human activity (Fig. 5c) is almost in-phase with (cycles 1-2 and 4-9) or probability in NE China. The ~500-yr cycle of changing human activity (Fig. 5c) is almost in-phase with (cycles 1-2 and 4-9) or (Lower Xiajiadian) and ~3000–2600 cal yr B.P. (Upper Xiajia- dian) (Fig. 5b). In general, the phases of prosperous prehistoric a b c 9 6 5 8 7 4 3 2 1 4 3 ? 9 8 7 6 5 2 1 3000 4000 5000 6000 7000 8000 –4 0 4 3000 4000 5000 6000 7000 8000 –0.1 0.0 0.1 Quercus Upper Xiajiadian N = 18 Lower Xiajiadian N = 60 Cultural 14C probability Xiaoheyan N = 6 Hongshan N = 19 Zhaobaogou N = 7 Xinglongwa N = 21 Summed 14C probability Age (cal yr B.P. ) Fig. 5 Comparison of the detrended Quercus record (EASM proxy) with detrended 14C probability. a 400–600-yr band-pass ﬁlter of the detrended Quercus record. The central frequencies and bandwidths of the Quercus percentages (EASM, c2-c5 component after HHT analysis) ﬁlters are 0.020 yr−1 (500-yr period), 0.017 yr−1 (600-yr period), and 0.025 yr−1 (400-yr period), respectively. b Density histogram of cultural 14C probability from prehistorical archeological sites; the 14C-dated sites belong to the Xinglongwa, Zhaobaogou, Hongshan, Xiaoheyan, Lower Xiajiadian and Upper Xiajiadian cultures. c 400–1250-yr band-pass ﬁlter of the detrended SCP record. NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications ARTICLE On the other hand, the collapse of a prehistoric culture may not represent a complete collapse, since the succeeding culture can inherit the older one to a certain extent. Archeological evidence shows that the Lower Xiajiadian culture adopted several aspects of the Hongshan culture43. For example, many attributes of the ritual sites and jade and ceramic manufacturing technology of the Lower Xiajiadian culture are derived from the Hongshan culture (Supplementary Fig. 16)43. The economic foundations of these cultures were associated with the gradual transition from a broad-spectrum subsistence strategy based on millet farming, pig husbandry, and hunting- gathering wild foods, to an intensiﬁed millet agriculture with pig domestication, and then to agro-pastoralist subsistence strategies, which were all highly dependent on a warm environment and adequate precipitation13,15,42. Warm and humid conditions favored the increased productivity of wild plants, cultivated crops, wild animals and livestock1,16. An abundant food supply would have promoted the growth of population, an expansion of the area of occupation, and overall prosperity1,16. The sites of the six cultures are distributed in the present EASM northern marginal region43,44. Among the six prehistoric cultures, the early Xinglongwa and Zhaobaogou, which were characterized by a subsistence economy dominated by hunting- gathering with a minor farming component, are closely correlated with 500-yr climatic cyclicity in the early Holocene when the EASM began to strengthen. ~500-yr ENSO cycle linkages between EASM and human activity. NE China is a marginal area affected by the EAM11,22, and its ecological environment is very sensitive to monsoon advance and retreat11. Among the various factors affecting the change of the EAM, ENSO has the greatest impact on NE China on the inter-annual and inter-decade scale48. Moreover, ENSO variance exhibits ~500-yr cyclic change during the entire Holo- cene (Supplementary Fig. 17) and regulates EAM changes on the centennial scale5,39,49,50. ENSO can drive the movement of the rainbelt caused by intensifying the interaction between the wes- tern Paciﬁc anticyclone and dipolar sea surface temperature anomalies in the Indo-Paciﬁc warm pool and the Northwest Paciﬁc48. It could also weaken the western Paciﬁc subtropical high and limit the extension of the rainbelt to NE China51. ARTICLE During the El Niño-like phases there was a weakening of the EASM and southward migration of the rain belt, resulting in reduced precipitation in NE China; and in La Niña-like (or normal) phases, a strengthening of the EASM triggered the northward movement of the rain belt, resulting in more rainfall in NE China5,11,39,52. g g At ~6500–5300 cal yr B.P. (cycles 3 and 4), the warmest and wettest period of the Holocene, the EASM marginal region migrated at least 150–200 km northwards22,41. This may have suppressed the signal of the 500-yr monsoon cycle in this area. The prosperity of the Hongshan culture during the middle Holocene generally corresponds to the occurrence of warm and humid conditions (Supplementary Fig. 14). However, during ~6500–4800 cal yr B.P., two strong peaks and a weak peak in human activity are evident (Fig. 5b, c: SCP 3, 4 and 5), described here as early, middle, and late Hongshan phases, respectively. The 14C dates from the large ritual centers at Niuheliang and Dongshanzui fall within the middle phase (~5800–5300 cal yr B. P., Supplementary Fig. 15), corresponding to the warmest and wettest period indicated by the trend of the Quercus record (Supplementary Fig. 14). However, the 14C dates of the late phase (~5100–4800 cal yr B.P.) are all derived from small sites which exhibit no clear evidence of large-scale ritual architecture15. g This pattern suggests that the rise and fall of the Hongshan complex society may have been related to a series of societal responses to environmental ﬂuctuations, including the intensi- ﬁcation of land use for agriculture, the construction of monumental architecture for religious ceremonies, and the development of elite ritual powers. Hongshan ritual activity (Supplementary Fig. 16) reached a peak during the middle phase (~5800–5300 cal yr B.P.), represented by Niuheliang and Dongshanzui (Supplementary Fig. 15), and ended at about 5300 cal yr B.P. However, the termination of elite activities at large ritual centers does not imply a total collapse of the Hongshan culture as previously thought; instead, it marked the disappearance of elite power with associated material symbols. The band pass ﬁlter and cross wavelet transform (XWT) results show that the ~500-yr cycle of ENSO and EASM exhibits large amplitudes and nearly in-phase oscillations from ~8000 to 6500 cal yr B.P. and from ~5500 to 2500 cal yr B.P. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12138-0 cultures coincide with the warm-humid phases of the ~500-yr monsoon climate cycles (Fig. 5a, b). The middle phase was followed by a period of several hundred years of continuous human occupation in small residential settlements during the late phase. However, the amplitudes of the ~500-yr periodicities in the Quercus record do not correspond exactly to either the periodic changes of SCP or the frequency distribution of cultural 14C probability (Fig. 5a, c and Supplementary Fig. 13). The most obvious difference is that the amplitudes of cycles 5 and 7 are relatively large in the Quercus record and small in the SCP record. In addition, the amplitudes of cycles 3 and 4 are small in the Quercus record and large in the SCP record. Moreover, from ~6500 to 5300 cal yr B.P., between cycles 3 and 4, three relatively weak warm-humid peaks are indicated by the Quercus record (Fig. 5a), but the record of human activity shows only two peaks (Fig. 5b, c). Interestingly, this period witnessed the most signiﬁcant social development in the region, known as the Hongshan culture; thus, the relationship between climatic conditions and human activities needs to be investigated in greater detail. The late phase ended at around 4800 cal yr B.P., which coincides with an episode of climatic deterioration caused by a superposition of ~500-yr cycles and the long-term climatic trend (Fig. 5 and Supplementary Fig. 14). If the rise and fall of the Hongshan elite was related, in part, to climatic ﬂuctuations such as recurrent droughts, the continuous presence of the Hongshan population during the late phase indicates that the human response to a worsened climate included the rejection of the elite (who were seemingly unable to deliver favorable weather as reliably as before), the modiﬁcation of social organization, and changing cultural values15. A similar scenario occurred in the case of the Classic Maya collapses in Mesoamerica45,46. y p However, as most archaeologists believe, prehistoric cultures possessed social resilience to climate change47. The Hongshan people’s strategy for coping with the deteriorating climate and environment included changes in settlement location and religion practices15. The late phase of the Niuheliang, Dongshanzui, Caomaoshan and Wudaowan (Supplementary Fig. 15) was characterized by small, sparse and scattered sites with few examples of ritual and public architecture15. ARTICLE The central frequencies and bandwidths of the SCP (c2-c5 component after HHT analysis) ﬁlters are 0.020 yr−1 (500-yr period), 0.008 yr−1 (1250-yr period), and 0.025 yr−1 (400-yr period), respectively. The two proxies (EASM and SCP) show nine ~500-yr cycles. The ~500-yr cycle of SCP is almost in-phase with ~500-yr oscillations of EASM. The density histogram of SCP likely reﬂects the ﬂourishing and decline of the Xinglongwa, Zhaobaogou, Hongshan, Xiaoheyan and Lower Xiajiadian and Upper Xiajiadian cultures. The light-red vertical bars show nine periods of intense human activity and ﬂourishing of prehistoric culture 6 NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications a b c 9 6 5 8 7 4 3 2 1 4 3 ? 9 8 7 6 5 2 1 3000 4000 5000 6000 7000 8000 –4 0 4 3000 4000 5000 6000 7000 8000 –0.1 0.0 0.1 Quercus Upper Xiajiadian N = 18 Lower Xiajiadian N = 60 Cultural 14C probability Xiaoheyan N = 6 Hongshan N = 19 Zhaobaogou N = 7 Xinglongwa N = 21 Summed 14C probability Age (cal yr B.P. ) Quercus Fig. 5 Comparison of the detrended Quercus record (EASM proxy) with detrended 14C probability. a 400–600-yr band-pass ﬁlter of the detrended Quercus record. The central frequencies and bandwidths of the Quercus percentages (EASM, c2-c5 component after HHT analysis) ﬁlters are 0.020 yr−1 (500-yr period), 0.017 yr−1 (600-yr period), and 0.025 yr−1 (400-yr period), respectively. b Density histogram of cultural 14C probability from prehistorical archeological sites; the 14C-dated sites belong to the Xinglongwa, Zhaobaogou, Hongshan, Xiaoheyan, Lower Xiajiadian and Upper Xiajiadian cultures. c 400–1250-yr band-pass ﬁlter of the detrended SCP record. The central frequencies and bandwidths of the SCP (c2-c5 component after HHT analysis) ﬁlters are 0.020 yr−1 (500-yr period), 0.008 yr−1 (1250-yr period), and 0.025 yr−1 (400-yr period), respectively. The two proxies (EASM and SCP) show nine ~500-yr cycles. The ~500-yr cycle of SCP is almost in-phase with ~500-yr oscillations of EASM. The density histogram of SCP likely reﬂects the ﬂourishing and decline of the Xinglongwa, Zhaobaogou, Hongshan, Xiaoheyan and Lower Xiajiadian and Upper Xiajiadian cultures. The light-red vertical bars show nine periods of intense human activity and ﬂourishing of prehistoric culture 6 Discussion I In summary, our results reveal an ~500-yr cyclicity in variations of EASM monsoon climate and human activity in NE China during the last 9260 yr, with a near synchronous relationship between them. Monsoon-induced ~500-yr cyclic changes in ecosystems in NE China evidently had major impacts on human activity and cultural change. A strengthened EASM would have promoted increased plant and animal productivity and hence material and cultural prosperity. We have reliably identiﬁed a closely coupled relationship between changes in human culture and in climate and environment, using a record of decadal resolution. We have also demonstrated that the coupled changes were periodic and on a centennial scale, in contrast to prior studies which focused on millennial-scale events. Overall, we are conﬁdent that our results constitute robust evidence that the ~500-yr cyclic variations in human activity and climate change were inﬂuenced by ENSO and AO/NAO variability. Radiocarbon dates from archeological sites in NE China. We screened uncali- brated 14C dates using the criteria described by Maher et al.57 and Wang et al.1. The following dates were eliminated: (1) dates with high error bars (1σ standard deviation >400 14C yr); (2) dates from shells, soils, unknown materials or other materials considered inappropriate for dating; and (3) dates derived from sites or materials that had weak associations with human occupation or settlement, such as ancient temples, pagodas or canoes. In cases where dates were derived from several sample materials (e.g. charcoal and shell, or charcoal and charred millet seed), both obtained in the same context, the most reliable dating material was chosen. The remainder of the database contained 550 dates, have errors of 84.1 yrs (see Sup- plementary Data 2). We used the R_Combine command within OxCal v4.2.3 to combine the redundant dates. This approach has two advantages: (1) reducing the standard deviation and increasing the accuracy of each site’s temporal assignments; and (2) reducing sampling bias created by sites/phases with numerous radiocarbon dates during statistical analyses58,59. We calibrated averaged dates (95.4% con- ﬁdence) and generated summed probability values for the regions of NE China using the Sum function in the CALIB 7.04 program23 and the IntCal13 calibration curve24: (1) the individual calibrated probability distribution is generated using the calibration command; and (2) summed probability values are calculated by the sum probabilities command. ARTICLE They also show that from ~6500 to 5500 cal yr B.P., the ~500-yr cycle of ENSO and EASM exhibits weak amplitudes and a large offset, with ENSO leading EASM39,49 (Supplementary Fig. 18a & b). The ~500-yr cycles of the EASM and SCP are nearly in phase from ~8000 to 6500 cal yr B.P. and from ~5500 to 2500 cal yr B.P., but they are out of phase from ~6500 to 5500 cal yr B.P. (Supplementary Fig. 18a, c). The out of phase relationship between SCP and EASM from ~6500 to 5500 cal yr B.P. may have been related to 7 TURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12138-0 social resilience15. This phenomenon implies a close relationship between ENSO variability and the ~500-yr cycle of EASM and SCP variations5,11,34. In addition, we have previously demon- strated that the ~500-yr climate cyclicity in the EAM region was affected by the Arctic Oscillation//North Atlantic Oscillation (AO/NAO) in the high-latitude climate system11. Thus, the ~500-yr cyclicity in cultural prosperity and development and EASM changes in NE China may be related to interactions of the high and low-latitude components of the climate system51. The ~500-yr cycles are both signiﬁcant components of ENSO in the low-latitude region5,39 and of AO/NAO53,54 in the high- latitude region, and their changes may be related to ﬂuctuations in solar activity. social resilience15. This phenomenon implies a close relationship between ENSO variability and the ~500-yr cycle of EASM and SCP variations5,11,34. In addition, we have previously demon- strated that the ~500-yr climate cyclicity in the EAM region was affected by the Arctic Oscillation//North Atlantic Oscillation (AO/NAO) in the high-latitude climate system11. Thus, the ~500-yr cyclicity in cultural prosperity and development and EASM changes in NE China may be related to interactions of the high and low-latitude components of the climate system51. The ~500-yr cycles are both signiﬁcant components of ENSO in the low-latitude region5,39 and of AO/NAO53,54 in the high- latitude region, and their changes may be related to ﬂuctuations in solar activity. its emissions at 662 keV. The unsupported 210Pb activity (210Pbunsup), obtained by subtracting 226Ra, was used to estimate average linear sedimentation rates for the core. Radiometric dates were calculated from the 210Pb and 137Cs records using the CRS model55. Radiocarbon dating. The radiocarbon chronology for the upper 387 cm of the core is reported elsewhere10. Received: 28 June 2018 Accepted: 23 August 2019 Received: 28 June 2018 Accepted: 23 August 2019 Received: 28 June 2018 Accepted: 23 August 2019 Discussion I We also applied the empirical model proposed by Surovell et al.60 to correct for taphonomic bias, since it is assumed that older dates may be underestimates due to natural destructive processes60. After correction, the data were standardized (normalized) as follows: Xi/Xmax, where Xi is each single value and Xmax is the maximum value in the series. y y Nevertheless, the relationship was not always straightforward, since humans responded to the superimposed climatic changes in different ways. In the case of the early-mid Hongshan culture, relatively weak ~500-yr cyclic monsoon oscillations, corre- sponding to the strongest period of the mid Holocene monsoon, were associated with very high levels of human activity. While the emergence of unparalleled ritual power acquired by the Hon- gshan elite may have been a human spiritual response to accommodate sub-optimal environmental conditions, the sub- sequent decline of elite culture likely reﬂected a different social strategy used by ordinary people to cope with worsened climatic ﬂuctuations. Overall, our ﬁndings reveal a close correspondence between ecosystem change and the archeological record in NE China, and they provide insights into the long-term inter- relationship between cyclical centennial-scale climate change and prehistoric human activities in the EAM region. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. ARTICLE The reservoir correction factor is 212 yr for the uppermost 20 cm of the core. Corrected 14C ages with 2σ-range were obtained using the IntCal13 data set24 from the CALIB 7.04 program23. Pollen preparation procedures. Samples of weight 0.3 ± 0.05 g at 1-cm intervals (average resolution of 25-yr) were prepared for pollen analysis. This interval was selected for thin section preparation for varve counting to ensure the best temporal correlation between the pollen and varve data. All samples were treated with KOH, HCl, HF and a hot acetolysis mixture56. Lycopodium spores (27637 per tablet) were added to each sample to calculate pollen concentrations. Sample residues were suspended in glycerine and analyzed using a Leica DM750 microscope at ×400 magniﬁcation. Pollen preservation was excellent and the pollen concentration was high. An average of 701 (range 585–949) terrestrial pollen grains was counted for each sample (Fig. 2 and Supplementary Fig. 19). A total of 378 pollen samples were analyzed, with an average temporal resolution of ~25 yr. TILIA 2.0.41 (ref. 26) was used to calculate pollen percentages and to plot the pollen diagram. Pollen per- centages were calculated on the sum of total terrestrial pollen, excluding the pollen of aquatic plants and fern spores. It should be noted that the ~500-yr cycles are superimposed on a long-term trend. The general climatic trend of the Holocene contributed to the pattern of stability and prosperity of prehistoric cultures, while the ~500-yr periodicities affected the intensity of prehistoric human activity on the centennial scale. Data availability All data generated or analyzed during this study are included in this published article (and its supplementary information ﬁles). References 1 W C Varve counting. The sediment cores were cut into slabs of 6.5-cm length, with a 1.5-cm overlap, and were then shock-frozen, vacuum-dried and impregnated with epoxy resin prior to prepare thin sections. Varves were identiﬁed and counted from thin sections at different magniﬁcations (×4, ×6.5, ×20) using a Leitz optical microscope. 1. Wang, C., Lu, H., Zhang, J., Gu, Z. & He, K. Prehistoric demographic ﬂuctuations in China inferred from radiocarbon data and their linkage with climate change over the past 50,000 years. Quat. Sci. Rev. 98, 45–59 (2014). 1. Wang, C., Lu, H., Zhang, J., Gu, Z. & He, K. Prehistoric demographic ﬂuctuations in China inferred from radiocarbon data and their linkage with climate change over the past 50,000 years. Quat. Sci. Rev. 98, 45–59 (2014). ( ) 2. Steinhilber, F. et al. 9,400 Years of cosmic radiation and solar activity from ice cores and tree rings. Proc. Natl Acad. Sci. USA 109, 5967–5971 (2012). 137Cs and 210Pb dating. The upper 19 cm of the core were sampled at a 1-cm interval for 137Cs and 210Pb dating (Supplementary Fig. 3). Vacuum-dried samples were measured for radionuclides in a well-type germanium detector (EGPC 100P- 15R). Each sample was packed in a 15-mm polyethylene tube and stored for 3 weeks in sealed containers to allow for radioactive equilibration, and then counted for 48 h. 210Pb was determined via its energy emission at 46.5 keV. 226Ra was determined by gamma spectrometry, based on measurements of 214Pb (241.9, 295.2 and 351.9 keV) and 214Bi (609.3 keV) photo peaks. 137Cs was measured via 137Cs and 210Pb dating. The upper 19 cm of the core were sampled at a 1-cm interval for 137Cs and 210Pb dating (Supplementary Fig. 3). Vacuum-dried samples were measured for radionuclides in a well-type germanium detector (EGPC 100P- 15R). Each sample was packed in a 15-mm polyethylene tube and stored for 3 weeks in sealed containers to allow for radioactive equilibration, and then counted for 48 h. 210Pb was determined via its energy emission at 46.5 keV. 226Ra was determined by gamma spectrometry, based on measurements of 214Pb (241.9, 295.2 and 351.9 keV) and 214Bi (609.3 keV) photo peaks. 137Cs was measured via g 3. Wu, W. & Liu, T. Possible role of the “Holocene Event 3” on the collapse of neolithic cultures around the Central Plain of China. Quatern Int. 117, 153–166 (2004). 4. Zhang, D. D. et al. MMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12138-0 37. Schulz, M. & Mudelsee, M. REDFIT: estimating red-noise spectra directly from unevenly spaced paleoclimatic time series. Comput. Geosci. 28, 421–426 (2002). 6. Mann, M. E., Cane, M. A., Zebiak, S. E. & Clement, A. Volcanic and solar forcing of the tropical Paciﬁc over the past 1000 years. J. Clim. 18, 447–456 (2005). 7. Hsu, K. J. Sun, climate, hunger, and mass migration. Sci. China Ser. D. 41, 449–472 (1998). 38. Torrence, C. & Compo, G. P. A practical guide to wavelet analysis. B Am. Meteor. Soc. 79, 61–78 (1998). 8. Lu, H. The cultural and climatic changes of grassland in north temperate zone since Neolithic age. Sci. Conserv. Archaeol. 3, 41–50 (1991). 39. Zhu, Z. et al. Holocene ENSO-related cyclic storms recorded by magnetic minerals in speleothems of central China. Proc. Natl Acad. Sci. USA 114, 852–857 (2017). 9. Catto, N. & Catto, G. Climate change, communities, and civilizations: driving force, supporting player, or background noise? Quatern Int. 123, 7–10 (2004). 40. Li, W. & Guo, Y. Analysis on precipitation change in Changchun city. Jilin Water Resour. 308, 33–34 (2008). pp g p y g 10. Chu, G. et al. Holocene cyclic climatic variations and the role of the Paciﬁc Ocean as recorded in varved sediments from northeastern China. Quat. Sci. Rev. 102, 85–95 (2014). y Ocean as recorded in varved sediments from northeastern China. Quat. Sci. Rev. 102, 85–95 (2014). 41. Yang, S. et al. Warming-induced northwestward migration of the East Asian monsoon rain belt from the Last Glacial Maximum to the mid-Holocene. Proc. Natl Acad. Sci. USA 112, 13178–13183 (2015). 11. Xu, D. et al. 500-year climate cycles stacking of recent centennial warming documented in an East Asian pollen record. Sci. Rep. 4, 3611 (2014). 42. Liu, X., Jones, M. K., Zhao, Z., Liu, G. & O’Connell, T. C. The earliest evidence of millet as a staple crop: new light on neolithic foodways in North China. Am. J. Phys. Anthr. 149, 283–290 (2012). 12. Chifeng Archaeology Team. Banzhijianhe Zhongyou Xian Qin Shiqi Yizhi (Pre-Qin Settlement Distribution in the Middle Banzhijian River Valley) (Science Press, 2002). y 43. The Institute of Archaeology CASS. Chinese Archaeology: Neolithic (China Social Sciences Press, 2010). 13. Project CICAR. Settlement Patterns in the Chifeng Region (Center for Comparative Archaeology, University of Pittsburgh, 2011). 44. Jia, X. et al. ARTICLE A contrast of the East Asian Summer Monsoon–ENSO relationship between 1962–77 and 1978–93*. J. Clim. 15, 3266–3279 (2002). 21. Chu, G. Q. et al. A 1600 year multiproxy record of paleoclimatic change from varved sediments in Lake Xiaolongwan, northeastern China. J. Geophys Res. Atmos. 114, D22108 (2009). 53. Stuiver, M. & Braziunas, T. F. Sun, ocean, climate and atmospheric 14CO2: an evaluation of causal and spectral relationships. Holocene 3, 289–305 (1993). 22. Zhao, Y., Yu, Z. C., Chen, F. H., Zhang, J. W. & Yang, B. Vegetation response to Holocene climate change in monsoon-inﬂuenced region of China. Earth Sci. Rev. 97, 242–256 (2009). 54. Hu, F. S. et al. Cyclic variation and solar forcing of Holocene climate in the Alaskan subarctic. Science 301, 1890–1893 (2003). 23. Stuiver, M. & Reimer, P. J. Extended 14C data base and revised CALIB 3.0 14C Age Calibration Program. Radiocarbon 35, 215–230 (1993). 55. Appleby, P. G. et al. 210Pb dating by low background gamma counting. Hydrobiologia 143, 21–27 (1986). 24. Reimer, P. J. et al. Intcal13 and Marine13 radiocarbon age calibration curves 0-50,000 years Cal Bp. Radiocarbon 55, 1869–1887 (2013). 56. Faegri, K., Kaland, P. E. & Krzywinski, K. Textbook of pollen analysis (John Wiley & Sons Ltd., 1989). 57. Maher, L. A., Banning, E. B. & Chazan, M. Oasis or Mirage? Assessing the role of abrupt climate change in the prehistory of the Southern Levant. Camb. Archaeol. J. 21, 1–30 (2011). 25. Bamforth, D. B. & Grund, B. Radiocarbon calibration curves, summed probability distributions, and early Paleoindian population trends in North America. J. Archaeol. Sci. 39, 1768–1774 (2012). 26. Grimm, E. C. Coniss - a Fortran-77 program for stratigraphically constrained cluster-analysis by the method of incremental sum of squares. Comput. Geosci. 13, 13–35 (1987). 58. Ward, G. K., Wilson, S. R., Ward, G. K. & Wilson, S. R. Procedures for comparing and combining radiocarbon age determinations: a critique. Archaeometry 20, 19–31 (1978). 27. Zhao, D. Vertical Vegetation Zone in Changbai Mountain (China Forestry Publishing House, 1980). 59. Selden, R. Z. Modeling regional radiocarbon trends: a case study from the East Texas Woodland Period. Radiocarbon 54, 239–265 (2012). 60. Surovell, T. A., Finley, J. B., Smith, G. M., Brantingham, P. J. & Kelly, R. 28. Sun, X. J., Luo, Y. L., Tian, J. & Wu, Y. S. ARTICLE Spatial and temporal variations in prehistoric human settlement and their inﬂuencing factors on the south bank of the Xar Moron River, Northeastern China. Front Earth Sci. 11, 137–147 (2017). 14. Li, X. Development of Social Complexity in the Liaoxi Area, Northeast China (Archaeopress, 2008). 45. Diamond, J. Collapse: How Societies Choose to Fail or Succeed (Penguin Books, London, 2005). 15. Liu, L. & Chen, X. The Archaeology of China: from the Late Paleolithic to the Early Bronze Age (Cambridge University Press, 2012). 16. Munoz, S. E., Gajewski, K. & Peros, M. C. Synchronous environmental and cultural change in the prehistory of the northeastern United States. Proc. Natl Acad. Sci. USA 107, 22008–22013 (2010). 46. Dunning, N. P., Beach, T. P. & Luzzadder-Beach, S. Kax and kol: collapse and resilience in lowland Maya civilization. Proc. Natl Acad. Sci. USA 109, 3652–3657 (2012). 17. Tallavaara, M., Luoto, M., Korhonen, N., Jarvinen, H. & Seppa, H. Human population dynamics in Europe over the Last Glacial Maximum. Proc. Natl Acad. Sci. USA 112, 8232–8237 (2015). 47. Redman, C. L. Resilience theory in archaeology. Am. Anthropol. 107, 70–77 (2005). 48. Wang, B., Li, J. & He, Q. Variable and robust East Asian monsoon rainfall response to El Niño over the past 60 years (1957–2016). Adv. Atmos. Sci. 34, 1235–1248 (2017). 18. Bevan, A. et al. Holocene ﬂuctuations in human population demonstrate repeated links to food production and climate. Proc. Natl Acad. Sci. USA 114, E10524–E10531 (2017). 49. Liu, Z. et al. Evolution and forcing mechanisms of El Niño over the past 21,000 years. Nature 515, 550 (2014). 19. Chu, G. et al. Dinocyst microlaminations and freshwater “red tides” recorded in Lake Xiaolongwan, northeastern China. J. Paleolimnol. 39, 319–333 (2008). y 50. Wu, J. et al. Shrinkage of East Asia Winter Monsoon associated with increased ENSO events since the mid‐Holocene. J. Geophys. Res. 124, 3839–3848 (2019). 20. Sun, Q. et al. Alkanes, compound-speciﬁc carbon isotope measures and climate variation during the last millennium from varved sediments of Lake Xiaolongwan, northeast China. J. Paleolimnol. 50, 331–344 (2013). 51. Sun, Q. et al. An oxygen isotope record from Lake Xiarinur in Inner Mongolia since the last deglaciation and its implication for tropical monsoon change. Glob. Planet Change 163, 109–117 (2018). g Xiaolongwan, northeast China. J. Paleolimnol. 50, 331–344 (20 g 52. Wu, R. & Wang, B. Acknowledgements This study was supported by SPRPCAS (XDA2007010103, XDB26000000), NSFC (41771237), NKPC (2017YFA0603403), NSFC (41430103, 41830322,41471166), NBRPC (2015CB953801) and YPIAT (2013HA024). We thank Dr. Xin Jia, Dr. Luo Wang, Dr. Keyang He, Dr. Jing Wu, Dr. Yonggang Sun, Dr. Zongmin Zhu and Dr. Jan Bloemendal for valuable discussions and help. Dr. Jan Bloemendal edited the English of this manuscript. This study was supported by SPRPCAS (XDA2007010103, XDB26000000), NSFC (41771237), NKPC (2017YFA0603403), NSFC (41430103, 41830322,41471166), NBRPC (2015CB953801) and YPIAT (2013HA024). We thank Dr. Xin Jia, Dr. Luo Wang, Dr. Keyang He, Dr. Jing Wu, Dr. Yonggang Sun, Dr. Zongmin Zhu and Dr. Jan Bloemendal for valuable discussions and help. Dr. Jan Bloemendal edited the English of this manuscript. 31. Zhang, B., Xu, J., Wu, H., Xiao, F. & Zhu, Y. Digital integration and pattern analysis of mountain altitudinal belts in China. J. Mt. Sci. 24, 144–149 (2006). 32. Xu, W., Zou, C. & Bu, J. The Inﬂuence of global warming on vegetation in Northeast China and measures be taken. Sci. Geogr. Sin. 16, 26–36 (1996). 33. Gansert, D. Treelines of the Japanese Alps – altitudinal distribution and species composition under contrasting winter climates. Flora 199, 143–156 (2004). 34. Stebich, M. et al. Holocene vegetation and climate dynamics of NE China based on the pollen record from Sihailongwan Maar Lake. Quat. Sci. Rev. 124, 275–289 (2015). ARTICLE Pollen record of surface sediments from vertical forest zones of Changbai Mountain, Northeast China and their relations to the modern vegetation. Acta Bot. Sin. 45, 910–916 (2003). 60. Surovell, T. A., Finley, J. B., Smith, G. M., Brantingham, P. J. & Kelly, R. Correcting temporal frequency distributions for taphonomic bias. J. Archaeol. Sci. 36, 1715–1724 (2009). Correcting temporal frequency distributions for taphonomic bias. J. Archaeol. Sci. 36, 1715–1724 (2009). 29. Li, Y. et al. Pollen inﬂux and surface pollen assemblage on the northern slope of Taibai Mountain. Geogr. Res. 27, 536–546 (2008). 30. Korner, C. A re-assessment of high elevation treeline positions and their explanation. Oecologia 115, 445–459 (1998). References 1 W C The pulse of imperial China: a quantitative analysis of long- term geopolitical and climatic cycles. Glob. Ecol. Biogeogr. 24, 87–96 (2015). 4. Zhang, D. D. et al. The pulse of imperial China: a quantitative analysis of long- term geopolitical and climatic cycles. Glob. Ecol. Biogeogr. 24, 87–96 (2015). 4. Zhang, D. D. et al. The pulse of imperial China: a quantitative analysis of long- term geopolitical and climatic cycles. Glob. Ecol. Biogeogr. 24, 87–96 (2015). 5. Park, J. Solar and tropical ocean forcing of late-Holocene climate change in coastal East Asia Regional. Palaeogeogr. Palaeocl. 469, 74–83 (2017). 5. Park, J. Solar and tropical ocean forcing of late-Holocene climate change in coastal East Asia Regional. Palaeogeogr. Palaeocl. 469, 74–83 (2017). NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications 8 Author contributions D.X. and H.L. conceived and designed the study. G.C. organized ﬁeld work with D.X. and H.L.; G.C. constructed the age-depth model of Lake XLW; D.X. identiﬁed and analyzed the pollen data. C.W., H.L. and D.X. compiled archeological radiocarbon dates. D.X., H.L., L.L. and C.S. wrote the paper. D.X., H.L., L.L., G.C., C.S., F.L., C.W. and N.W. contributed to the interpretation of the results and all the authors provided inputs to the manuscript. D.X. and H.L. conceived and designed the study. G.C. organized ﬁeld work with D.X. and H.L.; G.C. constructed the age-depth model of Lake XLW; D.X. identiﬁed and analyzed the pollen data. C.W., H.L. and D.X. compiled archeological radiocarbon dates. D.X., H.L., L.L. and C.S. wrote the paper. D.X., H.L., L.L., G.C., C.S., F.L., C.W. and N.W. contributed to the interpretation of the results and all the authors provided inputs to the manuscript. 35. Wu, Z. & Huang, N. E. Ensemble empirical mode decomposition: a noise- assisted data analysis method. Adv. Adapt. Data Anal. 01, 1–41 (2009). 35. Wu, Z. & Huang, N. E. Ensemble empirical mode decomposition: a noise- assisted data analysis method. Adv. Adapt. Data Anal. 01, 1–41 (2009). 36. Huang, N. E. et al. The empirical mode decomposition and the Hilbert spectrum for nonlinear and non-stationary time series analysis. Poc. R. Soc. A 454, 903–995 (1998). 36. Huang, N. E. et al. The empirical mode decomposition and the Hilbert spectrum for nonlinear and non-stationary time series analysis. Poc. R. Soc. A 454, 903–995 (1998). NATURE COMMUNICATIONS \| (2019) 10:4105 \| https://doi.org/10.1038/s41467-019-12138-0 \| www.nature.com/naturecommunications 9 Additional information 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. 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https://openalex.org/W2047027696	https://www.scielo.br/j/rbz/a/QNx9N3FMGBF8pC9SJ7kh8Rg/?lang=en&format=pdf	English	null	Evaluation of carcass traits, non-carcass components and 12th rib analysis of hair sheep supplemented with phosphorus	Revista Brasileira de Zootecnia	2,006	cc-by	3,749	Helder Louvandini1, Concepta McManus2, Bruno Steffano Dallago3, Bruno de Oliveira do3, Dalton Araujo Antunes4 1 Faculdade de Agronomia e Medicina Veterinária - FAV, UnB, Caixa Postal 04508 70901-970 – Brasília-DF, Brasil. 2 Faculdade de Agronomia e Medicina Veterinária - FAV. Pesquisadora CNPq. 3 Graduando em Medicina Veterinária, bolsistas PIBIC, FAV, UnB. 4 Mestrando, FAV, UnB. ABSTRACT - The aim of this study was to evaluate the performance of sheep supplemented with phosphorus (P) using carcass traits and analysis of 12th rib. Twenty male lambs weighing 13.88 ± 2.51 kg and grazing Andropogon gayanus pasture were divided in two treatments of 10 animals each. In group P, animals were supplemented with 3 g phosphorus/animal/day, while in group NP no P supplementation was given. After 82 days the animals were slaughtered and carcass measurements were taken. The 12th rib was also removed for analysis. No significant differences in body weight, carcass yield, or commercial cut weights were found between both treatments, except for liver and kidneys that were heavier in the supplemented group (p=0.0954). The 12th rib analysis showed that the group P had higher rib, bone, and muscle weights when compared to animals not supplemented with P. Other measurements such as carcass fatness and rib eye area were not significantly affected by treatments as well as contents of crude protein, ether extract, ash and P. It was shown that P supplementation improved performance in young sheep, including bone and muscle tissue and liver and kidney weights. Revista Brasileira de Zootecnia © 2006 Sociedade Brasileira de Zootecnia ISSN impresso: 1516-3598 ISSN on-line: 1806-9290 www.sbz.org.br Revista Brasileira de Zootecnia © 2006 Sociedade Brasileira de Zootecnia ISSN impresso: 1516-3598 ISSN on-line: 1806-9290 www.sbz.org.br R. Bras. Zootec., v.35, n.2, p.550-554, 2006 Correspondências devem ser enviadas para: concepta@unb.br Avaliação das características de carcaça, constituintes corporais e da 12a costela de ovinos deslanados suplementados com fósforo RESUMO - Este trabalho foi realizado com a finalidade de avaliar o desempenho de ovinos suplementados com fósforo (P), utilizando-se as análises das características de carcaça e da 12a costela. Foram utilizados 20 ovinos com 13,88 ± 2,51 kg de PV, mantidos em pastagem de Andropogon gayanus, distribuídos em dois tratamentos com dez animais: um com suplementação de 3g/fósforo/animal/dia e o outro sem suplementação. Após 82 dias, os animais foram abatidos, tomando-se as medidas da carcaça e da 12a costela, para posterior análise. Não foram encontradas diferenças significativas entre os valores de peso vivo, rendimento de carcaça, cobertura de gordura e cortes comerciais entre os dois tratamentos, exceto os pesos do fígado e dos rins, que foram superiores (p=0,0954) no grupo suplementado. Na análise da 12a costela, o grupo com suplementação demonstrou maior peso da costela, do osso e do músculo em comparação aos animais que não receberam P suplementar. As demais medidas, como gordura e área de olho-de-lombo, não foram afetadas, assim como os níveis de PB, EE, CIN e P. A suplementação de fósforo melhorou o desempenho, a formação dos tecidos ósseo e múscular e os pesos de fígado e rins. Palavras-chave: cordeiros, crescimento, deficiência, minerais, Santa Inês Key Words: lambs, deficiency, growth, mineral, Santa Inês Introduction latossoils with low levels of available P, despite their good physical properties. The deficiency of available P is aggravated by elevated levels of iron and aluminun oxides and hydroxides that react with P in the soil making it practically unavailable for more nutritionally demanding plants and plants with root systems unable to overcome aluminum toxicity (Carvalho et al., 2003). Small ruminants, especially sheep, are growing in importance for animal production worldwide. In Brazil, meat sheep production increased considerably in recent years mainly using native hair breeds at pasture (Veloso et al. 2004). Phosphorus (P) deficiency in tropical countries is more common in pasture-fed herds because most of the grasses are established on P deficient soils. For instance, 50% of the soils destined to animal production on these areas are Phosphorus is a key mineral for animal growth and energy metabolism. Moreover, it is an essential component in buffer systems in the blood and other organic fluids. Therefore, low intake of P by ruminants may cause a Louvandini et al. 551 reduction in voluntary consumption of feed, slow growth and weak bones (Underwood & Suttle, 1999). Pasture samples were collected on days 6, 42 and 69 of the experiment, and chemical analysis carried out according to the Association of Official Agricultural Chemists (AOAC, 1995) recommendations (Table 1). Carcass is an important factor for evaluating the ability of an animal to transform feed into edible product (meat). With increasing consumer demand for lamb, the standardization of product quality is necessary (Macedo et al., 2000). Animals were weighed on days 1, 8, 29, 43, 57 and 82 of the experiment. Parasites were counted (eggs per gram of faeces – EGF) weekly using modified Mc Master technique (Whitlock, 1948). Animals were treated as necessary using febendazole (Panacur oral). Generally, sheep carcasses may represent between 40 and 50% of the animal body weight depending on factors such as age, sex, birth weight, nutrition, management and others. Factors related to the carcass such as weight, length, fattening and conformation also influence dressing percentage (Pérez, 2002). After 82 days on experiment, lambs were weighed (slaughter body weight – SBW) and slaughtered after a 24 hour fast. Soon after bleeding, skin was removed and weighed. Viscera were also removed and thoracic (TCO – lung, heart and trachea) and abdominal cavity organs (ACO – liver and kidneys) weighed followed by measurement of hot carcass weight (HCW). Introduction Carcass traits were evaluated using the system proposed by Müller (1987) and Osório et al. (1998). Fat was measured subjectively on a 0.25 point increasing scale varying from score 1 (no visible subcutaneous fat on the carcass) to score 5 (high fatness – obese – carcass surface covered with subcutaneous fat). Carcass length (distance between base of the tail and base of the neck) was measured with a metric tape. The most important component in valorization of the carcass or cut is muscle. Therefore, the greater proportion of muscle, the higher commercial value of the animal (Fernandes, 1994). Fat offers protection to frozen meat, reducing loss and it also affects palatability (Deambrosis, 1972). Measurements that can be correlated with composition are very useful in avoiding costly total dissection of the carcass. The rib has been used for predicting the proportions of muscle, fat and bone in the carcass (Silva & Pires, 2000). In this study carcass, non-carcass components and 12th rib were analysed to evaluate the effect of P supplementation in young sheep. Half-carcasses were obtained by a longtitudinal cut on the vertebral column and the left half weighed (LHCW). A fraction of the rib cage was taken by transverse cuts at the 12th and 13th ribs to obtain the 12th rib cutting close to the posterior faces of the 11th rib and 12th, with ventral separation at the end of the 12th rib followed by weigh and composition (muscle, bone and fat – adapted from Hankins & Howe, 1946). The component rib tissues were minced together for freeze drying. Afterwards, samples were chopped before chemical analysis according to AOAC (1995). Results and Discussion Table 1 - Chemical composition of feeds offered to sheep Chemical composition Grass Concentrate (g/kg DM) Phosphorus No phosphorus DM 322.6 888.5 880.1 CP 61.7 238.0 232.0 EE 28.0 14.8 11.1 NDF 733.6 143.5 142.7 ADF 380.8 31.5 32.6 Ash 63.4 84.1 28.6 Phosphorus 2.1 16.4 0.9 0 5 10 15 20 Body weight (kg) 1 8 29 43 57 71 82 Days Phosphorus No phosphorus Studying the effect of four different forage types (hay of water-grass, hay of bagasse of pineapple, forage palm and corn silage) on the quality of meat in Santa Inês sheep, Madruga et al. (2002) apud Madruga (2003), found in the meat higher concentrations of lipids for animals fed water- grass hay, bagasse of pineapple hay, and corn silage. This was also due to higher energy levels in the diet. In the meat, levels of water content, protein, phosphorus, calcium and cholesterol were significantly greater for sheep fed with forage palm compared to the other treatments. Table 1 - Chemical composition of feeds offered to sheep Chemical composition Grass Concentrate (g/kg DM) Phosphorus No phosphorus Figure 1 - Body weight change in sheep with or without P supplementation. Table 1 - Chemical composition of feeds offered to sheep Chemical composition Grass Concentrate (g/kg DM) Phosphorus No phosphorus DM 322.6 888.5 880.1 CP 61.7 238.0 232.0 EE 28.0 14.8 11.1 NDF 733.6 143.5 142.7 ADF 380.8 31.5 32.6 Ash 63.4 84.1 28.6 Phosphorus 2.1 16.4 0.9 0 5 10 15 20 Body weight (kg) 1 8 29 43 57 71 82 Days Phosphorus No phosphorus Table 1 - Chemical composition of feeds offered to sheep Fat in the carcass has beneficial effects in terms of flavour, and reduces dehydration which occurs during the cooling process. This dehydration makes the meat dark and dry, undesirable to the customer (Sainz, 1996). Higher fat levels found in adult sheep are not desirable for sale purposes, because of poor texture and consistency, due to a high degree of saturated fatty acids which adhere to the palate when not served hot (Osório & Osório, 2003). 0 5 10 15 20 Body weight (kg) 1 8 29 43 57 71 82 Days Phosphorus No phosphorus Weights of the 12th rib, bone and muscle (p=0.0618) were higher for animals supplemented with P but no significant differences were observed for the remaining variables (P>0.10; Table 3). Material and Methods This trial was conducted in the sheep section of the Água Limpa farm of the Brasília University (UnB), located at 15o 47' latitude south and 47o 56' longtitude west. The regional climate is classified as AW by Köppen with mean annual temperature of 21.1oC varying from 16.0oC (minimum) to 34.0oC (maximum). Mean annual precipitation is 1578.5 mm and mean relative air humidity is 68%. Twenty Santa Inês male sheep averaging 13.8 ± 2.5 Kg of initial body weight and three months of age were maintained in a single group on Andropogon gayanus grass pasture. Pastures were not fertilized the 12 months before the experiment. The lambs were randomly allocated to two dietary treatments as follows: 16.66 g/animal/day of bicalcium phosphate providing 3.0 g phosphorus/ animal/day (group P) or no phosphorus supplementation (group NP). Eye muscle area was determined by drawing around the longissimus muscle exposed by the transverse cut of the 12th intercostal space using a standard transparent checked base (1cm2/cell). The halfcarcass was divided into six commercial cuts: leg, shoulder, back, rib, belly and neck (adapted from Santos, 1999) and then individually weighed. Dressing percentage of the hot carcass (DHC) was calculated using the formula suggested by Osório et al. (1998): DHC = (HCW/SBW) x 100 The experiment was analyzed as a completely randomized design with two treatments (dietary levels of P) with 10 replications and five repeated measures in time for body weight only. The program Statistical Analysis System (SAS, 1996) was used for data analysis. At the end of each day, the groups were separated in closed stables and received a concentrate containing 200 g cassava flour, 10 g urea and 9.57 g a mineral mixture (0.009 KI; 0.0008 CoSO4; 0.03 CuSO4; 1.61 MgO; 3.0 NaCl; 0.32 ZnSO4; 0.148 MnSO4; 0.457 FeSO4; 4.0 S). © 2006Sociedade Brasileira de Zootecnia Evaluation of carcass traits, non-carcass components and 12th rib analysis of hair sheep supplemented with phosphorus 552 Results and Discussion No significant differences were observed between treatments for carcass component weights (Table 2), except for liver and kidneys that were heavier in the supplemented group (p=0.0954). Although liver and kidneys are not carcass components, they have an aggregated economic interest for sale because can be consumed by lower income families in developing countries such as Brazil (Tonetto et al., 2004). Chemical composition of the feeds used in the experiment are presented in Table 1. No significant differences were observed in the body weight of the animals comparing both treatments throughout the experimental period (Figure 1). Winks (1990) observed that P deficiency was more severe in grazing ruminants on pastures with P level lower than 0.15%. In the present study, P content of the pasture was approximately 0.21%, which may be considered as a marginal deficiency for the NP sheep. Moreover, it can be seen that the 82-day period was not enough to significantly reduce weight gain of animals not supplemented with P. Carcass dressing percentage averaged 40% across treatments and was lower than those reported by Alves et al. (2003), which varied from 45 to 50%. However, these authors used heavier animals (14.5 – 16.5 kg carcass weight). Comparison of carcass traits should taking into account breed, sex, age, weight and nutrition of the animals in order to avoid inadequate conclusions. Sousa (2003) used Santa Inês sheep similar in weight and age to those in the present trial but a different feeding system (confined animals fed corn and sunflower silage). Carcass fatness (fat and ether extract of the 12th rib) was higher in Souza (2003) study, due to the greater energy density of the diet, but few differences were found when other carcass traits were compared between both experiments. Working with 215 kg Nellore calves, Nicodemo et al. (2000) found reduction in both intake and weight gain on animals receiving 5g P/day. However, some studies showed that body weight alone is not a good indicator for early diagnosis of P deficiency (Vitti et al., 1988; Villarroel et al., 1991). Figure 1 - Body weight change in sheep with or without P supplementation. © 2006Sociedade Brasileira de Zootecnia Results and Discussion Animals that received P supplementation had higher degree of development of the 12th rib, reflected by the greater amount of muscle and bone tissue. Muscle is edible and adds more economic value to the carcass. Nutrition is a key point in the standardization of lamb carcasses that is required to improve market value of the Figure 1 - Body weight change in sheep with or without P supplementation. 553 Louvandini et al. Table 2 - Slaughter and carcass weights and weights and proportions of commercial cuts of half carcasses and non-carcass components in sheep with or without P supplementation Carcass components Item Phosphorus No phosphorus Standard Error Body weight at slaughter (kg) 20.10 18.24 1.12 Carcass weight (kg) 8.08 7.51 0.52 Half carcass weight (kg) 4.09 3.90 0.27 Carcass dressing (%) 40.2 41.2 0.78 Carcass length (cm) 60.2 62.4 1.34 Carcass fatness (score 1 to 5) 2.3 2.2 0.1 Leg (kg) 1.33 1.21 0.08 %1 32.7 31.2 1.1 Shoulder (kg) 0.89 0.81 0.06 %1 21.8 20.9 1.0 Back (kg) 0.44 0.50 0.06 %1 10.7 12.7 1.1 Rib (kg) 0.44 0.41 0.01 %1 10.9 10.5 0.5 Belly (kg) 0.68 0.64 0.06 %1 16.5 16.3 0.5 Neck (kg) 0.39 0.34 0.0 %1 9.5 8.9 20.4 Non-carcass components Skin (kg) 1.17 1.19 0.07 %2 14.5 15.4 2.1 Lung, trachea and heart (kg) 0.46 0.42 0.03 %2 5.8 5.8 0.3 Liver and kidney (kg) 0.57b 0.49a 0.03 %2 7.2 6.7 0.3 Scrotum weight (kg) 0.21 0.20 0.04 %2 2.3 2.7 0.4 Scrotal circumference (cm) 19.5 19.0 1.65 a, b Means followed by different letters in the same row are significantly different (p<0.10) 1 Half carcass weight as reference and 2 Carcass weight as reference. Table 2 - Slaughter and carcass weights and weights and proportions of commercial cuts of half carcasses and non-carcass components in sheep with or without P supplementation Table 2 - Slaughter and carcass weights and weights and proportions of commercial cuts of half carca components in sheep with or without P supplementation and carcass weights and weights and proportions of commercial cuts of half carcasses and non-carcas s in sheep with or without P supplementation a, b Means followed by different letters in the same row are significantly different (p<0.10) 1 Half carcass weight as reference and 2 Carcass weight as reference. Literature Cited SILVA L.F.; PIRES C.C. Avaliações quantitativas e predição das proporções de osso, músculo e gordura da carcaça em ovinos. Revista Brasileira de Zootecnia, v.29, n.4, p.1253-1260. 2000. ALVES K.S.; CARVALHO F.F.R.; FEREIRA, M.A. et al. Níveis de energia em dietas para ovinos Santa Inês: características de carcaça e constituintes corporais. Revista Brasileira de Zootecnia, v.32, n. 6, supl. 2, p. 1927-1936, 2003. SILVA SOBRINHO, A.G.; GASTALDL, K.A.; GARCIA, C.A. et al. Diferentes dietas e pesos ao abate na produção de órgãos de cordeiros. Revista Brasileira de Zootecnia, v.32, n.6, supl.1, p.1792-1799, 2003. ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS - AOAC. Official methods of analysis of AOAC. 16.ed. Arlington: AOAC International, 1995. v.1, p.4/1-4/30. SOUSA, V.S. Desempenho e características de carcaça de ovinos deslanados alimentados com silagem de girassol Brasília: Universidade de Brasília, 2003. 47p. Dissertação (Mestrado em Ciências Agrárias) - Universidade de Brasília, 2003. CARVALHO, F.A.N.; BARBOSA, F.A.; MCDOWELL, L.R. Nutrição de bovinos a Pasto. 1.ed. Belo Horizonte: Papel Form Editora Ltda, 2003. 428p. STATISTICAL ANALISIS SYSTEM - SAS. SAS System for linear models. Cary: 211p. DEAMBROSIS, A. Producción de carne ovina. Crescimento. In: PRODUCCIÓN Y COMERCIALIZACIÓN DE CARNES, 2., 1972, Montevideo. Anais... Montevideo: Universidad de la República, 1972. TONETTO, J.C.; PIRES C.C.; MÜLLER,L. et al. Rendimentos de cortes da carcaça, carcterísticas da carne e componentes do peso vivo em cordeiros terminados em três sistemas de alimentação. Revista Brasileira de Zootecnia, v.33, n.1, p.234-241, 2004. FERNANDES, S. Peso vivo ao abate e características de carcaça de cordeiros da raça Corriedale e mestiços Ile de France X Corriedale recriados em confinamento. Botucatu: Universidade Estadual Paulista, 1994. 82p. Dissertação (Mestrado em Zootecnia) - Universidade Estadual Paulista, 1994. UNDERWOOD, E.J.; SUTTLE, N.F. The mineral nutrition of livestock. 3.ed. Wallingford: CAB International, 1999. p.180. HANKINS, O.G.; HOWE, P. E. Estimation of composition of beef carcasses and cuts. Washington, D.C: 1946. 20p. (Technical Bulletin , 926) VILLARROEL, A.B.S.; CATTANI, J.C.; FIGUEIRÓ, P.R.P. et al. Suplementação mineral em cordeiros desmamados. Pesquisa Agropecuária Brasileira, v.26, n.8, p.1275-1281, 1991. Suplementação mineral em cordeiros desmamados. Pesquisa Agropecuária Brasileira, v.26, n.8, p.1275-1281, 1991. VELOSO, C.F.M.; LOUVANDINI, H.; KIMURA, E.A. et al. Efeitos da suplementação protéica no controle da verminose e nas características de carcaça de ovinos Santa Inês. Ciência MADRUGA S.M. Fatores que afetam a qualidade da carne caprina e ovina In: SIMPÓSIO INTERNACIONAL SOBRE CAPRINOS E OVINOS DE CORTE, 2., 2003, João Pessoa. Anais... João Pessoa: 2003. Conclusion OSÓRIO J.C.S.; OSÓRIO M.T.M. Cadeia produtiva e comercial da carne de ovinos e caprinos – qualidade e importância dos cortes. In: SIMPÓSIO INTERNACIONAL SOBRE CAPRINOS E OVINOS DE CORTE, 2., 2003, João Pessoa. Anais... João Pessoa: 2003. (CD-ROM) Phosphorus supplementation can improve bone tissue and muscle deposition, as well as, kidney and liver weights in young sheep reared at pasture in the savannah region of Brazil. Therefore, it is important to feed mineral supplementation to animals, particularly P, in cases of P deficiency in pasture and soil. PÉREZ, J.R.O. Ovinocultura: alguns conceitos. In: SEVETUnB, 4., 2002, Brasília. Resumos... Brasília: 2002. p.152. SAINZ, R.D. Qualidade das Carcaças e da Carne Ovina e Caprina. In: REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE ZOOTECNIA, 33., SIMPÓSIO INTERNACIONAL SOBRE TÓPICOS ESPECIAIS EM ZOOTECNIA, SEMANA DA CAPRINOCULTURA E DA OVINOCULTURA BRASILEIRA, 2., 1996, Fortaleza, Anais... Fortaleza: Sociedade Brasileira de Zootecnia, 1996. p.3-14. Results and Discussion a, b Means followed by different letters in the same row are significantly different (p<0.10) 1 Half carcass weight as reference and 2 Carcass weight as reference. Table 3 - Weight, proportion and chemical composition of 12th rib and rib eye muscle area for sheep with or without P supplementation Treatments Phosphorus No phosphorus Standard error Rib weight (g) 55.72 b 43.37 a 3.77 Muscle weight (g) 35.94b 29.23a 2.43 % 65.0 67.4 2.3 Bone weight (g) 12.57 b 8.53a 1.30 % 22.3 19.5 1.3 Fat weight (g) 5.16 4.17 0.67 % 9.2 9.9 1.2 Eye muscle area (cm2) 8.1 8.3 0.6 12th rib chemical composition Dry matter % (DM) 32.9 33.6 0.88 Crude protein (%DM) 61.9 59.2 2.46 Ether extract (%DM) 17.2 19.3 1.60 Ash (%DM) 15.2 14.2 0.65 Phosphurus (%DM) 2.7 2.5 0.12 a, b Means followed by different letters in the same row are significantly different (p<0.10). on and chemical composition of 12th rib and rib eye muscle area for sheep with or without P supplementation Phosphorus No phosphorus Standard error Table 3 - Weight, proportion and chemical composition of 12th rib and rib eye muscle area for sheep with or wi Treatments Phosphorus No phosphorus Sta a, b Means followed by different letters in the same row are significantly different (p<0.10). score of carcasses are criteria that define quality and carcass finished with good conformation tends to have better prices at sale, especially in countries with tradition in lamb and mutton production (Silva Sobrinho et al., 2003). product and to attract consumers. Carcass must have a high percentage of muscle, uniform subcutaneous fat deposit and adequate fat levels to fulfill market requirements. All these traits are influenced by the degree of maturity of the genotype. Conformation and fattening © 2006Sociedade Brasileira de Zootecnia Evaluation of carcass traits, non-carcass components and 12th rib analysis of hair sheep supplemented with phosphorus 554 Acknowledgements SANTOS, C.L. Estudo do desempenho, das características da carcaça e do crescimento alométrico de cordeiros das raças Santa Inês e Bergamácia. Lavras: Universidade Federal de Lavras, 1999. 143p. Dissertação (Mestrado em Zootecnia) - Universidade Federal de Lavras, 1999. Literature Cited (CD-ROM) Animal Brasileira, v.5, n.3, p.131-139, 2004. VITTI, D.M.S.S.; ABDALA. A.L.; SILVA FILHO, J.C. Métodos para diagnóstico da deficiência de fósforo em ruminantes. Pesquisa Agropecuária Brasileira, v.23, n.6, p.645-651, 1988. MACEDO, F.A.F.; SIQUEIRA, E.R.; MARTINS, E.N. et al. Qualidade de carcaças de cordeiros Corriedale puros e mestiços terminados em pastagem e confinamento. Revista Brasileira de Zootecnia, v.29, n.5, p.1520-1527, 2000. MÜLLER, L. Normas para avaliação de carcaças e concurso de carcaças de novilhos. Santa Maria: Universidade Federal de Santa Maria, 1987. 31p. WHITLOCK, J.H. Some modifications of the Mc Máster helminth egg-counting technique and apparatus. Journal of Council for Scientific and Industrial Research, v.21, p.177-180, 1948. NICODEMO, M.L.F.; MORAES, S.S.; ROSA, I.V. et al. Uso de parâmetros ósseos, plasmáticos e fecais na determinação da deficiência de fósforo em bovinos. Revista Brasileira de Zootecnia. v.29, n.3, p.840-847, 2000. WINKS, L. Phosphorus and beef production in northern Australia. Responses to phosphorus by ruminants: a review. Tropical Grassland, v.24, p.140-158, 1990. OSÓRIO, J.C.S.; OSÓRIO, M.T.M.; JARDIM, P.O.C. et al. Métodos para avaliação da produção de carne ovina: in vivo na carcaça e na carne. Pelotas: Universidade Federal de Pelotas, 1998. 107p. Recebido: 03/02/04 Aprovado:08/07/05 © 2006Sociedade Brasileira de Zootecnia
https://openalex.org/W1811653118	https://europepmc.org/articles/pmc4613501?pdf=render	English	null	Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from <i>Necator americanus</i>	Biochemical journal	2,015	cc-by	13,417	Abbreviations: Ace-FAR-1, Ancylostoma ceylanicum fatty acid- and retinol-binding protein-1; Ac-FAR-1, Ancylostoma caninum fatty acid- and retinol- binding protein-1; Ac-SPI, serine protease inhibitor from Ancylostoma caninum; Bm-FAR, Brugia malayi fatty acid- and retinol-binding protein; Ce-FAR-7, Caenorhabditis elegans fatty acid- and retinol-binding protein-7; CRABP, cellular retinoic acid-binding protein family; CRBP, cellular retinol-binding protein; DAG, diacylglycerol; DAUDA, 11-(dansylamino)undecanoic acid; FA, fatty acid; FABP, fatty acid-binding protein; FAR, fatty acid- and retinol- binding protein; GC, gas chromatography; Gp-FAR-1, Globodera pallida fatty acid- and retinol-binding protein-1; Hp-FAR-1, Heligmosomoides polygyrus fatty acid- and retinol-binding protein-1; HPLC, high performance liquid chromatography; HSQC, heteronuclear single-quantum correlation spectroscopy; L3, third (infective) larval stage of a nematode; Mj-FAR-1, Meloidogyne javanica fatty acid- and retinol-binding protein-1; Na-FAR-1, Necator americanus fatty acid- and retinol-binding protein-1; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; NPA, nematode polyprotein antigen; Oo-FAR-1, Ostertagia ostertagi fatty acid- and retinol binding protein-1; Ov-FAR-1, Onchocerca volvulus fatty acid- and retinol-binding protein-1; PBS, phosphate buffered saline; PDB, protein structure database; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL, phospholipid; RDC, residual dipolar coupling; RMSD, root-mean-square deviation; TLC, thin layer chromatography. 1 Correspondence may be addressed to either of these authors (email Brian.Smith@glasgow.ac.uk or malcolm.kennedy@glasgow.ac.uk). Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus ¶Institute of Molecular, Cell & Systems Biology, University of Glasgow, Glasgow G12 8QQ, U.K. Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand- binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein–ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as conﬁrmed in the present study by our ﬁnding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male. Key words: fatty acid-binding protein, Necator americanus, nematode, nuclear magnetic resonance (NMR), parasite, protein structure, retinol-binding protein, X-ray. and also in the delivery of small signalling lipids to their destinations [1]. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. 403 403 Biochem. J. (2015) 471, 403–414 doi:10.1042/BJ20150068 INTRODUCTION There are as yet uncharted numbers of FAR isoforms in parasitic nematodes, but FARs have drawn attention because they are secreted by both plant- and animal-parasitic species and their encoding transcripts are relatively abundant [11–18]. They have also proven useful for serodiagnosis, have shown promise in experimental vaccines [19,20] and have been proposed to facilitate infection by manipulating host lipid-mediated defences [21,22]. At least one FAR has been shown to bind an anthelmintic drug, these drugs being typically hydrophobic and so may require carrier proteins to conduct them to their site of action within parasites [23]. The deﬁnitive role of FARs in parasitism is not known, but their presence in secretions of the worms, coupled with their ligand-binding propensities, suggests roles for them either in acquiring lipids from the hosts, or in delivering or in sequestering signalling lipids and so modifying the tissues the parasites occupy or the immune responses against them. Antiserum was tested by ELISA and Western blot analysis against the recombinant protein. To analyse the expression of Na-FAR-1 in the worm, soluble extracts of adult N. americanus, as well as recombinant proteins (each 100 ng) of homologues from Ancylostoma caninum FAR-1 (Ac-FAR- 1); Brugia malayi FARs (Bm-FAR-1 and Bm-FAR-2) and the unrelated protein, recombinant Ac-SPI (serine protease inhibitor from A. caninum) were separated on a 4%–20% gradient SDS precast polyacrylamide gel (Invitrogen) and subsequently electrotransferred on to a PVDF membrane (Millipore). Rabbit anti-Na-FAR-1 serum was diluted 1:5000 into PBS (phosphate buffered saline), pH 7.4, with 0.05% Tween-20 and incubated with transferred membrane for 2 h. Horseradish peroxidase- conjugated goat anti rabbit-IgG was used as secondary antibody and ECL was used to develop the reaction (GE). p To determine the tissue-speciﬁc localization of Na-FAR-1, adult N. americanus worms were prepared as previously described [29]. Brieﬂy, adult worms were collected from the intestines of hamsters infected with N. americanus L3 [third (infective) larval stage of a nematode] for 45 days and ﬁxed with 10% formalin. The ﬁxed worms were sectioned and mounted on glass slides. The non-speciﬁc binding sites on worm sections were blocked with 5% FBS in PBS for 1 h. The rabbit anti-Na-FAR-1 serum was applied (1:500 dilution) to each tissue section and incubated for 2 h at room temperature in a humidiﬁed chamber. Pre-immune rabbit serum at the same dilution was used as a negative control. INTRODUCTION Lipids such as acids (FAs) and retinoids are relatively insoluble in water, can be susceptible to oxidation and are potentially damaging to membranes in their free form. Consequently, they are usually transported within proteins or protein-mediated lipid aggregates. Examples of vertebrate FA and retinoid transporter proteins include serum albumins (∼64 kDa), which bind a range of compounds, lipocalins (∼20 kDa), which are found in many secretions and the proteins of the cytoplasmic FA-binding/cellular retinol-binding/cellular retinoic acid-binding protein (FABP/CRBP/CRABP) family (∼14 kDa) that are conﬁned to the cytoplasm, where they may bind FAs, retinol or retinoic acid, depending on the isoform. These proteins are variously involved in transport and storage of lipids Nematodes, both free-living and parasitic to plants and animals, exhibit several additional types of lipid-binding proteins that are not found in other phyla. Prominent examples include the nematode polyprotein antigens (NPAs), such as ABA-1, whose structure has previously been solved [2]. These are synthesized as large polypeptide precursors that are post- translationally processed down to multiple copies of small lipid- binding proteins of ∼14 kDa [3–7]. Other examples include the nemFABPs (nematode FABPs), which are similar to the intracellular FABP/CRBP/CRABP family of proteins, but in nematodes have structural modiﬁcations found in no other group of animals and are not conﬁned to the cytosol [8,9]. The subject of the present paper is an unusual class of lipid-binding protein, the FA and retinol-binding proteins (FARs). These occur in several M.F. Rey-Burusco and others 404 Antiserum was tested by ELISA and Western blot analysis against the recombinant protein. To analyse the expression of Na-FAR-1 in the worm, soluble extracts of adult N. americanus, as well as recombinant proteins (each 100 ng) of homologues from Ancylostoma caninum FAR-1 (Ac-FAR- 1); Brugia malayi FARs (Bm-FAR-1 and Bm-FAR-2) and the unrelated protein, recombinant Ac-SPI (serine protease inhibitor from A. caninum) were separated on a 4%–20% gradient SDS precast polyacrylamide gel (Invitrogen) and subsequently electrotransferred on to a PVDF membrane (Millipore). Rabbit anti-Na-FAR-1 serum was diluted 1:5000 into PBS (phosphate buffered saline), pH 7.4, with 0.05% Tween-20 and incubated with transferred membrane for 2 h. Horseradish peroxidase- conjugated goat anti rabbit-IgG was used as secondary antibody and ECL was used to develop the reaction (GE). isoforms of ∼20 kDa, eight having been found encoded within the genome of the free-living species Caenorhabditis elegans, each of which binds FAs and retinol to varying extents [10]. Protein expression and puriﬁcation Recombinant Na-FAR-1 (rNa-FAR-1) was expressed in BL21 (λDE3) Escherichia coli cells as described [27]. For native crystallographic studies, Na-FAR-1 was puriﬁed to homogeneity, as previously described [28], from cells grown in LB media. Selenomethionine-labelled protein was puriﬁed from B834 cells grown in M9 minimal medium supplemented with a cocktail of free amino acids (each 0.5 g·l −1) and selenomethionine (50 mg·l −1; Generon). For NMR studies, samples of unlabelled, 15N-labelled and 13C15N-labelled protein were puriﬁed by nickel-afﬁnity, size exclusion and reverse-phase chromatographies, as described [27], from cells grown in M9 minimal medium containing 15NH4Cl, [13C6]-glucose or their unlabelled equivalents. [13C6]-glucose or their unlabelled equivalents. Crystallization, data collection, processing and structure solution We have shown previously that Na-FAR-1 crystallizes in two crystal forms, one of which (form 2) shows signiﬁcant twinning [28]. Here, in order to obtain phasing information, selenomethionine-substituted protein was puriﬁed and crystallized, selecting only the cubic crystal form 1. Crystals were frozen in a stream of cool nitrogen gas (100 K) and brought to the Diamond Light Source, station I04 (DLS) for X-ray diffraction data collection. Data were collected at 0.7◦increments per image, for a total of 200 images [wavelength 0.9793 Å (1 Å = 0.1 nm)] and processed by the automatic processing routines fast_dp, which utilized XDS [30], POINTLESS and SCALA [31]. The structure was solved using the SAS protocol of Auto-Rickshaw [32]. The input diffraction data were prepared and converted for use in Auto- Rickshaw, using programs of the CCP4 suite [33]. FA values were calculated using the program SHELXC [34]. Based on an initial analysis of the data, the maximum resolution for sub-structure determination and initial phase calculation was set to 2.14 Å based on the scaling statistics and the increase in Rmeas in the highest resolution bin. All the four heavy atoms requested were found using the program SHELXD [35]. The correct handedness for the substructure was determined using the programs ABS [36] and SHELXE [34]. Initial phases were calculated after density modiﬁcation using the program SHELXE. 83.9% of the model was built using the program ARP/wARP [37]. Despite a solvent content above 70%, only one copy of the protein was observed in the asymmetric unit. The model was completed by hand using COOT [38] and iterative rounds of BUSTER [39]. Ligands and water were added using COOT. Based on its prevalence in the lipids co-purifying with recombinant Na-FAR-1, palmitate was ﬁtted into unoccupied electron density within the binding pocket. INTRODUCTION Sections were washed six times for 5 min each in PBS and probed with anti-rabbit Cy3-conjugated IgG (Rockland). Sections were viewed under a Nikon TE-2000 Inverted ﬂuorescence microscope using a 550 nm excitation ﬁlter block and emission at 565 nm. The structure of one FAR protein, C. elegans FAR-7 (Ce- FAR-7), has been solved by X-ray crystallography, revealing a helix-rich structure that is unlike any type of lipid-binding protein previously described [24]. The mRNA for Ce-FAR-7 does not encode a secretory signal peptide and its amino acid sequence indicates that it is in a different subfamily of FARs from those that are secreted from the synthesizing cell. We set out to conﬁrm the expression pattern of a secreted FAR protein from a parasite and characterize its structure and ligand- binding characteristics. The protein Na-FAR-1 derives from the blood feeding intestinal hookworm of humans, N. americanus. This parasite and the other hookworm of humans, Ancylostoma duodenale, together infect over 300 million people worldwide [25,26], causing considerable morbidity, together with adverse social and economic consequences. We have determined the structure of Na-FAR-1 by both X-ray crystallography and NMR spectroscopy in solution. We ﬁnd that it has a similar overall fold to Ce-FAR-7, indicating that FARs are structurally conserved despite considerable sequence diversity. But, in addition to the structural differences between Ce-FAR-7 and Na-FAR-1, we ﬁnd that their ligand-binding sites differ signiﬁcantly in position and form. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. Lipid extraction and analysis Total lipids were extracted according to the methodology described by [58] and [59], with minor modiﬁcations. For unstripped Na-FAR-1 (no RP-HPLC puriﬁcation), lipids were extracted from 15 mg of protein and compared with control apo-Na-FAR-1 (RP-HPLC puriﬁed protein). For comparison, approximately 3 ml of culture of E. coli BL21 (λDE3) cells were lysed by sonication. Each sample was mixed with 15 ml of CHCl3–CH3OH (2:1) and vigorously shaken for 15 min in an ice bath. The homogenate was washed with 250 μl of 2.9% (w/v) NaCl solution. After agitation, the phases were separated by centrifugation and the upper, aqueous phase discarded. The lower phase containing lipids was recovered and dried under a stream of N2 gas, re-dissolved in CHCl3 and stored at –20 ◦C under N2 gas until analysis. Ligand-binding analysis by chemical shift perturbation In order to examine Na-FAR-1’s ligand binding properties by NMR, the chemical shift changes induced in 15N HSQC spectra of the protein by the presence of increasing amounts of ligand were analysed. Double-labelled 13C-15N recombinant Na-FAR-1 (0.4 mM) was titrated by sequential addition of small volumes of unlabelled sodium oleate (Sigma–Aldrich) from a 125 mM stock solution in water (pH ∼9). Triple resonance experiments were recorded at selected points of the titration in order to assign the displaced cross-peaks. The titration started by addition of 0.5 molar equivalent of the ligand and was continued until turbidity due to the presence of insoluble oleic acid was observed. Structure calculations from NMR-based restraints Lipid classes bound to Na-FAR-1were analysed by TLC (thin layer chromatography) on silica gel plates Si250 (J.T.Baker) with the methodology and solvent systems described by [59]. Lipid samples obtained from holo-Na-FAR-1 and E. Coli lysates controls and standards were spotted on TLC plates (20 × 20 cm) previously activated at 100 ◦C for 30 min and developed with methyl acetate–isopropanol–chloroform–methanol–0.25% KCl (25:25:25:10:9, by volume) for polar lipids and hexane/diethyl- ether/acetic acid (80:20:1, by volume) for neutral lipids. Distance restraints were derived from NOESY (nuclear Overhauser effect spectroscopy) cross-peaks with the initial mapping from normalized intensity to distance and grouped in distance bins. NOE distance restraints were incorporated in restrained MD calculations using the ambiguous distance restraints formalism [47] using ARIA 2.3 [48] and CNS [49]. Loose backbone dihedral restraints for regions of regular secondary structure predicted based on secondary chemical shifts by DANGLE [50] were incorporated during the high temperature phases of the simulations but omitted during the ﬁnal cooling phase. RDC and hydrogen bond restraints were then introduced. The average RDC alignment tensor was estimated from the ensemble calculated using only NOEs with PALES [51] and used to incorporate the RDC restraints via the SANI potential [52] in square-well mode. The 20 structures that best satisfy the experimental restraints were chosen from 100 structures generated in the ﬁnal iteration and reﬁned in explicit water [53]. The quality of these structures was analysed using PROCHECK_nmr [54] and their co-ordinates deposited in the Protein Data Bank under accession code 4UET. Structure ﬁgures were generated using PyMOL (http://www.PyMOL.org). Non-esteriﬁed FA and phospholipid (PL) TLC spots were scraped and extracted from the silica with pure chloroform for FAs and chloroform–methanol–water for PLs respectively. FA composition was analysed by GC (gas chromatography)–MS of their methyl ester derivatives, prepared with BF3-methanol according to the method of Morrison and Smith [60] as described previously [61]. The individual FA methyl ester peaks were identiﬁed by comparison of their retention times with those of standards and by their mass spectra. NMR spectroscopy and analysis All spectra were recorded at 311 K on a Bruker AVANCE 600 MHz spectrometer equipped with TCI cryoprobe. Resonances were assigned, as described previously [27]. All spectra were processed in AZARA (Wayne Boucher, http://www.bio.cam.ac.uk/azara). Maximum entropy reconstruction [42] was used to enhance resolution of the indirect dimensions of 3D experiments. Spectra were analysed with CCPNmr analysis software [43]. Frequency-based methods were employed to measure 1DNH [44] and 1DCαHα [45] couplings from in-phase/antiphase 15N-HSQC (heteronuclear single-quantum correlation spectroscopy) spectra. Distance restraints for structure calculations were derived from 3D 15N-NOESY-HSQC [46] and 3D 13C-NOESY-HSQC spectra, each recorded with 100 ms mixing time. NMR relaxation measurements 15N-relaxation time constants, T1 and T2 were assessed using the method of Kay [55–57] at a ﬁeld strength of 14.1 T. Relaxation delays for assessment of T1 were 101, 601, 1001 and 1401 ms whereas those for T2 were 17, 34, 68, 102 and 136 ms. Selected time points in each series were repeated in order to estimate the inherent error in calculation of cross-peak intensities. Relaxation times T1 and T2 were calculated using non-linear least squares ﬁtting. Collection of 15N-HSQC-heteronuclear NOE experiments with and without saturation allowed extraction of 1H,15N NOE values. Both saturation and reference experiments were repeated for the purpose of error estimation. Preparation of NMR samples Apo-Na-FAR-1 samples were delipidated by reverse phase- HPLC (high performance liquid chromatography) as previously described [27] using a C8-silica stationary phase and a water/acetonitrile gradient in the presence of 0.1% triﬂuoroacetic acid as the mobile phase. Samples were lyophilized, reconstituted and concentrated to approximately 0.6 mM in 20 mM sodium phosphate, pH 7.20. 2H2O was added to a ﬁnal concentration of 5% (v/v). The sample used for residual dipolar coupling (RDC) measurements was prepared by partial alignment of the uniformly 13C15N-enriched Na-FAR-1 in a solution of magnetically aligned ﬁlamentous Pf1 bacteriophage [41] (ASLA biotech). The degree of alignment was evaluated by measuring the 2H quadrapolar splitting in the HDO resonance. After testing several conditions, a NaCl concentration of 300 mM with 9 mg·ml −1 of bacteriophage and 300 μM protein was selected. Western blotting and immunolocalization of Na-FAR-1 Antiserum prepared against recombinant Na-FAR-1 was raised in three rabbits by subcutaneous injection with 0.7 mg of puriﬁed recombinant Na-FAR-1 in Freund’s complete adjuvant. Structure of Necator americanus FAR protein 405 Further electron density that may represent additional ligand molecules was observed, but as the density was incomplete and not at full occupancy, it was left unmodelled. The geometry of the ﬁnished models was validated using Molprobity [40]. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. Na-FAR-1 Na-FAR-1 had been identiﬁed in a gene survey of the N. americanus transcriptome under the name N. americanus LBP- 20 (sequence ID NAC00128) [62]. LBP-20 proteins are currently named FAR due to their capacity for binding FAs and retinol and Na-FAR-1 was renamed accordingly [63]. More recently, the genome of N. americanus has been published and at least six FAR proteins have been recognized (Figure 1; Supplementary Figure S1), including Na-FAR-1, under the sequence name NECAME_14208 [64]. Na-FAR-1 cDNA predicts a 19364.57 Da protein with a 14 amino acid secretion signal peptide predicted by SignalP [65]. The post-translational removal of the leader peptide would yield a mature protein of 155 amino acids with a molecular mass of 17082.49 Da. blood-feeding stage [64]. Immunohistochemistry revealed that Na-FAR-1 protein occurs in the cloacal aperture and copulatory bursa of male worms (Figures 1c and 1d) and in the intestine (Figures 1f and 1g). The presence of Na-FAR-1 in the parasite’s intestinal cells may indicate involvement in transportation and storage of lipids derived from host blood and its location in copulatory bursa of male worms suggests its function may additionally be related to reproduction. Na-FAR-1 was detected in the intestinal cells but not the reproductive structures of females (Figure 1e). A phylogenetic tree was constructed to show the relationship between Na-FAR-1 and FARs from other species (Figure 1b). The tree was constructed with amino acid sequences omitting any leader peptides identiﬁed by SignalP. The sequences included those of C. elegans (Ce-FAR-1–8), the human parasitic nematodes Onchocerca volvulus FAR-1 (Ov-FAR-1), B. malayi (Bm- FAR-1), animal parasitic nematodes A. caninum (Ac-FAR-1 and Ac-FAR-2), Ancylostoma ceylanicum FAR-1 (Ace-FAR-1), Ostertagia ostertagi FAR-1 (Oo-FAR-1) and Heligmosomoides polygyrus FAR-1 (Hp-FAR-1) and the plant parasitic nematodes Globodera pallida FAR-1 (Gp-FAR-1) and Meloidogyne javanica FAR-1 (Mj-FAR-1). FAR transcript levels in some of these parasites are notably high, particularly in the parasitic stages of life cycles [11–18,62,66]. Figure 1 shows that most FAR proteins known from parasites group together, including those from N. americanus, and a subset of FARs from C elegans (Ce-FAR-1, 2 and 6). As noted above, Ce-FAR-7, the only FAR protein whose structure was previously known, falls in a subfamily of FARs dis- tant from all of the FAR proteins from parasites [10] (Figure 1b). 3D structure of Na-FAR-1 The 3D structure of Na-FAR-1 was determined by both protein X-ray crystallography and solution state NMR spectroscopy. Na- FAR-1, which had not been subjected to reverse phase HPLC puriﬁcation to strip out co-purifying ligands, crystallized as previously reported and diffracted to 2.14 Å [28]. Molecular replacement using the Ce-FAR-7 structure proved unsuccessful and we therefore obtained the crystal structure of Na-FAR-1 in complex with co-purifying ligands, hereafter referred to as holo-Na-FAR-1, by anomalous dispersion from crystals of Se- Met-labelled protein (Table 1). In contrast, NMR spectra of unstripped Na-FAR-1 in solution, like those of other FAR proteins we have tested, were characterized by broad signal peaks indicative of multiple conformations and/or conformational exchange. However, stripped Na-FAR-1 gave good solution NMR spectra and the structure of apo-Na-FAR-1 was determined from a total of 7289 NOE-derived distance restraints, 316 dihedral angle restraints and restraints derived from 177 RDCs (101 1DNH and Fluorescence spectroscopy Wilson B (A) 30.11 Number of Selenomethionines found 4 Reﬁnement Rwork (%) 20.5 Rfree (%) 22.4 Protein residues/atoms 155 (1213) Water molecules 139 Palmitic acid 1 RMSD bond lenghts (A˚)/angles (◦) 0.01/0.99 Average isotropic thermal parameters (A˚2) Main chain/side chain atoms 25.06/33.69 Water molecules 38.09 Palmitic acid 38.11 Ramachandran analysis % Favoured regions 98.05 % Outliers 0.65 c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. Fluorescence spectroscopy Fluorescence experiments were performed with a Fluorolog- 3 Spectroﬂuorometer (Horiba-Jobin Yvon). Buffer alone was 015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. M.F. Rey-Burusco and others 406 Table 1 Data collection, phasing and structure reﬁnement statistics for holo-Na-FAR-1 determined by X-ray crystallography (PDB:4XCP) Numbers in brackets indicate values in the highest resolution bin (2.27–2.14 A˚). Abbreviation: PDB, protein structure database. Data collection Space group P432 Unit cell a = b = c = 121.123 Wavelength (A˚) 0.9793 Resolution (A˚) 29.38-2.14 Observed reﬂections 502169 (17556) Unique reﬂections 17353 (1212) Completeness 99.91 (97.0) Multiplicity 28.9 (14.5) Rmeas (%) 14.2 (78.5) Rpim (%) 2.6 (20.3) Ranom (%) 5.2 (17.5) <I/σ(I)> 25.5 (4.2) Wilson B (A˚)2 30.11 Number of Selenomethionines found 4 Reﬁnement Rwork (%) 20.5 Rfree (%) 22.4 Protein residues/atoms 155 (1213) Water molecules 139 Palmitic acid 1 RMSD bond lenghts (A˚)/angles (◦) 0.01/0.99 Average isotropic thermal parameters (A˚2) Main chain/side chain atoms 25.06/33.69 Water molecules 38.09 Palmitic acid 38.11 Ramachandran analysis % Favoured regions 98.05 % Outliers 0.65 Table 1 Data collection, phasing and structure reﬁnement statistics for holo-Na-FAR-1 determined by X-ray crystallography (PDB:4XCP) used to correct for Raman and background scattering. RP- HPLC delipidated Na-FAR-1 was employed in all ligand-binding experiments. p The ligand-binding capacity of Na-FAR-1 was investigated with the ﬂuorescent ligands 11-(dansylamino)undecanoic acid (DAUDA) and retinol. Stock solutions (10 mM) were prepared in ethanol and then diluted in PBS for use in the assays. Retinol solutions were diluted in ethanol and added directly to the cuvette to minimize degradation. g The FA chain length preference of rNa-FAR-1 was tested by displacement of the ﬂuorescent ligand DAUDA as described [13]. Binding of non-ﬂuorescent ligands was detected by a reversal of the wavelength shift and a decrease in ﬂuorescence emission intensity on equal additions of test ligands to a DAUDA–rNa- FAR-1 complex recorded at the peak ﬂuorescence emission wavelength of DAUDA in the protein (470 nm). The concentration of Na-FAR-1 in the cuvette was 1.5 μM. DAUDA ethanol stock solution was diluted 1:10000 in PBS for use in the assays at 1 μM. Stock solutions of all the non-ﬂuorescent competitors were made to approximately 10 mM in ethanol, then diluted in PBS for use in the assays. Na-FAR-1 localization within the parasite americanus recently identiﬁed FAR proteins (NECAME_09996, NECAME_04475, NECAME_04474, NECAME_14206, NECAME_14205 and NECAME_14203), the free living nematode C. elegans (Ce-FAR-1 to Ce-FAR-8), the human parasitic nematodes O. volvulus (Ov-FAR-1), B. malayi (Bm-FAR-1), animal parasitic nematodes A. caninum (Ac-FAR-1 and Ac-FAR-2), A. ceylanicum (Ace-FAR-1), O. ostertagi (Oo-FAR-1) and H. polygyrus (Hp-FAR-1) and the plant parasitic nematodes G. pallida (Gp-FAR-1) and M. javanica (Mj-FAR-1). All the parasite proteins are coloured blue. See Supplementary Figure S1 for the multiple sequence alignment from which the tree was constructed. (c–g) Localization of Na-FAR-1 within adult male (c and d) and female (e–g) worms. Indirect immunoﬂuorescence localization with rabbit anti-Na-FAR-1 serum stains the intestinal cells of adult N. americanus worms. Na-FAR-1 was also detected on the copulatory bursa and cloacal aperture of male worms. (h) Control carried out using pre-immune serum. Scale bars represent 100 μm. 1 i l h li 3 ( id 3 5) 2 (8 11) d 3 (16 24) Ac-FAR-2 NECAME_09996 Oo-FAR-1 NECAME_14203 Ce-FAR-1 Ce-FAR-2 Ce-FAR-6 Av-FAR-1 Ov-FAR-1 Bm-FAR-1 Gp-FAR-1 Mj-FAR-1 NECAME_14205 Hp-FAR-1 Ce-FAR-3 Ce-FAR-5 Ce-FAR-4 Ce-FAR-7 Ce-FAR-8 cloacal aperture seminal duct (d) copulatory bursa Male worm spicules (c) Female worm (e) intestine (f) intestine oesophagus bucal capsule (g) Control (h) 64 50 36 16 6 Ce FAR 5 Ce-FAR-4 Ce-FAR-7 Ce-FAR-8 cloacal aperture seminal duct (d) copulatory bursa Male worm spicules (c) Female worm (e) intestine (f) intestine oesophagus bucal capsule (g) Control (h) (c) (e) (g) (f) Figure 1 FAR protein relationships and Na-FAR-1 expression (a) Western blot with anti-Na-FAR-1 serum that speciﬁcally recognizes native Na-FAR-1 in N. americanus extracts (lane 3, 0.5 μg) and excreted/secreted (ES) products (lane 4, 0.5 μg) at an approximate Mr of 14 kDa, but not in L3 extracts (lane 1) and L3 ES products (lane 2) at the same loading, indicating the speciﬁc expression of Na-FAR-1 in adult stage as a secreted protein. The antiserum also recognized the recombinant Na-FAR-1 at 16 kDa (with His-tag, lane 5, 20 ng). There was no cross-reaction with FAR homologues from dog hookworm A. caninum (Ac-FAR-1, lane 6); B. malayi (Bm-FAR-1/ lane 7, Bm-FAR-2/lane 8) and non-relevant recombinant protein Ac-SPI (lane 9) loaded at the same amount (20 ng). (b). Neighbour joining tree of Na-FAR-1 and other nematode FAR proteins. The tree was generated in jalview 2.8 [69] using the BLOSUM 62 matrix from a T-coffee WS sequence alignment [70] with FAR protein amino acid sequences of: N. Na-FAR-1 localization within the parasite Na-FAR-1-encoding mRNA had been detected in adult and larval L4 N. americanus life stages [62] and analysis of differences in gene expression between infective larvae (iL3) and adult parasitic stages shows that Na-FAR-1 is mainly expressed in the adult, 407 Structure of Necator americanus FAR protein Na-FAR-1 NECAME_14208 NECAME_04475v2 NECAME_14206 NECAME_04474 Ac-FAR-1 Ace-FAR-1 Ac-FAR-2 NECAME_09996 Oo-FAR-1 NECAME_14203 Ce-FAR-1 Ce-FAR-2 Ce-FAR-6 Av-FAR-1 Ov-FAR-1 Bm-FAR-1 Gp-FAR-1 Mj-FAR-1 NECAME_14205 Hp-FAR-1 Ce-FAR-3 Ce-FAR-5 Ce-FAR-4 Ce-FAR-7 Ce-FAR-8 (a) (b) cloacal aperture seminal duct (d) copulatory bursa Male worm spicules (c) Female worm (e) intestine (f) intestine oesophagus bucal capsule (g) Control (h) 1 2 3 4 5 6 7 8 9 MW (kDa) 98 64 50 36 16 6 Na-FAR-1 NECAME_14208 NECAME_04475v2 NECAME_14206 NECAME_04474 Ac-FAR-1 Ace-FAR-1 Ac-FAR-2 NECAME_09996 Oo-FAR-1 NECAME_14203 Ce-FAR-1 Ce-FAR-2 Ce-FAR-6 Av-FAR-1 Ov-FAR-1 Bm-FAR-1 Gp-FAR-1 Mj-FAR-1 NECAME_14205 Hp-FAR-1 Ce-FAR-3 Ce-FAR-5 Ce-FAR-4 Ce-FAR-7 (a) (b) 1 2 3 4 5 6 7 8 9 MW (kDa) 98 64 50 36 16 6 (b) (a) NECAME_14206 NECAME_04474 Ac-FAR-1 Ace-FAR-1 Ac-FAR-2 NECAME_09996 Oo-FAR-1 NECAME_14203 Ce-FAR-1 Ce-FAR-2 Ce-FAR-6 Av-FAR-1 Ov-FAR-1 Bm-FAR-1 Gp-FAR-1 Mj-FAR-1 NECAME_14205 Hp-FAR-1 Ce-FAR-3 Ce-FAR-5 Ce-FAR-4 Ce-FAR-7 Ce-FAR-8 cloacal aperture seminal duct (d) copulatory bursa Male worm spicules (c) Female worm (e) intestine (f) intestine oesophagus bucal capsule (g) Control (h) 1 2 3 4 5 6 7 8 9 MW (kDa) 98 64 50 36 16 6 Figure 1 FAR protein relationships and Na-FAR-1 expression (a) Western blot with anti-Na-FAR-1 serum that speciﬁcally recognizes native Na-FAR-1 in N. americanus extracts (lane 3, 0.5 μg) and excreted/secreted (ES) products (lane 4, 0.5 μg) at an approximate Mr of 14 kDa, but not in L3 extracts (lane 1) and L3 ES products (lane 2) at the same loading, indicating the speciﬁc expression of Na-FAR-1 in adult stage as a secreted protein. The antiserum also recognized the recombinant Na-FAR-1 at 16 kDa (with His-tag, lane 5, 20 ng). There was no cross-reaction with FAR homologues from dog hookworm A. caninum (Ac-FAR-1, lane 6); B. malayi (Bm-FAR-1/ lane 7, Bm-FAR-2/lane 8) and non-relevant recombinant protein Ac-SPI (lane 9) loaded at the same amount (20 ng). (b). Neighbour joining tree of Na-FAR-1 and other nematode FAR proteins. The tree was generated in jalview 2.8 [69] using the BLOSUM 62 matrix from a T-coffee WS sequence alignment [70] with FAR protein amino acid sequences of: N. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. Na-FAR-1 localization within the parasite americanus recently identiﬁed FAR proteins (NECAME_09996, NECAME_04475, NECAME_04474, NECAME_14206, NECAME_14205 and NECAME_14203), the free living nematode C. elegans (Ce-FAR-1 to Ce-FAR-8), the human parasitic nematodes O. volvulus (Ov-FAR-1), B. malayi (Bm-FAR-1), animal parasitic nematodes A. caninum (Ac-FAR-1 and Ac-FAR-2), A. ceylanicum (Ace-FAR-1), O. ostertagi (Oo-FAR-1) and H. polygyrus (Hp-FAR-1) and the plant parasitic nematodes G. pallida (Gp-FAR-1) and M. javanica (Mj-FAR-1). All the parasite proteins are coloured blue. See Supplementary Figure S1 for the multiple sequence alignment from which the tree was constructed. (c–g) Localization of Na-FAR-1 within adult male (c and d) and female (e–g) worms. Indirect immunoﬂuorescence localization with rabbit anti-Na-FAR-1 serum stains the intestinal cells of adult N. americanus worms. Na-FAR-1 was also detected on the copulatory bursa and cloacal aperture of male worms. (h) Control carried out using pre-immune serum. Scale bars represent 100 μm. 76 1DCaHa) observed in a sample that had been partially aligned in Pf1 ﬁlamentous bacteriophage (Table 2). Comparison of the structures of Na-FAR-1 and Ce-FAR-7 Ce-FAR-7 has previously been noted to lie in a distinct sub-group of FARs because it possesses two cysteines that may form a disulﬁde bond, has distinct lipid-binding characteristics, lacks a secretory leader peptide and because of the intron arrangement of its encoding gene [10,24]. The X-ray crystallographic structure of Ce-FAR-7 [24] displays similarities but several distinct differences to Na-FAR-1 that could relate to differences in their ligand binding and biological properties. A structure-based sequence alignment (Figure 3a) reveals sequence identity between the two proteins at 32 positions (∼21%). Among these are: hydrophobic residues, Leu77 and Ala81 (Leu78 and Ala82 in Ce-FAR-7) that help to determine the angle between α6 and α7; Leu33, Leu59 (Leu33 and Leu60 in Ce-FAR-7) together with Leu52, which is replaced by another hydrophobic residue Val53, which are involved in maintaining the orientation between α6 and helices α4 and α5; a salt bridge between Glu35 and Lys55 (Glu35 and Lys56 in Ce-FAR-7) that helps to maintain the relative orientation of α4 and α5; and another salt bridge between Lys73 (Arg74 Ce-FAR-7) and Glu17 that helps to maintain the relative orientation of α6 and α3. Na-FAR-1 has an insertion of four residues (107–110) that constitute an additional turn of helix at the N-terminal end of α8. Conserved prolines Pro7, Pro15 and Pro79 initiate helices α2, α3 and α7, corresponding to Pro7, Pro15 and Pro80 in Ce-FAR-7, as they seem likely to, in other FARs. Ce-FAR-7 has an additional residue in the loop between helices α4 and α5 and it is 14 residues shorter at the C-terminal region, thereby lacking helices α10 and α11. In Ce-FAR-7 α1 comprises a full turn of α-helix, whereas Na-FAR-1 has a 310 helix at this position. A comparison of the structures gives an RMSD of 1.711 Å (for a total of 636 atoms aligned) with the apo form and 1.646 Å (608 atoms aligned) with the holo form. The region comprising the α4–α5 joining loop, which might constitute part of the entrance to the cavity, was not modelled in the structure of Ce-FAR-7 due to insufﬁcient electron density. It is likely that the lack of good deﬁnition is indicative of dynamic processes occurring in the α4–α5 loop, as we found by NMR for Na-FAR-1 (Supplementary Figure S2). residues are on the external surface of the protein generating a predominately hydrophilic surface. 76 1DCaHa) observed in a sample that had been partially aligned in Pf1 ﬁlamentous bacteriophage (Table 2). In the holo structure the expanded cavity is lined by the same residues as in the apo structure, but with 21 additional, predominantly apolar, side chains contributing to the surface of the expanded cavity (Phe1, Ile6, Leu25, Lys30, Lys34, Val36, Ser48, Ile49, Leu52, Met69, Phe84, Ala85, Ile91, Ile95, Tyr99, Leu110, Ser113, Phe132, Phe136, Val142 and Ala148). In addition to the opening seen in the apo form, the cavity is also accessible to solvent via an opening between the α2–α3 and α9–α10 turns and α6. It is therefore possible that more than one region could be involved in ligand entrance or exit and the loop between α7 and α8, which exhibits a higher mobility than the average, could be a candidate region to participate in those processes. Within the cavity of the holo form, clear electron density was observed that is well ﬁtted by a single FA molecule, containing 16 carbon atoms (Figure 2d). Since the predominant FA found in E. coli lipids is palmitate, we ﬁtted this to the electron density. The palmitate appears to be curled into a C-shape around the side chain of Lys96 and contacts 11 other residues (Phe1, Ile6, Met14, Phe21, Leu22, Leu25, Leu33, Leu66, Ile95, Tyr99 and Tyr100). Additional electron density, possibly indicative of several other bound ligand molecules, is seen in the cavity of Na-FAR-1, but in light of the heterogeneous mixture of lipids associated with the protein and the poor quality of the density, we have not modelled them. 76 1DCaHa) observed in a sample that had been partially aligned in Pf1 ﬁlamentous bacteriophage (Table 2). terminal helices, 310 (residues 3–5), α2 (8–11) and α3 (16–24) are co-planar with the C-terminal helices α9 (126–135), α10 (137– 144) and α11 (147–152) and together with one of the longest helices, α6 (57–75), form one large face of the wedge. The other two long helices, α7 (79–99) and α8 (107–122), are co-planar with α5 (45–55) and form the other large face. These two faces enclose an internal cavity that is closed at the thick end of the wedge by α4 (29–37), which is almost perpendicular to the long axis of the molecule. Hydrophobic residues are located almost exclusively on the inward-facing sides of the helices with their side chains pointing towards the internal cavity. Most of the polar and charged The overall folds of the Na-FAR-1 structures in complex with ligands (holo) and without ligand (apo) are similar (Figure 2a–c). Superposition of the holo and apo structures gives a 1.814 Å co-ordinate RMSD (root-mean-square deviation) for all heavy atoms (1.579 Å for main chain heavy atoms only). Na-FAR-1 presents a wedge-shaped structure with two larger faces each of approximately 40 by 30 Å in area and of ∼17 Å in width at the wide end of the wedge. The fold is organized into 11 helices of various lengths, deﬁning an internal cavity (Figure 3). The N- M.F. Rey-Burusco and others 408 Table 2 Structural statistics for the ensemble of 20 apo-Na-FAR-1 structures determined by NMR spectroscopy Table 2 Structural statistics for the ensemble of 20 apo-Na-FAR-1 structures determined by NMR spectroscopy NOE distance restraints Total NOE 7097 Ambiguous 3443 Unambiguous 3654 Intra-residue 1548 Inter-residue 2106 Sequential (\|i −j\| = 1) 833 Short range (1 < \|i −j\| ⩽4) 756 Long range (\|i −j\| ⩾5) 517 Violations > 0.5 A˚ 0.3 Violations > 0.3 A˚ 1.6 Distance restraints RMSD (A˚) 0.016 Coordinate RMSD (A˚) Backbone heavy atoms 0.597 All heavy atoms 1.012 Parameter RMSD from idealized geometry (mean +−S.D.) Bond lengths (A˚) 0.00326 +−1.4 × 10 −4 Bond angles (◦) 0.595 +−0.018 Impropers (◦) 1.57 +−0.07 RDC restraints 1DNH 102 1DCαHα 76 RDC Q factor 0.164 Dihedral angles 312 Ramachandran statistics (%) Most favoured 92.9 Additionally allowed 6.0 Generously allowed 0.8 Disallowed 0.3 Val117 and Phe143. The cavity has a single opening located on one side of the wedge between α6 and α7 and the α4–α5 loop that is surrounded by charged residues. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. Figure 2 The structure of Na-FAR-1 (a) Stereoscopic view (wall-eyed) of the superimposed apo- and holo-Na-FAR-1 structures shown in cartoon form. The apo structure determined by NMR is coloured from blue at the N-terminus through to red at the C-terminus. The holo form determined by X-ray crystallography is shown in grey. (b) The ensemble of the 20 lowest energy structures of apo-Na-FAR-1 shown in two orientations related by a 90◦rotation. (c) The holo-Na-FAR-1 structure shown in b-factor putty representation with the palmitate shown as sticks. (d) Expanded view of the bound palmitate and its environment with 2Fo-Fc electron density within 4 A˚ of the ligand displayed as a mesh at 1σ and shaded yellow and Fo-Fc difference density displayed at +−3σ (green and red). (a) Stereoscopic view (wall-eyed) of the superimposed apo- and holo-Na-FAR-1 structures shown in cartoon form. The apo structure determined by NMR is coloured from blue at the N-terminus through to red at the C-terminus. The holo form determined by X-ray crystallography is shown in grey. (b) The ensemble of the 20 lowest energy structures of apo-Na-FAR-1 shown in two orientations related by a 90◦rotation. (c) The holo-Na-FAR-1 structure shown in b-factor putty representation with the palmitate shown as sticks. (d) Expanded view of the bound palmitate and its environment with 2Fo-Fc electron density within 4 A˚ of the ligand displayed as a mesh at 1σ and shaded yellow and Fo-Fc difference density displayed at +−3σ (green and red). (a) Stereoscopic view (wall-eyed) of the superimposed apo- and holo-Na-FAR-1 structures shown in cartoon form. The apo structure determined by NMR is coloured from blue at the N-terminus through to red at the C-terminus. The holo form determined by X-ray crystallography is shown in grey. (b) The ensemble of the 20 lowest energy structures of apo-Na-FAR-1 shown in two orientations related by a 90◦rotation. (c) The holo-Na-FAR-1 structure shown in b-factor putty representation with the palmitate shown as sticks. (d) Expanded view of the bound palmitate and its environment with 2Fo-Fc electron density within 4 A˚ of the ligand displayed as a mesh at 1σ and shaded yellow and Fo-Fc difference density displayed at +−3σ (green and red). Comparison of the structures of Na-FAR-1 and Ce-FAR-7 The notable exceptions are Ser88, Lys96 and Tyr100 whose side chains are located within the cavity, whereas His67 and Arg93 stand on either sides of the largest opening to the cavity. Despite their similarities, the structures of the apo and holo forms of Na-FAR-1 reveal signiﬁcant differences that reﬂect structural alterations upon ligand binding. The internal cavity is much larger for the holo protein, achieved by a global outward displacement of the surrounding helices by a little over 1 Å compared with the apo form, with the biggest changes seen at the C-terminal end of helix α7 (Figure 2a). For the apo structure, the central cavity has a volume of approximately 1220 Å3 accessible to a 1.4 Å radius probe (equivalent to a water molecule) or 940 Å3 accessible to a 1.925 Å probe (equivalent to a CH2 group). In the holo form, the cavity is more than twice as big, reaching 2570 and 2170 Å3 for probes of 1.4 and 1.925 Å respectively. The main part of the apo-Na-FAR-1 cavity is lined by the side chains of residues Leu13, Met14, Pro15, Ala18, Phe21, Leu22, Leu33, Phe37, Thr45, Val63, Leu66, His67, Val70, Ile89, Ala92, Arg93, Lys96, Tyr100 and Leu139, most of which are apolar, with polar groups con- tributed by the side chain of Tyr100 and backbone carbonyl of Leu13 and the positively charged Lys96. His67 and Arg93 are located at the entrance to the cavity. A further, less accessible volume is located beyond a pinch point between Ala92 and Leu139 and is lined by the predominately hydrophobic side chains of Tyr10, Ser88, Val114, Structure of Necator americanus FAR protein 409 Structure of Necator americanus FAR protein 409 ucture of Na-FAR-1 (wall-eyed) of the superimposed apo- and holo-Na-FAR-1 structures shown in cartoon form. The apo structure determined by NMR is coloured from blue at the N-terminus terminus. The holo form determined by X-ray crystallography is shown in grey. (b) The ensemble of the 20 lowest energy structures of apo-Na-FAR-1 shown in two orientations on. (c) The holo-Na-FAR-1 structure shown in b-factor putty representation with the palmitate shown as sticks. (d) Expanded view of the bound palmitate and its environment ensity within 4 A˚ of the ligand displayed as a mesh at 1σ and shaded yellow and Fo-Fc difference density displayed at +−3σ (green and red). Figure 2 The structure of Na-FAR-1 The estimated cavity for Ce-FAR-7 calculated with CASTp has a much smaller size than for both forms of Na-FAR-1, with a volume of 687.7 Å3 accessible to a probe equivalent to a water molecule and just to 390.0 Å3 for a probe equivalent to a CH2 group. In Ce-FAR-7, helices α2 and α3 approach α7 and α8 more closely, reducing the volume of the cavity and resulting in two distinct hydrophobic pockets. The majority of residues that line the Na-FAR-1 cavity and the Ce-FAR-7 P1 and P2 pockets are conserved or conservatively substituted. The exceptions are that the Na-FAR-1 cavity includes the side chains of more polar amino acids, such as Ser88, Arg93 and Lys96, which are found in positions occupied by Leu89, Thr94 and Leu97 respectively, in Ce-FAR-7. lipid retinol (Figures 4d and 4e). The degree of blue shift in DAUDA ﬂuorescence emission (from 543 nm in buffer to 480 nm) indicates Na-FAR-1 has a highly apolar binding site, as shown for other members of the FAR protein family [10]. It is also interesting to note that Ce-FAR-7 has shown no binding capacity for DAUDA [10] and binding to retinol showed no saturation [24]. The preference of Na-FAR-1 for FAs was investigated by the addition of FAs with different chain lengths to preformed protein–DAUDA complexes (Supplementary Figure S3), showing that DAUDA displacement was greatest with saturated FAs in the range C14:0–C19:0. In order to determine the lipid classes bound to Na-FAR-1 in a cellular environment, a Folch extraction of the lipidic fraction of Na-FAR-1 puriﬁed from E. coli and subsequent TLC analyses were performed. The bacterial cytoplasm is not the natural environment of Na-FAR-1, but this approach could contribute to the understanding of its binding preferences. The c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. Ligand binding Comparison of Na-FAR-1 apo (b and e) and holo (c and f) forms with Ce-FAR-7 (d and g) shown in cartoon representation and coloured from blue (N-terminus) to red (C-terminus) viewed from two orientations related by a 90◦rotation about the horizontal axis. Internal cavities accessible to a probe of 1.925 A˚, equivalent to a methylene group are shown as transparent surfaces with the surrounding sidechains shown as sticks. (PDB accession codes Na-FAR-1 apo 4UET and holo 4XCP; Ce-FAR-7, 29WY) lipid fraction bound to Na-FAR-1 exhibits heterogeneous lipid content (Figure 4a). Previous studies have shown that other members of the FARs bind retinol and FAs [10,13,15,19,21]. In addition, the present analysis reveals that the protein binds not only to FAs but also a broader range of lipid classes such as PLs. binds three ligands with high afﬁnity. Beyond this, additional chemical shift changes were observed in the fast exchange regime from a 1:4 protein–ligand ratio where only one cross peak was seen for each amide group. The titration was carried on to a maximum protein–ligand ratio of 1:10 where it was limited by the precipitation of excess, unbound ligand. These results suggest that the protein binds up to at least four oleate molecules, three of them with high afﬁnity in the sub micromolar range and a fourth with lower afﬁnity. Substantial chemical shift changes were observed for residues throughout the protein, consistent both with ligand binding affecting the local environment of residues within the helices surrounding the cavity and with the changes in conformation of residues in the loops required to allow the structure to expand. The qualitative analysis conducted suggests the presence of polar lipids such as phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Esteriﬁcation and analysis of the non-esteriﬁed FA and PL-derived FA methyl esters by GC–MS (Figures 4b and 4c) showed that in the lipids carried by Na-FAR-1, palmitate (16:0) was the most common FA. Examination of total E. coli lipids showed a similar FA composition. Protein–ligand interactions were further investigated by titration of Na-FAR-1 with sodium oleate followed using NMR. Ligand binding In ﬂuorescence-based ligand-binding assays, Na-FAR-1 bound the ﬂuorescent FA analogue DAUDA and the naturally ﬂuorescent c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3 M.F. Rey-Burusco and others 410 Figure 3 Na-FAR-1 cavities and comparison with Ce-FAR-7 (a) Sequence alignment of Na-FAR-1 and Ce-FAR-7. Secondary structure elements are indicated in boxes (α-helix) and lines (loops) coloured grey for Na-FAR-1 and yellow Ce-FAR-7. Comparison of Na-FAR-1 apo (b and e) and holo (c and f) forms with Ce-FAR-7 (d and g) shown in cartoon representation and coloured from blue (N-terminus) to red (C-terminus) viewed from two orientations related by a 90◦rotation about the horizontal axis. Internal cavities accessible to a probe of 1.925 A˚, equivalent to a methylene group are shown as transparent surfaces with the surrounding sidechains shown as sticks. (PDB accession codes Na-FAR-1 apo 4UET and holo 4XCP; Ce-FAR-7, 29WY) ities and comparison with Ce-FAR-7 FAR-1 and Ce-FAR-7. Secondary structure elements are indicated in boxes (α-helix) and lines (loops) coloured grey for Na holo (c and f) forms with Ce-FAR-7 (d and g) shown in cartoon representation and coloured from blue (N-terminus) to red t the horizontal axis. Internal cavities accessible to a probe of 1.925 A˚, equivalent to a methylene group are shown as DB i d N FAR 1 4UET d h l 4XCP C FAR 29WY) Figure 3 Na-FAR-1 cavities and comparison with Ce-FAR-7 (a) Sequence alignment of Na-FAR-1 and Ce-FAR-7. Secondary structure elements are indicated in boxes (α-helix) and lines (loops) coloured grey for Na-FAR-1 and yellow Ce-FAR-7. Comparison of Na-FAR-1 apo (b and e) and holo (c and f) forms with Ce-FAR-7 (d and g) shown in cartoon representation and coloured from blue (N-terminus) to red (C-terminus) viewed from two orientations related by a 90◦rotation about the horizontal axis. Internal cavities accessible to a probe of 1.925 A˚, equivalent to a methylene group are shown as transparent surfaces with the surrounding sidechains shown as sticks. (PDB accession codes Na-FAR-1 apo 4UET and holo 4XCP; Ce-FAR-7, 29WY) (a) Sequence alignment of Na-FAR-1 and Ce-FAR-7. Secondary structure elements are indicated in boxes (α-helix) and lines (loops) coloured grey for Na-FAR-1 and yellow Ce-FAR-7. Ligand binding The ﬁrst step of the titration was made by adding of 0.5 molar equivalents of sodium oleate (Figure 5), at which point two cross-peaks originating from each shifted amide group were detected, one remaining at the original shift that corresponds to the apo protein conformation and another cross-peak at a different shift resulting from the protein conformation in the presence of one ligand bound. At subsequent stages in the titration, further chemical shift changes were observed, in each case resulting in the appearance of a new cross-peak and disappearance of the original. The ligand binding process therefore exhibited slow exchange behaviour through the addition of 1, 2 and 3 molar equivalents of oleate, which would suggest that the protein c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. CONCLUSION FAs detected were: myristic (14:0), palmitic (16:0), palmitoleic (16:1), margarinic (17:0), heptadecenoic (17:1), stearic (18:0), oleic (18:19), vaccenic (18:111) and nonadecenoic acid (19:1). (c) FAs found in PLs isolated from puriﬁed Na-FAR-1 (dark grey) or found in E. coli extracts (light grey). (d and e) Fluorescent ligand binding by Na-FAR-1. (d) Fluorescence emission spectra of 1 μM DAUDA excited at 345 nm in buffer, on the addition of 1 μM Na-FAR-1 and after successive additions of oleic acid (81 nM, 810 nM and 8.1 μM). (e) Fluorescence emission spectra of 5 μl of 0.15 mM retinol in ethanol added to the ﬂuorescence cuvette and excited at 350 nm and on the addition of 1.5 μM Na-FAR-1, compared with 1.5 μM protein in buffer and buffer alone. (a) Lipid fractions bound to bacterially-expressed Na-FAR-1 detected by TLC. Lipids were extracted from the protein and fractions were analysed by TLC in conditions for resolving separately neutral (left panel) and polar (right panel) lipid classes. Standards and samples applied to TLC plates were: Hol, Na-FAR-1 puriﬁed without an HPLC step; Ec, extract from whole E. coli cells; LIV, standard mix of lipids from rat liver homogenate; STD, E. coli whole extract. CHO, cholesterol; TG, triglycerides; CHOe, cholesterol esters; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; CL, cardiolipin. (b) GC–MS analysis of non-esteriﬁed FAs isolated from lipids associated with Na-FAR-1 puriﬁed from E. coli (dark grey) or found in E. coli extracts (light grey). FAs detected were: myristic (14:0), palmitic (16:0), palmitoleic (16:1), margarinic (17:0), heptadecenoic (17:1), stearic (18:0), oleic (18:19), vaccenic (18:111) and nonadecenoic acid (19:1). (c) FAs found in PLs isolated from puriﬁed Na-FAR-1 (dark grey) or found in E. coli extracts (light grey). (d and e) Fluorescent ligand binding by Na-FAR-1. (d) Fluorescence emission spectra of 1 μM DAUDA excited at 345 nm in buffer, on the addition of 1 μM Na-FAR-1 and after successive additions of oleic acid (81 nM, 810 nM and 8.1 μM). (e) Fluorescence emission spectra of 5 μl of 0.15 mM retinol in ethanol added to the ﬂuorescence cuvette and excited at 350 nm and on the addition of 1.5 μM Na-FAR-1, compared with 1.5 μM protein in buffer and buffer alone. sequence diversity among FARs [19,24,67,68]. CONCLUSION Standards and samples applied to TLC plates were: Hol, Na-FAR-1 puriﬁed without an HPLC step; Ec, extract from whole E. coli standard mix of lipids from rat liver homogenate; STD, E. coli whole extract. CHO, cholesterol; TG, triglycerides; CHOe, cholesterol esters; PC, phosphatidylcholine; PS, phosphatidy phosphatidylinositol; CL, cardiolipin. (b) GC–MS analysis of non-esteriﬁed FAs isolated from lipids associated with Na-FAR-1 puriﬁed from E. coli (dark grey) or found in E. coli ext grey). FAs detected were: myristic (14:0), palmitic (16:0), palmitoleic (16:1), margarinic (17:0), heptadecenoic (17:1), stearic (18:0), oleic (18:19), vaccenic (18:111) and nonadec (19:1). (c) FAs found in PLs isolated from puriﬁed Na-FAR-1 (dark grey) or found in E. coli extracts (light grey). (d and e) Fluorescent ligand binding by Na-FAR-1. (d) Fluorescence spectra of 1 μM DAUDA excited at 345 nm in buffer, on the addition of 1 μM Na-FAR-1 and after successive additions of oleic acid (81 nM, 810 nM and 8.1 μM). (e) Fluorescence spectra of 5 μl of 0.15 mM retinol in ethanol added to the ﬂuorescence cuvette and excited at 350 nm and on the addition of 1.5 μM Na-FAR-1, compared with 1.5 μM protein in buffer alone. CONCLUSION The FAR proteins are unique to nematodes and we in the present study demonstrate the ﬁrst structure of a member of this family from a parasitic species. FARs are presentin the secretory products of parasites, which is apparent both for those that reside in the lumen of the gut and those that penetrate and reside in the host tissue of animals and plants [22,67]. This family of proteins comprises subfamilies that differ in their ligand-binding characteristics and in whether or not they are secreted from The FAR proteins are unique to nematodes and we in the present study demonstrate the ﬁrst structure of a member of this family from a parasitic species. FARs are presentin the secretory products of parasites, which is apparent both for those that reside in the lumen of the gut and those that penetrate and reside in the host tissue of animals and plants [22,67]. This family of proteins comprises subfamilies that differ in their ligand-binding characteristics and in whether or not they are secreted from Structure of Necator americanus FAR protein 411 Structure of Necator americanus FAR protein 350 400 450 500 550 600 650 700 0.0 0.2 0.4 0.6 0.8 1.0 1.2 + successive additions of oleic acid DAUDA + rNa-FAR-1 Relative fluorescence intensity Wavelength of emission (nm) DAUDA in buffer alone 350 400 450 500 550 600 650 0.0 0.2 0.4 0.6 0.8 1.0 Buffer + retinol + rNa-FAR-1 Relative fluorescence intensity Buffer + rNa-FAR-1 Buffer + retinol Buffer alone Wavelength of emission (nm) 0 5 10 15 20 25 30 35 40 14:0 m 16:0 p 16:1 po 17:0 m 17:1 h o 18:0 s 18:1 o 18:1 v 19:1 no Relative abundance % free FA Na FAR E.c.+IPTG 0 5 10 15 20 25 30 35 40 14:0 m 16:0 p 16:1 po 17:0 m 17:1 h o 18:0 s 18:1 o 18:1 v 19:1 no Relative abundance % FA from phospholipids Na FAR E.c.+IPTG Hol Ec Liv Std Hol Ec Ec Liv Oa (a) (b) (c) (d) (e) CL PC PS PI PE FFA TG CHOe PG PE CHOe TG FFA CHO Figure 4 Lipid binding by recombinant Na-FAR-1 (a) Lipid fractions bound to bacterially-expressed Na-FAR-1 detected by TLC. Lipids were extracted from the protein and fractions were analysed by TLC in conditions for resolving neutral (left panel) and polar (right panel) lipid classes. CONCLUSION Hol Ec Liv Std CL PC PS PI PE FFA TG CHOe PG PE (a) 0 5 10 15 20 25 30 35 40 14:0 m 16:0 p 16:1 po 17:0 m 17:1 h o 18:0 s 18:1 o 18:1 v 19:1 no Relative abundance % free FA Na FAR E.c.+IPTG 0 5 10 15 20 25 30 35 40 14:0 m 16:0 p 16:1 po 17:0 m 17:1 h o 18:0 s 18:1 o 18:1 v 19:1 no Relative abundance % FA from phospholipids Na FAR E.c.+IPTG Hol Ec Liv Std Hol Ec Ec Liv Oa (b) (c) (d) (e) CL PC PS PI PE PG PE TG FFA CHO 0 5 10 15 20 25 30 35 40 14:0 m 16:0 p 16:1 po 17:0 m 17:1 h o 18:0 s 18:1 o 18:1 v 19:1 no Relative abundance % free FA Na FAR E.c.+IPTG 0 5 10 15 20 25 30 35 40 14:0 m 16:0 p 16:1 po 17:0 m 17:1 h o 18:0 s 18:1 o 18:1 v 19:1 no Relative abundance % FA from phospholipids Na FAR E.c.+IPTG (b) (c) (d) (e) FA from phospholipids Na FAR E IPTG (d) (e) 350 400 450 500 550 600 650 0.0 0.2 0.4 0.6 0.8 1.0 Buffer + retinol + rNa-FAR-1 Relative fluorescence intensity Buffer + rNa-FAR-1 Buffer + retinol Buffer alone Wavelength of emission (nm) e) 350 400 450 500 550 600 650 700 0.0 0.2 0.4 0.6 0.8 1.0 1.2 + successive additions of oleic acid DAUDA + rNa-FAR-1 Relative fluorescence intensity Wavelength of emission (nm) DAUDA in buffer alone (d) Wavelength of emission (nm) Figure 4 Lipid binding by recombinant Na-FAR-1 (a) Lipid fractions bound to bacterially-expressed Na-FAR-1 detected by TLC. Lipids were extracted from the protein and fractions were analysed by TLC in conditions for resolving separately neutral (left panel) and polar (right panel) lipid classes. Standards and samples applied to TLC plates were: Hol, Na-FAR-1 puriﬁed without an HPLC step; Ec, extract from whole E. coli cells; LIV, standard mix of lipids from rat liver homogenate; STD, E. coli whole extract. CHO, cholesterol; TG, triglycerides; CHOe, cholesterol esters; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; CL, cardiolipin. (b) GC–MS analysis of non-esteriﬁed FAs isolated from lipids associated with Na-FAR-1 puriﬁed from E. coli (dark grey) or found in E. coli extracts (light grey). c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. AUTHOR CONTRIBUTION Florencia Rey-Burusco, Mads Gabrielsen, Kate Grifﬁths, Gisela Franchini Bin Zhan, Alan Cooper, Malcolm Kennedy, Betina C´orsico and Brian Smith performed the experiments. Florencia Rey-Burusco, Marina Ib´a˜nez-Shimabukuro, Mads Gabrielsen, Gisela Franchini Andrew Roe, Kate Grifﬁths, Bin Zhan, Alan Cooper, Malcolm Kennedy, Betina C´orsico and Brian Smith analysed the data. Florencia Rey-Burusco, Betina C´orsico, Malcolm Kennedy and Brian Smith wrote the paper. All authors have seen, made appropriate modiﬁcations to and approved of the manuscript. CONCLUSION 78 46 4 85 155 1 2 89 6 73 81 38 26 95 39 82 25 37 125 36 123 58 17 24N 54 86 72 12 24 9 106 128 145 56 23Q 102 44 94 40 60 153 83 133 101 43 3 146 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 105 110 115 120 125 δ15N (ppm) δ1H (ppm) 0 1 2 3 4 number of oleate bound Figure 5 Titration of Na-FAR-1 with sodium oleate followed by NMR spectroscopy 15N HSQC spectra of 0.4 mM Na-FAR-1 with successive additions of sodium oleate. The spectrum of the apo protein is shown in dark blue, with spectra coloured through purple to red at the ﬁnal protein–ligand ratio of 1:10. A subset of the backbone and side chain (lettered) assignments are shown and the trajectories of selected residues indicated with arrows. The inset shows an expanded view of the cross-peaks from Gly78 backbone amide with our interpretation of the protein–ligand stoichiometry responsible for each distinct cross-peak position. 15N HSQC spectra of 0.4 mM Na-FAR-1 with successive additions of sodium oleate. The spectrum of the apo protein is shown in dark blue, with spectra coloured through purple to red at the ﬁnal protein–ligand ratio of 1:10. A subset of the backbone and side chain (lettered) assignments are shown and the trajectories of selected residues indicated with arrows. The inset shows an expanded view of the cross-peaks from Gly78 backbone amide with our interpretation of the protein–ligand stoichiometry responsible for each distinct cross-peak position. although there was no change for FA. In both Ce-FAR-7 and Na- FAR-1, the amino acid side chain thought to be phosphorylated lies in a short unstructured region between two helices at one end of the molecule. In neither protein is the side chain of this residue oriented towards the internal cavity and nor in Ce-FAR-7 it is more distant from the internal surface than in Na-FAR-1. It is therefore not immediately clear how phosphorylation could affect the proteins’ ligand-binding characteristics unless structural changes result. and enzymes that transport or modify FAs and retinoids. But, it is entirely possible that we have yet to discover the true range of the ligand-binding propensities of FARs in nematodes. CONCLUSION An understanding of their structures and how they vary between those secreted by different species of parasites could illuminate their roles in parasitism and suggest possible targets for therapeutic interventions. g The differences in the size and shape of the internal cavities of Ce-FAR-7 and Na-FAR-1 are probably indicative of differences in their ligand selectivity. Both proteins bind retinol and FAs, but whether these compounds are relevant in vivo and whether other classes of ligand (such as PLs here found to be bound by Na-FAR- 1) are important, remains to be established. The fact that FARs are commonly found in the secretions of parasitic nematodes bolsters conﬁdence that they play an important role in parasitism. Parasites need to acquire nutrients from their hosts, but also need to defend themselves against immune defence reactions and some parasites induce modiﬁcations to the tissues they occupy. If FARs do bind signalling lipids, then it is conceivable that they are released by parasites in order to subvertor modify the hosts’ tissue and defence reactions. If so, then FARs would seem to be logical targets for vaccines, particularly against parasites that are embedded in host tissues. Development of drugs against them would need to take into account the possibility of interference with the hosts’ proteins c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. CONCLUSION This argues that this site may have some signiﬁcance in, for instance, altering the ligand-binding activity of the proteins or modifying interaction with other cellular factors such as cell surface receptors (host or parasite). There is currently no evidence that this site is phosphorylated naturally, though experiments with Ce-FAR-7 indicate that modiﬁcation of this site does alter ligand binding. The previous study found that direct phosphorylation rendered the protein unstable in some unknown way, but when phosphorylation was mimicked by substitution of the presumed target threonine by an aspartic acid, the afﬁnity of Ce-FAR-7 for retinol increased, the cells that synthesize them. Transcripts encoding Na-FAR- 1 are relatively abundant in the parasite from which it derives and the encoded polypeptide bears a secretory signal sequence. It falls into a new subfamily of the FARs that has not been found so far in any other group of nematodes, although the inventory of FARs is only comprehensive for those species for which genomic information is essentially complete and annotated (e.g. C. elegans). g It has been noted that all FAR protein sequences exhibit a consensus casein kinase II-type sequence that occurs in approximately the same place, despite the considerable degree of M.F. Rey-Burusco and others 412 78 46 4 85 155 1 2 89 6 73 81 38 26 95 39 82 25 37 125 36 123 58 17 24N 54 86 72 12 24 9 106 128 145 56 23Q 102 44 94 40 60 153 83 133 101 43 3 146 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 105 110 115 120 125 δ15N (ppm) δ1H (ppm) 0 1 2 3 4 number of oleate bound Figure 5 Titration of Na-FAR-1 with sodium oleate followed by NMR spectroscopy 15N HSQC spectra of 0.4 mM Na-FAR-1 with successive additions of sodium oleate. The spectrum of the apo protein is shown in dark blue, with spectra coloured through purple to red at the ﬁnal protein–ligand ratio of 1:10. A subset of the backbone and side chain (lettered) assignments are shown and the trajectories of selected residues indicated with arrows. The inset shows an expanded view of the cross-peaks from Gly78 backbone amide with our interpretation of the protein–ligand stoichiometry responsible for each distinct cross-peak position. PLoS Negl. Trop. Dis. 8, e2865 CrossRef PubMed 26 Pullan, R.L., Smith, J.L., Jasrasaria, R. and Brooker, S.J. (2014) Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasit. Vectors 7, 37 CrossRef PubMed 8 Mei, B., Kennedy, M.W., Beauchamp, J., Komuniecki, P.R. and Komuniecki, R. (1997) Secretion of a novel, developmentally regulated fatty acid-binding protein into the perivitelline ﬂuid of the parasitic nematode, Ascaris suum. J. Biol. Chem. 272, 9933–9941 CrossRef PubMed 27 Rey-Burusco, M.F., Ibanez-Shimabukuro, M., Cooper, A., Kennedy, M.W., Corsico, B. and Smith, B.O. (2014) 1H, 13C and 15N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus. Biomol. NMR Assign. 8, 19–21 CrossRef PubMed 9 Pleneﬁsch, J., Xiao, H., Mei, B.S., Geng, J.M., Komuniecki, P.R. and Komuniecki, R. (2000) Secretion of a novel class of iFABPs in nematodes: coordinate use of the Ascaris/Caenorhabditis model systems. Mol. Biochem. Parasitol. 105, 223–236 CrossRef PubMed 28 Gabrielsen, M., Rey-Burusco, M.F., Grifﬁths, K., Roe, A.J., Cooper, A., Smith, B.O., Kennedy, M.W. and Corsico, B. (2012) Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68, 835–838 CrossRef PubMed 10 Garofalo, A., Rowlinson, M.C., Amambua, N.A., Hughes, J.M., Kelly, S.M., Price, N.C., Cooper, A., Watson, D.G., Kennedy, M.W. and Bradley, J.E. (2003) The FAR protein family of the nematode Caenorhabditis elegans - differential lipid binding properties, structural characteristics, and developmental regulation. J. Biol. Chem. 278, 8065–8074 CrossRef PubMed 29 Williamson, A.L., Brindley, P.J., Abbenante, G., Prociv, P., Berry, C., Girdwood, K., Pritchard, D.I., Fairlie, D.P., Hotez, P.J., Dalton, J.P. and Loukas, A. (2002) Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host speciﬁcity. FASEB J 16, 1458–1460 PubMed 30 Kabsch, W. (2010) Xds. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 125–132 CrossRef 11 Jones, J.T., Kumar, A., Pylypenko, L.A., Thirugnanasambandam, A., Castelli, L., Chapman, S., Cock, P.J., Grenier, E., Lilley, C.J., Phillips, M.S. and Blok, V.C. (2009) Identiﬁcation and functional characterization of effectors in expressed sequence tags from various life cycle stages of the potato cyst nematode Globodera pallida. Mol. Plant. Pathol. 10, 815–828 CrossRef PubMed 31 Evans, P. (2006) Scaling and assessment of data quality. Acta Crystallogr. Sect. D Biol. Crystallogr. 62, 72–82 CrossRef 32 Pajinkar, S., Parthasarathy, V., Lamzin, V.S., Weiss, M.S. and Tucker, P.A. PLoS Negl. Trop. Dis. 8, e2865 CrossRef PubMed (2005) Auto-Rickshaw - an automated crystal structure determination platform as an efﬁcient tool for the validation of an X-ray diffraction experiment. Acta Crystallogr. Sect. D Biol. Crystallogr. 61, 760–763 12 Popeijus, M., Blok, V.C., Cardle, L., Bakker, E., Phillips, M.S., Helder, J., Smant, G. and Jones, J.T. (2000) Analysis of genes expressed in second stage juveniles of the potato cyst nematodes Globodera rostochiensis and G. pallida using the expressed sequence tag approach. Nematology 2, 567–574 CrossRef 33 Winn, M.D., Ballard, C.C., Cowtan, K.D., Dodson, E.J., Emsley, P., Evans, P.R., Keegan, R.M., Krissinel, E.B., Leslie, A.G., McCoy, A. et al. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 235–242 CrossRef 13 Prior, A., Jones, J.T., Blok, V.C., Beauchamp, J., McDermott, L., Cooper, A. and Kennedy, M.W. (2001) A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern. Biochem. J. 356, 387–394 CrossRef PubMed 34 Sheldrick, G.M. (2010) Experimental phasing with SHELXC/D/E: combining chain tracing with density modiﬁcation. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 479–485 CrossRef 14 Ranjit, N., Jones, M.K., Stenzel, D.J., Gasser, R.B. and Loukas, A. (2006) A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy. Int. J. Parasitol. 36, 701–710 CrossRef PubMed 35 Schneider, T.R. and Sheldrick, G.M. (2002) Substructure solution with SHELXD. Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 1772–1779 CrossRef 36 Hao, Q. (2004) ABS: a program to determine absolute conﬁguration and evaluate anomalous scatterer substructure. J. Appl. Crystallogr. 37, 498–499 CrossRef 15 Basavaraju, S.V., Zhan, B., Kennedy, M.W., Liu, Y., Hawdon, J. and Hotez, P.J. (2003) Ac-FAR-1, a 20 kDa fatty acid- and retinol-binding protein secreted by adult Ancylostoma caninum hookworms: gene transcription pattern, ligand binding properties and structural characterisation. Mol. Biochem. Parasitol. 126, 63–71 CrossRef PubMed 37 Perrakis, A., Morris, R. and Lamzin, V.S. (1999) Automated protein model building combined with iterative structure reﬁnement. Nat. Struct. Biol. 6, 458–463 CrossRef PubMed 38 Emsley, P., Lohkamp, B., Scott, W.G. and Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 486–501 CrossRef 16 McCarter, J.P., Mitreva, M.D., Martin, J., Dante, M., Wylie, T., Rao, U., Pape, D., Bowers, Y., Theising, B., Murphy, C.V. et al. (2003) Analysis and functional classiﬁcation of transcripts from the nematode Meloidogyne incognita. REFERENCES 1 Storch, J. and Corsico, B. (2008) The emerging functions and mechanisms of mammalian fatty acid-binding proteins. Annu. Rev. Nutr. 28, 73–95 CrossRef PubMed 1 Storch, J. and Corsico, B. (2008) The emerging functions and mechanisms of mammalian fatty acid-binding proteins. Annu. Rev. Nutr. 28, 73–95 CrossRef PubMed 20 Burbelo, P.D., Leahy, H.P., Iadarola, M.J. and Nutman, T.B. (2009) A four-antigen mixture for rapid assessment of Onchocerca volvulus infection. PLoS Negl. Trop. Dis. 3, e438 20 Burbelo, P.D., Leahy, H.P., Iadarola, M.J. and Nutman, T.B. (2009) A four-antigen mixture for rapid assessment of Onchocerca volvulus infection. PLoS Negl. Trop. Dis. 3, e438 CrossRef PubMed 2 Meenan, N.A, Ball, G., Bromek, K., Uhrin, D., Cooper, A., Kennedy, M.W. and Smith, B.O. (2011) Solution structure of a repeated unit of the ABA-1 nematode polyprotein allergen of ascaris reveals a novel fold and two discrete lipid-binding sites. PLoS Negl. Trop. Dis. 5, e1040 CrossRef PubMed 2 Meenan, N.A, Ball, G., Bromek, K., Uhrin, D., Cooper, A., Kennedy, M.W. and Smith, B.O. (2011) Solution structure of a repeated unit of the ABA-1 nematode polyprotein allergen of ascaris reveals a novel fold and two discrete lipid-binding sites. PLoS Negl. Trop. Dis. 5, e1040 CrossRef PubMed 21 Kennedy, M.W., Garside, L.H., Goodrick, L.E., McDermott, L., Brass, A., Price, N.C., Kelly, S.M., Cooper, A. and Bradley, J.E. (1997) The Ov20 protein of the parasitic nematode Onchocerca volvulus - a structurally novel class of small helix-rich retinol-binding proteins. J. Biol. Chem. 272, 29442–29448 CrossRef PubMed 3 Kennedy, M.W. (2000) The polyprotein lipid binding proteins of nematodes. Biochim. Biophys. Acta 1476, 149–164 CrossRef PubMed 3 Kennedy, M.W. (2000) The polyprotein lipid binding proteins of nematodes. Biochim. Biophys. Acta 1476, 149–164 CrossRef PubMed 22 Iberkleid, I., Vieira, P., de Almeida Engler, J., Firester, K., Spiegel, Y. and Horowitz, S.B. (2013) Fatty acid-and retinol-binding protein, Mj-FAR-1 induces tomato host susceptibility to root-knot nematodes. PLoS One 8, e64586 CrossRef PubMed 4 Kennedy, M.W., Allen, J.E., Wright, A.S., McCruden, A.B. and Cooper, A. (1995) The Gp15/400 polyprotein antigen of Brugia malayi binds fatty- acids and retinoids. Mol. Biochem. Parasitol. 71, 41–50 CrossRef PubMed 4 Kennedy, M.W., Allen, J.E., Wright, A.S., McCruden, A.B. and Cooper, A. (1995) The Gp15/400 polyprotein antigen of Brugia malayi binds fatty- acids and retinoids. Mol. Biochem. Parasitol. 71, 41–50 CrossRef PubMed 23 Lal, P.G. and James, E.R. (1996) Onchocerca retinol- and ivermectin-binding protein activity. FUNDING This work was supported by the Wellcome Trust [grant number 083625 (to M.W.K., A.C., B.O.S. and B.C.)]; the National Research Council of Argentina (to M.F.R.-B., M.I.-S., G.R.F. and B.C.O); the European Molecular Biology Organization short term fellowship [grant number ASTF 12-2012 (to M.F.R.-B.)]; and the Biotechnology and Biological Sciences Research Council [grant numbers (BB/G011389/1 (to A.J.R. and M.G.)]. This work was supported by the Wellcome Trust [grant number 083625 (to M.W.K., A.C., B.O.S. and B.C.)]; the National Research Council of Argentina (to M.F.R.-B., M.I.-S., G.R.F. and B.C.O); the European Molecular Biology Organization short term fellowship [grant number ASTF 12-2012 (to M.F.R.-B.)]; and the Biotechnology and Biological Sciences Research Council [grant numbers (BB/G011389/1 (to A.J.R. and M.G.)]. 18 Ramanathan, R., Varma, S., Ribeiro, J.M, Myers, T.G., Nolan, T.J., Abraham, D., Lok, J.B. and Nutman, T.B. (2011) Microarray-based analysis of differential gene expression between infective and noninfective larvae of Strongyloides stercoralis. PLoS Negl. Trop. Dis. 5, e1039 CrossRef PubMed ( ) gy g Research Council [grant numbers (BB/G011389/1 (to A.J.R. and M.G.)]. 19 Fairfax, K.C., Vermeire, J.J., Harrison, L.M., Bungiro, R.D., Grant, W., Husain, S.Z. and Cappello, M. (2009) Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum. Int. J. Parasitol. 39, 1561–1571 CrossRef PubMed ACKNOWLEDGEMENTS We gratefully acknowledge the Diamond Light Source (MX1229) for access to beamline I04. We thank Karl Burgess and ThermoScientiﬁc for the use of their LipidSearch software. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We declare that no competing interests exist. 413 Structure of Necator americanus FAR protein 17 Vanholme, B., Mitreva, M., Van Criekinge, W., Logghe, M., Bird, D., McCarter, J.P. and Gheysen, G. (2006) Detection of putative secreted proteins in the plant-parasitic nematode Heterodera schachtii. Parasitol. Res. 98, 414–424 CrossRef PubMed CrossRef PubMed 6 Kennedy, M.W., Britton, C., Price, N.C., Kelly, S.M. and Cooper, A. (1995) The DVA-1 polyprotein of the parasitic nematode Dictyocaulus viviparus - a small helix-rich lipid-binding protein. J. Biol. Chem. 270, 19277–19281 CrossRef PubMed 25 Hotez, P.J., Alvarado, M., Basanez, M.-G., Bolliger, I., Bourne, R., Boussinesq, M., Brooker, S.J., Brown, A.S., Buckle, G., Budke, C. M. et al. (2014) The Global Burden of Disease Study 2010: interpretation and implications for the neglected tropical diseases. PLoS Negl. Trop. Dis. 8, e2865 CrossRef PubMed 7 Moore, J., McDermott, L., Price, N.C., Kelly, S.M., Cooper, A. and Kennedy, M.W. (1999) Sequence-divergent units of the ABA-1 polyprotein array of the nematode Ascaris suum have similar fatty-acid- and retinol- binding properties but different binding-site environments. Biochem. J. 340, 337–343 CrossRef PubMed REFERENCES Parasitology 112 Pt 2, 221–225 CrossRef PubMed 5 Kennedy, M.W., Brass, A., McCruden, A.B., Price, N.C., Kelly, S.M. and Cooper, A. (1995) The ABA-1 allergen of the parasitic nematode Ascaris suum - fatty-acid and retinoid-binding function and structural characterization. Biochemistry 34, 6700–6710 CrossRef PubMed 24 Jordanova, R., Groves, M.R., Kostova, E., Woltersdorf, C., Liebau, E. and Tucker, P.A. (2009) Fatty acid- and retinoid-binding proteins have distinct binding pockets for the two types of cargo. J. Biol. Chem. 284, 35818–35826 CrossRef PubMed c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. PLoS Negl. Trop. Dis. 8, e2865 CrossRef PubMed Genome Biol. 4, R26 39 Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Vonrhein, C. and Womack, T.O. (2009) In BUSTER version 2.8.0, Global Phasing Ltd, Cambridge 39 Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Vonrhein, C. and Womack, T.O. (2009) In BUSTER version 2.8.0, Global Phasing Ltd, Cambridge uthors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0. 414 M.F. Rey-Burusco and others 55 Farrow, N.A., Zhang, O.W., Formankay, J.D. and Kay, L.E. (1994) A heteronuclear correlation experiment for simultaneous determination of N-15 longitudinal decay and chemical-exchange rates of systems in slow equilibrium. J. Biomol. NMR 4, 727–734 CrossRef PubMed 40 Davis, I.W., Leaver-Fay, A., Chen, V.B., Block, J.N., Kapral, G.J., Wang, X., Murray, L.W., Arendall, III, W.B., Snoeyink, J., Richardson, J.S. and Richardson, D.C. (2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 35, W375–W383 CrossRef PubMed 56 Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by N-15 inverse detected heteronuclear Nmr-spectroscopy - application to staphylococcal nuclease. Biochemistry 28, 8972–8979 CrossRef PubMed 41 Hansen, M.R., Mueller, L. and Pardi, A. (1998) Tunable alignment of macromolecules by ﬁlamentous phage yields dipolar coupling interactions. Nat. Struct. Biol. 5, 1065–1074 CrossRef PubMed 42 Laue, E.D., Mayger, M.R., Skilling, J. and Staunton, J. (1986) Reconstruction of phase-sensitive two-dimensional NMR spectra by maximum entropy. J. Magn. Reson. 68, 14–29 57 Kay, L.E., Xu, G.Y. and Yamazaki, T. (1994) Enhanced-sensitivity triple-resonance spectroscopy with minimal H2O saturation. J. Magn. Reson. A 109, 129–133 CrossRef 43 Vranken, W.F., Boucher, W., Stevens, T.J., Fogh, R.H., Pajon, A., Llinas, M., Ulrich, E.L., Markley, J.L., Ionides, J. and Laue, E.D. (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59, 687–696 CrossRef PubMed 58 Folch, J., Lees, M. and Sloane Stanley, G.H. (1957) A simple method for the isolation and puriﬁcation of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 PubMed 59 Obal, G., Ramos, A.L., Silva, V., Lima, A., Batthyany, C., Bessio, M.I., Ferreira, F., Salinas, G. and Ferreira, A.M. (2012) Characterisation of the native lipid moiety of Echinococcus granulosus antigen B. PLoS Negl. Trop. Dis. 6, e1642 CrossRef PubMed 44 Ottiger, M., Delaglio, F. and Bax, A. PLoS Negl. Trop. Dis. 8, e2865 CrossRef PubMed (1998) Measurement of J and dipolar couplings from simpliﬁed two-dimensional NMR spectra. J. Magn. Reson. 131, 373–378 CrossRef PubMed 60 Morrison, W.R. and Smith, L.M. (1964) Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron ﬂuoride–methanol. J. Lipid. Res. 5, 600–608 PubMed 45 Ball, G., Meenan, N., Bromek, K., Smith, B.O., Bella, J. and Uhr´ın, D. (2006) Measurement of one-bond 13Calpha-1Halpha residual dipolar coupling constants in proteins by selective manipulation of CalphaHalpha spins. J. Magn. Reson. 180, 127–136 CrossRef PubMed 61 Mat´e, S.M., Brenner, R.R. and Ves-Losada, A. (2004) Phosphatidyl choline fatty acid remodeling in the hepatic cell nuclei. Prostaglandins Leukot. Essent. Fatty Acids 70, 49–57 CrossRef PubMed 46 Marion, D., Driscoll, P.C., Kay, L.E., Wingﬁeld, P.T., Bax, A., Gronenborn, A.M. and Clore, G.M. (1989) Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta. Biochemistry 28, 6150–6156 CrossRef PubMed 62 Daub, J., Loukas, A., Pritchard, D.I. and Blaxter, M. (2000) A survey of genes expressed in adults of the human hookworm, Necator americanus. Parasitology 120 Pt 2, 171–184 CrossRef PubMed 63 Bradley, J.E., Nirmalan, N., Kl¨ager, S.L., Faulkner, H. and Kennedy, M.W. (2001) River blindness: a role for parasite retinoid-binding proteins in the generation of pathology? Trends Parasitol. 17, 471–475 CrossRef PubMed 47 Nilges, M. (1995) Calculation of protein structures with ambiguous distance restraints - automated assignment of ambiguous noe crosspeaks and disulﬁde connectivities. J. Mol. Biol. 245, 645–660 CrossRef PubMed 64 Tang, Y.T., Gao, X., Rosa, B.A., Abubucker, S., Hallsworth-Pepin, K., Martin, J., Tyagi, R., Heizer, E., Zhang, X., Bhonagiri-Palsikar, V. et al. (2014) Genome of the human hookworm Necator americanus. Nat. Genet. 46, 261–269 CrossRef PubMed 48 Habeck, M., Rieping, W., Linge, J.P. and Nilges, M. (2004) NOE assignment with ARIA 2.0: the nuts and bolts. Method Mol. Biol. 278, 379–402 65 Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997) Identiﬁcation of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1–6 CrossRef PubMed 49 Br¨unger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S. et al. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. Sect. D Biol. Crystallogr. y g ; p g Accepted Manuscript online 28 August 2015, doi:10.1042/BJ20150068 PLoS Negl. Trop. Dis. 8, e2865 CrossRef PubMed 54, 905–921 66 Cantacessi, C., Mitreva, M., Jex, A.R., Young, N.D., Campbell, B.E., Hall, R.S., Doyle, M.A., Ralph, S.A., Rabelo, E. M., Ranganathan, S. et al. (2010) Massively parallel sequencing and analysis of the Necator americanus transcriptome. PLoS Negl. Trop. Dis. 684 C R f P bM d 50 Cheung, M.S., Maguire, M.L., Stevens, T.J. and Broadhurst, R.W. (2010) DANGLE: a Bayesian inferential method for predicting protein backbone dihedral angles and secondary structure. J. Magn. Reson. 202, 223–233 CrossRef PubMed Received 16 January 2015/12 August 2015; accepted 27 August 2015 Received 16 January 2015/12 August 2015; accepted 27 August 2015 Accepted Manuscript online 28 August 2015, doi:10.1042/BJ20150068 Received 16 January 2015/12 August 2015; accepted 27 August 2015 Accepted Manuscript online 28 August 2015 doi:10 1042/BJ20150068 4, e684 CrossRef PubMed 67 Garofalo, A., Klager, S.L., Rowlinson, M.C., Nirmalan, N., Klion, A., Allen, J.E., Kennedy, M.W. and Bradley, J.E. (2002) The FAR proteins of ﬁlarial nematodes: secretion, glycosylation and lipid binding characteristics. Mol. Biochem. Parasitol. 122, 161–170 CrossRef PubMed 51 Zweckstetter, M. and Bax, A. (2000) Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. J. Am. Chem. Soc. 122, 3791–3792 CrossRef 52 Tjandra, N., Omichinski, J.G., Gronenborn, A.M., Clore, G.M. and Bax, A. (1997) Use of dipolar H-1-N-15 and H-1-C-13 couplings in the structure determination of magnetically oriented macromolecules in solution. Nat. Struct. Biol. 4, 732–738 CrossRef PubMed 68 Garofalo, A., Kennedy, M.W. and Bradley, J.E. (2003) The FAR proteins of parasitic nematodes: their possible involvement in the pathogenesis of infection and the use of Caenorhabditis elegans as a model system to evaluate their function. Med. Microbiol. Immunol. 192, 47–52 PubMed 53 Linge, J.P., Williams, M.A., Spronk, C.A., Bonvin, A.M.J.J. and Nilges, M. (2003) Reﬁnement of protein structures in explicit solvent. Proteins 50, 496–506 CrossRef PubMed 69 Waterhouse, A.M., Procter, J.B., Martin, D.M., Clamp, M. and Barton, G.J. (2009) Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 CrossRef PubMed 54 Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. and Thornton, J.M. (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 CrossRef PubMed 70 Notredame, C., Higgins, D.G. and Heringa, J. (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302, 205–217 CrossRef PubMed y g ; p g Accepted Manuscript online 28 August 2015, doi:10.1042/BJ20150068 c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution License 3.0.
https://openalex.org/W4383185187	https://dergipark.org.tr/tr/download/article-file/2416950	Turkish	null	Performance Analysis of Underwater Ad-hoc Networks	DergiPark (Istanbul University)	2,022	cc-by	5,987	ÖZ Sualtı tasarsız ağlar oldukça karmaşık bir ortama sahip olmakla birlikte, karasal radyo tabanlı ağlara göre hem fiziksel hem de teknolojik farklılıklar barındırmaktadır. Ayrıca birçok sorun barındırması nedeniyle bu tür karmaşık haberleşme ağlarını modellemek oldukça zordur. Bu alandaki çalışmalar kablosuz haberleşme teknolojilerindeki yeni gelişmelerden etkilenmektedir. Kablosuz haberleşme için sualtının doğası gereği var olan bazı sınırlılıklar bulunmaktadır. Bu sınırlılıkları aşmak için yeni teknikler ve yaklaşımlar önerilmektedir. Bu çalışmada, insansız sualtı araçlarının birbirleri ile haberleşmesinin başarım analizi gerçekleştirilmiştir. Benzetim programında daha gerçekçi bir haberleşme kanalı için akustik kanalın tüm özellikleri tasarlanmış ve performans analizleri yapılmıştır. Tasarsız ağ biçiminde CSMA/CA protokolü kullanılarak sualtı kanal modeli ile ortamda farklı sayıda insansız sualtı araçlarının haberleşmesinde önemli başarım parametrelerinden bit hata oranı, iş çıkarım oranı, çakışma durumu ve paket kayıp oranları detaylıca incelerek sualtı kanalının haberleşmeye etkileri gözlemlenmiştir. İki farklı senaryo Riverbed Modeler benzetim yazılımı üzerinde analiz edilmiş ve ortamdaki düğüm sayısının artması bit hata oranını yaklaşık 6 kat, paket çakışmalarını ise %30 oranında arttırdığı gözlemlenmiştir. Elde edilen sonuçlara göre düğüm sayısının su altı haberleşmesinde önemli bir parametre olduğu görülmüştür. Performance Analysis of Underwater Ad-hoc Networks Research Article ABSTRACT Article History: Received: 09.05.2022 Accepted: 24.10.2022 Published online: 05.07.2023 Although underwater Ad-hoc networks have a very co environment, they have physical and technological diffe compared to terrestrial radio-based networks. In addition, challenging to model such complex communication networks many problems. Studies in this field are influenced by developments in wireless communication technologies. There are limitations to wireless communication due to the nature of under New techniques and approaches are proposed to overcome limitations. In this study, the performance analysis o communication of unmanned underwater vehicles has been perfo In the simulation program, all the detail of the acoustic channe been implemented and performance analyses have been carried o a more realistic communication channel. By using the CSM Keywords: Underwater Ad-hoc networks Channel model Performance analysis Acoustic communication Performance Analysis of Underwater Ad-hoc Networks Performance Analysis of Underwater Ad-hoc Networks OKU Fen Bilimleri Enstitüsü Dergisi 6(2): 1079-1093, 2023 OKU Journal of The Institute of Science and Technology, 6(2): 1079-1093, 2023 Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi Osmaniye Korkut Ata University Journal of The Institute of Science and Technology S lt T Ağl B A li i OKU Fen Bilimleri Enstitüsü Dergisi 6(2): 1079-1093, 2023 OKU Journal of The Institute of Science and Technology, 6(2): 1079-1093, 2023 Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi Osmaniye Korkut Ata University Journal of The Institute of Science and Technology S lt T Ağl B A li i O 6 Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi Osmaniye Korkut Ata University Journal of The Institute of Science and Technology Murtaza CİCİOĞLU1, Ali ÇALHAN2 1Bursa Uludağ Üniversitesi, Mühendislik Fakültesi, Bilgisayar Mühendisliği Bölümü, Bursa 2Düzce Üniversitesi, Mühendislik Fakültesi, Bilgisayar Mühendisliği Bölümü, Düzce p g Sorumlu yazar: murtazacicioglu@uludag.edu.tr 1. Giriş İnsansız araçlara olan ilgi, gereklilik ve çalışmalar her geçen gün artmaktadır (Yılmaz ve Yılmaz, 2022). İnsansız araçların en önemli donanımı göreve özel çalışmaları ve bir insan tarafından uzaktan kontrol edildiği için haberleşme birimleridir (Genç ve Erciyes, 2020). İnsansız araçların bir çeşidi olan insansız sualtı araçları (İSA) sualtı gözlemlerinin yürütülmesi açısından oldukça önemlidir. Ancak özellikle sualtında haberleşme şartlarının zorluğundan dolayı çeşitli problemlerle karşılaşılmaktadır. İSA’ların haberleşmesini etkileyen birçok çevresel faktör bulunmaktadır. Bunların başında suyun derinliği, tuzluluk oranı, suyun akış hızı ve daha da önemli olan ortamın deniz, okyanus ya da tatlı su olmasıdır (Zhang ve ark., 2020). Literatürde haberleşme kanal modelleri incelendiğinde bu kanallardan modellenmesi en zor olanların su gibi yoğun ortamların olduğu görülmektedir (Han ve ark., 2015). Radyo frekans sinyalleri gibi elektromanyetik sinyaller su gibi yoğun ortamlarda hızla sönümlenmektedir. Bu nedenle uygun iletişim mesafelerinde haberleşmenin sürdürülebilmesi için verici cihazının çok yüksek güçlerde çalışması gerekmektedir. Bu durum hem maliyet hem de kullanılabilirlik açısından problemlere yol açmaktadır. Bu gibi nedenler kablosuz sualtı haberleşmesi için akustik, optik ve manyetik indüksiyon dahil olmak üzere farklı teknolojilerin kullanılmasına imkan sunmaktadır. İnsansız araçlara olan ilgi, gereklilik ve çalışmalar her geçen gün artmaktadır (Yılmaz ve Yılmaz, 2022). İnsansız araçların en önemli donanımı göreve özel çalışmaları ve bir insan tarafından uzaktan kontrol edildiği için haberleşme birimleridir (Genç ve Erciyes, 2020). İnsansız araçların bir çeşidi olan insansız sualtı araçları (İSA) sualtı gözlemlerinin yürütülmesi açısından oldukça önemlidir. Ancak özellikle sualtında haberleşme şartlarının zorluğundan dolayı çeşitli problemlerle karşılaşılmaktadır. İSA’ların haberleşmesini etkileyen birçok çevresel faktör bulunmaktadır. Bunların başında suyun derinliği, tuzluluk oranı, suyun akış hızı ve daha da önemli olan ortamın deniz, okyanus ya da tatlı su olmasıdır (Zhang ve ark., 2020). Literatürde haberleşme kanal modelleri incelendiğinde bu kanallardan modellenmesi en zor olanların su gibi yoğun ortamların olduğu görülmektedir (Han ve ark., 2015). Radyo frekans sinyalleri gibi elektromanyetik sinyaller su gibi yoğun ortamlarda hızla sönümlenmektedir. Bu nedenle uygun iletişim mesafelerinde haberleşmenin sürdürülebilmesi için verici cihazının çok yüksek güçlerde çalışması gerekmektedir. Bu durum hem maliyet hem de kullanılabilirlik açısından problemlere yol açmaktadır. Bu gibi nedenler kablosuz sualtı haberleşmesi için akustik, optik ve manyetik indüksiyon dahil olmak üzere farklı teknolojilerin kullanılmasına imkan sunmaktadır. İSA’lar için en yaygın kablosuz haberleşme yöntemi akustik dalgalardır. Bu dalgalar su altında çok verimli bir şekilde yayılmaktadır. Bu nedenle su altında İSA’lar sinyal göndermek ve almak için akustik modemleri kullanmaktadır. Yaygın kullanıma sahip akustik haberleşme tekniğinin bazı dezavantajları da bulunmaktadır. Havadaki elektromanyetik dalgalara nazaran sesin sudaki hızı oldukça yavaştır. ABSTRACT Although underwater Ad-hoc networks have a very complex environment, they have physical and technological differences compared to terrestrial radio-based networks. In addition, it is challenging to model such complex communication networks due to many problems. Studies in this field are influenced by new developments in wireless communication technologies. There are some limitations to wireless communication due to the nature of underwater. New techniques and approaches are proposed to overcome these limitations. In this study, the performance analysis of the communication of unmanned underwater vehicles has been performed. In the simulation program, all the detail of the acoustic channel have been implemented and performance analyses have been carried out for a more realistic communication channel. By using the CSMA/CA 1079 protocol in the form of an Ad-hoc network, the effects of the underwater channel on communication have been observed by examining in detail the bit error rate, throughput, collision and packet loss ratio, which are important performance parameters in the communication of different numbers of unmanned underwater vehicles in the environment with the underwater channel model. Two different scenarios are analysed on the Riverbed Modeler simulation software and the findings show that the increase in the number of nodes in the environment raised the bit error rate by about 6 times and packet collision by 30%. The research results indicate that the number of nodes is an important parameter in underwater communication. To Cite: Cicioğlu M., Çalhan A. Sualtı Tasarsız Ağlarının Başarım Analizi. Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi 2023; 6(2): 1079-1093. To Cite: Cicioğlu M., Çalhan A. Sualtı Tasarsız Ağlarının Başarım Analizi. Osmaniye Korkut Ata Üniversitesi Fen Bilimleri Enstitüsü Dergisi 2023; 6(2): 1079-1093. 1. Giriş Bu durum yüksek yayılım gecikmelerine ve düşük bant genişliğine sebep olmaktadır. Ayrıca İSA’lar su altında ilgili algılayıcı düğümlerden elde ettiği verileri toplayarak birbirine ya da birbiri üzerinden gönderebilmeli ve gerektiğinde de birbirleri ile çift yönlü şekilde haberleşip uyarılar yapabilmelidir. Çevresel faktörlerden kaynaklanan birçok gürültü kaynağı İSA’lar arasındaki haberleşmeyi olumsuz etkileyebilmektedir. Bu nedenle bit hata oranı (BER) metriğinin geliştirilen teknikler üzerinde incelenmesi oldukça önemlidir. Bu sayede sualtı akustik haberleşme sistemlerinin tasarımında verim 1080 ve doğruluk düzeyi ele alınabilmektedir. Ayrıca sualtı akustik haberleşmesinde mesafelerin de bant genişliği üzerindeki etkileri bulunmaktadır. Alıcı ve verici arasındaki mesafe arttıkça kullanılabilecek bant genişliği 5 kHz’e kadar düşebilmektedir. Uygun bir modülasyon tekniği sualtı akustik haberleşmesinde bant genişliğinden verimli yararlanma oranını önemli düzeyde iyileştirecektir. Bu nedenle alternatif modülasyon teknikleri, çok taşıyıcılı haberleşme ve benzeri yeni çözümler araştırmacıların ilgisini çekmektedir. Kablosuz yüksek hızda ve düşük güçte veri iletim platformu İSA’lar arası haberleşme için en önemli beklentiler arasındadır (Cicioğlu ve Çalhan, 2022). Şekil 1’de İSA haberleşmesine ait örnek bir görsel paylaşılmıştır. Özellikle İSA’lar tarafından toplanan önemli verilerin kayıpsız ve hatasız bir şekilde gönderilmesi kritik bir öneme sahiptir. Kaynak İSA’dan hedef İSA’ya gönderilen verilerin teslimi garanti edilmeli ve belirli başarım kriterleri sağlanmalıdır. Bu sebeplerden dolayı TCP/IP protokol kümesindeki fiziksel ve veri bağı katmanlarının tüm ayrıntıları ile modellenmesi ve ayrıca paket teslim onayının sağlanarak çakışmaların önlenmesi ya da en aza indirilmesi büyük önem arz etmektedir. Bu çalışmada yukarıda ifade edilen tüm ayrıntılar Riverbed Modeler benzetim programında modellenmiş ve İSA’lar için bir başarım analiz ortamı tasarlanmıştır. Geliştirilen ağ senaryosunun bit hata oranı, iş çıkarım oranı, paket kayıp oranı ve çakışma durumu parametreleri incelenmiştir. ve doğruluk düzeyi ele alınabilmektedir. Ayrıca sualtı akustik haberleşmesinde mesafelerin de bant genişliği üzerindeki etkileri bulunmaktadır. Alıcı ve verici arasındaki mesafe arttıkça kullanılabilecek bant genişliği 5 kHz’e kadar düşebilmektedir. Uygun bir modülasyon tekniği sualtı akustik haberleşmesinde bant genişliğinden verimli yararlanma oranını önemli düzeyde iyileştirecektir. Bu nedenle alternatif modülasyon teknikleri, çok taşıyıcılı haberleşme ve benzeri yeni çözümler araştırmacıların ilgisini çekmektedir. Kablosuz yüksek hızda ve düşük güçte veri iletim platformu İSA’lar arası haberleşme için en önemli beklentiler arasındadır (Cicioğlu ve Çalhan, 2022). Şekil 1’de İSA haberleşmesine ait örnek bir görsel paylaşılmıştır. Özellikle İSA’lar tarafından toplanan önemli verilerin kayıpsız ve hatasız bir şekilde gönderilmesi kritik bir öneme sahiptir. Kaynak İSA’dan hedef İSA’ya gönderilen verilerin teslimi garanti edilmeli ve belirli başarım kriterleri sağlanmalıdır. 1. Giriş Bu sebeplerden dolayı TCP/IP protokol kümesindeki fiziksel ve veri bağı katmanlarının tüm ayrıntıları ile modellenmesi ve ayrıca paket teslim onayının sağlanarak çakışmaların önlenmesi ya da en aza indirilmesi büyük önem arz etmektedir. Bu çalışmada yukarıda ifade edilen tüm ayrıntılar Riverbed Modeler benzetim programında modellenmiş ve İSA’lar için bir başarım analiz ortamı tasarlanmıştır. Geliştirilen ağ senaryosunun bit hata oranı, iş çıkarım oranı, paket kayıp oranı ve çakışma durumu parametreleri incelenmiştir. Çalışmanın geri kalanı şu şekilde organize edilmiştir. Bölüm 2'de sualtı tasarsız ağlar konusunda literatürde yapılmış çalışmalar incelenmiştir. Önerilen sistemin detayları Bölüm 3'te anlatılmıştır. Ardından, Bölüm 4'te benzetim parametreleri ve benzetim sonuçları verilmiştir. Son olarak, Bölüm 5'te çalışmanın sonuç kısmı verilmiştir. Şekil 1. İSA haberleşmesi için bir örnek senaryo İSA-1 İSA-2 İSA-3 İSA-4 İSA-5 Şekil 1. İSA haberleşmesi için bir örnek senaryo 2.1 İlgili Çalışmalar Kablosuz haberleşme teknolojileri birçok alanda olduğu sualtı haberleşme sistemlerine de katkılar sunmuştur. Deniz ve denizaltı iletişim sistemleri genellikle uzaktan takip, haritalama ve kontrol amaçlı konum bilgilerinin kullanılması gibi birçok amaca hizmet etmektedir. Sualtı ağlarında birçok ortam erişim kontrol (MAC) protokolü önerilmiştir. Sualtı ağları için özel bir MAC yerine başarım performansı yüksek CSMA/CA protokolünün kullanıldığı bazı çalışmalarda görülmüştür. Kablosuz haberleşme teknolojileri birçok alanda olduğu sualtı haberleşme sistemlerine de katkılar sunmuştur. Deniz ve denizaltı iletişim sistemleri genellikle uzaktan takip, haritalama ve kontrol amaçlı konum bilgilerinin kullanılması gibi birçok amaca hizmet etmektedir. Sualtı ağlarında birçok ortam erişim kontrol (MAC) protokolü önerilmiştir. Sualtı ağları için özel bir MAC yerine başarım performansı yüksek CSMA/CA protokolünün kullanıldığı bazı çalışmalarda görülmüştür. Jin ve Huang (2013) sualtı akustik kablosuz algılayıcı ağlar için pekiştirmeli öğrenme algoritması ve zaman dilimli (slot) CSMA protokolü kullanan enerji verimli bir mimari geliştirmişlerdir. Önerilen mimaride düğümler sualtı ortamına uyum sağlamak için pekiştirmeli öğrenme algoritmasını kullanarak parametrelerini optimize etmektedir. Benzetim sonuçlarına göre önerilen mimari ile ağın ömrünün önemli ölçüde arttırdığı görülmektedir. Jin ve Huang (2013) sualtı akustik kablosuz algılayıcı ağlar için pekiştirmeli öğrenme algoritması ve zaman dilimli (slot) CSMA protokolü kullanan enerji verimli bir mimari geliştirmişlerdir. Önerilen mimaride düğümler sualtı ortamına uyum sağlamak için pekiştirmeli öğrenme algoritmasını kullanarak parametrelerini optimize etmektedir. Benzetim sonuçlarına göre önerilen mimari ile ağın ömrünün önemli ölçüde arttırdığı görülmektedir. Alablani ve Arafah (2022) Sualtı Algılayıcı Ağlar için Enerji Verimli Protokol (EE-UWSN) olarak adlandırılan yeni bir MAC/yönlendirme protokolü geliştirerek enerji sorununa çözüm önermişlerdir. Çalışmada enerjisi tasarrufu için sonlu güç seviyelerini kullanma, çok atlamalı iletim, iletim kapsamını daraltma, uyku modunu uygulama ve enerji tüketimini dengeleme gibi çeşitli amaçları öne sürmüşlerdir. Benzetim sonuçları literatürde CSMA ve türevi protokolleri ile karşılaştırılmış ve önerilen protokolün ortalama enerji tüketimini %68,49'a kadar azalttığını göstermişlerdir. Zhang ve ark. (2021) sualtı akustik algılayıcı ağlarında dinamik kanaldaki zaman adaletsizliği erişim problemlerine Q-öğrenmeye dayalı bir geri çekilme algoritması önermişlerdir. Bu algoritmada çekişme penceresi makine öğrenmesi yardımıyla sualtı uyarlamalı şekilde geliştirilmiş ve yeni bir MAC protokolü geliştirmişlerdir. Benzetim sonuçlarına göre önerilen yaklaşımın düğüm erişim kanallarının adaletini etkin bir şekilde geliştirdiğini, veri çerçevelerinin çarpışma oranını azalttığını ve ağ verimini artırdığını göstermiştir. Khater ve ark. (2022) sualtı kablosuz algılayıcı ağları için Aloha protokolünün türevleri olan P-Aloha, S-Aloha, Aloha–CS, Aloha-AN, Buffered-Aloha, Slotted-CS-Aloha, VI-Aloha, L-Aloha, ST-Slotted- CS-Aloha, Modified-Slotted-Aloha protokollerini ortalama gecikme, enerji tüketim oranı ve iş çıkarım oranı gibi farklı performans ölçütlerine göre karşılaştırmış, öne çıkan özellikleri ile sorunları detaylıca incelemiş ve analiz etmiştir. Li ve ark. 2. Materyal ve Metot y Bu bölümde tasarsız sualtı ağlarında yapılmış çalışmalar ve önerilen mimari incelenecektir. 1081 2.1 İlgili Çalışmalar (2021) NS2/NS-Miracle benzetim yazılımında Dünya Okyanus Simülasyon Sistemi (WOSS) ile entegre bir benzetim sistemi üzerinde beş düğümlü merkezi bir sualtı algılayıcı ağını analiz etmişlerdir. Üç MAC protokolü (Aloha-CS, CSMA/CA ve DACAP) aynı koşullar altında karşılaştırılmış, ortalama iş çıkarım oranı, ortalama gecikme ve paket hata oranı gibi benzetim sonuçları incelenmiştir. Elde edilen sonuçlara göre, CSMA/CA protokolü bu tür ağlardaki diğer protokollere göre daha yüksek sonuç vermiştir. Gazi ve ark. (2022) Sualtı Akustik Ağı ortamında multimedya algılama için pekiştirmeli öğrenme makine öğrenmesi yaklaşımına dayalı bir MAC protokolü önermişlerdir. Algılayıcı düğümlerin hareketlilik durumları göz önünde bulundurularak çok atlamalı ağlarda ara düğümlerin verimli bir 1082 biçimde paket iletmelerini sağlayacak yeni teknikler üzerinde yoğunlaşmışlardır. Burada da çekişme penceresinin hesaplanması aşamasında makine öğrenmesinden yararlanılmıştır. Düşük maliyetli bir donanım üzerinde örnek bir uygulama verilmiş ayrıca NS-3 üzerinden detaylı benzetimler yapılmıştır. Elde edilen sonuçlar paket iletimi ve iş çıkarım oranlarında iyileşmeler olduğunu göstermiştir. Liu ve ark. (2021) sualtı akustik algılayıcı ağlarında iletim gecikmesini azaltmak için yeni bir zamanlama tabanlı MAC protokolü geliştirmişlerdir. Zaman dilimi yönetim sorununu çözmek için en uygun paket düzeyinde slot zamanlama (PLSS) ve sezgisel yaklaşık PLSS (PLSS-A) algoritması olmak üzere iki algoritma geliştirilmiştir. Önerilen çözümlerin performansları, farklı ağ ölçekleri, paket uzunlukları ve trafik yükleri ile analiz edilmiş, elde edilen sayısal sonuçlar, hem PLSS hem de PLSS-A'nın ortalama uçtan uca gecikme ve hizmet adaleti açısından başarılı sonuçlar gösterdiğini ve büyük ölçekli ağlar için ağ veriminde avantaja sahip olduğunu göstermiştir. biçimde paket iletmelerini sağlayacak yeni teknikler üzerinde yoğunlaşmışlardır. Burada da çekişme penceresinin hesaplanması aşamasında makine öğrenmesinden yararlanılmıştır. Düşük maliyetli bir donanım üzerinde örnek bir uygulama verilmiş ayrıca NS-3 üzerinden detaylı benzetimler yapılmıştır. Elde edilen sonuçlar paket iletimi ve iş çıkarım oranlarında iyileşmeler olduğunu göstermiştir. Liu ve ark. (2021) sualtı akustik algılayıcı ağlarında iletim gecikmesini azaltmak için yeni bir zamanlama tabanlı MAC protokolü geliştirmişlerdir. Zaman dilimi yönetim sorununu çözmek için en uygun paket düzeyinde slot zamanlama (PLSS) ve sezgisel yaklaşık PLSS (PLSS-A) algoritması olmak üzere iki algoritma geliştirilmiştir. Önerilen çözümlerin performansları, farklı ağ ölçekleri, paket uzunlukları ve trafik yükleri ile analiz edilmiş, elde edilen sayısal sonuçlar, hem PLSS hem de PLSS-A'nın ortalama uçtan uca gecikme ve hizmet adaleti açısından başarılı sonuçlar gösterdiğini ve büyük ölçekli ağlar için ağ veriminde avantaja sahip olduğunu göstermiştir. Song (2021) çalışmasında slotted-Aloha ortam erişim kontrol protokolüne dayalı üç boyutlu topolojiye sahip uygun maliyetli sualtı akustik algılayıcı ağ mimarisi geliştirmiştir. 2.1 İlgili Çalışmalar Bu çalışmada hizmet kalitesi gereksinimlerini karşılamak için Sualtı Nesnelerinin İnterneti ağ tasarımını optimize etmeyi amaçlamıştır. Morozs ve ark. (2020) kontrol yükü olmadan yeni algılayıcıların mevcut bir Sualtı Nesnelerinin İnterneti ağ topolojisine birleştirilmesini kolaylaştırmak için “JOIN” olarak adlandırılan yeni bir protokol önermişlerdir. Geliştirilen bu protokol ile algılayıcıların ağa ne zaman katılacağı yerel topoloji ve çizelge bilgilerine göre belirlenebilmektedir. Bu sayede beklenen paket kayıp sayısı düşürülmüştür. Krishnaraj ve ark. (2020) Sualtı Nesnelerinin İnterneti için görüntü sıkıştırmada ayrık dalgacık dönüşümü (DWT) tabanlı bir derin öğrenme modeli önerilmiştir. Daha iyi rekonstrüksiyon görüntü kalitesi ile etkili sıkıştırma elde etmek için, kodlama ve kod çözme tarafında evrişim sinir ağı (CNN) kullanılmıştır. Gül ve Leblebicioglu (2012) çalışmalarında İSA’ların otonom olarak çalışması için matematiksel model, hız, pitch ve yaw eksenlerinde oto pilot, hareket ve görev planlama algoritmalarının geliştirilmesi için çeşitli tanımlamalar yapmıştır. Önerilen matematiksel model, kontrol modeli, hareket ve görev planlama algoritmaları bilgisayar benzetim sonuçları ile analiz edilmiştir. Karadeniz Kartal ve ark. (2018) bir başka çalışmalarında MATLAB ortamında insansız bir sualtı araştırma aracı (SAGA) için doğrusal olmayan bir matematiksel model önermişlerdir. Bu matematiksel model için Newton-Euler denklemleri kullanılmıştır. Sağ, sol ve dikey iticilerin kombinasyonu kullanılarak üç boyutlu hareket kabiliyeti sağlanmıştır. Navigasyon sorunu ele alınmış, konum ve derinlik bilgileri için manyetik pusula ve derinlik algılayıcı cihazları kullanılmış ve kalman filtresi ile seyir iyileştirmeleri gerçekleştirilmiştir. Yılmaz ve Kılcı (2021) çalışmalarında otonom su altı araçları için gerekli tasarım kavramlarını ve ilkelerini detaylıca incelemişlerdir. Bayrakdar (2020) çalışmasında, sualtı kirliliğinin oluşumunu tespit eden ve alarm veren bir akıllı algılayıcı tabanlı izleme sistemi için bir benzetim modeli geliştirmiştir. 1083 Bu modelin uygun maliyetli bir sistem olabilmesi için ortam erişim kontrol protokolü olarak Aloha seçilmiştir. Çalışmada önerilen model test edilerek sistemin verimliliğinin mevcut insan gözetimini içeren izleme sürecine göre daha istikrarlı, uygun maliyetli ve yönetilebilir olduğu ifade edilmiştir. Mahmutoğlu ve ark. (2021) çalışmalarında Monte Carlo yaklaşımı kullanılarak sualtı kablosuz optik haberleşme kanal modelinin verici ile alıcı arasındaki mesafeye, verici için ışının yayılım açısına ve alıcının açıklık çapına göre SNR ve kanal kapasitesindeki değişimleri incelenmişlerdir. Liao ve Huang (2012) çalışmalarında sualtı akustik algılayıcı ağlar için uzamsal olarak adil bir çoklu erişim kontrol (SF-MAC) protokolü önermişlerdir. Çekişme pencereleri ile haberleşme öncesi el sıkışma paketleri olan CTS ve RTS paketleri kullanılarak paket çarpışmalarının azaltılması sağlanmıştır. Ayrıca bu paketlerin çoklu erişim durumlarında çekişme tabanlıdan ziyade adil olarak ortama erişilmesi için kullanılmıştır. Pompili ve ark. (2009) çalışmalarında sualtı akustik algılayıcı ağlar için tasarlanmış dağıtılmış bir MAC protokolü geliştirmişlerdir. 2.1 İlgili Çalışmalar Yazarlar en uygun iletim gücünü ve kod uzunluğunu birlikte ayarlamak için yeni bir kapalı döngü dağıtık algoritmayı içeren verici tabanlı bir Kod Bölmeli Çoklu Erişim (CDMA) protokolü önermişlerdir. Bu çalışmada okyanus derinliğinin 100 m'den fazla olduğu yerlerde yüksek ağ verimi, düşük kanal erişim gecikmesi ve düşük enerji tüketiminin sağlandığı gösterilmiştir. Bouabdallah ve ark. (2019) ise çalışmalarında su altı akustik algılayıcı ağlar için tasarlanmış çok kanallı bir MAC protokolü (MC-UWMAC) önermişlerdir. MC-UWMAC, bir göndericinin amaçlanan bir komşu alıcı ile el sıkışmak için ortak kontrol kanalı üzerindeki kendi tahsis edilmiş zaman dilimi kullanılarak çarpışmasız bir iletişim sağlamayı amaçlayan enerji açısından verimli bir MAC protokolüdür. 2.2 Sualtı Tasarsız Ağları Karasal ağlarda olduğu gibi sualtı ağlarında da altyapılı ve tasarsız ağlar olmak üzere ikiye ayrılabilmektedir. Altyapılı ağlarda bir merkezi düğüm sualtı düğümlerinden gelen verileri toplamakta ve bir su üstü birime bu verileri iletmektedir. Tasarsız sualtı ağlarında ise hareketlilik ve derinlik durumları göz önünde bulundurularak düğümler birbirleri ile doğrudan haberleşebilmektedir. Altyapılı ağlarda olduğu gibi bir baz istasyonu görevinde bulunan merkezi düğüm bulunmamaktadır. Özellikle İSA’ların hareketlilik, derinlik ve kablosuz haberleşme durumlarından dolayı tasarsız ağ yapısının daha uygun olduğu görülmektedir. Bu sayede veri paylaşımı sağlanarak özel görevlerde İSA’lar arasında koordinasyonun sağlanması amaçlanmaktadır. Kablosuz ortamlarda her an kullanıcıların ağa bağlanıp ayrılma durumları söz konusu olduğundan haberleşme kanallarına sabit kullanıcı ataması yapılması uygun değildir. Ayrıca kullanıcı sayıları sürekli farklılıklar gösterebilmektedir. Bu sebeplerden dolayı CSMA/CA ortam erişim kontrol protokolünün kullanımı diğer protokollere nazaran en uygundur. CSMA/CA protokolü kablosuz ağlar için tasarlanmış ve göndermeden önce kanalı dinleme tekniğine dayalı bir ortam erişim kontrol protokolüdür (Çalhan ve Cicioğlu, 2022). CSMA/CA kullanan her düğüm haberleşme kanalına veri göndermeden önce kanalı dinler, eğer kanalı kullanan başka bir düğüm yoksa verisini gönderir. 1084 Kanalda başka kullanıcı var ise belirli bir süre sonra kanalı dinleme işlemine tekrar başlar. Her gönderilen paketin hedefine ulaştığına dair bir bilgilendirme paketi gönderilir. Bilgilendirme paketi verici düğüme ulaşmaz ise iletimin gerçekleşmediği ortaya çıkar ve aynı paket tekrar aynı hedefe gönderilir. Ayrıca ortamın her meşgul olma durumunda geri çekilme algoritması çalıştırılarak CSMA/CA protokolünün çalışması tamamlanmış olur. Çalışmamızda özellikle CSMA/CA protokolünün seçilme nedeni literatürde sualtı kablosuz haberleşme çalışmalarında yapılan performans analizlerinde bu protokolün diğer protokollere (Aloha-CS, DACAP) göre daha başarılı olduğunu gösteren çalışmalardır (Li ve ark., 2021). Sualtı kablosuz algılayıcı ağlar için, pekiştirmeli öğrenme algoritması ile CSMA/CA protokolü kullanılarak enerji verimli mimarilerin geliştirilmesi (Jin ve Huang, 2013), UWSNs olarak adlandırılan enerji verimli ortam erişim kontrol ve yönlendirme algoritmasının tasarlanması (Alablani ve Arafah, 2022), VFFO optimizasyon algoritması kullanılarak enerji tüketimi ve performans sonuçlarının iyileştirilmesi (Zhang ve ark., 2017) gibi farklı yaklaşımlarla da karşılaşılmıştır. Sualtı haberleşme sistemleri kablosuz haberleşme için oldukça kötü şartlara sahiptir. Bu nedenle haberleşmenin verimli bir biçimde sürdürülebilmesi adına akustik sinyaller tercih edilmektedir. Ayrıca bant genişliği sınırlı ve düşük olduğundan akustik haberleşme düğümleri 10 ile 40 KHz arasındaki frekans kanallarını kullanmaktadır. Yol kaybı, doppler yayılımı, çok yollu sönümleme gibi çok hızlı değişen haberleşme kanalı durumları sualtındaki haberleşmeyi olumsuz etkilemektedir. Sonuç olarak yüksek bit hata oranı ve paket kaybı oluşumuna sebep olmaktadır. 2.2 Sualtı Tasarsız Ağları Daha öncede belirtildiği üzere su derinliği, su sıcaklığı, tuzluluk oranı ve sudaki diğer maddeler akustik sinyalin yayılımını etkileyebilmektedir (Senel ve ark., 2013; Qiao ve ark., 2017). Literatürdeki sualtı kablosuz haberleşme çalışmalarında çoğunlukla yaklaşık kanal modelleri (Beer-Lambert) (Mahmutoglu ve ark., 2020; Matta ve ark. 2019) ile yüksek doğruluk ve hassasiyet için Monte Carlo yaklaşımlı kanal modelleri (Tang ve ark., 2014; Zeng ve ark. 2017) tercih edilmiştir. Sualtı haberleşme kanalı doğası gereği oldukça değişken bir yapıya sahiptir. Ayrıca servis kalite gereksinimleri bu ortamlar için çeşitli su altı ortam faktörlerine bağlıdır. Akustik sinyal hızı, su sıcaklığına, su derinliğine ve tuzluluğa göre büyük ölçüde değişebilmektedir. Su altı haberleşmesi için gerekli fiziksel katman gereklilikleri Mackenzie formülüyle tanımlanmıştır. Sualtı haberleşmesi fiziksel kanallarının gerçekçi modellenmesi için Denklem 1’de verilen Mackenzie (1998) formülündeki tüm matematiksel modeller Riverbed Modeler benzetim yazılımında alıcı antenin tasarımına uygulanmıştır. Veri bağlantı katmanı olarak ikinci katmandaki akustik sinyaller çerçevelere dönüştürülmüştür. Çalışmamızda sualtı ortamında birkaç sualtı algılayıcı düğüm bulunmaktadır. Akustik kanal yapılandırması sualtı paket iletimi için CSMA/CA MAC protokolü ile gerçekleşmektedir. 𝑐(𝑑, 𝑆, 𝑇) = 1448.96 + 4.591 ∗ T − 5.304 ∗ 10 −2 ∗ T2 + 2.374 ∗ 10 −4 ∗ T3 + 1.340 ∗ (S −35) + 1.630 ∗ 10 −2 ∗ D + 1.675 ∗ 10 −7 ∗ D2 − 1.025 ∗ 10 −2 ∗ T ∗ (S − 35) − 7.139 ∗ 10 −13 ∗ T ∗ d3 (1) 𝑐(𝑑, 𝑆, 𝑇) = 1448.96 + 4.591 ∗ T − 5.304 ∗ 10 −2 ∗ T2 + 2.374 ∗ 10 −4 ∗ T3 + 1.340 ∗ (S −35) + 1.630 ∗ 10 −2 ∗ D + 1.675 ∗ 10 −7 ∗ D2 − 1.025 ∗ 10 −2 ∗ T ∗ (S − 35) − 7.139 ∗ 10 −13 ∗ T ∗ d3 (1 (1) 1085 Riverbed Modeler benzetim yazılımında TCP/IP’nin tüm katmanları modellenebilmektedir. Bu kapsamda karasal kablosuz haberleşme için fiziksel katmanda tanımlanan alıcı anten tipi ve özellikleri sualtı akustik haberleşme için yeniden düzenlenmiştir. Benzetim programında sualtı akustik haberleşme için yayılım gecikmesi (Propagation Delay), alıcı anten gücü (Receiver Power) ve arkaplan gürültü (Background Noise) dosyaları sırasıyla uygun bir şekilde ele alınmıştır. Yayılım gecikmesi için “dra _propdel.ps.c”, alıcı anten gücünü hesaplamak amacıyla “dra_power.ps.c” ve arkaplan gürültüsü için “uwa _bkgnoise.ps.c” isimli C tabanlı dosyalar bulunmaktadır. Bu dosyalar sualtı akusitik haberleşme kanal modeline göre revize edilmiştir. Şekil 2’de Riverbed Modeler benzetim yazılımı için alıcı ve verici antenlerinin yukarıda ifade edilen anten özellikleri gösterilmiştir. 2.2 Sualtı Tasarsız Ağları Mackenzie formülündeki notasyonlar şu şekilde ifade edilmektedir; T santigrat derece cinsinden sıcaklık (◦C), S binde (%o) olarak tuzluluk oranını ve d ise metre cinsinden derinlik olarak varsayılmıştır. Riverbed Modeler benzetim yazılımında TCP/IP’nin tüm katmanları modellenebilmektedir. Bu kapsamda karasal kablosuz haberleşme için fiziksel katmanda tanımlanan alıcı anten tipi ve özellikleri sualtı akustik haberleşme için yeniden düzenlenmiştir. Benzetim programında sualtı akustik haberleşme için yayılım gecikmesi (Propagation Delay), alıcı anten gücü (Receiver Power) ve arkaplan gürültü (Background Noise) dosyaları sırasıyla uygun bir şekilde ele alınmıştır. Yayılım gecikmesi için “dra _propdel.ps.c”, alıcı anten gücünü hesaplamak amacıyla “dra_power.ps.c” ve arkaplan gürültüsü için “uwa _bkgnoise.ps.c” isimli C tabanlı dosyalar bulunmaktadır. Bu dosyalar sualtı akusitik haberleşme kanal modeline göre revize edilmiştir. Şekil 2’de Riverbed Modeler benzetim yazılımı için alıcı ve verici antenlerinin yukarıda ifade edilen anten özellikleri gösterilmiştir. Mackenzie formülündeki notasyonlar şu şekilde ifade edilmektedir; T santigrat derece cinsinden sıcaklık (◦C), S binde (%o) olarak tuzluluk oranını ve d ise metre cinsinden derinlik olarak varsayılmıştır. Çalışmamızda yukarıda anlatılan tüm durumlar göz önüne alınarak İSA’ların fiziksel ve veri bağı katmanı protokolleri Riverbed Modeler ağ benzetim programında tasarlanmış ve başarım analizleri yapılmıştır (Riverbed Modeler Software, 2020). Riverbed Modeler, çeşitli ağ topolojilerinin davranışını ve performansını analiz eden ve akademik dünyada yaygın kabul gören bir ağ benzetim yazılımıdır. Bu yazılımın diğer benzetim programlarından temel farkı desteklediği haberleşme teknolojilerindeki çeşitliliğidir. Hem önceden oluşturulmuş protokol ve cihaz modellerini sunarken hem de yeni protokollerin tasarlanmasına imkan sunabilmektedir. Farklı ağ topolojileri oluşturulabilmesi ve benzetim yapılabilmesi nedeniyle bu çalışmada tercih edilmiştir. Bir sonraki bölümde İSA haberleşme sisteminin çeşitli parametrelere göre başarım analizleri gerçekleştirilmiştir. Şekil 2. Riverbed Modeler radyo alıcı-verici antenlerinin özellikleri Radyo vericisi için kullanılan özellikler Radyo alıcısı için kullanılan özellikler Radyo vericisi için kullanılan Şekil 2. Riverbed Modeler radyo alıcı-verici antenlerinin özellikleri 1086 1086 3. Bulgular ve Tartışma Tasarlanan İSA haberleşme ağı Riverbed Modeler benzetim yazılımında C yazılım dili kullanılarak programlanmıştır. Riverbed Modeler nesneye dayalı programlama özelliklerini barındıran ağ protokolleri için zengin kütüphanelere sahip uluslararası saygınlığı olan bir ağ benzetim yazılımıdır. Ağ protokolleri, ağ modelinin tüm katmanları düzeyinde programlanabilmesini sağlayan Proto-C tabanlı bir ağ benzetim yazılımıdır. Ağ başarım değerlendirme işlemleri için özellikle fiziksel ve veri bağı katmanlarının ayrıntılı ve doğru bir şekilde programlanması gerekmektedir. Önceki bölümde anlatılan tüm özellikler dikkate alınarak İSA-İSA haberleşmesi tasarsız ağ biçiminde CSMA/CA protokolü ve sualtı haberleşme kanal modeliyle birlikte programlanmıştır. Tablo 1’de senaryolar için Riverbed Modeler benzetim yazılımında kullanılan parametreler verilmiştir. Tablo 1. Benzetim Parametreleri Tablo 1. Benzetim Parametreleri Parametreler Değerler Benzetim süresi 3600 sn Frekans 20 - 25 kHz Düğüm sayısı 1 - 4 MAC protokolü CSMA/CA Model Mackenzie Öncelikler 0-8 (düşükten yükseğe) Bant genişliği 40 kHz Veri hızı 1024 bps Paket boyutu 100 Byte Derinlik 0 m /100 – 1000m Rüzgar hızı (w) 0 m/sn Sıcaklık 18 C Su tuzluluğu (S) 27 ppt Su Asitliği (pH) 7 İSA-İSA haberleşmesinin başarım analizinde iki senaryo tasarlanmış ve bit hata oranı, iş çıkarım oranı, çakışma durumu ve paket kayıp oranı parametreleri incelenmiştir. İlk senaryoda sualtı haberleşme ortamında Şekil 3’te gösterildiği gibi iki farklı İSA (İSA-1 ve İSA-2) bulunmaktadır. Her iki senaryo Tablo 1’de verilen benzetim parametrelerine göre düzenlenmiştir. İSA’lar birbirlerine iki yönlü olarak veri gönderebilmektedir. İSA-1’in yukarıda ifade edilen dört farklı başarım parametresi incelenmiştir. Ortam erişim kontrol protokolü olarak başarım performansı yüksek olan CSMA/CA tercih edilmiştir. Bu sayede oldukça kötü haberleşme ortamında başarımın uygun bir seviyede sürdürülmesi amaçlanmıştır. 1087 Şekil 3. İki İSA’dan oluşan birinci senaryonun gösterimi Şekil 3. İki İSA’dan oluşan birinci senaryonun gösterimi İlk senaryonun başarım sonuçları Şekil 4’te verilmiştir. Bit hata oranı ortalama 2.10-5, paket kayıp oranı ortalama 5.10-3, çakışma durumu 0,5 ve iş çıkarım oranı 1,15 civarında elde edilmiştir. Akustik haberleşme kanalları yüksek sinyal yayılım gecikmesine sebep olmaktadır. Ayrıca bu kanallardaki ses hızının yaklaşık 1,5103 m/sn ve düğümlerin genellikle 10-40 kHz'deki frekanslarda çalıştığı bilinmekledir. Bu senaryoda benzetimi gerçekleştirilen sualtı haberleşme kanalının sınırlı bant genişliği ve kablosuz algılayıcı düğümlerin aksine çok hızlı kanal değişimi gibi birçok sorun nedeniyle özellikle İSA-1 ve İSA-2 arasındaki iki yönlü haberleşmenin performansı incelenmiş ve elde edilen sonuçlar ışığında kaynak paketlerinin hedefe başarılı bir biçimde ulaştığı gözlemlenmiştir. a) Bit hata oranı b) Paket kayıp oranı b) Paket kayıp oranı a) Bit hata oranı 1088 c) Çakışma durumu d) İş çıkarım oranı Şekil 4. 3. Bulgular ve Tartışma Senaryo 1 için başarım sonuçları (İSA-1 İSA-2 ye veri gönderiyor / İSA-1 in sonuçları) d) İ k d) İş çıkarım oranı Şekil 4. Senaryo 1 için başarım sonuçları (İSA-1 İSA-2 ye veri gönderiyor / İSA-1 in sonuçları) Diğer bir taraftan ikinci senaryoda ise tasarsız sualtı haberleşme ortamında İSA-1 ve İSA-2 ile İSA-3 ve İSA-4 düğümleri konuşlandırılmıştır. Bu sayede akustik kanal modelinin her düğüm çifti arasındaki iki yönlü haberleşmeye olan etkisi incelenmektedir. Bu düğümler Şekil 5’te de gösterildiği gibi birbirlerine veri gönderebilmektedir. Her iki senaryoda da İSA-1’in bit hata oranı, iş çıkarım oranı, çakışma durumu ve paket kayıp oranı parametreleri ele alınmıştır. İSA-1 İSA-2’ye, İSA-3 ise İSA-4’e veri göndermektedir. Bu şartlar altında İSA-1’in başarım sonuçları Şekil 6’da verilmiştir. Şekil 5. Dört İSA’dan oluşan ikinci senaryonun gösterimi Şekil 5. Dört İSA’dan oluşan ikinci senaryonun gösterimi İkinci senaryonun bit hata oranı, paket kayıp oranı, çakışma durumu ve iş çıkarım oranına ait başarım sonuçları Şekil 6’da verilmiştir. Bit hata oranı ortalama 1,5.10-4, paket kayıp oranı ortalama 0,025, çakışma durumu 0,65 ve iş çıkarım oranı 1,15 civarında elde edilmiştir. İkinci senaryonun bit hata oranı, paket kayıp oranı, çakışma durumu ve iş çıkarım oranına ait başarım sonuçları Şekil 6’da verilmiştir. Bit hata oranı ortalama 1,5.10-4, paket kayıp oranı ortalama 0,025, çakışma durumu 0,65 ve iş çıkarım oranı 1,15 civarında elde edilmiştir. 1089 Akustik iletişim kanallarında büyük sinyal yayılma gecikmesi oluşur. Kablosuz kanaldaki elektromanyetik dalgalar ile sinyalin hızı yaklaşık 3108 m/sn iken akustik kanaldaki sesin hızı yaklaşık 1,5*103 m/sn'dir. Akustik iletişimdeki sensör düğümleri genellikle 10-40 kHz'deki frekansı kullanır (Han ve ark., 2015). İki senaryo karşılaştırıldığında ilk senaryoda iki düğüm olması sebebiyle bit hata oranı ve paket kayıp oranları düşük çıkmış ve çakışma durumu 0,5 civarındadır. İkinci senaryoda düğüm sayısı 2 katına çıktığı için bit hata oranı ve paket kayıp oranı artarak çakışma durumun da 0,65 civarına yükseldiği gözlemlenmiştir. Senaryo sonuçları paketlerin iş çıkarım oranı grafiğine göre incelendiğinde paketlerin hedeflerine teslim edildiği görülmektedir. Başarım sonuçları az sayıda kullanıcı olmasına rağmen kullanıcı sayısının artmasıyla kayıpların arttığını ve çakışmaların arttığını da göstermektedir. Bunun temel nedeni is sualtı haberleşme ortamının zorlu şartlarından kaynaklanmaktadır. Sualtı algılayıcı düğüm haberleşmesinde, yüksek yol kaybı, Doppler yayılımı, sınırlı bant genişliği ve kablosuz algılayıcı düğümlerin aksine çok hızlı kanal değişimi gibi birçok sorun performans sonuçlarına etki etmektedir. a) Bit hata oranı b) Paket kayıp oranı c) Çakışma durumu d) İş çıkarım oranı Şekil 6. 4. Sonuç İnsansız araçlar gün geçtikçe önemi ve kullanımı artan teknolojiler olarak hayatımızda yerini almaktadırlar. Bu sistemlerdeki en önemli bileşenlerden birisi de haberleşme sistemleridir. Araçların birbirleri ile haberleşme özelliğine sahip olması kaçınılmaz bir gerekliliktir. Kayıpsız ve artan kullanıcı sayılarında sualtında önemli problemlerle karşılaşılabilmektedir. İnsansız Sualtı araçlarının düşük frekans ve dar bant genişliklerinde kayıpsız bir şekilde iletimlerini gerçekleştirmeleri önemli görevlerde hayati önem arz etmektedir. Bu çalışmada insansız sualtı araçlarının birbirleri ile haberleşmesi ele alınmış ve bir ağ modeli ortaya konulmuştur. Önemli başarım parametreleri olan bir hata oranı, paket kayıp oranı, çakışma durumu ve iş çıkarım oranı iki farklı senaryo üzerinde incelenmiştir. İlk senaryonun başarım sonuçlarına göre İSA’lar arasındaki iki yönlü haberleşmenin başarılı bir biçimde gerçekleştiğini ancak akustik haberleşme kanallarının yüksek sinyal yayılım gecikmesine sebep olduğunu göstermiştir. İkinci senaryoda ise düğüm sayısındaki artışın bit hata oranı ve paket kayıp oranına etkisi incelenmiş ve özellikle çakışma durumun 0,65 civarına yükseldiği gözlemlenmiştir. Ancak buna rağmen paketlerin hedeflerine başarılı bir biçimde teslim edildiği de görülmüştür. Gelecek çalışmalar için Riverbed Modeler benzetim yazılımı üzerinde farklı kanal modelleri ile sürü halinde insansız sualtı araçları için ağ modellerinin tasarlanması planlanmaktadır. Bu modeller benzetim programında kullanılan 14 aşamalı Alıcı-Verici veri hattı üzerinde (aşama 5 yayılım gecikmesi, aşama 7 alıcı gücü ve aşama 9 arka plan gürültüsü) farklı modeller programlanarak farklı ağ topolojileri üzerinde çeşitli analizler yapılacaktır. Makine öğrenmesi yardımıyla farklı kanal modellerinin ortam koşullarına göre belirlenmesi problemi de çalışılması düşünülen gelecek konular arasındadır. Çıkar Çatışması Beyanı Makale yazarları aralarında herhangi bir çıkar çatışması olmadığını beyan ederler. Araştırmacıların Katkı Oranı Beyan Özeti Yazarlar makaleye eşit oranda katkı sağlamış olduklarını beyan ederler. 3. Bulgular ve Tartışma Senaryo 2 için başarım sonuçları a) Bit hata oranı b) Paket kayıp oranı c) Çakışma durumu d) İş çıkarım oranı Şekil 6. Senaryo 2 için başarım sonuçları 1090 Kaynakça Alablani IA., Arafah MA. EE-UWSNs: A joint energy-efficient MAC and routing protocol for underwater sensor networks. Journal of Marine Science and Engineering 2022; 10(4): 1-25. Alablani IA., Arafah MA. EE-UWSNs: A joint energy-efficient MAC and routing protocol for underwater sensor networks. Journal of Marine Science and Engineering 2022; 10(4): 1-25. Bouabdallah F., Chaima Z., Raouf B., Ahmed M. Collision avoidance energy efficient multi-channel MAC protocol for underwater acoustic sensor networks. IEEE Transactions on Mobile Computing 2019; 18(10): 2298–2314. Çalhan A., Cicioğlu M. Drone-assisted smart data gathering for pandemic situations. Computers & 1091 Electrical Engineering 2022; 98: 1-11. Çalhan A., Cicioğlu M. Performance analysis of cross-layer design for ınternet of underwater things. IEEE Sensors Journal 2022; 22(15): 15429-15434. Bayrakdar EM. Cost effective smart system for water pollution control with underwater wireless sensor networks: A simulation study. Computer Systems Science and Engineering 2020; 35(4): 283–292. Gazi F., Ahmed N., Sudip M., Wei W. Reinforcement learning-based MAC protocol for underwater multimedia sensor networks. ACM Transactions on Sensor Networks 2022; 18: 1-27. Genç YM., Erciyes E. İnsansız hava araçları (İHA) tehditleri ve güvenlik yönetimi. Turkish Journal of Unmanned Aerial Vehicles 2020; 2(2): 36-42. Gul UD., Leblebicioglu K. Autonomous underwater vehicle modelling, control, motion and mission planning design. In 20th Signal Processing and Communications Applications Conference (SIU), IEEE:2012; 1–4, Mugla, Turkey. Han G., Chenyu Z., Shu L., Rodrigues JPC. Impacts of deployment strategies on localization performance in underwater acoustic sensor networks. IEEE Transactions on Industrial Electronics 2015; 62(3): 1725-1733. Jin L., Defeng H. A slotted CSMA based reinforcement learning approach for extending the lifetime of underwater acoustic wireless sensor networks. Computer Communications 2013; 36(9): 1094- 1099. Karadeniz KS., Leblebicioglu MK., Ege E. Experimental test of the acoustic-based navigation and system identification of an unmanned underwater survey vehicle (SAGA). Transactions of the Institute of Measurement and Control 2018; 40(8): 2476–2487. Khater EM., El-Fishawy N., Tolba MB., Mahmoud D., Badawy MB. Review of ALOHA-based MAC protocols for underwater wireless sensor networks. Mansoura Engineering Journal 2022; 47(1): 12–25. Krishnaraj N., Elhoseny M., Thenmozhi M., Mahmoud M. S., Shankar K. Deep learning model for real-time image compression in internet of underwater things (IoUT). Journal of Real-Time Image Processing 2020; 17(6): 2097–2111. Li L., Jin X., Lu C., Wei Z., Li J. Modelling and simulation on acoustic channel of underwater sensor networks. Wireless Communications and Mobile Computing 2021; 2021: 1–12. Liao W., Chih-Chien H. Kaynakça SF-MAC: A spatially fair MAC protocol for underwater acoustic sensor networks. IEEE Sensors Journal 2012; 12(6): 1686–1694. Liu M., Zhuo X., Wei Y., Wu Y., Qu F. Packet-level slot scheduling mac protocol in underwater acoustic sensor networks. IEEE Internet of Things Journal 2021; 8(11): 8990–9004. Mackenzie KV. Nine‐term equation for sound speed in the oceans. The Journal of the Acoustical Society of America 1998; 70(3): 1-6. Mahmutoğlu Y., Cenk A., Kadir T. Değişik ortam ve sistem parametreleri için sualtı kablosuz optik 1092 haberleşme monte carlo kanal kapasitesinin incelenmesi. El-Cezeri Fen ve Mühendislik Dergisi 2021; 8(2): 567-581. Mahmutoglu Y., Kadir T., Cenk A. Investigation of underwater wireless optical communication channel capacity for different environment and system parameters. Hittite Journal of Science & Engineering 2020; 7(4): 279–285. Matta G., Monika A., Rajendar B. Channel capacity for underwater visible light communication systems. In OCEANS, IEEE:2019; 1-4, Marseille, France. Morozs N., Paul D. M., Roee D. Scalable adaptive networking for the internet of underwater things. IEEE Internet of Things Journal 2020; 7(10): 10023–10037. Pompili, D., Tommaso M., Ian FA. A CDMA-based medium access control for underwater acoustic sensor networks. IEEE Transactions on Wireless Communications 2009; 8(4): 1899–1909. Riverbed Modeler Software. https://www.riverbed.com/gb/products/steelcentral/steelcentral-riverbed- modeler.html (Erişim Tarihi: 24 Mayıs 2021), 2022. Senel F., Akkaya K., Yilmaz T. Autonomous deployment of sensors for maximized coverage and guaranteed connectivity in underwater acoustic sensor networks. In 38th Annual IEEE Conference on Local Computer Networks: 2013; 211–218, Sydney. Song Y. Underwater acoustic sensor networks with cost efficiency for internet of underwater things. IEEE Transactions on Industrial Electronics 2021; 68(2): 1707–1716. Tang S., Yuhan D., Xuedan Z. Impulse response modeling for underwater wireless optical communication links. IEEE Transactions on Communications 2014; 62(1): 226–234. Yılmaz G., Yılmaz S. İnsansız sualtı araçlarında (İSA) hidrodinamik sürüklenme ve kaldırma kuvvetlerinin derinlik ve hıza bağlı değişiminin had ile analizi. Uluslararası Mühendislik Araştırma ve Geliştirme Dergisi 2022; 14(1): 72–83. Yılmaz S., Kılcı BS. General design principles for autonomous underwater vehicles. Journal of the Institute of Science and Technology 2021; 11(1): 119–131. Ying Z., Yunlong Q., Haiyang Z. Coverage enhancing for underwater acoustic sensor networks based on virtual force and fruit fly optimization algorithm. Journal of Shanghai Jiaotong University 2017; 51(6): 7-15. Zeng Z., Fu S., Zhang H., Dong Y., Cheng J. A survey of underwater optical wireless communications. IEEE Communications Surveys & Tutorials 2017; 19(1): 204–238. Zhang W., Han G., Wang X., Guizani M., Fan K., Shu LA. Kaynakça Node location algorithm based on node movement prediction in underwater acoustic sensor networks. IEEE Transactions on Vehicular Technology 2020; 69(3): 3166–3178. Zhang W., Wang X., Han G., Peng Y., Guizani M., Sun JA. Load-adaptive fair access protocol for MAC in underwater acoustic sensor networks. Journal of Network and Computer Applications 2021; 173: 1-13. 1093
W3111753613.txt	https://www.mdpi.com/1424-8220/20/24/7128/pdf?version=1607763591	en		Recent Advances in Cell Adhesive Force Microscopy	Sensors	2,020	cc-by	9,269	sensors Review Recent Advances in Cell Adhesive Force Microscopy Ying Tu 1 and Xuefeng Wang 1,2, * 1 2 * Department of Physics and Astronomy, Iowa State University, Ames, IA 50011, USA; yingtu@iastate.edu Molecular, Cellular, and Development Biology Interdepartmental Program, Iowa State University, Ames, IA 50011, USA Correspondence: xuefeng@iastate.edu Received: 6 November 2020; Accepted: 7 December 2020; Published: 12 December 2020 Abstract: Cell adhesive force, exerting on the local matrix or neighboring cells, plays a critical role in regulating many cell functions and physiological processes. In the past four decades, significant efforts have been dedicated to cell adhesive force detection, visualization and quantification. A recent important methodological advancement in cell adhesive force visualization is to adopt force-to-fluorescence conversion instead of force-to-substrate strain conversion, thus greatly improving the sensitivity and resolution of force imaging. This review summarizes the recent development of force imaging techniques (collectively termed as cell adhesive force microscopy or CAFM here), with a particular focus on the improvement of CAFM’s spatial resolution and the biomaterial choices for constructing the tension sensors used in force visualization. This review also highlights the importance of DNA-based tension sensors in cell adhesive force imaging and the recent breakthrough in the development of super-resolution CAFM. Keywords: integrin; cell adhesion; cell adhesive force; cell traction force; integrin tension; tension sensor; DNA; cellular force imaging; super-resolution; mechanotransduction; mechanobiology 1. Introduction Cells are dynamic systems which constantly undergo biochemical and physical activities. Early cell biology studies have focused on understanding the biochemical aspects of cells, such as genetics, immunology, development, pathology, etc. The physical aspect received relatively less attention until recent decades. As cells live in a physical world, many biological motions in metazoan cells including cell adhesion, migration and division are driven by physical forces. These forces are critical for the normal functions and sustainability of cells. In particular, cell adhesive force, a result of cell–matrix and cell–cell biophysical interactions, has emerged as an important physical signal in regulating cell functions and maintaining tissue integrity. In the long term, cell adhesive force can also be converted to biochemical signals through mechanotransduction, and take effect on cell survival [1], proliferation [2], differentiation [3], cancer progression [4], biological development [5], immune response [6] and more. Hence, substantial research interests have been drawn to study cell adhesive force and its related biological functions in the recent four decades. To study a signal, one needs first to detect and quantify the signal. As cell adhesive force is spatially distributed across the cell–matrix or cell–cell interface, visualization of the force would provide critical information about the force distribution and dynamics. Cell adhesive force visualization had not been easily accessible due to the miniscule level of force (at the piconewton (pN)–nanonewton (nN) level) produced by cells and the invisible nature of force signals. To “see” the force, one needs to find an approach to convert the invisible force to a visible signal. Retrospectively speaking, cell adhesive force sensing methods can be categorized into two types: force-to-strain conversion adopted in traction force microscopy (TFM), and force-to-fluorescence conversion adopted by fluorescent tension Sensors 2020, 20, 7128; doi:10.3390/s20247128 www.mdpi.com/journal/sensors Sensors 2020, 20, 7128 Sensors 2020, 20, x FOR PEER REVIEW 2 of 14 2 of 14 the 1980s. Cell adhesive force was first visualized by plating cells on ultrathin elasticforce silicone sensors (FTSs). The prototypes of TFM were developed in the 1980s. Cell adhesive wasfilms first [7]. The cells contracted and produced wrinkles on the film which can be imaged by an optical visualized by plating cells on ultrathin elastic silicone films [7]. The cells contracted and produced microscope. following decades, a seriesby of an TFMs havemicroscope. been developed based on this decades, strategy wrinkles on In thethe film which can be imaged optical In the following of force-to-strain conversion [8–16]. The force–reporting substrates are generally elastic materials a series of TFMs have been developed based on this strategy of force-to-strain conversion [8–16]. embedded with fluorescent markers reporting the force-induced substrate strains (Figuremarkers 1A), or The force–reporting substrates are generally elastic materials embedded with fluorescent with micro-patterns whosesubstrate deformation be 1A), monitored 1B). With computational reporting the force-induced strainscan (Figure or with(Figure micro-patterns whose deformation modeling, the substrate strain maps are converted to cell stress maps, hence reporting lateral can be monitored (Figure 1B). With computational modeling, the substrate strain maps are the converted distribution of cell hence adhesive force.the TFM pioneered cell adhesive force visualization and has cell the to cell stress maps, reporting lateral distribution of cell adhesive force. TFM pioneered unique advantages in measuring whole cell force. adhesive force visualization and has the unique advantages in measuring whole cell force. Figure 1. Schematics Figure 1. Schematics of of techniques techniques for for cell cell adhesive adhesive force force imaging. imaging. (A) (A) Traction Traction force force microscopy microscopy (TFM) using an elastic polymer film doped with fluorescence beads or particles as the TFM (TFM) using an elastic polymer film doped with fluorescence beads or particles assubstrate. the substrate. converts cell adhesive forceforce to substrate deformation. (B)(B) TFM using TFM converts cell adhesive to substrate deformation. TFM usingmicropillar micropillararrays arraysas as the the substrate. substrate. TFM TFMrecords records the the bending bending of of the the micropillar micropillar arrays arrays and and computes computes the the cell cell adhesive adhesive force. force. (C) Fluorescent tension sensor (FTS) converts converts cell adhesive adhesive force force to fluorescence using an “entropic spring” molecule. molecule. (D) (D)FTS FTSbased based on binary material with distinct statesas such as doubleon binary material with distinct on/off on/off states such double-stranded stranded DNA (dsDNA) hairpin DNA. DNA (dsDNA) or hairpinorDNA. However, compared compared to to the cell structural imaging imaging that benefits benefits from fluorescent staining or However, fluorescent staining fluorescentproteins, proteins,strain-based strain-based TFM inferior resolution, is usually in theofrange of fluorescent TFM hashas inferior resolution, whichwhich is usually in the range microns microns [17]. As many force transmission structures in cells, such as podosomes, invadopodia, [17]. As many force transmission structures in cells, such as podosomes, invadopodia, focal complexes, focal complexes, filopodia, etc., are inor the size of microns or submicrons, techniques werethe in filopodia, etc., are in the size of microns submicrons, new techniques were innew demand to improve demand toofimprove the resolution of cellAadhesive forcefor imaging. A newwas strategy for force imaging resolution cell adhesive force imaging. new strategy force imaging developed in the recent was developed in the decade. A number of techniques adopted force-to-fluorescence decade. A number ofrecent techniques adopted force-to-fluorescence conversion to visualize cellconversion adhesive to visualize adhesive force. a strategy was first adopted sensors that areinside genetically force. Such acell strategy was first Such adopted by tension sensors that by aretension genetically expressed cells expressed inside cells [18,19]. The strategy was then adopted by extracellular tension sensors that are [18,19]. The strategy was then adopted by extracellular tension sensors that are immobilized on surfaces immobilized on surfaces and target specific force-transmitting receptors [20–24] on the cell membrane and target specific force-transmitting receptors on the cell membrane (Figure 1C,D).[20–24] These (Figure sensors 1C,D). These tension sensorsunits are force-activatable units that emittheir fluorescence or properties alter their tension are force-activatable that emit fluorescence or alter fluorescent fluorescent properties under tension. The onsite fluorescent signalmolecular responding to local molecular force under tension. The onsite fluorescent signal responding to local force events gives rise to events gives rise spatial to unprecedented resolutionin and sensitivity cell adhesive imaging. Here we unprecedented resolutionspatial and sensitivity cell adhesiveinforce imaging.force Here we collectively collectively dub these sensors as fluorescent tension sensors (FTSs). areseveral alreadyexcellent several excellent dub these sensors as fluorescent tension sensors (FTSs). There areThere already reviews reviews covering TFM andseparately FTSs, separately [17,25–28]. This review compares the performance of TFM covering TFM and FTSs, [17,25–28]. This review compares the performance of TFM and and FTSs, and focuses onrecent the recent advances in spatial resolutions of these methods. review FTSs, and focuses on the advances in spatial resolutions of these methods. This This review also also discusses biomaterial choices constructing FTSs and highlightsthe theperformance performanceofofDNA DNA and discusses biomaterial choices for for constructing FTSs and highlights DNA-like materials as the FTS constructs. Of note, there are another series of point-based single-molecule force techniques such as atomic force microscopy (AFM) [29], optical tweezers [30], magnetic tweezers [31] and biomembrane force These techniques techniques proactively proactively apply apply force force to to probe probe the mechanical mechanical properties properties of single probes [32]. These biomolecules or or molecular molecular bonds, bonds, and and are advantageous advantageous in in obtaining obtaining the force spectroscopy of biomolecules biomechanical events eventssuch suchas asprotein proteinunfolding, unfolding,bond bonddissociation, dissociation, etc. Because these techniques biomechanical etc. Because these techniques do not image force (AFM generally does not perform imaging when used as a force probing tool) and Sensors 2020, 20, 7128 3 of 14 do not image force (AFM generally does not perform imaging when used as a force probing tool) and the force is produced by the instruments as a probing signal, we do not categorize them into CAFM in this review as CAFM is developed to image cell-generated force. Readers interested in these single-molecule force techniques may refer to other review articles [33]. 2. Advances in Cell Adhesive Force Microscopy in Term of Spatial Resolution In this review, we focus on presenting and discussing the spatial resolutions offered by the two branches of CAFMs: TFM and FTS-enabled CAFM. Table 1 summarizes their spatial resolutions. Table 1. Force detection ranges and spatial resolutions of cell adhesive force microscopies. Detection Range Spatial Resolution References TFM ~1 nN 1–5 µm [7,34–38] Super-resolution TFM <1 nN 40–80 nm * [15,16] 0–20 pN 1–100 pN 7–14 pN 12–54 pN <1 µm <1 µm <1 µm <1 µm [22,23,39–41] [42,43] [24,40,44,45] [46–53] 12–54 pN 50 nm [54] 7–14 pN 25 nm [55] FTS-based cell adhesive force microcopy PEG peptide DNA hairpin dsDNA CFN tPAINT * Not force resolution, but accuracy of bead tracking. 2.1. Traction Force Microscopy Pioneered Cell Adhesive Force Imaging To detect and evaluate cell adhesive force, the prototype of traction force microscopy (TFM) was developed in the 1980s [7]. Subsequently, TFM was continuously improved and the algorithm was also refined for cell adhesive force computation. In TFM, cells are cultured on deformable polymer films [34] (Figure 1A) or micropillar arrays [10,35,36] (Figure 1B). The deformation of the substrates caused by cells is monitored by optical microscopy. To aid the deformation monitoring, the substrates are often embedded with fluorescent beads and the micropillar tips are fluorescently stained. By converting the displacements of the elastic substrates or micropillar arrays to traction stresses, TFM quantitatively maps force distribution at the interface between a cell and the extra-cellular matrix (ECM). TFM also has wide dynamic range and is adaptive to force measurement at different force levels. This can be achieved simply by altering substrate elasticity. However, because TFM computes cellular force from a strain map in an indirect manner, it had limited spatial resolution and sensitivity as an imaging technique. It is challenging for TFM to resolve forces in micron-sized cells (e.g., the small blood cell platelets) or subcellular structures (focal complexes, podosomes, filopodia, etc.). Figure 2A–C shows the representative TFM images of fibroblasts, keratocytes and platelets. Continuous efforts have been made to improve the resolution of TFM. A high-resolution TFM microscope compatible with spinning-disk confocal setting was developed in Waterman’s lab with a 10–20 µm thick fibronectin-coated polyacrylamide substrate [37]. The polymer substrates were embedded with a high density of fluorescent beads. By reconstructing the cell-induced bead displacements to cellular traction stress, a spatial resolution of ~1 µm was achieved using the Fourier-transform traction cytometry method. The improved spatial resolution was ascribed to high areal density (10 beads/µm2 ) of small-sized (40 nm) fluorescent beads in the polymer substrates. This high-resolution traction force microscopy revealed a robust biphasic relationship between F-actin polymerization speed and traction force [56], and showed force fluctuation in focal adhesions (FAs) [13]. Lately, super-resolved TFM microscopes have also been developed by increasing the accuracy in localizing fluorescent markers embedded in the substrate [15]. An improved fluctuation-based super-resolution (FBSR) traction force microscopy was reported by further increasing the bead density and only embedding fluorescent beads on the topmost layer of the gel [16]. Moreover, the FBSR algorithm Sensors 2020, 20, 7128 Sensors 2020, 20, x FOR PEER REVIEW 4 of 14 4 of 14 density and only embedding fluorescent beads on the topmost layer of the gel [16]. Moreover, the and two-step deconvolution led to a better localization of the fluorescent beads. These methods further FBSR algorithm and two-step deconvolution led to a better localization of the fluorescent beads. improved the accuracy in producing the strain map that is important for the reconstruction of the cell These methods further improved the accuracy in producing the strain map that is important for the adhesive force map with higher resolution. However, of note, the accuracy of bead tracking is different reconstruction of the cell adhesive force map with higher resolution. However, of note, the accuracy from the resolution of cell adhesive force imaging. Although these methods gained higher accuracy of bead tracking is different from the resolution of cell adhesive force imaging. Although these in tracking thegained beads,higher the cellaccuracy adhesiveinforce usually a resolution comparable methods tracking the cannot beads, be thereconstructed cell adhesive at force usually cannot be to reconstructed the accuracy level. The computation of to high-resolution traction also demands considerably at a resolution comparable the accuracy level. The force computation of high-resolution higher computation power and longer calculation time. traction force also demands considerably higher computation power and longer calculation time. Figure 2. 2.Advances ofofcell adhesive force imaging techniques. Figure Advancesininspatial spatialresolution resolution cell adhesive force imaging techniques.(A–C) (A–C)Force Forcemaps mapsof a fibroblast [57], a migatory keratocyte [58] and a stationary platelet [59] obtained by TFM. The resolution of a fibroblast [57], a migatory keratocyte [58] and a stationary platelet [59] obtained by TFM. The is resolution in the range 1–5 µm. of(D–F) Force maps of maps a fibroblast obtained by molecular tension sensor is inofthe range 1–5 μm. (D–F) Force of a fibroblast obtained by molecular tension (MTS) [43], a keratocyte obtainedobtained by integrative tension tension sensor (ITS) and[50] a platelet obtained sensor (MTS) [43], a keratocyte by integrative sensor[50] (ITS) and a platelet byobtained molecular fluorescence microscopy (MTFM) [60], respectively. The resolutions are at the by tension molecular tension fluorescence microscopy (MTFM) [60], respectively. The resolutions diffraction limit: 0.3–0.4 µm. (G–I) super-resolution force maps of a fibroblast obtained by MTS assisted are at the diffraction limit: 0.3–0.4 μm. (G–I) super-resolution force maps of a fibroblast obtained by with Bayesian computation [61], a keratocyte obtained by cellular force nanoscopy (CFN) [54] and a MTS assisted with Bayesian computation [61], a keratocyte obtained by cellular force nanoscopy platelet tPAINT [55], respectively. Resolutions were reported were as 50–100 nmas for50–100 the live (CFN)obtained [54] and aby platelet obtained by tPAINT [55], respectively. Resolutions reported cell samples. nm for the live cell samples. 2.2.2.2. Fluorescent Force Imaging ImagingtotoDiffraction DiffractionLimit Limit FluorescentTension TensionSensors SensorsBring Bringthe theResolution Resolution of of Force InIn thethe recent decade, FTSs have been developed to convert cellcell adhesive force to fluorescence onsite. recent decade, FTSs have been developed to convert adhesive force to fluorescence Immobilized on surfaces, these sensors are equipped with ligands that bind to target force-transmitting onsite. Immobilized on surfaces, these sensors are equipped with ligands that bind to target forcereceptors (mainly integrins) on the cell membrane. are plated and on sensor-grafted transmitting receptors (mainly integrins) on the cellCells membrane. Cells arecultured plated and cultured on sensor-grafted surfaces. The integrins bind and forcewhich to therespond sensors, with which respond with a surfaces. The integrins bind and transmit force totransmit the sensors, a fluorescent signal. fluorescent signal. While a variety of biomaterial molecules been adopted construct these While a variety of biomaterial molecules have been adoptedhave to construct these to sensors, as detailed detailed in another section, sensors share a similar force-sensing principle. The main in sensors, anotheras section, these sensors share these a similar force-sensing principle. The main constructs of the constructs of the sensors are deformable molecules to that are responsive to molecular tensions. The sensors are deformable molecules that are responsive molecular tensions. The constructs are labeled constructs labeled withpair a dye, dye–quencher pair orthe a construct dye–dye pair. When the isconstruct with a dye, aare dye–quencher or aadye–dye pair. When configuration altered by configuration is altered by force, the fluorescent activity of the sensor is consequently changed. force, the fluorescent activity of the sensor is consequently changed. Thus, cell adhesive forceThus, can be cell adhesive force can be reported by surfacegain fluorescence loss, fluorescence gain or(FRET) Förster[62] reported by surface fluorescence loss, fluorescence or Förster resonance energy transfer efficiency change. Note that the responsivity range of the sensor constructs should match the range of Sensors 2020, 20, 7128 5 of 14 the molecular force transmitted by single receptors (such as integrins). Empirically speaking, integrin tension has a broad range varying in 1–100 pN. With force-to-fluorescence conversion, the spatial resolution of cell adhesive force imaging was immediately brought to the diffraction limit. A series of fluorescent tension sensors such as MTS [43], TGT [21], MTFM sensor [60], TP [24] and ITS [49] have been developed to image cell adhesive forces. These sensors have been routinely applied to visualize cell adhesive forces in fibroblasts, platelets, keratocytes, T cells, etc. (Figure 2D–F). With these sensors, cell adhesive force has become as visible as cell structures, making the force-structure study highly convenient. A detailed discussion about these sensors is included in next section with an emphasis on biomaterials used in the sensor construction. Tension sensors report force with high resolution, but usually cannot measure the whole cell force. This is because tension sensor activation by force is stochastic at the molecular level, and not all cell adhesive force is transmitted onto sensors. In this regard, the tension sensor cannot replace TFM. However, the perk brought by tension sensors is not only the improved resolution, but also the capability of evaluating the force level of the molecular tension transmitted by single receptors. The molecular construct of the sensor can be tuned to be responsive to a designed force level, enabling the selective visualization of molecular tension in a certain range. Therefore, although these sensors do not evaluate bulk cell adhesive force, they have a unique advantage of assessing the cell adhesive force at the molecular tension level. 2.3. Super-Resolved Cell Adhesive Force Microscopies With FTSs, it seems naturally feasible to enable force imaging with resolution beyond the diffraction limit by integrating the sensors with super-resolution microscopies. The term “super-resolution” refers to the resolution beyond the diffraction limit of optical microscopy. Among the various techniques enabling super-resolution imaging, stochastic optical reconstruction microscopy (STORM) [63] and photoactivated localization microscopy (PALM) [64] are the representative ones. STORM and PALM share the similar principle of using single molecule imaging and localization to break the diffraction limit. The dyes in an ensemble are sparsely and stochastically turned on and off, to ensure the imaging of these dyes in a molecularly distinguishable manner. The center of the point-spread functions of fluorescence from each dye can be localized with ultra-high accuracy, which reaches the nanometer level. By imaging and localizing these dyes in many consecutive frames, one can assemble the frames and reconstruct the image of the dye-stained sample with a resolution beyond the diffraction limit. Since FTSs are able to convert force to fluorescence, the technical challenge to achieve super-resolution force imaging is about how to report force signals in a sparse and stochastic manner. Computational super-resolution force microscopy. Computational super-resolution force microscopy was developed with the combination of molecular tension sensors (MTSs) [61] and Bayesian localization microscopy [65]. In this method, cell adhesive force signals are recorded by MTS immobilized on a surface. The fluorescence activated by cell adhesive force is collected in ensemble, not at the molecular tension level. By inducing bleaching and blinking of the dyes in the consecutive frame, one can apply Bayesian analysis to extract spatial information of the sensor activation sites beyond the diffraction limit. It demonstrated the visualization of traction forces within FAs with about 100 nm spatial resolution (Figure 2G). This technique is the first attempt in the field to obtain cell adhesive force in a super-resolution manner. However, Bayesian analysis is computationally demanding, and it is difficult to acquire the cell adhesive force dynamics in real time. The resulting resolution is generally inferior to that provided by super-resolution microscopies based on single molecule localization. Super-resolution force microscopy based on single molecule imaging and localization. By the time of this review, two force microscopies based on molecule localization have emerged: cellular force nanoscopy (CFN) [54] and tPAINT [55]. Both techniques report integrin tensions in a stochastic and sparse manner, so that the fluorescent tension signals are distinguishable in each frame of imaging for the purpose of molecular localization. Single molecule localization is then performed to localize the tension events with ultra-high accuracy. By repeating this process to each frame, a force map Sensors 2020, 20, 20, 7128 Sensors 2020, x FOR PEER REVIEW of 14 6 of6 14 for the purpose of molecular localization. Single molecule localization is then performed to localize with super-resolution is obtained. Although both CFN and tPAINT use DNA-based tension sensors, the tension events with ultra-high accuracy. By repeating this process to each frame, a force map with there is distinct difference between them in CFN termsand of tPAINT how to achieve sparse and stochastic super-resolution is obtained. Although both use DNA-based tension sensors, tension there imaging. In CFN, a force-activatable emitter is used as the tension sensor. This emitter is conjugated is distinct difference between them in terms of how to achieve sparse and stochastic tension imaging. with quencher–dye pair, hence being darkasuntil activated by targeted integrin tension. with The dye In aCFN, a force-activatable emitter is used the tension sensor. This emitter is conjugated a of the emitter is bright and can be easily bleached. As a result, the fluorescence of tension quencher–dye pair, hence being dark until activated by targeted integrin tension. The dye ofevents the in each frame is collected bleached, to ensure sparsity required byofsingle molecule imaging emitter is bright and canand be easily bleached. As a the result, the fluorescence tension events in each andframe localization. Although the emitters are not activated and photobleached, theand high is collected and bleached, to ensure thereusable sparsityonce required by single molecule imaging 2 coating densityAlthough of sensors ) ensures sufficient cellphotobleached, adhesive forcethe imaging. localization. the(~2000/µm emitters are not reusable once sensors activatedfor and high 2) ensures sufficient coating density of sensors (~2000/μm sensors forare cellnot adhesive force imaging. Inor In tPAINT, the surface-immobilized DNA-hairpin tension sensors conjugated with dyes tPAINT, the surface-immobilized tension sensors are conjugated with adyes or quenchers. Instead, the sensor has aDNA-hairpin cryptic site that is openable by not force and provides docking Instead, the ssDNA-dye sensor has a cryptic sitein that openable solution. by force and provides docking site sitequenchers. for complementary diffusing theis ambient The sparse aand stochastic for complementary ssDNA-dye the ambient The sparse andthe stochastic tension imaging is enabled by thediffusing transientinbinding and solution. dissociation between dockingtension site and imaging is enabled by the and dissociation between the docking and ssDNAssDNA-dye. Both CFN andtransient tPAINTbinding have successfully achieved single integrinsite tension imaging, Both CFN and tPAINT successfully respectively, achieved single integrin tension imaging, and anddye. demonstrated 50 nm and 25have nm resolutions, in live cell adhesive force imaging demonstrated 50 nm and 25 nm resolutions, respectively, in live cell adhesive force imaging (Figure (Figure 2H,I). A potential limit of CFN originates from the irreversible dye bleaching, resulting in a 2H,I). A potential limit of CFN originates from the irreversible dye bleaching, resulting in a gradual gradual consumption of local sensors by cells. Still, in a practical test, CFN can provide up to 1000 consumption of local sensors by cells. Still, in a practical test, CFN can provide up to 1000 frames of frames of force images before the sensor density has an appreciable decrease. A potential limit of force images before the sensor density has an appreciable decrease. A potential limit of tPAINT could tPAINT could be due to the obstructed or reduced diffusion of ssDNA-dye to sensor location under be due to the obstructed or reduced diffusion of ssDNA-dye to sensor location under cell adhesion cell adhesion regions at the cell–substrate interface, therefore affecting the fidelity of reporting local regions at the cell–substrate interface, therefore affecting the fidelity of reporting local force density. force density. Another technical challenge shared by both CFN and tPAINT is the spontaneous DNA Another technical challenge shared by both CFN and tPAINT is the spontaneous DNA dissociation dissociation due energy to thermal energy yieldsfluorescent non-specific fluorescent signals to cell due to thermal which yieldswhich non-specific signals unrelated to cell unrelated adhesive force. adhesive force. In the future, a fluorescent tension sensor with higher thermal stability is desired In the future, a fluorescent tension sensor with higher thermal stability is desired to reduce or to reduce or eliminate such background false signals. eliminate such background false signals. 3. Biomaterial Choices Sensors 3. Biomaterial Choicesfor forConstructing ConstructingFluorescence Fluorescence Tension Tension Sensors AsAs FTSs areare in in thethe center at high high resolution, resolution,the thecritical critical part FTSs centerofofcell celladhesive adhesiveforce force imaging imaging at part of of anan FTSFTS construct is the force-responsive molecular biomaterial that changes conformation under tension. construct is the force-responsive molecular biomaterial that changes conformation under tension. This section reviews the materials Thesematerials materialsusually usually have This section reviews the materialsused usedfor fortension tensionsensor sensor construction. construction. These have well characterized mechanical for molecular molecularengineering. engineering.Figure Figure well characterized mechanicalproperties propertiesand andaccessible accessible chemistry chemistry for 3 3 presents these materials adoptedininconstructing constructing extracellular extracellular FTSs these presents these materials adopted FTSsin inaachronical chronicalorder. order.Among Among these materials, DNA and DNA-likematerials materialsare areparticularly particularly versatile ofof FTSs materials, DNA and DNA-like versatileto tobe beused usedasasthe theconstructs constructs FTSs because their programmableforce forcesensing sensingthresholds thresholds and sensor synthesis. because of of their programmable and convenient convenientchemistry chemistryfor for sensor synthesis. Figure 3. 3.Biomaterials fluorescenttension tensionsensors. sensors. Figure Biomaterialsfor forconstructing constructing extracellular extracellular fluorescent Sensors 2020, 20, 7128 7 of 14 Polyethylene glycol (PEG) as an entropy “spring” was applied in AFM to measure single molecular forces in 1999 [66]. PEG has accessible chemistry for modification, well-characterized mechanical properties and excellent biocompatibility and stability. Wiegand and colleagues reported the use of PEG tension sensors in quantitative analysis of a virus or particles uptake kinetics [39]. Stabley and colleagues constructed a PEG-based tension sensor that is labeled with a dye and immobilized on gold nanoparticle, which functions as a quencher [20]. The sensor successfully reported cell adhesive force by fluorescence turned on by the force. The sensor mapped the mechanical forces during the early stage of regulatory endocytosis of the ligand-activated epidermal growth factor receptor. With the same “spring” molecules conjugated with integrin ligand, Liu and colleagues visualized integrin-transmitted cell adhesive forces at the pN level at the cell–substrate interface. Integrin tension in the range from 1 to 15 pN was reported by quantitative characterization of different PEG molecules [22]. Peptide molecules are natural polymers and can function as entropic springs for force sensing. Intracellular tension sensors based on peptide were first developed in 2008 [18]. A peptide in an α-helix conformation coupled with a CFP–GFP pair was developed as a force-sensing cassette and inserted into alpha-actinin, non-erythrocyte spectrin and filamin A. Transfected cells successfully expressed these intracellular tension sensors, which reported cell adhesive force in cells. In 2011, Grashoff and colleagues used repetitive amino-acid motif derived from the spider flagelliform silk protein to replace the α-helix for force sensing [19]. The elastic property of such peptide was characterized by AFM-based force spectroscopy, showing broad force range from several pN to as high as 800–900 pN [67]. The “spring” coupled with two fluorescent proteins as a FRET pair was genetically inserted into a force-bearing protein vinculin to report intracellular force. The vinculin FRET sensor revealed that vinculin was required for stabilizing adhesions under force [19], and reported that tension across vinculin in stable focal adhesions was ~2.5 pN. Spider silk peptide was also adopted as biomaterial for the construction of an extracellular tension sensor. Morimatsu et al. developed a tension sensor consisting of a (GPGGA)8 peptide flanked with two organic dyes as a FRET-pair [23,42,43]. The peptide was decorated with biotin for surface immobilization and RGD peptide as integrin ligand. Integrin tensions were reported by the FRET value of the sensor. The dynamic range of the spider silk peptide-based sensor was 1–5 pN, therefore sensitive to the integrin tension variation at the lower level. In contrast, Galior et al. developed another type of peptide-based tension sensor for reporting integrin tensions at a very high level (>80 pN) [68]. In this design, immunoglobulin 27th (I27) domain of cardiac titin flanked with a fluorophore and gold nanoparticle was used as the main construct of the tension sensor. High-level tension (80−200 pN) [69] is required to expose the cryptic disulfide bonds in this peptide which is cleaved by redox reagent dithiothreitol in the cell culture medium. The disulfide bond cleavage leads to the extension of the peptide and the de-quenching of the fluorophore. As a result, this sensor reports molecular tension higher than 80 pN. It was shown that some integrin tensions are indeed able to activate this sensor, demonstrating the existence of high-level integrin tensions in live cells. dsDNA was initially adopted as a programmable tension sensor to evaluate the strength of molecular bonds such as antigen-antibody bonds [70,71]. Compared to other elastic polymers, DNA enjoys mature chemistry methods for synthesis and modification. Its tension responsivity can be conveniently adjusted by altering the DNA sequence or geometrical configuration (by adjusting the force application sites on the DNA backbones), hence being “programmable”. In 2013, a dsDNA-based tension sensor was first applied to a live cell force study [21]. Tension gauge tether (TGT) with dsDNA as the main construct was developed to quantitatively define the force transmitted by integrin molecules during cell adhesion [21]. TGT can be viewed as a molecular linker with a defined tension tolerance (Ttol ). Any integrin tensions higher than the Ttol value would rupture TGT and the tension becomes abolished, so that molecular tensions are restricted under a designed level. TGT defined the minimum tensions required for integrin and notch activations. Apart from determining the minimum force requirement for a force transmitting receptor activation, a fluorescently labeled TGT was also used to image the distribution of integrin molecular tensions in cells [46,48]. Two distinct levels of integrin tensions were Sensors 2020, 20, 7128 8 of 14 revealed by TGT: ~40 pN integrin tension caused by the cell membrane during initial cell adhesion, and >54 pN integrin tension in FAs due to the actomyosin contraction. DNA hairpin was soon adopted as another DNA construct for tension sensor construction [24,72]. A DNA hairpin is a single-stranded DNA forming intramolecular base pairs. Under a tension, the hairpin can be unzipped but the DNA strand remains intact. When the tension is removed, the hairpin is able to refold back. In contrast to dsDNA, DNA hairpin opening by force is reversible and the hairpin refolding negates the fluorescence signal, hence reporting cell adhesive force in a real-time manner. DNA hairpin-based tension sensors share a similar working mechanism as PEG-based tension sensors. The tension threshold can be more accurately defined by varying the GC content in the DNA sequence [72]. DNA hairpin-based molecular tension sensors were applied to uncover the role of cell adhesive force in cell initial adhesion and T cell activation [72,73]. The force distribution by integrin αIIb β3 in the process of platelet activation was spatially visualized by DNA hairpin–based tension sensors, revealing that platelets produced a force greater than 19 pN in the central zone but weaker forces (between 4.7 and 13.1 pN) in the cell edge (Figure 2F) [60]. DNA hairpin-based tension sensor was also applied to visualize the adhesive force at the cell-cell interface. Zhao et al. developed a tension sensor with one end labeled by cholesterol tags insertable to cell membrane, and the other end labeled with integrin ligand targeting the integrin, or labeled with cadherin targeting cadherin in neighboring cell surface [44]. This tension sensor successfully reported the force at the cell-cell interface. Nano-yoyo is a tension sensor based on a ssDNA-protein complex that is specialized in reporting low-level molecular tension (~4 pN) [74]. Both dsDNA-based and DNA hairpin-based tension sensors cannot report tensions lower than the DNA unzipping force which is at ~10 pN. In contrast, it was shown that the unbinding force between ssDNA and the SSB protein (ssDNA binding protein) is as low as ~4 pN. This ssDNA-SSB complex has a configuration similar to a yoyo, as the ssDNA wraps on the SSB with several turns, hence the name. The unique property of nano-yoyo is its good thermal stability. This is because of the long contact length between ssDNA and the SSB. In one complex, there are 65 nucleotides of DNA wrapping around the SSB tetramer, producing good thermal stability despite the low unbinding force, as the dissociation energy is determined by the product of force and length. A nano-yoyo-based tension sensor was applied to fathom the force required for Notch activation with higher sensitivity. It showed that the force requirement for Notch activation is as low as 4 pN, updating the previously reported value of <12 pN [21]. dsDNA labeled with a dye-quencher pair. Inspired by TGT, dsDNA labeled by a fluorophore-quencher pair, termed as integrative tension sensor (ITS) [49] or quenched TGT (qTGT) [47], was developed to image cell adhesive force with a positive fluorescence signal. The quenched dye on the ITS becomes fluorescent after the upper DNA strand with a quencher is removed by integrin tension, hence reporting cell adhesive force onsite (Figure 1D). The mechanical dissociation of dsDNA on the surface is irreversible, and therefore an ITS remains fluorescent after the force is released, leading to signal integration over time. The accumulated force signal offers higher sensitivity in cell adhesive force imaging, with the trade-off of non-real-time force imaging. However, real-time force mapping can still be achieved by recording the force map in a time-series manner and obtaining the newly produced force signal with the frame subtraction method. Figure 2E demonstrates the real-time force in a migrating keratocyte [50]. ITSs have been applied to study cell adhesive force in keratocytes and platelets. It was shown that high-level integrin tension (>54 pN) was generated at the cell rear margin to detach integrins off from the substrate, therefore facilitating cell rear retraction during fast cell migration [50]. ITSs also revealed the polarized force distribution in adherent platelets [49]. The spatial resolution of cell adhesive force imaging by ITS was calibrated to be 0.3–0.4 µm, approaching the diffraction limit of fluorescence microcopy. PNA/DNA hybrid was adopted to construct a tension sensor resisting the degradation from DNase on the cell membrane [75]. Many cancer cells such as mouse thyroid carcinoma cells and MDA-MB-231 breast cancer cells were reported to express substantial membrane-bound DNase [76]. Sensors 2020, 20, 7128 9 of 14 We also observed significant DNase activity on the cell membrane of macrophages and neutrophils (unpublished data). Such membrane-bound DNase easily destroys DNA-based tension sensors immobilized at the cell–substrate interface and separating the fluorophore-quencher pair without force involved. Thus, DNA-based tension sensors are potentially problematic in investigating adhesive forces of cancer cells and immune cells. In order to report cell adhesive force without disruption from DNase, Zhao et al. constructed tension sensors with PNA (peptide nucleic acid)/DNA hybrid duplexes [75]. The PNA/DNA hybrid structure shows strong resistance to DNase and retains the ability of reporting cell adhesive force. Moreover, PNA also enhances the fluorescence intensity of cyan dye by two-fold due to the PIFE effect (protein induced fluorescence enhancement), therefore increasing the signal-to-noise ratio in cell adhesive force imaging. PNA/DNA hybrid was used instead of PNA/PNA duplex (which likely has equal resistance to DNase if not better) because the DNA strand is more cost-effective and convenient for dye, quencher or integrin ligand conjugation. The PNA/DNA-based tension sensor broadens the application of FTSs in the study of cell adhesive force in the DNase-present environment. 4. Outlook Structural and biochemical studies in cells have historically preceded the force study because approaches are more available for visualizing and quantifying structural and biochemical signals. However, just like investigating how a car runs, one not only needs to know the parts constituting the car, but also needs to know how these parts synergistically produce and transmit the force driving the car. Similarly, force visualization and quantification are essential for the study of cell mechanobiology. Techniques were in demand to reveal when and where cell adhesive force is produced at cell–cell and cell–matrix interfaces. This review summarizes the recent development of techniques for cell adhesive force imaging, with a focus on the advances of spatial resolution and sensor constructs. TFM has pioneered the cell adhesive force visualization and measurement. Even now, it is advantageous in measuring the bulk cell forces, obtaining the local force direction and being applicable in 3-dimensional (3D) force mapping. In comparison, an FTS converts cell adhesive force to fluorescence onsite, greatly improving resolution and sensitivity in force imaging. FTS-based CAFM is also more convenient to implement in terms of assay preparation and data processing. Moreover, an FTS can selectively report receptor-transmitted molecular tensions at different force levels, as the tension sensor has pre-calibrated responsivity to molecular tensions. Overall, TFM and FTSs should be viewed as complementary techniques in cell adhesive force studies. However, in terms of spatial resolution, FTSs with onsite force-to-fluorescence conversion have apparent advantages in achieving high resolution and high sensitivity in cell adhesive force imaging. With FTSs, at present, cell adhesive force has become as visible as cell structures, and the force can even be imaged at the single molecular tension level. Currently, although no research has demonstrated simultaneous monitoring of both adhesive force and structural/biochemical signal at the molecular level in real time, there is no technical barrier in achieving such a feat. The comparable sensitivities and resolutions in cell adhesive force imaging and structure imaging provide great opportunities for the study of force–structure interplay in cells. We speculate that such force–structure imaging at the single molecule level would yield unprecedented insights into the mechanism of mechanotransduction in cells. The next challenge in cell adhesive force imaging is to generalize FTSs to the 3-dimensional (3D) context. To our knowledge, so far all FTSs were only used on 2D surfaces for imaging cell–matrix forces. There is great difficulty in applying current FTSs directly to 3D cell adhesive force imaging. This is because FTSs generally have non-zero fluorescence background, as the sensors are labeled with dyes (albeit in the proximity of quenchers). The fluorescence background is tolerable if FTSs are coated on 2D surfaces as a molecular monolayer. If grafted in the 3D matrix gel for 3D force imaging, the FTSs in the bulk gel would likely produce a bright background that submerges the cell force signal. We speculate that quenchers with extremely high quenching efficiency would be required for the application of FTSs in the 3D context. Another challenge in the application of FTS sensors comes from Sensors 2020, 20, 7128 10 of 14 their spontaneous activation due to thermal energy. The molecular sensors can stochastically overcome the energy barrier, therefore changing the sensor conformation and non-specifically activating the sensors, transiently or permanently. Such spontaneous activation causes false force signals which is especially problematic for the detection of single molecular tension events at a low rate of occurrence at the cell–matrix interface, such as the tethering force transmitted by selectin during leukocyte rolling, or the force transmitted by the von Willebrand factor receptor during platelet activation, etc. Therefore, lowering the fluorescence background and reducing the non-specific activation rate of FTSs would further improve the performance of FTSs in cell adhesive force imaging, especially at the molecular tension level. Until now, CAFM was mainly applied to image integrin-transmitted cellular force. Certainly, the central reason is that integrins are the major proteins mediating cell-matrix adhesion and playing vital roles in many cellular functions. In addition, integrins are clustered and form patterns in various cell adhesive units such as focal complexes, focal adhesions, filopodia, podosomes and invadosomes, making force imaging necessary to reveal the spatial dynamics of the integrin-transmitted force. Another reason is perhaps that integrins have a short peptide ligand (peptide RGD consisting of arginine, glycine, and aspartate) which is chemically stable and can tolerate the ligand-tension sensor conjugation process which could be chemically harsh to protein ligands. The small size of the integrin peptide ligand also facilitates FTS immobilization at a high density on a surface. Collectively, these factors make it attractive and feasible to image integrin-transmitted force in cells by FTSs. Still, as the force sensing module of FTSs is seeing steady advancement, the ligand part can use a better variety so that FTSs can be applied to the study of many other mechano-sensitive receptors on the cell membrane. Author Contributions: Conceptualization, Y.T. and X.W.; Writing—original draft preparation, Y.T. and X.W.; writing—review and editing, X.W.; visualization, Y.T. and X.W.; supervision, X.W.; funding acquisition, X.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Science Foundation (1825724) and the National Institute of General Medical Sciences (1R35GM128747). Conflicts of Interest: The authors declare no conflict of interest. Abbreviations CAFM CFN FTS ITS MTFM MTS TFM TGT TP t-PAINT Cell adhesive force microscopy Cellular force nanoscopy Fluorescent tension sensor Integrative tension sensor Molecular tension fluorescence microscopy Molecular tension sensor Traction force microscopy Tension gauge tether Tension probe tension-point accumulation in nanoscale topology References 1. 2. 3. 4. 5. Vogel, V.; Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 2006, 7, 265–275. [CrossRef] [PubMed] LeGoff, L.; Lecuit, T. Mechanical Forces and Growth in Animal Tissues. Cold Spring Harb. Perspect. Biol. 2015, 8, a019232. [CrossRef] [PubMed] Discher, D.E.; Janmey, P.; Wang, Y.-L. Tissue Cells Feel and Respond to the Stiffness of Their Substrate. Science 2005, 310, 1139–1143. [CrossRef] [PubMed] Kumar, S.; Weaver, V.M. Mechanics, malignancy, and metastasis: The force journey of a tumor cell. Cancer Metastasis Rev. 2009, 28, 113–127. [CrossRef] [PubMed] Geiger, B.; Spatz, J.P.; Bershadsky, A.D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 2009, 10, 21–33. [CrossRef] Sensors 2020, 20, 7128 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 11 of 14 Rossy, J.; Laufer, J.M.; Legler, D.F. Role of Mechanotransduction and Tension in T Cell Function. Front. Immunol. 2018, 9, 2638. [CrossRef] Harris, A.K.; Wild, P.; Stopak, D. Silicone rubber substrata: A new wrinkle in the study of cell locomotion. Science 1980, 208, 177–179. [CrossRef] Balaban, N.Q.; Schwarz, U.S.; Riveline, D.; Goichberg, P.; Tzur, G.; Sabanay, I.; Mahalu, D.; Safran, S.; Bershadsky, A.; Addadi, L.; et al. Force and focal adhesion assembly: A close relationship studied using elastic micropatterned substrates. Nat. Cell Biol. 2001, 3, 466–472. [CrossRef] Beningo, K.A.; Dembo, M.; Kaverina, I.; Small, J.V.; Wang, Y.-l. Nascent Focal Adhesions Are Responsible for the Generation of Strong Propulsive Forces in Migrating Fibroblasts. J. Cell Biol. 2001, 153, 881–888. [CrossRef] Tan, J.L.; Tien, J.; Pirone, D.M.; Gray, D.S.; Bhadriraju, K.; Chen, C.S. Cells lying on a bed of microneedles: An approach to isolate mechanical force. Proc. Natl. Acad. Sci. USA 2003, 100, 1484. [CrossRef] Legant, W.R.; Miller, J.S.; Blakely, B.L.; Cohen, D.M.; Genin, G.M.; Chen, C.S. Measurement of mechanical tractions exerted by cells in three-dimensional matrices. Nat. Methods 2010, 7, 969. [CrossRef] [PubMed] Franck, C.; Maskarinec, S.A.; Tirrell, D.A.; Ravichandran, G. Three-Dimensional Traction Force Microscopy: A New Tool for Quantifying Cell-Matrix Interactions. PLoS ONE 2011, 6, e17833. [CrossRef] [PubMed] Plotnikov, S.V.; Pasapera, A.M.; Sabass, B.; Waterman, C.M. Force Fluctuations within Focal Adhesions Mediate ECM-Rigidity Sensing to Guide Directed Cell Migration. Cell 2012, 151, 1513–1527. [CrossRef] [PubMed] del Álamo, J.C.; Meili, R.; Álvarez-González, B.; Alonso-Latorre, B.; Bastounis, E.; Firtel, R.; Lasheras, J.C. Three-Dimensional Quantification of Cellular Traction Forces and Mechanosensing of Thin Substrata by Fourier Traction Force Microscopy. PLoS ONE 2013, 8, e69850. [CrossRef] [PubMed] Colin-York, H.; Shrestha, D.; Felce, J.H.; Waithe, D.; Moeendarbary, E.; Davis, S.J.; Eggeling, C.; Fritzsche, M. Super-Resolved Traction Force Microscopy (STFM). Nano Lett. 2016, 16, 2633–2638. [CrossRef] Stubb, A.; Laine, R.F.; Miihkinen, M.; Hamidi, H.; Guzmán, C.; Henriques, R.; Jacquemet, G.; Ivaska, J. Fluctuation-Based Super-Resolution Traction Force Microscopy. Nano Lett. 2020, 20, 2230–2245. [CrossRef] Polacheck, W.J.; Chen, C.S. Measuring cell-generated forces: A guide to the available tools. Nat. Methods 2016, 13, 415–423. [CrossRef] Meng, F.; Suchyna, T.M.; Sachs, F. A fluorescence energy transfer-based mechanical stress sensor for specific proteins in situ. FEBS J. 2008, 275, 3072–3087. [CrossRef] Grashoff, C.; Hoffman, B.D.; Brenner, M.D.; Zhou, R.; Parsons, M.; Yang, M.T.; McLean, M.A.; Sligar, S.G.; Chen, C.S.; Ha, T.; et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 2010, 466, 263–266. [CrossRef] Stabley, D.R.; Jurchenko, C.; Marshall, S.S.; Salaita, K.S. Visualizing mechanical tension across membrane receptors with a fluorescent sensor. Nat. Methods 2012, 9, 64–67. [CrossRef] Wang, X.; Ha, T. Defining Single Molecular Forces Required to Activate Integrin and Notch Signaling. Science 2013, 340, 991–994. [CrossRef] [PubMed] Liu, Y.; Yehl, K.; Narui, Y.; Salaita, K. Tension sensing nanoparticles for mechano-imaging at the living/nonliving interface. J. Am. Chem. Soc. 2013, 135, 5320–5323. [CrossRef] [PubMed] Morimatsu, M.; Mekhdjian, A.H.; Adhikari, A.S.; Dunn, A.R. Molecular tension sensors report forces generated by single integrin molecules in living cells. Nano Lett. 2013, 13, 3985–3989. [CrossRef] [PubMed] Blakely, B.L.; Dumelin, C.E.; Trappmann, B.; McGregor, L.M.; Choi, C.K.; Anthony, P.C.; Duesterberg, V.K.; Baker, B.M.; Block, S.M.; Liu, D.R.; et al. A DNA-based molecular probe for optically reporting cellular traction forces. Nat. Methods 2014, 11, 1229–1232. [CrossRef] Jurchenko, C.; Salaita, K.S. Lighting up the Force: Investigating Mechanisms of Mechanotransduction Using Fluorescent Tension Probes. Mol. Cell. Biol. 2015, 35, 2570–2582. [CrossRef] Matellan, C.; Del Río Hernández, A.E. Where No Hand Has Gone Before: Probing Mechanobiology at the Cellular Level. ACS Biomater. Sci. Eng. 2019, 5, 3703–3719. [CrossRef] Ma, V.P.-Y.; Salaita, K. DNA Nanotechnology as an Emerging Tool to Study Mechanotransduction in Living Systems. Small 2019, 15, 1900961. [CrossRef] Yasunaga, A.; Murad, Y.; Li, I.T.S. Quantifying molecular tension-classifications, interpretations and limitations of force sensors. Phys. Biol. 2020, 17, 011001. [CrossRef] Binnig, G.; Quate, C.F.; Gerber, C. Atomic Force Microscope. Phys. Rev. Lett. 1986, 56, 930–933. [CrossRef] Sensors 2020, 20, 7128 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 12 of 14 Ashkin, A. Optical trapping and manipulation of neutral particles using lasers. Proc. Natl. Acad. Sci. USA 1997, 94, 4853–4860. [CrossRef] Smith, S.B.; Finzi, L.; Bustamante, C. Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science 1992, 258, 1122. [CrossRef] [PubMed] Evans, E.; Ritchie, K.; Merkel, R. Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces. Biophys. J. 1995, 68, 2580–2587. [CrossRef] Neuman, K.C.; Nagy, A. Single-molecule force spectroscopy: Optical tweezers, magnetic tweezers and atomic force microscopy. Nat. Methods 2008, 5, 491–505. [CrossRef] [PubMed] Dembo, M.; Wang, Y.L. Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophys. J. 1999, 76, 2307–2316. [CrossRef] Fu, J.; Wang, Y.K.; Yang, M.T.; Desai, R.A.; Yu, X.; Liu, Z.; Chen, C.S. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat. Methods 2010, 7, 733–736. [CrossRef] Pandey, P.; Hawkes, W.; Hu, J.; Megone, W.V.; Gautrot, J.; Anilkumar, N.; Zhang, M.; Hirvonen, L.; Cox, S.; Ehler, E.; et al. Cardiomyocytes Sense Matrix Rigidity through a Combination of Muscle and Non-muscle Myosin Contractions. Dev. Cell 2018, 44, 326–336.e323. [CrossRef] Sabass, B.; Gardel, M.L.; Waterman, C.M.; Schwarz, U.S. High resolution traction force microscopy based on experimental and computational advances. Biophys. J. 2008, 94, 207–220. [CrossRef] Hui, K.L.; Balagopalan, L.; Samelson, L.E.; Upadhyaya, A. Cytoskeletal forces during signaling activation in Jurkat T-cells. Mol. Biol. Cell 2015, 26, 685–695. [CrossRef] Wiegand, T.; Fratini, M.; Frey, F.; Yserentant, K.; Liu, Y.; Weber, E.; Galior, K.; Ohmes, J.; Braun, F.; Herten, D.P.; et al. Forces during cellular uptake of viruses and nanoparticles at the ventral side. Nat. Commun. 2020, 11, 1–13. [CrossRef] Glazier, R.; Brockman, J.M.; Bartle, E.; Mattheyses, A.L.; Destaing, O.; Salaita, K. DNA mechanotechnology reveals that integrin receptors apply pN forces in podosomes on fluid substrates. Nat. Commun. 2019, 10, 1–13. [CrossRef] Liu, Y.; Medda, R.; Liu, Z.; Galior, K.; Yehl, K.; Spatz, J.P.; Cavalcanti-Adam, E.A.; Salaita, K. Nanoparticle tension probes patterned at the nanoscale: Impact of integrin clustering on force transmission. Nano Lett 2014, 14, 5539–5546. [CrossRef] [PubMed] Tan, S.J.; Chang, A.C.; Anderson, S.M.; Miller, C.M.; Prahl, L.S.; Odde, D.J.; Dunn, A.R. Regulation and dynamics of force transmission at individual cell-matrix adhesion bonds. Sci. Adv. 2020, 6, 1–12. [CrossRef] [PubMed] Chang, A.C.; Mekhdjian, A.H.; Morimatsu, M.; Denisin, A.K.; Pruitt, B.L.; Dunn, A.R. Single Molecule Force Measurements in Living Cells Reveal a Minimally Tensioned Integrin State. ACS Nano 2016, 10, 10745–10752. [CrossRef] [PubMed] Zhao, B.; O’Brien, C.; Mudiyanselage, A.P.K.K.K.; Li, N.W.; Bagheri, Y.; Wu, R.; Sun, Y.B.; You, M.X. Visualizing Intercellular Tensile Forces by DNA-Based Membrane Molecular Probes. J. Am. Chem. Soc. 2017, 139, 18182–18185. [CrossRef] Blanchard, A.T.; Bazrafshan, A.S.; Yi, J.; Eisman, J.T.; Yehl, K.M.; Bian, T.; Mugler, A.; Salaita, K. Highly Polyvalent DNA Motors Generate 100+ pN of Force via Autochemophoresis. Nano Lett. 2019, 19, 6977–6986. [CrossRef] Wang, Y.; Wang, X. Integrins outside focal adhesions transmit tensions during stable cell adhesion. Sci. Rep. 2016, 6, 1–9. [CrossRef] Jo, M.H.; Cottle, W.T.; Ha, T. Real-Time Measurement of Molecular Tension during Cell Adhesion and Migration Using Multiplexed Differential Analysis of Tension Gauge Tethers. ACS Biomater. Sci. Eng. 2019, 5, 3856–3863. [CrossRef] Wang, X.; Sun, J.; Xu, Q.; Chowdhury, F.; Roein-Peikar, M.; Wang, Y.; Ha, T. Integrin Molecular Tension within Motile Focal Adhesions. Biophys. J. 2015, 109, 2259–2267. [CrossRef] Wang, Y.; LeVine, D.N.; Gannon, M.; Zhao, Y.; Sarkar, A.; Hoch, B.; Wang, X. Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging. Biosens. Bioelectron. 2018, 100, 192–200. [CrossRef] Zhao, Y.; Wang, Y.; Sarkar, A.; Wang, X. Keratocytes Generate High Integrin Tension at the Trailing Edge to Mediate Rear De-adhesion during Rapid Cell Migration. iScience 2018, 9, 502–512. [CrossRef] Sensors 2020, 20, 7128 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 13 of 14 Murad, Y.; Li, I.T.S. Quantifying Molecular Forces with Serially Connected Force Sensors. Biophys. J. 2019, 116, 1282–1291. [CrossRef] [PubMed] Wang, X.; Rahil, Z.; Li, I.T.S.; Chowdhury, F.; Leckband, D.E.; Chemla, Y.R.; Ha, T. Constructing modular and universal single molecule tension sensor using protein G to study mechano-sensitive receptors. Sci. Rep. 2016, 6, 21584. [CrossRef] [PubMed] Li, I.T.S.; Ha, T.; Chemla, Y.R. Mapping cell surface adhesion by rotation tracking and adhesion footprinting. Sci. Rep. 2017, 7, 44502. [CrossRef] [PubMed] Zhao, Y.; Pal, K.; Tu, Y.; Wang, X. Cellular Force Nanoscopy with 50 nm Resolution Based on Integrin Molecular Tension Imaging and Localization. J. Am. Chem. Soc. 2020, 142, 6930–6934. [CrossRef] Brockman, J.M.; Su, H.; Blanchard, A.T.; Duan, Y.; Meyer, T.; Quach, M.E.; Glazier, R.; Bazrafshan, A.; Bender, R.L.; Kellner, A.V.; et al. Live-cell super-resolved PAINT imaging of piconewton cellular traction forces. Nat. Methods 2020, 17, 1018–1024. [CrossRef] Gardel, M.L.; Sabass, B.; Ji, L.; Danuser, G.; Schwarz, U.S.; Waterman, C.M. Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed. J. Cell Biol. 2008, 183, 999–1005. [CrossRef] Legant, W.R.; Choi, C.K.; Miller, J.S.; Shao, L.; Gao, L.; Betzig, E.; Chen, C.S. Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions. Proc. Natl. Acad. Sci. USA 2013, 110, 881–886. [CrossRef] Barnhart, E.; Lee, K.C.; Allen, G.M.; Theriot, J.A.; Mogilner, A. Balance between cell-substrate adhesion and myosin contraction determines the frequency of motility initiation in fish keratocytes. Proc. Natl. Acad. Sci. USA 2015, 112, 5045–5050. [CrossRef] Schwarz Henriques, S.; Sandmann, R.; Strate, A.; Koster, S. Force field evolution during human blood platelet activation. J. Cell Sci. 2012, 125, 3914–3920. [CrossRef] Zhang, Y.; Qiu, Y.; Blanchard, A.T.; Chang, Y.; Brockman, J.M.; Ma, V.P.-Y.; Lam, W.A.; Salaita, K. Platelet integrins exhibit anisotropic mechanosensing and harness piconewton forces to mediate platelet aggregation. Proc. Natl. Acad. Sci. USA 2018, 115, 325. [CrossRef] Morimatsu, M.; Mekhdjian, A.H.; Chang, A.C.; Tan, S.J.; Dunn, A.R. Visualizing the Interior Architecture of Focal Adhesions with High-Resolution Traction Maps. Nano Lett. 2015, 15, 2220–2228. [CrossRef] [PubMed] Sahoo, H. Förster resonance energy transfer—A spectroscopic nanoruler: Principle and applications. J. Photochem. Photobiol. C Photochem. Rev. 2011, 12, 20–30. [CrossRef] Rust, M.J.; Bates, M.; Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 2006, 3, 793–795. [CrossRef] [PubMed] Betzig, E.; Patterson, G.H.; Sougrat, R.; Lindwasser, O.W.; Olenych, S.; Bonifacino, J.S.; Davidson, M.W.; Lippincott-Schwartz, J.; Hess, H.F. Imaging intracellular fluorescent proteins at nanometer resolution. Science 2006, 313, 1642–1645. [CrossRef] [PubMed] Cox, S.; Rosten, E.; Monypenny, J.; Jovanovic-Talisman, T.; Burnette, D.T.; Lippincott-Schwartz, J.; Jones, G.E.; Heintzmann, R. Bayesian localization microscopy reveals nanoscale podosome dynamics. Nat. Methods 2012, 9, 195–200. [CrossRef] Oesterhelt, F.; Rief, M.; Gaub, H.E. Single molecule force spectroscopy by AFM indicates helical structure of poly(ethylene-glycol) in water. New J. Phys. 1999, 1, 6. [CrossRef] Becker, N.; Oroudjev, E.; Mutz, S.; Cleveland, J.P.; Hansma, P.K.; Hayashi, C.Y.; Makarov, D.E.; Hansma, H.G. Molecular nanosprings in spider capture-silk threads. Nat. Mater. 2003, 2, 278–283. [CrossRef] Galior, K.; Liu, Y.; Yehl, K.; Vivek, S.; Salaita, K. Titin-Based Nanoparticle Tension Sensors Map High-Magnitude Integrin Forces within Focal Adhesions. Nano Lett. 2016, 16, 341–348. [CrossRef] Li, H.B.; Carrion-Vazquez, M.; Oberhauser, A.F.; Marszalek, P.E.; Fernandez, J.M. Point mutations alter the mechanical stability of immunoglobulin modules. Nat. Struct. Biol. 2000, 7, 1117–1120. Albrecht, C.; Blank, K.; Lalic-Mülthaler, M.; Hirler, S.; Mai, T.; Gilbert, I.; Schiffmann, S.; Bayer, T.; Clausen-Schaumann, H.; Gaub, H.E. DNA: A Programmable Force Sensor. Science 2003, 301, 367. [CrossRef] Jones, M.R.; Seeman, N.C.; Mirkin, C.A. Programmable materials and the nature of the DNA bond. Science 2015, 347, 6224. [CrossRef] [PubMed] Zhang, Y.; Ge, C.; Zhu, C.; Salaita, K. DNA-based digital tension probes reveal integrin forces during early cell adhesion. Nat. Commun. 2014, 5, 5167. [CrossRef] [PubMed] Sensors 2020, 20, 7128 73. 74. 75. 76. 14 of 14 Liu, Y.; Blanchfield, L.; Ma, V.P.-Y.; Andargachew, R.; Galior, K.; Liu, Z.; Evavold, B.; Salaita, K. DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity. Proc. Natl. Acad. Sci. USA 2016, 113, 5610. [CrossRef] [PubMed] Chowdhury, F.; Li, I.T.S.; Ngo, T.T.M.; Leslie, B.J.; Kim, B.C.; Sokoloski, J.E.; Weiland, E.; Wang, X.; Chemla, Y.R.; Lohman, T.M.; et al. Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo. Nano Lett. 2016, 16, 3892–3897. [CrossRef] Zhao, Y.; Sarkar, A.; Wang, X. Peptide nucleic acid based tension sensor for cellular force imaging with strong DNase resistance. Biosens. Bioelectron. 2020, 150, 111959. [CrossRef] Boutry, C.M.; Beker, L.; Kaizawa, Y.; Vassos, C.; Tran, H.; Hinckley, A.C.; Pfattner, R.; Niu, S.; Li, J.; Claverie, J.; et al. Biodegradable and flexible arterial-pulse sensor for the wireless monitoring of blood flow. Nat. Biomed. Eng. 2019, 3, 47–57. [CrossRef] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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https://openalex.org/W4392968663	https://www.obgynia.com/obgyn/index.php/obgynia/article/download/75/pdf	Indonesian	null	Perbedaan Morfologi dan Fragmentasi DNA Sperma sebelum dan sesudah Kriopreservasi dengan Metode Slow Cooling di Klinik Aster RSUP Dr. Hasan Sadikin Bandung	Obgynia	2,018	cc-by-sa	3,231	eISSN 2615-496X eISSN 2615-496X Perbedaan Morfologi dan Fragmentasi DNA Sperma sebelum dan sesudah Kriopreservasi dengan Metode Slow Cooling di Klinik Aster RSUP Dr. Hasan Sadikin Bandung Faizal Arif,1 Tono Djuwantono,2 Dian Tjahyadi, 2 Yusuf Sulaeman Effendi, 2 Anita Deborah Anwar, 2 Amillia Siddiq 2 1Rumah Sakit Ibu dan Anak Asyiyah Samarinda Kalimantan Timur 2 Departemen Obstetri dan Ginekologi Fakultas Kedokteran Universitas Padjadjaran Rumah Sakit Dr. Hasan Sadikin Bandung Korespondensi: Faizal Arif, Email: faizalarif1984@gmail.com Abstrak Tujuan: Penelitian ini bertujuan untuk mengetahui perbedaan parameter fragmentasi DNA, morfologi sperma pasca proses pembekuan dengan metode slow cooling. Metode: Penelitian ini merupakan penelitian obervasional analitik dengan pendekatan pre-post design. Subjek penelitian adalah sperma dengan hasil analisis yang normal sesuai dengan standar WHO (n=25). Penelitian dilakukan di Klinik Aster RSUP Dr. Hasan Sadikin Bandung pada bulan Juli hingga Agustus 2017. dilakukan di Klinik Aster RSUP Dr. Hasan Sadikin Bandung pada bulan Juli hingga Agustus 2017. Hasil: Setelah proses kriopreservasi, terdapat peningkatan fragmentasi DNA tiga kali lipat (nilai p<0,05) dan terdapat penurunan jumlah morfologi normal sebesar 50% (nilai p<0,05). Kesimpulan: Terdapat penurunan kualitas sperma pasca proses kriopreservasi dengan metode slow cooling g p gg g Hasil: Setelah proses kriopreservasi, terdapat peningkatan fragmentasi DNA tiga kali lipat (nilai p<0,05) dan terdapat penurunan jumlah morfologi normal sebesar 50% (nilai p<0,05). esimpulan: Terdapat penurunan kualitas sperma pasca proses kriopreservasi dengan metode slow cooling Kata kunci: Fragmentasi DNA, morfologi, slow cooling, spermatozoa Difference of DNA Fragmentation, Morphology Sperm before and after Cryopreservation with Slow Cooling Method in Aster Fertility Clinic Dr. Hasan Sadikin General Hospital Bandung Abstract Objective: Of this study was to compare sperm quality parameters including DNA fragmentation and morphology after cryopreservation with slow-cooling method. Key words: DNA fragmentation, morphology, slow cooling, spermatozoa Abstract Objective: Of this study was to compare sperm quality parameters including DNA fragmentation and morphology after cryopreservation with slow-cooling method. Method: This was an analytical observational study with pre and post design. Subjects were men whose sperm analysis met the WHO criteria of being normal (n=25). The study was conducted at the Aster Clinic of Dr. Hasan Sadikin General Hospital Bandung from July to August 2017. p g f y g Results: After cryopreservation, there was a three fold increase of DNA fragmentation (with p value <0.05) and a decrease in morphology 50% (with p value <0.05). p g f y g ts: After cryopreservation, there was a three fold increase of DNA fragmentation (with p value <0.05) and ase in morphology 50% (with p value <0.05). p gy ( p ) lusion: There is a decrease in sperm quality after cryopreservation with slow-cooling method. Key words: DNA fragmentation, morphology, slow cooling, spermatozoa 131 Obgynia, Volume 1 Nomor 2 September 2018 (slow cooling) pada berbagai penelitian sebelumnya dilaporkan memiliki hasil dan hasil yang bervariasi. Sinan dkk. melaporkan bahwa terdapat penurunan kualitas morfologi, sementara saat menilai fragmentasi DNA, dilaporkan bahwa kriopreservasi tidak menyebabkan kerusakan pada sel. Beberapa penelitian lain menunjukkan bahwa ada peningkatan fragmentasi DNA setelah kriopreservasi. Pattama dkk melaporkan bahwa motilitas dan fragmentasi DNA meningkat setelah kriopreservasi dengan metode pendinginan lambat.5-10 Perbedaan ini mungkin karena metode kriopreservasi yang berbeda, yaitu antara metode pendinginan lambat dan vitrifikasi. Diharapkan, penelitian ini dapat mengevaluasi perbedaan atau jumlah fragmentasi DNA setelah kriopreservasi sperma dengan metode pendinginan lambat. Akhirnya, penelitian ini berharap bisa membantu keluarga yang mengalami kemandulan dan ingin mendapatkan keturunan. Pendahuluan Infertilitas adalah ketika pasangan gagal mencapai kehamilan setelah setidaknya 12 bulan melakukan hubungan seksual reguler tanpa menggunakan kontrasepsi. Jenis infertilitas ini juga disebut infertilitas primer. Gangguan kesuburan dapat menyebabkan dampak yang besar bagi pasangan, tidak hanya mengenai masalah medis tetapi juga masalah ekonomi dan psikologis.1 Di Indonesia, terdapat 40% pasangan usia subur dan 10% di antara pasangan tersebut mengalami infertilitas. Penyebab infertilitas pada pasangan suami istri dapat diklasifikasikan menjadi tiga kelompok, dengan proporsi sebagai berikut: faktor wanita 45%, faktor pria 40% dan faktor idiopatik 15%. Salah satu metode untuk mengevaluasi etiologi infertilitas yang terkait dengan pria adalah dengan menilai kualitas semen, yaitu analisis sperma. Beberapa parameter analisis sperma yang dapat dievaluasi meliputi morfologi, konsentrasi, kromatin, dan fragmentasi DNA sperma.2 Faizal Arif: Perbedaan Morfologi dan Fragmentasi DNA Sperma puluh lima spesimen spermatozoa normal berdasarkan standar WHO diproses dan ditambahkan dengan larutan krioprotektan. Spesimen campuran kemudian diberi cryopreserved dengan metode pendinginan yang lambat. yang ditemukan pada hasil analisis sperma, dan 2) pasien memiliki alasan tertentu untuk tidak berpartisipasi sebagai subjek penelitian. Penelitian ini dilakukan setelah menerima kesepakatan dan rekomendasi dari Komite Etika Penelitian Kesehatan, Fakultas Kedokteran Universitas Padjadjaran, Rumah Sakit Umum Dr. Hasan Sadikin Bandung. Tabel 1 menyajikan perbandingan fragmentasi DNA di dalam spesimen sebelum dan sesudah kriopreservasi yang dihitung dengan menggunakan uji statistik Wilcoxon. Fragmentasi DNA ditemukan tiga kali lebih tinggi setelah kriopreservasi (p <0,05). Pasien yang memenuhi kriteria inklusi dan bersedia mengikuti penelitian ini setelah diberi penjelasan dan telah menandatangani informed consent tertulis akan diminta mengikuti prosedur berikut, yaitu kumpulan sampel sperma. Sampel sperma yang telah dianalisis dan ternyata normal akan menjalani pencucian dengan menggunakan centrifuge selama 5-10 menit. Setelah prosedur pencucian, spesimen akan dibagi menjadi 2, yaitu: 1). spesimen untuk penilaian parameter morfologi, dan 2). spesimen untuk penilaian fragmentasi DNA menggunakan metode Sperm Chromatin Disperse (SCD). Kedua spesimen spermatozoa tersebut akan menjalani kriopreservasi dengan metode pendinginan lambat. Spesimen spermatozoa beku akan disimpan dalam tabung khusus selama sebulan dan kemudian akan dicairkan. Tabel 2 menyajikan perbandingan morfologi sperma sebelum dan sesudah kriopreservasi dihitung dengan menggunakan uji paired-T. Kami menemukan bahwa rata-rata skor morfologi normal sebelum kriopreservasi lebih tinggi dibandingkan dengan kriopreservasi. Skor rata-rata sebelum kriopreservasi adalah 2,2(1,6), sedangkan nilai rata-rata setelah kriopreservasi adalah 1,4(1,1). Dengan demikian, dapat disimpulkan bahwa ada penurunan jumlah sperma yang signifikan secara statistik sebesar 50% dengan morfologi normal setelah kriopreservasi (p=0,05). Tabel 1 Perbandingan Fragmentasi DNA sebelum dan sesudah Kriopreservasi dengan Metode Pendinginan Lambat Fragmentasi DNA Pengukuran nilai-p* Pre Post Rerata (Std Deviasi) 0,1 (0,1) 0.2 (0,3) <0,001 Median 0,018 0,147 Kisaran 0,00− 0,243 0,009− 0,990 Catatan: * dihitung berdasarkan uji Wilcoxon, * pre: pre washing, post: post thawing Tabel 1 Perbandingan Fragmentasi DNA sebelum dan sesudah Kriopreservasi dengan Metode Pendinginan Lambat Fragmentasi DNA Pengukuran nilai-p* Pre Post Rerata (Std Deviasi) 0,1 (0,1) 0.2 (0,3) <0,001 Median 0,018 0,147 Kisaran 0,00− 0,243 0,009− 0,990 Catatan: * dihitung berdasarkan uji Wilcoxon, * pre: pre washing, post: post thawing Variabel spesimen sperma, yang akan diteliti dalam penelitian ini, meliputi sperma (diamati dari jumlah dan motilitas sperma yang layak), morfologi sperma, dan fragmentasi DNA sperma. Data yang dihitung kemudian diolah dengan analisis deskriptif dan analitik. Faizal Arif: Perbedaan Morfologi dan Fragmentasi DNA Sperma Analisis deskriptif terhadap data akan disajikan sebagai mean, standar deviasi, median, dan range. Uji statistik yang digunakan untuk membandingkan sperma sebelum dan sesudah prosedur dipasangkan uji T atau uji Wilcoxon jika datanya tidak terdistribusi normal. Hasil dianggap signifikan jika p <0,05. Catatan: * dihitung berdasarkan uji Wilcoxon, * pre: pre washing, post: post thawing Metode Salah satu cara untuk mengatasi masalah ketidaksuburan pada pria adalah dengan melestarikan spermatozoa. Metode yang paling umum digunakan untuk melestarikan spermatozoa adalah dengan kriopreservasi. Kriopreservasi adalah teknik untuk melestarikan bahan genetik dalam kondisi beku di bawah suhu rendah melalui pengurangan aktivitas metabolik tanpa mempengaruhi organel intraselular, atau fisiologis, biologi, dan morfologi sel. Teknik kriopreservasi dapat dikelompokkan menjadi metode pendinginan lambat (slow cooling) dan metode pendinginan cepat (vitrification). Teknik kriopreservasi yang paling sering dilakukan sampai saat ini adalah metode pendinginan lambat. Metode ini telah sering diterapkan untuk melestarikan berbagai sel, jaringan, serta organ tubuh manusia.3, 4 Penelitian ini merupakan penelitian analitik observasional dengan pendekatan desain studi pra-pasca dari bulan Juli−Agustus 2017 di Instalasi reproduksi berbantu umum Dr. Hasan Sadikin Bandung. Pengumpulan sampel dilakukan dengan menggunakan metode sampling berturut-turut dan subjek dialokasikan secara acak ke dalam salah satu dari kedua kelompok. Spesimen penelitian adalah spesimen sperma dengan hasil analisis normal berdasarkan standar WHO yang dikumpulkan dari pria usia subur yang datang ke klinik aster RSUP Dr. Hasan Sadikin Bandung. Kriteria inklusi dari penelitian ini meliputi: 1). bersedia berpartisipasi sebagai subjek penelitian, 2). pria usia subur 20−35 tahun, dan 3). memiliki hasil analisis sperma yang normal berdasarkan kriteria WHO. Sedangkan kriteria eksklusi dari penelitian ini meliputi: 1). Ada kelainan Proses kriopreservasi spermatozoa menggunakan metode pendinginan lambat 132 Hasil Penelitian ini dilakukan selama satu bulan dari bulan Juli sampai Agustus 2017. Dua 133 Obgynia, Volume 1 Nomor 2 September 2018 Tabel 2 Perbandingan Morfologi sebelum dan sesudah Kriopreservasi dengan Metode Pendinginan Lambat Morfologi Pengukuran nilai-p* Pre Post Rerata±Std Deviasi 2,2 (1,6) 1,4 (1,1) 0,005 Median 2 1 Kisaran 0,00–5.00 0,00–4,00 Catatan: nilai-p dihitung berdasarkan uji T berpasangan. Perbandingan Morfologi sebelum dan sesudah Kriopreservasi dengan Metode Pendinginan Lambat mengelilingi spermatozoa. Kristal es ini akan meningkatkan konsentrasi natrium, glukosa, dan protein. Pada kondisi ini, tekanan osmotik di luar sel akan meningkat secara bertahap, menyebabkan cairan intraselular bocor di luar, terutama dari kepala spermatozoa dan menyebabkan dehidrasi di dalam sel. Metode pendinginan yang lambat juga menyebabkan perubahan transisi fase lipid, peningkatan lipid peroxidase, dan produksi spesies oksigen reaktif (ROS). Semua proses ini dapat menurunkan parameter spermatozoa.5, 8, 12, 13 Secara keseluruhan, hasil dalam penelitian ini menunjukkan bahwa prosedur pendinginan dan pencairan dapat menyebabkan perubahan morfologi sperma. Sementara itu, pada analisis integritas DNA, kami menemukan peningkatan fragmentasi DNA pasca pendinginan dan prosedur pencairan. Faizal Arif: Perbedaan Morfologi dan Fragmentasi DNA Sperma meningkat sebesar 7,7(7,9%). Kriopreservasi juga dilaporkan mengubah kondensasi kromatin spermatozoa dalam penelitian oleh Boitrelle, yaitu sebesar 17% untuk pasien dengan sperma normal dan 24,5% untuk pasien dengan teratozoospermia. Di sisi lain, beberapa penelitian menemukan bahwa tidak ada kerusakan pada spermatozoa setelah kriopreservasi. Pattama dkk menyatakan bahwa motilitas dan fragmentasi DNA memiliki hasil yang lebih baik setelah kriopreservasi dengan metode pendinginan lambat. Penelitian lain oleh Vutyavanich dkk di Thailand menemukan bahwa integritas DNA sperma tidak dipengaruhi oleh prosedur pendinginan dan pencairan, yang tercermin dari perubahan DNA sperma dan ekor yang tidak signifikan selama penilaian komet. Dari data yang ada, dapat disimpulkan bahwa ada variabilitas fragmentasi DNA sperma yang signifikan setelah proses kriopreservasi, yang merupakan kejadian yang cukup tinggi pada kasus individu.5, 7, 9, 10, 18-21 mempengaruhi kualitas sperma dan status kesuburan pria. Sumber utama fragmentasi DNA adalah: cacat karena rekombinasi bahan genetik selama spermatogenesis, pematangan abnormal (protopati abnormal), apoptosis sel punca testis, dan stres oksidatif. Dari semua faktor tersebut, penyebab utama kerusakan DNA pada spermatozoa adalah stres oksidatif, yang dimediasi oleh beberapa variasi spesies oksigen reaktif (ROS), termasuk radikal bebas, seperti anion superoksida (O2- •), nitrit oxide (NO + ), atau radikal hidroksil (OH +) serta oksidan kuat seperti hidrogen peroksida (H2O2) atau perokinitrit (ONOO-). Spermatozoa sangat rentan terhadap stres oksidatif karena kapasitas defensif antioksidannya yang terbatas, karena hilangnya sebagian besar sitoplasma selama spermatogenesis dan penurunan jumlah antioksidan sitoplasma seperti katalase atau superoksida dismutase. Stres oksidatif terjadi pada spermatozoa karena produksi ROS mitokondria yang berlebihan, sebagai akibat dari jalur apoptosis intrinsik. Kondisi yang dapat menyebabkan hilangnya perlindungan antioksidan meliputi merokok, kebiasaan makan/pola makan yang buruk, atau inkubasi kultur yang berkepanjangan tanpa suplemen antioksidan. Kondisi ini juga menyulitkan stres oksidatif dan kerusakan DNA oksidatif pada populasi spermatozoa. Namun, sperma yang DNAnya sudah rusak masih bisa membuahi sel telur dan akhirnya menyebabkan mutasi pada embrio, yang sayangnya jarang didiagnosis sampai embrio mencapai stadium blastosis atau telah menjadi janin. mempengaruhi kualitas sperma dan status kesuburan pria. Sumber utama fragmentasi DNA adalah: cacat karena rekombinasi bahan genetik selama spermatogenesis, pematangan abnormal (protopati abnormal), apoptosis sel punca testis, dan stres oksidatif. Dari semua faktor tersebut, penyebab utama kerusakan DNA pada spermatozoa adalah stres oksidatif, yang dimediasi oleh beberapa variasi spesies oksigen reaktif (ROS), termasuk radikal bebas, seperti anion superoksida (O2- •), nitrit oxide (NO + ), atau radikal hidroksil (OH +) serta oksidan kuat seperti hidrogen peroksida (H2O2) atau perokinitrit (ONOO-). Pembahasan Kriopreservasi adalah teknik untuk melestarikan bahan genetik dalam kondisi beku di bawah suhu rendah atau melalui pengurangan aktivitas metabolik tanpa mempengaruhi organel di dalam sel, fungsi fisiologis dan biologis serta morfologi. Kriopreservasi sperma merupakan bagian penting dari banyak laboratorium yang menyediakan Assisted Reproductive Technology (ART), terutama untuk In Vitro Fertilization (IVF) dan Intracytoplasmic Sperm Injection (ICSI).11 Metode kriopreservasi sperma menginduksi beberapa perubahan dan kerusakan parameter sperma melalui berbagai metode, yang pada akhirnya dapat menyebabkan penurunan viabilitas sperma, penurunan motilitas, perubahan pada membran plasma, akrosomia, dan integritas DNA. Stres oksidatif dalam proses pendinginan menghasilkan radikal bebas, yang menyebabkan proses lipoperoksidase, yang akan menyebabkan hilangnya motilitas ireversibel, kebocoran enzim intraselular, kerusakan DNA sperma, dan penurunan kemampuan untuk menembus oosit dan juga fusi sperma-oosit.7 Terdapat peningkatan fragmentasi DNA rata-rata pasca kriopreservasi; yaitu dari 0,5(0,6) sampai 0,2(0,2). Kenaikan ini mencerminkan penurunan kualitas spermatozoa. Proses dehidrasi yang terjadi selama kriopreservasi menyebabkan kristalisasi intraselular, yang akan mempengaruhi fungsi membran seluler. Kerusakan akibat kristalisasi akan mengganggu kondensasi kromatin, protamin dan kompleks DNA, mengakibatkan kerusakan DNA sperma. Proses pendinginan dan pencairan menyebabkan kerusakan kromatin yang signifikan. Kelainan Chromatin Dalam penelitian ini, kami menggunakan metode pendinginan yang lambat. Dalam metode ini, selama proses pendinginan, saat suhu menjadi sangat dingin, kristal es akan terbentuk dari air ekstraselular yang 134 Faizal Arif: Perbedaan Morfologi dan Fragmentasi DNA Sperma Spermatozoa sangat rentan terhadap stres oksidatif karena kapasitas defensif antioksidannya yang terbatas, karena hilangnya sebagian besar sitoplasma selama spermatogenesis dan penurunan jumlah antioksidan sitoplasma seperti katalase atau superoksida dismutase. Stres oksidatif terjadi pada spermatozoa karena produksi ROS mitokondria yang berlebihan, sebagai akibat dari jalur apoptosis intrinsik. Kondisi yang dapat menyebabkan hilangnya perlindungan antioksidan meliputi merokok, kebiasaan makan/pola makan yang buruk, atau inkubasi kultur yang berkepanjangan tanpa suplemen antioksidan. Kondisi ini juga menyulitkan stres oksidatif dan kerusakan DNA oksidatif pada populasi spermatozoa. Namun, sperma yang DNAnya sudah rusak masih bisa membuahi sel telur dan akhirnya menyebabkan mutasi pada embrio, yang sayangnya jarang didiagnosis sampai embrio mencapai stadium blastosis atau telah menjadi janin. Persentase morfologi normal sperma sebelum dan sesudah kriopreservasi menurun dari 2,2(1,6) menjadi 1,4(1,1). Ada laporan hubungan antara morfologi dan fragmentasi DNA. Gambaran morfologi sperma adalah representasi status genom sperma. Beberapa penelitian melaporkan bahwa kerusakan fisik langsung pada struktur sperma berhubungan dengan pembentukan es dan tekanan osmotik tinggi selama pembekuan. Pembekuan meningkatkan konsentrasi kandungan terlarut, seperti sodium, glukosa, dan protein, sebagai respon terhadap peningkatan tekanan osmotik yang meningkat ini dan fakta bahwa kandungan air dalam spermatozoa membentuk kristal es lebih lambat daripada air dari medium sekitarnya karena tekanan osmotik di luar sel. lebih tinggi dari pada sel. Akibatnya, spermatozoa terkena dehidrasi dan kerusakan fisik; misalnya ekor yang digulung. Selama proses pendinginan yang lambat, dehidrasi spermatozoa dapat berlanjut ke titik ekuilibrium osmotik Perkembangan mengenai evaluasi fragmentasi DNA memerlukan penelitian lebih lanjut dengan pendekatan yang berbeda untuk mengevaluasi kerusakan DNA. Dalam kasus ini, hasil yang berlawanan dilaporkan dalam sumber literatur, dengan beberapa menunjukkan kerusakan DNA setelah kriopreservasi.14-17 Penelitian oleh Simonenko menemukan bahwa fragmentasi 135 Obgynia, Volume 1 Nomor 2 September 2018 antara ruang intraselular dan ekstraselular dengan dehidrasi seluler maksimum, yang tidak menguntungkan. Namun, peningkatan kecepatan proses pembekuan tidak akan menghalangi pembentukan es intraselular akibat dehidrasi yang lambat. Selain itu, proses pembekuan terlalu cepat dapat menyebabkan pembentukan es intraselular dan pecahnya membran plasma sperma, serta kerusakan organ intraselular. Selain itu, ada juga peningkatan risiko kerusakan sel mekanis akibat kompresi es ekstraselular, yang menyebabkan deformasi sel dan kerusakan membran lebih lanjut. Laju pendinginan sperma harus optimal untuk menurunkan kadar zat yang terlarut dan dehidrasi intraselular, untuk mengurangi penyusutan sel sperma. Penelitian oleh Sinan dkk juga melaporkan hasil yang serupa; Persentase morfologi normal menurun setelah kriopreservasi. Faizal Arif: Perbedaan Morfologi dan Fragmentasi DNA Sperma Studi oleh Vutyavanich dkk di Thailand juga menemukan bahwa metode pendinginan yang lambat menghasilkan perubahan morfologi spermatozoa.6, 7, 10, 12, 20-22 3. Storey K, Storey J. Frozen and alive. Scientific American. 1990;263(6):9-27. 4. Rocha V, Cornish J, Sievers E, Filipovich A, Locatelli F, C Peters ea. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood. 2001;97(10):2962-71. 5. Santo MD, Tarozzi N, Nadalini M, Borini A. Human sperm cryopreservation: update on techniques, effect on DNA integrity, and implications for ART. Adv Urol. 2012. 6. Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahuseyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J Assist Reprod Genet. 2008;25(8):403-11. 7. Rahiminia T, Hosseini A, Anvari M, Ghasemi-Esmailabad S, Talebi A. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity. Taiwan J Obstet Gynecol. 2017;56(4):472-6. 8. Mohamed M. Slow cryopreservation is not superior to vitrification in human spermatozoa; an experimental controlled study. Iran J Reprod Med. 2015;13(10):633. Penelitian selanjutnya diperlukan untuk mengevaluasi hubungan antara hasil kriopreservasi sperma dan hasil reproduksi pasca melahirkan (IVF). Temuan ini mungkin bermanfaat karena evaluasi terus menerus terhadap sperma cryopreserved dengan menggunakan prosedur reproduksi dibantu. Simpulan, fragmentasi DNA spermatozoa meningkat setelah kriopreservasi dengan metode pendinginan yang lambat. Morfologi sperma normal juga menurun setelah kriopreservasi dengan metode pendinginan lambat. 9. Boitrelle F, Albert M, Theillac C, Ferfouri F, Bergere M, F Vialard ea. Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation. Journal Androl. 2012;33(6):1371-8. 10. Tongdee P, Sukprasert M, Satirapod C, Wongkularb A, Choktanasiri W. Comparison of Cryopreserved Human Sperm between Ultra Rapid Freezing and Slow Programmable Freezing: Effect on Motility, Morphology and DNA Integrity. J Med Assoc Thai. 2015;98:S33-42. Daftar Pustaka 1. Mazur P. Principles of cryobiology: life in the frozen state.Edisi.: CRC Press; 2004. 1. Mazur P. Principles of cryobiology: life in the frozen state.Edisi.: CRC Press; 2004. 2. Lestari S, Sari T. Fragmentasi DNA Spermatozoa: Penyebab, Deteksi, dan Implikasinya pada Infertilitas Laki-Laki. eJournal Kedokteran Indonesia. 2015. 2. Lestari S, Sari T. Fragmentasi DNA Spermatozoa: Penyebab, Deteksi, dan Implikasinya pada Infertilitas Laki-Laki. eJournal Kedokteran Indonesia. 2015. 11. Virro M, Larson-Cook K, Evenson D. Sperm chromatin structure assay 136 Faizal Arif: Perbedaan Morfologi dan Fragmentasi DNA Sperma cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil Steril. 2010;93(1):159-66. (SCSA®) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertil Steril. 2004;81(5):1289-95. (SCSA®) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertil Steril. 2004;81(5):1289-95. 18. Gavriliouk D, Aitken R. Damage to sperm DNA mediated by reactive oxygen species: its impact on human reproduction and the health trajectory of offspring. Edisi.: Springer; 2015. 12. Lemma A. Effect of cryopreservation on sperm quality and fertility. Artificial insemination in farm animals. InTech. 2011. 19. Simonenko E, Garmaeva S, Yakovenko S, Grigorieva A, Tverdislov V, A Mironova ea. The influence of the storage temperature and cryopreservation conditions on the extent of human sperm DNA fragmentation. Biophysics. 2016;61(2):267-70. 13. Agarwal A, Tvrda E. Chapter 5 Slow Freezing of Human Sperm. Cryopreservation of Mammalian Gametes and Embryos. Methods and Protocols. 2017:67-78. 14. Orief Y, Schultze-Mosgau A, Dafopoulos K, 2005;10(3):171. SA-H. Vitrification: will it replace the conventional gamete cryopreservation techniques. Middle East Fertil Soc J. 2005;10(3):171. 20. Vutyavanich T, Lattiwongsakorn W, Piromlertamorn W, Samchimchom S. Repeated vitrification/warming of human sperm gives better results than repeated slow programmable freezing. Asian J Androl. 2012;14(6):850. 15. Thomson L, Fleming S, Barone K, Zieschang J-A, Clark A. The effect of repeated freezing and thawing on human sperm DNA fragmentation. Fertil Steril. 2010;93(4):1147-56. 21. Vutyavanich T, Piromlertamorn W, Nunta S. Rapid freezing versus slow programmable freezing of human spermatozoa. Fertil Steril. 2010;93(6):1921-8. 16. Paula Td, Bertolla R, Spaine D, Cunha M, Schor N, Cedenho A. Effect of cryopreservation on sperm apoptotic deoxyribonucleic acid fragmentation in patients with oligozoospermia. Fertil Steril. 2006;66(3):597-600. 22. Moskovtsev S, Lulat A-M, Librach C. Cryopreservation of human spermatozoa by vitrification vs. slow freezing: Canadian experience. Current Frontiers in Cryobiology. 2012. 17. Daftar Pustaka Zribi N, Chakroun N, Euch HE, Gargouri J, Bahloul A, Keskes L. Effects of 137
https://openalex.org/W3045882178	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09151117.pdf	English	null	Estimating Autism Severity in Young Children From Speech Signals Using a Deep Neural Network	IEEE access	2,020	cc-by	10,169	Estimating Autism Severity in Young Children From Speech Signals Using a Deep Neural Network Corresponding author: Marina Eni (marinamu@post.bgu.ac.il) This work was supported in part by the Israel Science Foundation under Grant 961/14 and by the BGU Research Authority ABSTRACT Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that involves difﬁculties in social communication. Previous research has demonstrated that these difﬁculties are apparent in the way ASD children speak, indicating that it may be possible to estimate ASD severity using quantitative features of speech. Here, we extracted a variety of prosodic, acoustic, and conversational features from speech recordings of Hebrew speaking children who completed an Autism Diagnostic Observation Schedule (ADOS) assessment. Sixty features were extracted from the recordings of 72 children and 21 of the features were signiﬁcantly correlated with the children’s ADOS scores. Positive correlations were found with pitch variability and Zero Crossing Rate (ZCR), while negative correlations were found with the speed and number of vocal responses to the clinician, and the overall number of vocalizations. Using these features, we built several Deep Neural Network (DNN) algorithms to estimate ADOS scores and compared their performance with Linear Regression and Support Vector Regression (SVR) models. We found that a Convolutional Neural Network (CNN) yielded the best results. This algorithm predicted ADOS scores with a mean RMSE of 4.65 and a mean correlation of 0.72 with the true ADOS scores when trained and tested on different sub- samples of the available data. Automated algorithms with the ability to predict ASD severity in a reliable and sensitive manner have the potential of revolutionizing early ASD identiﬁcation, quantiﬁcation of symptom severity, and assessment of treatment efﬁcacy. INDEX TERMS Audio signals, autism, autism diagnostic observation schedule, autism spectrum disorder, convolutional neural network, deep neural network, early detection, outcome measure, pitch, speech, symptom severity, treatment efﬁcacy, zero crossing rate. INDEX TERMS Audio signals, autism, autism diagnostic observation schedule, autism spectrum disorder, convolutional neural network, deep neural network, early detection, outcome measure, pitch, speech, symptom severity, treatment efﬁcacy, zero crossing rate. SPECIAL SECTION ON EMERGING DEEP LEARNING THEORIES AND METHODS FOR BIOMEDICAL ENGINEERING Received June 30, 2020, accepted July 13, 2020, date of publication July 28, 2020, date of current version August 10, 2020. Received June 30, 2020, accepted July 13, 2020, date of publication July 28, 2020, date of current version August 10, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3012532 sed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 8, 2020 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons I. INTRODUCTION The ADOS recording room at the National Autism Research Center. centers in Israel. The recordings were performed with one microphone (CHM99, AKG, Vienna), which was located 1- 2 m from the child (Fig. 1) and connected to a sound card (US-16 × 08, TASCAM, California). Each ADOS session lasted ∼40-minutes (41.6 ± 12.4 min) and was recorded at a sampling rate of 44.1 kHz (down sampled to 16 kHz). Of the 72 children included in the study, 56 had a diagnosis of ASD, 10 were referred with a suspicion of ASD, but received other diagnoses (e.g., language or developmental delays), and 6 were typically developing controls (Table 1). With this in mind, several recent studies have utilized automated speech processing algorithms to examine speech recordings of English-speaking children with ASD. Most prominent are those that have used the Language Environ- ment Analysis (LENA) system [11], a commercially available system that enables long audio recordings of the children in their natural surroundings for several days. These recordings are automatically segmented into speech segments that are grouped by speaker, and the child is identiﬁed by his rela- tively high pitch. Studies utilizing LENA have reported that children with ASD differ signiﬁcantly from typically devel- oping (TD) children in the degree of voicing [11], amount of vocalizations [12], length of syllables [11], clarity of formant transitions [11], and amount of vocal reciprocity (i.e., conver- sational turn-taking) [13]. All children completed a full clinical assessment according to DSM-5 [15] criteria as well as an ADOS (second edition) assessment using the toddler’s module (n=10), module 1 (n=19), module 2 (n=31), or module 3 (n=12). The selection of the module depends on the age and language capacity of the child. The ADOS is a semi-structured assessment where a clinician administers speciﬁc tasks, observes the behavior of the child, and scores their behavior. The total ADOS score is in the range of 0-30 with higher scores indicating more severe symptoms. The total ADOS score is composed of SA (0-22) and Restricted and Repetitive Behavior (RRB, 0-8) scores, which can be standardized into comparison scores that enable comparison of ADOS scores across multiple ages and ADOS modules [16]. While these ﬁndings are encouraging, LENA is not an open-source software and it is, therefore, not possible to further develop the LENA algorithms nor assess their utility in estimating ASD severity. I. INTRODUCTION develop normal vocabulary and syntax, but exhibit difﬁculties with the use of appropriate prosody and pragmatics [2]. This heterogeneity is apparent in the variable scores that children with ASD receive in standardized language assessments [1]. Early studies that have examined speech in children with ASD were based on small samples and manual analysis of short speech recordings. These studies have revealed that a considerable number of children with ASD exhibit expressive language delays often involving a prolonged pre-verbal stage [3]. Of those who do develop speech, many exhibit echolalia develop normal vocabulary and syntax, but exhibit difﬁculties with the use of appropriate prosody and pragmatics [2]. This heterogeneity is apparent in the variable scores that children with ASD receive in standardized language assessments [1]. Autism Spectrum Disorder (ASD) is a neuro-developmental disorder that is diagnosed by the presence of social communi- cation impairments, repetitive behaviors, and conﬁned inter- ests [1]. The vast majority of ASD children exhibit speech and expressive language abnormalities, which range from a total lack of speech (i.e., non-verbal children) to those who Early studies that have examined speech in children with ASD were based on small samples and manual analysis of short speech recordings. These studies have revealed that a considerable number of children with ASD exhibit expressive language delays often involving a prolonged pre-verbal stage [3]. Of those who do develop speech, many exhibit echolalia The associate editor coordinating the review of this manuscript and approving it for publication was Wei Wei . 139489 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN (i.e., repeating words or phrases for no apparent reason) [4], including children with ASD who are deaf and use sign lan- guage [5]. Abnormal prosody is also very common [6] includ- ing reports of increased pitch variability and pitch range [7], slower speech rate [8], and prolonged word production [9]. FIGURE 1. The ADOS recording room at the National Autism Research Center. Using automated speech-processing techniques to identify and quantify these and other speech abnormalities would be of great clinical utility. For example, such techniques could be used to assess early ASD risk, measure the severity of symptoms, and quantify their improvement or deteriora- tion over time (e.g., in response to treatment). Furthermore, early intervention can successfully target expressive language capabilities in children with ASD, with the goal of improving general outcome [10]. FIGURE 1. I. INTRODUCTION One recent study used a Deep Neural Network (DNN) algorithm to estimate ASD sever- ity from Autism Diagnostic Observation Schedule (ADOS) recordings of 33 English-speaking children [14]. They esti- mated the Social Affect (SA) scores of the ADOS, which speciﬁcally assesses the children’s social abilities, and suc- cessfully explained ∼40% of the variability in these scores (i.e., R2 = 0.4). These results suggest that it may be possible to predict ASD severity from relatively limited recordings of speech performed during the 1-hour ADOS assessment. TABLE 1. Children characteristics. TABLE 1. Children characteristics. Here, we compared the ability of six different algorithms (two regression and four DNN models) to estimate ADOS scores from recordings of ADOS assessments performed with Hebrew speaking children. We extracted a variety of prosodic and conversational speech features from each recording and used them to train and test each of the algorithms, using a balanced cross validation approach. II. EXPERIMENTAL SETUP II. EXPERIMENTAL SETUP A. MANUAL LABELING OF SPEECH INTERVALS B. AUTOMATIC DETECTION OF VOCAL SEGMENTS Intervals of speech often contained multiple vocal segments (e.g., multiple utterances) separated by silence. To more accurately isolate vocal segments of individual speakers, we performed the following steps. First, we removed the dc of each audio recording (i.e., entire session). Second, we divided the audio signal into 40 ms frames with 30 ms overlap (i.e., frame rate of 10 ms), and computed the energy (in dB) in each frame (1): E (i) = 10 · log10  1 N N X j=1 x (j)2   (1) FIGURE 2. Example of a child’s speech interval, which was segmented into two vocal segments. (a) The speech interval in the time domain. (b) The energy of the interval. Vertical lines in both panels mark the start (red dashed line) and end of individual vocal segments (blue dotted line). Horizontal lines mark the baseline energy level (dashed magenta line), and the two energy thresholds: th1 (dotted gray line) and th2 (dashed-dotted green line). (1) where i is the frame index, j represents the sample index in the ith frame, and N is the total number of samples in a frame. , p Third, we deﬁned a baseline energy level, Eb, (in dB), as the most frequent energy level (i.e., background noise) within the audio interval and its vicinity (± 20 s). Fourth, we deﬁned the start of each vocal segment, Segstart, as the frame where the energy level was 90% above Eb (th1 = 10 · log10(1.9) + Eb) for at least 50 ms (see Fig. 2). Fifth, we deﬁned the end of the vocal segment, Segend, as the frame where the energy level was 10% above Eb (th2 = 10 · log10(1.1) + Eb) for 50 ms. Segments that were shorter than 110 ms (too short to contain an utterance) or longer than 3 s (too long to contain a well-formed phrase) were excluded from further analysis as also performed by the LENA’s algorithm [11]. We found that <1.5% of vocal segments were excluded by these criteria. These steps and criteria allowed us to isolate individual vocal segments within each audio interval. 4. Mean minimum value of pitch across vocal segments. 4. Mean minimum value of pitch across vocal segments. 5. Variance of minimum pitch across vocal segments. 6. Mean maximum value of pitch across vocal segments. 7. Variance of maximum pitch across vocal segments. 8. Mean pitch across voiced segments only. B. AUTOMATIC DETECTION OF VOCAL SEGMENTS Voiced seg- ments were deﬁned as those where >60% of frames had pitch in the range of 60-1600 Hz [11]. 9. Variance of pitch across voiced segments only. 10. Variance of mean pitch across voiced segments only. 11. Mean autocorrelation value from frames with pitch across all vocal segments. 12. Variance of autocorrelation value across frames with pitch. Formants: The resonant frequencies, or formants, are mainly determined by the size and shape of the vocal tract, including the tongue, pharynx, and laryngeal, oral and nasal cavities [20]. Audible formant transitions occur when the vocal tract moves from a consonant closure to a vowel or vice versa [21]. The ﬁrst two formants, and their bandwidths, were calculated for each frame, and the following features were extracted: Formants: The resonant frequencies, or formants, are mainly determined by the size and shape of the vocal tract, including the tongue, pharynx, and laryngeal, oral and nasal cavities [20]. Audible formant transitions occur when the vocal tract moves from a consonant closure to a vowel or vice versa [21]. The ﬁrst two formants, and their bandwidths, were calculated for each frame, and the following features were extracted: A. MANUAL LABELING OF SPEECH INTERVALS We selected recordings of 72 ADOS sessions that were per- formed at the National Autism Research Center of Israel (www.autismisrael.org), a collaborative project between Ben- Gurion University of the Negev (BGU), Soroka University Medical Center (SUMC), and other universities and medical We developed in-house software with a Graphical User Inter- face (GUI) and performed manual labeling of audio inter- vals containing speech (and/or other sounds, such as: crying, yelling, and mumbling) of the child, therapist 1, therapist 139490 VOLUME 8, 2020 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN FIGURE 2. Example of a child’s speech interval, which was segmented into two vocal segments. (a) The speech interval in the time domain. (b) The energy of the interval. Vertical lines in both panels mark the start (red dashed line) and end of individual vocal segments (blue dotted line). Horizontal lines mark the baseline energy level (dashed magenta line), and the two energy thresholds: th1 (dotted gray line) and th2 (dashed-dotted green line). 2 (some sessions had two clinicians), parent, simultaneous speech, and noise (e.g., chair being moved). All remaining intervals were automatically labeled as silence (i.e., back- ground noise). Simultaneous speech was deﬁned as speech of more than one speaker at a time. C. FEATURE EXTRACTION We extracted 60 features from the vocal segments of each recording using custom written code and the PRAAT software for analysis of pitch and formants [17]. 45. Mean ZCR across unvoiced segments only. 21. Mean 2nd formant bandwidth across vocal segments. 46. Variance of ZCR across unvoiced segments only. 22. Variance of 2nd formant bandwidth across vocal seg- ments. 22. Variance of 2nd formant bandwidth across vocal seg- ments. 2) CONVERSATIONAL FEATURES Mean slope in 500-1500 Hz across voiced segments only. 56. Mean response time of the child (i.e., time from end of the therapist’s segment). 56. Mean response time of the child (i.e., time from end of the therapist’s segment). 30. Mean slope in 500-1500 Hz across unvoiced segments only. 57. Variance of the response time. 58. Mean number of turn-takings in one minute (conversa- tional rate). Jitter: Is a measure of the cycle-to-cycle variations of fun- damental frequency, which is commonly used for speaker identiﬁcation and voice pathology [25]. We calculated jitter for frames with a deﬁned pitch (F0 was not zero). 59. Variance of the conversational rate. #Segments: The amount of child vocalizations. 31. Mean jitter across frame of all vocal segments. 60. Total number of the child’s vocal segments. 32. Variance of jitter across frames of all vocal segments. Energy: Since speech energy (i.e., volume) can be inﬂuenced from the distance between the child and the microphone, we normalized the energy (i.e., all frames) by the maximum energy (i.e., frame with maximal energy) of each recording (Enorm). The delta energy (1Enorm) and the delta-delta energy (11Enorm) were calculated as well yielding the following eight energy features: 2) CONVERSATIONAL FEATURES Spectral slope: We ﬁt a liner function to the FFT magni- tude of two frequency ranges: 20-500 Hz and 500-1500 Hz [22], [23] for each frame in each vocal segment. These two frequency ranges were selected, because it was found that they contain information regarding voice quality and emotional states [22], [24]. This resulted in two spec- tral slopes per frame. The following features were then extracted: Spectral slope: We ﬁt a liner function to the FFT magni- tude of two frequency ranges: 20-500 Hz and 500-1500 Hz [22], [23] for each frame in each vocal segment. These two frequency ranges were selected, because it was found that they contain information regarding voice quality and emotional states [22], [24]. This resulted in two spec- tral slopes per frame. The following features were then extracted: Vocalization rate: We counted the number of child vocal segments per minute in each recording. 47. Mean vocalization rate. 48. Variance of vocalization rate. Duration: We computed the duration/length of each of the vocal segments. The following features were extracted: 49. Mean length of vocal segments. 23. Mean slope in 20-500 Hz across frame of vocal seg- ments. 50. Variance in the length of vocal segments 50. Variance in the length of vocal segments. 51. Mean length of voiced vocal segments. 24. Variance of slope in 20-500 Hz across vocal segments. 25. Mean slope in 500-1500 Hz across frames of vocal segments. 53. Mean length of unvoiced vocal segments. 54. Variance in the length of unvoiced vocal segments. 54. Variance in the length of unvoiced vocal segments. 26. Variance of slope in 500-1500 Hz across vocal seg- ments. 55. Ratio between the mean length of voiced and unvoiced segments. 55. Ratio between the mean length of voiced and unvoiced segments. 27. Mean slope in 20-500 Hz across voiced segments only. 27. Mean slope in 20-500 Hz across voiced segments only. Turn-taking: We deﬁned turn-taking as cases where a ther- apist’s vocal segment was followed, within 2 s, by a child’s vocal segment. This yielded the following features: Turn-taking: We deﬁned turn-taking as cases where a ther- apist’s vocal segment was followed, within 2 s, by a child’s vocal segment. This yielded the following features: 28. Mean slope in 20-500 Hz across unvoiced segments only. 28. Mean slope in 20-500 Hz across unvoiced segments only. 29. Mean slope in 500-1500 Hz across voiced segments only. 29. D. MODELS FOR PREDICTING ADOS SCORES We built six models for predicting individual ADOS scores: 1) PROSODIC AND ACOUSTIC FEATURES Pitch (F0): The fundamental frequency generated by the child’s vocal folds [18]. Pitch was calculated for each frame (40 ms length, 10 ms frame rate). Frames with pitch values below the voicing threshold were excluded from further anal- ysis (voicing threshold was set to 0.45, which is a default value for normałlaryngeal speech [19]). The following 12 features were calculated for each child/recording: 13. Mean ﬁrst formant across frame of all vocal segments. 14. Variance of ﬁrst formant across frames of vocal seg- ments. 15. Mean second formant across vocal segments. 16. Variance of second formant across vocal segments. 17. Mean absolute difference between the two formants across frames of all vocal segments. 1. Mean pitch across frame of all vocal segments. 18. Variance of absolute difference between the two for- mants across frames of all vocal segments. 2. Variance of pitch across frames of all vocal segments. 19. Mean bandwidth of 1st formant across vocal segments. 3. Variance of pitch divided by the mean pitch of the segment (i.e., pitch coefﬁcient of variation). Mean was computed across vocal segments. 20. Variance of 1st formant bandwidth across vocal seg- ments. VOLUME 8, 2020 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN 45. Mean ZCR across unvoiced segments only. 21. Mean 2nd formant bandwidth across vocal segments. d 3) FULLY CONNECTED DEEP NEURAL NETWORKS We built three Fully Connected (FC) DNN models that dif- fered in their input arrangement (feature arrangement, see below) and the structure of their input layer. The three FC models contained one input layer, 3 FC hidden layers, and a ﬁnal output FC layer that yielded the ADOS predictions for each of the recordings/children (Fig. 4). A dropout of 0.5 was set between each two FC layers, in order to minimize the risk of overﬁtting [31]. Model 1 - Feature Vector for Each Session: The input for this model was a single vector for each recording session containing the values of the 60 features described above (Table 2) as computed across all vocal segments (Fig. 4a). The model was trained for 2000 epochs using batches of 8 samples in each training iteration. The ﬁnal number of FC layers, convolutional layers, and number of units in each architecture were chosen by tun- ing the models with different combinations of parameters. In addition, we selected optimal batch size and learning rate parameters for each of the DNN models by testing all pairs of the following combinations: batch size ∈{4,8,16} for models 1,3 and CNN, and {32, 64} for model 2; learning rate ∈{1e- 5, 5e-5, 1e-4, 5e-4}. The ﬁnal selected parameters for each model are shown in Table 3. p g Model 2 - Feature Vector for a Combination of Vocal Seg- ments: DNN algorithms beneﬁt from the availability of more data samples. To increase the number of data samples we selected sub-groups of 10 sequential vocal segments (of the child) and computed a subset of the features (1-46 and 49-50) for these sub-groups. We performed this procedure 100 times, selecting random sub-groups of sequential segments from each recording. This increased the number of available train- ing samples from 51 (i.e., one per child in the training dataset) to 5100 (i.e., 100 per child) and enabled us to train the model with batches of 64 samples. The input to the DNN model was a vector containing the values of 48 features that were com- puted for each sub-group of 10 segments (Fig. 4b). The DNN was trained, for 4000 epochs, with individual vectors/samples Since our algorithm was developed to solve a regression problem, all four models were trained using the MSE loss function, and RMSProp (Root Mean Square Propagation) optimizer [37]. 2) SUPPORT VECTOR REGRESSION (SVR) SVR is another commonly used regression model for analyz- ing speech [28]–[30]. We estimated ADOS scores using SVR while applying either a linear, Gaussian, polynomial, or radial basis function kernel, and z-normalizing each of the features. The results of the SVR model with the linear kernel were superior to the other kernels; hence, we report only the results from the linear kernel. 1) MULTPLE LINEAR REGRESSION We divided the training dataset into 5 random groups (folds), trained the multiple linear regression model using 4 of the groups, and calculated the Root Mean Square Error (RMSE) between the true ADOS scores and the predicted scores in the left out group. We performed this procedure 5 times (i.e., 5-folds cross validation) and used a Sequential Forward Feature Selection (SFS) strategy [27] to rank the features according to their ability to reduce the RMSE between the true ADOS scores and the predicted scores. Hence, each iteration resulted with a ranking of the 60 features from best to worst. We then computed the RMSE as a function of the number of features included (for each of the 5 iterations) and calculated the mean RMSE across iterations. We found that, on average, M∗= 15 features yielded the lowest mean RMSE (Fig. 3). We then trained a ﬁnal model using the 15 most popular features across the 5 iterations/folds. We uti- lized the entire training dataset (70% of the original data) and tested the ability of the model to predict ADOS scores from the independent testing dataset (30% of the original data). 33. Mean of Enorm across vocal segments. 34. Mean change in energy across consecutive frames (i.e., ﬁrst derivative, 1Enorm), across vocal segments. 35. Mean second derivate (11Enorm) across vocal seg- ments. 36. Mean absolute value of 1Enorm across vocal segments. 37. Variance of Enorm across vocal segments. 38. Variance of 1Enorm across vocal segments. 39. Variance of 11Enorm across vocal segments. 40. Variance of absolute 1Enorm across vocal segments. norm g ZCR: We quantiﬁed the Zero Crossing Rate (ZCR) in each frame [26] in each vocal segment and extracted the following features: 41. Mean ZCR across vocal segments. 42. Variance of ZCR across vocal segments. 43. Mean ZCR across voiced segments only. 44. Variance of ZCR across voiced segments only. VOLUME 8, 2020 139492 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN FIGURE 3. Feature selection. Demonstration of the average RMSE value when including an increasing number of features in the model. The optimal number of features was 15 and is marked with the red star, achieving a minimum average RMSE of 2.68 in the training dataset. such that each sample was associated with the ADOS score of the relevant child. 1) MULTPLE LINEAR REGRESSION The 48 selected features excluded the conversational features and included only features pertaining to the child’s speech characteristics. The ﬁnal output layer of the DNN yielded the predicted ADOS score of each sample. To generate the predicted ADOS score per child we computed the mean ADOS score across the samples of each child. Model 3 - Feature Matrix for Combinations of Vocal Seg- ments: Here we applied the same logic as in Model 2 but combined the 100 selected samples into a single input matrix. This yielded an input matrix of 100 × 48, one matrix per child. This model was trained with 51 samples/matrices in batches of 8 for 4000 epochs. Note, however, that each sample was a matrix that contained information from 100 randomly selected sub-groups of 10 sequential vocal segments (i.e., increasing the amount of information available in each sam- ple). The ﬁnal output layer of the DNN yielded the predicted ADOS score of the session/child (Fig. 4c). FIGURE 3. Feature selection. Demonstration of the average RMSE value when including an increasing number of features in the model. The optimal number of features was 15 and is marked with the red star, achieving a minimum average RMSE of 2.68 in the training dataset. 4) CONVOLUTIONAL NEURAL NETWORK (CNN) We also built a CNN model, which has previously been shown to accurately identify different aspects of speech intonation and prosody [32]–[35]. As in DNN model 3, here we used an input feature matrix with a size of 100 × 48 for each child. The model composed of two one-dimensional convolutional layers (Fig. 4d), with 256 ﬁlters (f) and a kernel size of 3 (k). A one-dimensional max pooling layer with pooling size of 3 (p) was evaluated between the two convolutional layers in order to help remove variability in the time-frequency domain that exists due to speech variability within each recording [36]. Next, four FC hidden layers with reducing dimension- ality (1024-512-256-128 units) were used and followed by the output layer. We applied a ReLu activation function to the output of each hidden FC layers and the two convolutional layers, and a dropout of 0.5 after the ﬁrst two FC layers. This model was trained using batches of 4 samples for 400 epochs. 3) FULLY CONNECTED DEEP NEURAL NETWORKS In the training step, all DNN and CNN models received a feature vector/matrix from children in the training dataset and a target vector of their true ADOS scores. The scores were normalized by 30, to derive a target vector with a range of [0, 1]. After training was completed, the test dataset was evaluated, yielding the predicted ADOS scores (these were multiplied by 30 for comparison with the true ADOS scores). 139493 VOLUME 8, 2020 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN FIGURE 4. DNN architectures for ADOS estimation. (a) FC-DNN model 1 – a model with a single 60-dimensional feature vector as input. (b) FC-DNN model 2 – a model with a single 48-dimensional feature vector as input. (c) FC-DNN model 3 – a model with a 100 × 48-dimensional feature matrix as input. (d) CNN model – convolutional model with a 100 × 48-feature matrix as input, with 256 filters and kernel size of 3 in the convolutional layers, and a pooling size of 3. FIGURE 4. DNN architectures for ADOS estimation. (a) FC-DNN model 1 – a model with a single 60-dimensional feature vector as input. (b) FC-DNN model 2 – a model with a single 48-dimensional feature vector as input. (c) FC-DNN model 3 – a model with a 100 × 48-dimensional feature matrix as input. (d) CNN model – convolutional model with a 100 × 48-feature matrix as input, with 256 filters and kernel size of 3 in the convolutional layers, and a pooling size of 3. TABLE 2. Features used in each of the DNN models. TABLE 3. Hyper parameters of the DNN models. TABLE 2. Features used in each of the DNN models. TABLE 2. Features used in each of the DNN models. To ensure that both the train and test datasets retained the distribution of ADOS scores in the initial data we imple- mented a balanced cross validation procedure, by randomly creating train and test groups that fulﬁlled the following criteria: • Mean ADOS score of each group had to be within the range of −10% and +10% of the total sample mean. TABLE 3. Hyper parameters of the DNN models. • The standard deviation of ADOS scores in each group had to be within the range of −10% and +10% of the total sample standard deviation. 3) FULLY CONNECTED DEEP NEURAL NETWORKS • Kurtosis of the ADOS score distribution of each group had to be within the range of −10% and +10% of the kurtosis of the total sample distribution. • Skewness of the distribution of each group had to be between −0.3 and +0.3. E. DATA ANALYSIS 1) VALUE OF INDIVIDUAL FEATURES If one of the conditions was not met, we randomly selected another pair. We created 50 different train and test groups and tested each of the 6 models on all of them to demonstrate the generalizability of the ﬁndings across different data selec- tions. We computed Pearson correlation coefﬁcients [38] between each of the speech features and each of the child’s charac- teristics (age, total ADOS score, SA score, and RRB score). This revealed potential relationships between the magnitude of each feature (e.g., pitch variability) and the severity of autism symptoms. The prediction accuracy of each model was assessed for each of the 50 datasets by computing the RMSE and Pear- son’s correlation coefﬁcient between the true ADOS scores and the ADOS scores predicted by the model. 2) PREDICTION OF ADOS SCORES To predict ADOS scores we trained and tested the models described above on independent samples. We divided the 72 available recordings into a training dataset with 51 chil- dren (70%) and a testing dataset with 21 children (30%). We identiﬁed a total of 27,395 vocal segments in the record- ings of the 72 children, which were used in the following analyses: VOLUME 8, 2020 139494 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN FIGURE 5. Correlation coefficients between the feature set and children characteristics. (a) Features correlations with ADOS score. (b) Features correlations with ADOS-SA score. (c) Features correlations with ADOS-RRB score. (d) Features correlations with age. Each color represents different feature group. Asterisks indicate significant correlations (∗p-value < 0.05, ∗∗p-value ≤0.01, ∗3 p-value ≤0.001, ∗4 p-value ≤0.0001). FIGURE 5. Correlation coefficients between the feature set and children characteristics. (a) Features correlations with ADOS score. (b) Features correlations with ADOS-SA score. (c) Features correlations with ADOS-RRB score. (d) Features correlations with age. Each color represents different feature group. Asterisks indicate significant correlations (∗p-value < 0.05, ∗∗p-value ≤0.01, ∗3 p-value ≤0.001, ∗4 p-value ≤0.0001). TABLE 4. Children characteristics in initial train and test dataset. TABLE 4. Children characteristics in initial train and test dataset. 3) DNN MODELS There were considerable differences in the performance of the four examined DNN models (Fig. 6). The CNN model yielded the highest correlation (R = 0.82, p-value < 0.0001) and lowest RMSE (3.83) of all models. FC-DNN model 3 yielded a slightly weaker correlation (R = 0.81, p-value < 0.0001) and higher RMSE (3.97). FC-DNN model 1 yielded a weaker correlation (R = 0.77, p-value < 0.0001) and higher RMSE (4.49), and FC-DNN model 2 yielded the lowest correlation (R = 0.47, p-value < 0.05) and highest RMSE (6.12). 2) SVR An SVR model trained with all 60 features, yielded predicted ADOS scores that were strongly and signiﬁcantly correlated with the actual ADOS scores (R = 0.78, p-value < 0.0001), but with a relatively high RMSE of 5.56 (Fig. 6b). An SVR model trained with the 15 selected features in the multiple linear regression yielded poorer results (R = 0.42, p-value > 0.05, RMSE = 6.45). B. ADOS ESTIMATION We initially compared the performance of all six models using a single training (i.e., 51 recordings) and testing (i.e., 21 recordings) datasets. The behavioral characteristics and the age of the children in the training and testing datasets were intentionally matched (Table 4). A. VALUE OF INDIVIDUAL FEATURES Twenty-one out of the 60 examined features were signif- icantly correlated with the ADOS scores of the examined children (Fig. 5). Seventeen features were signiﬁcantly corre- lated with the ADOS SA scores, 31 features were correlated with the RRB scores, and 18 features were correlated with the age of the children. Note that there was little overlap between the speech features that were correlated with age and those that were correlated with ASD severity, indicating that distinct features carry information about these different child characteristics. 2) SVR 1) MULTIPLE LINEAR REGRESSION We performed the multiple linear regression analysis with the 15 most informative features (#6, 9, 10, 13, 18-21, 24, 26, 27, 29, 32, 40 and 50), as selected by the FS procedure described above (Fig. 3). The predicted ADOS scores (rounded to the closest number) were moderately correlated with the true ADOS scores (R = 0.43, p-value = 0.05) with an RMSE of 6.93 (Fig. 6a). 139495 VOLUME 8, 2020 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN FIGURE 6. Scatter plots of the predicted ADOS scores vs. actual ADOS scores for each of the 6 models when using one selection of training and testing datasets. (a) Linear regression. (b) SVR model. (c) FC-DNN model 1 (d) FC-DNN model 2. (e) FC-DNN model 3. (f) CNN model. The RMSE and correlation coefficient (R) are presented in each panel. ∗p-value < 0.05, ∗∗∗∗p-value < 0.0001. FIGURE 7. Histograms demonstrating the distribution of R values for each of the 6 models when tested on 50 different selections of balanced testing and training datasets. (a) Linear regression model. R = 0.355 ± 0.193, RMSE = 7.364 ± 1.298. (b) SVR model. R = 0.510 ± 0.166, RMSE = 5.853 ± 0.431. (c) FC-DNN model 1. R = 0.501 ± 0.134, RMSE = 6.148 ± 0.864. (d) FC-DNN model 2. R = 0.312 ± 0.253, RMSE = 6.393 ± 0.701. (e) FC-DNN model 3. R = 0.696 ± 0.091, RMSE = 4.952 ± 0.651. (f) CNN model. R = 0.718 ± 0.093, RMSE = 4.648 ± 0.592. FIGURE 6. Scatter plots of the predicted ADOS scores vs. actual ADOS scores for each of the 6 models when using one selection of training and testing datasets. (a) Linear regression. (b) SVR model. (c) FC-DNN model 1 (d) FC-DNN model 2. (e) FC-DNN model 3. (f) CNN model. The RMSE and correlation coefficient (R) are presented in each panel. ∗p-value < 0.05, ∗∗∗∗p-value < 0.0001. FIGURE 7. Histograms demonstrating the distribution of R values for each of the 6 models when tested on 50 different selections of balanced testing and training datasets. (a) Linear regression model. R = 0.355 ± 0.193, RMSE = 7.364 ± 1.298. (b) SVR model. R = 0.510 ± 0.166, RMSE = 5.853 ± 0.431. (c) FC-DNN model 1. R = 0.501 ± 0.134, RMSE = 6.148 ± 0.864. (d) FC-DNN model 2. 4) BALANCED CROSS VALIDATION of correlation values for both models was 0.09. In contrast, the other models were considerably less consistent in their performance across dataset selections, exhibiting large stan- dard deviations that were between 0.13 – 0.25. These results demonstrate the importance of selecting different training and testing samples for determining the robustness and consis- tency of performance. To further evaluate the generalizability of these ﬁndings to alternative selections of training and testing datasets, we ran- domly selected 50 balanced training and testing datasets (as described above) and re-tested each of the six models (two regression and 4 DNN models) with each selection. This yielded a histogram summarizing the performance of each model across the 50 selections (Fig. 7). The CNN model demonstrated the best performance across datasets with the highest correlations (mean R = 0.72 ± 0.09) and lowest RMSE values (mean = 4.65 ± 0.59). FC-DNN model 3 followed with lower correlations (mean R = 0.70 ± 0.09) and higher RMSE values (mean = 4.95 ± 0.65). Then the SVR model (mean R = 0.51 ± 0.17 and mean RMSE = 5.86 ± 0.43), FC-DNN model 1 (mean R = 0.50 ± 0.13 and mean RMSE = 6.15 ± 0.86), and the linear regression model (mean R = 0.36 ± 0.19 and mean RMSE = 7.36 ± 1.3). Finally, FC-DNN model 2 exhibited the poorest performance (mean R = 0.31 ± 0.25 and mean RMSE = 6.39 ± 0.7). 1) MULTIPLE LINEAR REGRESSION R = 0.312 ± 0.253, RMSE = 6.393 ± 0.701. (e) FC-DNN model 3. R = 0.696 ± 0.091, RMSE = 4.952 ± 0.651. (f) CNN model. R = 0.718 ± 0.093, RMSE = 4.648 ± 0.592. FIGURE 7. Histograms demonstrating the distribution of R values for each of the 6 models when tested on 50 different selections of balanced testing and training datasets. (a) Linear regression model. R = 0.355 ± 0.193, RMSE = 7.364 ± 1.298. (b) SVR model. R = 0.510 ± 0.166, RMSE = 5.853 ± 0.431. (c) FC-DNN model 1. R = 0.501 ± 0.134, RMSE = 6.148 ± 0.864. (d) FC-DNN model 2. R = 0.312 ± 0.253, RMSE = 6.393 ± 0.701. (e) FC-DNN model 3. R = 0.696 ± 0.091, RMSE = 4.952 ± 0.651. (f) CNN model. R = 0.718 ± 0.093, RMSE = 4.648 ± 0.592. FIGURE 6. Scatter plots of the predicted ADOS scores vs. actual ADOS scores for each of the 6 models when using one selection of training and testing datasets. (a) Linear regression. (b) SVR model. (c) FC-DNN model 1 (d) FC-DNN model 2. (e) FC-DNN model 3. (f) CNN model. The RMSE and correlation coefficient (R) are presented in each panel. ∗p-value < 0.05, ∗∗∗∗p-value < 0.0001. 4) BALANCED CROSS VALIDATION A. VALUE OF INDIVIDUAL FEATURES The results revealed that speciﬁc prosodic, acoustic, and conversational features from individual recordings were sig- niﬁcantly correlated with their ADOS scores (Fig. 5). Positive correlations were apparent with most of the pitch (1-12) and ZCR (41-46) features indicating that Hebrew speaking ASD children with more severe symptoms tend to speak with higher pitch, larger pitch variability, higher rates of zero crossing values, and larger variability of zero crossing values. These results are in line with previous studies demonstrating that ASD children speak with larger pitch variability in com- parison to controls. This was true for both Hebrew [40] and English [41], [42] speaking children. The six models differed in their architecture and the struc- ture of their input data. In FC-DNN model 1 we used each recording as a single sample while computing a single value for each speech feature across the entire recording (i.e., sin- gle input vector with the values of 60 features per record- ing/child). The performance of this model was better than that of a linear regression model and slightly worse than the SVR model. In an attempt to improve ADOS prediction we created FC-DNN model 2 where we changed the input data such that instead of having a single sample per recording/child, we now extracted 100 random samples of 10 consecutive vocal segments from each recording. We computed 48 of the 60 speech features for each of these samples/vectors, thereby yielding 100 samples per recording/child instead of just one. This new architecture utilized the fact that there were many child vocalizations in each recording, enabling us to create multiple samples from each recording. The disadvantage of this approach was that we had to limit the feature vector to the 48 acoustic and prosodic features that could be computed from child vocalizations only, without the 12 conversational features that require assessment of the entire recording. This approach, however, did not work well, yielding the poorest performance of all models. In contrast, conversational features of turn-taking (56-59) and the total number of spoken segments (60) were nega- tively correlated with ADOS scores. This indicates that ASD children with more severe symptoms speak less, participate in fewer conversational turns, as also reported in previous studies with English speaking children [43]. Surprisingly, our results showed that when ASD children with more severe symptoms did respond, they tended to do so more quickly (feature #56). A. VALUE OF INDIVIDUAL FEATURES This contradicts previous studies showing the opposite in older English speaking children [43]. Further research is necessary to assess the reproducibility of either ﬁnding. This new architecture utilized the fact that there were many child vocalizations in each recording, enabling us to create multiple samples from each recording. The disadvantage of this approach was that we had to limit the feature vector to the 48 acoustic and prosodic features that could be computed from child vocalizations only, without the 12 conversational features that require assessment of the entire recording. This approach, however, did not work well, yielding the poorest performance of all models. The magnitude of correlations with speciﬁc features of speech differed when separating the total ADOS scores into their SA and RRB components. RRB scores were more strongly correlated with pitch, formants, jitter, energy, and zero crossing features, while SA scores were more corre- lated with vocalization rate, turn-taking, and total number of vocalizations. This suggests that RRB symptoms tend to be more strongly associated with acoustic and prosodic features while SA symptoms tend to be more strongly associated with conversational features. The speciﬁcity of particular speech features to speciﬁc ASD symptom domains is critical for developing speech analysis tools with clinical utility, because children with ASD exhibit heterogeneous symptoms that can differ dramatically across ASD cases. In FC-DNN model 3 we combined the same 100 randomly selected samples of 10 vocal segments into a single input matrix (size: 100 × 48). This approach yielded considerably better performance in comparison to all other FC-DNN and regression models, despite the limitation of utilizing only 48 acoustic and prosodic features (i.e., without the conver- sational features). Finally, altering the architecture to a CNN model while using the same input structure as FC-DNN model 3, yielded an additional increase in performance. This suggests that there is a large performance beneﬁt to architectures that take advantage of input containing multiple samples of the recorded child’s speech (i.e., matrix with speech features as extracted from multiple combinations of speech seg- ments). Furthermore, the considerable improvement in per- formance of the CNN model and FC-DNN model 3, relative to the linear regression and support vector regression models, Another important ﬁnding is the clear dissociation between features that were associated with ASD severity (ADOS scores) and features that were associated with the age of the children. B. ADOS SEVERITY ESTIMATION The speech features described above were used to train the six models that were built to estimate ADOS severity. There were considerable differences in the performance of the six models such that the CNN model and FC-DNN model 3 out- performed the four other models in a robust and consistent manner (Fig. 7). V. DISCUSSION Our study demonstrates that DNN models with speciﬁc archi- tectures can be trained to predict ASD severity from speech recordings of children with remarkably high accuracy. When using the CNN model or FC-DNN model 3, the predicted ADOS scores were strongly and consistently correlated with the actual ADOS scores reported by the clinician (Fig. 7). The models were trained with values of speciﬁc speech fea- tures that were extracted from recordings of clinical ADOS assessments where each child with ASD interacted with a clinician for ∼40 min. While previous studies have attempted to use such recordings for separating ASD and typically developing children [39], only a few studies to date have utilized these speech features to predict the actual severity of Note that the variability of performance across datasets differed across models. The CNN model and FC-DNN model 3 exhibited the most consistent performance across the 50 dataset selections such that the standard deviation 139496 139496 VOLUME 8, 2020 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN izations. Most acoustic and prosodic features were not sig- niﬁcantly correlated with age. Note that the utility of speciﬁc speech features for assessment of ASD severity may change with age, requiring a longitudinal research approach that tests the utility of different speech features during multiple developmental periods. ASD symptoms (i.e., ADOS scores). Furthermore, this is the ﬁrst time that these speech features and deep learning tech- nique have been applied to recordings of Hebrew speaking children. These results highlight the utility of speech analyses for estimating ASD severity at very young ages, regardless of the child’s spoken language or their cultural environment. Further development of these algorithms has great potential for aiding clinicians in assessing early risk for ASD and for quantifying changes in ASD severity over time and in response to treatment. VOLUME 8, 2020 A. VALUE OF INDIVIDUAL FEATURES The age of participating children was most strongly correlated with only few extracted features: vocalization rate, the duration of vocalizations, and the total number of vocal- 139497 VOLUME 8, 2020 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN demonstrates that the optimal ADOS prediction model is not likely to be linear. [4] M. Mergl, C. Alves, and S. Azoni, ‘‘Echolalia’s types in children with Autism spectrum disorder,’’ Revista CEFAC, vol. 17, no. 6, pp. 2072–2080, 2015. Previous studies in this domain have mostly focused on utilizing speech features to classify children into ASD and typically developing groups. For example, Pokorny et al. [39] examined recordings of vocalizations from twenty 10-month- old children later diagnosed with ASD, and a matched TD group. They extracted 88 acoustic features of the children’s vocalizations and achieved 75% accuracy in classifying chil- dren into their respective groups using a BLSTM DNN algo- rithm. [5] A. Shield, F. Cooley, and R. P. Meier, ‘‘Sign language echolalia in deaf children with autism spectrum disorder,’’ J. Speech, Lang., Hearing Res., vol. 60, no. 6, pp. 1622–1634, Jun. 2017. [6] J. McCann and S. Peppé, ‘‘Prosody in autism spectrum disorders: A critical review,’’ Int. J. Lang. Commun. Disorders, vol. 38, no. 4, pp. 325–350, Jan. 2003. [7] R. Fusaroli, A. Lambrechts, D. Bang, D. M. Bowler, and S. B. Gaigg, ‘‘‘Is voice a marker for Autism spectrum disorder? A systematic review and meta-analysis,’’’ Autism Res., vol. 10, no. 3, pp. 384–407, 2017. [8] D. Bone, C.-C. Lee, M. P. Black, M. E. Williams, S. Lee, P. Levitt, and S. Narayanan, ‘‘The psychologist as an interlocutor in autism spec- trum disorder assessment: Insights from a study of spontaneous prosody,’’ J. Speech, Lang., Hearing Res., vol. 57, no. 4, pp. 1162–1177, Aug. 2014. To the best of our knowledge, only a few studies to date have attempted to estimate ASD severity from vocal record- ings of children. For example, one study [14] used a combined DNN and Random Forest algorithm to estimate Calibrated Severity SA Scores (CSS SA) from 33 recordings and was able to predict CSS SA scores yielding a correlation of ∼0.63 with the true CSS SA scores. Their approach utilized a DNN for speech activity detection and speaker diarization, and a synthetic random forest algorithm for ADOS score estimation. [9] R. B. Grossman, R. H. Bemis, D. A. VALUE OF INDIVIDUAL FEATURES Plesa Skwerer, and H. Tager-Flusberg, ‘‘Lexical and affective prosody in children with high-functioning autism,’’ J. Speech, Lang., Hearing Res., vol. 53, no. 3, pp. 778–793, Jun. 2010. [10] L. Schreibman, G. Dawson, A. C. Stahmer, R. Landa, S. J. Rogers, G. G. McGee, C. Kasari, B. Ingersoll, A. P. Kaiser, Y. Bruinsma, E. McNerney, A. Wetherby, and A. Halladay, ‘‘Naturalistic develop- mental behavioral interventions: Empirically validated treatments for autism spectrum disorder,’’ J. Autism Develop. Disorders, vol. 45, no. 8, pp. 2411–2428, Aug. 2015. Further research incorporating automatic speaker diariza- tion techniques, instead of manual annotation, will enable development of fully automated ASD severity estimation systems. Furthermore, additional research will be able to extend these techniques for use with longer home recordings as performed using the LENA system [11] rather than short recordings performed at the clinic. [11] D. K. Oller, P. Niyogi, S. Gray, J. A. Richards, J. Gilkerson, D. Xu, U. Yapanel, and S. F. Warren, ‘‘Automated vocal analysis of naturalistic recordings from children with autism, language delay, and typical devel- opment,’’ Proc. Nat. Acad. Sci. USA, vol. 107, no. 30, pp. 13354–13359, Jul. 2010. [12] J. Rankine, E. Li, S. Lurie, H. Rieger, E. Fourie, P. M. Siper, A. T. Wang, J. D. Buxbaum, and A. Kolevzon, ‘‘Language ENvironment analy- sis (LENA) in phelan-McDermid syndrome: Validity and suggestions for use in minimally verbal children with autism spectrum disor- der,’’ J. Autism Develop. Disorders, vol. 47, no. 6, pp. 1605–1617, Jun. 2017. VI. CONCLUSION [13] A. L. Harbison, T. G. Woynaroski, J. Tapp, J. W. Wade, A. S. Warlaumont, and P. J. Yoder, ‘‘A new measure of child vocal reciprocity in children with autism spectrum disorder,’’ Autism Res., vol. 11, no. 6, pp. 903–915, Jun. 2018. Our results demonstrate that a variety of prosodic, acoustic, and conversational features are informative of ASD sever- ity in young Hebrew speaking children. These features can be utilized by a CNN model to yield remarkably accurate prediction of ADOS scores when applying an architecture that utilizes multiple vocalization samples from each child in tandem. We suggest that this speech analysis algorithm may have considerable clinical utility in assessing early ASD risk and as a novel outcome measure for quantifying ASD severity changes over time and following treatments. [14] S. Sadiq, M. Castellanos, J. Mofﬁtt, M.-L. Shyu, L. Perry, and D. Messinger, ‘‘Deep learning based multimedia data mining for autism spectrum disorder (ASD) diagnosis,’’ in Proc. Int. Conf. Data Mining Workshops (ICDMW), Nov. 2019, pp. 847–854. [15] Diagnostic and Statistical Manual of Mental Disorders, 5th ed. American Psychiatric Association, Arlington, TX, USA, 2013. [16] K. Gotham, A. Pickles, and C. Lord, ‘‘Standardizing ADOS scores for a measure of severity in autism spectrum disorders,’’ J. Autism Develop. Disorders, vol. 39, no. 5, pp. 693–705, May 2009. [17] P. Boersma and V. van Heuven, ‘‘Speak and unSpeak with PRAAT,’’ Glot Int., vol. 5, nos. 9–10, pp. 341–347, 2001. [18] S. Schelinski and K. von Kriegstein, ‘‘The relation between vocal pitch and vocal emotion recognition abilities in people with autism spectrum disorder and typical development,’’ J. Autism Develop. Disorders, vol. 49, no. 1, pp. 68–82, Jan. 2019. [1] C. Lord, M. Elsabbagh, G. Baird, and J. Veenstra-Vanderweele, ‘‘Autism spectrum disorder,’’ Lancet, vol. 392, no. 10146, pp. 508–520, 2018. [2] H. Tager-Flusberg, ‘‘Deﬁning language phenotypes in autism,’’ Clin. Neu- rosci. Res., vol. 6, nos. 3–4, pp. 219–224, Oct. 2006. [3] I. Rapin and M. Dunn, ‘‘Update on the language disorders of individuals on the autistic spectrum,’’ Brain Develop., vol. 25, no. 3, pp. 166–172, Apr. 2003. [3] I. Rapin and M. Dunn, ‘‘Update on the language disorders of individuals on the autistic spectrum,’’ Brain Develop., vol. 25, no. 3, pp. 166–172, Apr. 2003. ACKNOWLEDGMENT The authors would like to thank Alex Gorodetski, Carmel Most, Reut Altman, and Liat Moyal for their support in performing this research. They would also like to thank the Israeli Ministry of Science and Technology for creating and funding the National Autism Research Center of Israel, which enabled the collection of the data analyzed in this study. [19] P. Jongmans, T. G. Wempe, H. van Tinteren, F. J. M. Hilgers, L. C. W. Pols, and C. J. van As-Brooks, ‘‘Acoustic analysis of the voiced- voiceless distinction in dutch tracheoesophageal speech,’’ J. Speech, Lang., Hearing Res., vol. 53, no. 2, pp. 284–297, Apr. 2010. [20] K. Daqrouq and T. A. Tutunji, ‘‘Speaker identiﬁcation using vow- els features through a combined method of formants, wavelets, and neural network classiﬁers,’’ Appl. Soft Comput., vol. 27, pp. 231–239, Feb. 2015. [1] C. Lord, M. Elsabbagh, G. Baird, and J. Veenstra-Vanderweele, ‘‘Autism spectrum disorder,’’ Lancet, vol. 392, no. 10146, pp. 508–520, 2018. [2] H. Tager-Flusberg, ‘‘Deﬁning language phenotypes in autism,’’ Clin. Neu- rosci. Res., vol. 6, nos. 3–4, pp. 219–224, Oct. 2006. REFERENCES [21] E. Patten, K. Belardi, G. T. Baranek, L. R. Watson, J. D. Labban, and D. K. Oller, ‘‘Vocal patterns in infants with autism spectrum disorder: Canonical babbling status and vocalization frequency,’’ J. Autism Develop. Disorders, vol. 44, no. 10, pp. 2413–2428, Oct. 2014. [22] L. Tamarit, M. Goudbeek, and K. Scherer, ‘‘Spectral slope measurements in emotionally expressive speech,’’ in Proc. ISCA Tutorial Res. Work- shop (ITRW) Speech Anal. Process. for Knowl. Discovery, 2008, pp. 1–4, Paper 007. 139498 139498 VOLUME 8, 2020 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN [23] F. Eyben, K. R. Scherer, B. W. Schuller, J. Sundberg, E. Andre, C. Busso, L. Y. Devillers, J. Epps, P. Laukka, S. S. Narayanan, and K. P. Truong, ‘‘The geneva minimalistic acoustic parameter set (GeMAPS) for voice research and affective computing,’’ IEEE Trans. Affect. Comput., vol. 7, no. 2, pp. 190–202, Apr. 2016. MARINA ENI received the B.Sc. degree in biomedical engineering from the Ben-Gurion Uni- versity of the Negev, Beersheba, Israel, in 2018, where she is currently pursuing the master’s degree. Her current research interests include audio and image processing, biomedical engineer- ing, pattern recognition, and artiﬁcial intelligence. [24] M. Guzman, S. Correa, D. Muñoz, and R. Mayerhoff, ‘‘Inﬂuence on spectral energy distribution of emotional expression,’’ J. Voice, vol. 27, no. 1, pp. 129.e1-129.e10, 2013. [25] F. Alías, J. Socoró, and X. Sevillano, ‘‘A review of physical and percep- tual feature extraction techniques for speech, music and environmental sounds,’’ Appl. Sci., vol. 6, no. 5, p. 143, May 2016. [26] L. G. Pillai and E. Sherly, ‘‘A deep learning based evaluation of articulation disorder and learning assistive system for autistic children,’’ Int. J. Natural Lang. Comput., vol. 6, no. 5, pp. 19–36, Oct. 2017. [27] J. Lee, D. Park, and C. Lee, ‘‘Feature selection algorithm for intrusions detection system using sequential forward search and random forest clas- siﬁer,’’ KSII Trans. Internet Inf. Syst., vol. 11, no. 10, pp. 5132–5148, 2017. [28] M. Kriboy, A. Tarasiuk, and Y. Zigel, ‘‘A novel method for obstruc- tive sleep apnea severity estimation using speech signals,’’ in Proc. IEEE Int. Conf. Acoust., Speech Signal Process. (ICASSP), May 2014, pp. 3606–3610. ILAN DINSTEIN received the B.Sc. degree in life sciences from Tel Aviv University and the Ph.D. degree in neuroscience from New York Univer- sity. REFERENCES He completed Postdoctoral Training with the Weizmann Institute of Science and Carnegie Mel- lon University. In 2012, he joined the Psychology Department, Ben-Gurion University of the Negev, where he is currently an Associate Professor and the Director of the National Autism Research Cen- ter of Israel. [29] S. Gillespie, E. Moore, J. Laures-Gore, M. Farina, S. Russell, and Y.-Y. Logan, ‘‘Detecting stress and depression in adults with aphasia through speech analysis,’’ in Proc. IEEE Int. Conf. Acoust., Speech Signal Process. (ICASSP), Mar. 2017, pp. 5140–5144. [30] J. Han, Z. Zhang, F. Ringeval, and B. Schuller, ‘‘Prediction-based learning for continuous emotion recognition in speech,’’ in Proc. IEEE Int. Conf. Acoust., Speech Signal Process. (ICASSP), Mar. 2017, pp. 5005–5009. [31] N. Srivastava, G. Hinton, A. Krizhevsky, I. Sutskever, and R. Salakhutdinov, ‘‘Dropout: A simple way to prevent neural networks from overﬁtting,’’ J. Mach. Learn. Res., vol. 15, no. 1, pp. 1929–1958, 2014. [32] M. Li, D. Tang, J. Zeng, T. Zhou, H. Zhu, B. Chen, and X. Zou, ‘‘An auto- mated assessment framework for atypical prosody and stereotyped idiosyn- cratic phrases related to autism spectrum disorder,’’ Comput. Speech Lang., vol. 56, pp. 80–94, Jul. 2019. [33] S. Gupta, K. De, D. A. Dinesh, and V. Thenkanidiyoor, ‘‘Emotion recog- nition from varying length patterns of speech using CNN-based segment- level pyramid match kernel based SVMs,’’ in Proc. Nat. Conf. Commun. (NCC), Feb. 2019, pp. 1–6. MICHAL ILAN received the M.A. degree in linguistics from Bar-Ilan University, in 2008. She is currently pursuing the Ph.D. degree with the Psychology Department, Ben-Gurion Univer- sity of the Negev, studying the effect of educa- tional settings on changes in Autism symptoms, in preschool children. She has been a Speech and Language Therapist, since 2005. She is specializes in working with children with Autism. She is cur- rently working with the Soroka University Medical Center, ASD Team, Pre-School Psychiatric Unit. [34] S. Kwon, ‘‘A CNN-assisted enhanced audio signal processing for speech emotion recognition,’’ Sensors, vol. 20, no. 1, p. 183, Dec. 2019. [35] T. N. Sainath, B. Kingsbury, G. Saon, H. Soltau, A.-R. Mohamed, G. Dahl, and B. Ramabhadran, ‘‘Deep convolutional neural networks for large-scale speech tasks,’’ Neural Netw., vol. 64, pp. 39–48, Apr. 2015. [36] J. Lyu and S. Sheen, ‘‘A channel-pruned and weight-binarized convolu- tional neural network for keyword spotting,’’ in Proc. Int. Conf. Comput. Sci., Appl. Math. Appl., 2019, pp. 243–254. [37] A. M. REFERENCES Taqi, A. Awad, F. Al-Azzo, and M. Milanova, ‘‘The impact of multi- optimizers and data augmentation on TensorFlow convolutional neural network performance,’’ in Proc. IEEE Conf. Multimedia Inf. Process. Retr. (MIPR), Apr. 2018, pp. 140–145. [38] J. Benesty, J. Chen, and Y. Huang, ‘‘On the importance of the pearson correlation coefﬁcient in noise reduction,’’ IEEE Trans. Audio, Speech, Lang. Process., vol. 16, no. 4, pp. 757–765, May 2008. [39] F. B. Pokorny, B. Schuller, P. B. Marschik, R. Brueckner, P. Nyström, N. Cummins, S. Bölte, C. Einspieler, and T. Falck-Ytter, ‘‘Earlier identiﬁcation of children with autism spectrum disorder: An auto- matic vocalisation-based approach,’’ in Proc. Interspeech, Aug. 2017, pp. 309–313. IDAN MENASHE received the B.Sc. degree in medical sciences from Ben-Gurion University and the M.Sc. and Ph.D. degrees in human genetics and bioinformatics from the Weizmann Institute of Science. He also had a Postdoctoral Training in biostatistics with the Division of Cancer Epi- demiology and Genetics (DCEG), National Can- cer Institute (NCI), USA, and then worked as a Senior Scientist with MindSpec Inc. In 2012, he joined the Public Health Department, Ben-Gurion University (BGU). His main research interest includes genetic epidemiology, where he is studying the effect of genetic and non-genetic factors on the risk of autism and other complex disorders. He also serves as the Scientiﬁc Director of the National Autism Research Center of Israel. [40] Y. S. Bonneh, Y. Levanon, O. Dean-Pardo, L. Lossos, and Y. Adini, ‘‘Abnormal speech spectrum and increased pitch variability in young autistic children,’’ Frontiers Hum. Neurosci., vol. 4, p. 237, Jan. 2011. [41] M. Sharda, T. P. Subhadra, S. Sahay, and C. Nagaraja, ‘‘Sounds of melody—Pitch patterns of speech in autism,’’ Neurosci. Lett., vol. 478, no. 1, pp. 42–45, 2010. [42] L. D. Shriberg, R. Paul, L. M. Black, and J. P. van Santen, ‘‘The hypothesis of apraxia of speech in children with autism spectrum disorder,’’ J. Autism Develop. Disorders, vol. 41, no. 4, pp. 405–426, Apr. 2011. Develop. Disorders, vol. 41, no. 4, pp. 405–426, Apr. 2011. [43] D. Bone, S. Bishop, R. Gupta, S. Lee, and S. Narayanan, ‘‘Acoustic- prosodic and turn-taking features in interactions with children with neurodevelopmental disorders,’’ Interspeech, vol. 2016, pp. 1185–1189, Sep. 2016. 139499 VOLUME 8, 2020 VOLUME 8, 2020 M. Eni et al.: Estimating Autism Severity in Young Children From Speech Signals Using a DNN GAL MEIRI received the M.D. and M.H.A. REFERENCES degrees from the Ben-Gurion University of the Negev. He did part of his residency and a Research Fellowship with the Oregon Health Sciences Uni- versity at Portland, Oregon. He is currently a Child Psychiatrist and the Head of the Soroka University Medical Center, Preschool Psychiatric Unit. He is an Associate Professor with the Faculty of Health Sciences, Ben-Gurion University of the Negev. He is involved in the community and serves in local and national committees. He was recently the President of the Israeli Asso- ciation of Infant Mental Health (Afﬁliated with the WAIMH). He serves as an Active Member of the National Committee of Experts on Autism in the Israeli Ministry of Health. He and his partners from Soroka and BGU established in 2014 the Negev Autism Center and in 2018 this center was announced by the Ministry of Sciences as the National Autism Research Center of Israel. YANIV ZIGEL (Senior Member, IEEE) was born in Tel-Aviv, Israel, in 1970. He received the B.Sc., M.Sc., (summa cum laude), and Ph.D. degrees in electrical and computer engineering from Ben- Gurion University, Beersheba, Israel, in 1992, 1998, and 2004, respectively. His M.Sc. thesis and Ph.D. research were in the ﬁelds of biomedical and speech signal processing. From 2003 to 2006, he held a position as a Senior DSP Algorithm Engi- neer with the Audio Analysis Group, NICE Sys- tems Ltd. From 2006 to 2007, he was the Speech Research Group Leader, PuddingMedia Ltd. Since 2007, he has been a Faculty Member with the Department of Biomedical Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, and the Head of the Biomedical Signal Processing Research Laboratory. His main interests include biomedical sig- nal processing, speech and audio analysis, and pattern recognition. 139500 VOLUME 8, 2020
https://openalex.org/W4386944024	https://journals.iucr.org/s/issues/2023/06/00/vl5011/vl5011.pdf	English	null	<i>In situ</i> synchrotron X-ray total scattering measurements and analysis of colloidal CsPb<i>X</i> <sub>3</sub> nanocrystals during flow synthesis	Journal of synchrotron radiation	2,023	cc-by	6,654	In situ synchrotron X-ray total scattering measurements and analysis of colloidal CsPbX3 nanocrystals during flow synthesis ISSN 1600-5775 ISSN 1600-5775 Matthew W. Greenberg,a Cheng-Hung Lin,b Shirish Chodankarb and Sanjit K. Ghoseb* Received 28 March 2023 Accepted 20 August 2023 aDepartment of Chemistry and Biochemistry, Bard College, 30 Campus Road, Annandale-on-Hudson, NY 12504, USA, and bNational Synchrotron Light Source II, Brookhaven National Laboratory, Upton, NY 11973, USA. *Correspondence e-mail: sanjitghose@gmail.com Edited by M. A. G. Aranda, University of Malaga, Spain In situ X-ray scattering measurements of CsPbX3 (X = Cl, Br, I) nanocrystal formation and halide exchange at NSLS-II beamlines were performed in an automated ﬂow reactor. Total scattering measurements were performed at the 28-ID-2 (XPD) beamline and small-angle X-ray scattering at the 16-ID (LiX) beamline. Nanocrystal structural parameters of interest, including size, size distribution and atomic structure, were extracted from modeling the total scattering data. The results highlight the potential of these beamlines and the measurement protocols described in this study for studying dynamic processes of colloidal nanocrystal synthesis in solution with timescales on the order of seconds. Keywords: perovskites; nanocrystals; small- angle X-ray scattering; X-ray total scattering; nucleation; pair distribution function. research papers research papers 1. Introduction Fully inorganic caesium lead halide perovskite nanocrystals have been explored as potential emissive materials in displays and LEDs due to their bright, tunable and narrow photo- luminescence (Protesescu et al., 2015; Akkerman et al., 2015; Zhang & Yin, 2018). In particular, the defect tolerant band structure and ability to tune the bandgap by halide substitu- tion make colloidal lead halide perovskite nanocrystals attractive as solution processible and tunable luminescent materials (Tao et al., 2019; Brandt et al., 2017). The synthesis of CsPbX3 (X = Cl, Br, I) nanocrystals from ionic metathesis of Cs+, Pb2+ and X salts is known to proceed within seconds even at room temperature (Koolyk et al., 2016; Li et al., 2016; Nedelcu et al., 2015), and is relatively facile compared with II–VI (Garcı´a-Rodrı´guez et al., 2013) and III–V (Tamang et al., 2016) semiconductor nanocrystal synthesis. These rapid formation kinetics alongside structural lability of lead halide perovskites make mechanistic study of the nanoscale forma- tion of these important luminescent materials a substantial experimental challenge (Kovalenko et al., 2017). Recently, ﬂow synthesis and in situ photophysical char- acterization have been applied towards high-throughput studies of semiconductor nanocrystal formation (Lignos et al., 2015; Abolhasani et al., 2015; Epps et al., 2017). Continuous- ﬂow reactors afford ﬁne control over reaction residence time and volumetric precursor ratios and can facilitate efﬁcient screening of the large parameter space (e.g. reaction time, reagent and surfactant concentrations, temperature) of a nanocrystal synthesis (Abdel-Latif et al., 2020). Several studies using ﬂow reactors with in-line UV–vis absorbance/emission data collection have advanced mechanistic understanding and synthetic condition optimization for obtaining desired 1092 J. Synchrotron Rad. (2023). 30, 1092–1099 1092 https://doi.org/10.1107/S1600577523007300 research papers photophysical properties from lead halide perovskites (Maceiczyk et al., 2017; Epps et al., 2020; Abdel-Latif et al., 2020). Figure 1 Flow reactor setup for in-line total X-ray scattering and UV–Vis measurements at XPD 28-ID-2 beamline. ) Although electronic spectroscopy can provide quantitative information on lead halide perovskite nanocrystal concen- tration and size (Maes, Balcaen et al., 2018), direct information on ensemble atomic structure during the nanocrystal forma- tion process is essential for mechanism-guided synthesis. Time-resolved in situ diffraction and scattering measurements of nanocrystal formation capable of providing this atomic structural information have been made possible by the bril- liance of X-ray light available at third-generation synchrotron radiation user facilities (Wu et al., 2019). 1. Introduction Stopped-ﬂow and continuous-ﬂow reactors have been applied for X-ray scattering measurements of nanocrystal formation on milli- second and second timescales in the study of gold nanocrystal formation using SAXS (Abe´cassis et al., 2007; Polte et al., 2010). Benchmarking similar reactors at synchrotron facilities for measuring X-ray total scattering of nanoparticles in solu- tion during synthesis will help reveal structural evolution at the atomic and nano size scales during these processes. tion dynamics and the inﬂuence of different precursor concentrations on ﬁnal nanocrystal structure. Stopped-ﬂow and continuous-ﬂow reactors have been applied for X-ray scattering measurements of nanocrystal formation on milli- second and second timescales in the study of gold nanocrystal formation using SAXS (Abe´cassis et al., 2007; Polte et al., 2010). Benchmarking similar reactors at synchrotron facilities for measuring X-ray total scattering of nanoparticles in solu- tion during synthesis will help reveal structural evolution at the atomic and nano size scales during these processes. Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals 1093 J. Synchrotron Rad. (2023). 30, 1092–1099 1. Introduction Total X-ray scattering has been especially important for structural studies of lead halide perovskite nanocrystals in order to distinguish closely related crystalline phases (cubic, tetragonal and ortho- rhombic), characterize the substantially labile surface struc- ture, and reveal nanoscale twinning effects (Cottingham & Brutchey, 2016; Bertolotti et al., 2017, 2019). y ) X-ray total scattering and real-space pair distribution function (PDF) analysis is a key approach for studying complex nanostructure as it is capable of measuring both average and local atomic structure (Banerjee et al., 2018; Christiansen et al., 2020). By Fourier transforming Bragg diffraction and diffuse scattering along a wide range of momentum transfer Q, a pair correlation function G(r) is produced which measures the deviation from the average number density at a pair distance r (Farrow & Billinge, 2009). This method can extract structural information from dilute nanocrystals in solution from a difference PDF method (Terban et al., 2015). In situ PDF analysis has been used for measuring atomic-scale structural information during the nanocrystal formation process in many studies of structural evolution in the solvothermal synthesis of metal oxides (Saha et al., 2014; Jensen et al., 2012; Dippel et al., 2016). Atomistic and virtual crystal modeling of real-space G(r) can be used to extract quantitative atomic structural data during prenuclea- tion and nucleation and growth stages of synthesis (Bøjesen & Iversen, 2016; Dippel et al., 2016; Bertolotti et al., 2018; Campos et al., 2022). Alongside the atomic local structure from the real-space PDF analysis, information on the nanocrystal shape, average size and size dispersity can be extracted from the Debye scattering equation (DSE) ﬁtting of total scattering in reciprocal space (Cervellino et al., 2015). This provides an alternative to separate transmission electron microscope (TEM) or small-angle X-ray scattering (SAXS) data collection as is most often used for establishing nanocrystal shape and size distribution information (Pyrz & Buttrey, 2008; Maes, Castro et al., 2018). Therefore, as a complementary analysis to real-space modeling of G(r), DSE ﬁtting of I(Q) data with high-reciprocal-space resolution can provide nanostructure information. g Flow reactor setup for in-line total X-ray scattering and UV–Vis measurements at XPD 28-ID-2 beamline. tion dynamics and the inﬂuence of different precursor concentrations on ﬁnal nanocrystal structure. research papers research papers 2013). Qmin was determined by the beamstop at 0.45 A˚ 1 and Qmax to reduce statistical noise at 20 A˚ 1. Small-box PDF modeling was performed using PDFgui (Farrow et al., 2007) and structures from the materials project library (mp-600089, mp-567629) (Jain et al., 2013). DSE method ﬁtting of the data in reciprocal space was performed using DEBUSSY (Cervel- lino et al., 2015). The instrumental resolution function for the DEBUSSY software was corrected by convoluting the calcu- lated DSE with a pseudo-Voigt function (Dengo et al., 2022) with parameters used from nickel metal powder standard from NIST using TOPAS v7.17 (Coelho, 2018). SAXS was performed at NSLS-II on the Life Sciences X-ray Scattering (LiX) beamline 16-ID. Data were collected at a wavelength of 0.819 A˚ using the detectors Pilatus 1M in air and Pilatus 900K in vacuum with a pixel size of 172 mm; both detectors record data simultaneously to cover a wide contiguous q range. This conﬁguration yielded an accessible scattering range of 0.006 < q < 3.0 A˚ 1, where q is the momentum transfer, deﬁned as q = 4sin()/ ( is the wavelength and 2 is the scattering angle). Calibration of the detectors was carried out using silver behenate, which has a lamellar structure with 5.8 nm spacing. second T-joint using an independent Harvard Apparatus DDS dual syringe pump. Braided FEP tubing micromixers were used to assure mixing of the precursor streams. The ﬁnal mixed solution was fed to a ﬂow cell, placed in the X-ray beam path for scattering data collection. In-line photoluminescence was recorded by excitation with a ThorLabs 365 nm LED (M365LP1) through ﬁber optic cables and an Ocean Insight QE Pro spectrometer with spectral range from 200 nm to 990 nm. UV–Vis and photoluminescence data were collected with a 75 ms integration time per scan. The UV–Vis ﬂow cell was a custom-designed aluminium ﬂow cell with SMA ﬁber optic connectors made to simultaneously monitor the absorption and emission spectrum during ﬂow. The reaction residence time could be controlled by tuning two parameters, one being the ﬂow rate and the other the reactor path length from mixer to the probe spot (X-ray and optical) in the ﬂow cell. For this synthesis of CsPbX3 nanocrystals we have demonstrated the consistent control of residence time using different ﬂow rates as well as mixer lengths. research papers X-ray total scattering measurements were performed at Brookhaven National Laboratory using the 28-ID-2 (XPD) high-energy X-ray powder diffraction beamline at the National Synchrotron Light Source II (NSLS-II). The fully automated ﬂow reactor setup is integrated into the controls system (EPICS), and data collection and inline data reduction and visualization is carried out using the Bluesky suite Python programming language-based protocols (Koerner et al., 2020; Allan et al., 2019; Bluesky Project, https://blueskyproject.io/). X-ray total scattering data were collected in rapid acquisition mode using a 2D PerkinElmer detector (2048 2048 pixels, 200 mm 200 mm per pixel) and a sample-to-detector distance of 254 mm. The incident energy of the X-rays was 67.13 keV ( = 0.1847 A˚ ). In situ synthesized samples were measured in 1.5 mm Kapton polyimide tubes in the X-ray ﬂow cell shown in Fig. 1. An Ni standard was used as a calibrant. Azimuthal integration from raw 2D detector intensities to the 1D I(Q) was performed using pyFAI (Ashiotis et al., 2015). Back- ground scattering subtraction, and normalization and correc- tions to generate the total scattering structure function F(Q) and its Fourier transform to the PDF G(r) were performed using PDFgetX3 in xPDFsuite (Yang et al., 2014; Juha´s et al., 2. Experimental In situ synthesis of CsPbX3 was performed in a modiﬁed ﬂow synthesis setup (Fig. 1) adapted from Epps et al. (2017). Stock solutions were prepared following a room-temperature synthesis of CsPbBr3 from reacting in situ generated lead(II) oleate and caesium oleate with tetraoctylammonium bromide (Wei et al., 2016). Stock solution concentrations were [Cs+] = [Pb2+] = [Br] = 30 mM in 1:5 oleic acid:toluene for collecting total X-ray scattering data. The precursor solutions were prepared at 6 mM for the SAXS measurements to limit nanocrystal aggregation. Zinc halide (Cl, I) solutions were formed from dissolving the zinc halide in 1:19 oleyl- amine:toluene at a concentration of 60 mM [Zn2+]. Steel syringes containing Cs+/Pb2+ and Br stock solutions were loaded into Harvard Apparatus PHD Ultra syringe pumps and injected into 0.040 inch inner-diameter ﬂuorinated ethylene propylene (FEP) tubing and then mixed by combining in poly(ether ether ketone) (PEEK) T-joints. For the halide exchange experiments, the zinc halide solution described above was combined with ﬂow-synthesized CsPbBr3 by a Here, we report total scattering measurements for colloidal lead halide perovskites during synthesis in a custom-designed continuous-ﬂow reactor (Fig. 1). The control of residence times and precursor ratios by adjusting the relative and absolute precursor ﬂow rates allows us to explore both reac- 1093 Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals J. Synchrotron Rad. (2023). 30, 1092–1099 1094 Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals research papers Figure 3 G(r) of ﬂow-synthesized CsPbBr3 nanocrystals from consecutive 1 min scans at constant ﬂow rates [250 (left) and 750 ml min1 (right)]. The black difference curve at the bottom of each plot is the difference between the ﬁnal and ﬁrst scan in the ﬁgure. Table 1 Reﬁned parameters from DEBUSSY and PDFgui ﬁtting. Reﬁned parameters from DEBUSSY and PDFgui ﬁtting. Reﬁned full-r (2–60 A˚ ) ﬁtting parameters and goodness of ﬁt rw from PDFgui ﬁts. 2 is a 1/r 2 peak sharpening factor accounting for correlated atomic motion (Jeong et al., 1999) used in the PDFgui ﬁts. Averaged diameter and GoF are from the reciprocal-space DEBUSSY ﬁtting. The same reﬁned ADPs and lattice parameters from the full-r PDFgui ﬁt are used in the DEBUSSY ﬁtting, and the crystal size distribution as described above in Fig. 5 is reﬁned. Space group Pnma Pm3m a (A˚ ) 8.262 5.844 b (A˚ ) 11.779 – c (A˚ ) 8.208 – 2 6.9 7.6 Cs U iso (A˚ 2) 0.080 0.104 Pb U iso (A˚ 2) 0.027 0.028 Br1 U iso (A˚ 2) 0.069 0.164 Br2 U iso (A˚ 2) 0.039 – rw 0.174 0.275 Average diameter (nm) 10.50 1.01 8.29 3.73 GoF 0.376 0.601 Figure 3 G(r) of ﬂow-synthesized CsPbBr3 nanocrystals from consecutive 1 min scans at constant ﬂow rates [250 (left) and 750 ml min1 (right)]. The black difference curve at the bottom of each plot is the difference between the ﬁnal and ﬁrst scan in the ﬁgure. and can also be ﬁt in reciprocal space, as is shown later in Figs. 4 and 5. As mentioned previously, the rapid kinetics associated with the solution formation of CsPbBr3 nanocrystals has made studying the structural evolution during these processes an experimental challenge (Koolyk et al., 2016). Flow chemistry alongside in-line UV–Vis data sampling has previously been used to map solution formation processes of perovskite materials, as residence time can be controlled by either adjusting ﬂow rates or measuring at different positions along the reactor (Lignos et al., 2020; Abdel-Latif et al., 2020). Using our experimental reactor, by varying the ﬂow rates and continuously collecting diffraction images we can measure different time points during the formation of the CsPbBr3 nanocrystals as is shown in Fig. 3. research papers The datasets shown in this ﬁgure consist of G(r) traces generated from 1 min scans under continuous ﬂow at two different ﬂow rates, 250 and 750 ml min1, with a 15 cm path length reactor. Under these conditions, the higher ﬂow rate and path lengths can be used to isolate reaction timepoints following nucleation during the nanocrystal growth process which is discussed in detail later. For each ﬂow rate, G(r) from consecutive scans shows a high degree of similarity as illustrated by the black difference plot between the ﬁrst and ﬁnal scan in the series. Therefore, we see here that the ﬂow reactor allows for continuous X-ray scat- tering data to be collected at a given residence time. Figure 4 Figure 4 g CsPbBr3 nanocrystals (black circles) alongside orthorhombic ﬁt (Pnma, red line) and cubic ﬁt (Pm3m, green line). Gdiff = (Gobs Gcalc) for each ﬁt is plotted in dashed lines offset below the data. The octahedral coordination environment of Pb(II) ions for Pnma and Pm3m structures are shown in the insets from left to right (Pb = gray, Br = yellow, Cs = purple). detailed in Table 1. The orthorhombic model appears to be a clearly superior model for the local structure of the nano- crystals as compared with the cubic, both by comparison of rw values and the slightly unphysically large atomic displacement parameter (ADP) values reﬁned for the Pm3m model. The rw ﬁt value of 0.174 for the preferred orthorhombic model is within the range of good ﬁts for small-box PDF modeling of nanocrystals (Banerjee et al., 2018). More detailed synchro- tron diffraction and real-space PDF studies of CsPbBr3 nanocrystals, like our results, have consistently assigned the structure of the nanocrystals as locally orthorhombic (Cottingham & Brutchey, 2016; Bertolotti et al., 2017). g g Real-space PDFs of ﬂow-synthesized nanocrystals are ﬁt using the ‘real space Rietveld’ type structural reﬁnement in PDFgui (Farrow et al., 2007). While bulk CsPbBr3 is known to adopt an orthorhombic crystal structure, nanocrystals of CsPbBr3 have been assigned as either cubic or orthorhombic based on assigning broad nanocrystal diffraction from laboratory X-ray diffraction (Protesescu et al., 2015; Cottingham & Brutchey, 2016). The difference between the two closely related structures can be visualized as distortions of the Pb coordination environment from octahedral symmetry in the lower symmetry crystal structure as is shown in Fig. 4. Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals 1095 3. Results and discussion Caesium lead bromide perovskite nanocrystals were synthe- sized in ﬂow by a previously reported room-temperature route from caesium lead oleate and tetraoctylammonium bromide precursors adapted for ﬂow chemistry (Epps et al., 2017). Combining 30 mM Cs+/Pb2+/Br solutions in the ﬂow reactor described above resulted in solutions of nanocrystals with clearly visible Bragg reﬂections in reciprocal space as is shown in Fig. 2 (left). The datasets shown in Fig. 2 are of a completed nanocrystal formation reaction collected with a 60 cm total reactor path length and 100 ml min1 ﬂow rate for both precursors. Collection of data at a given reaction path distance under continuous ﬂow may be performed to ensure good signal to noise in F(Q) of the nanocrystals extracted from subtracting the background solvent scattering as is shown in Fig. 2 (right). Under these conditions the G(r) generated from this F(Q) is ﬁt excellently to a CsPbBr3 nanocrystal model Figure 2 (Left) Azimuthally integrated intensity of ﬂow-synthesized CsPbBr3 nanocrystals in toluene/oleic acid (orange) with separately measured solvent scattering (blue). Each I(Q) curve is generated from a 10 min data acquisition. (Right) Normalized reduced total scattering structure function F(Q) of nanocrystals extracted from the ﬁgure on the left. g (Left) Azimuthally integrated intensity of ﬂow-synthesized CsPbBr3 nanocrystals in toluene/oleic acid (orange) with separately measured solvent scattering (blue). Each I(Q) curve is generated from a 10 min data acquisition. (Right) Normalized reduced total scattering structure function F(Q) of nanocrystals extracted from the ﬁgure on the left. J. Synchrotron Rad. (2023). 30, 1092–1099 1094 Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals research papers Figure 4 CsPbBr3 nanocrystals (black circles) alongside orthorhombic ﬁt (Pnma, red line) and cubic ﬁt (Pm3m, green line). Gdiff = (Gobs Gcalc) for each ﬁt is plotted in dashed lines offset below the data. The octahedral coordination environment of Pb(II) ions for Pnma and Pm3m structures are shown in the insets from left to right (Pb = gray, Br = yellow, Cs = purple). Table 1 Reﬁned parameters from DEBUSSY and PDFgui ﬁtting. Reﬁned full-r (2–60 A˚ ) ﬁtting parameters and goodness of ﬁt rw from PDFgui ﬁts. 2 is a 1/r 2 peak sharpening factor accounting for correlated atomic motion (Jeong et al., 1999) used in the PDFgui ﬁts. Averaged diameter and GoF are from the reciprocal-space DEBUSSY ﬁtting. 3. Results and discussion The same reﬁned ADPs and lattice parameters from the full-r PDFgui ﬁt are used in the DEBUSSY ﬁtting, and the crystal size distribution as described above in Fig. 5 is reﬁned. Space group Pnma Pm3m a (A˚ ) 8.262 5.844 b (A˚ ) 11.779 – c (A˚ ) 8.208 – 2 6.9 7.6 Cs U iso (A˚ 2) 0.080 0.104 Pb U iso (A˚ 2) 0.027 0.028 Br1 U iso (A˚ 2) 0.069 0.164 Br2 U iso (A˚ 2) 0.039 – rw 0.174 0.275 Average diameter (nm) 10.50 1.01 8.29 3.73 GoF 0.376 0.601 J. Synchrotron Rad. (2023). 30, 1092–1099 research papers Using the real-space difference PDFs of the nano- crystal structure we examined the agreement of the Pnma model, which is substantially better than the Pm3m model, and relevant structural parameters and goodness of ﬁt rw are This assignment is further corroborated by ﬁtting the total scattering data in reciprocal space using the DSE method as implemented in DEBUSSY, as shown in Fig. 5. The DSE model used to ﬁt the data in Fig. 5 corresponds to an ortho- rhombic structure with a parallelepiped shape. The paralle- lepiped deﬁnes two independent spatial directions as ﬁtting parameters, one along the ab plane and one along c. The simulated crystal size distribution is then summarized as an 1095 research papers inset in Fig. 5. The DSE ﬁtting results of the Pnma model and the Pm3m model are also summarized in Table 1. The reci- procal-space ﬁtting of particle shape and dispersity by the DSE method here is consistent with the morphology and size Figure 5 Total X-ray scattering data (gray circles) acquired at the XPD 28-ID-2 beamline and DSE best ﬁt (red line) of in situ synthesized CsPbBr3 colloidal nanocrystals in toluene solvent (green line). The difference between DSE simulation and experimental data is shown by the blue line. The inset describes the 3D representation of the bivariate log-normal crystal size distribution function. Table 2 Selected ﬁtting parameters from the PDF ﬁts shown in Fig. 6. Flow rate (ml min1) rw (high-r) rw (low-r) d (A˚ ) 1000 0.35 0.84 76.8 750 0.29 0.80 87.2 500 0.34 0.88 96.4 250 0.20 0.66 113.6 Figure 7 Table 2 Selected ﬁtting parameters from the PDF ﬁts shown in Fig. 6. Flow rate (ml min1) rw (high-r) rw (low-r) d (A˚ ) 1000 0.35 0.84 76.8 750 0.29 0.80 87.2 500 0.34 0.88 96.4 250 0.20 0.66 113.6 Figure 7 G(r) data for the CsPb(oleate)3 precursor (bottom red trace) and Gdiff for the CsPbBr3 virtual crystal model and data for the 1000 ml min1 reaction conditions shown in Fig. 6. Figure 5 Total X-ray scattering data (gray circles) acquired at the XPD 28-ID-2 beamline and DSE best ﬁt (red line) of in situ synthesized CsPbBr3 colloidal nanocrystals in toluene solvent (green line). The difference between DSE simulation and experimental data is shown by the blue line. The inset describes the 3D representation of the bivariate log-normal crystal size distribution function. research papers Figure 5 e 7 g Total X-ray scattering data (gray circles) acquired at the XPD 28-ID-2 beamline and DSE best ﬁt (red line) of in situ synthesized CsPbBr3 colloidal nanocrystals in toluene solvent (green line). The difference between DSE simulation and experimental data is shown by the blue line. The inset describes the 3D representation of the bivariate log-normal crystal size distribution function. inset in Fig. 5. The DSE ﬁtting results of the Pnma model and the Pm3m model are also summarized in Table 1. The reci- procal-space ﬁtting of particle shape and dispersity by the DSE method here is consistent with the morphology and size dispersity reported previously for this room-temperature synthesis by TEM measurements (Wei et al., 2016). In both real- and reciprocal-space ﬁtting of the total scattering data the orthorhombic structure provided a better ﬁt to experi- mental data than the higher-symmetry cubic structure for nanocrystals synthesized in our room-temperature ﬂow synthesis conditions. This is consistent with the previously mentioned synchrotron X-ray studies of CsPbBr3 nanocrystals synthesized from high-temperature hot injection syntheses (Cottingham & Brutchey, 2016; Bertolotti et al., 2017), which suggests that this preference for the lower symmetry ortho- rhombic crystal phase is likely general for nanocrystals of CsPbBr3. g G(r) data for the CsPb(oleate)3 precursor (bottom red trace) and Gdiff for the CsPbBr3 virtual crystal model and data for the 1000 ml min1 reaction conditions shown in Fig. 6. PDF studies on nanocrystal solution growth (Campos et al., 2022). Consistent with other studies, the virtual crystal model accounts only partially for the observed G(r) which contains signal from molecular precursors, solutes and nucleated crys- tals that all coexist during the initial stages of nanocrystal formation. We ﬁt low-r (0.23 up to 1 nm) and high-r (1–6 nm) regions separately, as the former region is most likely to contain real-space correlations from poorly ordered molecular precursors and solutes. The difference PDFs (green curves in Fig. 6) between the virtual crystal model and the data sets primarily show deviations below 1 nm in the low-r region (Table 2). This is presumably from the disordered Cs and Pb oleate molecular precursors and solute of CsPbBr3 that are present as the nanocrystals are formed. 1096 Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals J. Synchrotron Rad. (2023). 30, 1092–1099 research papers The increase in intensity as Q approaches zero and shift of the position of the decay of intensity to lower Q are both a consequence of the increasing size of nanocrystals and concentration of CsPbBr3 in nanocrys- tals as the reaction residence time increases. The uptick at low Q in all of the datasets in Fig. 8 represents rapid interparticle aggregation under all recorded conditions shown below. Finally, several sharp Bragg peaks consistent with CsPbBr3 seen in the high-Q region of the data between Q = 1 and 3 A˚ 1 corroborate the identity of the nanocrystalline material. Figure 8 g Small-angle X-ray scattering of CsPbBr3 nanocrystals formed in ﬂow with varied residence times achieved by varying the ﬂow rate of the precursors (left) at a constant reactor path length (15 cm) and by varying the reactor path length (right) at several different ﬂow rates. Finally, we sought to observe the effects of halide substi- tution into CsPbBr3 nanocrystals synthesized in ﬂow that could be observed by mixing a third concentrated precursor stream of ZnI2/ZnCl2 dissolved in oleylamine and toluene (Abdel-Latif et al., 2019). The halide exchange reaction is conﬁrmed by the shift of the perovskite photoluminescence (Fig. 9, left) that is also clearly visible by eye in the reactor. As expected from the relative atomic radii of the halogens, addition of I to the CsPbBr3 lattice shifts the nearest- neighbor correlation to higher r and likewise to lower r for the addition of Cl (Fig. 9, center). Interestingly, the structural coherence of the nanocrystals is more substantially affected by the addition of iodide than chloride at the same relative concentration of iodide as is seen by the diminished intensity of correlations at higher r (Fig. 9, right). This may be due to different amounts of partial phase segregation during the initial stage of halide substitution into CsPbBr3 with either Cl or I (Gratia et al., 2016; Zhang et al., 2019). A more detailed follow-up study on structural changes during the process of halide substitution into CsPbBr3 will be published elsewhere to examine different possible explana- high-r ﬁtting as the ﬂow rate is decreased at a given reactor length (Table 2). research papers A separate X-ray PDF measurement of the 30 mM molecular Cs and Pb oleate precursor solution shows a strong correlation that aligns with the major peak that the virtual crystal model fails to capture slightly above 4 A˚ in the early time experimental G(r) as shown in Fig. 7. Prior X-ray PDF studies of related fatty acid lead carboxylates in the literature assign the most prominent feature in these compounds that is seen in G(r) around this real-space distance as a Pb–Pb separation (Campos et al., 2022; Martı´nez-Casado et al., 2017). As expected, monotonic growth of the nanocrystals is seen in the increasing reﬁned crystallite size reﬁned in the d 250 ml min1 bottom nd difference (green). The crystalline fraction of CsPbBr3 during intermediate stages of nanocrystal formation can also be modeled using the virtual crystal model approach in PDFgui. These data are shown in Fig. 6 using 1000, 750, 500 and 250 ml min1 ﬂow rates. The growth of the nanocrystal can be seen by the appearance of increasingly intense peaks at higher pair distances, consistent with what has been reported in previous Figure 6 G(r) data (black) recorded at different precursor ﬂow rates (1000, 750, 500 and 250 ml min1 bottom to top with a 15 cm reactor length), alongside the Pnma nanocrystal ﬁt (red) and difference (green). The low-r ﬁt is shown on the left and high-r on the right. Figure 6 gu e 6 G(r) data (black) recorded at different precursor ﬂow rates (1000, 750, 500 and 250 ml min1 bottom to top with a 15 cm reactor length), alongside the Pnma nanocrystal ﬁt (red) and difference (green). The low-r ﬁt is shown on the left and high-r on the right. 1096 J. Synchrotron Rad. (2023). 30, 1092–1099 96 Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals research papers Figure 8 Small-angle X-ray scattering of CsPbBr3 nanocrystals formed in ﬂow with varied residence times achieved by varying the ﬂow rate of the precursors (left) at a constant reactor path length (15 cm) and by varying the reactor path length (right) at several different ﬂow rates. reactor length (Fig. 8, left), as well as the reactor length for several different ﬂow rates (Fig. 8, right). 4. Conclusions We have used X-ray total scattering at the NSLS-II XPD 28-ID-2 beamline to measure the atomic local structure by the real-space PDF method of dilute CsPbBr3 nanocrystals synthesized in ﬂow. We further demonstrated that these measurements could be carried out on the nucleation and growth processes of these nanocrystals, as well as during the process of halide exchange reactions. Analysis of reciprocal- space total scattering data using the Debye scattering equation can be used to acquire complimentary data on morphology and the size distribution function. These results demonstrate that time-resolved X-ray total scattering measurements of dilute nanocrystals in solution during nucleation and growth processes may be performed to generate PDFs with high real-space resolution by controlling the residence time at the measurement point to allow for extended data collection. This is even the case when the underlying kinetics of the crystal growth process are shorter than the measurement time needed to acquire good signal to noise in reciprocal space which is essential for application of the real-space PDF method. The ﬂow reactor principle explored in this work could therefore be of general interest for applying synchrotron X-ray total scat- tering measurements of atomic local structure on relatively fast nanocrystal nucleation and growth processes on the seconds to minutes timescales. Akkerman, Q. A., D’Innocenzo, V., Accornero, S., Scarpellini, A., Petrozza, A., Prato, M. & Manna, L. (2015). J. Am. Chem. Soc. 137, 10276–10281. Allan, D., Caswell, T., Campbell, S. & Rakitin, M. (2019). Synchrotron Radiat. News, 32(3), 19–22. Ashiotis, G., Deschildre, A., Nawaz, Z., Wright, J. P., Karkoulis, D., Picca, F. E. & Kieffer, J. (2015). J Appl. Cryst. 48, 510–519. Banerjee, S., Liu, C. H., Lee, J. D., Kovyakh, A., Grasmik, V., Prymak, O., Koenigsmann, C., Liu, H., Wang, L., Abeykoon, A. M. M., Wong, S. S., Epple, M., Murray, C. B. & Billinge, S. J. L. (2018). J. Phys. Chem. C, 122, 29498–29506. Bertolotti, F., Moscheni, D., Guagliardi, A. & Masciocchi, N. (2018). Eur. J. Inorg. Chem. 2018, 3789–3803. Bertolotti, F., Nedelcu, G., Vivani, A., Cervellino, A., Masciocchi, N., Guagliardi, A. & Kovalenko, M. V. (2019). ACS Nano, 13, 14294– 14307. Bertolotti, F., Protesescu, L., Kovalenko, M. V., Yakunin, S., Cervellino, A., Billinge, S. J. L., Terban, M. W., Pedersen, J. S., Masciocchi, N. & Guagliardi, A. (2017). ACS Nano, 11, 3819–3831. Bøjesen, E. D. & Iversen, B. B. (2016). CrystEngComm, 18, 8332– 8353. Brandt, R. E., Poindexter, J. research papers tions for the loss of structural coherence following iodide exchange seen here. Abdel–Latif, K., Epps, R. W., Kerr, C. B., Papa, C. M., Castellano, F. N. & Abolhasani, M. (2019). Adv. Funct. Mater. 29, 1900712. Abe´cassis, B., Testard, F., Spalla, O. & Barboux, P. (2007). Nano Lett. 7, 1723–1727. Abolhasani, M., Coley, C. W., Xie, L., Chen, O., Bawendi, M. G. & Jensen, K. F. (2015). Chem. Mater. 27, 6131–6138. 4. Conclusions R., Gorai, P., Kurchin, R. C., Hoye, R. L. Z., Nienhaus, L., Wilson, M. W. B., Polizzotti, J. A., Sereika, R., Zˇ altauskas, R., Lee, L. C., MacManus-Driscoll, J. L., Bawendi, M., Stevanovic´, V. & Buonassisi, T. (2017). Chem. Mater. 29, 4667– 4674. Campos, M. P., De Roo, J., Greenberg, M. W., McMurtry, B. M., Hendricks, M. P., Bennett, E., Saenz, N., Sfeir, M. Y., Abe´cassis, B., Ghose, S. K. & Owen, J. S. (2022). Chem. Sci. 13, 4555–4565. Cervellino, A., Frison, R., Bertolotti, F. & Guagliardi, A. (2015). J. Appl. Cryst. 48, 2026–2032. research papers Consistent with previous reports on CsPbBr3 nanocrystals, our measurements are consistent with a virtually immediate nucleation of crystalline CsPbBr3 following mixing of the precursor solutions followed by a rapid nanocrystal growth process that we can observe different stages of by varying the total reaction residence time at a given reactor length by varying the precursor solution ﬂow rates. Consistent with the known fast nucleation kinetics of CsPbBr3, we see that even at room temperature we cannot observe an initial prenucleation stage as nanocrystalline orthorhombic CsPbBbr3 is observed in G(r) even at our fastest pump rate (an approximate residence time of 4 s in the reactor). We sought to further benchmark our reactor appa- ratus in capturing the formation process of CsPbBr3 by performing SAXS measurements. SAXS experiments were run at slightly lower precursor concentrations (6 mM) in order to avoid excessive particle aggregation, and further conﬁrm the rapid formation of ﬁnal nanocrystals (Fig. 8) at room temperature. Representative datasets shown in Fig. 8 demonstrate the effect of altering the ﬂow rate at a given Figure 9 (Left) In-line photoluminescence spectroscopy of the Cl (blue) and I (red) exchanged CsPbBr3 (green) nanocrystals, along with local and long-range G(r) (center and right, respectively). Local ﬁrst nearest neighbor and full G(r) of CsPbX3 nanocrystals formed from halide exchange. The dotted lines show the shift in ﬁrst-nearest-neighbor correlation in going from Cl exchanged nanocrystals (blue), parent Br (green) and ﬁnally I exchanged nanocrystals (red). g e 9 (Left) In-line photoluminescence spectroscopy of the Cl (blue) and I (red) exchanged CsPbBr3 (green) nanocrystals, along with local and long-range G(r) (center and right, respectively). Local ﬁrst nearest neighbor and full G(r) of CsPbX3 nanocrystals formed from halide exchange. The dotted lines show the shift in ﬁrst-nearest-neighbor correlation in going from Cl exchanged nanocrystals (blue), parent Br (green) and ﬁnally I exchanged nanocrystals (red). Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals 1097 1097 J. Synchrotron Rad. (2023). 30, 1092–1099 Funding information Gratia, P., Grancini, G., Audinot, J., Jeanbourquin, X., Mosconi, E., Zimmermann, I., Dowsett, D., Lee, Y., Gra¨tzel, M., De Angelis, F., Sivula, K., Wirtz, T. & Nazeeruddin, M. K. (2016). J. Am. Chem. Soc. 138, 15821–15824. The work at BNL was supported by the BNL Laboratory Directed Research and Development (LDRD) projects 20- 031 ‘Intelligent Quantum Dot Growbot for high throughput targeted quantum materials discovery’ (to SKG). Jain, A., Ong, S. P., Hautier, G., Chen, W., Richards, W. D., Dacek, S., Cholia, S., Gunter, D., Skinner, D., Ceder, G. & Persson, K. A. (2013). APL Mater. 1, 011002. Acknowledgements Christiansen, T. L., Cooper, S. R. & Jensen, K. M. O. (2020). Nanoscale Adv. 2, 2234–2254. This research used beamlines 28-ID-2 and 16-ID of the National Synchrotron Light Source II, a US DOE Ofﬁce of Science user facility operated for the DOE Ofﬁce of Science by Brookhaven National Laboratory under contract number DE-SC0012704. The authors acknowledge R. W. Epps and M. Abolhasani for their guidance in designing the ﬂow cell reactor setup. MWG acknowledges support from the Bard Summer Research Institute. The 16-ID LiX beamline is part of the Center for BioMolecular Structure (CBMS), which is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a P30 Grant (P30GM133893), and by the DOE Ofﬁce of Biological and Environmental Research (KP1605010). LiX also received additional support from NIH Grant S10 OD012331. Coelho, A. A. (2018). J. Appl. Cryst. 51, 210–218. Cottingham, P. & Brutchey, R. L. (2016). Chem. Commun. 52, 5246– 5249. Dengo, N., Masciocchi, N., Cervellino, A., Guagliardi, A. & Bertolotti, F. (2022). Nanomaterials, 12, 1252. Dippel, A.-C., Jensen, K. M. Ø., Tyrsted, C., Bremholm, M., Bøjesen, E. D., Saha, D., Birgisson, S., Christensen, M., Billinge, S. J. L. & Iversen, B. B. (2016). Acta Cryst. A72, 645–650. Epps, R. W., Bowen, M. S., Volk, A. A., Abdel-Latif, K., Han, S., Reyes, K. G., Amassian, A. & Abolhasani, M. (2020). Adv. Mater. 32, e2001626. Epps, R. W., Felton, K. C., Coley, C. W. & Abolhasani, M. (2017). Lab Chip, 17, 4040–4047. p Farrow, C. L. & Billinge, S. J. L. (2009). Acta Cryst. A65, 232–239. Farrow, C. L., Juhas, P., Liu, J. W., Bryndin, D., Boˇzin, E. S., Bloch, J., Proffen, T. & Billinge, S. J. L. (2007). J. Phys. Condens. Matter, 19, 335219. Garcı´a-Rodrı´guez, R., Hendricks, M. P., Cossairt, B. M., Liu, H. & Owen, J. S. (2013). Chem. Mater. 25, 1233–1249. Funding information 1098 Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals Abdel-Latif, K., Bateni, F., Crouse, S. & Abolhasani, M. (2020). Matter, 3, 1053–1086. References Jensen, K. M. Ø., Christensen, M., Juhas, P., Tyrsted, C., Bøjesen, E. D., Lock, N., Billinge, S. J. L. & Iversen, B. B. (2012). J. Am. Chem. Soc. 134, 6785–6792. 1098 Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals J. Synchrotron Rad. (2023). 30, 1092–1099 J. Synchrotron Rad. (2023). 30, 1092–1099 research papers Jeong, I. K., Proffen, T., Mohiuddin-Jacobs, F. & Billinge, S. J. L. (1999). J. Phys. Chem. A, 103, 921–924. Nedelcu, G., Protesescu, L., Yakunin, S., Bodnarchuk, M. I., Grotevent, M. J. & Kovalenko, M. V. (2015). Nano Lett. 15, 5635–5640. Juha´s, P., Davis, T., Farrow, C. & Billinge, S. (2013). J Appl. Cryst. 46, 560–566. Polte, J., Erler, R., Thu¨nemann, A. F., Sokolov, S., Ahner, T. T., Rademann, K., Emmerling, F. & Kraehnert, R. (2010). ACS Nano, 4, 1076–1082. Koerner, L. J., Caswell, T. A., Allan, D. B. & Campbell, S. I. (2020). IEEE Trans. Instrum. Meas. 69, 1698–1707. Koolyk, M., Amgar, D., Aharon, S. & Etgar, L. (2016). Nanoscale, 8, 6403–6409. Protesescu, L., Yakunin, S., Bodnarchuk, M. I., Krieg, F., Caputo, R., Hendon, C. H., Yang, R. X., Walsh, A. & Kovalenko, M. V. (2015). Nano Lett. 15, 3692–3696. Kovalenko, M. V., Protesescu, L. & Bodnarchuk, M. I. (2017). Science, 358, 745–750. Pyrz, W. D. & Buttrey, D. J. (2008). Langmuir, 24, 11350–11360. Li, X., Wu, Y., Zhang, S., Cai, B., Gu, Y., Song, J. & Zeng, H. (2016). Adv. Funct. Mater. 26, 2435–2445. Saha, D., Jensen, K. M. Ø., Tyrsted, C., Bøjesen, E. D., Mamakhel, A. H., Dippel, A. C., Christensen, M. & Iversen, B. B. (2014). Angew. Chem. Int. Ed. 53, 3667–3670. Lignos, I., Maceiczyk, R. M., Kovalenko, M. V. & Stavrakis, S. (2020). Chem. Mater. 32, 27–37. Tamang, S., Lincheneau, C., Hermans, Y., Jeong, S. & Reiss, P. (2016). Chem. Mater. 28, 2491–2506. Lignos, I., Stavrakis, S., Kilaj, A. & deMello, A. J. (2015). Small, 11, 4009–4017. Tao, S., Schmidt, I., Brocks, G., Jiang, J., Tranca, I., Meerholz, K. & Olthof, S. (2019). Nat. Commun. 10, 2560. Maceiczyk, R. M., Du¨mbgen, K., Lignos, I., Protesescu, L., Kovalenko, M. V. & deMello, A. J. (2017). Chem. Mater. 29, 8433–8439. Terban, M. W., Johnson, M., Di Michiel, M. & Billinge, S. J. L. (2015). Nanoscale, 7, 5480–5487. Maes, J., Balcaen, L., Drijvers, E., Zhao, Q., De Roo, J., Vantomme, A., Vanhaecke, F., Geiregat, P. & Hens, Z. (2018). J. Phys. Chem. Lett. Matthew W. Greenberg et al. Analysis of colloidal CsPbX3 nanocrystals 1099 J. Synchrotron Rad. (2023). 30, 1092–1099 References 9, 3093–3097. Wei, S., Yang, Y., Kang, X., Wang, L., Huang, L. & Pan, D. (2016). Chem. Commun. 52, 7265–7268. Maes, J., Castro, N., De Nolf, K., Walravens, W., Abe´cassis, B. & Hens, Z. (2018). Chem. Mater. 30, 3952–3962. Yang, X., Juhas, P., Farrow, C. L. & Billinge, S. J. L. (2014). arXiv:1402.3163. Martı´nez-Casado, F. J., Ramos-Riesco, M., Rodrı´guez-Cheda, J. A., Martınez-Casado, F. J., Ramos-Riesco, M., Rodrıguez-Cheda, J. A., Redondo-Ye´lamos, M. I., Garrido, L., Ferna´ndez-Martı´nez, A., Garcı´a-Barriocanal, J., da Silva, I., Dura´n-Olivencia, M. & Poulain, A. (2017). Phys. Chem. Chem. Phys. 19, 17009–17018. Zhang, H., Fu, X., Tang, Y., Wang, H., Zhang, C., Yu, W. W., Wang, X., Zhang, Y. & Xiao, M. (2019). Nat. Commun. 10, 1088. Zhang, Q. & Yin, Y. (2018). ACS Cent Sci. 4, 668–679. J. Synchrotron Rad. (2023). 30, 1092–1099
https://openalex.org/W1971121205	https://www.biodiversitylibrary.org/partpdf/249041	English	null	Systematic Zoology: Its Progress and Purpose	Science	1,907	public-domain	10,870	* Address before the Section of Systematic Zool- ogy, Seventh International Zoological Congress. MSS. intended for publication and books, etc., intended for review should be sent to the Editor of ScreNcE, Garrison-on- UWudson, N. Y. Fripay, October 18, 1907 CONTENTS Systematie Zoology—Its Progress and Pur- fOSOS IP, Asi, Esa Ss oeoecooasn cose 489 Scientific Books :— Barker on Anatomical Terminology: J. P. McM. Gray on the Labyrinth of Animals: Proressor C. R. Bardeen. Stejneger’s Herpetology of Japan: R. L. Dirmars .... 505 Discussion and Correspondence :— A Plan of Publication for Agricultural BHaperiment Station Investigations: Pro- FEssoR H. J. WEBBER. On the Effects of Magnesium Sulphate on Plants: Dr. Oscar ILO IAG oe SCO Death pad Hols omen rca 509 Special Articles :— The Spark Chronoscope: Proressor C. E. SeasHorRE. A Vector Diagram: PROFESSOR Igamaae Wie IDA oa oooudceeous Boas obdoE 512 Quotations — Liwing on our Capital ................. 514 Abstracts for Evolutionists: Proressor T. D. PAGE COCKEREIM mrontsian-teisiet retreat cieinicyeene ¢ 515 Current Notes on Meteorology and Climatol- ogy :— Monthly Weather Review; Forests and Rainfall; Influence of Forests upon Wind Velocity; Thunderstorms and “ False Cir- rus”; Olimatology of South Africa; The Weather Bureau; Salt of Marine Origin in the Atmosphere: PRoressor R. DEC. Warp 517 The Distribution of Radium in the Rocks of the Simplon Tunnel: Proressor J. Joty 518 The Are of Peru: IsAAc WINSTON ........ 519 Report of the International Commission on Zoological Nomenclature: Dr. CH. WaAR- WAG SHMMONIS Go doponoeeacecoaUeOGaA GOS 520 Walbur Olin Atwater ee. cee hee ess 523 Scientific Notes and News ................ 524 University and Educational News ......... 526 CONTENTS Systematie Zoology—Its Progress and Pur- fOSOS IP, Asi, Esa Ss oeoecooasn cose 489 Scientific Books :— Barker on Anatomical Terminology: J. P. McM. Gray on the Labyrinth of Animals: Proressor C. R. Bardeen. Stejneger’s Herpetology of Japan: R. L. Dirmars .... 505 Discussion and Correspondence :— A Plan of Publication for Agricultural BHaperiment Station Investigations: Pro- FEssoR H. J. WEBBER. On the Effects of Magnesium Sulphate on Plants: Dr. Oscar ILO IAG oe SCO Death pad Hols omen rca 509 Special Articles :— The Spark Chronoscope: Proressor C. E. SeasHorRE. A Vector Diagram: PROFESSOR Igamaae Wie IDA oa oooudceeous Boas obdoE 512 Quotations — Liwing on our Capital ................. 514 Abstracts for Evolutionists: Proressor T. D. PAGE COCKEREIM mrontsian-teisiet retreat cieinicyeene ¢ 515 Current Notes on Meteorology and Climatol- ogy :— Monthly Weather Review; Forests and Rainfall; Influence of Forests upon Wind Velocity; Thunderstorms and “ False Cir- rus”; Olimatology of South Africa; The Weather Bureau; Salt of Marine Origin in the Atmosphere: PRoressor R. DEC. CONTENTS Ir is most fitting that in this year, when the scientific world is commemorating the natal centenaries of two naturalists who have been regarded as the chief systemat- ists of their times, consideration should be given to the subject and object of their old pursuits. Carl Linné, whose bicentenary has been celebrated, was the man who first provided an elaborate code of laws for the nomenclature of all the kingdoms of nature and set an example to others by provision of concise and apt diagnoses of the groups and species he recognized. Louis Agassiz, who was born during the centenary year of Linné, gave a grand impulse to the study of nature in his adopted country, raised it in popular esteem, taught new methods of work and directed to new lines of investigation. Of all the students of nature from the time of Aristotle to the century of Linné, none requires present notice as a system- atic zoologist except John Ray, who was the true scientific father of the Swede. Born in 1627, he flourished in England during the last quarter of the seventeenth century, and died only two years before the birth of Linné. Fripay, October 18, 1907 Warp 517 The Distribution of Radium in the Rocks of the Simplon Tunnel: Proressor J. Joty 518 The Are of Peru: IsAAc WINSTON ........ 519 Report of the International Commission on Zoological Nomenclature: Dr. CH. WaAR- WAG SHMMONIS Go doponoeeacecoaUeOGaA GOS 520 Walbur Olin Atwater ee. cee hee ess 523 Scientific Notes and News ................ 524 University and Educational News ......... 526 1Lankester, Edwin, “The Correspondence of John Ray,” 1848, p. 485. JOHN RAY It was long ago truly affirmed by Edwin Lankester that ‘‘Ray has been pronounced by Cuvier to be the first true systematist of the animal kingdom, and the principal SCIENCE 490 [N.S. Vou. XXVI. No. 668 there has been much misapprehension re- specting their benefits to natural science. The two were Carl Linné and Georges Cuvier; the one exercised dictatorship from the middle of the eighteenth century till some time after its close; the other was almost equally dominant from the first quarter of the last century to well into the third quarter. No other men approached either of these two in influence on the work of contemporaries or successors. The evil features, as well as the good, were trans- mitted to and adopted by later authors. Therefore, a notice of those features may help us to a correct judgment of the his- tory of our subject, and may help to show why the disciples of the great Swede, as well as the great Frenchman, complicated many problems they investigated. Suffi- cient time has elapsed to enable us to judge knowingly and impartially. guide of Linné in the department of na- ture.’’ He, indeed, made a pathway in the zoological field which Linné was glad to follow, and to some extent he anticipated the brightest thoughts of the great Swede. He, for example, in a dichotomous system- atie table of the animal kingdom,’ first combined the lunged fish-like aquatic and viviparous animals in a special category (Vivipara) in contrast with all the other vertebrates, leaving to Linné only the privilege of giving a name to the class. He recognized a group of lung-bearing animals distinguished by a heart with a single ventricle, including quadrupeds and serpents, and thus appreciated better than Linné the class which the latter named Amphibia. He likewise gave the anatom- ical characters, based on the heart, blood and lungs, which Linné used for his classes. 2 Synopsis Methodica Animalium Quadruped- um et Serpentini Generis,’ 1693, p. 53. 1Lankester, Edwin, “The Correspondence of John Ray,” 1848, p. 485. 2 Synopsis Methodica Animalium Quadruped- um et Serpentini Generis,’ 1693, p. 53. CARL LINNE The classes were the Mammalia or Mam- mals, Aves or Birds, Amphibia, Pisces, Insecta and Vermes. The first four of these classes mainly correspond with the Aves and nameless groups of Ray. During the Linnean period of activity the invertebrates were little understood, and his treatment of that enormous host, referred to his two classes Insecta and Vermes, contrasts rather than compares with that at the present time. Naturally, the vertebrates were much better compre- hended, and all such then known, with a single exception, were distributed among four classes just named, the Mammalia, Aves, Amphibia and Pisces. The solitary exception of exclusion of a true vertebrate from its fellows was the reference of the genus Myzxine to the Vermes, next to Te- redo, the ship-worm. The first two classes were adopted with the same limits they now have, but the Amphibia and Pisces were constituted in a truly remarkable manner. The class of Amphibia was a creation of Linné, and was simply con- trasted with his Pisces by having a lung of some kind (‘‘pulmone arbitrario’’), while the Pisces have exposed branchie (“‘branchtis externis’?). The Amphibia, thus defined, were made to include as orders: (1) Reptiles or Reptilia, having feet; (2) Serpentes, footless, and (3) Nantes, having fins. A great advance, too—an inspiration of * genius, indeed—was the segregation of the animals combined under the class of mam- mals. Popular prejudice was long uni- versal and is still largely against the idea involyed. Sacred writ and classical poetry were against it. It seemed quite unnatural to separate aquatic whales from the fishes which they resembled so much in form and associate them with terrestrial hairy quadrupeds. How difficult it was to accustom one’s self to the idea is hard for naturalists of the present day to appre- ciate. Linné himself was not reconciled to the idea till 1758, although Ray had more than hinted at it more than three- score years before. At last, however, in no uncertain terms, he promulgated it. It was a triumph of science over popular im- pressions; of anatomical consideration over superficial views. But mingled with the great benefactions were many views which long influenced naturalists, but which modern zoology has overthrown. Under the Nantes were first grouped the lampreys, the selachians, the anglers (Lophius), and the sturgeons (Acipenser). CARL LINNE Systematic zoology is a vast subject, and any address devoted to it must necessarily be very partial. It need only be partial for such an assemblage of masters in zool- ogy as I have the great honor to address, and I shall confine the present discourse to a review of some of the elements which have made systematic zoology what it now is. I will venture, too, to submit reasons why we may have to take a somewhat dif- ferent view of the achievements of some men than did our early predecessors. If in doing so I may appear to be dogmatic, I entreat you in advance to insert all the “fs’’ and ‘‘I thinks’’ and ‘‘perhaps’’ that you may deem to be necessary. For the present purpose, the work of two who ex- ercised, each for a considerable time, a paramount influence on opinion and pro- cedure, deserves notice, especially because Linné needs no eulogy this year, for his praises have resounded over the whole world. Born just two centuries ago (1707), he published the first edition of the ‘‘Systema Nature’’ in 1735, and his last (twelfth) in 1766. The various edi- tions mark to some extent the steps of man’s progress in the knowledge of nature during the time limited by the respective dates. Linné’s industry was great, his sym- pathies wide-spread, and his method in large part good. Compare the ‘“‘Systema Nature’’ and other publications of Linné with works published by earlier authors, and the reason for the active appreciation and esteem which greeted his work will be obvious. he typographical dress and the clearness of expression left no doubt as to what the author meant, and enabled the student to readily grasp his intentions. His boldness in giving expression to new ideas insured success when they deserved SCIENCE 491 OcroserR 18, 1907] it. Although Ray had already recognized four of the great groups or classes of verte- brates, he had not named them and there were vernacular terms only for the birds and fishes. Linné, for the first time, ap- plied names to the other groups, and ad- mirable ones they were. Mammalia and Amphibia were the coinage of Linné and are still retained; Mammalia or mammals by all; Amphibia or amphibians by the majority for one of the classes now adopted. mitted with equal rank, no category being recognized between the class and kingdom. CARL LINNE In the twelfth edition were added Cyclop- terus, Balistes, Ostracion, Tetraodon, Dio- don, Centriscus, Syngnathus and Pegasus. The Nantes were added to the Amphibia partly because of the assumption that the branchial pouches of the lampreys and the selachians were lungs and partly on the authority of Dr. Alexander Garden, * Classis et ordo est sapientie, genus et species Nature opus.—Linn. “Syst. Nat.,” I., 13. LINNZZAN CLASSES After the tentative arrangements pub- lished in the original first, second and sixth editions of the ‘‘Systema,’’ Linné thor- oughly revised his work, and first consist- ently applied the binomial method of nom- enelature to all species in the tenth edition, published in 1758. Six classes were ad- SCIENCE 492 [N.S. Von. XXVI. No. 668 of Charleston, S. C., who mistook the pe- culiar transversely expanded and partly double air-bladder of Diodon for a lung. With such errors of observation as a basis, Linné apparently assumed that all the as- sociated genera also had lungs. Gmelin, in his edition of the ‘‘Systema Nature’’ (generally called the thirteenth), corrected this error, and returned all the Nantes to the class of Pisces, thus reverting to the older view of Linné himself. The Pisces of Linné included only the genera left after the exclusion of those just named and also of Myzine, which last was re- ferred to the class of Vermes between the leeches (Hirudo) and the ship-worms (Teredo). coequal with the families of modern au- thors, but, as has been indicated, such is by no means the case. Other striking ex- ceptions to the generalization may be shown. Not a few of the genera of Vertebrates, although not of the superlative rank as several of the Invertebrates, were equiva- lent to orders of modern zoology; such were, in the main, Simia, Testudo, Vesper- tilio and Rana. Simia included all the, anthropoid Primates or monkeys except man; Vespertilio was equivalent to the order Chiroptera less the genus Noctilio; Testudo was exactly equal to the order Testudinata or Chelonia; Rana to the or- der Salientia or Anura. A number of other genera of one or few species known to Linné were also of ordinal or subordinal value. * Scorpena. ° Labrus. notata. Cobitis., Corpus vix ad caudam angustatum. Caput cirris adspersum. Pinna dorsalis ramento post spinas Cobitis., Corpus vix ad caudam angustatum. * Scorpena. ° Labrus. notata. Caput cirris adspersum. Pinna dorsalis ramento post spinas p ° Labrus. notata. p p Pinna dorsalis ramento post spinas LINNZAN GENERA The genera of Linné were intended and thought by him to be natural,? and natural groups some of the so-called genera were, but present opinion assigns to most of them a very different valuation from that given in the ‘‘Systema Nature.’’ Some of the genera of invertebrates were extremely comprehensive. For example, Asterias in- eluded all the members of the modern lasses of Stelleroidea or Asteroidea and Ophiuroidea; Echinus was coequal with the Echinoidea; Cancer, Scorpio, Aranea, Scolopendra and Julus were essentially coextensive with orders or even higher groups of the zoologists of the present time. Others were so heterogeneous that they can not be compared with modern groups. Thus Holothuria, in the last edition of the “«Systema,’’ was made to include four holo- thurians in the modern sense, a worm, a Physaliid, and three tunicates; in other terms, the so-called genus included repre- sentatives of four different classes, and even branches of the animal kingdom. In striking contrast with the range of variation of such genera were others, of which several, well represented in northern waters, may be taken as examples. Scor- pena was distinguished simply because it had skinny tags on the head;* Labrus be- cause it had free membranous extensions behind the dorsal spines;> and Cobitis be- cause it had the caudal peduncle of regular height® and scarcely constricted as usual in fishes. These characters are of such slight systematic importance that they have not been used in the diagnoses of the genera by modern ichthyologists. Further, use of them misled even Linné as well as his suc- cessors. Some of the consequences may be noticed. The close affinity of the ‘‘Norway had- dock’’ or Swedish Kungsfisk or Rédfisk (Sebastes marinus) to the typical Scor- pena was unperceived and that species It has been stated by various writers that the genera of Linné were essentially SCIENCE 493 OctoBer 18, 1907] referred to Perca and even confounded with a Serranus. but the ‘‘dragons,’”? or Agamoid lizards with expansible ribs, are set apart In an independent genus.’° The typical Labri of the northern seas do, indeed, have filiform processes of the fin membrane behind the dorsal spines, but most of the species referred by Linné to Labrus do not, and among them is a com- mon sunfish (auritus == Lepomis auritus) of America. LINNZAN GENERA The genus Coluber was intended to em- brace all the snakes, except those with a rattle or undivided abdominal and caudal scutes,’ and hence the vipers and copper- heads, so very closely related to the rattle- snakes, were combined with ordinary snakes instead of with their true rela- tions.?? The genus Cobitis was made to include Cyprinodonts of the genera Anableps and Fundulus, and thus were associated fishes differentiated from the Loaches by char- acters of immeasurably more importance than the trivial one which was the cause of their juxtaposition. Many of the genera of Linné, in fact, were very incongruous, and the great Swede not infrequently failed to interpret and apply their characters in the allocation of species. A few cases furnished by com- mon European or American fishes will illustrate what is meant. Another conspicuous instance of a trivial character used as generic, and contrasting with very important differentials of spe- “cies included under the same genus, is fur- nished by Esox. The essential Linnean diagnostic character is the protrusion of the lower jaw.’ “Nine species were referred to the genus which represent no less than eight distinct and, mostly, widely separated families of modern systematists.2 Several of the species do not have the prominent lower jaw, and one of them (Lepisosteus osseus of modern ichthyology) is especially distinguished by Linné himself on account of the shorter lower jaw.® Specimens of the common gunnell or butterfish were received by Linné at dif- ferent times and once referred to his genus Ophidion and at another time to the genus Blenmius, and the same species stands under both names in the last two editions of his ‘‘Systema.’’ The common toadfish of the Americans (Opsanus taw) was placed in the genus Gadus (taw) and a nearly related species of the Indian Ocean was referred to the genus Cottus (grunniens). The common ten-pounder of the Amer- iean coast served as the type and only species of the genus Hlops, and also as a second species of the genus Argentina, although the characters given were in de- cided discord with those used for the latter But the most marked cases of insignif- ‘ieance of characters used to differentiate by the side of those serving for combina- tion are found in the class Amphibia. © Lacerta. “Corpus (Testa Alisve) nudum, caudatum ” contrasting with Draco. “ Corpus Alis volatile.” S. N., 66, 349. 1 Qoluber. “Scuta abdominalia; squame cau- dales” contrasting with “Crotalus. Scuta ab- dominalia cum crepitaculo” and “Boa. Scuta abdominalia caudaliaque absque erepitaculo.” S. N., 766, 349. 2 As an example of Coluber a figure (tab. 3, fig. 2) of a snake with venom fangs is given. caudaliaque 8 The species are (1) Sphyrena (Sphyrenide), (2) osseus (Lepisosteide), (3) Vulpes (Albul- ide), (4) Synodus (Synodontide), (5) Lucius (Luciide), (6) belone (Hsocide), (7) hepsetus and (8) brasiliensis (Exocetide), and (9) gym- nocephalus (Chirocentride). 8S. N., 66, 513-517. ®Mandibula inferior brevior. S. N., ’66, 516. 7 Hsow. Mandibula inferior longior, punctata. S. N., °58; °66, 424. 2 As an example of Coluber a figure (tab. 3, fig. 2) of a snake with venom fangs is given. © Lacerta. “Corpus (Testa Alisve) nudum, caudatum ” contrasting with Draco. “ Corpus Alis volatile.” S. N., 66, 349. 1 Qoluber. “Scuta abdominalia; squame cau- dales” contrasting with “Crotalus. Scuta ab- dominalia cum crepitaculo” and “Boa. Scuta abdominalia caudaliaque absque erepitaculo.” S. N., 766, 349. caudaliaque LINN AN NOMENCLATURE The example thus set by the master was naturally followed by his disciples. Many felt at liberty to change names and range of genera as they thought best and great confusion resulted, which has continued more or less down to this year of grace, 1907. The code of nomenclature devised by Linné was in many respects admirable, but he did not provide sufficiently for the principle of priority in nomenclature. He set the example of changing a name given by himself or by others, when he thought a better one could be substituted; he also felt at liberty to change the intent of a genus. A few examples of many cases may illustrate. Many of the evils which have been the consequence could have been rectified if the British Association for the Advance- ment of Science had been logical in the code (often admirable) which it published in 1842. Instead, however, of accepting the edition of the ‘‘Systema Nature’’ (tenth) in which Linné first introduced the binomial nomenclature as the starting point, they preferred homage to an indi- vidual rather than truth to a principle, and insisted on the twelfth edition as the initial volume of zoological nomenclature. The unfortunate consequences have been manifold. Such consequences are the natural outcome of illogical and _ ill-con- sidered action and must always sooner or later follow. After these many years almost all naturalists have acceded to the adoption of the tenth edition. In 1756 the name Salacia was given to the Portuguese man-of-war; in 1758 the name Holothuria was substituted; in 1766 the latter name was retained, but with a very different diagnosis, and for the first time three holothurians in the modern sense of the word were introduced. In 1756 the names Cenchris and Cro- talophorus were used for genera, two years later renamed Boa and Crotalus. In 1756 Artedi’s name, Catodon, was retained for the sperm whale, and Artedi’s Phy- seter mainly for the killers (Orca) ; but in 1758 ‘Physeter was taken up for the sperm whale, for which it has been retained ever since, except by a very few naturalists. If the vertebrates were so much misun- derstood by Linné, it may naturally be supposed that the invertebrates were equally or still less understood. Only one interesting case, however, can be referred to. LINNZAN GENERA The genus Lacerta is made to inelude all but one of the pedate Lizards and the Crocodilians as well as the salamanders, 7 Hsow. Mandibula inferior longior, punctata. S. N., °58; °66, 424. SCIENCE [N. 8. Vor. XXVI. No. 668 494 for what is still known as the genus Ophidium. genus, and in perfect harmony with those employed for the distinction of the former genus. Indeed, it might be properly as- sumed that the aseription of the Argentina carolina to Argentina was simply a matter of misplacement of a manuscript leaf, and such it may be even now considered, al- though the error is continued in the twelfth edition, having escaped the notice of Linné. In 1756 and 1758 Trichechus was used for the manatee alone, while the walrus was correctly associated with the seals, but in 1766 the very retrograde step was taken of associating the walrus with the manatee and retaining for the two the name Trichechus. Many naturalists persist to the present day in keeping the name for the walrus alone. LINN AN NOMENCLATURE In the ninth edition of the ‘‘Systema Nature’’ Linné had a monotypie genus In 1756 and 1758 Ophidion was used for an acanthopterygian jugular fish—the common northern butterfish, or gunnell, now generally called Pholis—but in 1766, under the guise of Ophidiwm, it was trans- ferred to the Apodes and primarily used SCIENCE 495 OcroBeR 18, 1907] Salacia (p. 79) with a species named Physalis which was evidently a Physalia as long understood. In the tenth edition the name Holothuria was substituted for Salacia and no holothurians in the modern sense were recognized. In the twelfth edition all the species of the former edition were retained, but the diagnosis was altered and four holothurians of recent authors were added, and thus animals of different subkingdoms or branches were confounded in the genus. Now, if we accept the tenth edition of the “‘Systema’’ as the starting of our nomenclature, ob- viously Holothuria can not be used as it has been for these many years, and it must be revived in place of Physalia, notwith- standing the laments of those who are dis- tressed by such a change. The echino- derms now called holothurians must be re- named. We can imagine the clamor that will arise when some one attempts the change. the commencement of Linné’s_ career (1737), the learned Professor Dillenius, of Oxford, strongly protested against such misusage for plant genera, but the sinner persisted in the practise till the end. Naturally his scholars and later nomen- clators followed the bad example, and systematic zoology is consequently bur- dened with an immense number of the grossest and most misleading misapplica- tions of ancient names revolting to the celassicist and historian alike. The influence of Linné continued to be felt and his system to be adopted until a new century had for sometime run its course. Meanwhile, in France, a great zoologist was developing a new system which was published at length in 1817, and anew with many modifications a dozen years later (1829). GEORGES LEOPOLD CHRETIEN FREDERIC DAGOBERT CUVIER Georges Cuvier (born 1769) claimed?® that before him naturalists distributed all the invertebrates among two classes as by Linné. In 1795 he published an account of memorable anatomical investigations of the invertebrates and ranged them all under six classes: molluscs, crustaceans, Insects, worms, echinoderms and zoophytes. This was certainly a great improvement over previous systematic efforts, but from our standpoint crude in many respects. It was, however, necessarily crude, for natu- ralists had to learn how to look as well as to think. \| Another fault of less moment—indeed a matter of taste chiefly—was committed by Linné. Very numerous names of plants and animals occur in the writings of vari- ous ancient authors and were mostly un- identifiable in the time of Linné. He drew upon this store with utter disregard of the consequences for names of new genera. Most of the ancient names can now be identified and associated with the species to which they were of old applied, and the incongruity of the old and new usage is striking. For example, Dasypus, a Greek name of the hare, was perverted to the armadillos; Trochilus, a name of an Egyptian plover, was misused for the humming birds; Amia, a name for a tunny, was transferred to the bowfin of North America. There was not the slightest justification for such perversion of the names im analogy or fitness of any kind; there was no real excuse for it. At Cuvier later essayed to do for the animal. kingdom alone what Linné did for all the kingdoms of nature. So greatly had the number of known animals increased, how- ever, that he did not attempt to give diagnoses of the species, but merely named them, mostly in foot-notes. His superior knowledge of anatomy enabled him to in- %“ Réegne Animal,” 1817, I., 61. %“ Réegne Animal,” 1817, I., 61. SCIENCE 496 [N.S. Vor. XXVI. No. 668 stitute great improvements in the system. He also first recognized the desirability of combining in major groups classes concern- ing which a number of general proposi- tions could be postulated. who enjoys the greatest reputation for the time being that anticipates future conclu- sions. A Frenchman who held a small place in the world’s regard in comparison with Cuvier advanced far ahead of him in certain ideas. Henri Marie Ducrotay de Blainville was the man. GEORGES LEOPOLD CHRETIEN FREDERIC DAGOBERT CUVIER When Cuvier (1817) associated the marsupials in the same order as the true carnivores and the monotremes with the edentates, Blainville (1816) contrasted the marsupials and monotremes as Implacentals (‘‘Didel- phes’’) against the ordinary Placentals (‘‘Monodelphes’’). While later (1829) Cuvier still approximated the marsupials to the carnivores, but in a distinet order between the carnivores and the rodents, and still retained the monotremes as a tribe of the edentates, Blainville (1834) recog- nized the marsupials and monotremes as distinct subclasses of mammals and had proposed the names Monodelphes, Didel- phes and Ornithodelphes, still largely used by the most advanced of modern therolo- gists. It was in 1812 that Cuvier presented to the Academy of Sciences?! his celebrated memoir on a new association of the classes of the animal kingdom, proposing a special category which he called branch (em- branchment), and marshaling the classes recognized by him under four primary groups: (1) the Vertebrates or Animaux vertébrés; (2) the Mollusks or Animaux mollusques; (3) the Articulates or Ani- maux articulés, and (4) the Radiates or Animaux rayonnés. These were adopted jn the ‘‘Régne Animal.’’ In the first (1817) edition, as in the second (1829- 1830), nineteen classes were recognized, and in the latter too little consideration was given to the numerous propositions for the improvement of the system that had been suggested and urged meanwhile. It has been generally assumed that Cuvier’s work was fully up to the high mark of the times of publication, and for many years the classification which he gave was accepted by the majority of natural- ists as the standard of right. To such ex- tent was this the case that his classification of fishes and the families then defined was retained to at least the penultimate decade of the last century by the first ichthy- ologists of France. Nevertheless the work was quite backward in some respects and exercised a retardative influence in that the preeminent regard in which the great Frenchman was held and the proclivity to follow a leader kept many from paying any attention to superior work emanating from Cuvier’s contemporaries. “Ann, Museum Nat. Hist., Paris, 1812, 19, 73-84. GEORGES LEOPOLD CHRETIEN FREDERIC DAGOBERT CUVIER Against the action of Cuvier in ranging all the hoofed mammals in two orders, the pachyderms (including the elephants) and the ruminants, may be cited the philosoph- ical ideas of Blainville (1816), who com- bined the same in two very different orders, the Ongulogrades and the Gravi- grades (elephants), and distributed the normal Ongulogrades under two groups, those with unpaired hoofs (Imparidigi- tates) and those with paired hoofs (Pari- digitates), thus anticipating the classifica- tion of Owen and recent naturalists by very many years. Cuvier’s treatment of the amphibia of Linné equally contrasted with Blainville’s. As late as 1829 the great French naturalist still treated the batrachians as a mere order of reptiles of a single family, and the crocodilians as a simple family of It is by no means always the naturalist SCIENCE 497 Octoser 18, 1907] Saurians. On the other hand, as early as 1816 Blainville had given subclass rank to the naked amphibians with four orders, and also ordinal rank to the erocodilians, and a little later (1822) he raised the sub- classes to class rank. Still more, Blain- ville early (1816) recognized that the so- called naked serpents were true amphib- ians and gave satisfactory reasons for his assumption, though to the last Cuvier (1829) considered them to be merely a family of the ophidians. As Blainville claimed, he based his classification on an- atomical facts.1® manifestly allowed himself to be influenced by the sentiment prevalent in his time, that systematic zoology and comparative anatomy were different provinces. It may, indeed, seem strange to make the charge against the preeminent anatomist, that he failed because he neglected anat- omy, but it must become evident to all who carefully analyze his zoological works that such neglect was his prime fault. He, in fact, treated zoology and anatomy as distinct disciplines, or, in other words, he acted on the principle that animals should be considered independently from two points of view, the superficial, or those facts easily observed, and the deep-seated, or anatomical characters. Blainville, on the contrary, almost from the first, con- sidered animals in their entirety and would estimate their relations by a view of the entire organization. Yet the sentiment then prevalent was reflected by one who enjoyed a high reputation for a time as a “‘philosophical zoologist’’— William Swainson. In ‘‘A Treatise on the Geog- raphy and Classification of Animals’’ (1836, p. «Ses bases sont anatomiques et surtout tirées de la consideration du crane,’ Bull. Se. Soc. Philom., 1816, p. 111. * Agassiz, “Essay on Classification,” p. 286. GEORGES LEOPOLD CHRETIEN FREDERIC DAGOBERT CUVIER 173), the author complained that ‘‘Cuvier rested his distinctions... upon characters which, however good, are not always comprehensible, except to the anatomist. The utility of his system, for general use, is consequently much di- minished, and it gives the student an im- pression (certainly an erroneous one) that the internal, and not the external, struc- ture of an animal alone decides its place in nature.’’ It was long before such a mischievous opinion was discarded. Cuvier was regarded almost universally by his contemporaries, and long after- A pupil of Blainville, Ferdinand L’Her- minier of the island of Gaudeloupe, at the instance and following the lead of his master (1827), undertook the comparative study of the sternal apparatus of birds and thereby discovered a key to the natural relationship of many types which antici- pated by many years the views now cur- rent. For instance, L’Herminier first cor- rectly appreciated the differences of the ostriches and penguins from other birds, the difference between the passerines and swifts, the homogeneity of the former, and the affinity of the humming birds and the swifts. Meanwhile Cuvier, like Linné, was content to accept as the basis for his primary classification of birds, superficial modifications of the bill and feet (toes and nails) which led to many unnatural asso- ciations as well as separations, but which nevertheless have been persisted in even to our own day by many ornithologists. Now what could have been the under- lying idea which hindered the foremost comparative anatomist of his age from the recognition of what are now considered to be elementary truths and what enabled Blainville to forge so far ahead? Cuvier Cuvier was regarded almost universally by his contemporaries, and long after- wards, in the words of his intellectual suc- cessor, Louis Agassiz, as ‘“‘the greatest zoologist of all time.’ In view of the facts already cited and innumerable others * Agassiz, “Essay on Classification,” p. 286. SCIENCE 498 [N.S. Vor. XXVI. No. 668 that could be added, however, the con- temporary verdict must be somewhat modi- fied. GEORGES LEOPOLD CHRETIEN FREDERIC DAGOBERT CUVIER Cuvier was a very great man of most impressive personality, wide versa- tility, extraordinary industry, vast knowl- edge of zoological and anatomical details, an excellent historian, a useful critic, and of good judgment in affairs generally, but although a greater all-round man, as a systematic zoologist he was not the equal of a couple of his French contemporaries, Blainville or Latreille. We have either to admit this conclusion or confess that our now universally admitted views are wrong. Nevertheless, Cuvier’s work was of great importance, and he first brought to the aid of systematic zoology the new science of vertebrate paleontology. Professor Huxley, in his excellent ‘‘In- troduction to the Classification of Ani- mals’’ (published in 1869), in his first chapter, ‘‘On Classification in General,”’ concluded a consideration of Cuvier’s law of the correlation of structure with the following paragraphs: Cuvier, the more servile of whose imitators are fond of citing his mistaken doctrines as to the nature of the methods of paleontology against the conclusions of logic and of common sense, has put this so strongly that I can not refrain from quo- ting his words.” But I doubt if any one would have divined, if untaught by observation, that all ruminants have the foot cleft, and that they alone have it. I doubt if any one would have divined that there are frontal horns only in this class; that those among them which have sharp canines for the most part lack horns. However, since these relations are constant, they must have some sufficient cause; but since we are ignorant of it, we must make good the defect of the theory by means of observation; it enables us to establish empirical laws, which be- come almost as certain as rational laws, when they rest on sufficiently repeated observations; so that now, whoso sees merely the print of a cleft foot may conclude that the animal which left this impression ruminated, and this conclusion is as certain as any other in physics or morals. This footprint alone, then, yields to him who observes it, the form of the teeth, the form of the jaws, the form of the vertebre, the form of all the bones of the legs, of the thighs, of the shoulders, and of the pelvis of the animal which has passed by; it is a surer mark than all those of Zadig. ““ Ossemens fossiles,” ed. 4me, tome Ir, p. 184. CUVIER AND PALEONTOLOGY The animals, and especially the verte- brates, of past ages were practically un- known to the early zoologists, and when they had large collections, as did Volta of the fishes of Mount Bolea, they identified them with modern species, or, with Scheuchzer, might consider a giant sal- amander as a man witness of the deluge— ‘‘Homo diluvii testis’?! It was not until Cuvier, with superior knowledge of skeletal details, examined numerous bones un- earthed from the Tertiary beds about Paris, that the complete distinction of animals of ancient formations from living species was recognized. Then was afforded the first glimpse of extinct faunas destined to far outnumber the existing one, but so im- perfect was the great paleontologist’s fore- sight of what lay in store for the future that he enunciated a dogma which was long accepted as sacrosanct; he called it the law of correlation of structure. A striking and even amusing example of its exposition and its failure I have previously drawn attention to. The first perusal of these remarks would occasion surprise to some and immediately induce a second, more careful reading to ascertain whether they had not been mis- understood. Men much inferior in ¢@a- pacity to Cuvier or Huxley may at once recall living exceptions to the positive statements as to the coordination of the ‘“foot cleft’’ with the other characteristics specified. One of the most common of domesticated animals—the hog—may come up before the ‘‘mind’s eye,’’ if not the ““ Ossemens fossiles,” ed. 4me, tome Ir, p. 184. ““ Ossemens fossiles,” ed. 4me, tome Ir, p. 184. SCIENCE 499 OcroBEeR 18, 1907] actual eye at the moment, to refute any such correlation as was claimed. Never- theless, notwithstanding the fierce contro- versial literature centered on Huxley, I have never seen an allusion to the lapse. And yet every one will admit that the hog has the ‘‘foot cleft’’ just as any ruminant, but the ‘‘form of the teeth’’—and the form of some vertebre are quite different from those of the ruminants and of course the multiple stomach and adaptation for ru- mination do not exist in the hog. That any one mammalogist should make such a slip is not very surprising, but that a second equally learned should follow in his steps is a singular psychological curiosity. CUVIER AND ANATOMY The failure to sufficiently apply anatomy to systematic zoology was especially exem- plified in the treatment of the fishes which absorbed so much of Cuvier’s attention in later years. He, as well as his associate, gave accounts of the visceral anatomy and was led—often misled—to conclusions re- specting relations by his dissections, but he failed to receive enlightenment by exam- ination of the numerous skeletons he had made. Those skeletons, pregnant with sig- nificance for the future, had no meaning for Cuvier; he never learned how to utilize them for the fishes as he did those of the mammals. His colleague and successor, Valenciennes, in the great ‘‘ Histoire Nat- urelle des Poissons,’’ was equally unappre- ciative of the importance of comparative osteology for comprehension of the mutual relations of the groups of fishes. I need scarcely add that the law of cor- relation applied by Cuvier to the structures of ruminants entirely fails in the case of many extinct mammals discovered since Cuvier’s days. Zadig would have been completely nonplussed if he could have seen the imprint of an Agriocherid, a Uintatheriid, a Menodontid or a Chalico- theriid. CUVIER AND PALEONTOLOGY To make the case clearer to those not well ac- quainted with mammals, I may add that because the feet are cleft in the same man- ner in the hogs as in the ruminants, both groups have long been associated in the same order under the name Paridigitates or Artiodactyles, contrasting with another (comprising the tapirs, rhinocerotids and horses) called Imparidigitates and Perisso- dactyles. is inadequate to that end... . In this opin- ion I do not coincide.’’® The many mis- takes Owen made in attempting to apply the principle proves how well Blainville’s contrary opinion was justified. The numberless remains of past animals, rescued from the many formations which the animals themselves distinguished, have entailed constant revisions of systems and clearer comprehension of the development of the animal kingdom. Such revision, too, must continue for many generations yet to come. % Owen, Am. Journ: Sc. and Arts, XLV., 1848, 185. CUVIER’S SUCCESSORS The value of this law was long insisted upon by many. Some of the best anat- omists, as Blainville, protested against its universality, but one who ranked with Cuvier in skill and knowledge of anatomy, Richard Owen, long upheld Cuvier’s view. ““You may not be aware,’’ he wrote in 1843, ‘‘that Mr. DeBlainville contends that the ground—viz., a single bone or articular facet of a bone—on which Cuvier deemed it possible to reconstruct the entire animal, The same defect in method or logic that characterized Cuvier’s work was manifest- ed by his great English successor in range of Inowledge of comparative anatomy, Richard Owen. His families, for the most part, were the artificial assemblages brought together by zoologists on account of superficial characters and too often SCIENCE 500 (N.S. Von. XXVI. No. 668 without rigorous attention to the applica- bility of the characters assigned. Much better was the work of the greatest nat- uralist of all, Johannes Miiller, who ad- vanced our knowledge of the systematic relations of all classes of vertebrates as well as invertebrates. But all were unable to free themselves from the incubus of the popular idea that all branchiferous verte- brates formed a unit to be compared with birds and mammals. Several propositions to segregate, as classes, Amphioxus and the chondropterygians had been made, and Louis Agassiz deserves the credit of claim- ing class value for the myzonts or marsipo- branchs as well as the selachians. But it was left to Ernst Haeckel, a pupil of Miil- ler, still happily living, to divest himself entirely of ancient prejudices and appre- ciate the interrelationship of the primary sections of the vertebrate branch. He for the first time (1866) set apart the amphi- oxids in a group opposed to all other verte- brates, then docked off the marsipobranchs from all the rest, and collected the classes generally recognized in essentially the same manner as is now prevalent. We may differ from Haeckel as to his classes of fishes and dipnoans, but his correctness in the action just noticed will be conceded by most, if not all, systematic zoologists to-day. terized them in embryological terms—EHvo- lutio radiata, Evolutio contorta (molluscs), Evolutio gemina (articulates) and Evolu- tio bigemina (vertebrates). The last were successively differentiated on account of the embryonic changes from the fishes to the mammals. CUVIER’S SUCCESSORS ‘‘These Beitrage,’’ Louis Agassiz justly affirmed, ‘‘and the papers in which Cuvier characterized for the first time the four great types of the animal kingdom, are among the most important contributions to general zoology ever pub- lished.”’ One of the most notable results, so far as systematic zoology was involved, was the deduction forced on Kowalevsky by his investigation of the embryology of tunicates, that those animals, long asso- ciated with acephalous mollusks, were really degenerate and specialized protover- tebrates. This view early won general ac- ceptance. While embryology was very successfully used for the elucidation of systematic zool- ogy its facts were often misunderstood and perverted. For instance, the cetaceans were regarded as low because they had a primitive fish-like form, although it must be obvious to all logical zoologists of the present time that they are derived from a quadruped stock; snakes have been also regarded as inferior in the scale because no legs were developed, although it would be now conceded by every instructed her- petologist that they are descendants of footed or lizard-like reptiles. Ammocates was considered as higher than Petromyzon ““masmuch as the division of the lips indi- cates a tendency towards a formation of a distinct upper and lower jaw,’’ but we now know that Ammocetes is the larval form of Petromyzon. Innumerable still more pertinent examples might be adduced for the inferior systematic grades, orders, families, genera, species, ete. The words high and low were used when generalized PHILOSOPHICAL ZOOLOGY As knowledge of the various animal groups increased and countless new spe- cies were piling up, yearning arose to dis- cover principles underlying the enormous mass of accumulating details, and the ex- cogitations of various naturalists resulted in some curious speculation and expression in classificatory form. They called their outpourings philosophy or philosophical zoology, and philosophers they were called by others. Some of the philosophers grouped ani- mals according to supposed degrees of nerv- ous sensibility ;® some according to the relations of parts to a center or an axis;°° some under groups supposed to correspond with different systems of the body, as the alimentary, the vascular, the respiratory, the skeletal and the muscular,” and some would accord to each of the senses definite groups.”” Lamarck (1812) contended for three cate- gories of animals: (1) apathetic animals and (2) sensitive animals among the invertebrates, and (3) intelligent animals, equivalent to the verte- brates. : But it was the much-contemned hypoth- esis of descent with modifications that was destined at last to relieve biological science of the wild and irrational speculations and 2 Blainville (1816) proposed to divide the ani- mal kingdom into three subkingdoms: (1) the Artiomorphes, having a bilateral form, (2) the Actinomorphes, having a radiate form, and (3) the Héteromorphes (mainly sponges and proto- zoans), having an irregular form. resentation of the sense-organs, and that they: must be arranged in accordance with them. Thus, strictly speaking, there are only five animal classes: Dermatozoa (skin or touch animals), or the Invertebrata; Glossozoa (tongue animals), or the fishes . . .; Rhinozoa (nose animals), or the reptiles . . .; Otozoa (ear animals), or the birds; Ophthalmozoa (eye animals), or the Thricozoa (mammals)... . But since all vegetative systems are subordinate to the tegument or general sense of feeling, the Dermatozoa divide into just as many or corresponding divisions, which on ac- count of the quantity of their contents, may be for the sake of convenience also termed classes.” —Oken, El. Physiophilosophy, 1847, p. xi. For the many other assumptions on similar and di- vergent lines the reader must refer to the “ Ele- ments of Physiophilosophy ” (1847). * Oken (1802-47) gave expression to his vary- ing views in several differing classifications. In one scheme (El. resentation of the sense-organs, and that they: must be arranged in accordance with them. Thus, strictly speaking, there are only five animal classes: Dermatozoa (skin or touch animals), or the Invertebrata; Glossozoa (tongue animals), or the fishes . . .; Rhinozoa (nose animals), or the reptiles . . .; Otozoa (ear animals), or the birds; Ophthalmozoa (eye animals), or the Thricozoa (mammals)... . But since all vegetative systems are subordinate to the tegument or general sense of feeling, the Dermatozoa divide into just as many or corresponding divisions, which on ac- count of the quantity of their contents, may be for the sake of convenience also termed classes.” —Oken, El. Physiophilosophy, 1847, p. xi. For the many other assumptions on similar and di- vergent lines the reader must refer to the “ Ele- ments of Physiophilosophy ” (1847). EMBRYOLOGY While Cuvier was still flourishing, a school of investigators into the develop- mental changes of the individual in dif- ferent classes, and among them the verte- brates,; was accumulating new material which should be of use to the systematic zoologist. _ Chief of these was Karl Ernst von Baer. In various memoirs (1826 et seq.) he subjected the major classification of animals to a critical review from an embryological point of view, recognized, with Cuvier, the existence of four distinct plans which he called types and charac- SCIENCE OCTOBER 18, 1907] 501 and specialized were really meant and those words, pregnant with mischief, often led their users astray as well as the stu- dents to which they were addressed. Equally, if not more extravagant, views were entertained by many naturalists that creative power delighted in the symmetry of numbers and in circular arrangements. It was contended that all groups of animals represented analogous groups in successive- ly diminishing circles; that in a perfect system there were a definite number of subkingdoms, an equal number of classes in each subkingdom, of orders in each class of suborders, of families, of genera, of subgenera, ete. Some maintained that three was the regnant number, others up- held four, others seven, but the most nu- merous and influential school contended for five. Exactly what the philosophers thought they meant, or what strange visions they may have conjured up may never be known. But for a time (1822-42) the school of quinarians, as they were called, claimed most of the naturalists of Britain. The most zealous of the school (William Swainson) was especially displeased with the developmental hypothesis of Lamarck and characterized the ‘‘speculations’’ of the great Frenchman ‘‘not merely as fanciful, but absolutely absurd.’’ PHILOSOPHICAL ZOOLOGY Physiophilosophy, 1847, 511 et seq.) he claimed that there were five “circles” corresponding with the “animal systems”: (1) Intestinal animals (Protozoa and Radiates) ; (2) Vascular, sexual animals (Mollusks) ; (3) Respi- ratory, cutaneous animals (Articulates); (4) Sarcose animals (Vertebrates except mammals), and (5) Aistheseozoa, or animals “with all... organs of sense perfectly developed ” (mammals). 2Oken maintained (1802-47): “that the ani- 2Oken maintained (1802-47): “that the ani- mal classes are virtually nothing else than a rep- SCIENCE [N.S. Vor. XXVI. No. 668 502 perhaps the most natural course, and at least had the advantage of proceeding from the comparatively known to the almost un- known. A significant and noteworthy ex- ception to this mode of procedure among the old naturalists was afforded by La- marck. (1809 et seq.), the precursor in this respect as well as in recognition of descent, of the modern school. classifications of the nature-philosophers, physiophilosophers, circularians, quinari- ans, trinitarians, septenarians, and their like that flourished during the first half of the past century. HISTOLOGY One of the most noteworthy modifica- tions of systematic zoology was the fruit of histological research. In 1839 Theodor Schwann, incited by the brilliant results of Matthias Jacob Schleiden’s researches -(1838) in vegetal histology, and at the suggestion of Johannes Miller, undertook investigations which led him to consider that the animal frame was built up from innumerable cells variously modified to form the different systems and organs of which it is composed. Ultimately the ani- mals thus developed were segregated by Ernst Haeckel, and the animal kingdom was limited to them, while the simple uni- cellular animals which had been already designated as Protozoa were associated with DEVELOPMENT THEORY Although there had been previous indi- cations of belief that transmutation of spe- cies might have been a cause for the di- versity of animal life, Jean Baptiste Pierre Antoine de Monet de Lamarck (1809) first framed a hypothesis that had a logical basis, although weakened by unproved pos- tulates. In view of those weaknesses, it was easy to bring forth many facts that seemed to militate unanswerably against it, and such were well put forward by Cuvier; as the hypothesis, too, was very unpopular, it was for a long time stifled. In the meanwhile geological and paleonto- logical investigation, comparative morphol- ogy, physiology and embryology, as well as systematic zoology, were revealing innu- merable facts that pointed all in the same direction and were only explicable collect- ively by the assumption that they were the result of original community of origin and subsequent deviation by gradual changes from time to time. The facts were at length collocated with extreme skill by Charles Darwin (1859) and a rational ex- planation of their evolution by means of natural selection made the new develop- ment theory acceptable to well-informed naturalists and logical thinkers generally. When it became generally recognized that there had been always a progression and development from antecedent forms, naturally there was a change in the man- ner of exposition of a series, and the lowest forms were taken as the initial ones and followed by those successively higher in the seale of beings. Even when old prejudices were administered to and the highest ani- mals put first in a work, it was often done in a reversed series; that is, after the sup- posed natural ascensive series had been de- termined on, that series was simply re- versed in order that the highest should be the first and the lowest the last. Many of our text-books of zoology still have this characteristic, but are being rapidly re- placed by those exhibiting the phyletic series. GRADUAL DELIMITATION OF GENERA As has been already noted, the animal genera of Linné were mostly extremely comprehensive, answering, when natural groups, to families, superfamilies, and even orders or classes of modern naturalists. Such contrast, however, with others of the Linnean genera, and when this fact became recognized and it was discovered that the large genera embraced types exhibiting many differences in detail, the latter were subdivided; early in the past century, at first owing especially to French and Ger- man naturalists, the subdivision of old genera on approximately present lines was commenced and applied at different times to various classes. It is noteworthy that im some instances the authors of the new genera quite abruptly changed their minds regarding the nature of such groups. For example, Lacépéde, in 1798, in the closing lecture of his course at the Museum of Natural History, recognized only 51 genera of mammals, but a few months later (in 1799), im a “‘tableau,’’ admitted and de- fined 84 genera. SEQUENCE OF GROUPS It had been almost the universal custom from olden time, as well as during the Lin- nean era, to commence the enumeration -or catalogues of animals with the forms exhibiting most analogy with man and consequently the highest in the scale of organic nature. As long as species were assumed to be individually created this was SCIENCE 503 OcTOBER 18, 1907] unicellular plants under the general term Protista. One of the prominent features of this idea was accepted by Thomas Henry Huxley (1874) with, however, the very important modification of retaining the old name Protozoa as the collective name of the animals and taking a suggested name of Haeckel’s (Metazoa) for the multicel- lular animals. quired eleven genera of gulls to include those now congregated in one. About the same time, some herpetologists were equally radical. Leopold J. F. J. Fitzinger, in 1843, distributed species which are now combined by all in the genus Anolis among no less than fifteen genera. The genus Bufo, as now understood, was split by some herpetologists into a dozen or more. These are only samples of numberless analogous cases. THE OLD AND THE NEW A comparison of systematic zoology at its dawn with that of the present time is rather a contrast of different themes. The old naturalists believed that all species of animals were created as such by a divine fiat; the modern consider that all animals are derivatives from former ones and that their differences have been ac- quired during descent and development. The Linneans based their systems on superficial characteristics, and the moderns take into consideration the entire animal. The early systematists assumed that characters drawn from structures or parts most useful to the animals were the best guides to the relationship of the animals; the latest ones have learned to distrust the evidential value of similarity of structures unaccompanied by similarity of all parts. The former were guided mainly by physio- logical characters; the latter take morpho- logical ones. The Linneans based their systems on superficial characteristics, and the moderns take into consideration the entire animal. The Linneans confined their generaliza- tions to few ecategories—genera, orders, classes; the moderns exhibit the manifold modifications and -coordinations of all structural parts in many categories— genera, subfamilies, families, superfami- lies and various higher groups. It seems to be generally supposed that there has been an uninterrupted tendency among zoologists to refinement and increase of number of genera to the present time, ~ but such is by no means the case. Half a century ago and more some ornithologists subdivided old genera and made new ones to an extent to which none of the present time is prepared to go. For example, Charles Bonaparte, Prince of Canino, re- The old naturalists believed more or less in the existence of a regular chain of beings from high to low; the new ones SCIENCE [N.S. Von. XXVI. No. 668 504 recognize the boundless ramifications of all animal stocks. for the different classes of the animal king- dom. The difference is especially great be- tween usage for the birds and that for the fishes. For the former class, genera, fami- fies and orders are based on characters of a very trivial kind. For example, the family of Turdide, or thrushes, relieved of formal verbiage, has been distinguished from neighboring families solely because the young have spots on the breast, but even this distinction is now known to fail in some instances. THE OLD AND THE NEW Extremely few, if any, of the families of oscine birds are based on characters of a kind which would be regarded as of family value in other classes of vertebrates. On the other hand, many of the families and genera of fishes are made by some excellent authorities to inelude types separated by striking pecul- larities of the skeleton as well as the exterior. The mammals are a class whose treatment has been mostly intermediate be- tween that for the birds afid that for the fishes. Its divisions, inferior as well as comprehensive, have been founded on an- atomical characters to a greater extent than for any other class. Its students are numerous and qualified. Mammalogy might therefore well be accepted as a standard for taxonomy, and the groups adopted for it be imitated as nearly as the differing conditions will admit. The families of birds would then be much re- duced in number and those of fishes in- creased. All the active herpetologists and ichthyologists of the United States have subordinated their own beliefs and ideas as to what would have been most desirable, to a greater or less extent, to approximate the desirable reduction of the terms ad- mitted by them to a standard uniform with that adopted by mammalogists. If others would likewise sacrifice their own predilections, the lamentable inequality of The elders assumed certain forms as highest and ranged their series from high to low; the sons commence their series with the most generalized types and progress from the less generalized to the more spe- elalized. PROSPECTS AND NEEDS In these days of extreme specialization one of the greatest needs in our universi- ties is a professor of systematic zoology with whom conference may be held as to the propriety of any systematic modifica- tion resulting from special investigation of the anatomy of any organ or part, or of any group of animals. Such conference might prevent the publication of many propositions due to exclusive consideration of an isolated subject. Perhaps the designation of systematic morphology might better indicate the nature of the sug- gested course. The consummation, how- ever, it must be admitted, is more desirable than probable. In 1887 the pressing need of an authorita- tive revision of anatomical nomenclature was brought to the attention of the German An- atomical Society, then but recently organized, and in 1889 it established a commission to deal with the matter, appointing upon the: commission Professors von Ké6lliker (chair- man), O. Hertwig, His, Kollmann, Merkel, Schwalbe, Toldt, Waldeyer and von Barde- leben, Professor Krause being later selected: as editor-in-chief and representatives of Great Britain and other countries being also included. For six years the commission labored with the difficulties assigned for its consideration, and in 1895 it presented a report to the society, submitting a list of some 4,500 terms, care- fully selected from the 30,000 or more, prin- cipally synonyms, which may be collected from the various standard text-books. The. society received and adopted the commission’s: report at its meeting in Basel, a circumstance which has gained for the list the appellation of the Basel Anatomical Nomenclature or,. more briefly, the BNA, and the report, drawn up by Professor His, was published as a sup- plement number of the Archiv fiir Anatomie: und Physiologie. I have intentionally refrained from any consideration of the work of living zoologists. If I had undertaken this, the task of selection would have been very difficult, and at any rate the time demanded for proper consideration would have been much more than that requisite for the re- minder of past discoveries. The progress of systematic zoology during recent years has been in accelerated ratio, and not a few of these whose achievements have helped to put zoology at its present level are in Boston to-day. PROSPECTS AND NEEDS In numerous old systematic and descrip- tive works—but in many cases not very old—the skeleton and other anatomical de- tails were noticed in connection with the species described, but not seldom some of those details, if rightly interpreted, would be in contravention of the classification adopted. In fact, the anatomy was to all intents and purposes treated as an offering of curious but useless information. Such conceptions, happily, are mainly—but not entirely—of the past, and we may live to welcome the day when every animal will be treated as whole. Systematic zoology will then be regarded as the expression of our knowledge of the entire structure and as an attempted equation of the results obtained by investigations of all kinds. In fact, systematic zoology is simply an at- tempt to estimate the relative importance of all structural details and to correlate them so that their relative values shall be- come most evident. It is the scientific out- come of all anatomical or morphological knowledge and the aim is to arrange the animal groups in such a manner as to show best their genetic relations and the succes- sive steps of divergence from more or less generalized stocks. One consummation devoutly to be wished for is general acceptance of a standard for comparison and the use of terms with as nearly equal values as the circumstances admit of. There is a great difference in the use of taxonomic names SCIENCE 505 OcrToBer 18, 1907] nized, and in anatomy several attempts have been made to establish a terminology which would be acceptable to the great body of anat- omists and eliminate from anatomical nomen- clature the ponderous mass of synonyms with which it is burdened. Henle in his classic “ Handbook” accomplished much towards the desired end, and since 1880 Professor B. G. Wilder has labored assiduously for the cause. But it was not a matter for accomplishment by a single individual working independently; it required concerted action. And although endeavors had been made to enlist the sym- pathies of the American Association for the Advancement of Science and the Association of American Anatomists in the work, for one reason or another little definite progress was made. such congruity would be to the great ad- vantage of comparative taxonomy. PROSPECTS AND NEEDS It is from the sum- mit of the elevation they have enabled us to reach that we look back to the deeds of old masters and can determine, better than their contemporaries or immediate succes- sors, their relative merits. THro. GILu Gill, Theodore. 1907. "Systematic Zoology: its progress and purpose." Science 26, 489–505. View This Item Online: https://www.biodiversitylibrary.org/item/97796 Permalink: https://www.biodiversitylibrary.org/partpdf/249041 Sponsored by Biodiversity Heritage Library Smithsonian Libraries and Archives Smithsonian Libraries and Archives This file was generated 29 March 2024 at 23:35 UTC SCIENTIFIC BOOKS SCIENTIFIC BOOKS Anatomical Terminology with Special Refer- ence to the [BNA]. By Proressor Lrew- ELLYS F. Barker. Philadelphia, P. Blakis- ton’s Son & Co. 1907. ' The necessity for both exactness and sim- plicity in the nomenclature employed in the descriptive sciences has always been recog- Anatomical Terminology with Special Refer- ence to the [BNA]. By Proressor Lrew- ELLYS F. Barker. Philadelphia, P. Blakis- ton’s Son & Co. 1907. This is neither the time nor the place for: a discussion of the work of the commission;, suffice it to say that its results have been, widely accepted and that a uniformity of an-~ ' The necessity for both exactness and sim- plicity in the nomenclature employed in the descriptive sciences has always been recog- Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 29 March 2024 at 23:35 UTC
https://openalex.org/W4380520572	http://anapub.co.ke/journals/jebi/jebi_pdf/2022/jebi_volume_2-issue_4/JEBI202202022.pdf	English	null	Evaluation of Environmental Pollution and Waste Management Strategies on the Ecosystem	Journal of enterprise and business intelligence./Journal of enterprise and business intelligence	2,022	cc-by	9,035	ISSN: 2789-5181 ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) Article Info Journal of Journal of Enterprise and Business Intelligence (http://anapub.co.ke/journals/jebi/jebi.html) Doi: https://doi.org/10.53759/5181/JEBI202202022 Received 06 April 2022; Revised from 22 May 2022; Accepted 13 June 2022. Available online 05 October 2022. Journal of Journal of Enterprise and Business Intelligence (http://anapub.co.ke/journals/jebi/jebi.html) Doi: https://doi.org/10.53759/5181/JEBI202202022 Received 06 April 2022; Revised from 22 May 2022; Accepted 13 June 2022. Available online 05 October 2022. © 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 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 https://creativecommons.org/licenses/by/4.0/. Evaluation of Environmental Pollution and Waste Management Strategies on the Ecosystem Bolormaa Budjav Department of Environmental and Forest Engineering, National University of Mongolia, Ulaanbaatar 14200, Mongolia. budjavnum@hotmail.com Correspondence should be addressed to Bolormaa Budjav : budjavnum@hotmail.com Published by AnaPub Publications Abstract – This article presents a discussion on environmental pollution, and waste management strategies. Whether it has a direct or indirect link, everything in the immediate ecosystem has some kind of bearing on the natural world. Pollution in the environment is caused by more than just humans; other organisms and natural disasters (such as quakes and volcanic eruptions) contribute, as well. Although environmental degradation has always been an issue, it has recently become a major danger to human civilization. The main question that surfaced when we were preparing our scientific research include: "What is the impact of the waste management on the environment?" There is widespread concern about environmental pollution today because of the many ways in which we are exposed to it on a daily basis, including through the food we eat, air we breathe, radiation we are exposed to, and water we drink. The depletion of natural resources, the loss of animal and plant species, and disruptions in global ecosystems and biochemical processes are further indicators of the effects of environmental concerns. The research findings suggest that waste management likely has serious consequences for the natural world. ywords – Environmental Pollution, Environmental Degradation, Waste Management, Waste Managemen I. INTRODUCTION All respondents agreed that if Langkawi did not have any problems with solid waste, more people would visit. Nevertheless, 6.1% of the respondents (2 people) said that solid waste was a major problem. Sixty-six percent (20 people) said they would dispose of solid wastes in the right areas, while thirty-three percent (10 people) said they would try to cut down on their source consumption to help. The way in which garbage is handled has real-world repercussions on people's quality of life in their communities. Proper garbage management ensures that residents may live in peace. Dasar Alam Sekitar Negara is a crucial government initiative for trash management [2]. The policy's ultimate goal is to ensure that future generations may always enjoy a healthy, prosperous, and environmentally sound environment. Human activity produces this garbage. Waste management is an important issue for both urban and rural communities in developing nations. Waste is defined by the Akta Kualiti Alam Sekeliling 1974 [3] as any substance that may contribute to environmental contamination, whether it is solid, liquid, or gaseous, and whether it is released, released, or deposited in the environment. g p Solids within sewage system or proof of water polluting agents such as solids and silt in industrial waste are not included in the definition of wastes, which includes undesired objects and other waste materials from community activities, agriculture and trade garbage. Nonetheless, the accumulation of trash may be harmful to the ecosystem. Pollutants in the air and water will both increase due to improper garbage disposal. Several of the elements involved in waste management that might have an adverse effect on the environment have contributed to this. Aesthetic degradation is the most readily apparent consequence of solid waste in the environment. The inappropriate burning of garbage poses a greater threat since it pollutes not only the air, but also the water and land underneath. Landfills produce methane and trash fleets are major producers of carbon dioxide and nitrous oxide, therefore these activities all contribute to global warming, as stated in [4]. The issues of environmental contamination and improper garbage disposal will be addressed here. Everything of our material possessions has some kind of indirect or direct link to the natural world. Man isn't the only thing that has an impact on environmental degradation; other animals and natural disasters (earthquakes, volcanic eruptions) do, too. I. INTRODUCTION Due to rising populations and rising levels of industrial production, the landfill that collect our garbage are multiplying at an alarming rate, further degrading our planet. Large amounts of trash are generated daily in urban regions as well as rural and agricultural ones. For instance, every 10 million tons of oily products and 500 billion tons of industrial waste products are disposed into waterways. There are approximately a billion tons of ash and aerosols released into the atmosphere annually from industrial facilities and transportation. Garbage gets piled up at the dump for years. Seventy percent of all trash ends up in landfills. Waste bio-decomposition causes environmental damage via its biochemical processes. Insects, mice, and rats, all of which make a significant contribution to disease widespread, thrive on the municipal garbage that contaminates the vegetation and soil, surface water, ground water, and air in enormous amounts. The health of future generations is at risk because of this new predicament. As a result, proper waste management becomes an issue for ecological security. Industrial waste, municipal solid trash, agricultural waste, electronic waste, medical waste, and other mixed waste products are only few examples of the wide variety of waste types. Chemical, biological, and nuclear waste all have serious pollution effects and may have devastating effects on the environment and humans. In the 90s of the 20th century, the average solid waste output per person was between 100 and 330 kilograms in the European Union, 414 kilograms in North America, and 720 kilograms in Asia. Waste generation and waste generation per person are both on the rise around the globe [1]. The main issue is that instead of being recycled or 223 ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) composted, this trash ends up in massive landfills near cities, where it may pollute the air, water, and ground. If solid wastes are not effectively handled, it may have negative consequences on public health, air quality, odours, and hazardous gas emissions. According to the data in Fig. 1, 27.3% of the population (9 people) agrees that improper waste management may lead to water contamination. A total of 21.2% (7 people) said that solid wastes may contribute to air pollution, and 18.2% (6 people) said the same about odor pollution. Solid garbage thrown into drainage systems might lead to sewage obstruction, according to the opinions of 12.1% (4 respondents). I. INTRODUCTION Environmental pollution has been there since the beginning of life on Earth, but it has only recently become a major danger to humanity. This is how the remainder of the paper is structured: Section II focuses on a brief discussion of environmental pollution. This section provides a graphical evaluation of the Mean PM 2.5 levels in many polluted nations globally from 2020 to 2021. Section III introduces the concept of waste management providing statistical evaluations, and a discussion of disposal of solid waste in landfill, industrial waste management, hazardous waste management, biodegradation, and recycling. In Section IV, the strategies of waste management are discussed: waste prevention, recycling and reuse, waste minimization, and biological treatment. The final Section V provides final remarks on environmental pollution and waste management. II. ENVIRONMENTAL POLLUTION Climate change, ozone depletion, and desertification are all the outcome of environmental degradation in nations and places where pollution is the worst. The United Nations established the definition that "pollution is exogenous chemical compounds encountered on a suitable site, at the proper time, and in insufficient amounts" [5]. Analysis (conducted in the early 20th century) suggests that the atmosphere and the hydrosphere are the most contaminated regions on Earth. Likewise, the condition of the cosmos in our solar system is cause for grave worry. The fundamental ecological unit, defined by complex variables of living and inanimate nature, must be taken into account if we are to define the idea of the environment. Ecology is the name for this system. Man's effect on the natural world is substantial since he is a thinking entity. Among the population of children aged 0-19 years old, the World Health Organization (WHO) identifies about 25 risk factors to human health, some of which are environmental. Direct (immediate) and indirect (reaching out to others) synergy both describe possible types of environmental influence from economic and other activity. In terms of the environment, the impacts of pollution are often expressed using the words we've previously mentioned: degradation, destruction, and threat. Pollution has observable consequences on the environment and its inhabitants. There are both immediate and long-term consequences of pollution. Understanding the idea of a working environment and how it relates to the idea of environmental protection might be crucial when discussing environmental management. There's a plethora of motivations for doing this. To begin, the operation is connected to and has significance for the environmental problem because of the working environment's role in the environment. Workplace safety and health laws, as well as other rules in this field, include a number of clauses that shed light on the nature of the connection between the office and the natural world. Not only is it important to have a firm grasp on the concept of "workplace," but also of phrases like "hazardous materials," "danger," "dangerous phenomena," "risk," and "risk assessment," among others. This legislation establishes what is meant by "work environment," or the conditions, methods, and relationships inside a certain workplace in which 224 ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) work is carried out. Human-caused environmental degradation, including altered energy inputs and radiation levels, environment pollution's physicochemical and microbiological components may be identified. II. ENVIRONMENTAL POLLUTION Physiological and anatomical structure, growth, and metabolism processes at different phases of cellular organization (from microscopic and molecular levels, through populations and individuals, to ecosystems and biocenosis) are affected negatively by pollution. The average PM2.5 concentration in Bangladesh in 2021 was 76.9 g/m3 hence making it the most polluted nation in the world. There was over 20 g/m3 higher PM2.5 than the norm in India. Due to a building boom in the nation, Bangladesh's brick industry has mushroomed to the point that thousands of coal-fired kilns are spewing out dangerous pollutants, contributing to the country's already high levels of air pollution. A tiny particle with a diameter of 2.5 micrometers or less is referred to as PM2.5. At high quantities, these airborne contaminants pose a serious threat to human health despite their microscopic size and light weight. Annual mean PM2.5 emissions should be kept below 10 g/m3 according to WHO recommendations. The incineration of solid waste, automobile emissions and industrial boilers are all significant contributors to PM2.5 levels in the environment. Average PM2.5 levels in Delhi were 85 g/m3 in 2021, making it the most contaminated capital city in the world once again. Dhaka, the capital of Bangladesh, came next. Due to COVID-19 shutdowns and limitations, PM2.5 concentrations decreased in numerous cities across the globe in 2020, with levels in Delhi decreasing by almost 15 percent relative to the previous year. hi decreasing by almost 15 percent relative to the previous year. Fig 1. Mean PM 2.5 Levels in many Polluted Nations Globally from 2020 to 2021 0 10 20 30 40 50 60 70 80 90 2020 2021 Mean PM 2.5 levels Kuwait Iran Kazakhstan China Saudi Arabia Mongolia Armania Nigeria Indonesia Montenego United Arab Emirates Afghanistan Qatar Uzbakistan Sudan Nepal Iraq Bahrain Kyrgyzstan Oman India Tajikistan Pakistan Chad Bangladesh Fig 1. Mean PM 2.5 Levels in many Polluted Nations Globally from 2020 to 2021 Statistical Evaluations Every year, humans produce 2 billion tons of municipal trash, and approximately 32% of it is not managed in an environmentally friendly manner. From 0.11 to 4.54 kilos per day, the international mean of wastes generated for every human is approximately 0.73 kg. High-income nations produce 34% of the globe's garbage (680 million ton), even though having approximately 15% of the globe’s population. By 2050, the estimated amount of garbage produced worldwide will be 3 billion tons that is more than twice the projected increase in human population during that timeframe. As a whole, more money means more trash, thus it stands to reason that more money would also mean more recycling. The daily per capita waste production is projected to increase by approximately 19% in nations that are categorized as high-income by 2050, but it is projected to rise by approximately 40% or even more in middle- and low-income nations. The degree of rise in the production of waste relating to an increase in income is more for low-income individuals compared to their counterparts in high-income nations, at least during their initial phases of income development. By 2050, the amount of garbage produced in the world's poorest nations is projected to grow by more than a factor of three. Twenty- three percent of the world's trash is being produced in East Asia and the Pacific, with 6 percent coming from the North Africa and Middle East. By 2050, overall garbage creation is predicted to more than quadruple in Sub-Saharan Africa, 225 Journal of Enterprise and Business Intelligence 2(4)(2022) ISSN: 2789-5181 ISSN: 2789-5181 double in the North Africa and Middle East and double in South Asia. More than 50% of garbage is disposed in the open spaces in their continental regions, and the projected expansion in waste has serious consequences for human health, the natural world, and economic development. Fig 2 provides an illustration of the forecasted waste production (millions of tons per year) based on region. double in the North Africa and Middle East and double in South Asia. More than 50% of garbage is disposed in the open spaces in their continental regions, and the projected expansion in waste has serious consequences for human health, the natural world, and economic development. Fig 2 provides an illustration of the forecasted waste production (millions of tons per year) based on region. Fig 2. Forecasted Waste Production by Region Although though garbage collection is essential, rates vary greatly by economic level, with practically universal waste collection available in upper-middle and high-income nations [6]. Around 48% of garbage in different cities gets collected in low-income nations, but that number decreases dramatically to 26% outside of metropolitan areas. As a whole, Europe, Central Asia, and North America gather at least 90% of their garbage, but Sub-Saharan Africa only collects 44%. Fig 3 shows the rate of waste collection based on the income level of individuals. 0 100 200 300 400 500 600 700 800 2016 2030 2050 Fig 2. Forecasted Waste Production by Region 0 100 200 300 400 500 600 700 800 2016 2030 2050 Fig 2. Forecasted Waste Production by Region Although though garbage collection is essential, rates vary greatly by economic level, with practically universal waste collection available in upper-middle and high-income nations [6]. Around 48% of garbage in different cities gets collected in low-income nations, but that number decreases dramatically to 26% outside of metropolitan areas. As a whole, Europe, Central Asia, and North America gather at least 90% of their garbage, but Sub-Saharan Africa only collects 44%. Fig 3 shows the rate of waste collection based on the income level of individuals. Although though garbage collection is essential, rates vary greatly by economic level, with practically universal waste collection available in upper-middle and high-income nations [6]. Around 48% of garbage in different cities gets collected in low-income nations, but that number decreases dramatically to 26% outside of metropolitan areas. ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) The content of trash varies by economic level because of the various lifestyle choices people make at those levels. In high-income nations, dry trash such as glass, metals, paper, cardboard, and plastic accounts for approximately 50% of the overall garbage, where green and food waste account for just 32 percent. Food and waste account for approximately 53% of all garbage in low-income countries, whereas green waste accounts for 57%; the proportion of organic waste rises in developing nations. Only 20% of garbage in low-income nations is recyclable. There is not much variation in waste streams among areas, save those that correspond to economic levels. With the exemption of Central Asia, North America, and Europe, which yield larger amounts of dry wastes, all areas create at least half of their trash as organic. Fig 4 illustrates the composition of global wastes in percentage. Fig 4. Composition of Global Wastes 0 5 10 15 20 25 30 35 40 45 50 Composition of global wastes in % Composition of global wastes in % Composition of global wastes in % Fig 4. Composition of Global Wastes Many people wrongly believe that modern technology holds the key to halting the spread of garbage that is now out of control. Technology is hardly a silver bullet and is often just one of several considerations in solid waste management. If a country wants to move away from waste management practices like open dumping, it will have the best chance of doing so if it chooses solutions that are tailored to its own needs and circumstances. Nowadays, most of the landfills are the most typical method of garbage disposal worldwide. Around 37% of trash is being dumped in a landfill, with just 8% going to sanitary dumps that also include landfill gas collecting systems. Around 31% of trash is dumped illegally, 19% is collected for reuse or recycling, and 11% is burned. High- and upper-middle-income nations are the only ones with the resources to provide proper waste disposal or treatment, such as regulated landfills or more strictly managed facilities. More than ninety-three percent of trash is discarded in low-income nations, whereas only 2% is deposited in high- income nations. Most of the world's garbage is dumped in the open in three places: the North Africa, Middle East, South Asia, and Sub-Saharan Africa. ISSN: 2789-5181 As a whole, Europe, Central Asia, and North America gather at least 90% of their garbage, but Sub-Saharan Africa only collects 44%. Fig 3 shows the rate of waste collection based on the income level of individuals. Although though garbage collection is essential, rates vary greatly by economic level, with practically universal waste collection available in upper-middle and high-income nations [6]. Around 48% of garbage in different cities gets collected in low-income nations, but that number decreases dramatically to 26% outside of metropolitan areas. As a whole, Europe, Central Asia, and North America gather at least 90% of their garbage, but Sub-Saharan Africa only collects 44%. Fig 3 shows the rate of waste collection based on the income level of individuals. Fig 3. Rate of Waste Collection According to The Level of Income 0 20 40 60 80 100 120 High income Upper-middle income Lower-middle income Low income Waste Collection in % Waste Collection in % Waste Collection in % Waste Collection in % Fig 3. Rate of Waste Collection According to The Level of Income 226 ISSN: 2789-5181 ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) enforcement tends to vary greatly. Over 70% of waste services are managed directly by local public authorities, with the federal government only being involved in regulatory monitoring and financial transfers. At least half of all waste management services, including collection, sorting, disposal, and treatment, are issued by governmental agencies, while another 30% are issued through private-public partnerships [7]. The partnership with private sectors for operation and finance are not always the best choice, however, since they often only work under particular circumstances with proper incentive structures and enforcement mechanisms. Fig 5. Global Disposal and Treatment of Wastes 0 5 10 15 20 25 30 35 Global disposal and treatment in % Global disposal and treatment in % Global disposal and treatment in % Global disposal and treatment in % Fig 5. Global Disposal and Treatment of Wastes Costs associated with maintaining and operating a waste management system are difficult to budget for, and must be considered in advance of any capital expenditures being made. In high-income nations, the whole cost of managing garbage, from collection to transportation to treatment to disposal, may easily top $100 per ton. Although lower-income nations pay less overall on waste operations, at about $35 per tonne and often more, they have a considerably harder time recouping their investment. Management of waste requires a lot of manpower, and only the transportation expenses might add up to $20 - $50 a ton. Recovery of garbage service costs varies greatly depending on household income. User fees average about $36 per annum in countries categorized as low-income and approximately $171 per annum in countries categorized as high-income being the only ones where the payments cover the costs. A user's price structure might be either constant or variable, depending on their circumstances [8]. Usually, national federal subsidies and the private sector make up the other half of the investment expenses for waste systems, with local governments picking up the tab for half. ISSN: 2789-5181 It is no surprise that nations in the upper middle income range have the greatest rates of trash dumped in landfills (54 percent). In high-income countries, this figure falls to approximately 39%, with 36% of waste being diverted to composting and recycling and about 22% to incineration. Only countries with a higher capacity, limited land usage, and high revenue usage resort to incineration. Fig 5 illustrates an international disposal and treatment of wastes in percentage. p g Solid waste treatment and disposal was responsible for about 5% of worldwide emission of greenhouse gas in 2016, or 1.5 billion tons of carbon dioxide equivalents. This estimate is based on the amount of garbage produced, its composition, and how it is handled. The primary factor in this is the use of landfills and open dumps without landfill gas gathering frameworks for wastes. Approximately 50% of greenhouse gases originated from trashed food. If no transformations are considering in the industry, solid waste emissions are forecasted to increase to approximately 2.40 billion tons of carbon- dioxide-equivalent per annum by 2050. q p y Almost 70% of global countries have established firms with the mandate for the regulation and creation of policies in the waste management sector and most countries' solid waste management activities are primarily handled at the municipal level. Over 60% of countries have enacted certain solid waste management regulations and laws; however, their level of 227 Table 1: Drawbacks of Disposal of Solid Wastes in Landfill Groundwater contamination Groundwater contamination might occur if you choose the improper subterranean landfill. Low oxygen concentration There is no oxygen for the garbage to decompose when it is buried and situated in the midst of the nation. It produces biogas, a mixture of hydrocarbons, predominantly methane. When it spreads horizontally, biogas may enter building basements and, when it comes into touch with open flames, burn and explode. Worsening drooping areas When trash decomposes, its volume diminishes, causing drooping areas where it is impossible to construct structures and other infrastructure. Disposal of Solid Waste in Landfills To dispose of garbage, people use landfills. There are a number of different kinds of landfills, distinguished by the way trash is disposed of and the security measures in place to avoid air and water contamination. Putting trash in the open without any kind of containment system pollutes the air and water. Sanitary landfills are constructed to minimize the possibility of solid waste items interacting with the surrounding air. As an alternative to landfills, today's trash is disposed of deep below. Hence, both air pollution and the unwelcome spread of animals are prevented. There are drawbacks to this waste management strategy as seen in Table 1, Table 1: Drawbacks of Disposal of Solid Wastes in Landfill Groundwater contamination Groundwater contamination might occur if you choose the improper subterranean landfill. Low oxygen concentration There is no oxygen for the garbage to decompose when it is buried and situated in the midst of the nation. It produces biogas, a mixture of hydrocarbons, predominantly methane. When it spreads horizontally, biogas may enter building basements and, when it comes into touch with open flames, burn and explode. Worsening drooping areas When trash decomposes, its volume diminishes, causing drooping areas where it is impossible to construct structures and other infrastructure. r n Groundwater contamination might occur if you choose the improper subterranean landfill. 228 Journal of Enterprise and Business Intelligence 2(4)(2022) ISSN: 2789-5181 Strategies for controlling and building landfill Three techniques identified in Table 2 are used for building landfills: Table 2. Techniques used for Building Landfill Holding wastes based in their biological and chemical properties To prevent the spread of dangerous compounds into the environment, the waste is held on land with the necessary properties and capacity for self-purification. The only thing preventing the contaminating chemical in this situation is land. Land should thus be selected carefully to fulfill certain chemical and biological requirements (such as grain size, clay distribution). Controlling maximal permeability of contaminants Restricting, or limiting, the maximal permeability of contaminants from landfills to landfill coating materials, which are water resistant, minimizing the entry of air and surface water and resulting soil contamination; establishing a layer of insulation in the landfill bottom to inhibit water filtration; and launching a draining framework to collect water, which has permeated the top covers. This water is either repurified or returned to the soil. Concept of restriction The theory in [9] views a landfill as a form chemical reagent. The current strategy keeps the concept of restriction, but does it in a regulated way to create a managed chemical reactor. Strategies for controlling and building landfill Strategies for controlling and building landfill Three techniques identified in Table 2 are used for building landfills: Three techniques identified in Table 2 are used for building landfills: Table 2. Techniques used for Building Landfill Industrial Waste Management Technological and scientific endeavors generate vast quantities of trash, yet many of these materials may be recycled and reused. There are a variety of environmental protection concerns that go into the decision to recycle or reuse materials, including the desire to lessen the impact on natural resources like water and soil. To further understand the nature of industrial trash, we may break it down into its component parts: waste wood, scrap, waste plastics, and other industrial wastes. New technologies are being developed that are good for the environment and are all-encompassing, cutting down on the quantity of primary production. Innovations in non-waste technology should include: - the utilization of closed systems for industrial water supply - the manufacturing of products and daily trashing into new goods - development and manufacturing that prioritizes reuse. Hazardous Waste Management The United States Environmental Protection Agency considers any trash that is combustible, corrosive, reactive, or poisonous to be hazardous. Nowadays, hazardous trash is literally everywhere. Approximately 60% of all industrial hazardous waste comes from the chemical industry; 20% comes from the metal industry; 5% comes from the military; 2% comes from the pharmaceutical industry; 1% comes from clinical centers; 1% comes from households and 2% comes from small businesses, according to studies. Technology Biodegradation of Organic Waste gy g f g Due to microorganisms, which are essential to the biosphere's ecological processes because of the large quantities of oxygen they produce via their immense activity, the planet supports life. Biodegradation of organic waste by bacteria has occurred over many years. Humans have learnt to control this process, creating targeted communities of microbes. Knowing the chemical composition of waste components is essential for microbial decomposition of both vegetable and animal matter. The make-up of the trash differs according to the types of food products that are discarded. Organic food waste is mostly "provided" by the food processing sector, agricultural growers, and a few organizations. The following are some of the ways in which industrial garbage and food scraps of animals or plants are used; wastes from other industrial and municipal activities should be collected of in landfills; organic wastes are applied as manure on farmland; domestic animal corpses are buried; industrial waste is discharged into the city's sewage system; waste is disposed of in illegal landfills. There is no environmental or economic justification for the current practice of disposing of municipal garbage, which accounts for a significant portion of the overall amount of solid waste, via incineration, compaction, or landfill. There are always efficient and low-cost solutions to deal with garbage. The use of biodegradation technology is the answer to these issues. European nations make extensive use of biodegradation in their economic operations. It is a common practice in Western European farms, where it is used to produce both energy and natural fertilizers. Microbial degradation of organic waste is commonplace in Norway and Canada, both in homes and in factories. It may be stated that the biodegradation technique is cost-effective. Fertilizer is a valuable commodity that may be sold to generate revenue from its use. Hazardous Veterinary and Medical Waste Biodegradation is a common economic technique in many nations, serving several functions including protecting arable land and biodiversity, producing electricity, and getting high-quality organic fertilizer. for life and soil fertility. Food scraps account up a large percentage of MST. Both the density and the humidity are rather high. The method of biodegradation is presented to deal with the waste issue. Biodegradation realization is possible in medium-sized rural and urban settings, particularly on smaller farms and greenhouses. Biodegradation's ultimate products (biogas, water, and organic fertilizer) are environmentally pure, making them useful in both commercial and residential settings. Biodegradation is a common economic technique in many nations, serving several functions including protecting arable land and biodiversity, producing electricity, and getting high-quality organic fertilizer. Recycling Recycling refers to the process of collecting usable materials and separating them from trash for later use. Repurposing entails gathering discarded goods, sorting them, processing them, and making new products from the resulting raw materials. Recycling refers to the practice of reusing materials rather than discarding them. It is hard to conceive of a fully integrated waste management system without the widespread implementation of recycling in schools. While there are facilities elsewhere that use recycled materials to create new products, no such facilities exist in the United States. Paper service is one of the few places where people may recycle their old paper for cash. Glass recycling depots are nonexistent, leading to the annual waste of thousands of bottles. Strategic goals such as conserving natural resources and lowering pollution levels may be achieved by recycling (all materials come from limited supplies and are obtained from natural sources). Cost-cutting Energy Efficiency Measures (there is no wasted energy during the basic operations, nor during the transportation and subsequent procedures, and surplus energy may be produced by burning non-recyclable materials). Prevention of pollution and degradation of the natural environment involves decomposing garbage and recycled goods. f y g "Materials may be: - Recyclable (this lessens the damage mining does on the environment) (may be put back into production once recycling has taken place), - Non-recyclable (do not belong in the production cycle again, but are instead used to create energy or buried in a manner that does not harm the environment). - Dangerous (materials that might potentially harm people or the environment), Safe (substances that are safe for both people and the planet). It is possible to classify recycling as either "primary" (reuse of materials that have been cleaned and processed several times is one example of recycling) or "secondary" (recycling, whereby recyclables are processed in the traditional manner utilizing cutting-edge tools to maximize efficiency). Hazardous Veterinary and Medical Waste Hazardous Veterinary and Medical Waste y Medical-veterinary waste encompasses any and all trash produced by hospitals and vet clinics, regardless of its chemical make-up or point of origin. It's a wide variety of things including trash from the city, germs from a lab, old medicine, unused packaging, and leftovers from the pharmacy. The categories of trash in Table 3 make up the 14% of medical waste that is considered hazardous: Table 3. Categories of Trash Making Up Medical Wastes Infectious waste These include those generated by a microbiology lab's instruments, materials, and accessories, which make contact with infectious patients or blood used in surgical operations, hemodialysis, garbage-infested gloves and experimental animals. Sharp items Anything that may stab or cut, such as needles, syringes, scalpels, and other sharp items. Repulsive materials These feature discolored or otherwise unappealing human or animal body parts (for example, those removed following surgery), laboratory animals, or animal anatomy. Medicines and chemicals Medicines and chemicals used in the pharmaceutical industry that have either expired or been returned to the original departments where they were taken from are considered pharmaceutical industrial waste. Discarded chemicals from medical or laboratory Discarded chemicals from medical or laboratory usage, household cleaning, or disinfection are examples of chemical waste. Black and gray radioactive contaminated tools, supplies, solutions, animal cadavers, and test subjects. Thermometers, blood pressure monitors, and other medical gadgets all contain mercury, lead, and arsenic, among other heavy metals, as a byproduct of their manufacturing. g Biodegradation and composting have considerable promise for treating organic waste from urban sources. Based on its thermal capacity of approximately 24000 KJ/m3, biogas produced from processing a single ton of organic wastes could amount to approximately 500 m3. Bacteria and other forms of biota rely on it to survive. Both the number and variety of bacteria much exceed that of any other group of microorganisms. Nitrogen fixation is a bacterial process that is essential 229 ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) for life and soil fertility. Food scraps account up a large percentage of MST. Both the density and the humidity are rather high. The method of biodegradation is presented to deal with the waste issue. Biodegradation realization is possible in medium-sized rural and urban settings, particularly on smaller farms and greenhouses. Biodegradation's ultimate products (biogas, water, and organic fertilizer) are environmentally pure, making them useful in both commercial and residential settings. Recycling of Vehicles at the End of Life y g f f f An item of such complexity as an automobile is rare. Steel predominate and cast iron in the construction of the vehicle's many components. Over 80% of a vehicle's mass may be recovered with today's recycling infrastructure. The complexity of auto recycling stems from the wide range of materials used in its construction. The 3% of a vehicle's mass that is glass is not processed in glass factories due to its complicated chemical makeup but is instead chipped and utilized as a concrete component. Motor vehicle fluids, e.g., gasoline, engine oil, transmission fluid, and radiator fluid account for approximately 2% of a vehicle's total mass. Because of their poisonous chemical make-up and unique properties, these fluids provide a considerable challenge throughout the auto recycling process. The five percent of a vehicle's weight that comes from rubber is processed further into industrial applications to generate various items (protective fencing, flooring, and asphalt). "Used vehicle batteries pose a serious hazard to the environment; to mitigate this, it is necessary to move to a system that uses various kinds of filters based on the life cycle of the battery. Carefully and separately handling the car's many components is essential. "Now, two technologies of recycling the automobiles are utilized, each with its own unique approach to separating the various components. The first approach relies on optical separation, whereas the others combine many techniques (such as mulching, gravity, and other unique techniques). Car-battery Recycling Car battery Recycling To put it another way: "Landfills are being used to dump enormous quantities of random trash. The year-round disposal of used batteries takes place in landlls that have not been prepared for such use. This causes serious soil contamination from the battery's lead, acids, and other inorganic elements. It's important to sort out which parts may be recycled ahead of time, recycle those parts, and then properly dispose of the remaining garbage from recycled batteries. Reusing batteries is not a widely adopted practice despite its obvious environmental and financial benefits. Since it is so challenging to make a selection, only a limited number of firms engage in the collection of old batteries from uncontrollable landfills that are widespread and settle all types of waste products. Data shows that we dispose of one-fourth of all batteries produced. Lead is a crucial raw material derived from used batteries. Waste Prevention Zero waste is the optimum solution for managing garbage. The major purpose of any waste management strategy needs to be geared towards the avoidance of wastes. Wastes could be eliminated and pollution avoided or reduced through the employment of different technologies, which can be employed at any phase of the products’ life cycle. Some examples of green manufacturing practices include using less toxic or harmful materials, installing leak detection systems in storage facilities, developing new methods of chemical ozonation to lessen reactions, and implementing water conservation technologies to cut down on the amount of potable water used in production. Recycling of Plastic Waste The intrinsic inertia of plastic trash makes it a formidable composting opponent. Reusing old boxes helps cut down on garbage and pollution while also conserving resources and power. Overconsumption of plastic goods and subsequent waste disposal is a major contributor to the problem. There are a variety of methods for recycling plastics, but they all accomplish the same thing. There are seven steps involved in the technical process: First, trash is gathered, and then recyclables are identified and chosen, then milled, then rinsed, then spun, then dried, and finally stored. Another problem arises when plastic trash from homes gets mixed in with the regular garbage. Several containers (of glass, paper, plastic, etc.) are developed to address this issue. This system of collection and classification is widely used in most EU nations. Over 30 million tons of plastic trash is collected in the 21st century’s first decade within the United States. The difficulty in properly discarding this trash highlights the value of finding new uses for it. The average American consumes 86 kilograms of plastic per year, 27 kilograms of which is packaging. Over 80% of Germany's yearly plastic waste volume is recycled or roughly 800 tons. Although this may seem like a lot, consider that only roughly 200 tons of 230 ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) plastic are recycled in the United Kingdom. There has been a rise in the processing of plastic waste since the year 2000, with each piece of plastic being returned to a landfill. It's an unusable byproduct of processing. Plastics that degrade when exposed to microorganisms are a major research and development focus. They are the so-called "biodegradable" plastics. Biodegradable plastic is not a panacea, and it cannot be used to replace all plastics. In spite of our considerable capabilities, we are not yet doing enough to properly handle plastic trash. It's the treatment of trash whose origin and make-up are already recognized. Processing power is likewise rather low. IV. WASTE MANAGEMENT STRATEGY Workers in the trash business face several dangers over the course of their jobs, including those associated with garbage collection, transportation, storage, and treatment. Waste management refers to the systems put in place and the actions taken by those systems to reduce trash and encourage people to recycle and compost their garbage. Waste Prevention Biological Treatment Disposal of trash with high organic components in landfills is being more frowned upon in many nations, the United States included. In fact, numerous European nations have outlawed this kind of waste disposal. Several techniques have been evaluated since landfilling is not a considerable management option. In order to dispose of or reuse the inorganic waste percentage (known as residuals) that remains after biodegradable materials have been destroyed, waste treatment is one possibility. p y Wastes may be biodegraded by aerobic composting, anaerobic digestion, and mechanical biological treatment. Anaerobic digestion and aerobic composting could be applied in decomposing wastes and transforming them into useable composts in case organic components can be eliminated from inorganic materials. Composts, collected from organic wastes such as yard trimmings, animal manure and food scraps, may be used as a natural fertilizer if the circumstances are right. Aerobic composting integrates arranging organic wastes in predetermined proportions in containers, heaps, and rows, either indoor or outdoor facilities with gas treatment and collection systems. Wood chips as well as other bulk agents are integrated to garbage to accelerate the rate of organic components’ aerobic decomposition. In the end, the substance is cured, which is a process of stabilization and maturation during which harmful microorganisms are eliminated. Composting generates carbon dioxide gas, water, and useable compost as its final products. There is a wide range of possible uses for compost. Compost has several uses than just being a great soil additive for plant culture. It may be used to clean up polluted water sources including soil, groundwater, and even stormwater. Keeping the composting process under tight control is essential to ensuring high-quality results, but it may be a lot of work. Compost that is not fit for its projected use could be generating if its operational conditions are not critically managed and monitored. Irrespectively, composting is currently attracting significant attention, with approximately 80 million tons of wastes being saved from landfill trashing streams in 2008, up from about 14 million tons in 1979. This detour saved more than 177 million tons of carbon dioxide in 2008, which is the same as the annual emissions from 33 million cars [11]. For certain operations, oxygenated systems just cannot be used. However, anaerobic systems can be used in their place. The anaerobic digestion process integrates organic waste degradation in containers without the availability of oxygen. Recycling and Reuse Metals, paper, glass, wood and plastic are just some of the various recyclable materials, which could be salvaged from waste heaps and employed in the manufacturing of new products. Enhanced employed of recycled materials implies that less of raw materials have to be applied for similar purposes. In addition to reducing the need for mining natural resources, recycling also allows for the reuse and recovery of one-disposed products. By transforming earlier-used materials from landfill disposal and incineration, including by minimizing the overall usage of energy and greenhouse gas concentration, which contribute to worldwide climate change, repurposing/reusing has a direct positive effect on the conservation of natural resources, and reducing pollution effects. Recycling also has the ability to provide growth and new employment opportunities for the economy. Materials such as glass, plastic, paper, steel, aluminium, and wood are typically recycled. Masonry, asphalt, concrete, and reinforcing steel are some of the reusable construction materials. Reusable green materials are typically applied as fertilizer and mulch. Regenerating and refining solvents for future use is a typical practise in various industrial segments, as is the recovery of other byproducts. Acids may be recovered by spray roasting, ion exchange, or crystallization, copper and nickel can be extracted from metal operations, finishing, oils, plasticizers, and fats can be collected from filter media such as clays and activated carbons, and many more examples exist. On a daily basis, people see different cases of reuse and recycling programs that have been effective. It is not uncommon for recycled materials to play a crucial role in the production process, both as raw materials and as the final product. Recycling is often practiced in a variety of industries, such as the paper and metals industries. Sometimes, the processing of salvaged materials is minimal, if any at all. Instances of this include the production of wood chips from tree trimmings and the use of previously used bricks and other building materials into brand-new buildings. For recycling to be successful, several factors must come into play, including widespread public acceptance and marketing of recycled goods and uses, as well as efficient collecting and processing of recyclables. Waste Minimization It is sometimes not possible to completely remove wastes from many different types of operations. Yet, several options exist for controlling garbage production. The term "waste minimization," sometimes known as "source reduction," alludes to a collection of practices based on the production and design of goods and services with the goal of reducing the quantity and/or toxicity of waste produced. Most of the time, these initiatives are launched when researchers discover a pattern or product that is contributing to an already existing waste issue. Repurposing resources, translating to less toxic alternative materials, and transforming design and production parameters are all applicable strategies to minimize industrial wastes. Source reduction and waste minimization have several advantages, such as lowering carbon emissions and toxicity levels in landfill garbage. Strategies for reducing waste in production are widespread; not only does minimizing the consumption of materials helps to reserve scarce resources, but it also amounts to significant saving in manufacturing-related expenditures. Advancements in shaping packaging not only cut down on the usage of materials, but also on the use of fuels and pollutants that come from transporting goods. Additionally, the weight and bulk of a building might frequently be significantly decreased by employing engineered building materials, which are established using particular advantages 231 ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) features. Hence, less material may be purchased in bulk, and less can be thrown away during the manufacture of the component. Toxic waste reduction by product substitution is shown well in the drier cleansing sector. Perc or Trichloroethylene was the selected solvent for drier cleanshing agents for decades. Tetrachloroethylene represents an effectual solvent, but it is also somewhat poisonous [10]. In addition, it is modest to release it into the ecosystem, where its physical features make it significantly challenging to eradicate. Also, the process of degradation generates intermediate sister chemical, which is significantly dangerous to the environment and human health. The dry cleansing business has adapted novel processes and it drastically employing minimally hazardous substitute solutions, such as petroleum-centred compounds, such as environmental and toxicity effect of the chemicals formerly used. Also, carbon dioxide and other innocuous compounds are being integrated into modern technology. Some replacement goods have been accepted in part because of government regulation, but also because of customer desire and other market-based pressures. Recycling and Reuse Ethics Approval and Consent to Participate Ethics Approval and Consent to Participate The research has consent for Ethical Approval and Consent to participate. ISSN: 2789-5181 ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) (biosolids). Biogas may be utilized as a fuel source or to generate power, while the leftover material can be put to good use as a soil amendment or fertilizer. Wet wastes are better degraded by anaerobic digestion than dry wastes, which are better degraded by composting. The capacity to gather biogas that could then be put to effectual use is one of the main reasons why anaerobic digestion is favored over conventional waste disposal methods like landfilling. Anaerobic digestion is suitable strategy of waste degradation compared to landfill disposal. Mechanical biological treatment (MBT) [12] is an alternative strategy, despite the fact that it is not widely used in countries such as United States. The European market has embraced this option, though. This process involves putting trash through a series of mechanical and biological procedures that decompose organic matter inside to lower the trash's overall volume. Crushing, shredding, and sorting are all the different samples of mechanical activities, which get the wastes ready for the next phase, which could either be anaerobic digestion or aerobic composting, the biological treatment of last resort. When the waste material has been reduced via biological processes, it may be incinerated. Biological Treatment Carbon dioxide and methane, as well as their residuals are generated during the process of anaerobic breakdown 232 Competing Interests There are no competing interests. V. CONCLUSION The advent of the industrial revolution and the first widespread usage of fossil fuels have had a profound effect on the natural world. More and more people are giving some thought to this issue, and rightly so; we cannot let this business to expand at the expense of the natural world. Sustainable development is an idea that has emerged as a result of this style of thinking. The continuation of industrial growth while minimizing negative effects on the environment is implied by this idea. Altering an ecosystem may have far-reaching consequences for human health due to the negative effects of contaminants already existing in the environment. The industrial style of production that was designed to make man the ruler of nature instead threatens the existence of both humans and the natural world. Air, water, and land pollution have all reached worrisome levels. The world still contains landscapes and areas that are inaccessible to humans. The natural resources were recognized and safeguarded by law. The majority of a man's day is spent in an urban or industrial setting, and he spends just fleeting minutes at a time in a pristine natural setting. Certain wastes pose a particularly serious risk to both ecosystems and human health. This threat was not immediately appreciated by the firm. Regulating garbage disposal is an area that is currently lacking in many nations. Due to the widespread use of pesticides in agriculture and the production of industrial waste that contains poisonous and carcinogenic compounds, the volume of hazardous trash has expanded substantially in recent years. Loss of biodiversity, soil depletion, deforestation, and supplying of clean water are only some of the worldwide environmental challenges that have arisen as a result of the worsening environmental condition in nations and areas where environmental degradation is most acute. Sustainable waste management aspires to minimize contamination to the surrounding environment. One helpful technique for extracting as much value as possible from trash is recycling. Most nations choose recycling because it offers economic benefits in addition to trash reduction. The use of recycled raw materials also helps save energy. Data Availability No data was used to support this study. Data Availability No data was used to support this study. Conflicts of Interests Conflicts of Interests The author(s) declare(s) that they have no conflicts of interest. Conflicts of Interests The author(s) declare(s) that they have no conflicts of interest. The author(s) declare(s) that they have no conflicts of interest. Funding No funding was received to assist with the preparation of this manuscript. Funding Funding No funding was received to assist with the preparation of this manuscript. ( ), [7]. D. Lim and H. Kim, “The cost-effectiveness of the supporting policies for food waste-reducing equipment: Purchase subsidy vs. Rental subsidy,” J. Korea Soc. Waste Manag., vol. 39, no. 01, pp. 78–86, 2022. y \| aya and E. F. Cali, “Smart garbage collection system for smart cities,” in 2022 Innovations in Intelligent Systems and Applications e (ASYU), 2022. ] et al., “A rose bengal/graphene oxide/PVA hybrid hydrogel with enhanced mechanical properties and light-triggered antibacte ty for wound treatment,” Mater. Sci. Eng. C Mater. Biol. Appl., vol. 118, no. 111447, p. 111447, 2021. , , , pp , [9]. “Introduction of restrictions on the landfilling of food waste,” DAERA, 01-Oct-2015. [Online]. Available: https://www.daera- ni.gov.uk/consultations/introduction-restrictions-landfilling-food-waste. [Accessed: 21-Feb-2023]. g g [ ] [10]. R. O. Lima, A. S. Barbosa, M. H. F. Bettega, S. D. Sanchez, and G. M. Moreira, “Cross sections for elastic collisions of low-energy positrons with tetrachloroethylene (C2Cl4) molecule,” Chem. Phys., vol. 567, no. 111807, p. 111807, 2023. [8]. Z. Abbas, S. Utomo, and B. Budiyono, “Analysis of optimum garbage heaps age on recovery of landfills dominated by organic solid waste,” Inż. Ekol., vol. 21, no. 8, pp. 91–98, 2020. References [1]. “Trends in solid waste management,” Worldbank.org. [Online]. Available: https://datatopics.worldbank.org/wh solid_waste_management.html. [Accessed: 21-Feb-2023]. [2]. “Dasar Alam sekitar negara – department of environment,” Gov.my. [Online]. Available: https://www.doe.gov.my/en/national-policy-of-the- environment/. [Accessed: 21-Feb-2023]. [3]. “Akta Kualiti Alam Sekeliling 1974 – ACT 127 – Jabatan Alam Sekitar,” Gov.my. [Online]. Available: https://www.doe.gov.my/akta/akta- kualiti-alam-sekeliling-1974-act-127-100/. [Accessed: 21-Feb-2023]. g [ ] [4]. R. Harth, “Methane from waste should not be wasted: Exploring landfill ecosystems,” ASU News, 18-Apr-2022. https://news.asu.edu/20220418-methane-waste-should-not-be-wasted-exploring-landfill-ecosystems. [Accessed: 21-Feb-2 [5]. United Nations, “History of the united nations \| united nations.” . United Nations, “History of the united nations \| united nations.” [6]. M. Karakaya and E. F. Cali, “Smart garbage collection system for smart cities,” in 2022 Innovations in Intelligent Syste Conference (ASYU), 2022. ( ), [7]. D. Lim and H. Kim, “The cost-effectiveness of the supporting policies for food waste-reducing equipment: Purchase subsidy vs. Rental subsidy,” J. Korea Soc. Waste Manag., vol. 39, no. 01, pp. 78–86, 2022. ( ) [7]. D. Lim and H. Kim, “The cost-effectiveness of the supporting policies for food waste-reducing equipment: Purchase subsidy vs. Rental subsidy,” J. Korea Soc. Waste Manag., vol. 39, no. 01, pp. 78–86, 2022. 233 [Accessed: 21-Feb-2023]. [12]. Y. Li et al., “A rose bengal/graphene oxide/PVA hybrid hydrogel with enhanced mechanical properties and light-triggered antibacterial activity for wound treatment ” Mater Sci Eng C Mater Biol Appl vol 118 no 111447 p 111447 2021 [11]. What a waste: An updated look into the future of solid waste management, World Bank. [Online]. Available: https://www.worldbank.org/en/news/immersive-story/2018/09/20/what-a-waste-an-updated-look-into-the-future-of-solid-waste-management. [Accessed: 21-Feb-2023]. [12]. Y. Li et al., “A rose bengal/graphene oxide/PVA hybrid hydrogel with enhanced mechanical properties and light-triggered antibacterial Journal of Enterprise and Business Intelligence 2(4)(2022) t a waste: An updated look into the future of solid waste management,” World Bank. [Online]. Availab //www.worldbank.org/en/news/immersive-story/2018/09/20/what-a-waste-an-updated-look-into-the-future-of-solid-waste-managemen ssed: 21-Feb-2023]. ISSN: 2789-5181 ISSN: 2789-5181 Journal of Enterprise and Business Intelligence 2(4)(2022) pp ion of restrictions on the landfilling of food waste,” DAERA, 01-Oct-2015. [Online]. Available: https://www.daera- consultations/introduction-restrictions-landfilling-food-waste. [Accessed: 21-Feb-2023]. 234
https://openalex.org/W4212984946	https://www.qeios.com/read/OAIAXX/pdf	English	null	Plant extract	Definitions	2,021	cc-by	165	Qeios · Definition, August 24, 2021 Open Peer Review on Qeios Open Peer Review on Qeios Plant extract Addiction Ontology Addiction Ontology Open Peer Review on Qeios Source Addiction Ontology Definition: An extract from a plant. Qeios ID: OAIAXX · https://doi.org/10.32388/OAIAXX Definition: An extract from a plant. This definition was imported from the Addiction Ontology (see https://addictovocab.org/). Comments and suggestions for improvements are welcome using the Qeios review system. Definitions imported from the Addiction Ontology (AddictO) are what are known as ‘ontological definitions’. See this article in Qeios for an explanation https://www.qeios.com/read/YGIF9B. Ontological definitions can sometimes be hard to read. In those cases we also include an informal definition. Definitions also often require elaboration to make it clear how they should be used and what they include. In those cases we include a comment. Also, definitions sometimes require an explanation as to how they came about to help users understand how they relate to alternative definitions. In those cases we include a curator note. 1/1
https://openalex.org/W2891188642	https://www.mdpi.com/2072-6643/10/10/1354/pdf?version=1537536786	English	null	Magnesium Intake and Sleep Disorder Symptoms: Findings from the Jiangsu Nutrition Study of Chinese Adults at Five-Year Follow-Up	Nutrients	2,018	cc-by	7,037	Received: 13 August 2018; Accepted: 18 September 2018; Published: 21 September 2018 Abstract: (1) Background: In clinical trials, dietary magnesium use can improve insomnia symptoms. However, little is known about the association between dietary magnesium consumption and sleep disorder symptoms including daytime falling asleep, sleepiness and snoring at the population level. (2) Methods: We used data from 1487 adults aged 20 and above attending the Jiangsu Nutrition Study. At baseline in 2002, dietary magnesium was assessed by 3-day weighed food records. At follow-up in 2007, sleep disorder symptoms, including daytime falling asleep, sleepiness and snoring at night, were gathered using a sleep questionnaire. (3) Results: The mean intake of magnesium was 332.5 mg/day. In total, 5.3%, 13.2% and 35.7% of the subjects reported daytime falling asleep, daytime sleepiness, and snoring during sleep, respectively. Compared with the lowest quartile of magnesium intake, the highest quartile was associated with decreased likelihood of falling asleep (odds ratio (OR) 0.12 (0.02, 0.57)) in women but not in men after adjusting for demographic, anthropometric, lifestyle factors, hypertension, and overall dietary patterns. No associations were found between dietary magnesium intake and daytime sleepiness nor night snoring in either gender. (4) Conclusions: Dietary magnesium intake may have long-term beneﬁts in reducing the likelihood of daytime falling asleep in women. Keywords: dietary magnesium; daytime falling asleep; Chinese adults Magnesium Intake and Sleep Disorder Symptoms: Findings from the Jiangsu Nutrition Study of Chinese Adults at Five-Year Follow-Up Yingting Cao 1,, Shiqi Zhen 2, Anne W. Taylor 1, Sarah Appleton 3 , Evan Atlantis 1,4 and Zumin Shi 1,2,5 1 School of Medicine, University of Adelaide, SAHMRI, L7, North Terrace, Adelaide, SA 5000, Australia; anne.taylor@adelaide.edu.au (A.W.T.); E.Atlantis@westernsydney.edu.au (E.A.); zumin.shi@adelaide.edu.au (Z.S.) 1 School of Medicine, University of Adelaide, SAHMRI, L7, North Terrace, Adelaide, SA 5000, Australia; anne.taylor@adelaide.edu.au (A.W.T.); E.Atlantis@westernsydney.edu.au (E.A.); zumin.shi@adelaide.edu.au (Z.S.) 2 Jiangsu Provincial Centre for Disease Control and Prevention, Nanjing 210000, China; cdczsq@163.com 3 The Health Observatory, University of Adelaide, Queen Elizabeth Hospital Campus, Woodville, SA 5000, Australia; sarah.appleton@adelaide.edu.au 4 School of Nursing and Midwifery, Western Sydney University, Sydney, NSW 2000, Australia 5 Human Nutrition Department, Qatar University, Doha 00000, Qatar Correspondence: yingting.cao1985@gmail.com 1 School of Medicine, University of Adelaide, SAHMRI, L7, North Terrace, Adelaide, SA 5000, Australia; anne.taylor@adelaide.edu.au (A.W.T.); E.Atlantis@westernsydney.edu.au (E.A.); zumin.shi@adelaide.edu.au (Z.S.) 2 Jiangsu Provincial Centre for Disease Control and Prevention, Nanjing 210000, China; cdczsq@163.co 3 The Health Observatory, University of Adelaide, Queen Elizabeth Hospital Campus, Woodville, SA 5000, Australia; sarah.appleton@adelaide.edu.au 4 School of Nursing and Midwifery, Western Sydney University, Sydney, NSW 2000, Australia 5 Human Nutrition Department, Qatar University, Doha 00000, Qatar 3 The Health Observatory, University of Adelaide, Queen Elizabeth Hospital Campus, Woodville, SA 5000, Australia; sarah.appleton@adelaide.edu.au 4 School of Nursing and Midwifery, Western Sydney University, Sydney, NSW 2000, Australia 5 Human Nutrition Department, Qatar University, Doha 00000, Qatar * Correspondence: yingting.cao1985@gmail.com nutrients nutrients nutrients www.mdpi.com/journal/nutrients 1. Introduction Sleep and wake cycle is regulated by the suprachiasmatic nucleus in the brain, which also coordinates the circadian rhythms in other tissues through the body [1]. Poor quantity and quality of sleep have been suggested to be adversely associated with a range of metabolic and cardiovascular systems, as well as quality of life [2,3]. Impaired sleep quantity and quality of sleep may be manifested as sleep disorder symptoms including daytime sleepiness, daytime falling asleep, and snoring at night. An increasing number of studies has found the associations between dietary macronutrients consumption and sleep outcomes. For example, carbohydrate with high glycemic index facilitated sleep onset, while low ﬁber, high saturated fat and sugar intake are associated with lighter and less restorative sleep with more arousals [4–6]. Sleep duration is positively associated with consumption Nutrients 2018, 10, 1354; doi:10.3390/nu10101354 www.mdpi.com/journal/nutrients 2 of 11 2 of 11 Nutrients 2018, 10, 1354 Nutrients 2018, 10, x FOR of iron and zinc, but negatively associated with vitamin K and B12 [7]. However, the research of micronutrients and sleep is limited, and the ﬁndings are inconsistent. p g y [ ] the research of micronutrients and sleep is limited, and the findings are inconsistent. Magnesium, a cofactor involved in more than 300 enzyme systems, regulates diverse of iron and zinc, but negatively associated with vitamin K and B12 [7]. However, the research of micronutrients and sleep is limited, and the ﬁndings are inconsistent. p g y [ ] the research of micronutrients and sleep is limited, and the findings are inconsistent. Magnesium, a cofactor involved in more than 300 enzyme systems, regulates diverse Magnesium, a cofactor involved in more than 300 enzyme systems, regulates diverse biochemical reactions in the body [8]. Recently, magnesium has also been found to regulate cellular timekeeping in both animal and plant cells [9], thus it is beneﬁcial to maintain the normal circadian rhythms and ensure a quality sleep in humans. Magnesium supplement use improves insomnia symptom among older people in a double-blind placebo-controlled clinical trial [10]. Low dietary magnesium intake was found to be signiﬁcantly associated with depression [11]. It is known that depression is associated with poor sleep. However, the studies on dietary magnesium and sleep are limited. biochemical reactions in the body [8]. 1. Introduction Recently, magnesium has also been found to regulate cellular timekeeping in both animal and plant cells [9], thus it is beneficial to maintain the normal circadian rhythms and ensure a quality sleep in humans. Magnesium supplement use improves insomnia symptom among older people in a double-blind placebo-controlled clinical trial [10]. Low dietary magnesium intake was found to be significantly associated with depression [11]. It is known that depression is associated with poor sleep. However, the studies on dietary magnesium and sleep are limited. The major dietary sources of magnesium include green leafy vegetables, whole grains, nuts, and legumes. These foods products are relatively inexpensive in China where average daily intakes are high compared to Western countries. We hypothesized that high intake of magnesium is beneﬁcial for the prevention of sleep disorder symptoms including daytime falling asleep, sleepiness and snoring. Therefore, the present study aimed to investigate the associations between baseline dietary magnesium intake and sleep symptoms at follow-up using ﬁve-year longitudinal data from the Jiangsu Nutrition Study. The major dietary sources of magnesium include green leafy vegetables, whole grains, nuts, and legumes. These foods products are relatively inexpensive in China where average daily intakes are high compared to Western countries. We hypothesized that high intake of magnesium is beneficial for the prevention of sleep disorder symptoms including daytime falling asleep, sleepiness and snoring. Therefore, the present study aimed to investigate the associations between baseline dietary magnesium intake and sleep symptoms at follow-up using five-year longitudinal data from the Jiangsu Nutrition Study. 2.6. Covariates The following covariates were included in the study: cigarette smoking (yes or no); alcohol consumption (never, 1–2/week, 3–4/week and daily); education (low, medium and high); occupation (manual or non-manual); sedentary activity (<1 h/day, 1–2 h/day; 2–3 h/day, and ≥3 h/day); residence (urban and rural). 2.1. Subjects 2.1. Subjects Data from the Jiangsu Nutrition Study cohort were used in the current study. Detailed methodology has been described previously [12]. In brief, in 2002, 2849 adults at least 20 years old living in two cities and six rural areas in Jiangsu Province took part in the Chinese National Nutrition and Health Survey. In 2007, an attempt to recontact all 1628 original participants was made and 1492 participated undertook the follow-up interview. For the current analysis, we included only those subjects with baseline (in 2002) magnesium intake and sleep records at follow-up (in 2007) (n = 1487) (Figure 1). The study was conducted according to the guidelines in the Declaration of Helsinki, and all procedures were approved by the Jiangsu Provincial Centre for Disease Control and Prevention. Informed consent for participation was obtained from each participant. Data from the Jiangsu Nutrition Study cohort were used in the current study. Detailed methodology has been described previously [12]. In brief, in 2002, 2849 adults at least 20 years old living in two cities and six rural areas in Jiangsu Province took part in the Chinese National Nutrition and Health Survey. In 2007, an attempt to recontact all 1628 original participants was made and 1492 participated undertook the follow-up interview. For the current analysis, we included only those subjects with baseline (in 2002) magnesium intake and sleep records at follow-up (in 2007) (n = 1487) (Figure 1). The study was conducted according to the guidelines in the Declaration of Helsinki, and all procedures were approved by the Jiangsu Provincial Centre for Disease Control and Prevention. Informed consent for participation was obtained from each participant. Figure 1. Sample description from Jiangsu Nutrition Study. Figure 1. Sample description from Jiangsu Nutrition Study. Figure 1. Sample description from Jiangsu Nutrition Study. Figure 1. Sample description from Jiangsu Nutrition Study. 3 of 11 Nutrients 2018, 10, 1354 2.2. Data Collection and Measurements Participants were interviewed at their homes by trained health workers using a pre-coded questionnaire. Interviews took about 2 h to complete and included questions on diet, sociodemographic information, medical history, cigarette smoking, physical activity, and other lifestyle factors. 2.5. Anthropometric Measurement and Other Variables In both 2002 and 2007 anthropometry was conducted using standard protocols and techniques. Body weight was measured in light indoor clothing without shoes to the nearest 0.1 kg. Height was measured without shoes to the nearest mm using a stadiometer. Body mass index (BMI) was calculated as weight in kilogram divided by the square of height in meters. BMI was subsequently categorized into four groups underweight (<18.5 kg/m2), normal weight (18.5–23.9 kg/m2), overweight (24–27.9 kg/m2) and obese (≥28 kg/m2) respectively according to the guideline for Chinese adults [15]. Waist circumference was measured to the nearest mm midway between the inferior margin of the last rib and the crest of the ilium, in the mid-auxiliary line in a horizontal plane. Blood pressure was measured twice with a mercury sphygmomanometer on the right upper arm of the subject, who was seated for 5 min before the measurement. The mean of these two measurements was used in the analyses. Hypertension was deﬁned as a systolic blood pressure above 140 mmHg and/or a diastolic blood pressure above 90 mmHg or the use of antihypertensive drugs. 2.4. Sleep Measurement At baseline in 2002, only sleep duration was collected but both sleep duration and self-reported daytime falling asleep were collected at follow-up in 2007. Self-reported sleep disorder symptoms were determined by relevant questions. Daytime falling asleep was determined by the question “Do you fall asleep involuntarily during the day? (1) no; (2) yes; (3) don’t know”; Daytime sleepiness was assessed by the question “Do you feel sleepy during the day? (1) never; (2) occasionally; (3) sometimes; (4) often and (5) frequently”; and snoring during the night was determined by the question ”Does your partner complain that you snore during the night? (1) never; (2) occasionally; (3) sometimes; (4) often; (5) frequently”. 2.3. Dietary Measurements Dietary data including magnesium intake were obtained by food weighing plus consecutive individual 3-day food records (including one weekend) that recorded all foods consumed by everyone in the household [13]. At the beginning and end of the 3-day survey, investigators weighed all the food stocked in the household. All purchases, home production, and processed snack foods were weighed and recorded each day. Nutrient intake was calculated using the Chinese Food Composition Table [14]. 3. Results In total, 1487 participants had complete dietary intake data and sleep data in both 2002 and 2007. There was a decrease in sleep duration (mean = 0.36 h/day) on average over ﬁve years in the study population with an increased sleep duration among short sleepers and a decreased sleep duration among long sleepers. The prevalence of self-reported sleep disorder symptoms at follow-up (year 2007) were: daytime sleepiness 13.2%, falling asleep 5.3% and snoring 35.7%. The median intake of dietary magnesium was 289 mg/day (interquartile range (IQR) 233.7–366.1 mg/day) at baseline (year 2002, mean intake 332.5 mg/day). Sex-speciﬁc characteristics by quartiles of magnesium intake at baseline are presented in Table 1. Dietary magnesium intake seemed to decrease as income increased in both sexes. Subjects in manual occupation and from rural region were more likely to consume high level of dietary magnesium. However, non-alcohol consumers were more likely to be low magnesium consumers in men, while those who had more frequent alcohol consumption were more likely to have more dietary magnesium. While in women, compared with the lowest quartile (Q1) of magnesium intake, those who had the highest quartile (Q4) were more likely to have lower education level. With the increase of magnesium intake, the intake of carbohydrate, protein and fat increased signiﬁcantly in both genders. For sleep disorder symptoms at follow-up, there was no difference across quartiles of magnesium intake at baseline except sleep duration and falling asleep in women. Those in Q1 of magnesium intake were more likely to have short sleep duration (<7 h/day) and more likely to fall asleep during the day compared with those who had Q4 of magnesium intake (Table 2). Across quartiles of magnesium intake, the prevalence of falling sleep was 4.9%, 8.6%, 5.0% and 3.7% in men (p = 0.240); 8.7%, 4.4%, 5.3% and 1.9% in women (p = 0.018), respectively. The prevalence of snoring was similar across quartiles of magnesium in men and women, respectively. After adjusting for age and energy intake, magnesium intake was inversely associated with falling asleep in women but not in men. Across quartiles of magnesium intake, the odds ratio (OR) for falling asleep were 1.00, 0.34 (95% CI 0.14–0.87), 0.42 (95% CI 0.16–1.09) and 0.11 (95% CI 0.02–0.57), respectively in women; 1.00, 1.32 (0.69, 2.50), 1.39 (0.68, 2.83), 1.39 (0.68, 2.83), and 1.62 (0.69, 3.80) respectively in men in the ﬁnal model (Table 3). 2.7. Statistical Analysis Descriptive data are presented as the mean (SD) (95% (conﬁdence interval (CI)) or as percentage. Chi-square tests were used to compare the differences between categorical variables, and (Analysis of variance) ANOVA was performed to compare differences in continuous variables between groups. Magnesium intake (mg) was recoded into sex-speciﬁc quartiles. A sex-speciﬁc association between baseline quartiles of magnesium intake and self-reported sleep disorder symptoms at follow-up was assessed using multivariable logistic/Poisson regression models. A set of models were used: 4 of 11 Nutrients 2018, 10, 1354 model 1 adjusted for age, energy intake; model 2 further adjusted for income, education, region, smoking, alcohol consumption, and sedentary activity; model 3 further adjusted for BMI, hypertension, and diabetes; model 4 further adjusted for four dietary patterns. These four dietary patterns are: Factor 1 (‘macho’) included various kinds of animal foods and alcohol; Factor 2 (the ‘traditional’ pattern) loaded heavily on rice, fresh vegetables and inversely on wheat ﬂour; Factor 3 (‘sweet tooth’) contained cake, milk, yoghurt and drinks; and, Factor 4 (‘vegetable rich’ pattern) was characterized whole grains, fruits, root vegetables, fresh and pickled vegetables, milk, eggs and ﬁsh. The four factors explained 28.5% of the variance in intake (published earlier [16]). In sensitivity analyses, we further adjusted for anemia, and baseline sleep duration. Statistical signiﬁcance was considered when p < 0.05. All statistical analyses were performed with STATA 14 (Stata Corporation, College Station, TX, USA). 3. Results There were no associations between quartiles of magnesium consumption and other sleep related symptoms including snoring and daytime sleepiness in either men or women. Sensitivity analyses for further adjustment for anemia and sleep duration at baseline did not change the results (data not shown). Compared to model 1, the subjects lost in the ﬁnal model (model 4) had no differences in age, and BMI, but with a shorter sleep duration at baseline (p < 0.05, data not shown). 5 of 11 Nutrients 2018, 10, 1354 Table 1. Sex-speciﬁc characteristics of subjects by quartiles of magnesium consumption at baseline (2002) (n = 1487) 1. 3. Results Quartiles of Magnesium Consumption (mg/day) Men Women Factors Q1 (n = 163) Q2 (n = 163) Q3 (n = 163) Q4 (n = 162) p-Value Q1 (n = 209) Q2 (n = 209) Q3 (n = 209) Q4 (n = 209) p-Value Nutrients consumption * Magnesium consumption (mg/day) 219 (30.3) 288 (16.4) 350 (20.6) 505 (122) <0.001 186 (27.7) 244 (13.1) 301 (21.4) 462 (132) <0.001 (124–259) (259–315) (315–392) (392–1015) (79–222) (222–267) (267–339) (339–1157) Carbohydrate (g/day) 259 (62.1) 317 (63.0) 353 (86.3) 427 (101) <0.001 219 (56.5) 277 (60.7) 318 (65.9) 392 (102) <0.001 Protein (g/day) 61.3 (13.9) 73.5 (14.9) 84.3 (15.6) 101.0 (23.5) <0.001 51.5 (14.5) 60.9 (13.5) 69.1 (11.2) 85.5 (23.7) <0.001 Fat (g/day) 73.5 (26.7) 84.7 (32.1) 97.3 (36.4) 104.6 (43.6) <0.001 63.1 (23.3) 70.6 (26.9) 74.9 (28.1) 88.6 (42.5) <0.001 Energy consumption (kcal) 1968 (408) 2389 (437) 2700 (469) 3195 (599) <0.001 1639 (367) 1976 (342) 2211 (344) 2707 (596) <0.001 Age (years) 51.6 (14.1) 49.6 (13.7) 48.4 (13.0) 48.8 (13.0) 0.13 50.2 (14.8) 48.3 (13.9) 47.0 (12.1) 46.9 (12.2) 0.042 BMI (kg/m2) 23.8 (2.9) 23.6 (3.6) 23.1 (3.0) 22.7 (3.1) 0.013 23.5 (3.6) 23.3 (3.9) 23.9 (3.6) 23.7 (3.3) 0.29 Income (%) <0.001 <0.001 Low 20.4 15.5 18.9 37.9 14.9 21.7 20.3 46.6 Medium 32.7 39.8 32.1 27.3 38.0 38.2 38.6 24.8 High 46.9 44.7 49.1 34.8 47.1 40.1 41.1 28.6 Education (%) 0.19 0.004 Primary school 44.2 35.6 33.7 45.1 55.0 52.6 61.7 71.6 Junior middle school 38.7 47.2 45.4 40.1 31.1 36.4 27.8 23.1 Senior middle school 11.7 14.7 17.8 13.0 12.9 10.0 10.5 4.8 University 5.5 2.5 3.1 1.9 1.0 1.0 0.0 0.5 Smoking (%) 58.9 61.3 55.8 64.8 0.40 2.9 3.8 1.0 4.3 0.19 Alcohol consumption (%) 0.002 0.17 Never 62.0 50.3 43.8 40.4 95.7 97.1 94.7 91.9 1–2/week 11.7 14.1 8.0 12.4 1.4 1.9 1.4 1.9 3–4/week 7.4 7.4 8.6 8.7 1.0 1.0 1.9 1.4 Daily 19.0 28.2 39.5 38.5 1.9 0.0 1.9 4.8 Sedentary activity (%) 0.057 0.23 <1 h 11.0 6.7 8.6 16.7 14.4 19.6 19.1 23.0 1–2 h 23.9 30.1 24.5 32.1 27.8 30.1 33.5 34.0 2–3 h/day 48.5 47.9 47.2 39.5 43.1 39.7 36.8 33.5 ≥3 h/day 16.6 15.3 19.6 11.7 14.8 10.5 10.5 9.6 Manual occupation (%) 34.0 50.3 52.8 63.0 <0.001 28.2 50.7 54.1 66.0 <0.001 Region (%) <0.001 <0.001 Urban 36.2 18.4 12.3 9.3 34.4 17.2 9.1 8.1 Rural 63.8 81.6 87.7 90.7 65.6 82.8 90.9 91.9 1 Participants presented were those with magnesium measurements at baseline (n = 2002) and participated in the follow-up (2007). nge in each quartile; BMI, body mass index, SD, standard deviation. g ( ) p p p ( ) D) present magnesium consumption range in each quartile; BMI, body mass index, SD, standard deviation. ants presented were those with magnesium measurements at baseline (n = 2002) and participated in the follow-up (200 fter mean (SD) present magnesium consumption range in each quartile; BMI, body mass index, SD, standard deviation. nts at baseline (n = 2002) and participated in the follow-up (2007). Data are presented as mean (SD) for such values. * ed were those with magnesium measurements at baseline (n = 2002) and participated in the follow-up (2007). Data are D) i i i h il BMI b d i d SD d d d i i 3. Results Data are presented as mean (SD) for such values. * brackets after mean (SD) present magnesium consumption range in each quartile; BMI, body mass index, SD, standard deviation. Table 1. Sex-speciﬁc characteristics of subjects by quartiles of magnesium consumption at baseline (2002) (n = 1487) 1. 6 of 11 Nutrients 2018, 10, 1354 Table 2. Sex-speciﬁc sleep outcomes of subjects by quartiles of magnesium consumption at baseline (2002) (n = 1487) 1. Quartiles of Magnesium Consumption (mg/day) Men Women Sleep variables Q1 (n = 163) Q2 (n = 163) Q3 (n = 163) Q4 (n = 162) p-Value Q1 (n = 209) Q2 (n = 209) Q3 (n = 209) Q4 (n = 209) p-Value Sleep duration (%), (baseline) 7–8 h/day 64.2 77.0 68.3 64.2 0.19 71.2 69.9 66.7 67.0 0.11 <7 h/day 16.7 11.2 13.0 16.0 13.5 13.1 9.7 8.6 ≥9 h/day 19.1 11.8 18.6 19.8 15.4 17.0 23.7 24.4 Sleep duration (%), (follow-up) 0.27 0.039 7–8 h/day 74.2 65.4 70.6 73.5 65.1 63.5 65.6 72.2 <7 h/day 17.8 21.0 16.0 19.1 25.4 20.7 18.7 13.4 9–13 h/day 8.0 13.6 13.5 7.4 9.6 15.9 15.8 14.4 Falling sleep (%) 4.9 8.6 5.0 3.7 0.24 8.7 4.4 5.3 1.9 0.018 Daytime sleepiness (%) 12.3 14.7 14.3 13.6 0.92 15.9 13.0 9.6 12.9 0.29 Snore (%) 47.5 51.5 49.7 46.3 0.79 26.8 26.6 26.3 22.5 0.71 1 Participants presented were those with magnesium measurements at baseline (n = 2002) and participated in the follow-up (2007). Data are presented as mean (SD) for such values. Table 2. Sex-speciﬁc sleep outcomes of subjects by quartiles of magnesium consumption a 7 of 11 Nutrients 2018, 10, 1354 Table 3. The odds ratios (OR (95% CI)) of self-reported sleep disorder symptoms at follow-up (2007) according to magnesium consumption (mg) quartiles at baseline (2002) (n = 1487) 1. 3. Results Magnesium Consumption (mg/day) Sleep disorder symptoms Men Falling asleep n Q1 (n = 163) Q2 (n = 163) Q3 (n = 163) Q4 (n = 162) Model 1 645 1.00 1.89 (0.73, 4.86) 1.07 (0.35, 3.34) 0.80 (0.20, 3.22) Model 2 635 1.00 2.32 (0.86, 6.27) 1.32 (0.40, 4.34) 1.38 (0.33, 5.73) Model 3 607 1.00 2.53 (0.93, 6.90) 1.32 (0.40, 4.34) 1.44 (0.34, 6.04) Model 4 606 1.00 2.51 (0.91, 6.91) 1.20 (0.36, 4.06) 1.36 (0.31, 5.98) Women Q1 (n = 209) Q2 (n = 209) Q3 (n = 209) Q4 (n = 209) Model 1 831 1.00 0.40 (0.17, 0.94) * 0.43 (0.18, 1.05) 0.10 (0.02, 0.43) ** Model 2 795 1.00 0.35 (0.14, 0.87) * 0.41 (0.16, 1.04) 0.10 (0.02, 0.47) ** Model 3 795 1.00 0.32 (0.13, 0.81) * 0.37 (0.14, 0.97) * 0.09 (0.02, 0.43) ** Model 4 792 1.00 0.34 (0.14, 0.87) * 0.42 (0.16, 1.09) 0.11 (0.02, 0.57) ** Men Daytime sleepiness n Q1 (n = 163) Q2 (n = 163) Q3 (n = 163) Q4 (n = 162) Model 1 649 1.00 1.30 (0.70, 2.42) 1.34 (0.68, 2.63) 1.36 (0.61, 3.03) Model 2 639 1.00 1.33 (0.71, 2.49) 1.38 (0.69, 2.78) 1.54 (0.68, 3.49) Model 3 638 1.00 1.33 (0.70, 2.50) 1.41 (0.70, 2.83) 1.64 (0.72, 3.70) Model 4 637 1.00 1.32 (0.69, 2.50) 1.39 (0.68, 2.83) 1.62 (0.69, 3.80) Women Q1 (n = 209) Q2 (n = 209) Q3 (n = 209) Q4 (n = 209) Model 1 834 1.00 0.84 (0.50, 1.43) 0.64 (0.34, 1.17) 0.87 (0.43, 1.73) Model 2 825 1.00 0.83 (0.49, 1.42) 0.63 (0.34, 1.17) 0.98 (0.47, 2.02) Model 3 825 1.00 0.83 (0.48, 1.42) 0.62 (0.33, 1.16) 0.99 (0.48, 2.05) Model 4 821 1.00 0.84 (0.49, 1.44) 0.61 (0.33, 1.14) 1.00 (0.47, 2.08) Men Snoring n Q1 (n = 163) Q2 (n = 163) Q3 (n = 163) Q4 (n = 162) Model 1 645 1.00 1.12 (0.81, 1.55) 1.11 (0.78, 1.58) 1.06 (0.69, 1.61) Model 2 635 1.00 1.06 (0.76, 1.47) 1.07 (0.74, 1.55) 1.11 (0.72, 1.71) Model 3 607 1.00 1.05 (0.75, 1.47) 1.10 (0.76, 1.59) 1.15 (0.74, 1.80) Model 4 607 1.00 1.04 (0.75, 1.46) 1.08 (0.74, 1.56) 1.10 (0.70, 1.75) Women Q1 (n = 209) Q2 (n = 209) Q3 (n = 209) Q4 (n = 209) Model 1 834 1.00 0.95 (0.65, 1.40) 0.93 (0.61, 1.40) 0.71 (0.42, 1.20) Model 2 825 1.00 0.97 (0.65, 1.43) 0.94 (0.62, 1.44) 0.80 (0.46, 1.37) Model 3 825 1.00 1.00 (0.68, 1.49) 0.91 (0.59, 1.39) 0.85 (0.50, 1.46) Model 4 821 1.00 1.01 (0.68, 1.49) 0.91 (0.60, 1.39) 0.89 (0.52, 1.55) 1 Participants included in the model had magnesium consumption at baseline (2002) and participated at follow-up (2007) (n = 1487, men (n = 651)). 3. Results Q1–Q4 stands for quartiles of isoﬂavone consumption for each sex. Model 1 adjusted for age and energy consumption; Model 2 further adjusted for income, education, rural region, smoking, alcohol consumption and sedentary activity; Model 3 further adjusted for BMI, hypertension, and diabetes. Model 4 further adjusted for dietary patterns; Four dietary patterns (previously published) are: Factor 1 (‘macho’) included various kinds of animal foods and alcohol; Factor 2 (the ‘traditional’ pattern) loaded heavily on rice, fresh vegetables and inversely on wheat ﬂour; Factor 3 (‘sweet tooth’) contained cake, milk, yoghurt and drinks; and, Factor 4 (‘vegetable rich’ pattern) was characterized whole grains, fruits, root vegetables, fresh and pickled vegetables, milk, eggs and ﬁsh. The four factors explained 28.5% of the variance in intake (published earlier [16]). * p < 0.05, ** p < 0.01. 1 Participants included in the model had magnesium consumption at baseline (2002) and participated at follow-up (2007) (n = 1487, men (n = 651)). Q1–Q4 stands for quartiles of isoﬂavone consumption for each sex. Model 1 adjusted for age and energy consumption; Model 2 further adjusted for income, education, rural region, smoking, alcohol consumption and sedentary activity; Model 3 further adjusted for BMI, hypertension, and diabetes. Model 4 further adjusted for dietary patterns; Four dietary patterns (previously published) are: Factor 1 (‘macho’) included various kinds of animal foods and alcohol; Factor 2 (the ‘traditional’ pattern) loaded heavily on rice, fresh vegetables and inversely on wheat ﬂour; Factor 3 (‘sweet tooth’) contained cake, milk, yoghurt and drinks; and, Factor 4 (‘vegetable rich’ pattern) was characterized whole grains, fruits, root vegetables, fresh and pickled vegetables, milk, eggs and ﬁsh. The four factors explained 28.5% of the variance in intake (published earlier [16]). * p < 0.05, ** p < 0.01. 4. Discussion This may indicate a potential beneﬁt of increased dietary magnesium consumption on poor quality sleep including both short and long sleep. We also found that the number of short sleepers seemed to decrease across the quartile of dietary magnesium consumption at follow-up, particularly in women. A shortened sleep at night may lead to falling asleep during the day. However, when we further adjusted for short sleep, the association between dietary magnesium consumption and falling asleep in women remained, and even seemed to be stronger. This may suggest that the association is independent of short sleep. The inverse association between magnesium consumption and falling asleep is biologically plausible. In animal studies, researchers have demonstrated that magnesium deﬁciency could affect normal sleep-wake rhythm by increasing periods of wakefulness and reducing slow wave sleep in rats, and such effect was recovered when reintroducing magnesium in their diet [22]. Magnesium deﬁciency has been suggested to related to muscle cramps, which can contribute to a poor sleep and consequent sleepiness the day after. Having magnesium may reduce night time wakefulness and maintain a normal sleep structure, which may explain a beneﬁcial effect of preventing falling asleep during the day found in our study. In the study mentioned above that found a sleep improving effect of magnesium supplements in the elderly, the researchers also showed that magnesium supplementation increased the serum levels of melatonin and renin while reducing cortisol levels [10]. This may partly explain the beneﬁcial effect of magnesium consumption on sleep quality that is via increasing the secretion of melatonin and lowering the level of cortisol. In addition, magnesium consumption has been suggested to be inversely associated with depression in human studies including cross-sectional [11] and longitudinal [23] designs. In animals, removal of magnesium from the diet of mice appears to result in anxiety and depressive-related behavior [24]. An aid of anti-depressant effect of magnesium also guarantees a quality sleep, which reduces poor sleep related sleep disorder symptoms. The association between magnesium consumption and falling asleep was limited to women. This gender difference is intriguing. Increasing evidence has supported the solid sex differences in terms of biological and physiological differences between men and women (e.g., distinct hormonal and physical changes in women’s life span including puberty, pregnancy, and menopause), which may underlie the differences in sleep physiology and sleep disorders between sexes [25]. 4. Discussion Dietary magnesium consumption was found to be associated with decreased risk of falling asleep in women but not in men in the present study. No associations were found between dietary magnesium consumption and other sleep related symptoms in either sex. Dietary magnesium consumption was found to be associated with decreased risk of falling asleep in women but not in men in the present study. No associations were found between dietary magnesium consumption and other sleep related symptoms in either sex. Dietary magnesium consumption in the Chinese population has been studied previously. In the Shanghai Women’s Health Study, the median consumption of dietary magnesium was 267 mg/day, which is similar with our result (289 mg/day) in both sexes [17]. However, both values are below the recommended daily amount by China Nutrition Society (330 mg/day) [18]. In the study of 8 of 11 Nutrients 2018, 10, 1354 comparing nutrients consumption between Chinese and Italians, Japanese and Americans, mean dietary magnesium consumption in Chinese subjects was higher than the Japanese in the younger group and lower than the Americans adults and Italian children and adolescents [19]. The authors claimed that the Chinese diet has been shifting away from the traditional diet towards a diet that contains high-fat, low-carbohydrate, ﬁber and mineral nutrients. It is interesting that the magnesium consumption was inversely associated with income and positively associated with manual occupation and rural regions. This is consistent with the high dietary magnesium consumption in rural areas found in a middle-aged Chinese population (mean daily consumption 371.5 mg/day vs 332.5 mg/day in our study with mixed population) [20]. The possible explanation may be the easy access to green leafy vegetables in rural areas and relatively low cost of such foods compared to urban areas. p Our ﬁnding of an inverse association between magnesium consumption and daytime falling asleep in women was consistent with other population studies. For example, magnesium consumption has been suggested to improve insomnia in a double-blind placebo-controlled clinical trial in elderly [10]. Improved sleep included subjective sleep efﬁciency, sleep time, sleep onset latency, and early morning awakening, and increased serum melatonin and decreased serum cortisol concentration in experimental group (500 mg supplemental magnesium daily) compared with the placebo group. Data from National Health and Nutrition Examination Survey (NHANES)suggested a U-shape association between dietary magnesium consumption and sleep duration [21]. 4. Discussion In addition, sex differences in terms of nutrition and supplements have been suggested, in which women do not immediately response to a normal increase in dietary carbohydrate when expressed as a percentage of total energy consumption compared with men [26]. Indeed, it has been suggested that dietary consumptions are different in men and women [27], and a Mediterranean dietary intervention had 9 of 11 Nutrients 2018, 10, 1354 more pronounced beneﬁts in the long term in men than in women [28]. This evidence provides foundations for future gender speciﬁc dietary interventions. Depression has been discussed above as a potential mechanism of understanding the association between magnesium consumption and sleep disorder symptoms, but it remains to be explored whether gender difference in depression can explain the different effects of magnesium consumption on sleep remains to be explored. Earlier studies have reported that women had twice the lifetime risk of developing depression compared with men [29]. Another earlier ﬁnding demonstrated that women exhibited a higher prevalence of somatic depression (e.g., body aches, fatigue, and sleep disturbance) than men but not a higher prevalence of pure depression [30]. In a Chinese cross-sectional study that investigated the prevalence and correlates with sleep disturbances and depression in adolescents [31], a higher likelihood of sleep disturbance was found in girls, which was associated with depressive symptoms. These studies indicate a female are inclination for depression, which is particularly associated with somatic symptoms including sleep disturbances. Magnesium supplement use has been shown to be effectively reduce depressive symptoms in a randomized clinical trial [32]. As magnesium may have a potential anti-depressant effect, magnesium consumption in this particular depressed (including sleep problems) female group may be more beneﬁcial compared with men. Such difference may potentially explain the gender difference in the relationship of magnesium consumption with falling asleep found in our study. However, we do not have depression data, and further exploration of whether depression is a confounder or a mediator in terms of the association between magnesium consumption and daytime falling asleep cannot be conducted. In addition, it is noticed that in the study sample, we have previously shown that the magnesium consumption is inversely associated with anemia. The prevalence of anemia in women in our study population is almost double that of men [33], and magnesium consumption was also found to be inversely associated the risk of anemia in our study population. 4. Discussion Anemia has been shown to be associated with poor sleep quality [34], and the possible mechanism was partly due to the dopaminergic function of iron, which plays an important role in sleep regulation. Whether the gender difference found in our study is related with anemia which women are inclined to have is unknown. Further adjustment for anemia did not change the association between magnesium consumption and sleep disorder symptoms (data not shown). The strengths of this study include the large general population sample and consecutive three-day weighed food diaries that provide robust food consumption data. Several limitations though need to be acknowledged: (1) supplemental magnesium was not included, mainly because the difﬁculties of accurate assessment due to variety of brands, and different concentration etc. The number of participants using supplements is very small at baseline; (2) detailed dietary consumption using the same 3-day food record method at follow-up is not available; (3) sleep disorder symptoms were self-reported, which may have bias; however, objective measures such as overnight sleep studies are not feasible to implement in large sample sizes; (4) self-reported sleep disorder symptoms were not collected at baseline. Consequently, we cannot make a conclusion on a causal relationship due to study design. References 1. Moore, R.Y. Suprachiasmatic nucleus in sleep-wake regulation. Sleep Med. 2007, 8 (Suppl. 3), 27–33. [CrossRef] [PubMed] 1. Moore, R.Y. Suprachiasmatic nucleus in sleep-wake regulation. Sleep Med. 2007, 8 (Suppl. 3), 27–33. [CrossRef] [PubMed] 2. Cappuccio, F.P.; D’elia, L.; Strazzullo, P.; Miller, M.A. Quantity and quality of sleep and incidence of type 2 diabetes: A systematic review and meta-analysis. Diabetes Care 2010, 33, 414–420. [CrossRef] [PubMed] 2. Cappuccio, F.P.; D’elia, L.; Strazzullo, P.; Miller, M.A. Quantity and quality of sleep and incidence of type 2 diabetes: A systematic review and meta-analysis. Diabetes Care 2010, 33, 414–420. [CrossRef] [PubMed] 3. Reimer, M.A.; Flemons, W.W. Quality of life in sleep disorders. Sleep Med. Rev. 2003, 7, 335–349. [CrossRef] [PubMed] 3. Reimer, M.A.; Flemons, W.W. Quality of life in sleep disorders. Sleep Med. Rev. 2003, 7, 335–349. [CrossRef] [PubMed] 4. St-Onge, M.P.; Roberts, A.; Shechter, A.; Choudhury, A.R. Fiber and Saturated Fat Are Associated with Sleep Arousals and Slow Wave Sleep. J. Clin. Sleep Med. 2016, 12, 19–24. [CrossRef] [PubMed] 4. St-Onge, M.P.; Roberts, A.; Shechter, A.; Choudhury, A.R. Fiber and Saturated Fat Are Associated with Sleep Arousals and Slow Wave Sleep. J. Clin. Sleep Med. 2016, 12, 19–24. [CrossRef] [PubMed] 5. Cao, Y.; Wittert, G.; Taylor, A.W.; Adams, R.; Shi, Z. Associations between Macronutrient Intake and Obstructive Sleep Apnoea as Well as Self-Reported Sleep Symptoms: Results from a Cohort of Community Dwelling Australian Men. Nutrients 2016, 8, 207. [CrossRef] [PubMed] 5. Cao, Y.; Wittert, G.; Taylor, A.W.; Adams, R.; Shi, Z. Associations between Macronutrient Intake and Obstructive Sleep Apnoea as Well as Self-Reported Sleep Symptoms: Results from a Cohort of Community Dwelling Australian Men. Nutrients 2016, 8, 207. [CrossRef] [PubMed] g 6. Afaghi, A.; O’connor, H.; Chow, C.M. High-glycemic-index carbohydrate meals shorten sleep onset. Am. J. Clin. Nutr. 2007, 85, 426–430. [CrossRef] [PubMed] 6. Afaghi, A.; O’connor, H.; Chow, C.M. High-glycemic-index carbohydrate meals shorten sleep onset. Am. J. Clin. Nutr. 2007, 85, 426–430. [CrossRef] [PubMed] 7. Ji, X. The relationship between micronutrient status and sleep patterns: A systematic review. Public Health Nutr. 2017, 20, 687–701. [CrossRef] [PubMed] 7. Ji, X. The relationship between micronutrient status and sleep patterns: A systematic review. Public Health Nutr. 2017, 20, 687–701. [CrossRef] [PubMed] . Wacker, W.E.; Parisi, A.F. Magnesium metabolism. N. Engl. J. Med. 1968, 278, 658–663. [CrossRef] [PubM 9. References Feeney, K.A.; Hansen, L.L.; Putker, M.; Olivares-Yañez, C.; Day, J.; Eades, L.J.; Larrondo, L.F.; Hoyle, N.P.; O’Neill, J.S.; van Ooijen, G. Daily magnesium ﬂuxes regulate cellular timekeeping and energy balance. Nature 2016, 532, 375–379. [CrossRef] [PubMed] 10. Abbasi, B.; Kimiagar, M.; Sadeghniiat, K.; Shirazi, M.M.; Hedayati, M.; Rashidkhani, B. The effect of magnesium supplementation on primary insomnia in elderly: A double-blind placebo-controlled clinical trial. J. Res. Med. Sci. 2012, 17, 1161–1169. [PubMed] 11. Tarleton, E.K.; Littenberg, B. Magnesium intake and depression in adults. J. Am. Board Fam. Med. 2015, 28, 249–256. [CrossRef] [PubMed] 12. Shi, Z.; Luscombe-Marsh, N.D.; Wittert, G.A.; Yuan, B.; Dai, Y.; Pan, X.; Taylor, A.W. Monosodium glutamate is not associated with obesity or a greater prevalence of weight gain over 5 years: Findings from the Jiangsu Nutrition Study of Chinese adults. Br. J. Nutr. 2010, 104, 457–463. [CrossRef] [PubMed] 3. Shi, Z.; McEvoy, M.; Luu, J.; Attia, J. Dietary fat and sleep duration in Chinese men and women. Int. J. O 2008, 32, 1835–1840. [CrossRef] [PubMed] 14. Yang, Y.; He, M.; Pan, X. China Food Composition Table; China Institute of Nutrition and Food Safety, China CDC: Beijing, China, 2002. 15. Chen, C.M.; Lu, F.C. Department of Disease Control Ministry of Health, The guidelines for prevention and control of overweight and obesity in Chinese adults. Biomed. Environ. Sci. 2004, 17, 1–36. [PubMed] 16. Shi, Z.; Zhen, S.; Wittert, G.A.; Yuan, B.; Zuo, H.; Taylor, A.W. Inadequate riboﬂavin intake and anemia risk in a Chinese population: Five-year follow up of the Jiangsu Nutrition Study. PLoS ONE 2014, 9, e88862. [CrossRef] [PubMed] 17. Villegas, R.; Gao, Y.T.; Dai, Q.; Yang, G.; Cai, H.; Li, H.; Zheng, W.; Shu, X.O. Dietary calcium and magnesium intakes and the risk of type 2 diabetes: The Shanghai Women’s Health Study. Am. J. Clin. Nutr. 2009, 89, 1059–1067. [CrossRef] [PubMed] 18. China Light Industry Publishing House. China Nutrition Society. Chinese Dietary Reference Intakes; China Light Industry Publishing House: Beijing, China, 2000. 19. Zhang, R.; Wang, Z.; Fei, Y.; Zhou, B.; Zheng, S.; Wang, L.; Huang, L.; Jiang, S.; Liu, Z.; Jiang, J.; et al. The Difference in Nutrient Intakes between Chinese and Mediterranean, Japanese and American Diets. Nutrients 2015, 7, 4661–4688. [CrossRef] [PubMed] 20. Li, Y.; Ma, A.; Sun, Y.; Liang, H.; Wang, Q.; Yi, X.; Han, X. Magnesium status and dietary intake of mid-old people in a rural area of China. Magnes Res. Nutrients 2018, 10, 1354 Nutrients 2018, 10, 1354 Acknowledgments: The authors are grateful to the participating regional Centres for Disease Control and Prevention in Jiangsu Province, including the Nanjing, Xuzhou, Jiangyin, Taicang, Suining, Jurong, Sihong and Haimen centres, for the support in data collection. Conﬂicts of Interest: The authors declared that there are no conﬂicts of interest. 5. Conclusions In conclusion, dietary magnesium consumption was inversely associated with falling asleep during the day in women but not in men. No associations were found between dietary magnesium consumption and daytime sleepiness or snoring at night. A sex difference in sleep management may need to be considered in future research and practice. Author Contributions: Y.C. carried out the data analyses and wrote the manuscript; A.W.T., S.Z., S.A., E.A. and Z.S. critically reviewed the manuscript and approved the ﬁnal version. Funding: This research received no speciﬁc grant from any funding agency, commercial or not-for-proﬁt sec 10 of 11 References 2009, 22, 66–71. [PubMed] 11 of 11 Nutrients 2018, 10, 1354 21. Grandner, M.A.; Jackson, N.; Gerstner, J.R.; Knutson, K.L. Dietary nutrients associated with short and long sleep duration. Data from a nationally representative sample. Appetite 2013, 64, 71–80. [CrossRef] [PubMed] 22. Depoortere, H.; Francon, D.; Llopis, J. Effects of a magnesium-deﬁcient diet on sleep organization in rats. Neuropsychobiology 1993, 27, 237–245. [CrossRef] [PubMed] 23. Yary, T.; Lehto, S.M.; Tolmunen, T.; Tuomainen, T.P.; Kauhanen, J.; Voutilainen, S.; Ruusunen, A. Dietary magnesium intake and the incidence of depression: A 20-year follow-up study. J. Affect. Disord. 2016, 193, 94–98. [CrossRef] [PubMed] 24. Singewald, N.; Sinner, C.; Hetzenauer, A.; Sartori, S.B.; Murck, H. Magnesium-deﬁcient diet alters depression- and anxiety-related behavior in mice—Inﬂuence of desipramine and Hypericum perforatum extract. Neuropharmacology 2004, 47, 1189–1197. [CrossRef] [PubMed] 25. Mallampalli, M.P.; Carter, C.L. Exploring sex and gender differences in sleep health: A Society for Women’s Health Research Report. J. Womens Health (Larchmt) 2014, 23, 553–562. [CrossRef] [PubMed] 26. Tarnopolsky, M.A. Gender differences in metabolism; nutrition and supplements. J. Sci. Med. Sport 2000, 3, 287–298. [CrossRef] 27. Leblanc, V.; Bégin, C.; Corneau, L.; Dodin, S.; Lemieux, S. Gender differences in dietary intakes: What is the contribution of motivational variables? J. Hum. Nutr. Diet. 2015, 28, 37–46. [CrossRef] [PubMed] 28. Leblanc, V.; Bégin, C.; Hudon, A.M.; Royer, M.M.; Corneau, L.; Dodin, S.; Lemieux, S. Gender differences in the long-term effects of a nutritional intervention program promoting the Mediterranean diet: Changes in dietary intakes, eating behaviors, anthropometric and metabolic variables. Nutr. J. 2014, 13, 107. [CrossRef] [PubMed] 29. Kessler, R.C.; McGonagle, K.A.; Swartz, M.; Blazer, D.G.; Nelson, C.B. Sex and depression in the National Comorbidity Survey. I: Lifetime prevalence, chronicity and recurrence. J. Affect. Disord. 1993, 29, 85–96. [CrossRef] 30. Silverstein, B. Gender difference in the prevalence of clinical depression: The role played by depression associated with somatic symptoms. Am. J. Psychiatry 1999, 156, 480–482. [PubMed] 31. Guo, L.; Deng, J.; He, Y.; Deng, X.; Huang, J.; Huang, G.; Gao, X.; Lu, C. Prevalence and correlates of sleep disturbance and depressive symptoms among Chinese adolescents: A cross-sectional survey study. BMJ Open 2014, 4, e005517. [CrossRef] [PubMed] 32. Tarleton, E.K.; Littenberg, B.; MacLean, C.D.; Kennedy, A.G.; Daley, C. Role of magnesium supplementation in the treatment of depression: A randomized clinical trial. PLoS ONE 2017, 12, e0180067. [CrossRef] [PubMed] 33. Shi, Z.; Hu, X.; He, K.; Yuan, B.; Garg, M. References Joint association of magnesium and iron intake with anemia among Chinese adults. Nutrition 2008, 24, 977–984. [CrossRef] [PubMed] 34. Semiz, M.; Uslu, A.; Korkmaz, S.; Demir, S.; Parlak, I.; Sencan, M.; Aydin, B.; Uncu, T. Assessment of subjective sleep quality in iron deﬁciency anaemia. Afr. Health Sci. 2015, 15, 621–627. [CrossRef] [PubMed] © 2018 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/).
https://openalex.org/W2342846911	https://www.nature.com/articles/srep20169.pdf	English	null	Oral glutathione supplementation drastically reduces Helicobacter-induced gastric pathologies	Scientific reports	2,016	cc-by	10,330	Oral glutathione supplementation drastically reduces Helicobacter- induced gastric pathologies Ellen De Bruyne1, Richard Ducatelle1, Dennis Foss2, Margaret Sanchez2, Myrthe Joosten1, Guangzhi Zhang1, Annemieke Smet1, Frank Pasmans1, Freddy Haesebrouck1,* & Bram Flahou1,* received: 24 March 2015 accepted: 23 December 2015 Published: 02 February 2016 Ellen De Bruyne1, Richard Ducatelle1, Dennis Foss2, Margaret Sanchez2, Myrthe Joosten1, Guangzhi Zhang1, Annemieke Smet1, Frank Pasmans1, Freddy Haesebrouck1,* & Bram Flahou1,* Helicobacter (H.) suis causes gastric pathologies in both pigs and humans. Very little is known on the metabolism of this bacterium and its impact on the host. In this study, we have revealed the importance of the glutamate-generating metabolism, as shown by a complete depletion of glutamine (Gln) in the medium during H. suis culture. Besides Gln, H. suis can also convert glutathione (GSH) to glutamate, and both reactions are catalyzed by the H. suis γ-glutamyltranspeptidase (GGT). Both for H. pylori and H. suis, it has been hypothesized that the degradation of Gln and GSH may lead to a deficiency for the host, possibly initiating or promoting several pathologies. Therefore the in vivo effect of oral supplementation with Gln and GSH was assessed. Oral supplementation with Gln was shown to temper H. suis induced gastritis and epithelial (hyper)proliferation in Mongolian gerbils. Astonishingly, supplementation of the feed with GSH, another GGT substrate, resulted in inflammation and epithelial proliferation levels returning to baseline levels of uninfected controls. This indicates that Gln and GSH supplementation may help reducing tissue damage caused by Helicobacter infection in both humans and pigs, highlighting their potential as a supportive therapy during and after Helicobacter eradication therapy. Non-Helicobacter (H.) pylori Helicobacter (NHPH) species have been found colonizing the stomach of 0.2–6% of humans patients with severe gastric complaints1. Infection causes gastritis and peptic ulceration and the rela- tive risk of developing mucosa-associated lymphoid tissue (MALT) lymphoma has been described to be higher with NHPH than with H. pylori1,2. Several studies have shown that H. suis, a member of the H. heilmannii sensu lato (s.l.) group3, is the most prevalent gastric NHPH in humans, which has been described to account for 14% to 78.5% of NHPH infections4–6. Interestingly, experimental infection studies in rodent models of human gas- tric disease have confirmed that long-term H. suis infection can lead to the development of gastric MALT lym- phoma7–9. Besides humans, the majority of pigs worldwide are colonized by this bacterium, in which infection causes chronic gastritis and reduced average daily weight gain10. Transmission of H. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Carbohydrate concentrations in the supernatant of a liquid Helicobacter suis culture. Minimal Essential Medium (MEM) and Mueller Hinton (MH) medium. The y-axis represents the fold change of the different carbohydrates relative to the 2 hour measurement point. Figure 1. Carbohydrate concentrations in the supernatant of a liquid Helicobacter suis culture. Minimal Essential Medium (MEM) and Mueller Hinton (MH) medium. The y-axis represents the fold change of the different carbohydrates relative to the 2 hour measurement point. which ultimately leads to apoptosis or necrosis of gastric epithelial cells in vitro15. Depletion of Gln in vitro can lead to an impaired proliferation and dysfunction of T-lymphocytes16–18. y y y Gln and GSH have a broad range of functions in the host. Gln plays a crucial role in the energy supply of rapidly dividing cells, such as intestinal epithelial cells and cells of the immune system23. In addition, this amino acid plays a role in protein turn-over and purine and pyrimidine synthesis24,25. The tripeptide GSH (γ-L-glutamyl-L-cysteinylglycine) is synthesized intracellularly. This ubiquitous free thiol is not only impor- tant for anti− oxidative protection of the plasma membrane and organelles, but it also plays distinct roles in cell cycle regulation and apoptosis26,27. After being transported outside the cells, GSH is degraded by eukaryotic membrane-bound GGT which removes the γ-glutamyl moiety28. The degradation products can be translocated into the cell, were they can for instance be recycled for GSH synthesis28. y y y Both deamination of Gln and degradation of GSH by Helicobacter GGT generate glutamate15,16,20, which can be taken up in the bacterial cell by a Na+-dependent transporter20,29. For H. pylori, metabolic reconstruction has suggested that glutamate can subsequently be converted to α -ketoglutarate, which can be channeled into the Tricarboxylic Acid cycle (TCA cycle)30. In contrast to most other bacteria, the TCA cycle of H. pylori is an atypical, non-cyclic pathway containing both an oxidative and reductive branch31. Some genes, necessary for a traditional TCA cycle are missing in the H. pylori genome. However, alternative reactions have been identified that allow connectivity between the metabolites of the TCA cycle30. No information is currently available on the metabolism of H. suis, a gastric Helicobacter species which is even far more fastidious than the well-studied H. www.nature.com/scientificreports/ pylori.hi py The present study is the first to examine the relative importance of the glutamate-generating metabolism (and by extension the amino acid metabolism) for survival and growth of H. suis. Since degradation of GSH and Gln by GGT from gastric helicobacters largely contribute to the glutamate-generating metabolism, we investi- gated the effect of GGT substrate supplementation (Gln/GSH) on the outcome of an experimental H. suis infec- tion in Mongolian gerbils. By correcting for possible imbalances, we aimed at reducing infection-related gastric pathologies. Oral glutathione supplementation drastically reduces Helicobacter- induced gastric pathologies suis most likely occurs through contact between pigs and humans1,11. Presence of viable H. suis bacteria in pork, however, suggests that foodborne infection might also occur12.t g A persistent mild chronic gastritis can be observed in human patients after H. heilmannii s.l. eradication treatment with antibiotics and proton-pump-inhibitors11,13. For H. pylori, it has also been described that after successful eradication in humans, corpus gastritis and dysplasia improve, although they do not completely dis- appear. Especially antral lesions seem non-responsive after eradication therapy14. This emphasizes the need for supplements that promote health of the gastric mucosa.i Recently, we and others identified the γ-glutamyltranspeptidase (GGT) from H. suis and H. pylori as an impor- tant factor causing epithelial cell death and modulating lymphocyte responses15–18. For all gastric helicobacters, the mode of action of the GGT mostly depends on the breakdown of 2 substrates, glutathione (GSH) and glu- tamine (Gln), both leading to the production of glutamate8,19–22. Under certain conditions, GGT-mediated degra- dation of GSH facilitates the formation of reactive oxygen species, causing lipid peroxidation of cell membranes, 1Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. 2Zoetis, Kalamazoo, MI 49007 USA. These authors jointly supervised this work. Correspondence and requests for materials should be addressed to B.F. (email: bramflahou@hotmail. com) Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 1 Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Amino acid concentrations in the supernatant of a liquid Helicobacter suis culture. Shown are the relative fold changes of the different amino acid concentrations compared to the 2 hour measurement point. MEM = Minimal Essential Medium, MH = Mueller Hinton. Figure 2. Amino acid concentrations in the supernatant of a liquid Helicobacter suis culture. Shown are the relative fold changes of the different amino acid concentrations compared to the 2 hour measurement point. MEM = Minimal Essential Medium, MH = Mueller Hinton. H. pylori genes encoding for L-serine deaminase (Accession number: NC.000915.1, gen ID 900301), cystathio- nine γ -synthase (NC.000915.1, gene ID 898970), alanine racemase (NC.000915.1, gen ID 899470) and D-amino acid dehydrogenase (NC.000915.1, gen ID 899472) in the H. suis genome (respective locus tags: HSUHS1_ RS00280, HSUHS1_RS06050, HSUHS1_RS00415 and HSUHS1_0061)21. Another amino acid that was decreased to basal levels after 24 h incubation in MEM was proline (p = 0.001, Fig. 2). We hypothesize that, as described for H. pylori, proline is taken up by the bacteria from the extracellular medium—mediated by the proline per- mease PutP32 – and subsequently converted to glutamate inside the bacteria by the proline utilizing flavoenzyme A (PutA)32,33. A homologue of the putP gene of H. pylori (NC.00915.1, gen ID 899191) is indeed present in the genome of H. suis (locus tag HSUHS1_1153)21. No significant changes were detected for this amino acid when H. suis was incubated in MH-medium. Other substrates that decreased over time in the culture supernatant included malate and lactate. In MEM, lactate levels significantly decreased (p < 0.001). In MH-media, lactate, as well as malate concentrations decreased (resp. p = 0.001 and p < 0.001, Fig. 1). They showed up to a 7-fold reduction after H. suis culture, suggesting they may also be important carbon sources. A homologue for the H. pylori gene encoding for D-lactate dehydrogenase (NC.000915.1, gene ID 898825) is also present in the H. suis genome (locus tag HSUHS5_RS01825)21, allowing the conversion of lactate to pyruvate. y Pyruvate can subsequently be converted to acetyl-CoA by pyruvate synthase. For H. pylori, it has been described that the main route for pyruvate assimilation is via a pyruvate:flavodoxin oxidoreductase (POR) (NC.00915.1, gen ID 899646)34. ORF’s have been identified in the H. suis genome encoding a POR-type pyruvate synthase (pyruvate oxidoreductase: HSUSH1_RS06160, phosphoenolpyruvate synthase: HSUHS1_RS07385)21, suggesting that a similar mechanism is present in H. suis. Results Th l The glutamate-generating metabolism is vital for survival and growth of H. suis. Annotation of the H. suis genome identified no homologues for some important genes with a putative role in glucose metab- olism21, suggesting that H. suis is unable to utilize the glycolytic pathway for metabolism. The genome of H. suis appears to lack glucose permease and several enzymes of the classic Embden-Meyerhof-Parnas pathway, includ- ing glucokinase and phosphofructokinase21. Furthermore, most components of the Entner-Doudoroff pathway are absent in the H. suis genome, such as glucose 1-dehydrogenase and gluconate dehydratase21. Utilization of glucose was not observed during H. suis culture in both Minimal Essential medium (MEM) and Mueller Hinton (MH) medium (Fig. 1, Table S1), further suggesting this bacterium does not utilize glucose as a major energy source. Instead, we hypothesized that this bacterium has to rely on other sources for its energy production. Pyruvate occupies a central branch point in the energy metabolism of H. suis. As the glycolytic pathway is impaired, pyruvate is most likely generated from amino acids such as serine, alanine and cysteine. This hypothesis was confirmed when we found that incubation of H. suis for 24 hours in MEM significantly lowered the extracellu- lar concentrations of serine (p < 0.001), alanine (p < 0.001) and cysteine (p < 0.001). A decrease (up to 6-fold) of these amino acids was observed, probably due to the generation of pyruvate. In contrast, in MH-medium only cysteine concentrations significantly decreased (p < 0.001), whereas serine and alanine concentrations remained steady over time (Fig. 2). Genome annotation (using RAST) showed the presence of homologues for Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Production of ammonia during Helicobacter suis/H. pylori GGT-mediated degradation of glutamine. Figure 3. Production of ammonia during Helicobacter suis/H. pylori GGT-mediated degradation of glutamine. Figure 4. Degradation of GSH by Helicobacter suis GGT (HsGGT) and H. pylori GGT (HpGGT). After supplementing 2 μ g/ml HsGGT or HpGGT to Ham’s F12 containing 1600 µg/ml glutathione (GSH), a decrease in GSH concentration was observed. Figure 4. Degradation of GSH by Helicobacter suis GGT (HsGGT) and H. pylori GGT (HpGGT). After supplementing 2 μ g/ml HsGGT or HpGGT to Ham’s F12 containing 1600 µg/ml glutathione (GSH), a decrease in GSH concentration was observed. importance of the amino acid metabolism and Gln metabolism in particular for the growth and energy supply of H. suis. A more detailed report of the metabolites used by H. suis in both the MEM- and MH-medium can be found in supplementary Table S1. importance of the amino acid metabolism and Gln metabolism in particular for the growth and energy supply of H. suis. A more detailed report of the metabolites used by H. suis in both the MEM- and MH-medium can be found in supplementary Table S1. Degradation of Gln and GSH by Helicobacter suis GGT. H. suis GGT (HsGGT) and H. pylori GGT (HpGGT) were shown to have a very similar Gln- and GSH-degrading capacity. After 2 hours of incubation, 3.88 ± 0.01 μ g/ml and 4.23 ± 0.02 μ g/ml ammonia was generated by Gln degradation through 2 μ g/ml HsGGT and HpGGT, respectively. After 6 hours of incubation, slightly higher amounts of ammonia were generated (4.30 ± 0.06 μ g/ml and 4.95 ± 0.02 μ g/ml, respectively) (Fig. 3).tt μg μg p y g After 8 hours of incubation, the initial GSH concentration was reduced by 80%, which added up to 97% after 24 h. Comparable data were obtained for HpGGT. After 8 h, the GSH concentration was reduced by 75%, whereas after 24 h the GSH concentration was reduced by 95% (Fig. 4). GSH and Gln supplementation drastically reduces Helicobacter-induced inflammation and cell damage. Given the importance of the glutamate-generating metabolism for H. suis and the potential impact of the concurrent Gln and GSH degradation (with an important role for H. suis GGT in these processes), we investigated the effects of feed supplementation with both substrates to H. www.nature.com/scientificreports/ As described for H. bizzozeronii, the genome of H. suis also codes for phosphoenol pyruvate carboxylase (locus tag HSUHS1_RS00640)21, an enzyme that plays a role in the synthesis of oxaloacetate from phosphoenolpyruvate35.h Certain amino acids were nearly depleted from the medium of a 24 h culture of H. suis. The levels of aspar- agine decreased approximately 50-fold (p < 0.001, Fig. 2), while the level of Gln dropped below the detection limit (p < 0.001, Fig. 2). Metabolic reconstruction showed that homologs for HP-ansB/HP-dcuA and HP-ggt/ HP-gltS are present in the H. suis genome21. For H. pylori, these hydrolysis/transport systems have been shown to be responsible for the deamination of asparagine and Gln and subsequent uptake by the bacteria of aspar- tate and glutamate, respectively29. These amino acids not only serve as precursors to protein synthesis, they can also be metabolized as energy source via the TCA cycle. Indeed, a homologue for the H. pylori gene coding for NADP-dependent glutamate dehydrogenase (NC.000915.1, gen ID 898871) is present in the genome of H. suis (locus tag HSUHS1_RS07485)21. This enzyme can convert glutamate to 2-ketoglutarate, an intermediate metabo- lite of the TCA cycle, whereas aspartate can be channeled into the TCA cycle by aspartate ammonia-lyase or other multi-step pathways, such as adenylosuccinate synthase—adenylosuccinate lyase. In any case, this underlines the Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 3 www.nature.com/scientificreports/ suis infected Mongolian gerbils.ff f No differences in feed intake were observed between animals from different groups during the experiment (p > 0.05, Table S2). In addition, animals in different groups showed a similar weight gain during the course of the experiment (Table S2). All uninfected control animals tested negative for the presence of H. suis. In experi- mentally infected animals, the highest H. suis colonization rates were found in the antrum. Average colonization rates in animals receiving the standard diet, Gln-supplemented and GSH-supplemented diet were 2.49 × 104 Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 4 www.nature.com/scientificreports/ Figure 5. Average gastritis scores observed in the antrum of the stomach of H. suis infected animals shows a markedly decreased inflammation in the group receiving the glutathione diet (GSH). An depicts significantly lower values compared to the H. suis infected animals receiving the standard feed. Error bars represent the standard deviation. Figure 5. Average gastritis scores observed in the antrum of the stomach of H. suis infected animals shows a markedly decreased inflammation in the group receiving the glutathione diet (GSH). An depicts significantly lower values compared to the H. suis infected animals receiving the standard feed. Error bars represent the standard deviation. Figure 6. Proliferating lymphocytes in the antrum of control (a)and Helicobacter suis infected animals (b–d). Proliferation of lymphocytes, as well as the formation of lymphoid follicles with proliferating germinal centers, is observed in H. suis infected animals receiving the standard diet (b) and infected animals receiving the glutamine-supplemented diet (c). In contrast, only a limited amount of proliferating lymphocytes are observed in H. suis infected animals receiving the diet supplemented with glutathione (d). In control animals (a), only a limited number of cells are proliferating and no lymphocytic aggregates are present. Proliferating Ki-67 positive cells are brown. igure 6. Proliferating lymphocytes in the antrum of control (a)and Helicobacter suis infected animals Figure 6. Proliferating lymphocytes in the antrum of control (a)and Helicobacter suis infected animals (b–d). Proliferation of lymphocytes, as well as the formation of lymphoid follicles with proliferating germinal centers, is observed in H. suis infected animals receiving the standard diet (b) and infected animals receiving the glutamine-supplemented diet (c). In contrast, only a limited amount of proliferating lymphocytes are observed in H. suis infected animals receiving the diet supplemented with glutathione (d). www.nature.com/scientificreports/ In control animals (a), only a limited number of cells are proliferating and no lymphocytic aggregates are present. Proliferating Ki-67 positive cells are brown. (± 2.76 × 104), 1.13 × 104 (± 6.08 × 104) and 2.62 × 103 (± 5.53 × 103) bacteria/mg tissue, respectively. There were no statistically significant differences between different groups, although animals receiving the GSH diet tended to show lower colonization rates (Standard vs Gln p = 0.606; Standard vs GSH p = 0.129, Fig. S1). None of the control animals showed signs of gastritis. In H. suis infected animals, gastritis was mainly pres- ent in the antrum. There was a significant difference in gastritis scores between the different groups of infected animals (Fig. 5). A detailed overview of the observed inflammation scores can be found in the online supple- mentary data (Table S3). Infected animals belonging to the group receiving the GSH supplemented diet showed None of the control animals showed signs of gastritis. In H. suis infected animals, gastritis was mainly pres- ent in the antrum. There was a significant difference in gastritis scores between the different groups of infected animals (Fig. 5). A detailed overview of the observed inflammation scores can be found in the online supple- mentary data (Table S3). Infected animals belonging to the group receiving the GSH supplemented diet showed Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 5 www.nature.com/scientificreports/ Figure 7. Fold change in cytokine expression in control and Helicobacter suis infected animals. An indicates a significant difference in infected animals compared to the negative control animals receiving the standard diet. An °indicates a significant difference in expression compared to H. suis infected animals receiving the standard diet (p < 0.05). Std: animals receiving the standard diet; Gln: Glutamine-supplemented diet; GSH: Glutathione- supplemented diet. Figure 7. Fold change in cytokine expression in control and Helicobacter suis infected animals. An indicates a significant difference in infected animals compared to the negative control animals receiving the standard diet. An °indicates a significant difference in expression compared to H. suis infected animals receiving the standard Figure 7. Fold change in cytokine expression in control and Helicobacter suis infected animals. An indicates a significant difference in infected animals compared to the negative control animals receiving the standard diet. An °indicates a significant difference in expression compared to H. suis infected animals receiving the standard diet (p < 0.05). www.nature.com/scientificreports/ Std: animals receiving the standard diet; Gln: Glutamine-supplemented diet; GSH: Glutathione- supplemented diet. substantially lower inflammation scores (mean score 0.3 ± 0.4; Fig. 6D) compared to infected animals from the group receiving the purified standard diet (mean score 2.6 ± 0.4; p < 0.001; Fig. 6B). Seven out of 10 H. suis infected animals receiving the GSH-supplemented diet even showed no detectable inflammation. Although notably less lymphocytic infiltration and fewer lymphocytic aggregates were also observed in the infected ani- mals receiving the Gln supplemented diet (mean score 1.9 ± 0.9; Fig. 6C), this was not statistically significant (p = 0.064). In general, deep lymphocytic infiltrates consisted mainly of CD3-positive cells whereas superficial lymphoid follicles contained a majority of CD3-negative B lymphocytes (Fig. S2).f y p j y g y p y ( g ) Results of cytokine expression are summarized in Fig. 7. For most cytokines tested, no differences were observed between control groups receiving different diets. Surprisingly, however, uninfected control animals receiving the Gln-supplemented diet did show an upregulation of INF-γ expression (p = 0.012, fold = 3.01 ± 1.19). When comparing control and H. suis infected animals, both receiving the standard diet, H. suis infection was shown to provoke an upregulation of IL 1β INF γ and IL 10 expression In H suis infected animals receiving y p j y g y p y g Results of cytokine expression are summarized in Fig. 7. For most cytokines tested, no differences were observed between control groups receiving different diets. Surprisingly, however, uninfected control animals receiving the Gln-supplemented diet did show an upregulation of INF-γ expression (p = 0.012, fold = 3.01 ± 1.19). Results of cytokine expression are summarized in Fig. 7. For most cytokines tested, no differences were observed between control groups receiving different diets. Surprisingly, however, uninfected control animals receiving the Gln-supplemented diet did show an upregulation of INF-γ expression (p = 0.012, fold = 3.01 ± 1.19). When comparing control and H. suis infected animals, both receiving the standard diet, H. suis infection was shown to provoke an upregulation of IL-1β , INF-γ and IL-10 expression. In H. suis infected animals receiving the Gln-supplemented diet, only an upregulation of IL-1β was noted, whereas mRNA expression levels of other cytokines dropped back to the levels of uninfected control animals. www.nature.com/scientificreports/ Animals receiving the GSH diet even showed cytokine expression levels comparable to those in uninfected control animals.f When comparing control and H. suis infected animals, both receiving the standard diet, H. suis infection was shown to provoke an upregulation of IL-1β , INF-γ and IL-10 expression. In H. suis infected animals receiving the Gln-supplemented diet, only an upregulation of IL-1β was noted, whereas mRNA expression levels of other cytokines dropped back to the levels of uninfected control animals. Animals receiving the GSH diet even showed cytokine expression levels comparable to those in uninfected control animals.f No differences in epithelial cell proliferation were observed between the non-infected groups receiving dif- ferent diets. Marked differences in terms of gastric epithelial proliferation at the level of the antral pits were observed between H. suis infected groups receiving different diets (Figs 8 and 9). H. suis infected animals receiv- ing the standard diet showed the highest epithelial proliferation rate (8.2 ± 1.0 Ki67-positive cells per gland; p < 0.001). The proliferation rate was significantly lower in animals receiving the Gln- (6.2 ± 1.1, p = 0.010) or the GSH-supplemented diet (4.9 ± 1.6, p = 0.002) compared to infected animals receiving the non-supplemented diet. In the corpus, no differences in epithelial cell proliferation were observed between the different H. suis infected groups (Fig. 9). g g Besides their abundant presence in the corpus, a limited number of parietal cells can also be found in the antrum. An abrupt loss of parietal cells was observed in the transition zone between the corpus and antrum in the H. suis infected animals receiving the standard diet as well as the Gln-supplemented diet. In H. suis infected animals receiving the GSH-supplemented diet, this abrupt loss was absent, revealing an image comparable to that of uninfected control animals (Fig. S3). Discussioni Metabolic profiling and the absence of putative genes encoding important enzymes of the Embden-Meyerhof- Parnas and Entner-Doudoroff pathway suggest that, in contrast to the closely related H. pylori and H. bizzozero- nii, H. suis is not able to utilize glucose for its main energy supply. As a consequence, H. suis has to rely on other sources to fulfill its energy requirements. Metabolic reconstruction and analysis of metabolites in H. suis culture medium showed that H. suis tends to use amino acids, not only for protein synthesis, but most likely also as an energy source. The consumption of amino acids was obvious in cultures grown in both liquid media, but was most pronounced in MEM, which can be considered to be the most basic one of the two. This could indicate that H. suis relies even more on amino acids for its survival when facing stressful situations. g One of the preferred nutrients seems Gln, which is the most abundant free amino acid in both plants and ani- mals36–38. Our current and previous studies have shown that Gln is depleted from the medium during H. suis cul- ture due to the hydrolysis by H. suis GGT16. As described for H. pylori, the formed glutamate can be taken up and converted to 2-ketoglutarate by the bacteria and channeled into the TCA cycle21,29, an important metabolic path- way that provides precursors for a variety of cell components, as well as for supplying energy to the bacterium31. Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 8. Proliferating Ki67-positive (brown) epithelial cells and lymphocytes in the antrum of the stomach of control and Helicobacter suis infected animals. (a) Control animal receiving the standard diet; (b) H. suis infected animal receiving the standard diet; (c) H. suis infected animal receiving the glutamine- supplemented diet; (d) H. suis infected animal on the glutathione-supplemented diet. A more pronounced proliferation is observed in H. suis infected animals receiving the standard and glutamine-supplemented diet compared to H. suis infected animals receiving the glutathione-supplemented diet. Figure 8. Proliferating Ki67-positive (brown) epithelial cells and lymphocytes in the antrum of the stomach of control and Helicobacter suis infected animals. (a) Control animal receiving the standard diet; (b) H. suis infected animal receiving the standard diet; (c) H. suis infected animal receiving the glutamine- supplemented diet; (d) H. suis infected animal on the glutathione-supplemented diet. Discussioni In the present study, however, supplementing 0.8% GSH to the diet of H. suis infected gerbils revealed tremendously beneficial effects on the health of a Helicobacter-infected stom- ach. We observed a marked decrease of gastric epithelial cell proliferation rates, protection against parietal cell death and a 90% reduction of gastritis, accompanied by normalized expression levels for all (pro-inflammatory) cytokines, confirming the suppression of the host immune response elicited by Helicobacter infection. Only three animals had small lymphoid follicles in the antrum of the stomach, despite normal H. suis colonization rates. Helicobacter GGT has been shown to inhibit proliferation of T-lymphocytes, an effect that has been suggested to play a role in the immune escape by these bacteria16,49. Supplementing GSH to the cell medium has been shown to aggravate this effect16. Possibly, similar mechanisms are involved in the current in vivo study.hfl ggf y y The observed protective effects on infection-induced inflammation and, surprisingly, epithelial cell hyperpro- liferation may be explained by a dose-dependent effect of GSH. Intracellular concentrations of GSH are usually very high, ranging from 0.5 to 10 mM, whereas extracellular concentrations are found in the micromolar range50. Therefore, supplementing feed with higher concentration of GSH (35 mM) compared to the dosages used for pre- vious in vitro studies (5 mM)15 probably results in an excess quantity of protective extracellular GSH, most likely counterbalancing some potentially negative effects on gastric epithelial cell health by GGT-induced generation of pro-oxidative GSH degradation products15. It has indeed been shown that, when available for the host cells, GSH plays an important role in the protection of cells against oxygen radicals, detoxification and metabolisation of var- ious endo- and exogenous compounds, cell cycle regulation, cell signaling and apoptosis16,50–52. GSH deficiency makes cells more sensitive to oxidative stress and can cause degeneration of epithelial cells50,51. The remarkable beneficial effects of GSH on Helicobacter-induced inflammation and epithelial cell homeostasis suggest that GSH supplementation could be considered as a supportive therapy during and even after a Helicobacter eradication therapy. It should be noted, however, that only the short/medium-term effects of GSH supplementation were evaluated during this trial. A study of Liu et al.53 showed that GSH supplementation had beneficial effects on acute clinical signs of inflammatory bowel disease (IBD), whereas some adverse effects were observed in the chronic stages of IBD in this particular disease model. Discussioni y p g y p y In the present study, the beneficial effect of Gln supplementation on inflammation was only mild to moderate, which may be due to the shorter infection period compared to that described by Hagen et al.41. Nevertheless, H. suis infected animals receiving the Gln diet showed smaller gastric lymphoid follicles and less diffuse lymphoid infiltration compared to the animals receiving the standard diet. This decreased proliferation of lymphocyte pop- ulations, as well as the reduced expression of pro- and anti-inflammatory cytokines in H. suis infected animals receiving Gln-supplemented feed is somewhat in contrast to in vitro studies showing that Gln supplementation restores the impaired proliferation of H. suis GGT-treated Jurkat T-cells16. Other factors are most likely involved, such as a protective effect of Gln on the epithelial barrier.if pf p Indeed, the current study is the first to show a direct effect of Gln supplementation on the proliferation rate of gastric epithelial cells in the context of a Helicobacter infection, suggesting a protective effect of Gln on gastro-intestinal epithelial cells43–45. Most likely, hyperproliferation of epithelial cells found in H. suis infected animals receiving the standard diet is a secondary response to increased cell death induced by this bacterium8,45,46. Increased cell death may be induced by an increased oxidative stress burden or a modification of the redox bal- ance of gastric epithelial cells following Gln depletion15,47. Supplementing Gln may therefore assist in restoring the redox balance. Besides oxidative stress, H. suis infection causes a dramatic increase of ammonia concentrations, resulting from urease and GGT activity of the bacterium1,20,42,48. Adding Gln to the diet can stimulate arginase activity, causing a conversion of ammonia to the less toxic urea, and helping in clearing ammonia from the cells by stimulating its excretion23,42. Although it apparently has a beneficial effect on compensatory hyperproliferation of gastric epithelial cells, supplementing Gln to H. suis infected animals does not prevent the abrupt loss of parietal cells at the transition zone between the corpus and antrum8. p Besides a depletion of Gln, GGT from gastric helicobacters also causes degradation of GSH15,16. Previous in vitro research has revealed that supplementation of GSH at a concentration of 5 mM to H. suis GGT-treated epi- thelial cells can stimulate the formation of pro-oxidative GSH degradation products and extracellular oxygen rad- icals, leading to necrosis of damaged cells15. Discussioni A more pronounced proliferation is observed in H. suis infected animals receiving the standard and glutamine-supplemented diet compared to H. suis infected animals receiving the glutathione-supplemented diet. Figure 9. Proliferation of gastric epithelial cells. Ki67 staining of the stomach epithelium in the antrum and corpus of the stomach. Std: standard feed, Gln: glutamine-supplemented feed, GSH: glutathione-supplemented feed. An depicts significantly lower values compared to the H. suis infected animals receiving the standard feed. Error bars represent the standard deviation. Figure 9. Proliferation of gastric epithelial cells. Ki67 staining of the stomach epithelium in the antrum and corpus of the stomach. Std: standard feed, Gln: glutamine-supplemented feed, GSH: glutathione-supplemented feed. An depicts significantly lower values compared to the H. suis infected animals receiving the standard feed. Error bars represent the standard deviation. The hydrolysis of extracellular Gln by the GGT from gastric helicobacters can cause a depletion of this con- ditionally essential amino acid in the gastric juice of an infected host, which may eventually lead to epithelial cell damage and immunological dysfunction16,18,20. By supplementing Gln to the diet of Mongolian gerbils we aimed The hydrolysis of extracellular Gln by the GGT from gastric helicobacters can cause a depletion of this con- ditionally essential amino acid in the gastric juice of an infected host, which may eventually lead to epithelial cell damage and immunological dysfunction16,18,20. By supplementing Gln to the diet of Mongolian gerbils we aimed Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 7 www.nature.com/scientificreports/ at reducing inflammation-related as well as other Helicobacter-related gastric pathological changes. In addition to a local effect, dietary Gln supplementation has been described to cause an increase of the systemic Gln concen- tration, which may also sustain the health and function of immune and epithelial cells39. The role of Gln in gastric mucosal protection against H. pylori infection has been reported previously36–38,40–42. In these studies, an effect of Gln supplementation on gastric inflammation and cytokine expression was observed41. In addition, a recent study indicated that the depletion of Gln, as a result of high GGT activity, might actually be responsible for the strong inflammatory response following H. pylori infection, as shown by increased release of pro-inflammatory IL-8 from epithelial cells17. In contrast, Wüstner et al.18 described that H. pylori GGT activity leads to an impaired T-cell function by depriving activated lymphocytes from Gln. Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 Methods M t b li These pooled samples were injected throughout the platform day run and served as technical replicates. l d d fi d d l l f d d h In total, 702 media were identified and statistical analysis was performed to detect concentration changes during H. suis culture. Based on information derived from a draft genomic sequence21, a partial metabolic recon- struction of H. suis strains HS1 and HS5 (currently named H. suis strains HS1LP and HS5bLP54) was performed, mainly focusing on the carbon metabolism of this bacterium and attempting to confirm the results of metabolic profiling (Integrated Genomics Inc., Chicago, Illinois, US).i i In addition, the RAST (Rapid Annotation using Subsystem Technology) tool was used to confirm the pres- ence/absence of genes important for the metabolism of H. suis in the genome of H. suis strains HS1LP and HS5bLP21,54. Effects of H. suis and H. pylori GGT on Gln and GSH. The H. suis and H. pylori GGT were expressed and purified as described previously15. Both enzymes were compared to each other regarding their capacity to degrade 2 important substrates: Gln and GSH. Two μ g/ml of H. suis or H. pylori GGT was added to HBSS supplemented with 2 mM Gln and incubated at 37 °C for 2 hours and 6 hours. As a control, a solution of HBSS supplemented with 2 mM Gln was used. The concentration of produced ammonia was detected using an Ammonia Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions.f g g To evaluate the effect of HsGGT and HpGGT on GSH, 1600 µg/ml GSH was incubated for 8–24 hours at 37 °C with 2 μ g/ml HsGGT or HpGGT in Ham’s F12 medium. GSH concentration was measured using the Glutathione Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Bacterial strain used for in vivo experimentation. H. suis strain HS5bLP, isolated from the gastric mucosa of a sow55, was used for experimental infection of Mongolian gerbils. Culture was performed as described previously54. Ethics statement. All experimental procedures were approved by the Ethical Committee of the Faculty of Veterinary Medicine of the University of Ghent, Belgium (approval number EC2011-113), and carried out in accordance with the approved guidelines and regulations. Animals, housing, inoculation and sampling. Sixty female Mongolian gerbils (Charles River, France) arrived at our animal housing units at the age of 4 weeks. They were randomly divided in 6 groups with 10 animals each. Methods M t b li Metabolic Profiling of H. suis: Energy Metabolism. H. suis type strain HS1LP was grown in liquid Minimal Essential Medium (MEM) and Mueller Hinton (MH) broth. The sample preparation process was car- ried out using the automated MicroLab STAR® system (Hamilton Company, Reno, US). Recovery standards were added prior to the first step in the extraction process for Quality Control purposes. Sample preparation was conducted using a proprietary (Metabolon, Durham, North Carolina, USA) series of organic and aqueous extractions to remove the protein fraction while allowing maximum recovery of small molecules. The resulting extract was divided into two fractions, one for analysis by Liquid Chromatography (LC) and one for analysis by gas chromatography (GC). Samples were placed briefly on a TuboVap® (Zymark, Massachusetts, US) to remove the organic solvent. Each sample was then frozen and dried under vacuum. Samples were then prepared for the appropriate instrument, either LC/MS or GC/MS.hh The LC/MS portion of the platform was based on a Surveyor HPLC and a Thermo-Finnigan LTQ Fmass spectrometer (Thermo scientific, Waltham, Massachusettes, USA), which consisted of an electrospray ionization (ESI) source and linear ion-trap (LIT) mass analyzer. Positive and negative ions were monitored within a single analysis by consecutively alternating the ionization polarity of adjacent scans. The vacuum-dried sample was dis- solved in 100.0 μ l of an injection solvent that contained five or more injection standards at fixed concentrations. The chromatography has been standardized by Metabolon, Inc. and once a method was validated, no changes were made to it. The internal standards were used both to assure injection and chromatographic consistency. The chromatographic system used a binary solvent (water and methanol) system delivered as a gradient. The HPLC columns were washed and reconditioned after every injection.h t The samples destined for GC/MS analysis were re-dried under vacuum desiccation for a minimum of 24 hours prior to being derivatized under dried nitrogen using bistrimethyl-silyl-triflouroacetamide (BSTFA). The GC column was 5% phenyl and the temperature ramp ranged from 40° to 300 °C in a 16 minute period. Samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer using electron impact ionization. p At 2, 12, 18 and 24 hours media supernatants was analyzed. For each condition, 4 biological replicates were performed. A small aliquot of each experimental sample for a specific matrix was obtained and pooled. Discussioni More research is therefore needed to evaluate the long-term safety and effects of GSH supplementation and to make recommendations on the chronic use of glutathione in people suffering from gastric disease.h f g g Feed additives are commonly used in the pig industry to promote pig health. They are easily manufactured and provided to the animals. Both Gln and GSH can be considered as a possible feed additive since the con- centrations of both were shown to be stable in the feed pellets used in the present study (unpublished results). These supplements may therefore be helpful for decreasing both the prevalence and severity of gastric pathologies caused by H. suis infection in pigs. Indeed, up to 90% of H. suis infected pigs have been shown to develop gastritis, which most likely plays a role in the observed reduction of daily weight gain, a primordial production character- istic in the pig industry10. p g y In conclusion, we showed that H. suis preferably uses amino acids to meet its energy requirements. In particu- lar, the glutamate-generating metabolism seems essential, as shown by a complete depletion of Gln during H. suis culture. The H. suis GGT has previously been shown to be an important factor degrading Gln, but also GSH. Oral 8 Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 www.nature.com/scientificreports/ supplementation of H. suis infected animals with these two substrates, Gln or GSH, resulted in a marked reduc- tion of infection-related gastric pathologies, both related to inflammation and epithelial cell damage. GSH was shown to be superior to glutamine and may prove to be of therapeutic value for use in both humans and animals. Methods M t b li Animals from 1 group were divided in 2 filter-top cages (5 animals each), all held in the same stable under controlled environmental conditions. During the experiment, feed intake was monitored weekly and body weight was measured three times, twice at inoculation and once at euthanasia.hf Three different rodent diets (Table 1) were provided ad libitum to 20 animals each (Research Diets, Inc., New Brunswick, USA). The standard diet was a purified feed (AIN-93 G, Research Diets, Inc.). The other 2 diets were identical to the standard diet, with the exception of being supplemented with 5% Gln or 0.8% GSH and with a variable amount of corn starch, used to correct minor differences in the energy content. At the age of 6 weeks, the animals were briefly anaesthetized with 3% isoflurane and inoculated intragastrically twice, with a two-day Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 9 www.nature.com/scientificreports/ Standard diet Gln-supplemented diet GSH-supplemented diet Gram% Kcal% Gram% Kcal% Gram% Kcal% Protein 20 20 25 25 20 20 Carbohydrate 64 64 59 59 63 64 Fat 7 16 7 16 7 16 Gram Kcal Gram Kcal Gram Kcal Casein 200 800 200 800 200 800 L-Cystine 3 12 3 12 3 12 Supplemented L-Glutamine 0 0 50 200 0 0 Corn Starch 397.486 1590 374.486 1390 397.486 1590 Maltodextrin 132 528 132 528 132 528 Sucrose 100 400 100 400 100 400 Cellulose 50 0 50 0 50 0 Soybean oil 70 360 70 360 70 360 Mineral Mix 35 0 35 0 35 0 Vitamin Mix 10 40 10 40 10 40 Choline Bitartrate 2.5 0 2.5 0 2.5 0 Supplemented Glutathione 0 0 0 0 8.07 0 Total 1000 4000 1000.05 4000 1008.12 4000 Table 1. Composition of the 3 different diets used in this study. Table 1. Composition of the 3 different diets used in this study. Table 1. Composition of the 3 different diets used in this study. Methods M t b li Cytokine Primers Sequence (5′ → 3′) Reference IL-17 F: AGC TCC AGA GGC CCT CGG AC 59 R: AGG ACC AGG ATC TCT TGC TG TNF-α F: GCT CCC CCA GAA GTC GGC G 59 R: CTT GGT GGT TGG GTA CGA CA IL-10 F: GGT TGC CAA GCC TTA TCA GA 56 R: GCT GCA TTC TGA GGG TCT TC IL-1β F: GGC AGG TGG TAT CGC TCA TC Modified from59, R: CAC CTT GGA TTT GAC TTC TA IL-5 F: AGA GAA GTG TGG CGA GGA GAG ACG Present study R: ACA GGG CAA TCC CTT CAT CGG IL-6 F: CAA AGC CAG AGC CAT TCA GAG 56 R: GCC ATT CCG TCT GTG ACT CCA GTT TCT CC IL-12p40 F: GAC ACG ACC TCC ACC AAA GT 56 R: CAT TCT GGG ACT GGA CCC TA IFN-γ F: CCA TGA ACG CTA CAC ACT GCA TC 60 R: GAA GTA GAA AGA GAC AAT CTG G Β -actin F: TCC TCC CTG GAG AGG AGC TA 61 R: CCA GAC AGC ACT GTG TTG GC HPRT F: CTC ATG GAC TGA TTA TGG ACA G 61 R: AGC TGA GAG ATC ATC TCC ACC AAT Table 2. Forward (F) and reverse (R) primers used for cytokine-expression. Table 2. Forward (F) and reverse (R) primers used for cytokine-expression. interval. Ten animals from each feed group received 8 × 107 viable H. suis bacteria/dose. The remaining 10 mals/feed group were sham-inoculated with the culture medium of H. suis.til nterval. Ten animals from each feed group received 8 × 107 viable H. suis bacteria/dose. The remaining 10 ani- mals/feed group were sham-inoculated with the culture medium of H. suis.til Twelve weeks after the first inoculation, animals were anaesthetized with 5% (vol/vol) isoflurane followed by cervical dislocation. The stomach was opened along the greater curvature. A longitudinal strip was taken from the forestomach to the duodenum, fixed in 4% phosphate buffered formaldehyde and embedded in paraffin for histopathological examination. Biopsies from antrum and corpus were taken for DNA and RNA extraction. These tissues were immediately submerged in RNAlater (Qiagen, Hilden, Germany) and stored at −70 °C until further processing. DNA extraction and quantitative-PCR to determine the colonization rate of H. suis. DNA extraction and Quantitative-PCR were performed on the DNA samples as described previously56,57. Methods M t b li DNA Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 10 www.nature.com/scientificreports/ RNA extraction and Real-Time PCR analysis of cytokine expression. After separation of DNA and RNA, the latter was further purified using the RNeasy Mini kit (Qiagen) following the suppliers’ instructions. Synthesis of cDNA was done by using the iScriptTM cDNA synthesis kit (Bio-rad, Hercules, USA) as described by the manufacturer. Real Time− PCR and melting curve analysis were done as described before58. The primer sequences are summarized in Table 2. Primers for interleukin (IL)-5 were designed using primer3 software. Histopathology. Four sections of 5 μ m were sliced from the paraffin-embedded stomach tissue. The first section was used for a hematoxylin and eosin (HE) staining to score the intensity of infiltration with mononuclear and polymorphonuclear cells. A second tissue slide was stained using a mouse monoclonal anti-Ki67 antibody (Menarini Diagnostics, Firenze, Italy; dilution 1:50) and a biotinylated goat anti-mouse IgG antibody to iden- tify proliferating cells. Positive cells lining the lumen of the gastric pits were counted in five randomly chosen High Power Fields (magnification: x400) in the corpus and antrum. For each animal, an average of the positive cell count was determined for both stomach regions. A third section was used to stain acid-producing parietal cells of the corpus of the stomach by using an antibody directed against the H+K+ATP− ase β -subunit (Abcam, Cambridge, UK; dilution 1:25000). The fourth tissue slide was used for visualization of T-lymphocytes, by using an antibody directed against CD3 (Abcam, dilution 1:100). Statistical analysis. T-tests were used to analyze the data of the metabolomics study. T-test comparisons were performed between each experimental group and its control.f Differences in gastritis between experimentally infected animals and control animals receiving the standard diet were analyzed using a Mann-Whitney U non-parametric test. Comparison of the colonization rates, cytokine expression and epithelial proliferation rates between the different diet groups of infected animals was done by means of a one-way ANOVA (SPSS 21, IBM, New York, USA). P-values ≤ 0.05 were considered statistically sig- nificant. When multiple groups were compared, a Bonferroni correction was made. All data are expressed as mean ± standard deviation. References An experimental Helicobacter suis infection causes gastritis and reduced daily weight gain in pigs. Vet. Microbiol 160, 449–454 (2012). ( ) 11. Joosten, M. et al. Case report: Helicobacter suis infection in a pig veterinarian. Helicobacter 18, 392–396 (2013). . Joosten, M. et al. Case report: Helicobacter suis infection in a pig v 12. De Cooman, L. M. et al. Survival of Helicobacter suis bacteria in retail pig meat. Int. J. Food Microbiol. 1, 164–167 (2013). l l b h l d l d l h l ft 13. Morgner, A. et al. Helicobacter heilmannii-associated primary low-grade MALT lymphoma: complete remission after curing the infection. Gastroenterol 118, 821–828 (2000).hf 14. Kokkola, A. et al. The effect of Helicobacter pylori eradication on the natural course of atrophic gastritis with dysplasia. Aliment. Pharmacol. Ther. 16, 515–520 (2002). h ahou, B. et al. Gastric epithelial cell death caused by Helicobacter suis and Helicobacter pylori gamma-glutamyl transpeptidase is ainly glutathione degradation-dependent. Cell. Microbiol. 13, 1933–1955 (2011).f y g g p 16. Zhang, G. et al. Effects of Helicobacter suis γ - glutamyl transpeptidase on lymphocytes: modulation by glutamine and glutathione supplementation and outer membrane vesicles as a putative delivery route of the enzyme. PLoS One 10, e77966 (2013). 17. Rimbara, E., Mori, S., Kim, H. & Shibayama, K. Role of γ-glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection. Microbiol. Immunol. 57, 665–673 (2013). 8. Wüstner, S. et al. Helicobacter pylori γ-glutamyltranspeptidase impairs T-lymphocyte function by compromising metabolic adaption through inhibition of cMyc and IRF 4 expression. Cell. Microbiol. 17, 51–61 (2015).h g y 19. Chevalier, C., Thiberge, J. M., Ferrero, R. L. & Labigne, A. Essential role of Helicobacter pylori γ-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol. Microbiol. 31, 1359–1372 (1999). 19. Chevalier, C., Thiberge, J. M., Ferrero, R. L. & Labigne, A. Essential role of Helicobacte colonization of the gastric mucosa of mice. Mol. Microbiol. 31, 1359–1372 (1999). g 0. Shibayama, K. et al. Metabolism of glutamine and glutathione via γ-glutamyltranspeptidase and glutamate transport in Helicobacte l bl fi h h h l f h l b l ( ) 20. Shibayama, K. et al. Metabolism of glutamine and glutathione via γ-glutamyltranspeptidase and glutamate transport in Helicobacter pylori: possible significance in the pathophysiology of the organism. Mol. Microbiol. 64, 396–406 (2007). 20. Shibayama, K. et al. References e e e ces 1. Haesebrouck, F. et al. Gastric helicobacters in domestic animals and nonhuman primates and their significance for human health. Clin. Microbiol. Rev. 22, 202–223 (2009). 1. Haesebrouck, F. et al. Gastric helicobacters in domestic animals and nonhuman primates and their significance for human health. Clin. Microbiol. Rev. 22, 202–223 (2009). g y p pp 3. Haesebrouck, F. et al. Non-Helicobacter pylori Helicobacter species in the human gastric mucosa: a proposal to introduce the ter H. heilmannii sensu lato and sensu stricto. Helicobacter 16, 339–340 (2011).i g y p pp 3. Haesebrouck, F. et al. Non-Helicobacter pylori Helicobacter species in the human gastric mucosa: a proposal to introduce the term H. heilmannii sensu lato and sensu stricto. Helicobacter 16, 339–340 (2011).i 4. Trebesius, K., Adler, K., Vieth, M., Stolte, M. & Haas, R. Specific detection and prevalence of Helicobacter heilmannii-like organisms in the human gastric mucosa by fluorescent in situ hybridization and partial 16S ribosomal DNA sequencing. J. Clin. Microbiol. 39, 1510–1516 (2001). 5. De Groote, D. et al. Detection of non-Helicobacter pylori species in “Helicobacter heilmanni”-infected humans. Helicobacter 10 398–406 (2005).i 6. Van den Bulck, K. et al. Identification of non-Helicobacter pylori spiral organisms in gastric samples from humans, dogs, and cats J. Clin. Microbiol. 43, 2256–2260 (2005). 7. Nakamura, M. et al. “Candidatus Helicobacter heilmannii” from a cynomolgus monkey induces gastric mucosa-associated lymphoid tissue lymphomas in C57BL/6 mice. Infect. Immun. 75, 1214–1222 (2007). y p y p f 8. Flahou, B. et al. Helicobacter suis causes severe gastric pathology in mouse and mongolian gerbil models of human gastric disease PLoS One 5, e14083 (2010). 9. Nakamura, M. et al. Microcirculatory alteration in low-grade gastric mucosa-associated lymphoma by Helicobacter heilmannii infection: Its relation to vascular endothelial growth factor and cyclooxygenase-2. J. Gastroenterol. Hepatol. 23, S157–S160 (2008). l l l b f d d d d l h b l 9. Naka u a, . et al. c oc cu ato y a te at o ow g ade gast c ucosa assoc ated y p o a by elicobacter heilmannii infection: Its relation to vascular endothelial growth factor and cyclooxygenase-2. J. Gastroenterol. Hepatol. 23, S157–S160 (2008). 10. De Bruyne, E. et al. An experimental Helicobacter suis infection causes gastritis and reduced daily weight gain in pigs. Vet. Microbiol. 160, 449–454 (2012). g y yg J p , ( ) 10. De Bruyne, E. et al. Acknowledgements g This work was supported by the Research Fund of Ghent University, Belgium, grant BOF08/GOA/004 and BOF14/GOA/010 and Fonds Wetenschappelijk Onderzoek Vlaanderen (grant FWO14/PDO/067). Technical assistance of Sofie De Bruyckere, Nathalie Van Rysselberghe and Sophie Callens is greatly acknowledged. g This work was supported by the Research Fund of Ghent University, Belgium, grant BOF08/GOA/004 and BOF14/GOA/010 and Fonds Wetenschappelijk Onderzoek Vlaanderen (grant FWO14/PDO/067). Technical assistance of Sofie De Bruyckere Nathalie Van Rysselberghe and Sophie Callens is greatly acknowledged Author Contributions ut o Co t but o s F.H., R.D. and D.F. funding and study supervision. F.H., B.F., R.D. and D.F. study concept and design. E.D.B., B.F., M.J., G.Z. and M.S. acquisition of data. E.D.B., B.F. and D.F. analyses and interpretation of the data. E.D.B., B.F. and F.H. drafting of manuscript. R.D., M.J., G.Z., A.S. and F.P. critical revision of the manuscript. F.H., R.D. and D.F. funding and study supervision. F.H., B.F., R.D. and D.F. study concept and design. E.D.B., B.F., M.J., G.Z. and M.S. acquisition of data. E.D.B., B.F. and D.F. analyses and interpretation of the data. E.D.B., B.F. and F.H. drafting of manuscript. R.D., M.J., G.Z., A.S. and F.P. critical revision of the manuscript. References Isolation and characterization of Helicobacter suis sp. nov. from pig stomachs. Int. J. Syst. Evol. Microbiol. 58 1350–1358 (2008). 56. Joosten, M. et al. Diversity in bacterium-host interactions within the species Helicobacter heilmannii sensu stricto. Vet. Res. 4 (2013). ( ) 57. Vermoote, M. et al. Antimicrobial susceptibility pattern of Helicobacter suis strains. Vet. Microbiol. 153, 339–342 (2011). 8. Flahou, B. et al. The local immune response of mice after Helicobacter suis infection: strain differences and distinction with Helicobacter pylori. Vet. Res. 43, 75 (2012). Helicobacter pylori. Vet. Res. 43, 75 (2012). py 59. Sugimoto, M., Ohno, T., Graham, D. Y. & Yamaoka, Y. Gastric mucosal interleukin-17 and -18 mRNA expression in Helicobacter pylori-induced Mongolian gerbils. Cancer Sci. 100, 2152–2159 (2009). py g g 0. Crabtree, J. E. et al. Gastric mucosal cytokine and epithelial cell responses to Helicobacter pylori infection in Mongolian gerbils J. Pathol. 202, 197–207 (2004). 61. Chirgwin, S. R. et al. Infection outcome and cytokine gene expression in Brugia pahangi-infected gerbils (Meriones unguiculatus) sensitized with Brucella abortus. Infect. Immun. 70, 5938–5945 (2002). References & Hazell, S. L. The tricarboxylic acid cycle of Helicobacter pylori. Eur. J. Biochem. 1 258–267 (1999). 31. Pitson, S. M., Mendz, G. L., Srinivasan, S. & Hazell, S. L. The tricarboxylic acid cycle of Helicobacter pylori. Eur. J. Bioche 258–267 (1999). 2. Krishnan, N. & Becker, D. F. Oxygen reactivity of PutA from Helicobacter species and proline-linked oxidative species. J. Bacteriol 188, 1227–1235 (2006). ( ) 3. Nagata, K. et al. L-serine, D- and L-proline and alanine as respiratory substrates of Helicobacter pylori: correlation between in vitro and in vivo amino acid levels. Microbiology 149, 2023–2030 (2003). gy 34. Hughes, N. J., Clayton, C. L., Chalk, P. A. & Kelly, D. J. Helicobacter pylori porCDAB and oorDABC genes encode distinct pyruvate:flavodoxin and 2-oxoglutarate: acceptor oxidoreductases which mediate electron transport to NADP. J. Bacteriol. 180, 1119–1128 (1998). ( ) 35. Scott, T., Kondadi, P. K., Hänninen, M. & Rossi, M. Comparative Genomics of Helicobacter pylori and the human-derived Helicobacter bizzozeronii CIII-1 nature of non-pylori gastric Helicobacter infections in humans. BMC Genomics 12, 534 (2011). h l l l Th l l f l py g 36. Lam, H. M., Coschigano, K. T., Oliveira, I. C., MeloOliveira, R. & Coruzzi, G. M. The molecular-genetics of nitrogen assimilation into amino acids in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 569–593 (1996).f 37. Roth, E., Spittler, A. & Oehler, R. Glutamine: Effects on the immune system, protein metabolism and intestinal function. Klein. Wochensch. 108, 669–676 (1996). 38. Wu, G. et al. Important roles for L-glutamine in swine nutrition and production. J. Anim. Sci. 89, 2017–2030 (2011).f 38. Wu, G. et al. Important roles for L-glutamine in swine nutrition 38. Wu, G. et al. Important roles for L-glutamine in swine nutrition and production. J. Anim. Sci. 89, 2017–2030 (2011). 39 Vicarcio M et al Dietary glutamine affects mucosal functions in rats with mild DSS induced colitis J Nutrition 137 1 39. Vicarcio, M. et al. Dietary glutamine affects mucosal functions in rats with mild DSS-induced colitis. J. Nutrition. 137, 1931 (2007).f 0. Nakamura, E. et al. Prophylactic effect of glutamine and marzulene against Helicobacter pylori-related gastric diseases in Mongolian gerbils. Jpn. Pharmacol. Ther. 35, 479–488 (2007).l h 41. Hagen, S. et al. Inflammation and foveolar hyperplasia are reduced by supplemental dietary glutamine during Helicobacter pylori infection in mice. J. Nutr. 22, 912–918 (2009).f 2. Amagase, A. et al. References New frontiers in gut nutrient sensor research: prophylactic effect of glutamine against Helicobacter pylori-induced gastric diseases in Mongolian gerbils. J. Pharmacol. Sci. 112, 25–32 (2010). 43. Nakamura, E. & Hagen, S. Role of glutamine and arginase in protection against ammonia-induced cell death in gastric epithelial cells. Am. J. Physiol-Gastr. L. 283, G1264–G1275 (2002). 44. Evans, M. E., Jones, D. P. & Ziegler, T. R. Glutamine prevents cytokine-induced apoptosis in human colonic epithelial cells. J. Nutr. 133, 3065–3073 (2003). 45. Wagner, S. et al. Regulation of gastric epithelial cell growth by Helicobacter pylori: Evidence for a major role of apopt Gastroenterol. 113, 1836–1847 (1997). , ( ) 46. Shirin, H. & Moss, S. F. Helicobacter pylori induced apoptosis. Gut 45, 592–594 (1998). 46. Shirin, H. & Moss, S. F. Helicobacter pylori induced apoptosis. Gut 45, 592–594 (1998). 7. Gong, M., Ling, S. S. M., Lui, S. Y., Yeoh, K. G. & Ho, B. Helicobacter pylori γ-glutamyl transpeptidase is a pathogenic factor in the development of peptic ulcer disease. Gastroenterol. 139, 564–573 (2010). 48. Ricci, V., Giannouli, M., Romano, M. & Zarrilli, R. Helicobacter pylori gamma-glutamyl transpeptidase and its pathogenic World J. Gastroenterol. 20, 630–638 (2014). 49. Schmees, C. et al. Inhibition of T-cell proliferation by Helicobacter pylori γ-glutamyl transpeptidase. Gastroenterol. 132, 1820– (2007). 50. Meister, A. & Anderson, M. E. Glutathione. Ann. Rev. Biochem. 52, 711–760 (1983). , J., Jain, A. & Meister, A. Glutathione is required for intestinal func 51. Martensson, J., Jain, A. & Meister, A. Glutathione is required for intestinal function. Proc. Natl. Acad. Sci. 87, 1715 1719 (1990 52. Circu, M. L. & Aw, T. Y. Glutathione and apoptosis. Free Radical Res. 42, 689–706 (2008).hfl q 52. Circu, M. L. & Aw, T. Y. Glutathione and apoptosis. Free Radical Res. 42, 689–706 (2008).hf 52. Circu, M. L. & Aw, T. Y. Glutathione and apoptosis. Free Radical Res. 42, 689–706 (2008). l Th ff f d l h d h d f fl b l d 53. Liu, C. et al. The effect of dietary glutathione and coenzyme Q10 on the prevention and treatment of inflammatory bowel disea mice. Int. J. Vitam. Nutr. Res. 74, 74–85 (2004). 4. Liang, J. et al. Purification of Helicobacter suis strains from biphasic cultures by single colony isolation: influence on strain characteristics. Helicobacter 20, 206–216 (2015). characteristics. Helicobacter 20, 206–216 (2015). 5. Baele, M. et al. References Metabolism of glutamine and glutathione via γ glutamyltranspeptidase and glutamate transport in Helicobact pylori: possible significance in the pathophysiology of the organism. Mol. Microbiol. 64, 396–406 (2007). i 21. Vermoote, M. et al. Genome sequence of Helicobacter suis supports its role in gastric pathology. Vet. Res. 42, 51 (2011). 22. Rossi, M. et al. Evidence of conserved function of γ-glutamyltranspeptidase in Helicobacter Genus. PloS One 7, e30543 23. Aledo, C. J. Glutamine breakdown in rapidly dividing cells: waste or investment? Bioessays 26, 778–785 (2004). 23. Aledo, C. J. Glutamine breakdown in rapidly dividing cells: was 24. DeBerardinis, R. J. & Cheng, T. Q’s next: the diverse functions of glutamine in metabolism, cell biology and cancer. Oncogen 313–324 (2010).hf 5. Hsu, C. B., Huang, H. J., Wang, C. H., Yen, W. H. T. & Yu, B. The effect of glutamine supplement on small intestinal morphology and xylose absorptive ability of weaned piglets. Afr. J. Biotechnol. 9, 7003–7008 (2010). 25. Hsu, C. B., Huang, H. J., Wang, C. H., Yen, W. H. T. & Yu, B. The effect of glutamine supplement xylose absorptive ability of weaned piglets. Afr. J. Biotechnol. 9, 7003–7008 (2010). 6. Shibayama, K. et al. Apoptotic signaling pathway activated by Helicobacter pylori infection and increase of apoptosis-inducin activity under serum-starved conditions. Infect. Immun. 69, 3181–3189 (2001). y f 27. Zhang, H. & Forman, H. J. Redox regulation of gamma-glutamyl transpeptidase. Am. J. Resp. Cell. Mol. 41, 510–515 (2009). Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 11 www.nature.com/scientificreports/ 8. Drozdz, R. et al. Gamma-glutamyltransferase dependent generation of reactive oxygen species from a glutathione/transferrin system. Free Radical Bio. Med. 25, 786–792 (1998). y 29. Leduc, D., Gallaud, J., Stingl, K. & De Reuse, H. Coupled amino acid deamidase-transport systems essential for Helicobacter colonization. Infect. Immun. 78, 2782–792 (2010). f ( ) 30. Schilling, C. H. et al. Genome-scale metabolic model of Helicobacter pylori 26695. J. Bacteriol. 184, 4582–4593 (2002). d ll Th b l d l f l b l 30. Schilling, C. H. et al. Genome-scale metabolic model of Helicobacter pylori 26695. J. Bacteriol. 184, 4582–4593 (2002). 31. Pitson, S. M., Mendz, G. L., Srinivasan, S. & Hazell, S. L. The tricarboxylic acid cycle of Helicobacter pylori. Eur. J. Biochem. 1 30. Schilling, C. H. et al. Genome-scale metabolic model of Helicobacter pylori 26695. J. Bacteriol. 184, 4582–4593 (2002). 31. Pitson, S. M., Mendz, G. L., Srinivasan, S. Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 Additional Information upplementary information accompanies this paper at http://www.nature.com/srepi Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: De Bruyne, E. et al. Oral glutathione supplementation drastically reduces Helicobacter- induced gastric pathologies. Sci. Rep. 6, 20169; doi: 10.1038/srep20169 (2016). How to cite this article: De Bruyne, E. et al. Oral glutathione supplementation drastically reduces Helicobacter- nduced gastric pathologies. Sci. Rep. 6, 20169; doi: 10.1038/srep20169 (2016). Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 12 www.nature.com/scientificreports/ This work is licensed under a Creative Commons Attribution 4.0 International License. The imag or other third party material in this article are included in the article’s Creative Commons licens unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licens users will need to obtain permission from the license holder to reproduce the material. 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The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ www.nature.com/scientificreports/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:20169 \| DOI: 10.1038/srep20169 13
https://openalex.org/W2028652322	https://hal.science/hal-02457008/document	English	null	Maternal Effects on Anogenital Distance in a Wild Marmot Population	PloS one	2,014	cc-by	5,033	To cite this version: Timothée D Fouqueray, Denis Blumstein, Julien G. A. Martin. Maternal Effects on Anogeni- tal Distance in a Wild Marmot Population. PLoS ONE, 2014, 9 (3), pp.e92718. 10.1371/jour- nal.pone.0092718. hal-02457008 Introduction they might contribute to offspring defeminisation or masculinisa- tion. However, the importance of this maternal source has not been studied in the wild. Maternal effects represent the influence of a mother’s genotype or phenotype to her offspring’s phenotype independently of additive genetic effects [1] and have numerous implications in a wide range of species on prenatal (development, immunity, stress level) and postnatal life (social rank, dispersal, reproductive performance, etc.) [2–5]. Early exposure to androgens is morphologically evident by the anogenital distance (AGD), the distance between the anus and the genital papilla. AGD results from the elongation of the perineal tissue, which is triggered by testosterone during the early development, and thus, males have larger AGD than females [13,14]. AGD, at young ages, is thus a proxy of early exposure to androgens [15,16] and is relatively easy to quantify under field conditions. Females (of several species) with larger AGD are masculinized. They are less likely to survive and more likely to disperse [10], are less likely to become pregnant [17], are less preferred by males, and have smaller and male-biased litters [18]. Similar findings have been observed as well in humans, including positive correlations between AGD and fertility in men [19], and ovarian follicles numbers in women [20]. For mammals, the intrauterine environment is a potentially important source of maternal effects on a variety of life history traits [6]. Pre-natal exposure to sex steroid hormones emerges from the complex hormonal environment fetuses are exposed to. Each individual is exposed to androgens produced by its male siblings via transplacental and transamniotic diffusion [7]. Testosterone has organisational and activational effects on the central nervous system, masculinising and defeminising the behavior exhibited later in life [8,9]. For instance, female rats (Rattus norvegicus) exposed to neonatal testosterone engaged in more masculinised impulsive behavior [9]. Similarly, masculinised yellow-bellied female marmots (Marmota flaviventris) had lower survival, were more likely to disperse, and had reduced weaning success compared with females exposed to reduced levels of intrauterine testosterone [10]. Thus, testosterone is an important modulator of life-history traits [10]. We studied a population of individually marked wild yellow- bellied marmots for which litter effects but not maternal effects on anogenital distance were known: female yellow-bellied marmots’ morphology, behavior, survival, and dispersal are influenced by the proportion of males in the litter [4,10]. Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: marmots@ucla.edu Maternal Effects on Anogenital Distance in a Wild Marmot Population Timothe´e D. Fouqueray1,2, Daniel T. Blumstein1,3, Raquel Monclu´ s4, Julien G. A. Martin1,5* 1 Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America, 2 De´partement de Biologie, E´cole Normale Supee´rieure de Lyon, Lyon, France, 3 The Rocky Mountain Biological Laboratory, Crested Butte, Colorado, United States of America, 4 Departamento de Biologı´a, Universidad Auto´noma de Madrid, Madrid, Spain, 5 School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom Abstract In mammals, prenatal exposure to sex steroid hormones may have profound effects on later behavior and fitness and have been reported under both laboratory and field conditions. Anogenital distance is a non-invasive measure of prenatal exposure to sex steroid hormones. While we know that intra-uterine position and litter sex ratio influence anogenital distance, there are other, heretofore unstudied, factors that could influence anogenital distance, including maternal effects. We capitalized on a long-term study of wild yellow-bellied marmots (Marmota flaviventris) to study the importance of maternal effects on explaining variation in anogenital distance and found significant effects. The strength of these effects varied annually. Taken together, our data highlights the strong variability due to environmental effects, and illustrates the importance of additive genetic and maternal genetic effects on neonatal anogenital distance. We suspect that, as others apply recently popularised quantitative genetic techniques to study free-living populations, such effects will be identified in other systems. Citation: Fouqueray TD, Blumstein DT, Monclu´s R, Martin JGA (2014) Maternal Effects on Anogenital Distance in a Wild Marmot Population. PLoS ONE 9(3): e92718. doi:10.1371/journal.pone.0092718 Editor: Maria` Alemany, University of Barcelona, Faculty of Biology, Spain Editor: Maria` Alemany, University of Barcelona, Faculty of Biology, Spain Received December 4, 2013; Accepted February 25, 2014; Published March 20, 2014 Received December 4, 2013; Accepted February 25, 2014; Published March 20, 2014 Copyright: 2014 Fouqueray 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: 2014 Fouqueray et al. This is an open-access article distributed under the terms of the Creative Comm unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: DTB was supported by the National Geographic Society, UCLA (Faculty Senate and the Division of Life Sciences), a Rocky Mountain Biological Laboratory research fellowship, and by the NSF (IDBR-0754247 and DEB-1119660 to DTB, as well as DBI 0242960 and 0731346 to the Rocky Mountain Biological Laboratory). RM was supported by postdoctoral fellowships from the Spanish Ministerio de Innovacio´n y Ciencia and the Fulbright program. JGAM was supported by a FRQNT postdoctoral fellowship and the NSF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. March 2014 \| Volume 9 \| Issue 3 \| e92718 HAL Id: hal-02457008 https://hal.science/hal-02457008v1 Submitted on 27 Jan 2020 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. Results : Females taken as reference {: N males: N total DF: numerator, denominator degrees of freedom F-cond: conditional Wald F-test doi:10.1371/journal.pone.0092718.t001 Estimate (SE) DF F-cond P (Intercept) 4.899 (0.808) 1, 33.5 36.750 ,0.001 Sex [Male] 4.205 (0.185) 1, 468.1 514.300 ,0.001 Mass 0.010 (0.001) 1, 388.7 124.100 ,0.001 Litter sex-ratio{ 1.749 (0.452) 1, 243.6 15.000 ,0.001 Litter size 20.058 (0.063) 1, 187.5 0.856 0.356 Days since emergence 20.038 (0.038) 1, 453.7 0.963 0.327 Maternal Effects on Anogenital Distance variance components, to assess how much of the variance in AGD in neonates was due to maternal, genetic and environmental effects. By fitting different random effects that are linked or not linked to a pedigree, it is thus possible to decompose the variance of a trait into its genetic and environmental effects. Random effects linked to the pedigree provide information of additive genetic variance whereas random effects not linked to the pedigree provide environmental variance estimates. Since an animal model is a mixed effect model, additional fixed effects could also be included to correct for potential biases in the variance estimates. By fitting different random effects that are linked or not linked to a pedigree, it is thus possible to decompose the variance of a trait into its genetic and environmental effects. Random effects linked to the pedigree provide information of additive genetic variance whereas random effects not linked to the pedigree provide environmental variance estimates. Since an animal model is a mixed effect model, additional fixed effects could also be included to correct for potential biases in the variance estimates. Results As expected from previous analyses [10], we found a significant effect of litter sex ratio on AGD (Table 1), whereby pups in male- biased litters had greater AGDs. Male AGD was significantly greater than female AGD (Table 1) as previously [10,29]. In addition, larger animals had larger AGDs (Table 1). For our sample of animals trapped within 10 days of emergence, there was no significant effect of the number of days since emergence, nor was there a significant effect of litter size (Table 1). Starting in 2002, we took a hair sample from every individual for DNA parentage assignment using 12 microsatellite loci. This permitted us to assign (with 95% confidence) paternity and maternity (for further details about the procedure and the study population, see [21]). The average litter size for yellow-bellied marmots is about 3–4 pups but varies from 1 to 10 pups. Females give birth at most once a year in the burrows, where the pups stay during lactation. They emerge about 25 days later to start foraging by themselves [22]. Litter sex-ratio was estimated using all pups trapped. For AGD analysis, however, we only used those individuals with full parentage and that were trapped within their first 10 days following natal emergence because AGD at that age is not as biased by morphological differences between individuals as it would be for older individuals [23]. We thus used AGD records from 564 pups, from 183 different litters, that were produced by 91 different mothers between 2002 and 2010. This represented 67% of all pups observed during that period. There were significant maternal effects at the phenotypic level accounting for 5.8% of the remaining variance (Fig. 1, Table 2, model 1, LRT = 3.98, df = 1, P = 0.046). When we decomposed the variance into additive genetic (LRT = 1.51, df = 1, P = 0.219), Table 1. Estimates (with standard error) of the fixed effects on anogenital distance of juvenile yellow-bellied marmots studied at the Rocky Mountain Biological Laboratory. Estimate (SE) DF F-cond P (Intercept) 4.899 (0.808) 1, 33.5 36.750 ,0.001 Sex [Male]* 4.205 (0.185) 1, 468.1 514.300 ,0.001 Mass 0.010 (0.001) 1, 388.7 124.100 ,0.001 Litter sex-ratio{ 1.749 (0.452) 1, 243.6 15.000 ,0.001 Litter size 20.058 (0.063) 1, 187.5 0.856 0.356 Days since emergence 20.038 (0.038) 1, 453.7 0.963 0.327 Estimates significantly different from zero are in bold. Ethics Statement Free-living yellow-bellied marmots were studied under research protocol ARC 2001-191-01 as well as permits issued by the Colorado Division of Wildlife. The research protocol was approved by the UCLA Animal Care Committee on 13 May 2002 and renewed annually. Trapping, measuring, and marking marmots are routine techniques that have been conducted with no deleterious consequences for over 50 years in this population of marmots. By design, marmots were not harmed during the course of this study. As fixed effects, we included mass (to correct for differences in body size), the number of days since emergence at trapping (to take into account the morphological development of the perineal tissue), sex (to control for sexual dimorphism), proportion of males in the litter (to control for litter effects), and litter size (to control for the number of siblings producing androgens). We tested the significance of the fixed effects using conditional Wald tests. For random effects, we first fitted a model including only maternal identity (to estimate between mother variation), year (to assess the inter-annual environmental variation), and the identity of the litter. We then decomposed the maternal effects into its genetic and environmental components. Thus, we included pup identity linked to the pedigree (direct additive genetic effect), maternal identity (maternal environment), maternal identity linked to the pedigree (maternal genetic), year, and the identity of the litter. We tested the significance of random effects using a log- likelihood ratio test comparing the full model to a model without a specific random effect [27,28]. Variance ratios for random effects were estimated using the estimated phenotypic variance from the animal model (i.e., the sum of variance parameters in the model after accounting for the fixed effects [28]). Experimental Subjects Yellow-bellied marmots have been studied since 1962 in and around the Rocky Mountain Biological Laboratory, Gunnison County, CO, USA (elevation approx. 2890 m). Marmots are individually marked and followed throughout their lives. Natal emergence date was determined by daily observations of the colonies during the active season (mid-April to mid-September). As soon as they emerged from their natal burrow, pups were trapped, sexed, ear tagged, and fur marked with Nyanzol fur dye. Assigning marmots to sex is straightforward and was successfully repeated during numerous trapping sessions by different observers. Subjects were weighed using a digital scale (accurate to 25 g) and we measured the AGD using digital callipers (accuracy 1 mm). Every summer, new observers were trained to measure AGD accurately by making multiple AGD measurements on different individuals until they obtained measurements similar to trained observers. This training ensured consistency in our measurements across observers. Since all pups within a litter were not trapped and measured by the same observers, measurement error due to the observer should only increase the noise in the data and make it more difficult to detect significant effects. Introduction Given the strong relation between androgenization and life-history traits later in life, it seems crucial to disentangle the different sources of variation that might affect the offspring phenotype. For that we fitted an animal model, a statistical technique designed to decompose Apart from male siblings’ testosterone, androgens are secreted by the mother’s placenta, adrenal glands, and ovaries [11,12], and PLOS ONE \| www.plosone.org March 2014 \| Volume 9 \| Issue 3 \| e92718 1 March 2014 \| Volume 9 \| Issue 3 \| e92718 Statistical Analyses Using the asreml function [24] in the statistical package R, v.2.14.1 [25], we fitted an animal model [26] to decompose the variance of AGD into its additive genetic, maternal (environment and genetic), litter and year components. An animal model is a particular type of mixed model in which the different individuals are not considered independent but related to each other by a matrix of relatedness (most often obtained from a pedigree) [26]. March 2014 \| Volume 9 \| Issue 3 \| e92718 PLOS ONE \| www.plosone.org 2 Maternal Effects on Anogenital Distance Table 2. Estimates (with standard error) of variance components and proportion of variance of anogenital distance in yellow-bellied marmot pups. Variance component Variance ratio Model LL AG MG ME L Y P AG MG ME L Y 1. ME + L + Y 2704.49 - - 0.390 (0.227) 0.311 (0.236) 2.480 (1.387) 6.728 (1.447) - - 0.058 (0.036) 0.046 (0.036) 0.368 (0.220) 2. AG + MG +ME + L + Y 2701.56 0.613 (0.558) 0.287 (0.304) 0.000 (0.000) 0.252 (0.207) 2.450 (1.372) 6.800 (1.572) 0.090 (0.084) 0.042 (0.045) 0.000 (0.000) 0.037 (0.031) 0.360 (0.218) 3. AG +ME + L + Y 2702.14 0.960 (0.518) - 0.029 (0.207) 0.300 (0.236) 2.521 (1.407) 6.847 (1.574) 0.140 (0.082) - 0.004 (0.030) 0.043 (0.035) 0.368 (0.222) 4. MG +ME + L + Y 2702.32 - 0.542 (0.260) 0.000 (0.000) 0.274 (0.208) 2.409 (1.348) 6.742 (1.411) - 0.080 (0.042) 0.000 (0.000) 0.040 (0.032) 0.357 (0.213) AG: additive genetic; MG: maternal genetic; ME: maternal environment; L: litter; Y: year; P: phenotypic; LL: log-likelihood; Variance ratio of AG is the heritability, h2. The raw phenotypic variance of AGD was 13.272. The phenotypic variance of AGD after controlling for the fixed effects was estimated as 6.743 (1.411). Significant random effects are in bold. doi:10.1371/journal.pone.0092718.t002 maternal environment (LRT = 1.18, df = 1, P = 0.277), and maternal genetic (LRT ,0.001, df = 1, P.0.999) components, we found that none of them were significant (Table 2, model 2). However, the inclusion of both additive genetic and maternal genetic effects provided a marginally better fit than when they were excluded (Fig. 1, Table 2, model 1 vs. model 2: LRT = 5.56, df = 2, P = 0.053). Statistical Analyses In addition, additive genetic (LRT = 4.70, df = 1, P = 0.030) and maternal genetic (LRT = 4.39, df = 1, P = 0.036) effects were both found to be significant if only one of them was included in the model and their estimates were larger (Fig. 1, Table 2, models 3 and 4). This suggests that despite nearly a decade’s worth of data, we had insufficient power to conclusively isolate both effects. Nonetheless, it suggests that both effects could be important. When a maternal genetic effect was included in a model, the maternal environment explained ,0.001 of the variance, a finding that suggests that most of the variance between mothers were due to maternal genetic effects. Litter identity effects were not significant in any model (Fig. 1, Table 2, all P.0.141). Year was significant in all models (all P,0.001) and explained 36% of the remaining variance in AGD (Fig. 1, Table 2). References Meaney MJ, McEwen BS (1986) Testosterone implants into the amygdala during the neonatal period masculinize the social play of juvenile female rats. Brain Research 398: 324–328. 24. Gilmour A, Gogel B, Cullis, Thompson R (2009) ASReml user guide Release 3.0 URL. Available: http://wwwvsnicouk/downloads/asreml/release2/doc/ asreml-Rpdf. Accessed 2014 Feb 27. 9. Bayless DW, Darling JS, Daniel JM (2013) Mechanisms by which neonatal testosterone exposure mediates sex differences in impulsivity in prepubertal rats. Hormones and Behavior 64: 764–769. 25. R Development Core Team (2011) R: A language and environment for statistical computing R Foundation for Statistical Computing. Available: http:// wwwR-projectorg/. Accessed 2014 Feb 27. 10. Monclu´s R, Blumstein DT (2012) Litter sex composition affects life-history traits in yellow-bellied marmots. Journal of Animal Ecology 81: 80–86. 26. Kruuk LEB, Hadfield JD (2007) How to separate genetic and environmental causes of similarity between relatives. Journal of Evolutionary Biology 20: 1890– 1903. 11. Sridaran R, Basuray R, Gibori G (1981) Source and regulation of testosterone secretion in pregnant and pseudopregnant rats. Endocrinology 108: 855–861. 27. Pinheiro J, Bates D (2000) Mixed-Effects Models in S and S-PLUS. New York: Springer. 530 p. 12. Do¨hler KD, Jarzab B, Sickmo¨ller PM, Kokocinska D, Kaminski M, et al. (1991) Influence of neurotransmitters on sexual differentiation of brain structure and function. Experimental and Clinical Endocrinology 98: 99–109. 28. Wilson AJ, Re´ale D, Clements MN, Morrissey MM, Postma E, et al. (2010) An ecologist’s guide to the animal model. Journal of Animal Ecology 79: 13–26. p gy 13. vom Saal FS, Bronson FH (1980) Sexual characteristics of adult female mice are correlated with their blood testosterone levels during prenatal development. Science 208: 597–599. 29. Monclu´s R, Cook T, Blumstein DT (2011) Masculinized female yellow-bellied marmots initiate more social interactions. Biology Letters 8: 208–210. 30. Chawnshang C (2002) Androgens and Androgen Receptor: Mechanisms, Functions, and Clinical Applications. New York: Springer. 503 p. 14. Zehr JL, Gans SE, McClintock MK (2001) Variation in reproductive traits is associated with short anogenital distance in female rats. Developmental Psychobiology 38: 229–238. 31. vom Saal FS (1989) Sexual differentiation in litter-bearing mammals: influence of sex of adjacent fetuses in utero. Journal of Animal Science 67: 1824–1840. 15. Palanza P, Parmigiani S, vom Saal FS (1995) Urine marking and maternal aggression of wild female mice in relation to anogenital distance at birth. Physiology and Behavior 58: 827–835. 32. References 17. Drickamer LC (1996) Intra-uterine position and anogenital distance in house mice: consequences under field conditions. Animal Behaviour 51: 925–934. 1. Wolf JB, Wade MJ (2009) What are maternal effects (and what are they not)? Philosophical Transactions of the Royal Society Series B 364: 1107–1115. 18. Ba´nszegi O, Szenczi P, Dombay K, Bilko´ A, Altba¨cker V (2012) Anogenital distance as a predictor of attractiveness, litter size and sex ratio of rabbit does. Physiology and Behavior 105: 1126–1230. 2. Ro¨del HG, Starkloff A, Bruchner B, von Holst D (2008) Social environment and reproduction in female European rabbits (Oryctolagus cuniculus): Benefits of the presence of litter sisters. Journal of Comparative Psychology 122: 73–83. 19. Eisenberg ML, Hsieh MH, Walters RC, et al. (2011) The Relationship between Anogenital Distance, Fatherhood, and Fertility in Adult Men. PLoS ONE 6(5): e18973. 3. Duckworth RA (2009) Maternal effects and range expansion: a key factor in a dynamic process? Philosophical Transactions of the Royal Society Series B 364: 1075–1086. 20. Mendiola J, Roca M, Minguez-Alarcon L, Mira-Escolanao M-P, Lo´pez-Espin JJ, et al. (2012) Anogenital distance is related to ovarian follicular number in young Spanish women: a cross-sectional study. Environmental Health 11: 90. 4. Monclu´s R, Tiulim J, Blumstein DT (2011) Older mothers follow conservative strategies under predator pressure: The adaptive role of maternal glucocorticoids in yellow-bellied marmots. Hormones and Behavior 60: 660–665. 21. Blumstein DT, Lea AJ, Olson LE, Martin JGA (2010) Heritability of anti- predatory traits: vigilance and locomotor performance in marmots. Journal of Evolutionary Biology 23: 879–887. 5. Garnier R, Boulinier T, Gandon S (2012) Coevolution between maternal transfer of immunity and other resistance strategies against pathogens. Evolution 66: 3067–3078. 22. Armitage KB (2003) Marmots: Marmota monax and allies. In: Feldhamer GA, Thompson BC, Chapman JA, editors. Wild mammals of North America. The John Hopkins University Press, Baltimore, pp. 188–210. 6. Ryan BC, Vandenbergh JG (2002) Intrauterine position effects. Neuroscience and Biobehavioral Reviews 26, 665–678. 7. Even MD, Dhar MG, vom Saal FS (1992) Transport of steroids between fetuses via amniotic fluid in relation to the intrauterine position phenomenon in rats. Journal of Reproduction and Fertility 96: 709–716. 23. Vandenbergh J (2009) Effects of intrauterine position in litter-bearing mammals. In: Maestripieri D, Mateo JM, editors. Maternal Effects in Mammals. Chicago: The University of Chicago Press. pp. 203–226. 8. Acknowledgments We thank all the members of Team Marmot who helped collect data over the past decade. Maternal Effects on Anogenital Distance Nevertheless, further quantitative measurements of maternal and pup androgen levels, in addition to formal genetic analyses of maternal endocrinological traits are needed to formally evaluate the mechanism underlying the maternal genetic effect on AGD. Such studies, beyond the scope of our study of free-living animals, would greatly improve our understanding of the mechanistic origin of the maternal effect we identified. variation. Despite extensive training, personnel turnover between years could be responsible for some of this variation. However, other biologically relevant factors could also explain annual variation. For instance, the current environment (food availability, predation level, weather conditions) could also explain annual variation through its influence on maternal stress and body condition [4,10]. In conclusion, AGD is a widely used proxy of early exposure to testosterone, and it is related to numerous other life-history traits [4,10,16,38]. We showed that by decomposing sources of variation using the animal model, we were able to identify, for the first time in a wild population, both an additive genetic and a maternal genetic effect on AGD that is expected to also impact future life- history traits and fitness of the pups. When other researchers quantify the magnitude of maternal effects on AGD and its genetic basis, we will be in a better position to understand its general importance. Author Contributions While the existence of a significant litter composition fixed effect agrees with previous findings [10], it is noteworthy that the bulk of the remaining variation in AGD variance was explained by annual Conceived and designed the experiments: JGAM. Performed the experiments: TDF DTB RM JGAM. Analyzed the data: TDF DTB RM JGAM. Wrote the paper: TDF DTB RM JGAM. Conceived and designed the experiments: JGAM. Performed the experiments: TDF DTB RM JGAM. Analyzed the data: TDF DTB RM JGAM. Wrote the paper: TDF DTB RM JGAM. Discussion We have shown that 6% of the remaining variation in the AGD of marmot pups is explained by a maternal effect. Our results also suggest that AGD is heritable and influenced by maternal genetic and non-maternal environmental effects, thus indicating that a mother’s genotype produces specific developmental conditions for her offspring. The presence of a maternal genetic effect on AGD could result from various mechanisms during gestation or lactation. It could be due to genetic differences in maternal care, maternal milk content or maternal stress. A more promising avenue of explanation might reside in utero during development. AGD is widely used as a proxy for prenatal androgen hormone exposure in rodents [14]. Prenatal exposure to androgens may influence vertebrate life-history traits [29–32], because organiza- tional and activational events take place at this stage. This androgen-sensitive prenatal developmental window is thus a key period that may influence life-history traits. Here, a possible Table 2. Estimates (with standard error) of variance components and proportion o Figure 1. Variance ratio and components estimated for random effects from anogenital distance models of juvenile marmots. The random effects were additive genetic (AG), maternal genetic (MG), maternal environmental (ME), litter (L) and year (Y) effects. N indicates that an effect was not fitted in the model and a 0 indicates that the effect was fitted but estimated as zero. * indicates significant effects. doi:10.1371/journal.pone.0092718.g001 Figure 1. Variance ratio and components estimated for random effects from anogenital distance models of juvenile marmots. The random effects were additive genetic (AG), maternal genetic (MG), maternal environmental (ME), litter (L) and year (Y) effects. N indicates that an effect was not fitted in the model and a 0 indicates that the effect was fitted but estimated as zero. * indicates significant effects. doi:10.1371/journal.pone.0092718.g001 March 2014 \| Volume 9 \| Issue 3 \| e92718 March 2014 \| Volume 9 \| Issue 3 \| e92718 PLOS ONE \| www.plosone.org 3 Maternal Effects on Anogenital Distance 1. Wolf JB, Wade MJ (2009) What are maternal effects (and what are they not)? Philosophical Transactions of the Royal Society Series B 364: 1107–1115. Maternal Effects on Anogenital Distance explanation of the maternal effects on the AGD would be such a hormonal transfer from the mother to her pups. However, some studies in laboratory rats, Rattus norvegicus, have found no correlation between maternal testosterone levels and fetal testos- terone levels. However, they did find a negative relationship between maternal testosterone levels and the capacity of nutrient transport across the placenta, resulting in reduced fetal growth [33]. Therefore, maternal testosterone might indirectly affect offspring AGD by influencing the offspring mass. In primates, in utero exposure to androgens has been explored mainly through the study of the 2d:4d ratio. There is evidence that up to 66% of the variance is explained by additive genetic effects. In addition, there is evidence of strong environmental effects and for shared environment effects (including maternal environment and part of maternal genetic effects) [34–37]. Our results are thus in line with previous studies suggesting additive genetic, maternal environment and maternal genetic effects on in utero exposition to androgens. Nevertheless, further quantitative measurements of maternal and pup androgen levels, in addition to formal genetic analyses of maternal endocrinological traits are needed to formally evaluate the mechanism underlying the maternal genetic effect on AGD. Such studies, beyond the scope of our study of free-living animals, would greatly improve our understanding of the mechanistic origin of the maternal effect we identified. explanation of the maternal effects on the AGD would be such a hormonal transfer from the mother to her pups. However, some studies in laboratory rats, Rattus norvegicus, have found no correlation between maternal testosterone levels and fetal testos- terone levels. However, they did find a negative relationship between maternal testosterone levels and the capacity of nutrient transport across the placenta, resulting in reduced fetal growth [33]. Therefore, maternal testosterone might indirectly affect offspring AGD by influencing the offspring mass. In primates, in utero exposure to androgens has been explored mainly through the study of the 2d:4d ratio. There is evidence that up to 66% of the variance is explained by additive genetic effects. In addition, there is evidence of strong environmental effects and for shared environment effects (including maternal environment and part of maternal genetic effects) [34–37]. Our results are thus in line with previous studies suggesting additive genetic, maternal environment and maternal genetic effects on in utero exposition to androgens. 38. Kerin TK, Vogler GP, Blizard DA Stout JT, McClearn GE, et al. (2003) Anogenital distance measured at weaning is correlated with measures of blood chemistry and behaviors in 450–day-old female mice. Physiology and Behavior 78: 697–702. 37. Paul SN, Kato BS, Cherkas LF, Andrew T, Spector TD (2006) Heritability of the second to fourth digit ratio (2d:4d): A twin study. Twin Research and Human Genetics 9: 215–219. g ( ) J y p gy 36. Nelson E, Voracek M (2010) Heritability of digit ratio (2D:4D) in rhesus macaques (Macaca mulatta). Primates 51: 1–5. References Field EF, Whishaw IQ, Pellis SM, Watson NV (2006) Play fighting in androgen- insensitive tfm rats: evidence that androgen receptors are necessary for the development of adult playful attack and defense. Developmental Psychobiology 48: 111–120. 16. Vandenbergh JG, Huggett CL (1995) The anogenital distance index, a predictor of the intrauterine position effects on reproduction in female house mice. Laboratory Animal Science 45: 567–573. 33. Sathishkumar K, Elkins R, Chinnathambi V, Gao H, Hankins GD, et al. (2011) Prenatal testosterone-induced fetal growth restriction is associated with PLOS ONE \| www.plosone.org March 2014 \| Volume 9 \| Issue 3 \| e92718 4 March 2014 \| Volume 9 \| Issue 3 \| e92718 yg 35. Voracek M, Dressler SG (2009) Brief communication: Familial resemblance in digit ratio (2D:4D). American Journal of Physical Anthropology 140: 367–380. down-regulation of rat placental amino acid transport. Reproductive Biology and Endocrinology 9: 110. 34. Gobrogge KL, Breedlove SM, Klump KL (2008) Genetic and Environmental Influences on 2D:4D Finger Length Ratios: A Study of Monozygotic and Dizygotic Male and Female Twins. Archives of Sexual Behavior 37: 112–118. 35. Voracek M, Dressler SG (2009) Brief communication: Familial resemblance in digit ratio (2D:4D). American Journal of Physical Anthropology 140: 367–380. 36. Nelson E, Voracek M (2010) Heritability of digit ratio (2D:4D) in rhesus macaques (Macaca mulatta). Primates 51: 1–5. gy 34. Gobrogge KL, Breedlove SM, Klump KL (2008) Genetic and Environmental Influences on 2D:4D Finger Length Ratios: A Study of Monozygotic and Dizygotic Male and Female Twins. Archives of Sexual Behavior 37: 112–118. down-regulation of rat placental amino acid transport. Reproductive Biology and Endocrinology 9: 110. 37. Paul SN, Kato BS, Cherkas LF, Andrew T, Spector TD (2006) Heritability of the second to fourth digit ratio (2d:4d): A twin study. Twin Research and Human Genetics 9: 215–219. 38. Kerin TK, Vogler GP, Blizard DA Stout JT, McClearn GE, et al. (2003) Anogenital distance measured at weaning is correlated with measures of blood chemistry and behaviors in 450–day-old female mice. Physiology and Behavior 78: 697–702. Maternal Effects on Anogenital Distance March 2014 \| Volume 9 \| Issue 3 \| e92718 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5
W4386266779.txt	https://ijssr.ridwaninstitute.co.id/index.php/ijssr/article/download/131/239	en		Improving Mathematics Learning Outcomes Using Discovery Learning in Class XI Software Engineering 1 INFOKOM Bogor Vocational High School	International Journal of Social Service and Research	2,022	cc-by-sa	3,363	INTERNATIONAL JOURNAL OF SOCIAL SERVICE AND RESEARCH IMPROVING MATHEMATICS LEARNING OUTCOMES USING DISCOVERY LEARNING IN CLASS XI SOFTWARE ENGINEERING 1 INFOKOM BOGOR VOCATIONAL HIGH SCHOOL Putri Riandini Mathematics and Science Study Program, Faculty of Postgraduate, Universitas Indraprasta PGRI, South Jakarta, Indonesia Email: puputliebe34@gmail.com Abstract This study aims to determine how to improve mathematics learning outcomes by using the Discovery Learning model. This research is Classroom Action Research (CAR). This research was conducted in 2 cycles, namely, cycle 1 and cycle 2. The research was carried out at Vocational High School INFOKOM Bogor class XI Software Engineering (RPL) 1 with a total of 25 students, consisting of 17 male and 8 female students. In pre-cycle activities, the average score obtained by students was 59.4, with the number of students who completed 9 people (36%). In cycle 1 activities, the average value of students obtained increased to 70.6 with the number of students who completed increased to 14 people or by 56%. In cycle 2, the average score of students increased to 90.6, with the number of completed students increasing to 22 (88%). Referring to the results obtained in cycles 1 and 2, it can be concluded that the Discovery Learning model can improve mathematics learning outcomes in class XI Software Engineering 1 Vocational High School INFOKOM Bogor. Keywords: discovery learning; mathematics; learning outcomes Received 03 July 2022, Revised 14 July 2021, Accepted 22 July 2022 INTRODUCTION Mathematics is a compulsory subject that is taught from elementary to the college level as stated in the Regulation of the Minister of National Education of the Republic of Indonesia number 22 of 2006 (Depdiknas, 2006). Mathematical comprehension is the mental foundation for solving math and daily situations (Sari, Gistituati, & Syarifuddin, 2019). Mathematics subjects need to be given to all students starting from elementary school to equip students with logical, analytical, systematic, critical, and creative thinking skills as well as the ability to cooperate. These competencies are needed so that students can have the ability to https://ijssr.ridwaninstitute.co.id/index.php/ijssr/ obtain, manage, and utilize information to survive in conditions that are constantly changing, uncertain, and competitive. There are several competencies or abilities that students must learn and master during the mathematics learning process in the classroom, including thinking and reasoning mathematically, arguing and communicating mathematically, modelling, compiling and solving problems, representations, symbols, and using and utilizing tools and technology (Shadiq, 2007). Moreover, Numerical knowledge is essential for other mathematical learnings, and the necessity of mathematical processes for achieving greater performance in daily life is International Journal of Social, Service and Research, 2(7), 605-610 underlined (Coronata & Alsina, 2014). Therefore, learning mathematics is necessary to support learning success at a higher level or in the face of increasingly developing technology. Although several competencies or abilities must be mastered by students, in reality, students still experience difficulties learning mathematics at school. Many students still have difficulty solving problems to solve given math problems. Moreover, in Africa many children struggle with numbers and the four fundamental operations (Maruyama & Kurosaki, 2021). This has an impact on student learning outcomes are low. Pre-cycle student learning outcomes data shows the average value of students is 59.4. This can be seen from the data on student learning outcomes carried out in class XI RPL 1 in Mathematics with threedimensional material. From these data, it can be seen that there are still many low student scores, which are still below the KKM (Minimum Completeness Criteria). The KKM score that should be obtained is 75. However, from the number of students, as many as 25 people, only 9 students or about 36%, have the correct score and are above the KKM. The remaining 16 students, about 64%, are still below the KKM. Several factors affect learning outcomes. Factors that influence learning outcomes include internal factors consisting of physiological aspects (physical, eye, ear) and psychological aspects (intellective factors: talent, intelligence and nonintellectual factors: attitudes, interests, needs, motivation), external factors consisting of the social environment (family, teachers, community, and friends), and non-social environment (conditions of the home, school, equipment around, and nature), and learning approach factors (Syah, 2012). In addition, students' classroom learning will be more engaging and able to cultivate critical thinking abilities if they are taught how to conduct research beginning with simple ways to find a 605 conclusion or knowledge base (Fahmi, Setiadi, Elmawati, & Sunardi, 2019). One factor that influences learning outcomes is a teacher's role. A teacher plays an essential role in improving student learning outcomes in the teaching and learning process. Therefore, a teacher must develop strategies when teaching so that students do not feel bored and bored, can play an active role in the learning process, and can create a pleasant classroom atmosphere. If students are happy with the class atmosphere, it can also impact the material being taught. Learning strategy is tactic teachers use to achieve learning objectives, including approaches, methods, and learning techniques (Kusrini, 2015). In mathematics, each concept is related to another concept, and one concept is a prerequisite for another concept. Therefore, students should be given more opportunities to learn and explore their learning concepts independently. For this reason, a learning model is needed that can motivate students to explore the concept. One learning model that emphasizes this concept is Discovery Learning. Discovery Learning is one of the models in teaching cognitive theory by prioritizing the teacher's role in creating learning situations that involve students learning actively and independently. Discovery Learning is learning to find and find yourself. In this teaching and learning system, the teacher presents lesson materials that are not final (Widodo, Surabaya, Kampus, & Surabaya, 2015). However, students can seek and find their own using a problem-solving approach. Discovery learning or discovery learning models is also defined as learning to understand concepts, meanings, and relationships, through an intuitive process to conclude (Mulyana, 2016). Discovery occurs when individuals are involved, especially in using their mental processes to find some concepts and principles. Discovery is made through Improving Mathematics Learning Outcomes Using Discovery Learning in Class XI Software Engineering 1 INFOKOM Bogor Vocational High School 606 International Journal of Social, Service and Research, 2(7), 604-610 observation, classification, measurement, prediction, determination and inference. Bruner stated that discovery learning is a teaching model that emphasizes the importance of helping students understand a discipline's structure and key ideas, the need for active student involvement in learning, and authentic learning comes through discovery (Nurdyansyah & Fahyuni, 2016). According to Bruner, the goal of Discovery Learning is that teachers should allow their students to become problem solvers, scientists, historians, or mathematicians (Ozdem-Yilmaz & Bilican, 2020). Moreover, through these activities, students will master it, apply it, and find things that are useful for themselves (Kemendikbud, 2014). Based on the opinions of the experts above, it can be concluded that Discovery Learning is a learning model that emphasizes students to be actively involved in finding a concept independently, where the teacher is only a facilitator, motivator and mentor. The research was conducted to improve student learning outcomes based on the problems in learning mathematics in class XI RPL 1 INFOKOM Bogor Vocational School. Therefore, the researcher compiled a study entitled "Improving Mathematics Learning Outcomes by Using Discovery Learning in class XI RPL 1 INFOKOM Bogor Vocational School. METHOD The type of research carried out is Classroom Action Research (CAR) (Sugiyono, 2013). Classroom Action Research (CAR) is research conducted by teachers in their classrooms through self-reflection to improve their performance so that student learning outcomes increase (Wardani & Wihardit, 2018). The research was conducted at INFOKOM Bogor Vocational School, Sindang Barang Loji Village, West Bogor. The subjects of the Classroom Action Research that the researcher conducted were students of class XI RPL 1 with a total of 25 students, consisting of 17 male and 8 female students. The research was carried out in two cycles, namely, cycle 1 and cycle 2. However, before that, pre-cycle activities were carried out. Each cycle has 4 stages, namely 1) Planning Phase; 2) Implementation Phase; 3). Observation Phase, and 4) Reflection Stage. In this study, researchers used a written test as a post-test to collect data on student learning outcomes in class XI learning mathematics. In observing the learning process, researchers used observation sheets. Student learning outcomes were obtained from each lesson's final test (posttest). The data obtained were analyzed by using the percentage of the number of students divided by the data of children who were completed and not yet completed. The percentage calculation uses the formula: RESULTS AND DISCUSSION A. Results Pre-cycle activities are carried out at the beginning of learning with Three Dimensional material. A score of 75 is the KKM value (Minimum Completeness Criteria) determined by the Mathematics subject teacher at school. So if the value is above 75, it is considered complete, and if it is below 75, it is considered incomplete. Table 1 Mathematics Post Test Values in PreCycle Putri Riandini No Indicators 1 2 3 4 Lowest Score Highest Score Average Score Students who completed 5 Students who did not complete PreCycle 20 100 59.4 9 people (36%) 16 people (64%) International Journal of Social, Service and Research, 2(7), 605-610 The data in Table 1 shows that from a total of 25 students, only 9 scored above the KKM or around 36%. Therefore, improvement is needed. The results of the improvement in learning mathematics obtained using Discovery Learning include: 1. Results of Learning Improvements in Cycle 1 The results of improving learning in cycle 1 using Discovery Learning have increased. This can be seen in the table of post-test values for cycle 1 in table 2. Table 2 Mathematics Post Test Scores in Cycle 1 No 1 2 3 4 5 Indicator Lowest score Highest score Average score Students who completed Students who did not finish Cycle 1 30 100 70.6 14 people (56%) 11 people (44%) The data in table 2 shows that the number of students who completed was 14 people, or a percentage was 56%. Students who had not finished were 14 people or 44%. Compared to the pre-cycle activities that have been carried out, the actions of cycle 1 have increased, although only slightly. Figure 1. Student Learning Outcomes of Pre-Cycle - Cycle 1 To improve the results of precycle activities, improvements were made in cycle 1 activities, namely by using Discovery Learning. Moreover, it can be seen that after the first cycle of activities, 607 the number of students who scored above the KKM increased to 14 people or 56%. This means that there is an increase in the number of students who complete as many as 5 people or an increase of 20% from pre-cycle activities to cycle 1 activities. There are several successes and failures contained in the first cycle of action, including: a) Some students have started to discuss in their respective groups actively. b) There are still students who do not want to be involved in the discussion and rely on their group mates. This is because the student still does not understand the concept of threedimensional material. To follow up on the shortcomings in cycle 1, improvements are made, including: a) They were using learning media, namely teaching aids in the form of a cube frame, so that students get an idea about determining the distance between points and the distance from the point to the line from the frame. The cube. b) Stimulate students' thinking power by involving them in making cube frames and determining the distance between points and the distance from points to lines in three-dimensional material, and processing them. 2. Results of Learning Improvements in Cycle 2 Cycle 2 was carried out to follow up on student learning outcomes in cycle 1 actions that were less than optimal. Based on the results of the test in the second cycle of action carried out, it was found that the number of students who completed was 22 people or the percentage was 88%, and students who had not completed as many as 3 people or (12%) of the total number of students. The data are listed in Table 3. Improving Mathematics Learning Outcomes Using Discovery Learning in Class XI Software Engineering 1 INFOKOM Bogor Vocational High School 608 International Journal of Social, Service and Research, 2(7), 604-610 Table 3 Mathematics Post Test Scores in Cycle 2 No 1 2 3 4 5 Indicators Lowest Score Highest Score Average Score Students who completed Students who did not complete Cycle 2 50 100 90.6 22 people (88%) 3 people (12 %) The results of improving learning in cycle 2 using the Discovery Learning and learning media, namely teaching aids in the form of a cube frame, have increased from the action of cycle 1. Figure 2. Student Learning Outcomes Cycle 1 – Cycle 2 The graph above shows that the number of students who completed cycle 2 increased. From the beginning of cycle 1, the number of students who completed as many as 14 people, or 56%, increased to 22 people, or the percentage was 88%. The number of students who completed from cycle 1 to cycle 2 increased by 8 people or 32% of the number of students in the class. So from the pre-cycle activities to the improvement of cycle 2, there was an increase in the number of students who completed, namely 13 students or 52%. This shows that learning mathematics with Three Dimensional material using Discovery Learning and coupled with teaching aids in the form of a cube frame can be successful or effective. As seen in graph 4.3 below. Figure 3. Student Learning Outcomes Pre-Cycle - Cycle 1 - Cycle 2 The success in the action of cycle 2, among others: 1. Increased student learning outcomes. Students take an active role in their group discussions. This is due to Discovery Learning, which allows students to communicate and be invited to discuss when they cannot do the given questions. 2. Student motivation also increases, supported by the use of teaching aids that make students understand more about the concept of building space because students independently apply the problems they get into the framework of the cube they make. B. DISCUSSION The average value obtained by class XI RPL 1 shows that the improvement results from cycle 1 using the Discovery Learning increased from 59.4 in pre-cycle activities to 70.6. This is because, during the learning process in pre-cycle activities, students have not shown any motivation and learning activities. This is because the researcher's method is still unable to make students active in learning. The absence of group discussions caused students to be confused with working on the problems given by the teacher because there were no friends to discuss. Students are busy working on their respective questions, which impacts student learning outcomes and shows a low average score. Putri Riandini International Journal of Social, Service and Research, 2(7), 605-610 The increase in the average value that occurs from pre-cycle activities to cycle 1 is 11.2. In the learning process in cycle 1, student activities have begun to be seen. Students are involved in the group discussion process. The learning model used by researchers is one of the reasons for increasing student activity and motivation. By using Discovery Learning, students become more active in solving a problem in the discussion process. However, the results obtained are still not optimal because the average value of cycle 1 is still below the KKM. Therefore, the researcher continued the action of cycle 2 activities. The learning process in cycle 2 had shown a significant increase in results by obtaining an average student score of 90.6. There was an increase in the average value of 20% from the activity of cycle 1 to cycle 2. If it was calculated from the activity of pre-cycle to the action of cycle 2, it was obtained that the increase in the average value was 31.2. The average value obtained in the improvement of cycle 2 has exceeded the KKM value, so it can be said that the indicators of success in the action of cycle 2 have been achieved. This is due to the Discovery Learning model and the use of learning media as teaching aids that make students more active in discussion, independent, and critical thinking in the learning process and also understand concepts in spatial or three-dimensional structures. The following average values of pre-cycle, cycle 1 and cycle 2 are depicted in the graph below: 609 Figure 4. The average score of pre-cycle students - cycle 1 - cycle 2 However, from 100% of the number of students who are expected to complete all of them, there are 12 % or three students whose grades have not been completed or whose grades are still below the KKM. After the researchers paid attention, several factors caused the three students not to be able to work on the questions given. Some of these factors include: being lazy to read, often staying up late so that when they are at school, they are sleepy and cannot concentrate on receiving subject matter, and also rarely going to school, so they often miss lessons, which confuses when given questions. Therefore, there need to be further improvements. CONCLUSION (1) Discovery Learning, applied to Mathematics with Three Dimensional material, can improve students' mathematics learning outcomes, (2) By using Discovery Learning, students become more active and critical by arguing with each other and being active in learning (3) Student learning outcomes in learning Mathematics increase. Judging from the results of the tests carried out by students after the learning activities were completed, the average score in cycle 1 was 70.6, increasing to 90.6 in cycle 2, with the percentage of students' completeness from 56% in cycle 1 to 88% in cycle 2 activities, and (4) The use of teaching aids helps students to be more active, creative, Improving Mathematics Learning Outcomes Using Discovery Learning in Class XI Software Engineering 1 INFOKOM Bogor Vocational High School 610 International Journal of Social, Service and Research, 2(7), 604-610 and independent in solving problems in the learning process. Students become better at understanding the concept of the third dimension because they prove themselves solving the problems given. Coronata, C., & Alsina, Á. (2014). Evaluation of the mathematical processes in the practices of teaching and learning in childhood education. Procedia-Social and Behavioral Sciences, 141, 1320– 1323. Scopus Peraturan Menteri Pendidikan Nasional Republik Indonesia nomor 22 tahun 2006. Google Scholar Center. Google Scholar Science Practice Kementerian Pendidikan dan Kebudayaan. 2014. Modul Materi Pelatihan Implementasi Kurikulum 2013 Mata Pelajaran Matematika SMP/MTs. Jakarta : Kemendikbud. Maruyama, T., & Kurosaki, T. (2021). Do remedial activities using math workbooks improve student learning? Empirical evidence from scaled-up interventions in Niger. World Development, 148, 105659. Scopus in Theory 177–190). and Springer. Sari, G., Gistituati, N., & Syarifuddin, H. (2019). The effect of guided discovery learning method toward students’ ability in understanding math concept. International Journal of Educational Dynamics, 1(2), 54–60. Google Scholar Shadiq, F. (2007). Apa dan mengapa matematika begitu penting. Departemen Pendidikan Nasional Direktorat Jenderal Peningkatan Mutu Pendidik Dan Tenaga Kependidikan Pusat Pengembangan Dan Pemberdayaan Pendidik Dan Tenaga Kependidikan (PPPPTK) Matematika, 1– 10. Google Scholar (2014). Kusrini, et al. (2015). Strategi Pembelajaran Matematika. Tangerang Selatan: Universitas Terbuka. Education (pp. Google Scholar (2006). Fahmi, F., Setiadi, I., Elmawati, D., & Sunardi, S. (2019). Discovery learning method for training critical thinking skills of students. European Journal of Education Studies. Google Scholar Kemendikbud. Inovasi model pembelajaran sesuai kurikulum 2013. Nizamia Learning Ozdem-Yilmaz, Y., & Bilican, K. (2020). Discovery Learning—Jerome Bruner. In REFERENCES Depdiknas. Nurdyansyah, N., & Fahyuni, E. F. (2016). D. (2013). Metode penelitian pendidikan pendekatan kuantitatif, kualitatif dan R&D. Google Scholar Sugiyono, Syah, M. (2012). Psikologi Belajar. Jakarta: Raja Grafindo Persada. Wardani, I., & Wihardit, K. (2018). Materi penelitian tindakan kelas. Jakarta: Universitas Terbuka. Google Scholar Widodo, G., Surabaya, U. N., Kampus, J., & Surabaya, K. (2015). Pengembangan Dan Implementasi Perangkat Pembelajaran Berbasis Proyek. Mulyana, A. (2016). Model pembelajaran Discovery Learning. Retrieved from https://ainamulyana.blogspot.com/2016 /06/model-pembelajaran-discoverylearning.html Innovation of Vocational Technology Education, 11(1), 41–56. https://doi.org/10.17509/invotec.v11i1.4 837. Google Scholar © 2022 by the authors. It was submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY SA) license (https://creativecommons.org/licenses/by-sa/4.0/). Putri Riandini
https://openalex.org/W3139339857	https://ojrd.biomedcentral.com/counter/pdf/10.1186/s13023-021-01779-4	English	null	New paradigms for the treatment of lysosomal storage diseases: targeting the endocannabinoid system as a therapeutic strategy	Orphanet journal of rare diseases	2,021	cc-by	4,960	© The Author(s) 2021. 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. Abstract Over the past three decades the lysosomal storage diseases have served as model for rare disease treatment develop‑ ment. While these efforts have led to considerable success, important challenges remain. For example, no treatments are currently approved for nearly two thirds of all lysosomal diseases, and there is limited impact of the existing drugs on the central nervous system. In addition, the costs of these therapies are extremely high, in part due to the fact that drug development has focused on a “single hit” approach – i.e., one drug for one disease. To overcome these obstacles researchers have begun to focus on defining common disease mechanisms in the lysosomal diseases, particularly in the central nervous system, with the hope of identifying drugs that might be used in several lysosomal diseases rather than an individual disease. With this concept in mind, herein we review a new potential treatment approach for the lysosomal storage diseases that focuses on modulation of the endocannabinoid system. We provide a short introduc‑ tion to lysosomal storage diseases and the endocannabinoid system, followed by a brief review of data supporting this concept. Keywords: Lysosomal storage diseases, Endocannabinoids, Treatment molecules may be different in the individual LSDs, many of the resultant cellular pathologies are similar.h New paradigms for the treatment of lysosomal storage diseases: targeting the endocannabinoid system as a therapeutic strategy Edward H. Schuchman1* , Maria D. Ledesma2 and Calogera M. Simonaro1 Schuchman et al. Orphanet J Rare Dis (2021) 16:151 https://doi.org/10.1186/s13023-021-01779-4 Schuchman et al. Orphanet J Rare Dis (2021) 16:151 https://doi.org/10.1186/s13023-021-01779-4 Open Access Lysosomal storage diseasesh The lysosomal storage diseases (LSDs) represent a group of over 60 inherited rare disorders, mostly due to dys- functional lysosomal enzymes or transport proteins [1]. The result of these abnormalities is the accumulation of macromolecules, first within the lysosomes but eventu- ally extending to other cell compartments. Over time these anomalies lead to cell dysfunction and tissue dam- age, including but not limited to inflammation, fibrotic changes and cell death. Although the initiating storage The LSDs have been a model for rare disease treatment development since the late 1960s, when the pioneering work of Neufeld and colleagues first demonstrated the principle of enzyme “cross-correction” [2]. This led to a highly productive period of research where investigators defined the mechanisms by which enzymes were targeted to lysosomes, secreted, and then taken up by cells, lead- ing to the early development of enzyme replacement therapy (ERT) for these disorders [3]. The first com- mercial success of ERT for Gaucher disease in the early 1990s [4], coupled with the implementation of several incentive programs by regulators to develop drugs for rare diseases, focused intense academic and pharmaceu- tical interest on the LSDs over the ensuing three decades, *Correspondence: Edward.schuchman@mssm.edu 1 Department of Genetics and Genomic Sciences, Icahn School of Medicine At Mount Sinai, 1425 Madison Avenue, Room 14‑20A, New York, NY 10029, USA Full list of author information is available at the end of the article The endocannabinoid systemh The ECS consists of several endogenous lipid signal- ing molecules, the most abundant of which are ananda- mide (AEA) and 2-arachidonoylglycerol (2-AG), and two G-coupled protein receptors, CB1 and CB2 [17]. CB1 is the predominant ECS receptor in the CNS and plays an important role in mediating anxiety, pain and other neu- rologic responses [18, 19]. CB1 is also expressed at high levels on sensory nerves that innervate peripheral tissues. In contrast, CB2 is primarily expressed in non-neuronal immune cells (e.g., glial cells in the CNS; macrophages in other organs), and has been linked to the modulation of inflammation and many other cellular functions [20, 21]. It is notable that in most LSDs chronic inflammation in neural and non-neural tissues is an important compo- nent of the disease pathology.f i Despite the success of many of these therapies, signifi- cant challenges remain. For example, although ERTs are available for several diseases, in many cases the admin- istered proteins cannot reach important sites of patholo- gies effectively, including the skeletal and central nervous systems (CNS) [9]. Thus, for some diseases the ERTs are highly effective, but for others the clinical impact is lim- ited. In fact, over half of all LSDs have an important neu- rologic component, and the inability of the administered enzymes to cross the blood brain barrier (BBB) is a major limitation of all ERTs. To overcome this obstacle fusion enzymes are being developed that facilitate movement of the proteins across the BBB [10], and gene therapies are being developed that directly express the enzymes in the CNS [11]. In addition, small molecules (e.g., chaperones, substrate reduction drugs) are being developed to cross the BBB [12]. We now know that the neural and behavioral effects of exogenously administered cannabinoids (e.g., tetrahydro- cannabinol [THC], cannabidiol [CBD]) can be traced, at least in part, to the modulation of the ECS [22], although it was first described as an endogenous system con- trolling nervous system function. To elicit these broad effects, numerous downstream signaling events result from activation of the CB1/CB2 receptors by endogenous endocannabinoids. Based on this, manipulation of ECS signaling has been investigated for the treatment of many diseases, with the main goal being the identification of molecules that can modulate the system without the psy- choactive effects attributed to THC and other exogenous molecules [e.g., 17, 23, 24]. © The Author(s) 2021. 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. Schuchman et al. Orphanet J Rare Dis (2021) 16:151 Page 2 of 6 leading to many new therapies and dozens of companies engaged in drug development.f to patients. They also may complement the ERT and gene therapy strategies that are being used to replace the defective proteins in individual diseases. Here we propose one such new approach that targets the endo- cannabinoid system (ECS), and discuss the rationale underlying this approach for the LSDs. Most of these treatment efforts have focused on recov- ering the missing lysosomal enzyme function in patient cells, either by protein or gene replacement [5]. To date, regulatory authorities have approved fourteen ERTs (for ten individual LSDs), and several more are under devel- opment. Gene therapies have also been extensively stud- ied in LSD animal models since the early 1990s, but only recently have advanced to clinical trials as the technolo- gies have matured [6]. Both of these therapeutic strate- gies represent “single-hit” approaches, where one drug is developed for one disease. Other approved therapies include chaperone therapies directed towards recovery of misfolded lysosomal proteins [7], and substrate reduction therapy, which aims to slow the production of macromol- ecules using specific enzyme inhibitors [8]. The endocannabinoid systemh To date, dozens of such ECS modulator molecules have been developed and numer- ous clinical trials have been undertaken, mostly to allevi- ate pain or for the treatment of anxiety disorders or other neuropsychiatric diseases. Another important limitation of many of the existing or pending therapeutic approaches is their expense. For example, ERTs can cost anywhere from $250–650,000/ year per patient [13], and gene therapies are being esti- mated to cost millions of dollars that are likely to be paid out over several years. These high costs have been justi- fied by the research and development costs incurred by the drug companies, as well as the cost saving value of the treatments when compared to the medical costs of caring for these patients in the absence of these therapies. How- ever, another important factor is the very small commer- cial market for these disorders, especially when the drugs are being developed for individual diseases.h Of direct relevance to the LSDs, endocannabinoids are known to bind and activate their receptors by lateral dif- fusion within the lipid bilayer, rather than through direct interactions at the cell surface [25]. Moreover, both CB1 and CB2 receptors are integral membrane proteins that function within lipid raft structures of the membrane, and changes in sphingomyelin, cholesterol and other raft lipids (e.g., ceramide and sphingosine) have impor- tant effects on their expression, distribution and function [26–28]. In many of the LSDs, abnormal accumulation of these lipids occurs, either as the primary defect or as a secondary consequence of the lysosomal dysfunction, Thus, in recent years researchers have begun to turn towards therapies that target common disease mecha- nisms in the LSDs, including autophagy, inflamma- tion and others [e.g., 14–16]. Such approaches have the advantage of potentially being used in multiple diseases, thus reducing development costs and ultimately the cost Schuchman et al. Orphanet J Rare Dis (2021) 16:151 Page 3 of 6 resulting in the disruption of lipid raft signaling. Thus, it is likely that the function of CB1/CB2 receptors may be disrupted in many of the LSDs, as has been shown in a mouse model of Type C Niemann-Pick disease [29]. sphingomyelinase activity [32]. Although an ERT is in the final stages of clinical development for this disorder (Oli- pudase alfa®), it does not impact the CNS disease that occurs in about half of all ASMD patients [33]. The endocannabinoid system and treatment of the lysosomal storage diseases As noted above, CB1 receptors are primarily expressed in neurons, while CB2 receptors are primarily expressed in immune cells. A large number of small molecules have been developed to modulate the function of CB1/CB2 receptors, and many of these cross the BBB. In addition, several of these molecules have been evaluated in clini- cal trials and safety data has been established. One of the major gaps in current treatments for the LSDs is the lack of therapies for the CNS, and therefore repurposing these ECS molecules is a logical approach. Such ECS effec- tor molecules have been shown to reduce inflammation, slow cell death processes, improve mitochondrial, lyso- somal and other cell functions, and enhance other meta- bolic pathways that are commonly defective in the LSDs (Table 1). As an example, an early paper showed that activation of CB1 receptors with the endocannabinoid AEA or with THC, the main psychoactive compound in cannabis, led to sphingomyelin degradation in cultured astrocytes through activation of a neutral sphingomyelinase [30]. In the LSD acid sphingomyelinase deficient Niemann- Pick disease (i.e., types A and B Niemann-Pick disease, ASMD), sphingomyelin storage is the primary meta- bolic abnormality [31], suggesting that CB1 activation may be a reasonable approach for the treatment of this disorder. Importantly, although the sphingomyelin stor- age in ASMD begins in lysosomes, it rapidly extends to the plasma membrane, mitochondria and other cell com- partments, making it potentially accessible to the neutral We first evaluated the ability of three such FAAH inhib- itors to reduce sphingomyelin levels in neurons from ASMKO mice. Significant sphingomyelin reduction was observed, which could be prevented using an inhibitor of neutral sphingomyelinase [35]. We then treated ASMKO mice with one FAAH inhibitor, and found improve- ments in many pathologic markers, including reduction of sphingomyelin in the brain and other tissues. Impor- tantly, several neurological clinical endpoints also were improved, and lifespan was significantly extended as well. A schematic depiction illustrating the putative mecha- nism of action of FAAH inhibition in ASMD is shown in Fig. 1. We also found that treatment of cells from patients with type C Niemann-Pick disease, due to mutations in the NPC1 gene, led to reduction of sphingomyelin and, importantly, reduction in cholesterol storage. This is consistent with previous work showing that reduction of sphingomyelin in type C Niemann-Pick disease cells using recombinant acid sphingomyelinase led to correc- tion of cholesterol trafficking [36]. The endocannabinoid systemh We therefore decided to evaluate this approach in a mouse model of type A and B NPD (acid sphingomyeli- nase deficient mice, ASMKO) [34], and found the unex- pected downregulation of CB1 on the surface of neurons from these mice [35]. This was due to entrapment of the receptor within lysosomes, indicating that CB1 signaling was likely abnormal in this disorder and further support- ing the activation of CB1 as a therapeutic strategy. Since direct activation of CB1 with synthetic or natural ago- nists leads to psychotropic effects, we chose an indirect strategy using small molecules that inhibit the enzyme fatty acid amide hydrolase (FAAH), leading to an eleva- tion of endogenous endocannabinoids, including AEA, and several other bioactive lipids (e.g., palmitoylethan- olamide [PEA] and oleoylthanolamide [OEA]). AEA pri- marily acts on CB1 receptors and to a lesser extent CB2, while PEA and OEA act on the PPAR-α receptor. Many FAAH inhibitors have been developed that cross the BBB, and several have entered clinical trials [23].i The endocannabinoid system and treatment of the lysosomal storage diseases While these examples may be specific to diseases with significant sphingomy- elin storage, activation of CB1 receptors has many other beneficial effects that could impact all LSDs, including prevention of neurodegeneration, inhibition of pain, remyelination and others. Table 1 Common cell processes defective in LSDs that may be impacted by CB1/CB2 modulator drugs Cell function Reference example Autophagy [39] Calcium homeostasis [40] Cell survival/death [41] Endo/exocytosis (vesicular transport) [42] Endothelial cell function (BBB integrity) [43] Extracellular matrix production/fibrosis [44] Inflammation [21] Mitochondrial function/energy balance [45] Myelination [46] Synaptic function [47] Table 1 Common cell processes defective in LSDs that may be impacted by CB1/CB2 modulator drugs In contrast to CB1, CB2 receptors are primar- ily expressed on immune cells both in the periphery and CNS. Therefore, unlike CB1, direct agonists of CB2 receptors do not have major psychotropic effects, and have been safely used in animal models of various Schuchman et al. Orphanet J Rare Dis (2021) 16:151 Schuchman et al. Orphanet J Rare Dis Page 4 of 6 Fig. 1 Effect of FAAH inhibition in ASMD. All pathology in ASMD is initiated by sphingomyelin (SPM) build-up. FAAH inhibition leads to the elevation of AEA and other endocannabinoids (ECs), resulting in the activation of CB1. This, in turn, activates neutral sphingomyelinase (NSM), which slows or prevents SPM buildup and the resulting downstream pathology and disease Fig. 1 Effect of FAAH inhibition in ASMD. All pathology in ASMD is initiated by sphingomyelin (SPM) build-up. FAAH inhibition leads to the elevation of AEA and other endocannabinoids (ECs), resulting in the activation of CB1. This, in turn, activates neutral sphingomyelinase (NSM), which slows or prevents SPM buildup and the resulting downstream pathology and disease BBB and are active in the CNS, should facilitate these repurposing efforts. Preliminary evidence in several LSD animal models indicates the potential of this approach, but significant questions still remain. Among them is the fact that most studies to date using CB1/CB2 modulator drugs in animal models or clinical trials have evaluated them for relatively short periods of time (days or weeks). In the case of chronic diseases such as the LSDs, long- term treatment will be required, and the safety of these molecules must be established in this context, along with the proper dosing. The endocannabinoid system and treatment of the lysosomal storage diseases The psychotropic effects and potential for dependency attributed to some of these molecules also indicates the need for more evaluation in the LSD models, and could especially limit their use in young children. This is a specific concern for CB1 activation, although FAAH inhibition may overcome this obstacle.i diseases. The main effect of these direct CB2 agonists is to reduce inflammatory responses in autoimmune and other inflammatory diseases, including neuroinflam- matory diseases, although additional positive effects on prevention of cell death, repair of the BBB, and other cell abnormalities have been reported [20, 21]. To initially evaluate the role of CB2 in the LSDs, we chose mouse models for two diseases: acid ceramidase deficiency (Farber disease) and mucopolysaccharidosis type IIIA (Sanfilippo A; MPS IIIA) [37, 38]. Although the initiating storage material in these two diseases is distinct (the lipid ceramide and the glycosaminoglycan heparan sulfate, respectively), both of these disorders are charac- terized by early activation of inflammatory pathways in the periphery and CNS. Consistent with this inflamma- tory disease, CB2 expression was found to be markedly elevated in several tissues from these animals (Fig. 2). Treatment studies using a CB2 agonist are underway, but preliminary findings have already revealed a slow- ing of disease progression and extension of lifespan in the Farber disease animals, consistent with the reduc- tion of inflammation and other positive effects attributed to these molecules. Thus, CB2 receptors are not only a potential new target for treatment, but also may serve as a biomarker to indicate macrophage infiltration and inflammation in different LSDs. As the LSD field moves forward into the next decade, researchers and clinicians must develop a new paradigm for LSD drug development that builds upon the sub- stantial progress that has already been made. Such drug development must address the missing needs of the cur- rent therapies, such as targeting difficult to reach patho- logic organs including the CNS and skeletal system, and also must move away from the “single-hit” approach to drug development in an effort to make the process more efficient and ultimately the costs of these drugs less pro- hibitive. We are currently witnessing the early investiga- tion of these broader approaches, and expect that further studies of this nature, including investigation of the ECS system, will continue to offer new hope to LSD patients. Competing interests EHS, MDL and CMS are inventors on pending patents related to the treatment of the LSDs through modulation of the ECS. Abbreviations AEA: Anandamide; 2-AG: 2-Arachidonoylglycerol; ASMD: Acid sphingomyeli‑ nase deficient Niemann-Pick disease; ASMKO: Acid sphingomyelinase knock- out; BBB: Blood brain barrier; CBD: Cannabidiol; CNS: Central nervous system; ECS: Endocannabinoid system; ERT: Enzyme replacement therapy; FAAH: Fatty acid amide hydrolase; LSDs: Lysosomal storage diseases; MPS: Mucopolysac‑ charidosis; THC: Tetrahydrocannabinol. Availability of data and materials 2. Fratantoni JC, Hall CW, Neufeld EF. The defect in Hurler and Hunter syn‑ dromes. II. Deficiency of specific factors involved in mucopolysaccharide degradation. Proc Natl Acad Sci USA. 1969;64:360–6. y All data are included in this published article and reference [35]. All data are included in this published article and reference [35]. 3. Ries M. Enzyme replacement therapy and beyond—in memoriam. Roscoe O. Brady, M.D. (1923–2016). J Inherit Metab Dis. 2017;40:343–56. Acknowledgements 1 Department of Genetics and Genomic Sciences, Icahn School of Medicine At Mount Sinai, 1425 Madison Avenue, Room 14‑20A, New York, NY 10029, USA. 2 Centro Biologia Molecular Severo Ochoa, 28049 Madrid, Spain. The authors wish to acknowledge the members of their laboratories who have participated in this work. Received: 8 October 2020 Accepted: 16 March 2021 Received: 8 October 2020 Accepted: 16 March 2021 All authors contributed to the writing and review of this manuscript prior to submission. MDL and CMS contributed to the generation of data described in the manuscript. All authors read and approved the final manuscript. Concluding thoughts We propose that the ECS represents a new and poten- tially important target for the treatment of some LSDs. The fact that many molecules that modulate this system have already been developed, some of which cross the Schuchman et al. Orphanet J Rare Dis (2021) 16:151 Page 5 of 6 Fig. 2 CB2 expression in tissues from Farber and MPS IIIA mice. CB2 is markedly overexpressed in the liver and CNS of mice with Farber disease and MPS IIIA. WT, wildtype; FD, Farber. Brown color indicates staining with CB2 antibodies. Blue color indicates cell nuclei. Bars = 50 µm Fig. 2 CB2 expression in tissues from Farber and MPS IIIA mice. CB2 is markedly overexpressed in the liver and CNS of mice with Farber disease and MPS IIIA. WT, wildtype; FD, Farber. Brown color indicates staining with CB2 antibodies. Blue color indicates cell nuclei. Bars = 50 µm Authors’ contributions Received: 8 October 2020 Accepted: 16 March 2021 References The Wylder Nation Foundation provided funding for this wor 1. Platt FM, d’Azzo A, Davidson BL, Neufeld EF, Tifft CJ. Lysosomal storage diseases. Nat Rev Dis Primers. 2018;4:27. Availability of data and materials Declarations 4. Beutler E, Kay A, Saven A, Garver P, Thurston D, Dawson A, et al. Enzyme replacement therapy for Gaucher disease. Blood. 1991;78:1183–9. Ethics approval and consent to participate Not applicable. Schuchman et al. Orphanet J Rare Dis (2021) 16:151 Page 6 of 6 Schuchman et al. Orphanet J Rare Dis (2021) 16:151 5. Leal AF, Espejo-Mojica AJ, Sánchez OF, Ramírez CM, Reyes LH, Cruz JC, et al. Lysosomal storage diseases: current therapies and future alterna‑ tives. J Mol Med (Berl). 2020;98:931–46. 29. Oddi S, Caporali P, Dragotto J, Totaro A, Maiolati M, Scipioni L, et al. The endocannabinoid system is affected by cholesterol dyshomeostasis: Insights from a murine model of Niemann Pick type C disease. Neurobiol Dis. 2019;130:104531. 6. Pearse Y, Iacovino M. A cure for Sanfilippo syndrome? A summary of current therapeutic approaches and their promise. Med Res Arch. 2020. https://doi.org/10.18103/mra.v8i2.2045. 30. Sánchez C, Galve-Roperh I, Rueda D, Guzmán M. Involvement of sphin‑ gomyelin hydrolysis and the mitogen-activated protein kinase cascade in the Delta9-tetrahydrocannabinol-induced stimulation of glucose metabolism in primary astrocytes. Mol Pharmacol. 1998;54:834–43. p // g/ / 7. Suzuki Y. Chaperone therapy for molecular pathology in lysosomal g 7. Suzuki Y. Chaperone therapy for molecular patholog diseases. Brain Dev. 2020;S0387–7604(20):30179. 7. Suzuki Y. Chaperone therapy for molecular pathology in lyso diseases. Brain Dev. 2020;S0387–7604(20):30179. 8. Feriozzi S, Hughes DA. New drugs for the treatment of Anderson–Fabry disease. J Nephrol. 2020. https://doi.org/10.1007/s40620-020-00721-4. 8. Feriozzi S, Hughes DA. New drugs for the treatme 31. Schuchman EH, Desnick RJ. Types A and B Niemann-Pick disease. Mol Genet Metab. 2017;120:27–33. disease. J Nephrol. 2020. https://doi.org/10.1007/s40620-020-0072 9. Concolino D, Deodato F, Parini R. Enzyme replacement therapy: efficacy and limitations. Ital J Pediatr. 2018;44(Suppl 2):120. 32. Scandroglio F, Venkata JK, Loberto N, Prioni S, Schuchman EH, Chigorno V, et al. Lipid content of brain, brain membrane lipid domains, and neurons from acid sphingomyelinase deficient mice. J Neurochem. 2008;107:329–38. 10. Sato Y, Okuyama T. Novel enzyme replacement therapies for neuropathic mucopolysaccharidoses. Int J Mol Sci. 2020;21:400. 11. Iqubal A, Iqubal MK, Khan A, Ali J, Baboota S, Haque SE. Gene therapy, a novel therapeutic tool for neurological disorders: current progress, chal‑ lenges and future prospective. Curr Gene Ther. 2020. https://doi.org/10. 2174/1566523220999200716111502. 33. Thurberg BL, Diaz GA, Lachmann RH, Schiano T, Wasserstein MP, Ji AJ, et al. Declarations Long-term efficacy of olipudase alfa in adults with acid sphingomy‑ elinase deficiency (ASMD): Further clearance of hepatic sphingomyelin is associated with additional improvements in pro- and anti-atherogenic lipid profiles after 42 months of treatment. Mol Genet Metab. 2020. https://doi.org/10.1016/j.ymgme.2020.06.010.f 12. Blumenreich S, Yaacobi C, Vardi A, Barav OB, Vitner EB, Park H, et al. Sub‑ strate reduction therapy using Genz-667161 reduces levels of pathogenic components in a mouse model of neuronopathic forms of Gaucher disease. J Neurochem. 2020. https://doi.org/10.1111/jnc.15136. 34. Horinouchi K, Erlich S, Perl DP, Ferlinz K, Bisgaier CL, Sandhoff K, et al. Acid sphingomyelinase deficient mice: a model of types A and B Niemann– Pick disease. Nat Genet. 1995;10:288–93. 13. Clarke JT, Amato D, Deber RB. Managing public payment for high-cost, high-benefit treatment: enzyme replacement therapy for Gaucher’s disease in Ontario. CMAJ. 2001;165:595–6. 35. Bartoll A, Toledano-Zaragoza A, Casas J, Guzman M, Schuchman EH, Ledesma MD. Inhibition of fatty acid amide hydrolase prevents pathology in neurovisceral acid sphingomyelinase deficiency by rescuing defective endocannabinoid signaling. EMBO Mol Med. 2020; e11776. 14. Parker H, Bigger BW. The role of innate immunity in mucopolysaccharide diseases. J Neurochem. 2019;148:639–51. 15. Ren H, Wang G. Autophagy and lysosomal storage disorders. Adv Exp Med Biol. 2020;1207:87–102. 36. Devlin C, Pipalia NH, Liao X, Schuchman EH, Maxfield FR, Tabas I. Improve‑ ment in lipid and protein trafficking in Niemann-Pick C1 cells by correc‑ tion of a secondary enzyme defect. Traffic. 2010;11:601–15. 16. Stepien KM, Roncaroli F, Turton N, Hendriksz CJ, Roberts M, Heaton RA, et al. Mechanisms of mitochondrial dysfunction in lysosomal storage disorders: a review. J Clin Med. 2020;9:E2596. 37. Alayoubi AM, Wang JC, Au BC, Carpentier S, Garcia V, Dworski S, et al. Systemic ceramide accumulation leads to severe and varied pathological consequences. EMBO Mol Med. 2013;5:827–42. 17. Chanda D, Neumann D, Glatz JFC. The endocannabinoid system: over‑ view of an emerging multi-faceted therapeutic target. Prostaglandins Leukot Essent Fatty Acids. 2019;140:51–6. 38. Bhaumik M, Muller VJ, Rozaklis T, Johnson L, Dobrenis K, Bhattacharyya R, et al. A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome). Glycobiology. 1999;9:1389–96. y 18. Murphy T, Le Foll B. Targeting the endocannabinoid CB1 receptor to treat body weight disorders: a preclinical and clinical review of the therapeutic potential of past and present CB1 drugs. Biomolecules. 2020;10:855. 39. Salazar M, Carracedo A, Salanueva IJ, Hernández-Tiedra S, Lorente M, Egia A, et al. Declarations Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells. J Clin Invest. 2009;119:1359–72. 19. Russo S, De Azevedo WF. Advances in the understanding of the cannabi‑ noid receptor 1 - focusing on the inverse agonists interactions. Curr Med Chem. 2019;26:1908–19. 40. Fernández-Ruiz J, Romero J, Ramos JA. Endocannabinoids and neu‑ rodegenerative disorders: Parkinson’s disease, Huntington’s Chorea, Alzheimer’s disease, and others. Handb Exp Pharmacol. 2015;231:233–59. 20. Bie B, Wu J, Foss JF, Naguib M. An overview of the cannabinoid type 2 receptor system and its therapeutic potential. Curr Opin Anaesthesiol. 2018;31:407–14. 41. Wójcik P, Žarković N, Gęgotek A, Skrzydlewska E. Involvement of metabolic lipid mediators in the regulation of apoptosis. Biomolecules. 2020;10:402. 21. Hussain MT, Greaves DR, Iqbal AJ. The impact of cannabinoid receptor 2 deficiency on neutrophil recruitment and inflammation. DNA Cell Biol. 2019;38:1025–9. 42. Rovira-Esteban L, Péterfi Z, Vikór A, Máté Z, Szabó G, Hájos N. Morphologi‑ cal and physiological properties of CCK/CB1R-expressing interneurons in the basal amygdala. Brain Struct Funct. 2017;222:3543–65. 22. Cooray R, Gupta V, Suphioglu C. Current aspects of the endocannabinoid system and targeted THC and CBD phytocannabinoids as potential thera‑ peutics for Parkinson’s and Alzheimer’s Diseases: a review. Mol Neurobiol. 2020. https://doi.org/10.1007/s12035-020-02054-6. 43. Vendel E, de Lange EC. Functions of the CB1 and CB 2 receptors in neuroprotection at the level of the blood-brain barrier. Neuromol Med. 2014;16:620–42. 23. Fazio D, Criscuolo E, Piccoli A, Barboni B, Fezza F, Maccarrone M. Advances in the discovery of fatty acid amide hydrolase inhibitors: what does the future hold? Expert Opin Drug Discov. 2020;15:765–78. 44. Siegmund SV, Schwabe RF. Endocannabinoids and liver disease. II. Endo‑ cannabinoids in the pathogenesis and treatment of liver fibrosis. Am J Physiol Gastrointest Liver Physiol. 2008;294:G357–62. 24. Mallipeddi S, Janero DR, Zvonok N, Makriyannis A. Functional selectivity at G-protein coupled receptors: advancing cannabinoid receptors as drug targets. Biochem Pharmacol. 2017;128:1–11. 45. García S, Martín Giménez VM, Mocayar Marón FJ, Reiter RJ, Manucha W. Melatonin and cannabinoids: mitochondrial-targeted molecules that may reduce inflammaging in neurodegenerative diseases. Histol Histopathol. 2020;35:789–800. g g 25. Reggio PH. Endocannabinoid binding to the cannabinoid receptors: what is known and what remains unknown. Curr Med Chem. 2010;17:1468–86. 46. Mecha M, Yanguas-Casás N, Feliú A, Mestre L, Carrillo-Salinas F, Azcoitia I. The endocannabinoid 2-AG enhances spontaneous remyelination by targeting microglia. Brain Behav Immun. 2019;77:110–26. 26. Declarations Di Scala C, Fantini J, Yahi N, Barrantes FJ, Chahinian H. Anandamide revis‑ ited: how cholesterol and ceramides control receptor-dependent and receptor-independent signal transmission pathways of a lipid neurotrans‑ mitter. Biomolecules. 2018;8:31. 47. Piette C, Cui Y, Gervasi N, Venance L. Lights on endocannabinoid-medi‑ ated synaptic potentiation. Front Mol Neurosci. 2020;13:132. 47. Piette C, Cui Y, Gervasi N, Venance L. Lights on endocannabinoid-medi‑ ated synaptic potentiation. Front Mol Neurosci. 2020;13:132. 27. Paugh SW, Cassidy MP, He H, Milstien S, Sim-Selley LJ, Spiegel S, et al. Sphingosine and its analog, the immunosuppressant 2-amino-2-(2- [4-octylphenyl]ethyl)-1,3-propanediol, interact with the CB1 cannabinoid receptor. Mol Pharmacol. 2006;70:41–50. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 28. Rimmerman N, Bradshaw HB, Kozela E, Levy R, Juknat A, Vogel Z. Com‑ partmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveolin-1. Br J Pharmacol. 2012;165:2436–49.
https://openalex.org/W420740165	http://real.mtak.hu/61543/1/1-s2.0-S0048969715004854-main.pdf	English	null	Predicting the preservation of cultural artefacts and buried materials in soil	Science of the total environment	2,015	cc-by	14,211	H I G H L I G H T S • The preservation in soils of different materials and of stratigraphic evidence is reviewed. • A predictive framework for the preservation of materials in soil is proposed. • Preservation of materials and stratigraphic evidence in soils of the EU is predicted. • Soil performs an important cultural service by preserving anthropogenic artefacts. • Preservation of materials and stratigraphic evidence in soils of the EU is predicted. • Soil performs an important cultural service by preserving anthropogenic artefacts. ⁎ Corresponding author. E-mail address: gergely.toth@jrc.ec.europa.eu (G. Tóth). Predicting the preservation of cultural artefacts and buried materials in soil Mark Kibblewhite a, Gergely Tóth b,⁎, Tamás Hermann b Mark Kibblewhite a, Gergely Tóth b,⁎, Tamás Hermann b a Cranﬁeld University, Cranﬁeld, Bedford MK43 0AL, United Kingdom b European Commission, Joint Research Centre (JRC), Institute for Environment and Sustainability (IES), Via Enrico Fermi a Cranﬁeld University, Cranﬁeld, Bedford MK43 0AL, United Kingdom b European Commission, Joint Research Centre (JRC), Institute for Environment and Sustainability (IES), Via Enrico Fermi 2749, 21027 Ispra, VA, It a r t i c l e i n f o Article history: Received 2 March 2015 Received in revised form 9 April 2015 Accepted 11 April 2015 Available online xxxx Editor: Charlotte Poschenrieder Keywords: Soil Cultural services Archaeology Preservation Artefacts Burial Stratigraphy This study identiﬁes factors affecting the fate of buried objects in soil and develops a method for assessing where preservation of different materials and stratigraphic evidence is more or less likely in the landscape. The results inform the extent of the cultural service that soil supports by preserving artefacts from and information about past societies. They are also relevant to predicting the state of existing and planned buried infrastructure and the persistence of materials spread on land. Soils are variable and preserve different materials and stratigraphic evidence differently. This study identiﬁes the material and soil properties that affect preservation and relates these to soil types; it assesses their preservation capacities for bones, teeth and shells, organic materials, metals (Au, Ag, Cu, Fe, Pb and bronze), ceramics, glass and stratigraphic evidence. Preservation of Au, Pb and ceramics, glass and phytoliths is good in most soils but degradation rates of other materials (e.g. Fe and organic materials) is strongly inﬂuenced by soil type. A method is proposed for using data on the distribution of soil types to map the variable preservation capacities of soil for different materials. This is applied at a continental scale across the EU for bones, teeth and shells, organic materials, metals (Cu, bronze and Fe) and stratigraphic evidence. The maps produced demonstrate how soil provides an extensive but variable preservation of buried objects. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Science of the Total Environment 529 (2015) 249–263 Science of the Total Environment 529 (2015) 249–263 1. Introduction which materials is valuable for the management of heritage and buried infrastructure and may also inform assessments of the longer-term im- pact on soil of spreading of wastes to land. The range of buried objects is wide and includes: artefacts made from a variety of materials e.g., stone, ceramics, bone, metals, wood and other plant materials, skins and hides, glass and plastics; burial mounds, cultivation terraces, and other earthworks; stratigraphic evidence of past environments (Harris, 1989), land management and human activities; and contemporary distri- bution and communication infrastructure. The spatial distribution of ar- chaeological artefacts and landscape features reﬂects past occupation patterns and land uses and the actual presence of buried objects in soil and earthworks depends on many factors other than the soil type and its potential to preserve materials (Holden et al., 2006; Lillie and Smith, 2007). It is useful, however, to assess the preservation service that soils may or may not provide if objects are buried in them; such an assessment has potential to provide information for valuing the cultural and other http://dx.doi.org/10.1016/j.scitotenv.2015.04.036 0048-9697/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1.1. Background The rate of surface degradation in soil is strongly affected by the glass composition and not easily predicted (Van Giffen, 2014). The alkali type and content is critical: Roman and other ancient glass is generally more resistant to chemical attack than glass from the mediaeval period when wood ash containing potassium (K) replaced soda ash in its manufacture. Under acidic conditions and moderately al- kaline conditions (pH b 9) alkali ions are leached from the glass matrix, while under more alkaline conditions hydroxyl ions disrupt silicon–ox- ygen bonds within the silica structure (Melcher et al., 2010). At more al- kaline pH, laminar surface layers are more likely to form (Roemich et al., 2003) which may be iridescent. In all but the driest soils, surface coat- ings and other decoration on glass are expected to degrade quite quickly (b100 y). The strong dependence of corrosion rates of glass objects on material composition and manufacture leads to uncertainty in any pre- diction of the relative rates of surface degradation in different soils: cor- rosion is expected to be least in very dry soils; rates of corrosion may be moderated in well-drained and neutral soils in drier regions; highly al- kaline soils are anticipated to be the most corrosive. Human and animal bones and teeth are made of hydroxyapatite (CaCO3) and smaller amounts of protein (collagen) ﬁbres. Bones that still retain collagen have some elasticity but become more brittle with age as collagen degrades. The circumstances of burial and the immedi- ate post-burial environment inﬂuence the longer-term fate of buried bones (Baxter, 2004; Jans et al., 2004). Relevant factors are the burial location, depth and any containment. In the early phases of bone burial, biological action affects the ageing process which may continue for decades. Colonisation is initially dominated by bacteria followed by fungi (Child, 1995; Jans, 2008). Biological degradation continues until nitrogen (N) derived from collagen is exhausted; in parallel and subse- quently, physical degradation and chemical alteration and degradation occur. The solubility of hydroxyapatite rises with increasing acidity and the survival of bone and teeth correlates with the pH of soil and ground- water. Dissolution of bone results in a lower density material with more and larger pores and this progressively increases the bone area being actively dissolved and the rate of degradation. Alongside dissolution, ions in the soil solution can be incorporated into new minerals. 1.1. Background Soil contributes to a series of ecosystem services through its functions. Assessment and maintenance of these functions are central to the EU's thematic strategy for soil protection (European Commission, 2006a,b). Storage of buried heritage and providing a platform for the built environ- ment are the main soil functions identiﬁed in the strategy and further as- sessments are required to characterise these and describe their spatial variability. A wide range of archaeological and cultural heritage and bur- ied infrastructure is preserved in the soil environment and in landscape features that are formed from soil. Knowledge about which soils preserve M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 250 ecosystem services provided by soils and to inform decisions about the management of buried resources. The survival and condition of buried objects and stratigraphic evidence depend both on the particular soil en- vironment in which they are buried and the material from which they are formed (Cronyn, 1990) and, for anthropogenic artefacts, the nature of their manufacture. This study investigates the preservation of buried ob- jects in soil, how this is affected by their material nature and soil type and how information about the distribution of soils can be used to assess pres- ervation capacities spatially. It builds on existing guidance about which soil properties are important for the preservation of buried objects (Davidson and Wilson, 2006; Crow, 2008; English Heritage, 2008, 2011) and provides a commentary on the fate of different materials for different soil types deﬁned according to standard taxonomic classiﬁcation. It de- scribes how soil mapping data can be used to systematically map the preservation of different materials by soil and applies this to pre- dict this potential for soils across the European Union (EU). tend to have a more open pore structure allowing water to enter and cause degradation, including by subsequent frost-shattering. Glass is a relatively durable material in the buried environment (Jackson et al., 2012) and the morphology of solid glass objects and frag- ments often remain intact. However, surface corrosion of glass occurs in moist and wet soils leading to a loss of transparency and the formation of a surface crust rich in silica and depleted of basic ions. This process weakens the glass and this may accelerate shattering of thinner objects (Huisman et al., 2008). 1.4. Organic materials Organic materials buried in soil include plant material (e.g., wood, ﬁbres, fruits, seeds, and pollen), fungal spores, insects and their larvae, parasite eggs and the remains of animals and humans (e.g., skin, soft tissues). Immediately following their burial, organic materials may be recovered or at least disturbed by soil fauna, ranging from macrofauna including burrowing rodents to arthropods and their larvae. Subse- quently, the main degradation process for organic material is biological oxidation by the soil ecosystem and this usually leads to its complete destruction where aerobic and moist soil conditions prevail, whereas soil conditions that are anaerobic are preserving, although not com- pletely (Bjordal et al., 1999; Douterelo et al., 2010). In very dry soils microbial activity is restricted and this preserves organic materials. The least preserving hydrological conditions are expected to be those where soil is seasonally wet but dries in summer as this cycling of soil moisture levels encourages ‘ﬂushes’ of more intense microbial activity as the soil wets up. Any activity that disturbs the soil and re- distributes and releases soil organic matter, including tillage, is also likely to accelerate aerobic degradation. The rate of biological degrada- tion of organic materials in soil is affected by their molecular structure 1.1. Background Avian and mollusc shells are formed from calcite (CaCO3) which dissolves more readily in moist acid conditions than hydroxyapatite in bones and their fate is similar but accelerated compared to bone and teeth. While both ceramic and to a lesser extent glass materials are pre- served well in soil, they tend to shatter and the resulting shards may then become dispersed. Physical damage to ceramics and glass buried in soil can arise from static and dynamic forces. Static forces increase with depth and dynamic forces from the treading action of animals and people and vehicle movements (Dain-Owens et al., 2013) may propagate in to subsoil. Where expansive clay minerals are present, these will create potentially destructive mechanical forces during wetting-drying cycles. Soil stiffness, which is a measure of resistance to deformation, will affect the likelihood that brittle objects will be fractured. For example, a dry clay soil will be more resistant to deforma- tion and better protect objects from shattering than will a wet sandy soil. For most soils, however, the dominant factor determining shat- tering is likely to be land use and management rather than soil type. Opaline silica is deposited as phytoliths in plants that vary in form be- tween species and can provide evidence of past vegetative cover; they are highly resistant to degradation in soil and will be preserved in most soils, a possible exception being very wet and strongly alkaline soils. The dry conditions present in soils in arid and semi-arid regions preserve bones and teeth and shells. Bones and teeth and shells are preserved better in alkaline soil, while their degradation and eventual de- struction are quite rapid where the soil water is acidic and unsaturated, as in acid soils that are wet and free draining and formed on sands and acidic parent material in higher precipitation zones. Bones, teeth and shells are preserved better in soils that are permanently waterlogged by stagnant alkaline groundwater, as occurs in some lowland peat soils. Static pres- sures and surface loading to the soil e.g., during cultivation and by vehi- cles (Dain-Owens et al., 2013) may cause physical damage to buried bone material as may soil movement resulting from wetting and drying cycles in soils that contain expansive clay minerals. 1.2. Bones, teeth and shells 1.3. Ceramics, glass and phytoliths Many types of ceramics are preserved in soil, including tiles and bricks as well as ﬁgures, pots and other domestic items. Ceramic artefacts can survive in the buried environment for very long periods and a ceramic ﬁg- urine dated to 16,000 years before present (Vandiver and Vasil'ev, 2002) has been found. This longevity reﬂects the resistance of ceramics to bio- logical and chemical degradation processes. The material properties of ce- ramics vary depending on the clay and other materials used for their manufacture, e.g., carbonaceous or non-carbonaceous clay, with or with- out addition of calcite (Fabbri et al., 2014). Firing temperature affects ro- bustness: higher ﬁring temperatures produce stiffer objects that resist mechanical and other stresses better. Objects ﬁred at lower temperature M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 251 because this determines the net energetic gain to the soil ecosystem of using one organic material as an energy substrate compared to another. Starch and other polysaccharides yield a higher net energy than more intractable components; for example, cellulose is utilised preferentially over lignin and other poly-phenols present in wood. Acidity inﬂuences the soil ecology and the ratio of fungal to bacterial population sizes increases as pH decreases while rates of organic matter degradation are generally reduced by increasing soil acidity. Where the soil solution is high in dissolved organic matter this can react with buried organic material and this process may confer resistance to biological degra- dation, as in the natural ‘tanning’ process that occurs when skin and soft tissues are deposited in waterlogged peat. Organic material may also be protected by absorption and occlusion in the soil matrix and this is more likely in ﬁne textured clay soils than coarse sandy ones. Nutrient levels may affect the survival of organic materials; for example where intensive agriculture introduces higher levels of nutrients releasing microbial activity that has been limited by nutri- ent availability. chemical and biological degradation in soil. Flints and igneous and meta- morphic rock-derived minerals are expected to be more resistant (Karkanas, 2010) than some sedimentary rock material e.g., sandstones and chalk, especially in wet environments and where there are active freeze–thaw cycles. Plaster and mortar can be likened to a weakly struc- tured sedimentary rock material that loses its structural integrity when wet, containing carbonates and sulphates that are solubilised under acid conditions. 1.5. Metals The degree of preservation of metals in soil is speciﬁc to the type of metal. Au objects are resistant to corrosion and indeﬁnitely preserved in the buried environment, although more fragile ones may be damaged by static and dynamic pressures. Ag is less resistant to corrosion than Au but more so than Cu while Zn corrodes still faster. Cu artefacts may contain As and this element is also commonly a minor constituent of bronze (an alloy of Cu and Sn which is more resistant to corrosion than pure Cu). Fe is much more easily corroded than Cu, while Pb is resistant to corrosion in most ambient aqueous environments. Al forms a protective surface oxide coating that gives it some resistance to oxidative corrosion. The soil factors that affect the survival of buried metal objects have been studied (Tylecote, 1979; Johnson and Francis, 1980; Gerwin and Baumhauer, 2000; Nord et al., 2005; Neff et al., 2006; Réguer et al., 2007). In an aerobic, oxygenated aqueous environment, oxidation of metal and resulting corrosion is favoured thermodynamically and becomes more so with increasing acidity. The presence of chloride increases the rate of oxidation and resulting corrosion, especially of Fe. Depending on the metal type and the solutes present, initial corrosion processes may create a protective layer that slows corrosion further: these layers may include oxides (e.g., Al2O3), phosphates (e.g., FePO4) and carbonates (e.g., CuCO3). Under reducing conditions, biological activity may encourage the formation of sulphides that slow corrosion that is already being inhibited by a lack of free oxygen. 1.7. Stratigraphy Stratigraphy describes archaeological contexts that are interpretable from the chronological succession of layers of deposited material in soil (Harris, 1989). It supports the collection and interpretation of informa- tion about the burial context of buried objects and materials, such as their relation to other objects, age and the environmental conditions that prevailed in the burial period. Where the depth of the soil proﬁle is being increased, stratigraphic evidence will be better preserved than where soil is being eroded. Evidence is likely to be better preserved in soils that are receiving continuing inputs of parent material such as sediments during ﬂood events. Therefore soils that form in alluvial ﬂoodplains or that have gained colluvium material should preserve ev- idence better than those that are on slopes and those that have proper- ties that make them easily eroded. Soil hydrology and changes in water regime will affect the preservation of stratigraphic evidence. Wet anaer- obic conditions will preserve organic remnants that distinguish strata from each other. Drainage of wetland soils causes oxidation of organic material and in the case of peat soils it can dramatically reduce the depth of the soil proﬁle, destroying stratigraphic evidence. Soils are liv- ing systems and biological activity within them can perturb stratigraph- ic evidence. Lighter texture soils are favoured by mammals and birds that burrow in them, disturbing the soil proﬁle. Worm activity mixes the soil, sometimes to considerable depth, and worm casts left on the soil surface gradually alter the apparent height of the soil surface so that objects appear to sink in to the soil proﬁle. Worm type and behaviour as well as population size varies between soil types. Soil that is well- drained but moist and rich in fresh organic material is more favourable for worms than waterlogged or very dry soil with low inputs of organic material, while neutral soil is more favourable to worms than is acid soil. 1.8. Relevant soil properties Soil properties that inﬂuence the preservation of buried materials are as follows. Metals are preserved in the dry conditions present in arid climates. In moist climates preservation is worst in free-draining soils that have oxygenated water ﬂowing through the soil proﬁle. In these climates, ﬁne-textured clay soils with permanent or seasonal waterlogging are more preserving than those with coarse sandy textures. Corrosion is slowed in peats and other waterlogged soils that are permanently anaerobic, especially of the groundwater is alkaline. Preservation in alkaline soils formed from calcareous parent materials may be aug- mented by protective carbonate coatings. The presence of chloride (e.g., in naturally saline soils or from tidal ﬂooding, marine-affected atmospheric deposition, irrigation with saline water or spreading of salt) increases the corrosion rate of Fe. Soil formed mainly by human action can be a strongly corrosive environment where derived from wastes that contain chloride and sulphur. Strongly acidic soils in which corrosion of metals is rapid result when sulphide is oxidised, such when marine sediments are drained. 1. Hydrology (e.g., drainage), as affected by texture and proﬁle type and the combined inﬂuences of climate and landscape features and posi- tion, because this affects the levels of dissolved oxygen in the soil so- lution and the potential for dissolution of bones, teeth and shells, the corrosion of metals and the biological oxidation of organic materials. 2. Acidity and alkalinity, because these affect the rates of bone, teeth and shell dissolution, metal corrosion, oxidative degradation of organic material and the corrosion of glass. 3. Solute types and concentrations, because these affect secondary mineral formation within bones, the formation of protective coatings on metals and corrosion of glass. 4. Levels of dissolved organic matter in the soil solution because this can protect organic materials. 5. Vulnerability to erosion, because this affects the likelihood of surface exposure and loss of stratigraphic evidence. 6. Stiffness because this affects the physical protection of brittle objects from fracture, with texture being the main determining factor. 1.6. Stone and plaster 2.2. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ The cultural value of objects buried in soil is reduced as they degrade and if the object is destroyed this value is lost. Whereas some materials (bones, teeth, shells; copper, bronze and iron; organic materials) are destroyed in soil, albeit sometimes over very long periods, other mate- rials (ceramics, glass, gold) may be degraded but are not generally destroyed even after as long as 5 × 103 y. We develop a qualitative narrative describing how different STUs may affect those materials that are not destroyed. For those materials that are destroyed, however, 2. Materials and methods We then deﬁne preservation categories for materials in relation to endpoints of (i.e., complete destruction) within 102 y (poor), after 102 and before 2000 y (103.3 y) (fair) and after 103.3 y (good). For example, if we estimate that a metal object in a well-drained and moist soil will be destroyed within 102 y then we would assign that soil to the category (poor) for preservation of this metal, or, if we estimate that an object made of organic material buried in a soil with permanently waterlogged subsoil will not be destroyed within 103.3 y, we would assign that soil to the category ‘good’ for preservation of organic materials. Numeric values were assigned to each preservation category (poor = 1, fair = 2, good = 3). These values were assigned by relating the STU descrip- tion (e.g., soil proﬁle, diagnostic horizons and soil properties) to soil properties that drive material destruction (e.g., hydrology and associat- ed redox conditions, pH, base saturation, soil organic matter content, chloride levels). Table 1 Table 1 Properties relevant to preservation capacities of soils and their ranges and range descriptions. Property Ranges (and range descriptions) Drainage Free drainage Some restriction Seasonal or permanent waterlogging of at least subsoil Wetness Dry Moist Wet Organic matter N2% (high) b2 and N0.5% (medium) b0.5% (low) Base saturation N50% (high) b50% (low) pH N7.5 (alkaline) b7.5 and N6.0 (neutral) b6.0 (acidic) Chloride Below (low) or above (high) background concentration Stiffness (Low) (Medium) (High) Vulnerability to erosion (Low) (Medium) (High) Climate zone in which soil occurs (IPPC deﬁnitions) Warm temperate dry Warm temperate moist Cool temperate dry Cool temperate moist Boreal moist Note: other relevant soil properties to be considered (in particular in relation to the preservation of stratigraphy) are the degree of biological perturbation and the presence of swelling and shrinking due to the presence of expansive clay minerals and wetting and drying cycles. Table 1 Properties relevant to preservation capacities of soils and their ranges and range descriptions. Note: other relevant soil properties to be considered (in particular in relation to the preservation of stratigraphy) are the degree of biological shrinking due to the presence of expansive clay minerals and wetting and drying cycles. we develop a more quantitative analytical approach as follows. We deﬁne destruction as being where the object no longer has any recognisable morphology and its material has dispersed, at which time its cultural value has been lost. The time after burial at which destruction is complete represents the endpoint of a process of degradation. As degradation pro- ceeds its rate changes and in many cases will reduce progressively as less resistant material is removed. We assume that the degradation rate de- cays exponentially and that the percentage of remaining material versus log time (log10 t) can be represented by a linear relationship, as in Fig. 1. Table 1 summarises the key soil properties that affect the preserva- tion service of soil and the ranges over which these properties need to be considered when assessing the service level provided. It includes the climate zone in which the soil occurs as this inﬂuences the annual cycle of soil wetness and temperature which in turn affects material preservation. 2.1. Deﬁnition of soil types 2.1. Deﬁnition of soil types Taxonomic deﬁnitions within the Soil Geographical Database of Eurasia (SGDBE) (European Commission, 2003) were used to identify soil types (Soil Typological Units (STUs)). The SGDBE contains a list of STUs deﬁned using the legend for the Soil Map of the World (FAO, 1998). Tóth et al. (2008) provide a commentary on the STUs, their extent in Europe and a table correlating them to the key used by IUSS Working Group WRB (2006); additional information on the STUs is given in the Soil Atlas of Europe (European Commission, 2006a). For this study, STUs were identiﬁed to the second taxonomic level (Reference Soil Group and preﬁx). 2.2. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ 1.6. Stone and plaster 7. Factors that favour preservation of stratigraphic evidence include continuing inputs of soil forming materials and an absence of erosion, together with wetness as this assists the preservation of organic evidence and is less favourable to perturbing fauna. Buried stone artefacts include ﬂint artefacts, ﬁgures, stone hand tools and mortars and building materials. These are resistant to physical, M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 252 Table 1 Properties relevant to preservation capacities of soils and their ranges and range descriptions. 2.3. Mapping the preservation service of soils for buried materials SGDBE consists of (1) a geometric dataset at scale 1:1,000,000 and (2) a semantic dataset containing attribute ﬁles. The geometrical com- ponent of the database are polygons that form soil mapping units (SMUs) (EC, 2003). SMUs contain one or more STUs that form a discrete landscape unit with shared characteristics. The semantic dataset of the 0 1 10 100 1000 10000 Years since burial Destruction 0% 100% Good Fair Poor Fig. 1. Deﬁnition of categories of soils for preservation of material buried in soil. 0 1 10 100 1000 10000 Years since burial Destruction 0% 100% Good Fair Poor Fig. 1. Deﬁnition of categories of soils for preservation of material buried in soil. Fair Fig. 1. Deﬁnition of categories of soils for preservation of material buried in soil. M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 253 assessed as being poor due to the unconsolidated nature of many Andosol proﬁles. SGDBE describe the soil types present within the SMU and the percent- age of the SMU area occupied by each STU present within it. Thus mul- tiple attribute values are linked to the polygons of the geometrical dataset. We assigned the appropriate preservation numeric score to each STU in each SMU and calculated the spatially weighted mean of these scores for each SMU. Areas of artiﬁcial surfaces, permanent ice or snow cover and water bodies were excluded from the analysis. We then mapped the mean scores for the SMUs across the European Union after assigning colours to a continuous scale of mean preservation score. The percentage areas of soil across the European Union that pro- vide good (highest third of scale), fair and poor (lowest third of scale) preservation capacities for bones, teeth and shells; organic materials; and metals (Cu, bronze and Fe) were then calculated. Anthrosols are soils whose development has been profoundly inﬂu- enced by human activity, such as the addition of waste or other materials, or irrigation. They are azonal and occur in climate zones that may be wet, moist or dry. In Europe, the largest extent is of Plaggic Anthrosols in the cool moist temperate climate zone: these soils have open, well-drained surface horizons and are moist, with neutral or slight acidity, enhanced base saturation, good levels of organic matter and high levels of phos- phate (and potentially chloride) from addition of organic and other wastes. 3.1. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ Preservation of stratigraphy is assessed as good except where erosion has occurred. Acrisols occur in warm temperate moist climatic zones that are rare in Europe. They are moist, strongly acidic soils with a low base saturation and low subsoil organic matter contents. Their physical stiffness is assessed as being above average relative to other soil types due to high levels of weathered clay in subsoil horizons. The moist and acidic soil en- vironment in Acrisols will strongly degrade bones, teeth and shells, cor- rode metals, leach some glasses and degrade plaster. The warm and moist soil environment will encourage biological degradation of organic materials. The relative absence of roots in subsoil horizons may assist preservation of stratigraphy but these soils are vulnerable to erosion and are assessed as being poor for preservation of stratigraphy. Cambisols are widespread in Europe and are soils in which soil- forming processes remain active. The more acidic Dystric Cambisols with a low base saturation contrast with more alkaline or neutral Calcaric, Eutric and Mollic Cambisols that have a higher base satura- tion, but all are normally well-drained. The Calcaric Cambisols occur in drier regions. Gleyic and Vertic Cambisols have impeded drainage and are waterlogged at least seasonally. The more acidic Dystric Cambisols are likely to degrade bones, teeth, shells, some glasses, metals and plaster. The higher base saturation, alkaline pH and rela- tive dryness of the Calcaric Cambisols will assist the preservation of bones, teeth and shells, but where there are moist soil conditions (as is more typical for the Eutric and Mollic Cambisols) degradation of metals and plaster is expected. Organic materials are unlikely to survive in well-aerated Cambisols but this is more likely in wetter ones (Gleyic and Vertic). The preservation of stratigraphy is expect- ed to be average when compared to other soils. Albeluvisols occur in cool temperate moist and boreal moist climates. Their soil proﬁles indicate intermittent reducing conditions and some restricted drainage. They are wet and acidic with a low base saturation and a low subsoil organic matter content. Their physical stiffness is assessed as being average relative to other soil types. The wet and acidic soil environment in Albeluvisols will strongly degrade bones, teeth and shells, corrode metals, leach some glass and degrade plaster. The cool, wet and acidic soil environment may preserve some organic materials. The preservation of stratigraphy is assessed as fair in non-disturbed soil. 3.1. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ 3.1. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ Arenosols develop in sandy parent material and are azonal. They are well-drained and droughty even in moist climates and tend to be acidic with a low base saturation and low organic matter contents. Chloride levels are likely to be enhanced in those Arenosols formed in coastal dunes. These soils are vulnerable to erosion and where not conﬁned, for example on slopes, have a low stiffness. In moist climates, the freely drained and acidic soil environment in Arenosols will strongly degrade bones, teeth and shells, corrode metals and leach some glasses. However, some organic materials may be preserved where these soils are wet. In dry and more arid climates, bones, teeth and shells, metals, glass, plaster and organic materials will be preserved better. Preservation of stratigra- phy may be good where the landscape is dynamic and eroded material ac- cumulates over soil surfaces but will be poor where there is active erosion. A brief description follows of each soil type identiﬁed in the SGDBE, together with a summary of its properties and how these affect the preservation of buried materials and stratigraphy. This is followed by an assessment of the capacity of the soil type to preserve different bur- ied materials and stratigraphy. For some soil types it is only necessary to assess them at the Reference Soil Group (RSG) level but for other types with a wider range of characteristics, the assessment needs to be taken to the second taxonomic level according to the assigned preﬁx quali- ﬁers. The outputs from the description of soil properties are presented in Appendix A. The preservation service level for each STU is scored as Good, Fair or Poor, with the results presented in Appendix B. No assess- ment was made of capacity to preserve Au, Pb, ceramics, glass or stone as it was concluded that all types were similarly well-preserving of these materials. The potential inﬂuence of chloride was only noted for those soil types for which higher than background levels of chloride may be present. Calcisols occur in more arid parts of the warm dry temperate climate zone in Europe. These dry soils have a high base saturation and are alkaline. The dry alkaline soil conditions in Calcisols will preserve bones, teeth and shells and also metals and plaster. Some preservation of organic materials is possible in the driest of the Calcisols. 2.3. Mapping the preservation service of soils for buried materials Their physical stiffness is assessed as being average relative to other soil types. The Plaggic Anthrosols are of particular cultural impor- tance and contain fragments of ceramics and glass from added waste ma- terials. Bones, teeth and shells, as well as metals and some organic materials may be preserved to a limited extent. Stratigraphy is likely to be compromised by continuing mechanical cultivation. 3.1. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ The higher base saturation and more alkaline pH of the Calcaric and to a lesser extent the Eutric and Mollic Fluvisols will assist the preservation of bones, teeth and shells, but where moist soil conditions prevail this will encourage corrosion of metals which will be accelerated in environments with higher chloride levels. Degra- dation of organic materials is anticipated in moist and well aerated Fluvisols but these materials may be preserved better in wetter subsoil horizons and in Gleyic Fluvisols. Stratigraphy preservation is expected to be good in Fluvisols, except where river-bank and other erosion processes are active, and be very good where there are regular additions of fresh sediment in ﬂood events. and there is a greater ﬂow of water through the soil proﬁle, such as in moist and mountainous areas, this protection will be reduced and it will be less where the underlying rock is acidic, for example granite. As these are well-aerated soils, organic materials will not be preserved well in them except in the drier examples. Stratigraphy may be well-preserved but is likely to be less so on slopes, particular- ly where erosion processes are active. Luvisols have a clay-depleted topsoil and a clay-enriched subsoil. They occur in all the major climate zones present in Europe, general- ly in ﬂatter landscapes. They form in glacial till and other non- consolidated parent material in more northern and wetter land- scapes, but are present in many regions of Europe. Most Luvisols (for example: Albic, Arenic, Chromic, Dystric and most Haplic) are relatively free-draining and well-aerated but others are less so (Gleyic and some Haplic). Depending on the parent material, the topsoil may be more or less acidic or alkaline, with the pH generally higher in the subsoil reﬂecting a higher base saturation. As most Luvisols are free-draining and well-aerated, the soil is aerobic in these and the pH neutral or slightly acidic so that bone, teeth, shells, some glass, organic materials and metals will degrade, although this will be slowed in Gleyic Luvisols. Those Luvisols that contain expan- sive clay minerals (including but not only Vertic Luvisols) will frac- ture objects and stratigraphy will be more disrupted in these compared to most Luvisols where it should be preserved. Gleysols are poorly drained with permanent or seasonal water- logging by groundwater. Although azonal, they are most frequent in cool moist temperate and moist boreal climates. 3.1. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ The moist, aerated and acidic conditions in most Podzols will degrade bones, teeth, shells, some glass, metals and plaster, as well as organic materials. Those Podzols that have impeded drainage and are wetter (including Gleyic Podzols) and have more reducing conditions will be more conserving of organic materials and less degrading of metals but the acid conditions in all of these soils corrode metals and the conserva- tion of stratigraphy may be compromised by strong leaching. Histosols form where there is permanent waterlogging and have reducing conditions that slow the decomposition of organic matter. They are widespread in cold moist temperate and moist boreal climates but also occur in wetlands in other climates. Dystric Histosols have a lower base saturation and are more acidic than are the Eutric Histosols which form where groundwater is more alkaline. The strongly reducing conditions within Histosols preserve organic materials and metals. This will be enhanced where groundwater is more alkaline (Eutric Histosols) as will the preservation of bones, teeth and shells, with the latter being degraded by the more acidic conditions found in Dystric Histosols. Stratigraphy will be well-preserved in undisturbed Histosols. Kastanozems share many of the features and properties of Cherno- zems but occur in somewhat drier climates where less soil organic matter accumulates and the leaching of calcium is slower. Their ca- pacity to preserve buried materials and stratigraphy will be similar but slightly better than that of Chernozems. Regosols have a shallow soil development over poorly consolidated parent material such as gravels and rocky till. They occur widely in all cli- matic zones and most commonly in drier ones. The preservation of buried materials in Regosols will depend on their physical stability and soil depth. Regosols on slopes are prone to erosion processes which will de- grade and disperse buried objects and stratigraphy. In drier climates, es- pecially metal objects may survive well but in wetter ones the ﬂow of well-aerated water will be degrading of metals as well as bones, teeth, shells and organic materials, especially where the parent material is acidic (such as for Dystric Regosols). Leptosols are shallow soils with rocky parent material at shallow depth and are common throughout Europe, especially in upland and mountainous areas. Other than normally being well-drained, their properties are related to the nature of their parent material and prevailing climate. 3.1. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ The more acidic Dystric Gleysols have a low base saturation in contrast with more alkaline or neutral Calcaric, Eutric and Mollic Gleysols that have a higher base sat- uration, but all Gleysols are poorly-drained and have some reducing conditions in their subsoil horizons. Organic matter decomposition in topsoil as well as subsoil is likely to be slower in those Gleysols with organic horizons (Histic, Humic, Mollic). Thionic Gleysols form in ma- rine wetlands with sediments rich in sulphur and are highly acidic. The characteristic reducing conditions of Gleysols are partially preserv- ing of metals and organic materials, although the former will be more quickly degraded where base saturation and the pH's of soil and groundwater are lower (Dystric and especially Thionic Gleysols). The survival of bones, teeth and shells will be best in Calcaric and least ex- pected in Dystric Gleysols. Stratigraphy in general and especially that dependent on organic remnants will be well-preserved. Phaeozems share some similarities with Chernozems and Kastonozems but occur in somewhat moister climate conditions and consequently they have a lower base saturation. Their capacity to preserve buried materials and stratigraphy is similar but slightly less than that of Chernozems. Planosols have an impermeable subsoil that impedes drainage resulting in seasonal waterlogging and reducing conditions in the upper soil proﬁle. Dystric and Eutric Planosols have contrasting base saturations with the former being more acidic. The cycling of wet and reducing conditions and seasonal dryness in Planosols indi- cates that organic materials and metals will be degraded. In those Planosols that are neutral or more alkaline some preservation of bones, teeth and shells may occur. Stratigraphy preservation should be fair in non-disturbed proﬁles. Gypsisols occur in very arid climates and are uncommon in Europe. They are characterised by an accumulation of gypsum (calcium sul- phate) and have a high base saturation and an alkaline pH. The dry and alkaline conditions in Gypsisols are favourable for preserving all types of materials and where erosion is absent, stratigraphy. Podzols form under forest, moorland and heaths in sandy and other coarser parent material and on material formed from weathered acidic rock. They are most widespread in cool moist temperate and boreal moist climates, but also occur in somewhat drier conditions. They are acidic with a low base status and are characterised by a strongly leached subsoil horizon and accumulation of Fe and Al in lower horizons. 3.1. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ Chernozems are soils of the Steppe and form in cool dry temperate cli- mates in loess-rich parent materials. These well-drained soils are relative- ly dry during the summer months. They have deep proﬁles with good levels of organic matter and a high base saturation with a neutral to slight- ly alkaline pH. These freely drained soils are moist for part of the year and although they are not acidic some corrosion of metals is expected. Their high base saturation will assist preservation of bones, teeth and shells. The preservation of organic materials is uncertain but the well-aerated and warm moist soil conditions in early summer will encourage biological activity. These soils are subject to substantial perturbation by soil fauna to depth and this is likely to disturb and degrade stratigraphy. Andosols develop in volcanic parent material and are azonal, that is they are not conﬁned to a particular climate zone, occurring in both cold temperate and warm temperate climates. They are gener- ally acidic, have a low base saturation and have medium levels of or- ganic matter in subsoil. Their physical stiffness is assessed as being less than average relative to other soil types due to the high levels of organic matter in surface horizons and continuing weathering of parent material to give a relatively open and loose soil structure. The acidic soil environment in Andosols will strongly degrade bones, teeth and shells, corrode metals, leach some glasses and de- grade plaster. Their acidic and moist soil environment may partially preserve some organic materials. Preservation of stratigraphy is Fluvisols are formed in alluvial, lacustrine or recent marine material and are azonal. The more acidic Dystric Fluvisols with a low base satura- tion contrast with the neutral or more alkaline pH of the Calcaric, Eutric and Mollic Fluvisols that have a higher base saturation, but all are well- M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 254 drained. Gleyic Fluvisols have impeded drainage with at least seasonal waterlogging. Fluvisols formed in marine environments may have en- hanced chloride levels and strongly acidic Thionic Fluvisols form in drained sulphur-rich marine sediments. The more acidic Dystric Fluvisols are likely to degrade bones, teeth, shells, some glass, metals and plaster. The highly acidic Thionic Fluvisols are perhaps the least preserving soil type. 3.1. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ Calcaric and Eutric Leptosols form on calcar- eous and more basic rocks while Dystric Leptosols form on more acidic rock. Rendzic Leptosols are mainly located in moister climate zones on chalk and limestone and in their natural state have a char- acteristic highly organic surface horizon. Those Leptosols that are drier and calcareous and/or more alkaline will be most protective of bones, teeth, shells, metals and plaster. Where rainfall is higher Solonchak soils form in drier climates where rising shallow saline groundwater accumulates salts in the upper soil horizons and on the soil surface. Solonetz soils form under similar but less extreme conditions where groundwater is less saline and they have more clay in subsoil than Solonchak soils. Both soil types are alkaline. The presence of high M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 255 Vertisols form in warm moist climates with distinct dry and wet sea- sons and contain clay minerals that expand and contract over wetting and drying cycles. The result is continuing mixing of soil horizons. They have a medium base saturation and are neither strongly acidic nor basic. The dynamic physical conditions in Vertisols will be destructive and dis- persive of buried objects and stratigraphy. Chemical degradation of bones, teeth, shells, glass, metals and plaster will be supported by the wet- ting and drying cycles that are typical for these soils. Vertisols form in warm moist climates with distinct dry and wet sea- sons and contain clay minerals that expand and contract over wetting and drying cycles. The result is continuing mixing of soil horizons. They have a medium base saturation and are neither strongly acidic nor basic. concentrations of salt and associated chloride in both Solonchak and Solonetz soils will accelerate corrosion of metals and loss of calcium from bones, teeth, shells and plaster. The preservation of organic mate- rials is uncertain: strong alkaline solutions will solubilise some organic matter; reducing conditions that are protective of organic materials may occur depending on the extent of seasonal waterlogging. The dynamic physical conditions in Vertisols will be destructive and dis- persive of buried objects and stratigraphy. Chemical degradation of bones, teeth, shells, glass, metals and plaster will be supported by the wet- ting and drying cycles that are typical for these soils. 3.1. Assignment of Soil Typological Units (STUs) to ‘preservation categories’ Umbrisols form mainly in a cool moist temperate climate on acidic par- ent material under forest and have an organic surface horizon. In Europe they are found mainly in the southern parts of the western oceanic zone. The moist, acidic, well-drained conditions in Umbrisols will degrade bones, teeth, shells, some glass, metals and plaster. The presence of higher levels of soil organic matter in Umbrisols reﬂects continuing inputs from woody vegetation and is not indicative of a preserving environment for buried organic materials in the these well-drained and aerobic soils. 3.2. Mapping the preservation service of soils for buried materials This helped to discrimi- nate between soils with different hydrology and wetness but was limit- ed by a predominance of Haplic forms i.e., those typical of the RSG and for which no special features are prominent or noted. Azonal soil 3.2. Mapping the preservation service of soils for buried materials Figs. 2, 3, 4 and 5 present maps of the preservation capacities for bur- ied materials and stratigraphy provided by soils across the European Fig. 2. Soil-based preservation capacity for buried bones, teeth and shells across the EU. Fig. 2. Soil-based preservation capacity for buried bones, teeth and shells across the EU. 256 M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 Fig. 3. Soil-based preservation capacity for organic materials across the EU. Fig. 3. Soil-based preservation capacity for organic materials across the EU. Union, for bones and teeth, organic materials, metals (Cu, bronze and Fe) and stratigraphy respectively. service for different materials provided by different soil types, allows an assessment of how this service varies between soil types and its consequent variation across the EU. Some soil types are much more common than others and assessing the preservation services that soil provides generally and across the EU depends especially on assessment of these common soil types. Cambisols, Luvisols, Podzols and Leptosols cover more than half the area of the EU (26.71%, 14.74%, 13.67% and 10.51% respectively). The assessment for these soils was extended to the second taxonomic level by reference to preﬁx qualiﬁers, including those indicative of wetness (Gleyic), pH (Dystric and Eutric) and higher organic matter content (Histic, Humic, Mollic). This helped to discrimi- nate between soils with different hydrology and wetness but was limit- ed by a predominance of Haplic forms i.e., those typical of the RSG and for which no special features are prominent or noted. Azonal soil service for different materials provided by different soil types, allows an assessment of how this service varies between soil types and its consequent variation across the EU. Some soil types are much more common than others and assessing the preservation services that soil provides generally and across the EU depends especially on assessment of these common soil types. Cambisols, Luvisols, Podzols and Leptosols cover more than half the area of the EU (26.71%, 14.74%, 13.67% and 10.51% respectively). The assessment for these soils was extended to the second taxonomic level by reference to preﬁx qualiﬁers, including those indicative of wetness (Gleyic), pH (Dystric and Eutric) and higher organic matter content (Histic, Humic, Mollic). 4.1. Methodology 4.1. Methodology There are many combinations of soil types and materials and their interactions are complex. Consequently, predictions of the preservation of buried objects are uncertain, especially given that the properties of both individual soil types and materials extend over wide ranges. Another complication is that objects are often constructed from more than one material. Nonetheless, the approach used here of developing an interpretive narrative as a basis for categorising the preservation M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 257 Fig. 4. Soil-based preservation capacity for metals (Cu, bronze and Fe) across the EU. Fig. 4. Soil-based preservation capacity for metals (Cu, bronze and Fe) across the EU. types that occur in all climate regions (e.g., Arenosols, Andosols) may be dry or moist and this creates uncertainty when predicting the level of preservation provided. It was assumed that all soils in these RSGs are moist which would tend to under estimate preservation in drier ones. compare these to spatial data on soil properties. However, this approach appears unworkable at present. Spatial data is available for some but not all of the relevant soil properties across the EU but mainly for topsoil only, whereas preservation depends on subsoil properties as much as or more than topsoil ones. Importantly, the seasonal dynamics of the whole soil proﬁle and the processes within it and their impact on preservation need to be taken in to account and this may be more easily done by reviewing the proﬁle characteristics that are speciﬁc to STU descriptions. There is no meaningful timescale for destruction in soil of ceramics, glass, stone and plaster. Although glass becomes degraded aesthetically when basic ions (e.g., Na, K, Ca) are leached from it in moist soil, and the structural integrity of plaster objects may be degraded in wet and espe- cially acidic soils, the morphology of objects made from these materials survives. Therefore it was decided that attempting to map the preserva- tion service for these materials was not very informative. 4.2. Preservation of buried materials Therefore the methodology we have used can be readily applied to provide information at scales that are relevant to spatial planning and land management in general. Speciﬁc applications could include: preliminary evaluations of where artefacts may exist that need to be identiﬁed and managed to inform environ- mental impact assessments; evaluating where existing or planned buried infrastructure (e.g., iron pipes) is likely to be better or worse preserved. Many different waste materials are spread on land. This practice can support the beneﬁcial recycling of nutrients but may also introduce materials that may persist in the soil environment. This study indicates that the persistence of different materials is controlled by soil type and conditions and the results suggest that soil type is a factor that should be included when assessing the suitability of wastes and land for spread- ing, especially where the waste may contain bone, ceramic, glass or metal objects or fragments. The survival of some materials in some soils but not others may also be relevant to investigations of options for the long-term disposal of hazardous wastes, as ancient metal, ceram- ic and glass artefacts found in the buried environment can act as proxies for long-term trials of similar materials that could be used to encapsu- late wastes (Johnson and Francis, 1980; Neff et al., 2006). Our results conﬁrm that a persistently dry and alkaline soil environment is the most preserving one for almost all materials. The preservation capacity for bones, teeth and shells across the EU (Fig. 2) reﬂects the predominance of drier soils in the south, which are more preserving than the wetter soils in northern and western regions, excepting those with permanent waterlogging (e.g., in the Netherlands and Denmark) that have a neutral or slightly alkaline pH. The driest regions are those for which preservation is predicted to be greatest, notably parts of southern and eastern Spain and Greece. The wettest soils of Northern Europe and some very dry soils in Spain provide most capacity for preserving organic materials (Fig. 3). The apparently low preservation capacity for organic materials in Northern Sweden is anomalous and considered inaccurate as it reﬂects the quality of underlying soil data, which has a poorer spatial resolution across Northern Sweden than, for example, the data for Finland where the common occurrence of Histosols is represented better. 5. Conclusions Burial in soil preserves almost all objects for at least a limited time and in many cases beyond 103 y. While the preservation capacities of different soils for different materials and for stratigraphy are variable, they are predictable. Some materials such as Au, ceramics, glasses, Pb and stone can survive almost indeﬁnitely in most soil environments, albeit especially their surfaces may be altered. Bones, teeth and shells, organic materials and Al, Ag, bronze, Cu, Fe and Zn are not preserved in all soils, but their degradation and eventual destruction is slowed in those soils that provide a favourable burial environment. The methodol- ogy that has been developed can be readily applied at local to regional to national scales and the results interpreted to inform the management of buried objects, including contemporary infrastructure as well as cultural heritage. Table 2 shows the percentages of the EU area assessed as having good, fair and poor preservation capacities for some of these materials. Although b10% of the total area of soils in the EU are highly preserving of them, meaning that objects made of them are expected to survive in soil for at least 2000 y (103.3 y), soils in an additional 30% of the EU area are assessed as preserving of these materials for more than 102 y and potentially as much as 103.3 y. The service that soil provides by preserving buried objects and stratigraphic evidence is con- siderable and this study illustrates its distribution at a continental scale. The driest soils (e.g., Calcisols, Gypsisols) are expected to be most preserving of the surfaces of glass objects and the most degrading soils for these are the most alkaline (Solonetz and Solonchak). The structural integrity of glass (and also ceramic) objects is perhaps more dependent on land use and land management than on soil type, although shattering and dispersion will be accelerated in Vertisols and other soils with Vertic tendencies that contain expansive clay minerals causing swelling and shrinking during wetting and drying cycles. The good preservation capacity for stratigraphic evidence of Fluvisols is clearly identiﬁable in Fig. 5. These are widespread in the EU. Additionally, some areas of Histosols in the north and west stand out as preserving of stratigraphic evidence. The poor preservation capacity associated with Andosols (e.g., in Central France) and Regosols (e.g., in Southern Spain) is also clear. 4.2. Preservation of buried materials There are only a few soils in the EU that offer very good preservation of metals including iron (Fig. 3) and these are the driest soils, including the Calcisols and Gypsisols that are conﬁned to a small number of loca- tions in Southern Europe. Lowland peat soils (Eutric Histosols) with shallow groundwater in Northern Europe are also relatively preserving. Metals' preservation is compromised where soils are freely-drained and there is a plentiful supply of oxygenated water, which is common across much of the EU. This norm is moderated, however, by soil pH and the more alkaline soils formed on chalk and limestone that are widespread provide a medium level of metals' preservation. 4.2. Preservation of buried materials / Science of the Total Environment 529 (2015) 249–263 259 Table 2 Percentages of the European Union area with soils assessed as having good, fair or poor preservation capacities for buried materials and stratigraphic evidence. Preservation capacity Poor Fair Good Bones 55.6 39.7 4.7 Metals 68.2 31.5 0.3 Organics 59.3 32.4 8.2 Stratigraphic evidence 7.9 74.9 17.1 Table 2 Percentages of the European Union area with soils assessed as having good, fair or poor preservation capacities for buried materials and stratigraphic evidence. conditions due to waterlogging, should provide some preservation of bones, teeth, shells, organic materials and metals, depending on the per- manence of waterlogging. Preservation will be less where waterlogging is seasonal as is common with soils that have gleyic features, such as Gleyic Cambisols, Gleyic Fluvisols and Gleyic Luvisols. Fluvisols emerge as an important soil type for preservation of cultural materials and evidence. Although not especially preserving of bones, teeth and shells, organic materials or metals, they appear important for preservation of stratigraphic evidence of the cultural and environmental context of materials that are preserved in them, including ceramics, glass and stone. Although the level of preservation service provided by Cambisols and Luvisols will be variable, overall, these relatively freely drained soils are assessed as not especially preserving of bones, teeth and shells, or- ganic materials or metals. The Chernozem, Kastanozem and Phaeozem should support an intermediate level of preservation for bones, teeth, organic materials and metals. The characteristic perturbation that occurs in these soils due to burrowing mammals will compromise strat- igraphic evidence. Of the group of soil types that are poorly preserving of bones, teeth and shells, organic materials and metals, the Podzols cover the largest area in the EU (others are Acrisols, Albeluvisols, Andosols, Arenosols, Leptosols, Planosols, Regosols, Solonchak, Solonetz and Vertisols). Table 2 Percentages of the European Union area with soils assessed as having good, fair or poor preservation capacities for buried materials and stratigraphic evidence. Preservation capacity Poor Fair Good Bones 55.6 39.7 4.7 Metals 68.2 31.5 0.3 Organics 59.3 32.4 8.2 Stratigraphic evidence 7.9 74.9 17.1 on soil types are available. Data at national to regional scales (1: 50,000) is available in many countries and ﬁner scale data exists for many regions (FAO, 2015; Jones et al., 2005). 4.2. Preservation of buried materials Many materials including ceramics, glass, stone and Au are preserved in most soils. Focusing, however, on those materials that are ultimately destroyed in soil (bones, teeth, shells, organic materials and An alternative more quantitative approach to the qualitative one adopted in this study was considered. This would set deﬁnitive ranges for soil properties that are preserving of different material types and 258 M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 Fig. 5. Soil-based preservation capacity for stratigraphic evidence across the EU. Fig. 5. Soil-based preservation capacity for stratigraphic evidence across the EU. in these anoxic and alkaline soil environments bones, teeth, shells, metals and less tractable organic materials are expected to be well- preserved. The more acidic Dystric Histosols that cover relatively large areas of Northern and Western Europe in the cool moist temperate and moist boreal zones are expected to be less conserving of bones, teeth, shells and metals but will generally conserve organic materials well. Eutric and Calcic Leptosols formed from chalk and limestone parent materials are well-drained and aerobic which does not favour the preservation of organic materials but, as they are alkaline, they should be relatively preserving of bones, teeth, shells and to some extent metals including bronze, Cu and to a lesser degree Fe. These soils occur widely in Europe. Soils that have subsoil with gley features (including but not conﬁned to Gleysols), indicative of anaerobic some metals), the most preserving soils for these materials are the dri- est, including Calcisols, Gypsisols and some drier Leptosols formed on limestone. These are, however, relatively uncommon in the EU and largely conﬁned to small areas in Spain and Greece of relative aridity. Elsewhere in Europe, where precipitation is higher, soil hydrology is the dominant factor determining the preservation or degradation of these materials, moderated by the pH of the soil and associated ground- water and sometimes by the presence of chloride. The most favourable burial environment for bones, teeth, organic materials and Cu, bronze and Fe, other than a very dry one, is where the soil is permanently waterlogged and strongly anaerobic. The best preservation is anticipat- ed where the groundwater is alkaline and stagnant as is typical of Eutric Histosols in some lowland peat lands in Northern and Western Europe; M. Kibblewhite et al. This study was ﬁnanced by the European Commission, Joint Research Centre: contract number — CCR.IES.C391026.X0. Acknowledgement In this study, preservation capacities have been mapped using spatial data on the occurrence of soil types at a continental scale. This approach can, however, be applied at any scale for which spatial data This study was ﬁnanced by the European Commission, Joint Research Centre: contract number — CCR.IES.C391026.X0. M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 260 Appendix A. Soil properties descriptions for soil types Appendix A. Acknowledgement / Science of the Total Environment 529 (2015) 249–263 261 (continued) Appendix A (continued) (continued) Appendix A (continued) (continued) Soil type Typical climate Soil properties Reference group Name in WRB 1998 Name in WRB 2006 Drainage Wetness pH status Base satn Chloride Subsoil OM Stiffness Calcic Luvisol Calcic Luvisol Cool temperate moist Good Moist Alkaline High Low Medium Medium Chromic Luvisol Haplic Luvisol Cool temperate moist Good Moist Neutral Medium Low Medium Medium Dystric Luvisol Haplic Luvisol Warm temperate dry Good Moist Acid Low Low Medium Medium Ferric Luvisol Haplic Luvisol Warm temperate dry Good Moist Acid Low Low Medium Medium Gleyic Luvisol Gleyic Luvisol Cool temperate moist Good Moist Neutral Medium Low Medium Medium Haplic Luvisol Haplic Luvisol Cool temperate moist Good Most Neutral Medium Low Medium Medium Vertic Luvisol Vertic Luvisol Warm temperate dry Good Moist Neutral Medium Low Medium Medium Phaeozem Albic Phaeozem Phaeozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Calcaric Phaeozem Haplic Phaeozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Gleyic Phaeozem Gleyic Phaeozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Haplic Phaeozem Haplic Phaeozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Luvic Phaeozem Luvic Phaeozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Sodic Phaeozem Haplic Phaeozem Cool temperate dry Good Dry Neutral Medium High Medium Medium Planosol Dystric Planosol Haplic Planosol Cool temperate moist Poor Wet Neutral Medium Low Low Medium Eutric Planosol Haplic Planosol Cool temperate moist Poor Wet Neutral Medium Low Low Medium Haplic Planosol Haplic Planosol Cool temperate moist Poor Wet Neutral Medium Low Low Medium Podzol Carbic Podzol Haplic Podzol Cool temperate moist Good Moist Acid Low Low Medium Medium Entic Podzol Haplic Podzol Cool temperate moist Good Moist Acid Low Low Medium Medium Gleyic Podzol Gleyic Podzol Cool temperate moist Fair Moist Acid Low Low Medium Medium Haplic Podzol Haplic Podzol Cool temperate moist Good Moist Acid Low Low Medium Medium Leptic Podzol Leptic Podzol Cool temperate moist Good Moist Acid Low Low Medium Medium Placic Podzol Placic Podzol Cool temperate moist Good Moist Acid Low Low Medium Medium Rustic Podzol Haplic Podzol Cool temperate moist Good Moist Acid Low Low Medium Medium Umbric Podzol Umbric Podzol Cool temperate moist Good Moist Acid Low Low Medium Medium Regosol Calcaric Regosol Haplic Regosol Various Good Moist Alkaline High Low Low Low Dystric Regosol Haplic Regosol Cool temperate moist Good Moist Acid Low Low Low Low Eutric Regosol Haplic Regosol Warm temperate dry Good Moist Neutral High Low Low Low Haplic Regosol Haplic Regosol Various Good Moist Neutral Medium Low Low Low Solonchak Gleyic Solonchak Gleyic Solonchak Warm temperate dry Poor Moist Alkaline High High Low Medium Haplic Solonchak Haplic Solonchak Warm temperate dry Poor Moist Alkaline High High Low Medium Takyric Solonchak Haplic Solonchak Warm temperate dry Poor Moist Alkaline High High Low Medium Mollic Solonchak Mollic Solonchak Warm temperate dry Poor Moist Alkaline High High Medium Medium Solonetz Gleyic Solonetz Gleyic Solonetz Warm temperate dry Poor Moist Alkaline High High Low Medium Haplic Solonetz Haplic Solonetz Warm temperate dry Poor Moist Alkaline High High Low Medium Mollic Solonetz Mollic Solonetz Warm temperate dry Poor Moist Alkaline High High Medium Medium Umbrisol Arenic Umbrisol Arenic Umbrisol Cool temperate moist Good Moist Acid Low High Low Medium Gleyic Umbrisol Gleyic Umbrisol Cool temperate moist Poor Moist Acid Low High Low Medium Vertisol Chromic Vertisol Haplic Vertisol Warm temperate dry Fair Moist Neutral Medium Low Medium Medium Haplic Vertisol Haplic Vertisol Warm temperate dry Fair Moist Neutral Medium Low Medium Medium Pellic Vertisol Haplic Vertisol Warm temperate dry Fair Moist Neutral Medium Low Medium Medium pp ( ) Appendix B. Acknowledgement Kibblewhite et al. (continued on next page) Acknowledgement Soil properties descriptions for soil types Soil type Typical climate Soil properties Reference group Name in WRB 1998 Name in WRB 2006 Drainage Wetness pH status Base satn Chloride Subsoil OM Stiffness Acrisol Ferric Acrisol Haplic Acrisol Warm temperate moist Fair Moist Acidic Low Low Low Medium Gleyic Acrisol Gleyic Acrisol Warm temperate moist Fair Moist Acidic Low Low Low Medium Haplic Acrisol Haplic Acrisol Warm temperate moist Fair Moist Acidic Low Low Low Medium Humic Acrisol Humic Acrisol Warm temperate moist Fair Moist Acidic Low Low Low Medium Plinthic Acrisol Plinthic Acrisol Warm temperate moist Fair Moist Acidic Low Low Low Medium Alisol Plinthic Alisol Plinthic Alisol Warm temperate moist Fair Moist Acidic Low Low Low Medium Albeluvisol Endoeutric Albeluvisol Haplic Albeluvisol Cool temperate moist Poor High Acidic Low Low Low Medium Gleyic Albeluvisol Gleyic Albeluvisol Cool temperate moist Poor High Acidic Low Low Low Medium Haplic Albeluvisol Haplic Albeluvisol Cool temperate moist Poor High Acidic Low Low Low Medium Histic Albeluvisol Histic Albeluvisol Cool temperate moist Poor High Acidic Low Low Medium Medium Stagnic Albeluvisol Stagnic Albeluvisol Cool temperate moist Poor High Acidic Low Low Low Medium Umbric Albeluvisol Umbric Albeluvisol Cool temperate moist Poor High Acidic Low Low Low Medium Andosol Dystric Andosol Aluandic Andosol Varied climates Good Moist Acidic Low Low Low Low Humic Andosol Humic Andosol Varied climates Good Moist Acidic Low Low Medium Low Umbric Andosol Umbric Andosol Varied climates Good Moist Acidic Low Low Low Low Mollic Andosol Mollic Andosol Varied climates Good Moist Acidic Medium Low Low Low Vitric Andosol Vitric Andosol Varied climates Good Moist Acidic Low Low Low Low Anthrosol Anthrosol Anthrosol Varied climates Good Moist Acidic Low Low Low Low Plaggic Anthrosol Plaggic Anthrosol Cool temperate moist Good Moist Acidic Low Low Low Low Arenosol Albic Arenosol Albic Arenosol Moist Good Moist Acidic Low Low Low Low Haplic Arenosol Haplic Arenosol Moist Good Moist Acidic Low Low Low Low Protic Arenosol Protic Arenosol Moist Good Moist Acidic Low Low Low Low Haplic Arenosol Haplic Arenosol Dry Good Dry Acidic Low Low Low Low Protic Arenosol Protic Arenosol Dry Good Dry Acidic Low Low Low Low Calcisol Aridic Calcisol Aridic Calcisol Warm temperate dry Impeded Dry Basic High Low Low Medium Chernozem Calcic Chernozem Calcic Chernozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Chernozem Haplic Chernozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Gleyic Chernozem Gleyic Chernozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Haplic Chernozem Haplic Chernozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Luvic Chernozem Luvic Chernozem Cool temperate dry Good Dry Neutral Medium Low Medium Medium Cambisol Calcaric Cambisol Haplic Cambisol Temperate Good Moist Neutral Medium Low Low Medium Chromic Cambisol Haplic Cambisol Temperate Good Moist Neutral Medium Low Low Medium Dystric Cambisol Haplic Cambisol Temperate Good Moist Acid Low Low Low Medium Eutric Cambisol Haplic Cambisol Temperate Good Moist Neutral Medium Low Low Medium Gleyic Cambisol Gleyic Cambisol Temperate Poor Wet Neutral Medium Low Low Medium Haplic Cambisol Haplic Cambisol Temperate Good Moist Neutral Medium Low Low Medium Mollic Cambisol Mollic Cambisol Temperate Good Moist Neutral Medium Low Low Medium Vertic Cambisol Vertic Cambisol Warm temperate dry Fair Moist Neutral Medium Low Low Medium Fluvisol Calcaric Fluvisol Haplic Fluvisol Varied climates Good Moist Alkaline High Low Medium Medium Dystric Fluvisol Haplic Fluvisol Varied climates Good Moist Acid Low Low Medium Medium Eutric Fluvisol Haplic Fluvisol Varied climates Good Moist Neutral High Low Medium Medium Gleyic Fluvisol Gleyic Fluvisol Varied climates Fair Moist Neutral Medium Low Medium Medium Haplic Fluvisol Haplic Fluvisol Varied climates Good Moist Neutral Medium Low Medium Medium Histic Fluvisol Histic Fluvisol Varied climates Good Moist Neutral High Low Medium Medium Mollic Fluvisol Mollic Fluvisol Varied climates Good Moist Neutral High Low Medium Medium Salic Fluvisol Salic Fluvisol Varied climates Good Moist Neutral High High Medium Medium Thionic Fluvisol Thionic Fluvisol Varied climates Fair Moist Acid Low Low Medium Medium Gleysol Calcaric Gleysol Haplic Gleysol Cool temperate moist Poor Moist Alkaline High Low Medium Medium Dystric Gleysol Haplic Gleysol Cool temperate moist Poor Moist Neutral Low Low Medium Medium Eutric Gleysol Haplic Gleysol Cool temperate moist Poor Moist Neutral High Low Medium Medium Haplic Gleysol Haplic Gleysol Cool temperate moist Poor Moist Neutral Medium Low Medium Medium Histic Gleysol Histic Gleysol Cool temperate moist Poor Moist Neutral Medium Low Medium Medium Humic Gleysol Haplic Gleysol Cool temperate moist Poor Moist Neutral Medium Low Medium Medium Mollic Gleysol Mollic Gleysol Cool temperate moist Poor Moist Neutral High Low Medium Medium Thionic Gleysol Thionic Gleysol Cool temperate moist Poor Moist Acid Low Low Medium Medium Gypsisol Aridic Gypsisol Haplic Gypsisol Warm temperate dry Fair Dry Alkaline High Low Low High Histosol Dystric Histosol Hemic Histosol Cool temperate moist Poor Wet Acid Low Low High Low Eutric Histosol Hemic Histosol Cool temperate moist Poor Wet Neutral High Low High Low Fibric Histosol Histosol Cool temperate moist Poor Wet Neutral Medium Low High Low Gelic Histosol Histosol Cool temperate moist Poor Wet Neutral Medium Low High Low Sapric Histosol Histosol Cool temperate moist Poor Wet Neutral Medium Low High Low Kastanozem Calcic Kastanozem Calcic Kastanozem Cool temperate dry Good Dry Alkaline High Low Medium Medium Haplic Kastanozem Haplic Kastanozem Cool temperate dry Good Dry Alkaline High Low Medium Medium Luvic Kastanozem Luvic Kastanozem Cool temperate dry Good Dry Alkaline High Low Medium Medium Leptosol Calcaric Leptosol Haplic Leptsol Warm temperate dry Good Dry Alkaline High Low Medium High Dystric Leptosol Haplic Leptsol Warm temperate dry Good Dry Acid Low Low Medium High Eutric Leptosol Haplic Leptsol Warm temperate dry Good Dry Neutral High Low Medium High Haplic Leptsol Haplic Leptsol Cool temperate moist Good Moist Neutral Medium Low Medium High Humic Leptosol Haplic Leptsol Cool temperate moist Good Moist Neutral Medium Low Medium High Rendzic Leptosol Rendzic Leptosol Cool temperate moist Good Moist Alkaline High Low High High Lithic Leptosol Lithic Leptosol Warm temperate dry Good Dry Neutral Medium Low Low High Luvisol Albic Luvisol Albic Luvisol Cool temperate moist Fair Wet Neutral Low Low Medium Medium Arenic Luvisol Haplic Luvisol Cool temperate moist Good Moist Neutral Medium Low Medium Medium M. Acknowledgement Preservation capacities of different soil types for different buried materials and stratigraphy Soil type Reference group Name in WRB 1998 Bones etc. Organics Metals Stratigraphy Acrisol Ferric Acrisol Poor Poor Poor Poor Gleyic Acrisol Poor Poor Poor Poor Haplic Acrisol Poor Poor Poor Poor Humic Acrisol Poor Poor Poor Poor Plinthic Acrisol Poor Poor Poor Poor Alisol Plinthic Alisol Poor Poor Poor Poor Albeluvisol Endoeutric Albeluvisol Poor Fair Poor Fair Gleyic Albeluvisol Poor Fair Poor Fair Haplic Albeluvisol Poor Fair Poor Fair Histic Albeluvisol Poor Fair Poor Fair Stagnic Albeluvisol Poor Fair Poor Fair Umbric Albeluvisol Poor Fair Poor Fair Andosol Dystric Andosol Poor Poor Poor Poor Humic Andosol Poor Poor Poor Poor Umbric Andosol Poor Poor Poor Poor Mollic Andosol Poor Poor Poor Poor Vitric Andosol Poor Poor Poor Poor Anthrosol Anthrosol Poor Poor Poor Fair Plaggic Anthrosol Poor Fair Poor Fair Arenosol Albic Arenosol Poor Fair Poor Fair Haplic Arenosol Poor Fair Poor Fair (continued on next page) Appendix B. Preservation capacities of different soil types for different buried materials and stratigraphy M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 262 (continued) Appendix B (continued) (continued) Soil type Reference group Name in WRB 1998 Bones etc. Acknowledgement Organics Metals Stratigraphy Protic Arenosol Poor Fair Poor Fair Calcisol Aridic Calcisol Good Good Good Fair Chernozem Calcic Chernozem Fair Fair Fair Fair Chernozem Fair Fair Fair Fair Gleyic Chernozem Fair Fair Fair Fair Haplic Chernozem Fair Fair Fair Fair Luvic Chernozem Fair Fair Fair Fair Cambisol Calcaric Cambisol Fair Poor Fair Fair Chromic Cambisol Poor Poor Poor Fair Dystric Cambisol Poor Poor Poor Fair Eutric Cambisol Fair Poor Fair Fair Gleyic Cambisol Fair Fair Poor Fair Haplic Cambisol Poor Poor Poor Fair Mollic Cambisol Fair Fair Fair Fair Vertic Cambisol Poor Poor Poor Poor Fluvisol Calcaric Fluvisol Fair Poor Poor Good Dystric Fluvisol Poor Poor Poor Good Eutric Fluvisol Fair Poor Poor Good Gleyic Fluvisol Poor Fair Poor Good Haplic Fluvisol Poor Poor Poor Good Histic Fluvisol Poor Fair Poor Good Mollic Fluvisol Poor Fair Poor Good Salic Fluvisol Poor Poor Poor Good Thionic Fluvisol Poor Poor Poor Good Gleysol Calcaric Gleysol Fair Fair Fair Good Dystric Gleysol Poor Fair Poor Good Eutric Gleysol Fair Fair Fair Good Haplic Gleysol Fair Fair Poor Good Histic Gleysol Fair Good Poor Good Humic Gleysol Fair Good Poor Good Mollic Gleysol Fair Good Poor Good Thionic Gleysol Poor Poor Poor Good Gypsisol Aridic Gypsisol Good Good Good Good Histosol Histosol Fair Good Poor Good Dystric Histosol Poor Good Poor Good Eutric Histosol Good Good Fair Good Fibric Histosol Fair Good Poor Good Gelic Histosol Fair Good Poor Good Sapric Histosol Fair Good Poor Good Kastanozem Calcic Kastanozem Fair Fair Fair Fair Haplic Kastanozem Fair Fair Fair Fair Luvic Kastanozem Fair Fair Fair Fair Leptosol Calcaric Leptosol Good Fair Fair Fair Dystric Leptosol Poor Poor Poor Fair Eutric Leptosol Good Fair Fair Fair Haplic Leptsol Fair Fair Fair Fair Humic Leptosol Fair Fair Fair Fair Rendzic Leptosol Fair Fair Fair Fair Lithic Leptosol Fair Poor Fair Fair Luvisol Albic Luvisol Poor Poor Poor Fair Arenic Luvisol Poor Poor Poor Fair Calcic Luvisol Fair Poor Fair Fair Chromic Luvisol Poor Poor Poor Fair Dystric Luvisol Fair Poor Poor Fair Ferric Luvisol Fair Poor Poor Fair Gleyic Luvisol Fair Fair Poor Fair Haplic Luvisol Poor Poor Poor Fair Vertic Luvisol Poor Poor Poor Poor Phaeozem Albic Phaeozem Fair Fair Poor Fair Calcaric Phaeozem Fair Fair Fair Fair Gleyic Phaeozem Fair Fair Fair Fair Haplic Phaeozem Fair Fair Fair Fair Luvic Phaeozem Fair Fair Fair Fair Sodic Phaeozem Fair Fair Poor Fair Planosol Dystric Planosol Poor Fair Poor Fair Eutric Planosol Fair Fair Fair Fair Haplic Planosol Fair Fair Fair Fair Podzol Carbic Podzol Poor Poor Poor Fair Entic Podzol Poor Poor Poor Fair Appendix B (continued) ( ) Soil type Reference group Name in WRB 1998 Bones etc. Acknowledgement Org Protic Arenosol Poor Fai Calcisol Aridic Calcisol Good Goo Chernozem Calcic Chernozem Fair Fai Chernozem Fair Fai Gleyic Chernozem Fair Fai Haplic Chernozem Fair Fai Luvic Chernozem Fair Fai Cambisol Calcaric Cambisol Fair Poo Chromic Cambisol Poor Poo Dystric Cambisol Poor Poo Eutric Cambisol Fair Poo Gleyic Cambisol Fair Fair Haplic Cambisol Poor Poo Mollic Cambisol Fair Fair Vertic Cambisol Poor Poo Fluvisol Calcaric Fluvisol Fair Poo Dystric Fluvisol Poor Poo Eutric Fluvisol Fair Poo Gleyic Fluvisol Poor Fair Haplic Fluvisol Poor Poo Histic Fluvisol Poor Fair Mollic Fluvisol Poor Fair Salic Fluvisol Poor Poo Thionic Fluvisol Poor Poo Gleysol Calcaric Gleysol Fair Fair Dystric Gleysol Poor Fair Eutric Gleysol Fair Fair Haplic Gleysol Fair Fair Histic Gleysol Fair Goo Humic Gleysol Fair Goo Mollic Gleysol Fair Goo Thionic Gleysol Poor Poo Gypsisol Aridic Gypsisol Good Goo Histosol Histosol Fair Goo Dystric Histosol Poor Goo Eutric Histosol Good Goo Fibric Histosol Fair Goo Gelic Histosol Fair Goo Sapric Histosol Fair Goo Kastanozem Calcic Kastanozem Fair Fair Haplic Kastanozem Fair Fair Luvic Kastanozem Fair Fair Leptosol Calcaric Leptosol Good Fair Dystric Leptosol Poor Poo Eutric Leptosol Good Fair Haplic Leptsol Fair Fair Humic Leptosol Fair Fair Rendzic Leptosol Fair Fair Lithic Leptosol Fair Poo Luvisol Albic Luvisol Poor Poo Arenic Luvisol Poor Poo Calcic Luvisol Fair Poo Chromic Luvisol Poor Poo Dystric Luvisol Fair Poo Ferric Luvisol Fair Poo Gleyic Luvisol Fair Fair Haplic Luvisol Poor Poo Vertic Luvisol Poor Poo Phaeozem Albic Phaeozem Fair Fair Calcaric Phaeozem Fair Fair Gleyic Phaeozem Fair Fair Haplic Phaeozem Fair Fair Luvic Phaeozem Fair Fair Sodic Phaeozem Fair Fair Planosol Dystric Planosol Poor Fair Eutric Planosol Fair Fair Haplic Planosol Fair Fair Podzol Carbic Podzol Poor Poo Entic Podzol Poor Poo Gleyic Podzol Poor Fair Haplic Podzol Poor Poo Leptic Podzol Poor Poo Placic Podzol Poor Poo Rustic Podzol Poor Poo Umbric Podzol Poor Poo Bones etc. Acknowledgement Organics Metals Stratigraphy Poor Fair Poor Fair Good Good Good Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Poor Fair Fair Poor Poor Poor Fair Poor Poor Poor Fair Fair Poor Fair Fair Fair Fair Poor Fair Poor Poor Poor Fair Fair Fair Fair Fair Poor Poor Poor Poor Fair Poor Poor Good Poor Poor Poor Good Fair Poor Poor Good Poor Fair Poor Good Poor Poor Poor Good Poor Fair Poor Good Poor Fair Poor Good Poor Poor Poor Good Poor Poor Poor Good Fair Fair Fair Good Poor Fair Poor Good Fair Fair Fair Good Fair Fair Poor Good Fair Good Poor Good Fair Good Poor Good Fair Good Poor Good Poor Poor Poor Good Good Good Good Good Fair Good Poor Good Poor Good Poor Good Good Good Fair Good Fair Good Poor Good Fair Good Poor Good Fair Good Poor Good Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Good Fair Fair Fair Poor Poor Poor Fair Good Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Poor Fair Fair Poor Poor Poor Fair Poor Poor Poor Fair Fair Poor Fair Fair Poor Poor Poor Fair Fair Poor Poor Fair Fair Poor Poor Fair Fair Fair Poor Fair Poor Poor Poor Fair Poor Poor Poor Poor Fair Fair Poor Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Fair Poor Fair Poor Fair Poor Fair Fair Fair Fair Fair Fair Fair Fair Fair Poor Poor Poor Fair Poor Poor Poor Fair Poor Fair Poor Fair Poor Poor Poor Fair Poor Poor Poor Fair Poor Poor Poor Fair Poor Poor Poor Fair Poor Poor Poor Fair M. Kibblewhite et al. / Science of the Total Environment 529 (2015) 249–263 263 (continued) Soil type Reference group Name in WRB 1998 Bones etc. References Baxter, K., 2004. Extrinsic factors that affect the preservation of bone. Paper 62. Nebraska Anthropologist. IUSS Working Group WRB, 2006. World reference base for soil resources 2006. World Soil Resources Reports No. 103. FAO, Rome. p g Bjordal, C., Nilsson, T., Daniel, G., 1999. Microbial decay of waterlogged archaeological wood found in Sweden applicable to archaeology and conservation. Int. Biodeterior. Biodegrad. 43, 63–73. p Jackson, C., Greenﬁeld, D., Howie, L., 2012. An assessment of compositional and morpho- logical changes in model archaeological glasses in an acid burial matrix. Archaeometry 54 (3), 489–507. Child, A., 1995. Towards an understanding of the microbial decomposition of archaeolog- ical bone in the burial environment. J. Archaeol. Sci. 22 (2), 165–174. ( ) M., 2008. Microbial bioerosion of bone — a review. In: Wisshak, M., Jans, M., 2008. Microbial bioerosion of bone — a review. In: Wisshak, M., Tapanila, L (Eds.), Current Developments in Bioerosion. Springer, Berlin Heidelberg. J , , , , p , (Eds.), Current Developments in Bioerosion. Springer, Berlin Heidelberg. Cronyn, J.M., 1990. The Elements of Archaeological Conservation. Routledge, London. Jans, M., Nielsen-Marsh, C., Smith, C., Collins, M., Kars, H., 2004. Characterisation of micro- bial attack on archaeological bone. J. Archaeol. Sci. 31 (1), 87–95. Crow, P., 2008. Mineral weathering in forest soils and its relevance to the preservation of the buried archaeological resource. J. Archaeol. Sci. 35 (8), 2262–2273. Johnson Jr., A., Francis, B., 1980. Durability of Metals From Archaeological Objects, Metal Meteorites, and Native Metals. Battelle Paciﬁc Northwest Labs, Richland WA. Dain-Owens, A., Kibblewhite, M., Hann, M., Godwin, R., 2013. The risk of harm to archae- ological artefacts in soil from dynamic subsurface pressures generated by agricultural operations: experimental studies. Archaeometry 55 (6), 1175–1186. Jones, R.J.A., Houšková, B., Bullock, P., Montanarella, L. (Eds.), 2005. Soil resources of Europe, second edition . European Soil Bureau Research Report No. 9, EUR 20559 EN. Ofﬁce for Ofﬁcial Publications of the European Communities, Luxembourg (420 pp.). p p y ( ) Davidson, D.A., Wilson, C.A., 2006. An Assessment of Potential Soil Indicators for the Pres- ervation of Cultural Heritage Final Report to Defra University of Stirling Stirling Davidson, D.A., Wilson, C.A., 2006. An Assessment of Potential Soil Indicators for the Pres- ervation of Cultural Heritage — Final Report to Defra. University of Stirling, Stirling. g p y g, g Douterelo, I., Goulder, R., Lillie, M., 2010. References Soil microbial community response to land- management and depth, related to the degradation of organic matter in English wet- lands: implications for the in situ preservation of archaeological remains. Appl. Soil Ecol. 44 (3), 219–227. Karkanas, P., 2010. Preservation of anthropogenic materials under different geochemical processes: a mineralogical approach. Quat. Int. 214 (1–2), 63–69. Lillie, M., Smith, R., 2007. The in situ preservation of archaeological remains: using lysim- eters to assess the impacts of saturation and seasonality. J. Archaeol. Sci. 34 (9), 1494–1504. English Heritage, 2008. Investigative Conservation: Guidelines on How the Detailed Examination of Artefacts From Archaeological Sites can Shed Light on Their Manufac- ture and Use. English Heritage, Swindon. Melcher, M., Wiesinger, R., Schreiner, M., 2010. Degradation of glass artifacts: application of modern surface analytical techniques. Acc. Chem. Res. 43 (6), 916–926. g g English Heritage, 2011. Environmental Archaeology: A Guide to the Theory and Practice of Methods, from Sampling and Recovery to Post-excavation. second edition. English Heritage, Swindon. Neff, D., Dillmann, P., Descostes, M., Beranger, G., 2006. Corrosion of iron archaeological artefacts in soil: estimation of the average corrosion rates involving analytical tech- niques and thermodynamic calculations. Corros. Sci. 48 (10), 2947–2970. European Commission. Soil Geographical Database of Eurasia at scale 1:1,000,000 (SGDBE). 2003. http://eusoils.jrc.ec.europa.eu/esdb_archive/esdbv2/intro.htm#SGDBE (last accessed 16 January 2015). Nord, A., Mattsson, E., Tronner, K., 2005. Factors inﬂuencing the long-term corrosion of bronze artefacts in soil. Prot. Met. 41 (4), 309–316. Réguer, S., Dillmann, P., Mirambet, F., 2007. Buried iron archaeological artefacts: corrosion mechanisms related to the presence of Cl-containing phases. Corros. Sci. 49 (6), 2726–2744. European Commission, 2006a. Soil Atlas of Europe. Ofﬁce for Ofﬁcial Publications of the European Communities, Luxembourg. European Commission, 2006b. Communication from the Commission to the Council, the European Parliament, the European Economic and Social Committee of the regions. Thematic Strategy for Soil Protection. Commission of the European Communities, COM, Brussels (231 ﬁnal; 2006). Roemich, H., Gerlach, S., Mottner, P., Mees, F., Jacobs, P., Van Dyck, D., Doménech Carbó, T., 2003. Results from burial experiments with simulated medieval glasses. Mater. Res. Soc. Symp. Proc. 757, 97–108. Tóth, G., Montanarella, L., Stolbovoy, V., Máté, F., Bódis, K., Jones, A., Panagos, P., van Liedekerke, M., 2008. Soils of the European Union. EUR 23439 EN – Joint Research Centre – Institute for Environment and Sustainability. Ofﬁce for Ofﬁcial Publications of the European Communities, Luxembourg. Fabbri, B., Gualtieri, S., Shoval, S., 2014. Vandiver, P., Vasil'ev, S., 2002. A 16,000 year-old ceramic human-ﬁgurine from Maina, Russia. Mater. Res. Soc. Symp. Proc. 712, 421–431. Acknowledgement Organics Metals Stratigraphy Regosol Calcaric Regosol Fair Fair Poor Poor Dystric Regosol Poor Poor Poor Poor Eutric Regosol Poor Poor Poor Poor Haplic Regosol Poor Poor Poor Poor Solonchak Gleyic Solonchak Poor Poor Poor Fair Haplic Solonchak Poor Poor Poor Fair Takyric Solonchak Poor Poor Poor Fair Solonetz Gleyic Solonetz Poor Poor Poor Fair Haplic Solonetz Poor Poor Poor Fair Mollic Solonetz Poor Poor Poor Fair Umbrisol Arenic Umbrisol Poor Poor Poor Fair Gleyic Umbrisol Poor Fair Poor Fair Vertisol Chromic Vertisol Poor Poor Poor Poor Haplic Vertisol Poor Poor Poor Poor Pellic Vertisol Poor Poor Poor Poor Appendix B (continued) (continued) Appendix B (continued) Huisman, D., Pols, S.B., Joosten, I., van Os, B., Smit, A., 2008. Degradation processes in colourless Roman glass: cases from the Bocholtz burial. J. Archaeol. Sci. 35 (2), 398–411. References References The presence of calcite in archaeological ceramics. J. Eur. Ceram. Soc. 34 (7), 1899–1911. FAO, 1998. World reference base for soil resources. World Soil Resources Report No. 84. ISSS–ISRIC–FAO, Rome. Tylecote, R., 1979. The effect of soil conditions on the long-term corrosion of buried tin- bronzes and copper. J. Archaeol. Sci. 6 (4), 345–368. FAO, 2015. Regional and National Soil Maps and Datasets. FAO Soil Portal (http://www. fao.org/soils-portal/soil-survey/soil-maps-and-databases/regional-and-national-soil- maps-and-databases/en/, Last accessed 2 April 2015). Van Giffen, A., 2014. Weathered Archaeological Glass. Corning Museum of Glass, http:// www.cmog.org/article/weathered-archaeological-glass (last accessed 9 February 2015). Gerwin, W., Baumhauer, R., 2000. Effect of soil parameters on the corrosion of archaeolog- ical metal ﬁnds. Geoderma 96 (1), 63–80. Vandiver, P., Vasil'ev, S., 2002. A 16,000 year-old ceramic human-ﬁgurine from Maina, Russia. Mater. Res. Soc. Symp. Proc. 712, 421–431. Harris, E.C., 1989. Principles of Archaeological Stratigraphy. 2nd edition. Academic Press, London. Holden, J., West, L.J., Howard, A.J., Maxﬁeld, E., Panter, I., Oxley, J., 2006. Hydrological con- trols of in situ preservation of waterlogged archaeological deposits. Earth Sci. Rev. 78 (12), 59–83.
https://openalex.org/W3008454003	https://journals.iucr.org/s/issues/2020/02/00/wz5004/wz5004.pdf	English	null	The HARE chip for efficient time-resolved serial synchrotron crystallography	Journal of synchrotron radiation	2,020	cc-by	10,403	The HARE chip for efficient time-resolved serial synchrotron crystallography ISSN 1600-5775 ISSN 1600-5775 Pedram Mehrabi,a Henrike M. Mu¨ller-Werkmeister,a,b Jan-Philipp Leimkohl,c Hendrik Schikora,c Jelena Ninkovic,d Silvia Krivokuca,d Ladislav Andricˇek,d Sascha W. Epp,a Darren Sherrell,e Robin L. Owen,e Arwen R. Pearson,f Friedjof Tellkamp,c Eike C. Schulza* and R. J. Dwayne Millera,f,g* Pedram Mehrabi,a Henrike M. Mu¨ller-Werkmeister,a,b Jan-Philipp Leimkohl, Hendrik Schikora,c Jelena Ninkovic,d Silvia Krivokuca,d Ladislav Andricˇek,d Sascha W. Epp,a Darren Sherrell,e Robin L. Owen,e Arwen R. Pearson,f Friedjof Tellkamp,c Eike C. Schulza* and R. J. Dwayne Millera,f,g* Received 19 November 2019 Accepted 20 January 2020 aDepartment for Atomically Resolved Dynamics, Max-Planck-Institute for Structure and Dynamics of Matter, Luruper Chaussee 149, 22761 Hamburg, Germany, bInstitute of Chemistry – Physical Chemistry, University of Potsdam, Karl- Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany, cScientific Support Unit Machine Physics, Max-Planck- Institute for Structure and Dynamics of Matter, Luruper Chaussee 149, 22761 Hamburg, Germany, dHalbleiterlabor der Max-Planck-Gesellschaft, Otto-Hahn-Ring 6, D-81739 Munich, Germany, eDiamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK, fDepartment of Physics, Universita¨t Hamburg, Jungiusstrasse 9, 20355 Hamburg, Germany, and gDepartments of Chemistry and Physics, University of Toronto, 80 St George Street, Toronto, Ontario M5S 3H6, Canada. *Correspondence e-mail: eike.schulz@mpsd.mpg.de, dwayne.miller@mpsd.mpg.de Edited by M. Wang, Paul Scherrer Institute, Switzerland Keywords: data collection; sample delivery; fixed-target serial synchrotron crystallography; time-resolved crystallography; HARE; LAMA. Supporting information: this article has supporting information at journals.iucr.org/s Keywords: data collection; sample delivery; fixed-target serial synchrotron crystallography; time-resolved crystallography; HARE; LAMA. Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using speciﬁcally patterned silicon chips for sample delivery, the ‘hit-and-return’ (HARE) protocol allows for efﬁcient time-resolved data collection. The speciﬁc pattern of the crystal wells in the HARE chip provides direct access to many discrete time points. HARE chips allow for optical excitation as well as on-chip mixing for reaction initiation, making a large number of protein systems amenable to time-resolved studies. Loading of protein microcrystals onto the HARE chip is streamlined by a novel vacuum loading platform that allows ﬁne-tuning of suction strength while maintaining a humid environment to prevent crystal dehydration. To enable the widespread use of time-resolved serial synchrotron crystallography (TR-SSX), detailed technical descriptions of a set of accessories that facilitate TR-SSX workﬂows are provided. Supporting information: this article has supporting information at journals.iucr.org/s 1. Introduction then pumping a set number of crystal wells which correspond to a speciﬁc HARE number. The initial probe image serves as a reference. The system returns to the initial crystal well, then probes the pumped wells again to obtain the pumped (delayed) image. As the translation time between all wells is identical, all crystal wells have the same desired time delay. The attainable HARE numbers are dictated by the pattern of crystal wells on the chip. We initially demonstrated this approach by using a photocaged substrate and laser-triggered reaction initiation to follow an enzymatic reaction over almost 30 s (Mehrabi, Schulz, Dsouza et al., 2019). We also combined the HARE approach with our on-chip mixing technique LAMA (Mehrabi, Schulz, Agthe et al., 2019). To be most efﬁcient, the HARE approach requires a speciﬁc pattern of the crystal wells on the chip. We have therefore developed a dedicated HARE chip design that is described in detail here. viscosity extruders and ﬁxed-target solutions with greatly reduced sample consumption (Suga et al., 2019; Hunter et al., 2014; Weierstall et al., 2014; Martiel et al., 2019; Gru¨nbein & Nass Kovacs, 2019; Zarrine-Afsar et al., 2012; Mueller et al., 2015). , ) Serial crystallographic experiments can also be conducted at synchrotrons (serial synchrotron crystallography, SSX), approximating the low-damage XFEL structures by using the serial data-collection approach to minimize dose accumulation (Roedig et al., 2016; Gati et al., 2014; Stellato et al., 2014; Chapman et al., 2014; Ebrahim, Moreno-Chicano et al., 2019; Owen et al., 2017; Weinert et al., 2017). Initially, SSX experi- ments were conducted using a simple mesh-screening approach with microcrystals randomly positioned and frozen in a loop, or by pushing a crystal suspension through a capil- lary in the X-ray beam (Gati et al., 2014; Stellato et al., 2014). Following these initial proof-of-principle experiments, hybrid methods such as tape drive approaches as well as multiple ﬁxed-target solutions have been developed at synchrotrons for both cryo- and room-temperature crystallography (Martiel et al., 2019). These methods present an alternative to liquid-jet- based sample-delivery systems and have the advantage of increased sample integrity, reduced sample consumption and hit-rate optimization (Roedig et al., 2015, 2016, 2017; Martiel et al., 2019; Oghbaey et al., 2016; Zarrine-Afsar et al., 2012, 2010; Mueller et al., 2015; Sherrell et al., 2015; Owen et al., 2017). 1. Introduction In combination with a fast accurate translation-stage system, silicon-based chips, such as those described here, deliver high crystal-well densities on the chip, high hit-rates and allow serial sampling of 100 000 crystal wells per hour at room temperature (Owen et al., 2017; Wierman et al., 2019; Sherrell et al., 2015; Oghbaey et al., 2016; Ebrahim, Moreno-Chicano et al., 2019; Ebrahim, Appleby et al., 2019). Using next- generation light sources and detectors allows the collection of multiple images per crystal well, which signiﬁcantly increases the throughput (Ebrahim, Appleby et al., 2019; Tolstikova et al., 2019). We note, however, that the lifetime of the crystals is still limited by the applicable X-ray dose and therefore the number of images that can be obtained. A further important aspect in the preparation of any ﬁxed- target SSX experiment is mounting the crystals on the chip and achieving a high fraction of loaded crystal wells. Fragile protein crystals can suffer from physical stress as well as dehydration during the loading process. To mitigate these problems, we have recently substantially improved our previously described crystal-loading pipeline (Mueller et al., 2015; Oghbaey et al., 2016). The new chip-loading platform, presented here, ensures humidity control and limits the force acting on the individual crystals in order to minimize detri- mental effects during the loading process while maintaining compatibility with a variety of different crystallization mother liquors. We have already successfully applied the tools and proto- cols described above to carry out time-resolved serial synchrotron crystallography (TR-SSX) experiments for three different proteins (Mehrabi, Schulz, Dsouza et al., 2019; Schulz et al., 2018; Mehrabi, Schulz, Agthe et al., 2019). In this report, we describe the HARE chip and provide details of our current best-practice protocols for chip maintenance and sample loading. These protocols include simple cheap off-the-shelf options as well as more comprehensive bespoke solutions. We hope this is a useful resource that will enable widespread use of TR-SSX at synchrotron beamlines worldwide. While XFELs are uniquely suited to provide insight into ultrafast dynamics on the femtosecond scale, metastable reaction intermediates of most enzyme mechanisms occur on the microsecond to second time domain and this can conveniently be addressed at third- and fourth-generation synchrotron sources (Schlichting, 2015; Chapman, 2019; Bar- Even et al., 2011; Weinert et al., 2019; Schulz et al., 2018; Mehrabi, Schulz, Dsouza et al., 2019; Mehrabi, Schulz, Agthe et al., 2019). 1. Introduction Time-resolved crystallography is one of the few techniques that can provide simultaneous insight into structure and dynamics with near-atomic resolution (Moffat, 1989, 1998; Pai, 1992). This method has experienced a renaissance since the advent of high-brilliance X-ray free-electron laser (XFEL) sources. XFELs triggered the development of serial diffraction data-collection methods because a single high-intensity FEL pulse can lead to the destruction of the crystal under study (Chapman, 2019; Chapman et al., 2011). Serial approaches have the additional advantage of avoiding many of the difﬁ- culties traditionally associated with time-resolved studies of single crystals, i.e. reaction initiation, radiation damage and signal-to-noise. Taking advantage of the ultrashort time- resolutions accessible at XFELs, many of the initial FEL studies have probed sub-picosecond timescales using liquid-jet delivery systems (Martin-Garcia et al., 2016; Kupitz et al., 2017, 2014; Lee et al., 2018; Schlichting, 2015; Chapman, 2019; Tenboer et al., 2014; Barends et al., 2015). Liquid jets brought new challenges: collecting thousands of still diffraction patterns mandates high-velocity crystal exchange, which initially resulted in large sample consumption and waste. This made studying scarce, expensive or difﬁcult to crystallize systems unfeasible and led to the development of high- J. Synchrotron Rad. (2020). 27, 360–370 360 https://doi.org/10.1107/S1600577520000685 research papers then pumping a set number of crystal wells which correspond to a speciﬁc HARE number. The initial probe image serves as a reference. The system returns to the initial crystal well, then probes the pumped wells again to obtain the pumped (delayed) image. As the translation time between all wells is identical, all crystal wells have the same desired time delay. The attainable HARE numbers are dictated by the pattern of crystal wells on the chip. We initially demonstrated this approach by using a photocaged substrate and laser-triggered reaction initiation to follow an enzymatic reaction over almost 30 s (Mehrabi, Schulz, Dsouza et al., 2019). We also combined the HARE approach with our on-chip mixing technique LAMA (Mehrabi, Schulz, Agthe et al., 2019). To be most efﬁcient, the HARE approach requires a speciﬁc pattern of the crystal wells on the chip. We have therefore developed a dedicated HARE chip design that is described in detail here. viscosity extruders and ﬁxed-target solutions with greatly reduced sample consumption (Suga et al., 2019; Hunter et al., 2014; Weierstall et al., 2014; Martiel et al., 2019; Gru¨nbein & Nass Kovacs, 2019; Zarrine-Afsar et al., 2012; Mueller et al., 2015). 1. Introduction However, a signiﬁcant problem when utilizing serial approaches for these longer delay times is the inevitable increase in overall data-collection time for a standard pump– delay–probe experiment, which makes data acquisition at long time delays impractical within a standard 24 hour beam time. We recently demonstrated that this problem can be alleviated with a ‘hit-and-return’ (HARE) method. HARE allows collection of sufﬁcient SSX data for approximately one time point per hour irrespective of the delay time (Schulz et al., 2018). In brief, the data-collection sequence begins by probing Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography 36 J. Synchrotron Rad. (2020). 27, 360–370 3.1. The HARE chip for TR-SSX 3.1. The HARE chip for TR-SSX In contrast to ﬁrst-generation chip designs, the HARE chips have an increased outer dimension of 30 mm by 30 mm to accommodate a higher number of crystal wells per chip (Mueller et al., 2015; Zarrine-Afsar et al., 2012; Oghbaey et al., 2016). This is the same form factor as used in the Oxford photochip, which has already been described elsewhere (Ebrahim, Moreno-Chicano et al., 2019; Ebrahim, Appleby et al., 2019). The HARE chip design also allows for smaller crystal sizes and higher crystal- well densities. However, the main design goal of the HARE chip was to simplify and streamline time-resolved pump– probe applications. To this end the HARE-chip design is fourfold symmetric, simplifying motion control and chip alignment. re 1 g Design of the HARE chip. (a) Left: overview of the HARE chip, consisting of 6 6 compartments (A1–F6). Top right: an individual compartment is magniﬁed showing the individual features aa–xx. Bottom right: A cross section of four microwells displays the dimensions. (b) Side-by-side comparison of the HARE chip with the Oxford photochip. 3.1.1. HARE chip design. The HARE chip is divided into 6 6 compartments (A1–F6) of 3.6 mm 3.6 mm separated by 900 mm gaps in each direction. For best implementation of the HARE approach, each compartment is subdivided into 24 24 crystal wells, resulting in 20 736 crystal wells per chip. This provides a high composite number of crystal wells per row (24), allowing maximum ﬂexibility in speciﬁc delay times that can be addressed without any other modiﬁcation of the experimental parameters, without making the chip too fragile (36 wells per row is too many) (Schulz et al., 2018). The pitch of the crystal wells is 150 mm in both lateral directions. The nominal dimensions of the square crystal wells are thus 82 mm on the top side and 10 mm on the bottom side (Figs. 1 and S1 in the supporting information). The compartment area is lined with ﬁducial marks etched into the silicon between the junctions of the outer compartments at a pitch of 4350 mm. The nominal dimensions of the ﬁducial marks are 86 mm on the top and 20 mm on the bottom and they are situated 850 mm from the centre of the adjacent aperture. 2.1. Protein crystallization Protein crystallization was conducted as described previously (Mehrabi, Schulz, Agthe et al., 2019). Xylose isomerase crystals were obtained from Hampton Research (HR7-102) and dissolved in water. Redissolved crystals were concentrated to 80 mg ml1 in xylose isomerase buffer (10 mM Hepes/NaOH pH 7.5). To displace any remaining Tris-HCl from the storage condition the concen- trated protein solution was re-diluted and concentrated again for nine consecutive times. Vacuum-induced crystallization in XI crystallization buffer [35%(w/v) PEG3350, 200 mM lithium sulfate and 10 mM Hepes/NaOH pH 7.5] yielded the micro- crystals. 361 J. Synchrotron Rad. (2020). 27, 360–370 research papers Figure 1 Design of the HARE chip. (a) Left: overview of the HARE chip, consisting of 6 6 compartments (A1–F6). Top right: an individual compartment is magniﬁed showing the individual features aa–xx. Bottom right: A cross section of four microwells displays the dimensions. (b) Side-by-side comparison of the HARE chip with the Oxford photochip. 2.2. Serial X-ray diffraction experiments 2.2. Serial X-ray diffraction experiments All serial X-ray diffraction experi- ments were conducted at room temperature at EMBL beamline P14-2 at the PETRA III synchrotron at DESY, Hamburg (https://www.embl-hamburg. de/services/mx/P14_EH2/). For sample exchange the chips were mounted on the translation-stage holders as described below and connected to the SmarAct translation stages as described previously (Schulz et al., 2018; Sherrell et al., 2015). 362 Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography 3.1. The HARE chip for TR-SSX Detailed CAD drawings of the HARE chip, which can be used for mask development, can be found in the supporting infor- mation (supporting material 1). 3.1.2. Production of the HARE chips. The HARE chips are manufactured from conventional double side polished silicon- on-insulator wafers and are produced as follows. (a) The production process sequence starts with two layers of mono- crystalline silicon in [100] orientation. A 50 mm-thick top layer and 450 mm-thick bottom layer are separated by a thin (300 nm) layer of thermally grown SiO2 (BOX, bonding oxide) [Fig. 2(a)]. (b) A thermal oxide of 250 nm is grown on the wafers which acts as the hard mask for the etching in the following steps [Fig. 2(b)]. (c) Photoresist is spun on the oxidized wafers on both sides to structure the hard mask [Fig. 2(c)]. (d) The photoresist layer is exposed through a glass mask with UV light and the exposed regions on the wafer are then removed in a photoresist development step [Fig. 2(d)]. (e) The oxide is wet chemically removed from the wafers in the open regions of the photoresist [Fig. 2(e)] and (f) the photoresist layer is stripped [Fig. 2(f)]. (g) The wafers with the 362 J. Synchrotron Rad. (2020). 27, 360–370 research papers Figure 2 Sequence of the HARE chip production. The HARE chips are produced in eight consecutive protection and etching steps described in detail in the text. Dark/light grey = monocrystalline silicon of 450 and 50 mm thickness, respectively. Black = 300 nm layer of SiO2. Blue = 250 nm thermal oxide layer. Red = photoresist. Figure 2 plex Industries Inc. USA) are much more convenient for sealing the chips than cutting foils to size from a roll or larger sheets. g Crystal dehydration can also be prevented by humidiﬁed environment (Sanchez- Weatherby et al., 2009). For LAMA applica- tions, where a small droplet of ligand is added to each crystal on the chip, the top half of the holder is replaced with a tear-shaped stainless steel cover with no Mylar foil and the crystals are prevented from dehydrating using a humidiﬁed gas stream. To this end, a constant ﬂow of humid air is generated by directing the humidity stream from a low-cost room humi- diﬁer via a simple rubber hose. 3.1. The HARE chip for TR-SSX The tear shape of the LAMA holder channels any conden- sation from the humidiﬁed gas stream away from the surface of the chip and holder via gravity and can be collected in a simple plastic dish, thereby preventing leakage onto nearby electronic devices (Mehrabi, Schulz, Agthe et al., 2019). The supporting information includes CAD drawings of the chip holder and the tear-shaped cover extension required for LAMA (supporting material 2 and 3). ght consecutive monocrystalline Blue = 250 nm g Sequence of the HARE chip production. The HARE chips are produced in eight consecutive protection and etching steps described in detail in the text. Dark/light grey = monocrystalline silicon of 450 and 50 mm thickness, respectively. Black = 300 nm layer of SiO2. Blue = 250 nm thermal oxide layer. Red = photoresist. structured oxide layer are then immersed in a wet chemical bath with TMAH (tetramethyl ammonium hydroxide). The exposed silicon is dissolved while the oxide acts as a hard mask for the etching and the BOX acts as an etch stop for the process [Fig. 2(g)]. In this way the etch process is self-limiting and the etching of both sides can be done in one single step. The lateral etch rate is suppressed by the anisotropic etch rates of the TMAH solution. (h) As a last step, the hard mask including the BOX is removed by immersion into hydroﬂuoric acid [Fig. 2(h)] after which the chips can be cut out of the wafer. 3.3. Chip maintenance 3.3.1. Cleaning. The simplest method to clean the chips is extensive incubation in water, which dissolves most residues over time. However, the chips can be more effectively cleaned after each data collection using the following protocol. Directly after use the chips are incubated in a solution of J. Synchrotron Rad. (2020). 27, 360–370 3.2. The chip sample holder To mount the chips onto the translation stages used for SSX (Sherrell et al., 2015), new sample holders were designed to accom- modate the larger chip dimensions (Fig. 3). To simplify the mounting of chips onto the stages the sample holders include half of a kinematic mount (KBT25T/M, Thorlabs Inc.), while the other half (KBB25/M, Thorlabs Inc.) is permanently attached to the stages. This allows the holder to easily snap into position during sample exchange. To prevent dehy- dration of the protein crystals during data collection, the chips were sealed with 2.5 mm Mylar foils, which were integrated into the holder. Wrinkling of the Mylar foils was prevented by stretching them across the two half holders using tight-ﬁtting circular rings. The two halves of the holder were equipped with neodymium magnets of opposite polarity to simplify the alignment and closing of the holder. After the chip was placed on the Mylar covered holder, the lid was ﬁxed with two M3 screws. We found that 66 mm circular cut Mylar sheets (2.5 mm thickness) (Chem- Figure 3 The chip sample holder. The multi-component sample holder is important both for efﬁcient alignment and translation of the chip in the X-ray beam and, for LAMA experiments, to maintain a humid environment for the protein crystals. (a) A fully assembled chip holder containing a HARE chip. (b) All components of a chip holder in an exploded view. (c) A fully assembled chip holder with the tear-shaped cover for LAMA applications. Figure 3 g The chip sample holder. The multi-component sample holder is important both for efﬁcient alignment and translation of the chip in the X-ray beam and, for LAMA experiments, to maintain a humid environment for the protein crystals. (a) A fully assembled chip holder containing a HARE chip. (b) All components of a chip holder in an exploded view. (c) A fully assembled chip holder with the tear-shaped cover for LAMA applications. Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography 363 363 J. Synchrotron Rad. (2020). 27, 360–370 research papers Figure 4 The effect of glow discharging on HARE chips. 3.2. The chip sample holder Photographs of HARE chips: (a) bottom view showing the compartment support bars, (b) top view showing the 36 compartments, (c) top view showing 3.5 ml of ddH2O loaded onto each compartment before glow discharging clearly forms droplets, and (d) top view showing 3.5 ml of ddH2O loaded onto each compartment after glow discharging evenly spreads out across each compartment and avoids the hydrophilic areas in between. The effect of glow discharging on HARE chips. Photographs of HARE chips: (a) bottom view showing the compartment support bars, (b) top view showing the 36 compartments, (c) top view showing 3.5 ml of ddH2O loaded onto each compartment before glow discharging clearly forms droplets, and (d) top view showing 3.5 ml of ddH2O loaded onto each compartment after glow discharging evenly spreads out across each compartment and avoids the hydrophilic areas in between. 1%(w/v) TERGAZYME (Sigma–Aldrich) or a 1 M HCl solution for 1 h. Subsequently, the chips are rinsed under running hot tap water and then washed with ddH2O. Finally, the chips are washed with isopropanol before being left to dry on the bench for a few hours. To speed up drying, chips can be placed in a custom-built drying station, attached to the pres- sured air or nitrogen gas feed usually found at synchrotron beamlines, and exposed to pres- surized gas for 5 min [Fig. 4(a)]. The infor- mation needed to print the drying station using a 3D printer is available in supporting material 4. ment areas leads to a more homogeneous liquid-layer distri- bution on the compartments, while the bars between compartments are more hydrophobic and repel the crystal slurry (Figs. 4 and 5). Information needed to reproduce the glow-discharge holder using a 3D printer is available in supporting material 5. Figure 5 Chip maintenance and loading solutions. (a) The chip-drying station for quick drying of the chips after cleaning. (b) The glow-discharge holder, which holds Kapton masks and chips together during the glow-discharge process. (c) The storage box for safe and easy transportation of the fragile chips. (d) The chip-loading block allows efﬁcient loading of crystalline slurries by providing a convenient vacuum seal between the polished stainless steel surface and the chip. (e) The nine-channel pipette adapter connects a conventional 1000 ml micropipette with nine 200 ml pipette tips. This allows for an increased homogeneity of the dispensed crystal slurry on the chip surface. 364 Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography 3.2. The chip sample holder Should they be contaminated with stubborn residue, chips can be comprehensively cleaned using hot piranha solution [H2O2(30% w/w):H2SO4 in a ratio of 1:2.5 at 100C], which removes all organic contami- nants and leaves the chip surface hydrophilic. We note that piranha solution is extremely caustic and should only be used with appro- priate safety precautions and personal protective equipment. 3.3.2. Glow discharging. Alteration of the surface charge allows for a more even wetting of the chips, improving crystal-loading homogeneity. As an alternative to chemical treatments, a convenient solution to generate hydrophilic surfaces is glow discharging or plasma cleaning. For a simple surface treat- ment of the whole chip, individual chips can simply be placed in a glow discharger (Balzers CTA 010, Balzers Union, Switzerland) one at a time. Chips are then glow discharged for 60 s at 35 mA. To allow a selective wetting of the crystal-well containing compartments on the chip we designed a custom chamber which can hold four chips covered with laser-cut Kapton masks. These masks selectively cover all non- compartment areas of the chips. Closing the chamber presses the Kapton mask onto the chip. The selective wetting of the compart- Figure 5 g Chip maintenance and loading solutions. (a) The chip-drying station for quick drying of the chips after cleaning. (b) The glow-discharge holder, which holds Kapton masks and chips together during the glow-discharge process. (c) The storage box for safe and easy transportation of the fragile chips. (d) The chip-loading block allows efﬁcient loading of crystalline slurries by providing a convenient vacuum seal between the polished stainless steel surface and the chip. (e) The nine-channel pipette adapter connects a conventional 1000 ml micropipette with nine 200 ml pipette tips. This allows for an increased homogeneity of the dispensed crystal slurry on the chip surface. g Chip maintenance and loading solutions. (a) The chip-drying station for quick drying of the chips after cleaning. (b) The glow-discharge holder, which holds Kapton masks and chips together during the glow-discharge process. (c) The storage box for safe and easy transportation of the fragile chips. (d) The chip-loading block allows efﬁcient loading of crystalline slurries by providing a convenient vacuum seal between the polished stainless steel surface and the chip. (e) The nine-channel pipette adapter connects a conventional 1000 ml micropipette with nine 200 ml pipette tips. 3.4. The chip-loading platform 3.4.3. Vacuum control unit. The vacuum control unit we have developed allows us to set speciﬁc vacuum levels, monitored by an analogue pressure gauge at the front of the device, as well as to control pressure ramps during the chip- loading process (Fig. 5). An analogue electronics board (PCB) allows gentle ramping up and down of control voltages for the vacuum controller which, in turn, provides a corresponding vacuum level. The vacuum is generated by a 230 V membrane pump (Pfeiffer MVP-040–2, Germany). The vacuum can be switched on or off via a manual switch on the control panel, or by using a convenient foot pedal, but also allows for contin- uous pumping (Fig. 6). For new systems we typically start to test vacuum levels between 0.3 and 0.5 bar. Refer to supporting material 9 for detailed CAD drawings of the vacuum control unit. Crystal loading onto the chips can be simply achieved by applying a slurry of crystals using a pipette and a laboratory vacuum pump to suck the crystals into the crystal wells via a chip-loading block (Mueller et al., 2015; Oghbaey et al., 2016). However, without proper control of both the vacuum and local humidity, crystal damage as a result of physical stress and/or dehydration is possible. A cheap, off-the-shelf solution is to use a dial gauge to control the vacuum pressure and to place the whole mounting assembly into a disposable humidity tent (SoloLab; solocontainment, UK) containing a standard home room humidiﬁer. However, this does not allow the vacuum pressure or the humidity surrounding the crystals to be precisely maintained. We have therefore developed a low-cost humidity hood with an automatic humidity feedback control (Rotronic Hydroﬂex, Ettlingen, Germany), which provides a more accurate control over the humidity, as well as a vacuum control unit. We will also describe a custom-built multi- channel pipette adapter, which can further simplify chip loading. 3.4.4. Humidity hood. To prevent dehydration of the crys- tals during loading and transfer of the loaded chips into the Figure 6 The vacuum control unit. (a) View of the front panel. From left to right: mains switches for the control unit (S1) and the pump (S2), a connector for the foot paddle (P3), control switches for the vacuum (S3), and ﬁlter insertion to control the vacuum ramp up/down (S4). Vacuum level (Pot1) and ﬁlter insertion rate (Pot2) can be adjusted using analogue potentiometers. research papers 3.3.3. Storage. Chips can be simply stored in small plastic membrane boxes that are cheaply available (i.e. Agar Scien- tiﬁc Ltd, UK). However, we found that to streamline handling during beam time, increase chip longevity, and safeguard the delicate chips during transport and storage, a custom storage box is extremely helpful. In our design, as in a microscope ‘slide box’, the chips are stored vertically in three arrays of 19 chips each. In comparison with membrane boxes, this minimizes storage space and allows for safe and convenient transport of a large number of chips to the beamline (Fig. 5). Information to reproduce the storage box using a 3D printer is available in supporting material 6. 3.4.2. A multi-channel pipette adapter. Even loading of crystal slurry onto the chip is crucial for all SSX experiments. This can simply be carried out using a single-channel pipette to distribute the crystal slurry over the chip surface. However, to improve the homogeneity of the crystal slurry distribution over the chip we have developed a nine-channel pipette adapter (Fig. 5). The adapter is designed to ﬁt on a standard 1000 ml micropipette and to connect this to an array of nine 200 ml pipette tips. A dispenser plate glued into the 1000 ml pipette adapter allows for convenient ejection of used pipette tips. For chip loading a crystal slurry is transferred to a typical 96-well PCR plate prior to loading of the nine-channel pipette. The information needed to reproduce the nine-channel pipette adapter using a 3D printer is available in supporting material 8. J. Synchrotron Rad. (2020). 27, 360–370 3.2. The chip sample holder This allows for an increased homogeneity of the dispensed crystal slurry on the chip surface. Chip maintenance and loading solutions. (a) The chip-drying station for quick drying of the chips after cleaning. (b) The glow-discharge holder, which holds Kapton masks and chips together during the glow-discharge process. (c) The storage box for safe and easy transportation of the fragile chips. (d) The chip-loading block allows efﬁcient loading of crystalline slurries by providing a convenient vacuum seal between the polished stainless steel surface and the chip. (e) The nine-channel pipette adapter connects a conventional 1000 ml micropipette with nine 200 ml pipette tips. This allows for an increased homogeneity of the dispensed crystal slurry on the chip surface. J. Synchrotron Rad. (2020). 27, 360–370 research papers Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography 3 3.4. The chip-loading platform An outlet port with push-in ﬁtting (F3) connects the vacuum pump to the chip-loading block and an analogue pressure gauge (G1) aids in setting the desired backpressure. (b) View of the back panel. From left to right: 230 V power socket (P1), vent to atmosphere (F2), inlet port with push-in ﬁtting connecting to the vacuum pump (F1) and membrane pump power feed-through (P2). p y p g 3.4.1. Chip-loading block. Our original chip-loading solution utilized an aluminium block with a single outlet port connected to a vacuum pump. The top of the block was covered with a silicon slide to form a vacuum- tight seal with the chip (Oghbaey et al., 2016). The new chip-loading block design presented here consists of a polished stainless steel block that is mounted on a stainless steel base for stability (Fig. 5). The ﬂatness of the polished surface allows a tight seal between the chip and the loading block. To allow a more homogeneous distribution of the vacuum, the loading block has an 8 8 array of holes that funnel into a single outlet port that connects to the vacuum control device. Chips are held in place prior to application of the vacuum by steel spring clips. All tubing connections are via push-in ﬁttings (Sang-A, Landefeld, Germany). Supporting material 7 contains detailed CAD drawings of the chip- loading block that should allow for repro- duction in local machine shops. Figure 6 Figure 6 g The vacuum control unit. (a) View of the front panel. From left to right: mains switches for the control unit (S1) and the pump (S2), a connector for the foot paddle (P3), control switches for the vacuum (S3), and ﬁlter insertion to control the vacuum ramp up/down (S4). Vacuum level (Pot1) and ﬁlter insertion rate (Pot2) can be adjusted using analogue potentiometers. An outlet port with push-in ﬁtting (F3) connects the vacuum pump to the chip-loading block and an analogue pressure gauge (G1) aids in setting the desired backpressure. (b) View of the back panel. From left to right: 230 V power socket (P1), vent to atmosphere (F2), inlet port with push-in ﬁtting connecting to the vacuum pump (F1) and membrane pump power feed-through (P2). 365 J. Synchrotron Rad. (2020). 27, 360–370 research papers Figure 7 The humidity control hood. An 3D schematic showing the humidity control hood, with the humidiﬁer, humidity sensor and chip-loading block inside. 3.4. The chip-loading platform should be 10 mm to avoid them ﬂowing straight through the chip holes. A convenient and cost-effective way to quickly assess the concentration of protein microcrystal solutions during beam times is to use a simple benchtop light micro- scope and a Neubauer cell-counting chamber (NanoEnTek, Korea) into which 10 ml of crystal slurry are deposited. At 10–40 magniﬁcation it is possible to not only assess the concentration of the protein microcrystals but also to assess the homogeneity of the crystal size distribution (Fig. 8). In our hands, crystal concentrations of 1 106 crystals ml1 to 1.5 106 crystals ml1 yielded reasonable chip-loading levels that allowed us to collect sufﬁcient data for a complete dataset with good signal-to-noise and merging statistics in an hour (i.e. two chips) (Schulz et al., 2018; Mehrabi, Schulz, Dsouza et al., 2019; Mehrabi, Schulz, Agthe et al., 2019). Higher crystal concen- trations result in higher loading levels, but also in a much higher percentage of multiple lattices (i.e. two or more crystals per well). This is problematic for TR-SSX as it makes the degree of reaction initiation by either light or on-chip mixing harder to assess. Figure 7 The humidity control hood. An 3D schematic showing the humidity control hood, with the humidiﬁer, humidity sensor and chip-loading block inside. 3.5.2. Chip loading with microcrystals. Although the rules of thumb stated above are, in our experience, a good starting point, optimal crystal concentrations for each speciﬁc system should be determined by comparing the crystal concentration with the diffraction hit rate. This allows the suitable vacuum level during chip loading to be determined. For our previously used systems (lysozyme, xylose isomerase, ﬂuoroacetate dehalogenase), vacuum levels between 0.3 and 0.5 bar below atmospheric pressure resulted in efﬁcient loading with no apparent crystal damage, while stronger vacuum levels resulted in visible damage to the crystals and in split diffrac- tion spots (Schulz et al., 2018; Mehrabi, Schulz, Dsouza et al., 2019; Mehrabi, Schulz, Agthe et al., 2019). However, these crystal systems are rather robust and more sensitive crystals of, for example, large macromolecular assemblies require much lower values than 0.3 bar, and these must be deﬁned on a case- by-case basis. Importantly, regardless of the vacuum pressure used, to prevent dehydration of the crystals, suction should holders, the vacuum loading block is placed in a humidity control hood made of plexiglass (Fig. 7). 3.4. The chip-loading platform Humidity is main- tained at the desired level by connecting a low-cost commer- cial room humidiﬁer (Honeywell, USA) to a humidity control sensor (Testo, Germany) via a proportional integral derivative controller unit (KR3, Ascon Technologic, Italy). During loading the relative humidity in the hood is typically kept at 85%. Refer to supporting material 10 for detailed CAD drawings of the humidity hood and the humidity control unit. 3.5. Chip-loading protocols 366 Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography 4. Discussion In comparison with our previous design, both the Oxford photochip and the HARE chip represent a substantial advance over the previous version (Zarrine-Afsar et al., 2012; Oghbaey et al., 2016; Ebrahim, Appleby et al., 2019). With only a slight increase in the outer dimension the crystal density has been increased approximately twofold (Mueller et al., 2015; Oghbaey et al., 2016; Ebrahim, Appleby et al., 2019). This has the clear advantage of increased data rates, as the most time- consuming step during data collection is the sample exchange and the re-alignment of the chip in the beam path. Impor- tantly, since both chip designs share the same 30 30 mm form factor, all instrumentation and protocols described here are common to both chips. Other silicon chip designs with different form factors and much higher hole densities than the HARE or Oxford photochip have also been realized. These have been used for high-throughput data collections of static structures at cryogenic and room-temperature alike (Lieske et al., 2019). However, the HARE chip has been speciﬁcally designed for time-resolved experiments. g pp While both chip designs are suitable for time-resolved measurements, the advantage of the Oxford photochip is that it can accommodate a higher number of crystals (>25 000), enabling a greater amount of data to be collected per chip (Ebrahim, Appleby et al., 2019). In contrast, the HARE chips with their high composite number of crystal wells per compartment are designed to make best use of the HARE algorithm for efﬁcient time-resolved crystallographic data collection (Schulz et al., 2018). This increases ﬂexibility with respect to the resolvable time points without requiring more complicated rastering patterns during data collection. This directly addresses our aim of broadening the accessibility of time-resolved experiments, as it allows a user to conveniently resolve a large number of reaction intermediates without encountering experimental complications in designing the data-collection protocol. These practical implications become more apparent if one compares the time points that can be addressed for a whole chip (Fig. 9). Usually the in crystallo kinetics of an enzymatic reaction are unknown before conducting a time-resolved experiment. Under these circum- stances it is of great advantage to initially characterize the reaction intermediates on a logarithmic timescale, which provides a systematic overview with the least number of snapshots along the reaction coordinate. 3.5. Chip-loading protocols 3.5.1. Determining sample concentration for micro- crystals. In order to prevent under- or over-loading of the chip the concentration of the protein microcrystals should be assessed. We note that, for the chips described here, crystals Figure 8 Crystal size and homogeneity estimation using a Neubauer counting chamber. Xylose isomerase microcrystals are shown before and after optimization of the batch crystallization process for improved size homogeneity. Before optimization (a),(b) the sample shows high crystal size heterogeneity. While one of the strengths of the chip technology lies in its tolerance of variable crystal sizes, a homogeneous crystal size distribution is advantageous for both homogeneous reaction initiation in time-resolved experiments and for stability of scaling during data processing. (a) Overview of xylose isomerase microcrystals in a Neubauer cell-counting chamber (the scale bar is 500 mm). (b) Close-up view of the indicated area (the scale bar is 50 mm). (c) Xylose isomerase sample after optimization of the crystal size homogeneity (the scale bar is 100 mm). This crystal concentration (2.88 106 ml1) should be diluted by a factor of two for TR-SSX experiments with the vacuum loading approach (described below), to avoid overloading the chip. Figure 8 Figure 8 Crystal size and homogeneity estimation using a Neubauer counting chamber. Xylose isomerase microcrystals are shown before and after optimization of the batch crystallization process for improved size homogeneity. Before optimization (a),(b) the sample shows high crystal size heterogeneity. While one of the strengths of the chip technology lies in its tolerance of variable crystal sizes, a homogeneous crystal size distribution is advantageous for both homogeneous reaction initiation in time-resolved experiments and for stability of scaling during data processing. (a) Overview of xylose isomerase microcrystals in a Neubauer cell-counting chamber (the scale bar is 500 mm). (b) Close-up view of the indicated area (the scale bar is 50 mm). (c) Xylose isomerase sample after optimization of the crystal size homogeneity (the scale bar is 100 mm). This crystal concentration (2.88 106 ml1) should be diluted by a factor of two for TR-SSX experiments with the vacuum loading approach (described below), to avoid overloading the chip. J. Synchrotron Rad. (2020). 27, 360–370 research papers large pitch (distance) between crystal wells is not only required for optical reaction initiation. To circumvent the need for optical excitation we have recently extended the repertoire of possible reaction initiations to an on-chip mixing approach called LAMA. 3.5. Chip-loading protocols In the LAMA approach, droplets of soluble substrates are directly applied to each well of the chip (Mehrabi, Schulz, Agthe et al., 2019). To prevent leaking of solution into neighbouring features, the pitch of the crystal wells has to be on the same scale as is required for optical pump–probe experiments. In the Oxford and HARE chips, leakage is further prevented by the pyramidal shape of the crystal wells. In contrast to ﬂat-surface chips the pyramidal shaped crystal wells trap the applied droplet and guide it towards the crystals. Additionally, the well shape probably prevents preferred orientation problems of the crystals (Davy et al., 2019). The larger well-to-well pitch and the shape of the wells make the Oxford and HARE chips extremely well suited to TR-SSX applications using both laser light and on-chip mixing approaches. only be applied until the moment the crystallization mother liquor is removed from the chip surface. In summary, the crystal concentration determines how well a particular chip is loaded. Based on the sample type (crystal concentration, viscosity of the medium, fragility of the crystals, etc.) the vacuum level dictates how well the samples drain into the crystal wells. If the vacuum level is set too high, the crystals will break or dehydrate and thereby reduce the diffraction hit rate. Therefore, the chip-loading device allows careful opti- mization of loading parameters for each system, enabling effective loading and preventing dehydration and damage of crystals (Mehrabi, Schulz, Agthe et al., 2019). References Barends, T. R., Foucar, L., Ardevol, A., Nass, K., Aquila, A., Botha, S., Doak, R. B., Falahati, K., Hartmann, E., Hilpert, M., Heinz, M., Hoffmann, M. C., Koﬁnger, J., Koglin, J. E., Kovacsova, G., Liang, M., Milathianaki, D., Lemke, H. T., Reinstein, J., Roome, C. M., Shoeman, R. L., Williams, G. J., Burghardt, I., Hummer, G., Boutet, S. & Schlichting, I. (2015). Science, 350, 445–450. Bar-Even, A., Noor, E., Savir, Y., Liebermeister, W., Davidi, D., Tawﬁk, D. S. & Milo, R. (2011). Biochemistry, 50, 4402–4410. Chapman, H. N. (2019). Annu. Rev. Biochem. 88, 35–58. Bar-Even, A., Noor, E., Savir, Y., Liebermeister, W., Davidi, D., T ﬁk D S & Mil R (2011) Bi h i 50 4402 4410 , , ( ) y, , Chapman, H. N. (2019). Annu. Rev. Biochem. 88, 35–58. By providing here all the required information to reproduce the tools and protocols we have described here, we hope to aid beamline staff and users in establishing TR-SSX at other synchrotron sources. Clearly, a larger community engaged in such studies will aid in further reﬁning protocols to reduce crystal consumption and to make TR-SSX a routine method at synchrotrons that is easily accessible to the large number of non-expert users with burning biological questions that can be addressed by time-resolved structural studies. Chapman, H. N., Caleman, C. & Timneanu, N. (2014). Philos. Trans. R. Soc. Lond. Ser. B, 369, 20130313. Chapman, H. N., Fromme, P., Barty, A., White, T. A., Kirian, R. A., Aquila, A., Hunter, M. S., Schulz, J., DePonte, D. P., Weierstall, U., Doak, R. B., Maia, F. R. N. C., Martin, A. V., Schlichting, I., Lomb, L., Coppola, N., Shoeman, R. L., Epp, S. W., Hartmann, R., Rolles, D., Rudenko, A., Foucar, L., Kimmel, N., Weidenspointner, G., Holl, P., Liang, M., Barthelmess, M., Caleman, C., Boutet, S., Bogan, M. J., Krzywinski, J., Bostedt, C., Bajt, S., Gumprecht, L., Rudek, B., Erk, B., Schmidt, C., Ho¨mke, A., Reich, C., Pietschner, D., Stru¨der, L., Hauser, G., Gorke, H., Ullrich, J., Herrmann, S., Schaller, G., Schopper, F., Soltau, H., Ku¨hnel, K. U., Messerschmidt, M., Bozek, J. D., Hau-Riege, S. P., Frank, M., Hampton, C. Y., Sierra, R. G., Starodub, D., Williams, G. J., Hajdu, J., Timneanu, N., Seibert, M. M., Andreasson, J., Rocker, A., Jo¨nsson, O., Svenda, M., Stern, S., Nass, K., Andritschke, R., Schro¨ter, C. D., Krasniqi, F., Bott, M., Schmidt, K. References E., Wang, X., Grotjohann, I., Holton, J. M., Barends, T. R. M., Neutze, R., Marchesini, S., Fromme, R., Schorb, S., Rupp, D., Adolph, M., Gorkhover, T., Andersson, I., Hirsemann, H., Potdevin, G., Graafsma, H., Nilsson, B. & Spence, J. C. H. (2011). Nature, 470, 73–77. research papers (Bar-Even et al., 2011). Once the critical time points have been identiﬁed they can of course be re-addressed with a more ﬁne- sliced data-collection strategy. pipette adapter. ECS and PM established chip-loading and maintenance protocols. ECS, PM and ARP wrote the manu- script. RJDM initiated this project and use of the chip concept for time-resolved studies. All of the authors discussed and corrected the manuscript. Obviously, it would be desirable to further increase the number of crystal wells per compartment to address a much larger number of discrete time points. However, using proto- types with up to 53 53 crystal wells per compartment we found that the fragility of the compartment areas drastically increases. This prevents the compartments from withstanding even small vacuum levels and results in chips breaking during crystal loading. Different material properties and/or different aspect ratios of the compartment areas might mitigate this effect and it is therefore possible that higher HARE numbers can be realized with different chip designs or materials. Currently, however, the 24 24 crystal well per compartment design appears to be a sweet spot between chip resilience and time-point versatility. Notably, with a change in the translation frequency the HARE delay times change accordingly, providing access to arbitrary delay times for all chip types. Funding information Support was provided by the Max Planck Society and by the Cluster of Excellence ‘The Hamburg Centre for Ultrafast Imaging’ of the Deutsche Forschungsgemeinschaft (EXC 1074) project ID 194651731 (RJDM), the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program (FP7/2007–2013) under REA grant agreement No. 623994 (HMW) and the Joachim Herz Foun- dation (Biomedical Physics of Infection). PM was the recipient of an Alexander von Humboldt-Stiftung postdoctoral research award. AP received funding for development and the construction of the T-REXX SSX endstation from the BMBF (‘Verbundforschung’, 05K16GU1 and 05K19GU1). In summary, in order to match the HARE chip with a versatile loading solution we have developed a cost-effective modular chip-loading platform that can be easily implemented at any beamline. To address the requirement to load un- damaged crystals, under controlled humidity and for a wide variety of buffer systems a vacuum-based solution seems to provide the most versatile chip-loading approach. We can limit physical stress by adapting vacuum levels and evacuation time to both crystals and buffer system and conveniently control the humidity in the chip-loading environment to prevent dehydration of the crystals during the loading process. 4. Discussion On a logarithmic scale the delay times that can be addressed by the HARE chip are distributed more linearly, in particular up to 10 s of delay time, which encompasses the majority of enzymatic turnovers In an optical pump–probe experiment, a laser pulse is used for reaction initiation. For signal optimization the pumped volume needs to exceed the probed volume by a factor of two to ﬁve. To achieve a homogenous sample excitation of a 5– 10 mm probe area (X-ray microfocus beam) a UV laser pulse has to have a 50 mm diameter, assuming optimal beam align- ment and a typical Gaussian proﬁle. In practice, laser beam diameters are determined by the optics used and wavelength, but typically lie between 50 mm and 100 mm (FWHM) (Epp et al., 2017; Schulz et al., 2018). Thus, a pitch of the crystal wells smaller than 100–120 mm has the potential to lead to excitation of neighbouring crystals in high-density grids. However, a Figure 9 HARE delay times that can be addressed with the HARE chip and the Oxford photochip. The ﬁgure illustrates the discrete time points that can be addressed with the Oxford photochip (blue) and the HARE chip (orange) without changing any experimental parameter other than the time delay between reaction initiation pump and probe. Time delays are calculated for a translation-stage frequency of 25 Hz. Figure 9 Figure 9 HARE delay times that can be addressed with the HARE chip and the Oxford photochip. The ﬁgure illustrates the discrete time points that can be addressed with the Oxford photochip (blue) and the HARE chip (orange) without changing any experimental parameter other than the time delay between reaction initiation pump and probe. Time delays are calculated for a translation-stage frequency of 25 Hz. Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography 36 Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography 3 367 J. Synchrotron Rad. (2020). 27, 360–370 368 Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography Acknowledgements L., Barthelmess, M., Lieske, J., Oberthuer, D., Wator, E., Mohacsi, I., Wulff, M., Schmitt, B., Chapman, H. N. & Meents, A. (2019). IUCrJ, 6, 927–937. Mehrabi, P., Schulz, E. C., Agthe, M., Horrell, S., Bourenkov, G., von Stetten, D., Leimkohl, J., Schikora, H., Schneider, T. R., Pearson, A. R., Tellkamp, F. & Miller, R. J. D. (2019). Nat. Methods, 16, 979–982. , Weierstall, U., James, D., Wang, C., White, T. A., Wang, D., Liu, W., Spence, J. C., Bruce Doak, R., Nelson, G., Fromme, P., Fromme, R., Grotjohann, I., Kupitz, C., Zatsepin, N. A., Liu, H., Basu, S., Wacker, D., Won Han, G., Katritch, V., Boutet, S., Messerschmidt, M., Williams, G. J., Koglin, J. E., Marvin Seibert, M., Klinker, M., Gati, C., Shoeman, R. L., Barty, A., Chapman, H. N., Kirian, R. A., Beyerlein, K. R., Stevens, R. C., Li, D., Shah, S. T., Howe, N., Caffrey, M. & Cherezov, V. (2014). Nat. Commun. 5, 3309. Mehrabi, P., Schulz, E. C., Dsouza, R., Mu¨ller-Werkmeister, H. M., Tellkamp, F., Miller, R. J. D. & Pai, E. F. (2019). Science, 365, 1167–1170. Moffat, K. (1989). Annu. Rev. Biophys. Biophys. Chem. 18, 309– 332. Moffat, K. (1998). Acta Cryst. A54, 833–841. Mueller, C., Marx, A., Epp, S. W., Zhong, Y., Kuo, A., Balo, A. R., Soman, J., Schotte, F., Lemke, H. T., Owen, R. L., Pai, E. F., Pearson, A. R., Olson, J. S., Anﬁnrud, P. A., Ernst, O. P. & Miller, R. J. D. (2015). Struct. Dyn. 2, 054302. Mueller, C., Marx, A., Epp, S. W., Zhong, Y., Kuo, A., Balo, A. R., Soman, J., Schotte, F., Lemke, H. T., Owen, R. L., Pai, E. F., Pearson, A. R., Olson, J. S., Anﬁnrud, P. A., Ernst, O. P. & Miller, R. J. D. (2015). Struct. Dyn. 2, 054302. Oghbaey, S., Sarracini, A., Ginn, H. M., Pare-Labrosse, O., Kuo, A., Marx, A., Epp, S. W., Sherrell, D. A., Eger, B. T., Zhong, Y., Loch, R., Mariani, V., Alonso-Mori, R., Nelson, S., Lemke, H. T., Owen, R. L., Pearson, A. R., Stuart, D. I., Ernst, O. P., Mueller- Werkmeister, H. M. & Miller, R. J. D. (2016). Acta Cryst. D72, 944–955. Weinert, T., Olieric, N., Cheng, R., Bru¨nle, S., James, D., Ozerov, D., Gashi, D., Vera, L., Marsh, M., Jaeger, K., Dworkowski, F., Panepucci, E., Basu, S., Skopintsev, P., Dore´, A. S., Geng, T., Cooke, R. M., Liang, M. N., Prota, A. Acknowledgements (1992). Curr. Opin. Struct. Biol. 2, 821–827. , Sherrell, D. A., Tono, K., Ueda, K., Westermeier, F. & Roedig, P., Duman, R., Sanchez-Weatherby, J., Vartiainen, I., Burkhardt, A., Warmer, M., David, C., Wagner, A. & Meents, A. (2016). J. Appl. Cryst. 49, 968–975. Gati, C., Bourenkov, G., Klinge, M., Rehders, D., Stellato, F., Oberthu¨r, D., Yefanov, O., Sommer, B. P., Mogk, S., Duszenko, M., Betzel, C., Schneider, T. R., Chapman, H. N. & Redecke, L. (2014). IUCrJ, 1, 87–94. ( ) pp y Roedig, P., Ginn, H. M., Pakendorf, T., Sutton, G., Harlos, K., Walter, T. S., Meyer, J., Fischer, P., Duman, R., Vartiainen, I., Reime, B., Warmer, M., Brewster, A. S., Young, I. D., Michels-Clark, T., Sauter, N. K., Kotecha, A., Kelly, J., Rowlands, D. J., Sikorsky, M., Nelson, S., Damiani, D. S., Alonso-Mori, R., Ren, J., Fry, E. E., David, C., Stuart, D. I., Wagner, A. & Meents, A. (2017). Nat. Methods, 14, 805–810. g M., Betzel, C., Schneider, T. R., Chapman, H. N. & Redecke, L. (2014). IUCrJ, 1, 87–94. ( ) Gru¨nbein, M. L. & Nass Kovacs, G. (2019). Acta Cryst. D75, 178– 191. Hunter, M. S., Segelke, B., Messerschmidt, M., Williams, G. J., Zatsepin, N. A., Barty, A., Benner, W. H., Carlson, D. B., Coleman, M., Graf, A., Hau-Riege, S. P., Pardini, T., Seibert, M. M., Evans, J., Boutet, S. & Frank, M. (2014). Sci. Rep. 4, 6026. Roedig, P., Vartiainen, I., Duman, R., Panneerselvam, S., Stube, N., Lorbeer, O., Warmer, M., Sutton, G., Stuart, D. I., Weckert, E., Roedig, P., Vartiainen, I., Duman, R., Panneerselvam, S., Stube, N., Lorbeer, O., Warmer, M., Sutton, G., Stuart, D. I., Weckert, E., David, C., Wagner, A. & Meents, A. (2015). Sci. Rep. 5, 10451. Kupitz, C., Basu, S., Grotjohann, I., Fromme, R., Zatsepin, N. A., Rendek, K. N., Hunter, M. S., Shoeman, R. L., White, T. A., Wang, D., James, D., Yang, J. H., Cobb, D. E., Reeder, B., Sierra, R. G., Liu, H., Barty, A., Aquila, A. L., Deponte, D., Kirian, R. A., Bari, S., Bergkamp, J. J., Beyerlein, K. R., Bogan, M. J., Caleman, C., Chao, T. C., Conrad, C. E., Davis, K. M., Fleckenstein, H., Galli, L., Hau- Riege, S. P., Kassemeyer, S., Laksmono, H., Liang, M., Lomb, L., Marchesini, S., Martin, A. Acknowledgements 4, 044003. ( ) Suga, M., Akita, F., Yamashita, K., Nakajima, Y., Ueno, G., Li, H., Yamane, T., Hirata, K., Umena, Y., Yonekura, S., Yu, L. J., Murakami, H., Nomura, T., Kimura, T., Kubo, M., Baba, S., Kumasaka, T., Tono, K., Yabashi, M., Isobe, H., Yamaguchi, K., Yamamoto, M., Ago, H. & Shen, J. R. (2019). Science, 366, 334– 338. Xu, W., Tolstikova, A., Morgan, A., Metz, M., Martin-Garcia, J. M., Zook, J. D., Roy-Chowdhury, S., Coe, J., Nagaratnam, N., Meza, D., y y g Fromme, R., Basu, S., Frank, M., White, T., Barty, A., Bajt, S., U., Spence, J., Schwander, P., Pollack, L., Fromme, P., Ourmazd, A., 338. Tenboer, J., Basu, S., Zatsepin, N., Pande, K., Milathianaki, D., Frank, M., Hunter, M., Boutet, S., Williams, G. J., Koglin, J. E., Oberthuer, D., Heymann, M., Kupitz, C., Conrad, C., Coe, J., Roy-Chowdhury, S., Weierstall, U., James, D., Wang, D., Grant, T., Barty, A., Yefanov, O., Scales, J., Gati, C., Seuring, C., Srajer, V., Henning, R., Schwander, P., Fromme, R., Ourmazd, A., Moffat, K., Van Thor, J. J., Spence, J. C., Fromme, P., Chapman, H. N. & Schmidt, M. (2014). Science, 346, 1242–1246. Tenboer, J., Basu, S., Zatsepin, N., Pande, K., Milathianaki, D., Frank, M., Hunter, M., Boutet, S., Williams, G. J., Koglin, J. E., Oberthuer, Lee, J., Zatsepin, N. A. & Kim, K. H. (2018). BioDesign, 6, 15–22. Lieske, J., Cerv, M., Kreida, S., Komadina, D., Fischer, J., Barthelmess, M., Fischer, P., Pakendorf, T., Yefanov, O., Mariani, V., Seine, T., Ross, B. H., Crosas, E., Lorbeer, O., Burkhardt, A., Lane, T. J., Guenther, S., Bergtholdt, J., Schoen, S., To¨rnroth-Horseﬁeld, S., Chapman, H. N. & Meents, A. (2019). IUCrJ, 6, 714–728. D., Heymann, M., Kupitz, C., Conrad, C., Coe, J., Roy-Chowdhury, S., Weierstall, U., James, D., Wang, D., Grant, T., Barty, A., Yefanov, O., Scales, J., Gati, C., Seuring, C., Srajer, V., Henning, R., Schwander, P., Fromme, R., Ourmazd, A., Moffat, K., Van Thor, J. J., Martiel, I., Mu¨ller-Werkmeister, H. M. & Cohen, A. E. (2019). Acta Cryst. D75, 160–177. Spence, J. C., Fromme, P., Chapman, H. N. & Schmidt, M. (2014). Science, 346, 1242–1246. Martin-Garcia, J. M., Conrad, C. E., Coe, J., Roy-Chowdhury, S. & Fromme, P. (2016). Arch. Biochem. Biophys. 602, 32–47. Tolstikova, A., Levantino, M., Yefanov, O., Hennicke, V., Fischer, P., Meyer, J., Mozzanica, A., Redford, S., Crosas, E., Opara, N. Acknowledgements V., Messerschmidt, M., Milathianaki, D., Nass, K., Ros, A., Roy-Chowdhury, S., Schmidt, K., Seibert, M., Steinbrener, J., Stellato, F., Yan, L., Yoon, C., Moore, T. A., Moore, A. L., Pushkar, Y., Williams, G. J., Boutet, S., Doak, R. B., Weierstall, U., Frank, M., Chapman, H. N., Spence, J. C. & Fromme, P. (2014). Nature, 513, 261–265. Kupitz, C., Basu, S., Grotjohann, I., Fromme, R., Zatsepin, N. A., Rendek, K. N., Hunter, M. S., Shoeman, R. L., White, T. A., Wang, D., James, D., Yang, J. H., Cobb, D. E., Reeder, B., Sierra, R. G., Liu, Sanchez-Weatherby, J., Bowler, M. W., Huet, J., Gobbo, A., Felisaz, F., Sanchez-Weatherby, J., Bowler, M. W., Huet, J., Gobbo, A., Felisaz, F., Lavault, B., Moya, R., Kadlec, J., Ravelli, R. B. G. & Cipriani, F. (2009). Acta Cryst. D65, 1237–1246. Lavault, B., Moya, R., Kadlec, J., Ravelli, R. B. G. & Cipriani, F. (2009). Acta Cryst. D65, 1237–1246. ( ) Schlichting, I. (2015). IUCrJ, 2, 246–255. Schulz, E. C., Mehrabi, P., Mu¨ller-Werkmeister, H. M., Tellkamp, F., Jha, A., Stuart, W., Persch, E., De Gasparo, R., Diederich, F., Pai, E. F. & Miller, R. J. D. (2018). Nat. Methods, 15, 901–904. g y g Marchesini, S., Martin, A. V., Messerschmidt, M., Milathianaki, D., Sherrell, D. A., Foster, A. J., Hudson, L., Nutter, B., O’Hea, J., Nelson, S., Pare´-Labrosse, O., Oghbaey, S., Miller, R. J. D. & Owen, R. L. (2015). J. Synchrotron Rad. 22, 1372–1378. Stellato, F., Oberthu¨r, D., Liang, M., Bean, R., Gati, C., Yefanov, O., Barty, A., Burkhardt, A., Fischer, P., Galli, L., Kirian, R. A., Meyer, J., Panneerselvam, S., Yoon, C. H., Chervinskii, F., Speller, E., White, T. A., Betzel, C., Meents, A. & Chapman, H. N. (2014). IUCrJ, 1, 204–212. Kupitz, C., Olmos, J. L., Holl, M., Tremblay, L., Pande, K., Pandey, S., Oberthu¨r, D., Hunter, M., Liang, M., Aquila, A., Tenboer, J., Calvey, G., Katz, A., Chen, Y., Wiedorn, M. O., Knoska, J., Meents, A., Majriani, V., Norwood, T., Poudyal, I., Grant, T., Miller, M. D., Xu, W., Tolstikova, A., Morgan, A., Metz, M., Martin-Garcia, J. M., Zook, J. D., Roy-Chowdhury, S., Coe, J., Nagaratnam, N., Meza, D., Fromme, R., Basu, S., Frank, M., White, T., Barty, A., Bajt, S., Yefanov, O., Chapman, H. N., Zatsepin, N., Nelson, G., Weierstall, U., Spence, J., Schwander, P., Pollack, L., Fromme, P., Ourmazd, A., Phillips, G. N. & Schmidt, M. (2017). Struct. Dyn. Acknowledgements The authors would like to thank Drs M. Agthe, S. Horrell, G. Bourenkov, D. von Stetten and T. R. Schneider for their continuous support and helpful discussion in the imple- mentation and improvement of these instruments. All serial X-ray diffraction experiments were conducted at room temperature on the T-REXX endstation of the EMBL beamline P14 at the PETRA III synchrotron at DESY, Hamburg (https://www.embl-hamburg.de/services/mx/). ECS, PM, HMW, LA and SWE designed the HARE TRX chip; ECS, PM and FT designed the humidity hood; FT designed the vacuum control unit, ECS, PM and FT designed the chip- loading block, SE designed the chip storage box; and ECS, HMW, FT, DS and RO designed the translation-stage holders. JPL and PM designed the tear-drop cover. ECS, JPL and HS designed the glow discharge box, the drying box and the q g Grotjohann, I., Holton, J. M., Barends, T. R. M., Neutze, R., Marchesini, S., Fromme, R., Schorb, S., Rupp, D., Adolph, M., pp p Gorkhover, T., Andersson, I., Hirsemann, H., Potdevin, G., Graafsma, H., Nilsson, B. & Spence, J. C. H. (2011). Nature, 470, 73–77. Davy, B., Axford, D., Beale, J. H., Butryn, A., Docker, P., Ebrahim, A., Leen, G., Orville, A. M., Owen, R. L. & Aller, P. (2019). J. Synchrotron Rad. 26, 1820–1825. Ebrahim, A., Appleby, M. V., Axford, D., Beale, J., Moreno-Chicano, T., Sherrell, D. A., Strange, R. W., Hough, M. A. & Owen, R. L. (2019). Acta Cryst. D75, 151–159. Ebrahim, A., Moreno-Chicano, T., Appleby, M. V., Chaplin, A. K., Beale, J. H., Sherrell, D. A., Duyvesteyn, H. M. E., Owada, S., Tono, K., Sugimoto, H., Strange, R. W., Worrall, J. A. R., Axford, D., Owen, R. L. & Hough, M. A. (2019). IUCrJ, 6, 543–551. 368 Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography J. Synchrotron Rad. (2020). 27, 360–370 J. Synchrotron Rad. (2020). 27, 360–370 research papers Epp, S. W., Hada, M., Zhong, Y., Kumagai, Y., Motomura, K., Mizote, S., Ono, T., Owada, S., Axford, D., Bakhtiarzadeh, S., Fukuzawa, H., Hayashi, Y., Katayama, T., Marx, A., Mu¨ller-Werkmeister, H. M., Owen, R. L., Sherrell, D. A., Tono, K., Ueda, K., Westermeier, F. & Miller, R. J. D. (2017). Struct. Dyn. 4, 054308. Owen, R. L., Axford, D., Sherrell, D. A., Kuo, A., Ernst, O. P., Schulz, E. C., Miller, R. J. D. & Mueller-Werkmeister, H. M. (2017). Acta Cryst. D73, 373–378. Pai, E. F. Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography Acknowledgements E., Panneels, V., Nogly, P., Ermler, U., Schertler, G., Hennig, M., Steinmetz, M. O., Wang, M. T. & Standfuss, J. (2017). Nat. Commun. 8, 542. ( ) y Oghbaey, S., Sarracini, A., Ginn, H. M., Pare-Labrosse, O., Kuo, A., Marx, A., Epp, S. W., Sherrell, D. A., Eger, B. T., Zhong, Y., Loch, R., Mariani, V., Alonso-Mori, R., Nelson, S., Lemke, H. T., Owen, R. L., Pearson, A. R., Stuart, D. I., Ernst, O. P., Mueller- Werkmeister, H. M. & Miller, R. J. D. (2016). Acta Cryst. D72, 944–955. Weinert, T., Skopintsev, P., James, D., Dworkowski, F., Panepucci, E., Kekilli, D., Furrer, A., Bru¨nle, S., Mous, S., Ozerov, D., Nogly, P., Wang, M. & Standfuss, J. (2019). Science, 365, 61–65. 369 Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography 369 J. Synchrotron Rad. (2020). 27, 360–370 Zarrine-Afsar, A., Barends, T. R. M., Mu¨ller, C., Fuchs, M. R., Lomb, L., Schlichting, I. & Miller, R. J. D. (2012). Acta Cryst. D68, 321– 323. Zarrine-Afsar, A., Mu¨ller, C., Talbot, F. O. & Miller, R. J. (2010). Anal. Chem. 83, 767–773. Wierman, J. L., Pare´-Labrosse, O., Sarracini, A., Besaw, J. E., Cook, M. J., Oghbaey, S., Daoud, H., Mehrabi, P., Kriksunov, I., Kuo, A., Schuller, D. J., Smith, S., Ernst, O. P., Szebenyi, D. M. E., Gruner, S. M., Miller, R. J. D. & Finke, A. D. (2019). IUCrJ, 6, 305–316. 370 Mehrabi et al. The HARE chip for time-resolved serial synchrotron crystallography Zarrine-Afsar, A., Barends, T. R. M., Mu¨ller, C., Fuchs, M. R., Lomb, L., Schlichting, I. & Miller, R. J. D. (2012). Acta Cryst. D68, 321– 323. Zarrine-Afsar, A., Mu¨ller, C., Talbot, F. O. & Miller, R. J. (2010). Anal. Chem. 83, 767–773. research papers Wierman, J. L., Pare´-Labrosse, O., Sarracini, A., Besaw, J. E., Cook, M. J., Oghbaey, S., Daoud, H., Mehrabi, P., Kriksunov, I., Kuo, A., Schuller, D. J., Smith, S., Ernst, O. P., Szebenyi, D. M. E., Gruner, S. M., Miller, R. J. D. & Finke, A. D. (2019). IUCrJ, 6, 305–316. J. Synchrotron Rad. (2020). 27, 360–370
https://openalex.org/W3127754785	https://bmccancer.biomedcentral.com/track/pdf/10.1186/s12885-021-07864-y	English	null	A novel scoring system integrating molecular abnormalities with IPSS-R can improve the risk stratification in patients with MDS	BMC cancer	2,021	cc-by	5,816	Gu et al. BMC Cancer (2021) 21:134 https://doi.org/10.1186/s12885-021-07864-y Gu et al. BMC Cancer (2021) 21:134 https://doi.org/10.1186/s12885-021-07864-y Open Access A novel scoring system integrating molecular abnormalities with IPSS-R can improve the risk stratification in patients with MDS Siyu Gu, Jingya Xia, Yulu Tian, Jie Zi and Zheng Ge* * Correspondence: Janege879@hotmail.com Department of Hematology, Zhongda Hospital, School of Medicine, Southeast University, Institute of Hematology Southeast University, Nanjing 210009, China Abstract Background: The treatment strategies for Myelodysplastic Syndromes (MDS) are usually based on the risk stratification system. However, few risk signatures which integrate the revised international prognostic scoring system (IPSS-R) with gene mutations can be easily applied in the real world. Methods: The training cohort of 63 MDS patients was conducted at Zhongda Hospital of Southeast University from January 2013 to April 2020. The validation cohort of 141 MDS patients was obtained from GSE129828. The mutation scoring system was based on the number of mutations and a unique favorable prognostic factor, which is SF3B1 mutation. Univariate Cox, multivariate Cox, and LASSO regression analyses were used to determine the significant factors that influenced the overall survival. The receiver operating characteristic curve (ROC) was used to evaluate the efficiency of the prognostic model. Results: A novel risk scoring system we named “mutation combined with revised international prognostic scoring system (MIPSS-R)” was developed based on the results derived from multivariate analysis which assigned points to the IPSS-R and the mutation scores according to their relative statistical weight. Based on the quintile of the new scores, patients were divided into five risk levels. The Kaplan-Meier curves showed the superiority of MIPSS-R in separating patients from different groups, comparing with IPSS-R both in the training cohort (p = 1.71e-08 vs. p = 1.363e-04) and validation cohort (p = 1.788e-04 vs. p = 2.757e-03). The area under the ROC of MIPSS-R was 0.79 in the training cohort and 0.62 in the validation cohort. The retrospective analysis of our house patients showed that the risk levels of 57.41% of patients would adjust according to MIPSS-R. After changing risk levels, 38.71% of patients would benefit from treatment strategies that MIPSS-R recommends. Conclusion: A mutation scoring system was conducted based on the number of mutations and a unique favorable prognostic factor. MIPSS-R, the novel integral risk stratification system was developed by integrating IPSS-R and the mutation scores, which is more effective on prognosis and treatment guidance for MDS patients. Keywords: Myelodysplastic syndrome, Molecular abnormalities, Prognostic factors, Real-world study © The Author(s). 2021 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. Gu et al. BMC Cancer (2021) 21:134 Gu et al. BMC Cancer (2021) 21:134 Page 2 of 9 Page 2 of 9 Mutation risk stratification and MIPSS-R Mutation risk stratification and MIPSS R The mutation risk stratification was constructed by the number of mutated genes and only one favorable prognostic mutated gene. Patients with no mutant or with only SF3B1 mutation classified into the low-risk; with one mutant except SF3B1 classified into intermediate-1-risk; with two to four mutants classified into intermediate-2-risk; with five or more mutants clas- sified into high-risk. Patients in low, intermediate-1, intermediate-2, and high risk were assigned 0, 1, 2, and 3 points, respectively. The mutation combined with re- vised international prognostic scoring system (MIPSS-R) was developed based on the results derived from multi- variate analysis which assigned points to the IPSS-R and the mutation scores according to their relative statistical weight. Based on the quintile of the MIPSS-R scores, pa- tients were divided into very low-, low-, intermediate-, high-, and very high-risk groups, respectively. Cytogenetic and molecular biology determination Cytogenetic and molecular biology determination All patients in the training cohort submitted BM aspi- rates at the time of admission. Cytogenetic analysis was conducted by conventional G-banding technology and fluorescence in situ hybridization (FISH). Each sample with three or more abnormal genetic characteristic BM cells was considered a sample with abnormal clones after analyzing at least 20 metaphases. Taking the FISH exam- ination, each probe analyzed at least 200 cells. When the proportion of abnormal signal cells of a sample exceed the threshold, the sample considered with cytogenetic abnormality. By using DNA extracted from each aspir- ate, the mutational analysis was taken with an amplicon- based, NGS panel targeting the entire coding regions of 31 genes frequently mutated in MDS (supplement Table 1). Only mutations that have been previously re- ported to be pathogenic either in the Catalogue of Somatic Mutations in Cancer (COSMIC) ID or other da- tabases or in the literature were considered in the present study. Based on these concepts, we would like to build a novel prognostic system that integrated the mutations with IPSS-R. To address these issues and to expand the knowledge about predictive factors, the data of 63 pa- tients from our clinical center was utilized as a training cohort, and the data of 141 patients from GSE129828 was utilized as a validation cohort. This research aims to provide physicians with practical information, support them in choosing the best treatment plan, and provide consultations for patients. Background institutional review boards and by the Declaration of Helsinki. The prognostic risk stratifications of all pa- tients were according to the IPSS-R prognosis integral systems [5, 12]. g Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by inef- fective and dysplastic hematopoiesis that causes cytope- nia, which are also likely to progress to the development of acute myeloid leukemia (AML) [1, 2]. MDS is pre- dominantly diagnosed among older adults, and more than half of the patients exceed the age of 75 [3, 4]. The treatment strategies are usually based on the risk stratifi- cations like the revised International Prognostic Scoring System (IPSS-R), which consists with bone marrow (BM) cytogenetics, blast percentage, and peripheral blood (PB) cytopenia [5]. However, with the rapid development of high through-put technology like next- generation sequence (NGS), multiple mutations have been revealed as significant factors in MDS [6], and ap- proximately 90% of MDS patients have at least one mu- tation [7]. One large-scale genomic research showed that TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutations appeared in more than 10% of MDS cases, and many mutations were correlated to higher risk groups or high blast counts [6]. According to National Comprehensive Cancer Network (NCCN) guidelines, epigenetic mutations such as TET2, DNMT3A, ASXL1, IDH1/2, and EZH2 commonly occur in MDS; Splicing factor-related mutations such as SF3B1, SRSF2, U2AF1 and ZRSR2 are not specific mutations of MDS but occur more frequently in MDS than in other myeloid tumors. SF3B1 mutation predicts a good prognosis; SRSF2, RUNX1, U2AF1, ASXL1, and TP53 mutation predict high risks of progressing to AML [8]. It is well known that mutations have the prognostic effect in MDS [9, 10], but a perfect scoring system based on mutation or combined with IPSS-R has not yet appeared. Meanwhile, A total of 141 patients with treatment- naive MDS from dataset GSE129828 [13] were com- posed to a validation cohort, which was similar to the training cohort and was obtained from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). Clinical data to determine the IPSS-R scores, mutations and French-American-British (FAB) classification were available at the time of sample collection. Patient cohorts A total of 63 de novo MDS patients were collected in the department of hematology, Zhongda Hospital from January 2013 to April 2020, which were conducted as the training cohort. The diagnostic criteria and subclas- sified standards were referenced to the World Health Organization (WHO) in 2008 [11]. All samples were col- lected with patient consent under protocols approved by Gu et al. BMC Cancer (2021) 21:134 Gu et al. BMC Cancer (2021) 21:134 Page 3 of 9 Page 3 of 9 Statistical analysis differences between cohorts (Supplement Table 2). In terms of abnormal karyotypes, 5q- was the most com- mon one both in the training cohort (20.4%) and the val- idation cohort (9.3%). Complex karyotype was defined as more than or equal to three abnormal karyotypes, con- stituting 9.3% in the training cohort and 8.3% in the val- idation cohort. The expression of karyotype was similar between the two cohorts except for the 20q- (p = 0.007, Supplement Table 3). y The overall survival (OS) was defined as the time in days from the date of MDS diagnosis to the date of last follow-up or death, regardless of causes. The univariate, multivariate Cox and Least absolute shrinkage and selection operator (LASSO) regression models, receiver operating characteristic curve (ROC) analyses, and Kaplan-Meier (K-M) survival curve with Log Rank ana- lysis were performed using R studio (version 3.6.3). The univariate, multivariate Cox regression and K-M survival analyses were performed with the package of “survival” in R. The LASSO regression analysis was performed with R package of “glmnet” in R. The prediction ability of the model was assessed by the area under the curve (AUC) of ROC with the package of “survivalROC” in R. Quanti- tative data were exhibited as the mean ± standard devi- ation (SD). Mann-Whitney U test and Fisher exact test analyzed continuous variable and categorical variables respectively by using SPSS 26.0 software. All statistical tests were bilateral, with a p-value < 0.05 being statisti- cally significant. Survival analysis For the training cohort, with a median follow up of 13.5 months (range, 0.39–88.24 months), the median OS per IPSS-R scoring system was > 60, > 60, > 60, 11.34, and 5.92 months for very low-, low-, intermediate-, high-, and very high-risk, respectively, p = 5.759e-06 (Fig. 1 a). The median OS per mutation risk stratification was > 60, 38.9, 11.3, and 2.7 months for low-, intermediate-1-, intermediate-2-, and high-risk, respectively, p = 6.096e- 04 (Fig. 1 b). For the validation cohort, with a median follow up of 22.2 months (range, 0.66–139.51 months), the median OS per IPSS-R scoring system was 25.59, 20.93, 16.8, 9.73, and 4.7 months for very low-, low-, intermediate-, high-, and very high-risk, respectively, p = 2.757e-03(Fig. 1 c). The median OS per mutation risk stratification was 33, 37, 22, and 18.2 months for low-, intermediate-1-, intermediate-2-, and high-risk, respect- ively, p = 4.242e-03 (Fig. 1 d). Baseline characteristics The follow-up deadline of the training cohort was April 20, 2020. 9 out of 63 patients were removed due to los- ing follow-up. In the validation cohort, 33 out of 141 pa- tients were removed from the present study since lack of survival data. Finally, data of a total of 162 patients were analyzed, 54 patients in the training cohort and 108 pa- tients in the validation cohort. The clinical characteris- tics for each patient were summarized in Table 1. The baseline characters including the age, gender, BM blasts proportion, and mutation risk stratifications had no dif- ferences between the two cohorts. Most of the patients were low-risk ones according to the IPSS-R category, 38.9% of patients in the training cohort, and 28.7% in the validation cohort (p = 0.015). The most common subtypes in the training cohort were multilineage dysplasia (MLD, 42.6%) based on 2008 WHO classifica- tion, and in the validation cohort were refractory anemia with excess of blast (RAEB, 35.2%) based on FAB classification. y g To identify all independent factors for OS, we next performed univariate Cox proportional hazards regres- sion analysis and LASSO regression analysis in the train- ing cohort. Univariate analysis demonstrated that age, TP53 mutation, mutation risk stratifications, IPSS-R, progression to AML, + 8, −7/7q-, and complex karyo- type were the prognostic factors (supplement Fig. 1 a). LASSO regression analysis was performed to select fac- tors, and −7/7q-, IPSS-R, and mutation risk stratification were retained according to the optimal lambda value [log(lambda.min) = −1.64, supplement Fig. 1 b, c]. Next, in multivariate analysis, we confirmed the IPSS-R (p < 0.01) and mutation risk stratification (p < 0.001) as significant predictors for OS (supplement Fig. 1 d). Incorporating mutation risk stratification into IPSS-R In terms of the mutation risk stratification, most of the patients were in the intermediate-2 risk group, both in the training cohort (46.3%) and validation cohort (56.5%, Table 1). The most common mutations were ASXL1, TET2, TP53, SRSF2, and SF3B1, accounting for 31.5, 27.8, 18.5, 14.8, and 14.8%, respectively, in the training cohort; meanwhile, the most common muta- tions in the validation cohort were TET2, ASXL1, SF3B1, RUNX1, and SRSF2, making up 31.5, 27.8, 25, 17.6, and 16.7%, respectively. A total of 20 same mutated genes were detected in both two cohorts, and there was no Incorporating mutation risk stratification into IPSS R In the next step, we aimed for the development of a practical risk score based on the results derived from multivariate analysis. A novel risk scoring system, we named MIPSS-R was developed based on a linear com- bination of the mutation risk stratification score and the IPSS-R score multiplied by regression coefficients ob- tained from the multivariate analysis: MIPSS-R score = mutation score × 1.047 + IPSS-R × 0.641. Based on the quintile of the MIPSS-R scores (ranged from 1.28 to 8.59), patients were divided into very low- (1.28–2.24), Gu et al. BMC Cancer (2021) 21:134 Page 4 of 9 Table 1 Baseline patient characteristics between the training cohort and validation cohort Characteristics Total Training Validation p No. % No. % No. Incorporating mutation risk stratification into IPSS-R % Total 162 54 108 Age, years 0.075 Median 71 70 72 Range 24–91 24–88 48–91 Male sex 112 69.10% 33 61.10% 79 73.10% 0.149 ANC, 109/L 0.007 Median 1.4 1.18 1.6 Range 0.07–28.04 0.07–6.33 0.16–28.04 Hemoglobin, g/dL < 0.0001 Median 9.2 7.05 9.7 Range 3.3–15.2 3.30–13.4 5.80–15.20 Platelets, 109/L < 0.0001 Median 81.5 53 101 Range 2–987 2–310 6–987 Blasts % 0.655 Median 1 0.8 1 Range 0–28 0–18.8 0–28 IPSS-R Category 0.015 Very low 22 13.6 1 1.9 21 19.4 Low 52 32.1 21 38.9 32 28.7 Intermediate 31 19.1 13 24.1 18 16.7 High 35 21.6 11 20.4 24 22.2 Very high 22 13.6 8 14.8 14 13 Mutation risk stratification 0.156 Low 30 18.5 11 20.4 19 17.6 Intermediate-1 39 24.1 13 24.1 26 24.1 Intermediate-2 86 53.1 25 46.3 61 56.5 High 7 4.3 5 9.3 2 1.9 MIPSSR 0.956 Very low 31 19.1 10 18.5 21 19.4 Low 29 17.9 11 20.4 18 16.7 Intermediate 33 20.4 10 18.5 23 21.3 High 36 22.2 11 20.4 25 23.1 Very high 33 20.4 12 22.2 21 19.4 2008 WHO classification NA MDS-SLD 5 9.3 MDS-MLD 23 42.6 MDS-RS 7 13.0 MDS-EB1 8 14.8 MDS-EB2 8 14.8 MDS-U 3 5.6 Table 1 Baseline patient characteristics between the training cohort and validation cohort Gu et al. BMC Cancer (2021) 21:134 Page 5 of 9 Gu et al. BMC Cancer (2021) 21:134 Table 1 Baseline patient characteristics between the training cohort and validation cohort (Continued) Characteristics Total Training Validation p No. % No. % No. % FAB classification NA RA 36 33.3 RARS 20 18.5 RAEB 38 35.2 RAEB-T 6 5.6 RCUD 2 1.9 RCMD 2 1.9 MDS-U 1 0.9 CMML 3 2.8 Table 1 Baseline patient characteristics between the training cohort and validation cohort (Continued) Characteristics Total Training Validation p No. % No. % No. % FAB classification NA RA 36 33.3 RARS 20 18.5 RAEB 38 35.2 RAEB-T 6 5.6 RCUD 2 1.9 RCMD 2 1.9 MDS-U 1 0.9 CMML 3 2.8 Table 1 Baseline patient characteristics between the training cohort and validation cohort (Continued) Fig. 1 a, b The Kaplan-Meier curve of patients from the training cohort; c, d The Kaplan-Meier curve of patients from the validation cohort. a, c The Kaplan-Meier curves of patients in different IPSS-R risk stratifications. b, d The Kaplan-Meier curves of patients in different mutation risk stratifications Fig. Incorporating mutation risk stratification into IPSS-R 1 a, b The Kaplan-Meier curve of patients from the training cohort; c, d The Kaplan-Meier curve of patients from the validation cohort. a, c The Kaplan-Meier curves of patients in different IPSS-R risk stratifications. b, d The Kaplan-Meier curves of patients in different mutation risk stratifications Gu et al. BMC Cancer (2021) 21:134 Gu et al. BMC Cancer (2021) 21:134 Page 6 of 9 (0.620), but higher than the mutation scoring system (0.555, Fig. 2 d). low- (2.33–3.93), intermediate- (4.02–4.34), high- (4.57– 5.30), and very high-risk (5.62–8.59) with the median OS of > 60, > 60, 44.41, 11.68, and 5.92 months respectively (Fig. 2 a, p = 1.71e-08). We then calculated the MIPSS-R scores of the patients in the validation cohort. The OS was significantly different between groups (p = 1.788e- 04) with the median OS of 75.1, 34.5, 24.2, 24.2, and 16.3 months in very low-, low-, intermediate-, high-, and very high-risk, respectively (Fig. 2 b). Meanwhile, the AUC value of the MIPSS-R was 0.790, which was higher than IPSS-R (0.731) and mutation scoring system (0.672) alone in the training cohort (Fig. 2 c). MIPSS-R in the validation cohort had an equal AUC value to the IPSS-R Discussion MDS are a group of highly heterogeneous diseases which are commonly occurred in the elderly population, char- acterized by pancytopenia and a high risk of progressing to AML. Various factors including the BM blasts, pan- cytopenia, cytogenetic characteristics, and genetic muta- tions affect the prognosis of the disease [14]. The gold criteria for assessing conditions of MDS patients is IPSS- R which is the latest version of IPSS revised by the MDS Prognosis International Working Group in 2012 [5]. However, independent prognostic elements such as red blood cell transfusion dependence, genetic mutations are not included in the scoring system, especially the gene mutations that help to the accurate assessment [15]. The advance of modern technology has improved the genome-wide analysis of genetic mutations in MDS [6, 16]. Although the evolution of molecular technology has introduced new challenges, it is also leading to novel recognition of accurate diagnosis and therapy. g g py One large scale molecular research analyzed 994 MDS patients and revealed the genomic landscape of the dis- ease [6]. The most frequently mutated genes were TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1, that all accounted for more than 10% in these patients. Another whole-exome sequencing study of 699 patients revealed distinct patterns of clonal evolution in MDS [17]. The data showed that MDS patients with SF3B1 mutations were enriched in the low-risk group, but patients with GATA2, NRAS, KRAS, IDH2, TP53, RUNX1, STAG2, ASXL1, ZRSR2, and TET2 mutations were enriched in the high-risk group. Patients with FLT3, PTPN11, WT1, IDH1, IDH2, NPM1, and NRAS mutations were signifi- cantly correlated to the AML progression. Meanwhile, they also found that most of the patients have unique mutated patterns, leading to a great deal of heterogen- eity [6, 17]. Certainly, the specific effects of some mutations are commonly accepted, besides the mutated genes afore- mentioned utilized in multivariable models, DNMT3A, U2AF1, SRSF2, CBL, PRPF8, SETBP1, and KRAS have also been reported the association with decreased OS [6, 18, 21–23]. Only mutated SF3B1 is correlated to favor- able outcome [6, 24]. Dr. Bejar. argue that the mutation patterns of MDS are diverse and no two are the same [25]. Due to the heterogeneities of mutations in MDS patients, it is difficult to utilize the prognostic model with specific mutants to assess patients with mutants that with uncertain prognostic values. The clinical significance of the MIPSS-R To highlight the clinical significance of the MIPSS-R, we compared the risk stratifications changes in the training cohort (supplement Table 4). 27.78% (15/54) patients had a decreased risk, and 29.63% (16/54) patients had an elevated risk. Patients #49 and #58 were classified as intermediate-risk and low-risk based on IPSS-R and re- ceived demethylation therapy but died shortly owing to toxicity thereafter. These two patients would benefit from supportive care or other non-intensive therapy Fig. 2 a The Kaplan-Meier curves of patients in different MIPSS-R risk stratifications and c the receiver operating characteristic curves of different risk stratifications in the training cohort; b The Kaplan-Meier curves of patients in different MIPSS-R risk stratifications and d the receiver operating characteristic curves of different risk stratifications the validation cohort Fig. 2 a The Kaplan-Meier curves of patients in different MIPSS-R risk stratifications and c the receiver operating characteristic curves of different risk stratifications in the training cohort; b The Kaplan-Meier curves of patients in different MIPSS-R risk stratifications and d the receiver operating characteristic curves of different risk stratifications the validation cohort Page 7 of 9 Gu et al. BMC Cancer (2021) 21:134 Gu et al. BMC Cancer (2021) 21:134 described a mutation landscape of 439 MDS patients and screened out five mutations, TP53, EZH2, ETV6, RUNX1, and ASXL1, that could predict the poor overall survival of MDS patients independently [18]. Haferlach et al. utilized the predictors including age, gender, IPSS- R, and 14 mutations genes, building a novel prognostic model (model-1) and separating patients into four risk groups, which showed significantly different 3-year sur- vival rate of 95.2, 69.3, 32.8, and 5.3%, respectively [6]. Comparing with another model built by the 14 muta- tions alone (Model-2), and with IPSS-R, model-1 was more superior. Nazha et al. incorporated mutated EZH2, SF3B1, and TP53 with IPSS-R and improved the predict- ive ability in MDS [19]. Notably, MDS patients enrolled in the study were serial samples with different time points during their disease courses. The new model clas- sified patients into 4 risk groups with a median OS of 37.4, 23.2, 19.9, and 12.2 months, respectively. The new model also had a better C-index than IPSS-R. The re- sults of the paired samples also confirmed the new model had a dynamic prognostic potential. Hou et al. The clinical significance of the MIPSS-R built an integrated risk-stratification model consisting with the age, IPSS-R, and 5 mutations (CBL, IDH2, DNMT3A, ASXL1, and TP53) [20], diving patients into four risk groups, and the median OS of each group was 250.7, 38.4, 17, and 8.9 months respectively. They also showed that the new model could be well applied not only in the FAB-defined MDS patients but also those de- fined by WHO. Recently, Naqvi et al. also developed a new prognostic system incorporating 27-item Adult Co- morbidity Evaluation (ACE-27) and TP53 mutation with IPSS-R which improved outcome prediction in patients with MDS [7]. The C-index for the new model is 0.822, and the survival curves between risk groups of the new model were more well-separated than those of IPSS-R risk groups. The study also highlighted that clonal hematopoiesis of indeterminate potential (CHIP) associ- ated mutations were associated with a higher frequency of prior history of cardiovascular events and poor prog- nosis in patients with MDS based on MIPSS-R guidance. Moreover, patient #4, #18, and #41 were classified into low-risk and patients #36 and #44 were intermediate-risk based on IPSS-R, who eventually died as they only received supportive care. These patients might receive more intensive therapy and would expect a better prognosis based on MIPSS-R sug- gestion. Further, patients #5, #32, #34, #48, and #53 were grouped into low-risk based on IPSS-R but received de- methylation therapy or more intensive therapy instead of supportive therapy, who achieved better outcomes. Not- ably, patients #4, #40, #53, and #61 with elevated risk eventually developed to AML, which indicated the possi- bility of early identification of AML progression by MIPSS-R. References 1 T ff i A 1. Tefferi A, Vardiman JW. Myelodysplastic syndromes. N Engl J Med. 2009; 361(19):1872–85. Availability of data and materials Patients’ data that before December 2018 support the findings of this study are available on request from the Beijing Hyster Technology Co., Ltd. Patients’ data after December 2018 that support the findings of this study are available on request from the corresponding author. Data from GSE129828 was obtained from the Gene Expression Omnibus (https://www. ncbi.nlm.nih.gov/geo/). However, this study still has some limitations. First, our study was retrospective, so there may be some inherent biases. Secondly, the number of patients was limited and large-scale prospective researches are pros- pected. Third, the prognostic model needs to be verified in other cohorts, such as specific treatment strategies co- hort and paired cohort. Discussion Hence, we con- structed a more simple-to-use mutation scoring system which contained only one favorable factor and the In the past few years, some studies have integrated mutations with IPSS-R to improve the prognostic values for MDS patients. One of the researches by Bejar et al. Gu et al. BMC Cancer (2021) 21:134 Gu et al. BMC Cancer (2021) 21:134 Page 8 of 9 Page 8 of 9 writing of the manuscript. All authors have read and approved the manuscript. writing of the manuscript. All authors have read and approved the manuscript. number of mutations, assigning 0 to 3 points to patients respectively. Then, the novel prognostic scoring system, MIPSS-R, was constructed by the linear combination of the IPSS-R and the mutation scoring system. Although MIPSS-R would lose some specific information about mutated genes, it overcomes the heterogeneities of mu- tation patterns of MDS patients. The prognostic value was also verified in the validation cohorts. Moreover, retrospective analysis of our house patients showed that more than half of the patients would adjust the risk stratification based on the MIPSS-R. Through a compre- hensive analysis of the treatment strategies and out- comes of these patients, we found that part of the patients may obtain better prognosis under the guidance of MIPSS-R. writing of the manuscript. All authors have read and approved the manuscript. writing of the manuscript. All authors have read and approved the manuscript. Consent for publication Not Applicable. Consent for publication Not Applicable. Consent for publication Not Applicable. Conclusions In summary, by integrating IPSS-R and gene mutations, MIPSS-R, a novel risk stratification system for MDS pa- tients has been developed. This system is more effective in prognosis and will be helpful to reduce treatment- related deaths, to recognize MDS patients with high-risk or AML progression risk earlier. Funding This work is supported in part by The National Natural Science Foundation of China (81770172); Jiangsu Provincial Special Program of Medical Science (BE2017747); Jiangsu Province “333” project (BRA2019103); The Fundamental Research Funds for the Central Universities (2242019K3DZ02); Milstein Medical Asian American Partnership (MMAAP) Foundation Research Project Award in Hematology (2017); Key Medical of Jiangsu Province (ZDXKB2016020). The funders had no roles in study design, data collection, data analysis, and interpretation, or writing of the manuscript. Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 8 September 2020 Accepted: 1 February 2021 Received: 8 September 2020 Accepted: 1 February 2021 Additional file 1. Additional file 1. 5. Greenberg PL, Tuechler H, Schanz J, Sanz G, Garcia-Manero G, Sole F, Bennett JM, Bowen D, Fenaux P, Dreyfus F, et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood. 2012; 120(12):2454–65. Ethics approval and consent to participate The written informed consent was provided by all the patients by the Declaration of Helsinki before enrollment in the study. The Institutional Review Board of Zhongda Hospital Southeast University, Nanjing, China, approved the study with approval number: 2017ZDKYBA001.0, 2017ZDKYBA002.0, 2019ZDSYLL033-P02. Abbreviations MDS: Myelodysplastic Syndromes; IPSS-R: Revised International Prognostic Scoring System; ROC: The receiver operating characteristic curve; AML: Acute myeloid leukemia; BM: Bone marrow; PB: Peripheral blood; NGS: Next- generation sequence; NCCN: National Comprehensive Cancer Network; WHO: World Health Organization; FISH: Fluorescence in situ hybridization; COSMIC: Catalogue of Somatic Mutations in Cancer; OS: Overall survival; LASSO: Least absolute shrinkage and selection operator; K-M: Kaplan-Meier; AUC: Area under the curve; SD: Standard deviation; MDS-SLD: MDS with single lineage dysplasia; MDS-MLD: MDS with multilineage dysplasia; MDS- RS: MDS with ring sideroblasts; MDS-5q-: MDS with isolated del 5q; MDS- EB: MDS with excess blasts; MDS-U: MDS, unclassified; BM: Bone marrow; ANC Ab l hil Hb H l bi PLT Pl l MIPSS R Th 6. Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G, Schnittger S, Sanada M, Kon A, Alpermann T, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia. 2014; 28(2):241–7. 6. Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G, Schnittger S, Sanada M, Kon A, Alpermann T, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia. 2014; 28(2):241–7. 7. Naqvi K, Sasaki K, Montalban-Bravo G, Alfonso Pierola A, Yilmaz M, Short N, Assi R, Jabbour E, Ravandi F, Kadia T, et al. Clonal hematopoiesis of indeterminate potential-associated mutations and risk of comorbidities in patients with myelodysplastic syndrome. Cancer. 2019;125(13):2233–41. 7. Naqvi K, Sasaki K, Montalban-Bravo G, Alfonso Pierola A, Yilmaz M, Short N, Assi R, Jabbour E, Ravandi F, Kadia T, et al. Clonal hematopoiesis of indeterminate potential-associated mutations and risk of comorbidities in patients with myelodysplastic syndrome. Cancer. 2019;125(13):2233–41. 8. Greenberg PL, Stone RM, Al-Kali A, Barta SK, Bejar R, Bennett JM, Carraway H, De Castro CM, Deeg HJ, DeZern AE, et al. Myelodysplastic syndromes, version 2.2017, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2017;15(1):60–87. ANC: Absolute neutrophil count; Hb: Hemoglobin; PLT: Platelet; MIPSS-R: The mutation combined with revised international prognostic scoring system Supplementary Information h l l 2. Nimer SD. Myelodysplastic syndromes. Blood. 2008;111(10):4841–51. 3. Ma X, Does M, Raza A, Mayne ST. Myelodysplastic syndromes. Cancer. 2007; 109(8):1536–42. The online version contains supplementary material available at https://doi. org/10.1186/s12885-021-07864-y. 4. Rollison DE, Howlader N, Smith MT, Strom SS, Merritt WD, Ries LA, Edwards BK, List AF. Epidemiology of myelodysplastic syndromes and chronic myeloproliferative disorders in the United States, 2001-2004, using data from the NAACCR and SEER programs. Blood. 2008;112(1):45–52. Additional file 1. Acknowledgements N t A li bl 9. Papaemmanuil E, Gerstung M, Malcovati L, Tauro S, Gundem G, Van Loo P, Yoon CJ, Ellis P, Wedge DC, Pellagatti A, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013; 122(22):3616–99. 9. Papaemmanuil E, Gerstung M, Malcovati L, Tauro S, Gundem G, Van Loo P, Yoon CJ, Ellis P, Wedge DC, Pellagatti A, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013; 122(22):3616–99. 9. Papaemmanuil E, Gerstung M, Malcovati L, Tauro S, Gundem G, Van Loo P, Yoon CJ, Ellis P, Wedge DC, Pellagatti A, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013; 122(22):3616–99. Authors’ contributions SG and JZ were involved in the conception and design of the study. JX and YT were involved in the collection and assembly of the data. SG and ZG were involved in data analysis and interpretation and were involved in the 10. Steensma DP, Bejar R, Jaiswal S, Lindsley RC, Sekeres MA, Hasserjian RP, Ebert BL. Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood. 2015;126(1):9–16. Page 9 of 9 Gu et al. BMC Cancer (2021) 21:134 Gu et al. BMC Cancer (2021) 21:134 11. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, Harris NL, Le Beau MM, Hellstrom-Lindberg E, Tefferi A, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114(5): 937–51. 12. Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T, Oscier D, et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood. 1997;89(6):2079–88. 13. Reilly B, Tanaka TN, Diep D, Yeerna H, Tamayo P, Zhang K, Bejar R. DNA methylation identifies genetically and prognostically distinct subtypes of myelodysplastic syndromes. Blood advances. 2019;3(19):2845–58. 14. Montalban-Bravo G, Garcia-Manero G. Myelodysplastic syndromes: 2018 update on diagnosis, risk-stratification and management. Am J Hematol. 2018;93(1):129–47. 15. Ganguly BB, Kadam NN. Mutations of myelodysplastic syndromes (MDS): an update. Mutat Res Rev Mutat Res. 2016;769:47–62. 16. Lukackova R, Gerykova Bujalkova M, Majerova L, Mladosievicova B. Molecular genetic methods in the diagnosis of myelodysplastic syndromes. A review. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2014;158(3):339–45. 17. Makishima H, Yoshizato T, Yoshida K, Sekeres MA, Radivoyevitch T, Suzuki H, Przychodzen B, Nagata Y, Meggendorfer M, Sanada M, et al. Dynamics of clonal evolution in myelodysplastic syndromes. Nat Genet. 2017;49(2):204–12. 18. Bejar R, Stevenson K, Abdel-Wahab O, Galili N, Nilsson B, Garcia-Manero G, Kantarjian H, Raza A, Levine RL, Neuberg D, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med. 2011;364(26):2496–506. 19. Nazha A, Narkhede M, Radivoyevitch T, Seastone DJ, Patel BJ, Gerds AT, Mukherjee S, Kalaycio M, Advani A, Przychodzen B, et al. Incorporation of molecular data into the revised international prognostic scoring system in treated patients with myelodysplastic syndromes. Leukemia. 2016;30(11): 2214–20. 20. Hou H-A, Tsai C-H, Lin C-C, Chou W-C, Kuo Y-Y, Liu C-Y, Tseng M-H, Peng Y- L, Liu M-C, Liu C-W, et al. Authors’ contributions Incorporation of mutations in five genes in the revised international prognostic scoring system can improve risk stratification in the patients with myelodysplastic syndrome. Blood Cancer J. 2018;8(4):39. 21. Walter MJ, Ding L, Shen D. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia. 2011;25:1153–8. 22. Graubert TA, Shen D, Ding L. Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. 2012;44:53–7. 22. Graubert TA, Shen D, Ding L. Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. 2012;44:53–7. 23. Thol F, Kade S, Schlarmann C. Frequency and prognostic impact of mutations in SRSF2, U2AF1, and ZRSR2 in patients with myelodysplastic syndromes. Blood. 2012;119:3578–84. 23. Thol F, Kade S, Schlarmann C. Frequency and prognostic impact of mutations in SRSF2, U2AF1, and ZRSR2 in patients with myelodysplastic syndromes. Blood. 2012;119:3578–84. 24. Patnaik MM, Lasho TL, Hodnefield JM. SF3B1 mutations are prevalent in myelodysplastic syndromes with ring sideroblasts but do not hold independent prognostic value. Blood. 2012;119:569–72. 24. Patnaik MM, Lasho TL, Hodnefield JM. SF3B1 mutations are prevalent in myelodysplastic syndromes with ring sideroblasts but do not hold independent prognostic value. Blood. 2012;119:569–72. 25. Bejar R. Advances in Personalized Therapeutic Approaches in Myelodysplastic Syndromes. J Natl Compr Canc Netw. 2019;17(11.5):1444–7. 25. Bejar R. Advances in Personalized Therapeutic Approaches in Myelodysplastic Syndromes. J Natl Compr Canc Netw. 2019;17(11.5):1444–7. 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.
https://openalex.org/W4295850233	https://www.ijsciences.com/pub/pdf/V5201602908.pdf	English	null	Phenotypic Characterization and Comparative Study on ESBL-producing E. coli of Clinical Origin	Zenodo (CERN European Organization for Nuclear Research)	2,016	cc-by	3,941	Ugbo Emmanuel1, Anyamene Chris2, Orji Jerry1, Eluu Stanley1, Ukpai Grace1, Ogene Lilian1, Okata-Nwali Divinegift3 1Department of Applied Microbiology, Faculty of Science, Ebonyi State University, P.M.B. 053, Abakaliki, Ebonyi State, Nigeria. 2Department of Applied Microbiology and Brewery Science, Faculty of Bioscience, Nnamdi Azikiwe University, Awka, P.M.B. 5025, Anambra State, Nigeria. g 3Department of Biological Sciences, Faculty of Science and Technology, Federal University Ndufu-Alike, Ikwo, Ebonyi State, Nigeria. Abstract: Extended-spectrum β-lactamases (ESBLs) continue to be a major challenge in clinical setups worldwide, conferring resistance to the expanded-spectrum cephalosporins. The present study focused on the prevalence of ESBL-producing E. coli clinical isolates among patients diagnosed of Wound and Urinary Tract Infections attending Federal Teaching Hospital Abakaliki. A total of one hundred and ninety two clinical isolates of E. coli was studied for their susceptibility patterns to cephalosporin antibiotics and detection of ESBL producers was carried out by double disc synergy test (DDST) and Brilliance ESBL Agar. Of the 192 isolates tested for their antibiogram, 19(9.9%), 41(21.4%), 132(68.7%); 48(25.0%) and 144(75.0%) isolates were from wound, high virginal swab, urine, male and female respectively. The isolates showed higher susceptibility to cefepime (a 4th-generation cephalosporin) with percentage susceptibility of 78.9, 85.4 and 73.5 to the isolates from wound, HVS and urine respectively. Higher resistance was recorded among the 3rd-generation cephalosporins which include Cefotaxime (63.4%), Ceftriaxone (57.9%), and Cefpodoxime (73.7%). Among the resistant isolates of E. coli, 20 isolates were phenotypically confirmed ESBL producers by the DDST and Brilliance ESBL Agar methods. Ten (10) ESBL producing E. coli was confirmed using DDST method whereas 15 ESBL producing E. coli was confirmed using the Brilliance ESBL Agar. Brilliance ESBL Agar was found to be better than DDST in the detection of ESBLs. Continuous monitoring of drug resistance and regulating the use of cephalosporin drugs in our hospitals is vital for proper infectious disease management and treatment. Keywords: ESBLs, E. coli, Patients, DDST and Brilliance ESBL Agar. Keywords: ESBLs, E. coli, Patients, DDST and Brilliance ESBL Agar. existence of resistance genes and poor use of infection control measures in our hospitals. Extended spectrum cephalosporins (including the third generation cephalosporins) gained widespread clinical use in the early 1980s and were developed because of the increasing prevalence of ampicillin- hydrolyzing β-lactamases such as TEM-1, TEM-2 and SHV-1 enzymes in Enterobacteriaceae, notably in Escherichia coli (Mark and Paul, 2003). The production of beta-lactamase enzymes by Gram- negative bacteria is one of the most important mechanisms of resistance to beta-lactam antibiotics. Phenotypic Characterization and Comparative Study on ESBL-producing E. coli of Clinical Origin Ugbo Emmanuel1, Anyamene Chris2, Orji Jerry1, Eluu Stanley1, Ukpai Grace1, Ogene Lilian1, Okata-Nwali Divinegift3 Introduction Extended-spectrum β-lactamase (ESBL)–producing Gram-negative bacteria are emerging pathogens. Clinicians, microbiologists, infection control practitioners, and hospital epidemiologists are concerned about ESBL-producing bacteria because of the increasing incidence of such infections, the limitations of effective antimicrobial drug therapy, and adverse patient outcomes (Lautenbach et al., 2001; Paterson, 2006). The rapidity of the development and spread of resistance is a complex process that is influenced by selective pressure, pre- This article is published under the terms of the Creative Commons Attribution License 4.0 Author(s) retain the copyright of this article. Publication rights with Alkhaer Publications. Published at: http://www.ijsciences.com/pub/issue/2016-02/ DOI: 10.18483/ijSci.908; Online ISSN: 2305-3925; Print ISSN: 2410-4477 This article is published under the terms of the Creative Commons Attribution License 4.0 Author(s) retain the copyright of this article. Publication rights with Alkhaer Publications. Published at: http://www.ijsciences.com/pub/issue/2016-02/ DOI: 10.18483/ijSci.908; Online ISSN: 2305-3925; Print ISSN: 2410-4477  Ejikeugwu Chika (Correspondence)  ejikeugwu_chika@yahoo.com  +234(0)8097684562  Ejikeugwu Chika (Correspondence)  ejikeugwu_chika@yahoo.com  +234(0)8097684562  Ejikeugwu Chika (Correspondence)  ejikeugwu_chika@yahoo.com  +234(0)8097684562  Ejikeugwu Chika (Correspondence)  ejikeugwu_chika@yahoo.com  +234(0)8097684562 Phenotypic Characterization and Comparative Study on ESBL-producing E. coli of Clinical Origin Bacteriology: All the clinical samples of wound swabs and urine samples were inoculated on MacConkey agar (MAC), Chocolate agar (CA) and Cystein Lactose Electrolyte Deficient (CLED) medium (Oxoid, UK), and incubated at 37°C for 18- 24 hrs. Suspected bacterial colonies were sub- cultured onto freshly prepared MAC, CLED and CA for the isolation of pure cultures of the bacteria. E. coli isolates were identified based on colony morphology and biochemical reactions using standard microbiological conventional identification techniques (Cheesbrough, 2010; Stephan and William, 2006). Bacteriology: All the clinical samples of wound swabs and urine samples were inoculated on MacConkey agar (MAC), Chocolate agar (CA) and Cystein Lactose Electrolyte Deficient (CLED) medium (Oxoid, UK), and incubated at 37°C for 18- 24 hrs. Suspected bacterial colonies were sub- cultured onto freshly prepared MAC, CLED and CA for the isolation of pure cultures of the bacteria. E. coli isolates were identified based on colony morphology and biochemical reactions using standard microbiological conventional identification techniques (Cheesbrough, 2010; Stephan and William, 2006). ESBLs therefore, are acquired plasmid-mediated beta-lactamases that hydrolysis broader spectrum beta-lactam antibiotics including the oxyimino-3rd- generation cephalosporins and the penicillins (Paterson and Bonomo, 2005). Antimicrobial Screening for ESBL production: Bacterial isolates that showed decreased susceptibility (intermediate by CLSI criteria) to any of the third generation cephalosporins including ceftazidime and cefotaxime were suspected to produce ESBL and further tested for ESBL production phenotypically. According to CLSI guidelines, strains showing zone of inhibition of ≤ 22 mm for ceftazidime, ≤ 27 mm for cefotaxime, and ≤ 25 mm for ceftriaxone were selected for ESBL confirmatory test. Phenotypic determination of ESBL using Double Disc Synergy Test (DDST): The E. coli isolated colonies were inoculated in peptone water at 37°C for 2–6 h. The turbidity was adjusted to 0.5 McFarland standards and lawn culture was made on Mueller- Hinton agar using sterile swab stick. Amoxicillin plus Clavulanic acid disk (20 µg and 10 µg, respectively) was placed in the centre of plate. A disc of cefotaxime (30 μg) and ceftazidime (30 μg), were placed at a distance of 15 mm away from the centrally placed disc (Amoxicillin plus Clavulanic acid disk). The plate was incubated at 37°C overnight. An increase of ≥ 5 mm in the inhibition zone diameter for either of the cephalosporins (ceftazidime and cefotaxime) tested in combination with amoxycillin-clavulanic acid versus its zone when tested alone confirms ESBL production phenotypically (Pituot et al., 2004). Introduction Some data supported that foreign travel as well as household and community exposure may be important risk factors leading to colonization of the human intestinal tract by ESBL-producing E. coli (Laupland et al., 2008; Valverde et al., 2008). Person-to-person transmission and acquisition from a common source, probably related to food and poor hygiene clearly contribute to ESBL dissemination within families or community (Rodriguez-Bano et al., 2008). Similarly, other studies have reported a high prevalence of ESBL- producing bacteria in certain communities. The high prevalence of these bacteria in healthy carriers may be an important source of community-acquired infections, including urinary tract infections (UTIs). However, whether these ESBL-producing bacteria can be causative infectious agents in the carrier has not yet been confirmed (Niki et al., 2011). Clinical strains of Escherichia coli producing ESBLs have high prevalence of ESBL-producing Enterobacteriaceae in hospitals in African countries including Morocco, South Africa and Nigeria (Iabadene et al., 2008; Sekhsokh et al., 2008; Pitout et al., 1998; Aibinu et al., 2003). Arpin et al., (2009) detected a variety of β-lactamases among the isolates of E. coli and K. pneumoniae, namely SHV-, CTX- M-, OXA- and TEM-type enzymes. The CTX-M type was the most common ESBL in our setting. In Nigeria, these enzymes have been reported in Enterobacter species from human patients in Lagos (Aibinu et al., 2003). Chau and Obegbunam (2007) reported the presence of high resistant extended spectrum beta-lactamase producing Escherichia coli in Enugu State. ESBLs have been implicated in a variety of stubborn bacterial-related infections such as bacteraemia, pneumonia, UTIs and wound infections (Iroha et al., 2010; Ejikeugwu et al., 2013). Hence, the present study is designed to evaluate the prevalence of ESBL-producing strains of Escherichia coli in the hospital and compare the efficiency of Brilliance ESBL Agar and Double Disc Synergy Test in the detection of ESBLs. Antimicrobial Susceptibility Testing: Antimicrobial susceptibility testing was performed by the modified Kirby-Bauer disc diffusion method (Bauer et al., 1990). Various antimicrobial discs were used which include antimicrobials for screening of ESBL- producing bacteria. Single antibiotic disks of Ceftazidime (30 µg), Cefotaxime (30 µg), Ceftriaxone (30 µg), Cefpodoxime (10 µg), Aztreonam (30 µg), Cefepime (30 µg) and Amoxicillin plus Clavulanic acid 30) (Oxoid, UK) were used for determining the susceptibility profile of the test bacterium. The results were interpreted as per the Clinical Laboratory Standard Institute (CLSI) recommendations (CLSI, 2006). Materials and Methods This appearance is the characteristic for ESBL-producing bacteria due to the synergistic effect produced between amoxicillin-clavulanic acid (a beta- lactamase inhibitor) and the third generation cephalosporins such as ceftazidime and cefotaxime. %) were from female. Moreover, the site of isolation of the isolates are wound 19 (9.9 %), high virginal swab 41 (21.4 %) and urine 132 (68.7 %). The results of antimicrobial susceptibility test of the E. coli isolates to some commonly used cephalosporin antibiotics are shown in Table 2. The results showed that cefepime are most effective drugs against the E. coli isolates. Higher levels of resistance by the E. coli isolates were recorded for ceftazidime, cefotaxime, ceftriaxone, cefpodoxime and aztreonam. Phenotypic characterization of ESBLs- producing E. coli isolated from patient attending FETHA was shown in Table 3. Among the resistant isolates of E. coli, 15 were ESBL producers detected by DDST and Brilliance ESBL Agar. Ten ESBL producing E. coli was confirmed using DDST whereas 15 ESBL producing E. coli was confirmed using Brilliance ESBL Agar. Figure 1 shows a keyhole effect produced by E. coli isolate expressing ESBL using DDST method. This appearance is the characteristic for ESBL-producing bacteria due to the synergistic effect produced between amoxicillin-clavulanic acid (a beta- lactamase inhibitor) and the third generation cephalosporins such as ceftazidime and cefotaxime. Materials and Methods Study area: This prospective study was conducted in the Department of Applied Microbiology at Ebonyi State University, Abakaliki, Nigeria. A total of 192 isolates were obtained from clinical samples from October 2014 to October 2015. Among 192 isolates, 48 were from male patients and 144 isolates were from female patients diagnosed of wound and UTIs; and who attended the Federal Teaching Hospital Abakaliki, (FETHA) Ebonyi State for medical attention. http://www.ijSciences.com Volume 5 – February 2016 (02) 28 Volume 5 – February 2016 (02) http://www.ijSciences.com 28 Phenotypic Characterization and Comparative Study on ESBL-producing E. coli of Clinical Origin ESBL Testing using Brilliance ESBL Agar. Code: PO5302: Brilliance ESBL Agar (pale, off white, semi-opaque gel medium) is a chromogenic screening agar plate for detection of ESBL- producing organisms. The medium provides presumptive identification of ESBL- Producing E. coli direct from clinical samples or isolates in 24 hours. Brilliance ESBL Agar was inoculated directly with isolated colony of E. coli prepared as a liquid suspension approximately equivalent to 0.5 McFarland turbidity standards. The medium was allowed to warm to room temperature before inoculation. The inoculated plates were incubated for 18-24 hours at 37oC. Negative plates were re- incubated for additional 24 hours. Blue or Pink colonies were recorded as presumptive positive ESBL-producing E. coli (CLSI, 2006; Thermo Scientific Oxoid Microbiology, 2013). %) were from female. Moreover, the site of isolation of the isolates are wound 19 (9.9 %), high virginal swab 41 (21.4 %) and urine 132 (68.7 %). The results of antimicrobial susceptibility test of the E. coli isolates to some commonly used cephalosporin antibiotics are shown in Table 2. The results showed that cefepime are most effective drugs against the E. coli isolates. Higher levels of resistance by the E. coli isolates were recorded for ceftazidime, cefotaxime, ceftriaxone, cefpodoxime and aztreonam. Phenotypic characterization of ESBLs- producing E. coli isolated from patient attending FETHA was shown in Table 3. Among the resistant isolates of E. coli, 15 were ESBL producers detected by DDST and Brilliance ESBL Agar. Ten ESBL producing E. coli was confirmed using DDST whereas 15 ESBL producing E. coli was confirmed using Brilliance ESBL Agar. Figure 1 shows a keyhole effect produced by E. coli isolate expressing ESBL using DDST method. Results The distribution of E. coli isolated from patient attending FETHA are shown in the Table 1. Out of 192 clinical isolates of E. coli used for this study, 48 (25.0 %) were isolated from male, while 144 (75.0 Table 1: Distribution of E. coli isolated from patient attending FETHA Sample site No of Isolates (%) No +ve for Male (%) No +ve for Female (%) Wound Swab 19 (9.9) 7 (36.8) 12(63.2) High Virginal Swab 41(21.4) -(0.0) 41(100) Urine 132(68.7) 41(31.1) 91(68.9) Total 192 (100) 48(25.0) 144(75.0) Table 2: Antibiotic profile of E. coli isolated from patient attending FETHA Wound Swab High Virginal Swab Urine Antibiotics S (%) R (%) S (%) R (%) S (%) R (%) Cefepime 15(78.9) 4 (21.1) 35(85.4) 6 (14.6) 97(73.5) 35(26.5) Ceftazidime 12(63.2) 7 (36.8) 25(61.0) 16(39.0) 70(53.0) 62(47.0) Ceftriaxone 8 (42.1) 11(57.9) 19(46.3) 22(53.7) 92(69.7) 40(30.3) Cefpodoxime 5 (26.3) 14(73.7) 22(53.7) 19(46.3) 69(52.3) 63(47.7) Azetronam 9 (47.4) 10(52.6) 24(58.5) 17(41.5) 80(60.6) 52(39.4) Cefotaxime 7 (36.8) 12(63.2) 15(36.6) 26(63.4) 50(37.9) 82(62.1) Table 3: Phenotypic characterization of ESBLs- producing E. coli isolated from patient attending FETHA Sample site Brilliance ESBL Agar No +ve Male; No +ve Female Double Disk Synergy Test No +ve Male; No +ve Female Wound Swab - - - - High Virginal Swab - 4 - 3 Urine 4 7 2 5 Total 15 10 Table 1: Distribution of E. coli isolated from patient attending FETHA Sample site No of Isolates (%) No +ve for Male (%) No +ve for Female (%) Wound Swab 19 (9.9) 7 (36.8) 12(63.2) High Virginal Swab 41(21.4) -(0.0) 41(100) Urine 132(68.7) 41(31.1) 91(68.9) Total 192 (100) 48(25.0) 144(75.0) Table 2: Antibiotic profile of E. coli isolated from patient attending FETHA 15 10 http://www.ijSciences.com Volume 5 – February 2016 (02) 29 Volume 5 – February 2016 (02) coli among patients diagnosed of wound infections, high vaginal infections and UTIs in Nigeria. Overall, 10 ESBL producing E. coli was confirmed using the DDST method whereas 15 ESBL producing E. coli was confirmed using the Brilliance ESBL Agar. The false negative results in ESBL detection phenotypically observed using DDST is as a result of co-production of other related enzymes such as metallo-beta-lactamases (Walsh et al., 2005), plasmid-mediated and chromosomal inducible AmpC beta-lactamase in the test bacteria other than ESBLs (Jacoby et al., 2005). However, the brilliant result observed using Brilliance ESBL Agar is as a result of formulation with antibiotic and chromogenic mixture which inhibits every other enzymes present and allows the production of ESBL enzyme only to grow and produce colour pigments. In this present study, Brilliance ESBL Agar was found to be better than DDST in the phenotypic detection of ESBLs. Continuous monitoring of drug resistance, proper susceptibility studies and regulating the use of Volume 5 – February 2016 (02) Volume 5 – February 2016 (02) http://www.ijSciences.com 29 Phenotypic Characterization and Comparative Study on ESBL-producing E. coli of Clinical Origin Figure 1. Illustration of ESBL positive plate. Figure 1. Illustration of ESBL positive plate. ESBL-producing organisms (Lye et al., 2008; Walsh et al., 2005 and Paterson, et al., 2005). Higher resistance of the E. coli isolates were also recorded among the 3rd-generation cephalosporins which includes Cefotaxime (63.4%), Ceftriaxone (57.9%), and Cefpodoxime (73.7%). Higher levels of resistance of ESBL-producing E. coli isolates as obtainable in this study have also been previously reported (Bradford, 2001). Among the resistant isolates of E. coli, 15 were ESBL producers detected by DDST and Brilliance ESBL Agar. Our findings agreed with the research work done by Aibinu et al., 2003, Chau and Obegbunam, 2007 and Iroha et al., 2010, where they found ESBL-producing E. coli among patients diagnosed of wound infections, high vaginal infections and UTIs in Nigeria. Overall, 10 ESBL producing E. coli was confirmed using the DDST method whereas 15 ESBL producing E. coli was confirmed using the Brilliance ESBL Agar. The false negative results in ESBL detection phenotypically observed using DDST is as a result of co-production of other related enzymes such as metallo-beta-lactamases (Walsh et al., 2005), plasmid-mediated and chromosomal inducible AmpC beta-lactamase in the test bacteria other than ESBLs (Jacoby et al., 2005). However, the brilliant result observed using Brilliance ESBL Agar is as a result of formulation with antibiotic and chromogenic mixture which inhibits every other enzymes present and allows the production of ESBL enzyme only to grow and produce colour pigments. In this present study, Brilliance ESBL Agar was found to be better than DDST in the phenotypic detection of ESBLs. Continuous monitoring of drug resistance, proper susceptibility studies and regulating the use of ESBL-producing organisms (Lye et al., 2008; Walsh et al., 2005 and Paterson, et al., 2005). Higher resistance of the E. coli isolates were also recorded among the 3rd-generation cephalosporins which includes Cefotaxime (63.4%), Ceftriaxone (57.9%), and Cefpodoxime (73.7%). Higher levels of resistance of ESBL-producing E. coli isolates as obtainable in this study have also been previously reported (Bradford, 2001). Among the resistant isolates of E. coli, 15 were ESBL producers detected by DDST and Brilliance ESBL Agar. Our findings agreed with the research work done by Aibinu et al., 2003, Chau and Obegbunam, 2007 and Iroha et al., 2010, where they found ESBL-producing E. References 15. Mark, E. Rupp and Paul, D. Fey (2003). Extended Spectrum Beta-lactamase (ESBL)- producing Enterobacteriaceae, consideration for diagnosis, prevention and drug treatment. Drug, 63 (4): 353-365. 1. Aibinu, I. E., Ohaegbulam, V. C., Adenipekun, E. A., Ogunsola, F. T., Odugbemi, T. O and Mee, B. J. (2003). Extended-spectrum beta-lactamase enzymes in clinical isolates of Enterobacter species from Lagos, Nigeria. J. Clin. Microbiol., 41: 2197-2206. 16. Niki, M., Hirai, I., Yoshinaga, A., Ulzii-Orshikh, L., Nakata, A., Yamamoto, A., Yamamoto, M and Yamamoto, Y. (2011). Extended-spectrum b-lactamase-producing Escherichia coli strains in the feces of carriers contribute substantially to urinary tract infections in these patients. J. Infection, 39:467– 471. 2. Arpin, C., Quentin, C., Grobost, F., Cambau, E., Robert, J., Dubois, V., Coulange, L., André, C. & on behalf of the Scientific Committee of ONERB (2009). Nationwide survey of extended-spectrum β-lactamase-producing Enterobacteriaceae in the French community setting. J Antimicrob Chemother 63, 1205–1214. 17. Paterson, D. L. (2006). Resistance in gram-negative bacteria: Enterobacteriaceae. Am J Med. 119:20–8. 3. Bauer, A. W., Truck, H. and Sherries, J. C. (1990). “Antibiotic Susceptibility testing by standardized single disc method,” American Journal of Clinical Pathology, 45: 493– 496. 18. Paterson, D. L. and Bonomo, R. A. (2005). Extended Spectrum Beta-lactamases: A clinical update. Clinical Microbiology Review, 18:657-686. 19. Pitout, J. D. D., Thomson, K. S., Hanson, N. D and Ehrhardt, A. F. (2004). Beta-lactamase responsible for resistance to extended-spectrum cephalosporin in Klabsiella pnumoniae, Escherichia coli and Proteus mirabils isolates recovered from South Africa. Antimicrob. Agents Chemother., 42: 1350-1354. 4. Bradford, P. A. (2001). Extended Spectrum Beta-lactamases in the 21st century: Characterization, Epidemiology and Detection of this Important Resistance Threat. Clinical Microbiology Reviews, 14 (4):933-951. 5. Chau, K. F and Oboegbunam, S. I. (2007). Extended – spectrum beta lactamase production among ampicillin resistant Escherichia coli strains from chicken in Enugu State, Nigeria. Brazilian J. Microbiol., 38:1-4 20. Rodriguez-Bano, J., Lopez-Cerero, L. and Navarro, M. D. (2008). Faecal carriage of extended-spectrum b- lactamaseproducing Escherichia coli: prevalence, risk factors and molecular epidemiology. J. Antimicrob. Chemother. 62:1142-1149. g 6. Cheesbrough, M. (2010).District Laboratory Practice in Tropical Countries, Part two, 2nd edn. Cambridge University Press, UK. Pp 143-180. 21. Stephan, J. A and William, J. (2006). Eds., Koneman’s Color Atlas and Text book of Diagnostic Microbiology, Lipponcott Williams & Wilkins, 6th edition. 7. Clinical Laboratory Standards Institute (CLSI) (2006). Performance standards for antimicrobial susceptibility testing.Proceedings of the 16th International Supplement (M100-S16). Acknowledgment Authors are grateful to the staff and management of the medical microbiology laboratory department of Federal Teaching Hospital, Abakaliki (FETHA), Ebonyi State and Applied Microbiology Laboratory Unit, Ebonyi State University, Abakaliki, Nigeria for their technical support during the collection and analysis of the clinical samples used for this study. 13. Laupland, K.B., Church, D.L and Vidakovich, J. (2008). Community-onset extendedspectrum b-lactamase (ESBL) producing Escherichia coli: importance of international travel. J. Infect. 57:441-448. 14. Lye, D. C., Wijaya, L., Chan, J., Teng, C. P. and Leo, Y. S. (2008). Ertapenem for treatment of Extended Spectrum beta- lactamase producing and Multidrug resistant Gram negative Bacteriaemia. Ann. Acad. Med. Singapore, 37 (10): 831-4. Discussions Extended-spectrum β-lactamases (ESBLs) are a rapidly evolving group of β-lactamases which share the ability to hydrolyze third-generation cephalosporins and aztreonam yet are inhibited by clavulanic acid. Typically, they derive from genes for TEM and SHV-type beta-lactamases by mutations that alter the amino acid configuration around the active site of these earlier β-lactamases. This extends the spectrum of β-lactam antibiotics susceptible to hydrolysis by these enzymes. ESBLs are often located on plasmids that are transferable from strain to strain and between bacterial species. However, it can still be chromosomal mediated. There is no doubt that ESBL-producing organisms are of enormous clinical and microbiological significance. Such bacteria are associated with severe infections such as bacteraemia, intra-abdominal infection, wound infection, urinary tract infections (particularly in the community setting), and respiratory tract infections (Pituot et al., 2004). A total of one hundred and ninety two (192) isolates of E. coli was studied for their susceptibility patterns to cephalosporin antibiotics and detection of ESBL producers by double disc synergy test (DDST) and Brilliance ESBL Agar. Of the 192 isolates tested for their antibiogram, 19 (9.9%), 41 (21.4%), and 132 (68.7%) isolates were from wound, high vaginal swab, and urine samples respectively while 48 (25.0%) and 144 (75.0%) isolates were from male and female patients respectively. The E. coli isolates showed higher susceptibility to 4th-generation cephalosporins (cefepime). This is akin with previous works in which cefepime was reported as the drug of choice for the treatment of bacterial infections caused by Volume 5 – February 2016 (02) http://www.ijSciences.com 30 Phenotypic Characterization and Comparative Study on ESBL-producing E. coli of Clinical Origin Gram – negative bacteria from a university teaching hospital in Nigeria. Current Research in Bacteriology, 3(2):77-83. Gram – negative bacteria from a university teaching hospital in Nigeria. Current Research in Bacteriology, 3(2):77-83. cephalosporin drugs in our hospitals is important in the containment of the emergence and spread of ESBL-producing bacteria in the hospital environment. 11. Jacoby, G. A. and Munoz-Price, L. S. (2005). Mechanisms of Disease: The New Beta-lactamases. N Engl J Med, 352: 380- 91. 12. Lautenbach, E., Patel, J. B., Bilker, W. B., Edelstein, P. H. and Fishman, N. O. (2001). Extended-spectrum beta- lactamase-producing Escherichia coli and Klebsiella pneumoniae: risk factors for infection and impact of resistance on outcomes. Clin Infect Dis. 32:1162–71. http://www.ijSciences.com References Wayne, Pa, USA. 22. Thermo Scientific Oxoid Microbiology Products (2013). Prepared Media-Ready to-use prepared plates; Brilliance ESBL Agar, Code: PO5302. www.thermoscientific.com.oxoid. 8. Ejikeugwu C, Iroha I, Adikwu M and Esimone C (2013). Susceptibility and Detection of Extended Spectrum β- Lactamase Enzymes from Otitis Media Pathogens. American Journal of Infectious Diseases. 9(1):24-29. f 23. Valverde, A., Grill, F. and Coque, T. M. (2008). High rate of intestinal colonization with extended-spectrum-b- lactamaseproducing organisms in household contacts of infected community patients. J. Clin. Microbiol. 46:2796- 2799. 9. Health Protection Agency, HPA (2008). Laboratory detection and reporting of bacteria with extended spectrum Beta- lactamases. National Standard Method QSOP 51 Issue 2.2. 10. Iroha I.R., Amadi E.S., Oji A.E., Nwuzo A.C and Ejikeugwu P.C (2010). Detection of plasmid borne extended – spectrum beta – lactamase enzymes from blood and urine isolates of 24. Walsh, T. R., Toleman, M. A., Poirel, L and Nordmann, P. (2005). Metallo Beta-lactamases: the Quiet before the storm? Clinical Microbiology Review, 18(2): 306-325. http://www.ijSciences.com Volume 5 – February 2016 (02) 31 Volume 5 – February 2016 (02) http://www.ijSciences.com http://www.ijSciences.com 31
https://openalex.org/W4391168008	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_11_from_Treatment_Response_Tumor_Infiltrating_Lymphocytes_and_Clinical_Outcomes_in_Inflammatory_Breast_Cancer_Treated_with_Neoadjuvant_Systemic_Therapy/25054801/1/files/44212630.pdf	English	null	Supplementary Figure 11 from Treatment Response, Tumor Infiltrating Lymphocytes and Clinical Outcomes in Inflammatory Breast Cancer–Treated with Neoadjuvant Systemic Therapy	null	2,024	cc-by	246	Supplementary Figure 11. Association of RCB with DFS and OS. (A) Kaplan-Meier curves of DFS according to RCB class; (B-C) Forest plots showing the association of RCB class (B), or non-zero RCB score (C), and standard clinicopathological and treatment variables with DFS quantified by Cox regression. (D) Kaplan-Meier curves of OS according to RCB class; (E-F) Forest plots showing the association of RCB class (E), or non-zero RCB score (F), and standard clinicopathological and treatment variables with OS quantified by Cox regression. Supplementary Figure 11. Association of RCB with DFS and OS. (A) Kaplan-Meier curves of DFS according to RCB class; (B-C) Forest plots showing the association of RCB class (B), or non-zero RCB score (C), and standard clinicopathological and treatment variables with DFS quantified by Cox regression. (D) Kaplan-Meier curves of OS according to RCB class; (E-F) Forest plots showing the association of RCB class (E), or non-zero RCB score (F), and standard clinicopathological and treatment variables with OS quantified by Cox regression. Supplementary Figure 11. Association of RCB with DFS and OS. (A) Kaplan-Meier curves of DFS according to RCB class; (B-C) Forest plots showing the association of RCB class (B), or non-zero RCB score (C), and standard clinicopathological and treatment variables with DFS quantified by Cox regression. (D) Kaplan-Meier curves of OS according to RCB class; (E-F) Forest plots showing the association of RCB class (E), or non-zero RCB score (F), and standard clinicopathological and treatment variables with OS quantified by Cox regression.
https://openalex.org/W2807716729	https://europepmc.org/articles/pmc6155002?pdf=render	English	null	Identification of active and taxonomically diverse 1,4-dioxane degraders in a full-scale activated sludge system by high-sensitivity stable isotope probing	The ISME journal	2,018	cc-by	9,904	Abstract 1,4-Dioxane is one of the most common and persistent artiﬁcial pollutants in petrochemical industrial wastewaters and chlorinated solvent groundwater plumes. Despite its possible biological treatment in natural environments, the identity and dynamics of the microorganisms involved are largely unknown. Here, we identiﬁed active and diverse 1,4-dioxane- degrading microorganisms from activated sludge by high-sensitivity stable isotope probing of rRNA. By rigorously analyzing 16S rRNA molecules in RNA density fractions of 13C-labeled and unlabeled 1,4-dioxane treatments, we discovered 10 signiﬁcantly 13C-incorporating microbial species from the complex microbial community. 16S rRNA expression assays revealed that 9 of the 10 species, including the well-known degrader Pseudonocardia dioxanivorans, an ammonia-oxidizing bacterium and phylogenetically novel bacteria, increased their metabolic activities shortly after exposure to 1,4-dioxane. Moreover, high-resolution monitoring showed that, during a single year of operation of the full-scale activated sludge system, the nine identiﬁed species exhibited yearly averaged relative abundances of 0.001–1.523%, and yet showed different responses to changes in the 1,4-dioxane removal efﬁciency. Hence, the co-existence and individually distinct dynamics of various 1,4-dioxane-degrading microorganisms, including hitherto unidentiﬁed species, played pivotal roles in the maintenance of the biological system removing the recalcitrant pollutant. The ISME Journal (2018) 12:2376–2388 https://doi.org/10.1038/s41396-018-0201-2 ARTICLE ARTICLE Identiﬁcation of active and taxonomically diverse 1,4-dioxane degraders in a full-scale activated sludge system by high-sensitivity stable isotope probing Tomo Aoyagi1 ●Fumiaki Morishita2 ●Yutaka Sugiyama2 ●Daisuke Ichikawa2 ●Daisuke Mayumi3 ● Yoshitomo Kikuchi4 ●Atsushi Ogata1 ●Kenji Muraoka2 ●Hiroshi Habe1 ●Tomoyuki Hori1 R i d 26 O b 2017 / R i d 6 M 2018 / A d 9 M 2018 / P bli h d li 13 J 2018 Tomo Aoyagi1 ●Fumiaki Morishita2 ●Yutaka Sugiyama2 ●Daisuke Ichikawa2 ●Daisuke Mayumi3 ● Yoshitomo Kikuchi4 ●Atsushi Ogata1 ●Kenji Muraoka2 ●Hiroshi Habe1 ●Tomoyuki Hori1 Received: 26 October 2017 / Revised: 6 May 2018 / Accepted: 9 May 2018 / Published online: 13 June 2018 © The Author(s) 2018. This article is published with open access 4 Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu- Higashi, Sapporo 062-8517, Japan Introduction Recently, compared with that of SIP with terminal restriction fragment length polymorphism [35, 36], the detection sensitivity has been 500-fold enhanced by high-throughput Illumina sequencing of isopycnic cen- trifugation gradients, referred to as high-sensitivity rRNA- SIP [37]. Although further rise in the sensitivity may be possible with advent of new technology in future, this sensitive approach at present would unveil the ecophysiol- ogy of low-abundance microorganisms that can actively dissimilate but only marginally assimilate the 13C-labeled persistent pollutant. processes, can effectively decompose 1,4-dioxane within only a few hours [10, 11]. Biological treatments have attracted increasing attention as naturally occurring pro- cesses with low cost and low energy [12], and because they require none of the external special equipment that the other abiotic treatments require. 1,4-Dioxane was initially recognized as a non- biodegradable compound, due to its long persistence in wastewater treatment plants [13]. In fact, this compound has been shown to exhibit a high degree of resistance to microbial degradation due to its C–O–C ether linkage, which features a ring structure and high-energy C–O bonds (360 kJ mol–1) [14]. However, recent studies have shown that intrinsic microorganisms capable of degrading 1,4- dioxane are more widely distributed in natural environments than previously expected [15–17]. Moreover, a number of 1,4-dioxane-degrading microorganisms have been isolated as a result of intensive efforts made in the last two decades [12]. It has been reported that 10 isolates grow on 1,4- dioxane as a sole carbon and energy source (1,4-dioxane metabolism) [18], whereas 12 isolates utilize 1,4-dioxane only when an additional growth-supporting carbon source is introduced (1,4-dioxane co-metabolism) [19]. In the case of co-metabolism, however, the microorganisms can grow with the primary substrate but not with the compound co- metabolized [20]. In this study, high-sensitivity rRNA-SIP was ﬁrst used to identify 1,4-dioxane degraders in a full-scale activated sludge system that is used to effectively treat high con- centrations of 1,4-dioxane in petrochemical industrial was- tewaters. In addition, both the identiﬁed 1,4-dioxane degraders and the whole microbial communities were monitored at high resolution using an Illumina MiSeq platform [38, 39] over 1-year of operation of a full-scale treatment system in order to better understand their popu- lation dynamics under changing environmental conditions. It has been shown that the isolated 1,4-dioxane degraders exhibit high phylogenetic diversity. A few strains in the well-known degrader genera Pseudonocardia and Rhodo- coccus have 1,4-dioxane degradation abilities [21]. Introduction Tracing 16S ribosomal RNA (rRNA) genes has not been possible, because 1,4-dioxane degradation is a widespread trait in the microbial realm, having been detected in many bacterial, as well as fungal lineages [12, 22, 23]. Instead of a universal bio-signature, functional genes encoding monooxygenase enzymes have been proposed as biomarkers for 1,4-dioxane degradation [24], and subsequent studies attempted to improve the accuracy of these biomarkers [25, 26]. How- ever, although this biomarker approach is effective for monitoring the known microorganisms participating in monooxygenase-driven 1,4-dioxane degradation [27–29], it cannot discover hitherto unknown 1,4-dioxane degraders whose genomes lack the monooxygenase-encoding genes or include unusual types of the genes. In fact, the 1,4-dioxane- degrading Xanthobacter ﬂavus strain DT8 has been reported to have a monooxygenase-independent degradation path- way [30]. Due to these limitations, there is still only limited information about the phylogenic identity and diversity of the microorganisms involved in the degradation of 1,4- dioxane in natural and contaminated environments. Introduction The cyclic ether 1,4-dioxane is generated as a byproduct in petrochemical industrial processes and has been widely utilized as a stabilizer for chlorinated solvents [1–3]. The International Agency for Research on Cancer (IARC) and United States Environmental Protection Agency (US EPA) have indicated its possible carcinogenic effects to humans [4, 5]. Due to its toxicity and high mobility, groundwater contamination by 1,4-dioxane is a serious and signiﬁcant concern over a large region [6, 7]. For this reason, 1,4- dioxane concentrations in public and ground waters are strictly regulated in Japan at below 0.05 mg l−1 and those in the treated wastewater at below 0.5 mg l−1. These authors contributed equally: Tomo Aoyagi, Fumiaki Morishita, Tomoyuki Hori. These authors contributed equally: Tomo Aoyagi, Fumiaki Morishita, Tomoyuki Hori. Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41396-018-0201-2) contains supplementary material, which is available to authorized users. * Tomoyuki Hori hori-tomo@aist.go.jp * Tomoyuki Hori hori-tomo@aist.go.jp 1 Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan 1 Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan 2 NIPPON SHOKUBAI Co., Ltd., 14-1 Chidorichou, Kawasaki 210-0865, Japan 2 NIPPON SHOKUBAI Co., Ltd., 14-1 Chidorichou, Kawasaki 210-0865, Japan Physical, chemical and biological treatment technologies have been developed to remove 1,4-dioxane from the con- taminated environments [8]. The physical treatments include thermal destruction, air stripping, distillation and carbon adsorption, but their efﬁcacy is limited by the solubility, boiling point and vapor pressure of 1,4-dioxane [9]. Chemical treatments, that is, advanced oxidation 3 Institute for Geo-Resources and Environment, Geological Survey of Japan, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8567, Japan 4 Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu- Higashi, Sapporo 062-8517, Japan Identiﬁcation of 1,4-dioxane degraders by SIP 2377 Dioxane degraders, that is, the main target of this study, exhibit very low degradation rates and growth rates [3, 33, 34], and therefore would be expected to have low abun- dance in natural environments. Thus, a substantial improvement in the sensitivity of SIP is needed in order to unambiguously detect small amounts of heavily 13C- enriched nucleic acids stemming from 13C-labeled 1,4- dioxane degraders. RNA extraction and density gradient centrifugation RNA was extracted from 2 ml of each sludge sample from the 13C and unlabeled treatments done in triplicate after an 8-h incubation. Total nucleic acids extraction was per- formed using a direct lysis protocol involving bead beating [44]. Then, RNA was puriﬁed by DNA digestion with a DNase (RQ1; Promega, Japan). Total RNA was quantiﬁed using a RiboGreen RNA quantiﬁcation kit (Invitrogen, CA, USA) and a microplate reader (SH-900Lab; Corona, Japan). Five-hundred nanograms of RNA mixed with cesium tri- ﬂuoroacetate (CsTFA) solution (Wako) was subjected in triplicate to ultra-centrifugation with 128,000 g for >60 h at 20 °C [45]. Gradients of density-separated RNAs were fractionated, and the CsTFA buoyant density (BD) of each fraction was determined with a refractometer (AR200; Reichert, NY, USA) [45]. Reverse transcription (RT)-PCR, Illumina sequencing The heaviest (1H), second-heaviest (2H), third-heaviest (3H) and light (L) fractions of RNA with BDs of 1.803–1.808, 1.796–1.800, 1.788–1.793 and 1.769–1.771 g ml–1, respectively, were subjected to RT-PCR with a one- step ampliﬁcation system (Access Quick; Promega). The universal primer set 515f/806r targeting the V4 region of 16S rRNA genes was used. Both primers were modiﬁed to contain an Illumina adapter region and the reverse primer was encoded with 12-bp barcodes according to a previous report [46]. Thermal conditions of RT-PCR were the same as described previously [37], except that a total of 28 cycles were employed during PCR. The absence of DNA con- tamination was conﬁrmed by the absence of ampliﬁcation in the absence of reverse transcriptase. Illumina sequencing was conducted as shown in Supplementary Information (SI). Aerobic incubation of activated sludge microorganisms with 13C-labeled 1,4-dioxane An activated sludge was collected on December 2015 from an aeration tank of a full-scale treatment system of petro- chemical industrial wastewaters at NIPPON SHOKUBAI Co., Ltd. (Kawasaki, Japan). Twenty milliliters of the sludge were transferred into a 123-ml glass serum vial, which was then sealed with a butyl rubber septum. The gaseous phase was air and the headspace O2 concentrations at hour 0 were 0.191−0.210 atm. Three treatments were carried out as follows: (i) 13C treatment: non-autoclaved sludge microorganisms were supplemented with 13C- labeled 1,4-dioxane (1,4-dioxane-13C4, 99 atom % 13C; Sigma-Aldrich, Japan). (ii) Unlabeled treatment: non- autoclaved sludge microorganisms with unlabeled 1,4- dioxane (Wako, Japan). (iii) Heat-killed treatment: auto- claved sludge microorganisms with unlabeled 1,4-dioxane. Autoclave sterilization of the sludge microorganisms was A direct method to link the identity and function of hitherto unknown microorganisms is stable isotope probing (SIP) of rDNA [31], and particularly of rRNA [32]. 1,4- 2378 T. Aoyagi et al. performed three times at 121 °C for 1 h to kill the sludge microorganisms completely [40]. Before starting the incu- bation, the chemical oxygen demand (COD) was deter- mined to be 27 mg l−1 and 1,4-dioxane was not detectable because it was almost completely absent in the original sludge sample. 13C-labeled and unlabeled 1,4-dioxane were added at ﬁnal concentrations of 200 mg l−1 at the start of the incubations. In all, 200 mg l−1 of 1,4-dioxane corresponded to 424 mg l−1 of COD. The activated sludge microorgan- isms were aerobically incubated at 25 °C with agitation at 200 rpm for 8 h in the dark. The pH value was nearly constant at 8.0−8.3 throughout the incubation (data not shown). All treatments were run in triplicate. The sludge samples were stored at –80 °C for subsequent chemical and molecular analyses. was determined with a TOC analyzer (TOC-L; Shimadzu). COD of the sludge water was measured with a COD kit (TNT821; Hach, CO, USA) and a COD analyzer (DRB200; Hach). pH was measured by a compact pH meter (Laquatwin; Horiba, Japan). was determined with a TOC analyzer (TOC-L; Shimadzu). COD of the sludge water was measured with a COD kit (TNT821; Hach, CO, USA) and a COD analyzer (DRB200; Hach). pH was measured by a compact pH meter (Laquatwin; Horiba, Japan). Chemical analyses during the incubation of activated sludge microorganisms The headspace gas and sludge water were collected at hours 0, 2, 4, 6 and 8 from each vial of each set of the 13C and unlabeled treatments performed in triplicate. Mixed liquor suspended solid (MLSS), that is, total biomass, of the sludge was 10,100 mg l–1 at hour 0. Total CO2 and O2 in the headspace gas were analyzed by a gas chromatograph (GC- 14B; Shimadzu, Japan) equipped with a thermal con- ductivity detector and a packed column (ShinCarbon ST; Shinwa, Japan) [41]. The carbon isotopic composition of gaseous CO2 was measured with a gas chromatograph combustion isotope ratio mass spectrometer (GC-C-IRMS) consisting of a Trace GC Ultra, a GC IsoLink, a ConFlo IV and a DELTA V Plus IRMS system (Thermo Fisher Sci- entiﬁc, MA, USA) [42]. The isotopic data were obtained as δ values, that is, per mil derivation of the 13C/12C ratio relative to the Vienna Pee Belemnite (VPDB) standard, in the IRMS analysis and converted to atom % by calculating (1 + δ/1000) × 1.124. The isotopic composition of dis- solved bicarbonate was calculated as δHCO3[−] = (δCO2 − εHCO3[−])/(1 + εHCO3[−]/1000), where εHCO3(−) represents the constants of carbon isotopic fractionation between gaseous CO2 and dissolved bicarbonate (i.e., −7.93‰ at 25 °C) [43]. The concentration of 1,4-dioxane was determined with direct injection of the sludge water by a gas chromatograph (GC-2101plus; Shimadzu) equipped with a ﬂame ionization detector and a dimethylsiloxane column DB-1 (Agilent Technologies, Japan). The column temperature was held at 100 °C for 15 min, then increased to 290 °C (40 °C per min) and held at 290 °C for 20 min. Standard solutions of 1,4- dioxane were prepared at concentrations of 0, 5.8, 29.2 and 92.1 mg l−1. The detection limit was determined to be 0.17 mg l−1. For the heat-killed treatment, the 1,4-dioxane con- centration was determined after an 8-h incubation. The inorganic carbon (IC) concentration from the sludge water Sequence data processing The PhiX, low-quality (Q < 30) and chimeric sequences were removed and the paired-end sequences were assem- bled as described previously [37, 47]. The sequences in each library were characterized phylogenetically using the QIIME software package version 1.7.0 [48]. An operational taxonomic unit (OTU) was deﬁned using a cut-off of 97% sequence identity. Relative abundances in the OTUs were determined in both the 13C and the unlabeled treatments, 2379 Identiﬁcation of 1,4-dioxane degraders by SIP were measured by a TOC analyzer (TOC-4110; Shi- madzu). The 1,4-dioxane concentration and COD of the activated sludge water were determined as described above. Long-term monitoring was performed for approximately 1 year, from May 2015 to April 2016, during which the system operation was stopped from 20th August to 23rd September for periodic maintenance. The activated sludge samples were taken from the aeration tank and return line, and stored at −20 °C for the microbial community analysis. and the statistical signiﬁcance of their difference was cal- culated using the Student’s t-test. The OTUs that were more abundant in the heavy fractions of the 13C treatments than in the unlabeled treatment were phylogenetically identiﬁed with the BLAST program in the NCBI nucleotide sequence database (http://www.ncbi.nlm.nih.gov). 16S rRNA expression assays of 13C-incorporating OTUs throughout SIP incubation Total RNAs extracted at hours 0, 2, 4, 6 and 8 of the 13C treatment were used as templates for RT-qPCR and RT- PCR followed by Illumina sequencing. As for RT-qPCR, the total 16S rRNA copy numbers were determined using the universal primer set 515f/806r [46] with a GoTaq one- step RT-qPCR kit (Promega). Thermal conditions of RT- qPCR were the same as described previously [37]. Fluor- escence was detected at the end of each extension step. In order to obtain a standard curve for quantiﬁcation, 16S rRNA genes from Escherichia coli were ampliﬁed by PCR with the primer set B27f/B907r [49] and a serial dilution series (104 to 108 copies) of the plasmid-inserted PCR amplicon was prepared [50]. The procedures for RT-PCR, Illumina sequencing and data processing were as described above. The 16S rRNA molecules from the 13C- incorporating OTUs were estimated by the total bacterial 16S rRNA copy numbers (copies ml−1) and relative abun- dances (%) of the OTUs that were obtained by RT-qPCR and Illumina sequencing, respectively. Monitoring of the identiﬁed 1,4-dioxane degraders in a full-scale treatment system All the sequences of 16S rRNA molecules and genes obtained from high-sensitivity SIP and Illumina sequencing of microbial communities in the full-scale treatment system have been deposited at the DNA Data Bank of Japan (DDBJ) under the accession numbers DRA005557 (206 libraries) and DRA005982 (15 libraries), and 16S rRNA genes of the 10 13C-incorporating OTUs were deposited under accession numbers LC312382 to LC312391. Fine-scale phylogenetic analyses of microbial communities in the full-scale system According to the procedure mentioned above, total nucleic acids were extracted from 41 sludge samples in the aeration tank and 41 sludge samples in the return line. RNA was digested using an RNase (Type II-A; Sigma-Aldrich). The puriﬁed DNA was used as a template for PCR ampliﬁcation with a Q5 High-Fidelity DNA polymerase (New England Biolabs, Japan). The primer set 515f/806r for Illumina sequencing was utilized, as mentioned. Thermal conditions of PCR were the same as described previously [41, 51], except that a total of 28−35 cycles were employed for ampliﬁcation. Illumina sequencing was conducted as shown in SI. The sequence data processing was performed as described above, except for the alpha- and beta-diversity analysis whose procedure is described in SI. Operation and physicochemical analyses of the full-scale system The full-scale activated sludge system (Fig. S1) at NIP- PON SHOKUBAI Co., Ltd. (Kawasaki, Japan) consisted mainly of a 450 m3 aeration tank and treated nearly 450 m3 day−1 of petrochemical industrial wastewaters con- taining high concentrations of 1,4-dioxane, mono-ethylene glycol and alkanes (e.g., undecane, dodecane, tridecane and tetradecane). The concentrations of 1,4-dioxane were 5.0−69.1 mg l−1, corresponding to 0.87–12.12% of the inﬂuent total organic carbon (TOC). The average hydraulic retention time was 1 day and the solids retention time for this system was 28 days. Dissolved oxygen (DO) in the aeration tank was monitored with a DO meter (DO5509; FUSO, Japan). Temperature and pH were determined with a thermometer (Yokogawa, Japan) and a pH meter (pH200S; Yokogawa), respectively. MLSS was deter- mined with a MLSS meter (SS-5Z; Kasahara Chemical, Japan). TOC concentrations in the inﬂuent and efﬂuent Aerobic degradation of 1,4-dioxane by activated sludge The 3H fraction (BD: 1.790 g ml−1) of the 13C treatment showed 8.0-, 5.4-, 4.4-, 3.0- and 3.0-fold higher abundances of the OTUs 8385, 8474, 2230, 100 and 8532 (i.e., 0.081, 0.645, 0.093, 0.134 and 0.061%) than those of the unlabeled treatment (all signiﬁcant at P < 0.04, n = 3; Fig. 2d). Among them, the OTUs 2230, 5104 and 8474 were identiﬁed more than once to be signiﬁcantly enriched in 13C by the independent assays of the 1H, 2H and 3H fractions, afﬁrming the validity of our criteria for the high-sensitivity SIP. Fig. 1 Changes in chemical parameters during aerobic incubation of activated sludge microorganisms with 13C-labeled and unlabeled 1,4- dioxane. Concentration of 1,4-dioxane a, 13C atom percentage of CO2 (b: open symbols) and calculated concentration of 13CO2 (b: closed symbols). The colors red, blue and gray indicate the 13C, unlabeled and heat-killed treatments, respectively. The error bars indicate the stan- dard deviations of three replications treatments gradually increased after hour 2 to the con- centrations of 0.028−0.031 atm at hour 8 (Fig. S2C). The fate of 13C-labeled 1,4-dioxane was traced by monitoring the 13C atom percentage of the gaseous product CO2 over time (Fig. 1b). The 13C atom percentage of CO2 increased drastically after hour 2, reaching a maximum value of 8.6% at hour 8 in the 13C treatment, whereas it remained constant at low levels (i.e., <1.1%) in the unlabeled treatment. The calculated 13CO2 exhibited a rapid increase after hour 2 in the 13C treatment (Fig. 1b). IC concentrations in the 13C treatment were almost constant (i.e., 136.0−142.4 mg l−1) (Fig. S2B). Concentrations of the headspace O2 ranged from 0.186 to 0.211 atm during the incubations (Fig. S2D), suggesting that a sufﬁcient amount of O2 was provided for microbial degradation of 1,4-dioxane. These results indi- cated that the added 13C-labeled 1,4-dioxane was aero- bically degraded to 13CO2 by the metabolic activities of the sludge microorganisms. The 10 13C-incorporating OTUs were phylogenetically diverse (Fig. 2b–d), or widely distributed in the phyloge- netic trees (Fig. S3). In the case of the Actinobacteria spe- cies, the OTUs 2197 and 13856 were phylogenetically identical and similar, respectively, to the well-known 1,4- dioxane degrader P. dioxanivorans (accession no. NR074465; 100 and 96.9% sequence similarities). The OTU 2197 and P. dioxanivorans were located separately in the phylogenetic tree, likely due to the different lengths of the 16S rRNA sequences analyzed (Fig. S3A). Aerobic degradation of 1,4-dioxane by activated sludge In the 13C and unlabeled treatments, concentrations of 1,4- dioxane in the liquid phases decreased by 49−51 mg l−1 during the incubations (Fig. 1a), and these decreases were accompanied with decreases of 77−101 mg l−1 in COD (Fig. S2A). No signiﬁcant decrease in the 1,4-dioxane concentration was observed in the heat-killed treatment. Total CO2 in gaseous phases of the 13C and unlabeled 2380 T. Aoyagi et al. Fig. 1 Changes in chemical parameters during aerobic incubation of activated sludge microorganisms with 13C-labeled and unlabeled 1,4- dioxane. Concentration of 1,4-dioxane a, 13C atom percentage of CO2 (b: open symbols) and calculated concentration of 13CO2 (b: closed symbols). The colors red, blue and gray indicate the 13C, unlabeled and heat-killed treatments, respectively. The error bars indicate the stan- dard deviations of three replications unlabeled RNAs during ultra-centrifugation. The total number of 16S rRNA molecules obtained from 21 density fractions was around 1.34 million, corresponding to an average of 63,999 sequences per library (Table S1). A large quantity of Illumina sequence data is essential for the unambiguous detection of a small amount of 13C-labeled RNAs [37]. Phylum and class-level phylogenetic analysis indicated that the relative abundances of the phylum Acti- nobacteria of the heavy fractions were 2.7−3.3 times higher than those of the light fraction in the 13C treatment, but no other obvious changes accompanied the increase in BDs in either treatment (Fig. 2a). Nevertheless, our high-resolution OTU-level survey indicated that 10 microbial OTUs exhibited signiﬁcantly higher relative abundances in the 13C treatment than in the unlabeled treatment (Fig. 2b–d). Speciﬁcally, in the 1H fraction (BD: 1.805 g ml−1) of the 13C treatment, the relative abundances of the OTUs 2197, 12266, 5104, 8474 and 6825 were 20.038, 0.747, 0.660, 0.566 and 0.080%, which were 3.7-, 2.5-, 11.0-, 4.9- and 8.7-fold higher than those in the 2H fraction of the unla- beled treatment (all signiﬁcant at P < 0.03, n = 3; Fig. 2b). The OTUs 5104, 2230, 13856 and 8474 made up 0.782, 0.116, 0.025 and 0.834% of the total population in the 2H fraction (BD: 1.796 g ml−1) of the 13C treatment, and these percentages were 13.0-, 10.7-, 8.5- and 7.3-fold higher than those of the unlabeled treatment (all signiﬁcant at P < 0.03, n = 3; Fig. 2c). Aerobic degradation of 1,4-dioxane by activated sludge This situa- tion was also found for the phylogenic positions of the OTUs 6825 and 100 (Fig. S3A, S3C). The OTU 6825 was identical to Micrococcus luteus (LN998081; 100%). The OTU 2230 had a very low sequence similarity (88.6%) to the cultured relative Acidithrix ferrooxidans (KC208497) and fell into a speciﬁc clade with uncultured bacteria (e.g., DH092107_RO_07E [KC358648] and SSIM-F1v Phylogenetic identiﬁcation of the sludge microorganisms incorporating 1,4-dioxane-13C We phylogenetically characterized the 1H, 2H, 3H and L fractions of RNA from the 13C treatment by high- throughput Illumina sequencing. Only the 2H, 3H and L fractions were subjected to Illumina sequencing in the unlabeled treatment, because the 1H fraction could not be ampliﬁed by RT-PCR, due to the low concentrations of the template RNA, indicating that there was little migration of Identiﬁcation of 1,4-dioxane degraders by SIP 2381 Fig. 2 Identiﬁcation of the 13C- incorporating microorganisms during the incubation with 13C- labeled 1,4-dioxane. a The phylum- and class-level distribution of density-resolved RNAs (n = 3). Phylogenetic groups are indicated by colors and their taxonomies are shown at the right side of the graph. The examined RNA density gradients are indicated as the 1H (heaviest), 2H (second-heaviest), 3H (third-heaviest) and L (light) fractions. The CsTFA BDs of the RNA density fractions and the total sequence numbers in the Illumina sequence libraries are summarized in Table S1. N. A. indicates no ampliﬁcation product from RT-PCR. b–d The signiﬁcantly 13C-incorporating OTUs. The OTUs that exhibited signiﬁcantly high relative abundances in the 1H (b), 2H (c) and 3H (d) fractions of the 13C treatment compared with those in the corresponding fractions of the unlabeled treatment (n = 3, p < 0.05) are shown. The closest relatives of the OTUs are indicated. The colors red and blue indicate the 13C and unlabeled treatments, respectively. The error bars indicate the standard deviations of three replications was quite distant from all cultured relatives in the phylo- genetic tree (Fig. S3E). [FJ946539]) (Fig. S3B). Concerning the betaproteobacterial species, the OTU 12266 was the most related to Nitroso- monas ureae (AF272414; 97.6%) and was afﬁliated with the Nitrosomonas clade (Fig. S3D). The OTUs 5104 and 8474 were related to some extent to Methyloversatilis uni- versalis (KC577607; 94.9%) and Chitinimonas koreensis (AB682437; 94.1%), and together formed a distinctive cluster together with uncultured bacteria (e.g., Anxy6 [HQ343211] and 5–25 [JQ923516]). The alphaproteo- bacterial OTU 8385 was related to Blastochloris viridis (NR117911; 95.7%) but fell into a speciﬁc clade not with B. viridis but rather Methylocystaceae bacterium PKR-39 (KJ000026) and uncultured bacteria (e.g., QEDN4CG12 [CU92524] and J22 [HQ697480]), whereas the OTU 100 was identical to Rhodopseudomonas palustris (KT180194; 100%) (Fig. S3C). The cyanobacterial OTU 8532 had a very low sequence similarity (88.9%) to the cultured rela- tive Vampirovibrio chlorellavorus (NR104911), and in fact To address the metabolic activities of the 10 13C- incorporating OTUs, the quantity of their 16S rRNA molecules was monitored throughout the 13C treatment (Fig. 3). The 16S rRNA molecules from the OTUs 12266, 2197 and 8532 linearly increased from hour 2, whereas those from six other OTUs (i.e., 5104, 8474, 2230, 13856, 8385 and 100) showed a two-step increase, that is, they increased temporally during hours 2−4 and 6−8. However, no expression of 16S rRNA from the OTU 6825 was found. Although potentially different in terms of metabolic induction, the nine OTUs became metabolically active promptly after the supplementation of 1,4-dioxane as the sole substrate, indicating these OTUs could directly degrade 1.4-dioxane. The range of their estimated 16S rRNA molecules was 2.42 × 109−2.95 × 1011 copies ml−1, which was apparently not linked with their enrichment in 13C, that 2382 T. Aoyagi et al. Consequently, a total of nine OTUs, including the 1,4- dioxane-degrading P. dioxanivorans, an ammonia- oxidizing bacterium and phylogenetically novel bacteria, were heavily enriched with 1,4-dioxane-derived 13C and further exhibited an increased expression of 16S rRNAs from the beginning of the SIP incubation, indicating their involvement in the degradation of 13C-labeled 1,4-dioxane. Fig. 3 16S rRNA expression of the 13C-incorporating OTUs during the incubation with 13C-labeled 1,4-dioxane. a–d Estimated 16S rRNA molecules from the 13C-incorporating OTUs in the SIP experiment. Genera of the closest relatives of the OTUs are indicated in the par- entheses of explanatory notes. Dynamics of the physicochemical parameters and whole microbial communities in a full-scale treatment system The efﬁciency of 1,4-dioxane removal ﬂuctuated dramati- cally in parallel with changes in temperature and DO throughout the operation, although the TOC removal ratio and pH were rather stable (Fig. S5B−S5D). Illumina sequencing of 16S rRNA genes from 82 different activated sludge samples from the aeration tank and return line pro- duced a total of 5.9 million sequences, corresponding to an average of 72,170 sequences per library (Table S3). Alpha diversity indices indicated that the microbial richness and evenness were almost the same between the tank and line, and these indices randomly shifted in certain ranges during the operation (Chao1: 1,779−11,131; Shannon: 2.8−7.0; Simpson reciprocal: 1.9−37.2). A principal coordinate analysis (PCoA) plot based on weighted UniFrac distances revealed that the whole microbial communities in the tank and line exhibited similar transition patterns (Fig. S6). Speciﬁcally, the microbial communities changed progres- sively throughout the operation, but those just after the system shutdown (upper right of the plot: dates 2nd, 13th and 19th October) were distinguishable from those on the other dates. In accordance with the irregular ﬂuctuations in the α and β diversities, the phylum and class-level phylo- genetic distribution show that the microbial communities changed drastically over the year of operation (Fig. S7). The dominant phylogenetic groups, that is, the phyla Acid- obacteria, Actinobacteria, Armatimonadetes and Bacter- oidetes and the classes Alpha- and Beta-proteobacteria, ﬂuctuated but kept their relative abundances of >10%. These results implied that the roles of whole microbial communities in 1,4-dioxane removal could be limited, which in turn strongly suggested that the nine 1,4-dioxane- degrading OTUs identiﬁed earlier served as a functional guild in the activated sludge system. Fig. 3 16S rRNA expression of the 13C-incorporating OTUs during the incubation with 13C-labeled 1,4-dioxane. a–d Estimated 16S rRNA molecules from the 13C-incorporating OTUs in the SIP experiment. Genera of the closest relatives of the OTUs are indicated in the par- entheses of explanatory notes. 16S rRNA molecules from the OTUs were estimated by the total bacterial 16S rRNA copy numbers (copies ml−1) and relative abundances (%) of the OTUs that were determined by RT-qPCR (n = 3) and Illumina sequencing (n = 3), respectively. The details of the Illumina sequence libraries are summarized in Table S2 S2 16S rRNA molecules from the OTUs were estimated by the total bacterial 16S rRNA copy numbers (copies ml−1) and relative abundances (%) of the OTUs that were determined by RT-qPCR (n = 3) and Illumina sequencing (n = 3), respectively. The details of the Illumina sequence libraries are summarized in Table S2 High-resolution dynamics of the 1,4-dioxane degraders in relation to the treatment system reactor performance is, their relative abundances in the 1H, 2H and 3H fractions of the 13C treatment. This implied that the total metabolic activities and assimilative activities of 1,4-dioxane-13C differed from one species to another, possibly depending on the difference in the metabolic strategies employed. With the high-sensitivity SIP results providing the phylo- genetic identities of 10 OTUs incorporating 1,4-dioxane- 13C, we extracted the data on the relative abundances of Identiﬁcation of 1,4-dioxane degraders by SIP 2383 e e . hanges in the 1,4- related parameters and d 1,4-dioxane degraders ll-scale activated sludge a 1,4-dioxane-related ers, that is, ations of 1,4-dioxane mbols, inﬂuent; closed , efﬂuent) and 1,4- removal efﬁciency lor symbols). ative abundances of the d 1,4-dioxane degraders eration tank (closed and solid lines) and ne (open symbols and nes) of the full-scale t system. Genera of the elatives of the OTUs are d in the parentheses of ory notes. The ce of each OTU was ned by Illumina ng of 16S rRNA genes. ails of the Illumina e libraries are zed in Table S3 Fig. 4 Changes in the 1,4- dioxane-related parameters and identiﬁed 1,4-dioxane degraders in the full-scale activated sludge system. a 1,4-dioxane-related parameters, that is, concentrations of 1,4-dioxane (open symbols, inﬂuent; closed symbols, efﬂuent) and 1,4- dioxane removal efﬁciency (gray-color symbols). b–f Relative abundances of the identiﬁed 1,4-dioxane degraders in the aeration tank (closed symbols and solid lines) and return line (open symbols and dotted lines) of the full-scale treatment system. Genera of the closest relatives of the OTUs are indicated in the parentheses of explanatory notes. The abundance of each OTU was determined by Illumina sequencing of 16S rRNA genes. The details of the Illumina sequence libraries are summarized in Table S3 these OTUs from the Illumina sequence libraries. The OTU 6825 was not detected throughout the operation, indicating that this OTU was not a member of functional guild of 1,4- dioxane degraders, which is consistent with the results from 16S rRNA expression analysis of the SIP incubation. The other nine OTUs, 2197, 12266, 5104, 8474, 2230, 13856, 8385, 100 and 8532, were detected in the aeration tank at the yearly averaged relative abundances of 0.007, 0.021, 0.001, 0.074, 0.137, 0.002, 0.682, 1.523 and 0.001%, respectively. In addition, the individual OTUs showed similar successions in the aeration tank and return line (Fig. 4b–f). High-resolution dynamics of the 1,4-dioxane degraders in relation to the treatment system reactor performance Changes in the absolute abundances of the nine OTUs were determined based on MLSS, that is, total bio- mass, of the tank and line (Fig. S9B−S9F), which showed a quite similar trend to the relative abundance changes (Fig. 4b–f). Although 1,4-dioxane degradation efﬁciency concentrations of 1,4-dioxane (open symbols, inﬂuent; closed symbols, efﬂuent) and 1,4- dioxane removal efﬁciency (gray-color symbols). b–f Relative abundances of the identiﬁed 1,4-dioxane degraders in the aeration tank (closed symbols and solid lines) and return line (open symbols and dotted lines) of the full-scale treatment system. Genera of the closest relatives of the OTUs are indicated in the parentheses of explanatory notes. The abundance of each OTU was determined by Illumina sequencing of 16S rRNA genes. The details of the Illumina sequence libraries are summarized in Table S3 these OTUs from the Illumina sequence libraries. The OTU 6825 was not detected throughout the operation, indicating that this OTU was not a member of functional guild of 1,4- dioxane degraders, which is consistent with the results from 16S rRNA expression analysis of the SIP incubation. The other nine OTUs, 2197, 12266, 5104, 8474, 2230, 13856, 8385, 100 and 8532, were detected in the aeration tank at the yearly averaged relative abundances of 0.007, 0.021, 0.001, 0.074, 0.137, 0.002, 0.682, 1.523 and 0.001%, respectively. In addition, the individual OTUs showed similar successions in the aeration tank and return line (Fig. 4b–f). Changes in the absolute abundances of the nine OTUs were determined based on MLSS, that is, total bio- mass, of the tank and line (Fig. S9B−S9F), which showed a quite similar trend to the relative abundance changes (Fig. 4b–f). Although 1,4-dioxane degradation efﬁciency 2384 T. Aoyagi et al. 2nd February, in association with a variety of transition behaviors of the identiﬁed 1,4-dioxane degraders, that is, gradual increases for the OTUs 100 and 8474, maintenance at a constant level of the OTU 8385, ﬂuctuations for the OTUs 2197 and 13856 and transient appearances for the OTUs 2230 and 12266. Moreover, a moderate decrease in 1,4-dioxane removal efﬁciency (89.2%) on 8th February was quickly recovered, concurrent with transient increases in the OTUs 2230, 8474 and 12266. These three OTUs increased gradually during the successive stable operation from 15th February to 22nd March, then more rapidly in response to the lowered 1,4-dioxane removal efﬁciencies (69.5–71.6%) during the end of the operation from 28th March to 4th April. High-resolution dynamics of the 1,4-dioxane degraders in relation to the treatment system reactor performance Thereafter, the decrease in temperature to 30.0 °C, increase in DO to 5.5 mg l−1 and lowered inﬂuent TOC of 183.4 mg l−1 on 19th August again led to a high 1,4-dioxane removal efﬁciency (98.0%), and under these conditions the OTUs 100 and 2230 appeared to increase (Fig. S8B and S8D). For about 10 days after the system restarted on 24th September, the temperature, DO and TOC removal ratio were stabilized to values similar to those before the system shutdown. How- ever, despite the high DO levels (4.2–6.4 mg l−1) provided, the removal efﬁciencies of 1,4-dioxane were quite low (0–34.5%) from 5th to 19th October, during which the relative abundance of the OTU 2230 rapidly increased from 0.005% to 0.907%. In the days immediately after 19th October, a dramatic recovery from the performance decline was realized simultaneously with sharp increases in the OTUs 8385, 100 (Fig. S8A and S8B), 2197 and 13856. Due to the nearly constant temperatures (21.3 °C–30.2 °C) and sustained DO levels (3.7–5.9 mg l−1), the stable reactor performance with a high 1,4-dioxane removal efﬁciency of 95.4–99.4% continued from 16th November to High-resolution dynamics of the 1,4-dioxane degraders in relation to the treatment system reactor performance These results indicate that the nine 1,4- dioxane-degrading OTUs co-existed in the tank at yearly averaged relative abundances of 0.001–1.523%, but showed dynamic and individually distinct transitions in response to changes in the 1,4-dioxane removal efﬁciency under chan- ging conditions in the activated sludge system. did not represent the actual removal capability because of the non-constant concentrations of 1,4-dioxane in the inﬂuent, the dynamics of the nine identiﬁed OTUs in the tank were tightly associated with changes in the 1,4-dioxane removal efﬁciency (Fig. 4, S9 and Table S4). In particular, the removal efﬁciencies of 1,4-dioxane were high (98.7–99.2%) when the temperature was held nearly con- stant at <30.7 °C, and the DO levels were 2.7–6.4 mg l−1 from 17th May to 6th July (except on 25th May [83.7%] and 8th June [93.6%]), when relatively high abundances of the OTUs 8385 and 100 (0.184–5.120%) were noted. The decreases in performance on 25th May and 8th June may have been related to a temporal decrease in the inﬂuent TOC to 47.4 mg l−1 and the resultant high ratio (8.3%) of 1,4-dioxane to the TOC on 18th May . The OTUs 8385 and 100 were temporarily increased in response to the former decrease in efﬁciency and the OTUs 2230 and 8474 were temporarily increased in response to the latter decrease, which would have contributed to the rapid recovery of the removal efﬁciencies. From 6th to 21st July, a gradual increase in temperature to 33.0 °C and a sudden drop of DO to 1.1 mg l−1 coincided with lasting decreases in the OTUs 100, 8385 (Fig. S8A) and 2230 (Fig. S8C), but transient increases in the OTUs 8474, 8532 and 5104, thereby maintaining the high 1,4-dioxane removal efﬁciency (98.4%) on 21st July. However, from 27th July to 10th August, the inﬂows of 1,4-dioxane at 12.5−21.9 mg l−1 and TOC at 446.9–684.5 mg l−1, as well as the continued high temperatures (34.0 °C–35.4 °C) and low DO levels (0.9–1.7 mg l−1), resulted in low 1,4-dioxane removal efﬁciencies (0–68.2%). In this regard, the relative abundances of all the 1,4-dioxane degrader OTUs decreased to substantially low levels (0.001–0.079%) on 10th August. Discussion The BDs 1.796−1.805 g ml−1 of the 1H and 2H fractions correspond to those of the RNA density fractions in which even low amounts of 13C-labeled rRNAs are signiﬁcantly accumulated [37]. The BD 1.790 g ml−1 of the 3H fraction corresponds to the BD showing a moderate accumulation of 13C-labeled rRNAs [37]. High- throughput sequencing of these heavy fractions indicated that the 10 OTUs signiﬁcantly incorporated the 13C-labeled 1,4-dioxane, and thus were enriched in 13C (Fig. 2b–d). Among them, the OTU 6825 was ruled out from the func- tional guild as a result of 16S rRNA expression assays. Based on the principle of density separation, the 13C enrichment of 16S rRNA molecules should be higher in the 1H and 2H fractions than in the 3H fraction. We speculate that the OTUs 2197, 12266, 5104 and 13856, which were identiﬁed exclusively in the 1H and 2H fractions, may have metabolized only 13C-labeled 1,4-dioxane, whereas the OTUs 8385, 100 and 8532, which were identiﬁed solely in the 3H fraction, may have co-metabolized 13C-labeled 1,4- dioxane in the presence of the other unlabeled compounds (e.g., mono-ethylene glycol and alkanes) that were origin- ally contained in the activated sludge. Such a co- metabolism mechanism is supported by the previous ﬁnd- ing that several short-chain alkanes were the primary carbon sources supporting the growth of the 1,4-dioxane degrader Mycobacterium austroafricanum strain JOB5 during co- metabolism [53]. By selecting these two distinct metabolic modes, the other OTUs 8474 and 2230 might ﬂexibly adapt to the changing environmental conditions, for example, dynamic ﬂuctuation of the ratio of 1,4-dioxane to the inﬂuent TOC in a wide range of 0.87–12.12% (Fig. 4a and S5D). In addition, the assimilation of 13C-labeled 1,4- dioxane into 16S rRNA molecules of the moderately 13C- enriched OTUs 8385, 100, 8532, 8474 and 2230 should be noted. There is a possibility that, through the co-metabolism of these OTUs, 1,4-dioxane-derived 13C was utilized not for growth but for maintenance (i.e., for the generation of 16S RNA l l ) [54] O th th h d d i th operation of the full-scale activated sludge system, the putative 1,4-dioxane co-metabolizer OTUs 8385, 100, 8532, 8474 and 2230 showed high yearly averaged relative abundances (the mean: 0.483%, the range: 0.001–1.523%), whereas the abundances of the putative metabolizer OTUs 2197, 12266, 5104 and 13856 were rather low (the mean: 0.008%, the range: 0.001–0.021%) (Fig. 3). Discussion 1,4-Dioxane is one of the most recalcitrant artiﬁcial pollu- tants in petrochemical industrial wastewaters and chlori- nated solvent groundwater plumes [2]. As the ﬁrst application of high-sensitivity rRNA-SIP to environmental samples, we herein investigated the identity and diversity of the activated sludge microorganisms involved in the degradation of 1,4-dioxane. Using GC-C-IRMS in parallel with chemical analyses, we clariﬁed the fate of 13C-labeled 1,4-dioxane during SIP incubation across time. The envir- onmentally relevant concentration of 13C-labeled 1,4-diox- ane and short incubation time allowed the sludge microorganisms to incorporate the substrate-borne 13C under quasi-natural conditions. Although it is possible that some of the 13C-incorporating microorganisms could not degrade 13C-labeled 1,4-dioxane but rather utilized its degradative intermediates supplied by the other degraders, the increased 16S rRNA expression shortly after the addi- tion of 13C-labeled dioxane as the sole substrate conﬁrmed the key microorganisms that could directly metabolize 1,4- dioxane. Together, we identiﬁed a diverse range of active 1,4-dioxane degraders, although some degraders might be overlooked due to the controlled environmental variables in the SIP experiment. Furthermore, high-resolution phyloge- netic monitoring over 1-year of operation of a full-scale activated sludge system highlighted an apparent link between the dynamics of the identiﬁed degraders and 1,4- dioxane removal efﬁciency. During the SIP incubation, the concentrations of degra- ded 1,4-dioxane-13C and gaseous product 13C (i.e., 13CO2) were 0.0466 and 0.0144 mmol per vial, respectively 2385 Identiﬁcation of 1,4-dioxane degraders by SIP (Fig. 1). Given that IC is present mostly in the form of bicarbonate in pH 8.2, the produced dissolved inorganic 13C was calculated as 0.0201 mmol per vial (Fig. S2B). The carbon recovery in the 13C-labeled 1,4-dioxane treatment was 74.04%, which was consistent with the 14C-labeled 1,4- dioxane turnover data [52], in which more than half of the 1,4-dioxane was mineralized to CO2 by cultured 1,4-diox- ane degraders. The carbon not recovered was likely attri- butable to either the degradative intermediates of 1,4- dioxane remaining in the incubated sludge and/or the por- tion assimilated to the microbial biomass including rRNA, which became labeled with 13C. In spite of the relatively low concentration (i.e., 0.58 mmol l−1) of the 13C-labeled 1,4-dioxane converted, 13C-labeled RNAs, that is, the 1H, 2H and 3H fractions, were retrieved by ultra-centrifugation and fractionation (Table S1). Discussion It is tempting to imagine that the presumed metabolic modes of the identiﬁed 1,4-dioxane degraders affected their population size and dynamics in the system. However, the extent of 13C enrichment of 16S rRNA molecules is not yet conﬁrmed as an index of the versatility of carbon source utilization of the degraders. Future investigations will be needed to clarify the ecophysiological futures and metabolic strategies of the identiﬁed 1,4-dioxane-degrading microorganisms. It is noteworthy that the 1,4-dioxane removal efﬁciency of the full-scale treatment system was intrinsically linked with the dynamics of the identiﬁed 1,4-dioxane degraders (Fig. 4) but not with the dynamics of whole microbial communities (i.e., α-diversity indices, the PCoA plot based on weighted UniFrac distances, or phylum- and class-level phylogenetic data) (Figs. S6, S7 and Table S3). The heavily 13C-enriched OTUs 2197 and 13856 were phylogenetically identical and similar, respectively, to the well-known 1,4- dioxane degrader P. dioxanivorans (NR074465) (Fig. 2b, c and S3A). P. dioxanivorans can aerobically degrade 1,4- dioxane to CO2 as a sole carbon and energy source [55] and its degradation pathway has been proposed [56]. The transformation ability of P. dioxanivorans has been observed after the induction by 1,4-dioxane and its related compounds [25, 56, 57]. During the operation of the full-scale system, these OTUs showed similar annual transitions, especially with respect to their proliferation and continued existence under relatively stable aerobic conditions after 19th October (Fig. 4e, f and S5B). The detection of the conﬁrmed 1,4- dioxane-degrading bacterium solidiﬁed the reliability of the high-sensitivity SIP results in this study. Aside from P. dioxanivorans, the OTU 100 was phylogenetically identical to R. palustris (KT180194), which has been shown to exhibit benzoate degradation ability and to operate a variety of oxygen-dependent enzymes [58]. Notably, this OTU was located in a phylogenetic cluster containing the 1,4-dioxane- degrading Aﬁpia sp. strain D1 (accession no. AB586143) (Fig. S3C). The sequence similarity of OTU 100 and strain D1 was relatively high (96.4%). Whereas the other alpha- proteobacterial OTU 8385 showed 95.7% similarity to B. viridis (NR117911), which grows unimpaired in the pre- sence of dioxane [59], it was more closely related to and formed the same phylogenetic clade with Methylocystaceae bacterium PKR-39 (KJ000026; 96.5% similarity) (Fig. S3C), a relative of the obligate methanotroph Methylosinus trichosporium involved in the co-metabolism of 1,4-dioxane [24]. These two OTUs identiﬁed in the 3H fraction (Fig. 2d) 2386 T. Aoyagi et al. labeled 1,4-dioxane. Compliance with ethical standards Conﬂict of interest The authors declare that they have no conﬂict of interest. Discussion These ﬁve OTUs increased rapidly in response to transient decreases in 1,4-dioxane removal efﬁciencies, especially during the system restart after 24th September (for the OTU 2230) and the end of the operation from 28th March to 4th April (for the OTUs 8474 and 12266), thereby facilitating a recovery from the reduction in performance. Acknowledgements We thank Tomomi Ujiie, Yumiko Kayashima and Maki Yanagisawa of the National Institute of Advanced Industrial Science and Technology for their technical assistance. We also thank Dr. Takashi Narihiro for help with the phylogenetic tree analysis. This study was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI Grants Numbers 16K14816 (Grant-in-Aid for Challenging Exploratory Research) and 16H05886 (Grant-in-Aid for Young Scientists [A]) to TH. Discussion The subsequent high-resolution phylo- genetic analysis over the 1-year operation of a full-scale treatment system corroborated the presence and dynamics of these nine species as the functional guild that were tightly associated with the 1,4-dioxane degradation efﬁciency. The strategy we have taken is very effective for identiﬁcation and ecophysiological characterization of the elusive 1,4-dioxane degraders. Based on the extent of their 13C enrichment in the SIP incubation and their relative abundances in the actual system run, we showed that each of these 1,4-dioxane degraders may possess a distinct metabolic mode, that is, metabolism or co-metabolism, which would not otherwise be accessible, under changing environmental conditions. Consequently, the co-existence and individually distinct dynamics of various 1,4-dioxane degraders, including hitherto unidentiﬁed species, played pivotal roles in the maintenance of this biological system for removing 1,4- dioxane. The global distribution and ecophysiological roles of these 1,4-dioxane degraders should be investigated to clarify the microbiological mechanism underlying the nat- ural attenuation of the persistent pollutant. were the dominant degraders in the actual system run, showing yearly averaged relative abundances of 1.523% (for the OTU 100) and 0.682% (for the OTU 8385) (Fig. 3b). Their proliferation was associated with high removal efﬁ- ciencies of 1,4-dioxane. In this context, these aerobic organisms decreased remarkably under low DO conditions from 6th July to 10th August, triggering the decline in performance of the system. Further, the OTU 12266 belonged to Nitrosomonas ureae (AF272414; 97.6%), a chemolithotrophic ammonia-oxidizing bacterium [60]. It is vital to point out that the ammonia monooxygenase of the OTU 12266 would play a role in the ﬁrst step of 1,4-dioxane degradation, because some of the cultured bacteria that express various types of monooxygenases are also involved in the metabolism and co-metabolism of 1,4-dioxane [24– 26]. The OTUs 8474 and 5104 formed a distinctive phylo- genetic cluster comprised of only uncultured bacteria, such as Wu-C65 (KJ783141) and Anxy6 (HQ343211), found in activated sludges (Fig. S3D), indicating that the constituent members of this cluster were likely candidates for hitherto unknown 1,4-dioxane degraders. The OTUs 2230 and 8532 have quite low similarities (<88.9%) to any cultured organisms, which makes it impossible to estimate their physiological functions. Conclusion 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/. Due to the high phylogenetic diversity of 1,4-dioxane degraders, which renders the tracing of the functional guild rather difﬁcult, microbial transformation and degradation of 1,4-dioxane in natural environments are largely unknown while 1,4-dioxane degradation has been intensively studied by characterizing isolates and tracing biomarker genes [12]. In this study, high-sensitivity SIP was implemented to identify hitherto unknown but functionally important 1,4- dioxane degraders in natural environments. We discovered the 10 signiﬁcantly 13C-incorporating microbial species from the sludge microorganisms by rigorously analyzing 16S rRNA molecules in RNA density fractions of the 13C- labeled and unlabeled 1,4-dioxane treatments. By employing a 16S rRNA expression assay, we showed that 9 of the 10 species, including the 1,4-dioxane-degrading P. dioxa- nivorans and an ammonia-oxidizing bacterium, increased their metabolic activities promptly after the onset of the SIP incubation, strengthening the argument that these 13C- incorporating microorganisms could directly degrade 13C- 2. Mohr T. Environmental investigation and remediation:1,4-dioxane and other solvent stabilizers. Boca Roca, FL: CRC Press; 2010. References 1. Lanigan RS, Cosmetic Ingredient Review E. Addendum to the ﬁnal report on the safety assessment of polysorbates. Int J Toxicol. 2000;19:43–89. 2. Mohr T. Environmental investigation and remediation:1,4-dioxane and other solvent stabilizers. Boca Roca, FL: CRC Press; 2010. Identiﬁcation of 1,4-dioxane degraders by SIP 2387 3. Zenker MJ, Borden RC, Barlaz MA. Occurrence and treatment of 1,4-dioxane in aqueous environments. Environ Eng Sci. 2003;20:423–32. 23. Skinner K, Cuiffetti L, Hyman M. Metabolism and cometabolism of cyclic ethers by a ﬁlamentous fungus, a Graphium sp. Appl Environ Microbiol. 2009;75:5514–22. 4. IARC. Re-evaluation of some organic chemicals, hydrazine and hydrogen peroxide. Proceedings of the IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon, France, 17-24 February 1998. IARC Monogr Eval Carcinog Risks Hum. 1999;71(Pt 1):1–315. 24. Mahendra S, Alvarez-Cohen L. Kinetics of 1,4-dioxane biode- gradation by monooxygenase-expressing bacteria. Environ Sci Technol. 2006;40:5435–42. 25. Gedalanga PB, Pornwongthong P, Mora R, Chiang SY, Baldwin B, Ogles D, et al. Identiﬁcation of biomarker genes to predict biodegradation of 1,4-dioxane. Appl Environ Microbiol. 2014;80:3209–18. 5. USEPA. Integrated risk information system (IRIS) toxicological review of 1,4-dioxane. National Center for Environmental Assesment, Ofﬁce of Resarch and Development, Washington, DC. 2013. 26. Li M, Mathieu J, Yang Y, Fiorenza S, Deng Y, He Z, et al. Widespread distribution of soluble di-iron monooxygenase (SDIMO) genes in arctic groundwater impacted by 1,4-dioxane. Environ Sci Technol. 2013;47:9950–8. 6. Abe A. Distribution of 1,4-dioxane in relation to possible sources in the water environment. Sci Total Environ. 1999;227:41–7. 27. Gedalanga P, Madison A, Miao Y, Richards T, Hatton J, DiGuiseppi WH, et al. A multiple lines of evidence framework to evaluate intrinsic biodegradation of 1,4-dioxane. Remediat J. 2016;27:93–114. 7. Adamson DT, Mahendra S, Walker KL, Rauch SR, Sengupta S, Newell CJ. A multisite survey to identify the scale of the 1,4- dioxane problem at contaminated groundwater sites. Environ Sci Technol Lett. 2014;1:254–8. 8. DiGuiseppi W, Whitesides C, Treatment options for remediation of 1,4-dioxane in groundwater. Environ Eng Appl Res. 2007; Pract2:1–7. 28. Li M, Mathieu J, Liu Y, Van Orden ET, Yang Y, Fiorenza S, et al. The abundance of tetrahydrofuran/dioxane monooxygenase genes (thmA/dxmA) and 1,4-dioxane degradation activity are sig- niﬁcantly correlated at various impacted aquifers. Environ Sci Technol Lett. 2014;1:122–7. 9. Adams CD, Scanlan PA, Secrist ND. Oxidation and biodegrad- ability enhancement of 1,4-dioxane using hydrogen peroxide and ozone. Environ Sci Technol. 1994;28:1812–8. 29. References Zhang S, Gedalanga PB, Mahendra S. Biodegradation kinetics of 1,4-dioxane in chlorinated solvent mixtures. Environ Sci Technol. 2016;50:9599–607. 10. Kim B-Y, Weon H-Y, Yoo S-H, Chen W-M, Kwon S-W, Go S-J, et al. Chitinimonas koreensis sp. nov., isolated from greenhouse soil in Korea. Int J Syst Evol Microbiol. 2006;56:1761–4. 30. Chen D-Z, Jin X-J, Chen J, Ye J-X, Jiang N-X, Chen J-M. Intermediates and substrate interaction of 1,4-dioxane degradation by the effective metabolizer Xanthobacter ﬂavus DT8. Int Biod- eter Biodegr. 2016;106:133–40. 11. Stefan MI, Bolton JR. Mechanism of the degradation of 1,4- dioxane in dilute aqueous solution using the UV/hydrogen per- oxide process. Environ Sci Technol. 1998;32:1588–95. 12. Zhang S, Gedalanga PB, Mahendra S. Advances in bioremedia- tion of 1,4-dioxane-contaminated waters. J Environ Manag. 2017;15:765–74. 31. Radajewski S, Ineson P, Parekh NR, Murrell JC. Stable-isotope probing as a tool in microbial ecology. Nature. 2000;403:646–9. 32. Maneﬁeld M, Whiteley AS, Grifﬁths RI, Bailey MJ. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl Environ Microbiol. 2002;68:5367–73. 13. Zenker MJ, Borden RC, Barlaz MA. Mineralization of 1,4-diox- ane in the presence of a structural analog. Biodegradation. 2000;11:239–46. 33. Nam JH, Ventura JS, Yeom IT, Lee Y, Jahng D. Structural and kinetic characteristics of 1,4-dioxane-degrading bacterial consortia containing the phylum TM7. J Microbiol Biotechnol. 2016;26:1951–64. 14. White GF, Russell NJ, Tidswell EC. Bacterial scission of ether bonds. Microbiol Rev. 1996;60:216–32. 15. Chiang S-YD, Mora R, Diguiseppi WH, Davis G, Sublette K, Gedalanga P, et al. Characterizing the intrinsic bioremediation potential of 1,4-dioxane and trichloroethene using innovative environmental diagnostic tools. J Environ Monit. 2012;14:2317–26. 34. Zenker MJ, Borden RC, Barlaz MA. Biodegradation of 1,4- dioxane using trickling ﬁlter. J Environ Eng. 2004;130:926–31. 35. Hori T, Noll M, Igarashi Y, Friedrich MW, Conrad R. Identiﬁ- cation of acetate-assimilating microorganisms under methano- genic conditions in anoxic rice ﬁeld soil by comparative stable isotope probing of RNA. Appl Environ Microbiol. 2007;73:101–9. 16. Li M, Van Orden ET, DeVries DJ, Xiong Z, Hinchee R, Alvarez PJ. Bench-scale biodegradation tests to assess natural attenuation potential of 1,4-dioxane at three sites in California. Biodegrada- tion. 2015;26:39–50. 17. Sei K, Kakinoki T, Inoue D, Soda S, Fujita M, Ike M. Evaluation of the biodegradation potential of 1,4-dioxane in river, soil and activated sludge samples. Biodegradation. 2010;21:585–91. 36. Hori T, Muller A, Igarashi Y, Conrad R, Friedrich MW. References Carbon isotope fractionation between dissolved bicarbonate and gaseous carbon dioxide. Earth Planet Sci Lett. 1974;22:169–76. 53. Lan RS, Smith CA, Hyman MR. Oxidation of cyclic ethers by alkane-grown Mycobacterium vaccae JOB5. Remediat J. 2013;23:23–42. 44. Noll M, Matthies D, Frenzel P, Derakshani M, Liesack W. Suc- cession of bacterial community structure and diversity in a paddy soil oxygen gradient. Environ Microbiol. 2005;7:382–95. 54. Lever MA, Rogers KL, Lloyd KG, Overmann J, Schink B, Thauer RK, et al. Life under extreme energy limitation: a synthesis of laboratory- and ﬁeld-based investigations. FEMS Microbiol Rev. 2015;39:688–728. 45. Lueders T, Maneﬁeld M, Friedrich MW. Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ Microbiol. 2004;6:73–8. 55. Mahendra S, Alvarez-Cohen L. Pseudonocardia dioxanivorans sp. nov., a novel actinomycete that grows on 1,4-dioxane. Int J Syst Evol Microbiol. 2005;55:593–8. 46. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, et al. Ultra-high-throughput microbial community ana- lysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012;6:1621–4. 56. Grostern A, Sales CM, Zhuang WQ, Erbilgin O, Alvarez-Cohen L. Glyoxylate metabolism is a key feature of the metabolic degradation of 1,4-dioxane by Pseudonocardia dioxanivorans strain CB1190. Appl Environ Microbiol. 2012;78:3298–308. 47. Itoh H, Aita M, Nagayama A, Meng XY, Kamagata Y, Navarro R, et al. Evidence of environmental and vertical transmission of Burkholderia symbionts in the oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae). Appl Environ Microbiol. 2014;80:5974–83. 57. Sales CM, Grostern A, Parales JV, Parales RE, Alvarez-Cohen L. Oxidation of the cyclic ethers 1,4-dioxane and tetrahydrofuran by a monooxygenase in two Pseudonocardia species. Appl Environ Microbiol. 2013;79:7702–8. 58. Hanson JR, Ackerman CE, Scow KM. Biodegradation of methyl tert-butyl ether by a bacterial pure culture. Appl Environ Micro- biol. 1999;65:4788–92. 48. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335–6. 59. Darcy DL. Optimization and characterization of the growth of the photosynthetic bacterium Blastochloris viridis and a brief survey of its potential as a remediative tool. Doctor of Philosophy, University of Notre Dame: IN, USA. 2006. 49. Hori T, Akuzawa M, Haruta S, Ueno Y, Ogata A, Ishii M, et al. Involvement of a novel fermentative bacterium in acidiﬁcation in a thermophilic anaerobic digester. FEMS Microbiol Lett. 2014;361:62–7. 60. References Identi- ﬁcation of iron-reducing microorganisms in anoxic rice paddy soil by 13C-acetate probing. ISME J. 2010;4:267–78. 18. Suthersan SS. Remediation engineering: design concepts. LLc, Boca Raton, FL: Taylor & Francis Group; 2017. 37. Aoyagi T, Hanada S, Itoh H, Sato Y, Ogata A, Friedrich MW, et al. Ultra-high-sensitivity stable-isotope probing of rRNA by high-throughput sequencing of isopycnic centrifugation gradients. Environ Microbiol Rep. 2015;7:282–7. 19. Luo W, Zhu X, Chen W, Duan Z, Wang L, Zhou Y. Mechanisms and strategies of microbial cometabolism in the degradation of organic compounds—chlorinated ethylenes as the model. Water Sci Technol. 2014;69:1971–83. 38. Miller CS, Handley KM, Wrighton KC, Frischkorn KR, Thomas BC, Banﬁeld JF. Short-read assembly of full-length 16S ampli- cons reveals bacterial diversity in subsurface sediments. PLoS One. 2013;8:e56018. 20. Alvarez-Cohen L, McCarty PL. A cometabolic biotransformation model for halogenated aliphatic compounds exhibiting product toxicity. Environ Sci Technol. 1991;25:1381–7. 39. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci USA. 2011;108(Suppl 1):4516–22. 21. Inoue D, Tsunoda T, Sawada K, Yamamoto N, Saito Y, Sei K, et al. 1,4-Dioxane degradation potential of members of the genera Pseudonocardia and Rhodococcus. Biodegradation. 2016;27:277–86. 40. Navarro RR, Aoyagi T, Kimura M, Itoh H, Sato Y, Kikuchi Y, et al. High-resolution dynamics of microbial communities during dissimilatory selenate reduction in anoxic soil. Environ Sci Technol. 2015;49:7684–91. 22. Nakamiya K, Hashimoto S, Ito H, Edmonds JS, Morita M. Degradation of 1,4-dioxane and cyclic ethers by an isolated fun- gus. Appl Environ Microbiol. 2005;71:1254–8. 2388 T. Aoyagi et al. 51. Hori T, Aoyagi T, Itoh H, Narihiro T, Oikawa A, Suzuki K, et al. Isolation of microorganisms involved in reduction of crystalline iron (III) oxides in natural environments. Front Microbiol. 2015;6:386. 41. Aoyagi T, Kimura M, Yamada N, Navarro RR, Itoh H, Ogata A, et al. Dynamic transition of chemolithotrophic sulfur-oxidizing bacteria in response to amendment with nitrate in deposited marine sediments. Front Microbiol. 2015;6:426. 52. Mahendra S, Petzold CJ, Baidoo EE, Keasling JD, Alvarez-Cohen L. Identiﬁcation of the intermediates of in vivo oxidation of 1,4- dioxane by monooxygenase-containing bacteria. Environ Sci Technol. 2007;41:7330–6. 42. Mayumi D, Mochimaru H, Tamaki H, Yamamoto K, Yoshioka H, Suzuki Y, et al. Methane production from coal by a single methanogen. Science. 2016;354:222–5. 43. Mook WG, Bommerson JC, Staverman WH. References Purkhold U, Pommerening-Röser A, Juretschko S, Schmid MC, Koops H-P, Wagner M. Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys. Appl Environ Microbiol. 2000;66:5368–82. 50. Hori T, Haruta S, Ueno Y, Ishii M, Igarashi Y. Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester. Appl Environ Microbiol. 2006;72:1623–30.
https://openalex.org/W2899888562	https://europepmc.org/articles/pmc6226484?pdf=render	English	null	Diffusion-limited association of disordered protein by non-native electrostatic interactions	Nature communications	2,018	cc-by	13,756	ARTICLE OPEN ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y Since TAD is disordered, the FRET efﬁciency of unbound TAD is low com- pared to that of the bound state, in which the end-to-end distance is much shorter (Fig. 1a). IDPs are often highly charged, possibly to avoid aggregation5, and binding partners are oppositely charged. Since the net charges of TAD and NCBD are −10 and +6, respectively, a strong electrostatic attraction is expected, which should facilitate the association. Therefore, we ﬁrst investigated this effect by changing the ionic strength of the solution. Fig. 2a shows the binned ﬂuorescence trajectories, FRET efﬁciency (E) trajectories, and FRET efﬁciency histograms measured at different NaCl concentrations (10 mM Tris, pH 7) (see Supplementary Fig. 2 for the FRET efﬁciency histograms obtained from freely diffusing molecules). At 0 mM NaCl, the FRET efﬁciency trajectory shows clear transitions between two levels, E ~ 0.6 and E ~ 0.25, which correspond to the bound and unbound states, respectively, indicating NCBD keeps binding and dissociating. As the NaCl concentration is increased, the bound and unbound states become less clear and the two peaks in the FRET efﬁciency histogram merge into a single peak. A narrow single peak at high ionic strength indicates that binding and dissociation are fast and E is averaged by multiple transitions occurring during the bin time of 1 ms as observed for fast-folding, two-state proteins35–37. In order to obtain accurate parameters, we used a maximum likelihood method that extracts the FRET efﬁciencies of the bound and unbound states and apparent association and dissociation rates38 (see Supplementary Fig. 3 and Methods for the kinetic models and the details of analysis methods). Since photon trajectories are analyzed directly without binning, it is possible to extract accurate parameters even for the case that no transition is identiﬁable in the binned trajectory at the highest NaCl concentration (150 mM, Fig. 2a). In addition, frequent acceptor photoblinking on the microsecond time scale can also be modeled in the analysis, which is necessary for the accurate determination of the parameters. The results are summarized in Fig. 2c and Supplementary Table 1. The FRET efﬁciency of the unbound state (EU) gradually increases with the increasing NaCl concen- tration while that of the bound state (EB) remains unchanged, indicating that disordered TAD becomes more compact because of reduced electrostatic repulsion at higher ionic strength39. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y I ntrinsically disordered proteins (IDPs) play important roles in various dynamic cellular processes such as gene transcription and signal transduction1,2. Since IDPs are unstructured and ﬂexible, they can interact with multiple binding partners1–4. The kinetics of these interactions are, therefore, a key factor in the tight regulation of a complex binding network. The rates of association and dissociation of these proteins determine how fast a system can respond to external environmental changes2,5,6. The rate of a bi-molecular reaction in solution is limited by the relative diffusion of the two reacting molecules (i.e., the Smo- luchowski limit) when the reaction occurs as soon as the mole- cules make contact (isotropic reactivity)7. In the Smoluchowski limit, the association rate coefﬁcient (kA) ranges from 109 to 1010 M−1 s−1. However, this fast association is not possible for binding of macromolecules because the fraction of reactive areas (i.e., binding interfaces) is much smaller. Due to the orientational constraint, kA can be smaller by several orders of magnitude, 105–106 M−1 s−18,9. In IDP binding, the association is expected to be even slower because folding should occur during the binding process and an IDP may dissociate easily before folding even if it encounters a target protein. However, there are number of IDP binding systems that exhibit extremely fast association10–14. In this work, we investigated the molecular mechanism of such fast binding: association of the transactivation domain (TAD) of the tumor suppressor protein p53 and the nuclear coactivator bind- ing domain (NCBD) of CREB-binding protein (CBP)15 using single-molecule FRET spectroscopy (Fig. 1a). The transcription activity of p53 is controlled by binding of many proteins to the unfolded N-terminal TAD such as the E3 ubiquitin ligase MDM2 and various domains of the general transcriptional coactivator CBP including NCBD16. NCBD is also a ﬂexible protein like a molten-globule17–19 and interacts with multiple binding part- ners20 including TAD and the activation domain of SRC-3 (ACTR)21,22. I unlabeled NCBD in solution (Fig. 1a) (see Supplementary Fig. 1 and Methods for the sequences of the protein constructs and details of protein expression and puriﬁcation). The concentration of NCBD was varied to be near the equilibrium dissociation constant at each experimental condition so that the bound and unbound populations of TAD are comparable and both binding and dissociation transitions are frequently observed. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y Both dissociation (kD) and apparent association rates (kA,app) increase with the increasing NaCl concentration, consistent with the changes of the shape of the FRET efﬁciency histograms (Fig. 2a) and the increased relaxation rate (sum of the apparent association and dissociation rates) of the donor–acceptor cross-correlation function (Supplementary Fig. 4 and Supplementary Table 1). However, the association rate coefﬁcient (kA) of a bi-molecular reaction (the reaction is pseudo-ﬁrst order because [TAD] « [NCBD]), which is obtained by dividing kA,app by the NCBD concentration, actually decreases as the NaCl concentration is increased (Fig. 2c). ( ) Unbound TAD is largely disordered, but three short helices are formed and the chain wraps around NCBD during binding (Fig. 1a). Unlike the binding of two folded proteins, therefore, binding of TAD involves large conformational changes, and it resembles a spontaneous protein folding process. Most impor- tantly, binding pathways are expected to be highly diverse23,24 as transition paths in protein folding, which can be probed only by watching individual molecules25–27. Characterization of this heterogeneity (i.e., distribution of binding pathways) is the key to understanding the detailed binding mechanism. We deﬁne a transient complex (TC, also known as encounter complex28–31) as a representative state appearing in the collection of binding pathways that lead unbound (disordered) TAD to a bound complex. We aim to describe the mechanism of diffusion-limited association of TAD and NCBD in terms of the properties of the TC. So far, the average picture of intermediate species during binding including encounter complexes has been characterized by NMR spectroscopy32 such as relaxation dispersion33 and para- magnetic relaxation enhancement29,34. The analysis of photon trajectories shows that the lifetime of TC of TAD and NCBD binding is much longer than that of the association of two folded proteins. The long lifetime results from the stabilization of TC by non-native electrostatic interactions, which makes diffusion- limited association possible because disordered TAD can rear- range and fold rather than dissociating quickly. Diffusion-limited binding of TAD via formation of stable TC. The strong ionic strength dependence of TAD–NCBD binding is expected from the large opposite net charges of the two proteins because the charge screening effect at high ionic strength will reduce kA. However, it is unexpected that kA at 0 mM NaCl reaches 3.8 × 109 M−1 s−1 (Fig. 2c), which is the regime of the ARTICLE ARTICLE Jae-Yeol Kim 1, Fanjie Meng1, Janghyun Yoo1 & Hoi Sung Chung 1 Jae-Yeol Kim 1, Fanjie Meng1, Janghyun Yoo1 & Hoi Sung Chung 1 Intrinsically disordered proteins (IDPs) usually fold during binding to target proteins. In contrast to interactions between folded proteins, this additional folding step makes the binding process more complex. Understanding the mechanism of coupled binding and folding of IDPs requires analysis of binding pathways that involve formation of the transient complex (TC). However, experimental characterization of TC is challenging because it only appears for a very brief period during binding. Here, we use single-molecule ﬂuorescence spectroscopy to investigate the mechanism of diffusion-limited association of an IDP. A large enhancement of the association rate is observed due to the stabilization of TC by non-native electrostatic interactions. Moreover, photon-by-photon analysis reveals that the lifetime of TC for IDP binding is at least two orders of magnitude longer than that for binding of two folded proteins. This result suggests the long lifetime of TC is generally required for folding of IDPs during binding processes. 1 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892- 0520, USA. Correspondence and requests for materials should be addressed to H.S.C. (email: chunghoi@niddk.nih.gov) NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 1 Results Since this state with an intermediate FRET efﬁ- ciency is not always obviously detectable in a binned trajectory, we performed a maximum likelihood analysis of photon trajec- tories with the three-state model including the bound state, TC, and the unbound state (six states including the acceptor dark state, see Supplementary Fig. 3 and Methods) as shown in Fig. 3b. At 0, 10, 30, and 60 mM NaCl, there are peaks in the likelihood plot that are signiﬁcantly higher than the 95% conﬁdence level (Fig. 3c), and therefore, the lifetime of TC, tTC can be determined from the time at the maximum with high conﬁdence35. On the other hand, there is no peak above this conﬁdence level at 150 mM NaCl. In this case, only the upper bound of the lifetime can be determined35. At 90 mM, the peak is at the conﬁdence level, and we determined both the time at the maximum and the upper bound for comparison (Supplementary Table 2). (See Methods and Supplementary Fig. 5 to ﬁnd the measurable lifetimes given the amount of data and other parameters.) The analysis in Fig. 3c assumes that the FRET efﬁciency of TC (ETC) is equal to the average of those of the bound and unbound states (ETC = (EB + EU)/2). To check the validity of this assumption, we calculated the likelihood functions with various ETC values. Supplementary Smoluchowski limit. Despite the facilitation of binding by elec- trostatic attraction, this fast association is very unlikely unless a stable TC exists, in which two molecules are held together long enough for unfolded TAD to fold before separating from NCBD40. The lifetime of TC should be shorter than the bin time of 1 ms because the transitions appear instantaneous in Fig. 2a. For a better time resolution, we raised the laser illumination intensity by a factor of 5–10 and reduced the bin time to 100–200 μs (Fig. 3a)35,41,42. As indicated by yellow shades, there are several bins (200 μs bin time) with intermediate FRET efﬁciencies between the bound (E ~ 0.6) and unbound (E ~ 0.25) states, suggesting that an additional state exists between the bound and unbound states. Results Since this state with an intermediate FRET efﬁ- ciency is not always obviously detectable in a binned trajectory, we performed a maximum likelihood analysis of photon trajec- tories with the three-state model including the bound state, TC, and the unbound state (six states including the acceptor dark state, see Supplementary Fig. 3 and Methods) as shown in Fig. 3b. Figure 6 shows that the likelihood is higher at ETC lower than the midway value, suggesting that TAD is largely unstructured in the TC (0 mM NaCl). Therefore, we also determined tTC and ETC simultaneously that maximize the likelihood (see Methods for the analysis details). tTC determined in this way is generally longer as summarized in Fig. 3e. ETC increases with the increasing NaCl concentration similar to EU (Fig. 3e, inset), indicating unstruc- tured TAD in TC also becomes more compact at higher ionic strength. tTC in both analyses show a decreasing trend with increasing ionic strength (Fig. 3e). This result clearly indicates that TC is stabilized by electrostatic interactions, which makes diffusion-limited association possible. In this measurement, we particularly note that the lifetime of TC, 183 μs (or 630 μs when determined with ETC) at 0 mM NaCl is very long compared to transition path times (barrier crossing time) of protein folding, which has been measured so far to be between 2 and 12 μs depending on the presence of internal friction35,43,44. Given the analogy between the binding pathway and the transition path, we questioned if this long tTC could be a unique characteristic of coupled binding and folding of IDPs. y g g At 0, 10, 30, and 60 mM NaCl, there are peaks in the likelihood plot that are signiﬁcantly higher than the 95% conﬁdence level (Fig. 3c), and therefore, the lifetime of TC, tTC can be determined from the time at the maximum with high conﬁdence35. On the other hand, there is no peak above this conﬁdence level at 150 mM NaCl. In this case, only the upper bound of the lifetime can be determined35. At 90 mM, the peak is at the conﬁdence level, and we determined both the time at the maximum and the upper bound for comparison (Supplementary Table 2). (See Methods and Supplementary Fig. 5 to ﬁnd the measurable lifetimes given the amount of data and other parameters.) The analysis in Fig. Results Binding of TAD and NCBD depends strongly on ionic strength. In single-molecule FRET experiments, we immobilized TAD, which was labeled with a donor (Alexa 488) and an acceptor (Alexa 647) on a glass surface, and incubated with NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y A D A D + + A D A D TAD NCBD Barnase Barstar a b PEG PEG Biotin/ NeutrAvidin Biotin/ NeutrAvidin Fig. 1 Binding experiment of immobilized proteins. a Alexa 488 (D, donor) and Alexa 647 (A, acceptor) are attached to cysteine at the C-terminus and 4- acetylphenylalanine at the N-terminus of TAD, respectively. Dye-labeled molecules were immobilized on a polyethylene glycol (PEG)-coated glass surface via a biotin–NeutrAvidin linkage and incubated with unlabeled NCBD in solution. b Donor-labeled barnase was immobilized on a glass surface and incubated with acceptor-labeled barstar A D + TAD NCBD a PEG Bio Ne a Biotin/ NeutrAvidin + A A D Barstar EG Biotin/ NeutrAvidin + A D D Barnase Barstar b PEG Biotin/ NeutrAvidin b + D Barnase b PEG Biotin/ NeutrAvidin Fig. 1 Binding experiment of immobilized proteins. a Alexa 488 (D, donor) and Alexa 647 (A, acceptor) are attached to cysteine at the C-terminus and 4- acetylphenylalanine at the N-terminus of TAD, respectively. Dye-labeled molecules were immobilized on a polyethylene glycol (PEG)-coated glass surface via a biotin–NeutrAvidin linkage and incubated with unlabeled NCBD in solution. b Donor-labeled barnase was immobilized on a glass surface and incubated with acceptor-labeled barstar Smoluchowski limit. Despite the facilitation of binding by elec- trostatic attraction, this fast association is very unlikely unless a stable TC exists, in which two molecules are held together long enough for unfolded TAD to fold before separating from NCBD40. The lifetime of TC should be shorter than the bin time of 1 ms because the transitions appear instantaneous in Fig. 2a. For a better time resolution, we raised the laser illumination intensity by a factor of 5–10 and reduced the bin time to 100–200 μs (Fig. 3a)35,41,42. As indicated by yellow shades, there are several bins (200 μs bin time) with intermediate FRET efﬁciencies between the bound (E ~ 0.6) and unbound (E ~ 0.25) states, suggesting that an additional state exists between the bound and unbound states. Results 3c assumes that the FRET efﬁciency of TC (ETC) is equal to the average of those of the bound and unbound states (ETC = (EB + EU)/2). To check the validity of this assumption, we calculated the likelihood functions with various ETC values. Supplementary Lifetime of TC of barnase and barstar is extremely short. In order to answer the above question, we investigated the binding of barnase and barstar as a comparison (Fig. 1b). Binding of these two proteins is very similar to that of TAD and NCBD in that kA of the wildtype is close to that of the Smoluchowski limit and is very sensitive to the ionic strength of the solution45,46. The dif- ference is that both barnase and barstar are folded regardless of binding. Since there is very little conformational change during binding, a donor was attached to barnase and an acceptor was attached to barstar to monitor binding. Fig. Results 2b shows repre- sentative ﬂuorescence and FRET efﬁciency trajectories measured NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 3 ONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y 0 0.2 0.4 0.6 0.8 1 0 200 400 600 0 50 100 1 ms bin time 200 100 0 0.2 0.4 0.6 0.8 1 0 200 400 600 0 50 100 80 160 120 0 0.2 0.4 0.6 0.8 1 0 200 400 0 50 100 80 160 120 0 0.2 0.4 0.6 0.8 1 0 400 800 0 50 100 80 160 120 0 0.2 0.4 0.6 0.8 1 0 500 1000 0 50 100 80 160 120 0 mM 10 mM 30 mM 90 mM 150 mM [NaCl] 0 E 300 1 200 100 300 0 1 0 1 0 1 0 1 200 80 160 120 80 160 120 80 160 120 80 160 120 200 Acceptor Donor 0.025 μM [NCBD] 1.6 μM 8 μM 20 μM 0 0.2 0.4 0.6 0.8 1 0 200 400 0 50 100 100 180 140 60 mM 0 1 5 μM Time (ms) a 0 4 8 2 4 8 0 1 E 0 0.2 0.4 0.6 0.8 1 b 0 2 0 4 8 0 40 120 80 4 1 3 6 2 0 0 40 80 4 8 c d 0 mM 0 1 2 3 100 180 140 600 0 5000 10,000 20 ms bin time Photons/ms 6 Time (s) 2 4 8 6 Time (s) Time (ms) 0.1 μM FRET efficiency 20 nM Number of bins FRET efficiency Number of bins kA (× 109 M–1 s–1) [NaCl] (mM) kD (× 103 s–1) kA (× 107 M–1 s–1) [NaCl] (mM) kD (s–1) Photons/ms Fig. 2 Ionic strength dependence of binding kinetics. a Donor and acceptor ﬂuorescence trajectories (left), FRET efﬁciency trajectories (middle), and FRET efﬁciency histograms (right) of TAD/NCBD binding at various NaCl concentrations. Bin time is 1 ms and quoted numbers in the FRET efﬁciency histograms are NaCl (left) and NCBD (right) concentrations. Red arrows indicate photobleaching of the acceptor. b Donor and acceptor ﬂuorescence trajectories (left), FRET efﬁciency trajectory (middle), and FRET efﬁciency histogram (right) of binding of barnase and barstar (20 nM) at 0 mM NaCl. Bin time is 20 ms. Results Since there is no resonance energy transfer before binding, the FRET efﬁciency of the unbound state is the same as that of the donor-only state. (E is slightly higher than 0 because of the donor leak into the acceptor channel and direct excitation of the acceptor attached to barstar in solution.) In this case, acceptor photobleaching is not distinguishable from dis- sociation, and will reduce the apparent residence time in the bound state and result in the increased dissociation rate. There- fore, the two-state model parameters were determined using the maximum likelihood method with a correction for acceptor photobleaching (see Methods, Supplementary Fig. 7, and Sup- plementary Table 3 for the effect of acceptor photobleaching and its correction). kA at 0 mM NaCl is ~108 M−1 s−1, which is lowered by mutation and dye attachment compared to that of the wildtype. However, kA is very sensitive to the ionic strength while kD is unchanged (Fig. 2d), similar to the ionic strength depen- dence of the wildtype kinetics, suggesting that the binding mechanism is not altered. The most striking difference between the binding of barnase and barstar and binding of TAD and NCBD is found in the lifetime of TC (Fig. 3d). tTC of barnase and barstar binding is shorter than 2 μs (upper bound), which is at 0 mM NaCl, which exhibits multiple association and dis- sociation events. Since there is no resonance energy transfer before binding, the FRET efﬁciency of the unbound state is the same as that of the donor-only state. (E is slightly higher than 0 because of the donor leak into the acceptor channel and direct excitation of the acceptor attached to barstar in solution.) In this case, acceptor photobleaching is not distinguishable from dis- sociation, and will reduce the apparent residence time in the bound state and result in the increased dissociation rate. There- fore, the two-state model parameters were determined using the maximum likelihood method with a correction for acceptor photobleaching (see Methods, Supplementary Fig. 7, and Sup- plementary Table 3 for the effect of acceptor photobleaching and its correction). kA at 0 mM NaCl is ~108 M−1 s−1, which is lowered by mutation and dye attachment compared to that of the wildtype. However, kA is very sensitive to the ionic strength while kD is unchanged (Fig. Results c, d Association (red) and dissociation (blue) rate coefﬁcients of c TAD/NCBD and d barnase/barstar binding 0 50 100 1 ms bin time 200 100 0 50 100 80 160 120 0 50 100 80 160 120 0 50 100 80 160 120 0 50 100 80 160 120 300 200 Acceptor Donor 0 50 100 100 180 140 Time (ms) a Photons/ms 0 E 1 200 100 300 0 1 0 1 0 1 0 1 80 160 120 80 160 120 80 160 120 80 160 120 200 0 1 100 180 140 Time (ms) a 0 2 0 4 8 0 40 120 80 4 1 3 6 2 c kA (× 109 M–1 s–1) [NaCl] (mM) kD (× 103 s–1) c Number of bins 0 0 40 80 4 8 d 0 1 2 3 kA (× 107 M–1 s–1) [NaCl] (mM) kD (s–1) 0 0.2 0.4 0.6 0.8 1 0 400 800 0 0.2 0.4 0.6 0.8 1 0 500 1000 90 mM 150 mM 8 μM 20 μM 0 0.2 0.4 0.6 0.8 1 0 FRET efficiency d FRET efficiency 0 0.2 0.4 0.6 0.8 1 0 mM 0 5000 10,000 20 nM Number of bins FRET efficiency 0 4 8 2 4 8 b 20 ms bin time Photons/ms 6 Time (s) b 0 1 E 2 4 8 6 Time (s) Number of bins Fig. 2 Ionic strength dependence of binding kinetics. a Donor and acceptor ﬂuorescence trajectories (left), FRET efﬁciency trajectories (middle), and FRET efﬁciency histograms (right) of TAD/NCBD binding at various NaCl concentrations. Bin time is 1 ms and quoted numbers in the FRET efﬁciency histograms are NaCl (left) and NCBD (right) concentrations. Red arrows indicate photobleaching of the acceptor. b Donor and acceptor ﬂuorescence trajectories (left), FRET efﬁciency trajectory (middle), and FRET efﬁciency histogram (right) of binding of barnase and barstar (20 nM) at 0 mM NaCl. Bin time is 20 ms. c, d Association (red) and dissociation (blue) rate coefﬁcients of c TAD/NCBD and d barnase/barstar binding shorter than that of TAD and NCBD by two orders of magnitude (Fig. 3e). shorter than that of TAD and NCBD by two orders of magnitude (Fig. 3e). at 0 mM NaCl, which exhibits multiple association and dis- sociation events. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y a 0 25 50 101 102 103 101 102 103 0 25 50 116 0 25 50 121 122 121 122 0 25 50 131 132 132 0 25 50 134 136 75 134 136 0 25 50 116 Acceptor Donor 100 0 50 110 111 110 111 0 111 112 113 111 112 113 10 30 20 40 0 20 40 0 mM 30 mM 0 20 90 mM 0 0 4 8 –4 –8 150 mM 0 4 8 –4 –8 10 mM 40 80 0 mM 0 4 8 –4 –8 60 mM 0 4 8 –4 –8 Δ in L Δ in L b B TC U Unbound TC Bound tTC = 1/(2kTC) E c d 0 200 0 40 120 80 600 400 e ETC 0.4 0 40 [NaCl] (mM) 80 0.2 0.6 12 –12 114 116 115 115 114 131 114 115 116 114 115 Time (ms) Time (ms) Photons/200 μs 200 μs bin time E 0.6 0.2 E 0.6 0.2 E 0.6 0.2 E 0.6 0.2 E 0.6 0.2 E 0.6 0.2 E 0.6 0.2 E 0.6 0.2 Time kTC kTC kA,app′ kD′ Δ in L 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) tTC (μs) [NaCl] (mM) Fig. 3 Measurement of the lifetime of transient complex. a Selected donor and acceptor ﬂuorescence trajectories (left) and FRET efﬁciency trajectories (right) collected at high illumination intensity (200 μs bin time) that exhibit gradual changes in the ﬂuorescence intensities and FRET efﬁciency (yellow shade) during association and dissociation. The accurate lifetime of TC was determined using the maximum likelihood analysis of photon trajectories without binning (see e). b Three-state model to determine the lifetime of TC (tTC). The two rate coefﬁcients of the transitions from TC to the bound and unbound states are set to be equal (kTC) for the convenience of the analysis, which does not reﬂect the actual relative heights of the two barriers in Fig. 4c. c The difference of log-likelihood (Δln L) plots for binding of TAD and NCBD as a function of tTC at various NaCl concentrations (quoted numbers). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y Errors are standard deviations obtained from the diagonal elements of the covariance matrix calculated at the maximum of the likelihood function NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y ARTICLE B TC U Unbound TC Bound tTC = 1/(2kTC) E Time kTC kTC kA,app′ kD′ Unbound TC Bound tTC = 1/(2kTC) E Time b b a c 40 0 20 40 0 mM 30 mM 0 20 0 10 mM 40 80 c Δ in L 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) Photons/200 μs tTC (μs) tTC (μs) 90 mM 0 4 8 –4 –8 150 mM 0 4 8 –4 –8 60 mM 0 4 8 –4 –8 Δ in L 12 –12 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 tTC (μs) 90 mM 0 4 8 –4 –8 60 mM 0 4 8 –4 –8 Δ in L 12 –12 0.1 1 10 100 1000 tTC (μs) 0.1 1 10 100 1000 tTC (μs) 150 mM 0 4 8 –4 –8 0 0.1 1 10 100 1000 tTC (μs) 90 mM 0 4 8 –4 –8 0.1 1 10 100 1000 tTC (μs) 0 200 0 40 120 80 600 400 e ETC 0.4 0 40 [NaCl] (mM) 80 0.2 0.6 tTC (μs) [NaCl] (mM) 0 mM 0 4 8 –4 –8 Δ in L d 0.1 1 10 100 1000 tTC (μs) e d 110 111 111 112 113 116 114 115 Time (ms) E 0.6 0.2 E 0.6 0.2 d Fig. 3 Measurement of the lifetime of transient complex. a Selected donor and acceptor ﬂuorescence trajectories (left) and FRET efﬁciency trajectories (right) collected at high illumination intensity (200 μs bin time) that exhibit gradual changes in the ﬂuorescence intensities and FRET efﬁciency (yellow shade) during association and dissociation. The accurate lifetime of TC was determined using the maximum likelihood analysis of photon trajectories without binning (see e). b Three-state model to determine the lifetime of TC (tTC). The two rate coefﬁcients of the transitions from TC to the bound and unbound states are set to be equal (kTC) for the convenience of the analysis, which does not reﬂect the actual relative heights of the two barriers in Fig. 4c. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y c The difference of log-likelihood (Δln L) plots for binding of TAD and NCBD as a function of tTC at various NaCl concentrations (quoted numbers). Δln L = ln L(tTC) −ln L(0) compares the likelihood of the three-state model with a ﬁnite lifetime, tTC, with the model with an instantaneous transition (tTC = 0). The FRET efﬁciency of TC is assumed to be the average of the bound and unbound FRET efﬁciencies (ETC = (EB + EU)/2). When the peak of the likelihood is signiﬁcantly higher than the 95% conﬁdence level (Δln L = +3, upper dashed line), tTC can be determined from the time at the maximum. When there is no signiﬁcant peak, the upper bound of the lifetime can be determined from the time where Δln L crosses the lower 95% conﬁdence level (Δln L = −3, lower dashed line). d Δln L plotted for binding of barnase and barstar at 0 mM NaCl. e The dependence of tTC on the NaCl concentration. tTC of TAD and NCBD binding is determined from either the maximum of Δln L in c (blue) or by maximizing Δln L with ETC as a free parameter (red, see Methods). Fitted values of ETC are shown in the inset. Green ﬁlled squares are the upper bound of tTC at 90 and 150 mM NaCl. A purple ﬁlled square is the upper bound of tTC of barnase and barstar binding at 0 mM NaCl in d. Fitting parameters are also listed in Supplementary Table 2. Errors are standard deviations obtained from the diagonal elements of the covariance matrix calculated at the maximum of the likelihood function of binding in the simulation is still much shorter than that of TAD and NCBD. The native-like transition state and other transient intermediate states explain why the lifetime of TC of barnase and barstar binding is very short compared to that of TAD and NCBD. shows that the early intermediate states with different orienta- tions would not affect overall binding kinetics signiﬁcantly48. In the simulation, the lifetime of the transition state is 2 μs, which is close to the upper bound of the lifetime of TC. Although the lifetime of late intermediate states is ~10 μs, which is not detected as a signiﬁcant peak in the likelihood plots with different ETC values in our analysis (Supplementary Fig. Results 2d), similar to the ionic strength depen- dence of the wildtype kinetics, suggesting that the binding mechanism is not altered. The most striking difference between the binding of barnase and barstar and binding of TAD and NCBD is found in the lifetime of TC (Fig. 3d). tTC of barnase and barstar binding is shorter than 2 μs (upper bound), which is Non-native electrostatic interactions stabilize TC of TAD. The above results, similar sensitivity of the association rate coefﬁcient to the ionic strength but very different tTC of the two systems, can be explained by very different binding mechanisms. First, the binding interface of barnase and barstar comprises pre- dominantly electrostatic interactions between oppositely charged sidechains (Fig. 4a). Vijayakumar et al.47 have shown that increased kA compared to that of the basal rate (inﬁnite ionic strength) results from the reduced free energy of the transition state (i.e., barrier height) due to the electrostatic interactions. In addition, the conﬁgurations of the two molecules at the transition state are very close to the structure of the bound state. The two molecules are shifted away slightly to accommodate a layer of water molecules and the relative orientation of the two binding interfaces can be only slightly tilted (within 3°). In other words, when two molecules approach with different orientations, bind- ing would not happen (Fig. 4d). This interpretation is consistent with the recent MD simulation result by Plattner et al., which NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 4 4 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y Δln L = n L(tTC) −ln L(0) compares the likelihood of the three-state model with a ﬁnite lifetime, tTC, with the model with an instantaneous transition (tTC = 0). The FRET efﬁciency of TC is assumed to be the average of the bound and unbound FRET efﬁciencies (ETC = (EB + EU)/2). When the peak of the likelihood is signiﬁcantly higher than the 95% conﬁdence level (Δln L = +3, upper dashed line), tTC can be determined from the time at the maximum. When there is no signiﬁcant peak, the upper bound of the lifetime can be determined from the time where Δln L crosses the lower 95% conﬁdence level (Δln L = −3, lower dashed line). d Δln L plotted for binding of barnase and barstar at 0 mM NaCl. e The dependence of tTC on the NaCl concentration. tTC of TAD and NCBD binding is determined from either the maximum of Δln L in c (blue) or by maximizing Δln L with ETC as a free parameter (red, see Methods). Fitted values of ETC are shown in the inset. Green ﬁlled squares are the upper bound of tTC at 90 and 150 mM NaCl. A purple ﬁlled square is the upper bound of tTC of barnase and barstar binding at 0 mM NaCl in d. Fitting parameters are also listed in Supplementary Table 2. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y 8), the overall duration On the other hand, the binding interface of TAD and NCBD consists of hydrophobic interactions although both molecules are NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y Glu80 Arg59 Lys27 Arg83 Glu76 Asp53 Asp39 Arg87 [NaCl] Unbound TC Bound Free energy Reaction coordinate TAD \| NCBD TAD NCBD TAD + NCBD k+ k– kf a b c d Fig. 4 Binding mechanisms. a Electrostatic interactions in the binding interface of barnase and barstar. Blue and red sticks indicate positively charged side chains of barnase (cyan) and negatively charged side chains of barstar (orange), respectively. Dashed ellipses show two clusters of interacting residues. b Positively charged side chains (blue sticks) of NCBD (orange molecule) and negatively charged side chains (red sticks) of TAD (cyan molecule). Charged side chains are exposed to the solvent and do not interact except those in one salt bridge indicated by a black arrow. Binding interface is predominantly hydrophobic. c Kinetic scheme of the association via the formation of TC, and proposed free energy surface of TAD/NCBD binding with an arbitrary NCBD concentration at different NaCl concentrations. k+ and k−are the association and dissociation rate coefﬁcients of the formation of TC. kf is the folding rate of unfolded TAD in TC. kA = k+kf/(kf + k−). As [NaCl] is increased, the formation of TC becomes slower (reduced k+) due to the increased charge screening effect, and TC becomes less stable and dissociates more easily (increased k−, lowered dissociation barrier) before TAD folds to form a fully bound complex. Both effects decrease kA. d The transition state conﬁgurations (indicated by green O) of barnase (cyan) and barstar (orange) are very similar to that of the bound complex, and therefore, the conﬁgurations with unfavorable orientations will not result in association (indicated by red x). Positively and negatively charged side chains in the binding interface are colored in blue and red. On the other hand, the association can be initiated from different parts of NCBD (orange) via non-native interactions between charged residues (i.e., parallel binding pathways23,24, indicated by green O). Cyan coils represent disordered TAD. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y Dashed ellipses show two clusters of interacting residues. b Positively charged side chains (blue sticks) of NCBD (orange molecule) and negatively charged side chains (red sticks) of TAD (cyan molecule). Charged side chains are exposed to the solvent and do not interact except those in one salt bridge indicated by a black arrow. Binding interface is predominantly hydrophobic. c Kinetic scheme of the association via the formation of TC, and proposed free energy surface of TAD/NCBD binding with an arbitrary NCBD concentration at different NaCl concentrations. k+ and k−are the association and dissociation rate coefﬁcients of the formation of TC. kf is the folding rate of unfolded TAD in TC. kA = k+kf/(kf + k−). As [NaCl] is increased, the formation of TC becomes slower (reduced k+) due to the increased charge screening effect, and TC becomes less stable and dissociates more easily (increased k−, lowered dissociation barrier) before TAD folds to form a fully bound complex. Both effects decrease kA. d The transition state conﬁgurations (indicated by green O) of barnase (cyan) and barstar (orange) are very similar to that of the bound complex, and therefore, the conﬁgurations with unfavorable orientations will not result in association (indicated by red x). Positively and negatively charged side chains in the binding interface are colored in blue and red. On the other hand, the association can be initiated from different parts of NCBD (orange) via non-native interactions between charged residues (i.e., parallel binding pathways23,24, indicated by green O). Cyan coils represent disordered TAD. Red bars indicate negative charges in TAD and positively charged side chains of NCBD are colored in blue given the short chain length of TAD in this study (49 residues), the ﬂy-casting type enhancement of the association rate is expected50. This enhancement will also be reduced by increased ionic strength, which makes disordered TAD more compact39. highly charged15. All charged side chains except two participating in the native salt bridge (indicated by a black arrow in Fig. 4b) are located on the opposite side of the binding interface (Fig. 4b). In this case, the formation of the native interactions will be hardly affected by the ionic strength of the solution. Therefore, the electrostatic interactions must be involved in the formation of the TC and these interactions are non-native, although non-native hydrophobic interactions can also contribute33,49. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y The long lifetime of TC indicates that once non-native contacts are formed between charged side chains, two molecules would stick together so that TAD rearranges and folds on the surface of NCBD without dissociation (small k−in Fig. 4c). In this way, the association can be diffusion-limited. As the ionic strength is increased, the formation of TC will be slower (reduced k+ in Fig. 4c) due to the increased charge screening effect, and TC becomes less stable and dissociates more easily before TAD folds to form a fully bound complex (increased k−). Both effects will decrease the association rate (kA = k+kf/[kf + k−]) (Fig. 4c). Non- native interactions at low ionic strength also suggest slow diffusion along the reaction coordinate, which contributes to the increased tTC along with the stability of TC similar to the increased folding transition path time of a designed protein, α3D by non-native salt-bridge formation44. highly charged15. All charged side chains except two participating in the native salt bridge (indicated by a black arrow in Fig. 4b) are located on the opposite side of the binding interface (Fig. 4b). In this case, the formation of the native interactions will be hardly affected by the ionic strength of the solution. Therefore, the electrostatic interactions must be involved in the formation of the TC and these interactions are non-native, although non-native hydrophobic interactions can also contribute33,49. The long lifetime of TC indicates that once non-native contacts are formed between charged side chains, two molecules would stick together so that TAD rearranges and folds on the surface of NCBD without dissociation (small k−in Fig. 4c). In this way, the association can be diffusion-limited. As the ionic strength is increased, the formation of TC will be slower (reduced k+ in Fig. 4c) due to the increased charge screening effect, and TC becomes less stable and dissociates more easily before TAD folds to form a fully bound complex (increased k−). Both effects will decrease the association rate (kA = k+kf/[kf + k−]) (Fig. 4c). Non- native interactions at low ionic strength also suggest slow diffusion along the reaction coordinate, which contributes to the increased tTC along with the stability of TC similar to the increased folding transition path time of a designed protein, α3D by non-native salt-bridge formation44. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y Red bars indicate negative charges in TAD and positively charged side chains of NCBD are colored in blue Glu80 Arg59 Lys27 Arg83 Glu76 Asp53 Asp39 Arg87 [NaCl] Unbound TC Bound Free energy Reaction coordinate TAD \| NCBD TAD NCBD TAD + NCBD k+ k– kf a b c d b b a d Free energy Reaction coordinate Reaction coordinate Fig. 4 Binding mechanisms. a Electrostatic interactions in the binding interface of barnase and barstar. Blue and red sticks indicate positively charged side chains of barnase (cyan) and negatively charged side chains of barstar (orange), respectively. Dashed ellipses show two clusters of interacting residues. b Positively charged side chains (blue sticks) of NCBD (orange molecule) and negatively charged side chains (red sticks) of TAD (cyan molecule). Charged side chains are exposed to the solvent and do not interact except those in one salt bridge indicated by a black arrow. Binding interface is predominantly hydrophobic. c Kinetic scheme of the association via the formation of TC, and proposed free energy surface of TAD/NCBD binding with an arbitrary NCBD concentration at different NaCl concentrations. k+ and k−are the association and dissociation rate coefﬁcients of the formation of TC. kf is the folding rate of unfolded TAD in TC. kA = k+kf/(kf + k−). As [NaCl] is increased, the formation of TC becomes slower (reduced k+) due to the increased charge screening effect, and TC becomes less stable and dissociates more easily (increased k−, lowered dissociation barrier) before TAD folds to form a fully bound complex. Both effects decrease kA. d The transition state conﬁgurations (indicated by green O) of barnase (cyan) and barstar (orange) are very similar to that of the bound complex, and therefore, the conﬁgurations with unfavorable orientations will not result in association (indicated by red x). Positively and negatively charged side chains in the binding interface are colored in blue and red. On the other hand, the association can be initiated from different parts of NCBD (orange) via non-native interactions between charged residues (i.e., parallel binding pathways23,24, indicated by green O). Cyan coils represent disordered TAD. Red bars indicate negative charges in TAD and positively charged side chains of NCBD are colored in blue Fig. 4 Binding mechanisms. a Electrostatic interactions in the binding interface of barnase and barstar. Blue and red sticks indicate positively charged side chains of barnase (cyan) and negatively charged side chains of barstar (orange), respectively. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y isopropylthiogalactoside (IPTG) for pJ414-TAD and pJ411-BirA, and 1 mM arabinose for pEvol in the presence of 1 mM 4-acetylphenylalanine and 50 µM d- biotin (B4501, Sigma-Aldrich). After further incubation at 37 °C for 4 h, cells were collected by centrifugation at 8000g for 10 min and the cell pellet was re-suspended in appropriate lysis buffers: 20 mM Tris–HCl pH 7.5, 8 M Urea, 10 mM DTT for TAD and NCBD, and 20 mM Tris–HCl pH 7.5, 400 mM NaCl, 10 mM DTT for barnase and barstar. Cells were lysed by brief sonication and resting cycles and the lysates were centrifuged at 50,000g for 40 min to collect supernatants. orders of magnitude shorter because there is no folding step during binding (Fig. 3). Moreover, we found that TC of TAD and NCBD is stabilized by non-native electrostatic interactions, which is also required for fast association. q Sugase et al. have found that the association rate can be enhanced by non-native hydrophobic interactions for a much slower binding system (binding of the disordered phosphorylated kinase inducible activation domain (pKID) and the KIX domain of CBP) using NMR relaxation dispersion experiments33. ϕ-Value analysis has also shown the involvement of non-native interac- tions for the same system49. The enhancement of the association by non-native interactions have also been found for various systems in simulations23,24,52. Therefore, the formation of a stable TC via non-native interactions may be a general mechanism for IDP binding. Although non-native hydrophobic interactions can contribute33,49, we would like to point out that non-native elec- trostatic interactions should prevail since IDPs usually possess a large number of charged residues53 while their binding interfaces are hydrophobic54 (e.g., amphipathic helix formation). y g g p From the supernatant, 6His-GB1-Thb-Avi-UA-TAD-Cys was puriﬁed by Ni- NTA afﬁnity chromatography. Streptavidin Mutein column (03708152001, Roche Diagnostics) was also used to purify biotinylated proteins. The truncated protein without incorporation of UA (6His-GB1-Avi) was separated by size-exclusion chromatography using AKTA pure FPLC system with SuperdexTM75 10/300GL (GE Healthcare, Chicago, IL). The incorporation rate of UA was about 30%. Then, 6His-GB1 was cut by thrombin in 20 mM Tris–HCl pH 7.4, 100 mM NaCl, and removed using IgG Sepharose 6 Fast Flow column (GE Healthcare, Chicago, IL), which binds GB1 speciﬁcally. Avi-UA-TAD-Cys was recovered from the unbound ﬂow-through. ARTICLE In the case of diffusion-limited association at 0 mM NaCl, the lifetime of TC (several hundred microseconds) corre- sponds to the folding time of TAD (tTC = 1/[kf + k−] and k−« kf in Fig. 4c). This time is actually much longer than folding times of many single domain (α-helical) fast-folding proteins that can fold in several microseconds or even shorter57. The slow folding rate of TAD may result from the increased internal friction by non- native interactions as found for folding of a designed protein, α3D44. Between the two opposite effects of non-native interac- tions on the overall rate of diffusion-limited association (kA = k+kf/[kf + k−]), the enhancement by avoiding dissociation (reduced k−)23 is much larger than the reduction due to slower folding by a few fold (reduced kf)44. Single-molecule spectroscopy. Single-molecule FRET experiments were per- formed using a confocal microscope system (MicroTime200, Picoquant) with a 75 μm diameter pinhole, a beamsplitter (ZT405/488/635rpc, Chroma Technology), and an oil-immersion objective (UPLSAPO, NA 1.4, ×100, Olympus)61. Alexa 488 was excited by a 485 nm diode laser (LDH-D-C-485, PicoQuant) in the CW mode. Alexa 488 and Alexa 647 ﬂuorescence was split into two channels using a beamsplitter (585DCXR, Chroma Technology) and focused through optical ﬁlters (ET525/50m for Alexa 488 and E600LP for Alexa 647, Chroma Technology) onto photon-counting avalanche photodiodes (SPCM-AQR-16, PerkinElmer Optoelectronics). In contrast to binding of barnase and barstar with a native-like TC, binding pathways are expected to be more heterogeneous because binding of TAD can be initiated from various parts of NCBD by non-native interactions (Fig. 4d) as observed in simulations for other systems23,24. For a more fundamental understanding of the binding mechanism, it will be very impor- tant to further investigate the characteristics of the TC including the heterogeneity of binding pathways for various IDP systems. In the immobilization experiment, biotinylated TAD or barnase molecules were immobilized on a biotin-embedded, polyethylene glycol-coated glass coverslip (Bio_01, Microsurfaces Inc.) via a biotin (surface)-NeutrAvidin-biotin (protein) linkage51. The surface was initially incubated with NeutrAvidin (30 μg/mL) for 5 min and subsequently with the solution of TAD or barnase (80 pM) for 3 min. Then, immobilized molecules were incubated with unlabeled NCBD or barstar for binding. Molecules were illuminated at 3 and 0.3 μW for the measurement of the binding kinetics of TAD/NCBD and barnase/barstar systems, respectively. In the measurement of the lifetime of the TC, molecules were illuminated at 15–60 μW. ARTICLE NCBD, Avi-barnase, and Cys-barstar were puriﬁed similarly by performing Ni-NTA puriﬁcation and size exclusion chromatography followed by thrombin cleavage and removal of 6His-GB1. g Avi-UA-TAD-Cys was labeled with Alexa Fluor 488 maleimide (Alexa 488, A10254, Thermo Fisher Scientiﬁc) at the cysteine residue and Alexa Fluor 647 hydroxylamine (Alexa 647, A30632, Thermo Fisher Scientiﬁc) at UA. First, 100 µL of 100 µM Avi-UA-TAD-Cys in 50 mM Tris–HCl, pH 7.0, 6 M GdmCl was incubated with 1.5 mM TCEP for 2 h to reduce disulﬁde bonds, and 100 µg of Alexa 488 dissolved in 5 µL of DMSO was added. The reaction solution was incubated at room temperature for 4 h. Unreacted Alexa 488 dyes were removed by size-exclusion chromatography using superdexTMPeptide 10/300GL. Alexa 647 was attached to UA using the formation of oxime between ketone and hydroxylamine60. The Alexa 488 labeled Avi-UA-TAD-Cys was incubated with Alexa 647 in an oxime reaction buffer, 50 mM sodium acetate, pH 4.0, 100 mM NaCl, 4 M GdmCl for 20 h at room temperature because oxime reaction requires low pH and shows slow reaction rate compared to the cysteine–maleimide reaction60. The reaction was quenched by raising pH to 7 and the unreacted dyes were removed using size-exclusion chromatography. Barnase and barstar were labeled with Alexa 488 maleimide and Alexa 647 maleimide, respectively, and puriﬁed similarly. Avi-UA-TAD-Cys was labeled with Alexa Fluor 488 maleimide (Alexa 488, A10254, Thermo Fisher Scientiﬁc) at the cysteine residue and Alexa Fluor 647 hydroxylamine (Alexa 647, A30632, Thermo Fisher Scientiﬁc) at UA. First, 100 µL of 100 µM Avi-UA-TAD-Cys in 50 mM Tris–HCl, pH 7.0, 6 M GdmCl was incubated with 1.5 mM TCEP for 2 h to reduce disulﬁde bonds, and 100 µg of Alexa 488 dissolved in 5 µL of DMSO was added. The reaction solution was incubated at room temperature for 4 h. Unreacted Alexa 488 dyes were removed by size-exclusion chromatography using superdexTMPeptide 10/300GL. Alexa 647 was attached to UA using the formation of oxime between ketone and It is also worth noting that the enhancement of association by non-native interactions is an important mechanism for IDP binding, whereas in protein folding, non-native interactions do not affect the folding mechanism55, but slow the folding pro- cess44,56. ARTICLE All experiments were performed in 10 mM Tris buffer (pH 7) with 0–150 mM of NaCl. To reduce dye photobleaching and blinking, 2 mM cyclooctatetraene (COT), 2 mM 4-nitrobenzyl alcohol (NBA), 2 mM trolox62,63, 10 mM cysteamine, and 100 mM β-mercaptoethanol64 were used in addition to an oxygen scavenging system, 50 nM protocatechuate 3,4-dioxygenase (PCD, P8279-25UN, Sigma) and 2.5 mM 3,4-dihydroxybenzoic acid (PCA, 37580–25G-F, Sigma)65. Discussion As stated in the Introduction, the most crucial information for understanding binding mechanisms of IDPs is contained in binding pathways including the formation of TC, in which con- formational changes actually occur, analogous to the transition path in protein folding51. However, diversity in binding pathways and the short lifetime of TC make characterization very difﬁcult. As a ﬁrst step toward this direction, we employed photon-by- photon analysis for single-molecule FRET with high time reso- lution to systematically measure the lifetime of TC of TAD and NCBD as a function of ionic strength and describe the binding mechanism. We found that both binding kinetics and lifetime of TC are very sensitive to the ionic strength of the solution (Figs. 2, 3). More importantly at low ionic strength, the association is diffusion-limited, which is very unlikely to happen in IDP binding, while the lifetime of TC is unexpectedly long, i.e., several hundred microseconds. Our interpretation is that diffusion- limited association of TAD requires a long lifetime of TC, during which unstructured TAD in TC can fold without dissociation. In contrast, the lifetime of TC of barnase and barstar is at least two y g Since the unbound TAD is very extended due to the electrostatic repulsion as indicated by the low FRET efﬁciency NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y For this model, the rate matrix is given by44 ð3Þ K ¼ k′D kd kTC 0 kb 0 0 k′D 2kTC kd k′A;app 0 kb 0 0 kTC k′A;app kd 0 0 kb kd 0 0 k′D kb kTC 0 0 kd 0 k′D 2kTC kb k′A;app 0 0 kd 0 kTC k′A;app kb 0 B B B B B B B B @ 1 C C C C C C C C A ð6Þ K ¼ k′D kd kTC 0 kb 0 0 k′D 2kTC kd k′A;app 0 kb 0 0 kTC k′A;app kd 0 0 kb kd 0 0 k′D kb kTC 0 0 kd 0 k′D 2kTC kb k′A;app 0 0 kd 0 kTC k′A;app kb 0 B B B B B B B B @ 1 C C C C C C C C A ð6Þ where Ed is the FRET efﬁciency of the acceptor dark state, and kb (kd) is the rate coefﬁcient for the transition from the dark (bright) state to the bright (dark) state of the acceptor. kb is independent of the photon count rate. On the other hand, as the probability of the transition from the bright state to the dark state will increase linearly with the time spent in the excited state, kd is proportional to the photon count rate of each trajectory as kd = k0 (n/n0), where n is the average photon count rate of a photon trajectory and k0 is the rate coefﬁcient at the reference photon count rate, n0 = 100 ms−1. pb = kb/(kb + kd) is the equilibrium population of the acceptor bright state. ð6Þ In the analysis of TAD/NCBD binding, ﬁrst, the bound and unbound segments were assigned using the Viterbi algorithm67,68 for the two-state model, adapted for photon trajectories69. Since transitions with short residence times caused by acceptor blinking were frequently assigned due to the relatively low EU value, transitions with residence times longer than ~200 μs in both bound and unbound states were analyzed. When there are multiple transitions in a trajectory, each pair of segments with a single binding or dissociation transition was analyzed separately. For the binding kinetics of barnase and barstar, the effect of microsecond time scale acceptor blinking is negligible because the photon count rate is much lower and the binding kinetics is much slower37. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y The likelihood function of the three- and six-state models in Supplementary Fig. 3c and d for the jth photon trajectory with a single transition is35 where Nj is the number of photons in the jth trajectory, ci is the color of the ith photon (donor or acceptor), and τi is a time interval between the (i −1)th and ith photons. K is the rate matrix, the photon color matrix F depends on color c of a photon as F(acceptor) = E and F(donor) = I −E, where E is a diagonal matrix with the uncorrected (apparent) FRET efﬁciencies of the individual states on the diag- onal, I is the unity matrix, 1T is the unit row vector (T means transpose), and peq is the vector of equilibrium populations. The likelihood function was calculated by the diagonalization of K as described in ref. 38. Practically, the total log-likelihood function of all trajectories was calculated by summing individual log-likelihood functions as ln L ¼ Σ jln Lj. where Nj is the number of photons in the jth trajectory, ci is the color of the ith photon (donor or acceptor), and τi is a time interval between the (i −1)th and ith photons. K is the rate matrix, the photon color matrix F depends on color c of a photon as F(acceptor) = E and F(donor) = I −E, where E is a diagonal matrix with the uncorrected (apparent) FRET efﬁciencies of the individual states on the diag- onal, I is the unity matrix, 1T is the unit row vector (T means transpose), and peq is the vector of equilibrium populations. The likelihood function was calculated by the diagonalization of K as described in ref. 38. Practically, the total log-likelihood function of all trajectories was calculated by summing individual log-likelihood functions as ln L ¼ Σ jln Lj. Lj ¼ vT fin Y Nj i¼2 FðciÞ exp Kτi ð Þ ½ Fðc1Þvini ð4Þ ð4Þ where vini and vﬁn are the state vectors at the beginning and end of the trajectory, respectively. For the binding transition, for example, vini = [0 0 1]T and vﬁn = [1 0 0]T for the three-state model (Supplementary Fig. 3c) and vini = [0 0 pb 0 0 (1 −pb)] T and vﬁn = [1 0 0 1 0 0]T for the six-state model (Supplementary Fig. 3d). In the calculation of the likelihood function in Eq. Methods P t i Protein expression, puriﬁcation, and dye-labeling. TAD was engineered as a fusion protein, 6His-GB1-Thb-Avi-UA-TAD-Cys. The sequence of 6His-GB1-Avi- UA-TAD-Cys consists of MGSSHHHHHHSSGMQYKLILNGKTLKGETTTEAV- DAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (6His-56 residue long immunoglobulin-binding domain B1 of streptococcal protein G (GB1)), SSGLVPRGSGH MGMS (thrombin cleavage site ﬂanked by spacer residues), GLNDIFEAQKIEWHE (biotin acceptor peptide termed Avi, Avidity LLC), SSGLVAGGGGSGGGGSGGGGS (long spacer) and UPLSQETFSDLWKLL- PENNVLSPLPSQ AMDDLMLSPDDIEQWFTEDPGPDC (UA-TAD-Cys). UA- TAD-Cys denotes the TAD of the tumor suppressor protein p53 (residue 13–61)15 with the incorporation of an unnatural amino acid (UA, 4-acetylphenylalanine, SC- 35005, SynChem Inc.) and a cysteine residue at the N- and C-terminus, respec- tively. NCBD (residue 2059–2117)15,21, barnase, and barstar58 were also designed as fusion proteins, 6His-GB1-Thb-NCBD, 6His-GB1-Thb-Avi-barnase (G34C/ H102A), and 6His-GB1-Thb-Cys-barstar. The protein sequences are listed in Supplementary Fig. 1. All genes were cloned in pJ414 vector ﬂanked by Nde1 and BamH1 restriction sites (DNA 2.0 Inc.). y y g In the free diffusion experiment, 80 pM of TAD was mixed with NCBD at various NaCl concentrations. The same chemicals used for the immobilization experiment were added to reduce dye photobleaching and blinking. To prevent protein sticking to a glass surface, 0.01% Tween 20 was used66. The solution was illuminated at 22 μW and ﬂuorescence bursts were measured 10 μm above the glass surface. All experiments were performed at room temperature (22 °C). Maximum likelihood analysis. To determine the parameters for the two-state binding kinetics, we used the maximum likelihood method developed by Gopich and Szabo that analyzes photon trajectories directly without binning38. The like- lihood function for the jth photon trajectory with records of photon colors and arrival times is For the expression of TAD with biotinylation and incorporation of UA, three plasmids, pJ414-TAD, pJ411-BirA, and pEvol were cotransformed into Escherichia coli. BL21(DE3) (200131, Agilent Technologies). pJ411-BirA encodes biotin ligase for biotinylation and pEvol encodes the amber codon (TAG) suppressor tRNACUA and amino acetyl-tRNA synthetase for 4-acetylphenylalanine59. The transformed cells were grown in Luria-Bertani medium. Expression was induced at an absorbance of 0.7 monitored at 600 nm by adding ﬁnal concentration of 1 mM Lj ¼ 1T Y Nj i¼2 FðciÞ exp Kτi ð Þ ½ Fðc1Þpeq ð; 1Þ ð; 1Þ absorbance of 0.7 monitored at 600 nm by adding ﬁnal concentration of 1 mM 7 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y Therefore, we used the two-state model (Eq. (2)). However, because the FRET efﬁciency of the unbound state is the same as that of the donor-only state (only the donor is attached to immobilized barnase), acceptor photobleaching is indistinguishable from dissociation. In this case, the waiting time in the bound state will be underestimated, which will increase the apparent dissociation rate and decrease the bound fraction, pB. Therefore, we ﬁrst determined pB from the fraction of the trajectories that begin with the bound state (high FRET efﬁciency), and then determined other two-state parameters by ﬁxing pB (Eq. (2)). The accuracy of the parameters determined with and without ﬁxing the bound fraction is compared in Supplementary Fig. 7 for the trajectories recolored with experimental parameters. As expected, pB and kA,app decrease and kD increases as the acceptor bleaching rate, kbleach increases when the parameters are extracted with pB as a free ﬁtting parameter, whereas extracted rates are very accurate regardless of kbleach when pB is ﬁxed. p y In the six-state analysis, before calculating Δln L(tTC) as a function of tTC, the blinking parameters kb and pb and the average FRET efﬁciencies of the bound and unbound states (EB and EU) were ﬁrst determined by maximizing Δln L(0) (instantaneous transition model with acceptor blinking (kTC →∞)). With these parameters, Δln L(tTC) was calculated with ﬁxed ETC = (EB + EU)/2 as shown in Fig. 3c (upper block in Supplementary Table 2)44. In the analysis to determine ETC and tTC together (Fig. 3e, lower block of Supplementary Table 2), all six parameters (EB, EU, kb, pb, ETC, and tTC) were determined simultaneously by maximizing ln L (tTC). Simulation of photon trajectories. To validate the experimental measurement of the lifetime of TC, photon trajectories were simulated, re-analyzed, and the results of this analysis were compared with the experimental results (Supplementary Fig. 5). In this simulation, instead of generating completely new photon trajec- tories, the intervals between photons of the experimental photon trajectories are retained, and only the photon colors are erased and recolored. By generating photon trajectories in this way, it is possible to produce a simulated dataset that is most similar to the experimental data in terms of both the average and distribution of the detected photons per unit time, and the length of the trajectories. NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y At the illumination intensity to collect photon trajectories with a count rate of 50–100 ms 1 f h d f ll d k h f bl k In this model, tTC = 1/2kTC. EB and EU were calculated for each transition and the likelihood function was calculated with ETC = (EB + EU)/2 as a function of tTC as described in ref. 35 (Fig. 3d). ETC can be varied and the analysis with different ETC values is shown in Supplementary Fig. 8. We analyzed only the ﬁrst binding event in each molecular trajectory because it is not possible to distinguish dissociation from acceptor photobleaching or photoblinking on the millisecond time scale. It is also impossible to distinguish between rebinding and transition from the acceptor dark state to the bright state. −1 for the determination of millisecond kinetics, there is frequent blinking. Donor blinking does not affect the result, but acceptor blinking increases the rate coefﬁcients37. Therefore, we incorporated acceptor blinking in the model and used this four-state model (Supplementary Fig. 3b) to determine the parameters of binding of TAD and NCBD. In the four-state model, the matrices are given by36 E ¼ EB 0 0 0 0 EU 0 0 0 0 Ed 0 0 0 0 Ed 0 B B B @ 1 C C C A; peq ¼ pBpb ð1 pBÞpb pBð1 pbÞ ð1 pBÞð1 pbÞ 0 B B B @ 1 C C C A K ¼ kD kd kA;app kb 0 kD kA;app kd 0 kb kd 0 kD kb kA;app 0 kd kD kA;app kb 0 B B B @ 1 C C C A ð For the determination of tTC of TAD and NCBD binding, we used the six-state model (Supplementary Fig. 3d) that includes acceptor dark states because acceptor blinking on the microsecond time scale prevents the accurate determination of tTC or the upper bound. In this model, each of the bound, unbound, and TC exists in both bright (ﬂuorescing) and dark (non-ﬂuorescing) states. All rate coefﬁcients between three states in the acceptor dark state are assumed to be the same as those in the bright state. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y (4), we reduced the apparent j j For the two-state model (Supplementary Fig. 3a), there are four ﬁtting parameters: the apparent FRET efﬁciencies of the bound and unbound states, EB and EU, the apparent association rate, kA,app, and dissociation rate coefﬁcient, kD. The association rate coefﬁcient is kA = kA,app/[Protein], where [Protein] is the concentration of the binding partner, NCBD or barstar. The matrix of FRET efﬁciencies, the rate matrix, and the vector of the equilibrium populations are given by association and dissociation rates by a factor of 1000 to effectively eliminate the contribution from multiple transitions that are not resolvable, i.e., kD′ (=kD/1000) and kA,app′ (=kA,app/1000). This treatment is valid since we use the difference of the log-likelihood values (Δln L). g For the analysis of barnase/barstar binding, we used the three-state model (Supplementary Fig. 3c). The rate matrix is K ¼ k′D kTC 0 k′D 2kTC k′A;app 0 kTC k′A;app 0 B @ 1 C A ð5Þ E ¼ EB 0 0 EU ; K ¼ kD kA;app kD kA;app ! ; peq ¼ pB 1 pB ð2Þ ð2Þ ð5Þ pB = kA,app/(kA,app + kD) is the equilibrium population of the bound state. At the illumination intensity to collect photon trajectories with a count rate of 50–100 ms −1 for the determination of millisecond kinetics, there is frequent blinking. Donor blinking does not affect the result, but acceptor blinking increases the rate coefﬁcients37. Therefore, we incorporated acceptor blinking in the model and used this four-state model (Supplementary Fig. 3b) to determine the parameters of binding of TAD and NCBD. In the four-state model, the matrices are given by36 pB = kA,app/(kA,app + kD) is the equilibrium population of the bound state. At the illumination intensity to collect photon trajectories with a count rate of 50–100 ms −1 for the determination of millisecond kinetics, there is frequent blinking. Donor blinking does not affect the result, but acceptor blinking increases the rate coefﬁcients37. Therefore, we incorporated acceptor blinking in the model and used this four-state model (Supplementary Fig. 3b) to determine the parameters of binding of TAD and NCBD. In the four-state model, the matrices are given by36 pB = kA,app/(kA,app + kD) is the equilibrium population of the bound state. ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y photon interval found by the Viterbi algorithm (two-state model) in the analysis. The width of the TC region was randomly chosen (exponential distribution) with the mean value that is the same as the assumed lifetime of TC in the simulation. The two sides of the TC region were then assigned as bound and unbound states according to the states in the experimental trajectory, and the photons of each region were recolored using the FRET efﬁciencies of the three states: EB, ETC, and EU. The probabilities for observing a donor and an acceptor photon are (1 −E) and E, respectively. Finally, each simulated trajectory was divided into acceptor bright and dark state regions using the acceptor blinking kinetics parameters, and photons in the dark state were recolored using E = 0.06 (fraction of donor photons leaking into the acceptor channel). 17. Kjaergaard, M., Teilum, K. & Poulsen, F. M. Conformational selection in the molten globule state of the nuclear coactivator binding domain of CBP. Proc. Natl Acad. Sci. USA 107, 12535–12540 (2010). 18. Dogan, J., Toto, A., Andersson, E., Gianni, S. & Jemth, P. Activation barrier- limited folding and conformational sampling of a dynamic protein domain. Biochemistry 55, 5289–5295 (2016). y 19. Zosel, F., Mercadante, D., Nettels, D. & Schuler, B. A proline switch explains kinetic heterogeneity in a coupled folding and binding reaction. Nat. Commun. 9, 3332 (2018). 20. Dogan, J., Jonasson, J., Andersson, E. & Jemth, P. Binding rate constants reveal distinct features of disordered protein domains. Biochemistry 54, 4741–4750 (2015). Correlation analysis. The donor–acceptor cross-correlation function was calcu- lated as 21. Demarest, S. J. et al. Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators. Nature 415, 549–553 (2002). Å 22. Dogan, J., Schmidt, T., Mu, X., Engström, Å. & Jemth, P. Fast association and slow transitions in the interaction between two intrinsically disordered protein domains. J. Biol. Chem. 287, 34316–34324 (2012). CDAðτÞ ¼ NDðt þ τÞNAðtÞ h i ND h i NA h i 1 ð7Þ CDAðτÞ ¼ NDðt þ τÞNAðtÞ h i ND h i NA h i 1 ð7Þ ð7Þ 23. De Sancho, D. & Best, R. B. Modulation of an IDP binding mechanism and rates by helix propensity and non-native interactions: association of HIF1α with CBP. Mol. Biosyst. 8, 256–267 (2012). References 30. Schreiber, G., Haran, G. & Zhou, H.-X. Fundamental aspects of protein–protein association kinetics. Chem. Rev. 109, 839–860 (2009 1. Wright, P. E. & Dyson, H. J. Intrinsically unstructured proteins: re-assessing the protein structure–function paradigm. J. Mol. Biol. 293, 321–331 (1999). 1. Wright, P. E. & Dyson, H. J. Intrinsically unstructured proteins: re-assessing the protein structure–function paradigm. J. Mol. Biol. 293, 321–331 (1999). 31. Ubbink, M. The courtship of proteins: understanding the encounter complex. FEBS Lett. 583, 1060–1066 (2009). 2. Wright, P. E. & Dyson, H. J. Intrinsically disordered proteins in cellular signalling and regulation. Nat. Rev. Mol. Cell Biol. 16, 18–29 (2015). 2. Wright, P. E. & Dyson, H. J. Intrinsically disordered proteins in cellular signalling and regulation. Nat. Rev. Mol. Cell Biol. 16, 18–29 (2015). ( ) 32. Dyson, H. J. & Wright, P. E. Unfolded proteins and protein folding studied by NMR. Chem. Rev. 104, 3607–3622 (2004). g g g 3. Dunker, A. K., Cortese, M. S., Romero, P., Iakoucheva, L. M. & Uversky, V. N. Fl ibl Th l f i i i di d i i i i k 3. Dunker, A. K., Cortese, M. S., Romero, P., Iakoucheva, L. M. & Uversky, V. N. Flexible nets. The roles of intrinsic disorder in protein interaction networks. FEBS J. 272, 5129–5148 (2005). 33. Sugase, K., Dyson, H. J. & Wright, P. E. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021–1025 (2007). 4. Hsu, W.-L. et al. Exploring the binding diversity of intrinsically disordered proteins involved in one-to-many binding. Protein Sci. 22, 258–273 (2013). 34. Volkov, A. N., Worrall, J. A. R., Holtzmann, E. & Ubbink, M. Solution structure and dynamics of the complex between cytochrome c and cytochrome c peroxidase determined by paramagnetic NMR. Proc. Natl Acad. Sci. USA 103, 18945–18950 (2006). 5. Liu, Z. & Huang, Y. Advantages of proteins being disordered. Protein Sci. 23, 539–550 (2014). 35. Chung, H. S., McHale, K., Louis, J. M. & Eaton, W. A. Single-molecule ﬂuorescence experiments determine protein folding transition path times. Science 335, 981–984 (2012). 6. Zhou, H.-X. Intrinsic disorder: signaling via highly speciﬁc but short-lived association. Trends Biochem. Sci. 37, 43–48 (2012). 7. Von Smoluchowski, M. Versuch einer mathematischen theorie der koagulationskinetik kolloider Lösungen. Z. Phys. Chem. 92, 129–168 (1917). 36. Chung, H. S., Cellmer, T., Louis, J. M. & Eaton, W. A. References Measuring ultrafast protein folding rates from photon-by-photon analysis of single molecule ﬂuorescence trajectories. Chem. Phys. 422, 229–237 (2013). 8. Schlosshauer, M. & Baker, D. Realistic protein–protein association rates from a simple diffusional model neglecting long-range interactions, free energy barriers, and landscape ruggedness. Protein Sci. 13, 1660–1669 (2004). 37. Chung, H. S. & Gopich, I. V. Fast single-molecule FRET spectroscopy: theory and experiment. Phys. Chem. Chem. Phys. 34, 18644–18657 (2014). p gg 9. Qin, S., Pang, X. & Zhou, H.-X. Automated prediction of protein association rate constants. Structure 19, 1744–1751 (2011). 38. Gopich, I. V. & Szabo, A. Decoding the pattern of photon colors in single- molecule FRET. J. Phys. Chem. B 113, 10965–10973 (2009). 10. Sugase, K., Lansing, J. C., Dyson, H. J. & Wright, P. E. Tailoring relaxation dispersion experiments for fast-associating protein complexes. J. Am. Chem. Soc. 129, 13406–13407 (2007). 39. Müller-Späth, S. et al. Charge interactions can dominate the dimensions of intrinsically disordered proteins. Proc. Natl. Acad. Sci. USA 107, 14609–14614 (2010). 11. Huang, Y. & Liu, Z. Kinetic advantage of intrinsically disordered proteins in coupled folding–binding process: a critical assessment of the “ﬂy-casting” mechanism. J. Mol. Biol. 393, 1143–1159 (2009). 40. Zhou, H.-X. & Szabo, A. Enhancement of association rates by nonspeciﬁc binding to DNA and cell membranes. Phys. Rev. Lett. 93, 178101 (2004). 41. Campos, L. A. et al. A photoprotection strategy for microsecond-resolution single-molecule ﬂuorescence spectroscopy. Nat. Methods 8, 143–146 (2011). 12. Rogers, J. M., Steward, A. & Clarke, J. Folding and binding of an intrinsically disordered protein: fast, but not ‘diffusion-limited’. J. Am. Chem. Soc. 135, 1415–1422 (2013). 42. Liu, J. et al. Exploring one-state downhill protein folding in single molecules. Proc. Natl Acad. Sci. USA 109, 179–184 (2012). 13. Arai, M., Ferreon, J. C. & Wright, P. E. Quantitative analysis of multisite protein–ligand interactions by NMR: binding of intrinsically disordered p53 transactivation subdomains with the TAZ2 domain of CBP. J. Am. Chem. Soc. 134, 3792–3803 (2012). 43. Chung, H. S. & Eaton, W. A. Single-molecule ﬂuorescence probes dynamics of barrier crossing. Nature 502, 685–688 (2013). g 44. Chung, H. S., Piana-Agostinetti, S., Shaw, D. E. & Eaton, W. A. Structural origin of slow diffusion in protein folding. Science 349, 1504–1510 (2015). 14. Borgia, A. et al. Extreme disorder in an ultrahigh-afﬁnity protein complex. Nature 555, 61–66 (2018). 45. Schreiber, G. & Fersht, A. R. ARTICLE ND(t) and NA(t) are the number of donor and acceptor photons in a bin at time t, 〈…〉denotes an average in a given segment in a trajectory, and the upper bar indicates the average over segments that are longer than 20–95 ms. The results are shown in Supplementary Fig. 4. y p p y with CBP. Mol. Biosyst. 8, 256–267 (2012). 24. Ganguly, D., Zhang, W. & Chen, J. Electrostatically accelerated encounter and folding for facile recognition of intrinsically disordered proteins. PLoS Comput. Biol. 9, e1003363 (2013). 25. Chung, H. S. Transition path times measured by single-molecule spectroscopy. J. Mol. Biol. 430, 409–423 (2018). Received: 22 May 2018 Accepted: 28 September 2018 28. Pontius, B. W. Close encounters: why unstructured, polymeric domains can increase rates of speciﬁc macromolecular association. Trends Biochem. Sci. 18, 181–186 (1993). 29. Clore, G. M., Tang, C. & Iwahara, J. Elucidating transient macromolecular interactions using paramagnetic relaxation enhancement. Curr. Opin. Struct. Biol. 17, 603–616 (2007). Data availability p py 26. Chung, H. S. & Eaton, W. A. Protein folding transition path times from single molecule FRET. Curr. Opin. Struct. Biol. 48, 30–39 (2018). Data supporting the ﬁndings of this manuscript are available from the corre- sponding author upon reasonable request. p 27. Neupane, K. et al. Direct observation of transition paths during the folding of proteins and nucleic acids. Science 352, 239–242 (2016). proteins and nucleic acids. Science 352, 239–242 (2016). Received: 22 May 2018 Accepted: 28 September 2018 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y In the data collected at high illumination intensity for the measurement of tTC, trajectory segments that contained a single transition were recolored after dividing each segment into three regions: bound, TC, and unbound states. First, the region corresponding to TC was centered at the most probable instantaneous transition Simulation of photon trajectories. To validate the experimental measurement of the lifetime of TC, photon trajectories were simulated, re-analyzed, and the results of this analysis were compared with the experimental results (Supplementary Fig. 5). In this simulation, instead of generating completely new photon trajec- tories, the intervals between photons of the experimental photon trajectories are retained, and only the photon colors are erased and recolored. By generating photon trajectories in this way, it is possible to produce a simulated dataset that is most similar to the experimental data in terms of both the average and distribution of the detected photons per unit time, and the length of the trajectories. In the measurement of the lifetime of the TC, tTC, molecules were illuminated at higher laser intensity to collect photons at a count rate of 300–900 ms−1. This high photon count rate made it possible to reduce the bin time to 100–200 μs, with which a brief residence of several hundred microseconds in TC of TAD and NCBD can be visualized for selected trajectories (Fig. 3a). A short segment of photon trajectories near each transition (400 μs to 3 ms) was then analyzed using the likelihood method. The difference of log-likelihood, Δln L = ln L(tTC) −ln L(0) compares the likelihood of the three-state model consisting of the bound state (B), TC, and unbound state (U) with a ﬁnite lifetime, tTC, with that of the model with an instantaneous transition (tTC = 0, i.e., two-state model) (see Supplementary Fig. 3 and the likelihood function in Eq. (4)). Δln L = + 3 and −3 were used for the 95% conﬁdence levels for the determination of tTC or an upper bound of the lifetime, respectively35. In the data collected at high illumination intensity for the measurement of tTC, trajectory segments that contained a single transition were recolored after dividing each segment into three regions: bound, TC, and unbound states. First, the region corresponding to TC was centered at the most probable instantaneous transition NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 8 Competing interests: The authors declare no competing interests. 57. Kubelka, J., Hofrichter, J. & Eaton, W. A. The protein folding ‘speed limit’. Curr. Opin. Struct. Biol. 14, 76–88 (2004). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 58. Buckle, A. M., Schreiber, G. & Fersht, A. R. Protein–protein recognition: crystal structural analysis of a barnase–barstar complex at 2.0-A resolution. Biochemistry 33, 8878–8889 (1994). y 59. Young, T. S., Ahmad, I., Yin, J. A. & Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395, 361–374 (2010). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 60. Brustad, E. M., Lemke, E. A., Schultz, P. G. & Deniz, A. A. A general and efﬁcient method for the site-speciﬁc dual-labeling of proteins for single molecule ﬂuorescence resonance energy transfer. J. Am. Chem. Soc. 130, 17664–17665 (2008). 61. Chung, H. S. et al. Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET. Proc. Natl. Acad. Sci. USA 114, E6812–E6821 (2017). 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/. 62. Zheng, Q., Jockusch, S., Zhou, Z. & Blanchard, S. C. The contribution of reactive oxygen species to the photobleaching of organic ﬂuorophores. Photochem. Photobiol. 90, 448–454 (2014). 63. Zheng, Q. et al. Ultra-stable organic ﬂuorophores for single-molecule research. Chem. Soc. Rev. 43, 1044–1056 (2014). 64. Nettels, D. et al. Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins. Proc. Natl Acad. Sci. USA 106, 20740–20745 (2009). 65. Aitken, C. E. et al. An oxygen scavenging system for improvement of dye stability in single-molecule ﬂuorescence experiments. Biophys. J. 94, 1826–1835 (2008). 66. Merchant, K. A., Best, R. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06866-y 48. Plattner, N., Doerr, S., De Fabritiis, G. & Noé, F. Complete protein–protein association kinetics in atomic detail revealed by molecular dynamics simulations and Markov modelling. Nat. Chem. 9, 1005–1011 (2017). 67. Viterbi, A. Error bounds for convolutional codes and an asymptotically optimum decoding algorithm. IEEE Trans. Inf. Theory 13, 260–269 (1967). g g f y 68. Rabiner, L. R. A tutorial on hidden Markov models and selected applications in speech recognition. Proc. IEEE 77, 257–286 (1989). 68. Rabiner, L. R. A tutorial on hidden Markov models and selected applications in speech recognition. Proc. IEEE 77, 257–286 (1989). g 49. Dahal, L., Kwan, T. O. C., Shammas, S. L. & Clarke, J. pKID binds to KIX via an unstructured transition state with nonnative interactions. Biophys. J. 113, 2713–2722 (2017). 69. Chung, H. S. et al. Extracting rate coefﬁcients from single-molecule photon trajectories and FRET efﬁciency histograms for a fast-folding protein. J. Phys. Chem. A 115, 3642–3656 (2011). 69. Chung, H. S. et al. Extracting rate coefﬁcients from single-molecule photon trajectories and FRET efﬁciency histograms for a fast-folding protein. J. Phys. Chem. A 115, 3642–3656 (2011). 50. Shoemaker, B. A., Portman, J. J. & Wolynes, P. G. Speeding molecular recognition by using the folding funnel: the ﬂy-casting mechanism. Proc. Natl. Acad. Sci. USA 97, 8868–8873 (2000). Acknowledgements 51. Chung, H. S., Louis, J. M. & Eaton, W. A. Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories. Proc. Natl Acad. Sci. USA 106, 11837–11844 (2009). We thank W.A. Eaton, A. Szabo, I.V. Gopich, and R.B. Best for numerous helpful discussions and comments; P.G. Schultz for sharing the plasmid for the expression and incorporation of the unnatural amino acid; and J.M. Louis for advices and suggestions on protein expression and puriﬁcation. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, NIH. 52. Turjanski, A. G., Gutkind, J. S., Best, R. B. & Hummer, G. Binding-induced folding of a natively unstructured transcription factor. PLoS Comput. Biol. 4, e1000060 (2008). 53. Uversky, V. N., Gillespie, J. R. & Fink, A. L. Why are ‘natively unfolded’ proteins unstructured under physiologic conditions? Proteins 41, 415–427 (2000). Author contributions J.-Y.K. and H.S.C. designed research and wrote the manuscript. J.-Y.K. performed research and data analysis. J.-Y.K. and F.M. developed protein expression and labeling protocols. J.Y developed data analysis tools. All authors discussed results and commented on the manuscript. 54. Mészáros, B., Tompa, P., Simon, I. & Dosztányi, Z. Molecular principles of the interactions of disordered proteins. J. Mol. Biol. 372, 549–561 (2007). 55. Best, R. B., Hummer, G. & Eaton, W. A. Native contacts determine protein folding mechanisms in atomistic simulations. Proc. Natl Acad. Sci. USA 110, 17874–17879 (2013). Additional information 56. Gin, B. C., Garrahan, J. P. & Geissler, P. L. The limited role of nonnative contacts in the folding pathways of a lattice protein. J. Mol. Biol. 392, 1303–1314 (2009). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-06866-y. Competing interests: The authors declare no competing interests. Competing interests: The authors declare no competing interests. References Energetics of protein–protein interactions: analysis of the barnase–barstar interface by single mutations and double mutant cycles. J. Mol. Biol. 248, 478–486 (1995). 15. Lee, C. W., Martinez-Yamout, M. A., Dyson, H. J. & Wright, P. E. Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein. Biochemistry 49, 9964–9971 (2010). 46. Schreiber, G. & Fersht, A. R. Rapid, electrostatically assisted association of proteins. Nat. Struct. Biol. 3, 427–431 (1996). 47. Vijayakumar, M. et al. Electrostatic enhancement of diffusion-controlled protein–protein association: comparison of theory and experiment on barnase and barstar. J. Mol. Biol. 278, 1015–1024 (1998). 16. Joerger, A. C. & Fersht, A. R. Structural biology of the tumor suppressor p53. Annu. Rev. Biochem. 77, 557–582 (2008). 9 NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 9 © The Author(s) 2018 Competing interests: The authors declare no competing interests. B., Louis, J. M., Gopich, I. V. & Eaton, W. A. Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations. Proc. Natl Acad. Sci. USA 104, 1528–1533 (2007). NATURE COMMUNICATIONS \| (2018) 9:4707 \| DOI: 10.1038/s41467-018-06866-y \| www.nature.com/naturecommunications 10
https://openalex.org/W2581810488	https://journals.open.tudelft.nl/ejtir/article/download/3190/3375	English	null	An exploration of moving walkways as a transport system in urban centers	Deleted Journal	2,017	cc-by	9,284	1A: EPFL ENAC IIC TRANSP-OR Station 18 CH-1015 Lausanne Switzerland T: +41(0)216932408 F: +41(0)216938060 E: riccardo.scarinci@epﬂ.ch 2A: EPFL, ENAC, IA, LAB-U Station 16 CH-1015 Lausanne Switzerland T: +41(0)216936274 E: farzaneh.bahrami@epﬂ.ch 3A: EPFL-ENAC-INTER-CHOROS Batiment polyvalent Station 16 CH-1015 Lausanne Switzerland T: +41(0)216938002 F: +41(0)216933840 E: andre.ourednik@epﬂ.ch 4A: EPFL ENAC IIC TRANSP-OR Station 18 CH-1015 Lausanne Switzerland T: +41(0)216932408 F: +41(0)216938060 E: michel.bierlaire@epﬂ.ch Issue 17(2), 2017 pp. 191 −206 ISSN:1567 −7141 tlo.tbm.tudelft.nl/ejtir Issue 17(2), 2017 pp. 191 −206 ISSN:1567 −7141 tlo.tbm.tudelft.nl/ejtir EJTIR An exploration of moving walkways as a transport system in urban centers Riccardo Scarinci1 Transport and Mobility Laboratory TRANSP-OR, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Riccardo Scarinci1 Transport and Mobility Laboratory TRANSP-OR, School of Architecture, Civil and Environmen Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Farzaneh Bahrami2 Laboratory of Urbanism LAB-U, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Farzaneh Bahrami2 Laboratory of Urbanism LAB-U, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Farzaneh Bahrami2 Laboratory of Urbanism LAB-U, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Andr´e Ourednik3 Laboratory Chˆoros, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecol Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Andre Ourednik3 Laboratory Chˆoros, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Andre Ourednik Laboratory Chˆoros, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Michel Bierlaire4 Michel Bierlaire Transport and Mobility Laboratory TRANSP-OR, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Transport and Mobility Laboratory TRANSP-OR, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Transport and Mobility Laboratory TRANSP-OR, School of Architecture, Civil and Environmental Engineering ENAC, ´Ecole Polytechnique F´ed´erale de Lausanne EPFL, Switzerland. Moving walkways (MW) have been imagined as a possible means of transport since the late 19th century, and this system has fascinated urban planners and engineers ever since. Contrary to what has been imagined, moving walkways are only used in transportation hub corridors, and not as a main transport mode in city centers. Today however, MWs are receiving increasing attention as a possible solution to congestion and pollution, as well as a catalyst for soft mobility. This paper explores the role of moving walkways as a transport system, and it presents an optimized design of a network of MWs in a city center. We review historical MWs, current installations and future pos- sibilities, using different perspectives, from geography to urban planning to transport engineering. We discuss the way in which MWs inﬂuence how people inhabit the urban space, and we review this system in the context of history of urban planning. Then, we describe a technological devel- opment called accelerating moving walkways (AMW), i.e. MWs able to reach a higher speed than traditional ones. We develop an optimization framework to design a network of AMWs, and we apply it to a real case study. The results of the network design are a reference useful to discuss the feasibility of the system starting from an engineering perspective. We conclude that the use of MWs can facilitate the ﬂexibility and spontaneity typical of pedestrian movements, and this system could be integrated in the mix of urban transport modes in city centers. Keywords: moving walkway, accelerating moving walkway, pedestrian mobility, innovative transport sys- tem, sustainable transport system. EJTIR 17(2), 2017, pp. 191 −206 J ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 2 An exploration of moving walkways as a transport system in urban centers 1 Introduction Moving walkways (MW), also known as moving sidewalks, have been imagined as a system capa- ble of facilitating pedestrian movements on speciﬁc routes, or even as the primary transport mode in city centers. This concept is present in the history of transportation since the late 19th century, and it has fascinated science-ﬁction writers, urban planners and engineers ever since. The use of impossibly fast (more than 100 km/h) and massive moving walkways as the main transport system of future megalopolis was described in several science-ﬁction novels such as A Story of the Days To Come (Wells, 1897), The Roads Must Roll (Heinlein, 1940) and The Caves of Steel (Asimov, 1954). In the same period, real implementations of moving walkways were presented in exhibition events such as the World’s Columbian Exposition, Chicago, USA, 1893 (Badger, 1979), Exposition Universelle, Paris, France, 1900 (Avenel, 1900) and 42nd street, New York, USA, 1923 (NYPL, 1923). These MWs were composed of parallel lanes, each of them moving at a higher speed, reaching the maximum speed of 9 km/h. The users could access them laterally everywhere and reach the high-speed lane jumping from one belt to the adjacent one, see Figure 1(a). (a) (b) Figure 1. Moving walkways. (a) historical MW at the Exposition Universelle in Paris, 1900, composed of two parallel lanes with increasing speed. (b) modern MW installed in a transportation hub. Adapted from Avenel (1900) and Otis (2015). (a) (b) (b) (a) Figure 1. Moving walkways. (a) historical MW at the Exposition Universelle in Paris, 1900, composed of two parallel lanes with increasing speed. (b) modern MW installed in a transportation hub. Adapted from Avenel (1900) and Otis (2015). Nowadays, MWs consist of one lane only, and the lateral access is not allowed for safety reasons, see Figure 1(b). In addition, the maximum speed is limited to 3 km/h to avoid discomfort. They are used on individual routes with high demand, usually in large transportation hubs such as airports and metro stations. In a few cases, MWs are installed in cities, for example, the Central- Mid-Levels escalator and walkway system in Hong Kong (Cullinane, 2002), the Mechanical Ramps in Vitoria-Gasteiz (Helzel and Taylor, 2011), or in hilly urban environments to facilitate the access to elevated areas, for instance, Medellin, Colombia (Nakamichi and Nakamura, 2014) and Perugia, Italy (Ciufﬁni, 1983). 1 Introduction This allows us to analyze the characteristics of the system, and to examine its feasibility based on an engineering approach. To design a network of MWs, ﬁrst, we review the technological advances and modern implemen- tations of this system. An interesting development is the so-called accelerating moving walkway (AMW), which provides a higher top speed than traditional MWs. Using an optimization algo- rithm, we design the network of AMWs that minimizes the travel time of passengers. We identify the street where to install AMWs on a real case study for different budget constraints analyzing travel time and costs. The results of the optimization algorithm allow us to discuss the impacts of the system on the urban environment. In the current paper, we focus on urbanistic oriented considerations rather than transportation aspects. In the case study, we envision a Post-Car World scenario, where the use of private cars is limited in city centers (PCW, 2015), and the entire pas- senger demand is satisﬁed by AMWs or walking. We do not consider the interaction of AMWs with other modes of transport currently present in the case study. Moreover, the urban planning considerations are limited to an exploratory analysis of the system, and further study is needed to provide a more accurate scenario. Moving walkways and accelerated MWs have been studied, tested and implemented in several cities around the world. As we present in the remaining of the paper, technical analyses of the system have been done by researchers such as Kusumaningtyas (2009), Ikizawa et al. (2001) and Abe et al. (2001). The system has been experimented in city such as Chicago, Paris, Toronto and Hong Kong; and it has been evaluated by Badger (1979), Cote and Gempp (1998), Gonzalez Alemany et al. (2007) and Cullinane (2002), respectively. However, an inter-disciplinary review of the system, as well as an evaluation of the use of this system at a network level are missing. The contribution of this work goes exactly in this direction. As a result of the multi-disciplinary project where this research took place, we are able to investigate MWs from several perspectives, namely geographic, urbanistic and engineering. Moreover, we lay the foundation for evaluating the potential of AMWs as a transport system in urban center. The paper is structured as follows. 1 Introduction Section 2 discusses the signiﬁcation of MWs for the individuals moving through the space and the existential implications on the inhabiting space. In Section 3, we look at the re-consideration of MWs in the 20th century, following the critiques of the car and the attempts to seek futuristic mobility alternatives. Section 4 reviews the technical characteristics of accelerating moving walkways, as well as current installations. Section 5 investigates the possibility to use AMWs as an urban transport mode by designing a network of AMWs on a real case study. We draw the ﬁnal considerations in Section 6, and the main conclusions are reported in Section 7 1 Introduction Nowadays, MWs consist of one lane only, and the lateral access is not allowed for safety reasons, see Figure 1(b). In addition, the maximum speed is limited to 3 km/h to avoid discomfort. They are used on individual routes with high demand, usually in large transportation hubs such as airports and metro stations. In a few cases, MWs are installed in cities, for example, the Central- Mid-Levels escalator and walkway system in Hong Kong (Cullinane, 2002), the Mechanical Ramps in Vitoria-Gasteiz (Helzel and Taylor, 2011), or in hilly urban environments to facilitate the access to elevated areas, for instance, Medellin, Colombia (Nakamichi and Nakamura, 2014) and Perugia, Italy (Ciufﬁni, 1983). Contrary to what was presumed in the past, MWs are not used as a means of transport in city centers. The presence of private and public vehicular trafﬁc is still predominant, and pedestrian mobility plays a minor role. Due to congestion, pollution and urbanistic considerations, soft mo- bility is now being promoted in particular in dense urban environments (EC, 2011). MWs could facilitate walking, and incentivize the modal shift toward more sustainable means of transport. Moreover, the system is fully electric, and it has a low energy consumption and noise level. This paper explores the use of MWs as a transport mode, the limitations, opportunities, technolog- ical developments and cultural paradigm shifts necessary for the success of this system. To do so, we adopt a multi-disciplinary approach. At ﬁrst, we follow the geographic and urbanistic perspec- tives. We describe how MWs can inﬂuence the way in which individuals experience and move in the urban environment, and we empathize the opportunities offered by this means of transport. Then, we discuss the role of MWs in the broader context of the history of urban planning. After EJTIR 17(2), 2017, pp. 191 −206 J ( ), , pp Scarinci, Bahrami, Ourednik and Bierlaire J ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 3 , , n exploration of moving walkways as a transport system in urban centers An exploration of moving walkways as a transport system in urban centers this analysis, we shift the perspective completely, and we investigate the system from a transport engineering point of view. The idea is to design a network of MWs starting from transportation and practical considerations such as speed, cost and passenger demand. 3 The moving walkway in the broader context of the history of urban plan- ning The ecological consciousness, oil crisis and the spatial consequences of decades of car urbanism generated, during the 1960s and 1970s, an urge to seek alternatives systems to car mobility. With the conviction that “people will use automobiles as long as nothing better is available” (Gruen, 1964) a series of explorations started proposing new innovative possibilities for the future that will give even more delight and convenience to the user. Given the considerable amount of studies and experiments on mechanical means of moving people prior to the mass motorization era, up to 1920s, urban projects in this period reemployed many of the historical schemes and examples to develop the future with the objective of reducing the role of the private car. Among many transport systems imagined, such as mini-rails, self-driven mini taxis, automatic people movers, and other non-stop moving systems, MWs were a promising recurrent scheme. Applied mostly within transportation hubs and shopping malls, it was also imagined to be a viable transport means for intra-city movements, especially within the new developments. MWs, also called pedestrian conveyors in the literature, were thought not only as an aid to pedes- trian movements but also capable to provide a new experience, with a view over the city at a different speed, not necessarily faster than that of pedestrians. In an inventory of transport sys- tems proposed by Richards (1966, 1976) the average speed of pedestrians (4.8 km/h) was interest- ingly higher than that of pedestrian conveyor (3km/h). However, given the fact that they could be walked on, the speed as an advantage was not excluded. Such effortless, immobile walk of the pedestrian conveyors offered another vision of the city without regrets for the car. As described by Rouillard (2013), the city is like a permanent Exposition Universelle. Richards (1976), in his ex- ploratory surveys into the future, also introduces the idea of high-speed conveyors, called Speed- away, with the average speed of 12-16 km/h. The Speedaway was intended to run above London Bridge, one of the densest corridors of movement in London, see Figure 2. The proposal used twin high-speed conveyors, operating within an air-conditioned enclosure and connected at either end with elevated pedestrian decks linked with the underground stations close by. The system was above all considered as a complement to both existing and future modes of transport. 2 The moving walkway as a new way of inhabiting the urban space The private car is often regarded as a means of transport offering a great freedom of movement, seen as a major quality of this mode of transport. In fact, the use of the car implies accepting inhabiting the space in a tunnel perspective. The car limits the possible spatial behavior of its driver to a movement forward, and its interaction with the immediate environment to occasional honking or headlight ﬂashing (Ourednik, 2008). Especially in the urban context, it is by no means easy to park a car and to leave it in order to engage into more complex ways of inhabiting the space (Heidegger, 1954; Bollnow, 1967). As opposed to the coerced freedom of the car, pedestrian movements are characterized by ﬂexi- bility and spontaneity, providing opportunities for interactions. However, this ﬂexibility is only efﬁcient in the context of dense and diverse urban centers, where such opportunities, whether they consist of people or places, are close to each other. The experience of density itself, however, is also closely related to the speed of movements through a given environment (Ourednik, 2010). Even in an urban setting, the number of reachable elements remains limited by the speed of pedestrian EJTIR 17(2), 2017, pp. 191 −206 J ( ), , pp Scarinci, Bahrami, Ourednik and Bierlaire J ( ), , pp Scarinci, Bahrami, Ourednik and Bierlaire J ( ), , pp Scarinci, Bahrami, Ourednik and Bierlaire 4 arinci, Bahrami, Ourednik and Bierlaire n exploration of moving walkways as a transport system in urban centers , , An exploration of moving walkways as a transport system in urban centers movements. As the average topographic distances between objects of interest grow, only speed of movements can restore an experience of density. Conversely, this also means that urban density, understood as an interaction potential, can be augmented by accelerating the pedestrian move- ments. Being capable to reach more things, people and places, signiﬁes having more choice of action. It opens up individual’s existential perspectives: which is the very purpose of any city. The collateral effect of this acceleration, however, is cutting the individual off the immediate environ- ment of his/her movements. A compromise thus has to be found between speed and the potential of local interactions. Open vehicles, such as bikes or segways, constitute an answer to this problem. 2 The moving walkway as a new way of inhabiting the urban space Another original answer is given by MWs, especially when these are organized into a network where the individuals can choose the direction at each crossroads. The technical implications of the construction of such a network are by no means trivial and are discussed in the following of the present paper. , , An exploration of moving walkways as a transport system in urban centers 3 The moving walkway in the broader context of the history of urban plan- ning The critical challenge however, was the spatial arrangement, the insertion of the system within the existing width of streets. Therefore in many cases it was imagined as an elevated level, following examples like the London Pedways project (Hebbert, 1993). This project planned and partly implemented a network of elevated walkways throughout the city, re-proposing the traditional idea of separating pedestrians from motorized or wheeled trafﬁc. It is not irrelevant, proposes Rouillard (2013), to link the very concrete and persistent ideas and sketches of moving walkways and other innovative moving systems, with the more utopian imag- inaries of the time, which were focused on the idea of augmented pedestrian and ecstatic mobility. For example, Cook (1999) proposes a combination of cushion and vehicle called the Cushicle. It EJTIR 17(2), 2017, pp. 191 −206 J ( ), , pp Scarinci, Bahrami, Ourednik and Bierlaire ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 5 An exploration of moving walkways as a transport system in urban centers (b) (a) (b) (a) (b) (c) Figure 2. Speedaway system. (a) Plan showing the Speedaway system crossing the Thames connecting existing underground stations on the north bank with new developments on the south bank. (b) Interior view along the system. (c) Speedaway running above a car-free shopping street. Adapted from Richards (1976). (a) (a) (b) (b (c) (c) Figure 2. Speedaway system. (a) Plan showing the Speedaway system crossing the Thames connecting existing underground stations on the north bank with new developments on the south bank. (b) Interior view along the system. (c) Speedaway running above a car-free shopping street. Adapted from Richards (1976). was a sort of mobile structure in two parts: a chassis with appliances and personalized appara- tuses and an inﬂatable envelope. Like a car, the Cushicle was envisioned as eventually becoming part of an urban system of personalized enclosures, though it was conceived as usable in any en- vironment. From the Cushicle to ideas of individual air travel, they were all struggles to achieve new paradigms in transport in cities, hoping that, as Buchanan (1963) put it, “we are not at the end of our ingenuity for that matter”. Contrary to many of its contemporary ideas, moving walkways were actually applied in real contexts, and nowadays are installed in several environments. 4.1 Technical characteristics AMWs are divided into three sections: acceleration, constant speed, and deceleration. The accelera- tion section, as the name suggests, has an acceleration between 0.14 m/s2 and 0.43 m/s2, depending on the installations (Dembart, 2003; Gonzalez Alemany et al., 2007; Kusumaningtyas and Lodewi- jks, 2008, 2013). In this section, the speed increases from the entry speed between 2.1 km/h and 2.7 km/h (Donoghue, 1981; Fruin, 1992) to the top speed. Real implementations of AMWs show that the top speed can be up to 12 km/h (Kusumaningtyas, 2009). The constant speed section has zero acceleration, and passengers can walk. Thus, the walking speed is added to the top speed of the AMW. The walking speed depends on factors such as the age, gender and trip purpose of the individuals. However, as a reference, Young (1999) shows that the walking speed on MWs is on average 3.7 km/h. The ﬁnal section is the deceleration section. Here, the speed decreases to an exit speed equal to the entry speed for a safe disembarking. AMWs have a constant width along the entire length. The typical width of the internal space usable by passengers is between 0.8 and 1.6 meters, and, considering the required lateral installation, the corridor width ranges between 1.2 meters and 2.3 meters (Kusumaningtyas, 2009). The length is limited by the technology, and it ranges between 120 meters and 350 meters (Abe et al., 2001; Lechner, 2011). The capacity of an AMW, expressed in passengers per hour (pax/h) per direction, depends on the entry speed and the width. Typical installations have capacities ranging from 4,500 pax/h to 7,500 pax/h (Kusumaningtyas and Lodewijks, 2008). The capital cost of AMW, including the surround- ing structure, is between 34.8 million EUR/km and 54.4 million EUR/km, and the operational cost ranges from 0.08 EUR/pax-km to 0.42 EUR/pax-km, with a typical value of 0.13 EUR/pax-km (Kusumaningtyas, 2009). AMWs present a limited energy consumption of approximately 0.11 MJ/pax-km. They are fully electric, thus, reduce local emission of greenhouse gases. In addition, the system has a low noise level close to 54 dB(A) (ThyssenKrupp, 2004). In comparison with other urban means of transport such as buses and light rail metro services, AMWs have a competitive speed, operational cost and capacity with the advantage of a limited corridor width. However, AMWs present a greater capital cost, especially in comparison with busses (Kusumaningtyas and Lodewijks, 2008). 4 Review of accelerating moving walkway The main limitation of MWs is the low speed, and since the 1960s, technical solutions able to achieve higher speeds have been investigated (Kusumaningtyas, 2009). Some of these solutions were developed between the 1970s and early 1980s, and the ﬁrst prototypes of accelerating moving walkways (AMW) were made. The successful prototypes were built and tested in the late 1990s and early 2000s. Since then, AMWs have received increasing attention from researchers. Gonzalez Alemany and Cuello (2003), Ikizawa et al. (2001), Saeki (1996) and Shirakihara (1997) study the technological aspects of the system. Abe et al. (2001) propose a different design able to cover inclined and even curved paths. The integration of this innovative system with the urban infrastructure is studied by Rockwood and Garmire (2015). Kusumaningtyas et al., in several research papers focused on AMWs, make a comprehensive investigation of the system from a technical and transportation perspective (Kusumaningtyas and Lodewijks, 2008; Kusumaningtyas, 2009; Kusumaningtyas and EJTIR 17(2), 2017, pp. 191 −206 EJTIR 17(2), 2017, pp. 191 −206 J ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 6 An exploration of moving walkways as a transport system in urban centers In the following, we present the AMW characteristics, the different AMW types and two installation examples. In the following, we present the AMW characteristics, the different AMW types and two installation examples. 4.1 Technical characteristics Busses and light rails have higher top speeds than AMWs, however, the discontinuous nature of these systems decreases the average speed. Busses have an average speed between 15 km/h and 20 km/h, while light rail can reach speeds up to 45 km/h (Brand and Preston, 2003; KFH Group, 2013). AMWs can have maximum speeds up to 15-17 km/h thanks to the possibility of pedestrians to walk on the constant speed section. This speed is competitive with private cars during peak hours, which travel at an average speed of 15 km/h (Christidis and Rivas, 2012). AMWs are designed for a high trafﬁc demand, and they have a maximum capacity larger than buses which is between 1,000 pax/h and 4,500 pax/h, but lower than light rail services, which can serve up to 30,000 passengers per hour (Brand and Preston, 2003; KFH Group, 2013). Moreover, AMWs require a limited space for installation in comparison with the other means of transport. This is an advantage for the integration of the system in the urban environment. Busses and light rails have corridors width between 2.5 meters and 4.2 meters per direction, more than double the minimum width of an AMW (KFH Group, 2013). While the operational cost is similar among all means of transport, AMWs present the high capital cost. This cost is comparable with the one of light rails, but much higher than the bus cost, which maximum is around 6.7 million EUR/km (Kusumaningtyas and Lodewijks, 2008). AMWs consume less energy than busses and light rails, which consumption ranges from 0.30 MJ/pax-km to 2.50 MJ/pax-km. Also the noise level is lower than the other means of transport that are between 60 dB(A) and 84 EJTIR 17(2), 2017, pp. 191 −206 J ( ), , pp Scarinci, Bahrami, Ourednik and Bierlaire 7 An exploration of moving walkways as a transport system in urban centers db(A) (Brand and Preston, 2003). In order to study a network of AMWs and not only AMW on individual paths, we need to make hypotheses on the behavior of the system at junctions. Passengers have two options at intersections. Either the AMW where they are travelling spans over the intersection allowing them to travel at the top speed, or they disembark and re-embark the next one. We refer to AMWs that span over several roads without decelerating at intersections as expressways. Given the absence of MW junctions in real installations, we take inspiration from existing embarking/disembarking and intersection systems from other technologies, and we propose hypothetical designs. Inspiration is given by reviewing solutions implemented for conveyor belts used for goods (Alspaugh, 2008; Woodcock and Mason, 2012), and real life implementations and futuristic ideas of intersections for pedestrian and vehicular trafﬁc such as cross intersection, rotating platform, circular walkway (DfT, 2011). We envisage several designs, from a simple approach where everyone is slowed down, to a complex intersection with multiple expressways at different levels, see Figure 3. The intersection design itself and the necessary technology is not part of the present study. We refer to Rojanawisut (2015) for a review of possible intersections. We only assume the presence of expressways able to span across intersections without conﬂicting with other AMWs. (a) (b) Figure 3. Possible intersection designs: (a) single-layer intersection and (b) multiple-layer intersection with expressways. Adapted from Rojanawisut (2015). (b) (b) Figure 3. Possible intersection designs: (a) single-layer intersection and (b) multiple-layer intersection with expressways. Adapted from Rojanawisut (2015). 4.2 Types of accelerating moving walkways Based on the method used to achieve acceleration and deceleration, AMWs can be categorized into four types: in-line belts, sliding parallelograms, accelerating/decelerating rollers and sliding pallets. In-line belts A number of short MWs with slightly different speeds are placed one after another, end to end. The in-line belts have increasing speeds at each entrance, such that they accelerate passengers to a high-speed MW in the middle section. Approaching the exit, another series of short MWs with decreasing speeds decelerates passengers, see Figure 4(a). This mechanism was used in the Loder- way system (Loder, 1998), which was tested for public use in Brisbane Airport, Melbourne Airport and Degraves Street Subway in Melbourne, Australia. A similar system, named Speedmove, was also built by Fujitec (Kazuo et al., 2003). EJTIR 17(2), 2017, pp. 191 −206 J ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 8 An exploration of moving walkways as a transport system in urban centers Figure 4. Accelerating moving walkway types. (a) in-line belts, adapted from Loder (1998). (b) sliding parallelograms, adapted from Todd (1974). (c) accelerating/decelerating rollers, adapted from Cote and Gempp (1998). (d) sliding pallets, adapted from Gonzalez Alemany and Cuello (2003). Figure 4. Accelerating moving walkway types. (a) in-line belts, adapted from Loder (1998). (b) sliding parallelograms, adapted from Todd (1974). (c) accelerating/decelerating rollers, adapted from Cote and Gempp (1998). (d) sliding pallets, adapted from Gonzalez Alemany and Cuello (2003). Sliding parallelograms A series of parallelograms form a continuous tread way. At the entrance, the parallelograms are aligned along the long side, and they move at a low speed for a short distance. Then, while contin- uing to move forward, each parallelogram begins to rotate progressively sideways with regard to the parallelogram behind it. The result of the forward moving and the sideway rotation creates an acceleration zone, see Figure 4(b). The parallelograms stop rotating once they reach the target top speed. The method is reversed for deceleration at the exit. This mechanism was used in the Dun- lop Speedaway (Todd, 1974), which was installed for public trial at the Expo ’70 in Osaka, Japan, and at the Battelle Institute in Geneva, Switzerland. The Mitsubishi Speedwalk (Shirakihara, 1997), which was demonstrated at the Seaside Park in Fukuoka, Japan, also adopted this mechanism. This speciﬁc AMW had an S-shape, showing a possible solution for a curvy path. At the curves, the ro- tation and the forward moving of each parallelogram result in accelerating or decelerating zones adjusting the speed. In this mechanism, a “carpet” of metal rollers at the entrance accelerates passengers onto a high- speed belt conveyor. Approaching the exit, another series of metal rollers decelerates the passen- gers, see Figure 4(c). The mechanism was adopted by the Gateway system (Cote and Gempp, 1998), which was installed in 2002 for public demonstration in the Paris Montparnasse metro station. 4.3 Example of existing accelerating moving walkways 4.3 Example of existing accelerating moving walkways We review two examples of AMWs, underlining working characteristics and implementation prob- lems. We review two examples of AMWs, underlining working characteristics and implementation prob- lems. (a) (b) Figure 5. Accelerating moving walkway real implementations. (a) TurboTrack, Toronto airport, Canada, and (b) Gateway Paris subway, France. Adapted from Gonzalez Alemany et al. (2007), and Gautier (2000). (a) (b) (b) (a) Figure 5. Accelerating moving walkway real implementations. (a) TurboTrack, Toronto airport, Canada, and (b) Gateway Paris subway, France. Adapted from Gonzalez Alemany et al. (2007), and Gautier (2000). Sliding pallets There are a number of different variations in the way the pallets slide. One system accelerates and decelerates individual main pallets by sliding them over continuous auxiliary pallets (Ikizawa et al., 2001). Another design uses an array of pallets that slightly overlap one on top of the previous at the entrance, each of which then progressively slides forward to accelerate passengers (Abe et al., 2001). These two systems were only tested in the factory. A more successful variation is the TurboTrack system (Gonzalez Alemany and Cuello, 2003), which employs auxiliary pallets and main pallets, see Figure 4(d). At the entrance and exit, this AMW type looks like present-day metal-pallet MWs. The auxiliary pallets “hide” underneath the main pallets at the extremes, and progressively extend EJTIR 17(2), 2017, pp. 191 −206 ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 9 , , n exploration of moving walkways as a transport system in urban centers An exploration of moving walkways as a transport system in urban centers or retract to accelerate or decelerate passengers. In the low-speed zones, auxiliary pallets are non- visible, and main pallets have no space between them. In the high-speed zone, auxiliary pallets are visible and much longer than the main pallets. In the central zone, the auxiliary pallets are fully extended, forming a continuous tread way with the main pallets. This mechanism allows different speeds for the boarding, central and exiting zones. 4.3 Example of existing accelerating moving walkways TurboTrack, Toronto airport The TurboTrack is a successful installation of an AMW currently in service, Figure 5(a). It is located in Toronto Airport, Canada, between Terminals 1 and 2, and it was built in 2007 by ThyssenKrupp Elevator (Gonzalez Alemany et al., 2007). The technology of this AMW is based on sliding pallets. The entry speed is 2.34 km/h, which is like conventional moving walkways. This speed assures the same safety as MWs for boarding. In the middle area, the top speed is 7.2 km/h, three times faster than the entry speed. When pallets are fully extended, people can walk on the AMW to travel even faster. The length of the TurboTrack is 270 m, and one AMW for each direction is built. It allows people to travel that distance in only 140 seconds, while previous MWs took 415 seconds, thus reducing the travel time by two thirds. Acceleration and deceleration zones, i.e. the transition between low-speed and high-speed zones, measure around 13 m each. In about 10 seconds, the AMW reaches the top speed. This implies an acceleration of 0.14 m/s2. The TurboTrack has a width of 1.2 m, which is the most common width used for MWs as well. It allows two columns of passengers, which permit the overtaking of standing passengers. The system has a capacity close to 7,000 passenger per hour, as calculated using the empirical equation deﬁned by CEN (1998). An exploration of moving walkways as a transport system in urban centers 5 Network design of a transport system based on accelerating moving walk- ways In this section, we study the use of AMWs at a large scale. First, we describe the optimization framework developed for this scope, and then we apply it to a real case study. The network design aims to identify the set of AMWs that could best satisfy the passenger demand minimizing the total travel time in the city. A street network of interconnected roads, an operational budget and an origin-destination demand are the inputs of this problem. The result deﬁnes for each road, if it is equipped with an AMW, and if so, the speed proﬁle and capacity of the installed walkway. To identify the solution, we evaluate only the travel time and the operational costs to run the system on an hourly basis. Other factors such as accessibility, comfort and construction costs are not considered. To solve this problem, ﬁrst, we formulate the system mathematically based on the characteristics previously reviewed, then we use an algorithm with a structure similar to the ones used in the ﬁeld of road network design (Yang and Bell, 1998; Farahani et al., 2013). We deﬁne the problem on a directed graph describing the road network, where each node represents an intersection, an origin or a destination, and each arc represents a road. Roads with geometrical limitations, or reserved exclusively for public transport, vehicular, bike or pedestrian trafﬁc are excluded. We start with an initial network equipped with all feasible AMWs, which we iteratively transform by adding or removing AMW lanes on selected roads until the operational budget limit is reached. The opti- mization problem parameters are the technological characteristics of AMWs such as acceleration, entry/exit speed and top speed. The passenger demand is deﬁned by an origin-destination matrix that indicates the number of passengers going from an origin node to a destination node. The de- cision variables are the capacity of each installed AMW. The capacity of an AMW is determined by the number of parallel AMW lanes, all having the same standard width. The main factors consid- ered in the objective function are travel time, budget and payback period. To solve the optimization problem, we use a heuristic method with two main phases: trafﬁc assignment and network update. A trafﬁc assignment, based on the shortest path algorithm, calculates the ﬂows on the network with a deﬁned conﬁguration of AMWs. Gateway, Paris subway The one-lane Gateway AMW in Paris, France, at Montparnasse station, was inaugurated in July 2002 (Gautier, 2000), Figure 5(b). It was built to connect the subway station to the train station. The acceleration/deceleration rollers were subject to frequent maintenance due to breakdowns. The technology used for the acceleration section was considered not safe from many users, and it led to numerous passenger falls. Due to the presence of one lane only, it was perceived that the direction of travel was often in the wrong way. When tests were made before the Gateway opened for the public, the initial top speed was set to 10.8 km/h. However, it was reduced to 9 km/h due to safety problems. As the entire system speed and acceleration proﬁle are interdependent, the EJTIR 17(2), 2017, pp. 191 −206 J ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 10 arinci, Bahrami, Ourednik and Bierlaire n exploration of moving walkways as a transport system in urban centers An exploration of moving walkways as a transport system in urban centers entry speed was also reduced proportionally from 2.7 km/h to 2.2 km/h. The main problems were on the acceleration and deceleration zones. The most problematic issues were not the maximum speed in the constant speed section, but the acceleration itself. Initially, the acceleration was ﬁxed to 0.43 m/s2 (for high-speed of 10.8 km/h), however due to many problems related to feelings of unbalance or even falls, the value of 0.28 m/s2 was chosen. The acceleration and deceleration parts measured about 10m, and, as the TurboTrack, the Gateway width is 1.2 meters and the capacity is close to 7,000 passenger per hour. , , An exploration of moving walkways as a transport system in urban centers 5 Network design of a transport system based on accelerating moving walk- ways The following network update phase modiﬁes the current net- work with the aim of exploring new solutions as commonly done in Variable Neighborhood Search algorithms. The results of the optimization algorithm are useful for analyzing the system feasibil- ity. They give practical insight on the possible dimensioning of the system, the optimal network designs, and how these vary with a reduction of the available budget. We refer to Scarinci et al. (2016) for the details on the optimization framework. We apply the optimization algorithm to a real case study. The city of Geneva, Switzerland, presents a suitable trafﬁc demand and road network.The trafﬁc demand is represented by an OD matrix with twenty-six zones, seven of which in the city centers (DGT, 2015). It has 117,349 trips during the morning peak-hour. The city roads are classiﬁed into three categories: primary, secondary, and local roads (SITG, 2015), see Figure 6(a). We decide to evaluate the installation of AMWs on the primary roads only for their spatial characteristics. Primary roads present a larger road section adapted to car mobility that provides more space available for the insertion of the AMW system. In addition, primary roads present a reduced intensity of use by pedestrians compared to secondary and local ones in favor of vehicular trafﬁc. Moreover, having a higher speed is also more desired in primary EJTIR 17(2), 2017, pp. 191 −206 J ( ), , pp Scarinci, Bahrami, Ourednik and Bierlaire ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 11 arinci, Bahrami, Ourednik and Bierlaire n exploration of moving walkways as a transport system in urban centers An exploration of moving walkways as a transport system in urban centers roads compared to small local roads with dense and intense public spaces where “pausability” and spontaneity of movements are their constitutive values. Thus, the choice of big arteries is made to minimize a fundamental drawback of MW infrastructure (like many other infrastructures), that is the creations of edges (barriers) and the reduction of the network permeability and thus spontaneity of ﬂows. Gates are placed around the study area through which incoming and outgoing trafﬁc can ﬂow. Figure 6(b) shows the simpliﬁed road network, composed of 47 links, 37 intersections and 10 gates for a total length of 32 km. Roads Primary Intersections Gate (a) (b) Secondary Local Intersection Figure 6. Case study road network: (a) original road network and (b) simpliﬁed road network. , , An exploration of moving walkways as a transport system in urban centers 5 Network design of a transport system based on accelerating moving walk- ways Intersections Gate (b) Intersection Roads Primary (a) Secondary Local (a) (b) Figure 6. Case study road network: (a) original road network and (b) simpliﬁed road network. The optimal network design of AMWs is returned by the optimization framework. To investigate how this AMW network modiﬁes when the budget decreases, the optimal AMW network is com- puted for a maximum budget of 125,000 EUR. Notice that the budget constraints the operational cost to run the system during the morning peak-hour. Then, this budget is gradually reduced to 100,000 EUR, 75,000 EUR and 50,000 EUR. Although empirical data are used, we have no ambition of designing a ready-to-use network. The goal is to have possible designs of AMW networks based on transportation considerations, i.e. travel demand and travel time, on a case study, and to draw useful information for discussing the feasibility of this system. The resulting networks are presented in Figure 7, where the origin and destination node for each AMW are connected by a straight line. This allows a clear identiﬁcation of expressways, otherwise overlapping with individual AMWs on the same road. In all cases, the networks are composed by one lane AMW for each direction, and they are almost exactly symmetric. This means that AMWs would be installed in both directions on the equipped links. We remind that one lane is 1.2 meters wide, and it has a capacity close to 7,000 passengers per hour. In comparison, a one lane urban road has the minimum width between 2.5 to 3.5 meters and a capacity ranging from 750 to 1,800 vehicles per hour depending on the intersection down- stream (DfT, 2011). This means that it is possible to install two AMW lanes, one per direction, in a single vehicular trafﬁc lane. For example, on a standard two ways street, it could be possible to pre- serve one lane for the AMW system (both directions) and to modify the viability to the remaining transport modes to one-way. Alternatively, AMWs could be installed instead of on-street parking slots at the road side. This could allow the integration of AMWs while maintaining the traditional mobility. EJTIR 17(2), 2017, pp. 6 Discussion The results of the optimization framework allow us to analyze the use of AMWs at a network level, and to provide useful considerations to delineate the system feasibility. Given the high capacity of the system and the limited space required, AMWs are suitable for dense city centers with a high passenger demand. The travel time between two given points remains the main criterion of this technical solution, aimed at intra-urban commuters primarily. However, the solution also allows us to imagine a possible city without cars, dominated by pedestrian mobility. The resulting network cannot be considered a ready-to-use design of the AMW system. However, it is a valid starting point for discussions with town planners to evaluate the potential of AMWs as a possible transport mode in urban areas. The AMW system allows pedestrian movements to and from small local roads with public spaces where “pausability” and spontaneity of movements are needed. This is possible thanks to the network structure of the system. The presence of multiple nodes for accessing/exiting the AMW system makes it permeable. The pedestrians leaving this system of AMWs are directly liberated from the means of transport, i.e. they do not have to park the mobility device nor return back to it at the end of a journey. The experience of movement and the exposure to the surrounding environment is greater than in the closed space of a car. Interacting with other passengers during travel on the AMW is as in a public tramway or bus. This system could be a catalyst for a mobility paradigm shift. Nowadays, the transport system of the future is imagined being dominated by driver-less vehicles. However, AMWs, liberating the passengers by the vehicles, could incentive a future system composed by “vehicle-less” drivers, instead of “driver-less” vehicles. With this inversion of the paradigm, the individuals, and not the vehicles, are at the center of the mobility system. Running in an ongoing fashion, moreover, the system does not force individuals to wait at public transport stops and generates, thus, a feeling of continuous ﬂow similar to the pedestrian experi- ence of the space. This happens also at intersections, thanks to the connections between AMWs and the presence of expressways, that allows a direct crossing of the junction. 5 Network design of a transport system based on accelerating moving walk- ways 191 −206 J ( ), , pp Scarinci, Bahrami, Ourednik and Bierlaire ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 12 An exploration of moving walkways as a transport system in urban centers p g y p y (a) (b) (c) (d) 194 AMWs 125,000 EUR 44 AMWs 100,000 EUR 28 AMWs 75,000 EUR 15 AMWs 50,000 EUR Figure 7. Case study results. Road network arcs equipped with AMWs for different operational budgets. In order to give a better understanding of the presented solutions, we provide some details of an AMW network. The network design presented in Figure 7 (c) is composed of 28 AMWs, for a total 194 AMWs 125,000 EUR 44 AMWs 100,000 EUR (b) (d) 15 AMWs 50,000 EUR (a) (c) 28 AMWs 75,000 EUR (a) (b) (d) (c) gure 7. Case study results. Road network arcs equipped with AMWs for different operational budgets. Figure 7. Case study results. Road network arcs equipped with AMWs for different operational budg In order to give a better understanding of the presented solutions, we provide some details of an AMW network. The network design presented in Figure 7 (c) is composed of 28 AMWs, for a total length of 18.7 km. In this case, all installed accelerating moving walkways are elementary AMWs, no expressway spanning over several intersections is suggested. This solution leads to a reduction of the total travel time of 15% in comparison with the scenario where no AMWs are installed. A rough estimation of the capital cost to build this conﬁguration is 650 M Euro. Considering the travel time saving and the cost of time, the payback period of this investment is close to one year. The main disadvantage of the system is the high capital cost. For this reason, its application can be imagined in city centers with a high transport demand and a limited space. AMWs could be a substitute for other means of transport able to free valuable spaces for other purpose. EJTIR 17(2), 2017, pp. 191 −206 J ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 13 An exploration of moving walkways as a transport system in urban centers 6 Discussion The continuous ﬂow system lowers also the level of frustration experienced by drivers stuck in trafﬁc jams and by pedestrians facing red lights on a road crossing. Thus, while allowing a gain in speed, the system does not prevent from taking advantage of the urban space. It might possibly contribute to liberate our cities from their twitchy rhythm imposed by car trafﬁc into a space of continuous and uncon- strained ﬂow of social interactions. Replacing the car trafﬁc, it should boost a more dense and vivid public space. In addition, the combined effects of promoting walking, fully electric system, low energy and space consumption could lead to a reduction of greenhouse gas emission and a more sustainable transport system. The main limitations of the system are the architectural barriers that it creates inside the city center, the high capital cost, the acceptance of the system by the users and its accessibility. 7 Conclusions In this paper, we explore the possible use of moving walkways (MW) as a transport system. We use different perspectives, from geography to urban planning to transport engineering, and we analyze the system in different historical periods. We conclude that MWs and AMWs are a promising transport mode for city centers. This system can incentive the spontaneity and pausability of movements, as well as the concept of “vehicle- less” mobility. Moreover, unlike current public transport systems, MWs offer a continuous service. On the technical side, MWs are characterized by an high capacity using limited space. In addition, the low energy consumption favors the sustainability of the system. For these reasons, MWs could be part of the transport system in a car-free urban environment where pedestrians are the main actors of mobility. EJTIR 17(2), 2017, pp. 191 −206 EJTIR 17(2), 2017, pp. 191 −206 ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 14 , , n exploration of moving walkways as a transport system in urban centers An exploration of moving walkways as a transport system in urban centers A more accurate analysis of the system feasibility should be carried out in further work. In par- ticular, the integration of MWs in the urban environment and the costs of the system should be evaluated in more details. Acknowledgment This research is part of the project Post-Car World (PCW, 2015) with the goal of exploring the future of mobility through the role of the car, funded by the Swiss National Science Foundation (SNSF, 2015). We thank Iliya Markov, Guillaume Lopez, Bastien Rojanawisut, Rapha¨el L¨uthi for their contributions on this research. References 191 −206 ( ) pp Scarinci, Bahrami, Ourednik and Bierlaire 15 An exploration of moving walkways as a transport system in urban centers Fruin, J., 1992. Designing for pedestrians. volume HS-011 999. Public Transportation United State Gautier, F., 2000. Gateway: the high-speed walkway, in: Rail Transit Conference, 2000. Gonzalez Alemany, M., Soffritti, M., Behrend, M., Eisele, T., 2007. TurboTrack: making long distances shorter. ThyssenKrupp techforum , 68–75. Gonzalez Alemany, M.A., Cuello, M.A., 2003. Accelerating walkway. Technical Report. ThyssenKrupp techforum. Gruen, V., 1964. The Heart of our Cities: The Urban Crisis, Diagnosis and Cure. New York: Simon and Schuster. Hebbert, M., 1993. The City of London walkway experiment. Journal of the American Planning Association 59, 433–450. Heidegger, M., 1954. Bauen wohnen denken. Vortrge und Aufstze 151. Heidegger, M., 1954. Bauen wohnen denken. Vortrge und Aufstze 151. Heinlein, R.A., 1940. The Roads Must Roll. The Science Fiction Hall of Fame, Volume One, 1929-1964. Helzel, M., Taylor, I., 2011. PUBLIC SPACES-Stainless Steel in the Urban Environment. Building Series 16. Ikizawa, K., Nagai, H., ichiro Aoe, S., Sato, T., Okuno, R., Yanagisawa, T., 2001. Development of an accelerating walkway. NKK Technical Review 84, 66–70. Kazuo, A., Kenro, M., Masakuni, H., Katsumi, Y., Tomoyoshi, K., 2003. Feature and prospect of accelerating moving walks. Technical Report. KFH Group, 2013. Transit Capacity and Quality of Service Manual. Technical Report. Kittelson & Assoc, Inc Parsons Brinckerhoff, Inc KFH Group, Inc Texas A&M Transportation Institute Arup. Transportation Research Board, Washington. Kusumaningtyas, I., 2009. Mind Your Step: Exploring aspects in the application of long accelerating moving walkways. Ph.D. thesis. Delft University of Technology. Mechanical, Maritime and Materials Engineer- ing. Marine and Transport Technology. Kusumaningtyas, I., Lodewijks, G., 2008. Accelerating Moving Walkway: A review of the characteristics and potential application. Transportation research part A: policy and practice 42, 591–609. Kusumaningtyas, I., Lodewijks, G., 2013. On the application of accelerating moving walkways to sup- port passenger processes in Amsterdam Airport Schiphol. Transportation Planning and Technology 36, 617–635. Lechner, N., 2011. Plumbing, Electricity, Acoustics: Sustainable Design Methods for Architecture. Plumbing, Electricity, Acoustics: Sustainable Design Methods for Architecture, Wiley. Loder, J., 1998. The in-line accelerating moving walkway. Elevator World 46, 94–97. Nakamichi, K., Nakamura, F., 2014. Current state of the modern urban transport system in Medellin, Colombia. Journal of the City Planning Institute of Japan 49. NYPL, 1923. Belt Conveyor Moving Platform New York Diagram Passengers Transport. New York Public Library digital collection. References Y., Beniya, Y., Masuda, H., 2001. Accelerating Moving Walkway“ACCEL-LINER”. Bridges 10, 55 Alspaugh, A., 2008. Bulk Material Handling by Conveyor Belt 7. Society for Mining, Metallurgy, and Exploration. Asimov, I., 1954. Caves of Steel. Random House Publishing Group. Asimov, I., 1954. Caves of Steel. Random House Publishing Group. Avenel, H., 1900. Histoire de la presse francaise, depuis 1789 jusqu’a nos jours, rapport au ministere du com- merce: exposition universelle de 1900. E. Flammarion. Badger, R., 1979. The great American fair: The world’s Columbian exposition & American culture. Taylor Trade Publications. Bollnow, O., 1967. Lived-Space, in Lawrence, Nathaniel and O’Connor, Daniel (eds.) Readings in Exis- tential Phenomenology . Brand, C., Preston, J., 2003. Which technology for urban public transport?, in: Proceedings of the Insti- tution of Civil Engineers-Transport, pp. 201–210. Buchanan, C., 1963. Trafﬁc in towns: a study of the long-term problems of trafﬁc in urban areas. Penguin Books. CEN, 1998. NEN-EN 115:1998 safety rules for the construction and installation of escalators and passenger conveyors. Technical Report. Comite Europeen de Normalisation. Christidis, P., Rivas, J.N.I., 2012. Measuring road congestion. Technical Report. European Union. Institute for Prospective and Technological Studies, Joint Research Centre. Ciufﬁni, F., 1983. Escalators to facilitate pedestrian movement in Perugia. Casabella 47, 28–28. Cook, P., 1999. Archigram. Princeton Architectural Press. Cook, P., 1999. Archigram. Princeton Architectural Press. Cote, A., Gempp, A., 1998. The High Speed Passenger Conveyor Reﬂections on Comfort, in: 6th Inter- national Conference on Automated People Movers (APMs). Cullinane, S., 2002. The relationship between car ownership and public transport provision: a case study of Hong Kong. Transport Policy 9, 29–39. Dembart, L., 2003. International Traveler/Update: In Paris, The Concorde of All Sidewalks. Technical Report. International Herald Tribune. DfT, 2011. Design manual for roads and bridges. Department of Transport, Great Britain. DGT, 2015. Direction generale des transports. URL: http://ge.ch/mobilite/. Donoghue, E.A., 1981. Handbook on A17.1: Safety Code for Elevator and Escalators. Technical Report. The American Society of Mechanical Engineers. EC, 2011. White paper Roadmap to a Single European Transport Area - Towards a competitive and resource efﬁcient transport system - The Impact Assessment. Technical Report. European Commission. Farahani, R.Z., Miandoabchi, E., Szeto, W.Y., Rashidi, H., 2013. A review of urban transportation net- work design problems. European Journal of Operational Research 229, 281–302. EJTIR 17(2), 2017, pp. References Otis, 2015. The online architecture and design exhibition. ArchiEXPO. Ourednik, A., 2008. Conatus et le miel de Jean-Baptiste. Pour un r´ecit humain de l’automobilit´e. Es- pacesTemps.net. URL: http://www.espacestemps.net/document6273.html. Ourednik, A., 2010. L’habitant et la cohabitation dans les modeles de l’espace habite. Ph.D. thesis. Ecole Polytechnique Federale de Lausanne EPFL. PCW, 2015. Post-Car World: A Trans-Disciplinary Multi-Dimensional Stimulation. URL: http:// postcarworld.epfl.ch. Richards, B., 1966. New movement in cities. London; New York: Littlehampton Book Services Ltd. Richards, B., 1976. Moving in Cities. Westview Press. Richards, B., 1976. Moving in Cities. Westview Press. Rockwood, D., Garmire, D., 2015. A new transportation system for efﬁcient and sustainable cities: Development of a next generation variable speed moving walkway. Sustainable Cities and Society 14, 209–214. EJTIR 17(2), 2017, pp. 191 −206 J ( ), , pp Scarinci, Bahrami, Ourednik and Bierlaire pp Scarinci, Bahrami, Ourednik and Bierlaire 16 c , , O e e e n exploration of moving walkways as a transport system in urban centers An exploration of moving walkways as a transport system in urban centers Rojanawisut, B., 2015. Optimisation of the network design of a futuristic transport system based on accelerated moving walkways. Master Thesis. Ecole Polytechnique Federale de Lausanne EPFL, Section of Mathe- matics. Rouillard, D., 2013. La marche le marketing du corps. Marche et espace urbain de l’Antiquite a nos jours. Le Maire, Judith, Christophe Loir, and Anne Desprechins. Editions Mardaga , 149–166. Saeki, H., 1996. Mitsubishi-Speedwalk: Development of accelerating moving walk, in: Colloque inter- national sur les transports collectifs automatiques, pp. 571–581. Scarinci, R., Markov, I., Bierlaire, M., 2016. Network design of a transport system based on accelerating moving walkways, in: Ninth Triennial Symposium on Transportation Analysis TRISTAN IX. Shirakihara, T., 1997. Speedwalk - an Accelerating Moving Walkway, in: Proceedings of the 1997 6th International Conference on Automated People Movers, April 9-12 April, pp. 624–633. SITG, 2015. Systeme d’information du territoire a Geneve. URL: http://ge.ch/sitg/. SNSF, 2015. Swiss National Science Foundation. URL: http://www.snf.ch/. ThyssenKrupp, 2004. Questions and answers: How loud are escalators or walkways? Technical Report. ThyssenKrupp Fahrtreppen GmbH. Todd, J., 1974. Dunlop S-type Speedway: A high-speed passenger conveyor. Transportation Research Record 522, 65–75. Wells, H.G., 1897. A Story of the Days to Come. Horizon Publishing Group. Woodcock, C., Mason, J., 2012. Bulk solids handling: an introduction to the practice and technology. Springer Science & Business Media. Yang, H., Bell, M.G.H., 1998. References Models and algorithms for road network design: a review and some new developments. Transport Reviews 18, 257–278. Young, S.B., 1999. Evaluation of pedestrian walking speeds in airport terminals. Transportation Research Record: Journal of the Transportation Research Board 1674, 20–26.
https://openalex.org/W3197647116	https://www.frontiersin.org/articles/10.3389/fmed.2021.708316/pdf	English	null	Plasma Macrophage Migration Inhibitory Factor Predicts Graft Function Following Kidney Transplantation: A Prospective Cohort Study	Frontiers in medicine	2,021	cc-by	8,490	Citation: Ye Y, Han F, Ma M, Sun Q, Huang Z, Zheng H, Yang Z, Luo Z, Liao T, Li H, Hong L, Na N and Sun Q (2021) Plasma Macrophage Migration Inhibitory Factor Predicts Graft Function Following Kidney Transplantation: A Prospective Cohort Study. Front. Med. 8:708316. doi: 10.3389/fmed.2021.708316 Plasma Macrophage Migration Inhibitory Factor Predicts Graft Function Following Kidney Transplantation: A Prospective Cohort Study Yongrong Ye 1†, Fei Han 1†, Maolin Ma 1†, Qipeng Sun 1, Zhengyu Huang 1, Haofeng Zheng 1,2, Zhe Yang 1, Zihuan Luo 1,2, Tao Liao 1,2, Heng Li 1, Liangqing Hong 1, Ning Na 1 and Qiquan Sun 1,2* 1 Division of Kidney Transplantation, Organ Transplantation Research Institution, The Third Afﬁliated Hospital, Sun Yat-sen University, Guangzhou, China, 2 Department of Kidney Transplantation, Guangdong Provincial People’s Hospital, Guangzhou, China Background: Delayed graft function (DGF) is a common complication after kidney transplantation (KT) with a poor clinical outcome. There are no accurate biomarkers for the early prediction of DGF. Macrophage migration inhibitory factor (MIF) release during surgery plays a key role in protecting the kidney, and may be a potential biomarker for predicting post-transplant renal allograft recovery. Edited by: Rujun Gong, University of Toledo Medical Center, United States Edited by: Rujun Gong, University of Toledo Medical Center, United States j g University of Toledo Medical Center, United States Reviewed by: Brian Duncan Tait, Australian Red Cross Blood Service, Australia Xiaopeng Hu, Capital Medical University, China Correspondence: Qiquan Sun sunqiq@mail.sysu.edu.cn †These authors have contributed equally to this work and share ﬁrst authorship Reviewed by: Brian Duncan Tait, Australian Red Cross Blood Service, Australia Xiaopeng Hu, Capital Medical University, China Reviewed by: Brian Duncan Tait, Australian Red Cross Blood Service, Australia Xiaopeng Hu, Capital Medical University, China Correspondence: Qiquan Sun sunqiq@mail.sysu.edu.cn Methods: Recipients who underwent KT between July 2020 and December 2020 were enrolled in the study. Plasma MIF levels were tested in recipients at different time points, and the correlation between plasma MIF and DGF in recipients was evaluated. This study was registered in the Chinese Clinical Trial Registry (ChiCTR2000035596). Capital Medical University, China Correspondence: Qiquan Sun sunqiq@mail.sysu.edu.cn Correspondence: Qiquan Sun sunqiq@mail.sysu.edu.cn Results: Intraoperative MIF levels were different between immediate, slowed, and delayed graft function groups (7.26 vs. 6.49 and 5.59, P < 0.001). Plasma MIF was an independent protective factor of DGF (odds ratio = 0.447, 95% conﬁdence interval [CI] 0.264–0.754, P = 0.003). Combining plasma MIF level and donor terminal serum creatinine provided the best predictive power for DGF (0.872; 95%CI 0.795–0.949). Furthermore, plasma MIF was signiﬁcantly associated with allograft function at 1-month post-transplant (R2 = 0.42, P < 0.001). †These authors have contributed equally to this work and share ﬁrst authorship Specialty section: This article was submitted to Nephrology, a section of the journal Frontiers in Medicine Conclusion: Intraoperative MIF, as an independent protective factor for DGF, has excellent diagnostic performance for predicting DGF and is worthy of further exploration. Received: 28 May 2021 Accepted: 31 July 2021 Published: 01 September 2021 Keywords: kidney transplantation, delayed graft function, macrophage migration inhibitory factor, biomarker, graft function ORIGINAL RESEARCH published: 01 September 2021 doi: 10.3389/fmed.2021.708316 INTRODUCTION Kidney transplantation (KT) is the primary therapy for end-stage renal disease (ESRD), with a lower mortality rate and superior quality of life compared to dialysis (1, 2). However, early complications limit the beneﬁt of KT, such as delayed graft function (DGF), which is often deﬁned as requiring dialysis within 1 week following KT (3, 4). The rate of DGF in recipients is as high September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org Ye et al. A Prospective Cohort Study as 20–33% (5), and the strategy of using an extended criteria donor to extend the donor pools signiﬁcantly increases the rate of DGF (6–8). DGF increases the costs of hospitalization, prolongs the length of stay, increases the rate of acute rejection, and aﬀects the renal allograft prognosis (9–11). There are no accurate biomarkers for the early prediction of DGF, and there remains an urgent need for relevant indications. The study was approved by the ethics committee of the Third Aﬃliated Hospital of Sun Yat-sen University, and it complied with the Declarations of Helsinki and Istanbul (24). All enrolled recipients agreed to participate in this study and signed an informed consent form. The China Organ Transplant Response System completed organ allocation according to equitable and transparent principles (25). This study was registered in the Chinese Clinical Trial Registry (ChiCTR2000035596). g Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is rapidly released following various stimuli from cellular stores, compared with other cytokines that require mRNA transcription and protein synthesis before release (12, 13). MIF is a 12.5-kDa homotrimer that binds and activates various receptors (13, 14). Previous studies have shown that MIF has antioxidant properties (15–17), and its binding to CD74 exerts a protective role in cardiac ischaemia reperfusion injury (IRI) by antioxidant eﬀects through the CD74/CD44/AMP-activated protein kinase pathway (18, 19). A retrospective study also reported that elevated MIF levels were negatively correlated with cardiac dysfunction after a heart operation (20). In addition, the rate of acute kidney injury (AKI) was dramatically reduced in cardiac surgery patients with high circulating MIF at 12 h postoperatively (21). Similar to heart operations, transplant surgery with blockade of blood ﬂow is associated with tissue hypoxia and systemic oxidative stress, leading to elevated MIF in circulation (22). However, the relationship between elevated MIF during KT and post-transplant renal allograft function has not been studied. INTRODUCTION We hypothesized that elevated circulatory MIF during KT alleviates IRI of the graft, thereby reducing the rate of DGF and improving postoperative renal allograft function. Data Source and Immunosuppressive Regimen Donor data were acquired from the organ procurement organizations. Clinical transplant surgeons provided recipient data. We divided the recipients into three groups: immediate graft function (IGF), slowed graft function (SGF), and delayed graft function (DGF). The creatinine reduction ratio (CRR) was used to diagnose IGF and SGF. The deﬁnition of DGF was requiring dialysis within 1 week following KT. SGF was deﬁned as CRR < 70% and without the need for dialysis within 1 week after KT. IGF was deﬁned as CRR>70% in the week after KT and without the need for dialysis (9). Immunosuppressive induction regimens were performed by antithymocyte globulin (50 mg/day, 0–day 2) or basiliximab (20 mg/day, 0 and day 4). Methylprednisolone was usually administered intravenously at a dose of 500 mg/day during inducing therapy and administration of the ﬁrst dose was completed before kidney reperfusion. Calcineurin inhibitor, mycophenolate mofetil and prednisone were combined to maintain immunosuppression. The onset doses of oral tacrolimus or cyclosporine were 0.1–0.15 or 6–8 mg/kg/day on day 2–4, respectively. The doses were adjusted according to required blood concentrations. Mycophenolate mofetil was started post-transplant and was maintained at a dose of 1.5–2 g/day. Oral prednisone was administered at 30 mg/day after inducing therapy and was decreased by 5 mg/week to a maintenance daily dose of 10–15 mg. This study explored the correlation between plasma MIF levels and postoperative renal allograft function in recipients. It may provide transplant physicians with an early, non-invasive, and accurate means of predicting DGF. Abbreviations: ATG, rabbit anti-human thymocyte immunoglobulin; AKI, acute kidney injury; BMI, body mass index; CNI, calcineurin inhibitor; CI, conﬁdence interval; CRR, creatinine reduction ratio; DGF, delayed graft function; ESRD, end-stage renal disease; GN, glomerulonephritis; GCs, glucocorticoids; HD, hemodialysis; HLA, human leukocyte antigen; IGF, immediate graft function; IRI, ischaemia reperfusion injury; KT, kidney transplantation; MIF, macrophage migration inhibitory factor; NGAL, neutrophil gelatinase-associated lipocalin; OR, odds ratio; PD, peritoneal dialysis; PRA, panel reactive antibody; ROC, receiver operating characteristic; SGF, slowed graft function; Scr, serum creatinine; WD, without dialysis. Study Design We conducted a prospective and observational cohort study. This study enrolled 85 recipients who received allografts from patients declared brain death between July 2020 and December 2020 at the Third Aﬃliated Hospital of Sun Yat-sen University. Exclusion criteria for recipients were as follows: (1) without blood samples or with haemolysis of blood samples; (2) patients who were administered diﬀerent doses of glucocorticoids (GCs) during surgery due to GC-induced MIF release (23); (3) patients undergoing simultaneous multiple organ transplantation. All patients were followed up for 1 month after KT. Sample Collection and Detection p Blood samples were acquired from the ﬁrst 30 recipients at the following timepoints: after anesthesia and before skin incision (pre-operation), 5 min after kidney reperfusion (during operation), and the end of surgery (post-operation). The last 47 recipient blood samples were only collected once 5 min after kidney reperfusion (during operation). Samples were placed in an ice box and immediately sent to the laboratory to prepare the plasma. The blood samples were immediately centrifuged at 1,500 rpm/min for 10 min, and the supernatants were transferred to the cryotubes with a pipette, and then stored at the −80◦C until analysis. We used an enzyme-linked immunosorbent assay (Human MIF ELISA Kit; ABclonal Technology, Wuhan, China) to measure plasma MIF concentrations according to the manufacturer’s protocol. This assay employs the quantitative sandwich enzyme immunoassay technique wherein the MIF- speciﬁc monoclonal antibody is pre-coated on the microplate, and any MIF present in the standard and sample is bound by September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org 2 Ye et al. A Prospective Cohort Study FIGURE 1 \| Flow chart of kidney recipients enrolled in the study. FIGURE 1 \| Flow chart of kidney recipients enrolled in the study. the immobilized antibody. The MIF-speciﬁc detection antibody binds to the combination of the capture antibody-MIF in the sample. Adding enzyme conjugate and substrate successively leads to the formation of a colored product TMB. The reaction was terminated by adding acid, and the absorbance is measured. The intensity of the TMB was proportional to the amount of MIF present in the sample. Finally, the concentration of the MIF sample was determined by plotting a curve of the absorbance for the sample and standard. for DGF. The optimal cut-oﬀpoints for the ROC curves analyses were calculated using Youden’s J-statistic. Spearman’s correlation coeﬃcient was used to analyse the correlation between MIF and recipient serum creatinine (Scr). The correlation between MIF levels and Scr levels at 1-month post-transplant was evaluated by the multiple linear regression analysis. Statistical analysis was carried out using SPSS version 25.0. Frontiers in Medicine \| www.frontiersin.org Statistical Analysis y The Mann-Whitney U-test was used to compare the diﬀerences between diﬀerent groups for nonnormally distributed variables, and Student’s t-test was used to test whether there is a diﬀerence between diﬀerent groups on a continuous dependent variable. The parametric analysis of variance (ANOVA) and Kruskal- Wallis tests were used to analyse the diﬀerences in biomarker levels between diﬀerent groups. The diagnostic performance of MIF and other biomarkers for the prediction of DGF was evaluated by receiver operating characteristic (ROC) curve analyses. The independent relevant parameters of DGF were acquired with multivariate logistic regression analyses using a backward stepwise method, and the method of stepwise analysis was set as P < 0.05 for entry and P > 0.1 for removal. These independent relevant parameters obtained from the multivariate logistic regression analyses were used to build a predictive model Of the 85 recipients enrolled, 8 recipients were excluded: 2 without plasma samples, 1 with haemolysis of plasma sample, 4 were administered diﬀerent doses of GCs during surgery, and 1 had undergone combined liver-kidney transplantation (Figure 1). Demographic data of the 77 recipients and their donors are presented in Table 1. All recipients were divided into 3 groups according to allograft function: 39 had IGF, 16 had SGF, and 22 had DGF. Recipient age was diﬀerent between the IGF, SGF, and DGF groups (43 years vs. 40 and 45.5 years, P = 0.029). The cold ischaemic time of the kidney in the IGF group was signiﬁcantly lower than that in the DGF and SGF groups (3.5 h vs. 5 and 5 h, P < 0.001). Donor body mass index was signiﬁcantly diﬀerent between the IGF, SGF, and DGF groups (22.9 kg/m2 vs. 23.8 and 24.1 kg/m2, P = September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org 3 A Prospective Cohort Study Ye et al. TABLE 1 \| Summary of characteristics in recipient and donor, stratiﬁed by recipients’ s allograft function. Statistical Analysis Characteristic All (n = 77) IGF (n = 39) SGF (n = 16) DGF (n = 22) P-value Recipients Age, years 42 [34.5,51] 43 [35,51] 40 [36,51.5] 45.5 [31.8,54] 0.029 Male sex 46 (60) 22 (56) 7 (44) 17 (77) 0.096 Cause of ESRD 0.63 GN 16 (21) 8 (21) 2 (13) 6 (27) Hypertension 2 (3) 0 (0) 1 (6) 1 (5) Diabetes 2 (3) 1 (3) 0 (0) 1 (5) Others 57 (74) 30 (77) 13 (81) 14 (64) Mode of dialysis 0.628 HD 66 (86) 33 (85) 15 (94) 18 (82) PD 6 (8) 3 (8) 0 3 (14) WD 5 (6) 3 (8) 1 (6) 1 (4) Dialysis duration, mo 12 [3.5,22.5] 12 [4,25] 5.5 [1.1,59.8] 12 [4.8,30] 0.713 Cold ischaemic time, h 4 [3,5.5] 3.5 [3,4.5] 5 [4,6] 5 [4,6] <0.001 Number of HLA mismatches 5 [4,5] 5 [4,5] 5 [4.3,5.8] 5 [4,5] 0.362 Panel reactive antibody 0.358 0% 66 (86) 34 (87) 12 (75) 20 (91) 1–10% 11 (14) 5 (13) 4 (25) 2 (9) Induction regimen 0.381 ATG 51 (66) 28 (72) 11 (69) 12 (55) Basiliximab 26 (34) 11 (18) 5 (31) 10 (45) CNI 0.368 Cyclosporin 2 (3) 2 (5) 0 (0) 0 (0) Tacrolimus 75 (97) 37 (95) 16 (100) 22 (100) Steroid 77 39 16 22 Renal functions at post-transplant (mg/dL) Scr, at 1 day 9.13 [6.34,11.22] 8 [5.15,11.11] 9.2 [6.11,10.24] 10.68 [7.9,11.84] 0.064 Scr, at 1 week 2.87 [1.62,5.46] 1.63 [1.17,2.14] 4.32 [3.28,6.65] 6.32 [4.71,9.36] <0.001 Scr, at 1 month 1.75 [1.46,2.28] 1.5 [1.23,1.82] 1.92 [1.66,2.27] 2.71 [1.86,4.21] <0.001 Variation of Scr after transplantation (mg/dL) Absolute decrease 0 h−1 day 0.94 [−0.76,3.32] 3.04 [0.94,4.62] −0.49 [−1.19,0.76] −0.03 [−1.22,1.38] <0.001 Relative decrease (0 h−1 day)/1 day 0.12 [−0.08,0.34] 0.28 [0.11,0.41] −0.05 [−0.17,0.1] 0 [−0.11,0.14] <0.001 MIF levels during operation (ng/mL) 6.52 [5.59,7.45] 7.26 [6.28,8.39] 6.49 [5.52,7.21] 5.59 [4.36,6.03] <0.001 Donors Age, years 51 [38.5,54] 42 [38,54] 52 [41,55] 51.5 [46,54.3] 0.207 Male sex 52 (68) 23 (59) 12 (75) 17 (77) 0.264 BMI (kg/m2) 23.4 [20.8,24.8] 22.9 [19.4,24.1] 23.8 [22.5,25.5] 24.1 [22.8,26.5] 0.017 Hypertension 43 (56) 19 (49) 10 (63) 14 (64) 0.442 Cause of death 0.526 Head trauma 7 (9) 2 (5) 2 (13) 3 (14) Stroke 51 (66) 25 (64) 12 (75) 14 (64) Other 19 (25) 12 (31) 2 (13) 5 (23) Admission Scr, mg/dL 1.28 [0.85,2.1] 0.97 [0.7,1.28] 1.59 [1.1,2.43] 2.26 [1.43,3.34] <0.001 Terminal Scr, mg/dL 1.46 [0.87,2.02] 1.01 [0.7,1.48] 1.64 [1.11,2.41] 1.94 [1.69,4.83] <0.001 Use of any vasoactive drugs 71 (92) 35 (90) 15 (94) 21 (95) 0.703 No. DGF, delayed graft function; ESRD, end-stage renal disease; GN, glomerulonephritis; HD, hemodialysis; PD, peritoneal dialysis; WD, without dialysis; HLA, human leukocyte antigen; ATG, Rabbit Anti-Human Thymocyte Immunoglobulin; CNI, calcineurin inhibitor; Scr, serum creatinine; MIF, Macrophage migration inhibitory factor; BMI, body mass index; AKI, acute kidney injury; ANOVA, analysis of variance. Frontiers in Medicine \| www.frontiersin.org g sts were used to analyze continuous variables and χ2 test and Fisher’s exact test were used to analyze categorical variables. a e e p essed by tota u be s a d pe ce tages ANOVA or Kruskal–Wallis tests were used to analyze continuous variables and χ2 test and Fisher’s exact test were used to analyze categorical variables. DGF, delayed graft function; ESRD, end-stage renal disease; GN, glomerulonephritis; HD, hemodialysis; PD, peritoneal dialysis; WD, without dialysis; HLA, human leukocyte antigen; ATG, Rabbit Anti-Human Thymocyte Immunoglobulin; CNI, calcineurin inhibitor; Scr, serum creatinine; MIF, Macrophage migration inhibitory factor; BMI, body mass index; AKI, acute est, if P > 0.05 for continuous variables, the data are presented as mean ±SD; otherwise, the data are presented as median [P25, P75]; catego bers and percentages. are expressed by total numbers and percentages. ANOVA or Kruskal–Wallis tests were used to analyze continuous variables and χ2 test and Fisher’s exact test were used to analyze categorical variables. DGF, delayed graft function; ESRD, end-stage renal disease; GN, glomerulonephritis; HD, hemodialysis; PD, peritoneal dialysis; WD, without dialysis; HLA, human leukocyte antigen; ATG, Rabbit Anti-Human Thymocyte Immunoglobulin; CNI, calcineurin inhibitor; Scr, serum creatinine; MIF, Macrophage migration inhibitory factor; BMI, body mass index; AKI, acute kidney injury; ANOVA, analysis of variance. p y p g ANOVA or Kruskal–Wallis tests were used to analyze continuous variables and χ2 test and Fisher’s exact test were used to analyze categorical variables. Statistical Analysis of any vasoactive drugs 4 [2,8] 3 [2,6] 4.5 [2.8] 5.5 [2,9] 0.544 No. of kidneys transplanted 1 5 (6) 1 (3) 1 (6) 3 (14) 0.241 2 72 (94) 38 (97) 15 (94) 19 (86) According to Shapiro-Wilk test, if P > 0.05 for continuous variables, the data are presented as mean ±SD; otherwise, the data are presented as median [P25, P75]; categorical variables are expressed by total numbers and percentages. ANOVA or Kruskal–Wallis tests were used to analyze continuous variables and χ2 test and Fisher’s exact test were used to analyze categorical variables. DGF delayed graft function; ESRD end stage renal disease; GN glomerulonephritis; HD hemodialysis; PD peritoneal dialysis; WD without dialysis; HLA human leukocyte antigen; ANOVA or Kruskal–Wallis tests were used to analyze continuous variables and χ2 test and Fisher’s exact test were used to analyze categorical variables. DGF, delayed graft function; ESRD, end-stage renal disease; GN, glomerulonephritis; HD, hemodialysis; PD, peritoneal dialysis; WD, without dialysis; HLA, human leukocyte antigen; ATG, Rabbit Anti-Human Thymocyte Immunoglobulin; CNI, calcineurin inhibitor; Scr, serum creatinine; MIF, Macrophage migration inhibitory factor; BMI, body mass index; AKI, acute kidney injury; ANOVA, analysis of variance. September 2021 \| Volume 8 \| Article 708316 4 A Prospective Cohort Study Ye et al. FIGURE 2 \| (A) Distributions of plasma macrophage migration inhibitory factor (MIF) from recipient during operation, recipient’s serum creatinine (Scr) at 1-day post-transplant, absolute Scr decrease from 0-h to 1-day post-transplant, and donor terminal Scr in the recipient. (B–D) Contents of MIF, recipient’ Scr at 1-day post-transplant, absolute Scr decrease after transplant, and donor terminal Scr in immediate graft function (IGF; red), slowed graft function (SGF; green), and delayed graft function (DGF; blue) groups. P < 0.05, P < 0.01, *P < 0.001. FIGURE 2 \| (A) Distributions of plasma macrophage migration inhibitory factor (MIF) from recipient during operation, recipient’s serum creatinine (Scr) at 1-day post-transplant, absolute Scr decrease from 0-h to 1-day post-transplant, and donor terminal Scr in the recipient. (B–D) Contents of MIF, recipient’ Scr at 1-day post-transplant, absolute Scr decrease after transplant, and donor terminal Scr in immediate graft function (IGF; red), slowed graft function (SGF; green), and delayed graft function (DGF; blue) groups. P < 0.05, P < 0.01, *P < 0.001. 0.017). Statistical Analysis There were no diﬀerences in the mode of dialysis, dialysis duration, cause of ESRD, induction regimen, number of HLA mismatches, panel reactive antibody, donor cause of death, use of vasoactive drugs, or number of vasoactive drugs used between the three groups (Table 1). Supplementary Figure 1. There was no diﬀerence between DGF and non-DGF patients in baseline plasma MIF at pre- transplant timepoints (1.28 ng/mL vs. 1.17 ng/mL, P = 0.428). However, intraoperative and postoperative plasma MIF levels were signiﬁcantly higher in the non-DGF group than in the DGF group, and the intraoperative diﬀerence was more evident between groups (7.02 ng/mL vs. 5.18 ng/mL, P < 0.001; 6.25 ng/mL vs. 5.5 ng/mL, P = 0.028, respectively). Plasma MIF levels signiﬁcantly increased at intraoperative and postoperative timepoints compared to pre-operation (6.56 ng/mL vs. Differences in Plasma MIF in Perioperative Patients Undergoing Kidney Transplant g g The diﬀerences in plasma MIF between 30 patients with DGF or non-DGF during the perioperative period are shown in September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org 5 Ye et al. A Prospective Cohort Study TABLE 2 \| Univariate and multivariate logistic regression analyses for predicting delayed graft function. Univariate Multivariate OR 95%CI P-value OR 95%CI P-value Donor age (years) 1.03 0.989–1.072 0.157 Donor sex (women) 0.515 0.165–1.607 0.253 Donor BMI (kg/m2) 1.123 0.977–1.29 0.102 Donor cause of death 0.747 0.306–1.821 0.521 Donor terminal Scr (mg/dL) 1.857 1.261–2.736 0.002 1.648 1.108–2.449 0.014 Cold ischaemic time (h) 1.552 1.097–2.195 0.013 Recipient age (years) 0.998 0.952–1.046 0.921 Recipient sex (female) 0.328 0.106–1.014 0.053 Duration of dialysis before transplantation (mo) 1.005 0.992–1.018 0.474 HLA mismatch 0.897 0.524–1.537 0.693 PRA 0.511 0.101–2.582 0.417 Induction regimen 2.031 0.732–5.640 0.174 Plasma MIF (ng/mL) 0.396 0.242–0.649 0.001 0.447 0.264–0.754 0.003 Multivariate logistic regression analysis was executed with a backward selection procedure. HLA, human leukocyte antigen; PRA, panel reactive antibody; MIF, Macrophage migration inhibitory factor; BMI, body mass index; AKI, acute kidney injury; Scr, serum creatinine; CI, conﬁdence interval; OR, odds ratio. TABLE 2 \| Univariate and multivariate logistic regression analyses for predicting delayed graft function. Multivariate logistic regression analysis was executed with a backward selection procedure. HLA, human leukocyte antigen; PRA, panel reactive antibody; MIF, Macrophage migration inhibitory factor; BMI, body mass index; AKI, acute kidney injury; Scr, serum creatinine; CI, conﬁdence interval; OR, odds ratio. 0.71 ng/mL, P < 0.001; 5.86 ng/mL vs. 0.71 ng/mL, P < 0.001, respectively), but there was no diﬀerence between intraoperative and postoperative timepoints (Supplementary Figure 2). Therefore, we used the intraoperative period as the timepoint at which to detect MIF. 95%CI 1.261–2.736, P = 0.002; OR = 1.552, 95%CI 1.097–2.195, P = 0.013). From multivariate logistic regression analyses, we further concluded that plasma MIF and donor terminal Scr were independent relevant parameters for DGF (OR = 0.447, 95%CI 0.264–0.754, P = 0.003; OR = 1.648, 95%CI 1.108–2.449, P = 0.014; Table 2). Predictive Value of Plasma MIF for Delayed Graft Function We used ROC curve analysis to evaluate the diagnostic performance of plasma MIF, the recipient’s Scr at 1-day post- transplant, absolute decreased Scr after transplant, and donor terminal Scr for predicting DGF. The area under the ROC curve (AUROC) of plasma MIF and donor terminal Scr in predicting DGF was 0.816 (95%CI 0.712–0.92, P < 0.001) and 0.8 (95%CI 0.693–0.908, P < 0.001), respectively, which were superior to that of the recipient’s Scr at 1-day post-transplant (0.671; 95%CI 0.544–0.799, P = 0.019 or absolute decreased Scr after transplant (0.686; 95%CI 0.559–0.812, P = 0.011; Figures 3A–D). Meanwhile, we combined the relevant parameters that were acquired from multivariate logistic regression analyses to build a predictive model for DGF. The model including the reciprocal plasma MIF level and donor terminal Scr improved the AUROC to 0.872 (95%CI 0.795–0.949, P < 0.001; Figure 3E). Moreover, this model had higher sensitivity and moderate speciﬁcity at the optimum cut-oﬀpoint (sensitivity = 0.96, speciﬁcity = 0.66). The sensitivity, speciﬁcity, and optimal cut-oﬀpoint of these parameters for predicting DGF are shown in Table 3. The intraoperative plasma MIF in the IGF group was higher than that in the SGF and DGF groups (7.26 ng/mL vs. 6.49 ng/mL, P < 0.05; 7.26 ng/mL vs. 5.59 ng/mL, P < 0.001, respectively), and the SGF group was also higher than that in the DGF group (6.49 ng/mL vs. 5.59 ng/mL, P < 0.05) (Figure 2A). Recipient Scr at 1-day post-transplant was diﬀerent between the IGF and DGF groups (8 mg/dL vs. 10.68 mg/dL, P = 0.033; Figure 2B). A decrease in recipient Scr from 0-h to 1-day post-transplant and donor terminal Scr eﬀectively distinguished the IGF group from the SGF and DGF groups (3.04 mg/dL vs. −0.49 and −0.03 mg/dL, P < 0.001; 1.01 mg/dL vs. 1.64 and 1.94 mg/dL, P < 0.001, respectively), but neither indicators diﬀered between the SGF and DGF groups (Figures 2C,D). According to the intraoperative plasma MIF level distribution, the incidence of needing dialysis within 1 week after transplantation increased 6-fold when the levels were <5.8 ng/mL (67 vs. 11%, P < 0.001). MIF Is an Independent Protective Factor for Delayed Graft Function The relevant parameters for predicting DGF were acquired using univariate and multivariate logistic regression analyses. From the univariate analyses, we concluded that recipient intraoperative plasma MIF, donor terminal Scr, and kidney cold ischaemia time were signiﬁcantly correlated with DGF (odds ratio [OR] = 0.396, 95% conﬁdence interval [CI] 0.242–0.649, P = 0.001; OR = 1.857, MIF Is Related to Graft Function 1-Month Post-transplant The reciprocal plasma MIF, donor terminal Scr, and recipient Scr at 1-day post-transplant were positively related with recipient Scr at 1-month post-transplant (R2 = 0.42, P < 0.001; R2 September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org 6 Ye et al. A Prospective Cohort Study FIGURE 3 \| Receiver operating characteristic (ROC) curves for recipient and don macrophage migration inhibitory factor (MIF) from recipients during operation (A), and recipient’s serum creatinine (Scr) at 1-day post-transplant (D). The linear pre = 0.129, P < 0.001; R2 = 0.058, P < 0.001, respectively, Figures 4A–C). Univariate analyses showed that recipient Scr at 1-day post-transplant, plasma MIF, female donors, cause of donor death, donor terminal Scr and kidney cold ischaemic FIGURE 3 \| Receiver operating characteristic (ROC) curves for recipient and donor characteristics for predicting delayed graft function. The ROC curves of plasma macrophage migration inhibitory factor (MIF) from recipients during operation (A), donor terminal Scr (B), absolute Scr decrease from 0-h to 1-day post-transplant (C), and recipient’s serum creatinine (Scr) at 1-day post-transplant (D). The linear prediction model including the reciprocal plasma MIF level and donor terminal Scr (E). = 0.129, P < 0.001; R2 = 0.058, P < 0.001, respectively, Fi 4A C) U i i t l h d th t i i t S time were signiﬁcantly related to recipient Scr 1-month post- t l t (β 2 142 95%CI 0 008 0 226 P 0 035 β nor characteristics for predicting delayed graft function. The ROC curves of plasma donor terminal Scr (B), absolute Scr decrease from 0-h to 1-day post-transplant (C), diction model including the reciprocal plasma MIF level and donor terminal Scr (E). FIGURE 3 \| Receiver operating characteristic (ROC) curves for recipient and donor characteristics for predicting delayed graft function. The ROC curves of plasma macrophage migration inhibitory factor (MIF) from recipients during operation (A), donor terminal Scr (B), absolute Scr decrease from 0-h to 1-day post-transplant (C), and recipient’s serum creatinine (Scr) at 1-day post-transplant (D). The linear prediction model including the reciprocal plasma MIF level and donor terminal Scr (E). FIGURE 3 \| Receiver operating characteristic (ROC) curves for recipient and donor characteristics for predicting delayed graft function. DISCUSSION Our study investigated the role of perioperative MIF release in predicting delayed graft function after KT and illustrated that the intraoperative plasma MIF was signiﬁcantly lower in patients who developed DGF vs. patients with non-DGF. Moreover, the diagnostic value of intraoperative plasma MIF for predicting DGF was superior to that of the recipient’s Scr at 1-day post- transplant. In addition, the combination of plasma MIF and donor terminal Scr has a high diagnostic value for predicting DGF. This may provide transplant physicians with an early, non-invasive, and accurate means of predicting graft function following KT. MIF is a pleiotropic cytokine and its functions in AKI are not well understood. Previous studies have shown that MIF is a proinﬂammatory mediator of the innate immune system, and increased urinary MIF was related to the severity of kidney injury in glomerulonephritis or pyelonephritis (26, 27). Similarly, during sepsis or liver transplantation, increased MIF levels appear to aggravate renal damage (22, 28). However, recent studies have indicated that patients undergoing cardiac surgery with high circulating MIF had a signiﬁcantly lower risk of developing AKI (21, 29). Furthermore, they demonstrated that MIF-deﬁcient mice exhibited increased tubular cell damage and increased apoptotic tubular cells, and administering recombinant MIF ameliorated renal tubular injury and apoptosis in AKI mouse model (21). These ﬁndings suggested that in AKI induced by IRI, the protective eﬀect of MIF on cell death may be more eﬀective, surpassing the potential pro-inﬂammatory and chemokine-like eﬀects of MIF. In addition, studies showed that MIF could bind to various receptors, including CXCR2, CXCR4, and CD74. The response to stress will be diverse when interacting with diﬀerent receptors on diﬀerent cells at diﬀerent times. In our study, plasma MIF levels were elevated during the intraoperative period, and high MIF levels were associated with better graft recovery. Secondly, we found that plasma MIF yield a better predicting power compared with recipient Scr for DGF and improve the predictive power of donor terminal creatinine for DGF. DGF is caused by IRI and multiple contributing factors, including the characteristics of the donor and recipient (34). Early identiﬁcation and stratiﬁcation of high-risk patients with DGF may advance postoperative management and potentially improve short- and long-term outcomes of recipients undergoing transplantation (35). MIF Is Related to Graft Function 1-Month Post-transplant The ROC curves of plasma macrophage migration inhibitory factor (MIF) from recipients during operation (A), donor terminal Scr (B), absolute Scr decrease from 0-h to 1-day post-transplant (C), and recipient’s serum creatinine (Scr) at 1-day post-transplant (D). The linear prediction model including the reciprocal plasma MIF level and donor terminal Scr (E). time were signiﬁcantly related to recipient Scr 1-month post- transplant (β = 2.142, 95%CI 0.008–0.226, P = 0.035; β = −5.292, 95%CI −0.773 to −0.35, P = 0.001; β = −2.47, 95%CI −1.791 to −0.192, P = 0.016; β = −3.246, 95%CI −1.713 to = 0.129, P < 0.001; R2 = 0.058, P < 0.001, respectively, Figures 4A–C). Univariate analyses showed that recipient Scr at 1-day post-transplant, plasma MIF, female donors, cause of donor death, donor terminal Scr and kidney cold ischaemic time were signiﬁcantly related to recipient Scr 1-month post- transplant (β = 2.142, 95%CI 0.008–0.226, P = 0.035; β = −5.292, 95%CI −0.773 to −0.35, P = 0.001; β = −2.47, 95%CI −1.791 to −0.192, P = 0.016; β = −3.246, 95%CI −1.713 to September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org Ye et al. A Prospective Cohort Study administering glucocorticoids before kidney reperfusion and surgical stimulation may induce MIF release in innate immune cells for recipients (23). Second, kidney ischaemia during organ procurement may stimulate MIF synthesis of tubular epithelial cells in donor and the reperfusion of graft may increase MIF release in epithelial cells (18). We further analyzed the diﬀerence in intraoperative and postoperative plasma MIF levels in 30 patients with diﬀerent allograft function status and found that MIF was signiﬁcantly higher in non-DGF patients than in DGF patients. Moreover, our study found that the diﬀerence was more remarkable during the intraoperative period. These results suggest that intraoperative MIF may be a potential protective factor for DGF. Next, we assessed the diﬀerence in intraoperative MIF levels in 77 patients with diﬀerent allograft function statuses. Our results revealed that intraoperative MIF was signiﬁcantly diﬀerent between the IGF, SGF, and DGF groups, suggesting that it distinguishes between a more subtle allograft recovery pattern. Studies show that MIF has anti-inﬂammatory and anti-ﬁbrotic eﬀects in the kidney (30), and can alleviate IRI through cell protection and antioxidant mechanisms (18–21). We assumed that elevated MIF during transplantation protects the graft by alleviating the refusion injury of the graft. MIF Is Related to Graft Function 1-Month Post-transplant Our research results also showed that 78% of recipients had postoperative IGF when their plasma MIF level was >7.26 during the surgery. In addition, we speculated that MIF might be an endogenous protective factor in remote ischaemic preconditioning due to release from the ischaemic hypoxia issue and protection in remote organs, such as the heart and kidney. Consistent with our hypothesis, recent studies have reported that MIF was signiﬁcantly elevated after remote ischaemic conditioning was carried out and alleviated cardiac IRI. However, the protective eﬀect of MIF on the heart could be blocked by genetic or pharmacological blocking of MIF (31, 32). Most IRI damage begins in the reperfusion process (33), and the damage begins soon after transplanted kidney blood ﬂow is reopened during KT. We deemed that circulatory MIF levels within 5 min of reperfusion reached a high level and were more likely to reﬂect the real protective eﬀect of the graft than circulating MIF levels over a longer period after reperfusion. −0.410, P = 0.002; β = 3.334, 95%CI 0.15–0.596, P = 0.001; β = 2.926, 95%CI 0.113–0.598, P = 0.005, respectively). Multivariate linear regression analysis revealed that kidney cold ischaemic time, donor cause of death, and plasma MIF were signiﬁcantly associated with recipient Scr at 1-month post-transplant (β = 2.213, 95%CI 0.024–0.46, P = 0.03; β = −2.08, 95%CI −1.252 to −0.027, P = 0.041; β = −3.706, 95%CI −0.646 to −0.194, P = 0.001, respectively; Table 4). Frontiers in Medicine \| www.frontiersin.org DISCUSSION The diagnosis of DGF is still dependent on Scr and urine volume in recipient, which requires an understanding of both previous levels and changes, and is inﬂuenced by diuretics, which usually requires several days to conﬁrm (36). The delay in diagnosis has greatly hindered clinical eﬀorts to prevent and treat DGF. Our study conﬁrmed that the predictive value of intraoperative plasma MIF and donor terminal Scr for DGF is better than that of recipient Scr. Furthermore, the model including plasma MIF and donor terminal Scr yield a higher predictive value for DGF than any of the two. Compared with postoperative Scr in the recipient, which Firstly, our study showed that plasma MIF might be an independent protective parameter of DGF. We demonstrated that circulating MIF was increased during intraoperative and postoperative transplantation. MIF is rapidly released into the circulation from the aﬀected cell pool under ischaemia, oxidative stress, inﬂammation or glucocorticoid, and the kidneys are protected by raised MIF (12, 13, 21, 23, 30). Several clinical studies have shown that MIF is elevated during and after liver transplantation or cardiac surgery (20–22), in accordance with our study. We believe that the source of intraoperative elevated MIF may be composed of two components. First, September 2021 \| Volume 8 \| Article 708316 8 Ye et al. A Prospective Cohort Study TABLE 3 \| Sensitivity, speciﬁcity, and predictive values for predicting delayed graft function at optimum cut-off value of recipient and donor characteristic. Cut-off value Sensitivity Speciﬁcity PPV NPV Youden index AUC (95%CI) A—Recipient’s Scr at 1-day post-transplant (mg/dL) 6.88 0.91 0.38 0.37 0.91 0.29 0.671 (0.544–0.799) B—Plasma MIF level (ng/mL) 5.92 0.8 0.77 0.58 0.91 0.57 0.816 (0.712–0.92) C—Donor terminal Scr (mg/dL) 1.73 0.77 0.80 0.61 0.90 0.57 0.8 (0.693–0.908) D—The model combined reciprocal plasma MIF level and donor terminal Scr 0.162 0.96 0.66 0.53 0.98 0.62 0.872 (0.795–0.949) Scr, serum creatinine; PPV, positive predictive value; NPV, negative predictive values; AUC, area under curve; CI, conﬁdence interval. nd predictive values for predicting delayed graft function at optimum cut-off value of recipient and donor characteristic. DISCUSSION ction at optimum cut-off value of recipient and donor characteristic B—Plasma MIF level (ng/mL) 5.92 0.8 0.77 0.58 0.91 0.57 0.816 (0.712–0.92) C—Donor terminal Scr (mg/dL) 1.73 0.77 0.80 0.61 0.90 0.57 0.8 (0.693–0.908) D—The model combined reciprocal plasma MIF level and donor terminal Scr 0.162 0.96 0.66 0.53 0.98 0.62 0.872 (0.795–0.949) Scr, serum creatinine; PPV, positive predictive value; NPV, negative predictive values; AUC, area under curve; CI, conﬁdence interval. FIGURE 4 \| Spearman’s correlation between recipient’s serum creatinine at 1-month post-transplant and intraoperative plasma macrophage migration inhibitory factor (MIF) level (A), donor terminal serum creatinine (B), and recipient’s serum creatinine at 1-day post-transplant (C). FIGURE 4 \| Spearman’s correlation between recipient’s serum creatinine at 1-month post-transplant and intraoperative plasma macrophage migration inhibitory factor (MIF) level (A), donor terminal serum creatinine (B), and recipient’s serum creatinine at 1-day post-transplant (C). FIGURE 4 \| Spearman’s correlation between recipient’s serum creatinine at 1-month post-transplant and intraoperative plasma macrophage migration inhibitory factor (MIF) level (A), donor terminal serum creatinine (B), and recipient’s serum creatinine at 1-day post-transplant (C). FIGURE 4 \| Spearman’s correlation between recipient’s serum creatinine at 1-month post-transplant and intraoperative plasma macrophage migration inhibitory factor (MIF) level (A), donor terminal serum creatinine (B), and recipient’s serum creatinine at 1-day post-transplant (C). FIGURE 4 \| Spearman’s correlation between recipient’s serum creatinine at 1-month post-transplant and intraoperative p factor (MIF) level (A), donor terminal serum creatinine (B), and recipient’s serum creatinine at 1-day post-transplant (C). optimizing ﬂuid balance, timely appropriate dialysis, adjusting the dose of immunosuppressive agents, and avoiding the use of other nephrotoxic drugs (38, 39). Among the recently used AKI is the most commonly used clinical indicator of kidney damage (37), we believe that the model can help transplant physicians recognize DGF early and make early treatment plans, such as September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org 9 Ye et al. A Prospective Cohort Study TABLE 4 \| Univariate and multivariate linear regression analyses for predicting 1-month graft function. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed. 2021.708316/full#supplementary-material FUNDING This study was supported by the National Key R&D Program of China (2018YFA0108804), the National Natural Science Foundation of China (Nos. 81970650, 81770753, and 81970649), the Youth Program of National Natural Science Foundation of China (No. 81800661), the Fundamental Research Funds for the Central Universities (20ykpy34) and China Postdoctoral Science Foundation Funded Project (2020M683083). In conclusion, we believe that plasma MIF has a better predictive performance for DGF, which can help transplant physicians to make a preliminary judgement in the early postoperative period and provide individualized treatment plans for patients. ETHICS STATEMENT markers, kidney injury molecule-1 in tissues could not predict DGF (40), urinary neutrophil gelatinase-associated lipocalin (NGAL) and interleukin-18 have limited predictive value for DGF. Moreover, urine is not produced in most patients with DGF (39, 41). The value of these biomarkers in predicting DGF is limited. However, because of our results based a prospective cohort study with a small size and without validation cohort, these results should be interpreted with cautions. In order to study the exact role of MIF in protecting kidney and to validate the utility of plasma MIF levels as a biomarker for patients DGF prediction, further basic or clinical studies should be carried out. The studies involving human participants were reviewed and approved by the Ethics Committee of the Third Aﬃliated Hospital of Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study. AUTHOR CONTRIBUTIONS QiqS and YY were responsible for study design, analysis and interpretation of data, and preparation and revision of the article. FH and YY performed the analysis. MM, HZ, and ZY performed the laboratory assays. QipS, ZH, ZL, and TL performed data collection. The article was edited by HL, LH, and NN. All authors have approved the manuscript for submission. Finally, we found that the reciprocal plasma MIF level during the intraoperative period was positively correlated with Scr at 1-month post-transplant, indicating that a high reciprocal plasma MIF was closely correlated with worse short-term renal graft function. Moreover, compared with the Scr levels of recipients and donors, intraoperative plasma MIF levels are more closely related to renal allograft function in the ﬁrst month after transplantation. Studies have shown that urinary NGAL is correlated with renal allograft function at 3-weeks post-operation. However, it was not associated with long-term allograft function (39). Our follow-up time was short, and consequently, we were unable to assess the correlation between plasma MIF and long-term allograft function. DISCUSSION Univariate Multivariate βcoefﬁcient 95%CI P-value βcoefﬁcient 95%CI P-value Donor age (years) 0.824 −0.015 to 0.037 0.413 Donor sex (women) −2.47 −1.791 to −0.192 0.016 Donor BMI (kg/m2) 1.895 −0.005 to 0.205 0.062 Donor cause of death −3.246 −1.713 to −0.410 0.002 −2.08 −1.252 to −0.027 0.041 Donor terminal Scr (mg/dL) 3.334 0.15 to 0.596 0.001 Cold ischaemic time (h) 2.926 0.113 to 0.598 0.005 2.213 0.024 to 0.46 0.03 Recipient age (years) 0.387 −0.03 to 0.044 0.7 Recipient sex (female) −0.671 −1.058 to 0.525 0.504 Duration of dialysis before transplantation (mo) −0.38 −0.013 to 0.009 0.705 HLA mismatch −0.74 −0.583 to 0.267 0.462 PRA 0.592 −0.78 to 1.44 0.556 Recipient’s Scr at 1-day post-transplant (mg/dL) 2.142 0.008 to 0.226 0.035 Plasma MIF level (ng/mL) −5.292 −0.773 to −0.35 0.001 −3.706 −0.646 to −0.194 0.001 Multivariate linear regression analysis was executed using a stepwise variable selection procedure. HLA, human leukocyte antigen; PRA, panel reactive antibody; Scr, serum creatinine; MIF, Macrophage migration inhibitory factor; BMI, body mass index; CI, conﬁdence interval. TABLE 4 \| Univariate and multivariate linear regression analyses for predicting 1-month graft function. Frontiers in Medicine \| www.frontiersin.org REFERENCES 18. Miller EJ, Li J, Leng L, McDonald C, Atsumi T, Bucala R, et al. Macrophage migration inhibitory factor stimulates AMP-activated protein kinase in the ischaemic heart. Nature. (2008) 451:578–82. doi: 10.1038/nature06504 1. Wolfe RA, Ashby VB, Milford EL, Ojo AO, Ettenger RE, Agodoa LY, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a ﬁrst cadaveric transplant. N Engl J Med. (1999) 341:1725–30. doi: 10.1056/NEJM199912023412303 19. Koga K, Kenessey A, Powell SR, Sison CP, Miller EJ Ojamaa K. Macrophage migration inhibitory factor provides cardioprotection during ischemia/reperfusion by reducing oxidative stress. Antioxid Redox Signal. (2011) 14:1191–202. doi: 10.1089/ars.2010.3163 2. Abecassis M, Bartlett ST, Collins AJ, Davis CL, Delmonico FL, Friedewald JJ, et al. Kidney transplantation as primary therapy for end-stage renal disease: a National Kidney Foundation/Kidney Disease Outcomes Quality Initiative (NKF/KDOQITM) conference. Clin J Am Soc Nephrology. (2008) 3:471–480. doi: 10.2215/CJN.05021107 20. Stoppe C, Grieb G, Rossaint R, Simons D, Coburn M, Götzenich A, et al. High postoperative blood levels of macrophage migration inhibitory factor are associated with less organ dysfunction in patients after cardiac surgery. Mol Med. (2012) 18:843–50. doi: 10.2119/molmed.2012.00071 3. Schröppel B Legendre C. Delayed kidney graft function: from mechanism to translation. Kidney Int. (2014) 86:251–8. doi: 10.1038/ki.2014.18 21. Stoppe C, Averdunk L, Goetzenich A, Soppert J, Marlier A, Kraemer S, et al. The protective role of macrophage migration inhibitory factor in acute kidney injury after cardiac surgery. Sci Transl Med. (2018) 10:eaan4886. doi: 10.1126/scitranslmed.aan4886 4. Siedlecki A, Irish W Brennan DC. Delayed graft function in the kidney transplant. Am J Transplant. (2011) 11:2279– 2296. doi: 10.1111/j.1600-6143.2011.03754.x 22. Stefaniak J, Schiefer J, Miller EJ, Krenn CG, Baron DM Faybik P. Macrophage migration inhibitory factor as a potential predictor for requirement of renal replacement therapy after orthotopic liver transplantation. Liver Transpl. (2015) 21:662–9. doi: 10.1002/lt.24103 5. Halloran PF, Hunsicker LG. Delayed graft function: state of the art, November 10-11, 2000. Summit meeting, Scottsdale, Arizona, USA. Am J Transplant. (2001) 1:115–20. 23. Calandra T, Bernhagen J, Metz CN, Spiegel LA, Bacher M, Donnelly T, et al. MIF as a glucocorticoid-induced modulator of cytokine production. Nature. (1995) 377:68–71. doi: 10.1038/377068a0 6. Summers DM, Johnson RJ, Allen J, Fuggle SV, Collett D, Watson CJ, et al. Analysis of factors that aﬀect outcome after transplantation of kidneys donated after cardiac death in the UK: a cohort study. Lancet. (2010) 376:1303–11. doi: 10.1016/S0140-6736(10)60827-6 24. REFERENCES World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. (2013) 310:2191– 4. doi: 10.1001/jama.2013.281053 7. Han F, Lin MZ, Zhou HL, Li H, Sun QP, Huang ZY, et al. Delayed graft function is correlated with graft loss in recipients of expanded-criteria rather than standard-criteria donor kidneys: a retrospective, multicenter, observation cohort study. Chin Med J (Engl). (2020) 133:561–70. doi: 10.1097/CM9.000000000000 0666 25. Huang J, Wang H, Fan ST, Zhao B, Zhang Z, Hao L, et al. The national program for deceased organ donation in China. Transplantation. (2013) 96:5–9. doi: 10.1097/TP.0b013e3182985491 26. Brown FG, Nikolic-Paterson DJ, Hill PA, Isbel NM, Dowling J, Metz CM, et al. Urine macrophage migration inhibitory factor reﬂects the severity of renal injury in human glomerulonephritis. J Am Soc Nephrol. (2002) 13 Suppl 1:S7–13. doi: 10.1681/ASN.V13suppl_1s7 8. Keitel E, Michelon T, dos Santos AF, Bittar AE, Goldani JC, D’Almeida Bianco P, et al. Renal transplants using expanded cadaver donor criteria. Ann Transplant. (2004) 9:23–24. 9. Johnston O, O’Kelly P, Spencer S, Donohoe J, Walshe JJ, Little DM, et al. Reduced graft function (with or without dialysis) vs immediate graft function– a comparison of long-term renal allograft survival. Nephrol Dial Transplant. (2006) 21:2270–74. doi: 10.1093/ndt/gﬂ103 27. Hong MY, Tseng CC, Chuang CC, Chen CL, Lin SH Lin CF. Urinary macrophage migration inhibitory factor serves as a potential biomarker for acute kidney injury in patients with acute pyelonephritis. Mediators Inﬂamm. (2012) 2012:381358. doi: 10.1155/2012/381358 28. Payen D, Lukaszewicz AC, Legrand M, Gayat E, Faivre V, Megarbane B, et al. A multicentre study of acute kidney injury in severe sepsis and septic shock: association with inﬂammatory phenotype and HLA genotype. PLoS One. (2012) 7:e35838. doi: 10.1371/journal.pone.0035838 10. Yarlagadda SG, Coca SG, Formica RN, Jr., Poggio ED Parikh CR. Association between delayed graft function and allograft and patient survival: a systematic review and meta-analysis. Nephrol Dial Transplant. (2009) 24:1039–47. doi: 10.1093/ndt/gfn667 29. Stoppe C, Werker T, Rossaint R, Dollo F, Lue H, Wonisch W, et al. What is the signiﬁcance of perioperative release of macrophage migration inhibitory factor in cardiac surgery? Antioxid Redox Signal. (2013) 19:231–39. doi: 10.1089/ars.2012. 5015 11. Freedland SJ, Shoske DA. Economic impact of delayed graft function and suboptimal donors. Transplant Rev. (1999) 13:23–30. 12. Calandra T Roger T. Macrophage migration inhibitory factor: a regulator of innate immunity. Nat Rev Immunol. (2003) 3:791–800. doi: 10.1038/nri1200 13. Kang I Bucala R. DATA AVAILABILITY STATEMENT Supplementary Figure 1 \| Distributions of plasma macrophage migration inhibitory factor (MIF) from the recipient at preoperative, intraoperative, and postoperative periods. Contents of MIF in non-delayed graft function (non-DGF; red) and delayed graft function (DGF; blue) groups. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org 10 Ye et al. A Prospective Cohort Study Supplementary Table 1 \| Summary of characteristics in recipient and donor, stratiﬁed by recipients’s allograft function. Supplementary Figure 2 \| Variation of MIF from recipient during the perioperative period. Supplementary Figure 2 \| Variation of MIF from recipient during the perioperative period. Supplementary Table 1 \| Summary of characteristics in recipient and donor, stratiﬁed by recipients’s allograft function. REFERENCES The immunobiology of MIF: function, genetics and prospects for precision medicine. Nat Rev Rheumatol. (2019) 15:427– 37. doi: 10.1038/s41584-019-0238-2 30. Djudjaj S, Martin IV, Buhl EM, Nothofer NJ, Leng L, Piecychna M, et al. Macrophage Migration Inhibitory Factor Limits Renal Inﬂammation and Fibrosis by Counteracting Tubular Cell Cycle Arrest. J Am Soc Nephrol. (2017) 28:3590–604. doi: 10.1681/ASN.2017020190 14. Unruh M, Wagner B Hallows KR. MIF Matters: The Macrophage Migration Inhibitory Factor and Kidney Injury. Am J Kidney Dis. (2019) 73:429– 31. doi: 10.1053/j.ajkd.2018.07.003 31. Wang C, Zuo B Wu X. The Role of Macrophage Migration Inhibitory Factor in Remote Ischemic Postconditioning. Can J Cardiol. (2019) 35:501– 10. doi: 10.1016/j.cjca.2018.12.040 15. Pohl J, Hendgen-Cotta UB, Rammos C, Luedike P, Mull E, Stoppe C, et al. Targeted intracellular accumulation of macrophage migration inhibitory factor in the reperfused heart mediates cardioprotection. Thromb Haemost. (2016) 115:200–12. doi: 10.1160/TH15-05-0436 32. Ruze A, Chen BD, Liu F, Chen XC, Gai MT, Li XM, et al. Macrophage migration inhibitory factor plays an essential role in ischemic preconditioning-mediated cardioprotection. Clin Sci (Lond). (2019) 133:665–80. doi: 10.1042/CS20181013 16. Kers J, Leemans JC Linkermann A. An Overview of Pathways of Regulated Necrosis in Acute Kidney Injury. Semin Nephrol. (2016) 36:139– 52. doi: 10.1016/j.semnephrol.2016.03.002 33. Murphy E Steenbergen C. Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol Rev. (2008) 88:581– 609. doi: 10.1152/physrev.00024.2007 17. Gando S, Nishihira J, Kobayashi S, Morimoto Y, Nanzaki S Kemmotsu O. Macrophage migration inhibitory factor is a critical mediator of systemic inﬂammatory response syndrome. Intensive Care Med. (2001) 27:1187– 93. doi: 10.1007/s001340000818 34. Cavaillé-Coll M, Bala S, Velidedeoglu E, Hernandez A, Archdeacon P, Gonzalez G, et al. Summary of FDA workshop on ischemia reperfusion September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org 11 Ye et al. A Prospective Cohort Study function following kidney transplantation. Am J Transplant. (2006) 6:1639– 45. doi: 10.1111/j.1600-6143.2006.01352.x function following kidney transplantation. Am J Transplant. (2006) 6:1639– 45. doi: 10.1111/j.1600-6143.2006.01352.x function following kidney transplantation. Am J Transplant. (2006) 6:1639– 45. doi: 10.1111/j.1600-6143.2006.01352.x injury in kidney transplantation. Am J Transplant. (2013) 13:1134– 48. doi: 10.1111/ajt.12210 injury in kidney transplantation. Am J Transplant. (2013) 13:1134– 48. doi: 10.1111/ajt.12210 j 35. Mühlberger I, Perco P, Fechete R, Mayer B Oberbauer R. Biomarkers in renal transplantation ischemia reperfusion injury. Transplantation. (2009) 88:S14–19. REFERENCES doi: 10.1097/TP.0b013e3181af65b5 Conﬂict of Interest: 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. 36. Daly PJ, Power RE, Healy DA, Hickey DP, Fitzpatrick JM Watson RW. Delayed graft function: a dilemma in renal transplantation. BJU Int. (2005) 96:498–501. doi: 10.1111/j.1464-410X.2005.05673.x Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 37. Bellomo R, Kellum JA Ronco C. Deﬁning acute renal failure: physiological principles. Intensive Care Med. (2004) 30:33– 37. doi: 10.1007/s00134-003-2078-3 38. Perico N, Cattaneo D, Sayegh MH Remuzzi G. Delayed graft function in kidney transplantation. Lancet. (2004) 364:1814– 27. doi: 10.1016/S0140-6736(04)17406-0 Copyright © 2021 Ye, Han, Ma, Sun, Huang, Zheng, Yang, Luo, Liao, Li, Hong, Na and Sun. 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) and the copyright owner(s) 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. 39. Hollmen ME, Kyllönen LE, Inkinen KA, Lalla ML Salmela KT. Urine neutrophil gelatinase-associated lipocalin is a marker of graft recovery after kidney transplantation. Kidney Int. (2011) 79:89–98. doi: 10.1038/ki.2010.351 40. Schröppel B, Krüger B, Walsh L, Yeung M, Harris S, Garrison K, et al. Tubular expression of KIM-1 does not predict delayed function after transplantation. J Am Soc Nephrol. (2010) 21:536–42. doi: 10.1681/ASN.2009040390 39. Hollmen ME, Kyllönen LE, Inkinen KA, Lalla ML Salmela KT. Urine neutrophil gelatinase-associated lipocalin is a marker of graft recovery after kidney transplantation. Kidney Int. (2011) 79:89–98. doi: 10.1038/ki.2010.351 y y 40. Schröppel B, Krüger B, Walsh L, Yeung M, Harris S, Garrison K, et al. Tubular expression of KIM-1 does not predict delayed function after transplantation. J Am Soc Nephrol. (2010) 21:536–42. doi: 10.1681/ASN.2009040390 41. Parikh CR, Jani A, Mishra J, Ma Q, Kelly C, Barasch J, et al. Frontiers in Medicine \| www.frontiersin.org September 2021 \| Volume 8 \| Article 708316 REFERENCES Urine NGAL and IL-18 are predictive biomarkers for delayed graft September 2021 \| Volume 8 \| Article 708316 Frontiers in Medicine \| www.frontiersin.org 12
https://openalex.org/W2110143873	https://opus.bibliothek.uni-wuerzburg.de/files/12779/Heavy_Neutrinos_10052-012-2056-4.pdf	English	null	Search for heavy neutrinos and right-handed W bosons in events with two leptons and jets in pp collisions at $\sqrt{s} = 7~\mathrm{TeV}$ with the ATLAS detector	European physical journal. C, Particles and fields	2,012	cc-by	21,271	Search for heavy neutrinos and right-handed W bosons in events with two leptons and jets in pp collisions at √s = 7 TeV with the ATLAS detector The ATLAS Collaboration⋆ CERN, 1211 Geneva 23, Switzerland Received: 24 March 2012 / Revised: 17 May 2012 / Published online: 3 July 2012 © CERN for the beneﬁt of the ATLAS collaboration 2012. This article is published with open access at Springerlink.com Received: 24 March 2012 / Revised: 17 May 2012 / Published online: 3 July 2012 © CERN for the beneﬁt of the ATLAS collaboration 2012. This article is published Abstract This letter reports on a search for hypothetical heavy neutrinos, N, and right-handed gauge bosons, WR, in events with high transverse momentum objects which in- clude two reconstructed leptons and at least one hadronic jet. The results were obtained from data corresponding to an integrated luminosity of 2.1 fb−1 collected in proton–proton collisions at √s = 7 TeV with the ATLAS detector at the CERN Large Hadron Collider. No excess above the Stan- dard Model background expectation is observed. Excluded mass regions for Majorana and Dirac neutrinos are presented using two approaches for interactions that violate lepton and lepton-ﬂavor numbers. One approach uses an effective oper- ator framework, the other approach is guided by the Left– Right Symmetric Model. The results described in this letter represent the most stringent limits to date on the masses of heavy neutrinos and WR bosons obtained in direct searches. same generation, and mN is the mass of a new heavy neu- trino, N. If the see-saw mechanism were to explain the mas- ses of the known neutrinos, both the light and the heavy neutrinos would have to be Majorana particles. This would violate lepton number conservation, and yield a striking sig- nature of two leptons with the same charge at the LHC [7]. p g This letter reports on a search for new heavy neutrinos of either Majorana or Dirac type, with data corresponding to an integrated luminosity of 2.1 fb−1 recorded with the ATLAS detector at the LHC. Two approaches are employed. The ﬁrst approach aims at exploring possible sources of new physics predicting heavy neutrinos using a Lagrangian of effective operators (referred to as HNEO hereafter) [8]. The theory is built on effective four-fermion operators (q ¯q′ →Nℓ) with the N decaying promptly via a three-body decay, N →ℓjj. The second approach is based on the concept of Left–Right Symmetry [9–11] which extends the electroweak part of the SM by a new gauge group. Eur. Phys. J. C (2012) 72:2056 DOI 10.1140/epjc/s10052-012-2056-4 Letter Letter Search for heavy neutrinos and right-handed W bosons in events with two leptons and jets in pp collisions at √s = 7 TeV with the ATLAS detector Its force particles (WR and Z′ bosons) could be produced at LHC energies. A particular implementation of left–right symmetry breaking [12], the Left–Right Symmetric Model (LRSM) with doubly charged Higgs bosons [13, 14] is used in the present analysis. Ac- cording to this model, the heavy neutrinos would be pro- duced in the decays of a WR boson via q ¯q′ →WR →ℓN, with N decaying subsequently via N →ℓW ∗ R →ℓjj. Thus, the ﬁnal state signature for both models consists of two leptons and two jets with high transverse momenta (pT). Only electrons and muons are considered in this analy- sis. ⋆e-mail: atlas.publications@cern.ch 1ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point in the center of the detector and the z-axis coinciding with the axis of the beam pipe. The x-axis points from the interaction point to the center of the LHC ring, and the y-axis points upward. Cylindrical coordinates (r,φ) are used in the transverse plane, φ being the azimuthal angle around the beam pipe. The pseudorapidity is deﬁned in terms of the polar angle θ as η = lntan(θ/2). 3 Trigger and data The data used in this analysis were recorded between March and August 2011 at a center-of-mass energy of 7 TeV. The application of beam, detector, and data quality requirements results in a total integrated luminosity of 2.1 fb−1 with an estimated uncertainty of ±3.7 % [25, 26]. The data were recorded with single lepton (e or μ) triggers [27]. At the last stage of the trigger decision, the electron trigger selects candidate electrons with transverse energy ET > 20 GeV, satisfying shower-shape requirements and matching an ID track. For the last part of the dataset, corresponding to an integrated luminosity of 0.5 fb−1, the threshold was raised to 22 GeV. The muon trigger selects candidate muons with pT > 18 GeV and \|η\| < 2.4. These triggers reach full ef- ﬁciency for electrons with pT > 25 GeV and muons with pT > 20 GeV. The typical trigger efﬁciencies measured from data for leptons selected for ofﬂine analysis are 99 ± 1 % for electrons, and 74 % and 91 % for muons in the barrel (\|η\| < 1.05) and end-cap (1.05 < \|η\| < 2.4) regions, respectively, with an uncertainty of about ±1 %. Heavy neutrinos were previously searched for at LEP and excluded for masses up to ≈100 GeV [17–20]. The most stringent direct limits on WR bosons [21, 22] come from the Tevatron, where WR →tb decays were searched for. As- suming a branching ratio of 100 %, WR boson masses be- low 825 GeV are excluded at 95 % conﬁdence level (C.L.). Recently, the ATLAS collaboration published an inclusive search for new physics in the same-sign dilepton signature for an integrated luminosity of 34 pb−1 [23]. The 95 % C.L. limits presented exclude WR masses up to about 1 TeV for the LRSM model and Majorana neutrino masses around 460 GeV for the HNEO model. 2 The ATLAS detector The ATLAS detector [24] is a multipurpose particle physics apparatus with a forward-backward symmetric cylindrical geometry and nearly 4π coverage in solid angle.1 The in- ner tracking detector (ID) covers the pseudorapidity range \|η\| < 2.5 and consists of: a silicon pixel detector, provid- ing typically three measurements per track; a silicon mi- crostrip detector (SCT), which provides typically four to ﬁve measurements; and, for \|η\| < 2.0, a transition radi- ation tracker (TRT), giving typically 30 straw-tube mea- surements per track. The ID is surrounded by a thin su- perconducting solenoid providing a 2 T magnetic ﬁeld. A high-granularity liquid-argon (LAr) sampling electromag- netic calorimeter covers the region \|η\| < 3.2. An iron-scinti- llator tile calorimeter provides hadronic coverage in the cen- tral rapidity range of \|η\| < 1.7. The end-cap and forward regions, spanning 1.5 < \|η\| < 4.9, are instrumented with LAr calorimeters for both electromagnetic and hadronic measurements. The muon spectrometer (MS) surrounds the calorimeters and consists of a system of air-core super- conducting toroid coils, precision tracking chambers up 1 Introduction The discovery of neutrino oscillations [1, 2] unambiguously establishes that neutrinos have non-zero mass and provides clear evidence for physics beyond the Standard Model (SM). One possible explanation for the mass of light neutrinos is provided by theoretical models based on a Grand Uniﬁed Theory (GUT). Such models often introduce one or more additional neutrino ﬁelds, which manifest themselves as new heavy particles that could be directly observable at the Large Hadron Collider (LHC). In the framework of GUT mod- els, the mass of the light neutrinos could be explained via the see-saw mechanism [3–6]. This predicts mν ≈m2 D/mN, where, for each generation, mν is the mass of a known light neutrino, mD is the Dirac mass for charged fermions of the The N invariant mass can be fully reconstructed from the decay products in both approaches. Given the s-channel production in the LRSM, the WR mass, mWR, can also be reconstructed in this model. The reconstructed WR boson and N masses are used to perform the search in the con- text of the HNEO and LRSM models, respectively. Like the SM neutrinos, heavy neutrinos can mix if their masses are Page 2 of 22 Eur. Phys. J. C (2012) 72:2056 different. Both the scenarios of no mixing [15] and maxi- mal mixing [16] between two generations of lepton ﬂavors (electron and muon) are investigated assuming that the mass difference between the heavy neutrinos is much smaller than the experimental resolution of their reconstructed invariant mass. In the case of maximal mixing, a mass difference of 2 GeV is assumed. If the heavy neutrinos are of Majorana type, they would contribute to both the same-sign (SS) and opposite-sign (OS) channels, while heavy Dirac neutrinos would contribute solely to the OS channel. to \|η\| < 2.7, and detectors for triggering in the region of \|η\| < 2.4. to \|η\| < 2.7, and detectors for triggering in the region of \|η\| < 2.4. 5 Object reconstruction and event selection The tree-level-generated dimension-6 operator OV corresponds to duNe, while Os1 and Os2 correspond to QuNL and LNQd, respectively, and Os3 corresponds to QNLd (e, u, d and L, where n is the operator dimension, Λ is the scale of LNV interactions, αi are the coupling constants between the neu- trino N and the leptons, and Oi are the effective opera- tors [8]. The signal samples are produced for four effective operator hypotheses: the four-fermion vector operator, OV , and four-fermion scalar operators, Os1, Os2, and Os3. The tree-level-generated dimension-6 operator OV corresponds to duNe, while Os1 and Os2 correspond to QuNL and LNQd, respectively, and Os3 corresponds to QNLd (e, u, d and L, Q denote the right-handed SU(2) singlets and left-handed SU(2) doublets, respectively). The production via the effec- tive operators Os1 and Os2 have the same cross section and lead to identical event kinematics, which makes them indis- tinguishable. The production cross sections for the Majorana and Dirac neutrinos in the framework of the effective La- grangian are related to the energy scale of new physics and the coupling constant σ ≈α2/Λ4, such that the coupling can be varied to scan for new physics at different Λ scales. Muons are required to be identiﬁed in both the ID and the MS systems. The ID track is required to have at least one pixel hit, more than ﬁve SCT hits, and a number of TRT hits that varies with η. Muon tracks that pass through an ac- tive region of the innermost pixel detector are required to have a measurement in that layer. The curvatures, as mea- sured by the ID and MS systems, must have the same sign. Only muons with pT > 25 GeV and \|η\| < 2.4 are consid- ered. Selection criteria on the displacement of the muon rel- ative to the primary vertex, selected as the one with the high- est p2 T of associated tracks, are required. The longitudi- nal (z0) and transverse (d0) impact parameters must satisfy \|z0\| < 5 mm, \|d0\| < 0.2 mm, and \|d0/σd0\| < 5, where σd0 is the uncertainty on d0. These cuts reduce the cosmic ray muon background to a negligible level and also reduce the background from non-prompt muons.2 The LRSM signal MC samples are generated using an implementation of this model [14] in PYTHIA, with modiﬁed leading-order parton distribution functions MRST2008LO* [41]. 2Leptons from W, Z and τ decays are classiﬁed as prompt leptons, while leptons any hadron decays are classiﬁed as non-prompt leptons. 4 Monte Carlo simulation Fully simulated Monte Carlo (MC) event samples are used to develop and validate the analysis procedure, es- timate the detector acceptance and reconstruction efﬁ- ciency, and aid in the background determination. The sim- ulation of background processes is described in detail in Ref. [28]. For the major backgrounds, Z/γ ∗+ jets pro- duction and top quark pair production, ALPGEN [29] and MC@NLO [30–32] are used, respectively. The leading- order parametrization CTEQ6L1 [33] of the parton density functions (PDF) is used for the ALPGEN simulation, while the next-to-leading order parametrization CTEQ6.6 [33] is used for the MC@NLO simulation. Fragmentation and hadronization are performed in both cases with HERWIG [34–36], using JIMMY [37] for the underlying event mod- elling. Diboson (WW, WZ, and ZZ) event samples are gen- erated using HERWIG, while MADGRAPH [38] interfaced to PYTHIA [39] is used for Wγ and Zγ production. Single top- quark production is generated with MC@NLO. The produc- tion of W +W + arising from a t-channel gluon exchange, resulting in two jets in the ﬁnal state and two same-sign W bosons, are generated with MADGRAPH interfaced to PYTHIA. The associated production of a vector boson with a t ¯t pair (t ¯tW, t ¯tZ, t ¯tγ ) is simulated with MADGRAPH interfaced with PYTHIA. Eur. Phys. J. C (2012) 72:2056 Eur. Phys. J. C (2012) 72:2056 Page 3 of 22 taken into account by reweighting MC events to match the pile-up conditions measured in data. The HNEO signal MC samples are generated using CALCHEP [40] and the leading-order PDF CTEQ6L [33], and hadronization simulated with PYTHIA. All lepton com- binations of e, μ or τ leading to lepton number violating (LNV) signatures, which produce SS or OS dilepton events, are included. The model is implemented via a Lagrangian of effective operators deﬁned as 5 Object reconstruction and event selection The criteria for electron and muon identiﬁcation closely fol- low those described in Ref. [46]. Electrons are required to pass the “medium” selection criteria, with pT > 25 GeV and \|η\| < 2.47, excluding the electromagnetic calorimeter tran- sition region, 1.37 < \|η\| < 1.52 [47]. To improve the back- ground rejection for \|η\| > 2.0, more stringent requirements are placed on the track-cluster matching in η and shower shape. Electron tracks that pass through an active region of the innermost pixel detector are required to have a measure- ment in that layer in order to suppress electrons from photon conversions. Additionally, an electron whose track matches the ID track of a muon candidate is rejected. L = ∞ n=5 1 Λn−4 · i αiOi(n), (1) (1) where n is the operator dimension, Λ is the scale of LNV interactions, αi are the coupling constants between the neu- trino N and the leptons, and Oi are the effective opera- tors [8]. The signal samples are produced for four effective operator hypotheses: the four-fermion vector operator, OV , and four-fermion scalar operators, Os1, Os2, and Os3. The tree-level-generated dimension-6 operator OV corresponds to duNe, while Os1 and Os2 correspond to QuNL and LNQd, respectively, and Os3 corresponds to QNLd (e, u, d and L, Q denote the right-handed SU(2) singlets and left-handed SU(2) doublets, respectively). The production via the effec- tive operators Os1 and Os2 have the same cross section and lead to identical event kinematics, which makes them indis- tinguishable. The production cross sections for the Majorana and Dirac neutrinos in the framework of the effective La- grangian are related to the energy scale of new physics and the coupling constant σ ≈α2/Λ4, such that the coupling can be varied to scan for new physics at different Λ scales. where n is the operator dimension, Λ is the scale of LNV interactions, αi are the coupling constants between the neu- trino N and the leptons, and Oi are the effective opera- tors [8]. The signal samples are produced for four effective operator hypotheses: the four-fermion vector operator, OV , and four-fermion scalar operators, Os1, Os2, and Os3. 6 Background estimation Several processes have the potential to contaminate the sig- nal regions. The main background to the SS dilepton ﬁ- nal state, which is referred to as “fake lepton” background, arises from SM W + jets, t ¯t, and multi-jet production where one or more jets are misidentiﬁed as prompt isolated lep- tons. This background is measured using a data-driven tech- nique rather than using less accurate estimates from MC simulation. The other signiﬁcant background arises from charge misidentiﬁcation of a reconstructed electron as a re- sult of hard bremsstrahlung followed by asymmetric conver- sion (e± hard →e± softγhard →e± softe± softe∓ hard). This background is estimated with a combination of MC and data-driven tech- niques. Small contributions from diboson and single top- quark events are also accounted for using MC. For the e±e∓and μ±μ∓ﬁnal states, the dominant back- grounds are Z/γ ∗+jets and t ¯t events, with about equal con- tributions after all selection criteria are applied. The e±μ∓ ﬁnal state is dominated by t ¯t production. The backgrounds from t ¯t, single top-quark, and diboson production are es- timated from MC simulation, while the estimation of the Z/γ ∗+ jets background is extracted from a normalization to the data. The fake lepton background is estimated from data, using the same method as for the SS ﬁnal states. Events are preselected by requiring exactly two identiﬁed leptons with pT > 25 GeV originating from the primary ver- tex and at least one jet with pT > 20 GeV. At least one of the lepton candidates must match a triggered lepton at the last stage of the trigger selection. To reduce the number of background events from Drell–Yan production and misiden- tiﬁed leptons, the dilepton invariant mass, mℓℓ, is required to be greater than 110 GeV. The signal region is then sub- divided into SS and OS dilepton events. In the OS dilepton channels, further background reduction is achieved by re- quiring that the scalar sum of the transverse energies of the two leptons and the leading two jets with pT > 20 GeV, de- noted by ST, is greater than 400 GeV. This event selection is referred to hereafter as the baseline selection. As mentioned previously, the mass of the N can be reconstructed from its decay products of one lepton and two jets. In the case where A data-driven approach, similar to the one described in Refs. 5 Object reconstruction and event selection The coupling constants for the WR and left-handed W boson are assumed to be the same, including the CKM ma- trix for WR boson couplings to right-handed chiral quark components. It is assumed that there is no mixing between the WR boson and the SM W boson. The LRSM signal MC samples are generated constraining the decays to e or μ and with mN < mWR. The branching fractions used are the ones predicted by PYTHIA. When the mass difference between the WR and the N is large, the leptonic branching fractions are ≈8 %, and they decrease with decreasing mass differ- ence. To reduce the background due to leptons from decays of hadrons (including heavy-ﬂavor hadrons) produced in jets, requirements on the isolation of leptons are imposed. To evaluate the isolation energy for electrons, the transverse en- ergies deposited in the calorimeter towers in a cone in η–φ space of radius R = (φ)2 + (η)2 = 0.2 around the electron direction are summed and corrected for energy de- position from pile-up events. In addition, the transverse en- ergy of the electron, ET, corrected for energy leakage into the neighboring towers, is subtracted. The isolation trans- verse energy is required to be less than 15 % of the elec- tron ET. An equivalent quantity, ER=0.3 T , is calculated for the muon using a cone size of R = 0.3. If there is no jet with pT > 20 GeV within R < 0.4 of the muon, ER=0.3 T is required to be less than 15 % of the muon pT. Addition- ally, muons with pT < 80 GeV should have no other track Both Majorana and Dirac type heavy neutrinos are con- sidered, assuming that the total production cross section is the same for both cases. The leading-order theoretical cross sections are used. All signal and background samples are generated us- ing the ATLAS underlying event tunes [42, 43] and pro- cessed through the ATLAS detector simulation [44] based on GEANT4 [45]. The MC samples are produced includ- ing the simulation of multiple interactions per LHC bunch crossing (pile-up). Varying pile-up conditions and their de- pendence on the instantaneous luminosity of the LHC are Eur. Phys. J. C (2012) 72:2056 Page 4 of 22 the N is boosted, the hadronic decay products can be recon- structed as one jet due to their proximity to each other. 5 Object reconstruction and event selection For scenarios with a large mass splitting between the WR and N, up to half of the signal events have only one jet. The WR bo- son invariant mass is reconstructed from the leptons and the two highest pT jets in events with at least two jets, or a sin- gle jet in events with only one jet. Anti-kt jets are massive, and therefore, the jet four-momenta are used in calculating the invariant mass. For the LRSM, the WR boson invariant mass, mℓℓj(j), is required to be greater than 400 GeV for both SS and OS ﬁnal states. with pT > 1 GeV originating from the primary vertex within a cone of R = 0.3 around the muon. Otherwise, if the muon has a jet nearby, it must satisfy pT > 80 GeV and (ER=0.3 T /pT −3)/pT > −0.02 GeV−1. These isolation re- quirements are powerful in rejecting background muons and highly efﬁcient for selecting signal muons produced in the decays of heavy neutrinos and reconstructed near the signal jets in cases where the heavy neutrino is boosted. with pT > 1 GeV originating from the primary vertex within a cone of R = 0.3 around the muon. Otherwise, if the muon has a jet nearby, it must satisfy pT > 80 GeV and (ER=0.3 T /pT −3)/pT > −0.02 GeV−1. These isolation re- quirements are powerful in rejecting background muons and highly efﬁcient for selecting signal muons produced in the decays of heavy neutrinos and reconstructed near the signal jets in cases where the heavy neutrino is boosted. Jets are reconstructed using the anti-kt jet clustering al- gorithm [48, 49] with a radius parameter R = 0.4. The input to this algorithm is clusters of calorimeter cells seeded by cells with energies signiﬁcantly above the measured noise. to this algorithm is clusters of calorimeter cells seeded by cells with energies signiﬁcantly above the measured noise. The energies and momenta of jets are evaluated by perform- ing a four-vector sum over these clusters, treating each clus- ter as an (E, p) four-vector with zero mass. Jets are cor- rected for calorimeter non-compensation, upstream material and other effects using pT and η-dependent calibration fac- tors [50] obtained from MC simulation [51], and validated with test-beam and collision-data studies. Only jets with pT > 20 GeV and \|η\| < 2.8 are considered. 5 Object reconstruction and event selection To avoid dou- ble counting, the closest jet within R < 0.5 of an electron candidate is discarded. The selected jets must pass quality requirements based on their shower shape, and their calori- meter signal timing must be consistent with the timing of the beam crossings [52]. Events with any jet that fails the jet quality criteria are rejected. To suppress jets unrelated to the hard scattering of interest, at least 75 % of the summed pT of all reconstructed tracks associated with a jet with \|η\| < 2.8 must come from tracks originating from the selected pri- mary vertex. During a part of the data-taking period, cor- responding to an integrated luminosity of 0.9 fb−1, an elec- tronic failure in a small η–φ region of the LAr EM calorime- ter created a dead region. For this integrated luminosity, events in data and MC containing either an identiﬁed elec- tron or a jet, with pT > 40 GeV, satisfying −0.1 < η < 1.5 and −0.9 < φ < −0.5 are rejected, leading to a loss of sig- nal efﬁciency of about 10 % for this portion of the data. 6 Background estimation [23, 28], is used to estimate the fake lepton back- ground. The method uses “loose” leptons in addition to the candidate leptons. Loose muons are deﬁned using the same identiﬁcation criteria as the candidate muons, except for the isolation requirements, which are not applied. For the electrons, looser requirements on the shower shape vari- ables, track-cluster matching, and track quality are used, and the isolation requirement is not applied. The method uses the fractions of these loose fake leptons, Rfake, and loose prompt leptons, Rprompt, which also pass the can- didate lepton requirements. A 4 × 4 matrix is then em- ployed on the “loose–loose” and “loose–tight” dilepton sample to predict the total fake lepton background con- tributing to the SS and OS dilepton ﬁnal states. The Rfake Eur. Phys. J. C (2012) 72:2056 Page 5 of 22 fractions are measured using fake lepton enriched control regions containing a single loose lepton. Additional cri- teria are imposed to reduce the true lepton contamina- tion from electroweak processes to a negligible level. For events in the control regions, the transverse mass, mT = 2 · pℓ T · Emiss T · (1 −cosφ(ℓ,Emiss T )), is required to be less than 40 GeV. Emiss T is deﬁned as the missing trans- verse momentum based on the calorimeter information and the transverse momenta of muons within \|η\| < 2.7 [46], while φ(ℓ,Emiss T ) is the azimuthal angle separation be- tween the lepton and the Emiss T vectors. Additionally, the fol- lowing requirements are imposed: φ(jet or e,Emiss T ) < 0.1 for the electron control region and φ(μ,Emiss T ) < 0.5 for the muon control region. For the muon control region, an additional requirement of at least one jet is imposed. After these criteria are applied, the remaining background from electroweak processes is estimated to be less than 5 %. The Rprompt fractions are measured using Z boson events satis- fying 86 GeV < mℓℓ< 96 GeV via a method referred to as the “tag-and-probe” method. The “tag” lepton is required to satisfy all lepton selection criteria, while the unbiased op- posite charge “probe” lepton should satisfy the loose crite- ria. The Rprompt fractions are parametrized as a function of the lepton pT and range from 89 % to 98 % for muons and 96 % to 99 % for electrons. 6 Background estimation To improve the accuracy of the prediction, the fractions are parametrized as a function of kinematic variables separately for leptons that pass the anal- ysis trigger requirement and those that do not. The muon Rfake is measured separately for muons that originate from heavy ﬂavor jets and those that do not, where the jet ﬂavor is identiﬁed using a combination of the secondary vertex [53] and impact parameter-based [54] b-tagging algorithms. For muons, Rfake ranges from 5 % to 10 % (5 % to 40 %) for heavy ﬂavor (light ﬂavor) jets. For electrons, Rfake ranges from 45 % to 60 %. The fraction of reconstructed electrons with charge mis- identiﬁcation due to hard bremsstrahlung is measured from simulated Z/γ ∗+ jets events, by comparing the MC gener- ated charge of the electron originating from the Z to that of the reconstructed electron candidate. The fraction is parame- trized as a function of the electron ET and η and applied to Z/γ ∗→e+e−and t ¯t →e±ℓ∓b ¯b MC backgrounds to ob- tain their contributions to the SS dilepton ﬁnal state, thus beneﬁting from the large number of simulated OS events. Since the MC overestimates the charge misidentiﬁcation as observed in the Z/γ ∗→ee data sample, η-dependent scale factors between data and MC simulation are obtained us- ing Z/γ ∗→e±e± events with 80 GeV < mℓℓ< 100 GeV. Both electrons are required to be within the same η range and with the same charge. These factors are applied to the MC estimates. The rate of charge misidentiﬁcation due to tracking resolution is found to be negligible within the lep- ton transverse momentum range of interest and is well de- scribed by the MC simulation. All other backgrounds are estimated from MC simulation and found to be small, as shown in Table 2. In the SS ee and eμ channels, the dominant background arises from fake lep- tons. The next most signiﬁcant background is diboson pro- duction for the eμ channel and Z/γ ∗production for the ee channel. The SS μμ channel is dominated by the diboson background with a smaller contribution from fake leptons. 8 Results and interpretation The quoted uncertainties include statis- tical and systematic components, excluding the luminosity uncertainty of ±3.7 %. The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data Physics processes e±e± μ±μ± e±μ± Total Z/γ ∗+ jets 26.1 ± 5.6 0.0+1.6 −0 1.2 ± 0.7 27 ± 6 Diboson 12.7 ± 2.3 7.2 ± 1.7 18.8 ± 3.0 39 ± 6 Top 5.8 ± 1.3 0.7 ± 0.3 6.8 ± 1.6 13 ± 3 Fake lepton(s) 93.6 ± 35.7 3.1 ± 1.6 53.8 ± 20.3 151 ± 50 Total background 138.3 ± 36.5 11.0+2.9 −2.5 80.7 ± 20.8 230 ± 52 Observed events 155 14 99 268 mℓℓj(j) ≥400 GeV Total background 48.4 ± 16.1 4.4+2.1 −1.3 24.6 ± 7.6 77 ± 21 Ob d 59 8 39 106 Physics processes e±e∓ μ±μ∓ e±μ∓ Total Z/γ ∗+ jets 136.1 ± 12.5 173.2 ± 15.1 0.8 ± 0.8 310 ± 20 Diboson 4.3 ± 1.8 7.3 ± 1.9 5.9 ± 1.6 18 ± 3 Top 103.1 ± 12.3 100.9 ± 12.0 199.4 ± 23.3 403 ± 46 Fake lepton(s) 12.5 ± 8.1 −0.2 ± 0.7 6.1 ± 4.2 18 ± 9 Total background 256.0 ± 26.2 281.2 ± 27.9 212.3 ± 33.8 750 ± 78 Observed events 248 245 247 740 mℓℓj(j) ≥400 GeV Total background 254.8 ± 25.8 279.7 ± 27.6 210.9 ± 33.4 745 ± 77 Observed events 246 241 244 731 Table 2 Summary of the expected background yields and observed numbers of events for the SS dilepton channels. The top part of the table shows the numbers obtained for events with two leptons, ≥1 jet and mℓℓ> 110 GeV. The bottom part of the table shows the num- bers for the ﬁnal LRSM selection, where an additional requirement mℓℓj(j) ≥400 GeV is imposed. The quoted uncertainties include statis- tical and systematic components, excluding the luminosity uncertainty of ±3.7 %. The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data mℓℓj(j) ≥400 GeV is imposed. The quoted uncertainties include statis- tical and systematic components, excluding the luminosity uncertainty of ±3.7 %. 7 Systematic uncertainties The dominant contribution to the systematic uncertainties in the SS ee and eμ channels arises from the fake lep- ton background estimate. As a ﬁrst step in validating the parametrization of Rfake and Rprompt, a closure test is per- formed in data. Measurements of Rfake and Rprompt are ob- tained by randomly sampling half of the control regions. The predicted values are then compared with the values mea- sured in the other half of the data. The closure test yields an agreement for Rfake and Rprompt of, respectively, ±40 % and ±5 % for muons and, for electrons, ±5 % (±20 % for 1 < \|η\| < 1.9) and ±2 %, which are propagated to the fake lepton background estimate. To evaluate the uncertainty on the overall fake lepton background estimate, the robustness of the procedure is tested against variations across samples. The estimated fake lepton background is compared to the observed background in SS events passing the same selec- tion as events in the signal region but where the sub-leading lepton has a transverse momentum between 15 GeV and 25 GeV. The fake lepton background contributes between 65 % in e±e± to 87 % in e±μ± of the total background. This study tests the reliability of both the parametrization and the use of Rfake and Rprompt to extract the background predic- tion. In this sample, the total background prediction agrees A partially data-driven approach is adopted to estimate Z/γ ∗→ee and Z/γ ∗→μμ contributions to the OS dilepton channels. A control region is deﬁned requiring 80 GeV < mℓℓ< 100 GeV and ≥1 jets, where non-Z bo- son contributions are found to be negligible. Normaliza- tion factors between the observed number of events in data and the MC prediction are obtained as a function of jet multiplicity from this region and applied to the MC esti- mates in the signal region. All other backgrounds, including Z/γ ∗→ττ, are estimated from MC simulation. The con- tribution of Z/γ ∗→ττ is found to be negligible after all selection criteria are applied. Table 1 summarises the back- ground estimates for the OS channels. In the OS ee and μμ channels, Z/γ ∗+ jets and t ¯t backgrounds dominate, while the t ¯t production contributes more than 90 % in the eμ channel. Smaller contributions arise from diboson pro- duction and events with fake leptons. Eur. Phys. J. 7 Systematic uncertainties C (2012) 72:2056 Page 6 of 22 with the observed data within ±10 %. A ±30 % overall sys- tematic uncertainty is assigned to cover for the differences between the predicted and the observed mℓℓspectra. up in the 2011 dataset. MC modelling uncertainties for t ¯t production [28] are derived using different MC generators and varying, within their uncertainties, the parameters that control initial and ﬁnal state radiation. The resulting uncer- tainties are ±15 % and ±(5–7) % for t ¯t and diboson contri- butions, respectively. The uncertainties on the background due to the electron charge misidentiﬁcation arise from the limited number of MC events used to parametrize the rate and the scale factors used to correct the simulation for differences between data and MC and contribute ±13 % and ±12 %, respectively. Due to the limited knowledge of PDFs and αs, the uncer- tainties are evaluated using a range of current PDF sets with the procedure described in Ref. [57]. The ﬁnal uncertainty is taken from the outer bounds of the overall error bands. The PDF uncertainties are estimated to be ±9 % for the LRSM signal and ±12 % for the HNEO signals. The background and signal estimates derived from MC are affected by the jet energy scale (JES) calibration and the jet energy resolution (JER), theoretical and MC modelling uncertainties, and pT and η dependent uncertainties on the lepton identiﬁcation and reconstruction efﬁciencies (identi- ﬁcation ±(0.2–3.3) %, pT scale ±(0.2–2) % and resolution ±(0.4–10) %) [47, 55, 56]. The JES (±(2–6) %) and JER (±(5–12) %) uncertainties applied depend on jet pT and η and are measured from the 2010 dataset [52]. An additional contribution of ±(2–7) % to the JES uncertainty is added in quadrature to account for the effect of high luminosity pile- 8 Results and interpretation The expected and observed numbers of events in each dilep- ton ﬁnal state for the baseline selection and the LRSM se- lections are compared in Tables 1 and 2 for the OS and requirement mℓℓj(j) ≥400 GeV is imposed. The quoted uncertainties include statistical and systematic components, excluding the luminos- ity uncertainty of ±3.7 %. The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data requirement mℓℓj(j) ≥400 GeV is imposed. The quoted uncertainties include statistical and systematic components, excluding the luminos- ity uncertainty of ±3.7 %. The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data Table 1 Summary of the expected background yields and observed numbers of events for the OS dilepton channels. The top part of the table shows the numbers obtained for events with two leptons, ≥1 jet, mℓℓ> 110 GeV, and ST > 400 GeV. The bottom part of the table shows the numbers for the ﬁnal LRSM selection, where an additional Table 1 Summary of the expected background yields and observed numbers of events for the OS dilepton channels. The top part of the table shows the numbers obtained for events with two leptons, ≥1 jet, mℓℓ> 110 GeV, and ST > 400 GeV. The bottom part of the table shows the numbers for the ﬁnal LRSM selection, where an additional Physics processes e±e∓ μ±μ∓ e±μ∓ Total Z/γ ∗+ jets 136.1 ± 12.5 173.2 ± 15.1 0.8 ± 0.8 310 ± 20 Diboson 4.3 ± 1.8 7.3 ± 1.9 5.9 ± 1.6 18 ± 3 Top 103.1 ± 12.3 100.9 ± 12.0 199.4 ± 23.3 403 ± 46 Fake lepton(s) 12.5 ± 8.1 −0.2 ± 0.7 6.1 ± 4.2 18 ± 9 Total background 256.0 ± 26.2 281.2 ± 27.9 212.3 ± 33.8 750 ± 78 Observed events 248 245 247 740 mℓℓj(j) ≥400 GeV Total background 254.8 ± 25.8 279.7 ± 27.6 210.9 ± 33.4 745 ± 77 Observed events 246 241 244 731 Table 2 Summary of the expected background yields and observed numbers of events for the SS dilepton channels. The top part of the table shows the numbers obtained for events with two leptons, ≥1 jet and mℓℓ> 110 GeV. The bottom part of the table shows the num- bers for the ﬁnal LRSM selection, where an additional requirement mℓℓj(j) ≥400 GeV is imposed. 8 Results and interpretation The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data Physics processes e±e± μ±μ± e±μ± Total Z/γ ∗+ jets 26.1 ± 5.6 0.0+1.6 −0 1.2 ± 0.7 27 ± 6 Diboson 12.7 ± 2.3 7.2 ± 1.7 18.8 ± 3.0 39 ± 6 Top 5.8 ± 1.3 0.7 ± 0.3 6.8 ± 1.6 13 ± 3 Fake lepton(s) 93.6 ± 35.7 3.1 ± 1.6 53.8 ± 20.3 151 ± 50 Total background 138.3 ± 36.5 11.0+2.9 −2.5 80.7 ± 20.8 230 ± 52 Observed events 155 14 99 268 mℓℓj(j) ≥400 GeV Total background 48.4 ± 16.1 4.4+2.1 −1.3 24.6 ± 7.6 77 ± 21 Observed events 59 8 39 106 Page 7 of 22 Eur. Phys. J. C (2012) 72:2056 SS events, respectively. Additionally, the reconstructed ma- sses of the N and WR candidates, mℓj(j) and, mℓℓj(j) re- spectively, are examined in each dilepton channel. Figures 1 and 2 show those distributions for the OS and SS channels (ee, μμ, and eμ combined). the LRSM, Aϵ varies between 40 % and 65 % across the (mWR,mN) plane. The lowest Aϵ occurs for small mN. It should be noted that the difference in Aϵ between the two models is dominated by the fact the decays to τ leptons are included in generating the HNEO samples, while only de- cays to e and μ are included in the LRSM samples. Table 3 quotes the limits obtained for each channel, after the base- line selection. Given the good agreement between the data and the ex- pectations from SM processes, the results are used to set limits at 95 % C.L. on the visible cross section, σAϵ, where σ is the cross section for new phenomena, A is the accep- tance (i.e. the fraction of events passing geometric and kine- matic selection requirements at the particle level), and ϵ is the efﬁciency (i.e. the detector reconstruction and identiﬁca- tion efﬁciency). For the HNEO model, Aϵ is about 10 % for mN = 0.1 TeV and reaches a plateau value of about 28 % at around mN = 0.8 TeV, for all six dilepton channels. For The resulting limits for the interpretation of the data in terms of the HNEO and LRSM models are derived using as templates the reconstructed masses of the N and WR candi- dates in each dilepton channel. 8 Results and interpretation The baseline selection is used for the HNEO model, while the additional cut of mℓℓj(j) is applied for the LRSM model. Systematic uncertainties Fig. 1 Distributions of the reconstructed N invariant mass, mℓj(j), for OS (top) and SS (bottom) dilepton events with ≥1 jets and mℓℓ> 110 GeV. A selection criterion ST ≥400 GeV is used for the OS selection. The hypothetical signal distributions for mN = 0.3 TeV for OV and Λ/√α = 2 TeV are superimposed Fig. 2 Distributions of the reconstructed WR invariant mass, mℓℓj(j), for OS (top) and SS (bottom) dilepton events with ≥1 jets, mℓℓ> 110 GeV, and mℓℓj(j) ≥400 GeV. A selection criterion ST ≥400 GeV is used for the OS selection. The hypothetical signal distributions for mWR = 1.2 TeV and mN = 0.1 TeV (mWR = 1.5 TeV and mN = 0.8 TeV) for the case of maximal mixing, are superimposed to the OS (SS) distribution Fig. 2 Distributions of the reconstructed WR invariant mass, mℓℓj(j), for OS (top) and SS (bottom) dilepton events with ≥1 jets, mℓℓ> 110 GeV, and mℓℓj(j) ≥400 GeV. A selection criterion ST ≥400 GeV is used for the OS selection. The hypothetical signal distributions for mWR = 1.2 TeV and mN = 0.1 TeV (mWR = 1.5 TeV and mN = 0.8 TeV) for the case of maximal mixing, are superimposed to the OS (SS) distribution Fig. 1 Distributions of the reconstructed N invariant mass, mℓj(j), for OS (top) and SS (bottom) dilepton events with ≥1 jets and mℓℓ> 110 GeV. A selection criterion ST ≥400 GeV is used for the OS selection. The hypothetical signal distributions for mN = 0.3 TeV for OV and Λ/√α = 2 TeV are superimposed Eur. Phys. J. C (2012) 72:2056 Page 8 of 22 Fig. 4 Expected and observed 95 % C.L. upper limits on the heavy neutrino and WR masses for the Majorana (top) and Dirac (bottom) cases, in the no-mixing and maximal-mixing scenarios Table 3 Observed (obs) and expected (exp) 95 % C.L. upper limits on the visible cross section, ⟨σAϵ⟩95, for each OS and SS dilepton channel after the baseline selection , ⟨ ⟩ , p channel after the baseline selection Channels ⟨σAϵ⟩95 obs [fb] ⟨σAϵ⟩95 exp [fb] e±e∓ 28.6 31.0 μ±μ∓ 25.1 36.7 e±μ∓ 50.9 36.4 e±e± 37.6 29.6 μ±μ± 6.1 4.6 e±μ± 25.4 16.2 Fig. 3 Expected and observed 95 % C.L. 8 Results and interpretation upper limits on Λ/√α as a function of the mass of a heavy neutrino, for the operators OV , Os1/Os2, and Os3, using the formalism of Lagrangian of effective op- erators, for the Majorana (top) and Dirac (bottom) scenarios Channels ⟨σAϵ⟩95 obs [fb] ⟨σAϵ⟩95 exp [fb] e±e∓ 28.6 31.0 μ±μ∓ 25.1 36.7 e±μ∓ 50.9 36.4 e±e± 37.6 29.6 μ±μ± 6.1 4.6 e±μ± 25.4 16.2 Fig. 4 Expected and observed 95 % C.L. upper limits on the heavy neutrino and WR masses for the Majorana (top) and Dirac (bottom) cases, in the no-mixing and maximal-mixing scenarios on Λ/√α are shown in Fig. 3 for the Majorana and Dirac scenarios using various effective operator hypotheses. Fig- ure 4 shows the exclusion limits for the masses of heavy neutrinos and the WR boson in the LRSM interpretation, for the no-mixing and maximal-mixing scenarios between Ne and Nμ neutrinos, for both the Majorana and Dirac heavy neutrinos hypotheses. The above results are obtained with a Bayesian [58] ap- proach, where systematic uncertainties are treated as nui- sance parameters with a truncated Gaussian as a prior shape. The prior shape on the parameters of interest, σ× BR, is as- sumed to be ﬂat. Fig. 3 Expected and observed 95 % C.L. upper limits on Λ/√α as a function of the mass of a heavy neutrino, for the operators OV , Os1/Os2, and Os3, using the formalism of Lagrangian of effective op- erators, for the Majorana (top) and Dirac (bottom) scenarios from JES and JER are included as variations in the signal and background templates. The uncertainties on the mea- surement of Rfake and Rprompt are included as variations in the fake lepton background templates. All other uncertain- ties have no signiﬁcant kinematic dependence. Correlations of uncertainties between signal and background, as well as across channels, are taken into account. 9 Conclusions A dedicated search for hypothetical heavy Majorana and Dirac neutrinos, and WR bosons in ﬁnal states with two high-pT same-sign or opposite-sign leptons and hadronic jets has been presented. In a data sample corresponding to an integrated pp luminosity of 2.1 fb−1 at √s = 7 TeV, no signiﬁcant deviations from the SM expectations are ob- served, and 95 % C.L. limits are set on the contributions The 95 % C.L. exclusion limits on the mass of the heavy neutrino in the HNEO model and their dependence Page 9 of 22 Eur. Phys. J. C (2012) 72:2056 of new physics. Excluded mass regions for Majorana and Dirac neutrinos are presented for various operators of an ef- fective Lagrangian framework and for the LRSM. The latter interpretation was used to extract a lower limit on the mass of the gauge boson WR. For both no-mixing and maximal- mixing scenarios, WR bosons with masses below ≈1.8 TeV (≈2.3 TeV) are excluded for mass differences between the WR and N masses larger than 0.3 TeV (0.9 TeV). In the ef- fective Lagrangian interpretation, considering the vector op- erator and Majorana-type heavy neutrinos, the lower limit on Λ/√α ranges from ≈2.5 TeV to ≈0.7 TeV for heavy neu- trino masses ranging from 0.1 TeV to 2.7 TeV. Comparable limits are obtained for Dirac-type neutrinos in both models. The results described represent the most stringent limits to date on the masses of heavy neutrinos and WR boson ob- tained in direct searches. 7. W.Y. Keung, G. Senjanovic, Phys. Rev. Lett. 50, 1427 (1983) 8. F. Aguila, S. Bar-Shalom, A. Soni, J. Wudka, Phys. Lett. B 670, 399 (2009) 9. J.C. Pati, A. Salam, Phys. Rev. D 10, 275 (1974). Phys. Rev. D 11 703 (1975), Erratum 10. R. Mohapatra, J.C. Pati, Phys. Rev. D 11, 2558 (1975) 11. G. Senjanovic, R.N. Mohapatra, Phys. Rev. D 12, 1502 (1975) 12. R.N. Mohapatra, R.E. Marshak, Phys. Rev. Lett. 44 13. A. Ferrari et al., Phys. Rev. D 62, 013001 (2000) 14. K. Huitu et al., Nucl. Phys. B 487, 27 (1997) 15. V. Tello, M. Nemevsek, F. Nesti, G. Senjanovic, F. Vissani, Phys. Rev. Lett. 106, 151801 (2011) 16. A. Melfo, M. Nemevsek, F. Nesti, G. Senjanovic, Y. Zhang, Phys. Rev. D 85, 055018 (2012) 17. D. Buskulic et al. (ALEPH Collaboration), Phys. Lett. B 384, 439 (1996) 18. P. Abreu et al. (DELPHI Collaboration), Eur. Phys. cern.ch/record/1376384 27. ATLAS Collaboration, Eur. Phys. J. C 72, 1849 (2012) 28. ATLAS Collaboration, Eur. Phys. J. C 71, 1577 (2011) 28. ATLAS Collaboration, Eur. Phys. J. C 71, 1577 (2011) 28. ATLAS Collaboration, Eur. Phys. J. C 71, 1577 (2011) 29 M Mangano et al J High Energy Phys 0307 001 (2003) 29. M. Mangano et al., J. High Energy Phys. 0307, 001 (2003) 30. S. Frixione, B.R. Webber, J. High Energy Phys. 0206, 029 (2002) 31. S. Frixione, P. Nason, B.R. Webber, J. High Energy Phys. 0308, 007 (2003) 32. S. Frixione, E. Laenen, P. Motylinski, J. High Energy Phys. 0603, 092 (2006) 33. J. Pumplin et al., J. High Energy Phys. 0207, 012 (2002) 34. G. Marchesini, B.R. Webber, G. Abbiendi et al., Comput. Phys. Commun. 67, 465 (1992) 35. G. Corcella, I.G. Knowles, G. Marchesini et al., J. High Energy Phys. 0101, 010 (2001) The crucial computing support from all WLCG partners is ac- knowledged gratefully, in particular from CERN and the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (UK) and BNL (USA) and in the Tier-2 facilities worldwide. 36. G. Corcella et al., HERWIG 6.5 release note, CERN-TH/2002-270 (2005) [hep-ph/0210213v2] p p 37. J. Butterworth, J. Forshaw, M. Seymour, Z. Phys. C 72, 637 (1996) 38. J. Alwall, P. Demin, S. de Visscher et al., J. High Energy Phys. 0709, 028 (2007) Open Access This article is distributed under the terms of the Cre- ative Commons Attribution License which permits any use, distribu- tion, and reproduction in any medium, provided the original author(s) and the source are credited. 39. T. Sjöstrand, S. Mrenna, P.Z. Skands, J. High Energy Phys. 0605, 026 (2006) 40. Calchep generator. http://theory.sinp.msu.ru/~pukhov/calchep. html 41. A. Sherstnev, R.S. Thorne, Eur. Phys. J. C 55, 553 (2008) 42. ATLAS Collaboration, ATLAS-CONF-2010-031 (2010). https:// cdsweb.cern.ch/record/1277665 26. ATLAS Collaboration, A cern.ch/record/1376384 26. ATLAS Collaboration, ATLAS-CONF-201 6. A. Atre, T. Han, S. Pascoli, B. Zhang, J. High Energy Phys. 0905, 030 (2009) 5. A. Davidson, K.C. Wali, Phys. Rev. Lett. 60, 1813 (1988) 9 Conclusions J. C 8, 41 (1999) 19. P. Achard et al. (L3 Collaboration), Phys. Lett. B 517, 75 (2001) 20. G. Abbiendi et al. (OPAL Collaboration), Eur. Phys. J. C 14, 73 (2000) Acknowledgements We thank CERN for the very successful oper- ation of the LHC, as well as the support staff from our institutions without whom ATLAS could not be operated efﬁciently. 21. V.M. Abazov et al. (DØ Collaboration), Phys. Rev. Lett. 100, 211803 (2008) 22. T. Aaltonen et al. (CDF Collaboration), Phys. Rev. Lett. 103, 041801 (2009) We acknowledge the support of ANPCyT, Argentina; YerPhI, Ar- menia; ARC, Australia; BMWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIEN- CIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Repub- lic; DNRF, DNSRC and Lundbeck Foundation, Denmark; EPLANET and ERC, European Union; IN2P3-CNRS, CEA-DSM/IRFU, France; GNAS, Georgia; BMBF, DFG, HGF, MPG and AvH Foundation, Ger- many; GSRT, Greece; ISF, MINERVA, GIF, DIP and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; RCN, Norway; MNiSW, Poland; GRICES and FCT, Portugal; MERYS (MECTS), Romania; MES of Russia and ROSATOM, Russian Federation; JINR; MSTD, Serbia; MSSR, Slo- vakia; ARRS and MVZT, Slovenia; DST/NRF, South Africa; MICINN, Spain; SRC and Wallenberg Foundation, Sweden; SER, SNSF and Cantons of Bern and Geneva, Switzerland; NSC, Taiwan; TAEK, Turkey; STFC, the Royal Society and Leverhulme Trust, United King- dom; DOE and NSF, United States of America. 23. ATLAS Collaboration, J. High Energy Phys. 1110, 107 (2011) 24. ATLAS Collaboration, J. Instrum. 3, S08003 (2008) 25. ATLAS Collaboration, Eur. Phys. J. C 71, 1630 (201 26. ATLAS Collaboration, ATLAS-CONF-2011-116. https://cdsweb. cern.ch/record/1376384 References Phys. J. (2011). http:// arxiv.org/abs/1112.6426 57. M. Botje et al. (2011). http://arxiv.org/abs/1101.0538 53. ATLAS Collaboration, ATLAS-CONF-2010-042 (2010). http:// cdsweb.cern.ch/record/1277682 53. ATLAS Collaboration, ATLAS-CONF-2010-042 (2010). http:// cdsweb.cern.ch/record/1277682 53. ATLAS Collaboration, ATLAS-CONF-2010-042 (2010). http:// cdsweb.cern.ch/record/1277682 58. T. Junk, Nucl. Instrum. Methods Phys. Res., Sect. A, Accel. Spec- trom. Detect. Assoc. Equip. 434, 435 (1999) 58. T. Junk, Nucl. Instrum. Methods Phys. Res., Sect. A, Accel. Spec- trom. Detect. Assoc. Equip. 434, 435 (1999) G. Aad48, B. Abbott110, J. Abdallah11, A.A. Abdelalim49, A. Abdesselam117, O. Abdinov10, B. Abi111, M. Abolins87, O.S. AbouZeid157, H. Abramowicz152, H. Abreu114, E. Acerbi88a,88b, B.S. Acharya163a,163b, L. Adamczyk37, D.L. Adams24, T.N. Addy56, J. Adelman174, M. Aderholz98, S. Adomeit97, P. Adragna74, T. Adye128, S. Aefsky22, J.A. Aguilar- Saavedra123b,a, M. Aharrouche80, S.P. Ahlen21, F. Ahles48, A. Ahmad147, M. Ahsan40, G. Aielli132a,132b, T. Akdogan18a, T.P.A. Åkesson78, G. Akimoto154, A.V. Akimov93, A. Akiyama66, M.S. Alam1, M.A. Alam75, J. Albert168, S. Albrand55, M. Aleksa29, I.N. Aleksandrov64, F. Alessandria88a, C. Alexa25a, G. Alexander152, G. Alexandre49, T. Alexopoulos9, M. Alhroob20, M. Aliev15, G. Alimonti88a, J. Alison119, M. Aliyev10, B.M.M. Allbrooke17, P.P. Allport72, S.E. Allwood- Spiers53, J. Almond81, A. Aloisio101a,101b, R. Alon170, A. Alonso78, B. Alvarez Gonzalez87, M.G. Alviggi101a,101b, K. Amako65, P. Amaral29, C. Amelung22, V.V. Ammosov127, A. Amorim123a,b, G. Amorós166, N. Amram152, C. Anastopou- los29, L.S. Ancu16, N. Andari114, T. Andeen34, C.F. Anders20, G. Anders58a, K.J. Anderson30, A. Andreazza88a,88b, V. An- drei58a, M-L. Andrieux55, X.S. Anduaga69, A. Angerami34, F. Anghinolﬁ29, A. Anisenkov106, N. Anjos123a, A. Annovi47, A. Antonaki8, M. Antonelli47, A. Antonov95, J. Antos143b, F. Anulli131a, S. Aoun82, L. Aperio Bella4, R. Apolle117,c, G. Ara- bidze87, I. Aracena142, Y. Arai65, A.T.H. Arce44, S. Arfaoui147, J-F. Arguin14, E. Arik18a,, M. Arik18a, A.J. Armbruster86, O. Arnaez80, C. Arnault114, A. Artamonov94, G. Artoni131a,131b, D. Arutinov20, S. Asai154, R. Asfandiyarov171, S. Ask27, B. Åsman145a,145b, L. Asquith5, K. Assamagan24, A. Astbury168, A. Astvatsatourov52, B. Aubert4, E. Auge114, K. Aug- sten126, M. Aurousseau144a, G. Avolio162, R. Avramidou9, D. Axen167, C. Ay54, G. Azuelos92,d, Y. Azuma154, M.A. Baak29, G. Baccaglioni88a, C. Bacci133a,133b, A.M. Bach14, H. Bachacou135, K. Bachas29, M. Backes49, M. Backhaus20, E. Bade- scu25a, P. Bagnaia131a,131b, S. Bahinipati2, Y. Bai32a, D.C. Bailey157, T. Bain157, J.T. Baines128, O.K. Baker174, M.D. Baker24, S. Baker76, E. Banas38, P. Banerjee92, Sw. Banerjee171, D. Banﬁ29, A. Bangert149, V. Bansal168, H.S. Bansil17, L. Barak170, S.P. Baranov93, A. Barashkou64, A. Barbaro Galtieri14, T. Barber48, E.L. Barberio85, D. Barberis50a,50b, M. Barbero20, D.Y. Bardin64, T. Barillari98, M. Barisonzi173, T. Barklow142, N. Barlow27, B.M. Barnett128, R.M. Barnett14, A. Baron- celli133a, G. Barone49, A.J. Barr117, F. Barreiro79, J. Barreiro Guimarães da Costa57, P. Barrillon114, R. Bartoldus142, A.E. Barton70, V. Bartsch148, R.L. Bates53, L. Batkova143a, J.R. Batley27, A. Battaglia16, M. Battistin29, F. Bauer135, H.S. Bawa142,e, S. Beale97, T. Beau77, P.H. Beauchemin160, R. Beccherle50a, P. Bechtle20, H.P. Beck16, S. Becker97, M. Beckingham137, K.H. Becks173, A.J. Beddall18c, A. Beddall18c, S. Bedikian174, V.A. Bednyakov64, C.P. Bee82, M. Begel24, S. Behar Harpaz151, P.K. Behera62, M. Beimforde98, C. Belanger-Champagne84, P.J. Bell49, W.H. Bell49, G. Bella152, L. Bellagamba19a, F. Bellina29, M. Bellomo29, A. Belloni57, O. Beloborodova106,f, K. Belotskiy95, O. Bel- tramello29, S. Ben Ami151, O. Benary152, D. Benchekroun134a, C. Benchouk82, M. Bendel80, N. Benekos164, Y. Benham- mou152, E. Benhar Noccioli49, J.A. Benitez Garcia158b, D.P. Benjamin44, M. Benoit114, J.R. Bensinger22, K. Benslama129, S. Bentvelsen104, D. Berge29, E. Bergeaas Kuutmann41, N. Berger4, F. Berghaus168, E. Berglund104, J. Beringer14, P. Bernat76, R. Bernhard48, C. Bernius24, T. Berry75, C. Bertella82, A. Bertin19a,19b, F. Bertinelli29, F. Bertolucci121a,121b, M.I. Besana88a,88b, N. Besson135, S. Bethke98, W. Bhimji45, R.M. Bianchi29, M. Bianco71a,71b, O. Biebel97, S.P. Bieniek76, K. Bierwagen54, J. Biesiada14, M. Biglietti133a, H. Bilokon47, M. Bindi19a,19b, S. Binet114, A. Bingul18c, C. Bini131a,131b, C. Biscarat176, U. Bitenc48, K.M. Black21, R.E. Blair5, J.-B. Blanchard135, G. Blanchot29, T. Blazek143a, C. Blocker22, J. Blocki38, A. Blondel49, W. Blum80, U. Blumenschein54, G.J. Bobbink104, V.B. Bobrovnikov106, S.S. Bocchetta78, A. Bocci44, C.R. Boddy117, M. Boehler41, J. Boek173, N. Boelaert35, J.A. Bogaerts29, A. Bogdanchikov106, A. Bo- gouch89,, C. Bohm145a, V. Boisvert75, T. Bold37, V. Boldea25a, N.M. Bolnet135, M. Bona74, V.G. Bondarenko95, M. Bon- dioli162, M. Boonekamp135, C.N. Booth138, S. Bordoni77, C. Borer16, A. Borisov127, G. Borissov70, I. Borjanovic12a, M. Borri81, S. Borroni86, V. Bortolotto133a,133b, K. Bos104, D. Boscherini19a, M. Bosman11, H. Boterenbrood104, D. Bot- terill128, J. Bouchami92, J. Boudreau122, E.V. Bouhova-Thacker70, D. Boumediene33, C. Bourdarios114, N. Bousson82, References 43. ATLAS Collaboration, ATL-PHYS-PUB-2010-014 (2010). http:// cdsweb.cern.ch/record/1276548 43. ATLAS Collaboration, ATL-PHYS-PUB-2010-014 (2010). http:// cdsweb cern ch/record/1276548 1. W.M. Alberico, S.M. Bilenky, Phys. Part. Nucl. 35, 297 (2004), and references therein 2. T. Araki et al. (KamLAND Collaboration), Phys. Rev. Lett. 94, 081801 (2005) 3. R.N. Mohapatra, G. Senjanovic, Phys. Rev. Lett. 44, 912 (1980) 4. R.N. Mohapatra, G. Senjanovic, Phys. Rev. D 23, 165 (1981) 5. A. Davidson, K.C. Wali, Phys. Rev. Lett. 60, 1813 (1988) 6. A. Atre, T. Han, S. Pascoli, B. Zhang, J. High Energy Phys. 0905, 030 (2009) 1. W.M. Alberico, S.M. Bilenky, Phys. Part. Nucl. 35, 297 (2004), and references therein 44. ATLAS Collaboration, Eur. Phys. J. C 70, 823 (2010) 45. S. Agostinelli et al. (GEANT4 Collaboration), Nucl. Instrum. Methods Phys. Res., Sect. A, Accel. Spectrom. Detect. Assoc. Equip. 506, 250 (2003) 2. T. Araki et al. (KamLAND Collaboration), Phys. Rev. Lett. 94, 081801 (2005) 3. R.N. Mohapatra, G. Senjanovic, Phys. Rev. Lett. 44, 912 (1980) 3. R.N. Mohapatra, G. Senjanovic, Phys. Rev. Lett. 44, 912 (1980) 4. R.N. Mohapatra, G. Senjanovic, Phys. Rev. D 23, 165 (1981) 46. ATLAS Collaboration, J. High Energy Phys. 1012, 060 (2010) 4. R.N. Mohapatra, G. Senjanovic, Phys. Rev. D 23, 165 (1981) 4. R.N. Mohapatra, G. Senjanovic, Phys. Rev. D 23, 165 (1981) 5. A. Davidson, K.C. Wali, Phys. Rev. Lett. 60, 1813 (1988) 47. ATLAS Collaboration, Eur. Phys. J. (2011). doi:10.1140/epjc/ s10052-012-1909-1 http://arxiv.org/pdf/1110.3174 y 6. A. Atre, T. Han, S. Pascoli, B. Zhang, J. High Energy Phys. 0905, 030 (2009) 48. M. Cacciari, G.P. Salam, Phys. Lett. B 641, 57 (2006) 48. M. Cacciari, G.P. Salam, Phys. Lett. B 641, 57 (2006 Page 10 of 22 Eur. Phys. J. C (2012) 72:2056 49. M. Cacciari, G.P. Salam, G. Soyez, http://fastjet.fr/ 54. ATLAS Collaboration, ATLAS-CONF-2010-041 (2010). http:// cdsweb.cern.ch/record/1277681 49. M. Cacciari, G.P. Salam, G. Soyez, http://fastjet.fr/ 50. ATLAS Collaboration, Eur. Phys. J. C 71, 1512 (2011) 50. ATLAS Collaboration, Eur. Phys. J. C 71, 1512 (2011) 55. ATLAS Collaboration, ATLAS-CONF-2011-046 (2011). http:// cdsweb.cern.ch/record/1281296 51. E. Abat et al., Nucl. Instrum. Methods Phys. Res., Sect. A, Accel. Spectrom. Detect. Assoc. Equip. 621, 134 (2010) 51. E. Abat et al., Nucl. Instrum. Methods Phys. Res., Sect. A, Accel. Spectrom. Detect. Assoc. Equip. 621, 134 (2010) 56. ATLAS Collaboration, ATLAS-CONF-2011-063 (2011). http:// cdsweb.cern.ch/record/1345743 52. ATLAS Collaboration, Submitted to Eur. Phys. J. (2011). http:// arxiv.org/abs/1112.6426 52. ATLAS Collaboration, Submitted to Eur. The ATLAS Collaboration G. Aad48, B. Abbott110, J. Abdallah11, A.A. Abdelalim49, A. Abdesselam117, O. Abdinov10, B. Abi111, M. Abolins87, O.S. AbouZeid157, H. Abramowicz152, H. Abreu114, E. Acerbi88a,88b, B.S. Acharya163a,163b, L. Adamczyk37, D.L. Adams24, T.N. Addy56, J. Adelman174, M. Aderholz98, S. Adomeit97, P. Adragna74, T. Adye128, S. Aefsky22, J.A. Aguilar- Saavedra123b,a, M. Aharrouche80, S.P. Ahlen21, F. Ahles48, A. Ahmad147, M. Ahsan40, G. Aielli132a,132b, T. Akdogan18a, T.P.A. Åkesson78, G. Akimoto154, A.V. Akimov93, A. Akiyama66, M.S. Alam1, M.A. Alam75, J. Albert168, S. Albrand55, M. Aleksa29, I.N. Aleksandrov64, F. Alessandria88a, C. Alexa25a, G. Alexander152, G. Alexandre49, T. Alexopoulos9, M. Alhroob20, M. Aliev15, G. Alimonti88a, J. Alison119, M. Aliyev10, B.M.M. Allbrooke17, P.P. Allport72, S.E. Allwood- Spiers53, J. Almond81, A. Aloisio101a,101b, R. Alon170, A. Alonso78, B. Alvarez Gonzalez87, M.G. Alviggi101a,101b, K. Amako65, P. Amaral29, C. Amelung22, V.V. Ammosov127, A. Amorim123a,b, G. Amorós166, N. Amram152, C. Anastopou- los29, L.S. Ancu16, N. Andari114, T. Andeen34, C.F. Anders20, G. Anders58a, K.J. Anderson30, A. Andreazza88a,88b, V. An- drei58a, M-L. Andrieux55, X.S. Anduaga69, A. Angerami34, F. Anghinolﬁ29, A. Anisenkov106, N. Anjos123a, A. Annovi47, A. Antonaki8, M. Antonelli47, A. Antonov95, J. Antos143b, F. Anulli131a, S. Aoun82, L. Aperio Bella4, R. Apolle117,c, G. Ara- bidze87, I. Aracena142, Y. Arai65, A.T.H. Arce44, S. Arfaoui147, J-F. Arguin14, E. Arik18a,, M. Arik18a, A.J. Armbruster86, O. Arnaez80, C. Arnault114, A. Artamonov94, G. Artoni131a,131b, D. Arutinov20, S. Asai154, R. Asfandiyarov171, S. Ask27, B. Åsman145a,145b, L. Asquith5, K. Assamagan24, A. Astbury168, A. Astvatsatourov52, B. Aubert4, E. Auge114, K. Aug- sten126, M. Aurousseau144a, G. Avolio162, R. Avramidou9, D. Axen167, C. Ay54, G. Azuelos92,d, Y. Azuma154, M.A. Baak29, G. Baccaglioni88a, C. Bacci133a,133b, A.M. Bach14, H. Bachacou135, K. Bachas29, M. Backes49, M. Backhaus20, E. Bade- scu25a, P. Bagnaia131a,131b, S. Bahinipati2, Y. Bai32a, D.C. Bailey157, T. Bain157, J.T. Baines128, O.K. Baker174, M.D. Baker24, S. Baker76, E. Banas38, P. Banerjee92, Sw. Banerjee171, D. Banﬁ29, A. Bangert149, V. Bansal168, H.S. Bansil17, L. Barak170, S.P. Baranov93, A. Barashkou64, A. Barbaro Galtieri14, T. Barber48, E.L. Barberio85, D. Barberis50a,50b, M. Barbero20, D.Y. Bardin64, T. Barillari98, M. Barisonzi173, T. Barklow142, N. Barlow27, B.M. Barnett128, R.M. Barnett14, A. Baron- celli133a, G. Barone49, A.J. Barr117, F. Barreiro79, J. Barreiro Guimarães da Costa57, P. Barrillon114, R. Bartoldus142, A.E. Barton70, V. Bartsch148, R.L. Bates53, L. Batkova143a, J.R. Batley27, A. Battaglia16, M. Battistin29, F. Bauer135, H.S. Bawa142,e, S. Beale97, T. Beau77, P.H. Beauchemin160, R. Beccherle50a, P. Bechtle20, H.P. Beck16, S. Becker97, M. Beckingham137, K.H. Becks173, A.J. Beddall18c, A. Beddall18c, S. Bedikian174, V.A. Bednyakov64, C.P. Bee82, M. Eur. Phys. J. C (2012) 72:2056 Page 11 of 22 A. Boveia30, J. Boyd29, I.R. Boyko64, N.I. Bozhko127, I. Bozovic-Jelisavcic12b, J. Bracinik17, A. Braem29, P. Branchini133a, G.W. Brandenburg57, A. Brandt7, G. Brandt117, O. Brandt54, U. Bratzler155, B. Brau83, J.E. Brau113, H.M. Braun173, B. Bre- lier157, J. Bremer29, R. Brenner165, S. Bressler170, D. Britton53, F.M. Brochu27, I. Brock20, R. Brock87, T.J. Brodbeck70, E. Brodet152, F. Broggi88a, C. Bromberg87, J. Bronner98, G. Brooijmans34, W.K. Brooks31b, G. Brown81, H. Brown7, P.A. Bruckman de Renstrom38, D. Bruncko143b, R. Bruneliere48, S. Brunet60, A. Bruni19a, G. Bruni19a, M. Bruschi19a, T. Buanes13, Q. Buat55, F. Bucci49, J. Buchanan117, N.J. Buchanan2, P. Buchholz140, R.M. Buckingham117, A.G. Buck- ley45, S.I. Buda25a, I.A. Budagov64, B. Budick107, V. Büscher80, L. Bugge116, O. Bulekov95, M. Bunse42, T. Bu- ran116, H. Burckhart29, S. Burdin72, T. Burgess13, S. Burke128, E. Busato33, P. Bussey53, C.P. Buszello165, F. Butin29, B. Butler142, J.M. Butler21, C.M. Buttar53, J.M. Butterworth76, W. Buttinger27, S. Cabrera Urbán166, D. Caforio19a,19b, O. Cakir3a, P. Calaﬁura14, G. Calderini77, P. Calfayan97, R. Calkins105, L.P. Caloba23a, R. Caloi131a,131b, D. Calvet33, S. Calvet33, R. Camacho Toro33, P. Camarri132a,132b, M. Cambiaghi118a,118b, D. Cameron116, L.M. Caminada14, S. Cam- pana29, M. Campanelli76, V. Canale101a,101b, F. Canelli30,g, A. Canepa158a, J. Cantero79, L. Capasso101a,101b, M.D.M. Ca- peans Garrido29, I. Caprini25a, M. Caprini25a, D. Capriotti98, M. Capua36a,36b, R. Caputo80, C. Caramarcu24, R. Car- darelli132a, T. Carli29, G. Carlino101a, L. Carminati88a,88b, B. Caron84, S. Caron103, G.D. Carrillo Montoya171, A.A. Carter74, J.R. Carter27, J. Carvalho123a,h, D. Casadei107, M.P. Casado11, M. Cascella121a,121b, C. Caso50a,50b,, A.M. Castaneda Her- nandez171, E. Castaneda-Miranda171, V. Castillo Gimenez166, N.F. Castro123a, G. Cataldi71a, F. Cataneo29, A. Catinaccio29, J.R. Catmore29, A. Cattai29, G. Cattani132a,132b, S. Caughron87, D. Cauz163a,163c, P. Cavalleri77, D. Cavalli88a, M. Cavalli- Sforza11, V. Cavasinni121a,121b, F. Ceradini133a,133b, A.S. Cerqueira23b, A. Cerri29, L. Cerrito74, F. Cerutti47, S.A. Cetin18b, F. Cevenini101a,101b, A. Chafaq134a, D. Chakraborty105, K. Chan2, B. Chapleau84, J.D. Chapman27, J.W. Chapman86, E. Chareyre77, D.G. Charlton17, V. Chavda81, C.A. Chavez Barajas29, S. Cheatham84, S. Chekanov5, S.V. Chekulaev158a, G.A. Chelkov64, M.A. Chelstowska103, C. Chen63, H. Chen24, S. Chen32c, T. Chen32c, X. Chen171, S. Cheng32a, A. Chep- lakov64, V.F. Chepurnov64, R. Cherkaoui El Moursli134e, V. Chernyatin24, E. Cheu6, S.L. Cheung157, L. Chevalier135, G. Chiefari101a,101b, L. Chikovani51a, J.T. Childers29, A. Chilingarov70, G. Chiodini71a, A.S. Chisholm17, M.V. Chizhov64, G. Choudalakis30, S. Chouridou136, I.A. Christidi76, A. Christov48, D. Chromek-Burckhart29, M.L. Chu150, J. Chudoba124, G. Ciapetti131a,131b, K. Ciba37, A.K. Ciftci3a, R. Ciftci3a, D. Cinca33, V. Cindro73, M.D. Ciobotaru162, C. Ciocca19a, A. Ciocio14, M. Cirilli86, M. Citterio88a, M. Ciubancan25a, A. Clark49, P.J. Clark45, W. Cleland122, J.C. Clemens82, B. Clement55, C. Clement145a,145b, R.W. Clifft128, Y. Coadou82, M. Cobal163a,163c, A. Coccaro171, J. Cochran63, P. Coe117, J.G. Cogan142, J. Coggeshall164, E. Cogneras176, J. Colas4, A.P. Colijn104, N.J. Collins17, C. Collins-Tooth53, J. Col- lot55, G. Colon83, P. Conde Muiño123a, E. Coniavitis117, M.C. Conidi11, M. Consonni103, V. Consorti48, S. Constanti- nescu25a, C. Conta118a,118b, F. Conventi101a,i, J. Cook29, M. Cooke14, B.D. Cooper76, A.M. Cooper-Sarkar117, K. Copic14, T. Cornelissen173, M. Corradi19a, F. Corriveau84,j, A. Cortes-Gonzalez164, G. Cortiana98, G. Costa88a, M.J. Costa166, D. Costanzo138, T. Costin30, D. Côté29, R. Coura Torres23a, L. Courneyea168, G. Cowan75, C. Cowden27, B.E. Cox81, K. Cranmer107, F. Crescioli121a,121b, M. Cristinziani20, G. Crosetti36a,36b, R. Crupi71a,71b, S. Crépé-Renaudin55, C.-M. Cu- ciuc25a, C. Cuenca Almenar174, T. Cuhadar Donszelmann138, M. Curatolo47, C.J. Curtis17, C. Cuthbert149, P. Cwetanski60, H. Czirr140, P. Czodrowski43, Z. Czyczula174, S. D’Auria53, M. D’Onofrio72, A. D’Orazio131a,131b, P.V.M. Da Silva23a, C. Da Via81, W. Dabrowski37, T. Dai86, C. Dallapiccola83, M. Dam35, M. Dameri50a,50b, D.S. Damiani136, H.O. Daniels- son29, D. Dannheim98, V. Dao49, G. Darbo50a, G.L. Darlea25b, W. Davey20, T. Davidek125, N. Davidson85, R. Davidson70, E. Davies117,c, M. Davies92, A.R. Davison76, Y. Davygora58a, E. Dawe141, I. Dawson138, J.W. Dawson5,, R.K. Daya- Ishmukhametova22, K. De7, R. de Asmundis101a, S. De Castro19a,19b, P.E. De Castro Faria Salgado24, S. De Cecco77, J. de Graat97, N. De Groot103, P. de Jong104, C. De La Taille114, H. De la Torre79, B. De Lotto163a,163c, L. de Mora70, L. De Nooij104, D. De Pedis131a, A. De Salvo131a, U. De Sanctis163a,163c, A. De Santo148, J.B. De Vivie De Regie114, S. Dean76, W.J. Dearnaley70, R. Debbe24, C. Debenedetti45, D.V. Dedovich64, J. Degenhardt119, M. Dehchar117, C. Del Papa163a,163c, J. Del Peso79, T. Del Prete121a,121b, T. Delemontex55, M. Deliyergiyev73, A. Dell’Acqua29, L. Dell’Asta21, M. Della Pietra101a,i, D. della Volpe101a,101b, M. Delmastro4, N. Delruelle29, P.A. Delsart55, C. Deluca147, S. Demers174, M. Demichev64, B. Demirkoz11,k, J. Deng162, S.P. Denisov127, D. Derendarz38, J.E. Derkaoui134d, F. Derue77, P. Dervan72, K. Desch20, E. Devetak147, P.O. Deviveiros104, A. Dewhurst128, B. DeWilde147, S. Dhaliwal157, R. Dhul- lipudi24,l, A. Di Ciaccio132a,132b, L. Di Ciaccio4, A. 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Duﬂot114, M-A. Dufour84, M. Dunford29, H. Duran Yildiz3a, R. Duxﬁeld138, M. Dwuznik37, F. Dydak29, M. Düren52, W.L. Ebenstein44, J. Ebke97, S. Eckweiler80, K. Edmonds80, C.A. Edwards75, N.C. Edwards53, W. Ehrenfeld41, T. Ehrich98, T. Eifert142, G. Eigen13, K. Einsweiler14, E. Eisenhan- dler74, T. Ekelof165, M. El Kacimi134c, M. Ellert165, S. Elles4, F. Ellinghaus80, K. Ellis74, N. Ellis29, J. Elmsheuser97, M. Elsing29, D. Emeliyanov128, R. Engelmann147, A. Engl97, B. Epp61, A. Eppig86, J. Erdmann54, A. Ereditato16, D. Eriksson145a, J. Ernst1, M. Ernst24, J. Ernwein135, D. Errede164, S. Errede164, E. Ertel80, M. Escalier114, C. Esco- bar122, X. Espinal Curull11, B. Esposito47, F. Etienne82, A.I. Etienvre135, E. Etzion152, D. Evangelakou54, H. Evans60, L. Fabbri19a,19b, C. Fabre29, R.M. Fakhrutdinov127, S. Falciano131a, Y. Fang171, M. Fanti88a,88b, A. Farbin7, A. Farilla133a, J. Farley147, T. Farooque157, S.M. Farrington117, P. Farthouat29, P. Fassnacht29, D. Fassouliotis8, B. Fatholahzadeh157, A. Favareto88a,88b, L. Fayard114, S. Fazio36a,36b, R. Febbraro33, P. Federic143a, O.L. Fedin120, W. Fedorko87, M. Fehling- Kaschek48, L. Feligioni82, D. Fellmann5, C. Feng32d, E.J. Feng30, A.B. Fenyuk127, J. Ferencei143b, J. Ferland92, W. Fer- nando108, S. Ferrag53, J. Ferrando53, V. Ferrara41, A. Ferrari165, P. Ferrari104, R. Ferrari118a, D.E. Ferreira de Lima53, A. Ferrer166, M.L. Ferrer47, D. Ferrere49, C. Ferretti86, A. Ferretto Parodi50a,50b, M. Fiascaris30, F. Fiedler80, A. Fil- ipˇciˇc73, A. Filippas9, F. Filthaut103, M. Fincke-Keeler168, M.C.N. Fiolhais123a,h, L. Fiorini166, A. Firan39, G. Fischer41, P. Fischer20, M.J. Fisher108, M. Flechl48, I. Fleck140, J. Fleckner80, P. Fleischmann172, S. Fleischmann173, T. Flick173, A. Floderus78, L.R. Flores Castillo171, M.J. Flowerdew98, M. Fokitis9, T. Fonseca Martin16, D.A. Forbush137, A. Formica135, A. Forti81, D. Fortin158a, J.M. Foster81, D. Fournier114, A. Foussat29, A.J. Fowler44, K. Fowler136, H. Fox70, P. Fran- cavilla11, S. Franchino118a,118b, D. Francis29, T. Frank170, M. Franklin57, S. Franz29, M. Fraternali118a,118b, S. Fratina119, S.T. French27, F. Friedrich43, R. Froeschl29, D. Froidevaux29, J.A. Frost27, C. Fukunaga155, E. Fullana Torregrosa29, J. Fuster166, C. Gabaldon29, O. Gabizon170, T. Gadfort24, S. Gadomski49, G. Gagliardi50a,50b, P. Gagnon60, C. Galea97, E.J. Gallas117, V. Gallo16, B.J. Gallop128, P. Gallus124, K.K. Gan108, Y.S. Gao142,e, V.A. Gapienko127, A. Gaponenko14, F. Garberson174, M. Garcia-Sciveres14, C. García166, J.E. García Navarro166, R.W. Gardner30, N. Garelli29, H. Garitaonan- dia104, V. Garonne29, J. Garvey17, C. Gatti47, G. Gaudio118a, B. Gaur140, L. Gauthier135, I.L. Gavrilenko93, C. Gay167, G. Gaycken20, J-C. Gayde29, E.N. Gazis9, P. Ge32d, C.N.P. Gee128, D.A.A. Geerts104, Ch. Geich-Gimbel20, K. Geller- stedt145a,145b, C. Gemme50a, A. Gemmell53, M.H. Genest55, S. Gentile131a,131b, M. George54, S. George75, P. Ger- lach173, A. Gershon152, C. Geweniger58a, H. Ghazlane134b, N. Ghodbane33, B. Giacobbe19a, S. Giagu131a,131b, V. Giak- oumopoulou8, V. Giangiobbe11, F. Gianotti29, B. Gibbard24, A. Gibson157, S.M. Gibson29, L.M. Gilbert117, V. Gilewsky90, D. Gillberg28, A.R. Gillman128, D.M. Gingrich2,d, J. Ginzburg152, N. Giokaris8, M.P. Giordani163c, R. Giordano101a,101b, F.M. Giorgi15, P. Giovannini98, P.F. Giraud135, D. Giugni88a, M. Giunta92, P. Giusti19a, B.K. Gjelsten116, L.K. Gladilin96, C. Glasman79, J. Glatzer48, A. Glazov41, K.W. Glitza173, G.L. Glonti64, J.R. Goddard74, J. Godfrey141, J. Godlewski29, M. Goebel41, T. Göpfert43, C. Goeringer80, C. Gössling42, T. Göttfert98, S. Goldfarb86, T. Golling174, A. Gomes123a,b, L.S. Gomez Fajardo41, R. Gonçalo75, J. Goncalves Pinto Firmino Da Costa41, L. Gonella20, A. Gonidec29, S. Gonza- lez171, S. González de la Hoz166, G. Gonzalez Parra11, M.L. Gonzalez Silva26, S. Gonzalez-Sevilla49, J.J. Goodson147, L. Goossens29, P.A. Gorbounov94, H.A. Gordon24, I. Gorelov102, G. Gorﬁne173, B. Gorini29, E. Gorini71a,71b, A. Gorišek73, E. Gornicki38, S.A. Gorokhov127, V.N. Goryachev127, B. Gosdzik41, M. Gosselink104, M.I. Gostkin64, I. Gough Es- chrich162, M. Gouighri134a, D. Goujdami134c, M.P. Goulette49, A.G. Goussiou137, C. Goy4, S. Gozpinar22, I. Grabowska- Bold37, P. Grafström29, K-J. Grahn41, F. Grancagnolo71a, S. Grancagnolo15, V. Grassi147, V. Gratchev120, N. Grau34, H.M. Gray29, J.A. Gray147, E. Graziani133a, O.G. Grebenyuk120, T. Greenshaw72, Z.D. Greenwood24,l, K. Gregersen35, I.M. Gregor41, P. Grenier142, J. Grifﬁths137, N. Grigalashvili64, A.A. Grillo136, S. Grinstein11, Y.V. Grishkevich96, J.- F. Grivaz114, M. Groh98, E. Gross170, J. Grosse-Knetter54, J. Groth-Jensen170, K. Grybel140, V.J. Guarino5, D. Guest174, C. Guicheney33, A. Guida71a,71b, S. Guindon54, H. Guler84,n, J. Gunther124, B. Guo157, J. Guo34, A. Gupta30, Y. Gusakov64, V.N. Gushchin127, P. Gutierrez110, N. Guttman152, O. Gutzwiller171, C. Guyot135, C. Gwenlan117, C.B. Gwilliam72, A. Haas142, S. Haas29, C. Haber14, H.K. Hadavand39, D.R. Hadley17, P. Haefner98, F. Hahn29, S. Haider29, Z. Hajduk38, H. Hakobyan175, D. Hall117, J. Haller54, K. Hamacher173, P. Hamal112, M. Hamer54, A. Hamilton144b,o, S. Hamilton160, H. Han32a, L. Han32b, K. Hanagaki115, K. Hanawa159, M. Hance14, C. Handel80, P. Hanke58a, J.R. Hansen35, J.B. Hansen35, J.D. Hansen35, P.H. Hansen35, P. Hansson142, K. Hara159, G.A. Hare136, T. Harenberg173, S. Harkusha89, D. Harper86, R.D. Harrington45, O.M. Harris137, K. Harrison17, J. Hartert48, F. Hartjes104, T. Haruyama65, A. Harvey56, S. Hasegawa100, Y. Hasegawa139, S. Hassani135, M. Hatch29, D. Hauff98, S. Haug16, M. Hauschild29, R. Hauser87, M. Havranek20, B.M. Hawes117, C.M. Hawkes17, R.J. Hawkings29, A.D. Hawkins78, D. Hawkins162, T. Hayakawa66, T. Hayashi159, D. Hay- den75, H.S. Hayward72, S.J. Haywood128, E. Hazen21, M. He32d, S.J. Head17, V. Hedberg78, L. Heelan7, S. Heim87, B. Heinemann14, S. Heisterkamp35, L. Helary4, C. Heller97, M. Heller29, S. Hellman145a,145b, D. Hellmich20, C. Helsens11, R.C.W. Henderson70, M. Henke58a, A. Henrichs54, A.M. Henriques Correia29, S. Henrot-Versille114, F. Henry-Couannier82, Eur. Phys. J. C (2012) 72:2056 Page 13 of 22 C. Hensel54, T. Henß173, C.M. Hernandez7, Y. Hernández Jiménez166, R. Herrberg15, A.D. Hershenhorn151, G. Herten48, R. Hertenberger97, L. Hervas29, G.G. Hesketh76, N.P. Hessey104, E. Higón-Rodriguez166, D. Hill5,, J.C. Hill27, N. Hill5, K.H. Hiller41, S. Hillert20, S.J. Hillier17, I. Hinchliffe14, E. Hines119, M. Hirose115, F. Hirsch42, D. Hirschbuehl173, J. Hobbs147, N. Hod152, M.C. Hodgkinson138, P. Hodgson138, A. Hoecker29, M.R. Hoeferkamp102, J. Hoffman39, D. Hoff- mann82, M. Hohlfeld80, M. Holder140, S.O. Holmgren145a, T. Holy126, J.L. Holzbauer87, Y. Homma66, T.M. Hong119, L. Hooft van Huysduynen107, T. Horazdovsky126, C. Horn142, S. Horner48, J-Y. Hostachy55, S. Hou150, M.A. Houlden72, A. Hoummada134a, J. Howarth81, D.F. Howell117, I. Hristova15, J. Hrivnac114, I. Hruska124, T. Hryn’ova4, P.J. Hsu80, S.- C. Hsu14, G.S. Huang110, Z. Hubacek126, F. Hubaut82, F. Huegging20, A. Huettmann41, T.B. Huffman117, E.W. Hughes34, G. Hughes70, R.E. Hughes-Jones81, M. Huhtinen29, P. Hurst57, M. Hurwitz14, U. Husemann41, N. Huseynov64,p, J. Hus- ton87, J. Huth57, G. Iacobucci49, G. Iakovidis9, M. Ibbotson81, I. Ibragimov140, R. Ichimiya66, L. Iconomidou-Fayard114, J. Idarraga114, P. Iengo101a, O. Igonkina104, Y. Ikegami65, M. Ikeno65, Y. Ilchenko39, D. Iliadis153, N. Ilic157, M. Imori154, T. Ince20, J. Inigo-Golﬁn29, P. Ioannou8, M. Iodice133a, V. Ippolito131a,131b, A. Irles Quiles166, C. Isaksson165, A. Ishikawa66, M. Ishino67, R. Ishmukhametov39, C. Issever117, S. Istin18a, A.V. Ivashin127, W. Iwanski38, H. Iwasaki65, J.M. Izen40, V. Izzo101a, B. Jackson119, J.N. Jackson72, P. Jackson142, M.R. Jaekel29, V. Jain60, K. Jakobs48, S. Jakobsen35, J. Jakubek126, D.K. Jana110, E. Jankowski157, E. Jansen76, H. Jansen29, A. Jantsch98, M. Janus20, G. Jarlskog78, L. Jeanty57, K. Je- len37, I. Jen-La Plante30, P. Jenni29, A. Jeremie4, P. Jež35, S. Jézéquel4, M.K. Jha19a, H. Ji171, W. Ji80, J. Jia147, Y. Jiang32b, M. Jimenez Belenguer41, G. Jin32b, S. Jin32a, O. Jinnouchi156, M.D. Joergensen35, D. Joffe39, L.G. Johansen13, M. Johansen145a,145b, K.E. Johansson145a, P. Johansson138, S. Johnert41, K.A. Johns6, K. Jon-And145a,145b, G. Jones117, R.W.L. Jones70, T.W. Jones76, T.J. Jones72, O. Jonsson29, C. Joram29, P.M. Jorge123a, J. Joseph14, J. Jovicevic146, T. Jovin12b, X. Ju171, C.A. Jung42, R.M. Jungst29, V. Juranek124, P. Jussel61, A. Juste Rozas11, V.V. Kabachenko127, S. Kabana16, M. Kaci166, A. Kaczmarska38, P. Kadlecik35, M. Kado114, H. Kagan108, M. Kagan57, S. Kaiser98, E. Kajomovitz151, S. Kalinin173, L.V. Kalinovskaya64, S. Kama39, N. Kanaya154, M. Kaneda29, S. Kaneti27, T. Kanno156, V.A. Kantserov95, J. Kanzaki65, B. Kaplan174, A. Kapliy30, J. Kaplon29, D. Kar43, M. Karagounis20, M. Karagoz117, M. Karnevskiy41, K. Karr5, V. Kartvelishvili70, A.N. Karyukhin127, L. Kashif171, G. Kasieczka58b, R.D. Kass108, A. Kastanas13, M. Kataoka4, Y. Kataoka154, E. Katsouﬁs9, J. Katzy41, V. Kaushik6, K. Kawagoe66, T. Kawamoto154, G. Kawamura80, M.S. Kayl104, V.A. Kazanin106, M.Y. Kazarinov64, R. Keeler168, R. Kehoe39, M. Keil54, G.D. Kekelidze64, J. Kennedy97, C.J. Kenney142, M. Kenyon53, O. Kepka124, N. Kerschen29, B.P. Kerševan73, S. Kersten173, K. Kessoku154, J. Keung157, F. Khalil-zada10, H. Khandanyan164, A. Khanov111, D. Kharchenko64, A. Khodinov95, A.G. Kholodenko127, A. Khomich58a, T.J. Khoo27, G. Khoriauli20, A. Khoroshilov173, N. Khovanskiy64, V. Khovanskiy94, E. Khramov64, J. Khubua51b, H. Kim145a,145b, M.S. Kim2, S.H. Kim159, N. Kimura169, O. Kind15, B.T. King72, M. King66, R.S.B. King117, J. Kirk128, L.E. Kirsch22, A.E. Kiryunin98, T. Kishimoto66, D. Kisielewska37, T. Kittelmann122, A.M. Kiver127, E. Kladiva143b, J. Klaiber-Lodewigs42, M. Klein72, U. Klein72, K. Kleinknecht80, M. Klemetti84, A. Klier170, P. Klimek145a,145b, A. Klimentov24, R. Klingenberg42, J.A. Klinger81, E.B. Klinkby35, T. Klioutchnikova29, P.F. Klok103, S. Klous104, E.-E. Kluge58a, T. Kluge72, P. Kluit104, S. Kluth98, N.S. Knecht157, E. Kneringer61, J. Knobloch29, E.B.F.G. Knoops82, A. Knue54, B.R. Ko44, T. Kobayashi154, M. Kobel43, M. Kocian142, P. Kodys125, K. Köneke29, A.C. König103, S. Koenig80, L. Köpke80, F. Koetsveld103, P. Ko- evesarki20, T. Koffas28, E. Koffeman104, L.A. Kogan117, F. Kohn54, Z. Kohout126, T. Kohriki65, T. Koi142, T. Kokott20, G.M. Kolachev106, H. Kolanoski15, V. Kolesnikov64, I. Koletsou88a, J. Koll87, M. Kollefrath48, S.D. Kolya81, A.A. Komar93, Y. Komori154, T. Kondo65, T. Kono41,q, A.I. Kononov48, R. Konoplich107,r, N. Konstantinidis76, A. Kootz173, S. Koperny37, K. Korcyl38, K. Kordas153, V. Koreshev127, A. Korn117, A. Korol106, I. Korolkov11, E.V. Korolkova138, V.A. Korotkov127, O. Kortner98, S. Kortner98, V.V. Kostyukhin20, M.J. Kotamäki29, S. Kotov98, V.M. Kotov64, A. Kotwal44, C. Kourkoumelis8, V. Kouskoura153, A. Koutsman158a, R. Kowalewski168, T.Z. Kowalski37, W. Kozanecki135, A.S. Kozhin127, V. Kral126, V.A. Kramarenko96, G. Kramberger73, M.W. Krasny77, A. Krasznahorkay107, J. Kraus87, J.K. Kraus20, A. Kreisel152, F. Krejci126, J. Kretzschmar72, N. Krieger54, P. Krieger157, K. Kroeninger54, H. Kroha98, J. Kroll119, J. Kroseberg20, J. Krstic12a, U. Kruchonak64, H. Krüger20, T. Kruker16, N. Krumnack63, Z.V. Krumshteyn64, A. Kruth20, T. Kubota85, S. Kuday3a, S. Kuehn48, A. Kugel58c, T. Kuhl41, D. Kuhn61, V. Kukhtin64, Y. Kulchitsky89, S. Kuleshov31b, C. Kummer97, M. Kuna77, N. Kundu117, J. Kunkle119, A. Kupco124, H. Kurashige66, M. Kurata159, Y.A. Kurochkin89, V. Kus124, E.S. Kuw- ertz146, M. Kuze156, J. Kvita141, R. Kwee15, A. La Rosa49, L. La Rotonda36a,36b, L. Labarga79, J. Labbe4, S. Lablak134a, C. Lacasta166, F. Lacava131a,131b, H. Lacker15, D. Lacour77, V.R. Lacuesta166, E. Ladygin64, R. Lafaye4, B. Laforge77, T. Lagouri79, S. Lai48, E. Laisne55, M. Lamanna29, C.L. Lampen6, W. Lampl6, E. Lancon135, U. Landgraf48, M.P.J. Lan- don74, J.L. Lane81, C. Lange41, A.J. Lankford162, F. Lanni24, K. Lantzsch173, S. Laplace77, C. Lapoire20, J.F. Laporte135, T. Lari88a, A.V. Larionov127, A. Larner117, C. Lasseur29, M. Lassnig29, P. Laurelli47, V. Lavorini36a,36b, W. Lavrijsen14, P. Laycock72, A.B. Lazarev64, O. Le Dortz77, E. Le Guirriec82, C. Le Maner157, E. Le Menedeu9, C. Lebel92, T. LeCompte5, F. Ledroit-Guillon55, H. Lee104, J.S.H. Lee115, S.C. Lee150, L. Lee174, M. Lefebvre168, M. Legendre135, A. Leger49, g y ( ) B.C. LeGeyt119, F. Legger97, C. Leggett14, M. Lehmacher20, G. Lehmann Miotto29, X. Lei6, M.A.L. Leite23d, R. Leit- ner125, D. Lellouch170, M. Leltchouk34, B. Lemmer54, V. Lendermann58a, K.J.C. Leney144b, T. Lenz104, G. Lenzen173, B. Lenzi29, K. Leonhardt43, S. Leontsinis9, C. Leroy92, J-R. Lessard168, J. Lesser145a, C.G. Lester27, A. Leung Fook Cheong171, J. Levêque4, D. Levin86, L.J. Levinson170, M.S. Levitski127, A. Lewis117, G.H. Lewis107, A.M. Leyko20, M. Leyton15, B. Li82, H. Li171,s, S. Li32b,t, X. Li86, Z. Liang117,u, H. Liao33, B. Liberti132a, P. Lichard29, M. Licht- necker97, K. Lie164, W. Liebig13, R. Lifshitz151, C. Limbach20, A. Limosani85, M. Limper62, S.C. Lin150,v, F. Linde104, J.T. Linnemann87, E. Lipeles119, L. Lipinsky124, A. Lipniacka13, T.M. Liss164, D. Lissauer24, A. Lister49, A.M. Litke136, C. Liu28, D. Liu150, H. Liu86, J.B. Liu86, M. Liu32b, Y. Liu32b, M. Livan118a,118b, S.S.A. Livermore117, A. Lleres55, J. Llorente Merino79, S.L. Lloyd74, E. Lobodzinska41, P. Loch6, W.S. Lockman136, T. Loddenkoetter20, F.K. Loebinger81, A. Loginov174, C.W. Loh167, T. Lohse15, K. Lohwasser48, M. Lokajicek124, J. Loken117, V.P. Lombardo4, R.E. Long70, L. Lopes123a, D. Lopez Mateos57, J. Lorenz97, N. Lorenzo Martinez114, M. Losada161, P. Loscutoff14, F. Lo Sterzo131a,131b, M.J. Losty158a, X. Lou40, A. Lounis114, K.F. Loureiro161, J. Love21, P.A. Love70, A.J. Lowe142,e, F. Lu32a, H.J. Lu- batti137, C. Luci131a,131b, A. Lucotte55, A. Ludwig43, D. Ludwig41, I. Ludwig48, J. Ludwig48, F. Luehring60, G. Lui- jckx104, D. Lumb48, L. Luminari131a, E. Lund116, B. Lund-Jensen146, B. Lundberg78, J. Lundberg145a,145b, J. Lundquist35, M. Lungwitz80, G. Lutz98, D. Lynn24, J. Lys14, E. Lytken78, H. Ma24, L.L. Ma171, J.A. Macana Goia92, G. Maccar- rone47, A. Macchiolo98, B. Maˇcek73, J. Machado Miguens123a, R. Mackeprang35, R.J. Madaras14, W.F. Mader43, R. Maen- ner58c, T. Maeno24, P. Mättig173, S. Mättig41, L. Magnoni29, E. Magradze54, Y. Mahalalel152, K. Mahboubi48, G. Ma- hout17, C. Maiani131a,131b, C. Maidantchik23a, A. Maio123a,b, S. Majewski24, Y. Makida65, N. Makovec114, P. Mal135, B. Malaescu29, Pa. Malecki38, P. Malecki38, V.P. Maleev120, F. Malek55, U. Mallik62, D. Malon5, C. Malone142, S. Mal- tezos9, V. Malyshev106, S. Malyukov29, R. Mameghani97, J. Mamuzic12b, A. Manabe65, L. Mandelli88a, I. Mandi´c73, R. Mandrysch15, J. Maneira123a, P.S. Mangeard87, L. Manhaes de Andrade Filho23a, I.D. Manjavidze64, A. Mann54, P.M. Manning136, A. Manousakis-Katsikakis8, B. Mansoulie135, A. Manz98, A. Mapelli29, L. Mapelli29, L. March79, J.F. Marchand28, F. Marchese132a,132b, G. Marchiori77, M. Marcisovsky124, C.P. Marino168, F. Marroquim23a, R. Mar- shall81, Z. Marshall29, F.K. Martens157, S. Marti-Garcia166, A.J. Martin174, B. Martin29, B. Martin87, F.F. Martin119, J.P. Martin92, Ph. Martin55, T.A. Martin17, V.J. Martin45, B. Martin dit Latour49, S. Martin-Haugh148, M. Martinez11, V. Martinez Outschoorn57, A.C. Martyniuk168, M. Marx81, F. Marzano131a, A. Marzin110, L. Masetti80, T. Mashimo154, R. Mashinistov93, J. Masik81, A.L. Maslennikov106, I. Massa19a,19b, G. Massaro104, N. Massol4, P. Mastrandrea131a,131b, A. Mastroberardino36a,36b, T. Masubuchi154, P. Matricon114, H. Matsumoto154, H. Matsunaga154, T. Matsushita66, C. Mat- travers117,c, J.M. Maugain29, J. Maurer82, S.J. Maxﬁeld72, D.A. Maximov106,f, E.N. May5, A. Mayne138, R. Mazini150, M. Mazur20, M. Mazzanti88a, S.P. Mc Kee86, A. McCarn164, R.L. McCarthy147, T.G. McCarthy28, N.A. McCubbin128, K.W. McFarlane56, J.A. Mcfayden138, H. McGlone53, G. Mchedlidze51b, R.A. McLaren29, T. Mclaughlan17, S.J. McMa- hon128, R.A. McPherson168,j, A. Meade83, J. Mechnich104, M. Mechtel173, M. Medinnis41, R. Meera-Lebbai110, T. Me- guro115, R. Mehdiyev92, S. Mehlhase35, A. Mehta72, K. Meier58a, B. Meirose78, C. Melachrinos30, B.R. Mellado Gar- cia171, L. Mendoza Navas161, Z. Meng150,s, A. Mengarelli19a,19b, S. Menke98, C. Menot29, E. Meoni11, K.M. Mercurio57, P. Mermod49, L. Merola101a,101b, C. Meroni88a, F.S. Merritt30, H. Merritt108, A. Messina29, J. Metcalfe102, A.S. Mete63, C. Meyer80, C. Meyer30, J-P. Meyer135, J. Meyer172, J. Meyer54, T.C. Meyer29, W.T. Meyer63, J. Miao32d, S. Michal29, L. Micu25a, R.P. Middleton128, S. Migas72, L. Mijovi´c41, G. Mikenberg170, M. Mikestikova124, M. Mikuž73, D.W. Miller30, R.J. Miller87, W.J. Mills167, C. Mills57, A. Milov170, D.A. Milstead145a,145b, D. Milstein170, A.A. Minaenko127, M. Miñano Moya166, I.A. Minashvili64, A.I. Mincer107, B. Mindur37, M. Mineev64, Y. Ming171, L.M. Mir11, G. Mirabelli131a, L. Miralles Verge11, A. Misiejuk75, J. Mitrevski136, G.Y. Mitrofanov127, V.A. Mitsou166, S. Mitsui65, P.S. Miyagawa138, K. Miyazaki66, J.U. Mjörnmark78, T. Moa145a,145b, P. Mockett137, S. Moed57, V. Moeller27, K. Mönig41, N. Möser20, S. Mohapatra147, W. Mohr48, S. Mohrdieck-Möck98, A.M. Moisseev127,, R. Moles-Valls166, J. Molina-Perez29, J. Monk76, E. Monnier82, S. Montesano88a,88b, F. Monticelli69, S. Monzani19a,19b, R.W. Moore2, G.F. Moorhead85, C. Mora Her- rera49, A. Moraes53, N. Morange135, J. Morel54, G. Morello36a,36b, D. Moreno80, M. Moreno Llácer166, P. Morettini50a, M. Morgenstern43, M. Morii57, J. Morin74, A.K. Morley29, G. Mornacchi29, S.V. Morozov95, J.D. Morris74, L. Mor- vaj100, H.G. Moser98, M. Mosidze51b, J. Moss108, R. Mount142, E. Mountricha9,w, S.V. Mouraviev93, E.J.W. Moyse83, M. Mudrinic12b, F. Mueller58a, J. Mueller122, K. Mueller20, T.A. Müller97, T. Mueller80, D. Muenstermann29, A. Muir167, Y. Munwes152, W.J. Murray128, I. Mussche104, E. Musto101a,101b, A.G. Myagkov127, M. Myska124, J. Nadal11, K. Na- gai159, K. Nagano65, A. Nagarkar108, Y. Nagasaka59, M. Nagel98, A.M. Nairz29, Y. Nakahama29, K. Nakamura154, T. Naka- mura154, I. Nakano109, G. Nanava20, A. Napier160, R. Narayan58b, M. Nash76,c, N.R. Nation21, T. Nattermann20, T. Nau- mann41, G. Navarro161, H.A. Neal86, E. Nebot79, P.Yu. Nechaeva93, T.J. Neep81, A. Negri118a,118b, G. Negri29, S. Nektar- ijevic49, A. Nelson162, S. Nelson142, T.K. Nelson142, S. Nemecek124, P. Nemethy107, A.A. Nepomuceno23a, M. Nessi29,x, M.S. Neubauer164, A. Neusiedl80, R.M. Neves107, P. Nevski24, P.R. Newman17, V. Nguyen Thi Hong135, R.B. Nickerson117, The ATLAS Collaboration Begel24, S. Behar Harpaz151, P.K. Behera62, M. Beimforde98, C. Belanger-Champagne84, P.J. Bell49, W.H. Bell49, G. Bella152, L. Bellagamba19a, F. Bellina29, M. Bellomo29, A. Belloni57, O. Beloborodova106,f, K. Belotskiy95, O. Bel- tramello29, S. Ben Ami151, O. Benary152, D. Benchekroun134a, C. Benchouk82, M. Bendel80, N. Benekos164, Y. Benham- mou152, E. Benhar Noccioli49, J.A. Benitez Garcia158b, D.P. Benjamin44, M. Benoit114, J.R. Bensinger22, K. Benslama129, S. Bentvelsen104, D. Berge29, E. Bergeaas Kuutmann41, N. Berger4, F. Berghaus168, E. Berglund104, J. Beringer14, P. Bernat76, R. Bernhard48, C. Bernius24, T. Berry75, C. Bertella82, A. Bertin19a,19b, F. Bertinelli29, F. Bertolucci121a,121b, M.I. Besana88a,88b, N. Besson135, S. Bethke98, W. Bhimji45, R.M. Bianchi29, M. Bianco71a,71b, O. Biebel97, S.P. Bieniek76, K. Bierwagen54, J. Biesiada14, M. Biglietti133a, H. Bilokon47, M. Bindi19a,19b, S. Binet114, A. Bingul18c, C. Bini131a,131b, C. Biscarat176, U. Bitenc48, K.M. Black21, R.E. Blair5, J.-B. Blanchard135, G. Blanchot29, T. Blazek143a, C. Blocker22, J. Blocki38, A. Blondel49, W. Blum80, U. Blumenschein54, G.J. Bobbink104, V.B. Bobrovnikov106, S.S. Bocchetta78, A. Bocci44, C.R. Boddy117, M. Boehler41, J. Boek173, N. Boelaert35, J.A. Bogaerts29, A. Bogdanchikov106, A. Bo- gouch89,, C. Bohm145a, V. Boisvert75, T. Bold37, V. Boldea25a, N.M. Bolnet135, M. Bona74, V.G. Bondarenko95, M. Bon- dioli162, M. Boonekamp135, C.N. Booth138, S. Bordoni77, C. Borer16, A. Borisov127, G. Borissov70, I. Borjanovic12a, M. Borri81, S. Borroni86, V. Bortolotto133a,133b, K. Bos104, D. Boscherini19a, M. Bosman11, H. Boterenbrood104, D. Bot- terill128, J. Bouchami92, J. Boudreau122, E.V. Bouhova-Thacker70, D. Boumediene33, C. Bourdarios114, N. Bousson82, Eur. Phys. J. C (2012) 72:2056 Page 11 of 22 Eur. Phys. J. C (2012) 72:2056 Page 12 of 22 Eur. Phys. J. C (2012) 72:2056 Page 13 of 22 Page 14 of 22 Eur. Phys. J. C (2012) 72:2056 B.C. LeGeyt119, F. Legger97, C. Leggett14, M. Lehmacher20, G. Lehmann Miotto29, X. Lei6, M.A.L. Leite23d, R. Leit- ner125, D. Lellouch170, M. Leltchouk34, B. Lemmer54, V. Lendermann58a, K.J.C. Leney144b, T. Lenz104, G. Lenzen173, B. Lenzi29, K. Leonhardt43, S. Leontsinis9, C. Leroy92, J-R. Lessard168, J. Lesser145a, C.G. Lester27, A. Leung Fook Cheong171, J. Levêque4, D. Levin86, L.J. Levinson170, M.S. Levitski127, A. Lewis117, G.H. Lewis107, A.M. Leyko20, M. Leyton15, B. Li82, H. Li171,s, S. Li32b,t, X. Li86, Z. Liang117,u, H. Liao33, B. Liberti132a, P. Lichard29, M. Licht- necker97, K. Lie164, W. Liebig13, R. Lifshitz151, C. Limbach20, A. Limosani85, M. Limper62, S.C. Lin150,v, F. Linde104, J.T. Linnemann87, E. Lipeles119, L. Lipinsky124, A. Lipniacka13, T.M. Liss164, D. Lissauer24, A. Lister49, A.M. Litke136, C. Liu28, D. Liu150, H. Liu86, J.B. Liu86, M. Liu32b, Y. The ATLAS Collaboration Liu32b, M. Livan118a,118b, S.S.A. Livermore117, A. Lleres55, J. Llorente Merino79, S.L. Lloyd74, E. Lobodzinska41, P. Loch6, W.S. Lockman136, T. Loddenkoetter20, F.K. Loebinger81, A. Loginov174, C.W. Loh167, T. Lohse15, K. Lohwasser48, M. Lokajicek124, J. Loken117, V.P. Lombardo4, R.E. Long70, L. Lopes123a, D. Lopez Mateos57, J. Lorenz97, N. Lorenzo Martinez114, M. Losada161, P. Loscutoff14, F. Lo Sterzo131a,131b, M.J. Losty158a, X. Lou40, A. Lounis114, K.F. Loureiro161, J. Love21, P.A. Love70, A.J. Lowe142,e, F. Lu32a, H.J. Lu- batti137, C. Luci131a,131b, A. Lucotte55, A. Ludwig43, D. Ludwig41, I. Ludwig48, J. Ludwig48, F. Luehring60, G. Lui- jckx104, D. Lumb48, L. Luminari131a, E. Lund116, B. Lund-Jensen146, B. Lundberg78, J. Lundberg145a,145b, J. Lundquist35, M. Lungwitz80, G. Lutz98, D. Lynn24, J. Lys14, E. Lytken78, H. Ma24, L.L. Ma171, J.A. Macana Goia92, G. Maccar- rone47, A. Macchiolo98, B. Maˇcek73, J. Machado Miguens123a, R. Mackeprang35, R.J. Madaras14, W.F. Mader43, R. Maen- ner58c, T. Maeno24, P. Mättig173, S. Mättig41, L. Magnoni29, E. Magradze54, Y. Mahalalel152, K. Mahboubi48, G. Ma- hout17, C. Maiani131a,131b, C. Maidantchik23a, A. Maio123a,b, S. Majewski24, Y. Makida65, N. Makovec114, P. Mal135, B. Malaescu29, Pa. Malecki38, P. Malecki38, V.P. Maleev120, F. Malek55, U. Mallik62, D. Malon5, C. Malone142, S. Mal- tezos9, V. Malyshev106, S. Malyukov29, R. Mameghani97, J. Mamuzic12b, A. Manabe65, L. Mandelli88a, I. Mandi´c73, R. Mandrysch15, J. Maneira123a, P.S. Mangeard87, L. Manhaes de Andrade Filho23a, I.D. Manjavidze64, A. Mann54, P.M. Manning136, A. Manousakis-Katsikakis8, B. Mansoulie135, A. Manz98, A. Mapelli29, L. Mapelli29, L. March79, J.F. Marchand28, F. Marchese132a,132b, G. Marchiori77, M. Marcisovsky124, C.P. Marino168, F. Marroquim23a, R. Mar- shall81, Z. Marshall29, F.K. Martens157, S. Marti-Garcia166, A.J. Martin174, B. Martin29, B. Martin87, F.F. Martin119, J.P. Martin92, Ph. Martin55, T.A. Martin17, V.J. Martin45, B. Martin dit Latour49, S. Martin-Haugh148, M. Martinez11, V. Martinez Outschoorn57, A.C. Martyniuk168, M. Marx81, F. Marzano131a, A. Marzin110, L. Masetti80, T. Mashimo154, R. Mashinistov93, J. Masik81, A.L. Maslennikov106, I. Massa19a,19b, G. Massaro104, N. Massol4, P. Mastrandrea131a,131b, A. Mastroberardino36a,36b, T. Masubuchi154, P. Matricon114, H. Matsumoto154, H. Matsunaga154, T. Matsushita66, C. Mat- travers117,c, J.M. Maugain29, J. Maurer82, S.J. Maxﬁeld72, D.A. Maximov106,f, E.N. May5, A. Mayne138, R. Mazini150, M. Mazur20, M. Mazzanti88a, S.P. Mc Kee86, A. McCarn164, R.L. McCarthy147, T.G. McCarthy28, N.A. McCubbin128, K.W. McFarlane56, J.A. Mcfayden138, H. McGlone53, G. Mchedlidze51b, R.A. McLaren29, T. Mclaughlan17, S.J. McMa- hon128, R.A. McPherson168,j, A. Meade83, J. Mechnich104, M. Mechtel173, M. Medinnis41, R. Meera-Lebbai110, T. Me- guro115, R. Mehdiyev92, S. Mehlhase35, A. Mehta72, K. Meier58a, B. Meirose78, C. Melachrinos30, B.R. Mellado Gar- cia171, L. R. Nicolaidou135, L. Nicolas138, B. Nicquevert29, F. Niedercorn114, J. Nielsen136, T. Niinikoski29, N. Nikiforou34, A. Niki- forov15, V. Nikolaenko127, K. Nikolaev64, I. Nikolic-Audit77, K. Nikolics49, K. Nikolopoulos24, H. Nilsen48, P. Nilsson7, Y. Ninomiya154, A. Nisati131a, T. Nishiyama66, R. Nisius98, L. Nodulman5, M. Nomachi115, I. Nomidis153, M. Nordberg29, B. Nordkvist145a,145b, P.R. Norton128, J. Novakova125, M. Nozaki65, L. Nozka112, I.M. Nugent158a, A.-E. Nuncio-Quiroz20, G. Nunes Hanninger85, T. Nunnemann97, E. Nurse76, B.J. O’Brien45, S.W. O’Neale17,, D.C. O’Neil141, V. O’Shea53, L.B. Oakes97, F.G. Oakham28,d, H. Oberlack98, J. Ocariz77, A. Ochi66, S. Oda154, S. Odaka65, J. Odier82, H. Ogren60, A. Oh81, S.H. Oh44, C.C. Ohm145a,145b, T. Ohshima100, H. Ohshita139, T. Ohsugi177, S. Okada66, H. Okawa162, Y. Oku- mura100, T. Okuyama154, A. Olariu25a, M. Olcese50a, A.G. Olchevski64, S.A. Olivares Pino31a, M. Oliveira123a,h, D. Oliveira Damazio24, E. Oliver Garcia166, D. Olivito119, A. Olszewski38, J. Olszowska38, C. Omachi66, A. Onofre123a,y, P.U.E. Ony- isi30, C.J. Oram158a, M.J. Oreglia30, Y. Oren152, D. Orestano133a,133b, I. Orlov106, C. Oropeza Barrera53, R.S. Orr157, B. Osculati50a,50b, R. Ospanov119, C. Osuna11, G. Otero y Garzon26, J.P. Ottersbach104, M. Ouchrif134d, E.A. Ouel- lette168, F. Ould-Saada116, A. Ouraou135, Q. Ouyang32a, A. Ovcharova14, M. Owen81, S. Owen138, V.E. Ozcan18a, N. Oz- turk7, A. Pacheco Pages11, C. Padilla Aranda11, S. Pagan Griso14, E. Paganis138, F. Paige24, P. Pais83, K. Pajchel116, G. Palacino158b, C.P. Paleari6, S. Palestini29, D. Pallin33, A. Palma123a, J.D. Palmer17, Y.B. Pan171, E. Panagiotopoulou9, B. Panes31a, N. Panikashvili86, S. Panitkin24, D. Pantea25a, M. Panuskova124, V. Paolone122, A. Papadelis145a, Th.D. Pa- padopoulou9, A. Paramonov5, D. Paredes Hernandez33, W. Park24,z, M.A. Parker27, F. Parodi50a,50b, J.A. Parsons34, U. Parze- fall48, E. Pasqualucci131a, S. Passaggio50a, A. Passeri133a, F. Pastore133a,133b, Fr. Pastore75, G. Pásztor49,aa, S. Pataraia173, N. Patel149, J.R. Pater81, S. Patricelli101a,101b, T. Pauly29, M. Pecsy143a, M.I. Pedraza Morales171, S.V. Peleganchuk106, H. Peng32b, R. Pengo29, B. Penning30, A. Penson34, J. Penwell60, M. Perantoni23a, K. Perez34,ab, T. Perez Cavalcanti41, E. Perez Codina11, M.T. Pérez García-Estañ166, V. Perez Reale34, L. Perini88a,88b, H. Pernegger29, R. Perrino71a, P. Per- rodo4, S. Persembe3a, A. Perus114, V.D. Peshekhonov64, K. Peters29, B.A. Petersen29, J. Petersen29, T.C. Petersen35, E. Pe- tit4, A. Petridis153, C. Petridou153, E. Petrolo131a, F. Petrucci133a,133b, D. Petschull41, M. Petteni141, R. Pezoa31b, A. Phan85, P.W. Phillips128, G. Piacquadio29, E. Piccaro74, M. Piccinini19a,19b, S.M. Piec41, R. Piegaia26, D.T. Pignotti108, J.E. Pilcher30, A.D. Pilkington81, J. Pina123a,b, M. Pinamonti163a,163c, A. Pinder117, J.L. Pinfold2, J. Ping32c, B. Pinto123a, O. Pirotte29, C. Pizio88a,88b, M. Plamondon168, M.-A. Pleier24, A.V. Pleskach127, A. Poblaguev24, S. Poddar58a, F. Podlyski33, L. Pog- gioli114, T. Poghosyan20, M. Pohl49, F. Polci55, G. Polesello118a, A. Policicchio36a,36b, A. Polini19a, J. Poll74, V. Poly- chronakos24, D.M. Pomarede135, D. Pomeroy22, K. Pommès29, L. Pontecorvo131a, B.G. Pope87, G.A. Popeneciu25a, D.S. Popovic12a, A. Poppleton29, X. Portell Bueso29, C. Posch21, G.E. Pospelov98, S. Pospisil126, I.N. Potrap98, C.J. Pot- ter148, C.T. Potter113, G. Poulard29, J. Poveda171, V. Pozdnyakov64, R. Prabhu76, P. Pralavorio82, A. Pranko14, S. Prasad29, R. Pravahan7, S. Prell63, K. Pretzl16, L. Pribyl29, D. Price60, J. Price72, L.E. Price5, M.J. Price29, D. Prieur122, M. Pri- mavera71a, K. Prokoﬁev107, F. Prokoshin31b, S. Protopopescu24, J. Proudfoot5, X. Prudent43, M. Przybycien37, H. Przysiez- niak4, S. Psoroulas20, E. Ptacek113, E. Pueschel83, J. Purdham86, M. Purohit24,z, P. Puzo114, Y. Pylypchenko62, J. Qian86, Z. Qian82, Z. Qin41, A. Quadt54, D.R. Quarrie14, W.B. Quayle171, F. Quinonez31a, M. Raas103, V. Radescu58b, B. Radics20, P. Radloff113, T. Rador18a, F. Ragusa88a,88b, G. Rahal176, A.M. Rahimi108, D. Rahm24, S. Rajagopalan24, M. Ram- mensee48, M. Rammes140, A.S. Randle-Conde39, K. Randrianarivony28, P.N. Ratoff70, F. Rauscher97, T.C. Rave48, M. Ray- mond29, A.L. Read116, D.M. Rebuzzi118a,118b, A. Redelbach172, G. Redlinger24, R. Reece119, K. Reeves40, A. Rei- chold104, E. Reinherz-Aronis152, A. Reinsch113, I. Reisinger42, M. Relich162, C. Rembser29, Z.L. Ren150, A. Renaud114, M. Rescigno131a, S. Resconi88a, B. Resende135, P. Reznicek97, R. Rezvani157, A. Richards76, R. Richter98, E. Richter- Was4,ac, M. Ridel77, M. Rijpstra104, M. Rijssenbeek147, A. Rimoldi118a,118b, L. Rinaldi19a, R.R. Rios39, I. Riu11, G. Ri- voltella88a,88b, F. Rizatdinova111, E. Rizvi74, S.H. Robertson84,j, A. Robichaud-Veronneau117, D. Robinson27, J.E.M. Robin- son76, A. Robson53, J.G. Rocha de Lima105, C. Roda121a,121b, D. Roda Dos Santos29, D. Rodriguez161, A. Roe54, S. Roe29, O. Røhne116, V. Rojo1, S. Rolli160, A. Romaniouk95, M. Romano19a,19b, V.M. Romanov64, G. Romeo26, E. Romero Adam166, L. Roos77, E. Ros166, S. Rosati131a, K. Rosbach49, A. Rose148, M. Rose75, G.A. Rosenbaum157, E.I. Rosen- berg63, P.L. Rosendahl13, O. Rosenthal140, L. Rosselet49, V. Rossetti11, E. Rossi131a,131b, L.P. Rossi50a, M. Rotaru25a, I. Roth170, J. Rothberg137, D. Rousseau114, C.R. Royon135, A. Rozanov82, Y. Rozen151, X. Ruan32a,ad, I. Rubinskiy41, B. Ruckert97, N. Ruckstuhl104, V.I. Rud96, C. Rudolph43, G. Rudolph61, F. Rühr6, F. Ruggieri133a,133b, A. Ruiz-Martinez63, V. Rumiantsev90,, L. Rumyantsev64, K. Runge48, Z. Rurikova48, N.A. Rusakovich64, J.P. Rutherfoord6, C. Ruwiedel14, P. Ruzicka124, Y.F. Ryabov120, V. Ryadovikov127, P. Ryan87, M. Rybar125, G. Rybkin114, N.C. Ryder117, S. Rzaeva10, A.F. Saavedra149, I. Sadeh152, H.F-W. Sadrozinski136, R. Sadykov64, F. Safai Tehrani131a, H. Sakamoto154, G. Sala- manna74, A. Salamon132a, M. Saleem110, D. Salihagic98, A. Salnikov142, J. Salt166, B.M. Salvachua Ferrando5, D. Sal- vatore36a,36b, F. Salvatore148, A. Salvucci103, A. Salzburger29, D. Sampsonidis153, B.H. Samset116, A. Sanchez101a,101b, V. Sanchez Martinez166, H. Sandaker13, H.G. Sander80, M.P. Sanders97, M. Sandhoff173, T. Sandoval27, C. Sandoval161, R. Sandstroem98, S. Sandvoss173, D.P.C. Sankey128, A. Sansoni47, C. Santamarina Rios84, C. Santoni33, R. Santon- ico132a,132b H Santos123a J G Saraiva123a T Sarangi171 E Sarkisyan-Grinbaum7 F Sarri121a,121b G Sartisohn173 Page 16 of 22 Eur. Phys. J. C (2012) 72:2056 O. Sasaki65, N. Sasao67, I. Satsounkevitch89, G. Sauvage4, E. Sauvan4, J.B. Sauvan114, P. Savard157,d, V. Savinov122, D.O. Savu29, L. Sawyer24,l, D.H. Saxon53, L.P. Says33, C. Sbarra19a, A. Sbrizzi19a,19b, O. Scallon92, D.A. Scannic- chio162, M. Scarcella149, J. Schaarschmidt114, P. Schacht98, U. Schäfer80, S. Schaepe20, S. Schaetzel58b, A.C. Schaf- fer114, D. Schaile97, R.D. Schamberger147, A.G. Schamov106, V. Scharf58a, V.A. Schegelsky120, D. Scheirich86, M. Sch- ernau162, M.I. Scherzer34, C. Schiavi50a,50b, J. Schieck97, M. Schioppa36a,36b, S. Schlenker29, J.L. Schlereth5, E. Schmidt48, K. Schmieden20, C. Schmitt80, S. Schmitt58b, M. Schmitz20, A. Schöning58b, M. Schott29, D. Schouten158a, J. Schovan- cova124, M. Schram84, C. Schroeder80, N. Schroer58c, G. Schuler29, M.J. Schultens20, J. Schultes173, H.-C. Schultz- Coulon58a, H. Schulz15, J.W. Schumacher20, M. Schumacher48, B.A. Schumm136, Ph. Schune135, C. Schwanenberger81, A. Schwartzman142, Ph. Schwemling77, R. Schwienhorst87, R. Schwierz43, J. Schwindling135, T. Schwindt20, M. Schwo- erer4, W.G. Scott128, J. Searcy113, G. Sedov41, E. Sedykh120, E. Segura11, S.C. Seidel102, A. Seiden136, F. Seifert43, J.M. Seixas23a, G. Sekhniaidze101a, K.E. Selbach45, D.M. Seliverstov120, B. Sellden145a, G. Sellers72, M. Seman143b, N. Semprini-Cesari19a,19b, C. Serfon97, L. Serin114, L. Serkin54, R. Seuster98, H. Severini110, M.E. Sevior85, A. Sfyrla29, E. Shabalina54, M. Shamim113, L.Y. Shan32a, J.T. Shank21, Q.T. Shao85, M. Shapiro14, P.B. Shatalov94, L. Shaver6, K. Shaw163a,163c, D. Sherman174, P. Sherwood76, A. Shibata107, H. Shichi100, S. Shimizu29, M. Shimojima99, T. Shin56, M. Shiyakova64, A. Shmeleva93, M.J. Shochet30, D. Short117, S. Shrestha63, E. Shulga95, M.A. Shupe6, P. Sicho124, A. Sidoti131a, F. Siegert48, Dj. Sijacki12a, O. Silbert170, J. Silva123a, Y. Silver152, D. Silverstein142, S.B. Silverstein145a, V. Simak126, O. Simard135, Lj. Simic12a, S. Simion114, B. Simmons76, M. Simonyan35, P. Sinervo157, N.B. Sinev113, V. Sipica140, G. Siragusa172, A. Sircar24, A.N. Sisakyan64, S.Yu. Sivoklokov96, J. Sjölin145a,145b, T.B. Sjursen13, L.A. Skin- nari14, H.P. Skottowe57, K. Skovpen106, P. Skubic110, N. Skvorodnev22, M. Slater17, T. Slavicek126, K. Sliwa160, J. Sloper29, V. Smakhtin170, B.H. Smart45, S.Yu. Smirnov95, Y. Smirnov95, L.N. Smirnova96, O. Smirnova78, B.C. Smith57, D. Smith142, K.M. Smith53, M. Smizanska70, K. Smolek126, A.A. Snesarev93, S.W. Snow81, J. Snow110, J. Snuverink104, S. Snyder24, M. Soares123a, R. Sobie168,j, J. Sodomka126, A. Soffer152, C.A. Solans166, M. Solar126, J. Solc126, E. Soldatov95, U. Sol- devila166, E. Solfaroli Camillocci131a,131b, A.A. Solodkov127, O.V. Solovyanov127, N. Soni2, V. Sopko126, B. Sopko126, M. Sosebee7, R. Soualah163a,163c, A. Soukharev106, S. Spagnolo71a,71b, F. Spanò75, R. Spighi19a, G. Spigo29, F. Spila131a,131b, R. Spiwoks29, M. Spousta125, T. Spreitzer157, B. Spurlock7, R.D. St. Denis53, J. Stahlman119, R. Stamen58a, E. Sta- necka38, R.W. Stanek5, C. Stanescu133a, S. Stapnes116, E.A. Starchenko127, J. Stark55, P. Staroba124, P. Starovoitov90, A. Staude97, P. Stavina143a, G. Steele53, P. Steinbach43, P. Steinberg24, I. Stekl126, B. Stelzer141, H.J. Stelzer87, O. Stelzer- Chilton158a, H. Stenzel52, S. Stern98, K. Stevenson74, G.A. Stewart29, J.A. Stillings20, M.C. Stockton84, K. Stoerig48, G. Stoicea25a, S. Stonjek98, P. Strachota125, A.R. Stradling7, A. Straessner43, J. Strandberg146, S. Strandberg145a,145b, A. Strandlie116, M. Strang108, E. Strauss142, M. Strauss110, P. Strizenec143b, R. Ströhmer172, D.M. Strom113, J.A. Strong75,, R. Stroynowski39, J. Strube128, B. Stugu13, I. Stumer24,, J. Stupak147, P. Sturm173, N.A. Styles41, D.A. Soh150,u, D. Su142, HS. Subramania2, A. Succurro11, Y. Sugaya115, T. Sugimoto100, C. Suhr105, K. Suita66, M. Suk125, V.V. Sulin93, S. Sul- tansoy3d, T. Sumida67, X. Sun55, J.E. Sundermann48, K. Suruliz138, S. Sushkov11, G. Susinno36a,36b, M.R. Sutton148, Y. Suzuki65, Y. Suzuki66, M. Svatos124, Yu.M. Sviridov127, S. Swedish167, I. Sykora143a, T. Sykora125, B. Szeless29, J. Sánchez166, D. Ta104, K. Tackmann41, A. Taffard162, R. Taﬁrout158a, N. Taiblum152, Y. Takahashi100, H. Takai24, R. Takashima68, H. Takeda66, T. Takeshita139, Y. Takubo65, M. Talby82, A. Talyshev106,f, M.C. Tamsett24, J. Tanaka154, R. Tanaka114, S. Tanaka130, S. Tanaka65, Y. Tanaka99, A.J. Tanasijczuk141, K. Tani66, N. Tannoury82, G.P. Tappern29, S. Tapprogge80, D. Tardif157, S. Tarem151, F. Tarrade28, G.F. Tartarelli88a, P. Tas125, M. Tasevsky124, E. Tassi36a,36b, M. Tatarkhanov14, Y. Tayalati134d, C. Taylor76, F.E. Taylor91, G.N. Taylor85, W. Taylor158b, M. Teinturier114, M. Teixeira Dias Castanheira74, P. Teixeira-Dias75, K.K. Temming48, H. Ten Kate29, P.K. Teng150, S. Terada65, K. Terashi154, J. Ter- ron79, M. Testa47, R.J. Teuscher157,j, J. Thadome173, J. Therhaag20, T. Theveneaux-Pelzer77, M. Thioye174, S. Thoma48, J.P. Thomas17, E.N. Thompson34, P.D. Thompson17, P.D. Thompson157, A.S. Thompson53, L.A. Thomsen35, E. Thom- son119, M. Thomson27, R.P. Thun86, F. Tian34, M.J. Tibbetts14, T. Tic124, V.O. Tikhomirov93, Y.A. Tikhonov106,f, S. Timo- shenko95, P. Tipton174, F.J. Tique Aires Viegas29, S. Tisserant82, B. Toczek37, T. Todorov4, S. Todorova-Nova160, B. Togger- son162, J. Tojo65, S. Tokár143a, K. Tokunaga66, K. Tokushuku65, K. Tollefson87, M. Tomoto100, L. Tompkins30, K. Toms102, G. Tong32a, A. Tonoyan13, C. Topfel16, N.D. Topilin64, I. Torchiani29, E. Torrence113, H. Torres77, E. Torró Pastor166, J. Toth82,aa, F. Touchard82, D.R. Tovey138, T. Trefzger172, L. Tremblet29, A. Tricoli29, I.M. Trigger158a, S. Trincaz-Duvoid77, T.N. Trinh77, M.F. Tripiana69, W. Trischuk157, A. Trivedi24,z, B. Trocmé55, C. Troncon88a, M. Trottier-McDonald141, M. Trzebinski38, A. Trzupek38, C. Tsarouchas29, J.C-L. Tseng117, M. Tsiakiris104, P.V. Tsiareshka89, D. Tsionou4,ae, G. Tsipolitis9, V. Tsiskaridze48, E.G. Tskhadadze51a, I.I. Tsukerman94, V. Tsulaia14, J.-W. Tsung20, S. Tsuno65, D. Tsy- bychev147, A. Tua138, A. Tudorache25a, V. Tudorache25a, J.M. Tuggle30, M. Turala38, D. Turecek126, I. Turk Cakir3e, E. Turlay104, R. Turra88a,88b, P.M. Tuts34, A. Tykhonov73, M. Tylmad145a,145b, M. Tyndel128, G. Tzanakos8, K. Uchida20, I. Ueda154, R. Ueno28, M. Ugland13, M. Uhlenbrock20, M. Uhrmacher54, F. Ukegawa159, G. Unal29, D.G. Underwood5, A. Undrus24, G. Unel162, Y. Unno65, D. Urbaniec34, G. Usai7, M. Uslenghi118a,118b, L. Vacavant82, V. Vacek126, B. Va- chon84, S. Vahsen14, J. Valenta124, P. Valente131a, S. Valentinetti19a,19b, S. Valkar125, E. Valladolid Gallego166, S. Val- lecorsa151, J.A. Valls Ferrer166, H. van der Graaf104, E. van der Kraaij104, R. Van Der Leeuw104, E. van der Poel104, D. van der Ster29, N. van Eldik83, P. van Gemmeren5, Z. van Kesteren104, I. van Vulpen104, M. Vanadia98, W. Vandelli29, G. Vandoni29, A. Vaniachine5, P. Vankov41, F. Vannucci77, F. Varela Rodriguez29, R. Vari131a, E.W. Varnes6, D. Varouchas14, A. Vartapetian7, K.E. Varvell149, V.I. Vassilakopoulos56, F. Vazeille33, T. Vazquez Schroeder54, G. Vegni88a,88b, J.J. Veil- let114, C. Vellidis8, F. Veloso123a, R. Veness29, S. Veneziano131a, A. Ventura71a,71b, D. Ventura137, M. Venturi48, N. Ven- turi157, V. Vercesi118a, M. Verducci137, W. Verkerke104, J.C. Vermeulen104, A. Vest43, M.C. Vetterli141,d, I. Vichou164, T. Vickey144b,af, O.E. Vickey Boeriu144b, G.H.A. Viehhauser117, S. Viel167, M. Villa19a,19b, M. Villaplana Perez166, E. Viluc- chi47, M.G. Vincter28, E. Vinek29, V.B. Vinogradov64, M. Virchaux135,, J. Virzi14, O. Vitells170, M. Viti41, I. Vivarelli48, F. Vives Vaque2, S. Vlachos9, D. Vladoiu97, M. Vlasak126, N. Vlasov20, A. Vogel20, P. Vokac126, G. Volpi47, M. Volpi85, G. Volpini88a, H. von der Schmitt98, J. von Loeben98, H. von Radziewski48, E. von Toerne20, V. Vorobel125, A.P. Vorobiev127, V. Vorwerk11, M. Vos166, R. Voss29, T.T. Voss173, J.H. Vossebeld72, N. Vranjes135, M. Vranjes Milosavljevic104, V. Vrba124, M. Vreeswijk104, T. Vu Anh48, R. Vuillermet29, I. Vukotic114, W. Wagner173, P. Wagner119, H. Wahlen173, J. Wak- abayashi100, J. Walbersloh42, S. Walch86, J. Walder70, R. Walker97, W. Walkowiak140, R. Wall174, P. Waller72, C. Wang44, H. Wang171, H. Wang32b,ag, J. Wang150, J. Wang55, J.C. Wang137, R. Wang102, S.M. Wang150, A. Warburton84, C.P. Ward27, M. Warsinsky48, P.M. Watkins17, A.T. Watson17, I.J. Watson149, M.F. Watson17, G. Watts137, S. Watts81, A.T. Waugh149, B.M. Waugh76, M. Weber128, M.S. Weber16, P. Weber54, A.R. Weidberg117, P. Weigell98, J. Weingarten54, C. Weiser48, H. Wellenstein22, P.S. Wells29, T. Wenaus24, D. Wendland15, S. Wendler122, Z. Weng150,u, T. Wengler29, S. Wenig29, N. Wermes20, M. Werner48, P. Werner29, M. Werth162, M. Wessels58a, C. Weydert55, K. Whalen28, S.J. Wheeler-Ellis162, S.P. Whitaker21, A. White7, M.J. White85, S.R. Whitehead117, D. Whiteson162, D. Whittington60, F. Wicek114, D. Wicke173, F.J. Wickens128, W. Wiedenmann171, M. Wielers128, P. Wienemann20, C. Wiglesworth74, L.A.M. Wiik-Fuchs48, P.A. Wi- jeratne76, A. Wildauer166, M.A. Wildt41,q, I. Wilhelm125, H.G. Wilkens29, J.Z. Will97, E. Williams34, H.H. Williams119, W. Willis34, S. Willocq83, J.A. Wilson17, M.G. Wilson142, A. Wilson86, I. Wingerter-Seez4, S. Winkelmann48, F. Win- klmeier29, M. Wittgen142, M.W. Wolter38, H. Wolters123a,h, W.C. Wong40, G. Wooden86, B.K. Wosiek38, J. Wotschack29, M.J. Woudstra83, K.W. Wozniak38, K. Wraight53, C. Wright53, M. Wright53, B. Wrona72, S.L. Wu171, X. Wu49, Y. Wu32b,ah, E. Wulf34, R. Wunstorf42, B.M. Wynne45, S. Xella35, M. Xiao135, S. Xie48, Y. Xie32a, C. Xu32b,w, D. Xu138, G. Xu32a, B. Yabsley149, S. Yacoob144b, M. Yamada65, H. Yamaguchi154, A. Yamamoto65, K. Yamamoto63, S. Yamamoto154, T. Ya- mamura154, T. Yamanaka154, J. Yamaoka44, T. Yamazaki154, Y. Yamazaki66, Z. Yan21, H. Yang86, U.K. Yang81, Y. Yang60, Y. Yang32a, Z. Yang145a,145b, S. Yanush90, Y. Yao14, Y. Yasu65, G.V. Ybeles Smit129, J. Ye39, S. Ye24, M. Yilmaz3c, R. Yoosoofmiya122, K. Yorita169, R. Yoshida5, C. Young142, S. Youssef21, D. Yu24, J. Yu7, J. Yu111, L. Yuan32a,ai, A. Yurkewicz105, B. Zabinski38, V.G. Zaets127, R. Zaidan62, A.M. Zaitsev127, Z. Zajacova29, L. Zanello131a,131b, A. Zayt- sev106, C. Zeitnitz173, M. Zeller174, M. Zeman124, A. Zemla38, C. Zendler20, O. Zenin127, T. Ženiš143a, Z. Zinonos121a,121b, S. Zenz14, D. Zerwas114, G. Zevi della Porta57, Z. Zhan32d, D. Zhang32b,ag, H. Zhang87, J. Zhang5, X. Zhang32d, Z. Zhang114, L. Zhao107, T. Zhao137, Z. Zhao32b, A. Zhemchugov64, S. Zheng32a, J. Zhong117, B. Zhou86, N. Zhou162, Y. Zhou150, C.G. Zhu32d, H. Zhu41, J. Zhu86, Y. Zhu32b, X. Zhuang97, V. Zhuravlov98, D. Zieminska60, R. Zimmermann20, S. Zim- mermann20, S. Zimmermann48, M. Ziolkowski140, R. Zitoun4, L. Živkovi´c34, V.V. Zmouchko127,, G. Zobernig171, A. Zoc- coli19a,19b, Y. Zolnierowski4, A. Zsenei29, M. zur Nedden15, V. Zutshi105, L. Zwalinski29 The ATLAS Collaboration Mendoza Navas161, Z. Meng150,s, A. Mengarelli19a,19b, S. Menke98, C. Menot29, E. Meoni11, K.M. Mercurio57, P. Mermod49, L. Merola101a,101b, C. Meroni88a, F.S. Merritt30, H. Merritt108, A. Messina29, J. Metcalfe102, A.S. Mete63, C. Meyer80, C. Meyer30, J-P. Meyer135, J. Meyer172, J. Meyer54, T.C. Meyer29, W.T. Meyer63, J. Miao32d, S. Michal29, L. Micu25a, R.P. Middleton128, S. Migas72, L. Mijovi´c41, G. Mikenberg170, M. Mikestikova124, M. Mikuž73, D.W. Miller30, R.J. Miller87, W.J. Mills167, C. Mills57, A. Milov170, D.A. Milstead145a,145b, D. Milstein170, A.A. Minaenko127, M. Miñano Moya166, I.A. Minashvili64, A.I. Mincer107, B. Mindur37, M. Mineev64, Y. Ming171, L.M. Mir11, G. Mirabelli131a, L. Miralles Verge11, A. Misiejuk75, J. Mitrevski136, G.Y. Mitrofanov127, V.A. Mitsou166, S. Mitsui65, P.S. Miyagawa138, K. Miyazaki66, J.U. Mjörnmark78, T. Moa145a,145b, P. Mockett137, S. Moed57, V. Moeller27, K. Mönig41, N. Möser20, S. Mohapatra147, W. Mohr48, S. Mohrdieck-Möck98, A.M. Moisseev127,, R. Moles-Valls166, J. Molina-Perez29, J. Monk76, E. Monnier82, S. Montesano88a,88b, F. Monticelli69, S. Monzani19a,19b, R.W. Moore2, G.F. Moorhead85, C. Mora Her- rera49, A. Moraes53, N. Morange135, J. Morel54, G. Morello36a,36b, D. Moreno80, M. Moreno Llácer166, P. Morettini50a, M. Morgenstern43, M. Morii57, J. Morin74, A.K. Morley29, G. Mornacchi29, S.V. Morozov95, J.D. Morris74, L. Mor- vaj100, H.G. Moser98, M. Mosidze51b, J. Moss108, R. Mount142, E. Mountricha9,w, S.V. Mouraviev93, E.J.W. Moyse83, M. Mudrinic12b, F. Mueller58a, J. Mueller122, K. Mueller20, T.A. Müller97, T. Mueller80, D. Muenstermann29, A. Muir167, Y. Munwes152, W.J. Murray128, I. Mussche104, E. Musto101a,101b, A.G. Myagkov127, M. Myska124, J. Nadal11, K. Na- gai159, K. Nagano65, A. Nagarkar108, Y. Nagasaka59, M. Nagel98, A.M. Nairz29, Y. Nakahama29, K. Nakamura154, T. Naka- mura154, I. Nakano109, G. Nanava20, A. Napier160, R. Narayan58b, M. Nash76,c, N.R. Nation21, T. Nattermann20, T. Nau- mann41, G. Navarro161, H.A. Neal86, E. Nebot79, P.Yu. Nechaeva93, T.J. Neep81, A. Negri118a,118b, G. Negri29, S. Nektar- ijevic49, A. Nelson162, S. Nelson142, T.K. Nelson142, S. Nemecek124, P. Nemethy107, A.A. Nepomuceno23a, M. Nessi29,x, M.S. Neubauer164, A. Neusiedl80, R.M. Neves107, P. Nevski24, P.R. Newman17, V. Nguyen Thi Hong135, R.B. Nickerson117, Eur. Phys. J. C (2012) 72:2056 Page 15 of 22 Eur. Phys. J. C (2012) 72:2056 Page 16 of 22 Page 17 of 22 Eur. Phys. J. The ATLAS Collaboration C (2012) 72:2056 1University at Albany, Albany NY, United States of America 2 1University at Albany, Albany NY, United States of America 2Department of Physics, University of Alberta, Edmonton AB, Canada b 3(a)Department of Physics, Ankara University, Ankara; (b)Department of Physics, Dumlupinar University, Kutahya; ( ) (d) of Physics, Gazi University, Ankara; (d)Division of Physics, TOBB University of Economics and ( ) c)Department of Physics, Gazi University, Ankara; (d)Division of Physics, TOBB University of Econ Technology, Ankara; (e)Turkish Atomic Energy Authority, Ankara, Turkey Technology, Ankara; (e)Turkish Atomic Energy Authority, Ankara, Turkey 4LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States of America Department of Physics, University of Arizona, Tucson AZ, United States of America 7Department of Physics, The University of Texas at Arlington, Arlington TX, United States of America s Department, University of Athens, Athens, Gree 9Physics Department, National Technical University of Athens, Zografou, Greece 10Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan Azerbaijan Academy of Sciences, Baku, Azerbaij d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona and ICREA, 11Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona and ICREA, B l S i 11Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona and ICREA, Barcelona, Spain Page 18 of 22 Eur. Phys. J. The ATLAS Collaboration C (2012) 72:2056 12(a)Institute of Physics, University of Belgrade, Belgrade; (b)Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 12(a)Institute of Physics, University of Belgrade, Belgrade; (b)Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 13Department for Physics and Technology, University of Bergen, Bergen, Norway 14 14Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 5 14Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 5 15Department of Physics, Humboldt University, Berlin, Germany 16Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 18(a)Department of Physics, Bogazici University, Istanbul; (b)Division of Physics, Dogus University, Istanbul; (c)Department of Physics Engineering, Gaziantep University, Gaziantep; (d)Department of Physics, Istanbul Technical University, Istanbul, Turkey 19(a)INFN Sezione di Bologna; (b)Dipartimento di Fisica, Università di Bologna, Bologna, Italy 20Physikalisches Institut, University of Bonn, Bonn, Germany 21 20Physikalisches Institut, University of Bonn, Bonn, Germany 21 20Physikalisches Institut, University of Bonn, Bonn, Germany 21Department of Physics, Boston University, Boston MA, United States of America 21Department of Physics, Boston University, Boston MA, United States of America 22Department of Physics, Brandeis University, Waltham MA, United States of America 22Department of Physics, Brandeis University, Waltham MA, United States of America 23(a)Universidade Federal do Rio De Janeiro COPPE/EE/IF Rio de Janeiro; (b)Federal University of Juiz de Fora (UFJF) 22Department of Physics, Brandeis University, Waltham MA, United States of America 23(a)Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b)Federal University of Juiz de Fora (UFJF), ( ) (d) 23(a)Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b)Federal University of Juiz de Fora; (c)Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d)Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil sics Department, Brookhaven National Laboratory, Upton NY, United States of America 24Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 25(a)N ti l I tit t f Ph i d N l E i i B h t (b)U i it P lit h i 24Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 25(a)National Institute of Physics and Nuclear Engineering, Bucharest; (b)University Politehnica Bucharest, Bucharest; Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 25(a)National Institute of Physics and Nuclear Engineering, Bucharest; (b)University Politehnica Bu Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 25(a)National Institute of Physics and Nuclear Engineering, Bucharest; (b)University Politehnica Bucharest, Bucharest; ( ) a)National Institute of Physics and Nuclear Engineering, Bucharest; (b)University Politehnica Bucha (c)West University in Timisoara, Timisoara, Romania 26Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 27Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 28Department of Physics, Carleton University, Ottawa ON, Canada 30Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 31(a)D t t d Fi i P tiﬁi U i id d C tóli d Chil S ti (b)D Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 30Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 30Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 31( ) (b) 31(a)Departamento de Fisica, Pontiﬁcia Universidad Católica de Chile, Santiago; (b)Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile p , U v C C , S g ; p , U v Técnica Federico Santa María, Valparaíso, Chile 32( ) (b) 32(a)Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b)Department of Mo ( ) University of Science and Technology of China, Anhui; (c)Department of Physics, Nanjing University, Jiangsu; (d)School of Physics, Shandong University, Shandong, China 33Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Aubiere Cedex, France 34Nevis Laboratory, Columbia University, Irvington NY, United States of America 35 34Nevis Laboratory, Columbia University, Irvington NY, United States of America 35Niels Bohr Institute University of Copenhagen Kobenhavn Denmark 34Nevis Laboratory, Columbia University, Irvington NY, United States of Am 35Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 36(a)INFN Gruppo Collegato di Cosenza; (b)Dipartimento di Fisica, Università della Calabria, Arcavata di Rende, Italy 37AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland 38The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 39Physics Department, Southern Methodist University, Dallas TX, United States of America iewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 38The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow 39 38The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krak 39Physics Department, Southern Methodist University, Dallas TX, United States of America Physics Department, Southern Methodist University, Dallas TX, United States of America 40Physics Department, University of Texas at Dallas, Richardson TX, United States of America 41DESY, Hamburg and Zeuthen, Germany 42Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 43Institut für Kern- und Teilchenphysik, Technical University Dresden, Dresden, Germany 44 44Department of Physics, Duke University, Durham NC, United States of America 45SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 46Fachhochschule Wiener Neustadt, Johannes Gutenbergstrasse 3, 2700 Wiener Neustadt, Austria 51(a)E.Andronikashvili Institute of Physics, Tbilisi State University, Tbilisi; (b)High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia Page 19 of 22 Eur. The ATLAS Collaboration Phys. J. The ATLAS Collaboration C (2012) 72:2056 52II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 53SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 54II Ph ik li h I tit t G A t U i ität Götti G 54II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany 55Laboratoire de Physique Subatomique et de Cosmologie, Université Joseph Fourier and CNRS/IN2P3 and Institut National Polytechnique de Grenoble, Grenoble, France National Polytechnique de Grenoble, Grenoble, France 56Department of Physics, Hampton University, Hampton VA, United States of Amer 56Department of Physics, Hampton University, Hampton VA, United States of America 58(a)Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, 58(a)Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b)Physikalisches Institut, ( ) irchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b)Physikalische recht-Karls-Universität Heidelberg, Heidelberg; (c)ZITI Institut für technische Informatik, ( )Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; ( )Physikali Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c)ZITI Institut für technische Informatik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c)ZITI Institut für technische Informa Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany 59Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 60Department of Physics, Indiana University, Bloomington IN, United States of America 61Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 62University of Iowa, Iowa City IA, United States of America 63Department of Physics and Astronomy, Iowa State University, Ames IA, United States of Americ 63Department of Physics and Astronomy, Iowa State University, A 64Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 65KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 66Graduate School of Science, Kobe University, Kobe, Japan 67Faculty of Science, Kyoto University, Kyoto, Japan 68Kyoto University of Education, Kyoto, Japan 68Kyoto University of Education, Kyoto, Japan 69Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 70 70Physics Department, Lancaster University, Lancaster, United Kingdom 71(a)INFN Sezione di Lecce; (b)Dipartimento di Fisica, Università del Salento, Lecce, Italy 72Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 73Department of Physics, Jožef Stefan Institute and University of Ljubljana, Ljubljana, Slove 75Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 77Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, P France 78Fysiska institutionen, Lunds Universitet, Lund, Sweden 9 78Fysiska institutionen, Lunds Universitet, Lund, Sweden 79 79Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 80Institut für Physik, Universität Mainz, Mainz, Germany 80Institut für Physik, Universität Mainz, Mainz, Germany 81School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 81School of Physics and Astronomy, University of Manchester, Mancheste 82CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 82CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 83Department of Physics, University of Massachusetts, Amherst MA, United States of America 83Department of Physics, University of Massachusetts, Amherst MA, Un 84Department of Physics, McGill University, Montreal QC, Canada 84Department of Physics, McGill University, Montreal QC, Canada 85School of Physics, University of Melbourne, Victoria, Australia 85School of Physics, University of Melbourne, Victoria, Australia 86Department of Physics, The University of Michigan, Ann Arbor MI, United States of America Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States ) (b) 87Department of Physics and Astronomy, Michigan State University, East Lansing MI, United Stat 88(a)INFN Sezione di Milano; (b)Dipartimento di Fisica, Università di Milano, Milano, Italy p y y g y g a)INFN Sezione di Milano; (b)Dipartimento di Fisica, Università di Milano, Milano, Italy 89B.I. The ATLAS Collaboration Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of 90National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Republi 91Department of Physics, Massachusetts Institute of Technology, Cambridge MA, United States of Group of Particle Physics, University of Montreal, Montreal QC, Canada P.N. Lebedev Institute of Physics, Academy of Sciences, Moscow, Russia 94Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 95Moscow Engineering and Physics Institute (MEPhI), Moscow, Russia 97Fakultät für Physik, Ludwig-Maximilians-Universität München, München, G 98 99Nagasaki Institute of Applied Science, Nagasaki, Japan 100Graduate School of Science, Nagoya University, Nagoya, Japan 100Graduate School of Science, Nagoya University, Nagoya, Japan Page 20 of 22 Eur. Phys. J. C (2012) 72:2056 01(a)INFN Sezione di Napoli; (b)Dipartimento di Scienze Fisiche, Università di Napoli, Napoli, Italy p p p p y 102Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States of America 103Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 02Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United Sta Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States of America 103Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands p y y y q q 103Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands ional Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 04Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Neth 105Department of Physics, Northern Illinois University, DeKalb IL, United States of America 105Department of Physics, Northern Illinois University, DeKalb IL, United States of America 106Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 107Department of Physics, New York University, New York NY, United States of America 107Department of Physics, New York University, New York NY, United States of America 109Faculty of Science, Okayama University, Okayama, Japan 110 109Faculty of Science, Okayama University, Okayama, Japan 110Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of America 110Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of America 11Department of Physics, Oklahoma State University, Stillwater OK, United States of America 112Palacký University, RCPTM, Olomouc, Czech Republic 113Center for High Energy Physics University of Oregon Eugene OR United States of America 113Center for High Energy Physics, University of Oregon, Eugene OR, United States of America 114LAL, Univ. The ATLAS Collaboration Paris-Sud and CNRS/IN2P3, Orsay, France 115Graduate School of Science, Osaka University, Osaka, Japan 116Department of Physics, University of Oslo, Oslo, Norway 117Department of Physics, Oxford University, Oxford, United Kingdom 118(a)INFN Sezione di Pavia; (b)Dipartimento di Fisica Nucleare e Teorica, Università di Pavia, Pa INFN Sezione di Pavia; Dipartimento di Fisica Nucleare e Teorica, Università di Pavia, Pavia, 119Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 19Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 120Petersburg Nuclear Physics Institute, Gatchina, Russia 121(a)INFN Sezione di Pisa; (b)Dipartimento di Fisica E. The ATLAS Collaboration Fermi, Università di Pisa, Pisa, Italy 122Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of America 123(a)Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa, Portugal; (b)Departamento de Fisi 22Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of A 23(a) b i d i i i l d i l i b l (b) p y y, y g , g , 123(a)Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa, Portugal; (b)Departamento de Fisica 23(a)Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa, Portugal; (b)D Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada, Spain 124 Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada, Spain 124Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 124Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 125Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 126Czech Technical University in Prague, Praha, Czech Republic 127State Research Center Institute for High Energy Physics, Protvino, Russia 128 128Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom ics Department, Rutherford Appleton Laboratory, 128Particle Physics Department, Rutherford Appleton Laboratory, D 129Physics Department, University of Regina, Regina SK, Canada 129Physics Department, University of Regina, Regina SK, Canada 30Ritsumeikan University, Kusatsu, Shiga, Japan 131(a)INFN Sezione di Roma I; (b)Dipartimento di Fisica, Università La Sapienza, Roma, Italy 132( ) (b) 132(a)INFN Sezione di Roma Tor Vergata; (b)Dipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 133( ) (b) 133(a)INFN Sezione di Roma Tre; (b)Dipartimento di Fisica, Università Roma Tre, Roma, Italy 134(a)Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, Casablanca; (b)Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c)Faculté des Sciences S l li U i ité C di A d LPHEA M k h (d)F lté d S i U i ité M h d P i d 134(a)Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - U Casablanca; (b)Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c)Fa 134(a)Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, Casablanca; (b)Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c)Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d)Faculté des Sciences, Université Mohamed Premier and LPTPM Oujda; (e)Faculté des Sciences Université Mohammed V Agdal Rabat Morocco des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan a; (b)Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c)Faculté des Science , y q g , )Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c)Faculté des Sciences Université Cadi Ayyad, LPHEA-Marrakech; (d)Faculté des Sciences, Université Mohamed Premier a ( ) Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d)Faculté des Sciences, Université Mo LPTPM, Oujda; (e)Faculté des Sciences, Université Mohammed V- Agdal, Rabat, Morocco 135DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat a l’Energie Atomique), Gif-sur-Yvette, France 136Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, 13 stitute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, United States of Am 137Department of Physics, University of Washington, Seattle WA, United States of America 138 37Department of Physics, University of Washington, Seattle WA, United States of America 38 38Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, United Kingdom 138Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, United Kingd 139Department of Physics, Shinshu University, Nagano, Japan 139Department of Physics, Shinshu University, Nagano, Japan 140Fachbereich Physik, Universität Siegen, Siegen, Germany 140Fachbereich Physik, Universität Siegen, Siegen, Germany 41Department of Physics, Simon Fraser University, Burnaby BC, Canada 141Department of Physics, Simon Fraser University, Burnaby BC, Canada 142SLAC National Accelerator Laboratory, Stanford CA, United States of America 142SLAC National Accelerator Laboratory, Stanford CA, United States of America 143(a)Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b)Department of Subnuclear 143(a)Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b)Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 144(a)Department of Physics, University of Johannesburg, Johannesburg; (b)School of Physics, University of the Wi d J h b S h Af i y y y p Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 144(a)Department of Physics, University of Johannesburg, Johannesburg; (b)School of Physics, University of the Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 144(a)Department of Physics, University of Johannesburg, Johannesburg; (b)School of Physics, University of the Witwatersrand, Johannesburg, South Africa Witwatersrand, Johannesburg, South Africa Page 21 of 22 Eur. The ATLAS Collaboration Phys. J. C (2012) 72:2056 145(a)Department of Physics, Stockholm University; (b)The Oskar Klein Centre, Stockholm, Swed 146Physics Department, Royal Institute of Technology, Stockholm, Sweden 46Physics Department, Royal Institute of Technology, Stockholm, Sweden y p y gy 147Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 147Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 147Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 48Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 148Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 149School of Physics, University of Sydney, Sydney, Australia 150Institute of Physics, Academia Sinica, Taipei, Taiwan 151Department of Physics, Technion: Israel Inst. of Technology, Haifa 151Department of Physics, Technion: Israel Inst. The ATLAS Collaboration of Technology, Haifa, Israel Raymond and Beverly Sackler School of Physics a y y y y 153Department of Physics, Aristotle University of Thessaloniki, Thessalonik 153Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 154International Center for Elementary Particle Physics and Department of Physics, The Un 155Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 155Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 156Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 157Department of Physics, University of Toronto, Toronto ON, Canada 158(a)TRIUMF, Vancouver BC; (b)Department of Physics and Astronomy, York University, Toronto ON, Canada 157Department of Physics, University of Toronto, Toronto ON, Canada 158(a)TRIUMF, Vancouver BC; (b)Department of Physics and Astronomy, York University, Toron p y y y 159Institute of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan 159Institute of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibar 160Science and Technology Center, Tufts University, Medford MA, United States of America 161Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 162 161Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia Department of Physics and Astronomy, University of California Irvine, Irvine CA, United States of A 162Department of Physics and Astronomy, University of California Irvine, Irvine 163(a)INFN Gruppo Collegato di Udine, Udine; (b)ICTP, Trieste; (c)Dipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy 163(a)INFN Gruppo Collegato di Udine, Udine; (b)ICTP, Trieste; (c)Dipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy artment of Physics, University of Illinois, Urbana IL, United States of America 164Department of Physics, University of Illinois, Urbana IL, United States of America 165 165Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 166 artment of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 165Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 166 166Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 166Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 67Department of Physics, University of British Columbia, Vancouver BC, Canada 167Department of Physics, University of British Columbia, Vancouver BC, Canada 68Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 69 168Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 169Waseda University, Tokyo, Japan 171Department of Physics, University of Wisconsin, Madison WI, United States of America 171Department of Physics, University of Wisconsin, Madison WI, United States of America 72Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 172Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 173Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 173Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 74Department of Physics, Yale University, New Haven CT, United States of America 5 174Department of Physics, Yale University, New Haven CT, United States of Ameri 175Yerevan Physics Institute, Yerevan, Armenia 176Domaine scientiﬁque de la Doua, Centre de Calcul CNRS/IN2P3, Villeurbanne Cedex, France 176Domaine scientiﬁque de la Doua, Centre de Calcul CNRS/IN2 177Faculty of Science, Hiroshima University, Hiroshima, Japan 177Faculty of Science, Hiroshima University, Hiroshima, Japan aAlso at Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa, Portuga bAlso at Faculdade de Ciencias and CFNUL, Universidade de Lisboa, Lisboa, Portugal cAlso at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom dAlso at TRIUMF, Vancouver BC, Canada dAlso at TRIUMF, Vancouver BC, Canada eAlso at Department of Physics, California State University, Fresno CA, United States of America eAlso at Department of Physics, California fAlso at Novosibirsk State University, Novosibirsk, Russia gAlso at Fermilab, Batavia IL, United States of America gAlso at Fermilab, Batavia IL, United States of America hAlso at Department of Physics, University of Coimbra, Coimbra, Portugal iAlso at Università di Napoli Parthenope, Napoli, Italy iAlso at Università di Napoli Parthenope, Napoli, Italy jAlso at Institute of Particle Physics (IPP), Canada jAlso at Institute of Particle Physics (IPP), Canada kAlso at Department of Physics, Middle East Technical University, Ankara, Turkey kAlso at Department of Physics, Middle East Technical University, Ankara, Turke ech University, Ruston LA, United States of Amer mAlso at Department of Physics and Astronomy, University College London, London, United K mAlso at Department of Physics and Astronomy, University College London, London, United Kingdom nAlso at Group of Particle Physics, University of Montreal, Montreal QC, Canada oAlso at Department of Physics, University of Cape Town, Cape Town, South Afri pAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan Eur. The ATLAS Collaboration Paris-Sud and CNRS/IN2P3, Orsay, France aeAlso at Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, United Kingdom aeAlso at Department of Physics and Astronomy, University of She afAlso at Department of Physics, Oxford University, Oxford, United Kingdom agAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan agAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan ahAlso at Department of Physics, The University of Michigan, Ann Arbor MI, United States of America i Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and aiAlso at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Di aiAlso at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France * The ATLAS Collaboration Paris-Sud and CNRS/IN2P3, Orsay, France aeAlso at Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, United Kingdom afAlso at Department of Physics, Oxford University, Oxford, United Kingdom agAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan ahAlso at Department of Physics, The University of Michigan, Ann Arbor MI, United States of America aiAlso at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France Deceased qAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany qAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany rAlso at Manhattan College, New York NY, United States of America rAlso at Manhattan College, New York NY, United States of America sAlso at School of Physics, Shandong University, Shandong, China sAlso at School of Physics, Shandong University, Shandong, China tAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France uAlso at School of Physics and Engineering, Sun Yat-sen University, Guanzhou, China vAlso at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan vAlso at Academia Sinica Grid Computing, Institute of Physics, Aca Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan wAlso at DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Sacla l’Energie Atomique), Gif-sur-Yvette, France l’Energie Atomique), Gif-sur-Yvette, France xAlso at Section de Physique, Université de Genève, Geneva, Switzerland xAlso at Section de Physique, Université de Genève, Geneva, Switzerland yAlso at Departamento de Fisica, Universidade de Minho, Braga, Portugal yAlso at Departamento de Fisica, Universidade de Minho, Braga, Portugal yAlso at Departamento de Fisica, Universidade de Minho, Braga, Portugal zAlso at Department of Physics and Astronomy, University of South Carolina, Columbia SC, Unit p y y y aaAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary aaAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budap abAlso at California Institute of Technology, Pasadena CA, United States of America acAlso at Institute of Physics, Jagiellonian University, Krakow, Poland acAlso at Institute of Physics, Jagiellonian University, Krakow, Poland adAlso at LAL, Univ. Paris-Sud and CNRS/IN2P3, Orsay, France adAlso at LAL, Univ. The ATLAS Collaboration Phys. J. C (2012) 72:2056 Page 22 of 22 qAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany rAlso at Manhattan College, New York NY, United States of America sAlso at School of Physics, Shandong University, Shandong, China tAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France uAlso at School of Physics and Engineering, Sun Yat-sen University, Guanzhou, China vAlso at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan wAlso at DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat a l’Energie Atomique), Gif-sur-Yvette, France xAlso at Section de Physique, Université de Genève, Geneva, Switzerland yAlso at Departamento de Fisica, Universidade de Minho, Braga, Portugal zAlso at Department of Physics and Astronomy, University of South Carolina, Columbia SC, United States of America aaAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary abAlso at California Institute of Technology, Pasadena CA, United States of America acAlso at Institute of Physics, Jagiellonian University, Krakow, Poland adAlso at LAL, Univ. CNRS/IN2P3, Paris, France Deceased *Deceased
https://openalex.org/W4287178616	https://zenodo.org/records/4777677/files/Abe%20Leser.pdf	English	null	Abe Leser: What are Trusts and How to Use Them in Real Estate?	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	382	What are trusts and how to use them in real estate? What are trusts and how to use them in real estate? Knowing what trusts are and how to use them in real estate can be an alternative to ensure the achievement of specific objectives in the future. It is a contract in which all kinds of assets can be used for a legal and specific purpose for their own benefit or that of a third party within a specified period. Here, real estate executive Abe Leser shars all the details about this concept, keep reading! Parties that make up a trust Parties that make up a trust There are three figures that participate in a trust: Settlor. It is the natural or legal person who assigns the property or ownership of one or more of its assets and / or rights for a specific purpose. It is also who decrees the mode of operation of said patrimony and the designation of one or more beneficiaries. Trust. In general, Abe Leser shares that it is a financial institution, which is responsible for the administration of the entrusted asset, as well as carrying out the pertinent actions to achieve the stated objective. It will have the rights and responsibilities to carry out its task, as well as the limitations or regulations established by the settlor when the trust is constituted. According to Abe Leser, credit institutions, investment fund operating companies, credit unions and multi-purpose financial companies can perform this function in exchange for an administration fee. Trustee. He is the one who receives the benefits derived from the trust, as long as the conditions established in the contract have been met. Unlike an inheritance, the beneficiary can be the same settlor. What are trusts and how to use them in real estate? What are trusts and how to use them in real estate? According to Abe Leser, CEO of The Leser Group, a real estate investment trust is one in which a property is transferred. Its particularity is that it is put in the hands of the trustee so that he is in charge of generating interest, renting or selling the real estate to reach the agreed objective. The beneficiary will receive the result the work when the requirements set out are met.
https://openalex.org/W2140434685	https://europepmc.org/articles/pmc1906746?pdf=render	English	null	Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification	Virology journal	2,007	cc-by	13,057	BioMed Central BioMed Central BioMed Central Received: 23 March 2007 Accepted: 7 June 2007 Virology Journal 2007, 4:56 doi:10.1186/1743-422X-4-56 This article is available from: http://www.virologyj.com/content/4/1/56 © 2007 Pinnoji et al; licensee BioMed Central Ltd. j 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. Virology Journal Open Access Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification Rajeswara C Pinnoji1, Gautam R Bedadala1, Beena George1, Thomas C Holland2, James M Hill3 and Shao-chung V Hsia1 Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification Rajeswara C Pinnoji1, Gautam R Bedadala1, Beena George1, Thomas C Holland2, James M Hill3 and Shao-chung V Hsia1 Address: 1Department of Basic Pharmaceutical Sciences, College of Pharmacy, The University of Louisiana at Monroe, 700 University Avenue, Monroe, LA 71209 USA, 2Department of Immunology and Microbiology, School of Medicine, Wayne State University, 540 East Canfield Avenue, Detroit, MI 48201 USA and 3Department of Ophthalmology, Neuroscience, Pharmacology, and Microbiology LSU Eye Center and LSU Health Sciences Center, New Orleans, LA 70118 USA C Pinnoji - prajeshwarachary@yahoo.com; Gautam R Bedadala - Gautam_744@yahoo.com; Email: Rajeswara C Pinnoji - prajeshwarachary@yahoo.com; Gautam R Bedadala - Gautam_744@yahoo.com; Beena George - beenaq79@yahoo.com; Thomas C Holland - thomas.holland@wayne.edu; James M Hill - jhill@lsuhsc.edu; Shao- chung V Hsia* - hsia@ulm.edu * C di th Email: Rajeswara C Pinnoji prajeshwarachary@yahoo.com; Gautam R Bedadala Gautam_744@yahoo.com; Beena George - beenaq79@yahoo.com; Thomas C Holland - thomas.holland@wayne.edu; James M Hill - jhill@lsuhsc.edu; Shao- chung V Hsia* - hsia@ulm.edu eenaq79@yahoo.com; Thomas C Holland - thomas.holland@wayne.edu; James M Hill - jhill@lsuhsc.edu; Shao- hsia@ulm.edu * Corresponding author Received: 23 March 2007 Accepted: 7 June 2007 Published: 7 June 2007 Virology Journal 2007, 4:56 doi:10.1186/1743-422X-4-56 Received: 23 March 2007 Accepted: 7 June 2007 A putative RE-1/NRSE was found within the promoter of HSV-1 ICP22 We identified a putative HSV-1 RE-1/NRSE having 76% identity to the published consensus sequence [14] in the intergenic region between the ICP4 and ICP22 coding sequences (Fig. 1A). The location of various cis-acting ele- ments was shown according to literature [15]. The HSV-1 RE-1 core sequence exhibited 100% identity to the con- sensus RE-1/NRSE. This HSV-1 RE-1/NRSE is located immediately downstream of the TATA box of the ICP22 promoter and 660 bp upstream of the ICP4 transcription initiation site (Fig. 1B). We hypothesize that a repressive chromatin structure and specific neuronal transcription factors contribute to tran- scription inhibition during latency. We identified a Restrictive Element-1/Neuronal Restrictive Silencer Ele- ment (RE-1/NRSE) located between the promoters for the Immediate-Early ICP4 and ICP22 genes. RE-1/NRSE is the binding site of RE1-Silencing Transcription factor/Neuro- nal Restrictive Silencer Factor (REST/NRSF) [8]. REST/ NRSF is a zinc finger transcription factor originally defined as a silencer protein for the neuron-specific gene SCG10 [9]. Recent studies revealed that REST/NRSF exhib- its ubiquitous presence [10] and plays roles in neurogen- esis, neural plasticity, tumor suppression, and cancer progression through transcription regulation [11]. REST/ NRSF and its corepressor complex CoREST have not been linked to HSV-1 biology until recent studies showing that the HSV-1 IE protein ICP0 dissociates HDAC 1 and 2 from the REST/CoREST complex [12]. However, the putative role of REST/NRSF on HSV-1 transcription has not been elucidated. Background context. The histone deacetylase inhibitor trichostatin A was sufficient to reverse the inhibition of ICP4 in stable cells, indicating the role of histone deacetylation in REST/ NRSF mediated regulation in this system. ChIP assays revealed that CoREST was recruited to the proximity of HSV-1 RE-1/NRSE and that histone H4 acetylation was reduced in the presence of REST/NRSF. These results dem- onstrate the roles of REST/NRSF in the regulation of HSV- 1 IE transcription. ac g ou d Lytic infection by Herpes Simplex Virus Type-1 (HSV-1) typically occurs in epithelial cells [1]. During these infec- tions, HSV-1 expresses more than eighty genes in a sequential regulatory cascade [2]. Immediate-early (IE or α) gene products are the first group to be transcribed fol- lowed by early (E or β) and late (L or γ) gene expressions [3]. Expression of E and L genes depends on the availabil- ity of IE proteins, thus demonstrating their importance in the lytic cycle. During lytic infection, viral DNA is not associated with nucleosomes. HSV-1, like other alphavi- ruses, also establishes latent infections in sensory neurons of the peripheral nervous system [4,5]. In contrast to lytic infection, latency is distinguished by the absence of viral polypeptides and a highly restricted pattern of transcrip- tion [2,6]. Studies of HSV-1 latency in animal models have indicated that the majority of viral DNA is main- tained in a circular form and associated with nucleosomes in a regularly spaced chromatin pattern [7]. However, detailed studies of latent viral chromatin have been diffi- cult to conduct and the role of chromatin in viral latency remains to be defined. Abstract Background: During primary infection of its human host, Herpes Simplex Virus Type-1 (HSV-1) establishes latency in neurons where the viral genome is maintained in a circular form associated with nucleosomes in a chromatin configration. During latency, most viral genes are silenced, although the molecular mechanisms responsible for this are unclear. We hypothesized that neuronal factors repress HSV-1 gene expression during latency. A search of the HSV-1 DNA sequence for potential regulatory elements identified a Repressor Element-1/Neuronal Restrictive Silencer Element (RE-1/NRSE) located between HSV-1 genes ICP22 and ICP4. We predicted that the Repressor Element Silencing Transcription Factor/Neuronal Restrictive Silencer Factor (REST/ NRSF) regulates expression of ICP22 and ICP4. Results: Transient cotransfection indicated that REST/NRSF inhibited the activity of both promoters. In contrast, cotransfection of a mutant form of REST/NRSF encoding only the DNA- binding domain of the protein resulted in less inhibition. Stably transformed cell lines containing episomal reporter plasmids with a chromatin structure showed that REST/NRSF specifically inhibited the ICP4 promoter, but not the ICP22 promoter. REST/NRSF inhibition of the ICP4 promoter was reversed by histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Additionally, chromatin immuno-precipitation (ChIP) assays indicated that the corepressor CoREST was recruited to the proximity of ICP4 promoter and that acetylation of histone H4 was reduced in the presence of REST/NRSF. Conclusion: Since the ICP4 protein is a key transactivator of HSV-1 lytic cycle genes, these results suggest that REST/NRSF may have an important role in the establishment and/or maintenance of HSV-1 gene silencing during latency by targeting ICP4 expression. Page 1 of 11 (page number not for citation purposes) (page number not for citation purposes) Virology Journal 2007, 4:56 http://www.virologyj.com/content/4/1/56 REST/NRSF repressed ICP22 and ICP4 promoter activity in transient co-transfection 0 20 40 60 80 100% Normalized relative SEAP Activity 24.3 * 12.6 * 69.3 80.6 1 2 3 4 5 100 p< 0.05 Actin cDNA ICP4 cDNA pSG28 pH4-2 1 2 3 4 5 6 LAT cDNA ICP0 cDNA 100 2.5 1.2 * 66.0 83.5 0 20 40 60 80 100% Normalized relative SEAP Activity 1 2 3 4 5 p< 0.05 Control pFLAG-REST pCMVp73 Control pFLAG-REST pCMVp73 RT-PCR arbitrary Unit pH4-2 pSG28 ICP4 expression LAT expression ICP0 expression cotransfected with pFLAG-REST or pCMVp73 to analyze the effect of HSV-1 RE-1/NRSE on the gene regulation of ICP4. It is noted that both pSG28 and pH4-2 contain the complete open reading frame of ICP4, LAT, and ICP0. The RT-PCR results indicated that REST/NRSF and mutant p73 exhibited no major effect on the ICP4 promoter of pSG28, which does not have HSV-1 RE-1/NRSE (Fig. 2C). How- ever, the ICP4 promoter of pH4-2 (containing HSV-1 RE- 1/NRSE) was significantly repressed by REST/NRSF but not p73 which showed no inhibition at all (Lane 4 5 and REST/NRSF inhibits ICP22 and ICP4 promoter activity in transient cotransfection Figure 2 REST/NRSF inhibits ICP22 and ICP4 promoter activ- ity in transient cotransfection. A. Cotransfection of pICP22 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP22 promoter. 1. pICP22 and con- trol plasmid. 2. pICP22 and pFLAG-REST (1:1). 3. pICP22 and pFLAG-REST (1:2). 4. pICP22 and pCMVp73 (1:1). 5. pICP22 and pCMVp73 (1:2). The asterisk P values represent Stu- dent's t tests in pairwise comparisons to the Lane 1 pICP22 + Control plasmid. The error bars represent standard devia- tions. The data were calculated and graphed using Microsoft Excel. B. Cotransfection of pICP4 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP4 pro- moter. 1. pICP4 and control plasmid. 2. pICP4 and pFLAG- REST (1:1). 3. pICP4 and pFLAG-REST (1:2). 4. pICP4 and pCMVp73 (1:1). 5. pICP4 and pCMVp73 (1:2). The P values represent Student's t tests in pairwise comparisons to the Lane 1 pICP4 + Control plasmid. The error bars represent standard deviations. The data were calculated and graphed using Microsoft Excel. C. Plasmid pSG28 and pH4-2 were cotransfected with pFLAG-REST or pCMVp73 followed by RNA isolation and RT-PCR. 1. pSG28 and control plasmid (1:1). 2. pSG28 and pFLAG-REST (1:1). 3. REST/NRSF repressed ICP22 and ICP4 promoter activity in transient co-transfection TRL UL IRL IRS US TRS ICP4 ICP22 HSV-1 OriS HSV-1 RE-1/NRSE (132082-132103) ICP22 TATA Box 100 200 300 400 500 600 700 800 900 1000 1100 1200 ICP4 Transcript ICP22 Transcript ICP4 TATA Box TATA box Sp1 site TAATGARAT CCCGTTGG GCGGAA Oct-1/NF-III ICP22 TATA REST/NRSF inhibits ICP22 and ICP4 promoter activity in transient cotransfection Figure 2 REST/NRSF inhibits ICP22 and ICP4 promoter activ- ity in transient cotransfection. A. Cotransfection of pICP22 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP22 promoter. 1. pICP22 and con- trol plasmid. 2. pICP22 and pFLAG-REST (1:1). 3. pICP22 and pFLAG-REST (1:2). 4. pICP22 and pCMVp73 (1:1). 5. pICP22 and pCMVp73 (1:2). The asterisk P values represent Stu- dent's t tests in pairwise comparisons to the Lane 1 pICP22 + Control plasmid. The error bars represent standard devia- tions. The data were calculated and graphed using Microsoft Excel. B. Cotransfection of pICP4 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP4 pro- moter. 1. pICP4 and control plasmid. 2. pICP4 and pFLAG- REST (1:1). 3. pICP4 and pFLAG-REST (1:2). 4. pICP4 and pCMVp73 (1:1). 5. pICP4 and pCMVp73 (1:2). The P values represent Student's t tests in pairwise comparisons to the Lane 1 pICP4 + Control plasmid. The error bars represent standard deviations. The data were calculated and graphed using Microsoft Excel. C. Plasmid pSG28 and pH4-2 were cotransfected with pFLAG-REST or pCMVp73 followed by RNA isolation and RT-PCR. 1. pSG28 and control plasmid (1:1). 2. pSG28 and pFLAG-REST (1:1). 3. pSG28 and pCMVp73 (1:1). 4. pH4-2 and control plasmid (1:1). 5. pH4-2 and pFLAG-REST (1:1). 6. pH4-2 and pCMVp73 (1:1). D. The RT-PCR results were quantified by Kodak Gel-Logic 100 sys- tem to measure the sum intensity of each band representing the regulatory effect of REST/NRSF on ICP4 transcription. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1 2 3 4 5 6 A. B. C. D. REST/NRSF repressed ICP22 and ICP4 promoter activity in transient co-transfection HSV-1 genome and HSV-1 RE-1/NRSE sequence Figure 1 HSV-1 genome and HSV-1 RE-1/NRSE sequence. A. HSV-1 genome is composed of two covalently linked compo- nents, designated as UL (Unique Long) or US (Unique Short). Each component contains unique sequences bracketed by inverted and terminal repeats (TRL and IRL). The ICP22 gene is present in US and one of the two ICP4 is located at the junction of US and IRS since the genes that are encoded within the repeat sequences are present twice in the viral genome. The HSV-1 RE-1/NRSE is mapped from 132082 to 132103 according to the HSV-1 complete genome sequence accession number X14112. B. Putative HSV-1 RE-1/NRSE. The HSV-1 RE-1/NRSE sequence was identified to overlap ICP22 TATA box (Bold) compared to consensus sequence. The core sequence is underlined. The matching result indi- cated that the homology is 76%. W: A or T; N: any nucle- otide; S: G or C; Y: C or T; R: A or G. HSV-1 genome and HSV-1 RE-1/NRSE sequence Figure 1 HSV-1 genome and HSV-1 RE-1/NRSE sequence. A. HSV-1 genome is composed of two covalently linked compo- nents, designated as UL (Unique Long) or US (Unique Short). Each component contains unique sequences bracketed by inverted and terminal repeats (TRL and IRL). The ICP22 gene is present in US and one of the two ICP4 is located at the junction of US and IRS since the genes that are encoded within the repeat sequences are present twice in the viral genome. The HSV-1 RE-1/NRSE is mapped from 132082 to 132103 according to the HSV-1 complete genome sequence accession number X14112. B. Putative HSV-1 RE-1/NRSE. The HSV-1 RE-1/NRSE sequence was identified to overlap ICP22 TATA box (Bold) compared to consensus sequence. The core sequence is underlined. The matching result indi- cated that the homology is 76%. W: A or T; N: any nucle- otide; S: G or C; Y: C or T; R: A or G. cotransfected with pFLAG-REST or pCMVp73 to analyze the effect of HSV-1 RE-1/NRSE on the gene regulation of ICP4. It is noted that both pSG28 and pH4-2 contain the complete open reading frame of ICP4, LAT, and ICP0. The RT-PCR results indicated that REST/NRSF and mutant p73 exhibited no major effect on the ICP4 promoter of pSG28, which does not have HSV-1 RE-1/NRSE (Fig. 2C). REST/NRSF repressed ICP22 and ICP4 promoter activity in transient co-transfection 4. pICP4 and pCMVp73 (1:1). 5. pICP4 and pCMVp73 (1:2). The P values represent Student's t tests in pairwise comparisons to the Lane 1 pICP4 + Control plasmid. The error bars represent standard deviations. The data were calculated and graphed using Microsoft Excel. C. Plasmid pSG28 and pH4-2 were cotransfected with pFLAG-REST or pCMVp73 followed by RNA isolation and RT-PCR. 1. pSG28 and control plasmid (1:1). 2. pSG28 and pFLAG-REST (1:1). 3. pSG28 and pCMVp73 (1:1). 4. pH4-2 and control plasmid (1:1). 5. pH4-2 and pFLAG-REST (1:1). 6. pH4-2 and pCMVp73 (1:1). D. The RT-PCR results were quantified by Kodak Gel-Logic 100 sys- tem to measure the sum intensity of each band representing the regulatory effect of REST/NRSF on ICP4 transcription. REST/NRSF inhibits ICP22 and ICP4 promoter activity in transient cotransfection Figure 2 REST/NRSF inhibits ICP22 and ICP4 promoter activ- ity in transient cotransfection. A. Cotransfection of pICP22 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP22 promoter. 1. pICP22 and con- trol plasmid. 2. pICP22 and pFLAG-REST (1:1). 3. pICP22 and pFLAG-REST (1:2). 4. pICP22 and pCMVp73 (1:1). 5. pICP22 and pCMVp73 (1:2). The asterisk P values represent Stu- dent's t tests in pairwise comparisons to the Lane 1 pICP22 + Control plasmid. The error bars represent standard devia- tions. The data were calculated and graphed using Microsoft Excel. B. Cotransfection of pICP4 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP4 pro- moter. 1. pICP4 and control plasmid. 2. pICP4 and pFLAG- REST (1:1). 3. pICP4 and pFLAG-REST (1:2). 4. pICP4 and pCMVp73 (1:1). 5. pICP4 and pCMVp73 (1:2). The P values represent Student's t tests in pairwise comparisons to the Lane 1 pICP4 + Control plasmid. The error bars represent standard deviations. The data were calculated and graphed using Microsoft Excel. C. Plasmid pSG28 and pH4-2 were cotransfected with pFLAG-REST or pCMVp73 followed by RNA isolation and RT-PCR. 1. pSG28 and control plasmid (1:1). 2. pSG28 and pFLAG-REST (1:1). 3. pSG28 and pCMVp73 (1:1). 4. pH4-2 and control plasmid (1:1). 5. pH4-2 and pFLAG-REST (1:1). 6. pH4-2 and pCMVp73 (1:1). D. The RT-PCR results were quantified by Kodak Gel-Logic 100 sys- tem to measure the sum intensity of each band representing the regulatory effect of REST/NRSF on ICP4 transcription. REST/NRSF repressed ICP22 and ICP4 promoter activity in transient co-transfection TRL UL IRL IRS US TRS ICP4 ICP22 HSV-1 OriS HSV-1 RE-1/NRSE (132082-132103) ICP22 TATA Box 100 200 300 400 500 600 700 800 900 1000 1100 1200 ICP4 Transcript ICP22 Transcript ICP4 TATA Box TATA box Sp1 site TAATGARAT CCCGTTGG GCGGAA Oct-1/NF-III ICP22 TATA A. B. 0 20 40 60 80 100% Normalized relative SEAP Activity 24.3 12.6 * 69.3 80.6 1 2 3 4 5 100 p< 0.05 100 2.5 1.2 * 66.0 83.5 0 20 40 60 80 100% Normalized relative SEAP Activity 1 2 3 4 5 p< 0.05 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1 2 3 4 5 6 A. B. C. D. 0 20 40 60 80 100% Normalized relative SEAP Activity 24.3 12.6 * 69.3 80.6 1 2 3 4 5 100 p< 0.05 Actin cDNA ICP4 cDNA pSG28 pH4-2 1 2 3 4 5 6 LAT cDNA ICP0 cDNA 100 2.5 1.2 * 66.0 83.5 0 20 40 60 80 100% Normalized relative SEAP Activity 1 2 3 4 5 p< 0.05 Control pFLAG-REST pCMVp73 Control pFLAG-REST pCMVp73 RT-PCR arbitrary Unit pH4-2 pSG28 ICP4 expression LAT expression ICP0 expression C. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1 2 3 4 5 6 C. D. Actin cDNA ICP4 cDNA pSG28 pH4-2 1 2 3 4 5 6 LAT cDNA ICP0 cDNA Control pFLAG-REST pCMVp73 Control pFLAG-REST pCMVp73 RT-PCR arbitrary Unit pH4-2 pSG28 ICP4 expression LAT expression ICP0 expression D. B. REST/NRSF inhibits ICP22 and ICP4 promoter activity in transient cotransfection Figure 2 REST/NRSF inhibits ICP22 and ICP4 promoter activ- ity in transient cotransfection. A. Cotransfection of pICP22 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP22 promoter. 1. pICP22 and con- trol plasmid. 2. pICP22 and pFLAG-REST (1:1). 3. pICP22 and pFLAG-REST (1:2). 4. pICP22 and pCMVp73 (1:1). 5. pICP22 and pCMVp73 (1:2). The asterisk P values represent Stu- dent's t tests in pairwise comparisons to the Lane 1 pICP22 + Control plasmid. The error bars represent standard devia- tions. The data were calculated and graphed using Microsoft Excel. B. Cotransfection of pICP4 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP4 pro- moter. 1. pICP4 and control plasmid. 2. pICP4 and pFLAG- REST (1:1). 3. pICP4 and pFLAG-REST (1:2). REST/NRSF repressed ICP22 and ICP4 promoter activity in transient co-transfection f The regulatory effect of REST/NRSF on the ICP22 pro- moter was first measured by transient transfection assays. We performed cotransfection of pICP22 (containing the SEAP reporter gene under the control of the ICP22 pro- moter) and pFLAG-REST into 293HEK cells at the molar ratio of 1:1 or 1:2 (pICP22: pFLAG-REST). SEAP assays were performed three days post-transfection according to the manufacturer's protocols. The results indicated that the REST/NRSF repressed the ICP22 promoter activity to 23% and 9% of control levels at the ratio of 1:1 and 1:2, respectively (Fig. 2A). Similar co-transfections were done with pCMVp73, which expresses a truncated form of REST/NRSF containing only the protein's DNA binding domain. The ICP22 promoter retained 70–80% of its activity in the presence of REST/NRSF mutant p73 (Fig. 2A). Empty vector pREP-SEAP transfection was performed and exhibited very low basal activity (data not shown). We investigated the effect of REST/NRSF on HSV-1 IE tran- scription using an episomally replicating plasmid that associates with nucleosomes in a standard chromatin con- figuration in stably transfected cell lines [13]. Plasmids containing the secreted alkaline phosphatase (SEAP) reporter gene driven by either ICP4 or ICP22 promoter were characterized in transient transfections and in stably transformed cells. In transient transfections, REST/NRSF repressed the activity of both the ICP22 and ICP4 promot- ers. However, in stably transfected cells, REST/NRSF exhibited significant inhibition of the ICP4 promoter but only moderate reduction on ICP22 activity in chromatin The regulatory effect of REST/NRSF on the ICP4 promoter was investigated by the same strategy. Cotransfection of pICP4 and pFLAG-REST at the molar ratio of 1:1 or 1:2 (pICP4: pFLAG-REST) revealed that REST/NRSF inhibited the ICP4 promoter activity to 2.5% and 1.2%, respec- tively, (Fig. 2B). The mutant vector pCMVp73 exerted only a weak repressive effect on the promoter (Fig. 2B). These results indicated that REST/NRSF inhibited ICP22 and ICP4 promoter activity in transient transfections. To further confirm the regulatory effect of REST/NRSF on HSV-1 transcription, plasmid pSG28 and pH4-2 were Page 2 of 11 (page number not for citation purposes) Page 2 of 11 (page number not for citation purposes) http://www.virologyj.com/content/4/1/56 Virology Journal 2007, 4:56 HSV-1 RE-1 5’-TTATGTGCGCCGGAGAGACCC-3’ Consensus RE-1 5’-NNCAGCACCNCGGASAGNNNC-3’ * * * ************ A. B. REST/NRSF repressed ICP22 and ICP4 promoter activity in transient co-transfection pSG28 and pCMVp73 (1:1). 4. pH4-2 and control plasmid (1:1). 5. pH4-2 and pFLAG-REST (1:1). 6. pH4-2 and pCMVp73 (1:1). D. The RT-PCR results were quantified by Kodak Gel-Logic 100 sys- tem to measure the sum intensity of each band representing the regulatory effect of REST/NRSF on ICP4 transcription. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1 2 3 4 5 6 A. B. C. D. 0 20 40 60 80 100% Normalized relative SEAP Activity 24.3 * 12.6 * 69.3 80.6 1 2 3 4 5 100 p< 0.05 Actin cDNA ICP4 cDNA pSG28 pH4-2 1 2 3 4 5 6 LAT cDNA ICP0 cDNA 100 2.5 1.2 * 66.0 83.5 0 20 40 60 80 100% Normalized relative SEAP Activity 1 2 3 4 5 p< 0.05 Control pFLAG-REST pCMVp73 Control pFLAG-REST pCMVp73 RT-PCR arbitrary Unit pH4-2 pSG28 ICP4 expression LAT expression ICP0 expression HSV-1 genome and HSV-1 RE-1/NRSE sequence Figure 1 HSV-1 genome and HSV-1 RE-1/NRSE sequence. A. HSV-1 genome is composed of two covalently linked compo- nents, designated as UL (Unique Long) or US (Unique Short). Each component contains unique sequences bracketed by inverted and terminal repeats (TRL and IRL). The ICP22 gene is present in US and one of the two ICP4 is located at the junction of US and IRS since the genes that are encoded within the repeat sequences are present twice in the viral genome. The HSV-1 RE-1/NRSE is mapped from 132082 to 132103 according to the HSV-1 complete genome sequence accession number X14112. B. Putative HSV-1 RE-1/NRSE. The HSV-1 RE-1/NRSE sequence was identified to overlap ICP22 TATA box (Bold) compared to consensus sequence. The core sequence is underlined. The matching result indi- cated that the homology is 76%. W: A or T; N: any nucle- otide; S: G or C; Y: C or T; R: A or G. HSV-1 RE-1 5’-TTATGTGCGCCGGAGAGACCC-3’ Consensus RE-1 5’-NNCAGCACCNCGGASAGNNNC-3’ * * * ************ A. B. TRL UL IRL IRS US TRS ICP4 ICP22 HSV-1 OriS HSV-1 RE-1/NRSE (132082-132103) ICP22 TATA Box 100 200 300 400 500 600 700 800 900 1000 1100 1200 ICP4 Transcript ICP22 Transcript ICP4 TATA Box TATA box Sp1 site TAATGARAT CCCGTTGG GCGGAA Oct-1/NF-III ICP22 TATA A. REST/NRSF repressed ICP22 and ICP4 promoter activity in transient co-transfection Lane 7–12: plasmid-specific nucleosmal protected ladder detected by vector probe. Lane 13 and 14: Naked plasmid digestion control. B. Analysis of nucleosomal formation on episomal plasmids. The nuclei from stable 293HEK-pICP22 were subjected to MNase diges- tion followed by Southern blot hybridization. Lane 1–3: ethidium bromide staining of the genomic DNA nucleosomal protected ladder. Lane 4–6: episomal plasmid-specific nucle- osmal protected ladder detected by vector probe. C. Exami- nation of episomal status of plasmids in the stable cells harboring pICP4 by Southern hybridization. Lane 1. Total DNA purified from 1.28 × 106 stable cells; Lane 2: 10 pg plas- mid; Lane 3: 0.1 ng plasmid DNA; 4: 1 ng plasmid DNA; 5: 10 ng plasmid DNA. A. B. C. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 MN dilution Tetramer Trimer Dimer Monomer MN dilution Tetramer Trimer Dimer Monomer 1 2 3 4 5 6 1 2 3 4 5 11.2 kb (Linear vector) A. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 MN dilution Tetramer Trimer Dimer Monomer REST/NRSF repressed ICP22 and ICP4 promoter activity in transient co-transfection How- ever, the ICP4 promoter of pH4-2 (containing HSV-1 RE- 1/NRSE) was significantly repressed by REST/NRSF but not p73, which showed no inhibition at all (Lane 4, 5, and 6, Fig. 2C). The quantification analysis revealed that 62% of ICP4 promoter activity in pH4-2 was repressed by REST/NRSF, compared to 28% in pSG28 (Fig. 2D; black bar, lane 4 and 5). In addition, REST/NRSF exhibited no repression on LAT and ICP0 transcription in both plas- cotransfected with pFLAG-REST or pCMVp73 to analyze the effect of HSV-1 RE-1/NRSE on the gene regulation of ICP4. It is noted that both pSG28 and pH4-2 contain the complete open reading frame of ICP4, LAT, and ICP0. The RT-PCR results indicated that REST/NRSF and mutant p73 exhibited no major effect on the ICP4 promoter of pSG28, which does not have HSV-1 RE-1/NRSE (Fig. 2C). How- ever, the ICP4 promoter of pH4-2 (containing HSV-1 RE- 1/NRSE) was significantly repressed by REST/NRSF but not p73, which showed no inhibition at all (Lane 4, 5, and 6, Fig. 2C). The quantification analysis revealed that 62% of ICP4 promoter activity in pH4-2 was repressed by REST/NRSF, compared to 28% in pSG28 (Fig. 2D; black bar, lane 4 and 5). In addition, REST/NRSF exhibited no repression on LAT and ICP0 transcription in both plas- (1:1). 2. pSG28 and pFLAG-REST (1:1). 3. pSG28 and pCMVp73 (1:1). 4. pH4-2 and control plasmid (1:1). 5. pH4-2 and pFLAG-REST (1:1). 6. pH4-2 and pCMVp73 (1:1). D. The RT-PCR results were quantified by Kodak Gel-Logic 100 sys- tem to measure the sum intensity of each band representing the regulatory effect of REST/NRSF on ICP4 transcription. Page 3 of 11 (page number not for citation purposes) Page 3 of 11 (page number not for citation purposes) Virology Journal 2007, 4:56 http://www.virologyj.com/content/4/1/56 mids (Fig. 2C and 2D, white and gray bars). These results demonstrated that REST/NRSF required HSV-1 RE-1/ NRSE to exert its regulatory effect on ICP4 promoter. Episomal plasmids remained in extra-chromosomal form and associated with nucleosomes in stable cells Figure 3 Episomal plasmids remained in extra-chromosomal form and associated with nucleosomes in stable cells. A. Analysis of nucleosomal formation on transient trans- fected reporter plasmid. Plasmid pICP4 was cotransfected with control vector (Lane 1, 2, 7, and 8), pFLAG-REST (Lane 3, 4, 9, and 10), and pCMVp73 (Lane 5, 6, 11, and 12). Lane 1–6: Ethidium bromide staining. Episomal p associated Figure 3 i Episomal plasmids remained in extra-chromosomal form and associated with nucleosomes in stable cells Figure 3 Episomal plasmids remained in extra-chromosomal form and associated with nucleosomes in stable cells. A. Analysis of nucleosomal formation on transient trans- fected reporter plasmid. Plasmid pICP4 was cotransfected with control vector (Lane 1, 2, 7, and 8), pFLAG-REST (Lane 3, 4, 9, and 10), and pCMVp73 (Lane 5, 6, 11, and 12). Lane 1–6: Ethidium bromide staining. Lane 7–12: plasmid-specific nucleosmal protected ladder detected by vector probe. Lane 13 and 14: Naked plasmid digestion control. B. Analysis of nucleosomal formation on episomal plasmids. The nuclei from stable 293HEK-pICP22 were subjected to MNase diges- tion followed by Southern blot hybridization. Lane 1–3: ethidium bromide staining of the genomic DNA nucleosomal protected ladder. Lane 4–6: episomal plasmid-specific nucle- osmal protected ladder detected by vector probe. C. Exami- nation of episomal status of plasmids in the stable cells harboring pICP4 by Southern hybridization. Lane 1. Total DNA purified from 1.28 × 106 stable cells; Lane 2: 10 pg plas- mid; Lane 3: 0.1 ng plasmid DNA; 4: 1 ng plasmid DNA; 5: 10 ng plasmid DNA. proteins exerted only minor inhibitory effects on the ICP22 promoter in 293HEK-pICP22 cells (Fig. 4A). Pro- moter activity was mildly reduced to 63% and 78% of control levels, respectively, by these proteins. Nucleosomes are associated with episomal plasmids in stable cells To assess the effect of REST/NRSF on nucleosomal forma- tion during transient transfection, we performed MNase digestion on cells two days after the transfection of reporter plasmids and expression vectors followed by Southern blot hybridization using probes against the plas- mid. The hybridization results revealed an irregular pat- tern of nucleosomal ladder compared to the genomic ladder and naked digestion control, suggesting that his- tones were associated with the plasmid but not in a bona fide nucleosomal structure (Fig. 3A). Expression of REST/ NRSF or p73 did not affect the nucleosomal configura- tion. B. MN dilution Tetramer Trimer Dimer Monomer 1 2 3 4 5 6 C. B. 1 2 3 4 5 11.2 kb (Linear vector) To establish reporter plasmids that are assembled into chromatin, we subjected cells transfected with the episo- mally replicating pICP4 or pICP22 plasmids to hygromy- cin B selection. The stable cells containing pICP22 or pICP4 were established after 10 days of selection. To examine the chromatin structure of the episomal plas- mids, nuclei from parental and stable cells were again sub- jected to different concentrations of MNase digestion followed by Southern blot hybridization. Ethidium bro- mide staining of the agarose gel revealed the nucleosome protected ladder characteristic of genomic DNA (Fig. 3B, Lane 1–3), indicating that the protocol of MNase diges- tion was effective. Southern hybridization showed a plas- mid-specific nucleosome protected ladder resembling the genomic ladder (Fig. 3B, Lane 4 to 6). The nucleosomal ladder of Southern hybridization is not an artifact since the samples from parental cells exhibited no signal at all (data not shown). These results demonstrated that the plasmids are associated with nucleosomes in the stably transfected cells. To test for integration of plasmids in cells, total DNA purified from stable cells and plasmid DNA was digested with NcoI, which cuts the plasmid once, followed by Southern blot hybridization using vec- tor probe. The results detected a single band with the size of 11.2 kb, equivalent to the size of the original plasmid (Fig. 3C). The results concluded that the plasmids remained in an extra-chromosomal form since integrated plasmid digestion would exhibit different sizes. Episomal plasmids remained in extra-chromosomal form and associated with nucleosomes in stable cells Figure 3 Episomal plasmids remained in extra-chromosomal form and associated with nucleosomes in stable cells. A. Analysis of nucleosomal formation on transient trans- fected reporter plasmid. Nucleosomes are associated with episomal plasmids in stable cells Plasmid pICP4 was cotransfected with control vector (Lane 1, 2, 7, and 8), pFLAG-REST (Lane 3, 4, 9, and 10), and pCMVp73 (Lane 5, 6, 11, and 12). Lane 1–6: Ethidium bromide staining. Lane 7–12: plasmid-specific nucleosmal protected ladder detected by vector probe. Lane 13 and 14: Naked plasmid digestion control. B. Analysis of nucleosomal formation on episomal plasmids. The nuclei from stable 293HEK-pICP22 were subjected to MNase diges- tion followed by Southern blot hybridization. Lane 1–3: ethidium bromide staining of the genomic DNA nucleosomal protected ladder. Lane 4–6: episomal plasmid-specific nucle- osmal protected ladder detected by vector probe. C. Exami- nation of episomal status of plasmids in the stable cells harboring pICP4 by Southern hybridization. Lane 1. Total DNA purified from 1.28 × 106 stable cells; Lane 2: 10 pg plas- mid; Lane 3: 0.1 ng plasmid DNA; 4: 1 ng plasmid DNA; 5: 10 ng plasmid DNA. REST/NRSF repressed ICP4 but not ICP22 promoter activity in stable cell lines In contrast, we observed a significant inhibitory effect on the ICP4 promoter by REST/NRSF in stable cells harbor- ing pICP4 (293HEK-pICP4). SEAP assays showed that ICP4 promoter activity was reduced to 21% of control lev- els by REST/NRSF. On the other hand, ICP4 promoter activity was essentially unchanged (94%) by the mutant p73 (Fig. 4B). These results suggested that the REST/NRSF To study the regulatory effect of REST/NRSF in a chroma- tin context, we transfected stable cells harboring pICP22 (293HEK-pICP22) with pFLAG-REST or pCMVp73. The cells were harvested for SEAP assays 72 hours after trans- fection. These assays showed that the FLAG-REST and p73 Page 4 of 11 (page number not for citation purposes) http://www.virologyj.com/content/4/1/56 Virology Journal 2007, 4:56 REST/NRSF exhibited significant reduction on ICP4 pro- moter activity and the REST/NRSF-mediated repression was reversed by HDAC inhibitor TSA Figure 4 REST/NRSF exhibited significant reduction on ICP4 promoter activity and the REST/NRSF-mediated repression was reversed by HDAC inhibitor TSA. A. Stable 293HEK cells containing pICP22 (293HEK-pICP22) was transfected with control plasmid, pFLAG-REST, or pCMVp73 in the presence (white bar) or absence (black bar) of 100 nM TSA. 1. transfected with control plasmid. 2. trans- fected with 1 µg of pFLAG-REST. 3. transfected with 1 µg of pCMVp73. The P values represent Student's t tests in pair- wise comparisons to the control without TSA. B. Stable 293HEK cells containing pICP4 (293HEK-pICP4) was trans- fected with control plasmid, pFLAG-REST, or pCMVp73 in the presence (white bar) or absence (black bar) of 100 nM TSA. 1. transfected with control plasmid. 2. transfected with 1 µg of pFLAG-REST. 3. transfected with 1 µg of pCMVp73. The P values represent Student's t tests in pairwise compari- sons to the control without TSA. C. Effect of TSA on REST/ NRSF and p73 transcriptions in 293HEK-pICP4. M: 100 bp ladder. 1. Transfected with 1 µg control plasmid. 2. Trans- fected with 1 µg pFLAG-REST. 3. Transfected with 1 µg pCMVp73. 4. Transfected with 1 µg control plasmid with 100 nM TSA. 5. Transfected with 1 µg pFLAG-REST with 100 nM TSA. 6. Transfected with 1 µg pCMVp73 with 100 nM TSA. The cDNA from REST/NRSF and actin were marked by arrows. A. B. C. REST/NRSF interacted with HSV-1 RE-1/NRSE To confirm the expression of the fusion protein FLAG- REST in the transfected 293HEK cells, we performed West- ern Blotting using the anti-REST antibody. The results indicated that the cells transfected with 1 µg of pFLAG- REST showed significant increase of REST/NRSF expres- sion compared to the control (Fig. 5A). The anti-REST antibody (produced against amino acid residues 801– 1097 of human REST/NRSF) did not recognize mutant p73 (amino acids 73–545). To demonstrate the in vitro binding of REST/NRSF to the HSV-1 RE-1/NRSE, we carried out EMSA using DIG-11- ddUTP-labeled wild-type ds oligonucleotide (oligo) con- taining HSV-1 RE-1/NRSE and oligo with core sequence mutation. Extract isolated from cells transfected with pFLAG-REST, pCMVp73, or control plasmids were used for in vitro interaction. The results revealed that both REST/NRSF and mutant p73 yield strong, increased signal of shifted bands while wild type oligo was used, demon- strating that REST/NRSF and its DNA binding domain bound to HSV-1 RE-1/NRSE in vitro (Fig. 5B, compare lane 1 to 2 and 4). The mutant oligo showed no band shifting, indicating that core sequence of HSV-1 RE-1/NRSE is crit- ical for the interaction (Fig. 5B, lane 3). The competition analysis using unlabeled wild-type oligo abolished the shifted bands, indicating that the interaction is specific (Fig. 5B, lane 5 and 6). may cooperate with chromatin to inhibit ICP4 transcrip- tion and the C-terminus of REST/NRSF played critical roles in this directional repression. REST/NRSF repressed ICP4 but not ICP22 promoter activity in stable cell lines REST/NRSF or p73 cDNA Actin cDNA 1 2 3 4 5 6 M No TSA 100 nM TSA 0 20 40 60 80 100 120% Normalized relative SEAP activity 100 62.3 75.6 108.6 77.6 95.6 TSA 1 2 3 + TSA + TSA 100 21.3, p< 0.005 91.3 127.6 96.6 105.6 1 2 3 0 20 40 60 80 100 120% Normalized relative SEAP activity TSA + TSA + TSA A. 0 20 40 60 80 100 120% Normalized relative SEAP activity 100 62.3 75.6 108.6 77.6 95.6 TSA 1 2 3 + TSA + TSA terminal part of REST/NRSF was reported to recruit HDAC [11,16]. To assess the role of histone deacetylation in this system, 293HEK-pICP22 and 293HEK-pICP4 stable cell lines were transfected with the control plasmid, pFLAG- REST, or pCMVp73 with or without TSA. The HDAC inhibitor TSA (100 nM, Sigma, MO) was added to the media 24-hour after transfection. Cells were harvested for SEAP assays 72-hour after transfection. In 293HEK- pICP22 cells transfected with pFLAG-REST, very little effect (1.2-fold induction) was observed in the presence of TSA (Fig. 4A). However, in 293HEK-pICP4 cells trans- fected with pFLAG-REST, TSA treatment increased ICP4 promoter activity 4.8-fold (Fig. 4B). We observed that the ICP4 promoter activity was not affected by TSA in the absence of REST/NRSF (Fig. 4B). To confirm that TSA did not affect the expression of REST/NRSF, we isolated total RNA from the transfected cells and performed RT-PCR using primers against REST/NRSF and actin. The data revealed that the transcriptions of REST/NRSF and p73 remained in the same pattern, indicating that the reactiva- tion of ICP4 transcription by TSA is not due to the reduc- tion of REST/NRSF expression (Fig. 4C). These results indicated that histone acetylation has a critical role in ICP4 gene expression in this system and that REST/NRSF may induce histone deacetylation to repress transcription of ICP4 in the context of chromatin. B. 100 21.3, p< 0.005 91.3 127.6 96.6 105.6 1 2 3 0 20 40 60 80 100 120% Normalized relative SEAP activity TSA + TSA + TSA C. REST/NRSF repressed ICP4 but not ICP22 promoter activity in stable cell lines transfected with 1 µg of pFLAG-REST. 3. transfected with 1 µg of pCMVp73. The P values represent Student's t tests in pairwise compari- sons to the control without TSA. C. Effect of TSA on REST/ NRSF and p73 transcriptions in 293HEK-pICP4. M: 100 bp ladder. 1. Transfected with 1 µg control plasmid. 2. Trans- fected with 1 µg pFLAG-REST. 3. Transfected with 1 µg pCMVp73. 4. Transfected with 1 µg control plasmid with 100 nM TSA. 5. Transfected with 1 µg pFLAG-REST with 100 nM TSA. 6. Transfected with 1 µg pCMVp73 with 100 nM TSA. The cDNA from REST/NRSF and actin were marked by arrows. REST/NRSF repressed ICP4 but not ICP22 promoter activity in stable cell lines REST/NRSF or p73 cDNA Actin cDNA 1 2 3 4 5 6 M No TSA 100 nM TSA REST/NRSF exhibited significant reduction on ICP4 pro- moter activity and the REST/NRSF-mediated repression was reversed by HDAC inhibitor TSA Figure 4 REST/NRSF exhibited significant reduction on ICP4 promoter activity and the REST/NRSF-mediated repression was reversed by HDAC inhibitor TSA. A. Stable 293HEK cells containing pICP22 (293HEK-pICP22) was transfected with control plasmid, pFLAG-REST, or pCMVp73 in the presence (white bar) or absence (black bar) of 100 nM TSA. 1. transfected with control plasmid. 2. trans- fected with 1 µg of pFLAG-REST. 3. transfected with 1 µg of pCMVp73. The P values represent Student's t tests in pair- wise comparisons to the control without TSA. B. Stable 293HEK cells containing pICP4 (293HEK-pICP4) was trans- fected with control plasmid, pFLAG-REST, or pCMVp73 in the presence (white bar) or absence (black bar) of 100 nM TSA. 1. transfected with control plasmid. 2. transfected with 1 µg of pFLAG-REST. 3. transfected with 1 µg of pCMVp73. The P values represent Student's t tests in pairwise compari- sons to the control without TSA. C. Effect of TSA on REST/ NRSF and p73 transcriptions in 293HEK-pICP4. M: 100 bp ladder. 1. Transfected with 1 µg control plasmid. 2. Trans- fected with 1 µg pFLAG-REST. 3. Transfected with 1 µg pCMVp73. 4. Transfected with 1 µg control plasmid with 100 nM TSA. 5. Transfected with 1 µg pFLAG-REST with 100 nM TSA. 6. Transfected with 1 µg pCMVp73 with 100 nM TSA. The cDNA from REST/NRSF and actin were marked by arrows. REST/NRSF exhibited significant reduction on ICP4 pro- moter activity and the REST/NRSF-mediated repression was reversed by HDAC inhibitor TSA Figure 4 REST/NRSF exhibited significant reduction on ICP4 promoter activity and the REST/NRSF-mediated repression was reversed by HDAC inhibitor TSA. A. Stable 293HEK cells containing pICP22 (293HEK-pICP22) was transfected with control plasmid, pFLAG-REST, or pCMVp73 in the presence (white bar) or absence (black bar) of 100 nM TSA. 1. transfected with control plasmid. 2. trans- fected with 1 µg of pFLAG-REST. 3. transfected with 1 µg of pCMVp73. The P values represent Student's t tests in pair- wise comparisons to the control without TSA. B. Stable 293HEK cells containing pICP4 (293HEK-pICP4) was trans- fected with control plasmid, pFLAG-REST, or pCMVp73 in the presence (white bar) or absence (black bar) of 100 nM TSA. 1. transfected with control plasmid. 2. Page 5 of 11 (page number not for citation purposes) http://www.virologyj.com/content/4/1/56 Anti-FLAG ChIP Anti-acetyl H4 ChIP Anti-CoREST ChIP INPUT Control Anti-FLAG ChIP (HGB Control) REST/HSV-1 RE-1 Oligonucleotide Complex Unbound Oligonucleotide 1 2 3 4 5 6 pICP4 pICP4+REST 1 2 REST/NRSF D-Tubulin 1 2 3 A. B. C. Anti-FLAG ChIP Anti-acetyl H4 ChIP Anti-CoREST ChIP INPUT Control Anti-FLAG ChIP (HGB Control) REST/HSV-1 RE-1 Oligonucleotide Complex Unbound Oligonucleotide 1 2 3 4 5 6 pICP4 pICP4+REST 1 2 REST/NRSF D-Tubulin 1 2 3 A. B. C. Anti-FLAG ChIP Anti-acetyl H4 ChIP Anti-CoREST ChIP INPUT Control Anti-FLAG ChIP (HGB Control) REST/HSV-1 RE-1 Oligonucleotide Complex Unbound Oligonucleotide 1 2 3 4 5 6 pICP4 pICP4+REST 1 2 REST/NRSF D-Tubulin 1 2 3 A. B C. pICP4 pICP4+REST 1 2 REST/NRSF D-Tubulin B. REST/HSV-1 RE-1 Oligonucleotide Complex Unbound Oligonucleotide 1 2 3 4 5 6 F B. Anti-FLAG ChIP Anti-acetyl H4 ChIP Anti-CoREST ChIP INPUT Control Anti-FLAG ChIP (HGB Control) 1 2 3 REST/NRS NRSE and Figure 5 REST/NRSF recruited corepressor CoREST to HSV-1 RE-1/ NRSE and induced histone H4 deacetylation Figure 5 REST/NRSF recruited corepressor CoREST to HSV- 1 RE-1/NRSE and induced histone H4 deacetylation. A. Over-expression of REST/NRSF in 293HEK cells by trans- fection of pFLAG-REST. Lane 1: 293HEK cells transfected with pICP4. Lane 2: 293HEK cells transfected with pICP4 and pFLAG-REST. Immunoblot was performed using anti-REST antibody. B. EMSA using transfected cell extract. Lane 1. Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with control plasmid. Lane 2. Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 3: Labeled mutant oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 4: Labeled HSV-1 RE-1 ds oligo incu- bated with extract isolated from cells transfected with pCMVp73. Lane 5: Labeled wild-type ds oligo containing 10× unlabeled wild-type oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 6: Labeled wild-type ds oligo containing 25× unlabeled wild-type oligo incubated with extract isolated from cells transfected with pFLAG-REST. Noted that endogenous REST/NRSF produced a shifted band (Lane 1). C. Analysis of REST/NRSF binding, histone H4 acetylation alteration, and CoREST recruitment by ChIP. 293HEK-pICP4 cell line was transfected with pFLAG-REST and subjected to ChIP assay. Samples prior to the immuno-precipitation are used for input control. The samples were amplified by PCR and subjected to 1.2% agar- ose electrophoresis staining with ethidium bromide. 1. pICP4 + Control. 2. pICP4 + pFLAG-REST. 3. pICP4 + pCMVp73. The REST/NRSF-mediated ICP4 inhibition was released by HDAC inhibitor TSA We predicted that the directional repression of ICP4 by REST/NRSF is through histone deacetylation since the C- Page 5 of 11 (page number not for citation purposes) Page 5 of 11 (page number not for citation purposes) http://www.virologyj.com/content/4/1/56 http://www.virologyj.com/content/4/1/56 Virology Journal 2007, 4:56 To investigate the in vivo binding of REST/NRSF to the HSV-1 RE-1/NRSE, we performed ChIP using anti-FLAG® M2 Affinity Gel. The results showed strong PCR signal from pFLAG-REST IP sample compared to the control and pCMVp73, indicating that FLAG-tagged REST/NRSF was recruited to the minichromosomes (Fig. 5C, anti-FLAG ChIP). The interaction of mutant p73 was not detected due to the lack of FLAG-tag. In addition, only a very weak signal was detected using primers against hygromycin B resistance gene (Fig. 5C, HGB control), indicating that the binding of REST/NRSF was specific to the promoter region and the shearing of minichromosome was sufficient. These results indicated that REST/NRSF and mutant p73 bound to the HSV-1 RE-1/NRSE. REST/NRSF recruited corepressor CoREST to HSV-1 RE-1/ NRSE and induced histone H4 deacetylation Figure 5 REST/NRSF recruited corepressor CoREST to HSV- 1 RE-1/NRSE and induced histone H4 deacetylation. A. Over-expression of REST/NRSF in 293HEK cells by trans- fection of pFLAG-REST. Lane 1: 293HEK cells transfected with pICP4. Lane 2: 293HEK cells transfected with pICP4 and pFLAG-REST. Immunoblot was performed using anti-REST antibody. B. EMSA using transfected cell extract. Lane 1. Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with control plasmid. Lane 2. Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 3: Labeled mutant oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 4: Labeled HSV-1 RE-1 ds oligo incu- bated with extract isolated from cells transfected with pCMVp73. Lane 5: Labeled wild-type ds oligo containing 10× unlabeled wild-type oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 6: Labeled wild-type ds oligo containing 25× unlabeled wild-type oligo incubated with extract isolated from cells transfected with pFLAG-REST. Noted that endogenous REST/NRSF produced a shifted band (Lane 1). C. Analysis of REST/NRSF binding, histone H4 acetylation alteration, and CoREST recruitment by ChIP. 293HEK-pICP4 cell line was transfected with pFLAG-REST and subjected to ChIP assay. Samples prior to the immuno-precipitation are used for input control. The samples were amplified by PCR and subjected to 1.2% agar- ose electrophoresis staining with ethidium bromide. 1. pICP4 + Control. 2. pICP4 + pFLAG-REST. 3. pICP4 + pCMVp73. A. B. C. Page 6 of 11 (page number not for citation purposes) promoter p To analyze the participation of corepressor to HSV-1 RE- 1/NRSE, we performed ChIP using the anti-CoREST anti- body. The results showed a much stronger signal from the FLAG-REST transfected samples, indicating that CoREST is recruited to HSV-1 RE-1/NRSE through REST/NRSF (Fig. 5C, anti-CoREST ChIP). We further investigated the his- tone acetylation status by the same method using anti- body against acetylated histone H4. The results revealed that acetylation was reduced in the presence of REST/ NRSF compared to the control and p73 (Fig. 5C). These results indicated that CoREST is recruited to the HSV-1 RE- 1/NRSE via the interaction of REST/NRSF in a chromatin context and this interaction reduced the histone acetyla- tion of histone H4 in the proximity of HSV-1 IE promot- ers. http://www.virologyj.com/content/4/1/56 Histone modi- fication is suggested to regulate transcription through a mechanism of "histone code"[25]. Our ChIP analysis indicated that at least one type of histone modification (acetylation of histone H4) participated in the gene regu- lation. It is not clear why REST/NRSF failed to repress the ICP22 promoter activity in a chromatin context. However, other nearby cis-acting elements may modulate the effects of REST/NRSF. Sequence analysis indicates that the OriS, located between the ICP4 and ICP22 promoters, may fold into a stable hairpin [26]. A recent study suggested that the OriS stem-loop structure encodes a microRNA that may regulate HSV and host gene expression [27]. Our hypothesis is that a chromatin insulator-like element par- ticipated in the regulation of ICP22. Another recent study identified a cluster of CTCF motifs, designated CTRS3, in the intron of the ICP22 gene [28]. CTCF elements can function as insulators shielding genes in one region of a chromosome from the regulatory effects of another region [29]. Two classes of insulators have been reported, enhancer-blocking and boundary/barrier elements [28]. The former impedes the enhancer function and the latter prevents the spreading of repressive heterochromatin into the transcriptionally active area. Based on our results, we predict that CTRS3 and/or OriS act as a boundary/barrier element to prevent the repressive chromatin from spread- ing to the ICP22 promoter and thus aborted the inhibi- tory effect of REST/NRSF on ICP22 in the presence of nucleosomes. Further studies using neuronal models and promoter deletion are required to elucidate the complex regulatory mechanisms. In transiently transfected cells, the transfected plasmids do not associate with nucleosomes in a regular chromatin pattern and thus resemble viral DNA in lytically infected cells. REST/NRSF repressed both the ICP4 and ICP22 pro- moter in these cells. In these conditions, repression of transcription from the ICP22 promoter might be due to steric hindrance of the TATA box and/or the transcription initiation site. However, repression of the ICP4 promoter must be due to other repressive effects of REST/NRSF. ICP22 and ICP4 have fundamentally different roles in HSV-1 replication. ICP4 is essential for expression of E and L genes [17]. ICP22 is not essential, but is required for efficient replication [18] and transcription of HSV in cer- tain cell types [19]. http://www.virologyj.com/content/4/1/56 It is needed for maximal expression of the γ1 and γ2 genes, probably due to the fact that ICP22 induces phosphorylation on the large subunit of cellular RNA polymerase II [20]. The role of ICP22 in HSV-1 latency is not fully understood. No direct role for ICP22 in establishment or maintenance of latent infection has been demonstrated. However, given the transactivating effect of ICP4, it may be more important for the virus to effectively repress this promoter during these phases of latency, and this may be mediated by REST/NRSF. This study complements a recent report showing that CoREST, a component of corepressor complex REST/CoR- EST/HDACs, exhibits a sequence similarity at the amino terminus to HSV-1 IE protein ICP0, and that the HDACs may be dissociated from the corepressor complex by ICP0 in cells infected by wild-type viruses [12]. Thus, the authors predicted that the REST/CoREST/HDACs complex could cause HSV-1 gene silencing at low multiplicities of infection (MOI). Our results revealed that CoREST is recruited to HSV-1 RE-1/NRSE in the presence of REST/ NRSF, supporting the hypothesis that REST/CoREST com- plexes participated in the regulatory effect on HSV-1 IE genes. We predict this mechanism applies to HSV-1 latent http://www.virologyj.com/content/4/1/56 Consist- ent with this, ChIP analysis also showed that CoREST, which is able to recruit histone deacetylases, was also associated with the ICP4 promoter in pFLAG-REST trans- fected cells. This was further supported by the p73 data, which indicated that the REST/NRSF mutant lacking effec- tor region does not recruit CoREST to the promoter and failed to deacetylate histone H4 at the promoter. In con- trast to the ICP4 promoter, the ICP22 promoter was rela- tively insensitive to repression by REST/NRSF. Since the RE-1/NRSE is located approximately 660 bp upstream of the ICP4 promoter but is adjacent to the ICP22 TATA box, this may be due to directional or distance-dependent effects, or it may indicate that REST/NRSF is able to exert promoter-specific effects in a chromatin context. the amount of acetylated histone H4 associated with the ICP4 promoter in pFLAG-REST transfected cells. Consist- ent with this, ChIP analysis also showed that CoREST, which is able to recruit histone deacetylases, was also associated with the ICP4 promoter in pFLAG-REST trans- fected cells. This was further supported by the p73 data, which indicated that the REST/NRSF mutant lacking effec- tor region does not recruit CoREST to the promoter and failed to deacetylate histone H4 at the promoter. In con- trast to the ICP4 promoter, the ICP22 promoter was rela- tively insensitive to repression by REST/NRSF. Since the RE-1/NRSE is located approximately 660 bp upstream of the ICP4 promoter but is adjacent to the ICP22 TATA box, this may be due to directional or distance-dependent effects, or it may indicate that REST/NRSF is able to exert promoter-specific effects in a chromatin context. ect o s ce S / S s p ese t eu o s [ 0, ]. This is supported by studies showing that HSV mutants lacking functional ICP0 exhibit poor reactivation effi- ciency [22-24]. Furthermore, at low MOI, nonneuronal cells infected with ICP0 deletion mutants produce about 100-fold less virus compared to cells infected with wild- type virus. In these experiments, the lack of ICP0 may have resulted in the failure to disrupt the inhibitory effect of the REST/CoREST/HDACs complex on IE genes. Our results showed that REST/NRSF inhibited ICP4 promoter activity in a chromatin context, suggesting REST/NRSF and repressive chromatin could maintain gene silencing during the establishment of a transcriptionally silent state and could provide a possible mechanism for long-term persistence through histone modification. http://www.virologyj.com/content/4/1/56 http://www.virologyj.com/content/4/1/56 Virology Journal 2007, 4:56 infection since REST/NRSF is present in neurons [10,21]. This is supported by studies showing that HSV mutants lacking functional ICP0 exhibit poor reactivation effi- ciency [22-24]. Furthermore, at low MOI, nonneuronal cells infected with ICP0 deletion mutants produce about 100-fold less virus compared to cells infected with wild- type virus. In these experiments, the lack of ICP0 may have resulted in the failure to disrupt the inhibitory effect of the REST/CoREST/HDACs complex on IE genes. Our results showed that REST/NRSF inhibited ICP4 promoter activity in a chromatin context, suggesting REST/NRSF and repressive chromatin could maintain gene silencing during the establishment of a transcriptionally silent state and could provide a possible mechanism for long-term persistence through histone modification. Histone modi- fication is suggested to regulate transcription through a mechanism of "histone code"[25]. Our ChIP analysis indicated that at least one type of histone modification (acetylation of histone H4) participated in the gene regu- lation. It is not clear why REST/NRSF failed to repress the ICP22 promoter activity in a chromatin context. However, other nearby cis-acting elements may modulate the effects of REST/NRSF. Sequence analysis indicates that the OriS, located between the ICP4 and ICP22 promoters, may fold into a stable hairpin [26]. A recent study suggested that the OriS stem-loop structure encodes a microRNA that may regulate HSV and host gene expression [27]. Our hypothesis is that a chromatin insulator-like element par- ticipated in the regulation of ICP22. Another recent study identified a cluster of CTCF motifs, designated CTRS3, in the intron of the ICP22 gene [28]. CTCF elements can function as insulators shielding genes in one region of a chromosome from the regulatory effects of another region [29]. Two classes of insulators have been reported, enhancer-blocking and boundary/barrier elements [28]. The former impedes the enhancer function and the latter prevents the spreading of repressive heterochromatin into the transcriptionally active area. Based on our results, we predict that CTRS3 and/or OriS act as a boundary/barrier element to prevent the repressive chromatin from spread- ing to the ICP22 promoter and thus aborted the inhibi- tory effect of REST/NRSF on ICP22 in the presence of nucleosomes. Further studies using neuronal models and promoter deletion are required to elucidate the complex regulatory mechanisms. the amount of acetylated histone H4 associated with the ICP4 promoter in pFLAG-REST transfected cells. Discussion In this study, we identified a RE-1/NRSE site in the HSV-1 genome between the ICP4 and ICP22 Immediate Early promoters and showed that REST/NRSF exerted a chroma- tin state-dependant repressive effect on the activity of these promoters. In stably transformed cells, the plasmids pICP4 and pICP22, respectively containing the HSV-1 ICP4 and ICP22 promoters and the SEAP reporter gene, associated with nucleosomes in a regular chromatin array. Thus the chromatin structure of these promoters should resemble their structure in latently infected cells more closely than in any other available model system. Trans- fection of pFLAG-REST into these cells resulted in a sub- stantial decrease in ICP4 promoter activity. This effect required the effector domain of REST/NRSF since pCMVp73, which contains only the DNA-binding domain, had little effect on the ICP4 promoter. Repres- sion of the ICP4 promoter by REST/NRSF was reversed by Trichostatin A, suggesting that it was mediated, at least in part, by histone deacetylation. This was confirmed by ChIP analysis, which showed a significant reduction in Page 6 of 11 (page number not for citation purposes) Page 6 of 11 (page number not for citation purposes) Methods y 293HEK cells (2 × 10 6) were transfected with pFLAG- REST or pCMVp73. For the preparation of cell extracts, the monolayers were washed with ice-cold phosphate-buff- ered saline (PBS), and the cells were lysed by adding 5 ml of cell extract buffer (25 mM Tris-HCl, 50 mM β-glycerol phosphate, 1 mM EGTA, 0.5 mM EDTA, 5% glycerol, 1% Triton X-100, 0.1 mM Benzamidine, 0.5 M Na3VaO4, 0.5 M phenylmethylsulfonyl fluoride, and protease inhibitors cocktail tablets from Roche). Protein concentration was determined by the Bradford protein assay (Bio-Rad, Her- cules, CA). Proteins were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transfered onto nitrocellulose membranes. The blots were blocked using PBS with 5% (wt/vol) non-fat dry milk and washed in PBS. Anti-REST rabbit polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) was used at a dilution of 1:1,000. After overnight incubation primary antibody was washed off in 1× PBST (1× PBS + 0.05% Tween 20) followed by the addition of secondary anti- body (anti rabbit IgG-horseradish peroxidase conjugate, Amersham Bioscience, Piscataway, NJ) at a dilution of 1:2,000 in PBST for one hour at room temperature. The membranes were washed as before and visualized using enhanced-chemiluminescence reagents (Pierce) according to the manufacturer's protocol. Anti-α-Tubulin mouse antibody (Calbiochem, Cat#: CP06) was added at a dilu- tion of 1:10,000 in PBST, and the secondary antibody (goat anti mouse IgG – horseradish peroxidase conjugate, PerkinElmer Life Sciences, Wellesley, MA) was added at a dilution of 1:5,000 in PBST for one hour at room temper- ature. Construction of plasmids and PCR amplification The construction of episomal vector pICP4 and pICP22 were essentially described previously [30]. REST/NRSF expression vectors pFLAG-REST and pCMVp73 are gifts from Dr. Gail Mandel (SUNY, Stony Brook, NY). Plasmid pFLAG-REST expresses fusion protein REST/NRSF with a FLAG tag and pCMVp73 is a mutant REST/NRSF encoding 485 amino acids of the DNA-binding domain [31]. The plasmid pH4-2, based on pUC19, contains the Hind III restriction fragment (non-prototype structure) cover- ing the entire long and short internal repeats. Plasmid pSG28, based on pUC18, contains EcoR I fragment cover- ing HSV-1 sequence from 106785 to 131534. Both plas- mids contain open reading frame of ICP4, LAT, and ICP0. However, pH4-2 contains HSV-1 RE-1/NRSE and ICP22 promoter but pSG28 does not. Plasmid pGL3-basic (Promega, Madison, WI) was used as control for transfec- tion. http://www.virologyj.com/content/4/1/56 http://www.virologyj.com/content/4/1/56 chromatin methylation (Fig. 6). More experiments are underway to investigate the role of REST/NRSF on HSV-1 gene regulation using neuronal cells and animal models. hours after transfection and replated in T25 flasks in medium containing 200 µg/ml of hygromycin B (Invitro- gen, CA). Methods Cell culture, transfection and selection for stable cell lines The 293HEK cell is a human embryonic kidney cell line and was purchased from the American Type Culture Col- lection (ATCC) and maintained in DMEM medium sup- plemented with 10% fetal bovine serum (FBS). For transfection, cultures of cells were prepared for transfec- tion by plating 5 × 105 cells in 60 mm culture dishes. After overnight incubation, the cells were transfected with 5 µg plasmid DNA complexed with 20 µl Superfect reagent (Qiagen, Valencia, CA) according to the procedures rec- ommended by the manufacturer. To obtain stably trans- formed cell lines, the transfected cells were trypsinized 72 Electrophoretic mobility shift assay (EMSA) p y f y ( ) EMSA was performed using a DIG Gel shift Kit 2nd gener- ation (Roche applied science, Indianapolis, IN) essen- tially described in the manufacturer's protocol. Briefly, single-stranded oligonucleotides (oligo) 5'-GGC CTT TAT GTG CGC CGG AGA GAC CCG CCC-3' and its comple- mentary oligo were synthesized (Invitrogen, San Diego, CA) and annealed to make double-stranded (ds) REST oligo. The core sequence is underlined. The oligo 5'-GGC CTT TAT GTG CGC TTT TGA GAC CCG CCC-3' and its complementary oligo were synthesized and annealed as mutant control. The ds oligos were terminally-labeled with non-radioactive DIG-11-ddUTP by terminal trans- ferase and incubated with the protein extracts isolated from parental cells or cells transfected with REST/NRSF or mutant p73 expression vectors for 15 min at room tem- perature. In addition, 10× or 25× of wild-type unlabeled oligo were added to the labeled oligo for competition Proposed model of HSV-1 ICP4 and ICP22 regulation by REST/NRSF Figure 6 Proposed model of HSV-1 ICP4 and ICP22 regulation by REST/NRSF. REST/NRSF interacts with HSV-1 RE-1 in chromatin and represses ICP4 by recruiting CoREST and HDAC to induce hypoacetylation around the ICP4 pro- moter. ICP22 Transcription REST/CoREST Co-repressor complex ICP4 Transcription RE-1/NRSE Histone H4 deacetylation ICP22 Transcription REST/CoREST Co-repressor complex ICP4 Transcription RE-1/NRSE Histone H4 deacetylation Histone H4 deacetylation ICP22 Transcription Conclusion In summary, we have provided the first direct evidence indicating that REST/NRSF can regulate HSV-1 IE gene expression. We propose that during the establishment and maintenance of latent infection, REST/NRSF binds to the HSV-1 RE-1/NRSE in a chromatin context and recruits CoREST/HDAC complexes. As a result, the repressor com- plexes inhibit the ICP4 transcription and produce long- term repression via histone deacetylation, and possibly Page 7 of 11 (page number not for citation purposes) Page 7 of 11 (page number not for citation purposes) Virology Journal 2007, 4:56 http://www.virologyj.com/content/4/1/56 http://www.virologyj.com/content/4/1/56 analysis. The samples were electrophoresed on a 6% DNA Retardation Gel (Invitrogen, CA) at 80 V for 1 h followed by alkaline transfer to positive-charged nylon membrane and chemiluminescence detection. monolayer had formed, the medium was replaced with fresh DMEM medium and the flasks were incubated over- night. Fifteen µl of culture medium was collected and mixed with 45 µl of dilution buffer. The samples were incubated at 65°C for 30 min, after which 60 µl of assay buffer was added to the cooled samples. The reaction mix then was incubated at room temperature for 5 min fol- lowed by addition of 60 µl of 1.25 mM CSPD substrate according to the manufacturer's protocol. The chemilumi- nescent signal was measured at 420 nm by 20/20n Lumi- nometer (Turner Biosystems, Sunnyvale, CA) after 10 min incubation. Each construct was tested using a minimum of three replicates and the data were collected and normal- ized as SEAP units relative to the controls. Reverse transcriptase PCR (RT-PCR) p ( ) For RT-PCR, total RNA from cultured cells was isolated by Trizol reagent (Invitrogen). RT-PCRs were performed using Superscript One-Step RT-PCR (Invitrogen) with 0.5 µg of total RNA and two primer sets per reaction tube: one set for the actin as a control and another for the REST/ NRSF. The RT-PCR primers were designed to bind in dif- ferent exons to avoid unintentional amplification of potential genomic DNA contamination. Their sequences are as follows: Actin: 5'-ATT CCT ATG TGG GCG ACG AG- 3' and 5'-TGG ATA GCA ACG TAC ATG GC-3'; REST/ NRSF: 5'-TGT ATT TGA GGC ATC AGG TGC TC-3' and 5'- GTG TGG TGT TTC AGG TGT GCT G-3'; ICP4: 5'-CGA CAC GGA TCC ACG ACC C-3' and 5'-GAT CCC CCT CCC GCG CTT CGT CCG-3'; LAT: 5' CGG CGA CAT CCT CCC CCT AAG C 3' and 5' GAC AGA CGA ACG AAA CGT TCC G 3'; ICP0: 5'-TTC GGT CTC CGC CTG AGA GT-3' and 5'- GAC CCT CCA GCC GCA TAC GA-3'. The reverse tran- scription/PCR reaction was carried out at 45°C for 20 min followed by 25 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 30 s. The RT-PCR products were analyzed by 2% agarose gel electrophoresis. The Kodak Gel-Logic 100 sys- tem was utilized for quantification. Chromatin Immunoprecipitation (ChIP) assays p p ( ) y The protocol was essentially described in Hsia and Shi [32] with modification according to the manufacturer's manual. Briefly, the cell monolayer was treated with 1% formaldehyde solution for 10 min at room temperature. The monolayer was then scraped into 15 ml tubes and subjected to the nuclei isolation protocol described previ- ously. The samples were fragmented by MNase digestion (3.75 units/µl on ice for 1 h). The reaction was stopped by EDTA at the final concentration of 50 mM. The nuclei was pelleted and incubated with 400 µl of SDS lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl {pH 8.1}) containing proteinase inhibitor (Complete Mini, Roche) on ice for 10 min. The lysed samples were spun for 10 min at 13,000 g with a refrigerated Eppendorf microfuge at 4°C, and the supernatant was diluted 10-fold with dilu- Southern blot hybridization The DNA was subjected to gel electrophoresis (1.2% aga- rose gel, 6 hours at 40 volts). After electrophoresis, the gel was treated twice with denaturing solution (0.4 N NaOH solution) for 15 min at room temperature. The DNA was transferred for 6 h from the gel to a positively charged nylon membrane using an alkaline transfer protocol. Hybridization was performed overnight at 42°C. To make a whole plasmid probe, linear pREP-SEAP DNA was labeled using Prime-a-Gene® Labeling System (Cat. No: TB049) from Promega (Madison, WI) based on the ran- dom-primed method. Nuclei isolation and micrococcal nuclease (MNase) digestion g To partially digest cellular chromatin with MNase, a T75 flask of cells was harvested by trypsinization. The cells were washed once with Dulbecco's PBS, and then washed twice with 5 ml ice-cold tris buffered saline (TBS) (10 mM tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2). The cells were then washed with 2 ml of ice-cold CB buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 5 mM MgCl2 supplemented with 1 mM dithiothreitol on the day of use), and then washed again with 1 ml ice-cold CB. The cells were pelleted once more, and lysed by resuspending them in 0.5 ml ice-cold CB plus 0.5% Triton X-100. Nuclei were pelleted by cen- trifugation at 2000 × g for 5 min. The nuclei were resus- pended in 50 µl ice-cold CB and 9 µl aliquots of nuclei were added to 0.5 ml microfuge tubes containing 1 µl MNase freshly diluted in MNase digestion buffer (20 mM PIPES, pH 7.0, 1 mM MgCl2, 10 mM NaCl, 250 mM sucrose, supplemented with 1 mM CaCl2, and 5 mM 2- mercaptoethanol). A stock solution of MNase (Fermentas Cat#: EN0181) was purchased and stored at -20°C. Nuclei were digested with different concentrations of MNase at room temperature for 20 min. After the MN reaction, the nuclei were digested by addition of 225 µl proteinase K solution (20 µg proteinase K/ml in 0.5% SDS, 10 mM tris, pH 8.0, 5 mM EDTA) followed by overnight incubation at 37°C. DNA was precipated at -20°C for 1 h after addition of 25 µl 3.0 M sodium acetate, pH 5.2, and 500 µl propa- nol. After centrifugation, DNA was washed with 70% eth- anol prior to electrophoresis. Proposed REST/NRS Figure 6 Proposed model of HSV-1 ICP4 and ICP22 regulation by REST/NRSF. REST/NRSF interacts with HSV-1 RE-1 in chromatin and represses ICP4 by recruiting CoREST and HDAC to induce hypoacetylation around the ICP4 pro- moter. Page 8 of 11 (page number not for citation purposes) Virology Journal 2007, 4:56 Page 9 of 11 (page number not for citation purposes) Competing interests The author(s) declare that they have no competing inter- ests. ( ) 7. Deshmane SL, Fraser NW: During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure. J Virol 1989, 63(2):943-947. References 1. Roizman B, Knipe DM: Herpes Simplex Viruses and Their Rep- lication. In Fundamental Fields Virology 4th edition. Edited by: Howley PM, Knipe DM. Lippincott Williams & Wilkins; 2001:p2399-2460. 2. Wang QY, Zhou C, Johnson KE, Colgrove RC, Coen DM, Knipe DM: Herpesviral latency-associated transcript gene promotes assembly of heterochromatin on viral lytic-gene promoters in latent infection. Proc Natl Acad Sci USA 2005, 102(44):16055-16059. For each reaction, a 50-µl, 25-cycle PCR was carried out in the presence of 10 pmol of the primers. Each cycle con- sisted of 1 min at 94°C, 40 s at 50°C, and 1 min at 72°C. Each experiment was done at least twice with similar results. The ChIP PCR products were analyzed by 2% aga- rose gel electrophoresis. ( ) 3. Pesola JM, Zhu J, Knipe DM, Coen DM: Herpes simplex virus 1 immediate-early and early gene expression during reactiva- tion from latency under conditions that prevent infectious virus production. J Virol 2005, 79(23):14516-14525. 3. Pesola JM, Zhu J, Knipe DM, Coen DM: Herpes simplex virus 1 immediate-early and early gene expression during reactiva- tion from latency under conditions that prevent infectious virus production. J Virol 2005, 79(23):14516-14525. 4. Jones C: Herpes simplex virus type 1 and bovine herpesvirus 1 latency. Clin Microbiol Rev 2003, 16(1):79-95. 5 Mitchell BM Bloom DC Cohrs RJ Gilden DH Kennedy PG: Herpes p J ( ) 4. Jones C: Herpes simplex virus type 1 and bovine herpesvirus 1 latency. Clin Microbiol Rev 2003, 16(1):79-95. p J ( ) 4. Jones C: Herpes simplex virus type 1 and bovine herpesvirus 1 latency. Clin Microbiol Rev 2003, 16(1):79-95. y ( ) 5. Mitchell BM, Bloom DC, Cohrs RJ, Gilden DH, Kennedy PG: Herpes simplex virus-1 and varicella-zoster virus latency in ganglia. J Neurovirol 2003, 9(2):194-204. J , ( ) 6. Jackson SA, DeLuca NA: Relationship of herpes simplex virus genome configuration to productive and persistent infec- tions. Proc Natl Acad Sci USA 2003, 100(13):7871-7876. http://www.virologyj.com/content/4/1/56 http://www.virologyj.com/content/4/1/56 tion buffer (25 ml; 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl {pH 8.1}, and 167 mM NaCl) containing protease inhibitor as described above. Immunoprecipitation was then performed with a ChIP assay kit essentially as described by the manufacturer with an antibody against acetylated H4 (Cat#: 17-229, Upstate Biotechnology, Lake Placid, N.Y.). This is a polyclonal antibody generated by using peptide corresponding to amino acids 2–19 of Tetrahymena histone H4 [AGGAcKG- GAcKGM GAcKVGAAcKRHSC], acetylated on lysines 5, 8, 12 and 16. For immunoprecipitation of FLAG fusion pro- tein, EZview™ Red ANTI-FLAG® M2 Affinity Gel (Sigma- Aldrich Biotechnology, St. Louis, MO, Cat#: F2426) was utilized. Anti-CoREST, against human CoREST corre- sponding to residue 109–293, was purchased from Upstate Biotechnology (Cat#: 07-455). To analyze the DNA immuno-precipitated by the antibody or affinity gel, PCR amplification was performed on the precipitated DNA with primers (5'-TGG GGT GGG CGG GTC TTT C-3' and 5'-ACG AAC GAC GGG AGC GGC TG-3') against HSV-1 RE-1/NRSE. The primer sequences against hygro- mycin B gene (HGB) are 5'-TTG TTG GAG CCG AAA TCC G-3' and 5'-CAA ACT GTG ATG GAC GAC ACC G-3'. ern blot hybridization, directed all the experimental approaches, analyzed the preliminary data, supervised the work, and prepared the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Dr. Gail Mandel, HHMI investigator and faculty of SUNY Stony Brook, for the REST/NRSF expression vectors pFLAG-REST and pCMVp73. We thank Dr. Paul Sylvester and Dr. Yun-Bo Shi for thorough reading and helpful discussions. We thank Josephine Everly for manuscript editing. Supports from University of Louisiana, Monroe and IDeA Net- works of Biomedical Research Excellence (INBRE), from NCRR/NIH to SVH, RCP, GRB, and JMH are acknowledged. JMH is supported in part by NEI EY006311, Research to Prevent Blindness Senior Scientific Investigator Award, and LSU Eye Center Core Grant NEI EY02377. This publication was made possible by NIH Grant P20RR16456. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. Authors' contributions J ( ) 8. Chong JA, Tapia-Ramirez J, Kim S, Toledo-Aral JJ, Zheng Y, Boutros MC, Altshuller YM, Frohman MA, Kraner SD, Mandel G: REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell 1995, 80(6):949-957. RCP generated the reporter plasmids pICP4 and pICP22, performed the transient transfections, carried out the RT- PCR, performed the MNase digestion and Southern blot hybridization, generated the stable cell lines, and per- formed the EMSA, and ChIP. GRB assisted the preparation of reporter plasmids, performed the transfection experi- ments, confirmed the RT-PCR results, assisted the MNase digestion and Southern analysis, and maintained the sta- ble cell lines. BG performed the Western blot analysis and repeated the transfection experiments. TCH participated in the identification of the RE-1/NRSE, designed the reporter plasmids, participated in the experimental strat- egy, and helped the manuscript preparation. JMH pre- pared the expression vectors, designed the experimental strategy, discussed the experimental data, conceived the strategic plan, and participated in the manuscript prepara- tion. SVH initiated the project, identified the RE-1/NRSE, prepared the original reporter plasmids to make pICP4 and pICP22, performed the MNase digestion and South- 9. Mori N, Schoenherr C, Vandenbergh DJ, Anderson DJ: A common silencer element in the SCG10 and type II Na+ channel genes binds a factor present in nonneuronal cells but not in neuro- nal cells. Neuron 1992, 9(1):45-54. ( ) 10. Shimojo M, Hersh LB: Regulation of the cholinergic gene locus by the repressor element-1 silencing transcription factor/ neuron restrictive silencer factor (REST/NRSF). Life Sci 2004, 74(18):2213-2225. ( ) 11. Coulson JM: Transcriptional regulation: cancer, neurons and the REST. Curr Biol 2005, 15(17):R665-668. ( ) 12. Gu H, Liang Y, Mandel G, Roizman B: Components of the REST/ CoREST/histone deacetylase repressor complex are dis- rupted, modified, and translocated in HSV-1-infected cells. Proc Natl Acad Sci USA 2005, 102(21):7571-7576. ( ) 13. Yates JL, Warren N, Sugden B: Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 1985, 313(6005):812-815. ( ) 14. Kim CS, Hwang CK, Choi HS, Song KY, Law PY, Wei LN, Loh HH: Neuron-restrictive silencer factor (NRSF) functions as a repressor in neuronal cells to regulate the mu opioid recep- tor gene. J Biol Chem 2004, 279(45):46464-46473. g J ( ) 15. Nguyen-Huynh AT, Schaffer PA: Cellular transcription factors enhance herpes simplex virus type 1 oriS – dependent DNA replication. J Virol 1998, 72(5):3635-3645. Chemiluminescent SEAP assays Promoter activity was analyzed by measuring SEAP reporter gene activity using GreatEscape kit according to the manufacturer's protocol (BD Biosciences). Transiently or stably transformed cells were plated in 60-mm plates at 5 × 105 cells/plate. After 2 days when an evenly distributed Page 9 of 11 (page number not for citation purposes) Page 9 of 11 (page number not for citation purposes) Virology Journal 2007, 4:56 Authors' contributions g J ( ) 15. Nguyen-Huynh AT, Schaffer PA: Cellular transcription factors enhance herpes simplex virus type 1 oriS – dependent DNA replication. J Virol 1998, 72(5):3635-3645. Page 10 of 11 (page number not for citation purposes) Virology Journal 2007, 4:56 http://www.virologyj.com/content/4/1/56 http://www.virologyj.com/content/4/1/56 16. Andres ME, Burger C, Peral-Rubio MJ, Battaglioli E, Anderson ME, Grimes J, Dallman J, Ballas N, Mandel G: CoREST: a functional corepressor required for regulation of neural-specific gene expression. Proc Natl Acad Sci USA 1999, 96(17):9873-9878. 17. Compel P, DeLuca NA: Temperature-dependent conforma- tional changes in herpes simplex virus ICP4 that affect tran- scription activation. J Virol 2003, 77(5):3257-3268. p J ( ) 18. Bruni R, Roizman B: Herpes simplex virus 1 regulatory protein ICP22 interacts with a new cell cycle-regulated factor and accumulates in a cell cycle-dependent fashion in infected cells. J Virol 1998, 72(11):8525-8531. J ( ) 19. Post LE, Roizman B: A generalized technique for deletion of specific genes in large genomes: alpha gene 22 of herpes sim- plex virus 1 is not essential for growth. Cell 1981, 25(1):227-232. ( ) 20. Long MC, Leong V, Schaffer PA, Spencer CA, Rice SA: ICP22 and the UL13 protein kinase are both required for herpes sim- plex virus-induced modification of the large subunit of RNA polymerase II. J Virol 1999, 73(7):5593-5604. p y J ( ) 21. Donahue LM, Reinhart AJ: POU domain genes are differentially expressed in the early stages after lineage commitment of the PNS-derived stem cell line, RT4-AC. Brain Res Dev Brain Res 1998, 106(1–2):1-12. ( ) 22. Leib DA, Coen DM, Bogard CL, Hicks KA, Yager DR, Knipe DM, Tyler KL, Schaffer PA: Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J Virol 1989, 63(2):759-768. ( ) 23. Cai W, Astor TL, Liptak LM, Cho C, Coen DM, Schaffer PA: The herpes simplex virus type 1 regulatory protein ICP0 enhances virus replication during acute infection and reacti- vation from latency. J Virol 1993, 67(12):7501-7512. 24. Clements GB, Stow ND: A herpes simplex virus type 1 mutant containing a deletion within immediate early gene 1 is latency-competent in mice. J Gen Virol 1989, 70(Pt 9):2501-2506. ) 25. Wang Y, Fischle W, Cheung W, Jacobs S, Khorasanizadeh S, Allis CD: Beyond the double helix: writing and reading the histone code. Novartis Found Symp 2004, 259:3-17. discussion 17–21, 163– 169 26. Authors' contributions Aslani A, Olsson M, Elias P: ATP-dependent unwinding of a min- imal origin of DNA replication by the origin-binding protein and the single-strand DNA-binding protein ICP8 from her- pes simplex virus type I. J Biol Chem 2002, 277(43):41204-41212. p p yp J ( ) 27. Cui C, Griffiths A, Li G, Silva LM, Kramer MF, Gaasterland T, Wang XJ, Coen DM: Prediction and Identification of Herpes Simplex Virus 1-Encoded MicroRNAs. J Virol 2006, 80(11):5499-5508. 28. Amelio AL, McAnany PK, Bloom DC: A chromatin insulator-like element in the herpes simplex virus type 1 latency-associ- ated transcript region binds CCCTC-binding factor and dis- plays enhancer-blocking and silencing activities. J Virol 2006, 80(5):2358-2368. 29. Felsenfeld G, Burgess-Beusse B, Farrell C, Gaszner M, Ghirlando R, Huang S, Jin C, Litt M, Magdinier F, Mutskov V, et al.: Chromatin boundaries and chromatin domains. Cold Spring Harb Symp Quant Biol 2004, 69:245-250. 30. Bedadala GR, Pinnoji RC, Hsia SC: Early growth response gene 1 (Egr-1) regulates HSV-1 ICP4 and ICP22 gene expression. Cell Research 2007, 17(5):1-10. 31. Tapia-Ramirez J, Eggen BJ, Peral-Rubio MJ, Toledo-Aral JJ, Mandel G: A single zinc finger motif in the silencing factor REST represses the neural-specific type II sodium channel pro- moter. Proc Natl Acad Sci USA 1997, 94(4):1177-1182. 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 11 of 11 (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 32. Authors' contributions Hsia SC, Shi YB: Chromatin disruption and histone acetylation in regulation of the human immunodeficiency virus type 1 long terminal repeat by thyroid hormone receptor. Mol Cell Biol 2002, 22(12):4043-4052. Page 11 of 11 (page number not for citation purposes)
https://openalex.org/W2337111012	https://europepmc.org/articles/pmc4853932?pdf=render	English	null	Does Citrulline Have Protective Effects on Liver Injury in Septic Rats?	BioMed research international	2,016	cc-by	6,719	Bin Cai,1 Yu-long Luo,1 Shi-jun Wang,1 Wei-yuan Wei,2 Xue-hui Zhang,3 Wei Huang,1 Tong Li,1 Meng Zhang,1 Nan Wu,1 Gopaul Roodrajeetsing,4 and Sen Zhang1 Bin Cai,1 Yu-long Luo,1 Shi-jun Wang,1 Wei-yuan Wei,2 Xue-hui Zhang,3 Wei Huang,1 Tong Li,1 Meng Zhang,1 Nan Wu,1 Gopaul Roodrajeetsing,4 and Sen Zhang1 Bin Cai,1 Yu-long Luo,1 Shi-jun Wang,1 Wei-yuan Wei,2 Xue-hui Zhang,3 Wei Huang,1 Tong Li,1 Meng Zhang,1 Nan Wu,1 Gopaul Roodrajeetsing,4 and Sen Zhang1 1Department of Colorectal Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, China 2Department of Gastrointestinal Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, China 3Department of Nuclear Medicine, People’s Hospital of Beihai City, Beihai, Guangxi 536000, China 4Department of Neurosurgery, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, China Correspondence should be addressed to Sen Zhang; zs1770@126.com Received 16 January 2016; Revised 29 March 2016; Accepted 31 March 2016 Academic Editor: Anastasia Kotanidou Academic Editor: Anastasia Kotanidou Copyright © 2016 Bin Cai 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. Citrulline (Cit) supplementation was proposed to serve as a therapeutic intervention to restore arginine (Arg) concentrations and improve related functions in sepsis. This study explored whether citrulline had positive effects on liver injury and cytokine release in the early stages of sepsis. The cecal ligation and puncture (CLP) model was utilized in our study. Rats were divided into four groups: normal, Cit, CLP, and CLP+Cit. The CLP group and CLP+Cit group were separated into 6-, 12-, and 24-hour groups, according to the time points of sacrifice after surgery. Intragastric administration of L-citrulline was applied to rats in Cit and CLP+Cit groups before surgery. Serum AST and ALT levels and levels of MDA, SOD, NO, and iNOS in the liver tissues were evaluated. Plasma concentrations of Cit and Arg were assessed using HPLC-MS/MS. Serum concentrations of cytokines and chemokines were calculated by Luminex. Results showed SOD activities of CLP+Cit groups were significantly higher than that of CLP groups, contrasting with the MDA and NO levels which were significantly lower in CLP+Cit groups than in CLP groups. In addition, plasma concentrations of TNF-𝛼, IL-6, and IL-1𝛽were significantly lower in the CLP+Cit 6-hour group than in the CLP 6-hour group. Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 1469590, 8 pages http://dx.doi.org/10.1155/2016/1469590 Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 1469590, 8 pages http://dx.doi.org/10.1155/2016/1469590 Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 1469590, 8 pages http://dx.doi.org/10.1155/2016/1469590 1. Introduction may be due to arginine supplementation having generated a lot of nitric oxide (NO) in the role of inducible nitric oxide synthase (iNOS), leading to a decrease in blood pressure and an increased burden on heart blood vessels, eventually result- ing in a lack of oxygen supply in tissues and organs [7]. L- arginine can serve as a substrate for iNOS, generating NO and L-citrulline. However, the structures of L-citrulline and L- arginine are very similar. If the L-citrulline cannot be cleared in time, the accumulation of L-citrulline may competitively inhibit the enzyme activity center. Furthermore, L-citrulline can be recycled to L-arginine during reactions catalyzed by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). All of the above constitute the so-called “Cit- NO cycle” [8]. Therefore, citrulline supplementation was proposed to serve as a therapeutic intervention to restore arginine concentrations in sepsis. Prospective studies investigating arginine (Arg) metabolism revealed that plasma arginine concentrations decreased sig- nificantly in sepsis patients [1, 2]. A meta-analysis contain- ing 341 patients demonstrated that, compared with control group, plasma concentrations of arginine in sepsis patients decreased by 41% [3]. Therefore, some researchers put for- ward that arginine supplementation could be a logical ther- apeutic option to restore the decreased arginine concentra- tions in sepsis [4]. However, clinical randomized controlled trials showed arginine supplementation had no significant improvement on hospital mortality, infectious complications, or the length of ICU stays in sepsis patients [5]. Another study reported that, for patients with severe sepsis, arginine supple- mentation led to a significantly higher mortality rate [6]. This 2 BioMed Research International Sepsis-associated liver injury is one of the most common complications in sepsis. It has been reported that the inci- dence of sepsis-associated liver injury was 34.7%, and sepsis- associated liver injury is a significant predictive sign of poor prognosis in sepsis patients [9]. One piece of research from France including 206 ICUs showed that in the first 24 hours of ICU stay the incidence of liver damage and liver failure was 46.6% and 6.3%, respectively [10]. The liver is the major organ of substance and energy metabolism, as well as the major source of cytokines. During the early stages of sepsis, the body is at a proinflammatory stage accompanied by the release of a large number of proinflammatory cytokines such as TNF- 𝛼, IL-1𝛽, and IL-6. 2. Materials and Methods 2.1. Animals. 120 male Wistar rats, 7 to 10 weeks old and weighing 250∼300 g, were provided by the Experimental Animal Center of Guangxi Medical University. All rats were housed in animal care facilities and maintained under pathogen-free conditions. Rats were reared in strict accor- dance with standard operating procedures. Ambient temper- ature was kept at 22∼25∘C, humidity at 45∼55%, with a 12- hour light/12-hour dark light cycle. Timely water addition and feeding were managed daily and litter was cleaned once every two days. Rats were euthanized by an intraperitoneal injection of chloral hydrate (700 mg/kg) if they appeared to show agonal breathing or a body weight loss greater than 20%. Animals’ health was specifically monitored and recorded once every six hours. 2.5. MDA, SOD, NO, and iNOS Levels Evaluation. The enzymatic activities of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA) in the liver tissues were evaluated by a microplate reader (SpectraMax Plus 384 MD, USA) using commercial reagent kits from Nanjing Jiancheng Bioengineering Institute. NO levels and iNOS activities in the liver tissues were measured by NO colorimetric assay kit and NOS Assay Kit, respectively (Nanjing Jiancheng Bioengineering Institute, China). 2.5. MDA, SOD, NO, and iNOS Levels Evaluation. The enzymatic activities of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA) in the liver tissues were evaluated by a microplate reader (SpectraMax Plus 384 MD, USA) using commercial reagent kits from Nanjing Jiancheng Bioengineering Institute. NO levels and iNOS activities in the liver tissues were measured by NO colorimetric assay kit and NOS Assay Kit, respectively (Nanjing Jiancheng Bioengineering Institute, China). 2.6. HPLC-MS/MS Assay. Plasma concentrations of arginine and citrulline were assessed by the method of HPLC-MS/MS. All experimental operations were done by China National Analytical Center (Guangzhou, China). HPLC-MS/MS anal- ysis was performed using an Agilent 1200LC/6410B HPLC system (Agilent Technologies Inc., Wilmington, USA). The analytical column was Agilent Poroshell 120 HILIC (2.1 mm × 100 mm, 2.7 𝜇m). The column temperature was room temperature. The chromatographic run was carried out at 0.3 mL/min as the flow rate, with 0.2% aqueous formic acid as mobile phase A and acetonitrile as mobile phase B. The injection volume was 3 𝜇L. The ion source was electrospray ionization ion source (ESI) in the positive mode. 1. Introduction These proinflammatory cytokines could activate a cytokine cascade reaction and lead to “cytokine storm,” aggravating inflammatory and immune response [11]. In our previous research, we provided L-citrulline to cecal ligation and puncture- (CLP-) induced septic rats and found that citrulline supplementation reduced the reaction of proinflammatory mediators [12]. In the present study, through supplying L-citrulline to CLP rats, we tried to explore whether citrulline supplementation had positive effects on liver injury and the release of cytokines in the early stages of sepsis. and disinfecting the abdomen, a 1.5∼2 cm incision on the anterior abdominal wall was performed for exposing and extracting the cecum colon. After gently pushing cecum con- tents to the distal end of the cecum, the distal 1/3 of the cecum was ligated with a 4-0 silk suture and punctured through one time using a 12-gauge needle. A small amount of intestinal contents was gently squeezed from the punctured holes. After suturing the incision, sterile saline solution was offered to rats subcutaneously for resuscitation at a dose of 4 mL/100 g body weight. For the sham group, in addition to the cecal ligation and puncture, the remaining operations were the same as CLP modeling groups. The general condition and survival status of rats were observed once every 6 hours after surgery. 2.4. Sample Collection. For CLP groups and CLP+Cit groups, six surviving rats were randomly selected and sacrificed 6, 12, and 24 hours after surgery. After anesthesia, blood was collected via the abdominal aorta and liver tissues were removed. Tissues were immediately frozen at −80∘C for further analysis. Blood was used to extract serum and plasma. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using an automatic biochemical analyzer (Beckman DXC800, USA). Plasma concentrations of arginine and citrulline were assessed by the method of HPLC-MS/MS. 2. Materials and Methods The drying gas (N2) temperature was 350∘C, atomization gas (N2) pres- sure was 40 psi, drying gas (N2) flow was 10.0 L/min, and the electrospray ionization voltage was 4000 V. Scan mode was multiple reactions monitoring (MRM) mode. 2.2. Study Design. According to a random number table, rats were randomly divided into following groups: normal group (𝑛= 8), Cit group (𝑛= 8), CLP group (𝑛= 52), and CLP+Cit group (𝑛= 52). CLP group and CLP+Cit group were then separated into 6-, 12-, and 24-hour groups, according to the time points of sacrifice after CLP operation, that is, CLP 6 h group (𝑛= 12), CLP 12 h group (𝑛= 16), CLP 24 h group (𝑛= 24), CLP+Cit 6 h group (𝑛= 12), CLP+Cit 12 h group (𝑛= 16), and CLP+Cit 24 h group (𝑛= 24). Intragastric administration of L-citrulline (purity ≥99%, RUIBIO, Germany) was supplied to the rats in Cit group and CLP+Cit groups for 7 days before further operation (a dose of 200 mg kg−1⋅d−1). 2.7. Serum Cytokines and Chemokines Concentrations Mea- surement. Serum concentrations of cytokines and chemo- kines were measured by Milliplex MAP Rat Cytokine/Chemo kine Magnetic Bead Panel (RECYMAG65K27PMX, Milli- pore, USA) following the kit-specific protocols provided by 2.3. CLP Model. The procedure of CLP modeling was per- formed as previously described [12]. Rats were weighed and anesthetized by intraperitoneal injection with 10% chloral hydrate at a dose of 0.35 mL/100 g body weight. After shaving BioMed Research International 3 Table 1: Serum levels of ALT and AST in each group. g p Group ALT (U/L) AST (U/L) 6 h 12 h 24 h 6 h 12 h 24 h Normal 56 ± 8.32 244.17 ± 123.95 Cit 59 ± 9.53 316.67 ± 54.14 CLP 120.5 ± 41.37 169.67 ± 27.83 278.5 ± 84.28 433.00 ± 73.72 631.83 ± 111.96 1231.17 ± 220.86 CLP+Cit 102.67 ± 17.04 158.17 ± 43.72 240 ± 64.15 370.83 ± 167.36 456 ± 129.71∗ 788.83 ± 123.78∗∗ Comparisons between CLP groups and CLP+Cit groups at the same time point; ∗𝑃< 0.05, ∗∗𝑃< 0.01. Table 2: SOD activities and MDA levels in the liver tissues. 2. Materials and Methods Citrulline supplementation remarkably reduced the production of MDA and improved the SOD activities in septic rats. 2.8. Statistical Analysis. All values were showed as mean ± standard deviation (SD). Data was analyzed by chi-square test, Student’s 𝑡-test, and one-way ANOVA. All data was statistically analyzed using SPSS 16.0 software (SPSS, Chicago, USA). 𝑃< 0.05 was regarded as statistically significant. 2. Materials and Methods Group SOD (U/mgprot) MDA (nmol/mgprot) 6 h 12 h 24 h 6 h 12 h 24 h Normal 344.27 ± 26.42 1.09 ± 0.14 Cit 358.05 ± 18.42 1.05 ± 0.17 CLP 285.97 ± 65.10 303.93 ± 13.86 260.67 ± 60.01 1.55 ± 0.17 1.77 ± 0.11 1.97 ± 0.14 CLP+Cit 402.90 ± 30.08∗∗ 358.48 ± 35.83∗ 392.38 ± 37.06∗∗ 1.37 ± 0.26 1.42 ± 0.18∗∗ 1.53 ± 0.29∗∗ Comparisons between CLP groups and CLP+Cit groups at the same time point; ∗𝑃< 0.05, ∗∗𝑃< 0.01. Table 2: SOD activities and MDA levels in the liver tissues. 3.2. Serum Levels of AST and ALT. Table 1 shows the serum levels of ALT and AST in each group. There were no significant differences in serum levels of ALT and AST between the normal and Cit groups (𝑃> 0.05). ALT and AST levels of CLP and CLP+Cit groups were much higher than that of the normal group. In addition, at 6, 12, and 24 hours after surgery, there was no significant difference in serum ALT levels between CLP groups and CLP+Cit groups (all 𝑃> 0.05). However, serum AST levels in CLP+Cit 12- and 24-hour groups were significantly lower than that in CLP 12- and 24-hour groups (both 𝑃< 0.05). Millipore. The kit was used for the simultaneous quantifi- cation of the following cytokines and chemokines: EGF, Eotaxin, Fractalkine, G-CSF, GM-CSF, GRO/KC/CINC-1, IFN-𝛾, IL-1𝛼, IL-1𝛽, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, IL-17A, IL-18, IP-10, Leptin, LIX, MCP-1, MIP-1𝛼, MIP-2, RANTES, TNF-𝛼, and VEGF. 96-well plates were read using Luminex FLEXMAP 3D (Luminex, Austin) with xPONENT 4.2 software (Luminex, Austin). Data was analyzed using Milliplex Analyst software (Millipore, USA). A five-parameter logistic regression model with weighting was utilized to create standard curves. Two quality controls were executed with each assay. 3.3. SOD Activities and MDA Levels in the Liver Tissues. Table 2 shows the SOD activities and MDA levels in the liver tissues of each group. There were no significant differences in SOD activities and MDA levels between the normal group and Cit group (𝑃> 0.05). At 6, 12, and 24 hours after surgery, SOD activities of CLP+Cit groups were significantly greater than that of CLP groups (all 𝑃< 0.05). In contrast, MDA levels in CLP+Cit 12- and 24-hour groups were significantly lower than that in CLP 12- and 24-hour groups (both 𝑃< 0.05). 3. Results 3.1. General Performances and Intra-Abdominal Lesions of Rats. Six∼twelve hours after surgery, all rats from the CLP groups and CLP+Cit groups appeared apathetic and unre- sponsive, showed reduced activity, towering hair, prone and shrinking body, and sticky secretions around the nose and mouth, and were drinking less. Rats displayed drowsiness, indifferent responses to external stimuli, perianal purulent discharge, and hemorrhaging eyes 24 hours after surgery. Intraperitoneal bloody ascites was observed in the CLP model rats, accompanied with stench, intestinal inflation, and cecal ligation segment necrosis. The liver presented edema, congestion, and visible point flaky bleeding in the CLP model rats. The above symptoms were not observed in the normal and Cit groups. In addition, liver morphology was also normal with no pathological changes in the normal and Cit groups. 3.4. NO Levels and iNOS Activities in the Liver Tissues. Table 3 shows the NO levels and iNOS activities in the liver tissues of each group. The normal and Cit groups showed no significant differences in NO levels and iNOS activities (𝑃> 0.05). At 6, 12, and 24 hours after surgery, NO levels in CLP+Cit groups were significantly lower than that in CLP groups (all 𝑃< 0.05). However, at the three time points, there was no significant difference in iNOS activities between CLP groups and CLP+Cit groups (all 𝑃> 0.05). 3.5. Plasma Concentrations of Cit and Arg. As shown in Figure 1(a), after CLP surgery, plasma concentration of Arg BioMed Research International 4 Table 3: NO levels and iNOS activities in the liver tissues. Table 3: NO levels and iNOS activities in the liver tissues. Group NO (nmol/mgprot) iNOS (U/mgprot) 6 h 12 h 24 h 6 h 12 h 24 h Normal 1.53 ± 0.46 0.13 ± 0.04 Cit 1.63 ± 0.46 0.14 ± 0.04 CLP 3.90 ± 1.18 5.04 ± 1.99 5.55 ± 1.55 0.42 ± 0.11 0.41 ± 0.18 0.43 ± 0.18 CLP+Cit 1.93 ± 0.82∗ 2.73 ± 1.53∗∗ 3.41 ± 0.94∗∗ 0.34 ± 0.08 0.33 ± 0.11 0.38 ± 0.06 Comparisons between CLP groups and CLP+Cit groups at the same time point; ∗𝑃< 0.05, ∗∗𝑃< 0.01. 3. Results Plasma concentrations of Cit and Baseline (Cit group) 6h 12h 24h 0 50 100 150 200 250 300 350 400 450 Arg in CLP+Cit groups (mg/L) Cit Arg (b) Plasma concentrations of Cit and Baseline (Cit group) 6h 12h 24h 0 50 100 150 200 250 300 350 400 450 Arg in CLP+Cit groups (mg/L) Plasma concentrations of Cit Baseline (normal group) 6h 12h 24h 0 2 4 6 8 10 12 14 16 18 and Arg in CLP groups (mg/L) ∗∗ (b) (a) Figure 1: Plasma concentrations of Cit and Arg in CLP groups and CLP+Cit groups. (a) Plasma Arg concentration of CLP 24-hour group was significantly lower than the baseline (normal group) (∗∗𝑃< 0.01). (b) Plasma Arg concentration of CLP+Cit 24-hour group remained near the baseline (Cit group). VEGF, IL-13, and Fractalkine) peaked earlier in the CLP group compared with CLP+Cit group. Only two cytokines (GM-CSF and IP-10) peaked earlier in CLP+Cit group. The remaining five kinds of cytokines and chemokines (IL-1𝛼, GRO/KC/CINC-1, LIX, RANTES, and IL-18) reached peak at the same time point in CLP group and CLP+Cit group. This may be due to citrulline supplementation postponing the releases of cytokines and chemokines in the early stages of sepsis. In addition, it was noteworthy that concentrations of proinflammatory cytokines TNF-𝛼, IL-6, and IL-1𝛽were significantly lower in the CLP+Cit 6-hour group than in the CLP 6-hour group (all 𝑃< 0.05). Citrulline supplementation reduced the release of TNF-𝛼, IL-6, and IL-1𝛽in the early stages of sepsis (Figure 2). declined in CLP groups. At 24 hours after surgery, plasma Arg concentration of CLP 24-hour group was significantly decreased compared with the baseline (𝑃< 0.01). Interest- ingly, in the first 24 hours after surgery, when compared to the baseline plasma Cit concentrations increased slightly in CLP groups, but without statistical significance. In our study, citrulline supplementation remarkably increased plasma con- centrations of Arg and Cit in CLP+Cit groups. In contrast to CLP groups, Cit concentrations declined in CLP+Cit groups. In addition, plasma Arg concentration of CLP+Cit 24-hour group remained near the baseline 24 hours after surgery. Our results suggested that citrulline supplementation alleviated the lack of arginine in CLP-induced septic rats. 3.6. Serum Cytokines and Chemokines Concentrations. Table 4 shows the serum concentrations of cytokines and chemokines in each group. 3. Results Serum concentrations of all cytokines and chemokines were higher in the CLP 6-hour group than that in the normal group, wherein 21 kinds of cytokines and chemokines increased significantly (𝑃< 0.05), including EGF, Eotaxin, G-CSF, GM-CSF, GRO/KC/CINC, IFN-𝛾, IL-1𝛼, IL-1𝛽, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, IP-10, Leptin, MCP-1, MIP-1𝛼, MIP-2, RANTES, and TNF-𝛼, suggesting that our modeling was satisfactory. Using mean values as references, in the first 24 hours after surgery, concentrations of 20 kinds of cytokines and chemokines (EGF, Eotaxin, G-CSF, IL-1𝛽, IL-2, IL-4, IL-5, IL-6, IL-10, IL- 12(p70), IFN-𝛾, IL-17, Leptin, MCP-1, MIP-1𝛼, MIP-2, TNF-𝛼, 3. Results Plasma concentrations of Cit Baseline (normal group) 6h 12h 24h 0 2 4 6 8 10 12 14 16 18 and Arg in CLP groups (mg/L) Cit Arg ∗∗ (a) Plasma concentrations of Cit and Baseline (Cit group) 6h 12h 24h 0 50 100 150 200 250 300 350 400 450 Arg in CLP+Cit groups (mg/L) Cit Arg (b) Figure 1: Plasma concentrations of Cit and Arg in CLP groups and CLP+Cit groups. (a) Plasma Arg concentration of CLP 24-hour group was significantly lower than the baseline (normal group) (∗∗𝑃< 0.01). (b) Plasma Arg concentration of CLP+Cit 24-hour group remained near the baseline (Cit group). Group NO (nmol/mgprot) iNOS (U/mgprot) 6 h 12 h 24 h 6 h 12 h 24 h Normal 1.53 ± 0.46 0.13 ± 0.04 Cit 1.63 ± 0.46 0.14 ± 0.04 CLP 3.90 ± 1.18 5.04 ± 1.99 5.55 ± 1.55 0.42 ± 0.11 0.41 ± 0.18 0.43 ± 0.18 CLP+Cit 1.93 ± 0.82∗ 2.73 ± 1.53∗∗ 3.41 ± 0.94∗∗ 0.34 ± 0.08 0.33 ± 0.11 0.38 ± 0.06 Comparisons between CLP groups and CLP+Cit groups at the same time point; ∗𝑃< 0.05, ∗∗𝑃< 0.01. Group NO (nmol/mgprot) iNOS (U/mgprot) 6 h 12 h 24 h 6 h 12 h 24 h Normal 1.53 ± 0.46 0.13 ± 0.04 Cit 1.63 ± 0.46 0.14 ± 0.04 CLP 3.90 ± 1.18 5.04 ± 1.99 5.55 ± 1.55 0.42 ± 0.11 0.41 ± 0.18 0.43 ± 0.18 CLP+Cit 1.93 ± 0.82∗ 2.73 ± 1.53∗∗ 3.41 ± 0.94∗∗ 0.34 ± 0.08 0.33 ± 0.11 0.38 ± 0.06 Comparisons between CLP groups and CLP+Cit groups at the same time point; ∗𝑃< 0.05, ∗∗𝑃< 0.01. Plasma concentrations of Cit Baseline (normal group) 6h 12h 24h 0 2 4 6 8 10 12 14 16 18 and Arg in CLP groups (mg/L) Cit Arg ∗∗ (a) Plasma concentrations of Cit and Baseline (Cit group) 6h 12h 24h 0 50 100 150 200 250 300 350 400 450 Arg in CLP+Cit groups (mg/L) Cit Arg (b) Figure 1: Plasma concentrations of Cit and Arg in CLP groups and CLP+Cit groups. (a) Plasma Arg concentration of CLP 24-hour group was significantly lower than the baseline (normal group) (∗∗𝑃< 0.01). (b) Plasma Arg concentration of CLP+Cit 24-hour group remained near the baseline (Cit group). 4. Discussion Sepsis is a complex syndrome with a variety of clinical manifestations. The cecal ligation and puncture method used in our study has been recognized as the “gold standard” in sepsis research throughout the past three decades [13]. Inflammation can induce changes in the amino acid profile, which is pronounced in sepsis. A previous study measured the changes in the plasma amino acid concentrations of sepsis patients and found that citrulline, glutamate, and arginine showed the greatest decreases when compared with concentrations in healthy control subjects. The decreased degrees of citrulline, glutamate, and arginine were 56%, 48%, BioMed Research International 5 Table 4: Serum concentrations of cytokines and chemokines in each group (pg/mL). 4. Discussion At 6 hours after surgery, concentrations of TNF-𝛼, IL-6, and IL-1𝛽were significantly lower in CLP+Cit 6-hour group than CLP 6-hour group; ∗𝑃< 0.05, ∗∗𝑃< 0.01. used indicators of hepatocellular damage. The distributions of ALT and AST in the liver cells are different. ALT are mainly distributed in the cytoplasm of hepatocytes; however, AST are mainly distributed in the mitochondria. In our study, ALT and AST levels of CLP and CLP+Cit groups were significantly higher than that of the normal group, indicating that hep- atocytes were damaged in CLP modeling rats. There were no statistical differences in serum ALT levels between CLP and CLP+Cit groups, suggesting that the degree of damage to the integrity of hepatocytes in both groups was about the same. However, serum AST levels of CLP+Cit groups were significantly lower than that of CLP groups, indicating that the degree of damage to the integrity of mitochondria in CLP+Cit groups was much lighter than that in CLP groups. Citrulline supplementation reduced mitochondrial damage in septic rats. SOD is one of the common antioxidants in hepatocytes; our results showed SOD activities in CLP+Cit groups were significantly higher than that in CLP groups, suggesting that citrulline supplementation enhanced SOD activities in sepsis. MDA is one of the cell membrane lipid peroxidation end-products, which can reflect the degree of membrane lipid peroxidation and the damage extent of cell membranes. In our study, MDA levels in CLP+Cit groups were significantly lower than that in CLP groups, suggesting that citrulline supplementation reduced the cell membrane lipid peroxidation.hf IL-1𝛽 IL-6 TNF-𝛼 ∗ and IL-1𝛽in CLP 6h group and 1 10 100 1000 10000 100000 CLP+Cit 6h group (pg/mL) ∗∗ ∗∗ Serum concentrations of TNF-𝛼, IL-6 Figure 2: Serum concentrations of TNF-𝛼, IL-6, and IL-1𝛽in CLP+Cit 6-hour and CLP 6-hour groups. At 6 hours after surgery, concentrations of TNF-𝛼, IL-6, and IL-1𝛽were significantly lower in CLP+Cit 6-hour group than CLP 6-hour group; ∗𝑃< 0.05, ∗∗𝑃< 0.01. and 47%, respectively [2]. Arginine is a nonessential amino acid which can be derived from daily dietary intake, de novo synthesis from citrulline, and protein breakdown [4]. Arginine is involved in protein synthesis and secretion of NO, creatine, urea, and amines. In addition, arginine plays an important role in cell regeneration, wound healing, cellular immune function, protein conversion, and the maintenance of positive nitrogen balance [14]. 4. Discussion In sepsis, a decline in argi- nine intake, accompanied with an impaired arginine de novo synthesis and reduced protein intake and absorption, causes endogenous arginine production to only 30% of normal levels [15]. In addition, lipopolysaccharide can promote the release of iNOS and accelerate the consumption of arginine in the development of sepsis [16].h p p The liver has a large number of Kupffer cells, which account for about 80% of the total fixed macrophage pop- ulation [19]. Bacteria and endotoxins can activate Kupffer cells, inducing excessive releases of oxygen radicals, NO, and inflammatory cytokines such as IL-1, IL-6, and TNF-𝛼[20]. Oxygen radicals can attack cell membranes and induce lipid peroxidation, generate a large number of metabolites, and cause damage to the integrity of membranes, leading to cell and mitochondrial swelling and resulting in liver damage eventually [21]. NO is a freely diffusible small molecule. The half-life of NO is only a few seconds, but NO can react with superoxide anion fast, forming peroxynitrite which can cause damage to cells [22]. The mass production of NO in sepsis can lead to hepatic microcirculation disorders. NO can combine with mitochondrial cytochrome oxidase and inhibit its activity, blocking the electron transfer chain and resulting in the formation of superoxide [23]. Mitochondrial damage can induce the accumulation of neutrophils in the liver, forming a vicious cycle. In addition, NO can reduce the production rate of glycogen in hepatocytes and result in hypoglycemia in sepsis [24]. Our results showed NO levels of CLP+Cit groups were significantly lower than that of CLP groups, suggesting that citrulline supplementation reduced the production of NO in septic rats. In our study, there was no statistically significant difference in iNOS activities between CLP+Cit groups and CLP groups, but lower iNOS activities were detected in CLP+Cit groups compared with CLP groups. The iNOS activity values in CLP 6-, 12-, and 24-hour groups were 0.42 ± 0.11, 0.41 ± 0.18, and 0.43 ± 0.18, respectively. However, values in CLP+Cit 6-, 12-, and 24-hour groups were 0.34 ± 0.08, 0.33 ± 0.11, and 0.38 ± 0.06, respectively. This may partly explain why less NO was produced in CLP+Cit groups. There has been no doubt that citrulline supplementation could enhance plasma and tissue arginine concentrations. 4. Discussion Analyte Normal group Cit group CLP group CLP+Cit group 6 h 12 h 24 h 6 h 12 h 24 h G-CSF Undetected Undetected 8.75 ± 2.38 13.59 ± 2.01󳵳 13.08 ± 0.08 4.72 ± 3.92 4.03 ± 1.62 21.38 ± 0.71󳵳 Eotaxin 6.65 ± 2.83 3.78 ± 1.57 14.89 ± 4.78󳵳 12.24 ± 7.88 10.49 ± 2.99 12.22 ± 2.59 12.46 ± 5.30 17.35 ± 4.95󳵳 GM-CSF Undetected Undetected 98.58 ± 92.66 105.45 ± 86.76󳵳 72.70 ± 22.15 82.70 ± 53.41󳵳 63.39 ± 36.31 49.49 ± 23.77 IL-1𝛼 3.10 ± 2.19 Undetected 55.67 ± 26.83󳵳 23.86 ± 9.70 41.49 ± 24.61 53.31 ± 48.32󳵳 26.85 ± 22.37 51.55 ± 21.02 Leptin 2810.2 ± 1184.97 2123.75 ± 699.71 20809.5 ± 7712.10󳵳 15190.6 ± 7086.61 6007.75 ± 4217.84 8735.2 ± 1996.72 9794 ± 5442.86 18706.6 ± 13241.32󳵳 MIP-1𝛼 32.67 ± 11.55 26.36 ± 3.51 513.14 ± 287.45󳵳 127.97 ± 87.43 36.86 ± 12.72 205.86 ± 88.74 183.71 ± 193.66 251.80 ± 196.25󳵳 IL-4 Undetected Undetected 29.49 ± 22.55󳵳 19.71 ± 20.95 8.18 ± 12.72 24.60 ± 11.00 31.65 ± 21.72 39.54 ± 19.73󳵳 IL-1𝛽 49.28 ± 42.19 44.16 ± 14.61 652.95 ± 54.49󳵳 371.35 ± 289.02 301.4 ± 234.89 233.07 ± 45.24 152.92 ± 117.18 278.02 ± 185.33󳵳 IL-2 37.92 ± 17.30 25.92 ± 6.67 170.84 ± 75.04󳵳 71.37 ± 68.65 65.11 ± 25.37 63.95 ± 33.33 94.15 ± 2.50󳵳 92.90 ± 63.45 IL-6 120.95 ± 25.71 64.18 ± 43.70 30070 ± 20440.72󳵳 1954.90 ± 923.72 1293.21 ± 934.89 2097.75 ± 2727.34 1688.46 ± 1826.3 21660.26 ± 27160.17󳵳 EGF 0.71 ± 0.177 Undetected 2.22 ± 0.35󳵳 1.13 ± 1.88 1.59 ± 1.33 0.11 ± 0.08 0.85 ± 1.21 1.40 ± 1.33󳵳 IL-13 19.14 ± 3.90 10.29 ± 5.82 22.72 ± 12.89 22.83 ± 9.28󳵳 15.98 ± 3.83 24.38 ± 4.70 20.37 ± 9.06 31.31 ± 13.92󳵳 L-10 53.06 ± 23.80 62.83 ± 28.28 2254.67 ± 1025.29󳵳 2067.44 ± 2493.23 502.68 ± 208.96 321.36 ± 65.49 725.92 ± 613.66 2459.21 ± 1949.54󳵳 IL-12(p70) 77.31 ± 22.39 77.80 ± 0.76 193.73 ± 124.92󳵳 148.91 ± 88.35 58.43 ± 64.20 111.88 ± 34.80 108.50 ± 47.57 168.61 ± 56.16󳵳 IFN-𝛾 56.042 ± 23.74 43.32 ± 15.73 125.1 ± 52.58󳵳 102.26 ± 42.89 85.74 ± 37.04 87.24 ± 39.86 98.87 ± 52.01 120.82 ± 57.06󳵳 IL-5 58.71 ± 21.54 50.30 ± 18.33 127.96 ± 30.39 132.35 ± 28.57󳵳 107.46 ± 10.24 105.54 ± 16.63 122.15 ± 26.10 140.97 ± 18.23󳵳 IL-17A 8.35 ± 3.91 3.70 ± 2.09 36.44 ± 30.49󳵳 29.68 ± 20.33 14.53 ± 4.73 19.65 ± 6.78 15.37 ± 3.42 30.52 ± 13.88󳵳 IL-18 136.57 ± 42.88 157.14 ± 85.53 471.45 ± 354.16 436.92 ± 374.40 640.50 ± 669.11󳵳 453.14 ± 383.18 214.26 ± 197.21 459.29 ± 305.39󳵳 MCP-1 855.72 ± 309.68 750.93 ± 219.38 7939.5 ± 5748.21 8296.6 ± 7103.81󳵳 5456.4 ± 2145.50 5685.4 ± 2610.89 7344.4 ± 2128.33 7420 ± 3687.26󳵳 IP-10 291.16 ± 76.83 241.01 ± 62.06 921.03 ± 458.42 979.95 ± 352.73󳵳 601.56 ± 241.94 867.48 ± 143.13󳵳 653.79 ± 180.21 822.54 ± 274.09 GRO/KC 12.23 ± 8.76 17.15 ± 6.99 16361 ± 14266.49󳵳 5538.8 ± 5664.49 3875 ± 3330.03 6199.4 ± 4313.97󳵳 4605.4 ± 2235.93 3894.75 ± 3154.25 VEGF 52.39 ± 21.31 52.0 ± 8.25 139.35 ± 111.78󳵳 97.94 ± 71.38 111.59 ± 93.36 94.86 ± 21.80 63.03 ± 21.77 106.72 ± 57.30󳵳 Fractalkine 80.86 ± 31.82 60.21 ± 10.99 131.05 ± 80.13 134.01 ± 135.06󳵳 73.80 ± 31.21 118.60 ± 40.96 69.76 ± 19.78 128.01 ± 75.52󳵳 LIX 2671 ± 172.81 2458.5 ± 347.71 3629.25 ± 859.08󳵳 3116.4 ± 983.28 3111.4 ± 1181.79 3815.6 ± 1137.01󳵳 3286.4 ± 796.47 3307.8 ± 796.00 MIP-2 91.92 ± 31.31 65.76 ± 16.40 5966 ± 3067.43󳵳 621.04 ± 280.54 371.54 ± 380.89 1844.39 ± 1099.42 840.10 ± 547.80 3875.67 ± 2332.24󳵳 TNF-𝛼 8.76 ± 1.17 5.35 ± 3.98 46.40 ± 20.48󳵳 40.76 ± 48.68 13.26 ± 1.40 20.02 ± 5.13 25.33 ± 17.72 49.52 ± 38.76󳵳 RANTES 2235.8 ± 385.35 2161 ± 537.21 3981.25 ± 779.13󳵳 2488.4 ± 1003.48 2629.4 ± 876.18 3949 ± 1156.80󳵳 3259.2 ± 1015.13 2318.6 ± 1027.21 Serum concentrations of cytokines and chemokines were undetected if less than the minimum detectable concentration; 󳵳represented the concentration peaks of cytokines and chemokines within 24 hours after surgery in CLP group and CLP+Cit group BioMed Research International 6 IL-1𝛽 IL-6 TNF-𝛼 ∗ and IL-1𝛽in CLP 6h group and 1 10 100 1000 10000 100000 CLP+Cit 6h group (pg/mL) ∗∗ ∗∗ CLP+Cit CLP Serum concentrations of TNF-𝛼, IL-6, Figure 2: Serum concentrations of TNF-𝛼, IL-6, and IL-1𝛽in CLP+Cit 6-hour and CLP 6-hour groups. 4. Discussion At the beginning of the century, citrulline as a precursor amino acid of arginine had been given orally in a clinical trial, resulting in elevated plasma levels of arginine [17]. A subsequent study further demonstrated that oral citrulline supplementation raised plasma arginine concentrations in a dose-dependent manner [18]. In our previous study, we found that serum arginine concentrations were greater in the citrulline-supplemented rats compared to rats on a standard “rat chow” diet [12]. Therefore, citrulline supplementation has been seen as an effective method to improve arginine concentrations. In our study, plasma arginine concentrations decreased in CLP groups but remained at the baseline in CLP+Cit groups, suggesting that citrulline supplementation alleviated the lack of arginine in CLP-induced septic rats. Interestingly, citrulline concentrations in CLP groups slightly increased in the first 24 hours after surgery, but without statistical significances. This may be due to the monitoring period of our study, which was only 24 hours long.h The liver is one of the most vulnerable organs in sepsis. The mechanism of liver injury in sepsis is complex, involving hepatic microcirculation disorders, releases of inflammatory mediators, activation of Kupffer cells, induced apoptosis of liver cells, and so forth. ALT and AST are the most commonly 7 BioMed Research International BioMed Research International Proinflammatory cytokines TNF-𝛼, IL-1𝛽, and IL-6 are produced in the initial phase of sepsis. They can stimulate the innate or adaptive immune response, characterized by further production of many kinds of cytokines, exacerbating the inflammatory response [25]. In addition, TNF-𝛼and IL- 1𝛽can enhance the expressions of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), aggravating hepatic microcirculation disorders [26]. IL-1𝛽and TNF-𝛼can also increase integrin adhesiveness in neutrophils and promote their extravasation into tissues, causing organ damage [27]. Moreover, the combination of TNF-𝛼and TNF receptor-1 can induce apoptosis of hepatocytes through multiple apoptotic pathways [28]. In our study, concentrations of TNF-𝛼, IL- 6, and IL-1𝛽were significantly lower in the CLP+Cit 6-hour group than in the CLP 6-hour group. This suggested that citrulline supplementation reduced the release of TNF-𝛼, IL- 6, and IL-1𝛽in the early stages of sepsis. In addition, Luminex results showed concentrations of 20 kinds of cytokines and chemokines reached peak earlier in CLP groups compared to CLP+Cit groups, whereas only two cytokines peaked earlier in CLP+Cit groups. 5. Conclusions In conclusion, our study revealed that citrulline supplementa- tion reduced the damage to mitochondria in hepatocytes. In addition, citrulline supplementation reduced the cell mem- brane lipid peroxidation and enhanced the SOD activities in septic rats. In the early stages of sepsis, the production of NO was decreased and the release of TNF-𝛼, IL-6, and IL-1𝛽was inhibited by citrulline supplementation. [8] M. Zieli´nska, M. Obara-Michlewska, W. Hilgier, and J. Albrecht, “Citrulline uptake in rat cerebral cortex slices: modulation by Thioacetamide—induced hepatic failure,” Metabolic Brain Disease, vol. 29, no. 4, pp. 1053–1060, 2014. [9] H. Kobashi, J. Toshimori, and K. Yamamoto, “Sepsis-associated liver injury: incidence, classification and the clinical signifi- cance,” Hepatology Research, vol. 43, no. 3, pp. 255–266, 2013. [10] C. Brun-Buisson, P. Meshaka, P. Pinton, and B. Vallet, “EPISEP- SIS: a reappraisal of the epidemiology and outcome of severe sepsis in French intensive care units,” Intensive Care Medicine, vol. 30, no. 4, pp. 580–588, 2004. Ethical Approval This study was carried out in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technol- ogy of China. The experimental procedures were approved by the Ethics Committee of the Institute of Guangxi Medical University (Approval no. 2015 (K-Y-E-005)). [11] T. S. Blackwell and J. W. Christman, “Sepsis and cytokines: current status,” British Journal of Anaesthesia, vol. 77, no. 1, pp. 110–117, 1996. [12] T. Asgeirsson, S. Zhang, R. Nunoo et al., “Citrulline: a potential immunomodulator in sepsis,” Surgery, vol. 150, no. 4, pp. 744– 751, 2011. [13] K. A. Wichterman, A. E. Baue, and I. H. Chaudry, “Sepsis and septic shock—a review of laboratory models and a proposal,” The Journal of Surgical Research, vol. 29, no. 2, pp. 189–201, 1980. References [1] Z. Argaman, V. R. Young, N. Noviski et al., “Arginine and nitric oxide metabolism in critically ill septic pediatric patients,” Critical Care Medicine, vol. 31, no. 2, pp. 591–597, 2003. [2] Y. C. Luiking, M. Poeze, G. Ramsay, and N. E. P. Deutz, “Reduced citrulline production in sepsis is related to dimin- ished de novo arginine and nitric oxide production,” The American Journal of Clinical Nutrition, vol. 89, no. 1, pp. 142– 152, 2009. [3] J. S. Davis and N. M. Anstey, “Is plasma arginine concentration decreased in patients with sepsis? A systematic review and meta-analysis,” Critical Care Medicine, vol. 39, no. 2, pp. 380– 385, 2011. [4] K. A. P. Wijnands, T. M. R. Castermans, M. P. J. Hommen, D. M. Meesters, and M. Poeze, “Arginine and citrulline and the immune response in sepsis,” Nutrients, vol. 7, no. 3, pp. 1426– 1463, 2015. [5] G. Bertolini, G. Iapichino, D. Radrizzani et al., “Early enteral immunonutrition in patients with severe sepsis: results of an interim analysis of a randomized multicentre clinical trial,” Intensive Care Medicine, vol. 29, no. 5, pp. 834–840, 2003. [6] H. Kieft, A. N. Roos, J. D. E. Van Drunen, A. J. G. H. Bindels, J. G. Bindels, and Z. Hofman, “Clinical outcome of immunonutrition in a heterogeneous intensive care population,” Intensive Care Medicine, vol. 31, no. 4, pp. 524–532, 2005. [7] J. A. Lorente, M. A. Delgado, C. Tejedor et al., “Modulation of systemic hemodynamics by exogenous L-arginine in normal and bacteremic sheep,” Critical Care Medicine, vol. 27, no. 11, pp. 2474–2479, 1999. 4. Discussion We estimated that citrulline supplemen- tation postponed the release of cytokines and chemokines in the early stages of sepsis. The authors declare that they have no competing interests. The authors declare that they have no competing interests. [15] S. Villalpando, J. Gopal, A. Balasubramanyam, V. P. Bandi, K. Guntupalli, and F. Jahoor, “In vivo arginine production and intravascular nitric oxide synthesis in hypotensive sepsis,” The American Journal of Clinical Nutrition, vol. 84, no. 1, pp. 197–203, 2006. Competing Interests h [14] G. Wu, F. W. Bazer, T. A. Davis et al., “Arginine metabolism and nutrition in growth, health and disease,” Amino Acids, vol. 37, no. 1, pp. 153–168, 2009. Acknowledgments The authors thank all their colleagues working in the Department of Colorectal Surgery, First Affiliated Hospital of Guangxi Medical University, and thank Abi, Gopaul, and Adeepika for their help on the language issues. This paper was supported by the National Natural Science Foundation of China (Grant no. 81160235). [16] A. Husson, C. Brasse-Lagnel, A. Fairand, S. Renouf, and A. Lavoinne, “Argininosuccinate synthetase from the urea cycle to the citrulline-NO cycle,” European Journal of Biochemistry, vol. 270, no. 9, pp. 1887–1899, 2003. BioMed Research International 8 [17] W. H. Waugh, C. W. Daeschner III, B. A. Files, M. E. McConnell, and S. E. Strandjord, “Oral citrulline as arginine precursor may be beneficial in sickle cell disease: early phase two results,” Journal of the National Medical Association, vol. 93, no. 10, pp. 363–371, 2001. [18] E. Schwedhelm, R. Maas, R. Freese et al., “Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L- arginine: impact on nitric oxide metabolism,” British Journal of Clinical Pharmacology, vol. 65, no. 1, pp. 51–59, 2008. [19] H. Nakashima, Y. Ogawa, S. Shono et al., “Activation of CD11b+ Kupffer cells/macrophages as a common cause for exacerba- tion of TNF/Fas-ligand-dependent hepatitis in hypercholes- terolemic mice,” PLoS ONE, vol. 8, no. 1, Article ID e49339, 2013. [20] P. Walbrun, C. Hellerbrand, T. S. Weiss et al., “Characterization of rat and human Kupffer cells after cryopreservation,” Cryobi- ology, vol. 54, no. 2, pp. 164–172, 2007. [21] E. C. Rackow, M. E. Astiz, and M. H. Weil, “Cellular oxygen metabolism during sepsis and shock. The relationship of oxygen consumption to oxygen delivery,” The Journal of the American Medical Association, vol. 259, no. 13, pp. 1989–1993, 1988. [22] P. O’Reilly, J. M. Hickman-Davis, P. McArdle, K. R. Young, and S. Matalon, “The role of nitric oxide in lung innate immunity: modulation by surfactant protein-A,” Molecular and Cellular Biochemistry, vol. 234-235, no. 1-2, pp. 39–48, 2002. [23] G. C. Brown, “Nitric oxide and mitochondrial respiration,” Biochimica et Biophysica Acta, vol. 1411, no. 2-3, pp. 351–369, 1999. [24] S. Anavi, M. Hahn-Obercyger, R. Margalit, Z. Madar, and O. Tirosh, “A novel antihypoglycemic role of inducible nitric oxide synthase in liver inflammatory response induced by dietary cholesterol and endotoxemia,” Antioxidants & Redox Signaling, vol. 19, no. 16, pp. 1889–1901, 2013. [25] S. Zanotti, A. Kumar, and A. Kumar, “Cytokine modulation in sepsis and septic shock,” Expert Opinion on Investigational Drugs, vol. 11, no. 8, pp. Acknowledgments 1061–1075, 2002. [26] D. Coquerel, R. Neviere, E. Delile et al., “Gene deletion of protein tyrosine phosphatase 1B protects against sepsis- induced cardiovascular dysfunction and mortality,” Arterioscle- rosis, Thrombosis, and Vascular Biology, vol. 34, no. 5, pp. 1032– 1044, 2014. [27] M. Schouten, W. J. Wiersinga, M. Levi, and T. van der Poll, “Inflammation, endothelium, and coagulation in sepsis,” Jour- nal of Leukocyte Biology, vol. 83, no. 3, pp. 536–545, 2008. [28] W.-X. Ding and X.-M. Yin, “Dissection of the multiple mecha- nisms of TNF-𝛼-induced apoptosis in liver injury,” Journal of Cellular and Molecular Medicine, vol. 8, no. 4, pp. 445–454, 2004.
https://openalex.org/W4386001305	https://ijsr.internationaljournallabs.com/index.php/ijsr/article/download/1388/913	English	null	Scope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta	Journal of Social Research	2,023	cc-by-sa	2,813	SCOPE MANAGEMENT ANALYSIS OF CONSTRUCTION WORK FOR MIX-USED BUILDING PROJECTS IN THE CITY OF JAKARTA Nanang Hidayat1, Manlian R. A. Simanjuntak2* 1,2Universitas Pelita Harapan * hidnanang@gmail.com ** manlian.adventus@uph.edu , , , on eywords Scope management, project planning and implementation, scope of work control, project completion performance, ABC Building project. This article is licensed under CC BY-SA 4.0 This article is licensed under CC BY-SA 4.0 P-ISSN: 2827-9832 E-ISSN: 2828-335x http://ijsr.internationaljournallabs.com/index.php/ijsr SCOPE MANAGEMENT ANALYSIS OF CONSTRUCTION WORK FOR MIX-USED BUILDING PROJECTS IN THE CITY OF JAKARTA P-ISSN: 2827-9832 E-ISSN: 2828-335x http://ijsr.internationaljournallabs.com/index.php/ijsr P-ISSN: 2827-9832 E-ISSN: 2828-335x http://ijsr.internationaljournallabs.com/index.php/ijsr eywords Scope management, project planning and implementation, scope of work control, project completion performance, ABC Building project. INTRODUCTION Construction project work often faces problems in managing certain stages of the work process. Management of the scope of construction work in a construction project contract is very important, considering that it can affect the process of future project implementation and the results of the construction work. So from this, every process of carrying out construction work must be determined in detail, and also the distribution of persons in charge of each work item, because if there are additional items during the construction process it can result in delays in completing a job (Susila & Handoyo, 2019). Apart from these impacts, it is possible that there will be a domino effect occurs, namely a delay that occurs in a work item can cause other work items to also be delayed so that it requires a lot of additional time besides that it can also increase work costs when carrying out work. In this case, the contractor, namely the service provider, will be greatly disadvantaged by the additional costs that can occur during the implementation of construction work, besides that other parties, namely the project owner (owner) can also receive the impact of losses due to delays in completing construction project work (Lambropoulos, 2013). Determining the scope of work in detail in the process of preparing tender documents can help the actual process of carrying out construction work so that each stage of construction implementation remains appropriate and is still within the scope of the work contract that has been made (Azevedo et al., 2014; Sutrisna et al., 1970). Therefore, every detailed work item 3238 cope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta must be recorded in detail and can be understood by all parties involved in the project so as not to cause additional work items and deviate from the initial contract which can burden the project cost (Miri & Khaksefidi, 2015). The project, which has a total of 26 floors, applies a design and build contract system. The characteristics of this type of design and build contract offer a practical system in which the planning stages of a project are still running concurrently with the construction implementation process in the field (Iqbal et al., 2015). METHOD This research was conducted using quantitative and qualitative analysis methods with a retrieval system from several existing literature sources and there is a specific link to the research studies made, to determine significant variables in determining factors relevant to the management of the scope of construction work of building construction projects ABC of the actual implementation of work in the field so that it has an impact on the completion time of construction projects (Nduhura et al., 2021). INTRODUCTION This study refers to the basic knowledge in the scope management discussion science chapter contained in the PMBOK (Project Management Body of Knowledge) and refers to the FIDIC (International Federation of Consulting Engineers) construction contract book literature as reference material and considerations in preparing this research study(Kongsong, 2017; Schoonwinkel et al., 2016). In the preparation of this research, an analysis of the control of the scope of the construction work contract was carried out on the actual results of the construction work in the field by taking a case study on the construction project of one of the government-owned high-rise buildings in the city of Jakarta (Kazemi et al., 2018). In the analysis of the study in this study, it was carried out at each stage that was directly involved in the project life cycle process, the components in which were related to construction actors which were reviewed from the perspective of the owner and contractor. It can be estimated that changes to the scope of work may occur from the side of the owner, consultant or contractor, thereby affecting the initial project contract (Liu et al., 2020). This study aims to get answers to research questions and is expected to be a means for improvement in the future. So the purpose of this research is as follows, knowing how construction work management can be arranged and implemented in the implementation of the project being studied. Knowing what factors and variables are involved in making management of the scope of construction work on this project. Research Process The research process was carried out in several stages. This stage starts from the first activity in determining the topic of discussion to the final stage of the conclusion. The research process involves the instruments used in answering each research problem as follows: 1. Secondary Data, namely data that has been processed beforehand. Secondary data sources are journals, books, government publications, and other supporting sources. Secondary data is the source comes from previous researchers. 1. Secondary Data, namely data that has been processed beforehand. Secondary data sources are journals, books, government publications, and other supporting sources. Secondary data is the source comes from previous researchers. 2. In obtaining data on these factors and variables, journal data and previous literature relevant to the topic were first used, then statistical tests were analyzed using the SPSS application as the calculation. Vol.2, No.9, August 2023 3239 Scope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta Secondary Data Secondary data is data obtained through collection from existing sources such as books, reports, electronic media, and previous research results. In this study, secondary data related to the management of control over the scope of construction work as well as schedule control management in the implementation of the ABC building construction project in the city of Jakarta. Research Methods The research begins with problems regarding the process of planning and controlling the scope of construction work in multi-story building construction projects carried out in the field which is the basis of the research, then continued with the research hypothesis which explains that there are factors and variables that influence the preparation of work details in the planning documents for the scope of construction work (Rose & Indelicato, 2009). Research Instruments The research instrument is a tool used to measure the variables studied with the aim of producing accurate quantitative data, so each instrument must have a scale. Sources of data in this study used 2 types of data, namely primary data and secondary data. Primary data Primary data can be explained that primary sources are data sources that directly provide data to data collectors. Primary data collection in this study was done by collecting data-related information in the process of project implementation and conducting direct interviews with parties related to research, in this case, workers who are contractors and consultants on this project. The research respondents came from parties involved in the project with at least 2 years of experience. The questions asked in the interview process were also prepared based on literature studies, and existing journals that have almost the same rules as this research topic. Secondary Data Management of the scope of construction work in the project Management of the scope of construction work in the project In answering problem 1 in this study, it was used by taking secondary data, which is sourced directly from the original data and documents from the project under study. The case study in this research was carried out in the ABC building construction project located in the City of Jakarta area with the status of project ownership by a government-owned business entity with a design and build contract system. Projects with a value of more than 500 billion are carried out by one of the BUMN contractors and start from April 2021 with a work contract period of 2 years of construction and 1 year of maintenance. Until now, the performance of the implementation of project construction activities has reached a percentage of 75 percent as of December 2022 with a target for completion in April 2023. In the construction of The ABC building project in this study, it was built with a scope of work contract starting from design planning, lower and upper structural work, architectural and landscape work, as well as mechanical electrical, and plumbing work (Kongsong & Pooworakulchai, 2018). In more detail, it is shown in Table 1 the scope of the work contracts in the construction of this project has been approved by various project stakeholders, namely the project owner, MK consultant, and implementing contractor(Muhammad et al., 2019). Vol.2, No.9, August 2023 3240 Scope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta cope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta Table 1 Data on the Scope of Work for the Construction of the ABC Building c es , In the process of carrying out construction activities in the ABC building project, the implementing contractor as a construction service provider applies procedures in planning and controlling the scope of construction work through the process of analyzing activities related to all aspects of construction work items (Halou et al., 2019). In addition, identification and analysis of resource requirements to support project construction implementation so that the project delivery process can run smoothly in accordance with the work contract. Management of the scope of construction work in the project This has been described by the construction team into a Project Management Plan document in which all activity information and descriptions and process plans for the implementation of the construction project have been summarized from start to finish. Factors and Variables of Construction Work Scope Management Vol.2, No.9, August 2023 Vol.2, No.9, August 2023 3241 Scope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta Scope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta From the results of research through a survey of respondent data to parties involved in the construction of the ABC Building project in the city of Jakarta. It can be seen how the process of planning and controlling the management of the scope of construction work in the project under study is as follows: The project implementing party has carried out an identification process related to the type and activity of work items and the service provider has carried out the process of dividing the scope of work according to the sequence and implementation procedures in accordance with the construction work contract agreement. The process of identifying all work items in accordance with the implementation contract has been compiled in the "Project Management Plan" book which contains system information and procedures for all construction work items in the project under study. The process of controlling the scope of construction work is based on tracking the actual construction activities and matching them with the baseline documents listed in the "Project Management Plan". Furthermore, to find out the factors and variables for compiling the scope of construction work, therefore there is data that was successfully obtained from the process of distributing questionnaires to as many as 40 respondents and strengthened by reference analysis relevant to this research to produce data that can be seen that there are 6 factors and 50 variables which may affect the management of the scope of construction work for the ABC Building project. The following are strong variables influencing the management of construction work scope management. Table 4 Selected Variables Management of Construction Project Scope of Work X3 X24 X16 X27 X42 X45 Of the six variables formed in the results of data analysis modeling, having a contribution as a constituent of the scope of construction work is expected to help the process of implementing construction work on the ABC Building construction project in Jakarta City so that it can be completed in a timely manner without any changes in work that result in the performance of project completion being hampered. Scope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta Scope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta Based on the results of interviews with project stakeholders and direct observations, several factors and variables were found to be used in controlling the scope of construction work in high-rise building development projects in the City of Jakarta. In this study, a survey was carried out on the parties responsible for the implementation of high-rise building projects in the city of Jakarta. In this case, the intended participants are the workers in the contractor on this project. The research respondents came from parties involved in the project with at least 2 years of experience. The questions asked in the interview process were also prepared based on literature studies, and existing journals that have almost the same rules as this research topic. Can be seen in the table below is a summary of the number of participants or respondents who participated in surveys and interviews directly at the project site. Table 2 Respondent Information Data The following are the results of the analysis of factors and variables that are formed to influence the planning of control over the scope of construction work for high-rise building development projects in the City of Jakarta, as follows. Table 3 Factors and Variables of Construction Project Work Scope Management N P P I I Vol.2, No.9, August 2023 3242 Scope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta CONCLUSION e Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta Vol.2, No.9, August 2023 3243 REFERENCES Azevedo, R. C. de, Ensslin, L., & Jungles, A. E. (2014). A Review of Risk Management in Construction: Opportunities for Improvement. Modern Economy, 05(04). https://doi.org/10.4236/me.2014.54036 Azevedo, R. C. de, Ensslin, L., & Jungles, A. E. (2014). A Review of Risk Management in Construction: Opportunities for Improvement. Modern Economy, 05(04). https://doi.org/10.4236/me.2014.54036 Halou, M. I. K., Samin, R., & Ahmad, M. (2019). Impacts of change management on risk and cost management of a construction projects. Journal of Project Management. Halou, M. I. K., Samin, R., & Ahmad, M. (2019). Impacts of change management on risk and cost management of a construction projects. Journal of Project Management. https://doi.org/10.5267/j.jpm.2019.1.005 Vol.2, No.9, August 2023 3244 Scope Management Analysis of Construction Work for Mix-Used Building Projects in the City of Jakarta Iqbal, S., Choudhry, R. M., Holschemacher, K., Ali, A., & Tamošaitienė, J. (2015). Risk management in construction projects. Technological and Economic Development of Economy, 21(1). https://doi.org/10.3846/20294913.2014.994582 Kazemi, A., Katebi, A., & Kazemi, M.-H. (2018). Causes of delay in construction projects: The case of oil and gas projects. International Journal of Advanced Research in Engineering, 4(1). https://doi.org/10.24178/ijare.2018.4.1.11 Kongsong, W. (2017). The Reduction of the Impacts in the Contract Management for Government Construction Projects. Ripublication.Com, 12(23). Kongsong, W., & Pooworakulchai, C. (2018). Improving for construction contract management of government construction projects. International Journal of Civil Engineering and Technology, 9(7). Lambropoulos, S. (2013). Objective Construction Contract Award using Cost, Time and Durability Utility. Procedia - Social and Behavioral Sciences, 74. https://doi.org/10.1016/j.sbspro.2013.03.052 Liu, J., Yi, Y., & Wang, X. (2020). Exploring factors influencing construction waste reduction: A structural equation modeling approach. Journal of Cleaner Production, 276. https://doi.org/10.1016/j.jclepro.2020.123185 Miri, M., & Khaksefidi, M. (2015). Cost Management in Construction Projects: Rework and Its Effects. Mediterranean Journal of Social Sciences. https://doi.org/10.5901/mjss.2015.v6n6s6p209 Muhammad, K., Saoula, O., Issa, M. R., & Ahmed, U. (2019). Contract management and performance characteristics: An empirical and managerial implication for Indonesia. Management Science Letters, 9(8). https://doi.org/10.5267/j.msl.2019.4.012 Nduhura, A., Anyango, H., & Mugerwa, B. (2021). Contract Management and Performance of Road Construction Projects at Local Government Entity in Uganda. Journal of Social Sciences 香港社會科學學報第一的第, 57. Rose, K. H., & Indelicato, G. (2009). Book Review: A Guide to the Project Management Body of Knowledge (PMBOK® Guide), Fourth Edition. Project Management Journal, 40(2). https://doi.org/10.1002/pmj.20125 Schoonwinkel, S., Fourie, C. J., & Conradie, P. D. F. (2016). A risk and cost management analysis for changes during the construction phase of a project. REFERENCES Journal of the South African Institution of Civil Engineering, 58(4). https://doi.org/10.17159/2309- 8775/2016/v58n4a3 Susila, H., & Handoyo, S. (2019). Analisis Pengaruh Konflik Dalam Pelaksanaan Konstruksi Terhadap Kesuksesan Proyek. Kinabalu, 11(2). Sutrisna, N., Arya Thanaya, I. N., & Ketut Sudipta, I. G. (1970). ANALISIS REWORK PADA PROYEK KONSTRUKSI GEDUNG DI KABUPATEN BADUNG. Jurnal Spektran. https://doi.org/10.24843/spektran.2013.v01.i02.p03 3245 Vol.2, No.9, August 2023
https://openalex.org/W2945762163	https://www.nature.com/articles/s41467-019-09823-5.pdf	English	null	Yin Yang 1 sustains biosynthetic demands during brain development in a stage-specific manner	Nature communications	2,019	cc-by	20,093	1 Institute of Anatomy, University of Zurich, 8057 Zurich, Switzerland. 2 Institute of Molecular Life Sciences, University of Zurich, Zurich 8057, Switzerland. 3 Laboratory of Computational Biology, KU Leuven Center for Human Genetics, Leuven 3000, Belgium. 4 Institute of Molecular Systems Biology, ETH Zurich, Zurich 8093, Switzerland. 5Present address: Wallenberg Centre for Molecular Medicine (WCMM), Department of Clinical and Experimental Medicine (IKE), Linköping University, Linköping 58183, Sweden. Correspondence and requests for materials should be addressed to L.S. (email: lukas.sommer@anatomy.uzh.ch) ARTICLE ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Y protein expression was lost in single cells as early as embryonic day 10.5 (E10.5) and was completely absent in the dorsal cortex from E11.5 onwards (Supplementary Fig. 1d). Y Y1 syndrome is a rare intellectual disability disorder, which is caused by mutations in the human Yin Yang 1 (YY1) gene1. Deletions and point mutations of YY1 lead to a congenital haploinsufﬁciency syndrome characterized mainly by cognitive impairment, facial dysmorphisms, and developmental delay. Interestingly, in mice, a subset of embryos lacking one Yy1 allele (Yy1+/−heterozygous) exhibit exencephaly, pseudoven- tricles, and brain asymmetry2. Although this has not yet been experimentally addressed, the combined data are consistent with the hypothesis that Yy1 has a role in mammalian brain development. At E18.5, the forebrain of Yy1cKO mice was considerably smaller than that of control mice, which was reﬂected by signiﬁcantly reduced cortical thickness and number of cells per radial unit (RU = 100 µm) (Fig. 1b and Supplementary Fig. 1e–g). Importantly, mesenchymal craniofacial structures known to inﬂuence forebrain development18,19 and to be malformed in human YY1 syndrome1 were not affected, consistent with the known Emx1 expression pattern16. Throughout our analysis, Emx1-Cre Yy1lx/wt mice appeared morphologically normal and were viable. This is consistent with previous reports showing lack of haploinsufﬁciency in mice for a number of congenital diseases, which indicates differential sensitivity towards gene dosage in humans vs. rodents20,21. To investigate the cellular mechanisms underlying the reduction of cortical size in Yy1cKO mice, we analyzed a potential requirement of Yy1 for proliferation at early stages of corticogenesis (Fig. 1c, e and Supplementary Fig. 1h–k). Immunohistochemical analysis revealed reduced numbers of cells positive for the mitotic marker phosphorylated histone H3 (pHH3) in Yy1cKO brains at E12.5 as compared with the control (Fig. 1c, e), whereas the ratio of apical vs. basal pHH3+ was not changed (Fig. 1f). Likewise, at E12.5, Yy1cKO cortices displayed strongly decreased numbers of cells expressing CyclinD1, which is required for cells to progress through the G1 phase of the cell cycle (Fig. 1g, i). In contrast, expression of CyclinB1, important for the transition from G2 to M phase, was not changed upon conditional Yy1 ablation (Fig. 1j, l). p The gene product of Yy1 is a ubiquitously expressed transcrip- tion factor, which controls transcriptional activation and repression and has been implicated in enabling enhancer–promoter interactions3,4. ARTICLE To conﬁrm the requirement of Yy1 for cell cycle progression, we isolated NPCs from E11.5 cortices by microdissection to perform siRNA-mediated knockdown (KD) experiments (Fig. 1p–r and Supplementary Fig. 1o). CyclinD1 expression was decreased in siYy1 KD-cells, whereas CyclinB1 expression remained unchanged (Fig. 1q). Moreover, cell cycle proﬁle analysis by ﬂow cytometry showed that treatment with siYy1 RNAs retained the cells preferentially in G1, indicating a role of Yy1 in G1 phase progression (Fig. 1r and Supplementary Fig. 1p). f l f l f l ll l p In this report, we genetically ablated Yy1 speciﬁcally in the developing dorsal cortex of mice. Loss of Yy1 before the onset of neurogenesis resulted in microcephaly owing to the depletion of NPCs. We found that ablation of Yy1 induced transient G1/S phase cell cycle arrest and p53-dependent cell death at embryonic day 12.5 (E12.5). In contrast, deletion of Yy1 after the onset of neurogenesis demonstrated a continuously decreasing inﬂuence on proliferation and cell survival. At the molecular level, loss of Yy1 at early developmental stages impaired numerous biosyn- thetic pathways, notably inﬂuencing the expression of metabolic genes, metabolite abundance, and protein translation rate. Intri- guingly, at later stages of cortex development, Yy1 inactivation did not affect metabolic processes anymore and the rate of pro- tein synthesis was generally reduced in later stage NPCs, revealing stage-dependent demands for metabolism and protein translation in cortical development. ARTICLE Yy1 exhibits context-dependent roles during the development and homeostasis of many tissues. It has been shown to regulate muscle5,6, lung7,8, and cardiac development9 and intestinal stem cell development and homeostasis10,11. Despite its ubiquitous expression, Yy1 seems to regulate distinct steps during the development of these tissues. Depending on the cell type, Yy1 has been associated with various functions, including regulation of signaling molecules, survival signals, cell cycle regulators or metabolism5,7,11–13. In the brain, a recent study using short hairpin RNA (shRNA) against Yy1 suggested a role for Yy1 in promoting neural progenitor cell (NPC) differentiation at mid-neurogenesis14. Likewise, Yy1 has been shown to be required for proper differ- entiation of the oligodendrocytic lineage at postnatal stages in vivo15. Although it is still unclear how cell type-speciﬁc func- tions of such an ubiquitous factor are achieved, the central nervous system and craniofacial structures appear to be especially depen- dent on the activity of YY1 as evidenced by the phenotype of YY1 loss-of-function in human patients1. y ( g j ) To conﬁrm the requirement of Yy1 for cell cycle progression, we isolated NPCs from E11.5 cortices by microdissection to perform siRNA-mediated knockdown (KD) experiments (Fig. 1p–r and Supplementary Fig. 1o). CyclinD1 expression was decreased in siYy1 KD-cells, whereas CyclinB1 expression remained unchanged (Fig. 1q). Moreover, cell cycle proﬁle analysis by ﬂow cytometry showed that treatment with siYy1 RNAs retained the cells preferentially in G1, indicating a role of Yy1 in G1 phase progression (Fig. 1r and Supplementary Fig. 1p). Apart from proliferation, survival of cortical cells was strongly affected upon Yy1 inactivation. Indeed, immunostaining for cleaved Caspase 3 (cCasp3) revealed transiently increased apoptosis in the forebrain of Yy1cKO mice at E12.5 (Fig. 1m, o). Similarly, the number of Annexin V + cells was signiﬁcantly higher in siYy1-treated cortical cells than in control cells (Fig. 1s), demonstrating a role of Yy1 in promoting cell survival. In contrast, Yy1 is apparently not involved in neuronal differentia- tion as such. Although at E18.5 the numbers of neuronal subtypes normally found in distinct cortical layers were generally decreased upon Yy1 inactivation, consistent with the reduced cortical thickness at this stage, the relative abundance of Dcx + immature neurons in comparison with Sox2-expressing NPCs was similar in Yy1cKO and control forebrains at E12.5 (Supplementary Fig. 2a–g). Yin Yang 1 sustains biosynthetic demands during brain development in a stage-speciﬁc manner Luis Zurkirchen1, Sandra Varum1, Sonja Giger1, Annika Klug1, Jessica Häusel1, Raphaël Bossart1, Martina Zemke1, Claudio Cantù2,5, Zeynep Kalender Atak3, Nicola Zamboni4, Konrad Basler2 & Lukas Sommer1 The transcription factor Yin Yang 1 (YY1) plays an important role in human disease. It is often overexpressed in cancers and mutations can lead to a congenital haploinsufﬁciency syndrome characterized by craniofacial dysmorphisms and neurological dysfunctions, consistent with a role in brain development. Here, we show that Yy1 controls murine cerebral cortex development in a stage-dependent manner. By regulating a wide range of metabolic pathways and protein translation, Yy1 maintains proliferation and survival of neural progenitor cells (NPCs) at early stages of brain development. Despite its constitutive expression, however, the dependence on Yy1 declines over the course of corticogenesis. This is associated with decreasing importance of processes controlled by Yy1 during development, as reﬂected by diminished protein synthesis rates at later developmental stages. Thus, our study unravels a novel role for Yy1 as a stage-dependent regulator of brain development and shows that biosynthetic demands of NPCs dynamically change throughout development. 1 Institute of Anatomy, University of Zurich, 8057 Zurich, Switzerland. 2 Institute of Molecular Life Sciences, University of Zurich, Zurich 8057, Switzerland. 3 Laboratory of Computational Biology, KU Leuven Center for Human Genetics, Leuven 3000, Belgium. 4 Institute of Molecular Systems Biology, ETH Zurich, Zurich 8093, Switzerland. 5Present address: Wallenberg Centre for Molecular Medicine (WCMM), Department of Clinical and Experimental Medicine (IKE), Linköping University, Linköping 58183, Sweden. Correspondence and requests for materials should be addressed to L.S. (email: lukas.sommer@anatomy.uzh.ch) 1 NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/ Results Yy1 regulates NPC survival and proliferation. To study the role of Yy1 in cortex development, we started by determining the expression pattern of Yy1 at various developmental stages. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis and immunostaining demonstrated that Yy1 mRNA and protein were prominently expressed throughout cortical devel- opment, with a slight decrease in overall expression levels at late developmental stages (Supplementary Fig. 1a–c). Notably, Yy1 protein was detectable in virtually all Sox2+ NPCs and doublecortin (Dcx) + neuronal cells at all stages analyzed (Supplementary Fig. 1c). To address the in vivo requirements of Yy1 in the developing cortex, we conditionally ablated Yy1 by combining Emx1-Cre mice16 with a transgenic mouse line car- rying Yy1 alleles ﬂanked by loxP sites (Yy1lx/lx mice17) (Fig. 1a). In Emx1-Cre Yy1lx/lx mice (hereafter, referred to as Yy1cKO), Yy1 Thus, our data suggest that Yy1 controls proper cortex size by regulating proliferation and survival of cortical progenitor cells. Intriguingly, however, when analyzed at E15.5 rather than at E12.5, the amount of cortex cells expressing pHH3, CyclinD1, and cleaved Caspase 3, respectively, was not affected anymore in Yy1cKO embryos (Fig. 1d, e, f, h, i, k, l, n, o), pointing to a speciﬁc requirement of Yy1 at early stages of development. Stage-speciﬁc control of NPC behavior by Yy1. To assess potential stage-speciﬁc roles of Yy1 in cortical development, we made use of the CreERT2 loxP system, by which activation of CreERT2 recombinase can be induced at deﬁned stages of URE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 a mbryonic development upon tamoxifen (TM) injection of regnant females. To this end, Yy1lx/lx mice were mated with mx-CreERT2 Yy1lx/wt mice (Emx-CreERT2 from22) to obtain nimals suitable for inducible deletion of Yy1 in the developing ortex (hereafter referred to as Yy1iKO mice) (Supplementary ig. 3a). Non-inducible Emx1-Cre is known to be active in the developing cortex from around E9.5 onwards16. According TM-induced activation of Emx-CreERT2 in Yy1iKO mice at E temporarily decreased pHH3+ cells and CyclinD1 expression a increased apoptosis at E12.5 (Supplementary Fig. 3b–l), in a logy to the phenotype described above for Emx1-Cre-carry Yy1cKO mice (Fig. 1). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Interestingly, however, when Yy1 delet Dapi Cyclin D1 Yy1 CycD1 CycB1 β-Actin siCtrl siYy1 0 10 20 30 Control Yy1cKO ns E12.5 E15.5 CycD1+/Dapi p = 0.002 Control Yy1cKO E12.5 g h i j q Dapi Cyclin D1 E15.5 VZ SVZ IZ Control Yy1cKO * 0 20 40 60 80 100 siCtrl siYy1 G2/M S G1 * * Percentage of cells Dapi Cyclin B1 k l 0 10 20 30 CycB1+/Dapi ns ns Control Yy1cKO p Control Yy1cKO E12.5 Control Yy1cKO E12.5 E15.5 Dapi cCasp3 20 40 60 0 E12.5 E15.5 cCasp3+ cells/RU ns siCtrl siYy1 0 5 10 15 25 AnnexinV+ cells (%) 35 p < 0.001 p = 0.0018 m o s Control E15.5 Control Yy1cKO Yy1cKO: Emx1 IRES Cre Yy1 Dapi Cyclin B1 Dapi cCasp3 n Yy1lx/lx Emx1-Cre siRNA 48 h Dorsal cortex cells E11.5 Analysis r Dapi pHH3 Control Yy1cKO E12.5 c e pHH3+ cells 0 10 20 30 40 50 p = 0.004 ns E12.5 E15.5 d Dapi pHH3 Control E15.5 Yy1cKO Control Yy1cKO Control Yy1cKO Control Yy1cKO ctx ctx b E18.5 E12.5 Yy1cKO VZ SVZ E15.5 30 20 pHH3+ cells (%) 0 25 50 75 100 ns Basal Apical E12.5 E15.5 Basal Apical Control Yy1cKO ns f ATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications C e y y Dapi pHH3 Control Yy1cKO E12.5 c Control Yy1cKO ctx ctx b E18.5 e pHH3+ cells 0 10 20 30 40 50 p = 0.004 ns E12.5 E15.5 Control Yy1cKO f b c e f d Dapi pHH3 Control E15.5 Yy1cKO d pHH3+ cells (%) 0 25 50 75 100 ns Basal Apical E12.5 E15.5 Basal Apical Control Yy1cKO ns f h Dapi Cyclin D1 E15.5 VZ SVZ IZ Control Yy1cKO 0 10 20 30 Control Yy1cKO ns E12.5 E15.5 CycD1+/Dapi p = 0.002 i h Dapi Cyclin D1 Control Yy1cKO E12.5 g h i g k Control Yy1cKO Dapi Cyclin B1 VZ SVZ E15.5 j Dapi Cyclin B1 Control Yy1cKO E12.5 l 0 10 20 30 CycB1+/Dapi ns ns Control Yy1cKO E12.5 E15.5 l l k E15.5 Control Yy1cKO Dapi cCasp3 n 20 40 60 0 E12.5 E15.5 cCasp3+ cells/RU ns p = 0.0018 o Control Yy1cKO Dapi cCasp3 m Control E12.5 Yy1cKO m n o * 0 20 40 60 80 100 siCtrl siYy1 G2/M S G1 * * Percentage of cells r Yy1 CycD1 CycB1 β-Actin siCtrl siYy1 q * 0 20 40 60 80 100 siCtrl siYy1 G2/M S G1 * * Percentage of cells r p siRNA 48 h Dorsal cortex cells E11.5 Analysis r Yy1 CycD1 CycB1 β-Actin siCtrl siYy1 q siCtrl siYy1 0 5 10 15 25 AnnexinV+ cells (%) 35 p < 0.001 s 30 20 q p s embryonic development upon tamoxifen (TM) injection of pregnant females. NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Together, our data suggest that loss of Yy1 exerts a stage- speciﬁc effect on cell proliferation and survival in the developing brain, which cannot just be attributed to different sensitivities of distinct NPC subpopulations. Yy1 regulates metabolic pathways and protein translation. To identify further factors potentially mediating Yy1 activity in the developing forebrain, we evaluated the global gene expression patterns of control vs. Yy1cKO cortices at E11.5 using RNA- sequencing (RNA-seq) (Fig. 4a). We identiﬁed 1087 and 467 annotated mRNAs that were downregulated and upregulated, respectively, in Yy1cKO cells (\|log2 fold change\| > 0.32, p < 0.05, FDR < 0.01). To ﬁnd which pathways were speciﬁcally enriched, we performed gene ontology (GO) analysis and assembled a GO perturbation network, where GO terms, which share > 50% of the genes, are connected (Fig. 4b). GO terms enriched upon deletion of Yy1 revealed alterations in various metabolic pathways, such as fatty-acid oxidation, nucleic-acid metabolism, and biosynthetic processes (Fig. 4b, Supplementary Fig. 6 and Supplementary Data 1). Furthermore, mitochondria were identiﬁed as organelles pri- marily associated with Yy1-dependent changes in gene expres- sion, in agreement with previous studies on muscle development and intestinal stem cell homeostasis5,11. Yy1 promotes NPC survival through downregulation of p53. Yy1 has been shown to inhibit activation of the tumor-suppressor p53, a well-established regulator of cell cycle progression and apoptosis24–26. To address whether Yy1 might regulate p53 also in the developing CNS, we investigated p53 expression in the forebrain of control and Yy1cKO mice at E12.5. Consistent with the reported posttranscriptional regulation of p53 by Yy124,25, p53 protein but not p53 mRNA levels were upregulated upon loss of Yy1 (Fig. 3a, b). To address a potential involvement of p53 in Yy1-dependent neural progenitor control, we then crossed Yy1cKO mice with mice carrying alleles of Trp53 ﬂanked by loxP sites27 (Fig. 3c). The resulting Yy1 Trp53 double knockout (Yy1Trp53dKO) mice displayed a cortex with a size intermediate To validate these ﬁndings, we performed qRT-PCR analysis of selected genes and conﬁrmed that a plethora of factors involved in metabolic pathways including glycolysis, TCA cycle, lipid metabolism, and mitochondrial biogenesis were downregulated in the Yy1cKO forebrain (Fig. 4c, d). Moreover, the expression of several genes implicated in nucleotide metabolism, ribosome and tRNA synthesis, and mRNA to protein translation was altered upon Yy1 inactivation (Fig. 4e). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 2b, c), and neither cortical cell proliferation nor apoptosis were signiﬁcantly altered at E15.5 in these Yy1iKO embryos as compared with the control (Fig. 2l–q). Thus, despite constitutive expression of Yy1 at later stages of cortical development (Supplementary Fig. 1c), NPC pro- liferation and survival are becoming less dependent on Yy1 as the embryonic brain develops, revealing a stage-dependent role of Yy1. to control and Yy1cKO cortices at E18.5, indicating that loss of p53 could partially rescue the Yy1cKO phenotype (Fig. 3d, e). Of note, immunostaining for cleaved Caspase 3 demonstrated that the increased cell death observed upon Yy1 inactivation (Fig. 1m, o) was fully rescued in the absence of p53 (Fig. 3f). In contrast, loss of p53 did not rescue the proliferation phenotype seen in Yy1cKO mice, as shown by quantiﬁcation of pHH3 and CyclinD1 expression (Fig. 3g–k). To substantiate these data, we inactivated p53 signaling in Yy1cKO mice by administration of the pharmacological inhibitor Piﬁthrin-α (PFTα) (Supplementary Fig. 5a)28,29. Again, this approach counteracted cell death induced by conditional Yy1 deletion, albeit not as efﬁciently as genetic p53 inactivation (Supplementary Fig. 5b). However, the proliferation defect in the developing forebrain of Yy1cKO mice was still present in PFTα- treated animals (Supplementary Fig. 5c–g). Thus, regulation of cortical progenitor survival by Yy1 appears to be largely mediated by the activity of p53, whereas the control of proliferation by Yy1 is p53-independent. With advancing cortical development, the relative number of intermediate progenitor cells compared to apical NPCs increases23. To assess whether the phenotype seen in Yy1 mutant embryos can be attributed to different progenitor cell types, we performed immunohistochemistry for Pax6 and Tbr2 to distinguish apical NPCs (Pax6+), intermediate NPCs (Tbr2+), and apical NPCs transitioning to intermediate NPCs (Pax6+ Tbr2+). At E12.5 and E15.5, both the number of Pax6+ and Tbr2+ cells per RU were decreased in Yy1cKO embryos (Supplementary Fig. 4a, b, c, e, f). The relative abundance of these NPC subtypes, however, were not altered (Supplementary Fig. 4d, g). Consistent with our previous results, TM-induced ablation of Yy1 at E12.5 showed a milder phenotype where only the number of Pax6+ cells per RU at E14.5 was signiﬁcantly decreased (Supplementary Fig 4h–o). Further, deletion of Yy1 at E13.5 did neither affect the absolute nor relative number of Pax6, Tbr2, and Pax6+ Tbr2+ cells (Supplementary Fig. 4p–s). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Fig. 1 Yy1 maintains proliferation and cell survival at early stages of cortex development. a Genotype of Yy1cKO mice with conditional ablation of Yy1 in the dorsal cortex. b Deletion of Yy1 leads to decreased cortex (ctx) size at E18.5. c–f Loss of Yy1 decreases the number of pHH3+ cells at E12.5 (c, e). At E15.5, the number of mitotic cells is comparable to control embryos (d, e). The ratio of apical vs. basal pHH3+ cells does not change upon knockout of Yy1 (f). The number of pHH3+ cells is normalized to 600 μm ventricular zone length (E12.5 and E15.5) and normalized to cortical thickness (E15.5). g–i The percentage of CyclinD1+ cells decreases upon ablation of Yy1 at E12.5 (g, i), but not at E15.5 (h, i). j–l The percentage of CyclinB1+ cells is not affected in Yy1cKO embryos. m–o Immunohistochemistry for cleaved Caspase 3 (cCasp3) shows that the number of apoptotic cells transiently increases at E12.5 in Yy1cKO embryos. Radial unit (RU) = 100 µm. p Experimental outline for knockdown experiments. q Efﬁcient knockdown of Yy1 in isolated E11.5 cortical progenitor cells decreases CyclinD1 protein levels without affecting CyclinB1 protein levels. r Cell cycle analysis by ﬂow cytometry reveals that knockdown of Yy1 increases the number of cells in G1 cell cycle phase. s Flow cytometric analysis of the apoptotic marker Annexin V. Knockdown of Yy1 in isolated E11.5 cortical progenitor cells increases the number of Annexin V+ cells. Nuclei are counterstained with DAPI. Scale bars represent 1 mm (b), 100 μm (m, n), 50 μm (c, d), 20 μm (g, h, j, k). Comparisons were performed using the two-tailed unpaired Student’s t test. Data are the mean ± standard deviation. p < 0.05. ns = not signiﬁcant was induced at E10.5 or later, the effect on forebrain size was progressively decreasing when compared to Yy1cKO mice, despite efﬁcient ablation of Yy1 protein expression (Fig. 2a–c and Sup- plementary Fig. 3m–r). In agreement with this, ablation of Yy1 at E12.5 affected proliferation and cell death less prominently at E14.5 and both of these parameters were at control levels at E15.5 (Fig. 2d–k). Moreover, upon TM-induced recombination at E13.5, the size of Yy1iKO cortices at E18.5 was indistinguishable from that of control embryos (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 To this end, Yy1lx/lx mice were mated with Emx-CreERT2 Yy1lx/wt mice (Emx-CreERT2 from22) to obtain animals suitable for inducible deletion of Yy1 in the developing cortex (hereafter referred to as Yy1iKO mice) (Supplementary Fig. 3a). Non-inducible Emx1-Cre is known to be active in the developing cortex from around E9.5 onwards16. Accordingly, TM-induced activation of Emx-CreERT2 in Yy1iKO mice at E9.5 temporarily decreased pHH3+ cells and CyclinD1 expression and increased apoptosis at E12.5 (Supplementary Fig. 3b–l), in ana- logy to the phenotype described above for Emx1-Cre-carrying Yy1cKO mice (Fig. 1). Interestingly, however, when Yy1 deletion NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications 3 developing cortex from around E9.5 onwards16. Accordingly, TM-induced activation of Emx-CreERT2 in Yy1iKO mice at E9.5 temporarily decreased pHH3+ cells and CyclinD1 expression and increased apoptosis at E12.5 (Supplementary Fig. 3b–l), in ana- logy to the phenotype described above for Emx1-Cre-carrying Yy1cKO mice (Fig. 1). Interestingly, however, when Yy1 deletion embryonic development upon tamoxifen (TM) injection of pregnant females. To this end, Yy1lx/lx mice were mated with Emx-CreERT2 Yy1lx/wt mice (Emx-CreERT2 from22) to obtain animals suitable for inducible deletion of Yy1 in the developing cortex (hereafter referred to as Yy1iKO mice) (Supplementary Fig. 3a). Non-inducible Emx1-Cre is known to be active in the 3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 l e 0 10 20 30 40 cCasp3 per RU 0 10 20 30 40 50 p = 0.038 E12.5 Tamoxifen Analysis E14.5 pHH3+ cells ns a Yy1iKO: Emx1 CreERT2 Yy1 Yy1lx/lx Emx1-CreERT2 b E15.5 E14.5 E15.5 ns Control Yy1iKO Dapi cCasp3 Control Yy1iKO Dapi cCasp3 Control Yy1iKO ns E14.5 E15.5 Control Yy1iKO d g i j k E13.5 Tamoxifen Analysis E15.5 Dapi pHH3 Dapi pHH3 Control Control Yy1iKO E15.5 Yy1iKO E14.5 E15.5 E14.5 f m E10.5 E11.5 Control Yy1iKO E18.5 E12.5 E13.5 Tamoxifen injection: W L E9.5 E11.5 E12.5 E13.5 E10.5 Ctrl KO 0 1 2 3 4 5 Size in mm Cortex length Cortex width E18.5 E18.5 E18.5 E18.5 E18.5 c E10.5 Tamoxifen Analysis E11.5 E12.5 E13.5 E18.5 * ns * * ns 0 25 50 75 100 E14.5 E15.5 pHH3+ cells (%) Basal Apical Basal Apical Control Yy1iKO * * * * 0 5 10 15 20 cCasp3 per RU ns 0 10 20 30 40 50 pHH3+ cells ns E15.5 Control Yy1iKO Dapi cCasp3 Control Yy1iKO Control Yy1iKO E15.5 p n q 0 25 50 75 100 pHH3+ cells (%) E15.5 Basal Apical Basal Apical Control Yy1iKO E15.5 Dapi pHH3 Control Yy1iKO h o ns ns ns E15.5 a Yy1iKO: Emx1 CreERT2 Yy1 Yy1lx/lx Emx1-CreERT2 b E10.5 E11.5 Control E18.5 E12.5 E13.5 Tamoxifen injection: W L E10.5 Tamoxifen Analysis E11.5 E12.5 E13.5 E18.5 Tamoxifen a b E10.5 E11.5 Control E18.5 E12.5 E13.5 Tamoxifen injection: W L E10.5 E11.5 E12.5 E13.5 E18.5 b E12.5 next analyzed the direct target genes of Yy1 by gating genome-wide occupancy of Yy1 using chromatin oprecipitation followed by sequencing (ChIP-seq) of cells isolated from E12.5 wild-type embryos (Fig. 5a). ighest scoring motif corresponded to a known Yy1 consensus binding site containing the core sequence A (Fig. 5b)6,11,30. Consistent with previous reports6,11,30 binding events predominantly took place within close pro of the transcriptional start site (TSS) of promoter regions ( d). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Visualizing GO terms enriched for the genes with Yy1-b l e 0 10 20 30 40 cCasp3 per RU 0 10 20 30 40 50 p = 0.038 E12.5 Tamoxifen Analysis E14.5 pHH3+ cells ns E15.5 E14.5 E15.5 ns Control Yy1iKO Dapi cCasp3 Control Yy1iKO Dapi cCasp3 Control Yy1iKO ns E14.5 E15.5 Control Yy1iKO d g i j k E13.5 Tamoxifen Analysis E15.5 Dapi pHH3 Dapi pHH3 Control Control Yy1iKO E15.5 Yy1iKO E14.5 E15.5 E14.5 f m Yy1iKO E9.5 E11.5 E12.5 E13.5 E10.5 Ctrl KO 0 1 2 3 4 5 Size in mm Cortex length Cortex width E18.5 E18.5 E18.5 E18.5 E18.5 c * * ns * * ns 0 25 50 75 100 E14.5 E15.5 pHH3+ cells (%) Basal Apical Basal Apical Control Yy1iKO * * * * 0 5 10 15 20 cCasp3 per RU ns 0 10 20 30 40 50 pHH3+ cells ns E15.5 Control Yy1iKO Dapi cCasp3 Control Yy1iKO Control Yy1iKO E15.5 p n q 0 25 50 75 100 pHH3+ cells (%) E15.5 Basal Apical Basal Apical Control Yy1iKO E15.5 Dapi pHH3 Control Yy1iKO h o ns ns ns E15.5 COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications Yy1iKO E1 T d E9.5 E11.5 E12.5 E13.5 E10.5 Ctrl KO 0 1 2 3 4 5 Size in mm Cortex length Cortex width E18.5 E18.5 E18.5 E18.5 E18.5 c * * ns * * ns * * * * E12.5 Tamoxifen Analysis E14.5 E15.5 d d c 0 10 20 30 40 50 pHH3+ cells ns E14.5 E15.5 ns Control Yy1iKO g e e Dapi pHH3 Dapi pHH3 Control Control Yy1iKO E15.5 Yy1iKO E14.5 f 0 25 50 75 100 E14.5 E15.5 pHH3+ cells (%) Basal Apical Basal Apical Control Yy1iKO h ns ns h g g Dapi cCasp3 Control Yy1iKO i E14.5 j k m E15.5 Dapi pHH3 Control Yy1iKO l l E13.5 Tamoxifen Analysis E15.5 m 0 10 20 30 40 50 pHH3+ cells ns E15.5 Control Yy1iKO n Dapi cCasp3 Control Yy1iKO p E15.5 Dapi cCasp3 Control p E15.5 Yy1iKO .5 0 5 10 15 20 cCasp3 per RU ns Control Yy1iKO E15.5 q 0 25 50 75 100 pHH3+ cells (%) E15.5 Basal Apical Basal Apical Control Yy1iKO o ns n p o q consensus binding site containing the core sequence ATGGC (Fig. 5b)6,11,30. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Of note, genes associated with metabolism and translation, were not rescued by pharmacological inhibition of p53 signaling in Yy1cKO cortical cells at E12.5 (Supplementary Fig. 7a–c), indicating that Yy1-mediated regula- tion of these genes is p53 independent in the developing cortex. URE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Fig. 2 Ablation of Yy1 after E12.5 does not inﬂuence cortical development. a Genotype of mice and experimental strategy used to induce ablation of Yy1 at different developmental stages. b Later stage tamoxifen-induced ablation of Yy1 ameliorates the decrease in cortex size compared with Yy1cKO cortices (Fig. 1b). c Measurement of cortical length (L) and width (W) as indicated in b. For representative picture of E18.5 Yy1cKO cortex, see Fig. 1b. p < 0.05. d Experimental strategy to ablate Yy1 at E12.5 in Yy1iKO embryos (for Fig. 2e–k). e–h Immunostaining and quantiﬁcation for pHH3+ cells at E14.5 (e, g) and E15.5 (f, g) in E12.5-ablated Yy1iKO embryos. h depicts the ratio of apical vs. basal pHH3+ cells. The number of pHH3+ cells is normalized to 600 μm ventricular zone length. i–k Ablation of Yy1 at E12.5 elicits cleavage of Caspase 3 (cCasp3) at E14.5 (i, k) but not at E15.5 (j, k). Note that red signals visible at E15.5 are blood cells. l Experimental strategy to ablate Yy1 at E13.5 in Yy1iKO embryos (for Fig. 2m–q). m–o The total number and ratio of apical vs. basal pHH3+ cells remains unchanged upon late ablation of Yy1 at E13.5. The number of pHH3+ cells is normalized to 600 μm ventricular zone length. p, q Ablation of Yy1 at E13.5 only induces cell death in a minority of cells (indicated by arrows, remaining red signals are blood cells). Nuclei are counterstained with DAPI. Scale bars represent 1 mm (b), 50 µm (e, f, i, j, m, p). Comparisons were performed using ANOVA (Tukey’s multiple comparisons test) (c) and two-tailed unpaired Student’s t test (g, h, k, n, o, q). Data are the mean ± standard deviation. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 ns = not signiﬁcant 0 1.00 Ctrl Yy1cKO 0 1 2 3 4 5 Control Yy1cKO Control Trp53 Yy1Trp53dKO * * * * * Dapi Cyclin D1 Control Trp53 Yy1Trp53dKO b e f Dapi cCasp3 Control Trp53 Yy1Trp53dKO g j k 0 10 20 30 40 p = 0.011 pHH3+ cells h Dapi pHH3 Yy1Trp53dKO Control Trp53 ns 0 5 10 15 20 25 CycD1+ per Dapi Yy1Trp53dKO Emx1 IRES Cre Yy1 Trp53 c Yy1lx/lx Emx1-Cre Trp53lx/lx Yy1Trp53dKO Control Trp53 d Ctrl Trp53 Yy1Trp53 dKO Ctrl Trp53 Yy1Trp53 dKO W L Dapi p53 Dcx a Yy1cKO pHH3+ cells (%) 0 25 50 75 100 ns Basal Apical E12.5 Basal Apical Ctrl Trp53 Yy1Trp53dKO i * Control E12.5 p53 mRNA expression 1.25 0.75 0.50 0.25 E18.5 Size (mm) Cortex length Cortex width E12.5 E12.5 E12.5 p < 0.001 Fig. 3 Loss of Yy1 induces p53-dependent apoptosis. a Accumulation of p53 protein in Yy1cKO embryos at E12.5. b qRT-PCR of p53 mRNA levels reveals no difference upon loss of Yy1. c Genotype of Yy1 Trp53 double knockout (Yy1Trp53dKO). d, e Double knockout of Yy1 and Trp53 partially rescues cortical size at E18.5. Cortical length (L) and width (W) measurements from Control and Yy1cKO from Fig. 2c were reused for illustrative reasons. Representative images of control and Yy1cKO cortices are shown in Fig. 1b. p < 0.01. f Knockout of Trp53 in the context of Yy1 ablation completely abolishes emergence of cleaved Caspase 3+ cells. g–i Trp53 Yy1 double knockout does not restore the number of mitotic pHH3+ cells. The ratio of apical vs basal pHH3+ cell does not change (i). The numbers of pHH3+ cells are normalized to 600 μm ventricular zone length. j, k Loss of Trp53 does not rescue the number of CyclinD1+ cells in Yy1 mutant embryos. Nuclei are counterstained with DAPI. Scale bars resemble 1 mm (d), 50 µm (f, g), 20 µm (a, j). Comparisons were performed using the two-tailed unpaired Student’s t test (b, h, i, k) and ANOVA (Tukey’s multiple comparisons test) (e). Data are the mean ± standard deviation. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Consistent with previous reports6,11,30, Yy1- binding events predominantly took place within close proximity of the transcriptional start site (TSS) of promoter regions (Fig. 5c, d). Visualizing GO terms enriched for the genes with Yy1-binding We next analyzed the direct target genes of Yy1 by interrogating genome-wide occupancy of Yy1 using chromatin immunoprecipitation followed by sequencing (ChIP-seq) of cortical cells isolated from E12.5 wild-type embryos (Fig. 5a). The highest scoring motif corresponded to a known Yy1 We next analyzed the direct target genes of Yy1 by interrogating genome-wide occupancy of Yy1 using chromatin immunoprecipitation followed by sequencing (ChIP-seq) of cortical cells isolated from E12.5 wild-type embryos (Fig. 5a). The highest scoring motif corresponded to a known Yy1 5 5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Data are the mean ± standard deviation b < 0.0005 < 0.005 < 0.05 Term p value BP CC MF Geneset size > 1000 > 100 > 10 Ontology Vesicle-mediated transport Vesicle Cytoplasm Nucleus Nucleic acid metabolic process Nucleic acid binding Gene expression Cellular biosynthetic process Organelle organization Mitochondrion Organonitrogen compound metabolic process Oxidation–reduction process Fatty-acid oxidation –2.2 2.2 Dorsal cortex E11.5 RNA-seq Yy1cKO Ctrl a Row z-score b a Oxidation–reduction process –2.2 2.2 Dorsal cortex RNA-seq Yy1cKO Ctrl Control Yy1cKO Pgm1 Eno1 Pdhb Suclg2 Acdm Eci1 Ech1 Echs1 Ehhadh Acot2 Ndufaf6 Ndufb11 Ndufb4 Ndufa3 Ndufc1 Ppargc1a Ppargc1b Glycolysis TCA cycle Complex I Mitochondrial biogenesis * * * * * * * ns ns c Row z-score Napepld * * Lipid metabolism Uqcc2 Compl. III * * * * * * * * 0 1 2 3 4 Qrsl1 Eif4G2 Eif1ax Eif1b Yars2 Mrps27 Utp20 Tyw3 Rpl5 * * * ns * * * * ns Translation initiation Ribosomal proteins tRNA synthases Rib. biog. e < 0.0005 < 0.005 < 0.05 Term p value BP CC MF Geneset size > 1000 > 100 > 10 Ontology Vesicle-mediated transport Vesicle Cytoplasm Nucleus Nucleic acid metabolic process Nucleic acid binding Gene expression Cellular biosynthetic process Organelle organization Mitochondrion metabolic process process Fatty-acid oxidation 0 1.0 Yy1 * Dhodh Nme3 Nucleotide metabolism * * d Ctrl Yy1cKO Yy1cKO Control 1.2 0.2 0.4 0.6 0.8 0 1.0 1.2 0.2 0.4 0.6 0.8 rel. mRNA expression rel. mRNA expression Bscl2 rel. mRNA expression Fig. 4 Yy1 regulates the expression of metabolic genes. a RNA-seq comparing Yy1cKO versus control cortex tissue at E11.5 identiﬁed 1554 differentiall expressed genes (\|log2 fold change\| > 0.3, p < 0.05, FDR < 0.01). b Gene Ontology (GO) term network analysis on the basis of differentially regulated genes. Each node represents an enriched GO term (adjusted p value (Corrected with Bonferroni step down procedure) < 0.05). Nodes are interconnecte when the gene overlap is > 50%, based on the kappa score. BP, biological process; MF, molecular function; CC, cellular component. A fully labeled versio of the network is given in Supplementary Fig. 6. c–e qRT-PCR validation for differentially regulated genes comparing control vs Yy1cKO cortical tissue a E12.5 conﬁrms RNA-seq results. Comparisons were performed using the two-tailed unpaired Student’s t test. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 ns = not signiﬁcant 0 1.00 Ctrl Yy1cKO b ns p53 mRNA expression 1.25 0.75 0.50 0.25 Yy1Trp53dKO Emx1 IRES Cre Yy1 Trp53 c Yy1lx/lx Emx1-Cre Trp53lx/lx Dapi p53 Dcx a Yy1cKO Control E12.5 b c a d 0 1 2 3 4 5 Control Yy1cKO Control Trp53 Yy1Trp53dKO * * * * * * e * Size (mm) Cortex length Cortex width Yy1Trp53dKO Control Trp53 d W L E18.5 e f Dapi cCasp3 Control Trp53 Yy1Trp53dKO E12.5 0 10 20 30 40 p = 0.011 pHH3+ cells h Ctrl Trp53 Yy1Trp53 dKO g Dapi pHH3 Yy1Trp53dKO Control Trp53 E12.5 h g pHH3+ cells (%) 0 25 50 75 100 ns Basal Apical E12.5 Basal Apical Ctrl Trp53 Yy1Trp53dKO i i Dapi Cyclin D1 Control Trp53 Yy1Trp53dKO j E12.5 k 0 5 10 15 20 25 CycD1+ per Dapi Ctrl Trp53 Yy1Trp53 dKO p < 0.001 j Fig. 3 Loss of Yy1 induces p53-dependent apoptosis. a Accumulation of p53 protein in Yy1cKO embryos at E12.5. b qRT-PCR of p53 mRNA levels reveals no difference upon loss of Yy1. c Genotype of Yy1 Trp53 double knockout (Yy1Trp53dKO). d, e Double knockout of Yy1 and Trp53 partially rescues cortical size at E18.5. Cortical length (L) and width (W) measurements from Control and Yy1cKO from Fig. 2c were reused for illustrative reasons. Representative images of control and Yy1cKO cortices are shown in Fig. 1b. p < 0.01. f Knockout of Trp53 in the context of Yy1 ablation completely abolishes emergence of cleaved Caspase 3+ cells. g–i Trp53 Yy1 double knockout does not restore the number of mitotic pHH3+ cells. The ratio of apical vs basal pHH3+ cell does not change (i). The numbers of pHH3+ cells are normalized to 600 μm ventricular zone length. j, k Loss of Trp53 does not rescue the number of CyclinD1+ cells in Yy1 mutant embryos. Nuclei are counterstained with DAPI. Scale bars resemble 1 mm (d), 50 µm (f, g), 20 µm (a, j). Comparisons were performed using the two-tailed unpaired Student’s t test (b, h, i, k) and ANOVA (Tukey’s multiple comparisons test) (e). Data are the mean ± standard deviation. ns = not signiﬁcant NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Data are the mean ± standard deviation Fatty-acid oxidation Nucleus Control Yy1cKO Pgm1 Eno1 Pdhb Suclg2 Acdm Eci1 Ech1 Echs1 Ehhadh Acot2 Ndufaf6 Ndufb11 Ndufb4 Ndufa3 Ndufc1 Ppargc1a Ppargc1b Glycolysis TCA cycle Complex I Mitochondrial biogenesis * * * * * * * ns ns c Napepld * * Lipid metabolism Uqcc2 Compl. III * * * * * * * * 0 1.0 Yy1 * d Ctrl Yy1cKO 1.2 0.2 0.4 0.6 0.8 0 1.0 1.2 0.2 0.4 0.6 0.8 rel. mRNA expression rel. mRNA expression Bscl2 Control Yy1cKO Pgm1 Eno1 Pdhb Suclg2 Acdm Eci1 Ech1 Echs1 Ehhadh Acot2 Ndufaf6 Ndufb11 Ndufb4 Ndufa3 Ndufc1 Ppargc1a Ppargc1b Glycolysis TCA cycle Complex I Mitochondrial biogenesis * * * * * * * * ns ns c Napepld * * Lipid metabolism Uqcc2 Compl. III * * * * * * * * 2 3 4 ns * * * * ns e 0 1.0 Yy1 * d Ctrl Yy1cKO Yy1cKO Control 1.2 0.2 0.4 0.6 0.8 0 1.0 1.2 0.2 0.4 0.6 0.8 rel. mRNA expression rel. mRNA expression Bscl2 NA expression d cycle 0 1 2 3 4 Qrsl1 Eif4G2 Eif1ax Eif1b Yars2 Mrps27 Utp20 Tyw3 Rpl5 * * * ns * * * * ns Translation initiation Ribosomal proteins tRNA synthases Rib. biog. e Dhodh Nme3 Nucleotide metabolism * * Yy1cKO Control rel. mRNA expression e Fig. 4 Yy1 regulates the expression of metabolic genes. a RNA-seq comparing Yy1cKO versus control cortex tissue at E11.5 identiﬁed 1554 differentially expressed genes (\|log2 fold change\| > 0.3, p < 0.05, FDR < 0.01). b Gene Ontology (GO) term network analysis on the basis of differentially regulated genes. Each node represents an enriched GO term (adjusted p value (Corrected with Bonferroni step down procedure) < 0.05). Nodes are interconnected when the gene overlap is > 50%, based on the kappa score. BP, biological process; MF, molecular function; CC, cellular component. A fully labeled version of the network is given in Supplementary Fig. 6. c–e qRT-PCR validation for differentially regulated genes comparing control vs Yy1cKO cortical tissue at E12.5 conﬁrms RNA-seq results. Comparisons were performed using the two-tailed unpaired Student’s t test. Data are the mean ± standard deviation Fig. 4 Yy1 regulates the expression of metabolic genes. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 ARTICLE –2.2 2.2 Dorsal cortex E11.5 RNA-seq Yy1cKO Ctrl Control Yy1cKO Pgm1 Eno1 Pdhb Suclg2 Acdm Eci1 Ech1 Echs1 Ehhadh Acot2 Ndufaf6 Ndufb11 Ndufb4 Ndufa3 Ndufc1 Ppargc1a Ppargc1b Glycolysis TCA cycle Complex I Mitochondrial biogenesis * * * * * * * * ns ns a b c Row z-score Napepld * * Lipid metabolism Uqcc2 Compl. III * * * * * * * * 0 1 2 3 4 Qrsl1 Eif4G2 Eif1ax Eif1b Yars2 Mrps27 Utp20 Tyw3 Rpl5 * * * ns * * * * ns Translation initiation Ribosomal proteins tRNA synthases Rib. biog. e < 0.0005 < 0.005 < 0.05 Term p value BP CC MF Geneset size > 1000 > 100 > 10 Ontology Vesicle-mediated transport Vesicle Cytoplasm Nucleus Nucleic acid metabolic process Nucleic acid binding Gene expression Cellular biosynthetic process Organelle organization Mitochondrion Organonitrogen compound metabolic process Oxidation–reduction process Fatty-acid oxidation 0 1.0 Yy1 * Dhodh Nme3 Nucleotide metabolism * * d Ctrl Yy1cKO Yy1cKO Control 1.2 0.2 0.4 0.6 0.8 0 1.0 1.2 0.2 0.4 0.6 0.8 rel. mRNA expression rel. mRNA expression Bscl2 rel. mRNA expression Fig. 4 Yy1 regulates the expression of metabolic genes. a RNA-seq comparing Yy1cKO versus control cortex tissue at E11.5 identiﬁed 1554 differentially expressed genes (\|log2 fold change\| > 0.3, p < 0.05, FDR < 0.01). b Gene Ontology (GO) term network analysis on the basis of differentially regulated genes. Each node represents an enriched GO term (adjusted p value (Corrected with Bonferroni step down procedure) < 0.05). Nodes are interconnected when the gene overlap is > 50%, based on the kappa score. BP, biological process; MF, molecular function; CC, cellular component. A fully labeled version of the network is given in Supplementary Fig. 6. c–e qRT-PCR validation for differentially regulated genes comparing control vs Yy1cKO cortical tissue at E12.5 conﬁrms RNA-seq results. Comparisons were performed using the two-tailed unpaired Student’s t test. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 a b Wild-type E12.5 Highest scoring motif (homer) E12.5 Promoter TSS Intergenic 5′ UTR 3′ UTR Intron Exon Other Ech1 30 0 6 0 Napepld Uqcc2 10 0 40 0 6 0 10 kb 2 kb 2 kb Ndufc1 2 kb Bscl2 2 kb Eif4g2 8 0 10 kb Tyw3 7 0 10 kb Utp20 8 0 50 kb Dorsal cortex ChIP-seq e c f d Catalytic complex Nucleoplasm part Protein acetyltransferase complex Mitochondrial transport Mitochondrion organization Mitochondrial part Mitochondrial translation tRNA aminoacylation Aminoacyl-tRNA ligase activity tRNA metabolic process RNA binding RNA processing Ribosome Ribosome biogenesis Mitochondrial ribosome Ribonucleoprotein complex biogenesis Translation ncRNA metabolic process < 0.0005 < 0.005 < 0.05 Term p value BP CC MF Geneset size > 30 20–30 10–20 5–10 Ontology E12.5 Metabolism E12.5 Protein translation p value < 1e(–50) TSS 30 Mean density (tag/50 bp) 0 TSS g Yy1 binding at E12.5 –5 kb 5 kb Fig. 5 Yy1 directly binds to metabolic genes. a Genome-wide binding of Yy1 to DNA regions was analyzed by chromatin immunoprecipitation against Yy1 followed by sequencing (ChIP-seq) of cortex cells derived at E12.5. Statistical analysis of the enriched regions identiﬁed 464 binding events at E12.5 (FDR < 0.001), present in at least 1 of 2 replicas. b Homer motif discovery revealed a known Yy1-binding motif as the highest scoring motif (p < 1e-50). c Read cluster proﬁle reveals preferential binding of Yy1 close to the transcription start site (TSS) of target genes. d Distribution of binding sites to different genomic locations. Yy1 binds preferentially to the promoter region of the transcription start site (TSS) of genes. e Gene Ontology (GO) term network analysis on the basis of Yy1-bound target genes at E12.5. Each node represents an enriched GO term (adjusted p value (Corrected with Bonferroni step down procedure) < 0.05). Nodes are interconnected when the gene overlap is > 50%, based on the kappa score. BP, biological process; MF, molecular function; CC, cellular component. A fully labeled version of the network is given in Supplementary Fig. 8. f, g Genomic snapshots depicting Yy1-binding events at metabolic genes (g) and genes involved in protein translation (f). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 kb, kilo bases Catalytic complex Nucleoplasm part Protein acetyltransferase complex Mitochondrial transport Mitochondrion organization Mitochondrial part Mitochondrial translation tRNA aminoacylation Aminoacyl-tRNA ligase activity tRNA metabolic process RNA binding RNA processing Ribosome Ribosome biogenesis Mitochondrial ribosome Ribonucleoprotein complex biogenesis Translation ncRNA metabolic process < 0.0005 < 0.005 < 0.05 Term p value BP CC M Geneset size > 30 20–30 10–20 5–10 Ontolog a Wild-type E12.5 Dorsal cortex ChIP-seq Mitochondrial transport Mitochondrion organization Mitochondrial part Mitochondrial translation tRNA aminoacylation Aminoacyl-tRNA ligase activity tRNA metabolic process RNA binding RNA processing Ribosome Ribosome biogenesis Mitochondrial ribosome Ribonucleoprotein complex biogenesis Translation ncRNA metabolic process < 0.0 < 0.0 < 0.0 Term p va b a Catalytic complex Nucleoplasm part Protein acetyltransferase complex c TSS 30 Mean density (tag/50 bp) 0 TSS Yy1 binding at E12.5 –5 kb 5 kb c Ribosome biogenesis E12.5 Promoter TSS Intergenic 5′ UTR 3′ UTR Intron Exon Other Ech1 30 0 6 0 Napepld Uqcc2 10 0 40 0 6 0 10 kb 2 kb 2 kb Ndufc1 2 kb Bscl2 2 kb Eif4g2 8 0 10 kb Tyw3 7 0 10 kb Utp20 8 0 50 kb f d E12.5 Metabolism E12.5 Protein translation g Ech1 30 0 Uqcc2 6 0 2 kb 2 kb E12.5 Metabolism g E12.5 Promoter TSS Intergenic 5′ UTR 3′ UTR Intron Exon Other Eif4g2 8 0 10 kb Tyw3 7 0 10 kb Utp20 8 0 50 kb f d E12.5 Protein translation Ech1 30 0 2 kb E12.5 Metabolism g d 6 0 Napepld 40 0 10 kb Ndufc1 2 kb g f Eif4g2 0 Tyw3 7 0 10 kb Utp20 8 0 50 kb Napepld 10 0 Bscl2 2 kb Napepld Ech1 Uqcc2 6 0 2 kb Uqcc2 Bscl2 Fig. 5 Yy1 directly binds to metabolic genes. a Genome-wide binding of Yy1 to DNA regions was analyzed by chromatin immunoprecipitation against Yy1 followed by sequencing (ChIP-seq) of cortex cells derived at E12.5. Statistical analysis of the enriched regions identiﬁed 464 binding events at E12.5 (FDR < 0.001), present in at least 1 of 2 replicas. b Homer motif discovery revealed a known Yy1-binding motif as the highest scoring motif (p < 1e-50). c Read cluster proﬁle reveals preferential binding of Yy1 close to the transcription start site (TSS) of target genes. d Distribution of binding sites to different genomic locations. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 a RNA-seq comparing Yy1cKO versus control cortex tissue at E11.5 identiﬁed 1554 differentially expressed genes (\|log2 fold change\| > 0.3, p < 0.05, FDR < 0.01). b Gene Ontology (GO) term network analysis on the basis of differentially regulated genes. Each node represents an enriched GO term (adjusted p value (Corrected with Bonferroni step down procedure) < 0.05). Nodes are interconnected when the gene overlap is > 50%, based on the kappa score. BP, biological process; MF, molecular function; CC, cellular component. A fully labeled version of the network is given in Supplementary Fig. 6. c–e qRT-PCR validation for differentially regulated genes comparing control vs Yy1cKO cortical tissue at E12.5 conﬁrms RNA-seq results. Comparisons were performed using the two-tailed unpaired Student’s t test. Data are the mean ± standard deviation To functionally test whether the impaired expression of nuclear encoded mitochondrial genes affects mitochondrial bioenergetics, we assessed mitochondrial function by measuring the oxygen consumption rate (OCR) of control and Yy1cKO cells directly isolated from E12.5 cortices. Real-time measurements of OCR demonstrated that Yy1 inactivation led to impaired basal respiration, ATP-linked OCR, and maximal respiration capacities motifs in a functionally grouped network demonstrated an association of Yy1-target genes with metabolism and mitochon- drial functions as well as with RNA processing, ribosome biogenesis, and protein translation (Fig. 5e–g, Supplementary Fig. 8 and Supplementary Data 2). Thus, Yy1 directly controls expression of numerous gene sets implicated in metabolic and translational processes. 7 NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Yy1 binds preferentially to the promoter region of the transcription start site (TSS) of genes. e Gene Ontology (GO) term network analysis on the basis of Yy1-bound target genes at E12.5. Each node represents an enriched GO term (adjusted p value (Corrected with Bonferroni step down procedure) < 0.05). Nodes are interconnected when the gene overlap is > 50%, based on the kappa score. BP, biological process; MF, molecular function; CC, cellular component. A fully labeled version of the network is given in Supplementary Fig. 8. f, g Genomic snapshots depicting Yy1-binding events at metabolic genes (g) and genes involved in protein translation (f). kb, kilo bases Apart from general metabolic processes, the analysis of our RNA-seq and ChIP-seq data indicated an involvement of Yy1 in protein translation. To functionally validate these ﬁndings, we quantiﬁed global protein synthesis in cortical cells with either undisturbed or reduced Yy1 levels by measuring incorporation of O-propargyl-puromycin (OP-puro) into nascent proteins31. In cells isolated from E11.5 cortices and treated with siYy1 RNA, the relative protein synthesis rate was highly reduced as compared with control cells (Fig. 6e, f). Likewise, cells directly isolated from the dorsal cortex of Yy1cKO embryos at E12.5 displayed signiﬁcantly lower translation rates than measured in control cortical cells (Fig. 6i, j). To elucidate whether differences in protein translation might depend on the cell cycle stage, we performed ﬂow cytometric analysis of OP-puro incorporation intensity in combination with propidium of cortical cells (Fig. 6a, b). These data could not simply be explained by reduced amounts of mitochondria, as the ratio of mitochondrial over nuclear DNA (Mit1 and CytB vs intergenic region) was not altered (Fig. 6c and Supplementary Fig 9a). Likewise, we excluded that apoptosis contributed to this phenotype since the number of living cells (7AAD-negative) just before OCR measurement was not altered in control vs. Yy1cKO cells (Supplementary Fig 9b, c). To obtain a global overview of metabolic changes induced by loss of Yy1, we analyzed mass spectrometry-based metabolic proﬁles of control and siYy1 KD cortical cells derived from E11.5 embryos. Quantitative examina- tion of the untargeted metabolomics data showed downregulation of various metabolic processes including glucose, lipid, amino acid, and nucleotide metabolism upon Yy1 KD (Fig. 6d and Supplementary Data 3). of cortical cells (Fig. 6a, b). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 ARTICLE FCCP Antimycin A/ rotenone * * * * * * * 0 0.25 0.50 0.75 1.00 1.25 Rel. OCR 0 0.25 0.50 0.75 1.00 1.25 Rel. OCR ns ns ns ns Control Yy1cKO Basal resp. ATP linked Max. resp. 1 2 3 4 5 6 7 8 9 10 11 12 a b E12.5 Control Yy1cKO Control Yy1cKO E12.5 Mit1 vs. intg1 0 50 100 150 200 mtDNA/nDNA ratio c d 0 1.0 siCtrl siYy1 Rel. protein synthesis rate e f i siCtrl siYy1 j Glucose metabolism Fructose and mannose degrad. Galactose metab. Gluconeogenesis Glucose-alanine cycle Glycolysis Lactose degrad. Lactose synthesis Pyruvate metab. TCA and OxPhos Citric acid cycle Malate-aspartate shuttle Mito. electron transport chain Lipid metabolism β-oxidation of very long chain FA Carnitine synthesis FA biosynthesis FA elongation in mitochondria FA metabolism Glycerolipid metab. Mito. β-oxid. of long chain saturated FA Mito. β-oxid. of medium chain saturated FA Mito. β-oxid. of short chain saturated FA Oxid. of branched chain FA Transfer of acetyl groups into mitochondria Amino acid metabolism Alanine metab. Amino sugar metab. Arginine and proline metab. Aspartate metab. Beta-alanine metab. Betaine metab. Cysteine metab. D-arginine and D-ornithine metab. Glutamate metab. Glutathione metab. Glycine and serine metab. Histidine metab. Homocysteine degrad. Lysine degrad. Methionine metab. Phenylalanine and tyrosine metab. Threonine and 2-oxobutanoate degrad. Tryptophan metab. Tyrosine metab. Valine, leucine and isoleucine degrad. Nucleotide metabolism Folate metab. Nucleotide sugars metab. Pentose phosphate pathway Purine metab. Pyrimidine metab. Other Ammonia recycling Androgen and estrogen metab. Arachidonic acid metab. Bile acid biosynthesis Biotin metab. Butyrate metab. Caffeine metab. Catecholamine biosynthesis Degradation of superoxides DNA replication fork Ethanol degrad. Glycerol phosphate shuttle Inositol metab. Inositol phosphate metab. Ketone body metab. Nicotinate and nicotinamide metab. Pantothenate and CoA biosynthesis Phenylacetate metab. Phosphatidylinositol phosphate metab. Phospholipid biosynthesis Phytanic acid peroxisomal oxidation Plasmalogen synthesis Porphyrin metab. Propanoate metab. Pterine biosynthesis Pyruvaldehyde degrad. Retinol metab. Riboflavin metab. Selenoamino acid metab. Spermidine and spermine biosynthesis Sphingolipid metab. Starch and sucrose metab. Steroid biosynthesis Steroidogenesis Sulfate/sulfite metab. Taurine and hypotaurine metab. Thiamine metab. Transcription/translation Trehalose degradation Ubiquinone biosynthesis Urea cycle Vitamin B6 metab. Vitamin K metab. Down Up 0 13 –Log (p value) Enriched metabolic pathways siYy1 vs. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 siCtrl Regulation upon siYy1 siCtrl siYy1 siCtrl siYy1 Count OPP intensity Ctrl Yy1cKO OPPneg ctrl OPP intensity E12.5 g h l Ctrl Yy1cKO siCtrl siYy1 OPPneg ctrl OPP intensity OPP intensity Count k E12.5 G0/G1 G0/G1 G0/G1 S/G2/M S/G2/M G0/G1 S/G2/M S/G2/M Ctrl Yy1cKO Ctrl Yy1cKO Count Count Oligomycin Timepoint p = 0.002 p = 0.003 p = 0.005 ns 1.2 0.8 0.6 0.4 0.2 0 1.0 Rel. protein synthesis rate 1.2 0.8 0.6 0.4 0.2 0 1.0 Rel. protein synthesis rate 1.2 0.8 0.6 0.4 0.2 0 1.0 Rel. protein synthesis rate 1.2 0.8 0.6 0.4 0.2 Ctrl Yy1cKO p < 0.001 p < 0.001 p < 0.001 p = 0.001 p = 0.002 p = 0.001 Fig. 6 Yy1 controls cortical metabolism and protein translation rate. a, b Oxygen consumption rate (OCR) measurement of isolated cortical cells at E using a Seahorse Extracellular Flux Analyzer reveals impaired mitochondrial bioenergetics upon ablation of Yy1. Injection of electron transport chain inhibitors are indicated after measurement 3 (oligomycin, ATP synthase inhibitor), 6 (FCCP, mitochondrial uncoupler) and 9 (Antimycin A/rotenon complex III & I inhibitors). Parameters derived from a are indicated in b: basal respiration, ATP-linked OCR, and maximum respiration capacity. Dat represented relative to ﬁrst basal respiration measurement of controls and as a mean of n = 9 (control), n = 6 (Yy1cKO) error bars indicate standard e of the mean. c qRT-PCR for mitochondrial DNA content shows no difference between Yy1cKO and control cortex tissue. Graphs present mitochond (Mit1) versus nuclear (intergenic region, intg1) DNA ratio. d Metabolomic alterations in isolated E11.5 NPCs upon knockdown of Yy1 for 48 h. Heatm shows enrichment of metabolic pathways which are downregulated or upregulated upon treatment with siYy1. Abbreviations: oxidative phosphoryla (OxPhos), β-oxidation (β-Ox), fatty acid (FA), metabolism (metab.), mitochondrial (mito.), degradation (degrad.). e–h Knockdown of Yy1 reduces pro translation rate. OP-puro (OPP) intensity histogram of representative siRNA-treated samples pulsed with OPP for 30 min and OPP-negative control Quantiﬁcation of mean ﬂuorescent OPP intensity (f). OPP incorporation in siRNA-treated cortex cells in G0G1 (DNA content = 2c) and S/G2/M (D content > 2c) phases of the cell cycle (g, h). DNA content was determined using propidium iodide. i–l Reduced protein translation rate in Yy1cKO cel E12.5. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 OP-puro (OPP) intensity histogram of representative siRNA-treated samples pulsed with OPP for 30 min and OPP-negative control (e). Quantiﬁcation of mean ﬂuorescent OPP intensity (f). OPP incorporation in siRNA-treated cortex cells in G0G1 (DNA content = 2c) and S/G2/M (DNA content > 2c) phases of the cell cycle (g, h). DNA content was determined using propidium iodide. i–l Reduced protein translation rate in Yy1cKO cells at E12.5. OP-puro (OPP) intensity histogram of representative E12.5 control and Yy1cKO cells pulsed with OPP for 30 min and OPP-negative control (i). Quantiﬁcation of mean ﬂuorescent OPP intensity (j). OPP incorporation in cortical cells in G0G1 (DNA content = 2c) and S/G2/M (DNA content > 2c) phases of the cell cycle (k, l). DNA content was determined using propidium iodide. Comparisons were performed using the two-tailed unpaired Student’s t test. Data are the mean ± standard deviation (c, f, h, j, l) and ± standard error of the mean (a, b). p < 0.05. ns = not signiﬁcant Fig. 6 Yy1 controls cortical metabolism and protein translation rate. a, b Oxygen consumption rate (OCR) measurement of isolated cortical cells at E12.5 using a Seahorse Extracellular Flux Analyzer reveals impaired mitochondrial bioenergetics upon ablation of Yy1. Injection of electron transport chain inhibitors are indicated after measurement 3 (oligomycin, ATP synthase inhibitor), 6 (FCCP, mitochondrial uncoupler) and 9 (Antimycin A/rotenone, complex III & I inhibitors). Parameters derived from a are indicated in b: basal respiration, ATP-linked OCR, and maximum respiration capacity. Data represented relative to ﬁrst basal respiration measurement of controls and as a mean of n = 9 (control), n = 6 (Yy1cKO) error bars indicate standard error of the mean. c qRT-PCR for mitochondrial DNA content shows no difference between Yy1cKO and control cortex tissue. Graphs present mitochondrial (Mit1) versus nuclear (intergenic region, intg1) DNA ratio. d Metabolomic alterations in isolated E11.5 NPCs upon knockdown of Yy1 for 48 h. Heatmap shows enrichment of metabolic pathways which are downregulated or upregulated upon treatment with siYy1. Abbreviations: oxidative phosphorylation (OxPhos), β-oxidation (β-Ox), fatty acid (FA), metabolism (metab.), mitochondrial (mito.), degradation (degrad.). e–h Knockdown of Yy1 reduces protein translation rate. OP-puro (OPP) intensity histogram of representative siRNA-treated samples pulsed with OPP for 30 min and OPP-negative control (e). Quantiﬁcation of mean ﬂuorescent OPP intensity (f). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 OP-puro (OPP) intensity histogram of representative E12.5 control and Yy1cKO cells pulsed with OPP for 30 min and OPP-negative control (i Quantiﬁcation of mean ﬂuorescent OPP intensity (j). OPP incorporation in cortical cells in G0G1 (DNA content = 2c) and S/G2/M (DNA content > phases of the cell cycle (k, l). DNA content was determined using propidium iodide. Comparisons were performed using the two-tailed unpaired Stude t test. Data are the mean ± standard deviation (c, f, h, j, l) and ± standard error of the mean (a, b). p < 0.05. ns = not signiﬁcant 0 0.25 0.50 0.75 1.00 1.25 Rel. OCR O Basal resp. ATP linked Max. resp. b Control Yy1cKO E12.5 p = 0.002 p = 0.003 p = 0.005 FCCP Antimycin A/ rotenone * * * * * * * * 0 0.25 0.50 0.75 1.00 1.25 Rel. OCR ns ns ns ns 1 2 3 4 5 6 7 8 9 10 11 12 a Oligomycin Timepoint Control Yy1cKO Mit1 vs. intg1 0 50 100 150 200 mtDNA/nDNA ratio c ns b c a Co Yy1 d Glucose metabolism Fructose and mannose degrad. Galactose metab. Gluconeogenesis Glucose-alanine cycle Glycolysis Lactose degrad. Lactose synthesis Pyruvate metab. TCA and OxPhos Citric acid cycle Malate-aspartate shuttle Mito. electron transport chain Lipid metabolism β-oxidation of very long chain FA Carnitine synthesis FA biosynthesis FA elongation in mitochondria FA metabolism Glycerolipid metab. Mito. β-oxid. of long chain saturated FA o. β-oxid. of medium chain saturated FA Mito. β-oxid. of short chain saturated FA Oxid. of branched chain FA nsfer of acetyl groups into mitochondria Amino acid metabolism Alanine metab. Amino sugar metab. Arginine and proline metab. Aspartate metab. Beta-alanine metab. Betaine metab. Cysteine metab. D-arginine and D-ornithine metab. Glutamate metab. Glutathione metab. Glycine and serine metab. Histidine metab. Homocysteine degrad. Lysine degrad. Methionine metab. Phenylalanine and tyrosine metab. Threonine and 2-oxobutanoate degrad. Tryptophan metab. Tyrosine metab. Valine, leucine and isoleucine degrad. Nucleotide metabolism Folate metab. Nucleotide sugars metab. Pentose phosphate pathway Purine metab. Pyrimidine metab. Other Ammonia recycling Androgen and estrogen metab. Arachidonic acid metab. Bile acid biosynthesis Biotin metab. Butyrate metab. Caffeine metab. Catecholamine biosynthesis Degradation of superoxides DNA replication fork Ethanol degrad. Glycerol phosphate shuttle Inositol metab. Inositol phosphate metab. Ketone body metab. Nicotinate and nicotinamide metab. Pantothenate and CoA biosynthesis Phenylacetate metab. Phosphatidylinositol phosphate metab. Phospholipid biosynthesis Phytanic acid peroxisomal oxidation Plasmalogen synthesis Porphyrin metab. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 These data could not simply be explained by reduced amounts of mitochondria, as the ratio of mitochondrial over nuclear DNA (Mit1 and CytB vs intergenic region) was not altered (Fig. 6c and Supplementary Fig 9a). Likewise, we excluded that apoptosis contributed to this phenotype since the number of living cells (7AAD-negative) just before OCR measurement was not altered in control vs. Yy1cKO cells (Supplementary Fig 9b, c). To obtain a global overview of metabolic changes induced by loss of Yy1, we analyzed mass spectrometry-based metabolic proﬁles of control and siYy1 KD cortical cells derived from E11.5 embryos. Quantitative examina- tion of the untargeted metabolomics data showed downregulation of various metabolic processes including glucose, lipid, amino acid, and nucleotide metabolism upon Yy1 KD (Fig. 6d and Supplementary Data 3). NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunicatio 8 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Propanoate metab. Pterine biosynthesis Pyruvaldehyde degrad. Retinol metab. Riboflavin metab. Selenoamino acid metab. Spermidine and spermine biosynthesis Sphingolipid metab. Starch and sucrose metab. Steroid biosynthesis Steroidogenesis Sulfate/sulfite metab. Taurine and hypotaurine metab. Thiamine metab. Transcription/translation Trehalose degradation Ubiquinone biosynthesis Urea cycle Vitamin B6 metab. Vitamin K metab. Down Up 0 13 –Log (p value) Enriched metabolic pathways siYy1 vs. siCtrl Regulation upon siYy1 Timepoint d d siCtrl siYy1 h G0/G1 S/G2/M 0 1.0 Rel. protein synthesis rate 1.2 0.8 0.6 0.4 0.2 p = 0.002 p = 0.001 siCtrl siYy1 siCtrl siYy1 Count OPP intensity g G0/G1 S/G2/M 0 1.0 siCtrl siYy1 Rel. protein synthesis rate f T 1.2 0.8 0.6 0.4 0.2 p = 0.001 e M siCtrl siYy1 OPPneg ctrl OPP intensity Count h g e i Ctrl Yy1cKO OPPneg ctrl OPP intensity E12.5 OPP intensity Count j j l 0 1.0 Rel. protein synthesis rate 1.2 0.8 0.6 0.4 0.2 Ctrl Yy1cKO p < 0.001 j i l l Ctrl Yy1cKO G0/G1 S/G2/M 0 1.0 Rel. protein synthesis rate 1.2 0.8 0.6 0.4 0.2 p < 0.001 p < 0.001 y OPP intensity Count k E12.5 G0/G1 S/G2/M Ctrl Yy1cKO Ctrl Yy1cKO k Fig. 6 Yy1 controls cortical metabolism and protein translation rate. a, b Oxygen consumption rate (OCR) measurement of isolated cortical cells at E12.5 using a Seahorse Extracellular Flux Analyzer reveals impaired mitochondrial bioenergetics upon ablation of Yy1. Injection of electron transport chain inhibitors are indicated after measurement 3 (oligomycin, ATP synthase inhibitor), 6 (FCCP, mitochondrial uncoupler) and 9 (Antimycin A/rotenone, complex III & I inhibitors). Parameters derived from a are indicated in b: basal respiration, ATP-linked OCR, and maximum respiration capacity. Data represented relative to ﬁrst basal respiration measurement of controls and as a mean of n = 9 (control), n = 6 (Yy1cKO) error bars indicate standard error of the mean. c qRT-PCR for mitochondrial DNA content shows no difference between Yy1cKO and control cortex tissue. Graphs present mitochondrial (Mit1) versus nuclear (intergenic region, intg1) DNA ratio. d Metabolomic alterations in isolated E11.5 NPCs upon knockdown of Yy1 for 48 h. Heatmap shows enrichment of metabolic pathways which are downregulated or upregulated upon treatment with siYy1. Abbreviations: oxidative phosphorylation (OxPhos), β-oxidation (β-Ox), fatty acid (FA), metabolism (metab.), mitochondrial (mito.), degradation (degrad.). e–h Knockdown of Yy1 reduces protein translation rate. NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 p < 0.05 induced knockout of Yy1 (\|log2 fold change\| > 0.32, p < 0.05, FDR < 0.05). We then compared changes in gene expression of direct Yy1 targets at early and late stages. Although some genes with signiﬁcantly changed expression (p < 0.05, FDR < 0.05) at E11.5 were not changed at E15.5, the majority of genes were similarly deregulated at both early and late stages (Fig. 7h). Likewise, when considering both indirect and direct Yy1 targets, the majority of Yy1-dependent gene products localizing to mitochondria or involved in metabolism and protein translation were similarly deregulated at E11.5 and E15.5 (Supplementary Fig. 10a–e). Accordingly, metabolic processes and lipid metabolism were enriched among GO terms for downregulated genes in Yy1iKO embryos at E15.5 (Supplementary Fig. 10f), comparable to the situation at E11.5 (Fig. 4b). In agreement with the observation that Yy1 seems to regulate similar genes throughout cortical development, computational prediction of transcription factor binding by i-cisTarget32 to ﬁnd Yy1 co-factors revealed that both at E12.5 and E15.5, the highest enriched motifs were from E2f induced knockout of Yy1 (\|log2 fold change\| > 0.32, p < 0.05, FDR < 0.05). We then compared changes in gene expression of direct Yy1 targets at early and late stages. Although some genes with signiﬁcantly changed expression (p < 0.05, FDR < 0.05) at E11.5 were not changed at E15.5, the majority of genes were similarly deregulated at both early and late stages (Fig. 7h). Likewise, when considering both indirect and direct Yy1 targets, the majority of Yy1-dependent gene products localizing to mitochondria or involved in metabolism and protein translation were similarly deregulated at E11.5 and E15.5 (Supplementary Fig. 10a–e). Accordingly, metabolic processes and lipid metabolism were enriched among GO terms for downregulated genes in Yy1iKO embryos at E15.5 (Supplementary Fig. 10f), comparable to the situation at E11.5 (Fig. 4b). In agreement with the observation that Yy1 seems to regulate similar genes throughout cortical development, computational prediction of transcription factor binding by i-cisTarget32 to ﬁnd Yy1 co-factors revealed that both at E12.5 and E15.5, the highest enriched motifs were from E2f cortical development. The stage-dependent phenotype observed in Yy1cKO and Yy1iKO embryos (Figs. 1 and 2) raises the question of whether Yy1 functions might change during the course of cortical development. To address this we performed ChIP-seq of wild-type cortical cells at E15.5 (Fig. 7a). Similar to what we observed at E12.5 (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 OPP incorporation in siRNA-treated cortex cells in G0G1 (DNA content = 2c) and S/G2/M (DNA content > 2c) phases of the cell cycle (g, h). DNA content was determined using propidium iodide. i–l Reduced protein translation rate in Yy1cKO cells at E12.5. OP-puro (OPP) intensity histogram of representative E12.5 control and Yy1cKO cells pulsed with OPP for 30 min and OPP-negative control (i). Quantiﬁcation of mean ﬂuorescent OPP intensity (j). OPP incorporation in cortical cells in G0G1 (DNA content = 2c) and S/G2/M (DNA content > 2c) phases of the cell cycle (k, l). DNA content was determined using propidium iodide. Comparisons were performed using the two-tailed unpaired Student’s t test. Data are the mean ± standard deviation (c, f, h, j, l) and ± standard error of the mean (a, b). p < 0.05. ns = not signiﬁcant preparations, as well as in cycling progenitor cells in S/G2/M phase (DNA content > 2c) (Fig. 6g, h, k, l). preparations, as well as in cycling progenitor cells in S/G2/M phase (DNA content > 2c) (Fig. 6g, h, k, l). iodide-based measurements of DNA content in siYy1 and Yy1cKO cells and their respective control cell populations (Supplementary Fig. 9d). These experiments demonstrated that proper translation was dependent on Yy1 both in G0/G1 cells (DNA content = 2c), which comprise NPCs in G1 phase of the cell cycle and postmitotic neurons potentially present in the cell iodide-based measurements of DNA content in siYy1 and Yy1cKO cells and their respective control cell populations (Supplementary Fig. 9d). These experiments demonstrated that proper translation was dependent on Yy1 both in G0/G1 cells (DNA content = 2c), which comprise NPCs in G1 phase of the cell cycle and postmitotic neurons potentially present in the cell Stage-dependent requirements for biosynthesis in NPCs. Our study shows that Yy1 controls a broad spectrum of metabolic pathways together with global protein synthesis at early stages of TURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications 9 9 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 p < 0.05 Highest scoring motif (homer) p value < 1e(–50) b ********************************************************************************** **** * *** * ****** * * ** * * * * * **** * * ** ** 3 0 log2 fold change 30 0 –5 kb TSS Mean density (tag/50 bp) 5 kb E11.5 Yy1cKO E15.5 Yy1iKO –3 Yy1 Zfp37 AW209491 Prepl 1110034G24Rik Cnnm2 Ech1 Ehd3 Napepld Phf8 Cradd 9030624J02Rik Knstrn Thap2 Zfp764 Chd2 N4bp1 Zfp747 3110043O21Rik Dpagt1 Slmap Pcif1 Psmb5 Zfp563 Mettl3 Dedd Slx4ip 9130019O22Rik Morf4l1 Caml Tra2b Cuedc2 Fam193b Eif4g2 Gba Nelfa Ndufb5 Zbtb11 Cdk5rap2 Map2k5 Lemd2 Txndc15 Srsf3 Prr14 Fig4 Cxxc1 Use1 Dnajc7 Ndufs2 Poglut1 Mff Wdr46 AU040320 Mrps7 Wdr77 Ftsj3 Pik3c3 Psmd7 Hdlbp Actr8 Tomm40l Rpa2 Osbpl11 Rrm1 Tm9sf4 Rcan1 Tor1a 0610009B22Rik Wbscr16 Fiz1 Gtf3c6 Ndrg3 Usf1 Fbxo38 Slc39a7 Sdf4 Wdr18 Mrps27 Lrrc41 Akt1s1 Sumf1 Ptrhd1 Qrsl1 Utp20 d h TSS 5 kb Yy1 binding at E15.5 –5 kb c Dorsal cortex ChIP-seq Wild-type E15.5 a b c E15.5 Promoter TSS Intergenic 5′ UTR 3′ UTR Intron Exon Other e f f Prom1 FITC E12.5 E15.5 279 188 58 h e Dorsal cortex Prom1+ cells Tamoxifen: E15.5 E12.5 FITC SSC-A Unstained ctrl g g Prom1-FITC Fig. 7 Yy1 controls a similar gene set throughout cortex development. a Genome-wide binding of Yy1 to DNA regions was analyzed by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of cortex cells derived at E15.5. Statistical analysis of the enriched regions identiﬁed 246 binding events at E15.5 (FDR < 0.001), present in at least 1 of 2 replicas. b Homer motif discovery revealed the known Yy1-binding motif as the highest scoring motif (p < 1e-50). c–e Read cluster proﬁle and distribution of binding sites to different genomic locations at E15.5. Yy1 binds preferentially to the promoter region of the transcription start site (TSS) of genes. f Analysis of overlap between E12.5 and E15.5 ChIP-seq data. g RNA-seq comparing E15.5 Yy1iKO vs control (each n = 3 embryos) radial glia cells from the dorsal cortex which were isolated by microdissection and subsequent Prominin1-based (CD133-FITC) FACS. A total of 1080 genes were downregulated (660 genes) or upregulated (420 genes) upon induced knockout of Yy1 (\|log2 fold change\| > 0.32, p < 0.05, FDR < 0.05). h RNA-seq expression of genes at E11.5 and E15.5, which are bound by at E12.5 and E15.5, upon knockout of Yy1. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Highest scoring motif (homer) Dorsal cortex ChIP-seq p value < 1e(–50) Yy1iKO Ctrl –2.2 2.2 Row z-score Dorsal cortex Prom1+ cells Tamoxifen: E15.5 E12.5 FITC SSC-A Unstained ctrl Prom1-FITC RNA-seq E15.5 ********************************************************************************** **** * *** * ****** * * ** * * * * * **** * * ** ** 3 0 log2 fold change E12.5 E15.5 279 188 58 TSS 5 kb 30 0 –5 kb TSS E15.5 Promoter TSS Intergenic 5′ UTR Wild-type E15.5 Yy1 binding at E15.5 –5 kb Mean density (tag/50 bp) 5 kb 3′ UTR Intron Exon Other E11.5 Yy1cKO E15.5 Yy1iKO –3 Yy1 Zfp37 AW209491 Prepl 1110034G24Rik Cnnm2 Ech1 Ehd3 Napepld Phf8 Cradd 9030624J02Rik Knstrn Thap2 Zfp764 Chd2 N4bp1 Zfp747 3110043O21Rik Dpagt1 Slmap Pcif1 Psmb5 Zfp563 Mettl3 Dedd Slx4ip 9130019O22Rik Morf4l1 Caml Tra2b Cuedc2 Fam193b Eif4g2 Gba Nelfa Ndufb5 Zbtb11 Cdk5rap2 Map2k5 Lemd2 Txndc15 Srsf3 Prr14 Fig4 Cxxc1 Use1 Dnajc7 Ndufs2 Poglut1 Mff Wdr46 AU040320 Mrps7 Wdr77 Ftsj3 Pik3c3 Psmd7 Hdlbp Actr8 Tomm40l Rpa2 Osbpl11 Rrm1 Tm9sf4 Rcan1 Tor1a 0610009B22Rik Wbscr16 Fiz1 Gtf3c6 Ndrg3 Usf1 Fbxo38 Slc39a7 Sdf4 Wdr18 Mrps27 Lrrc41 Akt1s1 Sumf1 Ptrhd1 Qrsl1 Utp20 a b c d e f h g Fig. 7 Yy1 controls a similar gene set throughout cortex development. a Genome-wide binding of Yy1 to DNA regions was analyzed by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of cortex cells derived at E15.5. Statistical analysis of the enriched regions identiﬁed 246 bind events at E15.5 (FDR < 0.001), present in at least 1 of 2 replicas. b Homer motif discovery revealed the known Yy1-binding motif as the highest scoring mo (p < 1*e-50). c–e Read cluster proﬁle and distribution of binding sites to different genomic locations at E15.5. Yy1 binds preferentially to the promoter region the transcription start site (TSS) of genes. f Analysis of overlap between E12.5 and E15.5 ChIP-seq data. g RNA-seq comparing E15.5 Yy1iKO vs control (ea n = 3 embryos) radial glia cells from the dorsal cortex which were isolated by microdissection and subsequent Prominin1-based (CD133-FITC) FACS. A tota 1080 genes were downregulated (660 genes) or upregulated (420 genes) upon induced knockout of Yy1 (\|log2 fold change\| > 0.32, p < 0.05, FDR < 0.0 h RNA-seq expression of genes at E11.5 and E15.5, which are bound by at E12.5 and E15.5, upon knockout of Yy1. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 In addition, some genes involved in cell cycle progression were upregulated speciﬁcally at late stages. However, based on our ChIP-seq analysis, all but two of these genes were not bound and only indirectly regulated by Yy1, pointing to compensatory mechanisms active in E15.5 Yy1iKO NPCs. Moreover, the two bound genes, Cdk5rap2 and Cdc45, were regulated in the same direction at both developmental stages. In summary, our data suggest that the gene regulatory network dependent on Yy1 does not change overtly along development, raising the question of whether later stage cortical cells can cope better with the changes in gene expression induced by Yy1 inactivation. Conditional ablation of Yy1 at early stages of cortical devel- opment resulted in microcephaly owing to decreased cell pro- liferation and increased cell death. Emx1-Cre- and Emx1- CreERT2-dependent recombination leads to ablation of the ﬂoxed gene in NPCs as well as neurons16,22. However, the observed phenotype appears to be owing to a requirement for Yy1 in NPCs rather than secondary to defects in neurons, given the very low number of neurons present at stages displaying the most prominent phenotype and, vice versa, the absence of a pro- liferation phenotype at later stages, when many neurons are present. Moreover, neuronal differentiation as such was not affected by the loss of Yy1. In addition, siRNA-mediated knockdown experiments in isolated NPCs recapitulated the Yy1cKO phenotype, suggesting a cell-autonomous effect speciﬁ- cally in NPCs. g y y To functionally address this point, we assessed whether loss of Yy1 after the onset of neurogenesis inﬂuences mitochondrial bioenergetics. Intriguingly, basal respiration, ATP-linked OCR, and maximal respiration capacity at E15.5 were not impaired upon late Yy1 inactivation (Fig. 8a–c), unlike at E12.5 after early Yy1 deletion (Fig. 6a, b). Likewise, global protein synthesis rates were only mildly affected at E15.5, especially when taking into account proliferating progenitor cells in S/G2/M phase (Fig. 8d–g). Importantly, global protein synthesis rates of Prom1+ RG cells of Yy1iKO and control cells were indistinguishable (Fig. 8h, i). Thus, consistent with the decreasing disturbance of proliferation and survival observed when Yy1 was ablated at later develop- mental stages (Fig. 2), mitochondrial bioenergetics and protein translation were not affected upon Yy1 deletion after the onset of neurogenesis. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 5), ChIP-seq for Yy1 at E15.5 showed that a Yy1-binding element was the most sig- niﬁcantly enriched motif and that most ChIP-seq peaks were found in proximity to the TSS of the target genes (Fig. 7b–e). Surprisingly, binding events which were detected at least once in both ChIP-seq replicas for E12.5 and E15.5, overlapped to a large extent between the two developmental stages (Fig. 7f, Supple- mentary Fig. 9e, f), indicating that Yy1 exhibits a similar binding pattern at these stages. p g Next, we performed RNA-seq analysis of FACS-isolated Prominin1+ radial glia (RG) cells of Yy1iKO vs control embryos at E15.5 upon late ablation of Yy1 (Fig. 7g). A total of 1080 genes were downregulated (660 genes) or upregulated (420 genes) upon NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications 10 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Yy1 is crucial during early cortical development to sustain NPC proliferation and survival, in contrast to developmental stages after the ﬁrst waves of neurogenesis when the activity of Yy1 seems to be dispensable for cortex development. By using a combination of genome-wide DNA-binding site identiﬁcation, transcriptomics, metabolomics, and analysis of protein transla- tion rates we demonstrate that Yy1 is a key factor safeguarding biosynthetic demands speciﬁc for early-stage NPCs. Our study not only reveals that Yy1 controls the expression of metabolic genes, metabolite abundance and mitochondrial bioenergetics during corticogenesis, but also shows that Yy1 is functionally involved in the transcriptional regulation of global protein synthesis. Possibly, aberrations in these processes may contribute to the neurodevelopmental defects in humans with heterozygous YY1 loss-of-function mutations1, although this needs to be addressed in future studies. and Ets transcription factor family members (Supplementary Fig. 11a, b). Of note, both E2f and Ets family members have previously emerged as important metabolic regulators33–37. However, although many genes of metabolic and biosynthetic pathways seemed to be affected by Yy1 at both developmental stages, a small but signiﬁcant fraction of such genes were differentially regulated at E11.5 but not at E15.5. These included Ndufc1, Ndufb4, Ndufb5 (subunits of the electron transport chain), Ppargc1α (negative regulator of glycolysis, promotes mitochondrial gene expression), Napepld, Cpt1c (lipid metabo- lism), Eif1b, Rps13, Rpl5, and Akt1s1 (protein translation and ribosome biogenesis) (Fig. 7h and Supplementary Fig. 10a–e). Thus, it is possible that this latter group of genes contributes to the stage-dependent phenotype observed upon ablation of Yy1. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 y Increased apoptosis observed upon ablation of Yy1 was accompanied by accumulation of p53 protein, a tumor-sup- pressor, which integrates various stress signals and activates downstream effectors to promote the appropriate cellular response: either damage repair and cell survival or induction of apoptosis in irreparable cases42,43. Apoptosis can be induced by p53-dependent or -independent processes44. By using both genetic ablation and pharmacological inhibition of p53, we show that apoptosis induced by the loss of Yy1 is dependent on p53 and contributes to the microcephaly phenotype. Yy1 has been implicated in the direct regulation of p53 and the phenotype upon loss of Yy1 in thymocytes can be completely rescued by p53 deﬁciency26. In contrast, inhibition of p53 in Yy1cKO embryos neither ameliorated the proliferation phenotype, nor rescued expression of Yy1-dependent metabolic genes. Thus, apart from p53-mediated cell death, loss of Yy1 in the cortex induces a p53-independent stress response that is associated with impaired proliferation of NPCs. Surprisingly, a recent study using shRNA-mediated knockdown of Yy1 suggested that Yy1 limits NPC expansion and promotes differentiation via repression of Sox2 at E14.514. However, our approach to genetically delete Yy1 did neither reveal alterations of Sox2 protein expression nor regulation of Sox2 mRNA levels and did not increase proliferation. Although the reasons for these con- tradicting ﬁndings remain unclear, it could be explained by the efﬁciency in reducing Yy1 levels using shRNA compared with genetic deletion. Our data are compatible with the hypothesis that the dependency of cortical cells on Yy1 is decreasing at later stages of development, because the main processes regulated by Yy1 might have lost relative importance over time. To address this hypothesis, we measured overall protein synthesis rates at E10.5, E12.5 and E15.5 in wild-type embryos. Levels of OP-puro incorporation were highly reduced in E15.5 cortical cells as compared with E10.5 and E12.5 cells (Fig. 8j, k and Supplemen- tary Fig. 12a, b). Translation was also signiﬁcantly lower in late than in early-stage cortical cells, when assessing OP-puro incorporation in G0/G1 and cycling S/G2/M cells (Fig. 8l, m and Supplementary Fig. 12c, d). Finally, the signiﬁcant decrease in protein translation rate was also apparent when comparing Prom1+ RG cells at E15.5 vs E12.5 (Fig. 8n, o). Thus, the stage- dependent phenotype in survival and proliferation of NPCs seen upon Yy1 inactivation appears to reﬂect stage-dependent requirements for biosynthetic processes. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Similar to our ﬁndings in the embryonic brain, Yy1 exerts a pro-proliferative role in many types of cancer45,46, in lung epi- thelial cells7 and during cardiac development9. Despite the decreased number of CyclinD1-expressing cells and decrease in CyclinD1 mRNA expression in the Yy1cKO cortex, we did not detect direct binding of Yy1 to the promoters of cell cycle pro- gression genes by ChIP-Seq. This suggests that Yy1 does not directly regulate the cell cycle machinery of NPCs, but rather NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications Discussion Regulation of NPC proliferation and survival is essential to ensure correct brain development. Aberrations in cell cycle progression and survival of NPCs lead to decreased numbers of NPCs and lie at the heart of many neurodevelopmental disorders38–41. In this study, we report a previously unappreciated role for the tran- scription factor Yy1 in the regulation of brain size. We show that 11 Ctrl Yy1iKO OPP intensity Yy1iKO Ctrl OPPneg ctrl E15.5 e 0 0.2 0.4 1.2 E15.5 g f Count OPP intensity Ctrl Yy1iKO Ctrl Yy1cKO G0/G1 S/G2/M ns Count Basal resp. ATP linked Max. resp. 0 Rel. OCR ns E15.5 ns ns Rel. OCR Control Timepoint E15.5 0 0.25 0.50 0.75 1.00 1.25 1 2 3 4 5 6 7 8 9 10 11 12 ns ns ns ns E12.5 E15.5 Prominin1+ cells Prominin1+ cells ns Yy1iKO Prominin1+ cells Count Yy1iKO Ctrl OPPneg ctrl E15.5 OPP intensity Prominin1+ cells n o High Low Susceptibility to loss of Yy1 Protein synthesis rate Yy1 expression Metabolic genes Protein synthesis genes Yy1 Functional metabolism and protein translation p53 Proliferation Apoptosis Brain size 0 1 E15.5 Wild-type NMRI ns. Wild-type NMRI E12.5 ns. ns. Count OPP intensity E12.5 E15.5 E10.5 Wild-type NMRI Count OPP intensity E15.5 E12.5 OPPneg ctrl E10.5 m E15.5 E12.5 OPPneg ctrl OPP intensity Count 0.50 0.75 1.00 1.25 0.25 1.50 Yy1iKO Control Rel. Yy1 expression 1.5 0.5 Yy1iKO Control Antimycin A/ rotenone FCCP Oligomycin Yy1iKO 0.6 0.8 1.0 0 0.2 0.4 1.2 0.6 0.8 1.0 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate 0 0.2 0.4 1.2 0.6 0.8 1.0 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate Rel. protein synthesis rate Rel. protein synthesis rate Rel. protein synthesis rate p < 0.001 Yy1iKO Ctrl p = 0.012 G0/G1 S/G2/M Ctrl p < 0.001 E15.5 E12.5 E10.5 p < 0.001 p < 0.001 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate p < 0.001 G0/G1 S/G2/M G0/G1 S/G2/M a b c d i k j h l p E10.5 E15.5 E12.5 E15.5 E10.5 p = 0.036 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 0 1 E15.5 Rel. Yy1 expression 1.5 0.5 Yy1iKO Control p < 0.001 a Rel. OCR Control Timepoint E15.5 0 0.25 0.50 0.75 1.00 1.25 1 2 3 4 5 6 7 8 9 10 11 12 ns ns ns ns Antimycin A/ rotenone FCCP Oligomycin Yy1iKO b Basal resp. ATP linked Max. resp. 0 Rel. OCR ns E15.5 ns ns 0.50 0.75 1.00 1.25 0.25 1.50 Yy1iKO Control c b a c Ctrl Yy1iKO e p 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate p = 0.036 E15.5 f Count OPP intensity Ctrl Yy1iKO Ctrl Yy1cKO G0/G1 S/G2/M OPP intensity Yy1iKO Ctrl OPPneg ctrl E15.5 Count d 0 0.2 0.4 1.2 g ns 0.6 0.8 1.0 Rel. protein synthesis rate Yy1iKO Ctrl p = 0.012 G0/G1 S/G2/M f g d e ns Yy1iKO Prominin1+ cells 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate Ctrl i i Count Yy1iKO Ctrl OPPneg ctrl E15.5 OPP intensity Prominin1+ cells h i Wild-type NMRI Count OPP intensity E15.5 E12.5 OPPneg ctrl E10.5 j Wild-type NMRI ns. 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate p < 0.001 E15.5 E12.5 E10.5 k j h k Count E12.5 E15.5 Prominin1+ cells Prominin1+ cells n o E15.5 E12.5 OPPneg ctrl OPP intensity Count 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate p < 0.001 E E E Prominin1+ cells n E15.5 E12.5 OPPneg ctrl OPP intensity Count Count OPP intensity E12.5 E15.5 E10.5 G0/G1 S/G2/M l Wild-type NMRI E12.5 ns. ns. Count OPP intensity E12.5 E15.5 E10.5 m p < 0.001 p < 0.001 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate G0/G1 S/G2/M G0/G1 S/G2/M l E10.5 E15.5 Wild-type NMRI E12.5 ns. ns. m p < 0.001 p < 0.001 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate G0/G1 S/G2/M E10.5 E15.5 E12.5 E15.5 Prominin1+ cells o 0 0.2 0.4 1.2 0.6 0.8 1.0 Rel. protein synthesis rate p < 0.001 l o m n o Count Metabolic genes Protein synthesis genes Yy1 Functional metabolism and protein translation p53 Proliferation Apoptosis Brain size p High Low Susceptibility to loss of Yy1 Protein synthesis rate Yy1 expression E12.5 E15.5 E10.5 p inﬂuences other cellular pathways important for proper pro- liferation of early NPCs. ARTICLE Fig. 8 Stage-dependent requirements for biosynthesis in NPCs. a Efﬁcient reduction of Yy1 mRNA at E15.5 upon tamoxifen-induced (TM) ablation at E12.5. b, c Oxygen consumption rate (OCR) measurement of isolated cortical cells at E15.5 using a Seahorse Extracellular Flux Analyzer reveals that mitochondrial bioenergetics are not altered upon ablation of Yy1 at E12.5. Injection of electron transport chain inhibitors are indicated after measurement 3 (oligomycin, ATP synthase inhibitor), 6 (FCCP, mitochondrial uncoupler), and 9 (Antimycin A/rotenone, complex III & I inhibitors). Parameters derived from b are indicated in c: basal respiration, ATP-linked OCR and maximum respiration capacity. Data represented relative to ﬁrst basal respiration measurement of controls and as mean of n = 9 (control), n = 7 (Yy1iKO), error bars indicate standard error of the mean. d–g Protein translation rate is only mildly affected in Yy1iKO cells at E15.5 upon recombination at E12.5. OP-puro (OPP) intensity histogram of representative E15.5 control and Yy1iKO cells pulsed with OPP for 30 min and OPP negative control (d). Quantiﬁcation of mean ﬂuorescent OPP intensity (e). OPP incorporation in cortical cells in G0G1 (DNA content = 2c) and S/G2/M (DNA content > 2c) phases of the cell cycle (f, g). DNA content was determined using propidium iodide. Note that the protein translation rate of cycling progenitor cells is not signiﬁcantly altered between control and Yy1iKO embryos. h, i TM-induced ablation of Yy1 at E12.5 does not alter OPP incorporation in Prominin1+ RG cells at E15.5. j–o Reduced protein translation rate of E15.5 vs E12.5 and E10.5 wild-type cortical cells. OP-puro (OPP) intensity histogram of representative E10.5, E12.5, and E15.5 cells pulsed with OPP for 30 min and OPP-negative control (j). Quantiﬁcation of mean ﬂuorescent OPP intensity (k). OPP incorporation in cortical cells in G0G1 (DNA content = 2c) and S/G2/M (DNA content > 2c) phases of the cell cycle (l, m). DNA content was determined using propidium iodide. The reduction of OPP incorporation is also apparent in Prominin1+ RG cells of E15.5 vs E12.5 (n, o). Comparisons were performed using the two-tailed unpaired Student’s t test. Data are the mean ± standard deviation (a, e, g, i, k, m, o) and ± standard error of the mean (b, c). ns = not signiﬁcant. p Schematic drawing summarizing the results. The transcription factor Yy1 binds and regulates genes involved in metabolism and protein synthesis. ARTICLE Functional biosynthesis sustains proliferation and survival of NPCs and eventually leads to correct brain development. Despite constitutive expression of Yy1, cortical NPCs exhibit a stage- dependent susceptibility to loss of Yy1, which coincides with decreased protein translation rates at later developmental stages mitochondrial biogenesis (Ppargc1a63,64), ribosome subunit and activator of p53 (Rpl561) and inhibition of protein translation (Akt1s165). Judged by their function, other genes could poten- tially also inﬂuence cell behavior by modulating lipid metabolism (Napepld) and protein translation (Eif1b, Rps13). Therefore, it is conceivable that rescued expression of these genes in late-stage Yy1iKO cortices may contribute to the ameliorated metabolic phenotype observed. Surprisingly, many cell cycle progression genes, although not directly bound by Yy1, were upregulated speciﬁcally upon late ablation of Yy1. This further emphasizes stage-speciﬁc transcriptional responses to Yy1 inactivation that potentially inﬂuence the susceptibility of NPCs toward loss of Yy1 at different developmental stages. demonstrate that Yy1 regulates the expression of these genes and thereby functionally controls global protein synthesis rates. y y g p y Historically viewed as mere housekeeping functions, metabo- lism and protein translation have arisen as key players coordi- nating stem cell behavior. Metabolic processes have recently emerged as crucial factors controlling tissue development and homeostasis, in particular, of the nervous system49–53. During cortex development, NPCs have been shown to rely on high glycolytic rates in order to proliferate50 and to modulate their mitochondrial morphology upon differentiation51. In addition, upregulation of electron transport chain components and of oxidative phosphorylation is critical for fast proliferating inter- mediate progenitor cells during adult neurogenesis53. Although little is known about the importance of protein synthesis during brain development, adult hematopoietic54, hair follicle,55 and neural stem cells56 have been demonstrated to synthesize less protein compared with their direct, cycling progeny. Fast pro- liferating cells therefore seem to depend on increased protein translation rates, possibly to satisfy their enormous demand of resources for biomass production. Deregulation in both meta- bolism and protein translation are known to interfere with cell proliferation and survival. Cell cycle progression has been shown to be dependent on the metabolic and nutritional status of a cell57,58 and impaired mitochondrial bioenergetics alone are capable of inducing G1/S cell cycle arrest in Drosophila59. Fur- thermore, ribosome biogenesis stress and increased levels of the ribosomal protein L5 (Rpl5) could contribute to the accumulation of p53 protein in Yy1cKO cortices, similar to observations made in other systems60,61. ARTICLE Thus, the concurrent attenuation of several metabolic pathways as well as decreased protein translation is likely too harsh to cope with for fast proliferating NPCs and results in the upregulation of stress sensors, proliferation defects and p53-dependent cell death observed in Yy1cKO embryos. p g In the complex and highly dynamic environment of brain development, several cellular processes affect corticogenesis in a stage-dependent manner. Cell cycle length of NPCs generally decreases as the embryo develops66–68. As a result of their fast proliferative nature and their substantial biosynthetic demand, early NPCs are thought to be especially vulnerable to cellular stress. For example, genome integrity is vital for NPCs to give rise to healthy daughter cells, but the susceptibility of NPCs to genomic stress appears to alter during the course of development. Interference with genome integrity by ablation of the DNA replication factor TopBP1 resulted in more pronounced neuro- developmental defects when induced in early, compared with late NPCs, despite causing comparable degrees of DNA damage at both developmental stages69,70. Furthermore, mRNA expression proﬁling of RNA-binding proteins identiﬁed many genes to be differentially regulated with progressive cortex development. Consistent with the high protein-synthesis rate of early NPCs, the expression of a majority of genes involved in RNA binding, protein translation, and ribosomal proteins is higher at early stages of corticogenesis71. Conceivably, this reﬂects the high biosynthetic demand of fast proliferating, early progenitor cells in order to synthesize proteins and precursor molecules for biomass production. In analogy to the temporal difference in vulnerability to genotoxic stress, our study indicates that early NPCs are par- ticularly susceptible to metabolic and protein synthesis stress. The shorter cell cycle might render early NPCs less ﬂexible and increase their dependency on rapid accumulation of building blocks. Our ﬁndings thus show that temporal control of basic cellular processes is critical for brain development. In sum, Yy1 plays a central role in cortex development by inﬂuencing stage- dependent progenitor cell behavior through transcriptional reg- ulation of metabolism and protein synthesis. Despite its constitutive expression in NPCs and neurons, our results show that Yy1 becomes less important over the course of embryonic neurogenesis. Our data indicate that, overall, Yy1 binds to and regulates similar target genes throughout cortex development. In agreement, reconstruction of the Yy1 gene reg- ulatory network revealed that E2F and ETS family members were the co-factors with the highest enrichment score at both devel- opmental stages. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Indeed, our molecular analysis demon- strates that Yy1 is a key transcription factor controlling the expression of genes involved in central metabolic pathways, such as glycolysis, TCA cycle, lipid metabolism, nucleotide synthesis, and subunits of the electron transport chain. In addition, we observed decreased mitochondrial function in absence of Yy1, which can further inﬂuence the abundance of precursor mole- cules for biosynthesis47,48. Yy1 has previously been associated with the regulation of metabolic functions, thereby supporting the development and homeostasis of muscle cells and intestinal stem cells5,10,11. Going beyond these previous reports, our RNA-seq and ChIP-seq data sets revealed a very broad implication of Yy1 in various metabolic pathways and also identiﬁed several genes involved in ribosome biogenesis, tRNA synthesis, and protein translation to be direct targets of Yy1. Although binding of Yy1 to genes of the protein translation machinery has been observed before1,6,11,30, this ﬁnding has not been explored further. We now inﬂuences other cellular pathways important for proper pro- liferation of early NPCs. Indeed, our molecular analysis demon- strates that Yy1 is a key transcription factor controlling the expression of genes involved in central metabolic pathways, such as glycolysis, TCA cycle, lipid metabolism, nucleotide synthesis, and subunits of the electron transport chain. In addition, we observed decreased mitochondrial function in absence of Yy1, which can further inﬂuence the abundance of precursor mole- cules for biosynthesis47,48. Yy1 has previously been associated NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications 12 Methods Cells were washed with HBSS + 5% FBS and Prominin1-positive cells were sorted directly in cold RLT lysis buffer (Qiagen) + 1% 2-mercaptoethanol. Unstained cells and isotype-speciﬁc IgG-FITC labeled cells served as negative control for gating. Isolated RNA was sequenced at the iGE3 Genomics Platform (Geneva, Switzerland) using the Illumina 4000 platform. RNA-seq data sets are deposited at the European Nucleotide Archive (ENA) (accession number PRJEB21545 and PRJEB30271). For RNA-sequencing at E15.5, cells of control (n = 3) and Yy1iKO (n = 3) dorsal cortex were isolated, stained for Prominin1 (rat CD133-FITC 1:100, 11-1331, eBioscience) in HBSS (Gibco) supplemented with 5% FBS for 30 min at 4 °C. Cells were washed with HBSS + 5% FBS and Prominin1-positive cells were sorted directly in cold RLT lysis buffer (Qiagen) + 1% 2-mercaptoethanol. Unstained cells and isotype-speciﬁc IgG-FITC labeled cells served as negative control for gating. Isolated RNA was sequenced at the iGE3 Genomics Platform (Geneva, Switzerland) using the Illumina 4000 platform. RNA-seq data sets are deposited at the European Nucleotide Archive (ENA) (accession number PRJEB21545 and PRJEB30271). For the determination of mitochondrial versus genomic DNA content, E12.5 dorsal cortex tissue was dissected and total DNA was isolated with the QiaAmp DNA kit (Qiagen) following the manufacturer’s instructions. Relative copy numbers of mitochondrial DNA was determined by quantitative real-time PCR for mitochondrial and genomic genes (Primers: Supplementary Table 3). Immunohistochemistry. Embryo heads or E18.5 brains were dissected, washed in HBSS (Gibco) and ﬁxed overnight in 4 % paraformaldehyde at 4 °C, followed by dehydration in ethanol and parafﬁn embedding. Sagittal 5-μm parafﬁn sections were deparafﬁnized, high-pressure antigen retrieval in citrate buffer (pH 6) was performed, and sections were subsequently stained following standard protocols (washes: PBS or PBS-TritonX 0.2% (PBS-T); blocking solution: 1% bovine serum albumin (BSA) in PBS-T). Methods The following primary antibodies were used (diluted in blocking solution): anti-cleaved caspase 3 (rabbit Cell signaling 9661, 1:300), anti-Ctip2 (rat Abcam ab18465, 1:200), anti-CyclinB1 (rabbit Santa Cruz sc-752, 1:200), anti-CyclinD1 (mouse Santa Cruz sc-450, 1:50), anti-Dcx (guinea pig Millipore ab2253, 1:300), anti- p53 (rabbit Santa Cruz sc-6243, 1:50; mouse Cell Signaling, 1:300), anti-Pax6 (mouse DSHB, 1:50 and rabbit Covance PRB-278P, 1:200), anti-phospho Histone 3 (mouse PH3 Cell Signaling 9706, 1:300), anti-Reelin (mouse Novus Biological NB600-1081, 1:100), anti-Sox2 (rabbit Chemicon AB5603, 1:100 and mouse R&D MAB2018), anti- Tbr1 (rabbit Abcam ab31940, 1:200), anti-Tbr2 (rabbit Chemicon AB9618, 1:200), anti-Yy1 (rabbit sc1703 and mouse sc-7341, both Santa Cruz, 1:100). ChIP-sequencing. ChIP material was prepared as previously described72. In brief, chromatin from dissected dorsal cortex tissue of E12.5 and E15.5 NMRI mice was prepared and immunoprecipitated according to the manufacturer’s instructions (Magna A/G ChIP Kit, Millipore). Crosslinked protein–DNA complexes of ~ 300–700 bp size were immunoprecipitated using 5 μg of rabbit anti-Yy1 antibody (sc1703, Santa Cruz) or 5 μg of rabbit IgG antibody (Santa Cruz). After recovery of the DNA fragments, binding sites were determined by sequencing at the iGE3 Genomics Platform, Geneva, Switzerland, using the Illumina HISeq4000 platform. Data analysis–peak calling. All sequenced reads were mapped using Bowtie 2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) onto the UCSC mm10 reference mouse genome. The command “ﬁndPeaks” from the HOMER tool package (http://homer.salk.edu/homer/) was used to identify enriched regions in the Yy1 immunoprecipitation experiments using the “-style = factor” option (routinely used for transcription factors with the aim of identifying the precise location of DNA-protein contact). Input samples were used as enrichment- normalization control. Peak calling parameters were adjusted as following: L = 2 (ﬁltering based on local signal), F = 2 (fold change in target experiment over input control). The annotation of peaks’ position (i.e., the association of individual peaks to nearby annotated genes or genomic loci) was obtained by the all-in-one program called “annotatePeaks.pl”. Finally, the HOMER program “makeUCSCﬁle” was used to produce bedGraph formatted ﬁles that can be uploaded as custom tracks and visualized in the UCSC genome browser (http://genome.ucsc.edu). GO networks were constructed with ClueGO (Version 2.3.2) and Cytoscape (Version 3.4.0). Clusters with less than three nodes were omitted. Computational prediction of co- factors was performed by i-cisTarget32. ChIP-Seq data sets are deposited at the European Nucleotide Archive (ENA) (accession number PRJEB21635). ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Methods y g pp y For RNA-sequencing at E11.5, total RNA was isolated of control (n = 3, from two different litters) and Yy1cKO (n = 3, from two different litters) dorsal cortex tissue. Sequencing was performed at the Functional Genomics Center Zurich, Switzerland, using the Illumina HiSeq 2000 platform. The heatmap in Fig. 4a shows differentially expressed genes with \|log2 fold change\| > 0.3, p < 0.05, FDR < 0.01. GO networks were constructed with ClueGO (Version 2.3.2) and Cytoscape (Version 3.4.0). Clusters with less than 3 nodes were omitted. For RNA-sequencing at E11.5, total RNA was isolated of control (n = 3, from two different litters) and Yy1cKO (n = 3, from two different litters) dorsal cortex tissue. Sequencing was performed at the Functional Genomics Center Zurich, Switzerland, using the Illumina HiSeq 2000 platform. The heatmap in Fig. 4a shows differentially expressed genes with \|log2 fold change\| > 0.3, p < 0.05, FDR < 0.01. GO networks were constructed with ClueGO (Version 2.3.2) and Cytoscape (Version 3.4.0). Clusters with less than 3 nodes were omitted. Cell culture and siRNA transfection. NPCs from dorsal E11.5 cortex (NMRI mice) were isolated by microdissection of the dorsal cortex in Hank's Balanced Salt Solution (HBSS) (14170, Gibco) and digestion in HBSS (14170, Gibco) containing 0.35 mg/ml collagenase type 3 (M3D14157, Worthington) and 0.04% Trypsin (7001719, Life Technologies). The digestion mix was inactivated using soybean trypsin inhibitor (Sigma). Cells were grown on plates (Corning) coated with poly-L- lysine (Cultrex) and ﬁbronectin (Sigma) in Dulbecco's Modiﬁed Eagle Medium (DMEM)/F12+ Glutamax medium, supplemented with B-27, N2 (all from Gibco), 20 ng/ml basic ﬁbroblast growth factor (bFGF), and 20 ng/ml EGF (both Pepro- tech) (cortex medium). For RNA interference of Yy1, cells were transfected with control siRNA (Medium GC Duplex #2, Invitrogen) or siRNAs against Yy1 (Stealth RNAi, Invitrogen) (Supplementary Table 1) using JetPRIME transfection reagent (Polyplus). After 48 h of siRNA treatment, cells were harvested using Trypsin (Gibco) and soybean trypsin inhibitor (Sigma), washed with phosphate-buffered saline (PBS) (Gibco) and processed for further experiments. For RNA-sequencing at E15.5, cells of control (n = 3) and Yy1iKO (n = 3) dorsal cortex were isolated, stained for Prominin1 (rat CD133-FITC 1:100, 11-1331, eBioscience) in HBSS (Gibco) supplemented with 5% FBS for 30 min at 4 °C. ARTICLE However, a small subset of genes were Yy1- dependent at early stages but not altered upon late ablation of Yy1. Some genes of this group have already been implicated in processes contributing to the Yy1-dependent phenotype, includ- ing oxidative phosphorylation (Ndufc162, Ndufb462, Ndufb562), 13 NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications Methods Cytometry kit, Thermo Scientiﬁc) and the DNA content was visualized with propidium iodide. For all ﬂow cytometry experiments, 10,000–50,000 cells were recorded. All samples were analyzed on a BD FACS Canto II ﬂow cytometer. FlowJo v7.6.5. was used to process the obtained data. Animal models. All animal experiments were conducted in accordance with Swiss guidelines and approved by the Veterinary Ofﬁce of the Canton of Zurich, Swit- zerland. Previously described Yy1lx/lx mice17 were crossed to Emx1-Cre16 or Emx1- CreERT2 mice22 to ablate Yy1 function in the developing cortex (Emx1-Cre Yy1lx/lx, Emx1-CreERT2 Yy1lx/lx). The following genotypes were used as control animals: Emx1-Cre Yy1lx/wt, Emx1-Cre Yy1wt/wt, Yy1lx/lx, and Emx1-CreERT2 Yy1lx/wt, Emx1-Cre ERT2 Yy1wt/wt. To study the function of p53 in the context of Yy1 ablation, previously described Trp53lx/lx mice27 were crossed to Emx1-Cre Yy1lx/lx mice (Emx1-Cre Yy1lx/lx Trp53 lx/lx). Emx1-Cre Yy1lx/wt Trp53lx/lx served as control animals. All genotypes were present at Mendelian ratios and control animals showed no overt phenotype. Wild-type NMRI or Swiss mice (Janvier, France) were used for ChIP-seq, knockdown experiments, to determine mRNA levels and pro- tein translation rates at different developmental stages. To generate embryos of a certain developmental stage, mice were mated overnight and the next morning was deﬁned as E0.5. Timed ablation of Yy1 in Emx1-CreERT2 Yy1lx/lx mice was induced by intraperitoneal (i.p.) injection of TM (200 mg/kg body weight, Sigma) into pregnant females at indicated stages. Pharmacological inhibition of p53 was achieved by i.p. injection of pregnant females with 2.2 mg/kg body weight PFTα29 (Enzo Life Sciences) twice per day from E10.5–E12.5. Quantitative real-time PCR and RNA-sequencing. Dorsal cortex tissue was dissected and total RNA was isolated with the RNAeasy kit (Qiagen) and RNase- Free DNase Set (79254, Qiagen) following the manufacturer’s instructions. For quantitative real-time PCR, RNA was reverse transcribed with the Maxima First Strand cDNA Synthesis kit (Fermentas, K1641). Quantitative real-time PCR was carried out using LightCycler® SYBR Green I master mix (Roche, 4887352001) and was run on a LightCycler® 480 System (Roche). Each experiment was performed in a minimum of biological and technical triplicates. Obtained data were analyzed by the ΔCt method and normalized to the expression levels of β-actin for comparing control and Yy1cKO samples or normalized to the average expression of four housekeeping genes (β-actin, Mrps6, Tbp, Ywazh) for comparison of different embryonic stages. Primers used are listed in Supplementary Table 2. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 were adjusted for FDR according to the Benjamini–Hochberg procedure. All calcu- lations were done in Matlab (The Mathworks, Natick, MA). were adjusted for FDR according to the Benjamini–Hochberg procedure. All calcu- lations were done in Matlab (The Mathworks, Natick, MA). 2. Donohoe, M. E. et al. Targeted disruption of mouse Yin Yang 1 transcription factor results in targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality. Mol. Cell Biol. 19, 7237–7244 (1999). 3. Weintraub, A. S. et al. YY1 is a structural regulator of enhancer-promoter loops. Cell 171, 1573–1588.e28 (2017). Oxygen consumption measurement. Cells from dorsal E12.5 or E15.5 cortex were isolated and equal numbers of living cells (determined by Trypan Blue exclusion assay) were seeded into Seahorse XFp or XFe24 cell plates (Seahorse Bioscience). Cells were incubated for 3 h at 37 °C and 5% CO2 in cortex medium to allow attachment to the poly(L-lysine) (PLL)/ﬁbronectin-coated plates. To prepare the cells for OCR measurement, cells were incubated for 1 h at 37 °C with athmospheric CO2 levels in non-buffered XF Base DMEM minimal medium supplemented with 0.5 mM sodium pyruvate, 5.5 mM glucose, and 2 mM glutamine (Seahorse medium). Cell viability prior to measurement was ensured by staining for 7AAD (BD Bioscience) in an inde- pendent experiment. OCR was measured on Seahorse XFp or XFe24 extracellular ﬂux analyzers in Seahorse medium. Relative OCR was assessed upon the injection of 1 μM oligomycin, 1 µM FCCP and 0.5 μM antimycin A/rotenone (all from Seahorse bioscience) and normalized to protein content (bicinchoninic acid assay) and the ﬁrst OCR measurement of control cells. For each condition, at least six embryos from at least two different litters were measured (n ≥6). 4. Beagan, J. A. et al. YY1 and CTCF orchestrate a 3D chromatin looping switch during early neural lineage commitment. Genome Res. 27, 1139–1152 (2017). 5. Blättler, S. M. et al. Defective mitochondrial morphology and bioenergetic function in mice lacking the transcription factor Yin Yang 1 in skeletal muscle. Mol. Cell Biol. 32, 3333–3346 (2012). 6. Lu, L. et al. Genome-wide survey by ChIP-seq reveals YY1 regulation of lincRNAs in skeletal myogenesis. EMBO J. 32, 2575–2588 (2013). 7. Boucherat, O. et al. Epithelial inactivation of Yy1 abrogates lung branching morphogenesis. Development 142, 2981–2995 (2015). 8. Berube-Simard, F. A., Prudhomme, C. & Jeannotte, L. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 He, Y. et al. The transcription factor Yin Yang 1 is essential for oligodendrocyte progenitor differentiation. Neuron 55, 217–230 (2007). 16. Gorski, Ja et al. Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J. Neurosci. 22, 6309–6314 (2002). 17. Affar, E. B., Gay, F., Shi, Y. & Liu, H. Essential dosage-dependent functions of the transcription factor Yin Yang 1 in late embryonic development and cell cycle progression. Mol Cell Biol. 16, 163–97 (2006). g g incorporating dead cells. For each condition, cells from at least ﬁve embryos from at least two different litters were measured (n ≥5). 18. Creuzet, S. E. Neural crest contribution to forebrain development. Semin. Cell Dev. Biol. 20, 751–759 (2009). Imaging, quantiﬁcation and statistical analysis. Epiﬂuorescence and confocal micrographs were taken with a Leica DMI6000 B or a CLSM Leica SP8 upright microscope, processed with Adobe Photoshop or Imaris and quantiﬁed manually using ImageJ. For each quantiﬁcation, at least three mutants and three controls from at least two different litters, and at least three sections per animal at different rostro- caudal levels were analyzed. A radial unit was deﬁned as a 100 µm-wide stripe measured at the ventricular surface with perpendicular borders given by the direction of RG cell ﬁbers. Numbers of pHH3-positive cells were counted along the whole VZ length and normalized to 600 μm VZ length. Measurements of cortical thickness, length, and width were done with ImageJ. No statistical methods were used to pre- determine sample size. Animals were not randomized and researchers were not blinded during experiments and for quantiﬁcations. Statistical analysis of all experi- ments was performed in Microsoft Excel using unpaired, two-tailed Student’s t test, or in GraphPad Prism using analysis of variance Tukey’s multiple comparison test for comparing more than two groups. Statistical signiﬁcance cutoff threshold was set at p < 0.05. Results are shown as mean ± standard deviation, except where indicated. 19. Le Douarin, N. M., Couly, G. & Creuzet, S. E. The neural crest is a powerful regulator of pre-otic brain development. Dev. Biol. 366, 74–82 (2012). 20. Bartha, I., di Iulio, J., Venter, J. C. & Telenti, A. Human gene essentiality. Nat. Rev. Genet. 19, 51–62 (2018). 21. Andersen, T. A., Troelsen, K. D. L. L. & Larsen, L. A. Of mice and men: molecular genetics of congenital heart disease. Cell. Mol. Life Sci. 71, 1327–1352 (2014). 22. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 Kessaris, N. et al. Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage. Nat. Neurosci. 9, 173–179 (2006). 23. Götz, M. & Huttner, W. B. The cell biology of neurogenesis. Nat. Rev. Mol. Cell Biol. 6, 777–788 (2005). 24. Sui, G. et al. Yin Yang 1 is a negative regulator of p53. Cell 117, 859–872 (2004). 25. Grönroos, E., Terentiev, A. A., Punga, T. & Ericsson, J. YY1 inhibits the activation of the p53 tumor suppressor in response to genotoxic stress. Proc. Natl. Acad. Sci. USA 101, 12165–12170 (2004). 26. Chen, L., Foreman, D. P., SantAngelo, D. B. & Krangel, M. S. Yin Yang 1 promotes thymocyte survival by downregulating p53. J. Immunol. 196, 2572–2582 (2016). Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 27. Marino, S., Vooijs, M., Van Der Gulden, H., Jonkers, J. & Berns, A. Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. Genes Dev. 14, 994–1004 (2000). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 YY1 acts as a transcriptional activator of Hoxa5 gene expression in mouse organogenesis. PLoS ONE 9, e93989 (2014). 9. Beketaev, I. et al. Critical role of YY1 in cardiac morphogenesis. Dev. Dyn. 244, 669–680 (2015). 10. Kumar, N. et al. A YY1-dependent increase in aerobic metabolism is indispensable for intestinal organogenesis. Development 143, 3711–3722 (2016). https://doi.org/10.1242/dev.137992 Measurement of protein translation rate. Cells were directly isolated from dorsal E12.5 or E15.5 cortex and equal numbers of cells were seeded into PLL/ﬁbronectin- coated 96-well plates. Cells were incubated for 2 h at 37 °C in cortex medium to allow attachment to the plates. Alternatively, cells isolated from E11.5 cortex were treated with siRNA for 48 h as described above. For measurement of protein synthesis rates, cells were pulsed with OPP (20 μM, Thermo Scientiﬁc and Jena Bioscience) for 30 min, harvested and ﬁxed with 4 % paraformaldehyde. Staining for OPP incorporation was carried out as indicated by the manufacturer’s instructions (Click-It OPP Flow Cytometry kit, Thermo Scientiﬁc) and the DNA content was visualized with propidium iodide to distinguish between G0/G1 (DNA content = 2c) and S/G2/M (DNA content > 2c). For analysis of protein synthesis rates of Prom1+ cells, ﬁxed OPP-pulsed cells were stained for Prominin1 (rat CD133 1:250, 14-1331-80 eBioscience; Dylight488 anti-rat 1:500, 112-486-072, Thermo Scientiﬁc) in PBS + 1% BSA (each for 30 min at 4 °C) before OPP Click-It staining. The samples were analyzed on a BD FACS Canto II ﬂow cytometer. FlowJo (Version 7.6.5) was used to process the obtained data. The relative rate of protein translation was calculated by using the mean ﬂuorescent intensity relative to control samples after subtraction of background staining and non-OPP- incorporating dead cells. For each condition, cells from at least ﬁve embryos from at least two different litters were measured (n ≥5). p g 11. Perekatt, A. O. et al. YY1 is indispensable for Lgr5+intestinal stem ce renewal. Proc. Natl. Acad. Sci. USA 111, 7695–7700 (2014). 12. He, Y. & Casaccia-Bonneﬁl, P. The Yin and Yang of YY1 in the nervous system. J. Neurochem. 106, 1493–1502 (2008). 13. Zhang, Q., Stovall, D. B., Inoue, K. & Sui, G. The oncogenic role of Yin Yang 1. Crit. Rev. Oncog. 16, 163–97 (2011). 14. Knauss, J. L. et al. Long noncoding RNA Sox2ot and transcription factor YY1 co-regulate the differentiation of cortical neural progenitors by repressing Sox2. Cell Death Dis. 9, 799 (2018). 15. Data availability The sequencing data sets generated and analyzed during the current study are available in the European Nucleotide Archive (https://www.ebi.ac.uk/ena) and are accessible through the accession numbers PRJEB21545, PRJEB30271, and PRJEB21635. All relevant data that support the ﬁndings of this study are available from the corresponding author upon reasonable request. Source data for all ﬁgures are provided as a Source Data ﬁle. 28. Caprio, C. & Baldini, A. p53 Suppression partially rescues the mutant phenotype in mouse models of DiGeorge syndrome. Proc. Natl. Acad. Sci. USA 111, 13385–13390 (2014). 29. Komarov, P. G. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 285, 1733–1737 (1999). Received: 28 March 2018 Accepted: 26 March 2019 Received: 28 March 2018 Accepted: 26 March 2019 30. Kleiman, E., Jia, H., Loguercio, S., Su, A. I. & Feeney, A. J. YY1 plays an essential role at all stages of B-cell differentiation. Proc. Natl. Acad. Sci. 113, E3911–E3920 (2016). 31. Liu, J., Xu, Y., Stoleru, D. & Salic, a Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc. Natl. Acad. Sci. 109, 413–418 (2012). ARTICLE ARTICLE Methods Secondary isotype-speciﬁc Alexa488-, Alexa546-, and Alexa647-conjugated antibodies against mouse were from Invitrogen, secondary biotin anti-rabbit, steptavidin-, Dylight488-, and DyLight549- conjugated antibodies against other species were purchased from Jackson ImmunoResearch. Secondary antibodies were diluted 1:500 in blocking solution and incubated for 1 h at RT. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, 1:2000). Immunoblotting. Cells or cortex tissue were harvested and lysed in radio- immunoprecipitation buffer (RIPA) buffer (89900, Thermo Scientiﬁc) containing Halt Phosphatase and Protease Inhibitor Cocktail (78420, 87786, Thermo Scientiﬁc). Cortex tissues were homogenized and both, cells, and tissue samples were sonicated in RIPA buffer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 4–20% Mini-PROTEAN TGX Gels (456–1094, Bio Rad). The following primary antibodies (4 °C, O/N, in Odyssey blocking buffer (927–40000, LI- COR Biosciences)) were used: anti-β-actin (mouse Sigma a5316, 1:10000), anti- CyclinB1 (rabbit Santa Cruz sc-752, 1:500), anti-CyclinD1 (mouse Santa Cruz sc-450, 1:500), anti-Histone H3 (mouse Cell Signaling, 1:1000), anti-Yy1 (rabbit Santa Cruz sc1703, 1:500). Blots were stained with isotype-speciﬁc secondary antibodies in Odyssey blocking buffer for 1 h at room temperature. Blots were scanned and quantiﬁed with an Odyssey imaging system (LI-COR Biosciences). Metabolomics. Isolated NPCs from E11.5 cortices were transfected with siCtrl or siYy1 (n = 9, each). After 48 h, cells were harvested and washed twice with 75 mM ammonium carbonate, ph 7.4, and snap frozen in liquid nitrogen. Metabolites were extracted two times with cold acetonitrile-methanol-water (40:40:20 ratio, −20 °C). Extracted metabolites were analyzed by ﬂow injection–time of ﬂight mass spectro- metry on an Agilent 6550 QTOF instrument operated in the negative mode as previously described73. Detectable ions were putatively annotated by matching measured mass-to-charge ratios with theoretical masses of compounds listed in the human metabolome database v3.074 using a tolerance of 0.001 amu. Pathway deﬁ- nition of differentially abundant metabolites was performed with the Small Molecule Pathway Database75. P values were calculated by two-tailed, heteroscedastic t test and Flow cytometry. For assessment of cell death, cells were harvested, washed, and stained with Annexin V-Cy5 according to the manufacturer’s instructions (BD Bioscience). For cell cycle analysis, cells were pulsed with EdU (10 μM) for 1 h, harvested and ﬁxed with 4 % paraformaldehyde. Staining for EdU incorporation was carried out as indicated by the manufacturer’s instructions (Click-It EdU Flow 14 NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunicatio Competing interests: The authors declare no competing interests. g 54. Signer, Ra. J., Magee, Ja, Salic, A. & Morrison, S. J. Haematopoietic stem cells require a highly regulated protein synthesis rate. Nature 509, 49–54 (2014). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 55. Blanco, S. et al. Stem cell function and stress response are controlled by protein synthesis. Nature 534, 335–340 (2016). Journal peer review information: Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Journal peer review information: Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. 56. Llorens-bobadilla, E. et al. Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury. Cell Stem Cell 17, 329–340 (2015). 57. Fajas, L. Re-thinking cell cycle regulators: the cross-talk with metabolism. Front. Oncol. 3, 4 (2013). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 58. Cai, L. & Tu, B. P. Driving the cell cycle through metabolism. Annu. Rev. Cell Dev. Biol. 28, 59–87 (2012). 59. Mandal, S., Guptan, P., Owusu-Ansah, E. & Banerjee, U. Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila. Dev. Cell 9, 843–854 (2005). 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/. 60. Dai, M. S. & Lu, H. Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5. J. Biol. Chem. 279, 44475–44482 (2004). 61. Fregoso, O. I., Das, S., Akerman, M. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09823-5 33. Denechaud, P. D., Fajas, L. & Giralt, A. E2F1, a novel regulator of metabolism. Front. Endocrinol. (Lausanne). 8, 1–8 (2017). 65. Kazi, A. A. & Lang, C. H. PRAS40 regulates protein synthesis and cell cycle in C2C12 myoblasts. Mol. Med. 16, 359–371 (2010). 34. Blanchet, E. et al. E2F transcription factor-1 regulates oxidative metabolism. Nat. Cell Biol. 13, 1146–1154 (2011). 66. Calegari, F., Haubensak, W., Haffner, C. & Huttner, W. B. Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development. J. Neurosci. 25, 6533–6538 (2005). 35. Benevolenskaya, E. V. & Frolov, M. V. Emerging links between E2F control and mitochondrial function. Cancer Res. 75, 619–623 (2015). g p 67. Takahashi, T., Nowakowski, R. S. & Caviness, V. S. The cell cycle of the pseudostratiﬁed ventricular epithelium of the embryonic murine cerebral wall. J. Neurosci. 15, 6046–6057 (1995). 36. Verschoor, M. L., Wilson, L. A., Verschoor, C. P. & Singh, G. Ets-1 regulates energy metabolism in cancer cells. PLoS ONE 5, 1–9 (2010). energy metabolism in cancer cells. PLoS ONE 5, 1–9 (2010). 68. Caviness, V. S., Takahashi, T. & Nowakowski, R. S. Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model. Trends Neurosci. 18, 379–383 (1995). 37. Zhang, X. et al. ETS-1: A potential target of glycolysis for metabolic therapy by regulating glucose metabolism in pancreatic cancer. Int. J. Oncol. 50, 232–240 (2017). 38. Alcantara, D. & O’Driscoll, M. Congenital microcephaly. Am. J. Med. Genet. Part C. Semin. Med. Genet. 166, 124–139 (2014). 69. Lee, Y. et al. Neurogenesis requires TopBP1 to prevent catastrophic replicative DNA damage in early progenitors. Nat. Neurosci. 15, 819–826 (2012). 39. Li, C. et al. Zika Virus Disrupts Neural Progenitor Development and Leads to Microcephaly in Mice. Cell Stem Cell 1–7 (2016). https://doi.org/10.1016/j. stem.2016.04.017 Li, C. et al. Zika Virus Disrupts Neural Progenitor Developmen h l ll ll ( ) h d 70. McKinnon, P. J. Maintaining genome stability in the nervous system. Nat. Neurosci. 16, 1523–1529 (2013). 71. Deboer, E. M., Kraushar, M. L., Hart, R. P. & Rasin, M.-R. Post-transcriptional regulatory elements and spatiotemporal speciﬁcation of neocortical stem cells and projection neurons. Neuroscience 248C, 499–528 (2013). 40. Tang, H., Hammack, C., Ogden, S. C. & Jin, P. Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell 18, 587–590 (2016). 72. ARTICLE Draganova, K., Zemke, M., Gotz, M. & Sommer, L. Wnt/b‐catenin signaling regulates sequential fate decisions of murine cortical precursor cells. Stem Cells 33, 170–182 (2015). 41. Thornton, G. K. & Woods, C. G. Primary microcephaly: do all roads lead to Rome? Trends Genet. 25, 501–510 (2009). 42. Fridman, J. S. & Lowe, S. W. Control of apoptosis by p53. Oncogene 22, 9030–9040 (2003). 73. Fuhrer, T., Heer, D., Begemann, B. & Zamboni, N. High-throughput, accurate mass metabolome proﬁling of cellular extracts by ﬂow injection-time-of-ﬂight mass spectrometry. Anal. Chem. 83, 7074–7080 (2011). 43. Kruiswijk, F., Labuschagne, C. F. & Vousden, K. H. P53 in survival, death and metabolic health: a lifeguard with a licente to kill. Nat. Rev. Mol. Cell Biol 16, 393–405 (2015). 74. Wishart, D. S. et al. HMDB: the human metabolome database. Nucleic Acids Res. 35, 521–526 (2007). 44. Chipuk, J. E. & Green, D. R. Dissecting p53-dependent apoptosis. Cell Death Differ. 13, 994–1002 (2006). 75. Frolkis, A. et al. SMPDB: the small molecule pathway database. Nucleic Acids Res. 38, 480–487 (2009). 45. Gordon, S., Akopyan, G., Garban, H. & Bonavida, B. Transcription factor YY1: structure, function, and therapeutic implications in cancer biology. Oncogene 25, 1125–1142 (2006). Acknowledgements We thank Dr. Wolfgang Langhans and Dr. Rosmarie Clara from the ETH Zurich for their support with the Seahorse XPe24 Extracellular Flux Analyzer. We thank past and present members of the Lukas Sommer laboratory for critical comments. S.V. is sup- ported by the Zurich University Research Priority Program “Translational Cancer Research”. This work was supported by the Swiss National Science Foundation. g 46. Castellano, G. et al. The involvement of the transcription factor Yin Yang 1 in cancer development and progression. Cell Cycle 8, 1367–1372 (2009). p p g y 47. Birsoy, K. et al. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell 162, 540–551 (2015). 48. Desler, C., Lykke, A. & Rasmussen, L. J. The effect of mitochondrial dysfunction on cytosolic nucleotide metabolism. J. Nucleic Acids 2010, 1–9 (2010). Author contributions 49. Knobloch, M. et al. Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis. Nature 493, 226–230 (2013). L.Z. designed the study, conducted the experimental work, analyzed the data, and wrote the paper. S.V. helped designing the study, conducted experimental work, analyzed the data, and edited the manuscript. S.G., A.K., J.H., R.B., C.C., and N.Z. conducted L.Z. designed the study, conducted the experimental work, analyzed the data, and wrote the paper. S.V. helped designing the study, conducted experimental work, analyzed the data, and edited the manuscript. S.G., A.K., J.H., R.B., C.C., and N.Z. conducted experimental work and analyzed the data. M.Z. conducted experimental work and edited the manuscript. Z.K.A. analyzed the data. K.B. and N.Z. acquired funding resources. L.S. supervised the project, acquired funding resources and wrote the paper. 50. Lange, C. et al. Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis. EMBO J. 35, 1–18 (2016). experimental work and analyzed the data. M.Z. conducted experimental work and edited the manuscript. Z.K.A. analyzed the data. K.B. and N.Z. acquired funding resources. L.S. supervised the project, acquired funding resources and wrote the paper. 51. Khacho, M. et al. Mitochondrial dynamics impacts stem cell identity and fate decisions by regulating a nuclear transcriptional program. Cell Stem Cell 19, 1–16 (2016). 52. Homem, C. C. F. et al. Ecdysone and mediator change energy metabolism to terminate proliferation in drosophila neural stem cells. Cell 158, 874–888 (2014). Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 019-09823-5. 53. Beckervordersandforth, R. et al. Role of mitochondrial metabolism in the control of early lineage progression and aging phenotypes in adult hippocampal neurogenesis. Neuron 93, 1–14 (2017). Competing interests: The authors declare no competing interests. Competing interests: The authors declare no competing interests. References 32. Imrichová, H., Hulselmans, G., Atak, Z. K., Potier, D. & Aerts, S. I-cisTarget 2015 update: generalized cis-regulatory enrichment analysis in human, mouse and ﬂy. Nucleic Acids Res. 43, W57–W64 (2015). 1. Gabriele, M. et al. Yy1 haploinsufﬁciency causes an intellectual disability syndrome featuring transcriptional and chromatin dysfunction. Am. J. Hum. Genet. 100, 907–925 (2017). 15 TURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications Competing interests: The authors declare no competing interests. & Krainer, A. R. Splicing-factor oncoprotein SRSF1 stabilizes p53 via RPL5 and induces Cellularsenescence. Mol. Cell 50, 56–66 (2013). 62. Arroyo, J. D. et al. A genome-wide CRISPR death screen identiﬁes genes essential for oxidative phosphorylation. Cell Metab. 24, 875–885 (2016). 63. Ventura-Clapier, R., Garnier, A. & Veksler, V. Transcriptional control of mitochondrial biogenesis: the central role of PGC-1α. Cardiovasc. Res. 79, 208–217 (2008). 64. Scarpulla, R. C. Metabolic control of mitochondrial biogenesis through the PGC-1 family regulatory network. Biochim. Biophys. Acta - Mol. Cell Res. 1813, 1269–1278 (2011). © The Author(s) 2019 © The Author(s) 2019 16 NATURE COMMUNICATIONS \| (2019) 10:2192 \| https://doi.org/10.1038/s41467-019-09823-5 \| www.nature.com/naturecommunications
https://openalex.org/W4390747264	https://www.nature.com/articles/s41746-023-00985-7.pdf	English	null	Multimodal digital phenotyping of diet, physical activity, and glycemia in Hispanic/Latino adults with or at risk of type 2 diabetes	npj digital medicine	2,024	cc-by	11,744	ARTICLE OPEN Multimodal digital phenotyping of diet, physical activity, and glycemia in Hispanic/Latino adults with or at risk of type 2 diabetes Amruta Pai 1✉, Rony Santiago2, Namino Glantz3, Wendy Bevier2, Souptik Barua1, Ashutosh Sabhar Digital phenotyping refers to characterizing human bio-behavior through wearables, personal devices, and digital health technologies. Digital phenotyping in populations facing a disproportionate burden of type 2 diabetes (T2D) and health disparities continues to lag compared to other populations. Here, we report our study demonstrating the application of multimodal digital phenotyping, i.e., the simultaneous use of CGM, physical activity monitors, and meal tracking in Hispanic/Latino individuals with or at risk of T2D. For 14 days, 36 Hispanic/Latino adults (28 female, 14 with non-insulin treated T2D) wore a continuous glucose monitor (CGM) and a physical activity monitor (Actigraph) while simultaneously logging meals using the MyFitnessPal app. We model meal events and daily digital biomarkers representing diet, physical activity choices, and corresponding glycemic response. We develop a digital biomarker for meal events that differentiates meal events into normal and elevated categories. We examine the contribution of daily digital biomarkers of elevated meal event count and step count on daily time-in-range 54-140 mg/dL (TIR54–140) and average glucose. After adjusting for step count, a change in elevated meal event count from zero to two decreases TIR54–140 by 4.0% (p = 0.003). An increase in 1000 steps in post-meal step count also reduces the meal event glucose response by 641 min mg/dL (p = 0.0006) and reduces the odds of an elevated meal event by 55% (p < 0.0001). The proposed meal event digital biomarkers may provide an opportunity for non-pharmacologic interventions for Hispanic/Latino adults facing a disproportionate burden of T2D. npj Digital Medicine (2024) 7:7 ; https://doi.org/10.1038/s41746-023-00985-7 To the best of our knowledge, there is a lack of studies investigating multimodal digital phenotyping for T2D, let alone in Hispanic/Latino populations. Hispanic/Latino populations face a disproportionate burden of T2D, with the prevalence of both diagnosed and undiagnosed T2D nearly twice as high among the U.S., Mexican-origin Hispanic/Latino adults compared to non- Hispanic Whites10–12. Past work has shown that among Hispanic/ Latino adults living with or at risk of T2D, continuous glucose monitoring (CGM) can provide digital biomarkers beyond tradi- tional measures of time-in-range, average glucose, and glycemic variability13,14. Similarly, measuring the intensity and timing of physical activity, assessed by wearable physical activity trackers, has been demonstrated feasible for this population15. www.nature.com/npjdigitalmed 1Electrical and Computer Engineering, Rice University, Houston, TX, USA. 2Sansum Diabetes Research Institute, Santa Barbara, CA, USA. 3Santa Barbara County Education Ofﬁce, Children & Family Resource Services, Santa Barbara, CA, USA. 4Sutter Center for Health Systems Research, Santa Barbara, CA, USA. ✉email: amrutarice@gmail.com ARTICLE OPEN Multimodal digital phenotyping of diet, physical activity, and glycemia in Hispanic/Latino adults with or at risk of type 2 diabetes While the feasibility of continuous glucose monitors and wearable activity trackers have been demonstrated, simultaneous use of the modalities along with food logging has not been explored in the Hispanic/Latino population. Our primary objectives were: (1) Evaluate the feasibility of multimodal digital data collection from Hispanic/Latino adults with or at risk of T2D, (2) to model meal events and daily digital biomarkers of diet, physical activity, and glycemic response using multimodal digital data and, (3) assess cause–effect relationship between digital biomarkers. INTRODUCTION 2 Model meal event and daily digital biomarkers of diet, physical activity and glycemic response Hispanic/Latino adults At-risk Prediabetes Type 2 Diabetes Hipworn ActiGraph MyFitnessPal App Blinded continuous glucose monitor Data modalities Compute the cause effect relationships between digital biomarkers Event-driven self-monitoring and interventions Personalized reflection report Daily meal management goals detected Just-in-time interventions M T W 0 Step count (steps) 12 am 5 am 10 am 3 pm 8 pm 1am Glucose levels (mg/dL) 150 100 50 1000 Collect diet, phyical activity and glycemic response Duration Time Normal meal event Elevated meal event Post-meal step count Baseline glucose Post-meal step count Daily step count Duration Elevated meal event count Baseline glucose Individual’s biology Calories of a meal event Elevated/Normal meal event Time-in-range (54-140 mg/dL) Cohort Modeling Study Analysis Potential Impact Fig. 1 Research summary. Overall contributions of multimodal digital phenotyping in Hispanic/Latino adults with or at risk of non-insu treated T2D. Hispanic/Latino adults At-risk Prediabetes Type 2 Diabetes Hipworn ActiGraph MyFitnessPal App Blinded continuous glucose monitor Data modalities Collect diet, phyical activity and glycemic response Cohort Study Modeling Model meal event and daily digital biomarkers of diet, physical activity and glycemic response 0 Step count (steps) 12 am 5 am 10 am 3 pm 8 pm 1am Glucose levels (mg/dL) 150 100 50 1000 Duration Time Normal meal event Elevated meal event Post-meal step count Baseline glucose Normal meal event Compute the cause effect relationships between digital biomarkers Post-meal step count Daily step count Duration Elevated meal event count Baseline glucose Individual’s biology Calories of a meal event Elevated/Normal meal event Time-in-range (54-140 mg/dL) y Fig. 1 Research summary. Overall contributions of multimodal digital phenotyping in Hi treated T2D. Compute the cause effect relationships between digital biomarkers Personalized reflection report Daily meal management goals detected Just-in-time interventions M T W Personalized reflection report Elevated meal event count Fig. 1 Research summary. Overall contributions of multimodal digital phenotyping in Hispanic/Latino adults with or at risk of non-insulin treated T2D ntributions of multimodal digital phenotyping in Hispanic/Latino adults with or at risk of non-insulin Research summary. Overall contributions of multimodal digital phenotyping in Hispanic/Latino adults wit d T2D Fig. 1 Research summary. Overall contributions of multimodal digital phenotyping in Hispanic/Latino ad treated T2D. Table 1. Demographic and clinical measurements for the participant cohort. INTRODUCTION Beyond genetic and biological factors, behavioral and socio- cultural inﬂuences, including diet and physical activity choices, are essential factors in determining the risk of progression of type 2 diabetes (T2D)1,2. Wearable devices and mobile health smart- phone applications are promising digital data sources for diet, physical activity choices, and glycemic response to support more personalized management of T2D3,4. Digital phenotyping refers to continuous quantiﬁcation of an individual’s bio-behaviors using digital data5,6. Quantiﬁcation requires the development of digital biomarkers that represent objective data-driven physiological and behavioral measures. An important application of digital phenotyping is actionable insights for patients, e.g., the ability to determine which combination of diet and exercise is beneﬁcial for them. Towards that, current approaches have mainly analyzed continuous glucose data and focused on aggregate measures of glycemic control over longer durations, e.g., time-in-range and glycemic variability over 2 weeks7. While the aggregate measures have been demonstrated to have clinical relevance8,9, they provide little actionable beneﬁt to patients in making their daily diet and exercise decisions. To provide actionable recommendations, we need multimodal digital phenotyping allowing simultaneous capture of diet, exercise, and glycemic proﬁles. Additionally, digital biomarkers around speciﬁc events (e.g., meals, day-to-day) that provide granularity to understand the causal impact of diet and exercise actions on glycemic response have to be developed. Our work demonstrates the application of multimodal digital phenotyping, i.e., the simultaneous use of CGM, physical activity monitors, and meal tracking in Hispanic/Latino individuals with or at risk of T2D. The overall summary of the paper is displayed in Fig. 1. We conducted our study in 36 Hispanic/Latino adults with or at risk of T2D and observed high levels of adherence across all modalities. We compute digital biomarkers for meal events, such Published in partnership with Seoul National University Bundang Hospital A. Pai et al. INTRODUCTION Variable (n = 36 participants) Median [interquartile range] Age (years) 51 [39–59] Gender 28 female, 8 male Body mass index (kg/m2) 32.6 [29.6–35.2] HbA1c (%) 6.0 [5.5–6.9] Number of participants with known diabetes 14 (38%) Number of participants born in the United States 6 (17%) Number of participants with no insurance coverage 11 (31%) Diabetes status At-risk 13 (36%) Prediabetes 9 (25%) Type 2 Diabetes 14 (39%) as the meal event’s glucose response, post-meal step count, and calorie content of the meal event using self-reported food logs, step count data from the physical activity monitor, and CGM curves. Further, we developed a digital biomarker for meal events that differentiates meal events into normal and elevated categories using CGM and HbA1c data. We also propose digital biomarkers for daily monitoring, such as the elevated meal event count and daily step count. We compute the cause-effect relationships between our proposed daily digital biomarkers and known glycemic outcomes, such as time-in-range and average glucose, while controlling for confounders like baseline glucose at the start of the day, and duration of the day. Our ﬁndings indicate that the proposed meal event and daily biomarkers can be speciﬁc targets for adaptive lifestyle and non-pharmaceutical interven- tions to help achieve glycemic goals. Table 1. Demographic and clinical measurements for the participant cohort. Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2024) 7 Description of cohort and feasibility of study We conducted a study in a Hispanic/Latino cohort with varying diabetes status (Table 1). Information on occupation is included in Supplementary Table 10. The study described in methods was approved by the Rice University Institutional Review Board (IRB-FY 2021-54). The dataset captured food choices (via MyFitnessPal App), physical activity (via hipworn ActiGraph), and glucose excursions (via CGM) in free-living conditions of 36 Hispanic/ Latino participants with or at risk of type 2 diabetes (Fig. 2). Each food log consisted of the timing of the log, macronutrient composition, and meal occasion label. Physical activity measure was the number of steps (step count) taken every minute calculated using the Actilife software from raw accelerometer sensor data. Finally, glucose excursions were measured as interstitial ﬂuid glucose levels every 15 min. The median adherence in days for MyFitnessPal meal logging was 13 [interquartile range (IQR): 9–14]; for CGM, 13 [IQR: 13–13] days; and for ActiGraph, 13 [IQR: 9–14]. The median simultaneous Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2024) 7 Data example step count Modalities Hipworn ActiGraph Continuous Glucose Monitor MyFitnessPal App Fig. 2 Visualization of a snapshot of the multimodal data. Digital health devices were used to collect synchronous information on the meal, activity, and glucose excursions in free-living conditions. A. Pai et al. A. Pai et al. 3 Data example Data example Fig. 2 Visualization of a snapshot of the multimodal data. Digital health devices were used to collect synchronous information on the meal, activity, and glucose excursions in free-living conditions. Fig. 2 Visualization of a snapshot of the multimodal data. Digital health devices were used to collect syn activity, and glucose excursions in free-living conditions. shot of the multimodal data. Digital health devices were used to collect synchronous information on the me ns in free-living conditions. Log is delayed with respect to start of glucose rise and needs correction Log coincides with the start of glucose rise Glucose level (mg/dL) Glucose level (mg/dL) Glucose level (mg/dL) Glucose level (mg/dL) Time (hours) Time (hours) Time (hours) Time (hours) :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 :00 Self-reported log timing Added timing Corrected timing Segmented 3-hour CGM curve a b c d Fig. 3 Meal event annotations. Various examples of CGM proﬁles overlayed with meal timings across different participants are shown in (a–d). Description of cohort and feasibility of study The red vertical lines correspond to self-reported food log timings. The green lines adjacent to the red lines correspond to corrected timing. The yellow lines correspond to added timings. Glucose level (mg/dL) Time (hours) :00 :00 :00 :00 :00 b Glucose level (mg/dL) Time (hours) :00 :00 :00 :00 :00 a Glucose level (mg/dL) Time (hours) :00 :00 :00 :00 :00 c Glucose level (mg/dL) Time (hours) :00 :00 :00 :00 :00 d Glucose level (mg/dL) Fig. 3 Meal event annotations. Various examples of CGM proﬁles overlayed with meal timings across different participants are shown in (a–d). The red vertical lines correspond to self-reported food log timings. The green lines adjacent to the red lines correspond to corrected timing. The yellow lines correspond to added timings. Published in partnership with Seoul National University Bundang Hospital Meal events and daily digital biomarkers adherence was 11 [IQR: 5–13] days. We observed high adherence for individual modalities. The percentage of participants having at least 10 days of adherence to CGM, MyFitnessPal, and ActiGraph was 94%, 75%, and 75%, respectively. Simultaneous adherence is often difﬁcult to achieve in multimodal studies. Hence, we chose 50% as the threshold, i.e., if more than 50% of the participants had at least 10 days of simultaneous adherence, then using these modalities for data collection is feasible in this population. We found that 52% of our participants displayed at least 10 days of simultaneous adherence to all three modalities. Hence, we conclude that multimodal monitoring in the Hispanic/Latino population is feasible. adherence was 11 [IQR: 5–13] days. We observed high adherence for individual modalities. The percentage of participants having at least 10 days of adherence to CGM, MyFitnessPal, and ActiGraph was 94%, 75%, and 75%, respectively. Simultaneous adherence is often difﬁcult to achieve in multimodal studies. Hence, we chose 50% as the threshold, i.e., if more than 50% of the participants had at least 10 days of simultaneous adherence, then using these modalities for data collection is feasible in this population. We found that 52% of our participants displayed at least 10 days of simultaneous adherence to all three modalities. Hence, we conclude that multimodal monitoring in the Hispanic/Latino population is feasible. We used the self-reported meal log timings and CGM curves to identify the start time of meal events. We implemented a framework to correct the meal log timings and add meal timings as self-reported logs are error-prone (described in “Methods”). We used the self-reported meal log timings and CGM curves to identify the start time of meal events. We implemented a framework to correct the meal log timings and add meal timings as self-reported logs are error-prone (described in “Methods”). In total, 1467 self-reported meal logs were provided by participants. For each self-reported log, each annotator indepen- dently identiﬁed a corrected timing or tagged the log unknown as described in the methods. Further, each annotator independently added timings as discussed in the methods. A few examples of corrected and added timing are shown in Fig. 3. The breakdown of as self reported logs are error prone (described in Methods ). In total, 1467 self-reported meal logs were provided by participants. Daily elevated meal event count and step count have a signiﬁcant effect on time-in-range and average glucose Digital health represents the convergence of healthcare with technology (devices and software), such that wearable devices, information technology and communication tools come together to support people living with or at risk of developing diabetes. However, there is growing evidence that minority communities facing a disproportionate burden of diabetes and health disparities often lack access to currently available digital health technologies18–20. Our study demonstrated the feasibility and acceptability of multimodal digital phenotyping using digital health technologies in a U.S. Hispanic/Latino population with or at risk of T2D. Facilitating the use of digital health technologies for underserved communities may be one approach to help reduce the barriers to this underserved community participating in clinical research21. We computed the effect of daily elevated meal event count and daily step count on daily glycemic outcomes to evaluate the signiﬁcance of the proposed digital biomarkers for monitoring and event-driven recommendations. The outcomes describing glyce- mic control were time-in-range (54–140 mg/dL) and average glucose was measured using CGM values during the duration. First, we investigated the effect of a day’s elevated meal event count on glycemic outcomes while adjusting for confounders such as baseline glucose at the start of the day, daily step count, and duration of the day (explained in Methods). We had 369 daily samples across 36 participants. p p p Figure 6 demonstrates a negative association between the elevated meal event count and time-in-range with TIR54-140 decreasing with an increase in elevated meal event count across participants who are at risk of or with prediabetes or T2D. The decrease in TIR54–140 and increase in average glucose due to the increase in the elevated meal event count is summarized in Table 2. An increase in average glucose and a decrease in TIR54–140 for a change in elevated meal event count from 0 to 1 was not statistically signiﬁcant. However, the change from 0 to 2 and 0 to 3 was statistically signiﬁcant. In this study, we assessed the contribution of individual meals to a 3-h integrated area under the curve glucose responses in free- living conditions and demonstrated that an individual’s average meal event glucose response (across all their meals) did appear to be predictive of the HbA1c level. It could be used to monitor T2D progression as well as provide a more personalized target for pharmaceutical and non-pharmaceutical interventions. Post-meal step count to reduce meal event’s glucose response and risk of elevated classiﬁcation We also investigated if the post-meal step count of a meal event could potentially be used as a targeted treatment to reduce the meal event’s glucose response and prevent an elevated classiﬁca- tion. As our dataset was collected in free-living conditions, the size of the meal (calorie content) was not ﬁxed. Hence, we adjusted for calorie content in our statistical analysis. To investigate the impact of post-meal step count on a meal event’s glucose response, we analyzed a subset of meal events that satisﬁed two conditions. First, the self-reported meal log (with non-zero calorie content) corresponding to the meal event should have high agreement in the corrected timing among the annotators. Speciﬁcally, the log was chosen if three out of the four corrected timings provided for that log were tagged in the same group (meal event) by the DBSCAN algorithm. Second, the post-meal step count was recorded during the meal event. Second, we developed digital biomarkers to analyze each meal event, namely calorie content of the meal event, post-meal step count, meal event’s glucose response denoted as MGR3 h (incremental area under the 3-h CGM curve from the start time of the meal event14,16), and elevated/normal classiﬁcation of the meal event. We developed a new CGM-driven meal event characterization that uses an “elevated” or “normal” classiﬁcation of the meal by exploiting the mapping between MGR3 h and HbA1c observed across participants (explained in methods). We calculated MGR3 h for each of the total 1584 meal events and MGR3 h for each of the 36 participants. The line of ﬁt for the robust linear regression model between MGR3 h and HbA1c is shown in Fig. 4a. The association between true and ﬁtted values of HbA1c is shown in Fig. 4b. The correlation between true and ﬁtted values was signiﬁcant with a Pearson correlation coefﬁcient of 0.72 (p < 0.0001, beta distribution test). The mean absolute error was 0.80%. Error analysis after excluding the four samples of extremely high values of HbA1c (HbA1c > 10) resulted in a mean absolute error of 0.41% and Pearson’s correlation coefﬁcient of 0.88 (p < 0.0001, beta distribution test). We computed MGR3 h for each of 764 meal events and classiﬁed each of the meal events into elevated or normal categories using its MGR3 h. We found that post-meal step count had a signiﬁcant effect on the meal event’s glucose response after adjusting for calorie content. Meal events and daily digital biomarkers For each self-reported log, each annotator indepen- dently identiﬁed a corrected timing or tagged the log unknown as described in the methods. Further, each annotator independently added timings as discussed in the methods. A few examples of corrected and added timing are shown in Fig. 3. The breakdown of Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2024) 7 A. Pai et al. 4 the number of correction and addition annotations for each of the four annotators is presented in Supplementary Table 1. There were a total of 6725 annotated timings combined across all four annotators. We aggregated the annotated timings as described in methods and identiﬁed 1584 meal events. To investigate inter- annotator agreement, we analyzed the intra-class correlation of the corrected timings across 951 logs (logs for which all four annotators had been able to identify a corrected timing). The intra-class correlation was found to be 0.95. Additionally, the causal analysis indicated that daily step count had a statistically signiﬁcant effect on average glucose. An increase in one unit, i.e., 1000 steps, led to an absolute decrease of 0.3 mg/dL (p = 0.02, linear regression t-test) in average glucose. However, the effect on time-in-range was not signiﬁcant. the number of correction and addition annotations for each of the four annotators is presented in Supplementary Table 1. There were a total of 6725 annotated timings combined across all four annotators. We aggregated the annotated timings as described in methods and identiﬁed 1584 meal events. To investigate inter- annotator agreement, we analyzed the intra-class correlation of the corrected timings across 951 logs (logs for which all four annotators had been able to identify a corrected timing). The intra-class correlation was found to be 0.95. Post-meal step count to reduce meal event’s glucose response and risk of elevated classiﬁcation An increase of 1000 steps leads to a decrease of MGR3 h by 641 min mg/dL (p = 0.0006, linear regression t-test). An increase of 1000 steps in post-meal step count is associated with a 55% (p < 0.0001, linear regression t-test) decrease in the odds of elevated classiﬁcation of the meal event. This ﬁnding suggests that post-meal physical activity can be used as an intervention to reduce the risk of elevated meal events and reduce meal events’ glucose response. We used the model to characterize individual meal events as elevated or normal (from a glucose perspective). MGR3 h quantiﬁes a person’s average glycemic regulation to meals, and its mapping to their baseline HbA1c is provided by the model. The thresholds for elevated/normal classiﬁcation were based on the HbA1c deﬁnitions for at-risk, prediabetes, and T2D17. Third, we constructed daily digital biomarkers. We deﬁned a duration (analysis window) for each day and computed daily measures of the digital biomarkers, such as daily elevated meal event count that is the number of meal events that were classiﬁed as elevated in that duration. We determined the daily step count as the total steps taken within the duration. An overview of the steps of computation is shown in Fig. 5. Additionally, we computed baseline glucose at the start time of day. We demonstrated the effect of calorie content on the MGR3 h and elevated/normal classiﬁcation of the meal event after adjusting for post-meal step count. An increase in 100 kcal leads to an increase in MGR3 h by 183 min mg/dL (p < 0.0001, linear regression t-test) and an 8% (p = 0.002, linear regression t-test) increase in the odds of elevated classiﬁcation of the meal event. Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2024) 7 Published in partnership with Seoul National University Bundang Hospital Daily elevated meal event count and step count have a signiﬁcant effect on time-in-range and average glucose Further, if an elevated meal event is detected, a notiﬁcation to engage in some form of physical activity after subsequent meals can be provided. Current CGM metrics such as time-in-range characterize behavior across days so targets in time-in-range cannot be easily translated to event-driven recommendations. Biomarkers such as glycemic index that can be used to inform meal events are not suitable for free-living meal events that consist of multiple food items consumed together, often followed by physical activity16,22,23. In this study, to characterize meal events, we leveraged the function mapping average meal event glucose response to HbA1c for classifying each individual meal event into a simple “normal” or “elevated” glucose response category. We found that an increase in the elevated meal event count was associated with a signiﬁcant lowering of daily time-in-range between 54 and 140 mg/dL and an increase in average glucose after adjusting for covariates such as physical activity. Increasing the elevated meal event count of two or more each day had a signiﬁcant effect on daily time-in- range and average glucose. As a corollary, any effect on daily glycemic outcomes due to an increase in elevated meal event count from zero to one was insigniﬁcant. Generally, 70 mg/dL is a lower limit for type 2 diabetic individuals. For completeness, we also performed our analysis with a lower limit of 70 mg/dL and found consistent results. Our results with a lower limit of 70 mg/dL are presented in Supplementary Table 12. This ﬁnding suggests that, at an individual level, one elevated meal event may be acceptable, while two or more elevated meal events signiﬁcantly increase the potential for progression of dysglyce- mia. Daily elevated meal event count from CGM and meal logging may potentially be used as a glycemic target parameter by healthcare providers. The binary characterization of a meal event from CGM and diet logging could be used to provide personalized CGM-driven food reﬂection reports similar to calorie density color codes proposed in the prior work24. p y y q p The study has two limitations. First, the participants self- reported their meals in the MyFitnessPal App. While self- reported food diary data may be prone to inaccuracy, recent research analyzed large datasets collected through MyFitnes- sPal to derive insights about dietary behaviors29–31. In our study, we observed that participants often logged their meals later in the day. Daily elevated meal event count and step count have a signiﬁcant effect on time-in-range and average glucose Hence, the self-reported data is prone to inaccuracy, especially when more time has passed between the actual meal and entry into the app. We leveraged the synchronous CGM curves and prior knowledge that meals lead to a sharp increase in glucose values to combat the inaccuracies in self-reported timing. In our analysis, we implemented a meal timing correction and addition framework manually. However, manual annotations were subject to human error. To reduce bias, we aggregated annotations across four independent annotators. In the future, automated meal annotation tools can substitute for manual annotations. Although automated meal annotation tools have been developed for type 1 diabetes32, they need to be validated on datasets comprising of individuals with or at risk of T2D. In real-world deployment scenarios, a real-time meal detection tool could be used to remind participants to log or take a photo of their meals and provide targeted just-in-time recommendations33. p g j The second limitation is that the generalizability of the study is limited due to the small sample size (36 participants). The error of 0.8% is large, considering the HbA1c statistics in our dataset is 6.0 [IQR: 5.5–6.9]. However, we do show that most of the error arises due to the model’s inability to accurately ﬁt for high HbA1c (HbA1c > 10) values mainly because of the low representation of such high HbA1c values in our dataset. Excluding the four high HbA1c samples, the error reduces to 0.41%. We also tried a quadratic term ﬁt shown in Supplementary Fig. 1, and the error was reduced to 0.75% and 0.4% (excluding the HbA1c > 10% samples). The trends in our results remain unchanged, as shown in Supplementary Table 11. The model mapping average meal event glucose response to HbA1c is a population model and would be more accurate with a larger sample size and more balanced representation across HbA1c values. Causal effects estimated on observational data are a function of the population. Population behaviors may be widely different for other ethnic and racial groups, especially regarding diet and physical activity34,35. Hence, y p p p Participants also recorded their daily levels of physical activity in this study. Daily elevated meal event count and step count have a signiﬁcant effect on time-in-range and average glucose Previously, in a similar population of Hispanic/Latino adults living with or at risk of developing T2D, it was possible to also predict HbA1c from npj Digital Medicine (2024) 7 Fig. 4 Mapping between HbA1c and average meal event glucose response. The scatterplot on the left a displays the robust regression ﬁt between average meal event glucose response and HbA1c. The equation representing the best line-of-ﬁt is reported (solid black line). The scatterplot in b shows the correlation between true and ﬁtted HbA1c values. The dashed black line represents the 45° line. A. Pai et al. A. Pai et al. 5 Fig. 4 Mapping between HbA1c and average meal event glucose response. The scatterplot on the left a displays the robust regression ﬁt between average meal event glucose response and HbA1c. The equation representing the best line-of-ﬁt is reported (solid black line). The scatterplot in b shows the correlation between true and ﬁtted HbA1c values. The dashed black line represents the 45° line. the breakfast glucose response alone14. In that study, self-reported meal logs were not collected and only breakfast responses were studied because it was easiest to annotate manually. participants who were more active later during the day, as well as for overweight and younger individuals15. Taken together with this study, these ﬁndings suggest that targeted step count recommendations could be provided as a just-in-time adaptive intervention28. For example, after an individual logs their meal, a post-meal step count needed to prevent an elevated meal event is recommended. Another example is that if a low post- meal step count is detected after the meal, a notiﬁcation encouraging you to take a walk is sent. Further, if an elevated meal event is detected, a notiﬁcation to engage in some form of physical activity after subsequent meals can be provided. participants who were more active later during the day, as well as for overweight and younger individuals15. Taken together with this study, these ﬁndings suggest that targeted step count recommendations could be provided as a just-in-time adaptive intervention28. For example, after an individual logs their meal, a post-meal step count needed to prevent an elevated meal event is recommended. Another example is that if a low post- meal step count is detected after the meal, a notiﬁcation encouraging you to take a walk is sent. Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2024) 7 Published in partnership with Seoul National University Bundang Hospital Daily elevated meal event count and step count have a signiﬁcant effect on time-in-range and average glucose For people living with T2D, physical activity is encouraged as part of the management of glycemia and overall health based on evidence that regular physical activity can help to improve glycemic control as measured by changes in HbA1c levels and 24-h ambulatory glucose pro- ﬁles25,26. In addition, reducing the amount of sedentary time may also help prevent T2D for those at risk27. In this study, an increase in physical activity (represented as step count increase) within one hour after a meal event was associated with a signiﬁcantly lower meal event glucose response and risk of an elevated classiﬁcation. Previously15 we reported that, for free- living Hispanic/Latino adults with or at risk of T2D, there appears to be clustering of their daily levels of physical activity by intensity and time of day, which, in turn, may inﬂuence achieved HbA1c and BMI. In that study, the amount of physical activity was more impactful on the HbA1c achieved among npj Digital Medicine (2024) 7 Published in partnership with Seoul National University Bundang Hospital A. Pai et al. steps taken in methods. The CGM curve and meal logs are analyzed to segment meal events. T lucose response (MGR3 h), post-meal step count, and elevated or normal classiﬁcation of meal e asures of elevated meal count daily step count baseline glucose and duration are then calc 6 Fig. 5 Illustration of steps taken in methods. The CGM curve and meal logs are analyzed to segment meal events. The meal event measures are the meal event’s glucose response (MGR3 h), post-meal step count, and elevated or normal classiﬁcation of meal events computed for each meal event. Daily measures of elevated meal count, daily step count, baseline glucose, and duration are then calculated. Fig. 5 Illustration of steps taken in methods. The CGM curve and meal logs are analyzed to segment meal events. The meal event measures are the meal event’s glucose response (MGR3 h), post-meal step count, and elevated or normal classiﬁcation of meal events computed for each meal event. Daily measures of elevated meal count, daily step count, baseline glucose, and duration are then calculated. Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2024) 7 A. Pai et al. 7 Table 2. Effect of daily step count and elevated meal event count on glycemic outcomes. Daily elevated meal event count and step count have a signiﬁcant effect on time-in-range and average glucose The adherence measure of the CGM device was the number of days with no missing glucose values. ActiGraph adherence was the number of days 10 or more hours of step counts were recorded40. Past work41 has established that within 14 days, having at least 10 days (70%) of CGM data is required to measure usual glycemic patterns reliably. Hence, this study considers ten days of simultaneous adherence to MyFitnessPal, ActiGraph, and CGM. Simultaneous adherence days refer to days with no missing values of CGM, ActiGraph recorded ten or more hours of step counts, and at least two meals were logged on MyFitnessPal. the effects need to be validated with a larger sample size and compared to other populations with different ethnicities. Given that the U.S. Hispanic/Latino population faces a disproportionate burden of diabetes compared to the background population11, elucidating the association between diet, physical activity, and glycemic control has been the focus of our work. Our study suggests that the use of digital health technologies is both feasible and acceptable for this population and could potentially help achieve more equitable health outcomes36. p q In conclusion, we conducted a multimodal digital phenotyping study to investigate the impact of meal events and physical activity on daily glycemic control in Hispanic/Latino adults with or at risk of T2D. We proposed a CGM-driven binary characterization of a meal event that is the elevated/normal classiﬁcation of a meal event by exploiting the association found between participants’ average meal event glucose response and HbA1c. We introduced the concept of an elevated meal event count and showed that it signiﬁcantly contributed to time-in-range between 54 and 140 mg/dl and average glucose. In this study, we also found that the post-meal step count signiﬁcantly impacts the risk of elevated meal events. A speciﬁc focus of the application of digital phenotyping is the ability to visualize and interpret physiological data from digital health technologies that can potentially create opportunities for new therapeutic interventions at an increasingly personal level, beneﬁting the majority by focusing on the uniqueness of individuals. The ﬁndings of our study may offer the opportunity for non-pharmacologic interventions for popula- tions facing a disproportionate burden of T2D. Ethics approval Ethics approval was obtained from the Rice University Institutional Review Board (IRB-FY2021-54). Written informed consent was obtained from all participants. METHODS Study Thirty-six adult participants of self-reported Hispanic/Latino heritage with a diagnosis of T2D or at risk for developing T2D using the American Diabetes Association diabetes risk assessment tool37 provided written informed consent to be enrolled in a prospective, observational cohort study (ClinicalTrials.gov number: NCT04820348). The American Diabetes Association created a simple 7-question test to estimate someone’s risk of having diabetes. The test considers age, gender, history of gestational diabetes, family history of diabetes, high blood pressure, level of physical activity, and BMI. Each question is scored from 0 to 1 or more points. A total score of 5 or higher indicates the person is at high risk for having diabetes. The goal of the test is to identify those with modiﬁable risk factors and raise awareness so they can make lifestyle changes to prevent diabetes. Daily elevated meal event count and step count have a signiﬁcant effect on time-in-range and average glucose Glycemic Variable Time-in-range (54–140 mg/dL) Percentage of total time (%) Average glucose mg/dL Variable of interest Estimate (standard error, t-test p-value) Estimate (standard error, t-test p-value) Elevated meal event count 0–1 −1.1 (1.3, 0.41) 1.7 (1.2, 0.16) Elevated meal event count 0–2 −4.0 (1.3, 0.003) 5.8 (1.3, <0.0001) Elevated meal event count 0 to ≥3 −8.1 (1.5, <0.0001) 7.7 (1.4, <0.0001) Daily step count (1 unit = 1000 steps) 0.07 (0.1, 0.63) −0.3 (0.1, 0.02) Time-in-range (54–140 mg/dL) refers to the percentage of total time glucose values between 54 and 140 mg/dL. The effects of elevated meal event count were computed after adjusting for confounders, namely baseline glucose, duration and daily step count. Similarly, the effects of daily step count were computed after adjusting for confounders’ baseline, glucose, duration, and elevated meal event count. The sample size n = 369. Effect of daily step count and elevated meal event count on glycemic outcomes. Time-in-range (54–140 mg/dL) refers to the percentage of total time glucose values between 54 and 140 mg/dL. The effects of elevated meal event count were computed after adjusting for confounders, namely baseline glucose, duration and daily step count. Similarly, the effects of daily step count were computed after adjusting for confounders’ baseline, glucose, duration, and elevated meal event count. The sample size n = 369. Libre Pro) for 14 days after enrollment. During enrollment, the premium version of the MyFitnessPal (MyFitnessPal, Inc.) app was installed on each participant’s personal smartphone in the desired language (English/Spanish). Study staff trained the participants to use the MyFitnessPal app. Participants reported the timing; meal occasion (breakfast, lunch, dinner, or snacks); name; and quantity of the food item by searching the MyFitnessPal food database. Based on the quantity selected and available information about the food item in the database, the app calculated the macronutrient composition for each food item. For measurement of physical activity, the participants wore the ActiGraph wGT3X-BT (ActiGraph, Pensacola, Florida, USA) on their dominant hip with an elastic belt for 24 hours/day except when bathing, swimming, and sleeping for 14 days. The downloaded data were screened for wear time using the Choi algorithm based on previous work using the manufacturer’s software (ActiLife 6.13.3)38. Participants were asked to continue normal activities during the study period. The adherence measure of MyFitnessPal meal logging was the number of days the participant logged food for two or more different meals39. Published in partnership with Seoul National University Bundang Hospital Framework to correct and manually add meal log timings Framework to correct and manually add meal log timings While analyzing meal logs, we observed that participants entered food items consumed in a given meal in stages. For example, under breakfast for an example participant, oatmeal was logged at 10 am, and orange juice at 10:15 am. We combined food logs with the same meal occasion label (e.g., breakfast) and timing difference within 60 minutes into a single meal event. The meal log timing assigned to the meal event was the timing of the ﬁrst food log in that meal (10 am in the example above). For each day, we overlaid the meal log timings on the 24-h CGM curve. Based on visual inspection, the starting time point of a bell- shaped CGM curve segment (between 4 am and 11:59 pm) with sharp glucose rise closest to the meal log timing with minimal overlap with other 3-h CGM curve segments corresponding to other meal logs was selected as the corrected timing. In Fig. 3, the orange boxes highlight examples where the meal log timing is delayed with respect to the start of a sharp rise in glucose levels, and the green lines denote the corrected timing. The time difference between the corrected timing and self-report timing varies as seen in the time difference between the green and red lines in the orange boxes in Fig. 3a, c, d. The green boxes highlight examples on the same day where the meal log timing coincided perfectly with the sharp rise in glucose rise. For every participant, we calculated their average meal events’ glucose response denoted as MGR3 h. By aggregating meal events’ glucose responses, variations due to differences in food choices and physical activity around meals were smoothed out and the association with HbA1c, which is also an average metric, was more prominent. We built a robust linear regression model44 with HbA1c as response variable and MGR3 h as a predictor variable. The robust linear regression model was denoted as HbA1c ¼ fðMGR3 hÞ þ ϵ where ϵ 2 Nð0; σ2Þ and f is a linear function. We used the learned function to characterize individual meal events as elevated or normal (from a glucose perspective). The model is utilized to ﬁnd a hypothetical HbA1c mapping for a single meal’s MGR3 h. Framework to correct and manually add meal log timings The HbA1c values of a single meal signify the hypothetical scenario where if the individual ate that meal all the time and their average meal event glucose response was equal to that meal event’s glucose response, what would be their HbA1c? We use this hypothetical HbA1c to classify the meal event as normal or elevated. In some cases, participants logged multiple meal events with different meal occasion labels at the same time. For example, when a participant logged all the meals at the end of the day. In such scenarios, multiple bell-shaped CGM segments with a sharp glucose rise were visually identiﬁed for each meal event. The meal event timings were aligned in the order speciﬁed by the meal occasion label, breakfast followed by lunch, followed by dinner. Snacks were logged at the same time as other meals and tagged unknown. Meal logs for which a corrected timing could not be identiﬁed were tagged unknown. y We deﬁne elevated meal events differently for at-risk, predia- betes, and T2D participants. Thus, for at risk and prediabetes participants, the classiﬁcation of the meal event’s glucose response was elevated if fðMGR3 hÞ ≥5.7 and normal if fðMGR3 hÞ < 5.7. In the case of type 2 diabetes participants, the meal event’s glucose response was elevated if fðMGR3 hÞ ≥6.5 and normal if fðMGR3 hÞ < 6.5. With the above formulation, we proposed a new CGM-driven meal event characterization that can be used to classify all detected meal events into elevated versus normal categories. For example, consider a participant with prediabetes consumed a meal event with a mapped HbA1c of 6.0. A mapped HbA1c of 6.0 indicates that the particular meal event’s glucose response is similar to the average meal event’s glucose response of a participant with T2D. Then repeated consumption of that meal event is unsafe for the prediabetic participant and so the meal event receives an elevated classiﬁcation for the prediabetic participant. In cases where a timing in the self-reported log was missing or none of the meal events of the day were self-reported, we added the timings. Since we did not have the self-reported meal log timing as a reference, we visually identiﬁed bell-shaped CGM curve segments (between 4 am and 11:59 pm) with a sharp glucose rise. An example in which all meals were added is displayed in Fig. 3b. Framework to correct and manually add meal log timings Another example in which only one of the meal events was added is highlighted in Fig. 3d (yellow line). Meal event biomarkers Meal event’s glucose response. We deﬁned a meal event’s glucose response as the incremental area under the 3-h CGM curve from the start time of the meal event and denoted it as MGR3 h. Post-meal step count of the meal event. We represented the post- meal step count of the meal event as the step count in the 1-h time window after the start time of the meal event. We selected a one-hour window based on previous works that showed that physical activity 15–30 min after completion of the meal reduced postprandial glucose responses45,46. Calorie content of the meal event. This was calculated as the sum of the calorie content of individual food items in the self-reported meal log corresponding to the particular meal event. Aggregation of annotated timings for identiﬁcation of the start time of meal event For each day of each participant, we used Density-based spatial clustering of applications with noise (DBSCAN)42 algorithm to aggregate the annotated timings into groups. The Sklearn package in Python was used to implement DBSCAN with parameters eps ¼ 1:5 and minsamples ¼ 2. The parameters were chosen such that each group had at least 2 annotated timings and groups were at least 1.5 h apart. Annotated timings that were tagged as outliers by the algorithm were removed. Each group symbolized a meal event if the start time of the group (i.e., the annotated timing that had the largest incremental area under the 3-h CGM curve) had a maximum glucose rise ≥18.6 mg/dL. The threshold of 18.6 mg/dL was based on a recent report that 97.5% of post-meal glucose proﬁles in healthy adults have a maximum glucose rise greater than 18.6 mg/dL43. npj Digital Medicine (2024) 7 Segmentation of meal events We used the self-reported meal log timings and CGM curves to identify the start time of meal events. We segmented 3 h of the CGM curve from the start time as the meal event. Participants were asked to log their meals just before eating so that the meal log timing would align with the sharp rise in glucose levels seen in the CGM curve. However, after the study was completed, it became apparent that the meals were often logged variably after eating, with the meal log timing not always coinciding with the rise in glucose levels. Also, in some instances, participants forgot to log their meals. Hence, we implemented a framework to correct the meal log timings and add meal timings. y g p Baseline measurement of HbA1c was taken using point-of-care HbA1c testing (Alere Aﬁnion 2). Multimodal monitoring involves three digital health modalities: continuous glucose monitoring (CGM), a diet-tracking mobile application, and physical activity monitoring. Participants wore a blinded CGM (Abbott Freestyle npj Digital Medicine (2024) 7 A. Pai et al. 8 from the glucose values in the 3-h segment, and (iii) negative glucose values set to zero. The trapezoidal rule is used to calculate the area under the curve. The incremental area under the curve is widely used to characterize glucose responses to meals in previous works14,16. A characterization of meal events based on MGR3 h does not exist for individuals with or at-risk of diabetes. However, HbA1c thresholds for prediabetes (5.7–6.4%) and type 2 diabetes (>6.4%) are well established in the literature. Hence, we developed a new CGM-driven meal event characterization that uses an “elevated” or “normal” classiﬁcation of the meal by exploiting the mapping between MGR3 h and HbA1c observed across participants (explained in “Methods”). Daily biomarkers confounding covariates X is given as ei ¼ PðTi ¼ t=XiÞ and is calculated using a propensity score model. The inverse probability weights are given as wi ¼ 1=PðTi ¼ t=XiÞ. The inverse probability weights are then used as weights in a weighted regression between the variable of interest T and outcome variable Y. When the covariate balance is not completely achieved with the inverse probability weights, doubly robust approach that is ﬁtting the weighted outcome regression with both T and X as input variables are performed50. We used the estimated coefﬁcients of the outcome regression to quantify the effect. We also report the statistical signiﬁcance (p-value) of the estimated coefﬁcients. Duration of the day. The duration of the day was deﬁned as the time period between the start time and end time of our analysis window for that day. Our analysis focuses on the effect of meals and physical activity. Hence, the start time of our analysis window of a day is the earlier time between the start time of the ﬁrst meal and the earliest detected ActiGraph wear time after 4 AM. The end time is later, between 3 hours after the start time of the last meal event and the last detected ActiGraph wear time before 11:59 PM. We consider the start time of the last meal event so that we include the physiological response of the meal event in our analysis window. The duration of the day differs across days and individuals. We estimated four causal effects in our analysis. First, the causal effect of elevated meal event counts on glycemic outcomes. Second, the causal effect of daily step count on glycemic outcomes. Third, the effect of post-meal step count on glycemic response of meal events. Lastly, the effect of calorie content on the glycemic response of meal events. Elevated meal event count of the day. The elevated meal event count of a day summarizes the number of meal events that were classiﬁed as elevated (deﬁned in the previous section) on that day. Elevated meal event count was deﬁned as a multi-category variable with four categories: zero elevated meal event, one elevated meal event, two elevated meal events, and ≥3 elevated meal events. As our dataset consists of multiple data samples from multiple participants, we use multilevel regression models in our causal analysis. The multilevel structure was captured using the user ID. Daily biomarkers For continuous biomarkers such as daily step count, post-meal step count and calorie content, we used linear mixed effect models as the propensity score model and normal density to transform estimates into probability scale weights51. For multi- category biomarkers such as the elevated meal event count, we used the multinomial mixed effects model as the propensity score model. We assessed the covariate balance before and after inverse probability weighting using weighted correlations between variable of interest and covariates for continuous treatments52. An absolute correlation coefﬁcient of less than 0.1 is desired to assume covariate balance52. For multi-category treatments, we calculated the maximum standardized mean difference between all pairwise treatment group comparisons. A difference of less than 0.2 is desired for covariate balance53. Daily step count of the day. The step count of the day was the total steps taken during the duration of the day. Baseline glucose of the day. The baseline glucose of the day was deﬁned as the glucose value in the CGM curve at the start time of the day. Glycemic outcomes of the day. The daily glycemic outcomes considered were time-in-range (54–140 mg/dL), and average glucose was measured using CGM values during the duration of the day. Time-in-range (TIR54–140) was calculated as the percen- tage of time the glucose measurements were within 54–140 mg/ dl. A lower limit of 54 mg/dl was chosen because at-risk and prediabetic individuals can have non-pathologic glucose values below 70 mg/dL47. Further, the range of <54 mg/dL is deﬁned as clinically signiﬁcant hypoglycemia in nondiabetic individuals48. An upper limit of 140 mg/dL was chosen based on recent ﬁndings that daytime time-in-range of 140–180 mg/dl can be used as an early indicator of diabetes progression14. To compute the effect of elevated meal event count on glycemic outcomes, we considered the elevated meal event count as the variable of interest, with covariates being baseline glucose level, duration of the day, and daily step count and outcomes being TIR54–140 and average glucose. The regression model is summarized in Supplementary Tables 3 and 4, and the covariate balance is achieved in Supplementary Table 2. While computing the effect of daily step count, the covariates were baseline glucose level, elevated meal event count, and duration of the day. Supplementary Tables 6 and 7 summarize the regression model, and the covariate balance is displayed in Supplementary Table 5. Elevated/normal classiﬁcation of meal event We developed a new CGM-driven meal event characterization that uses an “elevated” or “normal” classiﬁcation of the meal. For each meal event, we computed incremental area under the 3-h CGM curve denoted as MGR3 h using: (i) the 3-h CGM curve segment beginning from the start time of the meal event, (ii) the baseline glucose value at the the start time of the meal event is subtracted Elevated/normal classiﬁcation of meal event. Classiﬁcation of the meal event based on its MGR3 h was into normal or elevated categories. We describe the elevated/normal classiﬁcation of meal events in detail below. npj Digital Medicine (2024) 7 Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital A. Pai et al. 9 Daily biomarkers We computed elevated meal event count, daily step count, baseline glucose, and duration for each day and participant. For daily measures, we excluded days where no meals were identiﬁed, the ActiGraph wear time for that day was less than 10 h (between 4 am and 11:59 pm), or the CGM data was incomplete (the ﬁrst and last day of the study). Additionally, we removed days where the CGM proﬁle showed measurements ≤40 mg/dL and/or ≥500 mg/dL (glucose values were beyond the sensor limits of the CGM device). Next, we considered post-meal step count as the variable of interest, the calorie content of the meal as the covariate and MGR3 h and likelihood of elevated meal event classiﬁcation as the outcome variables. Inverse probability weighting was not imple- mented as the correlation between the post-meal step count and the calorie content was −0.05, which was low enough to assume adequate covariate balance. Thus, we did multiple regression using a linear mixed effects model to estimate the effect of post- meal step count and calorie content on MGR3 h (Supplementary Table 8). We used a generalized mixed effects model to compute the effect of post-meal step count and calorie content on elevated/normal classiﬁcation of the meal event (Supplementary Table 9). Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2024) 7 REFERENCES 22. Dodd, H., Williams, S., Brown, R. & Venn, B. Calculating meal glycemic index by using measured and published food values compared with directly measured meal glycemic index. Am. J. Clin. Nutr. 94, 992–996 (2011). 1. Grifﬁn, S. Diabetes precision medicine: plenty of potential, pitfalls and perils but not yet ready for prime time. Diabetologia 65, 1913–1921 (2022). 1. Grifﬁn, S. Diabetes precision medicine: plenty of potential, pitfalls and perils but not yet ready for prime time. Diabetologia 65, 1913–1921 (2022). 23. Jenkins, D. J. et al. Glycemic index of foods: a physiological basis for carbohydrate exchange. Am. J. Clin. Nutr. 34, 362–366 (1981). 2. Spruijt-Metz, D., Cook, L., O’Reilly, G. A., Page, K. A. & Quinn, C. Behavioral con- tributions to the pathogenesis of type 2 diabetes. Curr. Diabetes Rep. 14, 475 (2014). 24. Toro-Ramos, T. et al. Mobile delivery of the diabetes prevention program in people with prediabetes: randomized controlled trial. JMIR mHealth uHealth 8, e17842 (2020). 3. Kerr, D., King, F. & Klonoff, D. C. Digital health interventions for diabetes: every- thing to gain and nothing to lose. Diabetes Spectr. 32, 226–230 (2019). 25. Umpierre, D. et al. Physical activity advice only or structured exercise training and association with HbA1c levels in type 2 diabetes: a systematic review and meta- analysis. J. Am. Med. Assoc. 305, 1790–1799 (2011). 4. Kerr, D., Axelrod, C., Hoppe, C. & Klonoff, D. C. Diabetes and technology in 2030: a utopian or dystopian future? Diabet. Med. 35, 498–503 (2018). 5. Huckvale, K., Venkatesh, S. & Christensen, H. Toward clinical digital phenotyping: a timely opportunity to consider purpose, quality, and safety. npj Digit. Med. 2, 1–11 (2019). 26. Munan, M. et al. Acute and chronic effects of exercise on continuous glucose monitoring outcomes in type 2 diabetes: a meta-analysis. Front. Endocrinol. 11, 495 (2020). 6. Yang, Q. et al. Digital phenotyping self-monitoring behaviors for individuals with type 2 diabetes mellitus: observational study using latent class growth analysis. JMIR mHealth uHealth 8, e17730 (2020). 27. Schellenberg, E. S., Dryden, D. M., Vandermeer, B., Ha, C. & Korownyk, C. Lifestyle interventions for patients with and at risk for type 2 diabetes: a systematic review and meta-analysis. Ann. Intern. Med. 159, 543–551 (2013). 7. Battelino, T. et al. Clinical TArgets for Continuous Glucose Monitoring Data Interpretation: Recommendations from the International Consensus on Time in Range. Diabetes Care 42, 1593–1603 (2019). 28. Nahum-Shani, I., Hekler, E. B. DATA AVAILABILITY 16. Zeevi, D. et al. Personalized nutrition by prediction of glycemic responses. Cell 163, 1079–1094 (2015). Data are available upon reasonable request. The investigators agree to share de- identiﬁed individual participant data that underlie the results reported in this article and the study protocol. Requests should be directed to wbevier@sansum.org. To gain access, data requestors will need to sign a data access agreement. 17. Understanding A1C \| ADA. https://diabetes.org/diabetes/a1c. 18. Kerr, D., Aram, M., Crosby, K. M. & Glantz, N. A persisting parallel universe in diabetes care within America’s capital. eClinicalMedicine 43 https:// www.ncbi.nlm.nih.gov/pmc/articles/PMC8718894/ (2022). 19. Jang, M., Johnson, C. M., D’Eramo-Melkus, G. & Vorderstrasse, A. A. Participa- tion of racial and ethnic minorities in technology-based interventions to self- manage type 2 diabetes: a scoping review. J. Transcult. Nurs. 29, 292–307 (2018). CODE AVAILABILITY The computational and statistical analysis tools developed for this study are available upon request. Requests should be directed to the corresponding author. The computational and statistical analysis tools developed for this study are available upon request. Requests should be directed to the corresponding author. 20. Odugbesan, O. et al. Implicit racial-ethnic and insurance-mediated bias to recommending diabetes technology: insights from T1D exchange multicenter pediatric and adult diabetes provider cohort. Diabetes Technol. Ther. 24, 619–627 (2022). Received: 10 April 2023; Accepted: 4 December 2023; 21. National Academies of Sciences, E. Improving Representation in Clinical Trials and Research: Building Research Equity for Women and Underrepresented Groups. https://doi.org/10.17226/26479 (2022). Reporting summary 14. Barua, S. et al. The northeast glucose drift: stratiﬁcation of post-breakfast dys- glycemia among predominantly Hispanic/Latino adults at-risk or with type 2 diabetes. eClinicalMedicine 43, 101241 (2022). Further information on research design is available in the Nature Research Reporting Summary linked to this article. 15. Kerr, D. et al. Patterns of timing and intensity of physical activity and HbA1c levels in hispanic/latino adults with or at risk of type 2 diabetes. J. Diabetes Sci. Technol. https://doi.org/10.1177/19322968221105531 (2022). 15. Kerr, D. et al. Patterns of timing and intensity of physical activity and HbA1c levels in hispanic/latino adults with or at risk of type 2 diabetes. J. Diabetes Sci. Technol. https://doi.org/10.1177/19322968221105531 (2022). Causal analysis 10 Elevated meal event count of the day sk b Prediabetes c Type 2 Diabetes 0 1 2 3 0 20 40 60 80 100 0 1 2 3 50 60 70 80 90 100 2 3 Elevated meal event count of the day Time-in-range (54-140 mg/dL) of the day a At-risk b Prediabetes c Type 2 Diabetes 0 1 2 3 0 20 40 60 80 100 0 1 2 3 50 60 70 80 90 100 0 1 2 3 70 75 80 85 90 95 100 Elevated meal event count of the day Fig. 6 Association between Daily TIR54–140 and elevated meal event count. Daily TIR54–140 decreases associated with an increase in elevated meal event count across participants a at risk of T2D, b with prediabetes, c with T2D. The ends of the box represent the lower and upper quartiles. The median (second quartile) is marked by a line inside the box. The data points adjacent to the box plot display the underlying data points where each data point corresponds to a day. Fig. 6 Association between Daily TIR54–140 and elevated meal event count. Daily TIR54–140 decreases associated with an increase in elevated meal event count across participants a at risk of T2D, b with prediabetes, c with T2D. The ends of the box represent the lower and upper quartiles. The median (second quartile) is marked by a line inside the box. The data points adjacent to the box plot display the underlying data points where each data point corresponds to a day. Causal analysis In real-world datasets, variables are often correlated with each other. Biology and behaviors of an individual have a complex interplay as shown in Fig. 1c. Our computed biomarkers are expected to be associated with each other. Hence to isolate the causal impact of a variable of interest, we need to account for other variables as confounding covariates and adjust for them in our analysis. Custom code was developed for statistical analyses in Python (version 3.9.13) and R (version 4.2.0). The RLM function from the statsmodel package in Python was used for robust linear regression. The linear mixed-effect regression was performed using the lmer function from lmerTest library in R. The mblogit function from mclogit library in R was used for multinomial mixed- effect regression. The covariate balance was assessed with the cobalt package in R54. All custom code written as a Jupyter notebook is available on request. Statistical signiﬁcance is deﬁned as a p-value < 0.01. Propensity score models are required to estimate the causal effect of a variable of interest on outcome variables from observational data. The covariate variables need to be balanced if the variable of interest and covariates are correlated to ensure there is no confounding. We used inverse probability weighting, a widely used propensity score weighting estimator for balancing the covariates and measuring the causal effects49. The generalized propensity score for a sample i, for a variable of interest T; and Published in partnership with Seoul National University Bundang Hospital A. Pai et al. Elevated meal event count of the day Time-in-range (54-140 mg/dL) of the day a At-risk b Prediabetes c Type 2 Diabetes 0 1 2 3 0 20 40 60 80 100 0 1 2 3 50 60 70 80 90 100 0 1 2 3 70 75 80 85 90 95 100 Fig. 6 Association between Daily TIR54–140 and elevated meal event count. Daily TIR54–140 decreases associated with an increase in elevated meal event count across participants a at risk of T2D, b with prediabetes, c with T2D. The ends of the box represent the lower and upper quartiles. The median (second quartile) is marked by a line inside the box. The data points adjacent to the box plot display the underlying data points where each data point corresponds to a day. 14. Barua, S. et al. The northeast glucose drift: stratiﬁcation of post-breakfast dys- glycemia among predominantly Hispanic/Latino adults at-risk or with type 2 diabetes. eClinicalMedicine 43, 101241 (2022). Published in partnership with Seoul National University Bundang Hospital ACKNOWLEDGEMENTS 34. Gavin, J. R., Fox, K. M. & Grandy, S. Race/ethnicity and gender differences in health intentions and behaviors regarding exercise and diet for adults with type 2 diabetes: a cross-sectional analysis. BMC Public Health 11, 533 (2011). The authors thank Abbott Diabetes Care for supplying the Freestyle Libre sensors and readers that provided continuous glucose monitoring for this study. Funding for the study was provided by the NSF Engineering Research Center for Precise Advanced Technologies and Health Systems for Underserved Populations (PATHS-UP) (#1648451) and the Elsevier Foundation. The funder played no role in the study design, data collection, analysis, and interpretation of data or the writing of this manuscript. 35. Sex and Race/Ethnicity Differences in Following Dietary and Exercise Recom- mendations for U.S. Representative Sample of Adults With Type 2 Diabetes. Joan A. Vaccaro, Fatma G. Huffman. https://journals.sagepub.com/doi/full/10.1177/ 1557988316681126 (2017). 36. Principles for virtual health care to deliver real equity in diabetes. Lancet Diabetes Endocrinol. https://www.thelancet.com/journals/landia/article/PIIS2213-8587(21) 00176-5/fulltext. AUTHOR CONTRIBUTIONS 37. Risk Test Today: https://diabetes.org/diabetes/risk-test. 38. Choi, L., Liu, Z., Matthews, C. E. & Buchowski, M. S. Validation of accelerometer wear and nonwear time classiﬁcation algorithm. Med. Sci. Sports Exerc. 43, 357–364 (2011). A.P. conceptualized the analysis pipeline, wrote software for computing the proposed measures, performed statistical analyses, created visualizations, and led the writing of the paper. AP was involved in the development of the protocol design, IRB approval, and data collection. A.S. supervised AP in the design of the study and analysis pipeline and contributed to the review and editing of the paper. N.G. and W.B. were involved in the development and execution of the protocol design, IRB approval, data collection, paper generation, and editing. R.S. was involved in the execution of the study protocol, including participant recruitment and retention, data capture, and paper editing. D.K., A.S., A.P. conceptualized the study. D.K. was the study’s Principal Investigator, edited the paper, and is the guarantor of the study. All authors contributed to the writing of the paper. A.P., A.S., and D.K. have veriﬁed the underlying data. All authors read and approved the ﬁnal paper. 39. Turner-McGrievy, G. M. et al. Deﬁning adherence to mobile dietary self- monitoring and assessing tracking over time: tracking at least two eating occasions per day is best marker of adherence within two different mobile health randomized weight loss interventions. J. Acad. Nutr. Diet. 119, 1516–1524 (2019). 40. Troiano, R. P. Large-scale applications of accelerometers: new frontiers and new questions. Med. Sci. Sports Exerc. 39, 1501 (2007). 41. Bergenstal, R. M. Understanding Continuous Glucose Monitoring Data. in Role of Continuous Glucose Monitoring in Diabetes Treatment (American Diabetes Asso- ciation, 2018). 42. Ester, M., Kriegel, H. P., Sander, J. & Xiaowei, X. A Density-Based Algorithm for Discovering Clusters in Large Spatial Databases with Noise. (1996). REFERENCES & Spruijt-Metz, D. Building health behavior models to guide the development of just-in-time adaptive interventions: a pragmatic framework. Health Psychol. 34, 1209–1219 (2015). 8. Beck, R. W. et al. Validation of time in range as an outcome measure for diabetes clinical trials. Diabetes Care 42, 400–405 (2018). 29. Althoff, T., Nilforoshan, H., Hua, J. & Leskovec, J. Large-scale diet tracking data reveal disparate associations between food environment and diet. Nat. Commun. 13, 267 (2022). 9. Beck, R. W. et al. The relationships between time in range, hyperglycemia metrics, and HbA1c. J. Diabetes Sci. Technol. 13, 614–626 (2019). 30. Pai, A. & Sabharwal, A. Food habits: insights from food diaries via computational recurrence measures. Sensors 22, 2753 (2022). 10. Chow, E. A., Foster, H., Gonzalez, V. & McIver, L. The disparate impact of diabetes on racial/ethnic minority populations. Clin. Diabetes 30, 130–133 (2012). 31. Pai, A. & Sabharwal, A. Calorie compensation patterns observed in app-based food diaries. Nutrients 15, 4007 (2023). 11. Umpierrez, G. E., Gonzalez, A., Umpierrez, D. & Pimentel, D. Diabetes mellitus in the hispanic/latino population: an increasing health care challenge in the United States. Am. J. Med. Sci. 334, 274–282 (2007). 32. Zheng, M., Ni, B. & Kleinberg, S. Automated meal detection from continuous glucose monitor data through simulation and explanation. J. Am. Med. Inform. Assoc. 26, 1592–1599 (2019). 12. Cusi, K. & Ocampo, G. L. Unmet needs in Hispanic/Latino patients with type 2 diabetes mellitus. Am. J. Med. 124, S2–S9 (2011). 33. Mortazavi, B. J. & Gutierrez-Osuna, R. A review of digital innovations for diet monitoring and precision nutrition. J. Diabetes Sci. Technol. https://doi.org/ 10.1177/19322968211041356 (2021). 13. Barua, S. et al. Dysglycemia in adults at risk for or living with non-insulin treated type 2 diabetes: insights from continuous glucose monitoring. EClinicalMedicine 35, 100853 (2021). 13. Barua, S. et al. Dysglycemia in adults at risk for or living with non-insulin treated type 2 diabetes: insights from continuous glucose monitoring. EClinicalMedicine 35, 100853 (2021). Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2024) 7 A. Pai et al. 11 ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41746-023-00985-7. 46. Aadland, E. & Hostmark, A. T. Very light physical activity after a meal blunts the rise in blood glucose and insulin ~ !2008-06-11 ~ !2008-10-16 ~ !2008-11-28 ~ ! TONUTRJ 2, 94–99 (2008). Correspondence and requests for materials should be addressed to Amruta Pai. 47. Dehghani Zahedani, A. et al. Improvement in glucose regulation using a digital tracker and continuous glucose monitoring in healthy adults and those with type 2 diabetes. Diabetes Ther. 12, 1871–1886 (2021). COMPETING INTERESTS 43. Freckmann, G. et al. Continuous Glucose Proﬁles in Healthy People With Fixed Meal Times and Under Everyday Life Conditions. J. Diabetes Sci. Technol. https:// doi.org/10.1177/19322968221113341 (2022). A.P., R.S., N.G., A.L., W.B., S.B., and A.S. declare no competing interest(s). D.K. has received research support from Abbott Diabetes Care and Novo Nordisk and is an advisor to Sanoﬁ, Glooko, SNAQ, and Hi.Health. 44. Huber, P. J. Robust regression: asymptotics, conjectures and Monte Carlo. Ann. Stat. 1, 799–821 (1973). 45. Pahra, D. et al. Impact of post-meal and one-time daily exercise in patient with type 2 diabetes mellitus: a randomized crossover study. Diabetol. Metab. Syndr. 9, 64 (2017). Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints 48. International Hypoglycaemia Study Group. Glucose concentrations of less than 3.0 mmol/L (54 mg/dL) should be reported in clinical trials: a joint position statement of the American Diabetes Association and the European Association for the Study of Diabetes. Diabetes Care 40, 155–157 (2016). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 49. Chesnaye, N. C. et al. An introduction to inverse probability of treatment weighting in observational research. Clin. Kidney J. 15, 14–20 (2022). 50. Funk, M. J. et al. Doubly robust estimation of causal effects. Am. J. Epidemiol. 173, 761–767 (2011). 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/. 51. Schuler, M. S., Chu, W. & Coffman, D. Propensity score weighting for a continuous exposure with multilevel data. Health Serv. Outcomes Res. Method 16, 271–292 (2016). 52. Zhu, Y., Coffman, D. L. & Ghosh, D. A boosting algorithm for estimating gen- eralized propensity scores with continuous treatments. J. Causal Inference 3, 25–40 (2015). 53. Lopez, M. J. & Gutman, R. Estimation of causal effects with multiple treatments: a review and new ideas. Stat. Sci. 32, 432–454 (2017). 54. Greifer, N. cobalt: Covariate Balance Tables and Plots. R package version 4.4.1 (2023). © The Author(s) 2024 Published in partnership with Seoul National University Bundang Hospital Published in partnership with Seoul National University Bundang Hospital npj Digital Medicine (2024) 7
https://openalex.org/W3010900053	https://www.frontiersin.org/articles/10.3389/fpsyg.2020.00418/pdf	English	null	A Contextual Behavioral Account of Culture: Example Implementation of a Functional Behavioral Approach to the Study of Cultural Differences in Social Anxiety	Frontiers in psychology	2,020	cc-by	10,708	Edited by: Andrew G. Ryder, Concordia University, Canada Reviewed by: Vinai Norasakkunkit, Gonzaga University, United States Eunsoo Choi, Korea University, South Korea Correspondence: Alexander Krieg xkrieg@gmail.com Edited by: Andrew G. Ryder, Concordia University, Canada Reviewed by: Vinai Norasakkunkit, Gonzaga University, United States Eunsoo Choi, Korea University, South Korea Correspondence: Alexander Krieg xkrieg@gmail.com Specialty section: This article was submitted to Cultural Psychology, a section of the journal Frontiers in Psychology Keywords: culture, functional behavioral assessment, contextual behavior science, social anxiety, cross-cultural differences Received: 10 July 2019 Accepted: 24 February 2020 Published: 10 March 2020 A Contextual Behavioral Account of Culture: Example Implementation of a Functional Behavioral Approach to the Study of Cultural Differences in Social Anxiety Alexander Krieg* Department of Global Communication, Kobe Gakuin University, Kobe, Japan Alexander Krieg* The current article proposes integrating a functional behavior approach to the study of culture. After describing culture from a contextual behavioral science framework, we outline a three-step process to perform a functional behavior analysis of culture: (1) identifying potential contingencies, (2) determining functional relationships, and (3) gathering supporting evidence. As an example, we present each of the three steps through a re-analysis of data related to cultural differences in social anxiety between Japanese and European Americans as well as describe a hypothetical experiment. The results demonstrate how implementing an alternative framework that focuses on the relationship between behavioral function and environmental adaptability leads to different conclusions compared to implementing frameworks that emphasize the form or degree of a behavior or belief in one group compared to another. For this particular example, in contrast to viewing social anxiety in Japanese as something stemming from innate beliefs about themselves and others (e.g., self-construal), the current study suggests that displaying social anxiety in some situations within a Japanese context is more functionally adaptive (e.g., more likely leads to desirable outcomes) than within a European American context. ORIGINAL RESEARCH published: 10 March 2020 doi: 10.3389/fpsyg.2020.00418 Citation: Contextual Behavior Science (CBS) is the functional analysis of behavioral variation, selection, and retention within a given context across various dimensions (Hayes et al., 2012, 2017; Zettle et al., 2016). One important contextual dimension is culture. Culture has been deﬁned by cultural psychologists as a set of shared values, beliefs, and practices that are inﬂuenced by the environment and transmitted to others (Kitayama and Cohen, 2007; Markus and Hamedani, 2007). According to this widely agreed-upon deﬁnition, culture characterizes both an individual’s private thoughts (i.e., values and beliefs) and public behaviors (i.e., practices) if they Krieg A (2020) A Contextual Behavioral Account of Culture: Example Implementation of a Functional Behavioral Approach to the Study of Cultural Differences in Social Anxiety. Front. Psychol. 11:418. doi: 10.3389/fpsyg.2020.00418 March 2020 \| Volume 11 \| Article 418 Frontiers in Psychology \| www.frontiersin.org 1 Contextual Behavioral Culture Krieg are (1) inﬂuenced by salient features of the physical or social ecology, (2) shared by a speciﬁc group, and (3) transmitted by the group to new culture members. impossible for humans to perform underwater). From a pool of possible behaviors, the environment inﬂuences the selection and retention of behavior either by generalizing a behavioral response across a variety of stimuli through associations (i.e., classical conditioning; Pavlov, 1902) or through providing appetitive or aversive outcomes toward a behavior exhibited in a certain context (i.e., operant conditioning; Skinner, 1938, 1963). Contextual Behavior Science focuses on predicting and inﬂuencing both public behavior (practices) as well as private internal behavioral events (thoughts, feelings, and values), and provides a compelling account for how these behaviors are shaped by contextual factors. Speciﬁcally, CBS addresses (1) how the features of the environment create the contingencies that promote or obstruct a behavior from occurring (e.g., behavioral antecedents), (2) how social learning can occur so that individuals in close proximity share similar private and public behavior (e.g., behavioral imitation, Epstein, 1984; Boyd and Richerson, 1985), and (3) how behavioral repertoires are transmitted to others via the processes of associative and operant learning (e.g., behavioral consequences). Taken together, CBS aims to functionally relate organisms’ behaviors with features of the environment. Several interventions were developed from CBS and have shown a high degree of clinical utility across a broad range of aﬀective and behavioral concerns. Citation: For example, Acceptance Commitment Therapy (ACT) is a modern, process-based contextual behavior therapy that has demonstrated eﬀectiveness in treating a wide range of psychological phenomena all over the world (Hayes et al., 2006; Powers et al., 2009; A-tjak et al., 2015). Similarly, Applied Behavior Analysis (ABA) is the gold standard treatment for treating children with developmental disabilities worldwide (e.g., Austin and Carr, 2000; Hayes and Bissett, 2000). Making use of its sensitivity to subtle idiographic factors impacting an individual’s behavior in a speciﬁc context, several attempts have been made to capitalize on the potential for cultural sensitivity it oﬀers (Hayes et al., 2011; Pasillas and Masuda, 2014; Sabucedo, 2017). These approaches to working with psychological and behavioral concerns have become invaluable tools within the modern clinical psychologist’s toolbox. Many contributions to the study of culture emphasize the form, frequency, or intensity of a certain groups’ behavior relative to other groups. Unfortunately, the relation between that groups’ behavior and the environment in which they are situated is often relegated to the discussion section of a manuscript if addressed at all (Ryder and Chentsova-Dutton, 2015). In contrast, CBS emphasizes function over form, highlighting the role of environmental contingencies (i.e., antecedents and consequences). To a behaviorist, the question is not limited to just “how much more frequent does this behavior need to occur to be representative of a given culture?” but rather “is this behavior being diﬀerentially reinforced by even a subsection of the population?” (Tourinho and Vichi, 2012; Glenn et al., 2016; Baum, 2017). If it is, that alone creates a suﬃcient basis for cultural inquiry. Frontiers in Psychology \| www.frontiersin.org Contextual Behavioral Ideas in Cultural Psychology y gy A lesser-known fact among non-behaviorists is that Skinner’s treatment of human behavior inherently stressed the importance of social and cultural variables (Skinner, 1981). This idea is so central to a behavioral understanding of culture that Skinner (1953) even deﬁned culture as contingencies “arranged by other people” (p. 419). Speciﬁcally, Skinner (Skinner, 1953; Ferster and Skinner, 1957) hypothesized that an individual’s behavior often constitutes a signiﬁcant portion of the controlling environment for the behavior of other individuals and deﬁned social behavior on the basis of these “interlocking behavioral contingencies” (IBCs). According to the theory, IBCs may give rise to cultural practices when the behaviors involved are learned by other individuals and maintained by similar contingencies (Muchon de Melo and de Rose, 2013). Taken together, the behavior of other group members provides the antecedents and consequences of cultural practices. The current study provides a brief CBS account of culture, as well as re-analyzes the results of a recent study on Japanese and European American diﬀerences in social anxiety (Krieg, 2018) from a CBS perspective. In doing so, we hope to provide a theoretical framework for the contextual inﬂuence of culture on individual behavior as well as expound a CBS-approach for future studies to examine and compare cultures beyond the form and frequency of cultural practices or beliefs such as “individualism and collectivism.” Step 1: Identifying Potential Contingencies Step 1: Identifying Potential Contingencies In order to develop a list of potential contingencies that may account for a speciﬁc behavior, researchers can ask questions related to the behavior’s variation, selection, and retention to better elucidate these exact mechanisms. Table 1 outlines a non- exhaustive list of possible questions. The ﬁrst set of questions relates to deﬁning the research question and behavior of interest in concrete behavioral terms. This is in order to avoid the diﬃcult task of attempting to use environmental contingencies to predict an entirely mentalistic concept. Next, it is recommended that the researcher takes a moment to identify alternatives to their behavior of interest. If other behaviors are not occurring in this context or some do at much lower base rates, this information is likely beneﬁcial in forming hypotheses about possible antecedents and consequences. It is possible that the de-emphasis of behavioral antecedents and consequences from the study of cultural psychology was a little premature. Recent developments in both CBS as well as evolutionary branches of cultural psychology may have made a way for more promising collaborations between the two ﬁelds. Whereas the methodological behaviorism of the 1960’s–1980’s oﬀered a less than convincing account of verbal behavior as well as individuals’ internal thoughts or feelings (Chomsky, 1959; Winton, 1986), advances in theories such as Relational Frame Theory (RFT; Hayes et al., 2001) have opened up verbal behavior and “private internal behavioral events” (i.e., thoughts and feelings) as not only legitimate but fruitful topics of inquiry using modern behavioral methodology. Likewise, in cultural psychology, there seems to be a resurgence of the notion that the physical and social environment can profoundly aﬀect the phylogenic development of people groups both on genetic and cultural levels (Cole and Hatano, 2007; Konner, 2007; Newson et al., 2007; Keller, 2008). From there, the remaining questions work to generate a list of potential antecedents and consequences that would contribute to both the selection of a certain behavior (over and above potential alternatives) as well as the retention of that behavior. Among the list of sample questions in Table 1, many fall under the theme of identifying common environmental features occurring in diﬀerent cultural contexts where similar behaviors are observed. There has been some evidence to suggest that similar contextual contingencies result in similar behaviors across large geographic divides (Henry, 2014). Contextual Behavioral Science Contextual Behavior Science claims academic heritage from Charles Darwin, B. F. Skinner, and Murray Sidman, all of whom emphasized the role of environment × organism interactions in the variation, selection, and retention of a given behavioral repertoire (Hayes et al., 2017). Rooted the philosophy of functional contextualism, CBS emphasizes the centrality of situated action and sets a pragmatic truth criterion, attempting to answer the question “what works in this context” (Hayes et al., 2012). It utilizes various behavior analysis methodology to develop a basic behavioral account for complex organism behavior (Blackledge, 2003). The idea of understanding culture in terms of its antecedents and consequences is not new to the ﬁeld of cultural psychology. Although a simpliﬁcation, it could be said that traditional culture- comparative research deﬁned culture as a collection of antecedent conditions that shaped behavior. If some diﬀerence in behavior was observed between two groups there should be an antecedent variable that is able to account for this. The presumption of invariant antecedent-behavior relationships across human populations in all cultures was reﬂected by the notion of universalism, a prevalent belief at the time (Triandis, 2007). However, as the assumption of the universality of humankind gave way to the apt criticism oﬀered by the indigenous and cultural psychology traditions (Stigler et al., 1990; Sinha, 1997), Behavior variation occurs somewhat randomly (Hayes et al., 2017), with the environment simply setting constraints on what behavior is possible in a certain context (e.g., singing is March 2020 \| Volume 11 \| Article 418 Frontiers in Psychology \| www.frontiersin.org 2 Contextual Behavioral Culture Krieg or the individual’s environment. This is usually described as the ABC’s of behavior analysis (Sturmey, 1996; Iwata, 2000), where behaviors are deﬁned by their functions in the following format. this approach fell out of favor and more mentalistic concepts such as individualism and collectivism (Triandis, 1995) and self- construal (Markus and Kitayama, 1991) became increasingly popular. Although these concepts demonstrated some utility in certain explanatory models, there are certain philosophical challenges associated with “invisible” mentalistic concepts. As outlined by Ryle (1984), logical problems occur when “categories of behavior” are conﬂated with the behaviors themselves. In his famous example of ‘team spirit,’ we can observe teammates shouting, patting, and hugging each other after winning a sporting event, but there is no “ghostly team spirit” running around the ﬁeld or possessing each of the players (Baum, 2017). Contextual Behavioral Science Perhaps unsurprisingly, methodological limitations followed this shift toward mentalistic concepts, as cultural scientists struggled to deﬁne, measure, and support cultural validity/equivalence for these new mentalistic constructs (e.g., Levine et al., 2003). The current paper’s author is reminded of an interaction he witnessed at the International Association of Cross-Cultural Psychology’s (IACCP) 2016 Conference in Nagoya, Japan where a prominent cultural psychologist asked a room full of conference attendees “No, really, we talk about self-construal, but do we even know what a ‘self’ is?” The room went silent. [Behavior] functions to [Consequence] when/among/during [Antecedent] [Behavior] functions to [Consequence] when/among/during [Antecedent] Example: [Nodding one’s head] functions to [keep a speaker talking] during [conversation.] In order to identify the components necessary to complete the above conclusion, the current paper recommends the following three-step process: (1) identifying potential contingencies, (2) determining functional relationships, and (3) gathering supporting evidence. These three steps are discussed in turn in the sections below. Step 1: Identifying Potential Contingencies This is likely due to the fact that nowhere in Markus and Hamedani’s (2007) deﬁnition of culture is geographic location an essential feature. Culture is multidimensional and spans geographic region through other common features such as religion, socio- economic status, and generation (Cohen, 2014). For example, the exploration of cultural themes of honor in Middle East and southern United States has demonstrated a high degree of similarities as oppose to diﬀerences (at least with this particular cultural value; Cohen et al., 1996; Mosquera et al., 2007; Uskul et al., 2015). Examining these particular commonalities may help generate possible antecedents and consequences that work to select and retain this particular cultural value as well as the practices surrounding it. Recent research on gene-culture coevolution (e.g., Boyd and Richerson, 1985; Laland et al., 2010) and cultural neuroscience (e.g., Chiao et al., 2008, 2013; Han and Humphreys, 2016) has supported the integration of natural and cultural science and emphasized the dynamic organism × environment interactions on both the individual and culture group levels (Kashima, 2014, 2016). Likewise, the modern CBS approach has increasingly integrated within a larger framework of evolutionary science (Hayes et al., 2012, 2017; Wilson et al., 2018), and wholeheartedly agrees with these propositions. Step 3: Gathering Supporting Evidence Step 3: Gathering Supporting Evidence As in a traditional functional behavioral assessment, testing out predictions made by the model in novel situation as well as examining the change in behavior frequency when manipulating the contingencies are essential to supporting the ABC explanation. If the extant evidence has shown that the behavior of interest occurs within one particular context over another, not only should this be replicable within the same culture group, but individuals in other culture groups should perform similarly if the underlying contingencies are manipulated. Likewise, the removal of the speciﬁc contingencies that form the functional relationships should reduce the degree to which the behavior is performed within a variety of contexts. Although a wide variety of methodology can be implemented, experimental studies provide an excellent avenue for further elucidating the exact mechanism of reinforcement as well as antecedent-sensitivity. of interest. What do they gain from reinforcing these behaviors? What other behaviors might they also be motivated to reinforce? It is also possible that some form of mutual reinforcement is involved. Is the person or group of people reinforcing a certain behavior in turn reinforced for their reinforcing behavior? What can this interaction or meta-reinforcement inform us of potential consequences? What other consequences would happen if people within the group stop reinforcing the behavior of interest? Through these near inﬁnite questions, the researcher can begin to develop a list of identiﬁed antecedents and consequences that potentially relate to the behavior of interest. A CBS Approach to Examining Culture As discussed in previous sections, a functional behavioral approach to culture emphasizes identifying and understanding the contingencies of a given behavior rather than overly focusing on the behavior itself. The goal is to understand the resulting function of a given behavior and how it impacts the individual Likewise, given our understanding of interlocking behavioral contingencies (IBS), it is important to consider who or what subgroup of the population is a primary reinforcer of the behavior March 2020 \| Volume 11 \| Article 418 Frontiers in Psychology \| www.frontiersin.org 3 Contextual Behavioral Culture Krieg TABLE 1 \| Questions to facilitate the identiﬁcation of potential cultural contingencies on behavior. Process Question Behavioral deﬁnition How would one deﬁne the current object of study in behavioral terms? Is it possible to divide this behavior into smaller units? Variation What alternative behaviors are possible? What relevant behaviors are occurring at a lower base rate? What relevant behaviors are not occurring at all? Selection antecedents In what social/physical ecology does this occur? Are there social/physical ecologies in which this does not occur? Does this social/physical ecology occur in other culture groups’ set of social/physical ecologies? If so, do similar behaviors emerge? Are there any features in the social/physical ecology that are sufﬁciently salient to be a potential stimulus control variable? Can the antecedents of the behavior of interest be manipulated to change the frequency of the behavior? Selection consequences What possible ways does the social/physical ecology reinforce or punish this behavior? Does the strength and reinforcement schedule reﬂect the frequency of the observed behavior? Does this type of reinforcement occur in other cultural contexts or geographical settings? If so, do similar behaviors emerge? Does the inﬂuence of the physical or social ecology outweigh a given individual’s unshared learning history? Can the consequences of the behavior of interest be manipulated to change the rate or frequency of the behavior? Retention antecedents What would need to change about the physical/social ecology that would precede changes to the behavioral repertoire? What features of the social/physical environment preceding the behavior would have to change for the frequency of the behavior to also change? Retention consequences What is the adaptive cost of behavior change in the social/physical ecology? What consequences of the social/physical environment following the behavior would have to change for the frequency of the behavior to also change? Frontiers in Psychology \| www.frontiersin.org A CBS Approach to Examining Culture TABLE 1 \| Questions to facilitate the identiﬁcation of potential cultural contingencies on behavior. correlation. Three pieces of information are needed: (1) an overall base rate of a behavior (i.e., cultural practice) occurring in a non-speciﬁed setting under non-speciﬁed reinforcement contingencies, (2) a list of potential antecedents or consequences occurring in proximity to the behavior (as generated in Step 1), and (3) the probabilities of the behavior occurring before or after a consequence or antecedent, respectively. By comparing the relative base rates of a behavior within a contextual contingency to its overall base rate, a functional relationship can be derived (Sturmey, 1996; Iwata, 2000; Baum, 2017). For example, imagine that people in both Culture A and Culture B perform Behavior X 25% of the time across all antecedent conditions. However, people in Culture A perform it 85% of the time in situation Y or 100% of the time after experiencing Consequence Z, whereas members of Culture B continue to perform Behavior X at around 25% of the time even in these conditions. This would be an example of a functional relationship between antecedents/consequences and a behavior that varies between culture group. In the case of identifying a diﬀerential functional relationship like the one described above, culture membership becomes an additional antecedent condition. There are many statistical methods that could be used to calculate this. A researcher could compare eﬀect sizes, odds ratios, complete a non-parametric analysis, or perform a formal Bayesian analysis. Examining Cultural Differences in Social Anxiety Between Japanese and European Americans In order to provide a case example of this approach, the current set of two studies re-analyze and re-interpret data from the current author’s research program examining cultural diﬀerences in social anxiety among people of East Asian Heritage and European Heritage. A brief summary of this ﬁeld will precede engaging in the three steps outlined above. Current Studies In a contextual behavioral account of culture, social anxiety would not be something “felt,” but rather behaviorally signaled to others. This might be in the form of a fearful expression, stuttered speech, averted eye-gaze, behavioral avoidance, or an endorsement of social anxiety on a questionnaire. All of these signals (among many more) are explicit behavioral attempts to convey social anxiety by the participant to others in their environment. Following the three steps outlined above, we aim to identify potential contingencies, determine functional relationships, and gather supporting evidence to better understand how signaling social anxiety functions between groups of Japanese participants and European-American participants. Although several important questions have already been answered by the extant literature, some compelling questions remain. For instance, what situations are considered ‘socially threatening?’ Do people of East Asian-heritage consider the same situations socially threatening as European-heritage people, or are the two groups responding to diﬀerent situations? Is expressing social anxiety more adaptive in one context than another? Do diﬀerences in the level of adaptability explain cultural group diﬀerences in social anxiety? Each of these questions contain elements of a functional relationship between social anxiety and its antecedents (e.g., the situations where it occurs) as well as its consequences (e.g., whether the outcomes are desirable or “adaptive”). Thus, the incorporation of a functional behavioral approach may help in answering these questions. Krieg et al. (2018) originally used the self-construal model to explain diﬀerences in threat appraisal that would then predict social anxiety. They hypothesized that viewing oneself as less independent and more interdependent from social others would increase the degree to which ambiguous social situations would be perceived as threatening, thereby increasing social anxiety. In contrast, Study 1 re-analyzes situation data collected as a part of Krieg et al. (2018) in order to identify contexts in which social anxiety was signaled diﬀerentially by one group over and above the typical culture group diﬀerences in social anxiety. We hypothesize that Japanese and European American participants will both be able to generate a number of diverse situations in which they experience social anxiety. We also hypothesize that the two culture groups will respond diﬀerently to some situations over and above the patterns of responding identiﬁed across the pool of situations. These situations may be able to be organized by the theme of social-support seeking in uncomfortable situations. Step 2: Determining Functional Relationships Determining functional relationships between a behavior and its possible contingencies for a cultural group would likely be a similar process as it is for an individual. Essentially, researchers would be looking for a mathematical relationship that would resemble a Bayesian analysis more than a Pearson Over the past 30 years, the extant research has shown that individuals of East Asian cultural heritage relative to European Americans or European Canadians report higher social anxiety March 2020 \| Volume 11 \| Article 418 Frontiers in Psychology \| www.frontiersin.org 4 Contextual Behavioral Culture Krieg understanding a behavior’s function within a speciﬁc context, it can be a good place to begin one’s investigation. symptoms with remarkable consistency (for meta-analyses see Krieg and Xu, 2015; Woody et al., 2015), with a moderate average eﬀect size of Cohen’s d = 0.36 (95%, CI:0.27, 0.44; Krieg and Xu, 2015). Furthermore, follow-up study by Krieg et al. (2018) showed that this cultural diﬀerence in social anxiety was not an artifact of non-equivalent measurement properties between the two groups by testing for measurement invariance across common social anxiety measures. There is some supporting evidence for the viability of this universal function in helping explain diﬀerences in the expression of social anxiety between groups of Japanese and European Americans. In a recently published preprint that examined Japanese and European American responses to a vignette of someone suﬀering from social anxiety, the Japanese responses were more positive and neutral, whereas the European American responses were more negative and judgmental (Krieg et al., 2019). The authors concluded displaying social anxiety facilitated a more positive and even sympathetic reaction among Japanese participants, who used words such as “sensitive,” “victimized by own thoughts,” and “tendency to care too much” to describe the character in the vignette as compared to European Americans who used words such as “awkward” and “insecure.” Therefore, it is plausible that social anxiety functions to facilitate social support in a social situation. Following the trends in cultural psychology, independent and interdependent self-construal were posited as mediators (Okazaki, 1997; Krieg and Xu, 2015, 2018), where viewing oneself as separate or independent from social others led to reduced social anxiety and viewing oneself as interconnected with social others as either increasing (Okazaki, 1997; Krieg and Xu, 2018) or unrelated to social anxiety (Krieg and Xu, 2015). Step 2: Determining Functional Relationships Employing a cultural neuroscience perspective, self- construal was understood act as a framework for a variety of cognitive functions (Han and Humphreys, 2016), including determining the emotional saliency and relevance to the self, and “threat appraisal” was added to the model (Krieg and Xu, 2018). As originally suggested by Okazaki (1997, 2000), Krieg and Xu (2018) demonstrated that patterns of interdependence (and relatively less independence) transform the perception of a variety of social situations as “high stakes” situations for members of one group more than the other. The results of this process likely contribute to the detection of social threat and subsequent phenomenological experience of social anxiety. An additional study currently in preparation expands the examination of social threat by examining its role as a mediator in a behavior-based, and quasi-experimental study across multiple culture groups. Frontiers in Psychology \| www.frontiersin.org Current Studies y g An important ﬁrst step of a functional behavioral approach is examining the potential consequences of a certain behavior in general before investigating its function in a particular context. Researchers studying the behavioral ecological ramiﬁcations of emotional displays among primates from an evolutionary perspective context that displays of fear or anxiety can function to convey the readiness to submit (Fridlund, 1994), communicate the intention to avoid threat (Keltner, 2003), and request assistance and support from social others (Eisenberg et al., 1989). Taken together, one of the key general functions of displaying social anxiety is to garner social support and sympathy. Although a general function is not always helpful when it comes to Following the results of Study 1, Study 2 simulates experiment data based on descriptive statistics collected from Krieg (2018) and Okazaki et al. (2002) in order to test the tentative conclusions drawn from Study 1. The simulated data will mirror prior ﬁndings as well as conform to typical experimental protocols to make it as realistic as possible. All in all, both of the studies March 2020 \| Volume 11 \| Article 418 5 Contextual Behavioral Culture Krieg Krieg STUDY 1 DISCUSSION When all of the variance from situations were collapsed into a single global category, a small-to-moderate culture group diﬀerence in social anxiety endorsements emerged, with Japanese participants scoring higher than European American participants. This particular eﬀect size corresponds with the ﬁndings from prior meta-analyses (Krieg and Xu, 2015; Woody et al., 2015). As identiﬁed and disseminated in the original Krieg (2018) manuscript, both Japanese and European Americans generated approximately the same number of socially anxiety provoking situations in the ﬁrst part of the situation sampling analysis. This result suggests that the saliency of social anxiety- provoking situations is approximately the same between the two culture groups. That said, the speciﬁc types of situations identiﬁed varied widely, with standardized culture group diﬀerences in social anxiety varying as a function of these situations. From a contextual behavioral perspective, it seems that the antecedents for social anxiety diﬀer between the two groups, with some antecedents holding more inﬂuence on whether social anxiety will be endorsed. In accordance with the situation sampling method, each participant in the current sample answered a unique set of randomly selected situations. We randomly selected 30 situations, 15 generated from each cultural group and asked the current participants to rate the degree to which they would experience social anxiety in this situation (“How anxious would you feel if this situation happened to you?”; 1 = None, 5 = Extremely Anxious). Please see Krieg (2018) for a detailed description of situation preparation, cleaning, and redistribution procedures. Most situations had eﬀect sizes that were unable to be diﬀerentiated from the overall eﬀect, with only three situations being identiﬁed as “above and beyond” the base rate. These three situations had eﬀect sizes showing Japanese participants endorsing more social anxiety than European Americans (n = 2) and vice versa (n = 1). When examining these situations for themes, it became apparent that in the two situations where Japanese participants expressed more social anxiety than European Americans contained elements of “having made a mistake in an oﬃcial situation” (e.g., admitting a fault and being in a workplace setting). In contrast, the situation where European Americans endorsed more social anxiety when being verbally attacked by another person. By examining the patterns of eﬀect sizes in these “over and above situations,” we gained some preliminary evidence supporting the help-seeking function Step 2: Determining Functional Relationships are designed to examine the predictive utility of the following functional statement: Signaling social anxiety functions to garner social support or assistance when in a particular situation among Japanese culture group members relative to European-American culture group members. In order to establish a base rate of culture group diﬀerences in endorsed social anxiety, we calculated a Cohen’s d eﬀect size for the anxiety scores overall, irrespective of each situation. The base rate for the culture group diﬀerences in social anxiety was Cohen’s d = 0.194 (95% CI: 0.152, 0.236). From this point, we calculated the eﬀect sizes for culture group diﬀerences in social anxiety for each situation. The conﬁdence intervals of these eﬀect sizes were compared in order to identify if any situation antecedents produced a culture group diﬀerence in social anxiety over and above the base rate. Among all 610 situations, only three situations were associated with eﬀect sizes that were either statistically signiﬁcantly above or below the base rate and not overlapping with 0 (no eﬀect). These six situations were being with a boss/colleague (d = 3.24; 95% CI = 0.77, 5.70), admitting a fault (d = 1.65; 95% CI = 0.30, 3.00), and being called names (d = −1.59; 95% CI = −2.99, −0.19). All of these situations were made by European-American participants (see Figure 1). Procedure In order to produce a repository of participant-generated social- anxiety provoking situations (e.g., antecedent conditions), we incorporated a situation sampling approach (Kitayama et al., 1997; Morling et al., 2002). First, a pilot study was ﬁrst conducted to identify social situations that were perceived by individuals of Japanese and European-American heritage as anxiety provoking. The pilot study recruited 30 Japanese nationals and 30 European Americans. To generate a pool of situations that are relatively salient to members of the three cultural groups in provoking their social anxiety, participants responded to an open-ended question online, “For the following categories, please create brief, speciﬁc situations where someone would feel socially anxious,” and generated a total of 610 unique social situations; 313 by Japanese participants and 297 by European American participants. Participants The sample from Krieg (2018) Study 1 was used to examine social anxiety across antecedent conditions. This sample consisted of 212 Japanese (116 females; Mage = 20.88; SD = 2.23) and 249 European Americans (180 females; Mage = 21.14; SD = 5.01). All participants signed an informed consent, and this study was approved by the University of Hawai‘i at M¯anoa’s Human Studies Program (CHS #22337). For more information, please see the original publication. Procedure Participants were tasked with giving a 5-min speech in which they admit to a recent mistake they made at their workplace. After receiving 5 min to prepare, half of the participants were randomly assigned to give their speech in front of a video camera (camera condition) while viewing a video of stock footage of a small classroom listening to a speech projected on a nearby wall. No one else was in the room while the speech was being video recorded. The other half gave their speech in the presence of seven confederates (research assistants matched to the participant’s cultural background). In the audience condition, the speech was also video recorded. Upon completing the speech, participants were escorted to a separate room where they completed questionnaires that measured social performance anxiety and perceived social support. Given that the study’s hypotheses and experimental conditions were concealed at the beginning of the study in order to reduce the impact of response bias. Participants were debriefed of the study’s hypothesis and conditions following the completion of the speech task by the principal investigator. European Americans. HYPOTHETICAL STUDY 2 In order to gather supporting evidence (Step 3) of our proposed functional statement, we conducted a quasi-experiment that directly tests the hypothesis through manipulating antecedent conditions. If the main diﬀerence between the two groups in terms of the functional consequences of social anxiety is its amenability to garner social support or assistance when having made a mistake in an oﬃcial situation, then the degree of social anxiety expressed should vary in conditions were social support is potentially available as compared to social situations where it is less available. Study 2 is comprised of a hypothetical experiment of a speech task designed to elucidate the necessary antecedent conditions for the desired consequences of social anxiety among Japanese as compared to European Americans. Step 1: Identifying Potential Contingencies In order to generate a list of antecedent conditions that would provoke social anxiety among members of each culture group, we used the ﬁrst part of the situation sampling procedure [described as ‘pilot study’ in Krieg’s (2018) Study 1 “Methods” section]. The results of the pilot study were 610 social situations (297 from Europeans American and 313 from Japanese participants). March 2020 \| Volume 11 \| Article 418 Frontiers in Psychology \| www.frontiersin.org 6 Krieg Contextual Behavioral Culture 3.24 1.65 1.59 Being with a boss/colleague Admitting a fault Being called names 4 3 2 1 0 1 2 3 4 Cohen's d FIGURE 1 \| Cohen’s d effect sizes of culture group differences in social anxiety under three antecedent conditions relative to overall base rate effect size. FIGURE 1 \| Cohen’s d effect sizes of culture group differences in social anxiety under three antecedent conditions relative to overall base rate effect size. t sizes of culture group differences in social anxiety under three antecedent conditions relative to overall base rate effect size. for these participants was 22.95 (SD = 2.10) and 21.82 (SD = 1.73) for each culture group, respectively. of social anxiety. Thus, we can generate a functional statement as follows: Displaying social anxiety [behavior] functions to garner social support or assistance [consequence] when having made a mistake in an oﬃcial situation [antecedent] among Japanese relative to European Americans. Displaying social anxiety [behavior] functions to garner social support or assistance [consequence] when having made a mistake in an oﬃcial situation [antecedent] among Japanese relative to European Americans. Mediation Modeling Given that the pattern of mean diﬀerences suggested that the experimental condition may predict social anxiety displays and perceived social support among Japanese but not necessarily European Americans, we used structural equation modeling (SEM) to examine the potential mediating eﬀect of social anxiety displays in the relationship between experimental condition and perceived social support. Participants The Social Phobia Scale Six-Item Version (SPS-6; Peters et al., 2011) is a short version of Mattick and Clarke’s (1998) Social Phobia Scale, and measures social performance anxiety on a The simulated sample included 200 participants, 100 Japanese (50 female) and 100 European Americans (50 female). The mean age March 2020 \| Volume 11 \| Article 418 Frontiers in Psychology \| www.frontiersin.org 7 Contextual Behavioral Culture Krieg TABLE 2 \| Means and standard deviations of simulated SPS-6, social support, and BASA data for each culture group and condition. Japanese (n = 100) European Americans (n = 100) Variable Condition Mean SD Mean SD SPS-6 Camera 3.33 0.76 2.98 0.89 Audience 3.48 0.66 2.69 0.85 Overall 3.41 0.71 2.84 0.88 Social support Camera 1.90 0.57 1.97 0.51 Audience 2.33 0.39 0.205 0.33 Overall 2.12 0.54 2.01 0.42 BASA Camera 32.28 6.74 34.11 6.05 Audience 37.53 5.53 34.86 6.41 Overall 34.90 6.68 34.49 6.21 SPS-6, Social Phobia Scale – Six item version; BASA, Behavioral Assessment of Speech Anxiety. TABLE 2 \| Means and standard deviations of simulated SPS-6, social support, and BASA data for each culture group and condition. 5-point Likert scale (0 = not at all characteristic or true of me; 4 = extremely characteristic or true of me). The six-item version was created by selecting items with the best psychometric properties using Item Response Theory. As a result, the SPS- 6 has excellent reliability, validity, and sensitivity to change properties (Peters et al., 2011). Krieg et al. (2019) found that the SPS-6 was scalar invariant between Japanese and European American samples, allowing means to be compared. The inter- item reliability within the current sample is 0.88 for Japanese and 0.93 for European Americans. 5-point Likert scale (0 = not at all characteristic or true of me; 4 = extremely characteristic or true of me). The six-item version was created by selecting items with the best psychometric properties using Item Response Theory. As a result, the SPS- 6 has excellent reliability, validity, and sensitivity to change properties (Peters et al., 2011). Krieg et al. (2019) found that the SPS-6 was scalar invariant between Japanese and European American samples, allowing means to be compared. The inter- item reliability within the current sample is 0.88 for Japanese and 0.93 for European Americans. Perceived Social Support In addition to measuring self-reported social anxiety, we administered a single-item question to participants asking, “how often did you feel that your audience was supporting you as you made your speech.” Responses were on a 5-point Likert scale with 0 representing “not at all” and 4 representing “all the time.” Social Anxiety Displays experiencing more social anxiety. There was no eﬀect found for experimental condition (B = −0.29, p = 0.72), and the results of the interaction eﬀect were also statistical signiﬁcance (B = 0.45, p = 0.049). Three bilingual research assistants (two Japanese and one European American) who were blind to experimental conditions assigned to participants, rated each participant’s behavioral displays of anxiety from the video recordings. Speciﬁcally, the research assistants used the Behavioral Assessment of Speech Anxiety (BASA; Mulac and Sherman, 1974), a standardized behavioral assessment scale, to rate speciﬁc behaviors associated with social anxiety. The BASA examined eighteen speciﬁc behaviors, e.g., ﬁdgeting, swallowing, breathing heavy, and each were coded using a 7-point Likert scale (1 = not at all, 7 = strong). Each rater coded all of the videos and inter-rater reliability was calculated as an intra-class correlation of 0.91 (CI: 0.88, 0.94). Final scores consisted of the rounded average. All eighteen ratings were summed together to generate a ﬁnal behavioral score (alpha = 0.83). The BASA has demonstrated evidence of internal consistency, inter-rater reliability, and concurrent validity with expert ratings of speech performance in prior Western studies (Mulac and Sherman, 1974; Heeren et al., 2012). Simulated means and standard deviations for each group can be found in Table 2. For perceived social support, our results demonstrated no main eﬀect for culture group (B = −0.07, p = 0.442), or experimental condition (B = 0.08, p = 383). However, the culture group x experimental condition interaction eﬀect was also statistically signiﬁcant (B = 0.35, p < 0.01), with Japanese participants scoring higher overall, but especially in the live audience condition. Finally, for social anxiety displays, as measured by the BASA, our analysis revealed no main eﬀect for either group (B = −1.83, p = 0.140) or experimental condition (B = 0.74, p = 0.550). However, the results did demonstrate a statistically signiﬁcant interaction eﬀect for culture group × experimental condition (B = 4.50, p = 0.011), with Japanese participants scoring higher in the live audience condition (see Figure 2). Frontiers in Psychology \| www.frontiersin.org Step 3: Gathering Supporting Evidence Mean Differences In order to gather additional support hypothesis that increased displays (endorsements) of social anxiety functioned primarily in situations where having made a mistake in an important situation, we ﬁrst explored mean diﬀerences between culture groups and experimental conditions. To this end, we implemented a multivariate general linear model that regressed social performance anxiety, perceived social support, and social anxiety displays on culture group, experimental condition, and the interaction between the two. Speciﬁcally, we constructed two path models, one for Japanese participants and one for European American participants. We speciﬁed direct paths from experimental condition (1 = live audience) and perceived social support, as well as between perceived social support and anxiety displays. Social performance anxiety was included as social anxiety display’s covariate. Each model was estimated using the maximum likelihood estimator implemented with the ‘lavaan’ package (Rosseel, 2012) with R (R Core Team, 2014). For social performance anxiety, as measured by the SPS-6, our analysis revealed a statistically signiﬁcant main eﬀect for culture group (B = 0.34, p = 0.033), with Japanese participants reporting The results of our analyses revealed that among Japanese participants, social anxiety displays fully mediated the Frontiers in Psychology \| www.frontiersin.org March 2020 \| Volume 11 \| Article 418 8 Krieg Contextual Behavioral Culture FIGURE 2 \| Results of hypothetical study depicting the culture group × experimental condition interaction’s inﬂuence on social anxiety scores. BASA, Behavioral Assessment of Speech Anxiety. tudy depicting the culture group × experimental condition interaction’s inﬂuence on social anxiety scores. BASA, Behavioral FIGURE 2 \| Results of hypothetical study depicting the culture group × experimental condition interaction’s inﬂuence on social anxiety scores. BASA, Behavioral Assessment of Speech Anxiety. relationship between experiment condition and perceived social support (CFI: 0.999, TLI: 0.998, RMSEA: 0.036, and SRMR: 0.023). However, for European Americans, neither path coeﬃcient was statistically signiﬁcant, and model ﬁt was low (CFI: 0.818, TLI: 0.453, RMSEA: 0.421, and SRMR: 0.192). See Figure 3 for a depiction of the model as well as speciﬁc details on path coeﬃcients. relationship between experiment condition and perceived social support (CFI: 0.999, TLI: 0.998, RMSEA: 0.036, and SRMR: 0.023). However, for European Americans, neither path coeﬃcient was statistically signiﬁcant, and model ﬁt was low (CFI: 0.818, TLI: 0.453, RMSEA: 0.421, and SRMR: 0.192). See Figure 3 for a depiction of the model as well as speciﬁc details on path coeﬃcients. Step 3: Gathering Supporting Evidence Mean Differences functions to garner social support or assistance when having made a mistake in an oﬃcial situation among Japanese relative to European Americans.” However, it is also important to note that behavior can have multiple functions within the same context. Because anxiety displays can function to foster social support in one group does not necessarily mean that it cannot also be a reaction to the threat of being scrutinized or criticized by peers as concluded in Okazaki (1997) and Krieg et al. (2018). That being said, this functional statement is fundamentally diﬀerent than statements previously made in the literature that associated social anxiety behavior with mentalistic constructs like independent and interdependent self-construal in that each aspect can be directly observed and requires no further explanation (e.g., “where does self-construal come from,” “where is it located,” “is it malleable across the lifespan,” and “what predicts self-construal,” etc.). It also positions culture in a larger context that is not limited to geography or nationality. For instance, if social anxiety functions to facilitate social support among Japanese people, are there other groups that seek social support in a similar way? What about a diﬀerent way? Would Frontiers in Psychology \| www.frontiersin.org March 2020 \| Volume 11 \| Article 418 GENERAL DISCUSSION In the current studies, we attempted to provide a simple example of implementing functional behavioral methodology in an established program of culture research. First, we generated a list of antecedent conditions and proposed functional consequences from the extant literature (Step 1). Then we determined functional relationships by examining diﬀerences in the eﬀect sizes of a behavior among diﬀerent antecedents relative to its overall base rate (Step 2). We then used a functional statement to generate testable hypotheses and explicitly sought evidence to support the statement (Step 3). Adapting culture-group ﬁndings to individual clients is not terribly diﬃcult. With this type of analysis already in place, updating the model to include information related to a client’s idiographic learning history is entirely plausible. A clinician can examine the degree to which known contingencies associated with a given behavior apply to their client and can structure questions and in-session activities to gather further evidence. Sharing functional analyses with clients can also be helpful in facilitating a new understanding on how concerning behavior is reinforced and may have been initially or occasionally adaptive. It is important to note that there are already many cultural psychologists doing work that would be proposed by this model, though maybe under a diﬀerent name. For instance, the socioecological framework also emphasizes antecedent conditions within a social ecology to predict diﬀerences in cultural practices (Talhelm and Oishi, 2019). For instance, Gelfand et al. (2011) examined the degree to which tight situational constraints in modern cultures could be explained by historical population density. Arguing that historical population density created a survival pressure in these cultures, their study showed a strong correlation between ecological antecedents and cultural behaviors. Their conclusion, however, is more of an explanatory statement (answering “why”) than a functional statement (answering “what for”). No approach is without its limitations, however, and one important limitation is that behavior analysis itself is a culture (Ruiz and Roche, 2007), and using behavioral language evokes a set of Western values associated with action, causation, health, and wellbeing. By emphasizing these constructs, there is the possibility of missing important information that doesn’t necessarily conform to a behavioral framework. Likewise, although in clinical practice, behavior analysts are focused on the ideographic saliency of a reinforcer for each client, that nuance seems to be lost when using the same model to compare behaviors among diﬀerent culture groups. STUDY 2 DISCUSSION The results of Study 2 demonstrate the impact of the antecedent situation on the cultural group diﬀerences in social anxiety. By changing one aspect of the antecedent (i.e., camera vs. live audience) the behavioral expression changed based on its intended function. Assuming that the hypothetical experiment replicates with real experimental data, Study 2 would also provide the additional evidence needed to support the functional statement derived from Study 1. We would have provided support for the functional statement, “displaying social anxiety March 2020 \| Volume 11 \| Article 418 Frontiers in Psychology \| www.frontiersin.org 9 Krieg Contextual Behavioral Culture FIGURE 3 \| Hypothetical path model and standardized coefﬁcients for each cultural group. ∗p < 0.05 and ∗∗p < 0.01. the diﬀerent group’s social support seeking strategies also work in a Japanese context? If not, what alternative consequences are generated instead? Have social support seeking strategies changed over time? Does this align with changing contingencies in the environment or simply a product of behavioral variation (e.g., cultural drift)? Many of these questions can be asked and answered within single sample studies. Furthermore, examining social anxiety through a CBS lens centers the examination on adaptability rather than pathology, reducing the stigma of certain behaviors in certain groups by explicitly stating how the behavior is reinforced by desirable consequences (or avoiding undesirable consequences.). This hypothetical study would posit a new conclusion is that social anxiety is more adaptive in a Japanese context than an European American context in terms of garnering social support, which is a very diﬀerent conclusion than suggesting that people of Japanese heritage being intrinsically more anxious than European Americans. The concept of diﬀerential adaptability is necessarily focused on the environment, rather than the person or group of people responding to it. FIGURE 3 \| Hypothetical path model and standardized coefﬁcients for each cultural group. ∗p < 0.05 and ∗∗p < 0.01. behavior or a behavioral syndrome between groups in a non- stigmatizing manner. Likewise, by having behavioral deﬁnitions for each construct of interest, we reduce the challenges associated with relying on mentalistic concepts (Ryle, 1984; Baum, 2017). Given that the main objective of CBS is to predict behavior and successfully intervene based on its function, once we have established and supported a functional statement, further hypotheses abound. STUDY 2 DISCUSSION We could expand the statement to include multiple consequences or a series of antecedents in order to better predict behavior in context as well as design eﬀective interventions. Frontiers in Psychology \| www.frontiersin.org REFERENCES Gelfand, M. J., Raver, J. L., Nishii, L. H., Leslie, L. M., Lun, J., Lim, B. C., et al. (2011). Diﬀerences between tight and loose cultures: a 33-nation study. Science 332, 1100–1104. doi: 10.1126/science.1197754 A-tjak, J. G. L., Davis, M. L., Morina, N., Powers, M. B., Smits, J. A. J., and Emmelkamp, P. M. G. (2015). A meta-analysis of the eﬃcacy of acceptance and commitment therapy for clinically relevant mental and physical health problems. Psychother. Psychosom. 84, 30–36. doi: 10.1159/00036 5764 Glenn, S. S., Malott, M. E., Amalia, M., Abib, P., Malott, M. E., Amalia, M., et al. (2016). Toward consistent terminology in a behaviorist approach to cultural analysis. Behav. Soc. Issues 27, 11–27. doi: 10.5210/bsi.v.25i0.6634 Han, S., and Humphreys, G. (2016). Self-construal: a cultural framework for brain function. Curr. Opin. Psychol. 8, 10–14. doi: 10.1016/j.copsyc.2015.09.013 Austin, J., and Carr, J. E. (2000). Handbook of Applied Behavior Analysis. Oakland, CA: Context Press. Hayes, S. C., and Bissett, R. T. (2000). “Behavioral psychotherapy and the rise of clinical behavior analysis,” in Handbook of Applied Behavior Analysis, eds J. Austin, and J. Carr (Reno, NV: Context Press). Baum, W. M. (2017). Understanding Behaviorism: Science, Behavior, and Culture. Chichester: John Wiley & Sons. Hayes, S. C., Barnes-Holmes, D., and Roche, B. (2001). Relational Frame Theory: A Post-Skinnerian Account of Human Language and Cognition. New York, NY: Springer Science & Business Media. Blackledge, J. T. (2003). An introduction to relational frame theory: basics and applications. Behav. Anal. Today 3, 421–433. doi: 10.1037/h009 9997 Boyd, R., and Richerson, P. J. (1985). Culture and the Evolutionary Process. Chicago, IL: University of Chicago Press. Hayes, S. C., Barnes-Holmes, D., and Wilson, K. G. (2012). Contextual behavioral science: creating a science more adequate to the challenge of the human condition. J. Contextual Behav. Sci. 1, 1–16. doi: 10.1016/j.jcbs.2012.09.004 Chiao, J. Y., Cheon, B. K., Pornpattananangkul, N., Mrazek, A. J., and Blizinsky, Chiao, J. Y., Cheon, B. K., Pornpattananangkul, N., Mrazek, A. J., and Blizinsky, K. D. (2013). Cultural neuroscience: progress and promise. Psychol. Inq. 24, 1–19. doi: 10.1080/1047840x.2013.752715 Hayes, S. C., Luoma, J. B., Bond, F. W., Masuda, A., and Lillis, J. (2006). Acceptance and commitment therapy: model, processes and outcomes. Behav. Res. Ther. 44, 1–25. doi: 10.1016/j.brat.2005.06.006 K. D. (2013). Cultural neuroscience: progress and promise. Psychol. Inq. 24, 1–19. doi: 10.1080/1047840x.2013.752715 Chiao, J. Y., Iidaka, T., Gordon, H. L., Nogawa, J., Bar, M., Aminoﬀ, E., et al. REFERENCES (2008). Cultural speciﬁcity in amygdala response to fear faces. J. Cogn. Neurosci. 20, 2167–2174. doi: 10.1162/jocn.2008.20151 Hayes, S. C., Muto, T., and Masuda, A. (2011). Seeking cultural competence from the ground up. Clin. Psychol. Sci. Pract. 18, 232–237. doi: 10.1111/j.1468-2850. 2011.01254.x j Chomsky, N. (1959). A review of B.F. Skinner’s verbal behavior. Language 35, 26–58. Hayes, S. C., Sanford, B. T., and Chin, F. T. (2017). Carrying the baton: evolution science and a contextual behavioral analysis of language and cognition. J. Contextual Behav. Sci. 6, 314–328. doi: 10.1016/j.jcbs.2017.01.002 Cohen, A. B. (2014). Culture Reexamined: Broadening our Understanding of Social and Evolutionary Inﬂuences. Washington, DC: American Psychological Association. Heeren, A., Reese, H. E., McNally, R. J., and Philippot, P. (2012). Attention training toward and away from threat in social phobia: eﬀects on subjective, behavioral, and physiological measures of anxiety. Behav. Res. Ther. 50, 30–39. doi: 10.1016/j.brat.2011.10.005 Cohen, D., Nisbett, R. E., Schwarz, N., and Bowdle, B. F. (1996). Insult, aggression, and the southern culture of honor: an “Experimental Ethnography”. J. Pers. Soc. Psychol. 70, 945–959. doi: 10.1037/0022-3514.70.5.945 Cole, M., and Hatano, G. (2007). “Cultural-historical activity theory: integrating phylogeny, cultural history, and ontogenesis in cultural psychology,” in Handbook of Cultural Psychology, ed. S. K. D. Cohen (New York, NY: Guilford Press), 109–135. Henry, P. J. (2014). “Culture and social class,” in Culture Reexamined: Broadening Our Understanding of Social and Evolutionary Inﬂuences, ed. A. B. Cohen (Washington, DC: American Psychological Association), 81–97. Iwata, B. A. (2000). “The functional analysis model of behavioral assessment,” in Handbook of Applied Behavior Analysis, eds S. W. Kahng, J. Austin, and J. Carr (Oakland, CA: New Harbinger Publications). de Melo, C., and de Rose, J. C. (2013). The concept of culture in skinnerian radical behaviorism: debates and controversies. Eur. J. Behav. Anal. 14, 321–328. doi: 10.1080/15021149.2013.11434464 Kashima, Y. (2014). How can you capture cultural dynamics? Front. Psychol. 5:995. doi: 10.3389/fpsyg.2014.00995 Eisenberg, N., Reno, R. R., Fabes, R. A., Miller, P. A., Mathy, R. M., Fultz, J., et al. (1989). Relation of sympathy and personal distress to prosocial behavior: a multimethod study. J. Pers. Soc. Psychol. 57, 55–66. doi: 10.1037/0022-3514.57. 1.55 Kashima, Y. (2016). Culture and psychology in the 21st century: conceptions of culture and person for psychology revisited. J. Cross Cult. Psychol. 47, 4–20. doi: 10.1177/0022022115599445 Epstein, R. (1984). Spontaneous and deferred imitation in the pigeon. Behav. Process. 9, 347–354. doi: 10.1016/0376-6357(84)90021-4 Keller, H. (2008). DATA AVAILABILITY STATEMENT The author conﬁrms being the sole contributor of this work and has approved it for publication. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualiﬁed researcher. ETHICS STATEMENT This paper was funded in part by the Fulbright-Hays Dissertation Development Research Award (United States Department of Education) as well as the Crown Prince Akihito Scholarship Foundation (Japan–America Society of Hawaii). The studies involving human participants were reviewed and approved by University of Hawaii Human Studies Program. The GENERAL DISCUSSION To this point, there is evidence to suggest that, in general, European Americans ﬁnd contingencies related to enhancing one’s inﬂuence on others more salient, whereas Japanese participants were more focused on contingencies that highlighted adjustment to others (Morling et al., 2002). Not every antecedent or consequence can be readily Some advantages of a CBS perspective include the emphasis on adapting to one’s context (de-pathologization), focus on directly measurable behaviors (observable behavioral outcomes), and the amenability to intervention development. By describing how certain behaviors “work” in a certain context, but not necessarily others, the emphasis shifts from pathology to adaptability. The idea of diﬀerential adaptability is especially important in potentially explaining functional diﬀerences in March 2020 \| Volume 11 \| Article 418 Frontiers in Psychology \| www.frontiersin.org 10 Krieg Krieg Contextual Behavioral Culture compared. Keeping these potential limitations in mind, cultural scientists or culture-clinical researchers can evoke the strengths of this approach to better understand culture’s inﬂuence on behavior in context. patients/participants provided their written informed consent to participate in this study. REFERENCES Culture and biology: the foundation of pathways of development. Soc. Pers. Psychol. Compass 2, 668–681. doi: 10.1111/j.1751-9004.2008.00092.x Ferster, C. B., and Skinner, B. F. (1957). Schedules of Reinforcement. East Norwalk, CT: Appleton-Century-Crofts. doi: 10.1037/10627-000 Keltner, D. (2003). Expression and the course of life. Ann. N. Y. Acad. Sci. 1000, 222–243. Fridlund, A. J. (1994). Human Facial Expression: An Evolutionary View. Santa Barbara, CA: Academic Press. Kitayama, S., and Cohen, D. (eds). (2007). Handbook of Cultural Psychology. New York, NY: Guilford Press. Frontiers in Psychology \| www.frontiersin.org March 2020 \| Volume 11 \| Article 418 11 Krieg Contextual Behavioral Culture Kitayama, S., Markus, H. R., Matsumoto, H., and Norasakkunkit, V. (1997). Individual and collective processes in the construction of the self: self- enhancement in the United States and self-criticism in Japan. J. Pers. Soc. Psychol. 72, 1245–1267. doi: 10.1037/0022-3514.72.6.1245 scale (SPS) using nonparametric item response theory: the SIAS-6 and the SPS-6. Psychol. Assess. 24, 66–76. doi: 10.1037/a0024544 Powers, M. B., Sive Vording, M. B., and Emmelkamp, P. M. G. (2009). Acceptance and commitment therapy: a meta-analytic review. Psychother. Psychosom. 78, 73–80. doi: 10.1159/000190790 Konner, M. (2007). “Evolutionary foundations of cultural psychology,” in Handbook of Cultural Psychology, ed. S. K. D. Cohen (New York, NY: Guilford Press), 77–105. R Core Team, (2014). R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing. g Rosseel, Y. (2012). lavaan: an R package for structural equation modeling. J. Stat. Softw. 48, 1–36. doi: 10.3389/fpsyg.2014.01521 Krieg, A. (2018). Understanding East-West Diﬀerences in Social Anxiety: The Roles of Culturally-Tuned Attentional Processes. Honolulu: University of Hawaii at Manoa. Ruiz, M. R., and Roche, B. (2007). Values and the scientiﬁc culture of behavior analysis. Behav. Anal. 30, 1–16. doi: 10.1007/BF03392139 analysis. Behav. Anal. 30, 1–16. doi: 10.1007/BF03392139 Krieg, A., and Xu, Y. (2015). Ethnic diﬀerences in social anxiety between individuals of Asian heritage and European heritage: a meta-analytic review. Asian Am. J. Psychol. 6, 66–80. doi: 10.1037/a0036993 Ryder, A. G., and Chentsova-Dutton, Y. E. (2015). “Cultural-clinical psychology from cultural scripts to contextualized treatments,” in Re-visioning Psychiatry: Cultural Phenomenology, Critical Neuroscience, and Global Mental Health, eds L. J. Kirmayer, R. Lemelson, and C. A. Cummings (Cambridge: Cambridge University Press), 400–433. doi: 10.1017/CBO9781139424745.022 Asian Am. J. Psychol. 6, 66–80. doi: 10.1037/a0036993 Krieg, A., and Xu, Y. (2018). From self-construal to threat appraisal: understanding cultural diﬀerences in social anxiety between Asian Americans and European Americans. Cultur. Divers. REFERENCES New York, NY: John Wiley & Sons. Markus, H. R., and Kitayama, S. (1991). Culture and the self: implications for cognition, emotion, and motivation. Psychol. Rev. 98, 224–253. doi: 10.1037/ 0033-295x.98.2.224 Talhelm, T., and Oishi, S. (2019). “Culture and ecology,” in Handbook of Cultural Psychology, 2nd Edn, eds D. Cohen, and S. Kitayama (New York, NY: Guilford Press), 119–143. Tourinho, E. Z., and Vichi, C. (2012). Behavioral-analytic research of cultural selection and the complexity of cultural Phenomena. Rev. Latinoam. Psicol. 44, 169–179. Mattick, R. P., and Clarke, J. C. (1998). Development and validation of measures of social phobia scrutiny fear and social interaction anxiety. Behav. Res. Ther. 36, 455–470. doi: 10.1016/s0005-7967(97)10031-6 Morling, B., Kitayama, S., and Miyamoto, Y. (2002). Cultural practices emphasize inﬂuence in the United States and adjustment in Japan. Pers. Soc. Psychol. Bull. 28, 311–323. doi: 10.1177/0146167202286003 Triandis, H. C. (1995). Individualism & Collectivism. Boulder, CO: Westview Press. Triandis, H. C. (2007). “Culture and psychology: a history of the study of their relationship,” in Handbook of Cultural Psychology, ed. S. K. D. Cohen (New York, NY: Guilford Press), 59–76. Mosquera, P. M. R., Manstead, A. S. R., and Fischer, A. H. (2007). Honor in the mediterranean and northern Europe. J. Cross Cult. Psychol. 33, 16–36. doi: 10.1177/0022022102033001002 Uskul, A. K., Cross, S. E., Günsoy, C., Gerçek-Swing, B., Alözkan, C., and Ataca, B. (2015). A price to pay: Turkish and northern American retaliation for threats to personal and family honor. Aggress. Behav. 41, 594–607. doi: 10.1002/ab.21598 Mulac, A., and Sherman, A. R. (1974). Behavioral assessment of speech anxiety. Q. J. Speech 60, 134–143. doi: 10.1080/00335637409383219 Wilson, D. S., Hayes, S. C., and Biglan, A. (2018). Evolution and Contextual Behavioral Science: an Integrated Framework for Understanding, Predicting, Inﬂuencing Human Behavior. Oakland, CA: New Harbinger Publications. Newson, L., Richerson, P. J., and Boyd, R. (2007). “Cultural evolution and the shaping of cultural diversity,” in Handbook of Cultural Psychology, ed. S. K. D. Cohen (New York, NY: Guilford Press), 454–476. Winton, W. M. (1986). The role of facial response in self-reports of emotion: a critique of Laird. J. Pers. Soc. Psychol. 50, 808–812. doi: 10.1037/0022-3514.50. 4.808 critique of Laird. J. Pers. Soc. Psychol. 50, 808–812. doi: 10.1037/0022-3514.50. 4.808 Okazaki, S. (1997). Sources of ethnic diﬀerences between Asian American and white American college students on measures of depression and social anxiety. J. Abnorm. Psychol. 106, 52–60. doi: 10.1037/0021-843x.106.1.52 Woody, S. REFERENCES Ethnic Minor. Psychol. 24, 477–488. doi: 10.1037/ cdp0000194 L. J. Kirmayer, R. Lemelson, and C. A. Cummings (Cambridge: Cambridge University Press), 400–433. doi: 10.1017/CBO9781139424745.022 y Ryle, G. (1984). Concept of the Mind. Chicago, IL: University of Chicago Press. Krieg, A., Xu, Y., and Cicero, D. C. (2018). Comparing social anxiety between Asian Americans and European Americans: an examination of measurement invariance. Assessment 25, 564–577. doi: 10.1177/1073191116656438 Sabucedo, P. (2017). The psychological ﬂexibility model from a cultural perspective: an interpretative analysis of two native American healing rituals. Int. J. Cult. Ment. Health 10, 367–375. doi: 10.1080/17542863.2017.1323935 Sinha, D. (1997). “Indigenizing psychology,” in Handbook of Cultural Psychology, Sinha, D. (1997). “Indigenizing psychology,” in Handbook of Cultural Psychology, 2nd Edn, eds J. W. Berry, Y. P. Poortinga, and J. Pandey (Needham Heights, MA: Allyn & Bacon), 129–169. Krieg, A., Xu, Y., Cicero, D. C., Miyamoto, S., and Krieg, H. (2019). Comparing Social Anxiety in Japanese and European Americans: Integrating Measurement Invariance and Prototype Analyses. Available online at: https://psyarxiv.com/ fvubx/ (accessed July 1, 2019). 2nd Edn, eds J. W. Berry, Y. P. Poortinga, and J. Pandey (Needham Heights, MA: Allyn & Bacon), 129–169. Skinner, B. F. (1938). The Behavior of Organisms: An Experimental Analysis. New York, NY: Appleton-Century-Crofts. Skinner, B. F. (1938). The Behavior of Organisms: An Experimental Analysis. New York, NY: Appleton-Century-Crofts. Skinner, B. F. (1953). Science and Human Behavior. New York, NY: Simon & Schuster. Laland, K. N., Odling-Smee, J., and Myles, S. (2010). How culture shaped the human genome: bringing genetics and the human sciences together. Nat. Rev. Genet. 11, 137–148. doi: 10.1038/nrg2734 Skinner, B. F. (1953). Science and Human Behavior. New York, NY: Simon & Schuster. Levine, T. R., Bresnahan, M., Park, H. S., Lapinski, M. K., Wittenbaum, G. M., Shearman, S. M., et al. (2003). Self−construal scales lack validity. Hum. Commun. Res. 29, 210–252. doi: 10.1016/j.appet.2015.05.037 kinner, B. F. (1963). Operant behavior. Am. Psychol. 18, 503–515. Skinner, B. F. (1981). Selection by consequences. Science 213, 501–504. Stigler, J. W., Shweder, R. A., and Herdt, G. E.eds (1990). “Cultural psychology: essays on comparative human development,” in Culture and Human Development (Cambridge: Cambridge University Press). Markus, H. R., and Hamedani, M. G. (2007). “Sociocultural psychology: the dynamic interdependence among self systems and social systems,” in Handbook of Cultural Psychology, eds S. Kitayama, and D. Cohen (London: Guilford Press), 3–40. Sturmey, P. (1996). Functional Analysis in Clinical Psychology. REFERENCES R., Miao, S., and Kellman-McFarlane, K. (2015). Cultural diﬀerences in social anxiety: a meta-analysis of Asian and European heritage samples. Asian Am. J. Psychol. 6, 47–55. doi: 10.1037/a0036548 Okazaki, S. (2000). Asian American and white American diﬀerences on aﬀective distress symptoms: do symptom reports diﬀer across reporting methods? J. Cross Cult. Psychol. 31, 603–625. doi: 10.1177/0022022100031005004 Zettle, R., Hayes, S. C., Barnes-Holmes, D., and Biglan, A. (2016). The Wiley Handbook of Contextual Behavioral Science. Chichester: Wiley-Blackwell. Okazaki, S., Liu, J. F., Longworth, S. L., and Minn, J. Y. (2002). Asian American- white American diﬀerences in expressions of social anxiety: a replication and extension. Cultur. Divers. Ethnic Minor. Psychol. 8, 234–247. doi: 10.1037/1099- 9809.8.3.234 Conﬂict of Interest: The author declares that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Pasillas, R. M., and Masuda, A. (2014). “Cultural competency and acceptance and commitment therapy,” in Mindfulness and Acceptance in Multicultural Competency: A Contextual Approach to Sociocultural Diversity in Theory and Practice, ed. A. Masuda (Oakland, CA: New Harbinger Publications), 109–125. Pasillas, R. M., and Masuda, A. (2014). “Cultural competency and acceptance and commitment therapy,” in Mindfulness and Acceptance in Multicultural Competency: A Contextual Approach to Sociocultural Diversity in Theory and Practice, ed. A. Masuda (Oakland, CA: New Harbinger Publications), 109–125. Pavlov, I. P. (1902). The work of the digestive glands. JAMA 1911, 1307-1309. doi: 10.1001/jama.1911.02560180043032 Copyright © 2020 Krieg. 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) and the copyright owner(s) 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. p y pp y y Practice, ed. A. Masuda (Oakland, CA: New Harbinger Publications), 109–125. Pavlov, I. P. (1902). The work of the digestive glands. JAMA 1911, 1307-1309. doi: 10.1001/jama.1911.02560180043032 Peters, L., Sunderland, M., Andrews, G., Rapee, R. M., and Mattick, R. P. (2011). Development of a short form social interaction anxiety (SIAS) and social phobia March 2020 \| Volume 11 \| Article 418 Frontiers in Psychology \| www.frontiersin.org 12
https://openalex.org/W2059148525	https://www.scielo.br/j/gmb/a/YMZNvH56K4rgB4Y95F6g3Wz/?lang=en&format=pdf	English	null	Comet assay comparison of different Corbicula fluminea (Mollusca) tissues for the detection of genotoxicity	Genetics and Molecular Biology	2,005	cc-by	3,691	Genetics and Molecular Biology, 28, 3, 464-468 (2005) Copyright by the Brazilian Society of Genetics. Printed in Brazil www.sbg.org.br Research Article Research Article Abstract The comet assay was used to study the sensitivity of the widely distributed freshwater bivalve mollusk Corbicula fluminea to the DNA-damaging alkylating-agent methylmethane sulfonate (MMS). This study was undertaken to ascertain if C. fluminea is a good bioindicator of pollutants in aquatic environments and identify which C. fluminea tissue is most effective and practical for genotoxicity studies. The mollusks were exposed to 0.6, 1.2 or 2.4 X 10 -4 M MMS for 40 min and their hemolymph, gill tissue and digestive gland tissue assessed for the level of DNA damage and the time needed for the tissues to recovery. Regression analysis showed a direct linear dose-response relationship between MMS concentration and the number of damaged cells for hemolymph and digestive gland tissue but a quadratic relationship for gill tissue, which made the interpretation the gill tissue results difficult. The basal level of DNA damage to gill tissue was very high, possibly because gill is the organs most directly exposed to environmental toxins and mutagenic agents. Although all three types of tissue produced useful results, hemolymph and digestive gland tissue produced more reproducible and reliable results. Hemolymph was the best sample type in that it was easy to obtain and handle, while gill tissue required more manipulation to obtain cell suspensions. Our results indicate that C. fluminea is an optimal bioindicator for the determination genotoxic contaminants in aquatic environments. Key words: Corbicula fluminea, hemolymph, gills, digestive gland, comet assay, biomonitoring. Received: August 13, 2004; Accepted: February 15, 2005. Key words: Corbicula fluminea, hemolymph, gills, digestive gland, comet assay, biomonitoring. Comet assay comparison of different Corbicula fluminea (Mollusca) tissues for the detection of genotoxicity Janaina Rigonato, Mário Sérgio Mantovani and Berenice Quinzani Jordão Janaina Rigonato, Mário Sérgio Mantovani and Berenice Quinzani Jordão U i id d E d l d L d i D d Bi l i G l L d i PR B il Janaina Rigonato, Mário Sérgio Mantovani and Berenice Quinzani Jordão Universidade Estadual de Londrina, Departamento de Biologia Geral, Londrina, PR, Brazil. Janaina Rigonato, Mário Sérgio Mantovani and Berenice Quinzani Jordão Universidade Estadual de Londrina, Departamento de Biologia Geral, Londrina, PR, Brazil. Universidade Estadual de Londrina, Departamento de Biologia Geral, Londrina, PR, Brazil. Introduction al., 1998; Narbonne et al., 1999) and histological (Dowling and Mothersill, 2001) responses. Over the last few decades a number of studies have been undertaken to develop tools to determine the impact of xenobiotics on aquatic invertebrates (Le Pennec and Le Pennec, 2001). Bivalves are widely distributed sessile filter feeders which are good bioindicators of aquatic pollutants (Venier et al., 1997; López-Barea and Pueyo, 1998; Le Pennec and Le Pennec, 2001) and have often been used as test organisms in studies of aquatic pollution (Wilson et al., 1998; Mitchelmore et al., 1998; Narbonne et al., 1999; Pavlica et al., 2001; Pruski and Dixon, 2002). The comet assay has been utilized to determine the ability of aquatic contaminants to induce DNA damage but its applicability has been the subject of intense investiga- tion (Pavlica et al., 2001). The comet assay derives its name from the comet-like shape that cells with damaged DNA acquire due to the migration of broken DNA fragments dur- ing electrophoresis, the size of the comet tail reflecting the extent of DNA damage. The comet assay is better than other tests because it requires only a very small number of cells and these cells do not need to be undergoing active di- vision, which means that any tissue can be evaluated. A fur- ther advantage of the comet assay is that it is a very sensitive method for determining damage in genetic mate- rial (Farbain et al., 1995; Klaude at al., 1996; Speit and Hartmann, 1999; Tice et al., 2000; Lee and Steinert, 2003). The freshwater bivalve mollusk Corbicula fluminea is native to Southeast Asia but was introduced into North America at the end of the 19th century and rapidly spread throughout the Americas to become an important part of the benthic community. These mollusks have been shown to be very good bioindicators of aquatic pollution able to accu- mulate pollutants and show differential physiological, chemical (Johns and Luoma, 1990; Roberts, 1996; Bilos et In the study reported in this paper we used the alka- line comet test to determine whether or not the bivalve mol- lusk Corbicula fluminea can be used as a reliable bioindicator of DNA damage. During this study we com- pared the sensitivity of different C. fluminea tissues (hemolymph, digestive gland and gill tissues) to the Send correspondence to Berenice Quinzani Jordão. DNA damage recovery test The mollusks were exposed to 2.4 X 10-4 M MMS for 1 h and then returned to an aquarium containing continuously aerated well-water. On days 0, 2, 7, 9 and 30 following treat- ment the hemolymph of five mollusks was sampled as de- scribed above and subjected to the comet test. This test was repeated five times for each specimen by using hemolymph sub-samples from each of the mollusks. Organisms exposed to water containing no MMS were used as controls and treated in the same way as those exposed to MMS. Hemolymph (Figure 3a) and digestive gland tissue (Figure 3b) which had not been exposed to MMS showed low background damage which made it easy to evaluate the response of the cells to MMS. There was a high number of class 1 and 2 cells in control gill tissue which had not been exposed to MMS (Figure 3c), such results indicating a high level of baseline damage in this tissue. Statistical analysis For the tissue sensitivity test data regression analysis was performed on the curves for the different MMS con- centrations. The DNA damage recovery test data for mol- lusks treated with MMS was compared to mollusks exposed to water only using the Kruskal-Wallis test at the 5% significance level. Results For all the tissues studied there was a tendency to- wards a direct relationship between MMS concentration and the number of damaged cells or the damage score, with regression analysis showing a linearly-correlated dose- response relationship between MMS concentration and the number of damaged cells for hemolymph and digestive gland tissue but a quadratic relationship for gill tissue (Figure 2). For all three tissues class 1 damage was the most frequent type and class 3 the least frequent (Figure 2). Tissue sensitivity test Methylmethane sulfonate (MMS, CAS: 66-27-3, Aldrich) solutions were prepared at concentrations of 0.6, 1.2 and 2.4 X 10-4 M and five C. fluminea were placed in each solution for 40 min, after which hemolymph was col- lected from the posterior adductor muscle of each mollusk using a syringe and transferred to 1.5 mL microtubes and centrifuged for 5 min at 268 g. After centrifugation the supernatant was discarded and all the resultant pellet uti- lized for the comet assay. The gills and digestive gland of each mollusk were also removed and separately macerated in calcium- and magnesium-free phosphate-buffered saline (PBS) to obtain cell suspensions. The gill tissue required more maceration due its resistant nature. These cell suspen- sions were then centrifuged for 5 min at 268 g. After centrifugation most of the supernatant was discarded ex- cept for about 0.5 mL which was used to re-suspend the pel- let, 10 L of this suspension being used for the comet assay. The tissue sensitivity test was repeated four times for each mollusk by using sub-samples of the tissues collected. Mol- lusks exposed to water containing no MMS were used as controls and treated in the same way as those exposed to MMS. Biological material We collected specimens of the bivalve mollusk Corbicula fluminea (2 to 3.5 cm long) were collected from a single site at the riverbed of the river Cambé near the town of Londrina in the Brazilian state of Paraná. The specimens were transported to the laboratory at the University of Londrina where they were detoxified by maintaining them for 30 days in a aquarium containing continuously aerated well-water. Introduction Universidade Estadual de Londrina, Centro de Ciências Biológicas, Departa- mento de Biologia Geral, Campus Universitário, Caixa Postal 6001, 86.051-990 Londrina, PR, Brasil. E-mail: berejordao@uel.br. 465 Rigonato et al. mixed with 120 L of 0.5% low-melting point (LMP) agarose at 37 °C and dropped on microscope slides previ- ously covered with 1.5% normal melting point (NMP) agarose. The slides were protected with coverslips and al- lowed to set at 4 °C for 15 min. After the careful removal of the coverslip, the rest of the comet assay was conducted ac- cording to the method of Speit and Hartmann (1999), briefly summarized as follows: 1 h of lysis followed by 20 min denaturation in electrophoresis buffer (300 mM NaOH and 1 mM EDTA - pH > 13,0, prepared from a stock solution of 10 N NaOH and 200 mM EDTA - pH 10.0) and then electrophoresis for 20 min at 25 V and 300 mA (1.6 V/cm) followed by neutralization, fixing and staining with 0.002 mg mL-1 ethidium bromide. The cells were ex- amined with a fluorescence microscope at 400 X, using a 420-490 nm excitation filter and a 520 nm emission filter. Fifty cells were scored per mollusk in the tissue sensitivity test and 100 cells in the DNA damage recovery test. These cells were classified according to the size of the comet tail: class 0, no tail; class 1, a small tail less than the head (nu- cleus) diameter; class 2, tail length equal to or up to twice the head diameter; and class 3, tail more than twice the head diameter (Figure 1). For each treatment damage scores were calculated by summing the number of cells in each class and then multiplying this total by the class value (0-3). Samples from mollusks exposed to water containing no MMS were used as controls and the same investigator scored all the slides in blind test. genotoxic direct-acting alkylating agent methylmethane sulfonate (a stressor agent) and assessed the possibility of using these tissues for biomonitoring environmental genotoxic agents. Comet assay elevated baseline levels of DNA damage which could mask the results and the quadratic relationship between cell dam- age and mutagenic agent dose makes interpretation of re- sults difficult. Hemolymph and digestive gland tissue were good indicators of genotoxicity because both showed low baseline damage and a direct linear response to mutagen (stressor agent) concentration, both of which simplify the interpretation of results when assessing the effects of harm- ful agents present in the environment. The fact that gill cells showed a very high basal level of DNA damage may have been because it was very diffi- culty to obtain a cell suspension from the gill and the large amount of manipulation required to obtain cell suspensions of this organ may have led to mechanical injury to the cells and increased DNA damage. Figure 1 - Hemolymph cells exposed to methylmethane sulfonate (MMS). Class 0: undamaged cells; classes 1, 2 and 3: from the less to more damaged cells. When hemolymph cells were allowed to recover for two days there was a gradual reduction in genotoxic dam- age as measured by the number of damaged cells and the damage score, this reduction peaking on the ninth day of re- covery and remaining constant until day 30 (Figure 4). In the comet assay the basal level of DNA damage in hemolymph cells appears to be about 9.6%. Figure 4 also indicates that for recovering cells there was a time-related shift in the type and frequency of damaged cells from the more damaged class 3 and 2 cells to less damaged class 1 or undamaged class 0 cells. Because they are constantly exposed to oxygen radi- cals and other oxidants (oxidative stress) aerobic organisms are subject to high levels of damage in all cellular compo- nents, DNA included, and this can be the cause of the high baseline comet damage found in Mytilus edulis (Wilson et al., 1998). Rank and Jensen (2003) performed comet assays on M. edulis hemolymph and gill cells exposed to MMS and found that, although both tissues were equally sensitive to MMS, hemolymph was better for in vivo studies because it was much simpler to collect and handle. When working with live organisms it should be born in mind that there is natural inter-individual genetic variation (Nacci et al., 1996; Mitchelmore and Chipman, 1998) and suitable con- trols are essential to guarantee successful studies. The fact that C. Comet assay For each assay, 10 L of hemolymph pellet suspen- sion or gill or digestive gland cell suspension were gently Comet assay of different Corbicula tissues 466 elevated baseline levels of D the results and the quadratic age and mutagenic agent do sults difficult. Hemolymph a good indicators of genotoxic baseline damage and a dire (stressor agent) concentratio interpretation of results when ful agents present in the env The fact that gill cells s of DNA damage may have b culty to obtain a cell suspens amo nt of manip lation req Figure 1 - Hemolymph cells exposed to methylmethane sulfonate (MMS). Class 0: undamaged cells; classes 1, 2 and 3: from the less to more damaged cells. Figure 2 - Cell damage scores for h gill tissue after treatment with me hemolymph (—), gill tissue (- - values for 4 replicates. Hemolymph gestive gland tissue y = 15.508x y = 12.216x2 - 19.33x + 41.645/ R2 Figure 2 - Cell damage scores for hemolymph, digestive gland tissue and gill tissue after treatment with methylmethane sulfonate (MMS). Key: hemolymph (—), gill tissue (- - -), digestive gland (I- - -I). Mean values for 4 replicates. Hemolymph y = 14.821x + 18.25/ R2 = 0.882. Di- gestive gland tissue y = 15.508x + 14.05/ R2 = 0.8959. Gill tissue y = 12.216x2 - 19.33x + 41.645/ R2 = 0.8799. Figure 1 - Hemolymph cells exposed to methylmethane sulfonate (MMS). Class 0: undamaged cells; classes 1, 2 and 3: from the less to more damaged cells. Figure 2 - Cell damage scores for hemolymph, digestive gland tissue and gill tissue after treatment with methylmethane sulfonate (MMS). Key: hemolymph (—), gill tissue (- - -), digestive gland (I- - -I). Mean values for 4 replicates. Hemolymph y = 14.821x + 18.25/ R2 = 0.882. Di- gestive gland tissue y = 15.508x + 14.05/ R2 = 0.8959. Gill tissue y = 12.216x2 - 19.33x + 41.645/ R2 = 0.8799. Figure 2 - Cell damage scores for hemolymph, digestive gland tissue and gill tissue after treatment with methylmethane sulfonate (MMS). Key: hemolymph (—), gill tissue (- - -), digestive gland (I- - -I). Mean values for 4 replicates. Hemolymph y = 14.821x + 18.25/ R2 = 0.882. Di- gestive gland tissue y = 15.508x + 14.05/ R2 = 0.8959. Gill tissue y = 12.216x2 - 19.33x + 41.645/ R2 = 0.8799. Comet assay fluminea can exhibit hermaphrodism, self- fertilization and hence decreased inter-individual variation favors the use of this mollusk in biomonitoring studies. Discussion In this work was found that C. fluminea is a good bioindicator of genotoxic agents, with the tissues studied showing a good correlation and a very clear dose-response effect between increased MMS concentration and in- creased DNA lesions. The gills appear to be the least appro- priate organ to study because cells from this organ show Rigonato et al. Figure 3 - Frequency of DNA damage in hemolymph (a), digestive gland (b) and gill (c) cells after treatment with methylmethane sulfonate (MMS). Mean values for 4 replicates. 467 Rigonato et al. Figure 4 - Frequency of DNA damage in hemolymph cells at different re- covery times after treatment with methylmethane sulfonate (MMS). For each point 500 cells from five replicates were examined. Figure 4 - Frequency of DNA damage in hemolymph cells at different re- covery times after treatment with methylmethane sulfonate (MMS). For each point 500 cells from five replicates were examined. would have a highly active cell replacement system to avoid injury to vital functions, but in our study the mortality index was low and no apoptotic cells were detected. The ability of C. fluminea to recover from damage when unexposed to mutagens support the notion that there should be a 30-day detoxification period in the laboratory before exposing this mollusk to genotoxic agents. Mitchelmore et al. (1998) reported that 20% of M. edulis digestive gland cells can be expected to exhibit low-level DNA damage (measurable by the comet assay) caused by cell-breakage during isolation and that digestive gland cells taken from M. edulis show many DNA breaks compared to other vertebrate cell types. Our study with C. fluminea showed that maximum recovery from DNA dam- age (9.6%) occurred and leveled off after nine days, indi- cating that C. fluminea could be recommended for the monitoring of aquatic environments in response to some type of ecological disaster and that this mollusk is capable of determining when the environment has returned to ac- ceptable conditions. Figure 3 - Frequency of DNA damage in hemolymph (a), digestive gland (b) and gill (c) cells after treatment with methylmethane sulfonate (MMS). Mean values for 4 replicates. The present study suggests that Corbicula fluminea is an optimal biomonitor of aquatic environments and it con- tains various tissues and organs capable of furnishing good responses to genotoxicity. Discussion Other studies (Venier et al., 1997; Klobuèar et al., 2003; Rank and Jensen, 2003) have shown that hemolymph is a good tissue for the in vivo evaluation of organisms exposed to environmental pollutantsfi. Also these findings suggests that C. fluminea hemolymph is a valuable target tissue because of its ease of manipulation and its effi- cient response to DNA-stressing compounds. It therefore seems that C. fluminea could be useful in establishing tests to determine genotoxicity in aquatic environments. Figure 3 - Frequency of DNA damage in hemolymph (a), digestive gland (b) and gill (c) cells after treatment with methylmethane sulfonate (MMS). Mean values for 4 replicates. In this study the C. fluminea cell types studied showed rapid reversal of the MMS-induced genotoxic damage with a reduction in the number of damaged cells occurring nine days after MMS exposure, this reversal possibly being due to apoptosis or repair systems in the damaged cells. If apoptosis was responsible for the elimination of cells C. fluminea References (Mytilus edulis L.) digestive gland cells using the “comet” assay. Ecotoxicol Environ Saf 41:51-58. (Mytilus edulis L.) digestive gland cells using the “comet” assay. Ecotoxicol Environ Saf 41:51-58. Bilos C, Colombo JC, Presa MJR (1998) Trace metals in sus- pended particles, sediments and Asiatic clams (Corbicula fluminea) of the Río de la Plata Estuary, Argentina. Environ Pollution 99:1-11. Nacci DF, Cayula S and Jackim F (1996) Detection of DNA dam- age in individual cells from marine organisms using the sin- gle cell gel assay. Aquatic Toxicol 35:197-210. Narbonne JF, Djomo JE, Ribeira, FV and Garrigues P (1999) Ac- cumulation kinetics of polycyclic aromatic hydrocarbons adsorbed to sediment by the mollusk Corbicula fluminea. Ecotoxicol Environ Saf 42:1-8. Dowling K and Mothersill C (2001) The further development of rainbow trout primary epithelial cell cultures as a diagnostic tool in ecogenotoxicology risk assessment. Aquatic Toxicol 53:279-289. Ecotoxicol Environ Saf 42:1-8. Pavlica M, Klobuèar GIV, Moja N, Erben R and Pape D (2001) Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutat Res 490:209-214. Farbain DW, Olive PL and O’Neil KL (1995) The comet assay: A comprehensive review. Mutat Res 339:37-59. Pruski AM and Dixon DR (2002) Effects of cadmium on nuclear integrity and DNA repair efficiency in the gill cells of Mytilus edulis L. Aquatic Toxicol 57:127-137. Johns C and Luoma SN (1990) Arsenic in benthic bivalves of San Francisco Bay and the Sacramento/San Joaquin River delta, The Science of the Total Environ 97/98:673-684. Rank J and Jensen K (2003) Comet assay on gill cells and hemocytes from the blue mussel Mitylus edulis. Ecotoxicol Environ Saf 54:323-329. Klaude M, Eriksson S, Nygren J and Ahnström G (1996) The comet assay: Mechanisms and technical considerations. Mutat Res 363:89-96. Roberts CA (1996) Selenium contamination in Corbicula trans- planted into agricultural drains in the Imperial Valley, Cali- fornia, Division of Environ. Contaminants, Carsbad Field Office U.S. Fish and Wildlife Service, August. Klobuèar GIV, Pavlica M, Erben R and Pape D (2003) Applica- tion of the micronucleus and comet assays to mussel Dreissena polymorpha haemocytes for genotoxicity moni- toring of freshwater environments. Aquatic Toxicol 64:15-23. Speit G and Hartmann A (1999) The comet assay (single-cell gel test). In Henderson DS (ed) Methods in Molecular Biology, v.113, DNA Repair Protocols: Eukaryotic Systems. Humana Press, Totowa, pp 203-212. Acknowledgments This study was supported by the Brazilian agency CAPES/MEC. 468 Comet assay of different Corbicula tissues References Le Pennec G and Le Pennec M (2001) Evaluation of the toxicity of chemical compounds using digestive acini of the bivalve mollusk Pecten maximus L. maintained alive in vitro. Aquat Toxicol 53:1-7. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC and Sasaki YF (2000) Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ Molec Muta- genesis 35:206-221. Lee RF and Steinert S (2003) Use of the single cell gel electropho- resis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat Res 544:43-64. Venier P, Maron S and Canova S (1997) Detection of micronuclei in gill cells and haemocytes of mussels exposed to benzo[a]pyrene. Mutat Res 390:33-44. López-Barea J and Pueyo C (1998) Mutagen content and meta- bolic activation of promutagens by molluscs as biomarkers of marine pollution. Mutat Res 399:3-15. Wilson JT, Pascoe PL, Parry JM and Dixon DR (1998) Evaluation of comet assay as a method for the detection of DNA dam- age in cells of a marine invertebrate, Mytilus edulis L. (Mollusca, Pelecipoda). Mutat Res 399:87-95. Mitchelmore CL and Chipman JK (1998) DNA strand breakage in aquatic organisms and the potential values of the comet as- say in environmental monitoring. Mutat Res 399:135-147. Mitchelmore CL, Birmelin C, Livingstone DR and Chipman JK (1998) Detection of DNA strand breaks in isolated mussel Associate Editor: Carlos F.M. Menck
https://openalex.org/W2896037912	https://www.mdpi.com/1996-1073/11/10/2818/pdf	English	null	Finite Element Analysis to the Effect of Thermo-Mechanical Loads on Stress Distribution in Buried Polyethylene Gas Pipes Jointed by Electrofusion Sockets, Repaired by PE Patches	Energies	2,018	cc-by	19,732	Received: 7 September 2018; Accepted: 8 October 2018; Published: 19 October 2018 Abstract: Polyethylene (PE) gas pipes can be jointed together by electrofusion PE ﬁttings, which have sockets that are fused onto the pipe. Additionally, electrofused PE patches can be used to repair defected pipes. When these pipelines are buried under the ground, they can experience sever local stresses due to the presence of pipe joints, which is superimposed on the other effects including the soil-structure interaction, trafﬁc load, soil’s column weight, a uniform internal pressure, and thermal loads imposed by daily and/or seasonal temperature changes. The present contribution includes two cases. At ﬁrst, stress variations in buried polyethylene gas pipe and its socket due to the aforementioned loading condition is estimated using ﬁnite element. The pipe is assumed to be made of PE80 material and its jointing socket material is PE100. Afterward, the effects of aforementioned thermo-mechanical loads on the stress distribution in patch repaired buried pipes are well investigated. The soil physical properties and the underground polyethylene pipe installation method are based on the American association of state highway and transportation ofﬁcials and American society for testing and material standards. The computer simulation and analysis of stresses are performed through the ﬁnite element package of ANSYS Software. Stress concentrations can be observed in both components due to the presence of the socket or the repair patch. According to the results, the electrofusion sockets can be used for joining PE gas pipes even in hot climate areas. The maximum values of these stresses happen to be in the pipe. Also, the PE100 socket is more sensitive to a temperature drop. Additionally, all four studied patch arrangements show signiﬁcant reinforcing effects on the defected section of the buried PE gas pipe to withstand applied loads. Meanwhile, the defected buried medium density polyethylene (MDPE) gas pipe and its saddle fused patch can resist the imposed mechanical and thermal loads of +22 ◦C temperature increase. Keywords: buried gas distribution pipes; electrofusion socket joints; patch repair; medium density polyethylene (MDPE); high density polyethylene (HDPE); Von Mises stress; ﬁnite element method; temperature variation Finite Element Analysis to the Effect of Thermo-Mechanical Loads on Stress Distribution in Buried Polyethylene Gas Pipes Jointed by Electrofusion Sockets, Repaired by PE Patches Reza Khademi-Zahedi 1,* and Pouyan Alimouri 2 Reza Khademi-Zahedi 1,* and Pouyan Alimouri 2 1 Institute of Structural Mechanics, Bauhaus-Universität Weimar, 99423 Weimar, Germany 2 Department of Mechanical Engineering, Shahid Chamran University of Ahvaz, Ahvaz, Iran; Alimouri_P@yahoo.com * Correspondence: reza.khademi.zahedi@uni-weimar.de * Correspondence: reza.khademi.zahedi@uni-weimar.de www.mdpi.com/journal/energies energies energies 1. Introduction Petroleum, natural gas, and condensates are naturally occurring substances which are discovered within the Earth’s crust, are thought to originate from decomposed animal and plant matter. Scientists believe the plants and animals died in the distant past, and were gradually buried by thick layers of sediments. Over a long period of time, and with pressure and temperature, the organic materials were transformed into the oil and gas which are found today. When oil and gas are removed from the ground Energies 2018, 11, 2818; doi:10.3390/en11102818 www.mdpi.com/journal/energies 2 of 24 25 Energies 2018, 11, 2818 Energies 2018 11 x they are sent to reﬁneries by pipelines. Then, many products from these materials, which potentially contain several chemicals called hydrocarbons, can be obtained including energy for power, motor oil, gasoline for cars, diesel fuel for trucks and trains, hi-octane fuels for planes, heating oil for houses etc. Several other materials also come from petroleum such as plastics, asphalt, grease, lubricating oil, materials for clothes, chemicals for everyday use, paints etc. Since oil has natural gas in it, when oil is produced often some gas is produced with the oil if natural gas is in the solution. The majority of oil is trapped in the tiny pore spaces between grains of rock or sand. Oil and gas are discovered in natural traps which consist of domes or faults within the earth. Impermeable rock above the trap that ﬂuid cannot pass through it stops the oil and gas from move up to the surface. Without traps, the oil and gas could migrate all the way to the surface and evaporate. Figure 1 shows an example of an oil reservoir with a gas cap. by thick layers of sediments. Over a long period of time, and with pressure and temperature, the organic materials were transformed into the oil and gas which are found today. When oil and gas are removed from the ground they are sent to refineries by pipelines. Then, many products from these materials, which potentially contain several chemicals called hydrocarbons, can be obtained including energy for power, motor oil, gasoline for cars, diesel fuel for trucks and trains, hi-octane fuels for planes, heating oil for houses etc. Several other materials also come from petroleum such as plastics, asphalt, grease, lubricating oil, materials for clothes, chemicals for everyday use, paints etc. 1. Introduction Finally, the gas and oil can be sold to power cars and heat houses. gas pipes. A pipeline network needs a long length pipe to dispatch natural gas. It is troublesome to produce long pipes, hence the needed lengths of pipe is assembled by connecting short pipes together. Additionally, in order to construct long pipe networks, it is essential to utilize joining methods such as butt-welding, electro-fusion, mechanical joining etc. Mainly, electro-fusion joint is extensively employed since it is empirically feasible to perform even in areas where access is restricted. Generally, buried pipes are divided into two categories: flexible pipes, where the pipe deforms under applied loads at least 2 percent of the pipe diameter without any effect to the pipe material, and rigid pipes, where the pipe structure changes (for example with crack creation) in the mentioned deformation [5]. PE pipes behave as flexible pipes when installed underground. In the underground application of PE pipes, the installation procedure and the design method are of great importance for natural gas distributor companies. The first step in buried pipe design is to determine the soil dead load and surcharge loads on the pipe and ensure the safe operation of the PE pipes for a long period of time. The load resulted from soil column weight, vehicle wheel, pipe internal pressure, and daily or seasonal temperature changes which induced horizontal and vertical High density (HD) and medium density (MD) polyethylene materials are often used to produce gas pipes. A pipeline network needs a long length pipe to dispatch natural gas. It is troublesome to produce long pipes, hence the needed lengths of pipe is assembled by connecting short pipes together. Additionally, in order to construct long pipe networks, it is essential to utilize joining methods such as butt-welding, electro-fusion, mechanical joining etc. Mainly, electro-fusion joint is extensively employed since it is empirically feasible to perform even in areas where access is restricted. Generally, buried pipes are divided into two categories: ﬂexible pipes, where the pipe deforms under applied loads at least 2 percent of the pipe diameter without any effect to the pipe material, and rigid pipes, where the pipe structure changes (for example with crack creation) in the mentioned deformation [5]. PE pipes behave as ﬂexible pipes when installed underground. 1. Introduction Since oil has natural gas in it, when oil is produced often some gas is produced with the oil if natural gas is in the solution. The majority of oil is trapped in the tiny pore spaces between grains of rock or sand. Oil and gas are discovered in natural traps which consist of domes or faults within the earth. Impermeable rock above the trap that fluid cannot pass through it stops the oil and gas from move up to the surface. Without traps, the oil and gas could migrate all the way to the surface and evaporate. Figure 1 shows an example of an oil reservoir with a gas cap. Figure 1. An example of an oil reservoir with a gas cap. Figure 1. An example of an oil reservoir with a gas cap. Figure 1. An example of an oil reservoir with a gas cap. Figure 1. An example of an oil reservoir with a gas cap. After geophysicists find reservoirs and process the data to construct pictures of what the earth looks like underground, drilling companies start to drill into the proposed reservoirs. A drilling rig is a package of special equipment put together that enables us to create a hole to a projected depth for producing oil or natural gas [1–4]. Finally, the well is produced into a pipeline, which takes it to production facilities on surface. The production facilities on surface separate out the gas, oil, and water into their separate phases. From there, the oil and gas may be refined further before being ready to market. Finally, the gas and oil can be sold to power cars and heat houses. High density (HD) and medium density (MD) polyethylene materials are often used to produce After geophysicists ﬁnd reservoirs and process the data to construct pictures of what the earth looks like underground, drilling companies start to drill into the proposed reservoirs. A drilling rig is a package of special equipment put together that enables us to create a hole to a projected depth for producing oil or natural gas [1–4]. Finally, the well is produced into a pipeline, which takes it to production facilities on surface. The production facilities on surface separate out the gas, oil, and water into their separate phases. From there, the oil and gas may be reﬁned further before being ready to market. 1. Introduction In the underground application of PE pipes, the installation procedure and the design method are of great importance for natural gas distributor companies. The ﬁrst step in buried pipe design is to determine the soil dead load and surcharge loads on the pipe and ensure the safe operation of the PE pipes for a long period of time. The load resulted from soil column weight, vehicle wheel, pipe internal pressure, and daily or seasonal temperature changes which induced horizontal and vertical deﬂections along with signiﬁcant stresses in the pipe wall. Consequently the soil showed reaction to 3 of 24 Energies 2018, 11, 2818 the aforementioned pipe action and will limit the pipe deﬂection. Several experimental and analytical methods have been proposed by researchers to calculate the applied loads, deﬂections, and stresses on the buried pipes that, although suitable for most underground applications, do not produce accurate results [6]. The modern buried-pipe design was proposed by Marston from Iowa University in the early 20thcentury [6,7]. Spangler continued theoretical studies on ﬂexible buried pipes by assuming soil and pipe as linear elastic materials and proposed Iowa’s modiﬁed formula to predict deﬂection in ﬂexible buried pipes [7]. Afterward ﬂexible pipe materials including aluminum, polyvinyl, and polyethylene were used in underground applications to improve pipe-soil behavior and reduce costs. Pipe-soil interaction is a combination system that signiﬁcant share of the applied load is carried by the soil around the pipe which makes it difﬁcult to calculate the induced stresses in the pipe material. One of the strongest numerical approaches to investigate underground structure responses to the applied loads is the ﬁnite element method. Abaqus and Ansys software has been used by several researchers for three-dimensional modeling of the aforementioned structures. In this research, the methods discussed in contributions on ﬁnite element modeling of buried polyethylene gas pipes which can be found in references [8–13] are used to further investigate the stress distribution in special cases of buried polyethylene gas pipe problems which are subjected to the local geometry changes compared to simple buried pipes. This study can be divided in two parts. In the ﬁrst part, the case of joining buried PE pipes by electrofusion socket joints is well discussed. In the second part of the research, four different patch arrangements used to repair defected buried pipes are investigated, and their reinforcing effects on damaged parts are discussed. 2. Basic Design Theory In engineering design, soil classiﬁcation systems for structural applications are based on soil mechanical and physical properties including grain sizes. Commonly, AASHTO and ASTM standards are used as guides for these purposes [5,14]. The investigation of structural behavior of underground systems starts with the determination of applied loads. The design of underground structures is in accordance with the principle of soil-pipe interaction. Estimating the loads on structures, including gas pipelines used in underground space, depends on the pipe installation procedure in the trench that can be found in ASTM D 2321 standard [15,16]. The mechanical properties of the backﬁll environment or embedment should meet the special structural application. The mechanical properties of the soil grades used for bedding, haunching, initial and ﬁnal backﬁll and also asphalt cover, along with installation dimensions are selected based on references [8–11]. Additionally similar loading conditions are introduced and imposed. 1. Introduction The pipe, socket/patch, the soil grades surrounding them, and the underground installation procedure and burial depth are all selected based on the standards commonly used in gas distribution companies. For both cases, the simultaneous effects of mechanical loads including pipe internal pressure, vehicle trafﬁc load, soil column load, and more critical thermal loads of temperature variations in PE pipe and its socket/patch on the induced stresses in PE80 pipe and PE100 socket/patch materials are investigated using Ansys software. In both cases when the model is imposed to the aforementioned thermo-mechanical loads, stress concentrations will appear in the pipe and its socket/patch due to local changes in the pipe geometry. Table 1. Geometrical and mechanical properties of PE100 socket. Table 1. Geometrical and mechanical properties of PE100 socket. Geometrical Properties Mechanical Properties SDR Outer Diameter (mm) Wall Thickness (mm) Modulus of Elasticity Yield Strength (MPa) Rupture Strength (MPa) Poisson’s Ratio Thermal Expansion Coefﬁcient ( 1 °C) 11 110 10 1034 24.8 35.1 0.4 0.0002 The type of the joining is electro fused socket where the related dimensions are presented in Figure 2. Table 1. Geometrical and mechanical properties of PE100 socket. Geometrical Properties Mechanical Properties SDR Outer Diameter(mm) Wall Thickness(mm) Modulus of Elasticity Yield Strength (MPa) Rupture Strength (MPa) Poisson’s Ratio Thermal Expansion Coefficient ( 𝟏 ℃) 11 110 10 1034 24.8 35.1 0.4 0.0002 The type of the joining is electro fused socket where the related dimensions are presented in Figure 2. Geometrical Properties Mechanical Properties SDR Outer Diameter (mm) Wall Thickness (mm) Modulus of Elasticity Yield Strength (MPa) Rupture Strength (MPa) Poisson’s Ratio Thermal Expansion Coefﬁcient ( 1 °C) 11 110 10 1034 24.8 35.1 0.4 0.0002 Table 1. Geometrical and mechanical properties of PE100 socket. Geometrical Properties Mechanical Properties SDR Outer Diameter(mm) Wall Thickness(mm) Modulus of Elasticity Yield Strength (MPa) Rupture Strength (MPa) Poisson’s Ratio Thermal Expansion Coefficient ( 𝟏 ℃) 11 110 10 1034 24.8 35.1 0.4 0.0002 The type of the joining is electro fused socket where the related dimensions are presented in Figure 2. The type of the joining is electro fused socket where the related dimensions are presented in Figure 2. The type of the joining is electro fused socket where the related dimensions are presented in Figure 2. The type of the joining is electro fused socket where the related dimensions are presented in Figure 2. Socket dimensions D (mm) T (mm) L (mm) H (mm) 90 58 118 130 Figure 2. Selected socket geometry and dimensions for the investigated PE pipe [18]. D: Socket inside diameter; T: Inner length; L: Socket length; H: Socket height. Figure 2. Selected socket geometry and dimensions for the investigated PE pipe [18]. D: Socket inside diameter; T: Inner length; L: Socket length; H: Socket height. Figure 2. Selected socket geometry and dimensions for the investigated PE pipe [18]. D: Socket inside diameter; T: Inner length; L: Socket length; H: Socket height. Figure 2. Selected socket geometry and dimensions for the investigated PE pipe [18]. D: Socket inside diameter; T: Inner length; L: Socket length; H: Socket height. Table 1. Geometrical and mechanical properties of PE100 socket. Generally, PE100 material is the proper option to join PE80 pipes. PE100 material has the minimum allowable stress of 10 MPa for a working life of 50 years in 20 °C design temperature. In contrast, the minimum allowable stress for PE80 material for a working life of 50 years in 20 °C design temperature is as low as 8 MPa. PE100 material shows better resistance against perforation than PE80 material. Additionally, PE100 material shows better resistance to fast crack propagation in comparison to PE80 material. This means if a crack initiates in a PE100 material, it will stop propagating in a short length. This advantage is of great importance especially for pipes loaded under high pressure values that fast crack propagation is of magnificent importance. PE100 material shows good behavior against creep rupture compared to PE80 material. Additionally, for two different PE100 and PE80 pipes with the same SDR, PE100 pipe material can be used for higher pressure values. That means with the same inside pressure for PE100 and PE80 pipe material, the wall thickness of PE100 pipe material can be chosen 19% less than that of PE80 pipe material [19]. To investigate the stress distribution in the socket the simultaneous effects of soil column Generally, PE100 material is the proper option to join PE80 pipes. PE100 material has the minimum allowable stress of 10 MPa for a working life of 50 years in 20 ◦C design temperature. In contrast, the minimum allowable stress for PE80 material for a working life of 50 years in 20 ◦C design temperature is as low as 8 MPa. PE100 material shows better resistance against perforation than PE80 material. Additionally, PE100 material shows better resistance to fast crack propagation in comparison to PE80 material. This means if a crack initiates in a PE100 material, it will stop propagating in a short length. This advantage is of great importance especially for pipes loaded under high pressure values that fast crack propagation is of magniﬁcent importance. PE100 material shows good behavior against creep rupture compared to PE80 material. Additionally, for two different PE100 and PE80 pipes with the same SDR, PE100 pipe material can be used for higher pressure values. That means with the same inside pressure for PE100 and PE80 pipe material, the wall thickness of PE100 pipe material can be chosen 19% less than that of PE80 pipe material [19]. 3. Stress Investigation in Socket Joint of Buried Polyethylene Gas Pipe The socket material for joining MDPE buried gas pipes was selected from the PE100. Based on references [8–11] PE80 material was selected for the pipe. In order to investigate the stress distribution in a pressurized PE gas pipe, the value of 11 was selected for pipe and its socket SDR (Standard dimension ratio) Geometrical and mechanical properties of PE100 socket material are presented in Table 1 [17]. 4 of 24 Energies 2018, 11, 2818 Table 1. Geometrical and mechanical properties of PE100 socket. 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe In this section various thermal and mechanical loadings applied 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe In this section, various thermal and mechanical loadings applied to the buried socket jointed polyethylene gas pipe during the operation period are considered to be software inputs, and the resulting induced stresses are evaluated and discussed using finite element method. The geometry and the installation procedure of the socket jointed polyethylene pipe are similar In this section, various thermal and mechanical loadings applied to the buried socket jointed polyethylene gas pipe during the operation period are considered to be software inputs, and the resulting induced stresses are evaluated and discussed using ﬁnite element method. to that of normally buried polyethylene pipe which is well described [8–12]. Therefore, the same temperature variations for the buried pipe impose to the socket too. The finite element results for maximum values of Von Mises and axial stresses under the i ulta eou effe t of the oil olu ei ht i e i te al e u e ehi le heel load a d The geometry and the installation procedure of the socket jointed polyethylene pipe are similar to that of normally buried polyethylene pipe which is well described [8–12]. Therefore, the same temperature variations for the buried pipe impose to the socket too. simultaneous effects of the soil column weight, pipe internal pressure, vehicle wheel load and temperature variations of +5 °C, −15 °C and 0 °C (no variation) in the buried pipe and its socket are calculated and presented in Table 2.The maximum axial stress and the maximum Von Mises stress values in the wall of buried socket joint of the gas distribution pipe under the simultaneous effect of mechanical loads including soil load, internal pressure, vehicle wheel load and thermal loads in the form of maximum temperature drop of 15 °C and increase of 5 °C are calculated and plotted in Figure 4. For Figure 4, the coordinate system is selected so that the origin of the horizontal axes represents the socket internal surface and the stress variations are plotted from the origin to the socket crown. As expected, the upper curves pertain to the temperature decrease. Table 1. Geometrical and mechanical properties of PE100 socket. weight, traffic wheel load, pipe and socket temperature changes, internal pressure are considered and implemented in ANSYS (V12, ANSYS, Pittsburgh, PA, USA) software. Figure 3 illustrates a typical finite element model of pipe and socket subjected to internal pressure. It should be noted that the dimension of the model upper surface is x = 1.5 m and z = 1.5 m. Furthermore, because of the identical effect of the concentrated and distributed wheel load in the selected installation depth, the effect of distributed wheel load on the pipe and its socket is investigated. Additionally the soil-pipe interaction is also incorporated in the model by using face-to-face contact elements (conta 172 and target 170). To investigate the stress distribution in the socket the simultaneous effects of soil column weight, trafﬁc wheel load, pipe and socket temperature changes, internal pressure are considered and implemented in ANSYS (V12, ANSYS, Pittsburgh, PA, USA) software. Figure 3 illustrates a typical ﬁnite element model of pipe and socket subjected to internal pressure. It should be noted that the dimension of the model upper surface is x = 1.5 m and z = 1.5 m. Furthermore, because of the identical effect of the concentrated and distributed wheel load in the selected installation depth, the effect of distributed wheel load on the pipe and its socket is investigated. Additionally the soil-pipe interaction is also incorporated in the model by using face-to-face contact elements (conta 172 and target 170). 5 of 24 25 Energies 2018, 11, 2818 Energies 2018 11 x 2018, 11, 2818 ergies 2018, 11, x 5 o Figure 3. Typical finite element model of pipe and socket subjected to internal pressure. Pipe Figure 3. Typical ﬁnite element model of pipe and socket subjected to internal pressure. 2018, 11, 2818 ergies 2018, 11, x 5 o Figure 3. Typical finite element model of pipe and socket subjected to internal pressure. Pipe Figure 3. Typical ﬁnite element model of pipe and socket subjected to internal pressure. Figure 3. Typical finite element model of pipe and socket subjected to internal pressure. Figure 3. Typical ﬁnite element model of pipe and socket subjected to internal pressure. 4 Evaluation of the Result 4. Evaluation of the Results As previously mentioned, the effect of traffic load, soil weight, temperature changes, internal pressure, and the presence of the local changes in the pipe geometry were all included to investigate their overall effects on the stress distribution in the buried PE80 pipe and its related socket joint from PE100 material. For the following section of this research, the value of 1.5 is selected for the design coefficient (factor of safety). Considering the operating temperature of 35 °C for underground pipes buried at a depth of 125 cm, based on climate conditions in hot areas and Ahvaz city [8–13,20], according to the reported minimum strength value for polyethylene PE100 material [21], the design stress (maximum allowable stress) for this research is 5.4 MPa. Additionally, this value of design stress is calculated to be 4.3 MPa for the PE80 pipe material. In all stages of the next section of this investigation, the mentioned values are considered as the maximum applicable stresses on the pipe. As previously mentioned, the effect of trafﬁc load, soil weight, temperature changes, internal pressure, and the presence of the local changes in the pipe geometry were all included to investigate their overall effects on the stress distribution in the buried PE80 pipe and its related socket joint from PE100 material. For the following section of this research, the value of 1.5 is selected for the design coefﬁcient (factor of safety). Considering the operating temperature of 35 ◦C for underground pipes buried at a depth of 125 cm, based on climate conditions in hot areas and Ahvaz city [8–13,20], according to the reported minimum strength value for polyethylene PE100 material [21], the design stress (maximum allowable stress) for this research is 5.4 MPa. Additionally, this value of design stress is calculated to be 4.3 MPa for the PE80 pipe material. In all stages of the next section of this investigation, the mentioned values are considered as the maximum applicable stresses on the pipe. 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe In this section various thermal and mechanical loadings applied 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe In this section various thermal and mechanical loadings applied 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe Maximum axial and Von Mises stress variations in the wall of buried socket joint (the simultaneous effect of soil load internal pressure vehicle wheel load and temperature variations) -1 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 8 9 10 Maximum axial stress (MPa) Socket wall thickness (mm) allowable stress for a working life of 50 years at 35 °C axial stress for temperature change = –15 °C axial stress for temperature change = 0 °C axial stress for temperature change = +5 °C Von Mises stress for temperature change = –15 °C Von Mises stress for temperature change = +5 °C Figure 4. Maximum axial and Von Mises stress variations in the wall of buried socket joint (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). Item Maximum Stress (MPa) ∆T = 0 ◦C ∆T = +5 ◦C ∆T = −15 ◦C Pipe Axial stress 0.56 0.21 1.86 Von Mises stress 2.19 2.30 2.45 Socket Axial stress 2.52 1.74 4.14 Von Mises stress 2.6 2.11 4.5 and temperature variations. Item Maximum Stress (MPa) ΔT = 0 °C ΔT = +5 °C ΔT = −15 °C Pipe Axial stress 0.56 0.21 1.86 Von Mises stress 2.19 2.30 2.45 Socket Axial stress 2.52 1.74 4.14 Von Mises stress 2.6 2.11 4.5 -1 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 8 9 10 Maximum axial stress (MPa) Socket wall thickness (mm) allowable stress for a working life of 50 years at 35 °C axial stress for temperature change = –15 °C axial stress for temperature change = 0 °C axial stress for temperature change = +5 °C Von Mises stress for temperature change = –15 °C Von Mises stress for temperature change = +5 °C 4 5 6 7 Socket wall thickness (mm) Figure 4. Maximum axial and Von Mises stress variations in the wall of buried socket joint (the i ulta eou effe t of oil load i te al e u e ehi le heel load a d te e atu e a iatio ) Figure 4. Maximum axial and Von Mises stress variations in the wall of buried socket joint (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe In this section various thermal and mechanical loadings applied 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe Additionally, the dashed curve indicates the allowable stress value for design temperature of 35 °C based on a 50-year The ﬁnite element results for maximum values of Von Mises and axial stresses under the simultaneous effects of the soil column weight, pipe internal pressure, vehicle wheel load and temperature variations of +5 ◦C, −15 ◦C and 0 ◦C (no variation) in the buried pipe and its socket are calculated and presented in Table 2.The maximum axial stress and the maximum Von Mises stress values in the wall of buried socket joint of the gas distribution pipe under the simultaneous effect of mechanical loads including soil load, internal pressure, vehicle wheel load and thermal loads in the form of maximum temperature drop of 15 ◦C and increase of 5 ◦C are calculated and plotted in Figure 4. For Figure 4, the coordinate system is selected so that the origin of the horizontal axes represents the socket internal surface and the stress variations are plotted from the origin to the socket crown. As expected, the upper curves pertain to the temperature decrease. Additionally, the dashed curve indicates the allowable stress value for design temperature of 35 ◦C based on a 50-year life expectancy for the pipe and socket material. Additionally the Von Mises stresses variations are plotted from the socket internal surface to its crown. 6 of 24 re Energies 2018, 11, 2818 life expectancy Table 2. The simultaneous effect of soil column pressure, pipe internal pressure, vehicle wheel load, and temperature variations. plotted from the socket internal surface to its crown. Table 2. The simultaneous effect of soil column pressure, pipe internal pressure, vehicle wheel load, a d te pe atu e a iatio s Item Maximum Stress (MPa) ∆T = 0 ◦C ∆T = +5 ◦C ∆T = −15 ◦C Pipe Axial stress 0.56 0.21 1.86 Von Mises stress 2.19 2.30 2.45 Socket Axial stress 2.52 1.74 4.14 Von Mises stress 2.6 2.11 4.5 and temperature variations. Item Maximum Stress (MPa) ΔT = 0 °C ΔT = +5 °C ΔT = −15 °C Pipe Axial stress 0.56 0.21 1.86 Von Mises stress 2.19 2.30 2.45 Socket Axial stress 2.52 1.74 4.14 Von Mises stress 2.6 2.11 4.5 Figure 4. 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe In this section various thermal and mechanical loadings applied 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe p p Figure 5 shows the simultaneous effect of the aforementioned mechanical and thermal loads on the maximum axial and the maximum Von Mises stresses along the socket length of the buried polyethylene gas pipe. The origin of the horizontal axis is supposed to be the socket center. As the result of the geometry and loading symmetry, only the stress values of half of the socket are presented. Similarly, the dashed line is for the allowable stress values for the material of PE100. Additionally, maximum axial stress variations and maximum Von Mises stress variations along the half of pipe length under the simultaneous effect of soil load, internal pressure, and vehicle wheel load and temperature variations are calculated and plotted as Figure 6. Figure 5 shows the simultaneous effect of the aforementioned mechanical and thermal loads on the maximum axial and the maximum Von Mises stresses along the socket length of the buried polyethylene gas pipe. The origin of the horizontal axis is supposed to be the socket center. As the result of the geometry and loading symmetry, only the stress values of half of the socket are presented. Similarly, the dashed line is for the allowable stress values for the material of PE100. Additionally, maximum axial stress variations and maximum Von Mises stress variations along the half of pipe length under the simultaneous effect of soil load, internal pressure, and vehicle wheel load and temperature variations are calculated and plotted as Figure 6. Energies 2018, 11, x 7 of 25 Figure 5. Maximum axial and Von Mises stress variations along the socket length (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). -1 1 3 5 0 10 20 30 40 50 60 Maximum axial stress (MPa) Socket length (mm) allowable stress for a working life of 50 years at 35 °C axial stress for temperature change = –15 °C axial stress for temperature change = 0 °C axial stress for temperature change = +5 °C Von Mises stress for temperature change = –15 °C Von Mises stress for temperature change = +5 °C Von Mises stress for temperature change = 0 °C Figure 5. Maximum axial and Von Mises stress variations along the socket length (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). Figure 5. 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe In this section various thermal and mechanical loadings applied 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe Considering the presented maximum stress results in Table 2 and Figures 4–6 it can be concluded that: Considering the presented maximum stress results in Table 2 and Figures 4–6 it can be concluded that: 1. A daily temperature rise of +5 °C decreases the maximum axial stresses in the socket joint from 2.52 MPa to 1.74 MPa which is about 31% and additionally decreases the maximum axial stresses in the pipe from 0.56 MPa to 0.21 MPa which is about 63%. 1. A daily temperature rise of +5 ◦C decreases the maximum axial stresses in the socket joint from 2.52 MPa to 1.74 MPa which is about 31% and additionally decreases the maximum axial stresses in the pipe from 0.56 MPa to 0.21 MPa which is about 63%. 1. A daily temperature rise of +5 °C decreases the maximum axial stresses in the socket joint from 2.52 MPa to 1.74 MPa which is about 31% and additionally decreases the maximum axial stresses in the pipe from 0.56 MPa to 0.21 MPa which is about 63%. 1. A daily temperature rise of +5 ◦C decreases the maximum axial stresses in the socket joint from 2.52 MPa to 1.74 MPa which is about 31% and additionally decreases the maximum axial stresses in the pipe from 0.56 MPa to 0.21 MPa which is about 63%. 1. A daily temperature rise of +5 °C decreases the maximum axial stresses in the socket joint from 2.52 MPa to 1.74 MPa which is about 31% and additionally decreases the maximum axial stresses in the pipe from 0.56 MPa to 0.21 MPa which is about 63%. 1. A daily temperature rise of +5 ◦C decreases the maximum axial stresses in the socket joint from 2.52 MPa to 1.74 MPa which is about 31% and additionally decreases the maximum axial stresses in the pipe from 0.56 MPa to 0.21 MPa which is about 63%. 2. A daily temperature rise +5 ◦C decreases maximum Von Mises stresses in the socket joint from 2.6 MPa to 2.11 MPa which is about 19% and additionally increases maximum Von Mises stresses in the pipe from 2.19 MPa to 2.30 MPa which is about 3.8%. 3. 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe In this section various thermal and mechanical loadings applied 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe A daily temperature drop of −15 ◦C increases maximum axial stresses in the socket joint from 2.52 MPa to 4.14 MPa which is about 64% and additionally increases maximum axial stresses in the pipe from 0.56 MPa to 1.86 MPa which is about 235%. 4. A daily temperature drop of −15 ◦C increases maximum Von Mises stresses in the socket joint from 2.60 MPa to 4.50 MPa which is about 73% and additionally increases maximum Von Mises stresses in the pipe from 2.19 MPa to 2.45 MPa which is about 12%. 5. According to the Figures 4–6, in all cases the maximum values for both axial and Von Mises stresses occurs at the middle of the socket internal surface while the minimum values of the aforementioned stresses in the socket occur where the internal surface of the socket joins the pipe outer surface. After investigating the effect of socket joints, in the next section the effect of patch repair is well studied by ﬁnite element method. After investigating the effect of socket joints, in the next section the effect of patch repair is well studied by ﬁnite element method. 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe In this section various thermal and mechanical loadings applied 4.1. Results of Finite Element Solution for the Socket Jointed Buried Pipe Maximum axial and Von Mises stress variations along the socket length (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). Figure 5. Maximum axial and Von Mises stress variations along the socket length (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). 7 of 24 Energies 2018, 11, 2818 Figure 5. M effect of so Figure 6. Maximum axial and Von Mises stress variations along the pipe length (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). -1 1 3 5 0 150 300 450 600 750 Maximum axial stress (MPa) Pipe length (mm) allowable stress for a working life of 50 years at 35 °C axial stress for temperature change = –15 °C axial stress for temperature change = 0 °C axial stress for temperature change = +5 °C Von Mises stress for temperature change = –15 °C Von Mises stress for temperature change = +5 °C Von Mises stress for temperature change = 0 °C Figure 6. Maximum axial and Von Mises stress variations along the pipe length (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). -1 1 3 5 0 150 300 450 600 750 Maximum axial stress (MPa) Pipe length (mm) allowable stress for a working life of 50 years at 35 °C axial stress for temperature change = –15 °C axial stress for temperature change = 0 °C axial stress for temperature change = +5 °C Von Mises stress for temperature change = –15 °C Von Mises stress for temperature change = +5 °C Von Mises stress for temperature change = 0 °C allowable stress for a working life of 50 years at 35 °C axial stress for temperature change = –15 °C axial stress for temperature change = 0 °C axial stress for temperature change = +5 °C Von Mises stress for temperature change = –15 °C Von Mises stress for temperature change = +5 °C Von Mises stress for temperature change = 0 °C Figure 6. Maximum axial and Von Mises stress variations along the pipe length (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). Figure 6. Maximum axial and Von Mises stress variations along the pipe length (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). 4.2. Results of Finite Element Solution for the Patch Repaired Pipe Engineering structures bear or transfer several loads and may be defected during their lifetime due to several reasons. An understanding of fracture mechanisms plays an important role in structural materials safe design. Therefore several numerical methods have been used to investigate and model fracture in materials [22–40]. The calculation of stress distribution in a damaged underground 8 of 24 Energies 2018, 11, 2818 polyethylene gas pipe using ﬁnite element modeling was well discussed in [9], where several circular and elliptical shaped defects with various circular hole diameter and ellipse hole diameter ratios a/b, were modeled at the pipe crown. Additionally, the effect of 4 types of polyethylene patch repair arrangements on the stress reduction in the aforementioned defected pipes was well investigated by the computational modeling and analysis tools of ANSYS software [10]. In the following, we continued the study in references [9,10] to investigate the buried PE pipe with more details and under more critical loading conditions. The ﬁnite element code is used for modeling and analysis of the pipe, patch and their surrounding and since stress distribution in the pipe wall is very important, 3D brick-type SOLID95 elements, available in the ANSYS software package elements library, are well employed to model the physical medium. The SOLID95 element has three degrees of freedom at each node (translations in the x, y, and z coordinate directions) that makes it very suitable for solving curved boundary problems. Additionally, to have the best mesh control with a minimal number of ﬁnite elements and to reduce the computational time and costs, the mapped (structured) ﬁnite element mesh which typically has a regular pattern, is performed to control the mesh efﬁciency. In this research we study the stress reduction effects on defected polyethylene gas pipes reinforced by polyethylene patches under sever thermo-mechanical loads for four various patch arrangements including semi-cylindrical, circular-partial, square-partial and saddle fusion patches to ﬁnd the optimum patch shape. For the purpose of ﬁnding an appropriate patch shape and geometry, identical thickness (4.763 mm), inside diameter (114.3 mm) and material properties (PE 100) are considered for all patch types. It is assumed that the pipe and patch materials behave as linear elastic and have isotropic properties. A typical pipe-patch arrangement (saddle fusion patch) and related dimensions can be found on Figure 7. Additionally, the pipe and patch material mechanical properties can be found on Table 3. 4.2. Results of Finite Element Solution for the Patch Repaired Pipe Energies 2018, 11, x 9 of 25 Figure 7. polyethylene pipe and patch arrangement. (a) Two dimensional view (dimensions in mm) of the cross section of the medium density polyethylene gas pipe. (b) Three dimensional model of patch repaired pipe showing patch dimensions [8–12,41]. Figure 7. Polyethylene pipe and patch arrangement. (a) Two dimensional view (dimensions in mm) of the cross section of the medium density polyethylene gas pipe. (b) Three dimensional model of patch repaired pipe showing patch dimensions [8–12,41]. Figure 7. polyethylene pipe and patch arrangement. (a) Two dimensional view (dimensions in mm) of the cross section of the medium density polyethylene gas pipe. (b) Three dimensional model of patch repaired pipe showing patch dimensions [8–12,41]. Figure 7. Polyethylene pipe and patch arrangement. (a) Two dimensional view (dimensions in mm) of the cross section of the medium density polyethylene gas pipe. (b) Three dimensional model of patch repaired pipe showing patch dimensions [8–12,41]. Table 3. Mechanical properties of the PE80 pipe and PE100 patch materials [8–12,41]. Table 3. Mechanical properties of the PE80 pipe and PE100 patch materials [8–12,41]. Table 3. Mechanical properties of the PE80 pipe and PE100 patch materials [8–12,41]. Table 3. Mechanical properties of the PE80 pipe and PE100 patch materials [8–12,41]. Table 3. Mechanical properties of the PE80 pipe and PE100 patch materials [8–12,41]. Material Mechanical Properties Elasticity Module (MPa) Yield Stress (MPa) Poisson Ratio Thermal Expansion Coefficient ( 𝟏 ℃) PE80 427 19.3 0.35 0.0002 PE100 1034 24.8 0.4 0.0002 Table 3. Mechanical properties of the PE80 pipe and PE100 patch materials [8–12,41]. Material Mechanical Properties Elasticity Module (MPa) Yield Stress (MPa) Poisson Ratio Thermal Expansion Coefﬁcient ( 1 °C) PE80 427 19.3 0.35 0.0002 PE100 1034 24.8 0.4 0.0002 In the next section we investigate the aforementioned models with more details by superimposing various thermal loads in the form of temperature variations to the mechanical loads including surcharge loads, soil column weight, soil-pipe interaction, and inside pressure of 4 bar applied to the repaired defected polyethylene pipe and its patch. The results of previous section show that the maximum Von Mises stress values are well below the allowable stresses (based on the design factor of 1.5) for working life of 50 years at 35 °C. Additionally lower values of safety factor (design factor) are applicable to design underground gas pipes. 4.2. Results of Finite Element Solution for the Patch Repaired Pipe By selecting the value of 1.25 for safety factor, the values of allowable stresses for working life of 50 years at 35 °C will be 5.2 MPa and 6.5 MPa for PE80 pipe and PE100 patch materials respectively. We prefer to use these values for the In the next section we investigate the aforementioned models with more details by superimposing various thermal loads in the form of temperature variations to the mechanical loads including surcharge loads, soil column weight, soil-pipe interaction, and inside pressure of 4 bar applied to the repaired defected polyethylene pipe and its patch. The results of previous section show that the maximum Von Mises stress values are well below the allowable stresses (based on the design factor of 1.5) for working life of 50 years at 35 ◦C. Additionally lower values of safety factor (design factor) are applicable to design underground gas pipes. By selecting the value of 1.25 for safety factor, the values Energies 2018, 11, 2818 9 of 24 of allowable stresses for working life of 50 years at 35 ◦C will be 5.2 MPa and 6.5 MPa for PE80 pipe and PE100 patch materials respectively. We prefer to use these values for the following section of the research. Before starting to investigate the complicated case of underground problem and soil-structure interaction for buried patch repaired polyethylene gas pipe, we perform veriﬁcation by ﬁnite element modeling of the patch repaired PE pipe not buried under the ground and only loaded to a ﬁnal internal pressure of 4 bars. The results are well described in the following section. 4.2.1. Finite Element Modeling and Solution for an Unburied Patch Repaired Pipe with Circular Wall Holes In this section, we use finite element modeling to evaluate stress distribution in a defected unburied MDPE gas pipe that is repaired by applying a 76.2 mm (3”) long saddle fusion patch to verify the applicability and accuracy of the computer simulation to solve patch repaired pipe problems and to find the optimum finite element model dimension for pipe length. Therefore, a 4-inch (nominal diameter) unburied MDPE pipe and its related patch, which is subjected only to a final pressure of 405,300 Pa (4 bar), is modeled computationally. 4.2. Results of Finite Element Solution for the Patch Repaired Pipe The modeled pipe had circular holes with various diameters (5, 7.5, 10, 12.5, 15, 17.5, 20 mm) at the pipe crown, which are repaired by a saddle fusion patch made of high-density polyethylene material. It is assumed that the related patch is electro fused to the pipe, which creates a strong continuous connection. A geometric computer model of the pipe and its related patch was created in ANSYS so that the center of the coordinate system aligned with the center of the pipe and its patch and the z-axis aligned with the axis of the pipe and its patch. Also, the appropriate boundary conditions selected for the finite elements in this section is as Equations (1) and (2): @ z = ± l 2; uz = 0 (1) @ x = 0; ux = 0 (2) @ z = ± l 2; uz = 0 (1) (1) @ x = 0; ux = 0 (2) (2) The plane strain constraint in the direction of the pipe axis (z-axis) is assumed for the long pipe where this constraint is applied to the nodes located at the related coordinates as seen in Equation (1). Additionally, Equation (2) is well applied because of the model and loading condition symmetry in y-z plane. buried Patch Repair Finite Element Model Evaluation Unburied Patch Repair Finite Element Model Evaluation The good performance of the present method is demonstrated in Table 4 by a number of numerical examples. Maximum values of hoop and Von Mises stresses in pipe and its related patch for different hole diameters and pipe lengths derived by the ﬁnite element method are presented. Table 4. Comparing maximum stress values in pipe and patch under internal pressure, investigating the pipe length size. Table 4. Comparing maximum stress values in pipe and patch under internal pressure, investigating the pipe length size. the pipe length size. Model Dimension Maximum Stress in Pipe Maximum Stress in Patch Hole Diameter (mm) Pipe Length (m) Number of Elements Von Mises Stress (MPa) Hoop Stress (MPa) Von Mises Stress (MPa) Hoop Stress (MPa) 5 0.5 17,520 2.94 2.88 3.4 3.75 1.5 18,889 2.93 2.87 3.39 3.74 24,732 2.92 2.86 3.4 3.73 7.5 0.5 20,968 3.01 2.92 3.16 3.54 1.5 22,337 3 2.91 3.16 3.57 30,056 3.02 2.93 3.14 3.56 10 0.5 19,591 3.04 2.93 3.36 3.61 1.5 20,960 3.03 2.92 3.36 3.61 24,231 3.02 2.91 3.37 3.6 10 of 24 Energies 2018, 11, 2818 Table 4. Cont. Model Dimension Maximum Stress in Pipe Maximum Stress in Patch Hole Diameter (mm) Pipe Length (m) Number of Elements Von Mises Stress (MPa) Hoop Stress (MPa) Von Mises Stress (MPa) Hoop Stress (MPa) 12.5 0.5 9,726 3 2.9 3.56 3.73 1.5 11,095 2.99 2.89 3.55 3.72 17,928 2.98 2.88 3.56 3.71 15 0.5 9,726 3 2.88 3.83 3.66 1.5 11,095 2.99 2.87 3.82 3.65 22,118 2.98 2.89 3.81 3.66 17.5 0.5 10,769 3.02 2.85 3.73 3.92 1.5 12,138 3.01 2.84 3.73 3.92 18,380 3.02 2.86 3.72 3.91 20.0 0.5 10,769 3.01 3.83 3.86 3.98 1.5 12,138 3 3.82 3.85 3.98 18,380 3.02 3.82 3.84 3.99 Maximum Stress in Pipe Additionally, to obtain accurate results, the postulated model was run based on different mesh conditions and number of elements. Considering the obtained solutions for the hoop and Von Mises stresses from the ﬁnite element solution it can be concluded that: 1. For the patch repaired pipe models and considering the same hole diameter it is clear that for models longer than 0.5 m, increasing the pipe length will not affect the maximum hoop and Von Mises stress values at the damage location in the pipe and also in the patch, signiﬁcantly. 1. Unburied Patch Repair Finite Element Model Evaluation For the patch repaired pipe models and considering the same hole diameter it is clear that for models longer than 0.5 m, increasing the pipe length will not affect the maximum hoop and Von Mises stress values at the damage location in the pipe and also in the patch, signiﬁcantly. 2. Increasing the hole diameter will increase the maximum value of hoop and Von Mises stresses in the patch. On the other hand increasing the hole diameter will not increase the maximum value of hoop and Von Mises stresses in the pipe signiﬁcantly because of the reinforcing effect of the patch. 3. Comparing the results of various number of elements shows that if the distribution of the implemented ﬁnite elements is done properly, increasing the number of elements will not affect the results in pipe and patch signiﬁcantly. 4. According to the results of ﬁnite element solution, seen in Figure 8, the maximum values of hoop and Von Mises stresses in the perforated pipe occur on the internal surface (inside) of the pipe and around the hole at the sides of the defect location. Based on these results, the selected mesh type, ﬁnite elements, loading conditions, and applied boundary conditions in this section are appropriate for carrying out the next stages of the research and can be used to perform ﬁnite element modeling of the other three types of patch arrangements which will be discussed in the next section. Additionally, in order to obtain accurate results the value of 1.5 m is used for the pipe length in the next modeling stages. 11 of 24 lue Energies 2018, 11, 2818 which will be d of 1 5 m is used nergies 2018, 11, 2818 11 of y, of 1.5 m is used for the pipe length in the next modeling stages. (a) (b) (c) Figure 8. Finite element modeling of sectioned pipe and socket to show Von Mises stress distribution. (a) Mesh generation and the application of internal pressure. (b) Stress distribution in PE pipe with 1″ diameter circular hole. (c) Stress distribution in PE pipe and patch. C i h R l f Diff P h A Figure 8. Finite element modeling of sectioned pipe and socket to show Von Mises stress distribution. (a) Mesh generation and the application of internal pressure. (b) Stress distribution in PE pipe with 1” diameter circular hole. Unburied Patch Repair Finite Element Model Evaluation (c) Stress distribution in PE pipe and patch. p p g g g (a) (a) (c) (b) (b) (c) Figure 8. Finite element modeling of sectioned pipe and socket to show Von Mises stress distribution. (a) Mesh generation and the application of internal pressure. (b) Stress distribution in PE pipe with 1″ diameter circular hole. (c) Stress distribution in PE pipe and patch. Figure 8. Finite element modeling of sectioned pipe and socket to show Von Mises stress distribution. (a) Mesh generation and the application of internal pressure. (b) Stress distribution in PE pipe with 1” diameter circular hole. (c) Stress distribution in PE pipe and patch. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Curves for maximum Von Mises stresses in various patch arrangements for unburied pipe d h Figure 9. Curves for maximum Von Mises stresses in various patch arrangements for unburied pipe and patch arrangement. Figure 9. Curves for maximum Von Mises stresses in various patch arrangements for unburied pipe d h Figure 9. Curves for maximum Von Mises stresses in various patch arrangements for unburied pipe and patch arrangement. and patch arrangement. Table 5. Number of finite elements used to model unburied pipe and its patch for various patch Table 5. Number of ﬁnite elements used to model unburied pipe and its patch for various patch arrangements. and patch arrangement. Table 5. Number of finite elements used to model unburied pipe and its patch for various patch Table 5. Number of ﬁnite elements used to model unburied pipe and its patch for various patch arrangements. and patch arrangement. Table 5. Number of finite elements used to model unburied pipe and its patch for various patch Table 5. Number of ﬁnite elements used to model unburied pipe and its patch for various patch arrangements. arrangements. Hole Diameter (mm) Number of Finite Elements Saddle Fusion Patch Semi-Cylindrical Patch Circular Partial Patch Square Partial Patch 5 18,889 18,044 8,198 6,823 7.5 22,.337 20,739 8,198 6,791 10 20,960 20,004 8,198 6,099 12.5 11,095 10,619 8,198 4,933 15 11,095 10,619 8,198 4,933 17.5 12,138 10,881 8,198 5,426 20 12 138 10 881 8 198 5 426 Hole Diameter (mm) Number of Finite Elements Saddle Fusion Patch Semi-Cylindrical Patch Circular Partial Patch Square Partial Patch 5 18,889 18,044 8,198 6,823 7.5 22,.337 20,739 8,198 6,791 10 20,960 20,004 8,198 6,099 12.5 11,095 10,619 8,198 4,933 15 11,095 10,619 8,198 4,933 17.5 12,138 10,881 8,198 5,426 20 12,138 10,881 8,198 5,426 Table 6. Maximum Von Mises stress reduction percentage in the holed pipe for various patch 17.5 12,138 10,881 8,198 5,426 20 12,138 10,881 8,198 5,426 Table 6. Maximum Von Mises stress reduction percentage in the holed pipe for various patch arrangements under internal pressure. Table 6. Maximum Von Mises stress reduction percentage in the holed pipe for various patch arrangements under internal pressure. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Before investigating and discussing the complicated problem of buried patch repaired pipelines, for the better understanding, we decide to evaluate the efficiency of the patch repair in the reinforcement of the defected unburied pipe. Four different patch arrangements in the form of semi-cylindrical, circular-partial, square-partial and saddle fusion patches are modeled and discussed. The same procedure described in previous section is followed and used to calculate the Before investigating and discussing the complicated problem of buried patch repaired pipelines, for the better understanding, we decide to evaluate the efﬁciency of the patch repair in the reinforcement of the defected unburied pipe. Four different patch arrangements in the form of semi-cylindrical, circular-partial, square-partial and saddle fusion patches are modeled and discussed. The same procedure described in previous section is followed and used to calculate the maximum stresses in the patch and pipe loaded to an internal gas pressure of 4 bar. The number of ﬁnite elements used in this study to model unburied pipe and its patch for various patch arrangements are presented in Table 5. The plotted curves in Figure 9 show the variation of maximum Von Mises stress in the defected patch repaired PE pipe for each kind of patch arrangement and also in unrepaired defected pipe, versus various hole diameters in the pipe. Additionally, the reduction percentage in maximum stress values resulted from the application of a special patch repair are shown and compared in Table 6. Energies 2018, 11, 2818 versus various hole values resulted from 12 of 24 0 2 4 6 8 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Before repair Allowable stress for a working life of 50 years at 35 °C Circular partial patch Square partial patch Semi-cylindrical patch Saddle-fusion patch Figure 9. Curves for maximum Von Mises stresses in various patch arrangements for unburied pipe Figure 9. Curves for maximum Von Mises stresses in various patch arrangements for unburied pipe and patch arrangement 0 2 4 6 8 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Before repair Allowable stress for a working life of 50 years at 35 °C Circular partial patch Square partial patch Semi-cylindrical patch Saddle-fusion patch Figure 9. Curves for maximum Von Mises stresses in various patch arrangements for unburied pipe and patch arrangement. Figure 9. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Hole Diameter (mm) Maximum Von Mises Stress Reduction in Pipe (%) Square Partial Patch Circular Partial Patch Saddle Fusion Patch Semi-Cylindrical Patch 5 12.5 7.9 48.6 46.5 7.5 17 15.9 50.9 48.9 10 20.4 20.5 53.2 51.1 12.5 23.4 25.1 55.4 53.5 15 26.3 29.7 57.1 54.8 Hole Diameter (mm) Maximum Von Mises Stress Reduction in Pipe (%) Square Partial Patch Circular Partial Patch Saddle Fusion Patch Semi-Cylindrical Patch 5 12.5 7.9 48.6 46.5 7.5 17 15.9 50.9 48.9 10 20.4 20.5 53.2 51.1 12.5 23.4 25.1 55.4 53.5 15 26.3 29.7 57.1 54.8 17.5 29.9 32.3 59.2 57 20 33.5 34.2 61.4 59.3 Table 6. Maximum Von Mises stress reduction percentage in the holed pipe for various patch arrangements under internal pressure. Hole Diameter (mm) Maximum Von Mises Stress Reduction in Pipe (%) Square Partial Patch Circular Partial Patch Saddle Fusion Patch Semi-Cylindrical Patch 17.5 29.9 32.3 20 33.5 34.2 It can be implied from Figure 9 and Table 6 that: 17.5 29.9 32.3 59.2 57 20 33.5 34.2 61.4 59.3 It can be implied from Figure 9 and Table 6 that: 1. All four patch arrangements have signiﬁcant effect on the reduction of the maximum Von Mises stresses in the reinforcement of the damaged section of the pipe. 1. All four patch arrangements have signiﬁcant effect on the reduction of the maximum Von Mises stresses in the reinforcement of the damaged section of the pipe. 2. The results of Table 6 show that the percentage of the maximum Von Mises stress reduction in the pipe increases for larger hole diameters. 2. The results of Table 6 show that the percentage of the maximum Von Mises stress reduction in the pipe increases for larger hole diameters. 3. The most effective patch in the reinforcement of the pipe is saddle fusion arrangement which reduces the maximum Von Mises stresses about 48%. For square-partial patch, although the effect is notable, it is less than the other 3 kinds of patches. This patch reduces the maximum Von Mises stresses by about 8%. 3. The most effective patch in the reinforcement of the pipe is saddle fusion arrangement which reduces the maximum Von Mises stresses about 48%. For square-partial patch, although the effect is notable, it is less than the other 3 kinds of patches. This patch reduces the maximum Von Mises stresses by about 8%. 4. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements The aforementioned curves indicate that semi-cylindrical patch and saddle fusion patch show similar trends. Additionally, the results of circular-partial patch and square-partial patches are close to each other. 4. The aforementioned curves indicate that semi-cylindrical patch and saddle fusion patch show similar trends. Additionally, the results of circular-partial patch and square-partial patches are close to each other. Energies 2018, 11, 2818 13 of 24 5. It can be implied that, the reinforcement effect of semi-cylindrical patch and saddle fusion patch are signiﬁcantly more than the other two patch arrangements. 5. It can be implied that, the reinforcement effect of semi-cylindrical patch and saddle fusion patch are signiﬁcantly more than the other two patch arrangements. 4.2.2. Finite Element Modeling and Solution for a Buried Patch Repaired Pipe with Circular Wall Holes 2. Finite Element Modeling and Solution for a Buried Patch Repaired Pipe with Circular Wall Holes In this section we continue to investigate the studies in [9,10] with more details and under more critical loading conditions by superimposing various thermal loads in the form of temperature variations and the mechanical loads including surcharge loads, soil column weight, soil-pipe interaction and inside pressure of 4 bar applied to the repaired defected polyethylene pipe and its patch. The trench dimensions and pipe surroundings are selected based on Figure 10. Also, soil grades which are selected based on ASTM standards along with model dimensions are depicted on the computer simulation model in Figure 10. The results of previous section show that the maximum Von Mises stress values are well below the allowable stresses (based on the design factor of 1.5) for working life of 50 years at 35 ◦C. Additionally lower values of safety factor (design factor) are applicable to design underground gas pipes. By selecting the value of 1.25 for safety factor, the values of allowable stresses for working life of 50 years at 35 ◦C will be 5.2 MPa and 6.5 MPa for PE80 pipe and PE100 patch materials respectively. We prefer to use these values for the following section of the research. In order to perform a proper ﬁnite element study on the underground structure, it is important to ﬁnd an appropriate model dimension which the obtained results not to be dependent on the model size. Therefore, the ﬁnite element simulation of various model sizes are calculated and compared in Table 7. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Finite Element Model Speciﬁcations Maximum Stress in Pipe Maximum Stress in Patch Hole Diameter (mm) Model Upper Surface Dimension z × x = A Number Finite Elements Von Mises Stress (MPa) Hoop Stress (MPa) Von Mises Stress (MPa) Hoop Stress (MPa) 5 0.50 m × 0.4143 m 38,986 3.99 4.09 3.39 3.77 0.50 m ×1.00 m 49,182 3.36 3.35 3.37 3.73 1.50 m × 1.00 m 57,594 3.35 3.34 3.37 3.73 0.50 m × 1.50 m 73,537 3.25 3.22 3.36 3.71 1.50 m × 1.50 m 81,068 3.24 3.22 3.35 3.7 20 0.50 m × 0.4143 m 27,036 4.01 3.89 3.95 4.11 0.50 m × 1.00 m 46,977 3.38 3.22 3.87 3.98 1.50 m × 1.00 m 62,011 3.37 3.22 3.86 3.98 0.50 m × 1.50 m 74,326 3.28 3.11 3.84 3.94 1.50 m × 1.50 m 82,994 3.27 3.1 3.38 3.95 The Simultaneous Effects of Thermo-Mechanical Loads on Semi-Cylindrical Patch Arrangement The Simultaneous Effects of Thermo-Mechanical Loads on Semi-Cylindrical Patch Arrangement Figure 11 shows the variations in the maximum values of Von Mises stresses in the buried PE80 pipe that is repaired by a 76.2 mm long semi-cylindrical patch arrangement, versus defect sizes in the form of circular holes under simultaneous effects of mechanical loads in the form of soil load, 4 bar internal pressure, vehicle wheel load and various thermal loads in the form of daily and seasonal temperature variations. Energies 2018, 11, x 15 of 25 0 2 4 6 8 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Before patch repair Pipe and patch temperature change = +22 °C Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = –22 °C Figure 11. Maximum Von Mises stress variations in the buried pipe for various size circular hole defects repaired by semi-cylindrical patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Energies 2018, 11, x 14 of 25 Figure 10. Finite element model of the patched pipe with surrounding soil subjected too internal pressure. GW: Well-graded gravels; SM: Silty sands [11]. Figure 10. Finite element model of the patched pipe with surrounding soil subjected too internal pressure. GW: Well-graded gravels; SM: Silty sands [11]. Figure 10. Finite element model of the patched pipe with surrounding soil subjected too internal pressure. GW: Well-graded gravels; SM: Silty sands [11]. Figure 10. Finite element model of the patched pipe with surrounding soil subjected too internal pressure. GW: Well-graded gravels; SM: Silty sands [11]. The results of maximum Von Mises and hoop stresses in the pipe and its patch show that the upper surface dimension of z × x = (1.5 m) × (1.5 m) is appropriate to perform finite element modeling of this research, since larger models will not affect the stress values significantly. The results of maximum Von Mises and hoop stresses in the pipe and its patch show that the upper surface dimension of z × x = (1.5 m) × (1.5 m) is appropriate to perform ﬁnite element modeling of this research, since larger models will not affect the stress values signiﬁcantly. 14 of 24 Energies 2018, 11, 2818 Table 7. Simultaneous effect of soil column weight and internal pressure on maximum hoop and Von Mises stresses in defected pipe and patch for various x and z values. Table 7. Simultaneous effect of soil column weight and internal pressure on maximum hoop and Von Mises stresses in defected pipe and patch for various x and z values. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements 0 2 4 6 8 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Before patch repair Pipe and patch temperature change = +22 °C Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = –22 °C Figure 11. Maximum Von Mises stress variations in the buried pipe for various size circular hole defects repaired by semi-cylindrical patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). 8 Figure 11. Maximum Von Mises stress variations in the buried pipe for various size circular hole defects repaired by semi-cylindrical patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). Figure 11. Maximum Von Mises stress variations in the buried pipe for various size circular hole defects repaired by semi-cylindrical patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). The upmost curve shows the values of maximum Von Mises stress for the defected pipe before the application of patch repair and indicates that increasing hole diameter increases the maximum Von Mises stress significantly. The comparison of this curve with allowable stress value for a working life of 50 years (dashed line) shows that the imposed stresses in defected pipe are significantly higher than allowable stress values. For the other four curves which show the maximum Von Mises stress values in the defected polyethylene pipe repaired by semi-cylindrical patch, the stress values are well below the results of unrepaired defected pipe and also well below the allowable stress value. This means the aforementioned patch repair can strengthen the defected part of the pipe as well to transfer the gas fuel. Additionally, for the investigated defects at a constant temperature variation, the maximum Von Mises stresses remain approximately constant even by increasing the hole size. The curves for −22 °C, −15 °C, 0 °C (no temperature changes), +5 °C, and +22 °C temperature changes shows approximately similar trend with hole diameter increase where for these cases the minimum Von Mises stresses are not increasing significantly for larger hole sizes. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Generally stating the maximum Von Mises stresses in the pipe increases for higher temperature The upmost curve shows the values of maximum Von Mises stress for the defected pipe before the application of patch repair and indicates that increasing hole diameter increases the maximum Von Mises stress signiﬁcantly. The comparison of this curve with allowable stress value for a working life of 50 years (dashed line) shows that the imposed stresses in defected pipe are signiﬁcantly higher than allowable stress values. For the other four curves which show the maximum Von Mises stress values in the defected polyethylene pipe repaired by semi-cylindrical patch, the stress values are well below the results of unrepaired defected pipe and also well below the allowable stress value. This means the aforementioned patch repair can strengthen the defected part of the pipe as well to transfer the gas fuel. Additionally, for the investigated defects at a constant temperature variation, the maximum Von Mises stresses remain approximately constant even by increasing the hole size. The curves for −22 ◦C, −15 ◦C, 0 ◦C (no temperature changes), +5 ◦C, and +22 ◦C temperature changes 15 of 24 Energies 2018, 11, 2818 shows approximately similar trend with hole diameter increase where for these cases the minimum Von Mises stresses are not increasing signiﬁcantly for larger hole sizes. Generally stating, the maximum Von Mises stresses in the pipe increases for higher temperature increases. That means the patch is more effective in reinforcing defected pipe for lower temperature changes. For example, for a ﬁxed hole diameter of 20 mm, the stress reduction percentage in the pipe wall are 53%, 53%, 46%, 44%, and 35% for the temperature changes of −22 ◦C, −15 ◦C, 0 ◦C, +5 ◦C, and +22 ◦C respectively. Also, the patch is more effective in reinforcing defected pipe for larger hole diameters. The maximum stress values are related to the seasonal temperature increase of +22 ◦C. Additionally, Figure 12 presents maximum values of Von Mises stresses in the 3” long semi-cylindrical patch arrangement versus pipe circular hole diameters under simultaneous effects of mechanical loads and various thermal loads in the form of daily and seasonal temperature variations. Based on the obtained results, the temperature variations have a signiﬁcant effect on the maximum Von Mises stresses in the semi-cylindrical patch itself. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements The lowest curve on Figure 12 which shows the lowest values of maximum Von Mises stresses belongs to the situation where no temperature change is imposed to the pipe and patch at the burial depth under the ground that means it is the case with the minimum valves of the induced maximum Von Mises stresses compared to the other temperature changes. Additionally, the uppermost curve ﬁts the data obtained for the maximum temperature change (+22 ◦C temperature increase, based on seasonal variations), which shows the case with highest values of induced maximum Von Mises stresses among others. For the semi-cylindrical PE100 patch material it can be implied that higher temperature changes (both temperature increase and temperature decrease) impose higher maximum Von Mises stresses. Also, for the cases of low temperature changes including 0 ◦C, +5 ◦C, the maximum Von Mises stresses in the patch show an increase trend by increasing the pipe hole diameter, while for the cases of higher values of temperature changes including −15 ◦C, −22 ◦C, +22 ◦C the maximum Von Mises stresses will remain approximately constant even for larger hole diameters. Based on the calculated results, the semi-cylindrical patch can reinforce the proposed circular hole modeled defects efﬁciently. The only problem is that for higher temperature changes, the maximum Von Mises stress values in the patch itself can be critical which requires more research and investigation on the other patch conﬁgurations. Energies 2018, 11, x 16 of 25 variations), which shows the case with highest values of induced maximum Von Mises stresses among others. For the semi-cylindrical PE100 patch material it can be implied that higher temperature changes (both temperature increase and temperature decrease) impose higher maximum Von Mises stresses. Also, for the cases of low temperature changes including 0 °C, +5 °C, the maximum Von Mises stresses in the patch show an increase trend by increasing the pipe hole diameter, while for the cases of higher values of temperature changes including −15 °C, −22 °C, +22 °C the maximum Von Mises stresses will remain approximately constant even for larger hole diameters. Based on the calculated results, the semi-cylindrical patch can reinforce the proposed circular hole modeled defects efficiently. The only problem is that for higher temperature changes, the maximum Von Mises stress values in the patch itself can be critical which requires more research and investigation on the other patch configurations. Figure 12. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Maximum Von Mises stress in the semi-cylindrical patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). 0 3 6 9 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Pipe and patch temperature change = +22 °C Pipe and patch temperature change = –22 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Figure 12. Maximum Von Mises stress in the semi-cylindrical patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). Figure 12. Maximum Von Mises stress in the semi-cylindrical patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads) Figure 12. Maximum Von Mises stress in the semi-cylindrical patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). Energies 2018, 11, 2818 16 of 24 16 of 24 The Simultaneous Effects of Thermo-Mechanical Loads on Circular Partial Patch Arrangement The Simultaneous Effects of Thermo-Mechanical Loads on Circular Partial Patch Arrangement In this section in order to more investigate to ﬁnd a proper patch, a circular partial patch arrangement is designed and its reinforcing effect on the damaged underground PE gas pipe ar being studied. The results of the FE simulation in the form of variations in the maximum values o Von Mises stresses in the buried PE80 pipe that is repaired by the circular partial patch arrangemen versus defect sizes in the form of circular hole under simultaneous effects of the previously mentioned mechanical loads and various thermal loads are depicted in Figure 13. Similar to the previous case, as expected the upmost curve is related to the values of maximum Von Mises stresses for the defected pipe prior to circular-partial patch application and shows that for larger hole diameters, the maximum Von Mises stress increases signiﬁcantly. Based on the results of the other ﬁve presented curves which shows the maximum Von Mises stresses in the defected polyethylene pipe repaired by circular-partial patch and comparing these curves with dashed line that indicates allowable stress values, the imposed stress values are well below the results of unrepaired defected pipe but slightly above the allowable stress values. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Considering the case of 20 mm hole diameter, the stress reduction percentage in the pipe wall are 31%, 29%, 26%, 24%, and 22% for the temperature changes of −22 ◦C, −15 ◦C, 0 ◦C, +5 ◦C, and +22 ◦C respectively. The results imply that for the application of the aforementioned patch repair to reinforce the damaged part of the pipe some stress relief mechanisms must be employed too. Additionally, for the investigated defects at a constant temperature variation, the maximum on Mises stresses remain approximately constant even by increasing the hole size. The curves for −22 ◦C, −15 ◦C, 0 ◦C, +5 ◦C, and +22 ◦C temperature changes shows approximately similar trend with hole diameter increase. The maximum stress values are related to the seasonal temperature increase of +22 ◦C. Energies 2018, 11, x 17 of 25 mm hole diameter, the stress reduction percentage in the pipe wall are 31%, 29%, 26%, 24%, and 22% for the temperature changes of −22 °C, −15 °C, 0 °C, +5 °C, and +22 °C respectively. The results imply that for the application of the aforementioned patch repair to reinforce the damaged part of the pipe some stress relief mechanisms must be employed too. Additionally, for the investigated defects at a constant temperature variation, the maximum on Mises stresses remain approximately constant even by increasing the hole size. The curves for −22 °C, −15 °C, 0 °C, +5 °C, and +22 °C temperature changes shows approximately similar trend with hole diameter increase. The maximum stress values are related to the seasonal temperature increase of +22 °C. Figure 13. Maximum Von Mises stress variations in the buried pipe for various size circular hole defects repaired by circular partial patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). 2 4 6 8 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Before patch repair Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = –22 °C Pipe and patch temperature change = +22 °C Figure 13. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Maximum Von Mises stress variations in the buried pipe for various size circular hole defects repaired by circular partial patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). Maximum Von Mises stress (MPa) Figure 13. Maximum Von Mises stress variations in the buried pipe for various size circular hole defects repaired by circular partial patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations) Figure 13. Maximum Von Mises stress variations in the buried pipe for various size circular hole defects repaired by circular partial patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). p Additionally, the maximum values of Von Mises stresses in the circular partial patch arrangement versus pipe circular hole diameters under simultaneous effects of aforementioned mechanical loads and various thermal loads in the form of daily and seasonal temperature variations are presented in Figure 14. According to the obtained results, the temperature variations have significant effect on the maximum Von Mises stress variations in the circular partial patch. The l Fi 14 b l h i i h h i i d h Additionally, the maximum values of Von Mises stresses in the circular partial patch arrangement versus pipe circular hole diameters under simultaneous effects of aforementioned mechanical loads and various thermal loads in the form of daily and seasonal temperature variations are presented in Figure 14. According to the obtained results, the temperature variations have signiﬁcant effect on the maximum Von Mises stress variations in the circular partial patch. The lowest curve on Figure 14 17 of 24 Energies 2018, 11, 2818 belongs to the situation where no temperature change is imposed to the pipe and patch at the burial depth under the ground that means it is the case with the minimum valves of the induced maximum Von Mises stresses. Additionally, the uppermost curve ﬁts the data obtained for the maximum temperature change (+22 ◦C temperature decrease, based on seasonal variations), which shows the case with highest values of induced maximum Von Mises stresses among others. For the circular partial PE100 patch material it can be implied that higher temperature changes (both temperature increase and temperature decrease) impose higher maximum Von Mises stresses. changes up to the mentioned values. The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement The same procedure which was discussed for semi-cylindrical and circular partial patches in the two previous sections is used to investigate square partial patch arrangement by finite element method. The results of Ansys simulation for the variations in the maximum values of Von Mises stresses in the buried PE80 pipe that is repaired by a square partial patch arrangement, versus defect sizes in the form of circular hole under simultaneous effects of mechanical loads in the form of soil load, 4 bar internal pressure, vehicle wheel load, and various thermal loads in the form of daily and seasonal temperature variations are depicted in Figure 15 The same procedure which was discussed for semi-cylindrical and circular partial patches in the two previous sections is used to investigate square partial patch arrangement by ﬁnite element method The results of Ansys simulation for the variations in the maximum values of Von Mises stresses in the buried PE80 pipe that is repaired by a square partial patch arrangement, versus defect sizes in the form of circular hole under simultaneous effects of mechanical loads in the form of soil load, 4 bar internal pressure, vehicle wheel load, and various thermal loads in the form of daily and seasonal temperature variations are depicted in Figure 15. seasonal temperature variations are depicted in Figure 15. Comparing the curves showing the results of maximum Von Mises stress values for various temperature changes and the upmost curve which is related to the defected unrepaired pipe, shows that square-partial patch arrangement plays an important role in decreasing maximum stress values and strengthening the defected part of the pipe. For more understanding, considering the case of the 20 mm hole diameter, the results show the stress reduction percentage in the pipe wall are 29.5%, 29.2%, 25.6%, 24.3%, and 21.7% for the temperature changes of −22 °C, −15 °C, 0 °C, +5 °C, and +22 °C respectively, which shows that patch has more reinforcing effects for lower temperature changes. Comparing the Results of Different Patch Arrangements Comparing the Results of Different Patch Arrangements Energies 2018, 11, x 18 of 25 Fi 14 M i V Mi t i th i l ti l t h f i i f i l h l 0 3 6 9 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Pipe and patch temperature change = –22 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Pipe and patch temperature change = +22 °C Figure 14. Maximum Von Mises stress in the circular partial patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). Maximum Von Mises stress (MPa) Figure 14. Maximum Von Mises stress in the circular partial patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). Figure 14. Maximum Von Mises stress in the circular partial patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). For higher temperature changes, including a +22 °C temperature increase and a −22 °C temperature decrease, the maximum Von Mises stresses are above the allowable stress which means applying circular partial patches in these areas cannot be suggested. For lower temperature changes including −15 °C, 0 °C, and +5 °C the maximum Von Mises stresses are well below the allowable stress limit, which means the circular partial patch is applicable in the areas with the temperature For higher temperature changes, including a +22 ◦C temperature increase and a −22 ◦C temperature decrease, the maximum Von Mises stresses are above the allowable stress which means applying circular partial patches in these areas cannot be suggested. For lower temperature changes including −15 ◦C, 0 ◦C, and +5 ◦C the maximum Von Mises stresses are well below the allowable stress limit, which means the circular partial patch is applicable in the areas with the temperature changes up to the mentioned values. changes up to the mentioned values. The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement E th h th ti l t h h i ifi t ff t i f i th d f t d i d Comparing the curves showing the results of maximum Von Mises stress values for various temperature changes and the upmost curve which is related to the defected unrepaired pipe, shows that square-partial patch arrangement plays an important role in decreasing maximum stress values and strengthening the defected part of the pipe. For more understanding, considering the case of the 20 mm hole diameter, the results show the stress reduction percentage in the pipe wall are 29.5%, 29.2%, 25.6%, 24.3%, and 21.7% for the temperature changes of −22 ◦C, −15 ◦C, 0 ◦C, +5 ◦C, and +22 ◦C respectively, which shows that patch has more reinforcing effects for lower temperature changes. Even 18 of 24 18 of 24 Energies 2018, 11, 2818 though, the square-partial patch has signiﬁcant effect on reinforcing the defected pipe and decreasing the maximum Von Mises stress values, but comparing the results with the dashed line of allowable stress shows that the induced maximum Von Mises stresses are slightly higher than allowable stress values. Therefore, if we decide to use this kind of patch, more researches should be conducted to obtain some stress relief mechanisms. Additionally, the curves show similar trends for different temperature changes. Furthermore, comparing these curves with the results obtained for circular-partial patch shows approximately similar trends between these two cases. Energies 2018, 11, x 19 of 25 conducted to obtain some stress relief mechanisms. Additionally, the curves show similar trends for different temperature changes. Furthermore, comparing these curves with the results obtained for circular-partial patch shows approximately similar trends between these two cases. Figure 15. Maximum Von Mises stress variations in the buried pipe for various sizes of circular hole defects repaired by square partial patch (the simultaneous effect of soil load, internal pressure, hi l h l l d d i i ) 2 4 6 8 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Before patch repair Pipe and patch temperature change = +22 °C Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = –22 °C Figure 15. changes up to the mentioned values. The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement Maximum Von Mises stress variations in the buried pipe for various sizes of circular hole defects repaired by square partial patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). Maximum Von Mises stress (MPa) Figure 15. Maximum Von Mises stress variations in the buried pipe for various sizes of circular hole defects repaired by square partial patch (the simultaneous effect of soil load, internal pressure, Figure 15. Maximum Von Mises stress variations in the buried pipe for various sizes of circular hole defects repaired by square partial patch (the simultaneous effect of soil load, internal pressure, vehicle wheel load and temperature variations). p ) Additionally, the maximum values of Von Mises stresses in the square partial patch arrangement versus pipe circular hole diameters under simultaneous effects of aforementioned mechanical loads and various thermal loads in the form of daily and seasonal temperature variations are presented in Figure 16. As can be implied, the temperature variations have a significant effect on the variation of the maximum Von Mises stresses in the square partial patch. The lowest curve on Figure 16 belongs to the situation where no temperature change is imposed to the pipe and patch at the burial depth under the ground that means it is the case with the minimum valves of the induced maximum Von Mises stresses. Additionally, uppermost curve fits the data obtained for the maximum temperature change (+22 °C temperature increase, based on seasonal variations), which shows the case with highest values of induced maximum Von Mises stresses among others. It is clear that for low temperature changes, including 0 °C and +5 °C, the maximum Von Mises stresses in the patch increases for larger hole diameters. For higher temperature changes including −15 °C, −22 °C, and +22 °C the results will remain approximately constant even by increasing hole diameter. For the square partial PE100 patch material it can be implied that higher temperature changes (both temperature increase and temperature decrease) impose higher maximum Von Mises stresses. The curves showing the data of patch stress results are below the allowable stress of the patch material except for the case of +22 °C temperature increase. changes up to the mentioned values. The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement Maximum Von Mises stress in the square partial patch for various sizes of circular hole Figure 16. Maximum Von Mises stress in the square partial patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). defects (the simultaneous effect of mechanical and thermal loads). The Simultaneous Effects of Thermo-Mechanical Loads on Saddle Fusion Patch Arrangement The Simultaneous Effects of Thermo-Mechanical Loads on Saddle Fusion Patch Arrangement In the previous parts, three patch repair configurations were introduced and discussed in details. Finite element solutions for investigating the stress distribution in the aforementioned patch repairs to find the effectiveness of the proposed patch arrangements gave us the knowledge that semi-cylindrical patch configuration can effectively reinforce the defected part of the pipe. For the sake of finding a more reliable patch configuration we decide to investigate a full-cylindrical (called saddle fusion) patch repair. In order to verify the finite element model dimension, in Section 4.2.2 the variation of maximum Von Mises stresses in the repaired defected buried polyethylene gas pipe and its 3 inch long, saddle fusion patch under mechanical loads is well discussed. Figure 17 shows the variations in the maximum values of Von Mises stresses in the buried PE80 pipe that is repaired by a saddle fusion patch arrangement, versus defect sizes in the form of circular hole under simultaneous effects of mechanical loads in the form of soil load, 4 bar internal pressure, vehicle wheel load, and various thermal loads in the form of daily and seasonal temperature variations. The comparison of the curves resulted from saddle fusion patch repair for various temperature changes of −22 °C, −15 °C, 0 °C, +5 °C, and +22 °C with the upmost curve which shows the maximum Von Mises stress values for defected pipe before repair shows that the saddle fusion patch repair effectively reinforces the damaged part of the pipe to reliably transfer the gas. Additionally, comparing the mentioned five curves with the dashed line that indicates allowable stress values for PE80 pipe material shows that the maximum Von Mises stress values are well below the allowable stress. For more clarification considering the case of 20 mm hole diameter, the results show the stress reduction percentage in the pipe wall are 54%, 56%, 45%, 43%, and 36% for the temperature changes of −22 °C, −15 °C, 0 °C, +5 °C, and +22 °C respectively. changes up to the mentioned values. The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement Additionally, the maximum values of Von Mises stresses in the square partial patch arrangement versus pipe circular hole diameters under simultaneous effects of aforementioned mechanical loads and various thermal loads in the form of daily and seasonal temperature variations are presented in Figure 16. As can be implied, the temperature variations have a signiﬁcant effect on the variation of the maximum Von Mises stresses in the square partial patch. The lowest curve on Figure 16 belongs to the situation where no temperature change is imposed to the pipe and patch at the burial depth under the ground that means it is the case with the minimum valves of the induced maximum Von Mises stresses. Additionally, uppermost curve ﬁts the data obtained for the maximum temperature change (+22 ◦C temperature increase, based on seasonal variations), which shows the case with highest values of induced maximum Von Mises stresses among others. It is clear that for low temperature changes, including 0 ◦C and +5 ◦C, the maximum Von Mises stresses in the patch increases for larger hole diameters. For higher temperature changes including −15 ◦C, −22 ◦C, and +22 ◦C the results will remain approximately constant even by increasing hole diameter. For the square partial PE100 patch material it can be implied that higher temperature changes (both temperature increase and temperature decrease) impose higher maximum Von Mises stresses. The curves showing the data of patch stress results are below the allowable stress of the patch material except for the case of +22 ◦C temperature increase. 19 of 24 f 25 19 of 24 f 25 Energies 2018, 11, 2818 Energies 2018, 11, x Figure 16. Maximum Von Mises stress in the square partial patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads) 0 2 4 6 8 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Pipe and patch temperature change = +22 °C Pipe and patch temperature change = –22 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Figure 16. Maximum Von Mises stress in the square partial patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). Figure 16. changes up to the mentioned values. The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement All the five curves show similar trends. In the previous parts, three patch repair conﬁgurations were introduced and discussed in details. Finite element solutions for investigating the stress distribution in the aforementioned patch repairs to ﬁnd the effectiveness of the proposed patch arrangements gave us the knowledge that semi-cylindrical patch conﬁguration can effectively reinforce the defected part of the pipe. For the sake of ﬁnding a more reliable patch conﬁguration we decide to investigate a full-cylindrical (called saddle fusion) patch repair. In order to verify the ﬁnite element model dimension, in Section 4.2.2 the variation of maximum Von Mises stresses in the repaired defected buried polyethylene gas pipe and its 3 inch long, saddle fusion patch under mechanical loads is well discussed. Figure 17 shows the variations in the maximum values of Von Mises stresses in the buried PE80 pipe that is repaired by a saddle fusion patch arrangement, versus defect sizes in the form of circular hole under simultaneous effects of mechanical loads in the form of soil load, 4 bar internal pressure, vehicle wheel load, and various thermal loads in the form of daily and seasonal temperature variations. The comparison of the curves resulted from saddle fusion patch repair for various temperature changes of −22 ◦C, −15 ◦C, 0 ◦C, +5 ◦C, and +22 ◦C with the upmost curve which shows the maximum Von Mises stress values for defected pipe before repair shows that the saddle fusion patch repair effectively reinforces the damaged part of the pipe to reliably transfer the gas. Additionally, comparing the mentioned ﬁve curves with the dashed line that indicates allowable stress values for PE80 pipe material shows that the maximum Von Mises stress values are well below the allowable stress. For more clariﬁcation considering the case of 20 mm hole diameter, the results show the stress reduction percentage in the pipe wall are 54%, 56%, 45%, 43%, and 36% for the temperature changes of −22 ◦C, −15 ◦C, 0 ◦C, +5 ◦C, and +22 ◦C respectively. All the ﬁve curves show similar trends. Energies 2018, 11, 2818 Energies 2018, 11, x 20 of 24 1 of 25 Figure 17. changes up to the mentioned values. The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement Maximum Von Mises stress in buried pipe for circular hole defects, saddle fusion patch repaired (simultaneous effects of soil load, internal pressure, vehicle load and temperature 0 2 4 6 8 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Before patch repair Pipe and patch temperature change = +22 °C Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = –22 °C Figure 17. Maximum Von Mises stress in buried pipe for circular hole defects, saddle fusion patch repaired (simultaneous effects of soil load, internal pressure, vehicle load and temperature variations). Figure 17. Maximum Von Mises stress in buried pipe for circular hole defects, saddle fusion patch i d ( i lt ff t f il l d i t l hi l l d d t t 0 2 4 6 8 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Before patch repair Pipe and patch temperature change = +22 °C Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = –22 °C Figure 17. Maximum Von Mises stress in buried pipe for circular hole defects, saddle fusion patch repaired (simultaneous effects of soil load, internal pressure, vehicle load and temperature variations). Figure 17. Maximum Von Mises stress in buried pipe for circular hole defects, saddle fusion patch repaired (simultaneous effects of soil load internal pressure vehicle load and temperature Figure 17. Maximum Von Mises stress in buried pipe for circular hole defects, saddle fusion patch repaired (simultaneous effects of soil load, internal pressure, vehicle load and temperature variations). variations). Figure 18 presents maximum values of Von Mises stresses in the 3 inch long, saddle fusion patch arrangement versus pipe circular hole diameters under simultaneous effects of previously mentioned mechanical and various discussed thermal loads. The obtained results show that, the temperature variations have significant effect on the maximum Von Mises stresses in the saddle fusion patch. changes up to the mentioned values. The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement similar to the 3 previously discussed patch arrangements the lowest curve on Figure 18 belongs to the situation where no temperature change is imposed to the pipe and patch at the burial depth under the ground that means it is the case with the minimum valves of the induced maximum Von Mises stresses. Additionally, the uppermost curve fits the data obtained for the maximum temperature change (+22 °C temperature increase, based on seasonal variations), which shows the case with highest values of induced maximum Von Mises stresses among others. For the saddle fusion PE100 patch material it can be implied that higher temperature changes (both temperature increase and temperature decrease) impose higher maximum Von Mises stresses. For all curves, the maximum Von Mises stress values in the patch will increase for larger hole diameters. For all cases, the maximum Von Mises stresses are well below the allowable stress limit for PE100 material which means the saddle fusion patch is well applicable to repair the proposed defects in even hot areas. Therefore the results show the application of 3” saddle fusion patch is advisable for the proposed Figure 18 presents maximum values of Von Mises stresses in the 3 inch long, saddle fusion patch arrangement versus pipe circular hole diameters under simultaneous effects of previously mentioned mechanical and various discussed thermal loads. The obtained results show that, the temperature variations have signiﬁcant effect on the maximum Von Mises stresses in the saddle fusion patch. similar to the 3 previously discussed patch arrangements the lowest curve on Figure 18 belongs to the situation where no temperature change is imposed to the pipe and patch at the burial depth under the ground that means it is the case with the minimum valves of the induced maximum Von Mises stresses. Additionally, the uppermost curve ﬁts the data obtained for the maximum temperature change (+22 ◦C temperature increase, based on seasonal variations), which shows the case with highest values of induced maximum Von Mises stresses among others. For the saddle fusion PE100 patch material it can be implied that higher temperature changes (both temperature increase and temperature decrease) impose higher maximum Von Mises stresses. For all curves, the maximum Von Mises stress values in the patch will increase for larger hole diameters. 5 C l i 5. Conclusions 5. Conclusions In the present contribution a simple and effective computational method is developed to treat actual industrial fully three-dimensional complicated problems of large underground space. The finite element method is used to investigate stress distribution in buried gas pipe subjected to thermo-mechanical loads and stress concentrations due to changes in the geometry. This study was divided in two parts. In the first case, stress values were calculated for the socket joint of the buried PE pipe so that by applying the proper pipe joints, the stress be reduced to levels below the allowable values. For the second part, the effects of thermo-mechanical loads on the stress distribution in patch repaired buried pipes are well investigated. For this purpose, in this research, 3D finite element modeling of buried gas pipe and its socket/patch is performed using ANSYS software. Stress variations in the buried MDPE gas pipe and high density polyethylene (HDPE) socket/patch were fully investigated in a hot climate region to determine the critical stress values caused by stress concentrations at the pipe and socket/patch, enabling us to find the applicable method for joining or repairing MDPE gas transportation piping in such areas. The optimum burial depth was found to be 1.25 m, while the maximum and minimum ground surface temperatures at this depth were calculated to be 35 °C and 13 °C, respectively. Furthermore, the soil column weight above the pipe, the surcharge loads in terms of traffic load, the gas pressure of 4 bar inside the pipe and the stress concentrations due to a local change in geometry (in the form of damage) were imposed on the pipe and its socket/patch resulting in the following conclusions: By considering the obtained plots for stress values in buried pipe and its socket and comparing In the present contribution a simple and effective computational method is developed to treat actual industrial fully three-dimensional complicated problems of large underground space. The ﬁnite element method is used to investigate stress distribution in buried gas pipe subjected to thermo-mechanical loads and stress concentrations due to changes in the geometry. This study was divided in two parts. In the ﬁrst case, stress values were calculated for the socket joint of the buried PE pipe so that by applying the proper pipe joints, the stress be reduced to levels below the allowable values. 5 C l i 5. Conclusions For the second part, the effects of thermo-mechanical loads on the stress distribution in patch repaired buried pipes are well investigated. For this purpose, in this research, 3D ﬁnite element modeling of buried gas pipe and its socket/patch is performed using ANSYS software. Stress variations in the buried MDPE gas pipe and high density polyethylene (HDPE) socket/patch were fully investigated in a hot climate region to determine the critical stress values caused by stress concentrations at the pipe and socket/patch, enabling us to ﬁnd the applicable method for joining or repairing MDPE gas transportation piping in such areas. The optimum burial depth was found to be 1.25 m, while the maximum and minimum ground surface temperatures at this depth were calculated to be 35 ◦C and 13 ◦C, respectively. Furthermore, the soil column weight above the pipe, the surcharge loads in terms of trafﬁc load, the gas pressure of 4 bar inside the pipe and the stress concentrations due to a local change in geometry (in the form of damage) were imposed on the pipe and its socket/patch resulting in the following conclusions: it with allowable stress values for the pipe, the correct joining method can be investigated. Based on the results, maximum Von Mises stresses occurs at the middle of the socket internal surface while the maximum values of the aforementioned stresses in the socket occur where the internal surface of the socket joins the pipe outer surface. In both pipe and socket the maximum values of the aforementioned stresses are well below the allowable stresses and therefore the introduced socket joint can be used under the described working condition. Additionally, for the problem of the patch repaired buried pipes, the results show that all four patch configurations have significant reinforcing By considering the obtained plots for stress values in buried pipe and its socket and comparing it with allowable stress values for the pipe, the correct joining method can be investigated. Based on the results, maximum Von Mises stresses occurs at the middle of the socket internal surface while the maximum values of the aforementioned stresses in the socket occur where the internal surface of the socket joins the pipe outer surface. In both pipe and socket the maximum values of the aforementioned stresses are well below the allowable stresses and therefore the introduced socket joint can be used under the described working condition. changes up to the mentioned values. The Simultaneous Effects of Thermo-Mechanical Loads on Square Partial Patch Arrangement For all cases, the maximum Von Mises stresses are well below the allowable stress limit for PE100 material which means the saddle fusion patch is well applicable to repair the proposed defects in even hot areas. Therefore, the results show the application of 3” saddle fusion patch is advisable for the proposed loading condition. 21 of 24 of 25 Energies 2018, 11, 2818 Energies 2018, 11, x Figure 18. Maximum Von Mises stress in 3 inch long, saddle fusion patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). 0 2 4 6 5 7.5 10 12.5 15 17.5 20 Maximum Von Mises stress (MPa) Hole diameter (mm) Allowable stress for a working life of 50 years at 35 °C Pipe and patch temperature change = +22 °C Pipe and patch temperature change = –22 °C Pipe and patch temperature change = –15 °C Pipe and patch temperature change = +5 °C Pipe and patch temperature change = 0 °C Figure 18. Maximum Von Mises stress in 3 inch long, saddle fusion patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). Maximum Von Mises stress (MPa) Figure 18. Maximum Von Mises stress in 3 inch long, saddle fusion patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads) Figure 18. Maximum Von Mises stress in 3 inch long, saddle fusion patch for various sizes of circular hole defects (the simultaneous effect of mechanical and thermal loads). 5. AASHTO Standard Speciﬁcations for Transportation Materials and Methods of Sampling and Testing, 15th ed.; American Association of State Highway and Transportation Ofﬁcials: Washington, DC, USA, 1990. 5 C l i 5. Conclusions Additionally, for the problem of the patch repaired buried pipes, the results show that all four patch conﬁgurations have signiﬁcant reinforcing effect on the defected section of the buried pipe under the aforementioned thermo-mechanical loads. Meanwhile, the maximum Von Mises stresses in both pipe and saddle fusion patch are well below the allowable 22 of 24 Energies 2018, 11, 2818 stress limit for PE material which means the saddle fusion patch is well applicable to repair the proposed defects in even hot areas. Therefore, the results show the application of introduced socket and also saddle fusion patch is advisable for the proposed loading condition. stress limit for PE material which means the saddle fusion patch is well applicable to repair the proposed defects in even hot areas. Therefore, the results show the application of introduced socket and also saddle fusion patch is advisable for the proposed loading condition. Author Contributions: The two authors contributed to perform this research. R.K.-Z. conceived of the presented idea, developed the theory, created the ANSYS models, calculated mechanical stress on the models and discussed the results. P.A. gave feedback in all steps of the research work, made a critical review of the paper and also applied standards to use in this project. Funding: This research received no external funding. Acknowledgments: The ﬁrst author highly appreciated prevalent help of his late friend Ali Nasirian for sharing his knowledge and experience in Finite Element modeling of underground space. His memory will be with me always. Conﬂicts of Interest: The authors declare no conﬂict of interest. Abbreviations C Design coefﬁcient Di Pipe inside diameter (mm) Do Pipe outside diameter (mm) E Elastic modulus (MPa) h Pipe thickness (mm) l Pipe length in the model (mm) MRS Maximum reduced strength (MPa) pi Pipe inside pressure (mm) SDR Standard dimension ratio (SDR = D h ) t Time (sec) T(y,t) Induced temperature in the soil (◦C) y Depth of the pipe (cm or mm) α Thermal diffusivity (cm2/s) at penetration depth ∆T Temperature change (◦C) ν Poisson’s ratio σ Normal stress in the pipe (MPa) σe Von Mises stress (MPa) σθθ Tangential stress in the pipe (MPa) σrr Radial stress in the pipe (MPa) σzz Longitudinal stress in the pipe (MPa) 4. Sampaio, J.H.B., Jr. Drilling Engineering Fundamentals. Master’s Thesis, Curtin University Technology, Bentley, Australia, 2007. References References 1. Khademi-Zahedi, R.; Makvandi, M.; Shishesaz, M. Technical analysis of the failures in a typical drilling mud pump during ﬁeld operation. In Proceedings of the 22nd Annual International Conference on Mechanical Engineering—ISME2014, Ahvaz, Iran, 22–24 April 2014; Available online: https://www.civilica.com/Paper- ISME22-ISME22_347.html (accessed on 18 October 2018). 1. Khademi-Zahedi, R.; Makvandi, M.; Shishesaz, M. Technical analysis of the failures in a typical drilling mud pump during ﬁeld operation. In Proceedings of the 22nd Annual International Conference on Mechanical Engineering—ISME2014, Ahvaz, Iran, 22–24 April 2014; Available online: https://www.civilica.com/Paper- ISME22-ISME22_347.html (accessed on 18 October 2018). 2. Khademi-Zahedi, R.; Makvandi, M.; Shishesaz, M. The applicability of casings and liners composite patch repair in Iranian gas and oil wells. In Proceedings of the 22nd Annual International Conference on Mechanical Engineering—ISME2014, Ahvaz, Iran, 22–24 April 2014; Available online: https://www.civilica.com/Paper- ISME22-ISME22_351.html (accessed on 18 October 2018). 3. Makvandi, M.; Bahmani, H.; Khademi-Zahedi, R. Technical analysis of the causes of blowout in BiBiHakimeh well No. 76. In Proceedings of the National Conference in New Research of Industry and Mechanical Engineering, Tehran, Iran, 17–18 September 2015; Available online: https://www.civilica.com/Paper- NRIME01-NRIME01_066.html (accessed on 18 October 2018). 4. Sampaio, J.H.B., Jr. Drilling Engineering Fundamentals. Master’s Thesis, Curtin University Technology, Bentley, Australia, 2007. 5. AASHTO Standard Speciﬁcations for Transportation Materials and Methods of Sampling and Testing, 15th ed.; American Association of State Highway and Transportation Ofﬁcials: Washington, DC, USA, 1990. 23 of 24 Energies 2018, 11, 2818 6. Moser, A.P.; Folkman, S. Buried Pipe Design; Book 2—Chapter 7; The McGraw-Hill Companies, Inc.: New York, NY, USA, 2001; 900p. 7. Goddard, J.B. Plastic Pipe Design; Technical Report 4.103; Advanced Drainage Systems, Inc.: Hilliard, OH, USA, 1994; pp. 1–30. 8. Khademi-Zahedi, R. Stress Distribution in Patch Repaired Polyethylene Gas Pipes. Master’s Thesis, Shahid Chamran University, Ahvaz, Iran, 2011. 9. Khademi-Zahedi, R. Application of the ﬁnite element method for evaluating the stress distribution in burried damaged polyethylene gas pipes. Undergr. Space 2018. [CrossRef] 10. Khademi-Zahedi, R.; Shishesaz, M. Application of a ﬁnite element method to stress distribution in buried patch repaired polyethylene gas pipe. Undergr. Space 2018. [CrossRef] 11. Nasirian, A. Investigating the Application of Polyethylene Gas Pipes for Gas Transportation. Master’s Thesis, Shahid Chamran University, Ahvaz, Iran, 2007. 12. Shishesaz, M.; Shishesaz, M.R. Applicability of medium density polyethylene Gas Pipes in Hot Climate Areas of South-West Iran. Iran. Polym. J. 2008, 17, 503–517. 13. Khademi-Zahedi, R.; Shishesaz, M.; Karamy-Moghadam, A.; Veisi-Ara, A. References Stress analysis in defected buried polyethylene pipes for gas distribution networks. In Proceedings of the National Conference in New Research of Industry and Mechanical Engineering, Tehran, Iran, 17–18 September 2015. 14. Plastic Pipe and Building Products. In ASTM Annual Book of ASTM Standards; American Society for Testing and Material: Philadelphia, PA, USA, 1991. 15. The Plastics Pipe Institute. Handbook of Polyethylene Pipe; Plastics Pipe Institute, Inc.: Washington, DC, USA, 2006; Chapter 6; pp. 157–260. 16. Corrugated Polyethylene Pipe Association. Structural Design Method for Corrugated Polyethylene Pipe; Corrugated Polyethylene Pipe Association: Washington, DC, USA, 2000. 17. European Standard. EN1555-2, Plastic Piping Systems for the Supply of Gaseous Fuels-Polyethylene (PE)-Part 2: Pipes, European Committee for Standardization. Available online: https://webstore.ansi.org/ RecordDetail.aspx?sku=DIN+EN+1555-2%3A2010 (accessed on 18 October 2018). RecordDetail.aspx?sku=DIN+EN+1555-2%3A2010 (accessed on 18 October 2018). 18. Plastic Pipe Institute. Polyethylene Gas Pipes Systems, Uponor Aldyl Company Installation Guide; Plastics Pipe Institute: Washington, DC, USA, 2004. 19. Egan, B.; Qenos, A.H. PE100, and Its Advantages When Used for Ploughing & Trenchless Technology. Available online: www.masterplumbers.com/plumbwatch/pipes99/pe100.doc (accessed on 18 October 2018). 20. Tabatabaii, A.; Ameri, M.; Behbahani, H. Prediction of Asphalt Cover Temperature in City of Ahvaz (Iran). J. Eng. 1999, 7, 22. (In Persian) 21. International Organization for Standardization. ISO12162, Thermo plastics Materials for Pipes and Fittings for Pressure Applications–Classiﬁcation and Designation–Overall Service (Design) Coefﬁcient. Available online: https://www.iso.org/standard/43865.html (accessed on 18 October 2018). 22. Amiri, F.; Milan, D.; Shen, Y.; Rabczuk, T.; Arroyo, M. Phase-ﬁeld modeling of fracture in linear thin shells. Theor. Appl. Fract. Mech. 2014, 69, 102–109. [CrossRef] 23. Areias, P.; Rabczuk, T. Finite strain fracture of plates and shells with conﬁgurational forces and edge rotation. Int. J. Numer. Methods Eng. 2013, 94, 1099–1122. [CrossRef] 4. Areias, P.; Rabczuk, T. Steiner-point free edge cutting of tetrahedral meshes with applications in frac Finite Elem. Anal. Des. 2017, 132, 27–41. [CrossRef] 25. Areias, P.; Msekh, M.A.; Rabczuk, T. Damage and fracture algorithm using the screened Poisson equation and local remeshing. Eng. Fract. Mech. 2016, 158, 116–143. [CrossRef] 26. Areias, P.; Rabczuk, T.; Msekh, M. Phase-ﬁeld analysis of ﬁnite-strain plates and shells including element subdivision. Comput. Methods Appl. Mech. Eng. 2016, 312, 322–350. [CrossRef] 27. Areias, P.M.A.; Rabczuk, T.; Camanho, P.P. Finite strain fracture of 2D problems with injected anisotropic softening elements. Theor. Appl. Fract. Mech. 2014, 72, 50–63. [CrossRef] 28. Chau-Dinh, T.; Zi, G.; Lee, P.S.; Song, J.H.; Rabczuk, T. References Phantom-node Method for Shell Models with Arbitrary Cracks. Comput. Struct. 2012, 92–93, 242–256. [CrossRef] 29. Ghorashi, S.; Valizadeh, N.; Mohammadi, S.; Rabczuk, T. T-spline based XIGA for Fracture Analysis of Orthotropic Media. Comput. Struct. 2015, 147, 138–146. [CrossRef] 29. Ghorashi, S.; Valizadeh, N.; Mohammadi, S.; Rabczuk, T. T-spline based XIGA for Fracture Analysis of Orthotropic Media. Comput. Struct. 2015, 147, 138–146. [CrossRef] 24 of 24 24 of 24 Energies 2018, 11, 2818 30. Hamdia, K.; Zhuang, X.; Silani, M.; He, P.; Rabczuk, T. Stochastic analysis of the fracture toughness of polymeric nanoparticle composites using polynomial chaos expansions. Int. J. Fract. 2017, 206, 215–227. [CrossRef] 31. KhademiZahedi, R.; Alimouri, P.; Nguyen-Xuan, H.; Rabczuk, T. Crack detection in a beam on elastic foundation using differential quadrature method and the Bees algorithm optimization. Proc. Int. Conf. Adv. Comput. Mech. 2017, 36, 439–460. 32. Nguyen-Thanh, N.; Kiendl, J.; Nguyen-Xuan, H.; Wuchner, R.; Bletzinger, K.U.; Bazilevs, Y.; Rabczuk, T. Rotation free isogeometric thin shell analysis using PHT-splines. Comput. Methods Appl. Mech. Eng. 2011, 200, 3410–3424. [CrossRef] 33. Nguyen-Thanh, N.; Valizadeh, N.; Nguyen, M.N.; Nguyen-Xuan, H.; Zhuang, X.; Areias, P.; Zi, G.; Bazilevs, Y.; De Lorenzis, L.; Rabczuk, T. An extended isogeometric thin shell analysis based on Kirchhoff-Love theory. Comput. Methods Appl. Mech. Eng. 2015, 284, 265–291. [CrossRef] 34. Nguyen-Thanh, N.; Zhou, K.; Zhuang, X.; Areias, P.; Nguyen-Xuan, H.; Bazilevs, Y.; Rabczuk, T. Isogeometric analysis of large-deformation thin shells using RHT-splines for multiple-patch coupling. Comput. Methods Appl. Mech. Eng. 2017, 316, 1157–1178. [CrossRef] 35. Rabczuk, T. Computational Methods for Fracture in Brittle and Quasi-Brittle Solids: State-of-the-art Review and Future Perspectives. ISRN Appl. Math. 2013, 2013, 849231. [CrossRef] 36. Rabczuk, T.; Areias, P.M.A.; Belytschko, T. A meshfree thin shell method for non- linear dynamic fracture. Int. J. Numer. Methods Eng. 2007, 72, 524–548. [CrossRef] 37. Rabczuk, T.; Gracie, R.; Song, J.H.; Belytschko, T. Immersed particle method for ﬂuid-structure interaction. Int. J. Numer. Methods Eng. 2010, 81, 48–71. [CrossRef] 38. Ren, H.; Zhuang, X.; Cai, Y.; Rabczuk, T. Dual-Horizon Peridynamics. Int. J. Numer. Methods Eng. 2016, 108, 1451–1476. [CrossRef] 39. Ren, H.; Zhuang, X.; Rabczuk, T. Dual-horizon peridynamics: A stable solution to varying horizons. Comput. Methods Appl. Mech. Eng. 2017, 318, 762–782. [CrossRef] 40. KhademiZahedi, R.; Alimouri, P. Finite element model updating of a large structure using multi-setup stochastic subspace identiﬁcation method and bees optimization algorithm. Front. Struct. Civ. Eng. 2018, in press. p 41. References J-M Manufacturing Company Inc. International MDPE Yellow Gas Pipe. Available online: http://www. jmeagle.com/sites/default/ﬁles/Products_MDPE%20Yellow%20Gas.pdf (accessed on 6 December 2017). © 2018 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/).
https://openalex.org/W4207047116	https://www.frontiersin.org/articles/10.3389/fimmu.2021.810223/pdf	English	null	Case Report: Endocapillary Glomerulopathy Associated With Large Granular T Lymphocyte Leukemia	Frontiers in immunology	2,022	cc-by	3,713	CASE REPORT published: 25 January 2022 doi: 10.3389/fimmu.2021.810223 CASE REPORT published: 25 January 2022 doi: 10.3389/fimmu.2021.810223 Large granular T lymphocyte leukemia (T-LGLL) is a rare indolent lymphocyte leukemia. The clonal proliferation of T cells, which is related to STAT3 gene mutation and abnormal Fas-mediated apoptosis pathway after cell activation, plays a major role in disease progression. Some studies have found that the exogenous and continuous stimulation of endogenous antigens, such as virus infection, is related to the pathogenesis of T-LGLL. The renal pathological manifestations of T-LGLL have rarely been described. In this study, we report a case of T-LGLL with kidney involvement as proteinuria, acute kidney injury, with the appearance of circulating T-LGL inﬁltrating intra-glomerular capillaries, and endocapillary glomerulopathy. We also summarize reported cases of renal injury associated with LGLL. Edited by: Michael Loran Dustin, University of Oxford, United Kingdom Reviewed by: Anastasios E. Germenis, University of Thessaly, Greece Yong Zhong, Central South University, China Correspondence: Tao Su tao.su@bjmu.edu.cn Edited by: Michael Loran Dustin, University of Oxford, United Kingdom Edited by: Michael Loran Dustin, University of Oxford, United Kingdom Reviewed by: Anastasios E. Germenis, University of Thessaly, Greece Yong Zhong, Central South University, China Reviewed by: Anastasios E. Germenis, University of Thessaly, Greece Yong Zhong, Central South University, China Reviewed by: Anastasios E. Germenis, University of Thessaly, Greece Yong Zhong, Central South University, China Correspondence: Tao Su tao.su@bjmu.edu.cn Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology Received: 06 November 2021 Accepted: 24 December 2021 Published: 25 January 2022 Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology Keywords: large granular lymphocyte (LGL) leukemia, acute kidney injury, endocapillary glomerulonephritis, histology, immunohistochemistry INTRODUCTION Large granular T lymphocyte leukemia (T-LGLL) is a rare type of lymphoproliferative disorder characterized by the clonal expansion of large granular T lymphocytes (LGLs) (1, 2). The abnormal proliferation of LGLs is usually associated with hematologic disorders and autoimmune diseases. Kidney disease can be rarely recognized in T-LGLL patients. Here we described a case of acute kidney injury associated with T-LGLL. A diagnosis of endocapillary glomerulopathy was conﬁrmed by histology analysis, revealing the endocapillary inﬁltration of LGLs. Keywords: large granular lymphocyte (LGL) leukemia, acute kidney injury, endocapillary glomerulonephritis, histology, immunohistochemistry CASE REPORT published: 25 January 2022 doi: 10.3389/fimmu.2021.810223 DISCUSSION T-LGLL is a common type of LGL leukemia. However, the renal involvement of T-LGLL is rare. In the present study, we reported a case of T-LGLL-induced endocapillary proliferation. The current diagnosis of T-LGLL is mainly based on clinical manifestations, cell morphology, immunophenotype, and T cell clonality (3, 4). The typical clonal proliferating T lymphocytes (T-LGL) are mostly killer effector cytotoxic T lymphocytes, leading to tissue damage. Their abnormal proliferation causes an imbalance of lymphocyte subsets and secretes inﬂammatory cytokines (such as FasL and IL-18) (5, 6), leading to renal injury. Ribes D et al. (5) reported that the supernatant of cultured peripheral blood mononuclear cells, including clonal T cells, triggered the phenotype switch of the HK-2 cell line from a quiescent to a pro-inﬂammatory state, characterized by a pro- inﬂammatory state by NF-kB nuclear translocation and overexpression of inﬂammatory cytokines or chemokines. This suggests that circulating T-LGL could directly activate tubular epithelial cells or stimulate the immunoreactivity of T/B cell populations, which could contribute to kidney injury. Orman et al. (7) reported a case of T-LGLL-associated nephrotic syndrome, of which the clinical behavior favored minimal-change glomerulopathy (though a kidney biopsy was not undertaken), suggesting that cytotoxic T-LGL could disrupt the normal anionic charge of glomerular capillary walls independent of heavy local inﬁltration. Audemard et al. reported three among 10 cases of T-LGLL with cryoglobulin-associated vasculitis, renal insufﬁciency, and membranoproliferative glomerulonephritis without renal inﬁltration of T-LGL (8). They all had systemic The renal pathological ﬁndings showed that the glomeruli were negative for IgG, IgA, C1q, FRA, ALB, IgG1, IgG2, IgG3, and IgG4, with IgM+ and C3+++ being positive and granularly depositing in the mesangial area by immunoﬂuorescence analysis. Under light microscope observation, there were 21 global ischemic sclerosis of a total of 59 glomeruli, which were mainly distributed beneath the renal capsule. The non-sclerotic glomeruli had an appearance of diffuse endocapillary proliferation. Most heteromorphic lymphocytes and a small number of neutrophils were observed in the intra-glomerular capillaries (Figure 1). No structure of glomerular crescent and arteriolitis were detected. There was focal tubular necrosis, multifocal lymphocytes, and mononuclear cells inundates with interstitial ﬁbrosis; however, tubulitis was not found. Under electron microscope observation, segmental low-density electron-dense deposition was disclosed limitedly in the mesangial area with the extensive fusion of podocyte foot processes, and no crystal or special microstructure was observed. Citation: Moreover, considering the history of recent respiratory infection, increased ASO, and C3 deposition in the mesangial area, infection-related glomerulonephritis could not be excluded. The extremely decreased FeNa indicated intra- glomerular hypoperfusion, which could contribute to aggravated tubular injury and kidney function loss. In addition, lymphocyte and monocyte (Figure 1C and Figure 2) interstitial inﬁltration was revealed in histology, proving that tubulointerstitial injury might also be involved in the pathogenesis of the kidney injury. y On admission, the patient complained of anorexia. Upon physical examination, it was found that the blood pressure was at 180/100 mmHg. There was severe edema, no rash, no palpable superﬁcial lymph nodes, and no sign of livedo reticularis indicating cryoglobulinemia. The laboratory examination revealed acute kidney injury in that SCr was rapidly elevated to 327 mmol/L, with blood urea nitrogen (BUN) at 63.69 mmol/L, BUN to SCr ratio ≈60:1, uric acid at 714 mmol/L, and albumin at 30.4 g/L (40–55). The complete blood count results were as follows: WBC, 5.4 × 109/L; Hb, 96 g/L; reticulocyte, 176.3 × 109/L (24–84); platelet, 76 × 109/L; neutrophil, 10.7%, 0.6 × 109/L (1.8–6.3); and lymphocyte, 84.8%, 6.8 × 109/L (1.1–3.2). The urinalysis found microscopic hematuria at 4–7 cells/HPF, the 24-h urinary protein was at 10.85 g, the 24-h urinary sodium was at 12.75 mmol, and the ﬁltered sodium excretion fraction was at 0.2%. The tests for antinuclear, antineutrophil cytoplasmic, anti-GBM antibodies, hypocomplements (C3 and C4), and cryoglobulin did not detect their presence; the anti-cyclic citrulline polypeptide antibody was at 140 RU/ml (<5), and antistreptolysin O (ASO) was 235 IU/ml (<220). However, the systemic inﬂammatory response was mild (erythrocyte sedimentation rate, 28 mm/h; hypersensitive C-reactive protein, 13.2 mg/L). The CT scan showed bilateral pleural effusion, pericardial effusion, enlarged kidneys, and spleen as well as a large amount of peritoneal effusion. Citation: Zhao T, Hu N, Yu X and Su T (2022) Case Report: Endocapillary Glomerulopathy Associated With Large Granular T Lymphocyte Leukemia. Front. Immunol. 12:810223. doi: 10.3389/fimmu.2021.810223 A 63-year-old man was admitted to our Department of Nephrology for further investigation of massive proteinuria. He had a 2-year history of rheumatoid arthritis (RA) and splenomegaly. At January 2022 \| Volume 12 \| Article 810223 Frontiers in Immunology \| www.frontiersin.org T-LGLL Showing Endocapillary Glomerulopathy Zhao et al. 1 month before admission, the patient was diagnosed with T- LGLL accidentally when he was experiencing an exacerbation of upper respiratory tract infection (fever of 38.2°C with cough and sputum), with a lymphocyte count of 5.68 × 109/L showing the presence of speciﬁcally large lymphocytes containing granules and a ﬂow cytometry analysis showing a CD2+/CD3+/CD4−/ CD7+/CD8+/CD57+/TCRab+ phenotype. The bone marrow TCR gene analysis showed a rearrangement of TCRB and TCRG gene clones. At that time, the serum creatinine (SCr) was 90 mmol/L, whereas he developed progressive edema and proteinuria 10 days later. demonstrating a cytotoxic T cell immunophenotype (Figure 2). The above-mentioned ﬁndings of renal pathology suggested endocapillary proliferative glomerulonephritis associated with T-LGLL. Moreover, considering the history of recent respiratory infection, increased ASO, and C3 deposition in the mesangial area, infection-related glomerulonephritis could not be excluded. The extremely decreased FeNa indicated intra- glomerular hypoperfusion, which could contribute to aggravated tubular injury and kidney function loss. In addition, lymphocyte and monocyte (Figure 1C and Figure 2) interstitial inﬁltration was revealed in histology, proving that tubulointerstitial injury might also be involved in the pathogenesis of the kidney injury. Supportive therapy was initiated after the patient was admitted to our hospital, including hemodialysis and transfusion of platelet and plasma, while the levels of SCr and BUN continued to increase as shown in Figure 3. The peripheral levels of platelet (68–93 × 109/L) and neutrophil (0.4–1.4 × 109/L) remained low. The coagulation function test revealed prolonged activated partial thromboplastin time (35–40 s) and decreased ﬁbrinogen (1.65–2 g/L), strongly suggesting the occurrence of disseminated intravascular coagulation. Immunosuppressive therapy was denied by the patient after the ﬁnal diagnosis was conﬁrmed. He chose to transfer to a local hospital where supportive treatment was sustained and unfortunately died a few days later. demonstrating a cytotoxic T cell immunophenotype (Figure 2). The above-mentioned ﬁndings of renal pathology suggested endocapillary proliferative glomerulonephritis associated with T-LGLL. Frontiers in Immunology \| www.frontiersin.org DISCUSSION Furthermore, the immunohistochemistry staining veriﬁed that the inﬁltrated heterotypic lymphocyte intra- glomerular capillaries expressed CD20-/CD3+/CD4-/CD8+/ TCRb-/TIA1+/GranzymeB+/CD2-/CD7+/CD56-/Ki67 5%, January 2022 \| Volume 12 \| Article 810223 Frontiers in Immunology \| www.frontiersin.org 2 Zhao et al. T-LGLL Showing Endocapillary Glomerulopathy FIGURE 1 \| Light and electron microscopy analysis. (A–C) Light microscopy using hematoxylin–eosin staining showing diffuse endocapillary proliferation composed of heteromorphic lymphocytes and a small number of neutrophils (A, B) and multifocal interstitial inﬁltration of lymphocytes and monocytes with focal tubular necrosis (C) [magniﬁcation, ×100 (A); magniﬁcation, ×400 (B, C)]. (D) Electron microscopy showing endocapillary proliferation of heteromorphic lymphocytes (red arrows) and low-density electron-dense deposition in the mesangial area (blue arrows) with the extensive fusion of podocyte foot processes. FIGURE 1 \| Light and electron microscopy analysis. (A–C) Light microscopy using hematoxylin–eosin staining showing diffuse endocapillary proliferation composed of heteromorphic lymphocytes and a small number of neutrophils (A, B) and multifocal interstitial inﬁltration of lymphocytes and monocytes with focal tubular necrosis (C) [magniﬁcation, ×100 (A); magniﬁcation, ×400 (B, C)]. (D) Electron microscopy showing endocapillary proliferation of heteromorphic lymphocytes (red arrows) and low-density electron-dense deposition in the mesangial area (blue arrows) with the extensive fusion of podocyte foot processes. of the factors for acute kidney injury (AKI). The clinical and pathological characteristics of similar cases reported in the literature are summarized in Tables 1 and 2. involvement, including purpura, polyneuritis, and arthritis. Abnormal B-cell activities in LGLL patients could also induce kidney impairment. Zhang M et al. reported crescentic glomerulonephritis induced by anti-GBM disease in a patient of T-LGLL (9). There is also a case report of AH renal amyloidosis type (g1) with T-LGLL (10). More recently, Pierre I et al. reported a case of natural killer cell LGL-induced glomerulonephritis, which showed global endocapillary proliferation with a marked predominance of circulating and interstitial inﬁltrating NK cells, IgM, C3 deposit, tubulitis, and tubular necrosis (11). Our current patient had similar pathological features of endocapillary proliferative glomerular changes, which originated from a cytotoxic T lymphocyte subset. These activated lymphocytes express cytotoxic granule protein (TIA-1 and GranzymeB) and had strong cytotoxic effects. Previous genetic testing found that IL1B gene was silenced in CD4-/CD8+ T cells of normal individuals but activated in LGL-T cells (5). IL-1b, as an effective proinﬂammatory cytokine, is known to be associated with tumor necrosis factor a, playing a central role in tissue injury of RA accompanied by T-LGL leukemia. Frontiers in Immunology \| www.frontiersin.org DISCUSSION Interestingly, the glomerular inﬁltrating clonal T cells of the patient in this report showed an immunophenotype consistent with T-LGL, but with low aggressiveness, and his renal biopsy revealed polymorphic inﬂammatory cell interstitial inﬁltrates. Even so, interstitial inﬂammation also remained one However, the exact risk factors attributing to AKI in this T- LGLL patient were complicated. As we know, secondary neutrophil deﬁciency is a disadvantageous factor of innate immunity, which makes patients susceptible to infection. Infection-related nephritis would also be considered if patients presented with nephritis syndrome. We noticed that the patient had an extremely elevated BUN-to-SCr ratio and decreased FeNa. The renal biopsy revealed ischemic glomerulosclerosis, involving 35.6% of the glomeruli. We speculated that the intra- glomerular hypoperfusion was derived from severe endocapillary proliferative changes. All these could lead to peri-tubular capillary ischemia and tubular injury. Our patient has a 2-year history of splenomegaly and RA with a positive anti-CCP antibody. Splenic involvementisalmostuniversalin LGLL patients, manifested as splenomegaly and thrombocytopenia, whichiscloselyrelatedtoautoimmunediseases.RAismostcommonly described in 11–36% of patients, also including Felty syndrome, scleroderma, polymyositis, various types of vasculitis, Sjogren’s syndrome, and Behcet’s disease (12). The clonal T-LGL was reported to be detected in 3.6% of RA. The onset of LGLL is indolent; thus, diagnosis and speciﬁc treatment are easily delayed. Experts recommend the initiation of a speciﬁc treatment for T-LGLL with January 2022 \| Volume 12 \| Article 810223 Frontiers in Immunology \| www.frontiersin.org 3 Zhao et al. T-LGLL Showing Endocapillary Glomerulopathy A B FIGURE 2 \| Immunohistochemistry analysis. (A) Immunochemistry analysis showing CD20-/CD3+/CD8+/GranzymeB+ phenotype of the inﬁltrating endocapillary glomerular cells (magniﬁcation, ×200). (B) Immunochemistry analysis on tubulointerstitial compartment showing CD20+/CD3+/CD8+ cells inﬁltrating the interstitium (magniﬁcation, ×200). A B A B FIGURE 2 \| Immunohistochemistry analysis. (A) Immunochemistry analysis showing CD20-/CD3+/CD8+/GranzymeB+ phenotype of the inﬁltrating endocapillary glomerular cells (magniﬁcation, ×200). (B) Immunochemistry analysis on tubulointerstitial compartment showing CD20+/CD3+/CD8+ cells inﬁltrating the interstitium (magniﬁcation, ×200). FIGURE 3 \| Changes of serum creatinine and blood urea nitrogen during the disease course. \| FIGURE 3 \| Changes of serum creatinine and blood urea nitrogen during the disease course. Frontiers in Immunology \| www.frontiersin.org January 2022 \| Volume 12 \| Article 810223 T-LGLL Showing Endocapillary Glomerulopathy Zhao et al. TABLE 1 \| Clinical features of reported cases of renal injury associated with large granular lymphocyte leukemia. DISCUSSION Patient Age/ sex Subtype LGLs Peripheral LGLa Hematological abnormalities Symptoms Autoimmune disorders 1 57/F T-LGL 73% L, N, Fatigue, nausea, choking, neuropathic symptoms M-protein shown in SPEP and UPEP 2 78/M NK-LGL 72% L NS NS 3 74/F T-LGL 0.45 × 109/L Hb Purpura, polyneuritis Cyroglobulinemia mixed type 2 with IgM 4 59/F T-LGL 0.63 × 109/L Within normal range Purpura, arthritis Cyroglobulinemia 5 69/F T-LGL 0.45 × 109/L N, Purpura, arthritis Cyroglobulinemia 6 37/M T-LGL 16% (0.32 × 109/ L) NS NS Anti-GBM disease 7 51/M T-LGL 75% L, PLT “B” symptoms, hepatosplenomegaly NS 8 63/M T-LGL 72.7% L, N, Hb, PLT Splenomegaly, fever Rheumatoid arthritis L, lymphocyotosis (lymphocyte >4 × 109/L); N, neutropenia (neutrophil <1.5 × 109/L); Hb, anemia (hemoglobin >11 g/dL); PLT, thrombocytopenia (platelet <150 × 109/L); NS, not stated. aPresented as ratio or absolute count. 8 is our currently reported case. TABLE 1 \| Clinical features of reported cases of renal injury associated with large granular lymphocyte leukemia. L, lymphocyotosis (lymphocyte >4 × 109/L); N, neutropenia (neutrophil <1.5 × 109/L); Hb, anemia (hemoglobin >11 g/dL); PLT, thrombocytopenia (platelet <150 × 109/L); NS, not stated. aPresented as ratio or absolute count. 8 is our currently reported case. TABLE 2 \| Characteristic of renal disease in reported cases of renal injury associated with large granular lymphocyte leukemia. Patient Time of diagnosis of renal injury compared to LGL Clinical course SCr (µmol/L) Proteinuria Renal histology 1 14 years after Progressive proteinuria (0.5–1.5 g/h in 17 months) 74.26 UTP: 2.5 g/ 24 h AH amyloid (mostly restricted to glomeruli), moderate chronic changes (including glomerulosclerosis) 2 3 years after AKI 220 ACR: 2 g/g Endocapillary glomerulonephritis and tubulitis with LGL NK cell inﬁltration seen in both areas 3 Synchronous NS NS NS MPGN without LGL inﬁltration 4 6 years before NS NS NS NS 5 2 years before NS NS NS Endocapillary glomerulonephritis 6 1 year before AKI 295 UTP: 6.92 g/24 h Crescentic glomerulonephritis and segmental membranous nephropathy, with strong linear GBM staining on direct immunoﬂuorescence 7 Synchronous Nephrotic syndrome 132.6 UTP: 8.9 g/ 24 h Presumptive minimal-change nephropathy suggested by clinical behavior 8 1 month after AKI 327 UTP: 10.85 g/24 h Endocapillary glomerulonephritis with heterotypic lymphocyte inﬁltration, probable post-infection glomerulonephritis NS, Not Stated. TABLE 2 \| Characteristic of renal disease in reported cases of renal injury associated with large granular lymphocyte leukemia. relevant autoimmune diseases (13). DISCUSSION However, our patient presented rapid deterioration of kidney function with poor prognosis. One explanation was that the T-LGLL might have occurred a few years priortothediagnosisofsplenomegalyandRA.Insufﬁcientattentionto the history of splenomegaly might result in delayed diagnosis of T- LGLL and absence of timely treatment, consequently leading to disease progression. aim to arouse more attention to the kidney presentation of this clonal T cell proliferative disease. The signiﬁcance lies in the fact that early recognition of T-LGLL-associated organ-speciﬁc injury and timely initiation of therapy would be beneﬁcial to improve the disease outcome. Our report contained several limitations. Firstly, the reasons for the death of the patient need more analysis. However, clinical information concerning follow-up in the local hospital was lacking. Thus, it was hard to ﬁnd out the exact cause of his death. Secondly, to further evaluate the pathogenesis of peripheral T-LGL, it is necessary to measure the serum inﬂammatory cytokines produced by the activated cytotoxic T cells, such as FasL and IL- 18. Unfortunately, the patient died within a few days after the diagnosis, and we have not obtained consent from him. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author. REFERENCES 9. Fu J, Lee LX, Zhou P, Fogaren T, Varga C, Comenzo RL. A Case of T-Cell Large Granular Lymphocytic Leukemia and Renal Immunoglobulin Heavy Chain Amyloidosis. Am J Case Rep (2019) 20:43–7. doi: 10.12659/ AJCR.912282 1. Yang J, Liu X, Nyland SB, Zhang R, Ryland LK, Broeg K, et al. Platelet- Derived Growth Factor Mediates Survival of Leukemic Large Granular Lymphocytes via an Autocrine Regulatory Pathway. Blood (2010) 115 (1):51–60. doi: 10.1182/blood-2009-06-223719 10. Zhang M, Guan N, Zhu P, Chen T, Liu S, Hao C, et al. Recurrent Anti-GBM Disease With T-Cell Large Granular Lymphocytic Leukemia: A Case Report. Med (Baltimore) (2019) 98(31):e16649. doi: 10.1097/MD.0000000000016649 2. Sokol L, Agrawal D, Loughran TP Jr. Characterization of HTLV Envelope Seroreactivity in Large Granular Lymphocyte Leukemia. Leuk Res (2005) 29 (4):381–7. doi: 10.1016/j.leukres.2004.08.010 11. Isnard P, Linster C, Bruneau J, Thervet E, Duong Van Huyen JP, Rossignol J, et al. Natural Killer Cell Large Granular Lymphocyte Leukemia-Induced Glomerulonephritis.KidneyIntRep(2021)6(4):1174–7.doi:10.1016/j.ekir.2021.01.024 3. Steinway SN, LeBlanc F, Loughran TP Jr. The Pathogenesis and Treatment of Large Granular Lymphocyte Leukemia. Blood Rev (2014) 28(3):87–94. doi: 10.1016/j.blre.2014.02.001 12. Bareau B, Rey J, Hamidou M, Donadieu J, Morcet J, Reman O, et al. Analysis of a French Cohort of Patients With Large Granular Lymphocyte Leukemia: A Report on 229 Cases. Haematologica (2010) 95(9):1534–41. doi: 10.3324/ haematol.2009.018481 12. Bareau B, Rey J, Hamidou M, Donadieu J, Morcet J, Reman O, et al. Analysis of a French Cohort of Patients With Large Granular Lymphocyte Leukemia: A Report on 229 Cases. Haematologica (2010) 95(9):1534–41. doi: 10.3324/ haematol.2009.018481 4. Siebert JD, Mulvaney DA, Potter KL, Fishkin PA, Geoffery FJ. Relative Frequencies and Sites of Presentation of Lymphoid Neoplasms in a Community Hospital According to the Revised European-American Classiﬁcation. Am J Clin Pathol (1999) 111(3):379–86. doi: 10.1093/ajcp/ 111.3.379 13. Lamy T, Loughran TP Jr. How I Treat LGL Leukemia. Blood (2011) 117 (10):2764–74. doi: 10.1182/blood-2010-07-296962 13. Lamy T, Loughran TP Jr. How I Treat LGL Leukemia. Blood (2011) 117 (10):2764–74. doi: 10.1182/blood-2010-07-296962 Conﬂict of Interest: 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. 5. Ribes D, Casemayou A, El Hachem H, Laurent C, Guilbeau-Frugier C, Vegez F, et al. Asymptomatic Circulating T-Cell Clone Cause Renal Polymorphic Inﬂammatory Fibrosis. Clin Exp Nephrol (2017) 21(5):781–6. Concluding Remarks Written informed consent was obtained from the individual(s) for the publication of any potentially identiﬁable images or data included in this article. In conclusion, this case is a rare report of T-LGLL-induced AKI presenting endocapillary proliferative glomerulonephritis. We January 2022 \| Volume 12 \| Article 810223 Frontiers in Immunology \| www.frontiersin.org 5 T-LGLL Showing Endocapillary Glomerulopathy Zhao et al. FUNDING NH collected data and reviewed the literature. TZ and NH drafted the manuscript. The diagnosis was conﬁrmed clinically by TS and NH. This research was supported by the National Science and Technology Major Projects for major new drugs innovation and development (Grant 2017ZX09304028 to TS), and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (No. 2019-I2M-5-046). This research was supported by the National Science and Technology Major Projects for major new drugs innovation and development (Grant 2017ZX09304028 to TS), and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (No. 2019-I2M-5-046). XY made the pathological diagnosis and generated Figures 1 and 2. TS, NH, and TZ reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version. January 2022 \| Volume 12 \| Article 810223 REFERENCES doi: 10.1007/ s10157-016-1373-6 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 6. Saitoh T, Matsushima T, Kaneko Y, Yokohama A, Handa H, Tsukamoto N, et al. T Cell Large Granular Lymphocyte (LGL) Leukemia Associated With Behcet’s Disease: High Expression of Sfasl and IL-18 of CD8 LGL. Ann Hematol (2008) 87(7):585–6. doi: 10.1007/s00277-008-0438-3 7. Orman SV, Schechter GP, Whang-Peng J, Guccion J, Chan C, Schulof RS, et al. Nephrotic Syndrome Associated With a Clonal T-Cell Leukemia of Large Granular Lymphocytes With Cytotoxic Function. Arch Intern Med (1986) 146(9):1827–9. doi: 10.1001/archinte.146.9.1827 Copyright © 2022 Zhao, Hu, Yu and Su. 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) and the copyright owner(s) 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. 8. Audemard A, Lamy T, Bareau B, Sicre F, Suarez F, Truquet F, et al. Vasculitis Associated With Large Granular Lymphocyte (LGL) Leukemia: Presentation and Treatment Outcomes of 11 Cases. Semin Arthritis Rheum (2013) 43 (3):362–6. doi: 10.1016/j.semarthrit.2013.07.002 January 2022 \| Volume 12 \| Article 810223 Frontiers in Immunology \| www.frontiersin.org
https://openalex.org/W4362684257	https://ejournal.iaida.ac.id/index.php/Tarbiyatuna/article/download/1938/1187	Indonesian	null	IMPLEMENTASI MANAJEMEN SUMBERDAYA MANUSIA DALAM MENINGKATKAN MUTU GURU DI SMA FAVORIT NU TEGALDLIMO	Tarbiyatuna	2,023	cc-by-sa	6,611	48 48 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Abstract The purpose of this study was to determine how the implementation of human resource management (HR) at SMA Faforit NU Tegaldlimo Banyuwangi. Second, what is the quality of teachers at SMA Faforit NU Tegaldlimo Banyuwangi. Third, how is the implementation of human resource management (HR) in improving the quality of teachers at SMA Faforit NU Tegaldlimo Banyuwangi. This research uses a qualitative descriptive method, the problems in this study are formulated as follows: (1) How is the implementation of human resource management (HR) at SMA Faforit NU Tegaldlimo Banyuwangi (2) How is the quality of teachers at SMA Faforit NU Tegaldlimo Banyuwangi; (3) How is the implementation of human resource management (HR) in improving the quality of teachers at SMA Faforit NU Tegaldlimo Banyuwangi. Keywords: Human Resource Management, Improving Teacher Quality Abstrak Tujuan penelitian ini adalah untuk mengetahui Bagaimana implementasi manajemen sumberdaya manusia (SDM) di SMA Faforit NU Tegaldlimo Banyuwangi. Kedua, Bagaimana mutu guru di SMA Faforit NU Tegaldlimo Banyuwangi. Ketiga, Bagaimana implementasi manajemen sumberdaya manusia (SDM) dalam meningkatkan mutu guru di SMA Faforit NU Tegaldlimo Banyuwangi. Penelitian ini menggunakan metode deskriptif kualitatif, permasalahan dalam penelitian ini dirumuskan sebagai berikut: (1) Bagaimana implementasi manajemen sumberdaya manusia (SDM) di SMA Faforit NU Tegaldlimo Banyuwangi; (2) Bagaimana mutu guru di SMA Faforit NU Tegaldlimo Banyuwangi; (3) Bagaimana implementasi manajemen sumberdaya manusia (SDM) dalam meningkatkan mutu guru di SMA Faforit NU Tegaldlimo Banyuwangi. IMPLEMENTASI MANAJEMEN SUMBERDAYA MANUSIA DALAM MENINGKATKAN MUTU GURU DI SMA FAVORIT NU TEGALDLIMO Mamlukhah1, Heru Susanto2 e-mail: mamlukhah@iaida.ac.id1, heruisman94@gmail.com Program Studi Manajemen Pendidikan Islam Institut Agama Islam Darussalam Blokagung Banyuwangi A. Pendahuluan Sekolah sebagai suatu institusi atau lembaga pendidikan melakukan sarana untuk proses pendidikan. Sesungguhnya pendidikan memiliki peran penting dalam kehidupan bangsa dan negara yakni menciptakan sumber daya manusia SDM yang berkualitas. Baik buruknya mutu pendidikan di perguruan tinggi berpegang pada SDM yang mengelolanya. Maka perlu adanya manajemen SDM dalam lembaga pendidikan tersebut untuk bias mengelola suatu kinerja secara optimal sehingga Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 49 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) menghasilkan lembaga yang bermutu dan memperoleh mutu pendidikan yang baik sesuai dengan visi, misi dan target yang direncanakan. Menyadari peran strategis pendidikan tersebut, pemerintah Indonesia senantiasa mendukung ide yang menempatkan Sektor pendidikan, sebagai prioritas dalam pembangunan Naasional. Untuk itu pemerintah menyelenggarakan suatu sistem pendidikan Naasional sebagaimana tercantum dalam Undang-Undang Nomor 20 Tahun 2003 tentang Sistem Pendidikan Naasiona, guru sebagai mikro penentu dominan mutu bendidikan harus bermutu dan berkinerja baik dalam era globalisasi dengan berusaha menguasai berbagai teknologi informasi dan komunikasi, karena salah satu aspek yang mengalami perubahan dasyat dalam era globalisasi adalah kemajuan teknologi informasi dan komunikasi. Manajemen sumber daya manusia adalah suatu penanganan berbagai masalah pada ruang lingkup karyawan, pegawai, buruh, manajer dan tenaga kerja, untuk dapat meunjang aktivitas organisasi atau perusahaan demi mencapai tujuan yang telah ditentukan (Simamora Henry, 2004:170). Sedangkan dalam pendapat lain mendefinisikan sumber daya manusia merupakan keseluruhan penentuan dan pelaksanaan beberapa aktivitas, policy, dan program yang bertujuan untuk mendapatkan tenaga kerja, pengembangan dan pemeliharaan dalam usaha meningkatkan dukungannya terhadap peningkatan efektivitas organisasi dengan cara yang secara etis dan sosIial dapat dipertanggungjawabkan (Siagian, 1994:128). Dengan demikian dapat ditarik kesimpulan Manajemen sumber daya manusia adalah suatu penanganan berbagai masalah pada ruang lingkup karyawan, pegawai, buruh, manajer dan tenaga kerja bertujuan untuk mendapatkan tenaga kerja, pengembangan dan pemeliharaan dalam usaha meningkatkan dukungannya terhadap peningkatan efektivitas organisasi dengan cara yang secara etis dan sosIial dapat dipertanggungjawabkan Fungsi manajemen sumberdaya manusia terdapat beberapa macam fungsi utama manajemen sumber daya manusia (Marwansyah, 2016:228), diantaranya sebagai berikut: Pertama, Perencanaan Untuk Kebutuhan SDM. Fungsi perencanaan kebutuhan manusia meliputi dua kegiatan utama yaitu sebagai berikut: (1) Perencanaan dan peramalan permintaan tenaga kerja organisasi baik dalam Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 50 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) jangka pendek maupun jangka panjang; (2) Analisis jabatan dalam organisasi untuk menentukan tugas, tujuan, keahlian, pengetahuan dan kemampuan yang dibutuhkan; (3) Staffing Sesuai Dengan Kebutuhan Organisasi. Setelah kebutuhan SDM ditentukan, langkah selanjutnya adalah mengisiformasi yang tersedia. Dalam tahaoan pengisian staf ini terdapat dua kegiatan yang diperlukan, yaitu: (1) Penarikan (rekrutmen) calon atau pelamar pekerjaan; (2) Pemilihan (seleksi) para calon atau pelamar yang dinilai telah memenuhi syarat yang ditetapkan. jangka pendek maupun jangka panjang; (2) Analisis jabatan dalam organisasi untuk menentukan tugas, tujuan, keahlian, pengetahuan dan kemampuan yang dibutuhkan; (3) Staffing Sesuai Dengan Kebutuhan Organisasi. Setelah kebutuhan SDM ditentukan, langkah selanjutnya adalah mengisiformasi yang tersedia. Dalam tahaoan pengisian staf ini terdapat dua kegiatan yang diperlukan, yaitu: (1) Penarikan (rekrutmen) calon atau pelamar pekerjaan; (2) Pemilihan (seleksi) para calon atau pelamar yang dinilai telah memenuhi syarat yang ditetapkan. Kedua, Penilaian Kerja. Kegiatan ini dilakukan setelah calon atau pelamar dipekerjakan dalam kegiatan organisasi. Dalam penilaian kinerja ini dilakukan dua kegiatan utama, yaitu: (1) Penilaian dan pengevaluasian perilaku pekerja; (2) Analisiss dan pemberian mtivasi perilaku pekerja; (3) Perbaikan Kualitas Pekerja Dan Lingkungan Kerja. Saat ini pusat pemerhatian MSDM mengarah pada tiga kegiatan strategis, yaitu: (1) Menentukan, merancang dan mengimplementasikan program pelatihan dan pengembangan SDM guna meningkatkan kemampuan dan kinerja karyawan; (2) Memperbaiki kualitas lingkungan kerja, khususnya melalui kualitas kehidupan kerja dan program-program perbaikan produktifitas dan Memperbaiki kondisi fisik kerja guna memaksimalkan kesehatan dan keselamatan pekerja. Ketiga, Tujuan sumber daya manusia, yakni: (1) Tujuan Sosial manajemen sumber daya manusia adalah agar organisasi atau perusahaan dapat bertanggung jawab secara sosial dan etis terhadap kebutuhan dan tantangan masyarakat dapat meminimalkan dampak negatifnya; (2) Tujuan Organisasional, adalah sasaran formal yang dibuat untuk membantu organisasi mencapai tujuannya; (3) Tujuan Fungsional adalah sasaran formal untuk mempertahankan kontribusi department sumber daya manusia pada tingkat yang sesuai dengan kebutuhan organisasi; (4) Tujuan Individual adalah tujuan pribadi dari tiap anggota organisasi atau perusahaan yang hendak mencapai melalui aktivitasnya dalam organisasi. Prosedur Manajemen Sumber Daya Manusia terdapat program dan aktivitas yang terdiri atas beberapa bagian yaitu: (1) Perencanaan sumber daya manusia; (2) Rekrutmen sumber daya manusia. Sedangkan seleksi Sumber Daya Manusia merupakan tindak lanjut dari proses. Hasil rekrutmen yaitu berupa surat lamaran yang siap untuk diseleksi. Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Proses seleksi menentukan siapa yang layak diangkat jadi Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 51 51 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Standar mutu tenaga pendidik Dalam PP No 19 Tahun 2005 pasal 2. bahwa: “Standar Nasional pendidikan terdiri atas standar isi, proses, kompetensi lulusan, tenaga pendidik dan kependidikan, sarana dan prasarana, pengelolaan, pembiayaan, dan penilaian pendidikan yang harus ditingkatkan secara berencana dan berkala”. Lembaga pendidikan yang di maksud adalah SMA Favorit nu tegaldlimo banyuwani. Dengan berbagai kegiatan yang direncanakan oleh pihak sekolah diharapkan mampu menciptakan siswa siswi yang berkualitas. dengan kualitas peserta didik yang baik merupakan lembaga pendidikan yang memperhatikan pada kualitas pendidikannya. Usaha yang dilakukan untuk mempertahankan sekaligus meningkatkan kualitas pendidikan, yaitu dengan diadakannya pelatihan-pelatihan oleh pihak sekolah terhadap para pendidik dan tenaga kependidikan, sehingga memang diakui oleh masyarakat luas bahwa mutu atau kualitas pendidikan di SMA Faforit NU Tegaldlimo ini memang benar-benar memenuhi keinginan pelanggan. Dari gambaran tersebut peneliti merasa perlu untuk menemukan dan mengkaji lebih mendalam terkait dengan pelaksanaan manajemen sumber daya manusia di SMA Favorit nu Tegaldlimo Banyuwani, mengingat SMA Favorit NU Tegaldlimo Banyuwani beberapa tahun belakangan ini terus mengalami peningkatan prestasi dan menjadi sekolah yang cukup diminati oleh masyarakat. Adapun tujuan peneltian yang ditetapkan dalam penelitian ini untuk mendeskripsikan : (1) Implementasi manajemen sdm di SMA Favorit NU Tegaldlimo Banyuwangi; (2) Mutu guru di SMA Faforit NU Tegaldlimo Banyuwangi; (3) Implementasi manajemen sdm dalan meneingkatkan mutu guru di SMA Favorit NU Tegaldlimo Banyuwangi Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) karyawan dan siapa yang tidak. Kemudian orientasi dan penempatan. Orientasi adalah program upaya pelatihan dan pengembangan awal bagi para pegawai baru untuk dapat menyesuaikan diri dan juga memberi mereka informasi mengenai perusahaan, jabatan, dan kelompok kerja (French, 2012:65), Pelatihan dan pengembangan sumber daya manusia juga perlu dilakukan bertujuan untuk meningkatkan kualitas profesionalisme dan keterampilan para pegawai atau karyawan untuk melaksanakan tugas dan fungsinya secara optimal Pada konteks pendidikan, pengertian mutu mengacu pada masukan, proses, keluaran dan dampaknya. Mutu masukan dapat dilihat dari berbagai sisi. Pertama, kondisi baik atau tidaknya masukan sumber daya manusia seperti kepala sekolah, guru, staf tata usaha, dan siswa. Kedua, memenuhi atau tidaknya kriteria masukan material berupa alat peraga, buku-buku kurikulum, prasarana dan sarana sekolah. Ketiga, memenuhi atau tidaknya kriteria masukan yang berupa alat lunak,seperti peraturan struktur organisasi, deskripsi kerja, dan struktur organisasi. Keempat, mutu masukan yang bersifat harapan dan kebutuhan seperti visi, motivasi, ketekunan dan cita-cita. (Sudarwan, 2003:53) Menurut Undang-Undang No. 14 tahun 2005 pasal 1 yang dimaksud “guru yaitu pendidik profesional dengan tugas utama mendidik, mengajar, membimbing, mengarahkan, melatih, menilai, dan mengevaluasi, peserta didik pada pendidikan usia dini jalur pendidikan formal, pendidikan dasar, dan pendidikan menengah”. Pada pasal 2, menyatakan bahwa “guru mempunyai kedudukan sebagai tenaga profesional pada jenjang pendidikan dasar, pendidikan menengah, dan pendidikan anak usia dini pada jalur pendidikan formal yang diangkat sesuai dengan peraturan perundang-undangan Standar yang dijadikan parameter atau ukuran tinggi rendahnya mutu atau kualitas guru dalam kinerja ataupun produktivitasnya adalah “kom petensi guru (Tim Redaksi Fokusmedia, Standar Nasional Pendidikan, 2005:5). Sedangkan menurut Undang-undang Nomor 14 tahun 2005 tentang Guru dan Dosen pasal 10 dikemukakan bahwa kompetensi guru mencakup empat hal yaitu: (1) kompetensi pedagogic; (2) kompetensi kepribadian; (3) kompetensi sosial; (4) kompetensi professional. Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 52 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Penelitian ini digunakan untuk mendeskripsikan tentang segala sesuatu yang berkaitan dengan. implementasi manajemen sumberdaya manusia (SDM) dalam meningkatkan mutu guru di SMA Favorit NU Tegaldlimo Banyuwangi Pendekatan ini menggunakan pendekatan kualitataif metode deskriptif, dapat diartikan sebagai prosedur pemecah masalah yang diselidiki dengan menggambarkan keadaan objek penelitian pada saat sekarang berdasarkan fakta- fakta atau apa adanya, metode deskriptif memusatkan perhatian pada menemukan fakta-fakta sebagaimana keadaan sebenarnya (Moleong, 2009:14). Sumber data dalam penelitian kualitatif adalah kata-kata dan tindakan sedangkan dokumen dan lain-lain dapat dijadikan sebagai data tambahan. Moleong (2009:112) Sumber data dalam penelitian kualitatif ini adalah: a. Kepala Sekolah SMA Favorit NU Tegaldlimo a. Kepala Sekolah SMA Favorit NU Tegaldlimo b. Waka kurikulum SMA Favorit NU Tegaldlimo Data yang dikumpulkan bisa lewat instrumen maupun non instrumen yang nantinya akan menghasilkan informasi, baik informasi berupa keterangan langsung dalam arti hasil kegiatannya sendiri atau pengalamannya responden maupun informasi yang didapat merupakan keterangan langsung yang bukan kegiatan sendiri atau bukan pengalamannya sendiri dari responden yang bersangkutan. Subagyo (2011:86) Teknik pengumpulan data merupakan langkah yang paling utama dalam penelitian, karena tujuan utama dari penelitian adalah mendapatkan data. Tanpa mengetahui teknik pengumpulan data, maka peneliti tidak akan mendapatkan data yang memenuhi standar data yang ditetapkan. Sugiono (2010:308) yaitu: wawancara, observasi dan dokumentasi. Teknik pemeriksaan keabsahan data menggunakan triangulasi dan teknik analisis data menggunakan reduksi data, penyajian data dan penarikan kesimpulan. B. Metode Penelitian Penelitian ini adalah penelitian kualitatif. Penelitian kualitatif (Qualitative Research) adalah suatu penelitian yang ditujukan untuk mendeskripsikan dan menganalisis fenomena, peristiwa, aktivitas sosial, sikap, kepercayaan, persepsi, pemikiran orang secara individual maupun kelompok. Syaodih (2010:60) Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 53 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Favorit NU berdasarkan kebutuhan sekolah. melalui rapat atau musyawarah di awal tahun hal ini dapat dibuktikan dalam wawancara penulis dengan kepala sekolah H. Takiyudin Nazah, M.Si (12 juni 2022) “Dalam hal perencanaan Sumber Daya Manusia ada beberapa langkah yang kami lakukan dalam tahap perencanaan yaitu: 1) Menetapkan secara jelas kualitas dan kuantitas SDM yang dibutuhkan. 2) Mengumpulkan data dan informasi tentang SDM yang dib 3) Mengelompokkan data dan informasi serta menganalisisnya. 4) Menetapkan beberapa alternative maksudnya disini adalah apakah bisa ditutupi dari SDM internal atau harus kita merekrut tenaga baru. 5) Memilih yang terbaik dari alternatif yang ada menjadi rencana 6) Menginformasikan rencana kepada para pegawai untuk direalisasikan”. Juga dibenarkan oleh wakil kepala Sekolah Bpk. Hanif Setiawan S.Pd pada saat wawancara pada hari (12 juni 2022). “Dalam hal manajemen guru dan karyawan agar mutu pendidikan terjaga maka dalam hal karyawan dan guru kami menyesuaikan dengan kebutuhan terutama dalam hal guru baru dan bagaimana mengatur guru dan karyawan”. C. Hasil Penelitian 1. Manjemen Sumberdaya Manusia Di SMA Favorit NU 1. Manjemen Sumberdaya Manusia Di SMA Favorit NU 1. Manjemen Sumberdaya Manusia Di SMA Favorit NU a. Perencanaan Manajemen Sumber Daya Manusia Di SMA Favorit NU Tegaldlimo. Upaya perencanaan atau pengadaan sumber daya manusia di SMA Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 54 b. Penarikan sumber daya manusia (recruitmen) Menurut kepala sekolah SMA Favorit NU H. Takiyudin Nazah, M.Si (12 juni 2022) dalam merekrut tenaga pendidik atau karyawan telah ditetapkan beberapa persyaratan, yaitu: “dalam hal pelamar kami punya beberapa syarat yakni;1) Berhaluan Ahlus Sunnah Waljama’ah diutamakan yang memiliki jiwa santri. 2) Memilki ijazah minimal S1 Pendidikan sesuai dengan bidangnya, 3) Memiliki KARTANU, 4) Pengalaman lebih diutamakan, 5) Nilai IP minimal 3.00, 6)Memiliki jiwa Islami dan siap berjuang” Hal ini membuktikan bahwa SMA Favorit NU apabila membutuhkan tenaga baru melakukannya dengan jika masih ada SDM yang memenuhi spesifikasi pekerjaan, mengambil dari dalam atau sumber internal khususnya untuk jabatan manajerial (para wakil kepala) c. Seleksi Setelah SMA Favorit NU Tegaldlimo mendapatkan beberapa pelamar kemudian di adakan tes dan wawancara terhadap pelamar yang berminat. Tes Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) dan wawancara dilakukan oleh kepala sekolah atau siapa yang ditunjuk oleh kepala Sekolah Bentuk tesnya adalah tes tulis dan wawancara. dan wawancara dilakukan oleh kepala sekolah atau siapa yang ditunjuk oleh kepala Sekolah Bentuk tesnya adalah tes tulis dan wawancara. Hal itu dibenarkan dalam pendapat kepala Sekolah H. Takiyudin Nazah, M.Si (13 juni 2022). “Dalam proses selekssi ini kami melakukannya dengan melihat kelangkapan syarat administrasi dulu setelah itu baru dilakukan tes tulis dan wawancara yang bertujuan untuk meliat kualitas dan kapasitasnya”. Jika pelamar telah lolos seleksi atau diterima sebagai tenaga pendidik atau pegawai, maka dikeluarka Surat Keterangan (SK) pertama setelah masa training kerja selama 3 bulan baik bagi dari eksternal atau pegawai baru. Hal ini bertujuan untuk melihat dedikasi dari pelamar tersebut kepada lembaga, hal ini seperti yang dikatakan waka kurikulum Lukman Hakim, S. Kom (13 Juli 2022). “Setelah diterima sebagai guru atau karyawan kami mengadakan training selama 3 bulan sebelum SK dari sekolah dikeluarkan hal ini untuk meliaht dia secara pribadi dan secara pekerjaan hal ini juga saya alami setelah saya terpilih menjadi waka kurikulum 10 bulan yang lalu”. c. Seleksi Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 55 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) 55 dan wawancara dilakukan oleh kepala sekolah atau siapa yang ditunjuk oleh kepala Sekolah Bentuk tesnya adalah tes tulis dan wawancara. Hal itu dibenarkan dalam pendapat kepala Sekolah H. Takiyudin Nazah, M.Si (13 juni 2022). “Dalam proses selekssi ini kami melakukannya dengan melihat kelangkapan syarat administrasi dulu setelah itu baru dilakukan tes tulis dan wawancara yang bertujuan untuk meliat kualitas dan kapasitasnya”. Jika pelamar telah lolos seleksi atau diterima sebagai tenaga pendidik atau pegawai, maka dikeluarka Surat Keterangan (SK) pertama setelah masa training kerja selama 3 bulan baik bagi dari eksternal atau pegawai baru. Hal ini bertujuan untuk melihat dedikasi dari pelamar tersebut kepada lembaga, hal ini seperti yang dikatakan waka kurikulum Lukman Hakim, S. Kom (13 Juli 2022). “Setelah diterima sebagai guru atau karyawan kami mengadakan training selama 3 bulan sebelum SK dari sekolah dikeluarkan hal ini untuk meliaht dia secara pribadi dan secara pekerjaan hal ini juga saya alami setelah saya terpilih menjadi waka kurikulum 10 bulan yang lalu”. d. Pengorganisasian Sumberdaya Manusia Di SMA Favorit NU Tegaldlimo Penempatan fungsi pengorganisasian setelah fungsi perencanaan merupakan hal yang logis karena tindakan pengorganisasian menjembatani kegiatan perencanaan dengan pelaksanaannya sebagaimana diungkapakan kepala sekolah kepala Sekolah H. Takiyudin Nazah, M.Si (13 juni 2022). “Dalam pengorganisaian ini kami berusaha mengusahakan hubungan- hubungan kelakuan yang efektif antara orang-orang, sehingga mereka dapat bekerja sama secara efisien, dan memperoleh kepuasan pribadi dalam melaksanakan tugas-tugas tertentu, dalam kondisi lingkungan t t t i t j t t t t k i d 1) Orientasi dan penempatan Untuk tahap selanjutnya yakni penempatan, lebih detail ada dua cara yang dilakukan manajemen sekolah yakni seperti yang dijelaskan kepala sekolah; “Pertama, dengan melakukan penempatan atau pengisian jabatan/pekerjaan bagi guru dan pegawai baru. Kedua, dengan memberikan penugasan kembali kepada guru atau pegawai lama atas tugas atau jabatan baru yang berbeda, tetapi tetap memperhatikan skill dan kemampuannya”. Lebih jauh menambahkan tentang bagaimana proses untuk promosi atau penempatan pegawai baru kepala sekolah menjelaskan “Saya itu promosi jabatan setelah minimal setelah 2 tahun guru atau tenaga kependidikan bekerja setelah itu baru bisa promosi ke jabatan yang lebih tinggi hal ini juga melihat kinerjnya seperti dikatakan pak taufik kita itu yang terpilih professional dan demokratis artinya semua dipilh oleh elemen yang ada disini dan diketahui yayasan”. Tegaldlimo Penempatan fungsi pengorganisasian setelah fungsi perencanaan merupakan hal yang logis karena tindakan pengorganisasian menjembatani kegiatan perencanaan dengan pelaksanaannya sebagaimana diungkapakan kepala sekolah kepala Sekolah H. Takiyudin Nazah, M.Si (13 juni 2022). “Dalam pengorganisaian ini kami berusaha mengusahakan hubungan- hubungan kelakuan yang efektif antara orang-orang, sehingga mereka dapat bekerja sama secara efisien, dan memperoleh kepuasan pribadi dalam melaksanakan tugas-tugas tertentu, dalam kondisi lingkungan tertentu guna mencapai tujuan atau sasaran tertentu yakni dengan memberiakn job dan tugas sesuai hasil rapt dan ditanya kesiapannya”. Pengorganisasian terjadi karena pekerjaan yang perlu dilaksanakan itu merupakan pola yang membutuhkan antar SDM yang ada dan terbentuklah suatu kerja yang efektif antar SDM yang ada. Hal lain juga diungkapkan Bpk. kepala Sekolah H. Takiyudin Nazah, M.Si (13 juni 2022). “Pengorganisasian yang kami lakukan adalah pembagian tugas agar tugas dapat dilaksanakan dengan penuh tanggungjawab diharapkan Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 56 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) dengan adanya pembagian anggota organisasi dapat meningkatkan keterampilannya secara khusus (spesialisasi) dalam menangani tugas- tugas yang dibebankan.hal ini juga dilakukan stelah mereka bekerja 2 tahun untuk yang promosi jabatan”. e. Pelaksanaan Sumberdaya Manusia di SMA Favorit NU Tegaldlimo 2022). “Pelatihan tidak serta merta dilakukan setiap saat, pelatihan dilakukan apalagi Adanya karyawan baru Adanya penerapan sistem dan kurikulum atau informasi baru Adanya Prestasi kerja dibawah standard juga setiap pelatihan ini juga ada evaluasi dan pelatihan ini sangat membantu kami dalam hal pembaruan keilmuan” “Pelatihan tidak serta merta dilakukan setiap saat, pelatihan dilakukan apalagi Adanya karyawan baru Adanya penerapan sistem dan kurikulum atau informasi baru Adanya Prestasi kerja dibawah standard juga setiap pelatihan ini juga ada evaluasi dan pelatihan ini sangat membantu kami dalam hal pembaruan keilmuan” 2. Mutu guru Salah satu indikator tenaga pendidik yang bermutu adalah memenuhi kualifikasi akademik yaitu minimal D-3 atau Strata s-1. Berdasarkan studi dokumen yang peneliti lakukan didapati secara keseluruhan tenaga pendidik di SMA Favorit NU Tegaldlimo belum memenuhi kualifikasi akademik yaitu S1 dengan latar belakang tetapi sudah menempuh pendidikannya lagi untuk mendapatkan kualifikasi S1. Peneliti melakukan wawancara kepada Bpk. H. Takiyudin Nazah, M.Si (13 juni 2022) selaku kepala sekolah, sebagai berikut: “Alhamdulillah 70% guru di SMA Favorit NU Tegaldlimo ini sudah mempunyai sertifikat pendidik. Kualifikasi pendidikan Alhamdulillah sudah S1 walaupun masih sebagian masih menempuh kualifikasi S1 dikarena tidak sesuai dengan peraturan pemerintah, Karena tidak ingin berbenturan dengan Undang- undang No. 14 tahun 2005 tentang guru dan dosen, yang menyebutkan bahwa kualifikasi pendidikan guru SMA/SMK yaitu minimal Diploma Strata 1. Yang belum berualifiasi S1 maka saya tuntut harus berualifikasi S1agar sesuai dengan UUD yang di tetapkan oleh pemerintah. Dalam hal pembelajaran, tenaga pendidik SMA Favorit NU Tegaldlimo melaksanakan tugasnya sesuai dengan kompetensi yang dimilikinya, diantaranya 1) Kompetensi pedagogik 2) Pelatihan dan pengembangan Berhubung pentingnya hal ini SMA Favorit NU Tegaldlimo menjadikan pelatihan sebagai program unggulan di sekolah tersebut. Dengan pelatihan yang berkala, hal ini seperti didkatakn Bpk. kepala Sekolah H. Takiyudin Nazah, M.Si . “Pelatihan pengetahuan, keterampilan, kedisiplinan, dilaksanakan tiap tanggal 5 saat penerimaan bisyarah agara motivasi guru semakin tinggi, hal tersebut berdampak pada pengembangan dirinya maupun karier kedepanny karena kalau tidak musyawarah za evaluasi kalau tidak kadang kami isi dengan pelatihan”. Pelatiahn dan pengembangan Semua itu menjadi kebutuhan organisasi yang diwujudkan melalui pendekatan dari strategi sekolah dengan memperhatikan lingkungan saat ini dan juga bersifat melihat situasi yang ada suatu kebutuhan SDM yang ada sebagaimana yang dikatakan salah seorang guru bapak Irham Maulana pada tanggal (13 Juli Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 57 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) 2) Kompetensi professional Tenaga pendidik (guru) SMA Favorit NU Tegaldlimo mampu untuk mengajar mata pelajarann. seperti yang di sampaikan oleh Bpk. Abd. Rahman, S.Pd (14 juni 2022) selaku guru mata pelajaran Pendidikan Kewarganegaraan sebagai berikut: “ada seorang guru yang latar belakang ilmunya PKN seperti saya, tapi memegang mata pelajaran seni. berbeda antara latar belakang disiplin ilmu dengan tugas mengajar, tapi kebetulan yang bersangkutan menyukai dan mempunyai kemampuan di bidang tersebut karena beliau juga menekuni bidang tersebut.” 3) Kompetensi kepribadian Kepribadian yang mantap dan dewasa memiliki indikator esensial memiliki konsistensi dalam bertindak sesuai dengan norma, dan memiliki etos kerja sebagai guru. Tenaga pendidik di SMA Favorit NU Tegaldlimo konsisten dalam menjalankan tugasnya sebagai pendidik professional. Hal ini disampaikan oleh wakil kepala sekolah Bapak Hanif Setiawan S.Pd (14 juli 2022) berikut: “pengajar di SMA Favorit NU Tegaldlimo ini secara umum semua taat asas atau peraturan dalam mengajar. Kasus tidak mengajar sama sekali itu tidak ada, kalaupun ada yang tidak masuk dikarenakan sakit, pengajar disini tetap memberi tugas kepada anak-anak didik” 1) Kompetensi pedagogik Kompetensi pedagogik adalah kemampuan dalam mengelola pembelajaran peserta didik yang meliputi pemahaman guru terhadap peserta didik, perancangan dan pelaksanaan pembelajaran, evaluasi hasil belajar dan mengembangkan potensi yang dimiliki oleh peserta didik. Dengan mengetahui perkembangan kognitif peserta didik, pendidik (guru) akan lebih mudah dalam mengembangkan pola belajar di kelas. Hal ini sudah dilakukan oleh Bpk. H. Takiyudin Nazah, M.Si (13 juni 2022) selaku kepala sekolah, sebagai berikut:: Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 58 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) “…Iya tentu, awalnya harus di identifkasi karakteristik peserta didik yang memiliki keragaman dari latar belakang keluarga kemudian intelektual atau IQ nya kemudian dari Nem SMA nya. Hal ini bertujuan untuk mengetahui kemampuan awal peserta didik. Agar saya sebagai guru bisa menentukan dari mana pelajaran harus dimulai”. “…Iya tentu, awalnya harus di identifkasi karakteristik peserta didik yang memiliki keragaman dari latar belakang keluarga kemudian intelektual atau IQ nya kemudian dari Nem SMA nya. Hal ini bertujuan untuk mengetahui kemampuan awal peserta didik. Agar saya sebagai guru bisa menentukan dari mana pelajaran harus dimulai”. Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Dari pemaparan diatas dapat disimpulkan bahwa pendidik di di SMA Favorit NU Tegaldlimo telah melakukan komunikasi efektif baik dengan peserta didik maupun dengan warga sekolah lainnya. Komunikasi yang efektif di sekolah sangat dibutuhkan agar tercapainya peningkatan kualitas pendidikan. 4) Kompetensi sosial Setiap pagi tenaga pendidik atau guru di SMA Favorit NU Tegaldlimo menyambut peserta didik yang baru datang di depan gerbang sekolah untuk melaksanakan kegiatan salaman pagi. Kegiatan ini adalah salah satu bentuk komunikasi antara guru dan peserta didik. Mampu berkomunikasi dan bergaul secara efektif dengan sesama pendidik dan tenaga kependidikan terlihat pula dari bagaimana cara mereka berdiskusi atau bersenda gurau saat istirahat berlangsung. Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 59 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) perkembangan guru tersebut dan keadaan sekolah, dan peran guru disekolah sebagai pembimbing, Korektor, Inspirator, dan lain-ain. Selain itu guru juga mengawasi siswa baik dalam kegiatan proses belajar mengajar maupun diluar jam KBM. Yang mana pada akhirnya akan menentukan pada kualitas lulusannya. Jika mutu guru bagus, maka kualitas pembelajaran pun menjadi meningkat. Hal inilah yang pada gilirannya nanti akan dihasilkan mutu lulusan yang lebih baik. perkembangan guru tersebut dan keadaan sekolah, dan peran guru disekolah sebagai pembimbing, Korektor, Inspirator, dan lain-ain. Selain itu guru juga mengawasi siswa baik dalam kegiatan proses belajar mengajar maupun diluar jam KBM. Yang mana pada akhirnya akan menentukan pada kualitas lulusannya. Jika mutu guru bagus, maka kualitas pembelajaran pun menjadi meningkat. Hal inilah yang pada gilirannya nanti akan dihasilkan mutu lulusan yang lebih baik. 1. Perencanaan Manajemen Sumber Daya Manusia di SMA Favorit NU Perencanaan sumberdaya amnesia Dalam meningkatkatkan mutu guru telah melakukan dengan penyesuaian rencana dengan visi misi yakni kesesuain dengan kebutuhan termasuk dalam manajemen sumber daya manusia agar sesuai dengan visi misi. Hal ini sesuai dengan yang diungkapkan hasibuan Perencanaan SDM (human resources planning) adalah perencanaan tenaga kerja secara efektif serta efisien agar sesuai dengan kebutuhan perusahaan dalam membantu terwujudnya tujuan. Perencanaan dilakukan dengan menetapkan program kepegawaian. SMA Favorit NU Tegaldlimo Dalam sebuah wawancara dilakukan pada tangal 21 juli 2022 kepada H. Takiyudin Nazah, SE selaku kepala sekolah mengatakan: “Kriteria guru yang profesional baru bisa dikatakan ketika dia melaksanakan tugas secara profesional dan bertanggung jawab. Guru selain harus profesional juga harus memiliki kualifikasi akademik serta memiliki kecakapan hidup untuk mewujudkan tujuan lembaga pendidikan/sekolah khususya dan tujuan pendidikan nasional pada umumnya” y Dari wawancara di atas dapat penulis simpulkan bahwa guru yang professional dan bermutu dalam memenuhi tanggung jawabnya ialah guru yang mampu kompeten dan kreatif dalam mengajar Dan memenuhi standar kualifikasi S1, sehingga akan menjadikan siswa memiliki daya saing sebagai upaya mencapai visi, misi dan tujuan sekolah. Dalam sebuah wawancara yang peneliti lakukan pada tangal 21 juli 2022 kepada H. Takiyudin Nazah, SE selaku kepala sekolah mengatakan: ”Kompetensi pedagogik mutlak harus dimiliki oleh seorang guru yang profesional untuk menunjang tercapainya tujuan instruksional umum dan khusus. Dengan demikian guru harus mampu menempatkan berbagai substansi perbedaan pengalaman belajar, perbedaan bahasa dan budaya, gaya belajar, talenta, danintelegensi sebagai dasar dalam melaksanakan berbagai strategi pengajaran yang dipilihnya” Dalam sebuah wawancara dilakukan pada tangal 21 juli 2022 kepada Lukman Hakim, S.Kom selaku waka kurikulum mengatakan: Lukman Hakim, S.Kom selaku waka kurikulum mengatakan: “Tersertifikasi adalah salah satu bukti seorang guru bisa dikatakan bermutu atau berkualitas ketika memiliki surat izin mengajar yang biasa disebut akta empat” Jadi dapat disimpulkan bahwa bahwa Kepala sekolah di SMA Favorit NU Tegaldlimo juga berperan dalam peningkatan mutu guru, mengawasi guru, Jadi dapat disimpulkan bahwa bahwa Kepala sekolah di SMA Favorit NU Tegaldlimo juga berperan dalam peningkatan mutu guru, mengawasi guru, dalam aktifitas kesehariannya kepala sekolah keliling untuk melihat dalam aktifitas kesehariannya kepala sekolah keliling untuk melihat Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 60 a. Orientasi dan penempatan Manajemnen SMA Favorit NU Tegaldlimo mensosialisasikan personelnya pada pekerjaan baru melalui kegiatan orientasi untuk meningkatkan dukungan yang lebih efektif. Hal ini berarti kepala sekolah dan seluruh elemen pimpinan melakukan pantauan kinerja sebelum dilkukan penempatan yang merupakan hasil seleksi pantauan selama 3 bulan: Menurut Sastrohadiwiryo mengemukakan bahwa :Penempatan kerja adalah proses pemberian tugas dan pekerjaan kepada karyawan yang lulus seleksi untuk dilaksanakan sesuai ruang lingkup yang telah ditetapkan, serta mampu mempertanggungjawabkan segala resiko dan kemungkinan-kemungkinan yang terjadi atas tugas dan pekerjaan, wewenang serta tanggung jawab. b. Pelatihan dan pengembangan Dalam hal ini Kepala sekolah selalu mengupayakan agar para guru selalu mengikuti perkembangan teknologi dan informasi, dengan cara kepala Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 61 61 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) sekolah aktif mengikutkan tenaga kependidikan mupun guru dalam berbagai acara pelatihan pendidikan. Hal itu sebagaimana yang diungkapkan baharudin Manajemen sumber daya manusia (SDM) adalah teknik atau prosedur yang berhubungan dengan pengelolaan dan pendayagunaan personalia sekolah/madrasah atau instansi (SDM), baik tenaga edukatif maupun tenaga administratif secara efektif dan efisien banyak tergantung pada kemampuan kepala sekolah baik sebagai manager dan pemimpin pada lembaga pendidikan tersebut sekolah aktif mengikutkan tenaga kependidikan mupun guru dalam berbagai acara pelatihan pendidikan. Hal itu sebagaimana yang diungkapkan baharudin Manajemen sumber daya manusia (SDM) adalah teknik atau prosedur yang berhubungan dengan pengelolaan dan pendayagunaan personalia sekolah/madrasah atau instansi (SDM), baik tenaga edukatif maupun tenaga administratif secara efektif dan efisien banyak tergantung pada kemampuan kepala sekolah baik sebagai manager dan pemimpin pada lembaga pendidikan tersebut Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) berkomunikasi dan bergaul secara efektif dengan peserta didik, sesama pendidik, tenaga kependidikan, orang tua/wali peserta didik, dan masyarakat sekitar. Tenaga pendidik adalah makhluk sosial, yang dalam kehidupannya tidak terlepas dari kehidupan social masyarakat dan lingkungannya. . Hal ini Sesuai dengan teori Trianto dan Titik (2006:3). Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 3. Mutu guru di SMA Favorit NU Tegaldlimo Dalam hal pembelajaran, tenaga pendidik SMA Favorit NU Tegaldlimo melaksanakan tugasnya sesuai dengan kompetensi yang dimilikinya, diantaranya: a. Kompetensi pedagogik Dalam hal ini SMA Favorit NU Tegaldlimo Banyuwangi sudah menerapkan kompetensi pedagogik untu kemampuan dalam mengelola pembelajaran peserta didik yang meliputi pemahaman guru terhadap peserta didik, perancangan dan pelaksanaan pembelajaran, evaluasi hasil belajar dan mengembangkan potensi yang dimiliki oleh peserta didik. Hal ini sesuai dengan teori Trianto dan Titik (2006:3) b. kompetensi professional Dalam hal ini SMA Favorit NU Tegaldlimo Banyuwangi sudah menerapkan kompetensi profesional dapat dilihat dari penguasaan materi pembelajaran secara luas dan mendalam. Hal ini Sesuai dengan teori Trianto dan Titik (2006:3) b. kompetensi professional Dalam hal ini SMA Favorit NU Tegaldlimo Banyuwangi sudah menerapkan kompetensi profesional dapat dilihat dari penguasaan materi pembelajaran secara luas dan mendalam. Hal ini Sesuai dengan teori Trianto dan Titik (2006:3) c. Kompetensi kepribadian Dalam hal ini SMA Favorit NU Tegaldlimo Banyuwangi suda menerapakn Kompetensi kepribadian mencerminkan pribadi guru yang dapat dijadikan panutan bagi para peserta didik. Hal ini Sesuai dengan teori Trianto dan Titik (2006:3) c. Kompetensi kepribadian Dalam hal ini SMA Favorit NU Tegaldlimo Banyuwangi suda menerapakn Kompetensi kepribadian mencerminkan pribadi guru yang dapat dijadikan panutan bagi para peserta didik. Hal ini Sesuai dengan teori Trianto dan Titik (2006:3) d. Kompetensi sosial Dalam hal ini SMA Favorit NU Tegaldlimo Banyuwangi suda menerapakn Kompetensi sosial merupakan kemampuan guru untuk d. Kompetensi sosial Dalam hal ini SMA Favorit NU Tegaldlimo Banyuwangi suda menerapakn Kompetensi sosial merupakan kemampuan guru untuk Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 62 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Dalam melaksanakan pembinaan kompetensi sosial, kepala sekolah SMA Favorit NU Tegaldlimo mengadakan pembinaan keakraban para guru. Hal ini dilakukan agar sesama pendidik saling terbuka dan saling membantu apabila mengalami kesulitan 1. Sumberdaya Manusia Di SMA Favorit NU Di SMA Favorit NU Tegal delimo mengadakan perencanaan sumber daya manusia, perencanaan sumber daya manusia terdiri dari Perencanaan program kerja atau program kegiatan tahunan yang dilakukan Di SMA Favorit NU Tegaldlimo merupakan program kerja atau program kegiatan yang dihasilkan melalui rapat pengurus di SMA Favorit NU Tegaldlimo diawal tahun kepengurusannya. Perencanaan sumber daya manusia ini menurut penulis sangatlah perlu dilakukan karena perencanaan sumber daya manusia juga akan mempengaruhi penetapan penentuan jumlah guru baru yang akan diterima. Selanjutnya penentuan jumlah guru yang diterima di SMA Favorit NU Tegaldlimo. SMA Favorit NU Tegaldlimo Dalam rangka pembinaan tenaga pendidik (guru), kepala sekolah SMA Favorit NU Tegaldlimo berperan sebagai sumberdaya manusia mempunyai beberapa strategi dalam meningkatkan kompetensi tenaga pendidiknya yaitu: a. Pembinaan kompetensi pedagogic Dengan Kerjasama dengan Lembaga Penjamin Mutu Pendidikan (LPMP) Jawa Timur agar memberikan Pendidikan dan Latihan (Diklat) terhadap tenaga pendidik (guru) kemudian tindak lanjut dari LPMP yaitu meninjau langsung ke SMA Favorit NU Tegaldlimo untuk mengetahui tingkat keberhasilan Pendidikan dan Latihan (Diklat) tersebut. untuk meningkatkan kompetensi pedagogiknya yaitu melaksanakan pembinaan organisasi dan melaksanakan evaluasi pembelajaran. b. Pembinaan kompetensi professional Kepala sekolah SMA Favorit NU Tegaldlimo memberikan beasiswa kepada tenaga pendidik (guru) ketika mendapatkan tugas belajar untuk meningkatkan jenjang akademis. Hal ini dilakukan untuk meningkatkan kompetensi atau mutu tenaga pendidik dibidang akademik. b. Pembinaan kompetensi professional Kepala sekolah SMA Favorit NU Tegaldlimo memberikan beasiswa kepada tenaga pendidik (guru) ketika mendapatkan tugas belajar untuk meningkatkan jenjang akademis. Hal ini dilakukan untuk meningkatkan kompetensi atau mutu tenaga pendidik dibidang akademik. c. Pembinaan kompetensi kepribadian Melakukan percakapan pribadi dengan tenaga pendidik (guru) Kepala sekolah SMA Favorit NU Tegaldlimo selalu mengadakan percakapan pribadi dengan tenaga pendidiknya untuk mengetahui apakah ada permasalahan yang dihadapi oleh tenaga pendidik (guru) terhadap siswa, maupun terhadap sesama pendidik ataupun masalah pembelajaran. d. Pembinaan kompetensi sosial 63 E. Kesimpulan Berdasarkan hasil penelitian yang telah penulis lakukan tentang implementasi manajemen sumberdaya manusia (SDM) dalam meningkatkan mutu guru di SMA Favorit NU Tegal delimo Banyuwangi, dapat diambil kesimpulan sebagai berikut: 1. Sumberdaya Manusia Di SMA Favorit NU Daftar Pustaka Barthos, Basir 2012. Manajemen Sumber Daya Manusia: Suatu Pendekatan Makro, Jakarta: Bumi Aksara Emzir 2010, Analisis Data: Metodologi Penelitian Kualitatif, (Jakarta: RajawaliPers,) Henry 2004. Simamora. Manajemen Sumber Daya Manusia Edisi III,Yogyakarta: Sekolah Tinggi Ilmu Ekonomi YKPI Joko Subagyo 2011, Metode penelitian dalam teori dan praktik, (Jakarta: PT Rineka Cipta L J M l 2009 M t d l i P liti K lit tif Barthos, Basir 2012. Manajemen Sumber Daya Manusia: Suatu Pendekatan Makro, Jakarta: Bumi Aksara Emzir 2010, Analisis Data: Metodologi Penelitian Kualitatif, (Jakarta: RajawaliPers,) Henry 2004. Simamora. Manajemen Sumber Daya Manusia Edisi III,Yogyakarta: Sekolah Tinggi Ilmu Ekonomi YKPI Joko Subagyo 2011, Metode penelitian dalam teori dan praktik, (Jakarta: PT Rineka Cipta Lexy J. Moeleong 2009. Metodologi Penelitian Kualitatif Malayu S.P. 2004. Hasibuan, Manajemen Sumber Daya Manusia, Dasar dan Kunci Keberhasilan, (Jakarta: PT Toko Gunung Agung Mangkuprawira 2003. Manajemen sumber daya manusia Strategik. Jakarta: Ghalia Indonesia Marwansyah 2016. Manajemen Sumber Daya Manusia, Bandung: Alfabeta Nawawi, Hadari 2008. Manajemen Sumber Daya Manusia Untuk Bisnis yang Kompetitif,Yogyakarta: Gadjah Mada University Press Nawawi, Ismail 2013. Budaya Organisasi Kepemimpinn dan Kinerja, Jakarta: Prenada Media Group Sugiono 2010, Metode Penelitian Pendidikan: Pendekatan Kuantitatif, Kualitatif dan R&D, (Bandung: ALFABETA,). Suharsimi Arikunto 2010, Prosedur Penelitian Pendekatan Praktek, (Jakarta: Rineka Cipta,). Syaodih Sukmadinata 2010, Metode Penelitian Pendidikan, (Remaja Rosdakarya:). Uzer, Usman 2006 , Menjadi Guru Profesional, (Bandung: PT Remaja Rosdakarya Yonder, Dale 2004. Memahami Good Goverment Dalam Persfektif Sumber Daya Manusia, Jakarta: GavaMedia Barthos, Basir 2012. Manajemen Sumber Daya Manusia: Suatu Pendekatan Makro, Jakarta: Bumi Aksara Emzir 2010, Analisis Data: Metodologi Penelitian Kualitatif, (Jakarta: RajawaliPers,) Henry 2004. Simamora. Manajemen Sumber Daya Manusia Edisi III,Yogyakarta: Sekolah Tinggi Ilmu Ekonomi YKPI Joko Subagyo 2011, Metode penelitian dalam teori dan praktik, (Jakarta: PT Rineka Cipta Lexy J. Moeleong 2009. Metodologi Penelitian Kualitatif Malayu S.P. 2004. Hasibuan, Manajemen Sumber Daya Manusia, Dasar dan Kunci Keberhasilan, (Jakarta: PT Toko Gunung Agung Mangkuprawira 2003. Manajemen sumber daya manusia Strategik. Jakarta: Ghalia Indonesia Marwansyah 2016. Manajemen Sumber Daya Manusia, Bandung: Alfabeta Lexy J. Moeleong 2009. Metodologi Penelitian Kualitatif Malayu S.P. 2004. Hasibuan, Manajemen Sumber Daya Manusia, Dasar dan Kunci Keberhasilan, (Jakarta: PT Toko Gunung Agung Mangkuprawira 2003. Manajemen sumber daya manusia Strategik. Jakarta: Ghalia Indonesia Marwansyah 2016. Manajemen Sumber Daya Manusia, Bandung: Alfabeta Nawawi, Hadari 2008. Manajemen Sumber Daya Manusia Untuk Bisnis yang Kompetitif,Yogyakarta: Gadjah Mada University Press Nawawi, Ismail 2013. SMA Favorit NU Tegal delimo Dalam rangka pembinaan tenaga pendidik (guru), kepala sekolah SMA Favorit NU Tegaldlimo berperan sebagai sumberdaya manusia mempunyai beberapa strategi dalam meningkatkan kompetensi tenaga pendidiknya yaitu: (1) Kerjasama dengan Lembaga Penjamin Mutu Pendidikan (LPMP) Jawa Timur; (2) Pembinaan rutin yang dilakukan oleh kepala sekolah; (3) Pengalokasian anggaran untuk meningkatkan mutu tenaga pendidik; (4) Pembinaan kompetensi professional; (5) Pembinaan kompetensi kepribadian; (6) Melakukan percakapan pribadi dengan tenaga pendidik (guru); (7) Pembinaan rutin dari sekolah; dan (8) Pembinaan kompetensi sosial. 3. Implementasi Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegal delimo 3. Implementasi Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegal delimo 2. Mutu Guru di SMA Favorit NU Tegaldlimo Di SMA Favorit NU Tegaldlimo. selalu mengutamakan mutu atau kualitas tenaga pendidiknya yang paling utama dalam merekrut tenaga pendidik. Dalam seleksi penerimaan tenaga pendidik dilakukan dengan cara yang terbaik agar mendapatkan tenaga pendidik (guru) yang berkualitas atau bermutu. Tenaga pendidik (guru) yang berada di SMA Favorit NU Tegaldlimo. tahun 2022 sudah memenuhi standar kualifikasi S1, sebanyak 11 guru berstrata S1 dari jumlah keseluruhan guru yaitu 19 orang. Apabila ada tenaga pendidik yang masih belum kualifikasi S1 maka harus menempuh pendidikannya lagi untuk mendapatkan kualifikasi S1. Hal ini dilakukan karena ingin memperoleh tenaga pendidik (guru) yang berkualitas atau bermutu agar tujuan pendidikan dapat tercapai. Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto 64 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Daftar Pustaka Budaya Organisasi Kepemimpinn dan Kinerja, Jakarta: Prenada Media Group Sugiono 2010, Metode Penelitian Pendidikan: Pendekatan Kuantitatif, Kualitatif dan R&D, (Bandung: ALFABETA,). Suharsimi Arikunto 2010, Prosedur Penelitian Pendekatan Praktek, (Jakarta: Rineka Cipta,). Syaodih Sukmadinata 2010, Metode Penelitian Pendidikan, (Remaja Rosdakarya:). Uzer Usman 2006 Menjadi Guru Profesional (Bandung: PT Remaja Rosdakarya Syaodih Sukmadinata 2010, Metode Penelitian Pendidikan, (Remaja Rosdakarya:). Syaodih Sukmadinata 2010, Metode Penelitian Pendidikan, (Remaja Rosdakarya:). Uzer, Usman 2006 , Menjadi Guru Profesional, (Bandung: PT Remaja Rosdakarya Yonder, Dale 2004. Memahami Good Goverment Dalam Persfektif Sumber Daya Manusia, Jakarta: GavaMedia Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA Favorit NU Tegaldlimo Mamlukhah, Heru Susanto Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) 65 Yunarsih, Tjutju. dan Suwatno 2008, Manajemen Sumber Daya Manausia,Bandung: Alfabeta 65 Jurnal Tarbiyatuna, Vol. 3, No. 2, Desember 2022 ISSN: 2774-5724 (media Online) Yunarsih, Tjutju. dan Suwatno 2008, Manajemen Sumber Daya Manausia,Bandung: Alfabeta Implementasi Manajemen Sumberdaya Manusia Dalam Meningkatkan Mutu Guru Di SMA
https://openalex.org/W4322825337	https://nottingham-repository.worktribe.com/file/17938098/1/Division%20algebras%20and%20MRD%20codes%20from%20skew%20polynomials	English	null	Division algebras and MRD codes from skew polynomials	arXiv (Cornell University)	2,021	cc-by	17,057	1. Introduction Rank distance codes are important both in coding theory and cryptography. One of the best-known maximum rank distance (MRD) codes is probably the Gabidulin code [10] which was mentioned already by Delsarte [8]. In coding theory, MRD codes are well suited to correct errors [6, 31]. In cryptography, they are used to design public-key cryptosystems, see for instance [9, 12]. y g p y yp y MRD codes over general (non-ﬁnite) ﬁelds, in particular number ﬁelds, were already studied in [2] and later touched on in [34]. Rank metric codes over both cyclic and more general Galois exten- sions were considered in [3, 31, 32]. Although rank metric codes have been also constructed over ﬁnite principal ideal rings [19] and discretely valued rings [21], to our knowledge they have not yet been studied over noncommutative rings. In this paper, we also consider MRD codes in Mk(B), where B is a noncommutative division algebra. We construct these MRD codes using skew polynomials. Skew polynomials have been successfully used in constructions of both division algebras (mostly semiﬁelds) and linear codes [2, 4, 5, 13, 26–28], in particular building space-time block codes (STBCs) [29] and MRD codes [33, 34]. Our codes can be seen as generalizations of both the classical and twisted Gabidulin codes in [10], resp., [33]. We put Sheekey’s construction [34] in a broader context which helps to understand it better and potentially allows other ways to generalize MRD coding using skew polynomials. The drawback is that rather early on we have to rigorously restrict the choice of the polynomials f we can employ and that the construction remains rather theoretical. Sheekey [34] only considers skew polynomials f ∈K[t; σ] with coeﬃcients in cyclic Galois ﬁeld extensions for his construction and limits himself to the case that the minimal central left multiple of f has maximal degree. He misses out on codes (with matrix entries both in a noncommutative division algebra, and with entries in ﬁelds) and algebras that can be obtained by employing skew polynomials with coeﬃcients in a noncommutative division algebra. He also misses out on constructions using f ∈D[t; δ]. We construct both new division algebras and MRD codes with entries in a noncommutative division algebra, and with entries in ﬁelds. The ﬁrst ﬁve Sections of the paper contain the preliminaries (Section 1) and theoretical background needed to obtain the main results (Sections 2–5). Abstract Let D be a division algebra, ﬁnite-dimensional over its center, and R = D[t; σ, δ] a skew polynomial ring. Using skew polynomials f ∈R, we construct division algebras and maximum rank distance codes consisting of matrices with entries in a noncommutative division algebra or ﬁeld. These include Jha Johnson semiﬁelds, and the classes of classical and twisted Gabidulin codes constructed by Sheekey. RESEARCH ARTICLE RESEARCH ARTICLE C⃝The Author(s), 2023. Published by Cambridge University Press in association with Glasgow Mathematical Journal Trust. 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/S001708952300006X Published online by Cambridge University Press D. Thompson1 and S. Pumplün2 128 Coral Lane Newhall Swadlincote DE11 0XU, United Kingdom 2School of Mathematical Sciences, University of Nottingham University Park, Nottingham NG7 2RD, United Kingdom E-mails: thompson.danjames@gmail.com, susanne.pumpluen@nottingham.ac.uk Received: 22 June 2021; Revised: 3 February 2023; Accepted: 27 February 2023 Keywords: skew polynomial ring, skew polynomials, division algebras, MRD codes 2020 Mathematics Subject Classiﬁcation: Primary - 16S36 128 Coral Lane Newhall Swadlincote DE11 0XU, United Kingdom 2School of Mathematical Sciences, University of Nottingham University Park, Nottingham NG7 2RD, United Kingdom E-mails: thompson.danjames@gmail.com, susanne.pumpluen@nottingham.ac.uk Received: 22 June 2021; Revised: 3 February 2023; Accepted: 27 February 2023 Keywords: skew polynomial ring, skew polynomials, division algebras, MRD codes 2020 Mathematics Subject Classiﬁcation: Primary - 16S36 Received: 22 June 2021; Revised: 3 February 2023; Accepted: 27 February 2023 Keywords: skew polynomial ring, skew polynomials, division algebras, MRD codes 2020 Mathematics Subject Classiﬁcation: Primary - 16S36 Glasgow Mathematical Journal (2023), 1–21 doi:10.1017/S001708952300006X RESEARCH ARTICLE Division algebras and MRD codes from skew polynomials D. Thompson1 and S. Pumplün2 D. Thompson1 and S. Pumplün2 D. Thompson1 and S. Pumplün2 1. Introduction Let D be a division algebra of degree d over its center, D. Thompson and S. Pumplün 2 and f ∈R = D[t; σ, δ] a monic irreducible skew polynomial with a bound that lies in the center C(R) of R. and f ∈R = D[t; σ, δ] a monic irreducible skew polynomial with a bound that lies in the center C(R) of R C(R) While developing the theory, we point out how the choice of D and the polynomial f has to be restricted in order to construct both division algebras and MRD codes out of f, a scalar ν ∈D and a suitable ρ ∈Aut(D). Apart from Section 9, we ﬁx the following general assumptions unless speciﬁed otherwise: R = D[t; σ], where σ is an automorphism of D of ﬁnite order n modulo inner automorphisms, i.e. σ n = iu for some inner automorphism iu(z) = uzu−1, and F = C ∩Fix(σ). Choose ρ ∈Aut(D), such that F/F′ with F′ = Fix(ρ) ∩F is ﬁnite-dimensional. Let ν ∈D×. Let f ∈R be monic and irreducible of degree m > 1, and h the minimal central left multiple of f, so that R/Rh ∼= Mk(B) for some division algebra B (Theorem 3). Let l < k be a positive integer. Deﬁne Sn,m,l(ν, ρ, f) = {a + Rh \| a ∈P} ⊂R/Rh with the set P = {d0 + d1t + · · · + dlm−1tlm−1 + νρ(d0)tlm \| di ∈ D}. Let La : R/Rf →R/Rf be the left multiplication map La(b + Rf) = ab + Rf. We have well-deﬁned maps Sn,m,l(ν, ρ, f) −→EndB(R/Rf) −→Mk(B), a →La →Ma, where Ma is the matrix representing La with respect to a right B-basis of R/Rf. The image Cn,m,l = {Ma \| a ∈Sn,m,l(ν, ρ, f)} of Sn,m,l(ν, ρ, h) in Mk(B) is an F′-linear rank metric code. If Cn,m,l has distance dC = k −l + 1, then Cn,m,l is called a maxi- mum rank distance code in Mk(B). We will usually deal with the case that deg(h) = dmn, so that B is a ﬁeld. The most general results are contained in Section 6: If P does not contain a polynomial of degree lm, whose irreducible factors are all similar to f, then Cn,m,l is an F′-linear MRD code in Mk(B) with minimum distance k −l + 1 (Theorem 19). 1. Introduction Furthermore, let D = (E/C, γ , a) be a cyclic division algebra such that σ\|E ∈Aut(E) and γ ◦σ\|E = σ\|E ◦γ , and σ n(z) = u−1zu for some u ∈E. Let f(t) = m i=0 aiti ∈E[t; σ] be a monic irreducible polyno- mial of degree m, such that deg(h) = dmn, and such that all monic fi similar to f lie in E[t; σ]. Then, the algebra Sn,m,1(ν, ρ, f) is a division algebra, if one of the following holds: (i) ν ̸∈E and ρ\|E ∈Aut(E); (ii) ν ∈E× and ρ\|E ∈Aut(E), such that NE/F′(a0)NE/F′(ν) ̸= 1 (Theorem 16). MRD codes are canonically obtained from the matrices representing the left multiplication of these division algebras. In Section 7, the nuclei of the algebras and codes are investigated. We give some examples of algebras obtained from our construction employing f(t) = tn −θ ∈K[t; σ] in Section 8. We conclude with a brief look at the constructions using a diﬀerential polynomial f ∈D[t; δ], where the center of D is a ﬁeld of characteristic p, in Section 9. We conclude with a brief look at the constructions using a diﬀerential polynomial f ∈D[t; δ], where the center of D is a ﬁeld of characteristic p, in Section 9. The fact that we are using f ∈D[t; σ], respectively f ∈D[t; γ ], means we have a larger choice of skew polynomials to build codes that Sheekey does, who only considers f with coeﬃcients in a cyclic ﬁeld extension. This work is part of the second author’s PhD thesis [35]. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 2.1. Nonassociative algebras Let F be a ﬁeld. We call A an algebra over F if there exists an F-bilinear map A × A →A, (x, y) →x · y, denoted simply by juxtaposition xy, the multiplication of A. An algebra A is called unital if there is an element in A, denoted by 1, such that 1x = x1 = x for all x ∈A. We will only consider unital algebras. A nonassociative algebra A ̸= 0 is called a division algebra if for any a ∈A, a ̸= 0, the left multiplication with a, La(x) = ax, and the right multiplication with a, Ra(x) = xa, are bijective. If A is ﬁnite-dimensional as an F-vector space, then A is a division algebra if and only if A has no zero divisors. The left nucleus of A is deﬁned as Nucl(A) = {x ∈A \| [x, A, A] = 0}, the middle nucleus of A is Nucm(A) = {x ∈A \| [A, x, A] = 0}, and the right nucleus of A is Nucr(A) = {x ∈A \| [A, A, x] = 0}, where [x, y, z] = (xy)z −x(yz) is the associator. Nucl(A), Nucm(A), and Nucr(A) are associative subalgebras of A. Their intersection Nuc(A) = {x ∈A \| [x, A, A] = [A, x, A] = [A, A, x] = 0} is the nucleus of A. Nuc(A) is an associative subalgebra of A, and x(yz) = (xy)z whenever one of the elements x, y, z is in Nuc(A). The center of A is C(A) = {x ∈Nuc(A) \| xy = yx for all y ∈A}. 3 Glasgow Mathematical Journal Let A be a ﬁnite-dimensional central simple associative algebra over F of degree d and let F denote the algebraic closure of F. Then, A ⊗F F ∼= Md(F), so that we can ﬁx an embedding A −→Md(F) and view every a ∈A as a matrix in Md(F). The characteristic polynomial Let A be a ﬁnite-dimensional central simple associative algebra over F of degree d and let F denote the algebraic closure of F. Then, A ⊗F F ∼= Md(F), so that we can ﬁx an embedding A −→Md(F) and view every a ∈A as a matrix in Md(F). The characteristic polynomial ma(X) = Xd −s1(a)Xd−1 + s2(a)Xd−2 −· · · + (−1)dsd(a), of a ∈A has coeﬃcients in F and is independent of the choice of the embedding. dC = min{d(X, Y) \| X, Y ∈C, X ̸= Y}. dC = min{d(X, Y) \| X, Y ∈C, X ̸= Y}. r knowledge, such codes C ⊂Mn,m(B) have not previously been considered in the literatu To our knowledge, such codes C ⊂Mn,m(B) have not previously been considered in the literature. 2.1. Nonassociative algebras The coeﬃcient NA(a) = sd(a) is called the reduced norm of a [20]. Let K/F be a cyclic Galois extension of degree d with Galois group Gal(K/F) = ⟨γ ⟩and norm NK/F. Let c ∈F×. An associative cyclic algebra (K/F, γ , c) of degree d over F is a d-dimensional K-vector space (K/F, γ , c) = K ⊕eK ⊕e2K ⊕· · · ⊕ed−1K, with multiplication given by the relations ed = c, le = eσ(l), for all l ∈K. (K/F, γ , c) is a division algebra for all c ∈F×, such that cs ̸∈NK/F(K×) for all s which are prime divisors of d, 1 ≤s ≤d −1. d(X, Y) = colrank(X −Y), for all X, Y ∈Mn,m(B), where colrank is the column rank of A (the rank of the right B-module generated by the columns of A). A matrix in Mn,m(B) has column rank at most m; any matrix which attains this bound is said to have attained full column rank. The minimum distance of a rank metric code C ⊂Mn,m(B) is deﬁned as https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 2.2. MRD codes Let K be a ﬁeld. A code is a set of matrices C ⊂Mn,m(K). Let L ⊂K be a subﬁeld, then C is L-linear if C is a vector space over L. A rank metric code is a code C ⊂Mn,m(K) equipped with the rank distance function d(X, Y) = rank(X −Y). Deﬁne the minimum distance of a rank metric code C as dC = min{d(X, Y) \| X, Y ∈C, X ̸= Y}. dC = min{d(X, Y) \| X, Y ∈C, X ̸= Y}. An L-linear rank metric code C satisﬁes the Singleton-like bound dimL(C) ≤n(m −dC + 1)[K:L], where dimL(C) is the dimension of the L-vector space C [2, Proposition 6]. An L-linear rank metric code attaining the Singleton-like bound is called a maximum rank distance code or MRD code (for MRD codes over cyclic ﬁeld extensions see [2]). If now B is a not necessarily commutative division algebra then more generally, we again deﬁne a code as a set of matrices C ⊂Mn,m(B). Let B′ ⊂B be a subalgebra, then C is B′-linear (or simply linear), if C is a right B′-module. g A rank metric code C ⊂Mn,m(B) is a code together with the distance function d(X, Y) = colrank(X −Y), 2.3. Skew polynomial rings A skew polynomial f ∈R is bounded if there exists a nonzero polynomial f ∗∈R such that Rf ∗is the largest two-sided ideal of R contained in Rf. The polynomial f ∗is uniquely determined by f up to scalar A skew polynomial f ∈R is bounded if there exists a nonzero polynomial f ∗∈R such that Rf ∗is the largest two-sided ideal of R contained in Rf. The polynomial f ∗is uniquely determined by f up to scalar multiplication by elements of D× and is called a bound of f. If f ∈R has degree m, then for all g ∈R of degree l ≥m, there exist uniquely determined r, q ∈R with deg(r) < deg(f), such that g = qf + r. Let modrf denote the remainder of right division by f. The skew polynomials Rm = {g ∈R \| deg(g) < m} of degree less that m canonically represent the elements of the left R-modules R/Rf. Furthermore, Rm together with the multiplication g ◦h = gh modrf is a unital nonassociative algebra Sf = (Rm, ◦) over F0 = {a ∈D \| ah = ha for all h ∈Sf} = Comm(Sf) ∩D, called a Petit algebra. When the context is clear, we simply use juxtaposition for multiplication in Sf. Note that C(D) ∩Fix(σ) ∩Const(δ) ⊂F0. For all a ∈D×, we have Sf = Saf; thus, without loss of generality we can assume f is monic when working with Petit algebras Sf. If f has degree 1 then Sf ∼= D. Lemma 1. Let R be a ring with no zero divisors. For all g ∈C(R), every right divisor of g in R also divides g on the left. Proof. Suppose γ is a right divisor of g. Then, g = δγ for some δ ∈R. As g lies in the center of R, we have δg = gδ = δγ δ. This rearranges to 0 = δg −δγ δ = δ(g −γ δ). As R contains no zero divisors and δ ̸= 0, it follows that g = γ δ. 2.4. The minimal central left multiple of f ∈D[t; σ] From now on let, σ be an automorphism of D of ﬁnite order n modulo inner automorphisms, i.e. σ n = iu for some inner automorphism iu(z) = uzu−1. Then, the order of σ\|C is n. W.l.o.g., we choose u ∈Fix(σ). Let R = D[t; σ] and deﬁne F = C ∩Fix(σ). R has center C(R) = F[u−1tn] = k i=0 ai(u−1tn)i \| ai ∈F ∼= F[x] with x = u−1tn [18, Theorem 1.1.22]. All polynomials f ∈R are bounded. For any f ∈R = D[t; σ] with a bound in C(R), we deﬁne the minimal central left multiple mclm(f) of f in R to be the unique polynomial of minimal degree h ∈C(R) = F[u−1tn] such that h = gf for some g ∈R, and such that h(t) = ˆh(u−1tn) for some monic ˆh(x) ∈F[x]. Deﬁne Eˆh = F[x]/(ˆh(x)). If f has nonzero constant term, then f ∗∈C(R) [11, Lemma 2.11]). From now on, we assume that f has nonzero constant term and denote by h ∈C(R), h(t) = ˆh(u−1tn), the minimal central left multiple of f. Then, h equals the bound of f up to a scalar multiple from D. If f is irreducible in R, then ˆh(x) is irreducible in F[x]. If ˆh ∈F[x] is irreducible, then f = f1 · · · fr for irreducible fi ∈R such that fi ∼fj for all i, j ([23], cf. [36]). https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 2.3. Skew polynomial rings In the following, let D be a central simple division algebra of degree d over its center C, σ a ring endomorphism of D and δ : D →D a left σ-derivation, i.e. an additive map such that δ(ab) = σ(a)δ(b) + δ(a)b for all a, b ∈D. The skew polynomial ring D[t; σ, δ] is the set of skew polynomials g(t) = a0 + a1t + · · · + antn with ai ∈D, with term-wise addition and multiplication deﬁned via ta = σ(a)t + δ(a) for all a ∈D [22]. Deﬁne Fix(σ) = {a ∈D \| σ(a) = a} and Const(δ) = {a ∈D \| δ(a) = 0}. If δ = 0, deﬁne D[t; σ] = D[t; σ, 0]. If σ = id, deﬁne D[t; δ] = D[t; id, δ]. For f(t) = a0 + a1t + · · · + antn ∈R = D[t; σ, δ] with an ̸= 0, we deﬁne the degree of f as deg(f) = n and deg(0) = −∞. A skew polynomial f ∈R is irreducible if it is not a unit and it has no proper factors, 4 D. Thompson and S. Pumplün i.e if there do not exist g, h ∈R with 1 ≤deg(g), deg (h) < deg(f) such that f = gh [18, p. 2 ﬀ.]. We call f ∈R right-invariant if Rf is a left and a right ideal in R, and a two-sided maximal element, if f is right- invariant and Rf is a nonzero maximal ideal in R (equivalently, if f ̸= 0 and R/Rf is a simple ring) [18, p. 13]. Two nonzero skew polynomials f1, f2 ∈R are similar, written f1 ∼f2, if R/Rf1 ∼= R/Rf2 [18, p. 11]. i.e if there do not exist g, h ∈R with 1 ≤deg(g), deg (h) < deg(f) such that f = gh [18, p. 2 ﬀ.]. We call f ∈R right-invariant if Rf is a left and a right ideal in R, and a two-sided maximal element, if f is right- invariant and Rf is a nonzero maximal ideal in R (equivalently, if f ̸= 0 and R/Rf is a simple ring) [18, p. 13]. Two nonzero skew polynomials f1, f2 ∈R are similar, written f1 ∼f2, if R/Rf1 ∼= R/Rf2 [18, p. 11]. that is [Sf : Nucr(Sf)] = k. If f is not right-invariant which is equivalent to Sf being not associative, which in turn is equivalent to k > 1, then s ̸= dn looking at the degree of h. Note that deg(h) = dnm is the largest possible degree of h. All of the above applies in particular to the special case that D is a ﬁnite ﬁeld extension K of C of degree n, and σ ∈Aut(K) has order n. Then, R = K[t; σ] has center C(R) = F[tn] = {k i=0 ai(tn)i \| ai ∈ F} = F[x] where F = Fix(σ) [18, Theorem 1.1.22]. Lemma 2. Let f ∈R. (i) If f ∈R is irreducible, then every g ∈R similar to f has h as its minimal central left multiple. (ii) Suppose that ˆh ∈F[x] is irreducible. Then, f = f1 · · · fr for irreducible fi ∈R such that fi ∼fj for all i, j. This follows easily from [7, p. 9, Corollary 2] and [18, Theorem 1.2.9]. ˆ The quotient algebra R/Rh has center C(R/Rh) ∼= F[x]/(ˆh(x)), cf. [11, Lemma 4.2]. Deﬁne Eˆh = F[x]/(ˆh(x)). Suppose that ˆh(x) ̸= x and that ˆh is irreducible in F[x]. Then, h generates a maximal two- sided ideal Rh in R [18, p. 16] and R/Rh is simple over its center Eˆh. The quotient algebra R/Rh has center C(R/Rh) ∼= F[x]/(ˆh(x)), cf. [11, Lemma 4.2]. Deﬁne Eˆh = F[x]/(ˆh(x)). Suppose that ˆh(x) ̸= x and that ˆh is irreducible in F[x]. Then, h generates a maximal two- sided ideal Rh in R [18, p. 16] and R/Rh is simple over its center Eˆh. oi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 5 Glasgow Mathematical Journal Theorem 3 [23]. Let f ∈R = D[t; σ] be monic and irreducible of degree m > 1 with minimal central left multiple h(t) = ˆh(u−1tn). Then„ Nucr(Sf) is a central division algebra over Eˆh of degree s = dn/k, where k is the number of irreducible factors of h in R, and R/Rh ∼= Mk(Nucr(Sf)). In particular, this means deg(ˆh) = dm s , deg(h) = km = dnm s , and In particular, this means deg(ˆh) = dm s , deg(h) = km = dnm s , and [Nucr(Sf) : F] = s2 · dm s = dms. Moreover, s divides gcd(dm, dn). If f is not right-invariant, then k > 1 and s ̸= dn. We know that [Sf : F] = [Sf : C][C : F] = d2m · n. Since Nucr(Sf) is a subalgebra of Sf, comparing dimensions we obtain that d2mn = [Sf : F] = [Sf : Nucr(Sf)] · [Nucr(Sf) : F] = k · dms, 3. Constructing sets of matrices employing irreducible f∈D[t; σ] For all x, y ∈F[u−1tn], we have G(x + Rf) + G(y + Rf) = (x + Rh) + (y + Rh) = (x + y) + Rh = G(x + y + Rf), G(1 + Rf) = 1 + 8952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 3. Constructing sets of matrices employing irreducible f∈D[t; σ] Let R = D[t; σ] be as in Section 2.3 and f ∈R be an irreducible monic polynomial of degree m > 1 with nonzero constant term and minimal central left multiple h(t) = ˆh(u−1tn). Let Ef = {z(t) + Rf \| z(t) = ˆz(u−1tn) ∈F[u−1tn]} ⊂R/Rf. Ef = {z(t) + Rf \| z(t) = ˆz(u−1tn) ∈F[u−1tn]} ⊂R/Rf. Together with the multiplication (x + Rf) ◦(y + Rf) = (xy) + Rf for all x, y ∈F[u−1tn], Ef becomes an F-algebra. Together with the multiplication (x + Rf) ◦(y + Rf) = (xy) + Rf for all x, y ∈F[u−1tn], Ef becomes an F-algebra. Lemma 4. (i) For each z(t) = ˆz(u−1tn) ∈F[u−1tn] with ˆz ∈F[x], we have z ∈Rf if and only if z ∈Rh. (ii) (Ef, ◦) is a ﬁeld isomorphic to Eˆh. Proof. (i) As h = gf for some g ∈R, each z ∈Rh also lies in Rf. Proof. (i) As h = gf for some g ∈R, each z ∈Rh also lies in Rf. Conversely, let z(t) = ˆz(u−1tn) ∈F[u−1tn] with ˆz ∈F[x] be such that z ∈Rf. By the Euclidean division algorithm in F[x], there exist unique ˆq(x), ˆr(x) ∈F[x] such that ˆz = ˆqˆh + ˆr, where deg(ˆr) < deg(ˆh) = s or ˆr = 0. If ˆr ̸= 0, then ˆr = ˆz −ˆqˆh, i.e. we found q(t) = ˆq(u−1tn), r(t) = ˆr(u−1tn) ∈F[u−1tn], such that r(t) = z(t) −q(t)h(t) ∈Rf. Let ˆr′(x) = r−1 0 ˆr(x) ∈F[x], where r0 ∈F× is the leading coeﬃcient of ˆr(x), then r′(t) = ˆr′(u−1tn) is monic by deﬁnition. As r′(t) = ˆr′(u−1tn) ∈Rf, too, there exists a(t) ∈R such that r′(t) = a(t)f(t). Thus, r′(t) ∈F[u−1tn] is a monic polynomial of degree less than s which is right divisible by f. This contradicts the deﬁni- tion of h as the minimal central left multiple of f. Thus, we conclude that r = 0 and z = qh ∈Rh, as required. (ii) Ef is a commutative associative ring with identity 1 + Rf. Deﬁne the map G : Ef →Eˆh, G(z + Rf) = z + Rh for all z ∈F[u−1tn]. G is well-deﬁned and surjective. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 6 D. Thompson and S. Pumplün Rh, and G(x + Rf)G(y + Rf) = (x + Rh)(y + Rh) = xy + Rh = G(xy + Rf), yielding that G is an isomor- phism. To check injectivity, we note that G(x + Rf) = 0 + Rh if and only if x ∈Rh. By Lemma 4 (i), this implies x ∈Rf and so x + Rf = 0 + Rf. Let B = Nucr(Sf) and k be the number of irreducible factors of h(t) in R. Lemma 5. The left R-module R/Rf is a right B-module of rank k via the scalar multiplication R/Rf × B −→R/Rf, (a + Rf)(z + Rf) = az + Rf for all z ∈F[u−1tn] and a ∈R. We can identify R/Rf with Bk via a canonical basis. Lemma 5. The left R-module R/Rf is a right B-module of rank k via the scalar multiplication R/Rf × B −→R/Rf, (a + Rf)(z + Rf) = az + Rf for all z ∈F[u−1tn] and a ∈R. We can identify R/Rf with Bk via a canonical basis. Proof. Since the Petit algebra Sf = R/Rf with its multiplication ab = ab modr f is a nonassociative unital algebra with right nucleus B, R/Rf is a right B-module via the given scalar multiplication. As R/Rf is a vector space of dimension d2mn over F, R/Rf is free of rank k over B. Let ν ∈D× and ρ ∈Aut(D), and deﬁne F′ = Fix(ρ) ∩F. We assume in the following that F/F′ is ﬁnite-dimensional. Let s be the degree of B over Eˆh. We assume f is not right-invariant, i.e. k > 1. g h f g Let l < k = dn/s be a positive integer. Deﬁne the set Sn,m,l(ν, ρ, f) = {a + Rh \| a ∈ g h f g Let l < k = dn/s be a positive integer. Deﬁne the set Sn,m,l(ν, ρ, f) = {a + Rh \| a ∈P} ⊂R/Rh, where P = {d0 + d1t + · · · + dlm−1tlm−1 + νρ(d0)tlm \| di ∈D} ⊂D[t; σ]. Sn,m,l(ν, ρ, f) is a vector space over F′ of dimension d2nml[F : F′]. R/Rf is a right B-module of rank k, as shown above. Let La : R/Rf →R/Rf be the left multiplication map La(b + Rf) = ab + Rf. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press Then, La is B-linear, as we have a(xα) = (ax)α for all α ∈B, a, x ∈R/Rf, and therefore, La(xα) = La(x)α for all α ∈B. Thus, La ∈EndB(R/Rf) and R/Rh ∼= Mk(B) ∼= EndB(Bk) = EndB(R/Rf) by Theorem 3. Hence, we have well-deﬁned maps L : Sn,m,l(ν, ρ, f) →EndB(R/Rf), a →La, λ : Sn,m,l(ν, ρ, f) →Mk(B), a →La →Ma, where Ma is the matrix representing La with respect to a B-basis of R/Rf. We denote the image of Sn,m,l(ν, ρ, h) in Mk(B) by where Ma is the matrix representing La with respect to a B-basis of R/Rf. We denote the image of Sn,m,l(ν, ρ, h) in Mk(B) by Cn,m,l = {Ma \| a ∈Sn,m,l(ν, ρ, f)}. The code C = Cn,m,l is F′-linear by construction, and a generalized rank metric code. If C has minimum distance dC, the Singleton-like bound canonically generalizes to the bound dimF′(C) ≤k(k −dC + 1)[B : F′], with [B : F′] = s[F : F′]. If dC = k −l + 1, then dimF′(Sn,m,l(ν, ρ, f)) = d2nml/dms[B : F′] = d2mnl [F : F′] = lk[B : F′] = lkdms[F : F′]. Thus, if dC = k −l + 1, then C attains this bound and C is a maximum rank distance code in Mk(B). We will usually deal with the case that deg (h) = dmn, so that B = Eˆh is a ﬁeld, s = 1, and Cn,m,l ⊂ Mdn(Ef). Note that if l = 1 and dC = k, this generalized Singleton-like bound is achieved trivially: we obtain examples of MRD codes in Mk(B). This arises when we look at division algebras Sn,m,1(ν, ρ, f) and the matrices representing their left multiplication, cf. Remark 17 and Corollary 18. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 4. The rank of the matrix that corresponds to the element a + Rh Let R = D[t; σ] be as in Section 3, and f ∈R be an irreducible monic polynomial of degree m > 1 with minimal central left multiple h. Let B = Nucr(Sf). We have deg(ˆh) = km and R/Rh ∼= Mk(B) as Eˆh- algebras by Theorem 3. Let : R/Rh →Mk(B), (a + Rh) = Ma, be this isomorphism. For Ma ∈Mk(B), Let R = D[t; σ] be as in Section 3, and f ∈R be an irreducible monic polynomial of degree m > 1 with minimal central left multiple h. Let B = Nucr(Sf). We have deg(ˆh) = km and R/Rh ∼= Mk(B) as Eˆh- algebras by Theorem 3. Let : R/Rh →Mk(B), (a + Rh) = Ma, be this isomorphism. For Ma ∈Mk(B), oi.org/10.1017/S001708952300006X Published online by Cambridge University Press 7 7 Glasgow Mathematical Journal consider the right B-linear map LMa : Mk(B) →Mk(B), LMa : X →MaX. Then, we obtain the following generalization of [34], Proposition 7 (which was only proved for f with coeﬃcients in a ﬁnite ﬁeld, i.e. for the special case that deg(h) = nm is maximal): Theorem 6. Let deg(h) = km. Then Ma ∈Mk(B) and dimB(im(LMa)) = k2 −k mdeg(gcrd(a, h)), colrank(Ma) = k −1 mdeg(gcrd(a, h)) for all a + Rh ∈R/Rh. In particular, if deg(h) = dmn, then Ma ∈Mn(Eˆh), and rank(Ma) = dn −1 mdeg(gcrd(a(t), h(t)). dimB(im(LMa)) = k2 −k mdeg(gcrd(a, h)), colrank(Ma) = k −1 mdeg(gcrd(a, h)) rank(Ma) = dn −1 mdeg(gcrd(a(t), h(t)). Proof. For each Ma ∈Mk(B), deﬁne Annr(Ma) = {N ∈Mk(B) \| MaN = 0}. Then, Annr(Ma) is the kernel of the endomorphism LMa : Mk(B) →Mk(B). By the Rank-Nullity Theorem for free right B-modules of ﬁnite rank [16, ch. IV, Cor. 2.14], it follows that k2 = dimB(im(LMa)) + dimB(Annr(Ma)). We conclude that dimB(im(LMa)) = k2 −dimB(Annr(Ma)). Now for each b + Rh, MaMb = 0 if and only if (a + Rh) (b + Rh) = 0. As is multiplicative, this is true if and only if ((a + Rh)(b + Rh)) = 0. This means (a + Rh)(b + Rh) = 0. Hence, it is clear that Annr(Ma) ∼= Annr(a), where Annr(a) = {b + Rh ∈R/Rh \| (a + Rh)(b + Rh) = 0 + Rh}, so dim(Annr(Ma)) = dim(Annr(a)). Let γ = gcrd(a, h) so h = δγ for some δ ∈R. 4. The rank of the matrix that corresponds to the element a + Rh As h ∈C(R) and R is a domain, we also have h = γ δ by Lemma 1. Let b ∈R be the unique element such that a = bγ . Then, gcrd(b, δ) = 1, else γ is not the greatest common right divisor of a and h.Let v ∈R. By the left Euclidean division algorithm, there exist unique u, w ∈R such that v = δu + w where deg(w) < deg(δ) and gcld(w, δ) = 1. It follows that av = aδu + aw = bγ δu + bγ w = bhu + bγ w, and therefore, av + Rh = bγ w + Rh. Suppose bγ w ≡0modrh. As gcrd(b, δ) = 1, there exist c, d ∈R such that cb + dδ = 1, so cbγ + dδγ = γ . As δγ = h, this implies cbγ ≡γ : modrh. Hence, γ w ≡cbγ w ≡0 : modrh. However, deg(w) < deg(δ) so deg(γ w) < deg(γ δ) = deg(h); due to this, γ w ≡0 : modrh implies that γ w = 0. As γ ̸= 0 and R is a domain, we conclude that w = 0.Hence, (a + Rh)(v + Rh) = 0 + Rh if and only if v = δu where deg(u) < deg(γ ). As δ is uniquely deﬁned by a and h, every element of Annr(a) is determined by u ∈R such that deg(u) < deg(γ ). Thus, so dim(Annr(Ma)) = dim(Annr(a)). Let γ = gcrd(a, h) so h = δγ for some δ ∈R. As h ∈C(R) and R is a domain, we also have h = γ δ by Lemma 1. Let b ∈R be the unique element such that a = bγ . Then, gcrd(b, δ) = 1, else γ is not the greatest common right divisor of a and h.Let v ∈R. By the left Euclidean division algorithm, there exist unique u, w ∈R such that v = δu + w where deg(w) < deg(δ) and gcld(w, δ) = 1. It follows that av = aδu + aw = bγ δu + bγ w = bhu + bγ w, and therefore, av + Rh = bγ w + Rh. Suppose bγ w ≡0modrh. As gcrd(b, δ) = 1, there exist c, d ∈R such that cb + dδ = 1, so cbγ + dδγ = γ . 4. The rank of the matrix that corresponds to the element a + Rh As δγ = h, this implies cbγ ≡γ : modrh. Hence, γ w ≡cbγ w ≡0 : modrh. However, deg(w) < deg(δ) so deg(γ w) < deg(γ δ) = deg(h); due to this, γ w ≡0 : modrh implies that γ w = 0. As γ ̸= 0 and R is a domain, we conclude that w = 0.Hence, (a + Rh)(v + Rh) = 0 + Rh if and only if v = δu where deg(u) < deg(γ ). As δ is uniquely deﬁned by a and h, every element of Annr(a) is determined by u ∈R such that deg(u) < deg(γ ). Thus, Annr(a) = {v + Rh ∈R/Rh \| (a + Rh)(v + Rh) = 0 + Rh} = {δu \| u ∈R, deg(u) < deg(γ )} ∼= Rdeg(γ ) = {g ∈R \| deg(g) < deg(γ )}. As {1, t, . . . , tdeg(γ )−1} is a D-basis for the free left D-module Rγ, it follows that dimD(Annr(a)) = deg(γ ), so dimF(Annr(a)) = deg(γ )d2n. Since dimEˆh(B) = s2 = d2n2/k2 and [Eˆh : F] = km/n, we obtain dimF(B) = d2mn/k. Hence, we get dimB(Annr(a)) = deg(γ )d2nk d2mn = deg(γ )k m , dimB(Annr(a)) = deg(γ )d2nk d2mn = deg(γ )k m , and so and so dimB(im(LA)) = k2 −dimB(Annr(Ma)) = k2 −k mdeg(γ ). Let ci, respectively ri, denote the columns, and rows of Ma and xi denote the columns of X. Computing the matrix using dot product notation, we have MaX = ⎛ ⎜⎝ r1 · x1 . . . r1 · xk ... ... ... rk · x1 . . . rk · xk ⎞ ⎟⎠ https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press D. Thompson and S. Pumplün 8 The ith column of MaX is equal to The ith column of MaX is equal to ⎛ ⎜⎝ r1 · xi ... rk · xi ⎞ ⎟⎠= c1λ1 + · · · + ckλk for some λj ∈B. Hence, the dimension of the right B-module generated by the ith column of MaX is exactly the column rank of Ma. As there are k columns of MaX, it follows that dimB(im(LMa)) = k colrank(Ma). 5.1. The algebra (D(x), σ, ux) Let C/F be a ﬁnite cyclic ﬁeld extension of degree n with Gal(C/F) = ⟨σ⟩. Let D be a ﬁnite-dimensional division algebra of degree d with center C and suppose that σ extends to a C-algebra automor- phism of D that we call σ, too. Let R = D[t; σ] as in Section 3. Then, there exists u ∈D× such that σ n = iu and σ(u) = u. These two relations determine u up to multiplication with elements from F× [25, Lemma 19.7]. The quotient algebra (D, σ, a) = D[t; σ]/(tn −a)D[t; σ], where f(t) = tn −a ∈D[t; σ] with d ∈F×, is called a generalized cyclic algebra. The special case where D = C yields the cyclic algebra (C/F, γ , a) [18, p. 19]. Let D(t; σ) = {f/g \| f ∈D[t; σ], g ∈C(D[t; σ])} be the ring of central quotients of D[t; σ]. Let σ denote the extension of σ to D(x) that ﬁxes x [14, Lemma 2.1]. Then, C(D(t; σ)) = Quot(C(D[t; σ])) = F(x), x = u−1tn, is the center of D(t; σ), where Quot(U) denotes the quotient ﬁeld of an integral domain U. More precisely, D(t; σ) ∼= (D(x),σ, ux) is a generalized cyclic algebra of degree dn over its center F(x) and a division algebra [14, Theorems 2.2, 2.3]. Let N be the reduced norm of (D(x),σ, ux). 4. The rank of the matrix that corresponds to the element a + Rh ) All of the above applies in particular to the special case that K/F is a ﬁeld extension and σ ∈AutF(K) of ﬁnite order n, R = K[t; σ] and C(R) = F[tn] ∼= F[x]. Let f ∈R be a monic irreducible polynomial of degree m > 1, B = Nucr(Sf), and h(t) = ˆh(tn) its minimal central left multiple, deg(ˆh) = km. Then, : R/Rh →Mk(B), (a + Rh) = Ma is an Ef-algebra isomorphism. For each Ma ∈Mk(B), we have the endomorphism LMa : Mk(B) →Mk(B) by LMa : X →MaX. Analogously to Theorem 6, we can prove: Theorem 7 (for fnite ﬁelds and thus deg(h) = nm maximal, cf. [34], Proposition 7). Suppose that deg(h) = km, then for all a + Rh ∈R/Rh we have dimB(im(LMa)) = k2 −k mdeg(gcrd(a, h)), colrank(Ma) = k −1 mdeg(gcrd(a, h)). In particular, if deg(h) = mn then Ma ∈Mn(Eˆh) and rank(Ma) = n −1 mdeg(gcrd(a, h)). This generalizes [34, Remark 6]. From now on, we assume that Lemma 8. Let f ∈R. If N(f) is irreducible in F[x], then f is irreducible in R. Proof. If f = gp for g, p ∈R then N(f) = N(g)N(p) is reducible in F[x], since both N(g) and N(p) lie in F[x], which immediately yields the assertion. From now on, we assume that From now on, we assume that D = (E/C, γ , a) is a cyclic division algebra over C of degree d, σ\|E ∈Aut(E) such that γ ◦σ = σ ◦γ and u ∈E. D = (E/C, γ , a) is a cyclic division algebra over C of degree d, σ\|E ∈Aut(E) such that γ ◦σ = σ ◦γ and u ∈E. D = (E/C, γ , a) is a cyclic division algebra over C of degree d, σ\|E ∈Aut(E) such that γ ◦σ = σ ◦γ and u ∈E. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 9 Glasgow Mathematical Journal Glasgow Mathematical Journal Then, σ\|E has order n. Write m = kn + r for some 0 ≤r < n. Let f = m i=0 aiti ∈R be a polynomial such that a0 ̸= 0 and h ∈R be the minimal central left multiple of f in R. Theorem 9 [36]. For f ∈E[t; σ] ⊂D[t; σ], we have N(f(t)) = NE/F(a0) + · · · + (−1)dr(n−1)NE/F(am)NE/C(u)mxdm. Theorem 9 [36]. For f ∈E[t; σ] ⊂D[t; σ], we have N(f(t)) = NE/F(a0) + · · · + (−1)dr(n−1)NE/F(am)NE/C(u)mxdm. if h0 denotes the constant term of ˆh. if h0 denotes the constant term of ˆh. Proof. (iii) By Theorem 9, we have deg(N(f)) = dmn in R. N(f) is a two-sided multiple of f in R; therefore, the bound f ∗of f divides N(f) in R. Since (f, t)r = 1, f ∗∈C(R) and therefore f ∗equals h up to some factor in F×. Thus, h(t) = ˆh(u−1tn) must divide N(f) in R. Write N(f) = g(t)h(t) for some g ∈R. Comparing degrees in R, we obtain degN(f) = deg(g(t)) + dmn = dmn, which implies deg(g) = 0, i.e. g(t) = a ∈A×. This implies that N(f) = ah(t) = aˆh(u−1tn). Comparing highest coeﬃcients of N(f) and aˆh yields that a = (−1)dr(n−1)NE/F(am)NE/C(u)m by Theorem 9, so that comparing constant terms we get that NE/F(a0) = (−1)dr(n−1) NE/F(am)NE/C(u)mh0, if h0 is the constant term of ˆh(x). Theorem 11. Let f ∈E[t; σ] ⊂R be monic and irreducible of degree m. Let deg(ˆh) = dm and suppose that all the monic polynomials similar to f lie in E[t; σ]. If g is a monic divisor of h in R of degree lm, then NE/F(g0) = NE/F(a0)l. Proof. We know that h(t) = ˆh(u−1tn), with ˆh(x) irreducible in F[x], since f is irreducible. Thus, h is a t.s.m. element in Jacobson’s terminology [18] and the irreducible factors f1(t), . . . , fk(t) of any decom- position of h(t) are all similar and are all similar to f, as f must be one of them by the deﬁnition of h. Now, g(t) is a monic divisor of h. Thus, we can decompose g(t) into a product of irreducible factors and up to similarity the irreducible factors of g will be the same as suitably chosen irreducible factors of h by [18, Theorem 1.2.9.]. Hence, w.l.o.g. g = f1f2 · · · fl, where the fi are irreducible in R and fi is similar to f for all i = 1, 2, . . . , l [18, Theorem 1.2.19]. Thus by Lemma 2, the minimal central left multiple of each fi is equal to h. Since f is monic, we may assume w.l.o.g. that all fi are monic. By Theorem 10 and since all fi ∈E[t; σ] by our assumption, this implies that NE/F(fi(0)) = (−1)dm(n−1)NE/C(u)mh0 = NE/F(a0). if h0 denotes the constant term of ˆh. As the constant term of g is equal to l i=1 fi(0), we see that NE/F(g0) = l i=1 NE/F(fi(0)) = [(−1)dm(n−1)NE/C(u)mh0]l = (−1)ldm(n−1)NE/C(u)lmhl 0 = NE/F(a0)l. We are not able to say if the assumptions on the fi’s in the above result are empty or trivial. We are not able to say if the assumptions on the fi’s in the above result are empty or trivial. We are not able to say if the assumptions on the fi’s in the above result are empty or trivial. Theorem 10. Suppose that deg(h) = dmn. (i) [36, Theorem 14 (i)] If ˆh is irreducible in F[x], then f is irreducible in R. (i) [36, Theorem 14 (i)] If h is irreducible in F[x], then f is irreducible in R. (ii) [36, Theorem 14 (ii)] If f is irreducible, then N(f) is irreducible in F[x]. (iii) If f ∈E[t; σ] then N(f) = ( 1)dr(n−1)N (a )N (u)mˆh and ( ) [ , ( )] f [ ], f (ii) [36, Theorem 14 (ii)] If f is irreducible, then N(f) is irreducible in F[x]. (iii) If f E[ ] h N(f) ( 1)dr(n 1)N ( )N ( )mˆh d ii) [36, Theorem 14 (ii)] If f is irreducible, then N(f) is irreducible in F ii) If f ∈E[t; σ], then N(f) = (−1)dr(n−1)NE/F(am)NE/C(u)mˆh and (iii) If f ∈E[t; σ], then N(f) = (−1)dr(n−1)NE/F(am)NE/C(u)mˆh and NE/F(a0) = (−1)dr(n−1)NE/F(am)NE/C(u)mh0, 5.2. The algebra (K(x)/F(x), σ, x) Let K/F be a cyclic ﬁeld extension of degree n with Gal(K/F) = ⟨σ⟩, R = K[t; σ] and x = tn. We now look at the cyclic algebra (K(x)/F(x),σ, x) (this case corresponds to D = C in the previous Section). https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 10 D. Thompson and S. Pumplün Let N be the reduced norm of (K(x)/F(x),σ, x) over F(x) (cf. also [18, Proposition 1.4.6]). We have σ\|K = σ, and N is a nondegenerate form of degree n. Let f = m i=0 aiti ∈R be a polynomial of degree m such that a0 ̸= 0 and h ∈R be the minimal central left multiple of f in R. Then N(f(t)) = NK/F(a0) + · · · + (−1)m(n−1)NK/F(am)xm [36, Theorem 3]. Theorem 12. Suppose that deg(h) = mn. (i) [36, Theorem 6 (i)] If ˆh is irreducible in F[x], then f is irreducible in R. (i) [36, Theorem 6 (i)] If ˆh is irreducible in F[x], then f is irreducible in R. (ii) [36, Theorem 6 (ii)] If f is irreducible, then N(f) is irreducible in F[x]. ˆ (ii) [36, Theorem 6 (ii)] If f is irreducible, then N(f) is irreducible in F[x]. (iii) NK/F(a0) = (−1)m(n−1)h0, if h0 denotes the constant term of ˆh. (iii) NK/F(a0) = (−1)m(n−1)h0, if h0 denotes the constant term of ˆh. Theorem 12 (iii) is proved analogously as Theorem 10 (iii). 6.1. The case that f ∈D[t; σ] Let f ∈R = D[t; σ] be a monic polynomial of degree m. Let ρ ∈Aut (D), ν ∈D and F′ = Fix(ρ) ∩F where F = C ∩Fix(σ). Let b(t), c(t) ∈Rm = {g ∈R \| deg(g) < m} and b0 be the constant term of b(t). Then, the multiplication deﬁned via b(t) ◦c(t) = (b(t) + νρ(b0)tm)c(t) modrf, b(t) ◦c(t) = (b(t) + νρ(b0)tm)c(t) modrf, makes Rm into a non-unital nonassociative ring (Rm, ◦). When the context is clear, we will drop the ◦ notation and simply use juxtaposition. (Rm, ◦) is an algebra over F′. Example 14. If f(t) = t −c ∈D[t; σ] for some c ∈D, ν ̸= 0, then (Rm, ◦) has the multiplication a ◦b = (a + νρ(a)t)b) modrf = ab + νρ(a)σ(b)t modrf = ab + νρ(a)σ(b)c, Example 14. If f(t) = t −c ∈D[t; σ] for some c ∈D, ν ̸= 0, then (Rm, ◦) has the multiplication a ◦b = (a + νρ(a)t)b) modrf = ab + νρ(a)σ(b)t modrf = ab + νρ(a)σ(b)c, = ab + νρ(a)σ(b)t modrf = ab + νρ(a)σ(b)c, for all a, b ∈D. This generalizes the algebras studied in [30]. If R = K[t; σ] for some ﬁnite ﬁeld extension K/F; this is the multiplication of Albert’s twisted semiﬁelds [1]. If F/F′ is ﬁnite and (Rm, ◦) is not a division algebra, a ◦b = 0 for some nonzero a, b ∈D, if and only if ab = −νρ(a)σ(b)c. Taking norms of both sides and canceling ND/F′(ab) on both sides, we obtain that ND/F′(−νc) = (−1)d2n[F : F′]ND/F′(νc) = 1. Thus, if F/F′ is ﬁnite and ND/F′(νc) ̸= (−1)d2n[F : F′] then (Rm, ◦) is a division algebra. From now on for the rest of the paper, we again assume that f is an irreducible monic polynomial of degree m > 1, (f, t)r = 1, and that h is the minimal central left multiple of f. Let F/F′ be ﬁnite- dimensional, and P = {d0 + d1t + · · · + dlm−1tlm−1 + νρ(d0)tlm \| di ∈D} ⊂D[t; σ]. Theorem 12 (iii) is proved analogously as Theorem 10 (iii). Theorem 12 (iii) is proved analogously as Theorem 10 (iii). Theorem 13 (cf. [34, Theorem 5] for ﬁnite ﬁelds, the proof is the same). Suppose that f is not right- invariant. If deg(h) = mn and g is a monic divisor of h(t) in R of degree ml, then NK/F(g0) = NK/F(a0)l. Theorem 13 (cf. [34, Theorem 5] for ﬁnite ﬁelds, the proof is the same). Suppose that f is not right- invariant. If deg(h) = mn and g is a monic divisor of h(t) in R of degree ml, then NK/F(g0) = NK/F(a0)l. Note that f may be right-invariant. Note that f may be right-invariant. Proof. Suppose that there are b(t) = b0 + b1t + · · · + bm−1tm−1, c(t) ∈Rm, such that b(t) ◦c(t) = (b(t) + νρ(b0)tm)c(t)modrf = 0. Then, there exists g ∈Rm such that (b(t) + νρ(b0)tm)c(t) = g(t)f(t). Since f is irreducible and of degree m, while deg(c) < m, f must be similar to an irreducible factor of b(t) + νρ(b0)tm, because of the uniqueness of an irreducible decomposition in R up to similarity. But b(t) + νρ(b0)tm has degree at most m, so f is similar to b(t) + νρ(b0)tm. Thus, b(t) + νρ(b0)tm must have degree m and be irreducible as well. Hence if b(t) + νρ(b0)tm is not similar to f then b(t) + νρ(b0)tm is not a left zero divisor in (Rm, ◦). This happens for instance, if ν = 0 or if b(t) + νρ(b0)tm is reducible. Moreover, (Rm, ◦) is a division algebra if P does not contain any polynomial similar to f. We are again not able to say if the assumptions on the fi’s in the following result are empty or trivial. Theorem 16. Let D = (E/C, γ , a) be a cyclic division algebra over C of degree d such that σ\|E ∈Aut(E) and γ ◦σ\|E = σ\|E ◦γ . Suppose that σ n(z) = u−1zu with u ∈E. Theorem 16. Let D = (E/C, γ , a) be a cyclic division algebra over C of degree d such that σ\|E ∈Aut(E) and γ ◦σ\|E = σ\|E ◦γ . Suppose that σ n(z) = u−1zu with u ∈E. Let f(t) = m i=0 aiti ∈E[t; σ] ⊂D[t; σ] be monic and irreducible, and let deg(h) = dmn. Suppose that all monic fi similar to f lie in E[t; σ]. Then, (Rm, ◦) is a division algebra over F′, if one of the following holds: (i) ν ̸∈E and ρ\|E ∈Aut(E). (ii) ν ∈E× and ρ\|E ∈Aut(E), such that Let f(t) = m i=0 aiti ∈E[t; σ] ⊂D[t; σ] be monic and irreducible, and let deg(h) = dmn. Suppose that all monic fi similar to f lie in E[t; σ]. Then, (Rm, ◦) is a division algebra over F′, if one of the following holds: (i) ν ̸∈E and ρ\|E ∈Aut(E). (ii) ν ∈E× and ρ\|E ∈Aut(E), such that NE/F′(a0)NE/F′(ν) ̸= 1. Note that f may be right-invariant. Note that our global assumption that σ n(z) = u−1zu for all z ∈D, so that σ n(e) = u−1eu = e for all e ∈E, forces (σ\|E)n = id. Proof. By Theorem 15, (Rm, ◦) is a division algebra, if the set P with l = 1 does not contain any polynomial similar to f. All polynomials similar to f are irreducible factors of h(t), so (Rm, ◦) is a division algebra, if P does not contain any irreducible factor of h(t). Suppose that P contains an irreducible factor g of h with constant term g0. Then, g has degree m as it is similar to f. Let gmtm be its highest coeﬃcient, so that g−1 m g is a monic divisor of h. m By Theorem 10 and since g ∈E[t; σ] by assumption, this implies NE/F(g0g−1 m ) = (−1)m(n−1)h0 = NE/F(a0), and in particular, that g0 and gm are both nonzero. Since g ∈P, we also have gm = νρ(g0). Suppose ν ̸∈E and ρ(E) ⊂E. Since the coeﬃcients of the fi all lie in E, we have gm ̸= νρ(g0) which yields a contradiction. Hence, there is no divisor g of h in P and S is a division algebra. Suppose that ν ∈E× and ρ(E) ⊂E. Substituting gm = νρ(g0) into the above equation yields NE/F(g0) = NE/F(a0)NE/F(νρ(g0)). Applying NF/F′ to both sides implies that NE/F′(g0) = NF/F′(NE/F(a0))NE/F′(νρ(g0)). Now NE/F′(ρ(g0)) = NE/F′(g0), so we can cancel the nonzero term NE/F′(g0) to obtain 1 = NE/F′(a0) NE/F′(ν). Now NE/F′(ρ(g0)) = NE/F′(g0), so we can cancel the nonzero term NE/F′(g0) to obtain 1 = NE/F′(a0) NE/F′(ν). Remark 17. Let S = Sn,m,1(ν, ρ, f) = {a + Rh \| a ∈P}. We can use C(S) ⊂Mk(B) to deﬁne a multipli- cation on Bm. As dimF(D) = d2n and dimF(B) = d2mn/k, there exists an F-vector space isomorphism between Dm and Bk. Similarly, there exists an isomorphism G : Vf →Bk, G(a + Rf) = a. Deﬁne ∗: Bk × Bk →Bk by Remark 17. Let S = Sn,m,1(ν, ρ, f) = {a + Rh \| a ∈P}. We can use C(S) ⊂Mk(B) to deﬁne a multipli- cation on Bm. As dimF(D) = d2n and dimF(B) = d2mn/k, there exists an F-vector space isomorphism between Dm and Bk. Similarly, there exists an isomorphism G : Vf →Bk, G(a + Rf) = a. Theorem 15. Let l = 1. Then: Theorem 15. Let l = 1. Then: (i) Let b(t) ∈Rm with constant coeﬃcient b0. If b(t) + νρ(b0)tm ∈P is reducible in R, then b(t) is not a left zero divisor in (Rm, ◦). (i) Let b(t) ∈Rm with constant coeﬃcient b0. If b(t) + νρ(b0)tm ∈P is reducible in R, then b(t) is not a left zero divisor in (Rm, ◦). https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 11 Glasgow Mathematical Journal (ii) If ν = 0, then (Rm, ◦) is a division algebra over F′, which for m ≥2 is a Petit algebra. ii) If ν = 0, then (Rm, ◦) is a division algebra over F′, which for m ≥2 is a Petit algebra. (ii) If ν = 0, then (Rm, ◦) is a division algebra over F′, which for m ≥2 is a Petit algebra. (ii) If ν 0, then (Rm, ◦) is a division algebra over F , which for m ≥2 is a Petit algebra. (iii) If P does not contain any polynomial similar to f, then (Rm, ◦) is a division algebra over F′ (iii) If P does not contain any polynomial similar to f, then (Rm, ◦) is a division algebra over F′. Note that f may be right-invariant. Note that f may be right-invariant. Then, B is a division algebra over Eˆh and S(ν, ρ, f) deﬁnes an F′-linear MRD code in Mk(B) with minimum distance k, if one of the following holds: Let f = m i=0 aiti ∈R be monic and irreducible of degree m. Then, B is a division algebra over Eˆh and S(ν, ρ, f) deﬁnes an F′-linear MRD code in Mk(B) with minimum distance k, if one of the following holds: (i) ν = 0. Then, S(ν, ρ, f) is a (unital) Petit algebra. (ii) P does not contain any polynomial similar to f. (iii) Suppose ρ\|E ∈Aut(E), f = m i=0 aiti ∈E[t; σ] ⊂R, deg(h) = dmn, all the monic polynomials similar to f lie in E[t; σ], and one of the following holds: (a) ν ̸∈E, (b) NE/F′(ν)NE/F′(a0) ̸= 1. Then, we get an F′-linear MRD code in Mdn(Eˆh) with minimum distance dn. The case ν = 0 produces the MRD codes which are associated with the unital Petit algebras. They can be viewed as generalized Gabidulin codes. The case ν = 0 produces the MRD codes which are associated with the unital Petit algebras. They can be viewed as generalized Gabidulin codes. More generally, we can also construct MRD codes for l > 1. Let f ∈R not be right-invariant, and let l < k be a positive integer. More generally, we can also construct MRD codes for l > 1. Let f ∈R not be right-invariant, and let l < k be a positive integer. Theorem 19. Suppose that P does not contain any polynomial of degree lm, whose irreducible factors are all similar to f. Then, the set Sn,m,l(ν, ρ, f) deﬁnes an F′-linear MRD code in Mk(B) with minimum distance k −l + 1. In particular, if deg(h) = dmn, then this code is an F′-linear MRD code in Mdn(Eˆh) with minimum distance dn −l + 1. Theorem 19. Suppose that P does not contain any polynomial of degree lm, whose irreducible factors are all similar to f. Then, the set Sn,m,l(ν, ρ, f) deﬁnes an F′-linear MRD code in Mk(B) with minimum distance k −l + 1. In particular, if deg(h) = dmn, then this code is an F′-linear MRD code in Mdn(Eˆh) with minimum distance dn −l + 1. We are not able to say if the assumption on P can be satisﬁed in this general setup. Note that f may be right-invariant. Deﬁne ∗: Bk × Bk →Bk by https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 12 D. Thompson and S. Pumplün a ∗b = Ma · b for all a, b ∈Bk, where Ma ∈C(S) is the representation of the map La(t)+νρ(a0)tm ∈EndB(R/Rf) induced by G. (Each a ∈Rm corresponds to a map La(t)+νρ(a0)tm. As EndB(R/Rf) ∼= Mk(B) and dim(Rm) = dim(C(S)), there is a canonical bijection between La(t)+νρ(a0)tm and Ma.) As Ma represents La ∈EndB(R/Rf), (Bk, ∗) is isomorphic to R/Rf equipped with the multiplication (a + Rf)(b + Rf) = La(t)+νρ(a0)tm(b + Rf). Thus, (Rm, ◦) and (Bk, ∗) are isomorphic algebras and Sn,m,1(ν, ρ, f) is the same algebra as (Rm, ◦). for all a, b ∈Bk, where Ma ∈C(S) is the representation of the map La(t)+νρ(a0)tm ∈EndB(R/Rf) induced by G. (Each a ∈Rm corresponds to a map La(t)+νρ(a0)tm. As EndB(R/Rf) ∼= Mk(B) and dim(Rm) = dim(C(S)), there is a canonical bijection between La(t)+νρ(a0)tm and Ma.) As Ma represents La ∈EndB(R/Rf), (Bk, ∗) is isomorphic to R/Rf equipped with the multiplication (a + Rf)(b + Rf) = La(t)+νρ(a0)tm(b + Rf). Thus, (Rm, ◦) and (Bk, ∗) are isomorphic algebras and Sn,m,1(ν, ρ, f) is the same algebra as (Rm, ◦). If l = 1, we write S(ν, ρ, f) = Sn,m,1(ν, ρ, f) for (Rm, ◦). S(ν, ρ, f) is a division algebra if and only if every matrix in Cn,m,1 has full column rank. It then canonically deﬁnes an F′-linear MRD code in Mk(B), B = Nucr(Sf). Therefore, we obtain from all of the above results: Corollary 18. Let D = (E/C, γ , a) be a cyclic division algebra over C of degree d such that σ\|E ∈Aut(E) and γ ◦σ = σ ◦γ . Suppose that σ n(z) = u−1zu with u ∈E. Corollary 18. Let D = (E/C, γ , a) be a cyclic division algebra over C of degree d such that σ\|E ∈Aut(E) and γ ◦σ = σ ◦γ . Suppose that σ n(z) = u−1zu with u ∈E. Let f = m i=0 aiti ∈R be monic and irreducible of degree m. 6.2. The case R = K[t; σ] Let f = m i=0 aiti ∈R = K[t; σ] be an irreducible monic polynomial of degree m with minimal cen- tral left multiple h. Suppose throughout this section that F/F′ is a ﬁnite ﬁeld extension, ν ∈K, and ρ ∈Aut(K). Let 1 < l < k and Sn,m,l(ν, ρ, f) = {a + Rh \| a ∈P} ⊂R/Rh, where P = {d0 + d1t + · · · + dlm−1tlm−1 + νρ(d0)tlm \| di ∈K}. Then, we obtain the following results: Note that f may be right-invariant. It is satisﬁed in the case considered in [34, Theorem 7]. Proof. We have to show that the minimum column rank of the matrix corresponding to a nonzero element in Sn,m,l(ν, ρ, f) is k −l + 1. By Theorem 6, this is equivalent to ﬁnding an element g ∈A such that the greatest common right divisor of g and h has degree at most (l −1)m. Suppose towards a con- tradiction that there exists g ∈A such that deg(gcrd(g, h)) = lm; since deg(g) ≤lm, it follows that g must be a divisor of h. As any divisor of h is a product of irreducible polynomials similar to f, g must be a product of polynomials similar to f. This contradicts our assumption, so any matrix has rank at least k −l + 1. Theorem 20 (for f ∈K[t; σ], K a ﬁnite ﬁeld, this is [34, Theorem 7]). Let f = m i=0 aiti ∈E[t; σ] ⊂R = D[t; σ] be monic irreducible, and let deg(h) = dmn. Suppose that all monic fi similar to f lie in E[t; σ]. Then, Sn,m,l(ν, ρ, f) deﬁnes an F′-linear MRD code in Mdn(Eˆh) with minimum distance dn −l + 1, if one of the following holds: https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 13 Glasgow Mathematical Journal (i) ν = 0 (ii) ν ̸∈E and ρ\|E ∈Aut(E). (iii) ν ∈E, ρ\|E ∈Aut(E) and NE/F′(ν)NE/F′(a0)l ̸= 1. The proof is straightforward. Theorem 21. Let l = 1. (i) Let b(t) ∈Rm with constant coeﬃcient b0. If b(t) + νρ(b0)tm ∈P is reducible in R, then b(t) is not a left zero divisor in S(ν, ρ, f). ν = 0, then S(ν, ρ, f) is a division algebra over F′, a unital Petit algebra. (ii) If ν = 0, then S(ν, ρ, f) is a division algebra over F′, a unital Petit algebra. (iii) If P does not contain any polynomial similar to f, then S(ν, ρ, f) is a division algebra over F′. (iii) If P does not contain any polynomial similar to f, then S(ν, ρ, f) is a division algebra over F′. ii) If P does not contain any polynomial similar to f, then S(ν, ρ, f) is a division algebra The proof is analogous to the one of Theorem 15. Note that f may be right-invariant here. Using Theorems 10 and 13, we obtain (for ﬁnite ﬁelds, cf. [34], the proof is analogous): Theorem 22. Suppose that deg(h) = mn. Then, S(ν, ρ, f) is a division algebra over F′ if NK/F′(a0)NK/F′(ν) ̸= 1. Theorem 22. Suppose that deg(h) = mn. Then, S(ν, ρ, f) is a division algebra over F′ if NK/F′(a0)NK/F′(ν) ̸= 1. Corollary 23. B = Nucr(Sf) is a division algebra and the left multiplication of the algebra S(ν, ρ, f) deﬁnes an F′-linear MRD code in Mk(B) with minimum distance k, if one of the following holds: Corollary 23. B = Nucr(Sf) is a division algebra and the left multiplication of the algebra S(ν, ρ, f) deﬁnes an F′-linear MRD code in Mk(B) with minimum distance k, if one of the following holds: (i) ν = 0. (i) ν = 0. (ii) P does not contain any polynomial similar to f. (iii) deg(h) = mn and ν ∈K such that NK/F′(ν) ̸= 1/NK/F′(a0). In this case, the algebra S(ν, ρ, f) deﬁnes an F′-linear MRD code in Mn(Eˆh) with minimum distance n. Note that the condition on f in (iii) is satisﬁed for all f if gcd(m, n) = 1 or if n is prime. Note that the condition on f in (iii) is satisﬁed for all f if gcd(m, n) = 1 or if n is prime. We now look at the case that 1 < l < k and also assume that f is not right-invariant. (i) ν = 0 Theorem 21. Let l = 1. Note that the condition on f in (iii) is satisﬁed for all f if gcd(m, n) = 1 or if n is prime. ote that the condition on f in (iii) is satisﬁed for all f if gcd(m, n) = 1 or if n is prime. Note that the condition on f in (iii) is satisﬁed for all f if gcd(m, n) = 1 or if n is prime. We now look at the case that 1 < l < k and also assume that f is not right-invariant. We now look at the case that 1 < l < k and also assume that f is not right-invariant. Theorem 24 (for ﬁnite ﬁelds, cf. [34, Theorem 7]). If deg(h) = mn, then the set Sn,m,l(ν, ρ, f) deﬁnes an F′-linear MRD code in Mn(Eˆh) with minimum distance n −l + 1 for any ν ∈K such that NK/F′(ν) ̸= 1/NK/F′(a0)l. Note that k = n here since deg(h) = mn. Note that k = n here since deg(h) = mn. Corollary 25. The set Sn,m,l(ν, ρ, h) deﬁnes an F′-linear MRD code in Mn(Eˆh) with minimum distance n −l + 1, if one of the following holds: (i) deg(h) = mn and ν = 0, (ii) n is prime or gcd(m, n) = 1, and 1 ̸= NK/F′(ν)NK/F′(a0)l (iii) deg(h) = mn and NK/F′(ν) ̸∈(F′×)l. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 14 D. Thompson and S. Pumplün The codes Sn,m,l(0, ρ, h) generalize the Gabidulin codes constructed in [10] that go back to [8]. Note that NK/F′(ν) ̸∈(F′×)l implies NK/F′(ν) ̸= NK/F′(a0)l for any f. Thus, (ii) implies (iii) above. The codes Sn,m,l(0, ρ, h) generalize the Gabidulin codes constructed in [10] that go back to [8]. Note that NK/F′(ν) ̸∈(F′×)l implies NK/F′(ν) ̸= NK/F′(a0)l for any f. Thus, (ii) implies (iii) above. Theorem 26. Suppose that P does not contain any polynomial of degree lm, whose irreducible factors are all similar to f. Then the set Sn,m,l(ν, ρ, f) deﬁnes an F′-linear MRD code in Mk(B) with minimum distance k −l + 1. In particular, if deg(h) = mn then Sn,m,l(ν, ρ, f) deﬁnes an F′-linear MRD code in Mn(Eˆh) with minimum distance n −l + 1. Proof. We have to show that the minimum column rank of the matrix corresponding to a nonzero element in Sn,m,l(ν, ρ, f) is k −l + 1. 7. Nuclei Let M = M(A) = {La \| a ∈A} ⊆EndF(A) be the spread set of an F-algebra A, where La is the left multiplication map in A. We deﬁne the left and right idealizers of M as Let M = M(A) = {La \| a ∈A} ⊆EndF(A) be the spread set of an F-algebra A, where La is the left multiplication map in A. We deﬁne the left and right idealizers of M as Il(M) = { ∈EndF(A) \| M ⊆M}, respectively, Ir(M) = { ∈EndF(A) \| M ⊆M}. The centrali er of M is deﬁned as Cent(M) { ∈End (A) \| M M ∀M ∈M} We call Il(M) = { ∈EndF(A) \| M ⊆M}, respectively, Ir(M) = { ∈EndF(A) \| M ⊆M}. Il(M) = { ∈EndF(A) \| M ⊆M}, respectively, Ir(M) = { ∈EndF(A) \| M ⊆M}. The centralizer of M is deﬁned as Cent(M) = { ∈EndF(A) \| M = M ∀M ∈M}. We call Z(M) = Il(M) ∩Cent(M) the center of M. The centralizer of M is deﬁned as Cent(M) = { ∈EndF(A) \| M = M ∀M ∈M}. We call Z(M) = Il(M) ∩Cent(M) the center of M. Theorem 27 (cf. [34, Proposition 5] for ﬁnite ﬁelds). Let A be a unital division algebra and M be the spread set of A. Let M∗be the spread set associated with the opposite algebra Aop. Then Theorem 27 (cf. [34, Proposition 5] for ﬁnite ﬁelds). Let A be a unital division algebra and M be the spread set of A. Let M∗be the spread set associated with the opposite algebra Aop. Then Nucl(A) ∼= Il(M), Nucm(A) ∼= Ir(M), Nucr(A) ∼= Cent(M∗), C(A) ∼= Z(M). The proof from [34] holds verbatim in our general setting. The proof from [34] holds verbatim in our general setting. The above results can now be applied to determine the nuclei and center of the non-unital algebras S = Sn,m,l(ν, ρ, f). The above results can now be applied to determine the nuclei and center of the non-unital algebras S = Sn,m,l(ν, ρ, f). In the following, let R = D[t; σ]. We use the assumptions on D, respectively K, and σ from Section 6. Let f ∈R be an irreducible monic polynomial of degree m, and let h be the minimal central left multiple of f. We assume throughout that f is not right-invariant, so that k > 1. Remark 28. Theorem 21. Let l = 1. By Theorem 7, this is equivalent to ﬁnding an element g ∈P such that the greatest common right divisor of g and h has degree at most (l −1)m. Suppose towards a contradiction that deg(gcrd(g, h)) = lm; since deg(g) ≤lm, it follows that g must be a divisor of h. As any divisor of h is a product of irreducible polynomials similar to f, g must be a product of polynomials similar to f. This contradicts our assumption, so any matrix has rank at least k −l + 1. (iii) Cent(M) ∼= Eˆh, Z(M) ∼= F′ (ii) Ir(M) ∼= Fix(ρ−1 ◦σ lm) ⊂D, 7. Nuclei The algebras Sn,m,l(0, ρ, f) are unital Petit algebras and hence have left nucleus Nucm(S) = D, and their right nucleus {g ∈Rm \| fg ∈Rf} is the eigenspace of f. If Sn,m,l(0, ρ, f) is not associative, then {d ∈D \| dg = gd for all g ∈S} is their center [26]. Theorem 29. Let R = D[t; σ] and deg(h) = dmn. Suppose l ≤dn/2, n > 1 and lm > 2. Let S = Sn,m,l(ν, ρ, f) and M be the image of S in EndEf (R/Rf), that means the corresponding rank metric code lies in Mn(Eˆh). If ν ̸= 0, we have (i) Il(M) ∼= {g0 ∈D \| g0ν = νρ(g0)} ⊂D (in particular, Il(M) ∼= Fix(ρ) if ν ∈C), (ii) Ir(M) ∼= Fix(ρ−1 ◦σ lm) ⊂D, (iii) Cent(M) ∼= Eˆh, Z(M) ∼= F′. (i) Il(M) ∼= {g0 ∈D \| g0ν = νρ(g0)} ⊂D (in particular, Il(M) ∼= Fix(ρ) if ν ∈C), 1 l ρ (iii) Cent(M) ∼= Eˆh, Z(M) ∼= F′. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press Glasgow Mathematical Journal 15 If ν = 0, we have (iv) Il(M) ∼= D, Ir(M) ∼= D, (v) Cent(M) ∼= Eˆh, Z(M) ∼= F. Much of the proof works identically to the proof of [34, Theorem 9]. We sketch the proof to highlight the main diﬀerences in this more general case. The lm = 2 case has to be considered separately, and we have only been able to solve that for F = R. Proof. Let M = {La ∈EndEf (R/Rf) \| a ∈P} be the image of S in EndEf (R/Rf) ⊂EndF(R/Rf). In the following, we identify each element in M with the element g ∈S that induces it. Analogously to the proof of [34, Theorem 9], {g ∈Il(M) \| deg(g) ≤lm} = {g0 ∈D \| g0ν = νρ(g0)}. If ν = 0, then 1 ∈M so Il(M) ⊂M so all g ∈Il(M) have degree at most lm. Consider ν ̸= 0. To check there are no elements g ∈ Il(M) of degree higher than lm, we follow the approach of [34, Theorem 9] and consider gt mod ˆh(u−1tn). Recalling deg(h) = dm, we have h(t) = (u−1tn)dm + · · · = u−dm(tn + h′ dm−1t(dm−1)n + · · · + h′ 0) so gt mod h(t) = dmn−1 i=0 gi−1ti −gdmn−1udm dm−1 j=0 h′ jtnj . 7. Nuclei As g ∈Il(M), this implies gt mod h ∈M, so for all i ∈{lm + 1, . . . , dmn −1}, we have gi−1 = 0 for i ̸≡0 mod n gdmn−1udmh′ i/n for i ≡0 mod n (1) (1) where h′ i/n = 0 if i/n is not an integer. We will show that gdmn−1 = 0 and thus deg(g) ≤lm −1. As lm > 2, this follows verbatim from [34, Theorem 9]. where h′ i/n = 0 if i/n is not an integer. We will show that gdmn−1 = 0 and thus deg(g) ≤lm −1. As lm > 2, this follows verbatim from [34, Theorem 9]. The same holds for Ir(M) following Sheekey’s proof with the appropriate amendments made for D[t; σ]. The results for Cent(M) and Z(M) hold verbatim from [34, Theorem 9]. Corollary 30. Let R = D[t; σ] and deg(h) = dmn. Suppose n > 1, m > 2 and S = Sn,m,1(ν, ρ, f) with ν ̸= 0 be a division algebra. Then, (i) Nucl(S) ∼= {g0 ∈D \| g0ν = νρ(g0)} ⊂D, so in particular Nucl(S) = Fix(ρ) ⊂D, if ν ∈C. (ii) Nuc (S) ∼= Fix(ρ−1 ◦σ m) ⊂D (i) Nucl(S) ∼= {g0 ∈D \| g0ν = νρ(g0)} ⊂D, so in particular Nucl(S) = Fix(ρ) ⊂D, if ν ∈C. (ii) Nucm(S) ∼= Fix(ρ−1 ◦σ m) ⊂D. (iii) C(S) = Fix(ρ) ∩F = F′ (i) Nucl(S) ∼= {g0 ∈D \| g0ν = νρ(g0)} ⊂D, so in particular Nucl(S) = Fix(ρ) ⊂D, if ν ∈C. (ii) Nucm(S) ∼= Fix(ρ−1 ◦σ m) ⊂D. (iii) C(S) = Fix(ρ) ∩F = F′. (iii) C(S) = Fix(ρ) ∩F = F′. ρ v) dimF′Nucr(S) = dimF′(Eˆh) = deg(ˆh)[F : F′] = [F : F′]dm. (iv) dimF′Nucr(S) = dimF′(Eˆh) = deg(ˆh)[F : F′] = [F : F′]dm. Theorem 31. Let R = K[t; σ] and deg(h) = mn. Suppose l ≤n/2, n > 1 and lm > 2. Let S = Sn,m,l(ν, ρ, f) with ν ̸= 0 and M be the image of S in EndEf (R/Rf), so that the corresponding rank metric code lies in Mn(Eˆh). Then, (i) Il(M) ∼= Fix(ρ) ⊂K, Ir(M) ∼= Fix(ρ−1 ◦σ lm) ⊂K, (i) Il(M) ∼= Fix(ρ) ⊂K, Ir(M) ∼= Fix(ρ−1 ◦σ lm) ⊂K, ρ ρ (ii) Cent(M) ∼= Eˆh, Z(M) ∼= F′. If ν = 0, we have (ii) Cent(M) ∼= Eˆh, Z(M) ∼= F′. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 8.1. K = F(θ) Let K = F(θ) be an extension of prime degree n. Let f(t) = tn −θ ∈K[t; σ]. We now compute the rank metric code associated with the F′-algebra Sn,n,1(ν, ρ, f). Note that f(t) = t3 −θ ∈K[t; σ] is irreducible if and only if θ ̸= σ 2(z)σ(z)z for all z ∈K. If F contains a primitive nth root of unity, then f(t) is irreducible if and only if θ ̸= σ n−1(z) · · · σ(z)z for all z ∈K. We assume that f is irreducible. Deﬁne h(t) = (tn −θ)(tn −σ(θ)) · · · (tn −σ n−1(θ)) = (tn)n + · · · + (−1)nNK/F(θ), then h(t) = mclm(f): as tn −σ i(θ) ∈K[tn], the factors of h(t) all commute and h(t) ∈ K[tn]. Since σ(h(t)) = (tn −σ(θ)) · · · (tn −σ n−1(θ))(tn −θ) = h(t), we know that h(t) ∈Fix(σ)[t] = F[t] so h(t) ∈F[t] ∩K[tn] = F[tn] = C(R). Hence h(t) = ˆh(tn) with ˆh(x) = xn + (−1)nNK/F(θ) ∈F[x]. Thus f divides h both from the left and the right by Lemma 1. As n is prime, the minimal central left multiple of f must have degree n in F[x] by Theorem 3; thus, h(t) = mclm(f), and hence, ˆh(x) = xn + (−1)nNK/F(θ) also is an irreducible polynomial in F[x]. As a ﬁeld, Ef = {z + Rf \| z ∈F[tn]} is generated by {1 + Rf, tn + Rf, t2n + Rf, . . . , tn(n−1) + Rf} = {1 + Rf, θ + Rf, θ 2 + Rf, . . . , θ n−1 + Rf} over F. As K is generated by {1, θ, . . . , θ n−1}, there is a canonical isomorphism Ef −→K, x + Rf →x. f It is clear that {1 + Rf, t + Rf, . . . , tn−1 + Rf} is an Ef-basis for R/Rf. Let a = a0 + a1t + · · · + an−1tn−1 + νρ(a0)tn ∈S(ν, ρ, h). In order to determine Ma, we consider how Laiti acts on the basis elements of R/Rf. As left multiplication is distributive, i.e. La+b(x) = La(x) + Lb(x), it follows that La = n i=0 Laiti, where an = νρ(a0). 7. Nuclei If ν = 0, we have h (iii) Il(M) ∼= K, Ir(M) ∼= K, ( ) l( ) r( ) (iv) Cent(M) ∼= Eˆh, Z(M) ∼= F. Again, the proof is analogous to the one of [34], Theorem 9 (it does not use the fact that for ﬁnite ﬁelds the right nucleus of Sf is Eˆh, it only uses that R/Rh has center Eˆh). Corollary 32. Let R = K[t; σ] and deg(h) = mn. Suppose that n > 1, m > 2 and that S = (S(ν, ρ, f), ◦) is a division algebra with ν ̸= 0. Then, (i) Nucl(S) = Fix(ρ) ⊂K, (ii) Nucm(S) = Fix(ρ−1 ◦σ m) ⊂K, https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 16 D. Thompson and S. Pumplün (iii) C(S) = Fix(ρ) ∩F = F′. (iii) C(S) = Fix(ρ) ∩F = F′. (iii) C(S) = Fix(ρ) ∩F = F′. (iv) dimF′Nucr(S) = dimF′(Eˆh) = deg(ˆh)[F : F′] = [F : F′]m. ) ( ) (ρ) v) dimF′Nucr(S) = dimF′(Eˆh) = deg(ˆh)[F : F′] = [F : F′]m. ( ) ρ (iv) dimF′Nucr(S) = dimF′(Eˆh) = deg(ˆh)[F : F′] = [F : F′]m. Theorems 29, 31 and Corollaries 30, 32 generalize [34, Theorem 9, Corollary 1] which were proved for semiﬁelds. 8.1. K = F(θ) For each i, we have: Laiti(1 + Rf) =aiti + Rf = (ti + Rf)(σ n−i(ai) + Rf) Laiti(t + Rf) =aiti+1 + Rf = (ti+1 + Rf)(σ n−i−1(ai) + Rf) Laiti(tn−i + Rf) =aitn + Rf = aiθ + Rf = (1 + Rf)(aiθ + Rf) Laiti(tn−i+1 + Rf) =aitn+1 + Rf = aixθ + Rf = (t + Rf)(σ(ai)θ + Rf) Laiti(tn−1 + Rf) =aiti−1θ + Rf = (ti−1 + Rf)(σ n−i+1(ai)θ + Rf). Thus, the matrix representing Laiti is given by https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press Thus, the matrix representing Laiti is given by For n > 2 and ν ̸= 0, Corollary 32 yields Nucl(S) = Nucm(S) = Fix(ρ) ⊂K, C(S) = F′, dimF′Nucr(S) = [F : F′]m. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press Thus, the matrix representing Laiti is given by Thus, the matrix representing Laiti is given by Maiti = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝ 0 0 · · · 0 σ n−i(ai) 0 · · · 0 0 0 · · · 0 0 σ n−(i+1)(ai) · · · 0 ... ... ... ... 0 ... σ(am) aiθ ... 0 0 σ n−1(ai)θ ... ... ... ... 0 0 · · · σ n−(i−1)(ai)θ 0 0 · · · 0 ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ . https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press Glasgow Mathematical J 17 Glasgow Mathematical Journal As Ma = n i=0 Maiti, we obtain Ma = (mi,j)i,j where As Ma = n i=0 Maiti, we obtain Ma = (mi,j)i,j where mi,j = ⎧ ⎪⎨ ⎪⎩ σ n+1−i(a0) + σ n+1−i(νρ(a0))θ for i = j, σ n+1−i(ai−j) for i > j, σ n+1−i(an+i−j)θ for i < j. mi,j = ⎧ ⎪⎨ ⎪⎩ σ n+1−i(a0) + σ n+1−i(νρ(a0))θ for i = j, σ n+1−i(ai−j) for i > j, σ n+1−i(an+i−j)θ for i < j. This yields Cn,n,1 = {Ma \| ak ∈K for k = 0, 1, . . . , n −1} ⊂Mn(K) as the matrix spread set of the n2[F : F′]-dimensional F′-algebra Sn,n,1(ν, ρ, f). The algebra associated with this spread set is a divi- sion algebra if NK/F′(θ)NK/F′(ν) ̸= 1 (Theorem 22). In that case, the spread set will be an MRD code. In particular, for ν = 0 this condition is satisﬁed for any irreducible f(t) = tn −θ. This is the well known result that for irreducible f, the Petit algebra Sf is a division algebra and so are all its isotopes. For n > 2 and ν ̸= 0, Corollary 32 yields This yields Cn,n,1 = {Ma \| ak ∈K for k = 0, 1, . . . , n −1} ⊂Mn(K) as the matrix spread set of the n2[F : F′]-dimensional F′-algebra Sn,n,1(ν, ρ, f). The algebra associated with this spread set is a divi- sion algebra if NK/F′(θ)NK/F′(ν) ̸= 1 (Theorem 22). In that case, the spread set will be an MRD code. In particular, for ν = 0 this condition is satisﬁed for any irreducible f(t) = tn −θ. This is the well known result that for irreducible f, the Petit algebra Sf is a division algebra and so are all its isotopes. 8.2. Real division algebras of dimension 4 Then, (i) Nucl(S) = Nucm(S) = Fix(ρ), (ii) C(S) = R, (i) Nucl(S) = Nucm(S) = Fix(ρ), (ii) C(S) = R, (ii) C(S) = R, (ii) C(S) = R, (ii) C(S) = R, (iii) dimR(Nucr(S)) = dimR(R[t2]) = 2. Proof. We have h(t) = t4 + 1 ∈R[t2]. Suppose g + Rh ∈Il(M) for some g(t) = g0 + g1t + g2t2 + g3t3 ∈R. Then, ga ∈S(ν, ρ, h) for all a ∈S. Direct and laborious computation yields g2 = 0, g3 = −g1ν, and νρ(g0a0 + g1νa1) = g0νρ(a0) + g1a1. This is satisﬁed for all a0, a1 ∈C if and only if νρ(g0) = g0ν and g1 = νρ(g1ν). If ρ = id, it follows that either g1 = 0 or NC/R(ν) = 1; as S is a division algebra, Theorem 13 (or [34, Theorem 4]) forces g1 = 0 and so g = g0 for some g0 ∈C. Thus Il(M) = C.If ρ = , then g1 = ν2g1 so either g1 = 0 or ν = ±1. As NC/R(ν) ̸= 1 [34, Theorem 4], this forces g1 = 0 so g = g0 for some g0 ∈R. In this case, Il(M) = R. 8.2. Real division algebras of dimension 4 Over a ﬁnite ﬁeld F, all division algebras of dimension 4 over F which have F as their center and a nucleus of dimension 2 over F can be constructed as algebras Sn,m,1(ν, ρ, f) for suitable parameters [34]. Let us now look at some real division algebras we obtain with our construction. If ν = 0, then any choice of an irreducible f ∈C[t; ] will yield an algebra isotopic to a real Petit division algebra. If ν ̸= 0, any choice of irreducible f ∈C[t; ] where NC/R(a0) ̸= 1/NC/R(ν) also yields a division algebra (Theorem 22). ˆ ˆ Let b ∈R and f(t) = t2 −bi ∈C[t; ]. Then, h(t) = ˆh(t2), ˆh(x) = x2 + b2 ∈R[x], is the minimal cen- tral left multiple of f, as h(t) = t4 + b2 = (t2 + bi)(t2 −bi). For all b > 0, f(t) = t2 −bi is irreducible in C[t; ]. Let b ∈R and f(t) = t2 −bi ∈C[t; ]. Then, h(t) = ˆh(t2), ˆh(x) = x2 + b2 ∈R[x], is the minimal cen- tral left multiple of f, as h(t) = t4 + b2 = (t2 + bi)(t2 −bi). For all b > 0, f(t) = t2 −bi is irreducible in C[t; ]. [ ; ] For every irreducible f(t) = t2 −bi, and ν ∈C such that NC/R(ν) ̸= 1 b2 , we obtain a four-dimensional real division algebra S2,2,1(ν, ρ, f) and an MRD code given by its matrix spread set For every irreducible f(t) = t2 −bi, and ν ∈C such that NC/R(ν) ̸= 1 b2 , we obtain a four-dimensional real division algebra S2,2,1(ν, ρ, f) and an MRD code given by its matrix spread set C2,2,1 = z0 + νρ(z0)bi z1bi z1 z0 + νρ(z0)bi \| z0, z1 ∈C , where ρ is either the identity or the complex conjugation. As mentioned in Theorem 7, [34, Theorem 9] uses results to deal with the case when lm = 2 that are valid over ﬁnite ﬁelds, but can be extended to R = C[t; ], for instance for f(t) = t2 −i: Theorem 33. Let f(t) = t2 −i ∈C[t; ]. Suppose S = S2,2,1(ν, ρ, f) is a division algebra for some ν ̸= 0 and ρ ∈AutR(C). https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 18 D. Thompson and S. Pumplün The computations for Ir(M) follow analogously and Cent(M) and Z(M) follow from the proof of [34, Theorem 4]. We obtain the ﬁnal result on the nuclei using Theorem 27 to relate the idealizers and centralizer of M to the nuclei of the algebra S. Example 34. If f(t) = t2 −i, we obtain division algebras S for all ν ∈C such that NC/R(ν) ̸= 1. If ν ̸= 0 and S is a division algebra, then C(S) = R and dimRNucr(S) = 2. Since therefore Nucr(S) is a two-dimensional division algebra over R, Nucr(S) is an Albert isotope of C and can be found in the classiﬁcation in [15, Theorem 1]: it must be C, C( , ), C(1+L(v) , ), C( ,1+L(v) ), or C(1+L(v) ,1+L(w) ), with u, v ∈C suitably chosen. If additionally ρ = id, then Nucl(S) = Nucm(S) = C, and if ρ = then Nucl(S) = Nucm(S) = R. Note that the four-dimensional algebras in the ﬁrst class are all isotopes of nonassociative quaternion algebras. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 9. Constructing algebras and codes using irreducible f∈R = D[t; δ] We now consider the same construction using diﬀerential polynomial rings. Let C a ﬁeld of characteristic p and D be a ﬁnite-dimensional division algebra with center C. Let R = D[t; δ], where δ is a derivation of D, such that δ\|C is algebraic with minimum polynomial g(t) = tpe + c1tpe−1 + · · · + cet ∈F[t] of degree pe, with F = C ∩Const(δ). (This includes the special case where d = 1, that is R = K[t; δ], and δ is an alge- braic derivation with minimum polynomial g.) Then, g(δ) = idd0 is an inner derivation of D. W.l.o.g. we choose d0 ∈F, so that δ(d0) = 0 [18, Lemma 1.5.3]. Then, C(D[t; δ]) = F[x] = {k i=0 ai(g(t) −d0)i \| ai ∈ F} with x = g(t) −d0. The two-sided f ∈R are of the form f(t) = uc(t) with u ∈D and c(t) ∈C(R) [18, Theorem 1.1.32]. All polynomials f ∈R are bounded. For every f ∈R, the minimal central left multiple of f in R is the unique polynomial of minimal degree h ∈C(R) = F[x] such that h = gf for some g ∈R, and such that h(t) = ˆh(g(t) −d0) for some monic ˆh(x) ∈F[x]. The bound f ∗of f is the unique minimal central left multiple of f up to some scalar. From now on, let f ∈R = D[t; δ] be a monic irreducible polynomial of degree m > 1 and let h(t) = ˆh(g(t) −d0) be its minimal central left multiple. Then, ˆh(x) is irreducible in F[x] and h generates a maximal two-sided ideal Rh in R [18, p. 16]. We have C(R/Rh) ∼= F[x]/F[x]ˆh(x) [17, Proposition 4], and deg(h) = pedeg(ˆh). Deﬁne Eˆh = F[x]/F[x]ˆh(x) and let k be the number of irreducible factors of h in R. Theorem 35 [23]. Nucr(Sf)is a central division algebra over Eˆh of degree s = dpe/k, and R/Rh ∼= Mk(Nucr(Sf)). R/Rh ∼= Mk(Nucr(Sf)). R/Rh ∼= Mk(Nucr(Sf)). In particular, this means that deg(ˆh) = dm s , deg(h) = km = dpem s , and [Nucr(Sf) : F] = s2 · dm s = dms. [Nucr(Sf) : F] = s2 · dm s = dms. Moreover, s divides gcd(dm, dpe). If f is not right-invariant, then k > 1 and s ̸= dpe. Moreover, s divides gcd(dm, dpe). If f is not right-invariant, then k > 1 and s ̸= dpe Moreover, s divides gcd(dm, dpe). If f is not right-invariant, then k > 1 and s ̸= dpe. The proof is analogous to the one of Theorem 3. In particular, [Sf : F] = [Sf : C]pe = d2m · pe. Comparing dimensions, we obtain again that [Sf : Nucr(Sf)] = k, and if f is not right-invariant, k > 1. The proof is analogous to the one of Theorem 3. In particular, [Sf : F] = [Sf : C]pe = d2m · pe. Comparing dimensions, we obtain again that [Sf : Nucr(Sf)] = k, and if f is not right-invariant, k > 1. For each z(t) = ˆz(g(t) −d0) ∈F[g(t) −d0] with ˆz ∈F[x], we have z ∈Rf if and only if z ∈Rh. Let Comparing dimensions, we obtain again that [Sf : Nucr(Sf)] k, and if f is not right invariant, k > 1. For each z(t) = ˆz(g(t) −d0) ∈F[g(t) −d0] with ˆz ∈F[x], we have z ∈Rf if and only if z ∈Rh. Let Ef = {z(t) + Rf \| z(t) = ˆz((g(t) −d0)) ∈F[(g(t) −d0)]} ⊂R/Rf. Together with the multiplication (x + Rf) ◦(y + Rf) = (xy) + Rf for all x, y ∈F[(g(t) −d0)], Ef is a ﬁeld extension of F of degree deg (ˆh) isomorphic to Eˆh. Let B = Nucr(Sf), then B has degree s over Eˆh, and Together with the multiplication (x + Rf) ◦(y + Rf) = (xy) + Rf for all x, y ∈F[(g(t) −d0)], Ef is a ﬁeld extension of F of degree deg (ˆh) isomorphic to Eˆh. Let B = Nucr(Sf), then B has degree s over Eˆh, and https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 19 Glasgow Mathematical Journal R/Rf is a free right B-module of dimension k via R/Rf × B −→R/Rf, (a + Rf)(z + Rf) = az + Rf. We assume f is not right-invariant which yields k > 1. For ρ ∈Aut(D) deﬁne F′ = Fix(ρ) ∩F. We assume that F/F′ is ﬁnite-dimensional. Let ν ∈D and 1 ≤l < k = dpe/s. Deﬁne the set Spe,m,l(ν, ρ, f) = {a + Rh \| a ∈P} ⊂R/Rh, where P = {a0 + a1t + · · · + alm−1tlm−1 + νρ(a0)tlm \| ai ∈D} ⊂D[t; δ]. P = {a0 + a1t + · · · + alm−1tlm−1 + νρ(a0)tlm \| ai ∈D} ⊂D[t; δ]. Spe,m,l(ν, ρ, f) is a vector space over F′ of dimension d2pem[F : F′]. for all x, y ∈D. Theorem 37. Let f ∈D[t; δ] be irreducible and deg(h) = km. For all a + Rh ∈R/Rh, Theorem 37. Let f ∈D[t; δ] be irreducible and deg(h) = km. For all a + Rh ∈R/Rh, dimB(im(LMa)) = k2 −k mdeg(gcrd(a, ˆh(g(t) −d0))), colrank(Ma) = k −1 mdeg(gcrd(a, ˆh(g(t) −d0)). In particular, if deg(h) = dmpe then Ma ∈Mpe(Eˆh) and rank(Ma) = dpe −1 mdeg(gcrd(a, ˆh(g(t) −d0))). dimB(im(LMa)) = k2 −k mdeg(gcrd(a, ˆh(g(t) −d0))), colrank(Ma) = k −1 mdeg(gcrd(a, ˆh(g(t) −d0)). In particular, if deg(h) = dmpe then Ma ∈Mpe(Eˆh) and rank(Ma) = dpe −1 deg(gcrd(a, ˆh(g(t) −d0))). colrank(Ma) = k −1 mdeg(gcrd(a, ˆh(g(t) −d0)). In particular, if deg(h) = dmpe then Ma ∈Mpe(Eˆh) and rank(Ma) = dpe −1 mdeg(gcrd(a, ˆh(g(t) −d0))). colrank(Ma) = k −1 mdeg(gcrd(a, ˆh(g(t) −d0)). Thus, Spe,m,1(ν, ρ, f) is a division algebra if and only if there are no divisors of h in Spe,m,1(ν, ρ, f). More generally for l > 1, the above result means: Theorem 38. Suppose that P does not contain any polynomial of degree lm, whose irreducible factors are all similar to f. Then, the set Spe,m,l(ν, ρ, f) deﬁnes an F′-linear MRD code in Mk(B) with minimum distance k −l + 1. In particular, if deg(h) = dmpe, then this code is an F′-linear MRD code in Mdpe(Eˆh) with minimum distance dpe −l + 1. Moreover, s divides gcd(dm, dpe). If f is not right-invariant, then k > 1 and s ̸= dpe. We identify each element of Spe,m,l(ν, ρ, f) with a map in EndB(R/Rf) as follows: For each a ∈Spe,m,l(ν, ρ, f), let La : R/Rf →R/Rf be the left multiplication map La(b + Rf) = ab + Rf. Let Ma be the matrix in Mk(B) representing La with respect to a B-basis of R/Rf and denote the image of S = Spe,m,l(ν, ρ, f) in Mk(B) by Cpe,m,l = {Ma \| a ∈Spe,m,l(ν, ρ, f)}. Cpe,m,l = {Ma \| a ∈Spe,m,l(ν, ρ, f)}. Cpe,m,l = {Ma \| a ∈Spe,m,l(ν, ρ, f)}. For l = 1, this construction again yields algebras over F′: deﬁne a multiplication on the F′-vector space Rm = {g ∈R \| deg(g) < m} via a(t) ◦b(t) = (a(t) + νρ(a0)tm)b(t) modr(f). a(t) ◦b(t) = (a(t) + νρ(a0)tm)b(t) modr(f). a(t) ◦b(t) = (a(t) + νρ(a0)tm)b(t) modr(f). For m > 1, (Rm, ◦) is isomorphic to S(ν, ρ, f) = Spe,m,1(ν, ρ, f). Therefore, we also denote (Rm, ◦) by S(ν, ρ, f) = Spe,m,1(ν, ρ, f). Example 36. Let R = D[t; δ] and f(t) = t + c for some c ∈D. For ν ∈D× and ρ ∈Aut(D), Spe,1,1(ν, ρ, f) = (D, ◦) has the multiplication x ◦y = (x + νρ(x)t)y) : modrf = xy + νρ(x)yt + νρ(x)δ(y) : modrf = xy + νρ(x)(δ(y) −yc) x ◦y = (x + νρ(x)t)y) : modrf = xy + νρ(x)yt + νρ(x)δ(y) : modrf = xy + νρ(x)(δ(y) −yc) for all x, y ∈D. Corollary 39. Suppose that l = 1. (i) If a(t) + νρ(a0)tm ∈P is reducible, then a(t) is not a left zero divisor of (Rm, ◦). (iii) If P does not contain any polynomial similar to f, then (Rm, ◦) is a division algebra over F′. The proofs are all identical to their analogues where f ∈D[t; σ]. The proofs are all identical to their analogues where f ∈D[t; σ]. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press 20 D. Thompson and S. Pumplün Remark 40. One can also use the reduced norm N of the central simple algebra D(t; δ) in this setting: given a central simple algebra D with a maximal subﬁeld E and R = D[t; δ], take the ring of central quotients D(t; δ) = {f/g \| f ∈R, g ∈C(R)} of R. It has center C(D(t; δ)) = Quot(C(R)) = F(x), where x = g(t) = d0. Let ˜δ be the extension of δ to D(x) such that ˜δ = idt\|D(x). Then, D(t; δ) is a central simple F(x)- algebra, more precisely D(t; δ) ∼= (D(x), ˜δ, d0 + x), i.e. D(t; δ) is a generalized diﬀerential algebra. Remark 40. One can also use the reduced norm N of the central simple algebra D(t; δ) in this setting: given a central simple algebra D with a maximal subﬁeld E and R = D[t; δ], take the ring of central quotients D(t; δ) = {f/g \| f ∈R, g ∈C(R)} of R. It has center C(D(t; δ)) = Quot(C(R)) = F(x), where x = g(t) = d0. Let ˜δ be the extension of δ to D(x) such that ˜δ = idt\|D(x). Then, D(t; δ) is a central simple F(x)- algebra, more precisely D(t; δ) ∼= (D(x), ˜δ, d0 + x), i.e. D(t; δ) is a generalized diﬀerential algebra. Let N be the reduced norm of D(t; δ). For all f ∈R, N(f) ∈F[x] and f divides N(f). Let ω : D →Md(E) be the left regular representation of D. For any f ∈R of degree m, N(f) = ±det(ω(am))pexdm + . . . . In particular, N(f) has degree dm [36]. As the bound of f has degree dm in F[x], it follows that N(f) is equal to the bound of f. Thus if deg(ˆh) = dm, we conclude that ˆh(x) = ±N(f). There is more work to be done, for example, to determine the constant term of N(f(t)). This may lead to criteria on how to obtain division algebras using our construction. Additionally, the nuclei of both the algebras and the codes need to be calculated. For instance, consider the special case where d = 1, i.e. R = K[t; δ] for some ﬁeld extension K/F. The proofs are all identical to their analogues where f ∈D[t; σ]. If f(t) = a0 + a1t + · · · + amtm ∈R = K[t; δ] has degree m, then N(f(t)) = (−1)m(pe−1)ape m xm + . . . [36, Thm 18(ii)] Thus, N(f(t)) = ape m xm + . . . N(f(t)) = ape m xm + . . . To ﬁnd the constant term of N(f) is diﬃcult. It is possible to compute special cases though, for example, for f(t) = g(t) + a ∈K[t; δ], N(f(t)) = (x + a)pe [36]. N(f(t)) = ap m xm + . . . To ﬁnd the constant term of N(f) is diﬃcult. It is possible to compute special cases though, for example, for f(t) = g(t) + a ∈K[t; δ], N(f(t)) = (x + a)pe [36]. To ﬁnd the constant term of N(f) is diﬃcult. It is possible to compute special cases though, for example, for f(t) = g(t) + a ∈K[t; δ], N(f(t)) = (x + a)pe [36]. Acknowledgement. We would like to thank J. Sheekey for several helpful discussions on the subject, and the referee, whose comments greatly improved our paper. References [1] A. A. Albert, Modern higher algebra (Dover Publications Inc., Mineola, NY, 2018). [2] D. Augot, P. Loidreau and G. Robert, Generalized Gabidulin codes over ﬁelds of any characteristic, Design Code Cryptogr. 86(8) (2018), 1807–1848. DOI 10.1007/s10623-017-0425-6. [3] D. Augot, A. Couvreur, J. Lavauzelle and A. Neri, SIAM J. Appl. Algebra Geom. 5(2) (2021), 165–199. DOI 10.1137/20M1348583. [4] D. Boucher and F. Ulmer, Linear codes using skew polynomials with automorphisms and derivations, Design Code Cryptogr. 70(3) (2014), 405–431. DOI 10.1007/s10623-012-9704-4. [5] M. Boulagouaz and A. Leroy, (s, d)-codes, Adv. Math. Commun. (AMC) 7(4) (2013), 463–474. DOI 10.3934/amc.2013.7.463. [6] M. Blaum and R. J. McEliece, Coding protection for magnetic tapes: A generalization of the Patel-Hong code, IEEE Trans. Inform. Theory 31(5) (1985), 690–693. DOI 10.1109/TIT.1985.1057098. [7] J. Carcanague, Idéaux bilatères d’un anneau de polynˆomes non commutatifs sur un corps, J. Algebra 18(1) (1971), 1–18. DOI 10.1016/0021-8693(71)90125-6. [8] P. Delsarte, Bilinear forms over a ﬁnite ﬁeld, with applications to coding theory, J. Comb. Theory, Ser 226–241. DOI 10.1016/0097-3165(78)90015-8. [9] C. Faure and P. Loidreau, A new public-key cryptosystem based on the problem of reconstructing p-polynomials, Coding and Cryptography (Springer, 2006), 304–315, Available at: https://perso.univ-rennes1.fr/pierre.loidreau/articles/wcc [10] E. M. Gabidulin, Theory of codes with maximum rank distance, Probl. Inf. Transm. 21 (1985), 1–12. [11] J. Gòmez-Torrecillas, F. J. Lobillo and G. Navarro, Computing the bound of an Ore polynomial. Applications to factorization, J. Symb. Comput. 92 (2019), 269–297. DOI 10.1016/j.jsc.2018.04.018. [12] E. M. Gabidulin, A. V. Paramonov and O. V. Tretjakov, Ideals over a noncommutative ring and their application in cryptology, in Advances in cryptology - EUROCRYPT 91 (D. W. Davies, Editor), vol. 547 (LNCS, Springer, 1991), 482–489. [13] N. Fogarty and H. Gluesing-Luerssen, A circulant approach to skew-constacyclic codes, Finite Fields Appl. 35 (2015), 92–114. DOI 10.1016/j.ﬀa.2015.03.008. [14] T. Hanke, A direct approach to noncrossed product division algebras, Dissertation, Universität Potsdam, Naturwissenschaftliche Fakultät. DOI 10.48550/arXiv.1109.1580. [15] M. Hübner and H. P. Petersson, Two-dimensional real division algebras revisited, Available fernuni-hagen.de/MATHEMATIK/ALGGEO/Petersson/Separata/Two-dimensonal%2BWidmung.pdf. [16] T. W. Hungerford, Algebra, Graduate texts in mathematics, vol. 73 (Springer Verlag, New York, 1980). [17] S. Ikehata, Purely inseparable ring extensions and Azumaya algebras, Math. J. Okayama Univ. 41 (1999), 63–69, Available at: https://www.math.okayama-u.ac.jp/mjou/mjou41/ https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press Glasgow Mathematical Journal 21 [18] N. Jacobson, Finite-dimensional division algebras over ﬁelds. (Springer Verlag, Berlin-Heidelberg-New York, 1996). [19] H. T. Kamche and C. Mouaha, IEEE Transactions of Information Theory 65(12) (2019), 7718–7735. DOI 10.1109/TIT.2019.2933520. [20] M.-A. Knus, A. Merkurjev, M. Rost and J.-P. Tignol, The book of involutions, vol. 44 (AMS Society. Colloquium Publications, 1998). [21] Y. E. Maazouz, M. A. Hahn, A. Neri and M. Stanojkovski, (2021), DOI 10.48550/arXiv.2104.03216. j ( ) [22] O. Ore, Theory of noncommutative polynomials, Ann. Math. 34(3) (1933), 480–508. DOI 10.2307/1968173. h h l f l b f h 55 6 [22] O. Ore, Theory of noncommutative polynomials, Ann. Math. 34(3) (1933), 480–508. DOI 10.2307/19 23] A. Owen, On the right nucleus of Petit algebras (University of Nottingham, 2022). DOI 10.48550/arXiv.2 [24] S. Pai and B. S. Rajan, A Singleton bound for generalized ferrer’s diagram rank metric codes, Online at arxiv: 1506.05558 [cs.IT]. DOI 10.48550/arXiv.1506.05558. [25] R. S. Pierce, Associative algebras. (Springer-Verlag, New York, 1982). rce, Associative algebras. (Springer-Verlag, New York, [26] J.-C. Petit, Sur certains quasi-corps généralisant un type d’anneau-quotient, Séminaire Dubriel. Algèbre et théorie des nombres 20(1966–67), 1–18. ( ) [27] S. Pumplün, Finite nonassociative algebras obtained from skew polynomials and possible applications to (f, σ, δ)-codes, Adv. Math. Commun. (AMC) 11(3) (2017), 615–634. DOI 10.3934/amc.2017046. ün, Finite nonassociative algebras obtained from skew polynomials and possible applications to (f, σ, δ)-codes, h C (AMC) 11(3) (2017) 615 634 DOI 10 3934/ 2017046 [28] S. Pumplün, How to obtain lattices from (f, σ, δ)-codes via a generalization of Construction A, Appl. Algebra Eng. Commun. Comput. 29(4) (2018), 313–333. DOI 10.1007/s00200-017-0344-9. [29] S. Pumplün, Quotients of orders in algebras obtained from skew polynomials with applications to coding theory, Commun. Algebra 46(11) (2018), 5053–5072. DOI 10.1080/00927872.2018.1461882. [29] S. Pumplün, Quotients of orders in algebras obtained from skew polynomials with applications to coding theory, Commun. Algebra 46(11) (2018), 5053–5072. DOI 10.1080/00927872.2018.1461882. [30] S. Pumplün, Albert’s twisted ﬁeld construction using algebras with a multiplicative norm, J. Food Pro 246–248. DOI 10.48550/arXiv.1504.00188. 31] R. M. Roth, Maximum-rank array codes and their application to crisscross error correction, IEEE Trans. I 37(2) (1991), 328–336. DOI 10.1109/18.75248. [32] R. M. Roth, Tensor codes for the rank metric, IEEE Trans. Inform. Theory 72(6) (1996), 2146–2157. DOI 10.1109/ISIT.1995.535754. [32] R. M. Roth, Tensor codes for the rank metric, IEEE Trans. Inform. Theory 72(6) (1996), 2146–2157. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press DOI 10.1109/ISIT.1995.535754. [33] J. Sheekey, A new family of linear maximum rank distance codes, Adv. Math. Commun. (AMC) 10(3) (2016), 475–488. DOI 10.3934/amc.2016019. [33] J. Sheekey, A new family of linear maximum rank distance codes, Adv. Math. Commun. (AMC) 10(3) (2016), 475–488. DOI 10.3934/amc.2016019. [34] J. Sheekey, New semiﬁelds and new MRD codes from skew polynomial rings, J. LMS 101(1) (September 2019), 432–456. DOI 10.1112/jlms.12281. [34] J. Sheekey, New semiﬁelds and new MRD codes from skew polynomial rings, J. LMS 101(1) (September 2019), 432–456. DOI 10.1112/jlms.12281. [35] D. Thompson, New classes of nonassociative division algebras and MRD codes, PhD Thesis (University of Nottingham, 2021). Available at: http://eprints.nottingham.ac.uk/64396/~ [35] D. Thompson, New classes of nonassociative division algebras and MRD codes, PhD Thesis (University of Nottingham, 2021). Available at: http://eprints.nottingham.ac.uk/64396/~ [36] D. Thompson and S. Pumplün, The norm of a skew polynomial, J. Algebra Represent. Theory 25(4) (2022), 869–887. DOI 10.1007/s10468-021-10051-z. [36] D. Thompson and S. Pumplün, The norm of a skew polynomial, J. Algebra Represent. Theory 25(4) (2022), 869–887. DOI 10.1007/s10468-021-10051-z. https://doi.org/10.1017/S001708952300006X Published online by Cambridge University Press
https://openalex.org/W4313185705	https://baas.aas.org/pub/2020i0320/download/pdf	English	null	William A. Dent (1938–2020)	Bulletin of the AAS	2,020	cc-by	648	Bulletin of the AAS • Vol. 52, Issue 2 William A. Dent (1938– 2020) Bulletin of the AAS • Vol. 52, Issue 2 Bulletin of the AAS • Vol. 52, Issue 2 William Irvine1 License: Creative Commons Attribution 4.0 International License (CC-BY 4.0) Bulletin of the AAS • Vol. 52, Issue 2 William A. Dent (1938–2020) Bill Dent with students in 1969. William A. Dent died on Wednesday the 18th of March, 2020 The Astronomy Department at the University of Massachusetts, Amherst, is saddened to report the passing of retired Full Professor William A. (Bill) Dent. Professor Dent was a key founder of the observational aspect of the Astronomy Program at UMass Amherst. As a graduate student at the University of Michigan, Bill made the amazing discovery that the brightness of what were called quasi-stellar radio sources (quasars) varied with time. This required that the physical sizes of these objects be vastly smaller than had been postulated; many years later we have learned that quasars contain supermassive black holes, often with masses of billions of times the mass of our Sun. Bill Dent with students in 1969. Bill Dent with students in 1969. The new astronomy program at UMass began in 1966 with the hiring of three theoretical astrophysicists. Clearly an astronomy program needed an observational component, and just as clearly New England was not a good location for traditional optical astronomy. Radio astronomy, where at most wavelengths the atmosphere is relatively transparent, was a possibility, if suitable faculty could be recruited. In 1968 two radio astronomers were hired, Bill Dent and Richard Huguenin. These two went in different directions in their research. Huguenin founded the Five College Radio Astronomy Observatory on the Prescott Peninsula in the Quabbin Reservoir, which has ultimately led to UMass participation in the Large Millimeter Telescope in Mexico. Bill Dent instead took advantage of the university, becoming a charter member of the Northeast Radio Observatory Corporation (NEROC), founded in 1967. After its original goal of building a new, extremely large radio telescope was not successful, NEROC took over from the Air Force the operation of the 37-meter-diameter Haystack radio telescope in Westford, Massachusetts. Of the original 13 university members of NEROC, the most active universities using the telescope for many years were Harvard, MIT, and the University of Massachusetts (primarily because of the research programs 2 Bulletin of the AAS • Vol. 52, Issue 2 William A. Dent (1938–2020) of Bill Dent). In addition to the Haystack Observatory, Bill also utilized the National Radio Astronomy Observatory’s 36-foot telescope at Kitt Peak and the now-defunct 300- ft telescope at Greenbank in his frontline studies of the variations in brightness of quasars, now commonly known as Active Galactic Nuclei (AGN). William A. Dent died on Wednesday the 18th of March, 2020 He continued to publish significant research results in the major astronomical journals throughout his career, normally with funding from federal science agencies. Bill was a member of the International Astronomical Union and was active in three of its Divisions. As a professor at UMass, Bill trained graduate students who have gone on, for example, to tenured faculty positions at American and Canadian universities and to important positions at the US National Science Foundation and the Space Telescope Science Institute. He was considered a great motivator of the graduate students, helping them relax via local swing dancing and once taking several canoeing through the Minnesota Boundary Waters. Bill was the chair of the committee for the student who was UMass’s first Black astronomy PhD recipient. Bill’s colleagues also recall his trips to very exotic places and the very adventurous ways he traveled, including bus trips in Bolivia, a trek across northern Australia by all- terrain truck, hiking the Tonto Plateau of the Grand Canyon, a far-off-the-beaten-track portion of Canyonlands National Park in mid-summer, and a month-long trek to the Mount Everest region of Nepal. 3
https://openalex.org/W1975633363	https://europepmc.org/articles/pmc3848915?pdf=render	English	null	A Community of One: Social Cognition and Auditory Verbal Hallucinations	PLoS biology	2,013	cc-by	3,989	Vaughan Bell* King’s College London, London, United Kingdom and, second, they are primarily experi- enced as social actors the hearers can relate to and interact with [6]. internalised social actors. To illustrate the extent of this neglect, a recent consensus statement that described an integrated cognitive model of auditory verbal hallu- cinations [1] included only vague men- tions of ‘‘perceptual expectations’’, ‘‘top down influences’’, and ‘‘emotion’’ to address how voices become distinguishable as social identities, without any specific suggestions for how these experiences take a social form. Abstract: Auditory verbal halluci- nations have attracted a great deal of scientific interest, but despite the fact that they are fundamen- tally a social experience—in es- sence, a form of hallucinated communication—current theories remain firmly rooted in an individ- ualistic account and have largely avoided engagement with social cognition. Nevertheless, there is mounting evidence for the role of social cognitive and social neuro- cognitive processes in auditory verbal hallucinations, and, conse- quently, it is proposed that prob- lems with the internalisation of social models may be key to the experience. Phenomenological studies have report- ed that between 30% and 69% of people who hear voices experience them as having specific personal identities (e.g., members of the family, God, celebrities) [5,7–9]. The biggest study to date reported that 31% of 199 voice hearers with psychiatric diagnosis did not experience anonymous voices, 32% experienced a mix of known and unknown voices, and only 37% experienced purely anonymous voices [5]. However, these figures are likely to be a minimum estimate of whether voices are experienced as having social identities, in terms of being able to reliably distinguish them from other voices by personal characteristics, because voices may have social identities but still be anonymous (e.g., identified only as an ‘‘unknown old woman’’ or ‘‘a man with a deep voice’’, as in Leudar et al. [7], who name them ‘‘incognito’’ voices). There is evidence to support this from studies that have specifically investigated this aspect of the experience. Nayani and David [9] reported that 61% of psychiatric voice hearers knew the identity of their voices, but an additional 15% had voices that were familiar but unknown. McCarthy- Jones et al. [5] found that 70% of voice hearers reported voices that, regardless of specific identity, were ‘‘like’’ people they had spoken to in the past. Introduction Auditory verbal hallucinations, the ex- perience of ‘‘hearing voices’’, present us with an interesting paradox: the experi- ences are generated from within a single individual but are typically experienced as a social phenomenon—that is, a form of communication from another speaker. Current theories attempt to explain auditory verbal hallucinations as alter- ations to individualistic information pro- cessing—namely, misattributions of inter- nal thoughts as external phenomena due to biases in cognitive monitoring [1]. The fact that voices stem from an internal source is, of course, clear, but the typical experience of ‘‘hearing voices’’ is not that thoughts seem to be ‘‘spoken aloud’’ but that hallucinated voices have a social identity with clear interpersonal relevance [2]. In other words, voices are as much hallucinated social identities as they are hallucinated words or sounds. Essay Essays articulate a specific perspective on a topic of broad interest to scientists. Vaughan Bell* In a qualitative study of 50 psychiatric voice hearers, Beavan [10] reported that characterising voice identity, regardless of citing a specific personal identity, was a major theme of the experience of hallucinating voices. In This clear omission is all the more surprising given the significant advances in the field of social cognitive neuroscience. Although early theories in this field focussed largely on the development of abilities (like ‘‘theory of mind’’ and ‘‘mentalising’’), more recent work high- lights the importance of developing inter- nalised models of social actors for both ‘‘inner’’ social reasoning and ‘‘live’’ social interaction (e.g., [3,4]). Here it is argued that alongside the well-established difficul- ties with source and intention monitoring that lead to misattribution, auditory verbal hallucinations may also involve a change in the neurocognitive processes that sup- port our internal social models of people and how they behave. Copyright: 2013 Vaughan Bell. 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: Bell V (2013) A Community of One: Social Cognition and Auditory Verbal Hallucinations. PLoS Biol 11(12): e1001723. doi:10.1371/journal.pbio.1001723 A Community of One: Social Cognition and Auditory Verbal Hallucinations Vaughan Bell* The Social Cognition of Hearing Voices Although, at its root, all speech is a social phenomenon, hallucinatory voices have clear interpersonal characteristics above and beyond the social basis on which they rest. First, hallucinated voices are usually experienced as having identi- ties and making coherent communicative speech acts [5] (see example in Box 1), Nevertheless, neurocognitive theories have largely ignored how people who ‘‘hear voices’’ acquire what amounts to Published December 3, 2013 Published December 3, 2013 Published December 3, 2013 Box 1. Hallucinated Social Identities ‘‘I mean, there are two voices—Simon and Jeremy. Simon’s…um…like a demon really. He’s very demonic and he…says people read my mind and they know I’m evil…. I’ve got a year to live, that if I don’t do as I’m told the horrible horseman of the apocalypse will come and get me and kill me and Armageddon will come and the world will be destroyed. And then Jeremy—he’s just a little boy, he’s just full of fun, you know, he’ll tell me things like—um—‘Move the food from the cupboard and put it in mum’s chest of 7 drawers.’ Just stupid things like that. It’s funny but it’s—it is annoying really.’’ —Lucy from Knudson and Coyle [33] —Lucy from Knudson and Coyle [33] identity or through resemblance [5]), it seems likely that intense emotion within a relationship is associated with both the formation and content of hallucinated voices. other words, regardless of whether a voice is associated with a personal identity in the outside world, the process of recognising it as a distinct social identity is likely to be a key experience for many voice hearers. Similarly, numerous studies have now found that voice hearers understand their connection with the voices in terms of relationships and interact with their voices in ways that ‘‘share many properties with interpersonal relationships within the so- cial world’’ [6]. Most obvious in this regard is the fact that over 80% of people who experience auditory verbal hallucina- tions have reported that they are able to engage in interactive conversations with their voices [7,8]. Judgements about the identity of hallucinated voices rely on perceptual features similar to those re- quired to judge identity when listening to the voices of other speakers, with per- ceived identity being an important medi- ator of distress [11]. These results suggest that the experi- ence of auditory verbal hallucinations is, for most voice hearers, primarily a social one, with social environment through the lifespan having a specific effect on the presence and form of voices. The fact that most voices are perceived as having social identities with which the hearer interacts in ways verifiably similar to external social relationships suggests that voices often function as internal models of social actors. Towards a Social Cognitive Approach to Voices With regard to the effect of social environment on voices, relationships with the hallucinated voices are experienced in terms of social power, while voice hearers’ perception of the external social world is mirrored in their relationship with hallu- cinated voices—similarly a significant me- diator of distress [6,12]. Studies on psychiatric voice hearers have indicated that the social environment plays a role not only in the formation of hallucinations but also the maintenance of the experience [12,13]. Although the risk of psychosis is raised after the experience of trauma in general, childhood sexual and emotional abuse—but not physical abuse—predict the presence of auditory verbal hallucina- tions in adulthood [14,15], suggesting a specific link with early relationship trau- ma. In adult life, the death of someone in a long-term, loving relationship will com- monly lead to hallucinations of that specific person, and hallucinated voices are among the most common experiences [16]. In line with phenomenological evi- dence that voices are typically associated with known people (either directly through There is good evidence that auditory verbal hallucinations are usually experi- enced as social entities with which voice hearers have dynamic relationships and which are associated with specific changes to brain networks used to support social cognition. It is surprising, therefore, that cognitive and neurocognitive theories of hallucinated voices have been almost entirely individualistic in their approach. Neuroimaging studies have linked audi- tory hallucinations to functional and structural differences in speech and lan- guage areas—most notably the superior and middle temporal gyri and the inferior frontal gyrus (Broca’s area). In addition, a wider network of non-sensory areas is implicated. These include areas typically described in the auditory verbal halluci- nation literature as linked to cognitive monitoring—namely, the dorsolateral pre- frontal cortex, anterior cingulate, and cerebellum—and areas typically linked to emotion and affect regulation—namely, the anterior insula, hippocampal and parahippocampal regions, and the orbito- frontal cortex [17,18]. However, these areas typically described as monitoring and emotion areas are also key compo- nents in social neurocognitive networks that make up the ‘‘social brain’’ [19,20]. Perhaps one exception is Fernyhough’s [28] developmental theory that draws on Vygotsky’s account of inner speech. Ac- cording to Vygotsky [29], children learn language through interaction and dialogue with others. Auditory Verbal Hallucinations and Social Cognitive Neuroscience The majority of studies on the cognitive neuroscience of auditory verbal hallucina- tions have not looked at ‘‘voices’’ as specifically social phenomena, but there is evidence that the neural networks involved in supporting these experiences have significant overlap with areas that play a key role in social neurocognition. Box 1. Hallucinated Social Identities Neuroimaging studies that have directly compared the experience of auditory hallucinations to imagined inner voices have found remarkably similar brain activ- ity, indicating that voices are not likely to be just ‘‘misidentified thoughts’’ but specifical- ly ‘‘misidentified voice images’’, potentially experienced as unintentional because of an altered sequence of activation in the supplementary motor area and auditory perceptual areas [25,26]. Such voice imag- es have been found to have dissociable neural substrates for spatial and social identity properties [27]. Published December 3, 2013 Funding: The author received no specific funding for this work. Competing Interests: The author has declared that no competing interests exist. December 2013 \| Volume 11 \| Issue 12 \| e1001723 PLOS Biology \| www.plosbiology.org 1 studies of auditory verbal hallucinations is the temporoparietal junction, now clearly identified as having a key role in verbal working memory and social cogni- tion [20] and, according to Saxe [21], a central role in representing others’ mental states. Using electroencephalography, this area has been found to be active in the second before the onset of auditory verbal hallucinations [22], and 1-Hz repetitive transcranial magnetic stimulation (used to dampen down neural excitability) applied to this area reduces hallucination intensity [23]. In the only study that has directly stimulated the temporoparietal junction (increasing neural excitability in the area), Arzy et al. [24] induced a clear experience of social imagery in non-voice-hearing, non-psychiatric participants in the form of a ‘‘sensed presence’’. Towards a Social Cognitive Approach to Voices The dialogue format is then used, first out loud, to problem-solve (children literally talk to themselves), and later becomes internal as we learn to internalise speech as thoughts, with thoughts retaining dialogic qualities and having elements of ‘‘inner self-talk’’. Fer- nyhough [27] uses this to explain how the ‘‘misattribution of thought’’ model of auditory verbal hallucinations could pro- duce speech-like experiences. However, Of particular interest in the network commonly identified in neuroimaging December 2013 \| Volume 11 \| Issue 12 \| e1001723 PLOS Biology \| www.plosbiology.org 2 models of other people and generate associated imagery of their voices is a normal developmental process. Longitudi- nal studies looking at developmental psychology should see that this ability develops in line with other social cognitive processes, and that people with difficulties in internalising predictive models of other individuals should have marked social difficulties. Secondly, it implies that chang- es to the normal functioning of this system play a causal role in many voice-hearing experiences. Although typically experi- enced as social, voice-hearing experiences can range from simple repetitive syllables to conversations between the hearer and several hallucinated vocal identities. Drawing on the work of Hassabis et al. [30], the more a voice is experienced as having a social identity, the more it should involve processes that support the creation of and prediction from internalised per- sonality models at the cognitive and neurobiological level. from being associated with specific identi- fiable persons in the outside world (as opposed to voices with distinguishable personalities who remain ‘‘anonymous’’ or ‘‘incognito’’) are still lacking. Further- more, little attention has been paid to how voices evolve over time [32], and knowing when voices become ‘‘social’’ may be key to understanding the role of social cogni- tion in the aetiology of auditory verbal hallucinations. this account still does not explain why hallucinated voices are typically experi- enced as distinct social identities with which the hearer has a relationship. In essence, it still strips the social phenome- nology—and, therefore, it could be ar- gued, key aspects of social cognitive processing—from the experience. Considering the evidence that auditory verbal hallucinations involve a misattribu- tion of internal phenomena as external due to biases in cognitive monitoring [1], we need to be clear about the nature of the internal phenomena that are being misat- tributed. Towards a Social Cognitive Approach to Voices It would be most parsimonious to assume that these phenomena stem from our normal ability to internalise models of people we know and their voices, rather than auditory hallucinations involving a de novo generation of persistent and inter- nally vocal social identities. Accounts including internalised models of social actors suggest that we internalise others’ voices and personalities so that we can predict what someone would say or do in any given situation [30]. These internal models can be for specific people, so I can imagine how my spouse might respond in a hypothetical conversation, or for generic stereotypes, so I can imagine how a policeman or shopkeeper might respond. Over the last decade, psychological- level research has focussed on the link between social cognition and auditory verbal hallucinations and has amassed a significant amount of evidence as a result. However, researchers working in cognitive neuroscience, who are specifically looking to make links with neurobiology, have only occasionally engaged with studies that have investigated the social neurocogni- tion of hearing voices. Despite some provocative results, they have not yet used paradigms that would disentangle the extent to which the ‘‘social brain’’ is part of the hallucinatory experience. This is clearly an area where more targeted research needs to be completed. Similarly, more effort needs to be put into develop- ing theories that include the socially relevant evidence, as this has been largely ignored in both cognitive and neurocog- nitive accounts. Furthermore, considering that one of the key experiences of voice hearing is the lack of agentive control over the voices, and that there is a link between social stress, trauma, and auditory hallucina- tions, the internal social models of indi- viduals associated with intense traumatic or emotional experiences should be less predictable—and resultant imagery more intrusive—than for individuals not associ- ated with such experiences. In addition, recent evidence has emerged that psychi- atric voice hearers are much more likely to identify voices as specific living people than non-clinical voice hearers [31], suggesting that social cognitive factors may differ depending on the level of disability associated with the experience. Neuropsychological models of voice hearing [17,18] suggest there is an impor- tant component involved in the generation of heard speech (changes to activity in the language system) and an important com- ponent involved in difficulty distinguishing internal from external phenomena (altered cognitive monitoring). 5. McCarthy-Jones S, Trauer T, Mackinnon A, Sims E, Thomas N, et al. (2012) A new phenomenolog- ical survey of auditory hallucinations: evidence for subtypes and implications for theory and practice. Schizophr Bull. E-pub ahead of print. 6. Hayward M, Berry K, Ashton A (2011) Applying interpersonal theories to the understanding of and therapy for auditory hallucinations: a review of the literature and directions for further research. Clin Psychol Rev 31: 1313–1323. Towards a Social Cognitive Approach to Voices The hypothesis suggested here is that, in addition to these well-established factors, there is an alter- ation to the social cognitive or social neurocognitive systems that support inter- nal models of social actors and their associated voice imagery, to explain why voices are typically experienced as having an identity and acting socially. As one of our most enigmatic experi- ences, ‘‘hearing voices’’ is at once both individual and social. There is a clear need to understand it in terms of the individual mind and brain, and a clear opportunity for it to shed light on the social world that lives within us. To further clarify these issues there is a clear need for more fundamental evidence to be gathered about the nature of voice- hearing experiences. Studies on how voices are distinguished as ‘‘individuals’’, which social characteristics they are perceived to have, and how this is distinct Acknowledgments Many thanks to Charles Fernyhough for useful and stimulating discussions during the writing of this article. It is worth examining what this implies. First, it implies that an ability to internalise 7. Leudar I, Thomas P, McNally D, Glinski A (1997) What voices can do with words: pragmatics of verbal hallucinations. Psychol Med 27: 885–898. 5. McCarthy-Jones S, Trauer T, Mackinnon A, Sims E, Thomas N, et al. (2012) A new phenomenolog- ical survey of auditory hallucinations: evidence for subtypes and implications for theory and practice. Schizophr Bull. E-pub ahead of print. 6. Hayward M, Berry K, Ashton A (2011) Applying interpersonal theories to the understanding of and therapy for auditory hallucinations: a review of the literature and directions for further research. Clin Psychol Rev 31: 1313–1323. 7. Leudar I, Thomas P, McNally D, Glinski A (1997) What voices can do with words: pragmatics of verbal hallucinations. Psychol Med 27: 885–898. 8. Garrett M, Silva R (2003) Auditory hallucina- tions, source monitoring, and the belief that ‘‘voices’’ are real. Schizophr Bull 29: 445–457. 3. Brown EC, Bru¨ne M (2012) The role of prediction in social neuroscience. Front Hum Neurosci 6: 147. 8. Garrett M, Silva R (2003) Auditory hallucina- tions, source monitoring, and the belief that ‘‘voices’’ are real. Schizophr Bull 29: 445–457. 12. Paulik G (2012) The role of social schema in the experience of auditory hallucinations: a systematic review and a proposal for the inclusion of social schema in a cognitive behavioural model of voice hearing. Clin Psychol Psychother 19: 459– 472. 4. Gallotti M, Frith CD (2013) Social cognition in the we-mode. Trends Cogn Sci 17: 160–165. References 9. Nayani TH, David AS (1996) The auditory hallucination: a phenomenological survey. Psychol Med 26: 177–189. 1. Waters F, Allen P, Aleman A, Fernyhough C, Woodward TS (2012) Auditory hallucinations in schizophrenia and nonschizophrenia populations: a review and integrated model of cognitive mechanisms. Schizophr Bull 38: 683–693. 10. Beavan V (2011) Towards a definition of ‘‘hearing voices’’: a phenomenological approach. Psychosis 3: 63–73. 2. Larøi F, Sommer IE, Blom JD, Fernyhough C, Ffytche DH, et al. (2012) The characteristic features of auditory verbal hallucinations in clinical and nonclinical groups: state-of-the-art overview and future directions. Schizophr Bull 38: 724–733. 11. Badcock JC, Chhabra S (2013) Voices to reckon with: perceptions of voice identity in clinical and non-clinical voice hearers. Front Hum Neurosci 7: 114. 12. Paulik G (2012) The role of social schema in the experience of auditory hallucinations: a systematic review and a proposal for the inclusion of social schema in a cognitive behavioural model of voice hearing. Clin Psychol Psychother 19: 459– 472. 3. Brown EC, Bru¨ne M (2012) The role of prediction in social neuroscience. Front Hum Neurosci 6: 147. 3. Brown EC, Bru¨ne M (2012) The role of prediction in social neuroscience. Front Hum Neurosci 6: 147. 4. Gallotti M, Frith CD (2013) Social cognition in the we-mode. Trends Cogn Sci 17: 160–165. 4. Gallotti M, Frith CD (2013) Social cognition in the we-mode. Trends Cogn Sci 17: 160–165. December 2013 \| Volume 11 \| Issue 12 \| e1001723 PLOS Biology \| www.plosbiology.org 3 13. Goldstone E, Farhall J, Ong B (2012) Modelling the emergence of hallucinations: early acquired vulnerabilities, proximal life stressors and mal- adaptive psychological processes. Soc Psychiatry Psychiatr Epidemiol 47: 1367–1380. 20. Rushworth MF, Mars RB, Sallet J (2013) Are there specialized circuits for social cognition and are they uniquetohumans?CurrOpinNeurobiol23:436–442. topographies for working memory maintenance of voice identity and location. Cereb Cortex 14: 768–780. topographies for working memory maintenance of voice identity and location. Cereb Cortex 14: 768–780. 21. Saxe R (2006) Uniquely human social cognition. Curr Opin Neurobiol 16: 235–239. 28. Fernyhough C (2004) Alien voices and inner dialogue: towards a developmental account of auditory verbal hallucinations. New Ideas Psychol 22: 49–68. y p 14. Bentall RP, Wickham S, Shevlin M, Varese F (2012) Do specific early-life adversities lead to specific symptoms of psychosis? A study from the 2007 the Adult Psychiatric Morbidity Survey. Schizophr Bull 38: 734–740. 22. References Line P, Silberstein RB, Wright JJ, Copolov DL (1998) Steady state visually evoked potential correlates of auditory hallucinations in schizo- phrenia. Neuroimage 8: 370–376. 29. Vygotsky L (1934) Thought and language. Cam- bridge (Massachusetts): MIT Press. 23. Slotema CW, Aleman A, Daskalakis ZJ, Sommer IE (2012) Meta-analysis of repetitive transcranial magnetic stimulation in the treatment of auditory verbal hallucinations: update and effects after one month. Schizophr Res 142: 40–45. 30. Hassabis D, Spreng RN, Rusu AA, Robbins CA, Mar RA, et al. (2013) Imagine all the people: how the brain creates and uses personality models to predict behavior. Cereb Cortex. E-pub ahead of print. p 15. Daalman K, Diederen KM, Derks EM, van Lutterveld R, Kahn RS, et al. (2012) Childhood trauma and auditory verbal hallucinations. Psychol Med 42: 2475–2484. 24. Arzy S, Seeck M, Ortigue S, Spinelli L, Blanke O (2006) Induction of an illusory shadow person. Nature 443: 287. 16. Keen C, Murray C, Payne S (2013) Sensing the presence of the deceased: a narrative review. Ment Health Relig Cult 16: 384–402. 31. Daalman K, van Zandvoort M, Bootsman F, Boks M, Kahn R, et al. (2011) Auditory verbal hallucinations and cognitive functioning in healthy individuals. Schizophr Res 132: 203– 207. g 17. Allen P, Modinos G, Hubl D, Shields G, Cachia A, et al. (2012) Neuroimaging auditory hallucinations in schizophrenia: from neuroanatomy to neuro- chemistry and beyond. Schizophr Bull 38: 695–703. 25. Linden DE, Thornton K, Kuswanto CN, John- ston SJ, van de Ven V, et al. (2011) The brain’s voices: comparing nonclinical auditory hallucina- tions and imagery. Cereb Cortex 21: 330–337. 32. Hartigan N, McCarthy-Jones S, Hayward M (2013) Hear today, not gone tomorrow? An exploratory longitudinal study of auditory verbal hallucinations (hearing voices). Behav Cogn Psychother 19: 1–7. tions and imagery. Cereb Cortex 21: 330–337. 18. Allen P, Larøi F, McGuire PK, Aleman A (2008) The hallucinating brain: a review of structural and functional neuroimaging studies of halluci- nations. Neurosci Biobehav Rev 32: 175–191. 26. Shergill SS, Bullmore E, Simmons A, Murray R, McGuire P (2000) Functional anatomy of audi- tory verbal imagery in schizophrenic patients with auditory hallucinations. Am J Psychiatry 157: 1691–1693. y 33. Knudson B, Coyle A (2002) The experience of hearing voices: an interpretative phenomenolog- ical analysis. Existent Anal 13: 117–134. 19. Kennedy DP, Adolphs R (2012) The social brain in psychiatric and neurological disorders. Trends Cogn Sci 16: 559–572. 27. References Ra¨ma¨ P, Poremba A, Sala JB, Yee L, Malloy M, et al. (2004) Dissociable functional cortical December 2013 \| Volume 11 \| Issue 12 \| e1001723 PLOS Biology \| www.plosbiology.org PLOS Biology \| www.plosbiology.org 4
https://openalex.org/W2567063872	https://europepmc.org/articles/pmc5363822?pdf=render	English	null	The <i>Pseudomonas putida</i> T6SS is a plant warden against phytopathogens	The ISME journal	2,017	cc-by	12,932	Correspondence: P Bernal or A Filloux, MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, Flowers Building, 1st Floor South Kensington Campus, London SW7 2AZ, UK. E-mail: p.bernal@imperial.ac.uk or a.filloux@imperial.ac.uk Received 13 July 2016; revised 8 October 2016; accepted 20 October 2016; published online 3 January 2017 ORIGINAL ARTICLE The Pseudomonas putida T6SS is a plant warden against phytopathogens Patricia Bernal1,2, Luke P Allsopp1, Alain Filloux1 and María A Llamas2 1MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK and 2Department of Environmental Protection, Estación Experimental del Zaidín-Consejo Superior de Investigaciones Científicas, Granada, Spain Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines have an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyze the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, 10 T6SS effector–immunity pairs were found, including putative nucleases and pore-forming colicins. We show that the K1-T6SS is a potent antibacterial device, which secretes a toxic Rhs-type effector Tke2. Remarkably, P. putida eradicates a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens, which demonstrates that the T6SS is instrumental to empower P. putida to fight against competitors. Furthermore, we observed a drastically reduced necrosis on the leaves of Nicotiana benthamiana during co-infection with P. putida and Xanthomonas campestris. Such protection is dependent on the activity of the P. putida T6SS. Many routes have been explored to develop biocontrol agents capable of manipulating the microbial composition of the rhizosphere and phyllosphere. Here we unveil a novel mechanism for plant biocontrol, which needs to be considered for the selection of plant wardens whose mission is to prevent phytopathogen infections. p p y p g e ISME Journal (2017) 11, 972–987; doi:10.1038/ismej.2016.169; published online 3 January 2017 The ISME Journal (2017) 11, 972–987 © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej The ISME Journal (2017) 11, 972–987 © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej OPEN OPEN Introduction have now been assigned clear functions (Figure 1a). TssB and TssC form a contractile sheath that encases a tube formed by rings of Hcp hexamers (Kudryashev et al., 2015). A puncturing device made up of a trimeric VgrG spike is placed on top of the Hcp tube and crowned with a PAAR protein (Cascales and Cambillau, 2012; Shneider et al., 2013). The cyto- solic part of the T6SS docks onto a membrane complex (TssLMJ) probably by interacting with a phage baseplate-like structure (Durand et al., 2015; Filloux and Freemont, 2016; Planamente et al., 2016). On contraction of the TssBC sheath, the T6SS effectors are propelled out of the bacterium together with the Hcp and VgrG proteins and delivered into prey cells (Basler and Mekalanos, 2012). Finally, the ClpV ATPase binds the contracted sheath for disassembly and recycling, enabling another round of injection (Kapitein et al., 2013; Kube et al., 2014). The T6SS is usually quite modular and can accommodate different combina- tions of VgrG/PAAR proteins to form the tip. This modularity allows the delivery of a great variety of effectors (Shneider et al., 2013; Hachani et al., 2014; Whitney et al., 2014). Alternatively, the effectors can also be ushered in and delivered by the tube-forming Hcp protein (Silverman et al., 2013). Thus, a wide variety of effectors with a broad range of activities can be secreted by a single T6SS. T6SS effectors have have now been assigned clear functions (Figure 1a). TssB and TssC form a contractile sheath that encases a tube formed by rings of Hcp hexamers (Kudryashev et al., 2015). A puncturing device made up of a trimeric VgrG spike is placed on top of the Hcp tube and crowned with a PAAR protein (Cascales and Cambillau, 2012; Shneider et al., 2013). The cyto- solic part of the T6SS docks onto a membrane complex (TssLMJ) probably by interacting with a phage baseplate-like structure (Durand et al., 2015; Filloux and Freemont, 2016; Planamente et al., 2016). On contraction of the TssBC sheath, the T6SS effectors are propelled out of the bacterium together with the Hcp and VgrG proteins and delivered into prey cells (Basler and Mekalanos, 2012). Finally, the ClpV ATPase binds the contracted sheath for disassembly and recycling, enabling another round of injection (Kapitein et al., 2013; Kube et al., 2014). Introduction The T6SS is usually quite modular and can accommodate different combina- tions of VgrG/PAAR proteins to form the tip. This modularity allows the delivery of a great variety of effectors (Shneider et al., 2013; Hachani et al., 2014; Whitney et al., 2014). Alternatively, the effectors can also be ushered in and delivered by the tube-forming Hcp protein (Silverman et al., 2013). Thus, a wide variety of effectors with a broad range of activities can be secreted by a single T6SS. T6SS effectors have The type VI secretion system (T6SS) is found in more than 25% Gram-negative bacteria and used to inject toxic proteins into prokaryotic or eukaryotic cells (Ho et al., 2013). Initially, the T6SS was assigned a role in virulence and eukaryotic cell manipulation (Ma and Mekalanos, 2010; Miyata et al., 2011). Further analyses showed that this system has a key role in inter-bacterial competition (Ho et al., 2013). It provides selective advantages to producer strains by annihilating competitors either in an indiscriminate manner or in response to danger signals (Hood et al., 2010; Basler et al., 2013; Ho et al., 2013; Hachani et al., 2014). The T6SS toxins are usually produced together with immunity proteins that prevent self- intoxication. In molecular terms, the T6SS displays structural similarities with the tail and the punctur- ing device of the bacteriophage T4 (Leiman et al., 2009; Filloux, 2011; Cascales and Cambillau, 2012). It is composed by 13 core components of which some P. putida T6SS is a biocontrol weapon P Bernal et al Figure 1 T6SS clusters in P. putida KT2440. (a) Schematic representation of the T6SS structure. (b) Distribution of the K1-, K2- a K3-T6SS clusters (blue), and the vgrG (yellow) and hcp (purple) genes in the KT2440 genome. (c–e) Genomic organization of t P. putida T6SSs cluster, including K1 (c), K2 and K3 (d) or the vgrG and hcp orphan clusters (e). The color code of the genes correlat with the color code shown in a. The asterisk () in the tssC2, vgrG2 and hcp4 genes indicates that these genes contain premature st codons. 973 Figure 1 T6SS clusters in P. putida KT2440. (a) Schematic representation of the T6SS structure. (b) Distribution of the K1-, K2- and K3-T6SS clusters (blue), and the vgrG (yellow) and hcp (purple) genes in the KT2440 genome. (c–e) Genomic organization of the P. Plasmids and cloning l d d b Plasmids and cloning l d d b g Plasmids are described in Supplementary Table S1 and primers listed in Supplementary Table S2. PCR amplifications were performed using Phusion Hot Start High-Fidelity (Finnzymes, ThermoFisher Scientific, Loughborough, UK), KOD Hot Start (EMD Millipore, Watford, UK) or Taq (Roche, Burgess Hill, UK) DNA polymerases. Recombinant plasmids were sequenced and transferred to P. putida by electroporation (Choi et al., 2006) or conjugation (Ramos-Gonzalez et al., 1991). , ) The T6SS was first identified in two pathogenic bacteria, Vibrio cholerae and Pseudomonas aerugi- nosa (Mougous et al., 2006; Pukatzki et al., 2006) and analyzed later in many other pathogens (Suarez et al., 2008; de Pace et al., 2010; Burtnick et al., 2011; Murdoch et al., 2011; Rosales-Reyes et al., 2012; Lin et al., 2013). However, analytical description of T6SS in non-pathogenic bacteria is underrepresented in the literature (Bladergroen et al., 2003; Marchi et al., 2013), despite an even distribution in both classes of organisms (Boyer et al., 2009). Pseudomonas putida is a saprophytic soil bacterium that has the capacity to colonize the root of crop plants (Espinosa-Urgel et al., 2000; Molina et al., 2000). It is a well- established biocontrol agent that provides growth advantages to the plant (Weller, 2007). In this study we identified and characterized the P. putida T6SS, which exhibits great variety and complexity both in terms of apparatus and secreted toxins. We showed that this secretion system is used by the bacterium to drive killing of resilient phytopathogens and appears to be a major factor in its biocontrol portfolio. Bioinformatic analyses Pseudomonas sequences were obtained from the Pseudomonas Genome database (Winsor et al., 2016). BLASTP analyses were performed at the NCBI website (Boratyn et al., 2013) and amino acid sequence searches using SMART (Letunic et al., 2015) and Pfam (Finn et al., 2016). The Protein Homology/analogy Recognition Engine (Phyre2) server was used to perform structural-base homology prediction (Kelley et al., 2015). The PyMOL Molecular Graphics System (Version 1.8 Schrondin- ger, LLC, Cambridge, UK) was used to build structural alignments. The phylogenetic tree was constructed using MEGA6 (Tamura et al., 2013). PSORTb software and SOSUI GramN server were used to predict sub-cellular location of proteins (Imai et al., 2008; Yu et al., 2010), TMHMM software to predict transmembrane domains (Krogh et al., 2001), and SignalP and SOSUIsignal to predict signal peptides (Gomi et al., 2004; Petersen et al., 2011). Plasmids and cloning l d d b Synteny was analyzed using the CoGe’s Genome Evolution tool (Lyons and Freeling, 2008). The UGENE bioinformatics software was used to identify open reading frames (orfs; Okonechnikov et al., 2012). Introduction putida T6SSs cluster, including K1 (c), K2 and K3 (d) or the vgrG and hcp orphan clusters (e). The color code of the genes correlates with the color code shown in a. The asterisk () in the tssC2, vgrG2 and hcp4 genes indicates that these genes contain premature stop codons. Figure 1 T6SS clusters in P. putida KT2440. (a) Schematic representation of the T6SS structure. (b) Distribution of the K1-, K2- and K3-T6SS clusters (blue), and the vgrG (yellow) and hcp (purple) genes in the KT2440 genome. (c–e) Genomic organization of the P. putida T6SSs cluster, including K1 (c), K2 and K3 (d) or the vgrG and hcp orphan clusters (e). The color code of the genes correlates with the color code shown in a. The asterisk (*) in the tssC2, vgrG2 and hcp4 genes indicates that these genes contain premature stop codons. The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al 974 been classified into specialized and cargo effectors (Cianfanelli et al., 2016). Specialized effectors are domains, usually at the C-terminus of specific T6SS structural components that are coined as ‘evolved’ VgrG, PAAR or Hcp proteins. In contrast, cargo effectors interact non-covalently with ‘canonical’ VgrG, PAAR or Hcp proteins (Durand et al., 2014). Several cargo effectors carry a motif named MIX (marker for type six effectors) that is proposed to be required for recognition and direct interaction with VgrG or PAAR proteins (Salomon et al., 2014). Specific adaptors such as Tap/Tec and EagR proteins are likely to facilitate the interaction between the structural components of the T6SS tip and the effectors (Alcoforado Diniz and Coulthurst, 2015; Liang et al., 2015; Unterweger et al., 2015). Finally, accessory elements (named tag from T6SS accessory genes) are required to modulate the assembly of the system and/or confer additional functions (Boyer et al., 2009). (HA)-tagged Hcp1 protein was introduced on the chromosome using the miniCTX transposon (Hoang et al., 2000). Insertions and gene replacements were confirmed by PCR. All strains were grown in lysogeny broth (LB; Sambrook et al., 1989). For secretion assays, tryptone soya broth medium (Oxoid, ThermoFisher Scientific, Loughborough, UK) was used. Escherichia coli was incubated at 37 °C, and P. putida and the phytopathogens at 25–30 °C. Antibiotics were used at (μg ml−1): ampicillin, 100; gentamycin, 20; kanamycin, 50; piperacillin, 25; rifampicin, 20; streptomycin, 100; tetracycline, 50. Results Most of the genes fall into three clusters that we named K1-, K2- and K3-T6SS (Figures 1b–d and Supplementary Tables S4 and S5). Several hcp and vgrG orphan genes were also found scattered on the chromosome (Figures 1b and e, and Supplementary Table S6). Phylogenetic analysis showed that the K2- and K3-T6SSs are related (group 1.2, Figure 2), whereas the K1 cluster clades separately (group 4B, Figure 2). Interbacterial competition assays In vitro competition assays were performed on LB plates as previously described (Hachani et al., 2013). Bacterial overnight cultures were adjusted to OD600 of 1 in PBS and mixed in a 1:1 ratio (P. putida-prey). Bacteria were co-cultured at 30 °C for 5 h (E. coli) or 24 h (phytopathogens). The competition was quanti- fied by counting colony-forming units on antibiotic selection. At least three biologically independent experiments were performed. In planta competition assays were carried out by infiltration of bacteria into Nicotiana benthamiana leaves as described before (Ma et al., 2014). Overnight cultures of P. putida and Xanthomonas campestris were adjusted to OD600 of 0.1 in PBS and mixed in a 1:1 ratio. Approximately 100 μl volume was infiltrated on the reverse of a 1-month-old leaf and the infiltration area marked. After 24 h of incubation in a plant chamber (23 °C, 16 h light), colony-forming units were determined. A section of the leaf from the infiltration area was cut out, homogenized in PBS and subsequently serially diluted. The leaves were visualized by fluorescence microscopy using a Leica M206FA stereomicro- scope. Imaging was performed at room temperature with a × 1 objective. The evaluation of necrosis was based on the coloration of the leaves following previous standard evaluation of virulence that goes from no visible effects to changes in the tissue color of the leaf, which can shift from green to yellowish (chlorosis), yellowish to brownish and blackening of the leaf (necrosis), up to complete rotting of the leaf at later stages (Katzen et al., 1998). In our assay, the circled zones point at deep brown color area. g K2 and K3 consist of two divergently transcribed gene clusters that contain 12 of the 13 genes encoding core T6SS components (Figure 1d). The missing core gene, clpV, encodes the ATPase required for disassembling the sheath (Kapitein et al., 2013; Kube et al., 2014), which is absent in all clusters belonging to group 1.2 (data not shown). Results SDS–polyacrylamide gel electrophoresis containing 8% or 15% (w/v) acrylamide and electro-transferred to nitrocellulose membranes. Immunodetection was performed using monoclonal antibodies directed against the influenza HA epitope (HA.11, Covance, Biolegend, London, UK) or the paramyxovirus of simian V5 epitope (Invitrogen, ThermoFisher Scien- tific). A monoclonal antibody against the β-subunit of the RNA polymerase (Neoclone, Biolegend, London, UK) was also used. The secondary antibody, horse- radish peroxidase-conjugated rabbit anti-mouse (Sigma Aldrich, Gillingham, UK), was detected using the SuperSignal West Pico Chemiluminescent Sub- strate (Thermo Scientific, Loughborough, UK). Blots were scanned and analyzed using the Image Reader LAS-3000 (Fuji, GE Healthcare, Little Chalfont, UK). Genome-wide screening for T6SSs in P. putida species In silico analyses of forty P. putida strains revealed that all encode T6SS genes and 90% of them have at least one cluster encoding a full set of T6SS components (Supplementary Table S3). The number of T6SS clusters in a single strain ranged from zero in P. putida BIRD-1 or H8234 to four in P. putida PA14H7, whereas most strains contained one or two clusters (Supplementary Table S3). In total, we identified 66 complete T6SS gene clusters distributed in three main phylogenetic clades (Figure 2). Following previous nomenclature (Boyer et al., 2009; Barret et al., 2011), we referred to these three groups as 1.2, 2 and 4B. Eighty percent of the clusters belong to group 1.2 or 4B, whereas 10% are found in group 2 (Figure 2). Each of these groups contains distinguishable genetic architecture and features (Supplementary Figure S1), as described in the next section. Growth inhibition assays Overnight LB cultures of E. coli DH5α harboring the vectors pNDM220 (Gotfredsen and Gerdes, 1998), pBAD33 (Guzman et al., 1995) or derivatives encod- ing Tke2 or Tki2 were adjusted to OD600 of 0.1. Expression of tke2 and tki2 was induced with 0.2% (w/v) L-arabinose and 1 mM isopropyl β-D-1-thioga- lactopyranoside, respectively. The reference strain P. putida KT2440 is equipped with three T6SSs The reference strain P. putida KT2440 is equipped with three T6SSs three T6SSs We used the strain KT2440 to perform in-depth genomic analysis. In this strain, only five T6SS- related genes, that is, the hcp genes PP2615 and PP4082 or the vgrG genes PP2614, PP3386 and PP4049 are annotated (http://pseudomonas.com/). Using bioinformatics approaches (for example, BLASTP, Ugene or SMART) we identified a large number of T6SS-related orfs (Supplementary Tables S4–S6). Materials and methods Bacterial strains and growth conditions Bacterial strains are listed in Supplementary Table S1. P. putida mutants were constructed by allelic exchange as described previously (Vasseur et al., 2005). Briefly, 750 bp DNA fragments upstream and downstream the gene to be deleted were amplified using KT2440 genomic DNA. Mutator fragments were obtained by overlapping PCR, cloned into pCR-BluntII-TOPO (Invitrogen, ThermoFisher Scien- tific, Loughborough, UK), sequenced and subcloned into the pKNG101 suicide vector (Kaniga et al., 1991). A similar approach was used to replace the wild-type tke2 gene with tke2-V5 encoding a C-terminally virus 5 (V5)-tagged Tke2 protein. The hcp1-HA gene encoding a C-terminal hemagglutinin Secretion assays Bacterial strains were grown in tryptone soya broth for 5 h at 30 °C and the extracellular fraction obtained and analyzed as previously described (Hachani et al., 2011). The proteins in the culture supernatants were precipitated with trichloroacetic acid and resuspended in 1 M of Tris-base and 4 × Laemmli buffer. Proteins were separated by The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al 975 Results Using the ‘CoGe's Genome Evolution Analysis’ tool, we observed a synteny among the K2 and K3 clusters (Figure 1d). The identity of the corresponding proteins encoded within each of these clusters was remarkably high, ranging from 64 to 99% (Supplementary Table S5). These observations indi- cate that the two clusters may have arisen from a duplication event. p The K1 system is not related to K2 and K3, and belongs to the plant-related group (group 4B, Figure 2; Boyer et al., 2009). This cluster comprises two putative operons and an ‘intermediate’ region The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al P. putida T6SS is a biocontrol weapon P Bernal et al 976 Figure 2 Phylogenetic distribution of T6SS clusters in P. putida species. Maximum likelihood tree with 1000 bootstrap replicates were built with Mega 6 for the core component protein TssB. T6SS cluster nomenclature (Boyer et al., 2009; Barret et al., 2011) is used to show the major phylogenetic clusters. Three main groups are clearly distinguishable: group 1.2 (green), group 2 (red) and group 4B (blue). P. aeruginosa and A. tumefaciens T6SSs loci are included into the phylogenetic tree to illustrate all the subgroups: 1.1 (P. aeruginosa H2), 1.2 (P. putida K2-K3), 2 (P. putida W619), 3 (P. aeruginosa H1), 4A (P. aeruginosa H3), 4B (P. putida K1) and 5 (A. tumefacines). Figure 2 Phylogenetic distribution of T6SS clusters in P. putida species. Maximum likelihood tree with 1000 bootstrap replicates were built with Mega 6 for the core component protein TssB. T6SS cluster nomenclature (Boyer et al., 2009; Barret et al., 2011) is used to show the major phylogenetic clusters. Three main groups are clearly distinguishable: group 1.2 (green), group 2 (red) and group 4B (blue). P. aeruginosa and A. tumefaciens T6SSs loci are included into the phylogenetic tree to illustrate all the subgroups: 1.1 (P. aeruginosa H2), 1.2 (P. putida K2-K3), 2 (P. putida W619), 3 (P. aeruginosa H1), 4A (P. aeruginosa H3), 4B (P. putida K1) and 5 (A. tumefacines). Figure 2 Phylogenetic distribution of T6SS clusters in P. putida species. Maximum likelihood tree with 1000 bootstrap replicates were built with Mega 6 for the core component protein TssB. T6SS cluster nomenclature (Boyer et al., 2009; Barret et al., 2011) is used to show the major phylogenetic clusters. Results We readily detected Hcp1 in the supernatant of wild- type cultures but not in an isogenic tssA1 mutant (Figure 3a), thus establishing that the K1-T6SS is a functional secretion machine. Several characterized T6SSs have anti-bacterial activity, resulting from the injection of T6SS toxins into bacterial preys (Russell et al., 2014; Cianfanelli et al., 2016). We performed competition assays using E. coli K12 as prey and P. putida wild type or T6SS mutants as predators. The E. coli prey harbors a plasmid that confers blue color to the colony in the presence of X-gal (Figure 3b). In a mixed culture, the P. putida wild-type strain was able to annihilate E. coli, whereas mutants in any of the K1-T6SS structural genes (tssA1, tssL1, tssK1, tssG1, tssF1 or tssE1) were no longer outcompeting E. coli (Figure 3b). In contrast, mutants in the K2- or K3-T6SS clusters, P. putida ΔtssM2 and ΔtssM3, respectively, still efficiently annihilated E. coli (data not shown). We concluded that K1 is the most active KT2440 T6SS in vitro, as under the laboratory conditions used here, and that its antibacterial activity may result from the secretion of T6SS effectors. VgrG linked effectors. Downstream vgrG1 and vgrG2 in the K1 and K2 clusters, respectively, putative effector genes, tke2 and tke4, and EagR adaptor genes, eagR1a-eagR1b and eagR2, were found (Figures 1c and d). Tke2 and Tke4 proteins share a similar structure, both containing an N-term- inal PAAR motif (Cascales and Cambillau, 2012; Shneider et al., 2013) and a conserved Rhs domain (Busby et al., 2013) limited by specific RVxxxxxxxxG and PxxxxDPxGL motifs (Figures 4a and b). PAAR proteins have been shown to be located at the tip of the VgrG trimer, sharpening the T6SS spike and/or creating an interface for T6SS effectors and adaptors (Whitney et al., 2015). The C-terminal region of Tke2 or Tke4 (110 and 102 amino acid long, respectively) carries a cytotoxic domain. This domain is similar in both proteins and belongs to the HNH superfamily of nucleases, for example, colicin E7 and pyocin S1 (Figures 4a and 5a; Huang and Yuan, 2007), although Tke4 domain contains a specific SHH signature (Figures 4a and 5b). Genes encoding putative effectors were also found downstream vgrG3, vgrG4 Results Three main groups are clearly distinguishable: group 1.2 (green), group 2 (red) and group 4B (blue). P. aeruginosa and A. tumefaciens T6SSs loci are included into the phylogenetic tree to illustrate all the subgroups: 1.1 (P. aeruginosa H2), 1.2 (P. putida K2-K3), 2 (P. putida W619), 3 (P. aeruginosa H1), 4A (P. aeruginosa H3), 4B (P. putida K1) and 5 (A. tumefacines). (Figure 1c). The first operon contains 15 genes, 12 of which encode T6SS core components, and was named the structural operon (Figure 1c). The last core component gene, vgrG, is located within the second operon that was therefore named the VgrG1 operon (Figure 1c). Within the structural operon we found a previously undefined orf, PP3090.1 encod- ing the accessory protein TagF1 (Supplementary Table S4). An ortholog of this protein was repor- ted to function as a posttranscriptional regulator (Silverman et al., 2011). Another accessory gene encodes TagP1 (Supplementary Table S4), a TssM derivative whose C-terminal periplasmic portion carries a peptydoglycan-binding domain (pfam00691; Aschtgen et al., 2010). Finally, our analysis identified a novel T6SS feature represented by the first gene in the K1-T6SS structural operon, PP3101.1, tagX1 (Figure 1c and Supplementary Table S4). The protein encoded by this gene has no homologs or recognizable features. It has not been assigned a role in the T6SS but is exclusively present in all clusters belonging to the 4B group (that is, P. putida and Pseudomonas syringae). The K1-T6SS is functional and anti-bacterial Hcp release is dependent on the T6SS and is a reliable marker for assessing functionality of the system (Pukatzki et al., 2006). Therefore, we engi- neered P. putida strains producing an HA-tagged version of Hcp1 to assess K1-T6SS activity. TssA is a core baseplate component of the T6SS, is essential The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al 977 Hachani et al., 2014; Ma et al., 2014; Liang et al., 2015; Unterweger et al., 2015). Our in-silico analyses identified a total of 10 potential EI pairs, most of them encoded in the vicinity of vgrG/hcp genes and in some cases near genes encoding Tap or EagR adaptors (Figures 1c–e and Supplementary Tables S4–S6). These EI pairs have been named Tke and Tki for Type six KT2440 effector and immunity, respec- tively (Figure 4a). for T6SS activity (Planamente et al., 2016) and we used a tssA mutant to disable the P. putida K1-T6SS. P. putida KT2440 encodes a wealth of T6SS bacterial effectors ff Genes encoding putative T6SS effectors and cognate immunity proteins (effector–immunity (EI) pairs) are often linked to hcp, vgrG genes and/or genes encoding chaperones/adaptors (Dong et al., 2013; Figure 3 Functionality of the P. putida K1-T6SS. (a) Production and secretion of Hcp1 in the P. putida KT2440 wild type and the ΔtssA1 mutant strains. The HA-tagged Hcp1 protein was detected by western blot analysis using an anti-HA antibody. Detection of the β-subunit of the RNA polymerase (β-RNAP) was used as control. The position of the molecular size marker (in kDa) is indicated. (b) Competition assay between P. putida and a lacZ-encoding E. coli strain. Blue patches on X-gal-containing LB plates indicate E. coli survival. The top row shows the growth of E. coli, P. putida KT2440 wild-type strain and a battery of P. putida mutants in K1-T6SS genes. The bottom row shows the growth of mixed E. coli/P. putida cultures after 5 h of co-incubation. Figure 3 Functionality of the P. putida K1-T6SS. (a) Production and secretion of Hcp1 in the P. putida KT2440 wild type and the ΔtssA1 mutant strains. The HA-tagged Hcp1 protein was detected by western blot analysis using an anti-HA antibody. Detection of the β-subunit of the RNA polymerase (β-RNAP) was used as control. The position of the molecular size marker (in kDa) is indicated. (b) Competition assay between P. putida and a lacZ-encoding E. coli strain. Blue patches on X-gal-containing LB plates indicate E. coli survival. The top row shows the growth of E. coli, P. putida KT2440 wild-type strain and a battery of P. putida mutants in K1-T6SS genes. The bottom row shows the growth of mixed E. coli/P. putida cultures after 5 h of co-incubation. The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al Figure 4 P. putida KT2440 T6SS effectors. (a) The domain organization of the putative effectors is shown, with PAAR motifs indicated in orange, MIX motifs in blue, Rhs domains in green, HNH nuclease motifs (Tox-HNH and Tox-SHH) in purple, colicin motifs in yellow and the Tox-61 domain in pink. Multiple sequence alignments of the PAAR (b) and MIX (c) protein motifs are represented. The KT2440 T6SS effectors identified in this work are indicated in blue. The sequence of known T6SS effectors containing these motifs was retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). Conservation logos of the motifs are indicated above the alignment. P. putida KT2440 encodes a wealth of T6SS bacterial effectors Conserved residues are highlighted according to the amino acid characteristic: hydrophobic (black), small (pink), positive (blue), negative (yellow) and polar (purple, light blue, red). 978 Figure 4 P. putida KT2440 T6SS effectors. (a) The domain organization of the putative effectors is shown, with PAAR motifs indicated in orange, MIX motifs in blue, Rhs domains in green, HNH nuclease motifs (Tox-HNH and Tox-SHH) in purple, colicin motifs in yellow and the Tox-61 domain in pink. Multiple sequence alignments of the PAAR (b) and MIX (c) protein motifs are represented. The KT2440 T6SS effectors identified in this work are indicated in blue. The sequence of known T6SS effectors containing these motifs was retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). Conservation logos of the motifs are indicated above the alignment. Conserved residues are highlighted according to the amino acid characteristic: hydrophobic (black), small (pink), positive (blue), negative (yellow) and polar (purple, light blue, red). Figure 4 P. putida KT2440 T6SS effectors. (a) The domain organization of the putative effectors is shown, with PAAR motifs indicated in orange, MIX motifs in blue, Rhs domains in green, HNH nuclease motifs (Tox-HNH and Tox-SHH) in purple, colicin motifs in yellow and the Tox-61 domain in pink. Multiple sequence alignments of the PAAR (b) and MIX (c) protein motifs are represented. The KT2440 T6SS effectors identified in this work are indicated in blue. The sequence of known T6SS effectors containing these motifs was retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). Conservation logos of the motifs are indicated above the alignment. Conserved residues are highlighted according to the amino acid characteristic: hydrophobic (black), small (pink), positive (blue), negative (yellow) and polar (purple, light blue, red). Figure 4 P. putida KT2440 T6SS effectors. (a) The domain organization of the putative effectors is shown, with PAAR motifs indicated in orange, MIX motifs in blue, Rhs domains in green, HNH nuclease motifs (Tox-HNH and Tox-SHH) in purple, colicin motifs in yellow and the Tox-61 domain in pink. Multiple sequence alignments of the PAAR (b) and MIX (c) protein motifs are represented. The KT2440 T6SS effectors identified in this work are indicated in blue. The sequence of known T6SS effectors containing these motifs was retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). Conservation logos of the motifs are indicated above the alignment. Conserved residues are highlighted according to the amino acid characteristic: hydrophobic (black), small (pink), positive (blue), negative (yellow) and polar (purple, light blue, red). P. putida KT2440 encodes a wealth of T6SS bacterial effectors orphan operons (hcp4, hcp5 and hcp6; Figure 1e and Supplementary Table S6). These effectors have similarities with pore-forming colicins (that is, colicin S4; Figures 4a and 5c, and Supplementary Table S6). The tke7 and tke8 genes are not genetically associated with vgrG or T6SS adaptor genes. These hcp-linked T6SS effectors could be delivered by docking into the lumen of the Hcp ring, instead of being attached at the VgrG tip, as observed with the P. aeruginosa Tse2 effector (Silverman et al., 2013). In contrast to tke7 and tke8, tke6 is not located within the hcp operon but 5 kb upstream of the hcp4 gene. Interestingly, hcp4 has a premature stop codon and might not be functional (Figure 1e and Supplementary Table S6), whereas Tke6 has an N-terminal PAAR domain (Figures 4a and b). Thus, in contrast to Tke7 and Tke8 that lack PAAR or MIX domains, the delivery of Tke6 could be and vgrG5 (Figures 1d and e). The tke5 and tke9 genes within the K3 and vgrG4 operons, respectively, are linked to genes encoding Tap adaptors (tap3 and tap4; Figures 1d and e). No recognizable features were found in Tke5 or Tke9, except for a conserved N-terminal MIX motif considered a marker for T6SS effectors (Salomon et al., 2014; Figures 4a and c). This motif is also present in the effector-encoded downstream vgrG5, Tke10, which is predicted to be a restriction endonuclease (Figures 1e and 4c). In addition, tke5 and tke10 are linked to genes encod- ing a PAAR-motif (named tsp for type six paar; tsp5 and tsp10; Figures 1d, e and 4b, and Supplementary Table S6). and vgrG5 (Figures 1d and e). The tke5 and tke9 genes within the K3 and vgrG4 operons, respectively, are linked to genes encoding Tap adaptors (tap3 and tap4; Figures 1d and e). No recognizable features were found in Tke5 or Tke9, except for a conserved N-terminal MIX motif considered a marker for T6SS effectors (Salomon et al., 2014; Figures 4a and c). This motif is also present in the effector-encoded downstream vgrG5, Tke10, which is predicted to be a restriction endonuclease (Figures 1e and 4c). In addition, tke5 and tke10 are linked to genes encod- ing a PAAR-motif (named tsp for type six paar; tsp5 and tsp10; Figures 1d, e and 4b, and Supplementary Table S6). Effectors encoded in proximity to hcp genes. Figure 5 P. putida KT2440 T6SS nucleases. (a, b) Multiple sequence alignments of the C-terminal domains of Tke2 (a) and Tke4 (b) effectors (blue) with known proteins of the family (black). Conservation logos of the motifs HNH (a) and SHH (b) are indicated above the alignment. Conserved residues are indicated with the color code used in Figure 4. A representation of the structural model of the C-terminal domain of the Tke2 effector (magenta) superimposed on the colicin E7 structure (blue; PDB: 2JB0) is shown on the right of a. Side chains of the active site residues are shown. (c) Multiple sequence alignment of T6SS colicin effectors (blue) with known proteins of the family (black). The secondary structure prediction (ssp) for effector Tke7 is shown above the alignment. A structural alignment of the Tke7 effector model (magenta) with the colicin S4 (blue, PDB: 3FEW) is shown on the right. mediated by a VgrG protein through a PAAR-VgrG interaction. mediated by a VgrG protein through a PAAR-VgrG interaction. TRIM20 (Weinert et al., 2015; Supplementary Figure S2b and Supplementary Table S4). In summary, we identified 10 potential T6SS effectors in the KT2440 genome. Three of them Tke2, Tke4 and Tke6 have an N-terminal PAAR domain (Figures 4a and b) and are therefore considered ‘specialized’ effectors. The others are not fused to any T6SS component and their domain architecture suggests they are ‘cargo’ effectors. Orphan effectors. We found two additional poten- tial EI pairs (tke1-tki1 and tke3-tki3) within the K1- T6SS cluster, both lacking PAAR or MIX motifs. Tke1 is an ortholog of the P. aeruginosa Tse6, which presents a C-terminal region carrying a toxic domain known as Toxin_61 (Figure 4a and Supplementary Figure S2a; Hachani et al., 2014; Whitney et al., 2014) and degrades NAD(P)(+) in target cells (Whitney et al., 2015). In case of Tke3, a Phyre2 analysis suggests that the C-terminal domain resem- bles the B30.2 fragment from the human protein Tke2/Tki2 is a P. putida K1-T6SS effector/immunity pair P. putida KT2440 encodes a wealth of T6SS bacterial effectors The potential effector genes tke6, tke7 and tke8 were found within or in the vicinity of the three hcp The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al Figure 5 P. putida KT2440 T6SS nucleases. (a, b) Multiple sequence alignments of the C-terminal domains of Tke2 (a) and Tke4 (b) effectors (blue) with known proteins of the family (black). Conservation logos of the motifs HNH (a) and SHH (b) are indicated above the alignment. Conserved residues are indicated with the color code used in Figure 4. A representation of the structural model of the C-terminal domain of the Tke2 effector (magenta) superimposed on the colicin E7 structure (blue; PDB: 2JB0) is shown on the right of a. Side chains of the active site residues are shown. (c) Multiple sequence alignment of T6SS colicin effectors (blue) with known proteins of the family (black). The secondary structure prediction (ssp) for effector Tke7 is shown above the alignment. A structural alignment of the Tke7 effector model (magenta) with the colicin S4 (blue, PDB: 3FEW) is shown on the right. 979 Tke2/Tki2 is a P. putida K1-T6SS effector/immunity pair p We have shown that the K1 system is functional, and that the corresponding gene cluster encodes several The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al Figure 6 Toxicity and secretion of the Tke2 effector. (a) The growth of E. coli K12 cells harboring the pTke2-CT and pTki2 plasmids containing the C-terminal toxin domain of the tke2 effector and the tki2 immunity genes, respectively, was deter- mined by measuring the OD at 600 nm. At time zero, either 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and/or 0.02% (w/v) arabinose were added to the LB medium, to induce expression of the tke2-CT or/and tki2 genes, respectively. (b) Western blot analyses using an anti-V5 or anti-HA mono- clonal antibody to detect the Tke2-CT-V5 or Tki2-HA-tagged proteins. Proteins were prepared from E. coli K12 cells grown during 10 h in presence (+) or absence ( −) of 1 mM IPTG and/or 0.02% (w/v) arabinose. (c) The indicated P. putida KT2440 strains bearing a tke2-V5-tagged gene were grown in tryptone soya broth (TSB) medium for 5 h. Tke2-V5 was detected in the whole cell and supernatant fractions using a monoclonal anti-V5 antibody. Detection of the β-subunit of the RNA polymerase (β- RNAP) d t l Th iti f th l l i 980 EI pairs including Tke2/Tki2 (Figures 1c, 4a and 5a, and Supplementary Table S4). To assess the func- tionality of this EI pair, the tke2 and tki2 genes were cloned into compatible plasmids and transformed into E. coli K12. Expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (tke2) or arabinose (tki2). On induction of the effector gene tke2, E. coli growth was significantly impaired (Figures 6a and b) but growth could readily be rescued on co-expression of the putative Tki2 immunity protein (Figures 6a and b). This suggests that Tke2/Tki2 is a genuine EI pair. We assessed whether Tke2 is secreted in a K1- T6SS-dependent manner. The corresponding gene was replaced on the KT2440 chromosome with a version encoding a C-terminally V5-tagged protein. However, Tke2-V5 production was only weakly detected when using this strain (Supplementary Figure S3). In contrast to bacterial killing, which is a highly sensitive assay, detection of secreted T6SS toxins by western blot may need higher level of T6SS expression (Cianfanelli et al., 2016). Tke2/Tki2 is a P. putida K1-T6SS effector/immunity pair It has been described in other bacteria that several global regulators are involved in T6SS expression, including the alternative sigma factor RpoN (Bernard et al., 2010, 2011; Sana et al., 2013). The tke2-V5 chimeric gene was introduced into an rpoN mutant and in this strain Tke2 production was considerably increased as compared with the wild-type P. putida (Supplementary Figure S3). We thus used this genetic background to analyze Tke2 secretion. Tke2 was produced in both the rpoN strain and the isogenic T6SS mutant (rpoNΔtssA1), but was only found in the supernatant of the strain with an intact T6SS (Figure 6c). Our results show that Tke2 is an effector of the K1-T6SS and its activity is antago- nized by the Tki2 immunity protein. P. putida outcompetes plant pathogens in a T6SS-dependent manner P. putida is an efficient biocontrol agent (Amer and Utkhede, 2000; Validov et al., 2007) and we hypothesized that it uses the T6SS to kill ecologi- cally relevant competitors. To test this we selected four plant pathogens, P. syringae, X. campestris, Pectobacterium carotovorum and Agrobacterium tumefaciens, which are leading causes of deadly diseases in several economically important crops (Mansfield et al., 2012). The various T6SSs are likely to be differentially expressed in vitro, in vivo, in planta or in the presence of different competitors (Ma et al., 2014). To assess whether the T6SS in general is required for outcompeting plant pathogens and thus involve in plant protection we used a triple T6SS mutant (ΔtssA1ΔtssM2ΔtssM3, also named ΔT6SS), so that none of the K1, K2 or K3 system is at play. First, a competition assay between KT2440 or the triple mutant and the phytopathogens was performed. The P. putida wild-type strain caused a 10-fold decrease in survival of A. tumefaciens and Figure 6 Toxicity and secretion of the Tke2 effector. (a) The growth of E. coli K12 cells harboring the pTke2-CT and pTki2 plasmids containing the C-terminal toxin domain of the tke2 effector and the tki2 immunity genes, respectively, was deter- mined by measuring the OD at 600 nm. At time zero, either 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and/or 0.02% (w/v) arabinose were added to the LB medium, to induce expression of the tke2-CT or/and tki2 genes, respectively. (b) Western blot analyses using an anti-V5 or anti-HA mono- clonal antibody to detect the Tke2-CT-V5 or Tki2-HA-tagged proteins. Proteins were prepared from E. Tke2/Tki2 is a P. putida K1-T6SS effector/immunity pair coli K12 cells grown during 10 h in presence (+) or absence ( −) of 1 mM IPTG and/or 0.02% (w/v) arabinose. (c) The indicated P. putida KT2440 strains bearing a tke2-V5-tagged gene were grown in tryptone soya broth (TSB) medium for 5 h. Tke2-V5 was detected in the whole cell and supernatant fractions using a monoclonal anti-V5 antibody. Detection of the β-subunit of the RNA polymerase (β- RNAP) was used as control. The position of the molecular size marker (in kDa) is indicated. Figure 6 Toxicity and secretion of the Tke2 effector. (a) The growth of E. coli K12 cells harboring the pTke2-CT and pTki2 plasmids containing the C-terminal toxin domain of the tke2 effector and the tki2 immunity genes, respectively, was deter- mined by measuring the OD at 600 nm. At time zero, either 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and/or 0.02% (w/v) arabinose were added to the LB medium, to induce expression of the tke2-CT or/and tki2 genes, respectively. (b) Western blot analyses using an anti-V5 or anti-HA mono- clonal antibody to detect the Tke2-CT-V5 or Tki2-HA-tagged proteins. Proteins were prepared from E. coli K12 cells grown during 10 h in presence (+) or absence ( −) of 1 mM IPTG and/or 0.02% (w/v) arabinose. (c) The indicated P. putida KT2440 strains bearing a tke2-V5-tagged gene were grown in tryptone soya broth (TSB) medium for 5 h. Tke2-V5 was detected in the whole cell and supernatant fractions using a monoclonal anti-V5 antibody. Detection of the β-subunit of the RNA polymerase (β- RNAP) was used as control. The position of the molecular size marker (in kDa) is indicated. The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al P. putida T6SS is a biocontrol weapon P Bernal et al 981 Target cells Survival (Log10 CFU) 4 5 6 7 Target KT2440 ∆T6SS KT2440 ∆T6SS - - - - KT2440 ∆T6SS KT2440 ∆T6SS Attacker P.putida X. campestris A. tumefaciens P. carotovorum P. syringae Figure 7 Bactericidal activity of P. putida KT2440 against a panel of phytopathogens. X. campestris, A. tumefaciens, P. carotovorum and P. syringae pv. tomato strains harbor the pRL662-gfp plasmid that confers gentamycin resistance. The P. putida KT2440 wild type (WT) and its isogenic ΔtssA1ΔtssM2ΔtssM3 triple mutant (ΔT6SS) were co-incubated with the phytopathogens for 24 h. Colony-forming unit (CFU) quantifications were performed on gentamycin selection. The average ± s.d. Tke2/Tki2 is a P. putida K1-T6SS effector/immunity pair from at least three biological replicates is plotted. P. caratovorum, and a 1000-fold decrease in the survival of X. campestris and P. syringae (Figure 7). The P. putida T6SS mutant had barely any impact on the survival of any of these bacteria (Figure 7). Our results indicate that KT2440 outcompetes all chal- lenged phytopathogens in a T6SS-dependent man- ner and suggest a role for this secretion system in biocontrol. P. caratovorum, and a 1000-fold decrease in the survival of X. campestris and P. syringae (Figure 7). The P. putida T6SS mutant had barely any impact on the survival of any of these bacteria (Figure 7). Our results indicate that KT2440 outcompetes all chal- lenged phytopathogens in a T6SS-dependent man- ner and suggest a role for this secretion system in biocontrol. T6SS-active P. putida protects plants from pathogen’s attack To assess the ability of P. putida to kill phytopatho- gens in an ecologically relevant set-up, we devel- oped an in planta competition assay. We selected X. campestris as the pathogen and N. benthamiana as the plant model. Leaves were co-infected with X. campestris and either P. putida wild type or the isogenic ΔT6SS mutant. X. campestris was tagged with a green fluorescent protein to monitor in situ colonization. X. campestris-induced halos of necro- sis on the leaves were observed 5 days post infection, whereas inoculation with P. putida resulted in healthy-looking leaves (Figure 8a). Remarkably, co- infiltration of X. campestris and P. putida wild-type strain considerably reduced the necrotic areas produced by X. campestris (circled in Figure 8b lower panel). This is not observable with the P. putida ΔT6SS mutant and we concluded that interference with X. campestris colonization is T6SS dependent (Figure 8b). The protection conferred by P. putida is due to reduced survival of X. campestris in the leaves (~2.5-fold reduction), as qualitatively observed by fluorescence microscopy (Figure 8b upper panel) and quantitatively measured by colony-forming unit counting (Figure 8c). Our results show that P. putida outcompetes X. campestris during plant colonization and this process involves the bactericidal properties of the T6SS. Figure 7 Bactericidal activity of P. putida KT2440 against a panel of phytopathogens. X. campestris, A. tumefaciens, P. carotovorum and P. syringae pv. tomato strains harbor the pRL662-gfp plasmid that confers gentamycin resistance. The P. putida KT2440 wild type (WT) and its isogenic ΔtssA1ΔtssM2ΔtssM3 triple mutant (ΔT6SS) were co-incubated with the phytopathogens for 24 h. Tke2/Tki2 is a P. putida K1-T6SS effector/immunity pair Alterna- tively, related VgrGs (that is, VgrG3, VgrG4 and VgrG5; Supplementary Figure S4) and TssC proteins (that is, TssC3; Supplementary Table S5) could be shared between different T6SSs. Tke2/Tki2 is a P. putida K1-T6SS effector/immunity pair Colony-forming unit (CFU) quantifications were performed on gentamycin selection. The average ± s.d. from at least three biological replicates is plotted. this secretion machine has an important role in P. putida physiology and fitness. The P. putida T6SS clusters clade within three phylogenetic groups, group 1.2, 2 or 4B (Figure 2). Remarkably, P. putida is the only Pseudomonas species encoding T6SSs from group 1.2, whereas T6SSs from group 4B are only present in P. putida and P. syringae (Barret et al., 2011). The P. putida KT2440 strain contains two clusters from group 1.2 (K2 and K3) and one cluster from group 4B (K1). The K2-T6SS cluster contains two orfs, vgrG2 and tssC2, which present premature stop codons (Supplementary Table S5), implying that this system is not functional. Prema- turely interrupted T6SS genes have been identified in functional T6SSs of Citrobacter rodentium and Yersinia pseudotuberculosis (Gueguen et al., 2014). In these cases, a transcriptional frameshifting caused by a poly-A tract allows the production of functional TssM variants (Gueguen et al., 2014). However, this is unlikely to be the case in KT2440, as poly-A tracts are not found either in tssC2 or in vgrG2. Alterna- tively, related VgrGs (that is, VgrG3, VgrG4 and VgrG5; Supplementary Figure S4) and TssC proteins (that is, TssC3; Supplementary Table S5) could be shared between different T6SSs. this secretion machine has an important role in P. putida physiology and fitness. The P. putida T6SS clusters clade within three phylogenetic groups, group 1.2, 2 or 4B (Figure 2). Remarkably, P. putida is the only Pseudomonas species encoding T6SSs from group 1.2, whereas T6SSs from group 4B are only present in P. putida and P. syringae (Barret et al., 2011). The P. putida KT2440 strain contains two clusters from group 1.2 (K2 and K3) and one cluster from group 4B (K1). The K2-T6SS cluster contains two orfs, vgrG2 and tssC2, which present premature stop codons (Supplementary Table S5), implying that this system is not functional. Prema- turely interrupted T6SS genes have been identified in functional T6SSs of Citrobacter rodentium and Yersinia pseudotuberculosis (Gueguen et al., 2014). In these cases, a transcriptional frameshifting caused by a poly-A tract allows the production of functional TssM variants (Gueguen et al., 2014). However, this is unlikely to be the case in KT2440, as poly-A tracts are not found either in tssC2 or in vgrG2. Discussion The T6SS was discovered in the bacterial pathogens V. cholerae (Pukatzki et al., 2006) and P. aeruginosa (Mougous et al., 2006). Since then, an increasing number of studies has provided details on the function and structure of this original bacterial secretion system (Russell et al., 2014; Zoued et al., 2014; Cianfanelli, et al., 2016; Hachani et al., 2016). However, although the presence of the T6SS in non- pathogenic strains is evident (Boyer et al., 2009), little work has been done to understand its relevance in this category of bacteria (Bladergroen et al., 2003; Mougous et al., 2006; Pukatzki et al., 2006; Aschtgen et al., 2008). The K2 and K3 clusters do not encode a ClpV protein, the ATPase responsible for disassembling the T6SS sheath. Yet, orphan clpV genes can be used. There are three Clp ATPase-encoding genes in Phylogeny and genetic structure of the P. putida T6SS clusters Phylogeny and genetic structure of the P. putida T6SS clusters Phylogeny and genetic structure of the P. putida T6SS clusters In this study we have identified a total of 66 T6SS clusters among P. putida strains, which suggests that The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al re 8 In planta competition assay between the biocontrol strain P. putida KT2440 and the phytopathogen X. campestris. (a enthamiana 24 h (upper panel) and 5 days (lower panel) after being infiltrated with X. campestris (pRL662-gfp; expressing ded green fluorescence protein), the P. putida KT2440 wild type (WT), or its isogenic ΔtssA1ΔtssM2ΔtssM3 triple mutan eaves of N. benthamiana 24 h (upper panel) and 5 days (lower panel) after co-infiltration of X. campestris (pRL662-gfp ated P. putida strain. In upper panel a and b, the leaves were visualized by fluorescence microscopy using a Leic omicroscope. The necrotic areas resulting from X. campestris infection are marked. The deep brown zone of necrosis is s portion of the leave (right panel), while such spread is far more restricted when the phytopathogen is co-inoculated w ive P. putida strain (left panel). (c) Quantification of X. campestris (pRL662-gfp) colony-forming units (CFUs) recov enthamiana leaves after 24 h of co-infiltration with the indicated P. putida strain. X. campestris CFU were quantified after g selection. Graphs represent mean +s.d., of at least five biological replicates with two technical replicates per experiment ficance is indicated t-test Po0.001. Discussion Instead, recently discovered phage-like protein translocation structures are encoded within gene clusters that also carry a clpV homolog (Kube and Wendler, 2015). This type of structure may have evolved divergently with some of the T6SS subgroups and acquired ClpV from ancestral systems. adaptors has not been analyzed yet, it is possible that both function together to assist Tke2 secretion. The recurrent association between PAAR/Rhs effectors and EagR adaptors is furthermore con- firmed by the association of tke4, encoding another P. putida PAAR/Rhs effector (Figures 1c and 4a, and Supplementary Figure S6), with an eagR gene (eagR2). Biocontrol properties of the P. putida T6SS It is becoming increasingly obvious that the anti- microbial properties of the T6SS could be instru- mental for the control of polymicrobial populations in excluding foes from natural and ecologically relevant environments. For instance, a clear correla- tion between activation of T6SS, enhanced fitness and subsequent antagonism against other bacteria has been observed with Vibrio parahaemolyticus in marine niches (Salomon et al., 2013). This suggested that T6SSs are key for survival and persistence of specialized species in specific habitats. In the lungs of cystic fibrosis patients, P. aeruginosa can persist for years, while the diversity of species that primarily colonizes this environment decreases over time (Marshall et al., 2015). P. aeruginosa isolates from cystic fibrosis patients have highly active T6SSs (Mougous et al., 2006; Moscoso et al., 2011), which suggests that T6SSs contribute to the coloni- zation advantage of P. aeruginosa over other species. In agreement with these observations, the T6SS has been proposed to be crucial in the establishment/ evolution of the gut microbiome (Russell, et al., 2014; Cianfanelli, et al., 2016). Half of the human- associated Bacteroidetes, the dominant phyla in the human gut, not only encode T6SSs (Coyne et al., 2016) and possess a wide range of T6SS effectors (Chatzidaki-Livanis et al., 2016) but accumulate immunity genes against other T6SS effectors (Wexler et al., 2016). This strongly supports that T6SS is a selective mechanism involved in the establishment of gut communities. These remarkable properties of the T6SS are obviously useful in the development of biocontrol strains. The T6SS was originally discovered in Rhizobium leguminosarum and involved in pea nodulation (Bladergroen et al., 2003), but barely any studies have demonstrated the potential that such system may have in the context of the plant microbiome. Discussion P Bernal et al 982 Figure 8 In planta competition assay between the biocontrol strain P. putida KT2440 and the phytopathogen X. campestris. (a) Leaves of N. benthamiana 24 h (upper panel) and 5 days (lower panel) after being infiltrated with X. campestris (pRL662-gfp; expressing a plasmid- encoded green fluorescence protein), the P. putida KT2440 wild type (WT), or its isogenic ΔtssA1ΔtssM2ΔtssM3 triple mutant (ΔT6SS). (b) Leaves of N. benthamiana 24 h (upper panel) and 5 days (lower panel) after co-infiltration of X. campestris (pRL662-gfp) with the indicated P. putida strain. In upper panel a and b, the leaves were visualized by fluorescence microscopy using a Leica M205FA stereomicroscope. The necrotic areas resulting from X. campestris infection are marked. The deep brown zone of necrosis is spread on a large portion of the leave (right panel), while such spread is far more restricted when the phytopathogen is co-inoculated with a T6SS positive P. putida strain (left panel). (c) Quantification of X. campestris (pRL662-gfp) colony-forming units (CFUs) recovered from N. benthamiana leaves after 24 h of co-infiltration with the indicated P. putida strain. X. campestris CFU were quantified after gentamycin (Gm) selection. Graphs represent mean +s.d., of at least five biological replicates with two technical replicates per experiment, statistical significance is indicated t-test Po0.001. The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al 983 the KT2440 genome (that is, PP0625, PP3316 and PP4008), but none encodes a protein from the ClpV family (Supplementary Figure S5). They are ClpA and ClpB members, which are phylogenetically distant from ClpV (Schlieker et al., 2005). Alterna- tively, the ClpV1 component within the K1 cluster could be shared between the systems but possibly a ClpV component may not be necessary for the function of the group 1.2 T6SS, as some P. putida strains (that is, S12, B001, SJ3 and S610) exclusively contain a group 1.2 cluster (Supplementary Table S3). In fact, functional T6SSs lacking the clpV gene have been identified in other bacteria (Chow and Mazmanian, 2010; Bröms et al., 2012). Furthermore, the clpV gene of V. cholerae can be deleted without a total loss of T6SS function (Zheng et al., 2011). After all, other nanomachines structurally comparable to the T6SS such as the contractile-tailed phages or R-type pyocins, do not use a ClpV homolog for their function. Discussion A parallel can be made between the gut and the rhizosphere, as both are eukaryotic-based environments hosting a symbiotic relationship with a complex microbial community (Stone, 2016). Both animals and plants depend on their microbiome to protect themselves against pathogens and to help assimilate necessary nutrients (Carmody et al., 2015; Haney and Ausubel, 2015; Haney et al., 2015). As a defence strategy, many plant species promote the development of a specific microbiome in the rhizosphere, which has antag- onistic activity against soil-borne pathogens (Cook et al., 1995; Weller et al., 2002; Lebeis et al., 2015). Antibacterial activity of the P. putida KT2440 T6SS The main role of the T6SS is to inject effectors into eukaryotic or prokaryotic prey cells (Alcoforado Diniz et al., 2015; Hachani et al., 2016). We identified an impressive battery of 10 potential T6SS effectors in P. putida KT2440. This is not unique but suggests that P. putida is primed to fight a wide range of competing organisms. At least three EI pairs are encoded within the K1-T6SS cluster (that is, tke1-tki1, tke2-tki2 and tke3-tki3), which belongs to the uncharacterized plant-related group (group 4B, Figure 2). A remarkable characteristic of the system is the presence of a conserved accessory gene, tagX, systematically absent from other T6SS groups and which is a hallmark for group 4B systems. Here we show that suitable preys for the K1-T6SS are bacterial cells, and that the Tke2 toxin contributes to the antibacterial activity. Tke2 contains a canoni- cal Rhs-effector domain organization, which includes an N-terminal PAAR motif, a central domain of conserved Rhs-repeats and a C-terminal toxic domain. Although the function of the Rhs domain is still unknown, it has been suggested that it forms a shell structure that encapsulates the C-term- inal region of effectors (Busby et al., 2013; Supplementary Figure S6). Furthermore, a specific adaptor named EagR (after ‘effector-associated gene’) that contains the DUF1795 domain, has been involved in the secretion of PAAR/Rhs effectors (Alcoforado Diniz and Coulthurst, 2015). Two different proteins containing DUF1795 domains are encoded immediately upstream tke2 (eagR1a and eagR1b, Figure 1c). Although the function of these The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al 984 Although the mechanisms for pathogen suppression are not completely understood, they include the production of bioactive metabolites such as anti- biotics, bacteriocins and siderophores (Weller, 2007). Discussion However, these mechanisms fail to account for the full level of protection conferred by the biocontrol organism (Matilla et al., 2010). Here we report for the first time that the T6SS might be a primary mechanism for phytopathogen control. Indeed, we demonstrate that the crop protection agent P. putida KT2440 readily outcompetes a panel of economically important phytopathogens and that the efficient destruction of the pest is mostly T6SS dependent. This property can likely be transferred to the field, as this effect was observed in vitro but also in vivo by demonstrating that P. putida protects plant leaves from the deleterious effect of X. campestris. effector-associated accessory protein. J Bacteriol 197: 2350–2360. Alcoforado Diniz J, Liu Y-CC, Coulthurst SJ. (2015). Molecular weaponry: diverse effectors delivered by the Type VI secretion system. Cell Microbiol 17: 1742–1751. Amer GA, Utkhede RS. (2000). Development of formula- tions of biological agents for management of root rot of lettuce and cucumber. Can J Microbiol 46: 809–816. Aschtgen M-SS, Bernard CS, De Bentzmann S, Lloubès R, Cascales E. (2008). SciN is an outer membrane lipoprotein required for type VI secretion in enteroag- gregative Escherichia coli. J Bacteriol 190: 7523–7531. Aschtgen M-SS, Thomas MS, Cascales E. (2010). Anchor- ing the type VI secretion system to the peptidoglycan: TssL, TagL, TagP... what else? Virulence 1: 535–540. Barret M, Egan F, Fargier E, Morrissey JP, O’Gara F. (2011). Genomic analysis of the type VI secretion systems in Pseudomonas spp.: novel clusters and putative effec- tors uncovered. Microbiology 157: 1726–1739. p In our study we have used a laboratory setup and further trials in crop plants are needed so that in- depth investigation of the impact of KT2440 in the rhizosphere can be assessed. Nevertheless, our finding shows that the T6SS can be used by environmental strains to protect plants from the attack of bacterial pathogens and can thus be considered as a plant health warden. This opens new possibilities in the selection of biocontrol agents used for biotechnological applications. Noticeably, the poor specificity of the T6SS (Hood et al., 2010) may allow such biocontrol organism to also fight eukaryotic pathogens belonging to different king- doms including nematodes and fungi. gy Basler M, Ho BT, Mekalanos JJ. (2013). Tit-for-tat: type VI secretion system counterattack during bacterial cell- cell interactions. Cell 152: 884–894. Basler M, Mekalanos JJ. (2012). Type 6 secretion dynamics within and between bacterial cells. Science 337: 815. The authors declare no conflict of interest. The authors declare no conflict of interest. Acknowledgements Boyer F, Fichant G, Berthod J, Vandenbrouck Y, Attree I. (2009). Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics 10: 104. We thank Milagros Lopez (IVIA, Spain) for providing X. campestris and P. carotovorum, Martin Buck (Imperial College London, UK) for P. syringae and Ehr Min Lai (Academia Sinica, Taiwan) for A. tumefaciens and the pRL662-gpf. We thank Tom Wood for kindly providing the T6SS scheme shown in Figure 1a. PB is supported by the Spanish Ministry of Economy through Juan de la Cierva grant (JCI-2010-06615), by the Andalusian Knowledge Agency through a Talent Hub grant (TAHUB-010) and by an EMBO short-term fellowship. MAL is supported by the Spanish Ministry of Economy through a Ramon&Cajal grant (RYC2011-08874). AF is supported by a BBSRC grant (BB/N002539/1). LA is supported by a BBSRC grant (BB/ N002539/1) and a Marie curie Fellowship (PIIF-GA-2012- 328261). Bröms JE, Meyer L, Sun K, Lavander M, Sjöstedt A. (2012). Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacroph- age infection. PLoS ONE 7: e50473. Burtnick MN, Brett PJ, Harding SV, Ngugi SA, Ribot WJ, Chantratita N et al. (2011). The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei. Infect Immun 79: 1512–1525. Busby JN, Panjikar S, Landsberg MJ, Hurst MR, Lott JS. (2013). The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device. Nature 501: 547–550. Carmody RN, Gerber GK, Luevano JM, Gatti DM, Somes L, Svenson KL et al. (2015). Diet dominates host genotype in shaping the murine gut microbiota. Cell Host Microbe 17: 72–84. Discussion Bernard CS, Brunet YR, Gavioli M, Lloubès R, Cascales E. (2011). Regulation of type VI secretion gene clusters by sigma54 and cognate enhancer binding proteins. J Bacteriol 193: 2158–2167. Bernard CS, Brunet YR, Gueguen E, Cascales E. (2010). Nooks and crannies in type VI secretion regulation. J Bacteriol 192: 3850–3860. Bladergroen MR, Badelt K, Spaink HP. (2003). Infection- blocking genes of a symbiotic Rhizobium legumino- sarum strain that are involved in temperature-dependent protein secretion. Mol Plant Microbe Interact 16: 53–64. Conflict of Interest Boratyn GM, Camacho C, Cooper PS, Coulouris G, Fong A, Ma N et al. (2013). BLAST: a more efficient report with usability improvements. Nucleic Acids Res 41: W29–W33. References p Haney CH, Samuel BS, Bush J, Ausubel FM. (2015). Associations with rhizosphere bacteria can confer an adaptive advantage to plants. Nat Plants 1: 15051. Coyne MJ, Roelofs KG, Comstock LE. (2016). Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements. BMC Genomics 17: 58. p g p Ho B, Dong T, Mekalanos J. (2013). A view to a kill: the bacterial type VI secretion system. Cell Host Microbe 15: 9–21. de Pace F, Nakazato G, Pacheco A, de Paiva JB, Sperandio V, da Silveira WD. (2010). The type VI secretion system plays a role in type 1 fimbria expression and pathogenesis of an avian pathogenic Escherichia coli strain. Infect Immun 78: 4990–4998. Hoang TT, Kutchma AJ, Becher A, Schweizer HP. (2000). Integration-proficient plasmids for Pseudomonas aeru- ginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 43: 59–72. Hood R, Singh P, Hsu F, Güvener T, Carl M, Trinidad R et al. (2010). A type VI secretion system of Pseudomo- nas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7: 25–37. f Dong TG, Ho BT, Yoder-Himes DR, Mekalanos JJ. (2013). Identification of T6SS-dependent effector and immu- nity proteins by Tn-seq in Vibrio cholerae. Proc Natl Acad Sci USA 110: 2623–2628. Huang H, Yuan HS. (2007). The conserved asparagine in the HNH motif serves an important structural role in metal finger endonucleases. J Mol Biol 368: 812–821. Durand E, Cambillau C, Cascales E, Journet L. (2014). VgrG, Tae, Tle, and beyond: the versatile arsenal of type VI secretion effectors. Trends Microbiol 22: 498–507. Imai K, Asakawa N, Tsuji T, Akazawa F, Ino A, Sonoyama M et al. (2008). SOSUI-GramN: high performance predic- tion for sub-cellular localization of proteins in gram- negative bacteria. Bioinformation 2: 417–421. Durand E, Nguyen VS, Zoued A, Logger L, Péhau-Arnaudet G, Aschtgen M-SS et al. (2015). Biogenesis and structure of a type VI secretion membrane core complex. Nature 523: 555–560. Kaniga K, Delor I, Cornelis GR. (1991). A wide-host-range suicide vector for improving reverse genetics in gram- negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109: 137–141. Espinosa-Urgel M, Salido A, Ramos JL. (2000). Genetic analysis of functions involved in adhesion of Pseudo- monas putida to seeds. J Bacteriol 182: 2363–2369. Kapitein N, Bönemann G, Pietrosiuk A, Seyffer F, Hausser I, Locker JK et al. References Alcoforado Diniz J, Coulthurst SJ. (2015). Intraspecies competition in serratia marcescens is mediated by type VI-secreted Rhs effectors and a conserved Cascales E, Cambillau C. (2012). Structural biology of type VI secretion systems. Philos Trans R Soc Lond B Biol Sci 367: 1102–1111. The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al 985 rodentium type VI secretion by the production of two length variants from the prematurely interrupted tssM gene. PLoS Genet 10: e1004869. Chatzidaki-Livanis M, Geva-Zatorsky N, Comstock LE. (2016). Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species. Proc Natl Acad Sci USA 113: 3627–3632. g Guzman LM, Belin D, Carson MJ, Beckwith J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177: 4121–4130. Choi K-HH, Kumar A, Schweizer HP. (2006). A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64: 391–397. Hachani A, Allsopp LP, Oduko Y, Filloux A. (2014). The VgrG proteins are ‘A la carte’ delivery systems for bacterial type VI effectors. J Biol Chem 289: 17872–17884. Chow J, Mazmanian SK. (2010). A pathobiont of the microbiota balances host colonization and intestinal inflammation. Cell Host Microbe 7: 265–276. Hachani A, Lossi N, Hamilton A, Jones C, Bleves S, Albesa-Jové D et al. (2011). Type VI secretion system in Pseudomonas aeruginosa secretion and multi- merization of VgrG proteins. J Biol Chem 286: 12317–12327. Cianfanelli FR, Alcoforado Diniz J, Guo M, De Cesare V, Trost M, Coulthurst SJ. (2016). VgrG and PAAR proteins define distinct versions of a functional type VI secretion system. PLoS Pathog 12: e1005735. Hachani A, Lossi NS, Filloux A. (2013). A visual assay to monitor T6SS-mediated bacterial competition. J Vis Exp 73: e50103. Cianfanelli FR, Monlezun L, Coulthurst SJ. (2016). Aim, load, fire: the type VI secretion system, a bacterial nanoweapon. Trends Microbiol 24: 51–62. p Hachani A, Wood TE, Filloux A. (2016). Type VI secretion and anti-host effectors. Curr Opin Microbiol 29: 81–93. Cook RJ, Thomashow LS, Weller DM, Fujimoto D, Mazzola M, Bangera G et al. (1995). Molecular mechanisms of defense by rhizobacteria against root disease. Proc Natl Acad Sci USA 92: 4197–4201. p Haney CH, Ausubel FM. (2015). MICROBIOME. Plant microbiome blueprints. Science 349: 788–789. References A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312: 1526–1530. Lebeis SL, Paredes SH, Lundberg DS, Breakfield N, Gehring J, McDonald M et al. (2015). PLANT MICRO- BIOME. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa. Science 349: 860–864. Murdoch SL, Trunk K, English G, Fritsch MJ, Pourkarimi E, Coulthurst SJ. (2011). The opportunistic pathogen Serratia marcescens utilizes type VI secretion to target bacterial competitors. J Bacteriol 193: 6057–6069. Leiman PG, Basler M, Ramagopal UA, Bonanno JB, Sauder JM, Pukatzki S et al. (2009). Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc Natl Acad Sci USA 106: 4154–4159. Okonechnikov K, Golosova O, Fursov M. (2012). Unipro UGENE: a unified bioinformatics toolkit. Bioinfor- matics 28: 1166–1167. Petersen TN, Brunak S, von Heijne G, Nielsen H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8: 785–786. Letunic I, Doerks T, Bork P. (2015). SMART: recent updates, new developments and status in 2015. Nucleic Acids Res 43: D257–D260. Planamente S, Salih O, Manoli E, Albesa-Jové D, Freemont PS, Filloux A. (2016). TssA forms a gp6-like ring attached to the type VI secretion sheath. EMBO J 35: 1613–1627. Liang X, Moore R, Wilton M, Wong MJ, Lam L, Dong TG. (2015). Identification of divergent type VI secretion effectors using a conserved chaperone domain. Proc Natl Acad Sci USA 112: 9106–9111. yp Pukatzki S, Ma AT, Sturtevant D, Krastins B, Sarracino D, Nelson WC et al. (2006). Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci USA 103: 1528–1533. Lin J-SS, Ma L-SS, Lai E-MM. (2013). Systematic dissection of the agrobacterium type VI secretion system reveals machinery and secreted components for subcomplex formation. PLoS One 8: e67647. Ramos-Gonzalez MI, Duque E, Ramos JL. (1991). Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids. Appl Environ Microbiol 57: 3020–3027. Lyons E, Freeling M. (2008). How to usefully compare homologous plant genes and chromosomes as DNA sequences. Plant J 53: 661–673. Ma AT, Mekalanos JJ. (2010). In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation. Proc Natl Acad Sci USA 107: 4365–4370. Rosales-Reyes R, Skeldon AM, Aubert DF, Valvano MA. (2012). References The type VI secretion system of Burkholderia cenocepacia affects multiple Rho family GTPases disrupting the actin cytoskeleton and the assembly of NADPH oxidase complex in macrophages. Cell Microbiol 14: 255–273. Ma L-SS, Hachani A, Lin J-SS, Filloux A, Lai E-MM. (2014). Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host Microbe 16: 94–104. Russell AB, Peterson SB, Mougous JD. (2014). Type VI secretion system effectors: poisons with a purpose. Nat Rev Microbiol 12: 137–148. Russell AB, Wexler AG, Harding BN, Whitney JC, Bohn AJ, Goo YA et al. (2014). A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism. Cell Host Microbe 16: 227–236. Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P et al. (2012). Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13: 614–629. Marchi M, Boutin M, Gazengel K, Rispe C, Gauthier J-PP, Guillerm-Erckelboudt A-YY et al. (2013). Genomic analysis of the biocontrol strain Pseudomonas fluor- escens Pf29Arp with evidence of T3SS and T6SS gene expression on plant roots. Environ Microbiol Rep 5: 393–403. Salomon D, Gonzalez H, Updegraff BL, Orth K. (2013). Vibrio parahaemolyticus type VI secretion system 1 is activated in marine conditions to target bacteria, and is differentially regulated from system 2. PLoS One 8: e61086. Salomon D, Kinch LN, Trudgian DC, Guo X, Klimko JA, Grishin NV et al. (2014). Marker for type VI secretion system effectors. Proc Natl Acad Sci USA 111: 9271–9276. Marshall B, Elbert A, Petren K, Rizvi S, Fink A, Ostrenga J et al. (2015). Cystic Fibrosis Foundation Patient Registry 2014 Annual Data Report. 2015 Cystic Fibrosis Foundation: Bethesda, Maryland, USA. Sambrook J, Maniatis T, Fritsch EF. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory: Cold Spring Harbor, NY. Matilla M, Ramos J, Bakker P, Doornbos R, Badri D, Vivanco J et al. (2010). Pseudomonas putida KT2440 causes induced systemic resistance and changes in Arabidopsis root exudation. Environ Microbiol Rep 2: 381–388. Sana TG, Soscia C, Tonglet CMM, Garvis S, Bleves S. (2013). Divergent control of two type VI secretion systems by RpoN in Pseudomonas aeruginosa. PLoS One 8: e76030. Schlieker C, Zentgraf H, Dersch P, Mogk A. (2005). ClpV, a unique Hsp100/Clp member of pathogenic proteobac- teria. Biol Chem 386: 1115–1127. Miyata ST, Kitaoka M, Brooks TM, McAuley SB, Pukatzki S. (2011). References (2013). ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion. Mol Microbiol 87: 1013–1028. p Filloux A. (2011). Protein secretion systems in Pseudomo- nas aeruginosa: an essay on diversity, evolution, and function. Front Microbiol 2: 155. Filloux A, Freemont P. (2016). Structural biology: baseplates in contractile machines. Nat Microbiol 1: 16104. Katzen F, Ferreiro DU, Oddo CG, Ielmini MV, Becker A, Pühler A et al. (1998). Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynth- esis and plant virulence. J Bacteriol 180: 1607–1617. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL et al. (2016). The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44: D279–D285. p Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10: 845–858. Gomi M, Sonoyama M, Mitaku S. (2004). High perfor- mance system for signal peptide prediction: SOSUI- signal. Chem Bio Info J 4: 142–147. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567–580. g f Gotfredsen M, Gerdes K. (1998). The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family. Mol Microbiol 29: 1065–1076. Kube S, Kapitein N, Zimniak T, Herzog F, Mogk A, Wendler P. (2014). Structure of the VipA/B type VI Gueguen E, Wills NM, Atkins JF, Cascales E. (2014). Transcriptional frameshifting rescues Citrobacter The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al P. putida T6SS is a biocontrol weapon P B l t l 986 secretion complex suggests a contraction-state-specific recycling mechanism. Cell Rep 8: 20–30. greenhouse and environmental conditions. Soil Biol Biochem 32: 315–321. secretion complex suggests a contraction-state-specific recycling mechanism. Cell Rep 8: 20–30. Moscoso JA, Mikkelsen H, Heeb S, Williams P, Filloux A. (2011). The Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling. Environ Microbiol 13: 3128–3138. y g p Kube S, Wendler P. (2015). Structural comparison of contractile nanomachines. AIMS Biophys 2: 88–115. Kudryashev M, Wang RY, Brackmann M, Scherer S, Maier T, Baker D et al. (2015). Structure of the type VI secretion system contractile sheath. Cell 160: 952–962. Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M, Gifford CA et al. (2006). References Vibrio cholerae requires the type VI secretion system virulence factor VasX to kill Dictyostelium discoideum. Infect Immun 79: 2941–2949. Shneider MM, Buth SA, Ho BT, Basler M, Mekalanos JJ, Leiman PG. (2013). PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500: 350–353. f Molina L, Ramos C, Duque E, Ronchel MC, Garcia JM, Wyke L et al. (2000). Survival of Pseudomonas putida KT2440 in soil and in the rhizosphere of plants under The ISME Journal P. putida T6SS is a biocontrol weapon P Bernal et al 987 inject and neutralize antibacterial toxins to per- sist in the gut. Proc Natl Acad Sci USA 113: 3639–3644. Silverman JM, Agnello DM, Zheng H, Andrews BT, Li M, Catalano CE et al. (2013). Haemolysin coregulated protein is an exported receptor and chaperone of type VI secretion substrates. Mol Cell 51: 584–593. Whitney JC, Beck CM, Goo YA, Russell AB, Harding BN, De Leon JA et al. (2014). Genetically distinct pathways guide effector export through the type VI secretion system. Mol Microbiol 92: 529–542. Silverman JM, Austin LS, Hsu F, Hicks KG, Hood RD, Mougous JD. (2011). Separate inputs modulate phosphorylation-dependent and -independent type VI secretion activation. Mol Microbiol 82: 1277–1290. Whitney JC, Quentin D, Sawai S, LeRoux M, Harding BN, Ledvina HE et al. (2015). An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells. Cell 163: 607–619. Stone M. (2016). Root and gut microbiomes are strikingly similar. Microbe 11: 107–110. Suarez G, Sierra JC, Sha J, Wang S, Erova TE, Fadl AA et al. (2008). Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila. Microb Pathog 44: 344–361. Winsor GL, Griffiths EJ, Lo R, Dhillon BK, Shay JA, Brinkman FS. (2016). Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res 44: D646–D653. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. (2013). MEGA6: molecular evolutionary genetics ana- lysis version 6.0. Mol Biol Evol 30: 2725–2729. Ö Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R et al. (2010). PSORTb 3.0: improved protein subcellular localization prediction with refined localization sub- categories and predictive capabilities for all prokar- yotes. Bioinformatics 26: 1608–1615. Unterweger D, Kostiuk B, Ötjengerdes R, Wilton A, Diaz-Satizabal L, Pukatzki S. (2015). n accompanies this paper on The ISME Journal website (http://www.nature.com/ismej) References Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae. EMBO J 34: 2198–2210. y f Zheng J, Ho B, Mekalanos JJ. (2011). Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae. PLoS One 6: e23876. Validov S, Kamilova F, Qi S, Stephan D, Wang JJ, Makarova N et al. (2007). Selection of bacteria able to control Fusarium oxysporum f. sp. radicis-lycopersici in stonewool substrate. J Appl Microbiol 102: 461–471. Zoued A, Brunet YR, Durand E, Aschtgen M-SS, Logger L, Douzi B et al. (2014). Architecture and assembly of the Type VI secretion system. Biochim Biophys Acta 1843: 1664–1673. pp Vasseur P, Vallet-Gely I, Soscia C, Genin S, Filloux A. (2005). The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation. Microbiology 151: 985–997. gy Weinert C, Morger D, Djekic A, Grütter MG, Mittl PR. (2015). Crystal structure of TRIM20 C-terminal coiled- coil/B30.2 fragment: implications for the recognition of higher order oligomers. Sci Rep 5: 10819. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/ g g p Weller DM. (2007). Pseudomonas biocontrol agents of soilborne pathogens: looking back over 30 years. Phytopathology 97: 250–256. Weller DM, Raaijmakers JM, Gardener BB, Thomashow LS. (2002). Microbial populations responsible for specific soil suppressiveness to plant pathogens. Annu Rev Phytopathol 40: 309–348. y p Wexler AG, Bao Y, Whitney JC, Bobay L-MM, Xavier JB, Schofield WB et al. (2016). Human symbionts formation accompanies this paper on The ISME Journal website (http://www.nature.com/is The ISME Journal
https://openalex.org/W1887231280	https://hal.archives-ouvertes.fr/hal-02328022/file/Pierre2015.pdf	English	null	Modeling vegetation and wind erosion from a millet field and from a rangeland: Two Sahelian case studies	Aeolian research	2,015	cc-by	15,044	Modeling vegetation and wind erosion from a millet field and from a rangeland: two Sahelian case studies Caroline PIERRE1,2, Laurent KERGOAT1, Gilles BERGAMETTI2, Éric MOUGIN1, Christian BARON3, Amadou ABDOURHAMANE TOURE4, Jean-Louis RAJOT2,5, Pierre HIERNAUX1, Béatrice MARTICORENA2, Claire DELON6 Caroline PIERRE1,2, Laurent KERGOAT1, Gilles BERGAMETTI2, Éric MOUGIN1, Christian BARON3, Amadou ABDOURHAMANE TOURE4, Jean-Louis RAJOT2,5, Pierre HIERNAUX1, Béatrice MARTICORENA2, Claire DELON6 1 Géosciences Environnement Toulouse (GET), CNRS/IRD/Université de Toulouse 14 Avenue Edouard Belin, 31400 Toulouse, France laurent.kergoat@get.obs-mip.fr eric.mougin@get.obs-mip.fr pierre.hiernaux@get.obs-mip.fr 2Laboratoire Interuniversitaire des Systèmes Atmosphériques (LISA), Universités Paris Est Créteil & Paris Diderot, UMR CNRS 7583 61 Avenue du Général de Gaulle, 94000 Créteil, France caroline.pierre@lisa.u-pec.fr (corresponding author, phone number: +33 1 45 17 15 79) gilles.bergametti@lisa.u-pec.fr beatrice.marticorena@lisa.u-pec.fr 6 Laboratoire d’Aérologie (LA) 14 Avenue Edouard Belin, 31400 Toulouse, France claire.delon@aero.obs-mip.fr Abstract Quantifying wind erosion and dust emissions in the semi-arid Sahel remains challenging because of the large seasonal and interannual dynamics of surface properties. The increasing conversion of rangelands into croplands raises issues for quantifying wind erosion over these two contrasted surfaces. Whereas wind erosion models have been so far applied to these two surface types separately, this study proposes a common modeling approach to represent the horizontal flux from Sahelian rangelands and croplands. Pair simulations of both typical Sahelian land surface types investigate the horizontal flux due to wind erosion over a 3-year period for two instrumented sites in Mali and Niger. Two different vegetation models simulate the specific phenology and growth of a rangeland grass and a millet crop. These models also account for the local cropping and pastoral practices. Compared to field measurements, the vegetation cover is satisfyingly simulated by the models, especially the strong seasonal dynamics. Specific parameterizations of the aerodynamic surface roughness length (Z0) as a function of vegetation variables are established using measurements from the two sites. The simulated horizontal flux turns out to be higher for a cropland than for a rangeland by approximately a factor 1.5, implying that increasing Sahelian cropped areas would increase dust emissions from the Sahel. This difference is mainly due to the time shift between grass and millet growth: the latter starts growing about 2 to 3 weeks later than annual grass. The amount of dry vegetation remaining during late dry season is also important for Sahelian wind erosion. Keywords: wind erosion, Sahel, modeling, cropland, rangeland. 1. Introduction Dust represents about 40% of the total amount of aerosols injected each year into the atmosphere (about 1.5 Mt.yr-1) (IPCC, 2007). It has strong radiative impacts (Tegen et al., 1997), both "direct" by absorption and scattering of the solar and telluric radiations, and "indirect" when acting as cloud condensation nuclei (Haywood and Boucher, 2000). When deposited, dust can provide significant amounts of nutrients such as phosphorus and iron to remote oligotrophic oceanic areas (Jickells et al., 2005) or like phosphorus and potassium in continental environments (Swap et al., 1992; Hermann et al., 2010; Breuning-Madsen et al., 2015). In the semi-arid dust source regions, wind erosion can also decrease soil fertility of already poor soils (Lyles and Allison, 1976; Bielders et al., 2002), and damage crops through abrasion (Sterk, 2003). At a global scale, the link between human activities and dust emissions - which result from wind erosion - is now recognized (Boucher et al., 2013). Especially, changes in land use influence dust emissions in populated semi-arid areas like the Sahel, where the number of inhabitants has more than doubled over the past 50 years. Meanwhile, according to many authors, Sahelian dust emissions increased during the early 20th century and started decreasing from the late 1980’s on (Moulin and Chiapello, 2006; Foltz and McPhaden 2008; Mahowald et al. 2010; Cowie et al, 2013; Chin et al, 2014; Doherty et al, 2014; Ridley et al, 2014). Recently, Stanelle et al. (2014) inferred from simulations that dust emissions from the agricultural areas of Northern Africa (including the Sahel) had increased from 2.8 Tg.yr-1 in 1880 to 8 Tg.yr-1 today. At a quasi-continental scale, Mulitza et al. (2010) hypothesized that the sudden increase in dust deposition off the West African coast about two centuries ago was linked to the onset of commercial agriculture in the Sahel. However, the relative roles played by climate change and human activities on Sahelian dust emissions are still debated, and no precise quantification of the dust emissions from this region is available (Tegen and Fung, 1995; Tegen et al., 2004; Mahowald et al., 2004). At a more local scale, studies based on field measurements performed in the Sahel have provided estimates of wind erosion in croplands (Bielders et al., 2001a; Sterk, 2003; Bielders et al., 2004), and rangelands (Tidjani et al., 2009). Highlights: Sediment horizontal fluxes from a cropland and a rangeland in the Sahel are simulated Simulated vegetation and horizontal fluxes are in good agreement with measurements Horizontal mass flux is greater for cropland than rangeland by about a factor 1.5 1 1 2.1. Study sites and data The Sahel is characterized by a short rainy season lasting from approximately June to October, and a long dry season during the rest of the year (Lebel and Ali, 2009). Annual precipitation ranges fom about 100 to 600 mm, allowing the growth of seasonal vegetation. Most of this rain is provided by a few mesoscale convective events, commonly accompanied by strong winds caused by systems outflows. The duration of these gusts ranges from a few minutes to more than one hour. Although the monsoon wind blows from South-West during the rainy season, the convective outflows are most often easterly winds, because convection mixes the African Easterly Jet down to the surface (Guichard et al., 2009). During the dry season, the Harmattan wind blows from North-East, while most plants wilt and progressively degrade. The main Sahelian land use is cultivation, usually with an alternance of cropping and fallowing periods over several years (e.g. Hiernaux and Turner, 2002), and grazing in the rangelands. After harvest, fields are open to grazing too. Ideally, experimental sites would provide a full set of wind erosion measurements, meteorological data and soil description for pairs of cropped / non-cropped plots. Yet, experimental sites where both vegetation characteristics and wind erosion horizontal fluxes have been monitored are scarce in the Sahel, and no paired-experiment (monitoring vegetation and wind erosion over a cropland and a rangeland in the same area) has been documented so far. Given these constraints, two sites where at least some of these data were acquired have been selected (Table 1). The first site is the Agoufou rangeland (15.3°N, 1.5°W), a study site of about 1 km² located in the Gourma area in Mali (e.g. Mougin et al., 2009). Agoufou is located in a typical pastoral Sahel area with annual rainfall averaging 350 mm. The dominant land use is extensive grazing. Numerous measurements were performed at this site during the AMMA campaign (Multidisciplinary Analysis of the African Monsoon): meteorological variables from a station and a rain gauge (wind speed, rain amount, temperature, and solar radiation), soil texture, and soil moisture at the same location. Wind speed has been measured at 3.5 m height using a cup anemometer. Wind data provided by a 3D sonic anemometer were available during part of this period. 1. Introduction Several studies have suggested that wind erosion was significantly increased by cropping (Rajot, 2001; Bielders et al, 2002; Ikazaki et al. 2011), but studies on traditionally-managed crop fields are still scarce (Bielders et al., 2001b; Abdourhamane Touré et al., 2011). In addition, cropping (Sterk, 2003) and pastoral practices (Tidjani et al., 2009) can modulate wind erosion. Until now, wind erosion modeling approaches have been developed for croplands and rangelands separately (Li et al., 2014). However, a common approach would help estimating wind erosion on these typical Sahelian surfaces, and their differences in terms of amounts and temporal dynamics. Another issue consists in representing the impacts of pastoral and cropping practices on wind erosion. The objective of this study is to model the horizontal fluxes of aeolian sediment of Sahelian croplands and rangelands with consistent modeling tools, and to discuss the differences of the simulated fluxes between these two surface types. Section 2 describes the data and the models development and setup. Results of the simulations are shown and evaluated in section 3 and further discussed in section 4. 2 2.1. Study sites and data Grass measurements have been performed along 1 km lines at about 15 dates each year of 2006-2008, and livestock observations have been collected at the nearby (2.5 km) permanent pond in 2010-2011 (Diawara, 2015). For this study, years 2006 to 2008 are retained because of their good data sampling and their contrasted rainfall conditions. The second site is a rectangular traditionally-managed millet field (about 100 m by 150 m) located in the Fakara region in Niger, in the vicinity of the village of Banizoumbou (13.5°N, 2.7°E). This site is fairly representative of agropastoral Sahel and receives 500 mm of annual rain on average. The millet field is embedded in a typical crop/fallow/rangeland mosaic, where fields are protected from grazing during the rainy season and open to grazing during the dry season. On this site, meteorological data (wind speed, wind direction, rain amount, air temperature), millet height during growth, crop residues cover fraction, and horizontal fluxes have been monitored from 2006 to 2008 (Abdourhamane Touré et al., 2011). Wind speed has been monitored at 4 heights (0.35, 0.70, 1.20, and 2.50 m). Solar radiation was not monitored at Banizoumbou but was provided by the Atmospheric Radiation Measurement (ARM) Mobile Facility in Niamey for year 2006. No measurements being available for 2007 and 2008, the 2006 measurements are used for the 3 years of simulation. This may not impact significantly the simulations since plant growth in the Sahel is 3 mainly limited by precipitation. Horizontal fluxes have been measured with BSNE sand traps (Fryrear, 1986) on a daily basis. Pierre et al. (2014a) performed horizontal flux simulations for this site. mainly limited by precipitation. Horizontal fluxes have been measured with BSNE sand traps (Fryrear, 1986) on a daily basis. Pierre et al. (2014a) performed horizontal flux simulations for this site. The two study sites have sandy soils that are typical of the Sahelian area. Information on the dry grain size distribution is available only at Banizoumbou, indicating a silt and clay proportion of about 5% together (Sabre et al., 1997). At Agoufou, the sand content of the top soil layer is about 95% (De Rosnay et al., 2009, Table 2), thus indicating the dominance of sand, like at Banizoumbou. Numerical tests (not shown) showed that for such fine sand the impact of a precise grain size distribution on the simulated horizontal flux is very low. 2.1. Study sites and data It was not taken into account in this study. Table 1: Available datasets and their characteristics (temporal coverage, time resolution) for Agoufou and Banizoumbou (U: wind velocity; u: wind friction velocity) Agoufou (Mali) Banizoumbou (Niger) wind U at 3.5 m 2006 to 2008 15 minutes u part of 2005, 2006, 2007 5 minutes U at 4 heights (0.35, 0.70, 1.20, 2.50 m) 2006 to 2008 5 minutes wind direction 2006 to 2008 5 minutes rain 2006 to 2008 15 minutes 2006 to 2008 5 minutes air temperature solar radiation 2006 15 minutes soil moisture No vegetation grass mass 2006 to 2008 43 dates millet height 2006 to 2008 39 dates during rainy season horizontal flux No 2006 to 2008 daily Available datasets and their characteristics (temporal coverage, time resolution) for Agoufou izoumbou (U: wind velocity; u: wind friction velocity) 2.2. Modeling approach 2.3. Vegetation modeling Two vegetation growth models are used: the STEP model (Sahelian Transpiration Evaporation and Productivity model; Mougin et al., 1995; Tracol et al., 2006) for the rangeland grass, and the SarraH model (Système d’Analyse Régionale des Risques Agroclimatiques – Version 3.2; Dingkuhn et al., 2003) for millet crops. These models run on a daily time step and provide the vegetation mass and Leaf Area Index (LAI) over the whole annual cycle. The mass values are simulated for green vegetation, standing straws, and litter by both models. The STEP model has been specifically designed to simulate the growth of annual grass under Sahelian conditions, and it exhibits good skills over the Sahel when compared to satellite-based indices (Lo Seen et al., 1995; Jarlan et al., 2002; Pierre et al., 2011). The SarraH model has been widely tested and used for several millet breeds for Sahelian sites in Senegal, Mali and Niger (Baron et al., 2005; Kouressy et al., 2008; Traoré et al., 2011; Marteau et al., 2011). The main differences between these two models are the representation of the phenological stages during the vegetation cycle, and the mass partition between leaves, stems and grain, for millet. Recently, these two models have been extended to simulate the vegetation during the dry season, taking into account grazing and trampling by livestock (Delon et al., 2015), and cropping practices in millet fields (see Appendix). To simulate grazing for the rangeland, livestock density is provided by field observations in 2010- 2011 around the Agoufou pond (Diawara, 2015). In agreement with field observations, a lower soil fertility (and hence a lower light-use-efficiency parameter in the STEP simulations) is assumed for the rangeland in Banizoumbou compared to Agoufou. For millet simulations, grazing pressure is set at 5 TLU.km-² (Tropical Livestock Unit), which is typical of moderately grazed areas in the Sahel (Hiernaux and Turner, 2002). Every year, the remaining standing millet straws are laid down into litter on January 1st (hereafter referred to as field clearing), a date considered as typical according to local observations. The Banizoumbou experimental plot was been clean-weeded, as commonly done for millet in the Sahel, thus it is assumed that no weeds grow in millet fields. 2.2. Modeling approach In order to estimate the respective wind erosion horizontal fluxes from a cropland and a rangeland under identical climatic and soil conditions, simulations are performed to represent the vegetation cycle of rangeland and millet over three years for each of the two selected sites. For that purpose, different models are used: a model describing the seasonal dynamics of the rangeland grass, a crop model simulating the millet growth cycle, and a wind erosion model. In other words, the two monitored cases of Agoufou rangeland and Banizoumbou millet field are simulated, as well as two additional cases (not monitored) of a millet field at Agoufou and a rangeland at Banizoumbou. Horizontal fluxes are simulated for both sites and both vegetation types. Combining the vegetation and wind erosion models requires parameterizing the seasonal dynamics of the surface properties, like the aerodynamic surface roughness length (Z0), as a function of vegetation characteristics. 4 2.3. Vegetation modeling or green grass, fcv is computed by the STEP model from the simulated green LAIg as follows: 2.3. Vegetation modeling Total grass cover fraction is the sum of the contributions of green grass, straws and litter. Before field clearing, the millet cover fraction is the sum of the contribution of laying litter and standing straws. This latter contribution is estimated by Eq. (2), with LAI being computed as the product of the Specific Leaf Area (SLA) at harvest by the dry leaves mass, thus considering that stems have a negligible LAI. The millet cover fraction for litter is estimated by the regression proposed by Abdourhamane Touré et al. (2011, Figure 8): (5) (5) BMlit the millet litter mass (in g.m-2). After field clearing, the cover fraction is only due to litter. with BMlit the millet litter mass (in g.m ). After field clearing, the cover fraction is only due to litter. According to preliminary work (not shown), millet height appeared to be a relevant variable to derive Z0, while vegetation mass is the most relevant one to estimate Z0 for grass (in agreement with Tidjani et al., 2009). In the simulations, millet height is estimated from the modeled phenological stages with a parameterization based on the millet heights measurements performed on the Banizoumbou field. According to these measurements, millet height follows two successive linear evolutions in time, with slopes corresponding to two distinct periods of the SarraH simulations: first, the vegetative growth from germination to flower initiation, and second, the reproductive growth from tiller elongation to grain filling. During grain drying (i.e. the last stage) millet height no longer increases. The following relations are obtained: According to preliminary work (not shown), millet height appeared to be a relevant variable to derive Z0, while vegetation mass is the most relevant one to estimate Z0 for grass (in agreement with Tidjani et al., 2009). In the simulations, millet height is estimated from the modeled phenological stages with a parameterization based on the millet heights measurements performed on the Banizoumbou field. According to these measurements, millet height follows two successive linear evolutions in time, with slopes corresponding to two distinct periods of the SarraH simulations: first, the vegetative growth from germination to flower initiation, and second, the reproductive growth from tiller elongation to grain filling. During grain drying (i.e. the last stage) millet height no longer increases. 2.3. Vegetation modeling Consitently with local observations (Abdourhamane Touré et al., 2011), the millet sowing date is searched for by the crop model from June 7th on, the same millet variety (“Haini Kirey”) is represented for both sites, and a slight quantity of manure is applied in the case of Banizoumbou (see Schlecht et al., 2004), but none at Agoufou. This difference is expressed by a conversion efficiency parameter, which is used in the SararaH model to compute the dry matter produced by photosynthesis according to water stress and radiation, and takes into account the effect of manure. Its value is set to 4 g.MJ-1 for Banizoumbou against 3.6 g.MJ-1 for Agoufou. Models are spun up in 2005 to provide dry season vegetation variables in early 2006. During the dry season, only vegetation masses are computed by the SarraH model. Two additional values have to be derived from vegetation models outputs in order to simulate wind erosion, namely the erodible surface fraction (E), i.e. the fraction of the surface on which wind erosion can occur, and the aerodynamic surface roughness length (Z0). Indeed, it has been shown for both rangeland and millet field that Z0 adequately accounts for the role of the surface properties on the wind shear stress that controls wind erosion (Pierre et al., 2014a, b). The vegetation cover fraction (fcv) is required to determine E: (1) E = 1 - fcv E = 1 - fcv For green grass, fcv is computed by the STEP model from the simulated green LAIg as follows: For green grass, fcv is computed by the STEP model from the simulated green LAIg as follows: 5 (2) with K=0.431 (Mougin et al., 2014). For standing millet, the same parameterization is applied to the LAI simulated by the SarraH model, with K=0.45 according to field trials. For grass straws and litter, the STEP model provides vegetation cover fraction based on the following parameterizations respectively: (3) (3) where LAIt (in m².m-2) is the total LAI (for green grass and straws) and LAIs is the straws LAI, and here LAIt (in m².m-2) is the total LAI (for green grass and straws) and LAIs is the straws LAI, and: (4) (4) for litter, where BMlit is the litter mass (in g.m-2). Total grass cover fraction is the sum of the contributions of green grass, straws and litter. for litter, where BMlit is the litter mass (in g.m-2). Agoufou site: At Agoufou, wind velocity and wind friction velocity are used at 30-minute time resolution to retrieve Z0 by solving Eq. (7). Wind friction velocity is derived from the sonic anemometer measurements following Walker (2005). The purpose being to describe a typical seasonal cycle for rangeland Z0, years 2005, 2006 and 2007 are used. An instrumental drift has occurred after 2007 that prevent the derivation of Z0 for 2008. A set of criteria proposed by Marticorena et al. (2006) is adapted to filter wind measurements. To select the wind measurements performed in near-neutral conditions, only the data acquired between 6am and 8am and between 6pm and 8pm in local time, and leading to a Monin-Obukov length such as \| z / L \| < 0.02 are retained. To discard the possible remaining free convection, only periods during which U > 1 m.s-1 and u > 0.2 m.s-1 are retained. Outliers are removed by keeping only u* < 1 m.s-1 and Z0 < 0.12 m, these limits being consistent with the Z0 values found for grass in the literature (e.g. Marticorena et al., 2006; Tidjani et al., 2009). Finally, Z0 are estimated each year by a 30-days sliding median to overcome remaining data scarcity and to remove outliers. From March to October (i.e. the period during which measurements are available), Z0 values averaged over the 3 years show a decrease of Z0 from March (~ 0.015 m) to June (~ 0.007 m), and a rapid growth from July to October (~ 0.050 m) (Figure 1). These values are in agreement with the literature for this cover type, as reviewed by Marticorena et al. (2006, Table 7): Pasquill (1950) obtained a value of 0.030 to 0.070 m for ‘long grass’. Similarly, Z0 values for ‘short grass’ vary from 0.002 m (Deacon, 1953) to 0.011 m (Saugier and Ripley, 1978), depending on the authors. More recently, Tidjani et al. (2009) obtained a Z0 value of 0.050 m at the peak of vegetation for a rangeland site located in southeastern Niger. 2.3. Vegetation modeling The following relations are obtained: H= 0.0076 Δj (first period) H= 0.0378 Δj + Hc (second period) (6) H= 0.0076 Δj (first period) H= 0.0378 Δj + Hc (second period) (6) where H is the plant height (in m) and is truncated to 2.3 m (according to Banizoumbou field observations), Δj is the number of days since the beginning of the considered period, and Hc is the millet height at the last day of the first period, which ensures continuity. Millet height after harvest is estimated as the product of its maximum height (at harvest) by the ratio of stem mass at the considered date to stem mass at harvest. The clearing of the field being set on January 1st, the millet height becomes nil at that date, until the next rainy season. where H is the plant height (in m) and is truncated to 2.3 m (according to Banizoumbou field observations), Δj is the number of days since the beginning of the considered period, and Hc is the millet height at the last day of the first period, which ensures continuity. Millet height after harvest is estimated as the product of its maximum height (at harvest) by the ratio of stem mass at the considered date to stem mass at harvest. The clearing of the field being set on January 1st, the millet height becomes nil at that date, until the next rainy season. 6 2.4. Surface characteristics estimates 2.4. Surface characteristics estimates Z0 is first estimated from measurements on the two sites. Then, these measurements are used to establish relations between the Z0 values and measured vegetation characteristics in order to determine parameterizations that can be used to derive Z0 from the vegetation model outputs. The estimation of Z0 is based on selected wind measurements assuming a logarithmic wind profile (Priestley, 1959): (7) (7) with k the von Karman constant (k= 0.4), U the wind speed (in m.s-1), u* the wind friction velocity (in m.s-1), z the height and Z0 the aerodynamic surface roughness length (z and Z0 are in the same length unit). Because of the different experimental setups in the two sites, Z0 values are estimated from friction velocity estimates at Agoufou and from wind profile measurements at Banizoumbou. A typical seasonal cycle of Z0 is derived from the pluriannual measurements performed at both sites. Banizoumbou site: 30-minute averaged values are used to retrieve Z0 from linear regression of the wind profile described by Eq. (5). For that purpose, several criteria are also adapted from Marticorena et al. (2006). To select near neutral conditions, only wind speeds recorded between 6am and 8am and between 6pm and 8pm in local time are retained. In order to avoid free convection, regression is only 7 calculated if wind speed at all heights is greater than 1 m.s-1, and the result of the regression is retained only if u* from the regression is greater than 0.2 m.s-1. Additionally, the result of the regression is retained if the difference between measured and fitted wind velocities is lower than 5% at all heights. Finally, a last selection is made on wind direction to keep the values corresponding to the four corners of the field, i.e. to the maximal fetch of the measurements: values are retained in four angles of 12° (field corners +/- 6°). From January to the end of July, measurements from the 4 heights can be used. As millet reaches the lowest sensor around the beginning of the rainy season, only the 3 upper ones are used afterwards. Millet further reaches the second sensor at 0.7 m (in early August 2006 and 2007, around mid-July 2008), causing Z0 values obtained after these dates to be less accurate. The Z0 estimates corresponding to millet heights up to 1 m (rather than 0.7 m like in Abourhamane Touré et al., 2011) are retained here, in order to capture the growing period in terms of roughness length time series and to allow high values to be used in the parametrizations. Erosion does not occur when millet is fully developped, so the accuracy of Z0 is not critical at this period of the year. Finally, Z0 is estimated for each year by a 30-days sliding median. The resulting seasonal cycle of Z0, averaged over the 3 years (Figure 1), reaches approximately 0.005 m in the late dry season (March to June). It increases from July on, until reaching about 0.020 m at the beginning of August. Since millet stems remain standing until field clearing on January 1st, no Z0 values are derived from about mid-July or August – depending on the year – to the end of December. Agoufou site: Both standing straws and litter, which masses are respectively noted BMstd and BMlit, inhibit wind erosion, although their respective effects may differ (Siddoway et al., 1965; Nandinstetseg and Shinoda, 2015). Consequently, the Z0 parameterization should take into account these two types of obstacles separately. Monthly means of Z0 estimates are regressed against monthly means of the measured vegetation mass. Separate linear regressions are computed depending on data availability for the dry (March to June) and wet (July to September) seasons, giving respectively the coefficients for litter (R²=0.63, n=3) and for standing vegetation (R²=0.74, n=4) of the final parameterization. In this way, the coefficients performing best for the dry season and the beginning of the growing season are estimated. The following parameterization is obtained for grass: Z0 = 0.0003 BMstd + 0.0007 BMlit (8) Z0 = 0.0003 BMstd + 0.0007 BMlit with BM expressed in g.m-² and Z0 in m. This regression is used for Z0 values greater than the aerodynamic surface roughness of the bare surface z0s = 9.7 10-5 m, a typical value for such a sandy site (see Pierre et al., 2014a). Banizoumbou site: Figure 1: Mean annual cycle of the aerodynamic surface roughness length (Z0) estimated from measurements of wind velocity and wind friction velocity, for the Agoufou rangeland and the Banizoumbou millet field over 2006-2008. Figure 1: Mean annual cycle of the aerodynamic surface roughness length (Z0) estimated from measurements of wind velocity and wind friction velocity, for the Agoufou rangeland and the Banizoumbou millet field over 2006-2008. The mean cycles imply that the rangeland Z0 is greater than the millet Z0 until mid-August. The marked increase in Z0 corresponding to the start of the vegetation growth occurs earlier and is sharper for the rangeland (beginning of July) compared to the millet field (mid-July), despite the fact that the millet field is located about 2° to the South, with on average an earlier rainy season (Frappart et al. 2009). Thus, the time shift between the Z0 cycles may reflect the phenological offset 8 between these two cover types, already observed for similar sites in Niger (Boulain et al., 2009; Velluet et al., 2014). Although these Z0 estimates do not cover the whole year, they cover the period which is critical for wind erosion, i.e. the end of the dry season and the beginning of vegetation growth (e.g. Rajot, 2001; Abdourhamane Touré et al., 2011). Banizoumbou site: Daily values of Z0, interpolated from monthly means to each millet height measurement day, are regressed against measured millet height (2006 to 2008). Bare soil value being the intercept of these regressions, the smallest intercept values among the three years is selected. Because of Z0 data scarcity, the slope is obtained from the regression over year 2008 only (R²=0.94, n=10), which exhibits a marked dynamics in millet heights and Z0 estimates. The following parametrization is obtained for millet height lower than 1 m: (9) Z0=0.016H + 0.003 where Z0 and H are in m. Bielders et al. (2004) reported maximum values of 0.1 m for Z0 in a similar millet field at Banizoumbou for years 1996 to 1998. Thus, for millet higher than 1 m, Z0 is parameterized so that it reaches 0.1 m for a millet height of 2.3 m: (10) Z0=0.062H - 0.043 with Z0 and H expressed in m. When only litter is left on the field (from early January to millet germination), Z0 is adapted from the relation proposed by Abdourhamane Touré et al. (2011, Figure 11) to ensure continuity of Z0 values: 9 Z0=0.0025 ln(fcv) + 0.006 (11) with Z0 expressed in m. At the beginning of millet growth, the maximum Z0 between standing millet (Eq. (9)) and crop residues (Eq. (11)) is retained. with Z0 expressed in m. At the beginning of millet growth, the maximum Z0 between standing millet (Eq. (9)) and crop residues (Eq. (11)) is retained. 2.6. Wind erosion modeling 2.6. Wind erosion modeling The wind erosion model has been described in Pierre et al. (2014b). The main principles are summarized hereafter. Wind erosion occurs when the wind friction velocity exceeds a threshold value that depends on the presence of non erodible elements – such as vegetation – on the surface (Bagnold, 1941; Gillette, 1979) and on the topsoil layer characteristics (e.g., surface soil moisture). Water in the soil strengthens the cohesive forces linking the soil grains together, increasing the threshold wind friction velocity. This effect is estimated through the empirical relationship proposed by Fécan et al. (1999): 1 if w < w0 M(w) = (12) [1 + 1.21(w-w0)0.68 ] 0.5 if w > w0 1 if w < w0 M(w) = (12) [1 + 1.21(w-w0)0.68 ] 0.5 if w > w0 1 (12) where w is the gravimetric soil moisture and w0 is the residual gravimetric soil moisture (in % g.g-1), which depends on the soil texture. w0 is derived from the soil clay content following Fécan et al. (1999): w0 = 0.0014 (%clay)2 + 0.17 (%clay) (13) w0 = 0.0014 (%clay)2 + 0.17 (%clay) (13) This yields a residual soil moisture close to zero for very sandy soils like the ones of Banizoumbou and Agoufou. This parameterization was applied to the Agoufou site, where soil moisture was monitored at 0.05 m depth. An estimate of soil moisture at 0.01 m depth is derived from these measurements, based on rainfall dynamics following Pierre et al. (2014b). The soil moisture estimate at 0.01 m depth is assumed to be equal to the soil moisture at 0.05 m depth during a rain event, and then to decrease to a minimum value 36 hours later. Since soil moisture is not available at Banizoumbou, the simulated wind erosion for this site is inhibited as soon as rain exceeds 1 mm in 1 hour, and during 24 hours afterwards, as in Pierre et al. (2014a). Because of the non erodible elements, the friction velocity exerted on the surface between obstacles (us) is less than the total friction velocity of the wind (u). Marticorena and Bergametti (1995) defined the effective fraction (feff) as the ratio of us* over u. The following expression results from the correction proposed by these authors, as stated in King et al. 2.6. Wind erosion modeling (2005): (14) 10 where z0s is the aerodynamic roughness of the bare soil, Z0 is the aerodynamic roughness length of the surface (including the obstacles) and X is tied to the height of the internal boundary layer that develops between the obstacles (z0s, Z0 and X are in the same length unit). where z0s is the aerodynamic roughness of the bare soil, Z0 is the aerodynamic roughness length of the surface (including the obstacles) and X is tied to the height of the internal boundary layer that develops between the obstacles (z0s, Z0 and X are in the same length unit). The parameter X is set to X = 0.4 m, in agreement with Pierre et al. (2014a). The threshold friction velocity (ut) is obtained from the effective fraction and the effect of soil moisture, assuming a threshold friction velocity for the bare soil of uts= 0.22 m.s-1, as in Pierre et al. (2014b): (15) (15) The friction velocity is computed with Eq. (7) from the simulated Z0 and the measured wind speed for each cover type. Wind erosion is computed at the time step of the wind measurements, i.e. 15- minute at Agoufou and 5-minute at Banizoumbou. When the threshold friction velocity is exceeded, particle entrainment occurs, and the particle mass flux depends on a power function of the wind friction velocity. As in Marticorena and Bergametti (1995), the horizontal flux G (in kg.m-1.day-1) – also called saltation flux – is estimated following Kawamura (1951) and White (1979), taking into account the fraction of erodible surface E: (16) (16) where ρa is the density of air (equal to 1.227 kg.m-3). where ρa is the density of air (equal to 1.227 kg.m-3). where ρa is the density of air (equal to 1.227 kg.m-3). The horizontal flux is computed over 2006 to 2008. For the Banizoumbou millet case, the results are compared to the 10 highest values of the monitoring sand traps, as in Pierre et al. (2014a). Indeed, the wind erosion model is designed to simulate a steady state flux, which is not reached at all locations of the instrumented plot. For consistency, measured horizontal flux is not considered when gaps in wind data preclude simulations. Agoufou site: The outputs of the two vegetation models show differences in vegetation masses (Figure 2): the mass of millet is larger than that of grass during most of the dry seasons of 2006-2007 and 2007- 2008. This is a direct consequence of higher millet production at the end of the rainy season, for which annual rainfall tallied 376 and 287 mm in 2006 and 2007 respectively. However, millet mass becomes smaller than grass mass at the very end of the dry season (from March on). The seasonal dynamics also differs: growth starts and vegetation mass reaches a maximum about 1 month later for millet than for rangeland in 2006 and 2007. Yet, the simulated germination occurs at the same period for both grass and millet, but the simulated millet mass during the first phenological stages remains very low. After harvest, millet leaves – but not stems – disappear quickly (not shown), eaten and trampled by livestock. At field clearing (January 1st), remaining standing millet stems are turned into litter, which causes litter mass to peak. 11 Figure 2: Green, straw and litter mass at Agoufou from 2006 to 2008, for rangeland grass according to the STEP model (solid lines) and for millet according to the SarraH model (dotted lines). STEP simulations are adapted from Delon et al. (2015) Figure 2: Green, straw and litter mass at Agoufou from 2006 to 2008, for rangeland grass according to the STEP model (solid lines) and for millet according to the SarraH model (dotted lines). STEP simulations are adapted from Delon et al. (2015) The decrease in rainfall over the 3 years has a strong impact on the simulated vegetation. Year 2008 is dry (227 mm) and the SarraH model does not simulate any millet growth. This is reasonable since Agoufou is at the northern boundary of millet crops in Mali, and millet growth and yield are very variable. Moreover, rainfall events are more scattered over time in 2008, the rainy season lasting 165 days (starting on April 30th), against 132 days (May 27th) and 137 days (June 6th) in 2006 and 2007. Dryspells interrupt plant growth by inducing water stress, particularly at the beginning of the rainy season: in 2008, there is no rain during 24 days after the first rain events. Agoufou site: Millet maximum occurs rather late in 2007 (around mid-October), but this date is still consistent with field observations (indicating a maximum between early September and mid-October). There is a good agreement between STEP simulations and field measurements at the Agoufou rangeland site (Figure 3): green vegetation mass and dynamics are well reproduced, as well as the dry season dynamics. The simulated cover fraction is also in good agreement with measurements (not shown). There might be a slight overestimation of peak vegetation mass, although in situ measurements may not fit exactly the date of maximum mass, and a slight underestimation of litter mass by the model from January to May in 2007 and 2008. 12 12 Figure 3: Green, straw and litter mass for rangeland grass at Agoufou from 2006 to 2008, according to the STEP model (solid lines) and to measurements (stars). Adapted from Delon et al. (2015). Figure 3: Green, straw and litter mass for rangeland grass at Agoufou from 2006 to 2008, according to the STEP model (solid lines) and to measurements (stars). Adapted from Delon et al. (2015). Figure 3: Green, straw and litter mass for rangeland grass at Agoufou from 2006 to 2008, according to the STEP model (solid lines) and to measurements (stars). Adapted from Delon et al. (2015). Banizoumbou site: Banizoumbou annual rainfall is 503 mm (2006), 456 mm (2007) and 700 mm (2008): thus rainfall in the two first years is close to average, while year 2008 is above average (see 2.1). Interannual variability over the 3 years is different at Banizoumbou and Agoufou, which is expected given the 500 km distance between the two sites and the spatial variability of Sahelian rainfall (Balme et al., 2006). Although the annual rainfall at Banizoumbou are greater than at Agoufou, they lead to lower simulated biomass at Banizoumbou in 2006 and 2007 (Figure 4). This mainly results from the distribution of precipitation during the rainy season. Indeed, the rainy season at Banizoumbou lasts 127 days (and starts on May 27th), 130 days (May 7th) and 144 days (May 21st) in years 2006, 2007 and 2008 respectively. A one week long dryspell occurs from June 10th in 2006, while three other dryspells occur between the first rain events in 2007 and two at the beginning of the rainy season in 2008. In 2006, the cumulated rainfall in June is very low, triggering an important water stress. In addition, the simulated grain filling occurs relatively early this year, which prevents millet to take full advantage of the whole rainy season. Conversely, ample precipitation in 2008 lead to a peak biomass in the upper range of what moderately manured fields can produce (e.g. Rockstrom and de Rouw, 1997; Schlecht et al., 2004). The lower soil fertility for rangeland in Banizoumbou compared to Agoufou (see 2.3) contributes to lower the simulated grass mass in Banizoumbou compared to Agoufou. As for the Agoufou site, there is a time shift in growth between the millet field and the rangeland, millet growth being delayed compared to that of grass. There is also a lower biomass maximum for grass than for millet, as expected for this area (Dardel et al 2014). During the dry season, particularly from March on, and as for the Agoufou site, less crop residues remain on the field compared to straws and litter on the rangeland. 13 Figure 4: Green, straw and litter mass at Banizoumbou from 2006 to 2008, for rangeland grass according to the STEP model (solid lines) and for millet according to the SarraH model (dotted lines). Agoufou site: Z0 estimates from Eq. (8) and the STEP model outputs are in good agreement with measured Z0 (Figure 6). Over 2006 and 2007, Z0 estimates and parameterized values correlate with a coefficient R²=0.74 (n=307), with RMSE=0.008 m. Z0 dynamics during the late dry season are well reproduced, as well as the onset of growth at the beginning of the rainy season. During the rest of the year, the Z0 dynamics corresponds to what is expected: a peak in September is followed by a rapid decrease due to senescence and to trampling and intake by livestock. The slope of this decrease is in good agreement with measurements performed from March to May 2006 and during June 2007. Z0 are not as well reproduced late in the rainy season in 2007, but this is not critical because wind erosion is negligible during this part of the year. Additionally, the minimum and maximum values of the aerodynamic roughness length are also in good agreement with the existing literature (Marticorena et al., 2006; Tidjani et al., 2009). Except at vegetation maximum, the parameterized Z0 for the rangeland is significantly higher than the one parameterized for the millet field. A well marked time shift of growth onset of about one month is simulated between the two cover types. As the SarraH model does not simulate any millet growth in 2008, the corresponding Z0 is only due to the degradation of the litter from the previous year. Figure 6: Rangeland measurements (stars) and rangeland and millet parameterized Z0 based on vegetation mass simulated by the STEP model and based on millet height according to the SarraH model at Agoufou from 2006 to 2008. Figure 6: Rangeland measurements (stars) and rangeland and millet parameterized Z0 based on vegetation mass simulated by the STEP model and based on millet height according to the SarraH model at Agoufou from 2006 to 2008. The vegetation cover fraction – taking into account green and dry vegetation – is similar for both cover types in 2006 and 2007, with slightly higher values for rangeland (Figure 7). While millet can reach height greater than 2 m, it is usually planted in clusters (or “hills”) one meter apart in average, yielding a lower cover fraction than for the low grass patches. However, the rangeland is not homogeneous, with bare soil patches varying in size during the season (Pierre et al., 2014b). Banizoumbou site: As the SarraH model does not simulate any growth in 2008, the corresponding Z0 is only due to the degradation of the litter from the pr year. Banizoumbou site: Figure 4: Green, straw and litter mass at Banizoumbou from 2006 to 2008, for rangeland grass according to the STEP model (solid lines) and for millet according to the SarraH model (dotted lines). Applying Eq. (6) to the SarraH model outputs results in simulated millet height that fits measured millet height satisfyingly (Figure 5). However, the simulated growth onset is slightly delayed in 2008 and slightly advanced in 2006 compared to observations. The impact of this simulation delay on wind erosion simulations will be discussed below. Applying Eq. (6) to the SarraH model outputs results in simulated millet height that fits measured millet height satisfyingly (Figure 5). However, the simulated growth onset is slightly delayed in 2008 and slightly advanced in 2006 compared to observations. The impact of this simulation delay on wind erosion simulations will be discussed below. Figure 5: Measured (stars) and parameterized (line) millet height according to the phenological phases simulated by the SarraH model at Banizoumbou from 2006 to 2008. Figure 5: Measured (stars) and parameterized (line) millet height according to the phenological phases simulated by the SarraH model at Banizoumbou from 2006 to 2008. 14 3.2. Surface characteristics Agoufou site: Z0 estimates from Eq. (8) and the STEP model outputs are in good agreement with measu (Figure 6). Over 2006 and 2007, Z0 estimates and parameterized values correlate with a coef R²=0.74 (n=307), with RMSE=0.008 m. Z0 dynamics during the late dry season are well reprodu well as the onset of growth at the beginning of the rainy season. During the rest of the year, dynamics corresponds to what is expected: a peak in September is followed by a rapid decrea to senescence and to trampling and intake by livestock. The slope of this decrease is in agreement with measurements performed from March to May 2006 and during June 2007. Z0 a as well reproduced late in the rainy season in 2007, but this is not critical because wind ero negligible during this part of the year. Additionally, the minimum and maximum values aerodynamic roughness length are also in good agreement with the existing literature (Marti et al., 2006; Tidjani et al., 2009). Except at vegetation maximum, the parameterized Z0 for the rangeland is significantly highe the one parameterized for the millet field. A well marked time shift of growth onset of abo month is simulated between the two cover types. Agoufou site: The maximum cover value of 40% for millet is in agreement with measurements reported by Levy and The vegetation cover fraction – taking into account green and dry vegetation – is similar for both cover types in 2006 and 2007, with slightly higher values for rangeland (Figure 7). While millet can reach height greater than 2 m, it is usually planted in clusters (or “hills”) one meter apart in average, yielding a lower cover fraction than for the low grass patches. However, the rangeland is not homogeneous, with bare soil patches varying in size during the season (Pierre et al., 2014b). The maximum cover value of 40% for millet is in agreement with measurements reported by Levy and 15 Jarvis (1999) for a millet field in Niger. The seasonal dynamics again show an earlier growth for the rangeland than for the millet field. The millet cover fraction shows a discontinuity on January 1st because remaining millet straws are laid down for field clearing. Figure 7: Vegetation cover fraction estimated from the outputs of the STEP and SarraH models at Agoufou from 2006 to 2008. Figure 7: Vegetation cover fraction estimated from the outputs of the STEP and SarraH models at Agoufou from 2006 to 2008. Banizoumbou site: Z0 derived from Eq. (9) to (11) applied to the modeled millet at Banizoumbou shows a reasonable agreement with measurements at the end of the dry season in all years, and at the beginning of the rainy season in 2008 (May to July) (Figure 8). For the 3 years, Z0 estimates and parameterized values correlate with a coefficient R²=0.21 (n=555) and a RMSE=0.011 m. This rather poor agreement is due to the low variability of Z0 estimates over this period, except for year 2008. When considering year 2008 only, the agreement between parameterized and estimated Z0 reaches a correlation coefficient of R²=0.91 (n=220) and RMSE=0.003 m. This suggests that Eq. (9) allows to reproduce reasonably Z0 values in the late dry season, and its increase at the beginning of vegetation growth. There is a tendency to overestimate Z0 in the early growing season (2006, 2007) but not in 2008, which reflects the early simulated germination these years. The sudden decrease of Z0 on January 1st is due to field clearing. The slope change observed in August corresponds to a change in millet phenology according to the SarraH model. The millet Z0 values exhibit slightly larger values than in Abdourhamane Touré et al. (2011) and Pierre et al. (2014a). This is mainly due to the fact that the present study considers a longer period of the year. Z0 derived from Eq. (9) to (11) applied to the modeled millet at Banizoumbou shows a reasonable agreement with measurements at the end of the dry season in all years, and at the beginning of the rainy season in 2008 (May to July) (Figure 8). For the 3 years, Z0 estimates and parameterized values correlate with a coefficient R²=0.21 (n=555) and a RMSE=0.011 m. This rather poor agreement is due to the low variability of Z0 estimates over this period, except for year 2008. When considering year 2008 only, the agreement between parameterized and estimated Z0 reaches a correlation coefficient of R²=0.91 (n=220) and RMSE=0.003 m. This suggests that Eq. (9) allows to reproduce reasonably Z0 values in the late dry season, and its increase at the beginning of vegetation growth. There is a tendency to overestimate Z0 in the early growing season (2006, 2007) but not in 2008, which reflects the early simulated germination these years. The sudden decrease of Z0 on January 1st is due to field clearing. Banizoumbou site: The slope change observed in August corresponds to a change in millet phenology according to the SarraH model. The millet Z0 values exhibit slightly larger values than in Abdourhamane Touré et al. (2011) and Pierre et al. (2014a). This is mainly due to the fact that the present study considers a longer period of the year. 16 16 Figure 8: Millet measurements (stars) and rangeland and millet parameterized Z0 based on vegetation mass simulated by the STEP model and based on millet height according to the SarraH model at Banizoumbou from 2006 to 2008. Figure 8: Millet measurements (stars) and rangeland and millet parameterized Z0 based on vegetation mass simulated by the STEP model and based on millet height according to the SarraH model at Banizoumbou from 2006 to 2008. As in Agoufou, the rangeland Z0 is slightly higher than that of millet field during the dry season, but the difference is smaller. Similarly, there is a time shift in plant growth between the two cover types, except in 2006 because of an earlier millet simulated germination. In this year, according to the SarraH model, the first millet plants that germinate survive the early season water stress, whereas STEP simulates a second germination after the stress period. The rangeland Z0 in Banizoumbou are lower than in Agoufou rangeland because of a lower grass mass simulated by the STEP model. Conversely, the Agoufou millet Z0 does not reach the maximum value of 0.1 m (Figure 6), because the simulated millet cycle at Agoufou is shorter (96 and 89 days in 2006 and 2007) than at Banizoumbou (115, 110 and 116 days for 2006, 2007 and 2008). This mainly reflects the meteorological difference between the two sites (higher temperature and lower rainfall in Agoufou than in Banizoumbou, and different length of the photoperiodic stage due to difference in latitude between the two sites). This difference is less sensitive in the case of the rangeland because degree-days are not considered in the STEP model. 17 17 Figure 9: Vegetation cover fraction estimated from the outputs of the STEP and SarraH models at Banizoumbou from 2006 to 2008. Figure 9: Vegetation cover fraction estimated from the outputs of the STEP and SarraH models at Banizoumbou from 2006 to 2008. Vegetation cover fractions in Banizoumbou show similar patterns than those in Agoufou, with larger values for rangeland than for millet – except at vegetation maximum. Agoufou site: The simulated threshold friction velocities (ut) exhibit strong seasonal dynamics (Figure 10). For both millet and grass, except for the failed millet crop in 2008, wind erosion is completely inhibited from approximately August to November because of an important vegetation cover and roughness, combined with less frequent strong winds and a more frequently wet soil. During rainfall events, which occur from June to September, soil moisture induces abrupt and brief increases of the threshold friction velocity by about a factor of 2 compared to baseline values. Over the 3-year period, ut* is usually higher in the rangeland than in the cropland. ut* reaches minimum values of about 0.48 m.s-1 in the rangeland and 0.45 m.s-1 in the millet field, with significant interannual variability: in the rangeland, the annual minimum threshold is reached in June (8th) for year 2006 (0.62 m.s-1), and in July (11th and 2nd) for years 2007 (0.54 m.s-1) and 2008 (0.48 m.s-1). These values are higher than the values of 0.26 to 0.49 m.s-1 reported by Tidjani et al. (2009) on a heavily grazed dune, probably because the Agoufou site is less grazed than their study site. In the millet field, the annual minimum threshold is reached in July (7th and 25th) for years 2006 (0.48 m.s-1) and 2007 (0.47 m.s-1), thus 2 to 4 weeks later than for the rangeland. In 2008, no millet growth is simulated and the threshold friction velocity in the millet field is only influenced by litter degradation. 18 Figure 10: Threshold wind friction velocity and wind friction velocity for the rangeland and the millet field at Agoufou from 2006 to 2008, based on vegetation simulations. Figure 10: Threshold wind friction velocity and wind friction velocity for the rangeland and the millet field at Agoufou from 2006 to 2008, based on vegetation simulations. In line with previous studies dealing with wind erosion on Sahelian surfaces (Rajot, 2001; Abourhamane Touré et al., 2011; Ikazaki et al., 2011), most of the horizontal flux occurs during the late dry and early rainy seasons, for both cover types (Figure 11). At this period of the year, the vegetation is mainly composed of standing straws and litter in the rangeland, and of litter in the millet field. The lower ut* of the millet field leads to greater and more frequent wind erosion events than for the rangeland. Agoufou site: The interannual variability is also linked to the frequency of strong winds: respectively 1.4 %, 1.2 % and 1.7 % of the wind speed data (at 3.5 m height) are higher than 7 m.s-1 in 2006, 2007 and 2008. Overall, these results are consistent with the measurements performed in Niger by Rajot (2001, Table 1), who obtained a maximum horizontal flux of 600 kg.m-1.yr-1 for a millet field. Cumulative horizontal fluxes over the 3 years are 1217 kg.m-1 for the rangeland and 1749 kg.m-1 for the millet field (Table 2). Each year, the horizontal flux is greater in the millet field than in the rangeland. These flux values exhibit a strong interannual variability, with the largest horizontal fluxes occurring during the driest year, 2008. Simulated straw and litter masses in June depend on the productivity of the previous year. Peak biomass is lower in 2007 than in 2005 (not shown) and 2006, therefore soil protection is lower in June for the year 2008. The interannual variability is also linked to the frequency of strong winds: respectively 1.4 %, 1.2 % and 1.7 % of the wind speed data (at 3.5 m height) are higher than 7 m.s-1 in 2006, 2007 and 2008. Overall, these results are consistent with the measurements performed in Niger by Rajot (2001, Table 1), who obtained a maximum horizontal flux of 600 kg.m-1.yr-1 for a millet field. Table 2: Simulated horizontal fluxes (kg.m-1.yr-1) for the rangeland and the millet field at Agoufou from 2006 to 2008. 2006 2007 2008 total Millet field 376 288 1085 1749 Rangeland 93 219 905 1217 Simulated horizontal fluxes (kg.m-1.yr-1) for the rangeland and the millet field at Agoufou 06 to 2008 Table 2: Simulated horizontal fluxes (kg.m-1.yr-1) for the rangeland and the millet from 2006 to 2008. Agoufou site: During the 3 years, there are 17 days with a horizontal flux greater than 1 kg.m-1 in the rangeland, against 40 in the millet field. The largest events are caused by gust fronts from deep convection occurring nearby and usually bringing rainfall. Each year, the largest event occurring in the rangeland contributes to 40% to 85% in mass of the annual horizontal flux. During these events, wind speed, averaged over 15 minutes, reaches values of about 16 m.s-1 at 3.5 m above the surface. Such events occur on June 10th in 2006, on July 4th in 2007 and on July 7th in 2008. These dates also correspond to the annual largest events simulated in the millet field with similar absolute values but a lesser contribution to the annual horizontal flux (only 20 to 44% in mass depending on the year). The events of July 4th, 2007 and July 7th, 2008, correspond to high wind speed from a gust front not followed by rain. After the rainy season in 2008, saltation still occurs on the millet field because no millet has grown. However, these saltation events are very low (34 kg.m-1 from August to December) compared to the horizontal flux simulated for the May-July period (1052 kg.m-1), because of the inhibition effect of soil moisture during the late rainy season and less frequent strong winds during the early dry season. 19 19 Figure 11: Simulated daily horizontal flux for the rangeland and the millet field at Agoufou from 2006 to 2008. Figure 11: Simulated daily horizontal flux for the rangeland and the millet field at Agoufou from 2006 to 2008. Figure 11: Simulated daily horizontal flux for the rangeland and the millet field at Agoufou from 2006 to 2008. Cumulative horizontal fluxes over the 3 years are 1217 kg.m-1 for the rangeland and 1749 kg.m-1 for the millet field (Table 2). Each year, the horizontal flux is greater in the millet field than in the rangeland. These flux values exhibit a strong interannual variability, with the largest horizontal fluxes occurring during the driest year, 2008. Simulated straw and litter masses in June depend on the productivity of the previous year. Peak biomass is lower in 2007 than in 2005 (not shown) and 2006, therefore soil protection is lower in June for the year 2008. Banizoumbou site: As in Agoufou, the threshold friction velocities are usually greater for the rangeland than for the cropland, and wind erosion is completely inhibited at vegetation maximum (Figure 12). ut* does not take soil moisture into account because it was not monitored at Banizoumbou, thus the corresponding curve (Figure 12) is smoother than for Agoufou (Figure 10). However, in the simulations, wind erosion is inhibited after rainfall (see 2.6). Threshold friction velocities show a slight interannual variability, with minimum values of 0.49 m.s-1, 0.44 m.s-1 and 0.50 m.s-1 being reached in the rangeland on June 5th and 20th, and on May 25th for years 2006, 2007 and 2008 respectively. In the millet field, minimum ut* of 0.49 m.s-1, 0.46 m.s-1 and 0.49 m.s-1 are respectively reached on June 10th, June 17th and June 9th for years 2006, 2007 and 20 2008, thus several days later than in the rangeland, except in 2007. Values for millet are slightly higher than those reported by Abdourhamane Touré et al. (2011). These authors estimated values of 0.46 m.s-1 in April to 0.36 m.s-1 in July from measurements of soil particles impacts, while the present study yields approximately 0.56 m.s-1 to 0.48 m.s-1 for the same periods. Figure 12: Threshold wind friction velocity and wind friction velocity for the rangeland and the millet field at Banizoumbou from 2006 to 2008, based on vegetation simulations. Figure 12: Threshold wind friction velocity and wind friction velocity for the rangeland and the millet field at Banizoumbou from 2006 to 2008, based on vegetation simulations. Also in line with previous studies, most of the erosion takes place from May to July (Figure 13), when friction velocity can exceed threshold friction velocity. As in Agoufou, wind erosion events are more frequent in the millet field – particularly in May – than in the rangeland, but the largest events occur at the same dates for both surfaces. Over the 3 years, there are 38 days with a simulated horizontal flux greater than 1 kg.m-1 in the rangeland, against 47 in the millet field, to be compared to 60 according to the measurements performed in the millet field (Abdourhamane Touré et al., 2011). In 2006, the largest event in the rangeland (July 5th) or the two largest events in the millet field (July 5th and June 15th) contribute more than 50% in mass to the annual horizontal flux. Banizoumbou site: Similarly, the strongest modeled event takes place on May 26th for both surfaces in 2007, while in 2008 the simulation yields a strongest daily horizontal flux on July 4th in the millet field and on May 20th in the rangeland. This high horizontal flux on July 4th corresponds to wind speed ranging between 10 m.s-1 and 18 m.s-1 at 2.5 m height and lasting approximately 1 hour until the rain starts. For this event, the threshold friction velocity is much stronger in the rangeland (where the vegetation growth has already started) than in the millet field. Each year, at least the two largest events simulated in the millet field are among the 5 largest measured horizontal fluxes. In other words, simulations are consistent with the dynamics of the main erosion events. The difference between horizontal flux from the rangeland and from the millet field is mainly due to the time shift between the two vegetation cover types (see Figure 4), which illustrates the high sensitivity of wind erosion to the vegetation dynamics at this period. 21 21 Figure 13: Simulated daily horizontal flux for the rangeland and the millet field at Banizoumbou from 2006 to 2008. Figure 13: Simulated daily horizontal flux for the rangeland and the millet field at Banizoumbou from 2006 to 2008. In terms of annual cumulated mass, the agreement between measured and modeled horizontal fluxes in the millet field is satisfying (Figure 13 and Table 3). The annual amounts increase through years, but the strong value simulated for the millet in 2008 is mainly due to the large event on July 4th. This simulated event is not caused by the underestimation of millet height at this period (Figure 5), since a simulation performed with larger millet heights yields an important event too: the horizontal flux is 226 kg.m-1 with a millet height of 0.35 m (taken from the measurements), compared to the original 253 kg.m-1 with a millet height of 0.20 m (parameterized from vegetation model outputs). Total horizontal fluxes in 2006 are sensibly lower than in the other years: strong winds (>10 m.s-1) are indeed less frequent in 2006 (0.08%) than in 2007 and 2008 (0.11% for both years). The total simulated horizontal flux from the millet field is higher than that from the rangeland over the 3- year period. 4. Concluding remarks This study proposes a modeling approach to quantify and compare sediment horizontal fluxes from Sahelian croplands and rangelands, using two vegetation growth models and a wind erosion model. Cropping and pastoral practices have been taken into account, as well as the phenological differences between annual grass and millet. Simulation results reproduce satisfyingly the available measurements. As a main result, the horizontal flux appears to be higher in croplands than in rangelands by approximately a factor 1.5 in mass, on average over 3 years. It suggests that land use and land use change may have an impact on Sahelian dust emissions, both because of the expansion of cropped land and because of evolving farming practices (e.g. collection of millet stover). The difference between the two surface types is mainly due to the delayed increase in vegetation mass of about 2 to 3 weeks in millet fields compared to annual grass in rangelands, and to a weaker soil surface protection in the cropped fields at the end of the dry season. The time shift between grass and millet is also partly due to the scattering of millet plants, which are sown in hills about a meter apart from each other, and to the weeding which is systematically carried out once or twice a year for millet fields. These practices result in a rather low vegetation mass (per surface unit) at the early stages of millet growth. This delay in vegetation growth occurs when (i) the surface is the least protected against wind erosion and (ii) high wind speeds are frequently recorded. The importance of the amount of dry vegetation (standing straws and litter laying down) at the end of the dry season for wind erosion was also pointed in Mongolian grasslands (Nandintsetseg and Shinoda, 2015), where the rainy season growth has a strong influence on the wind erosion occurring during the following dry season. As a consequence, cropping and pastoral practices and the changes thereof have played in the recent past, and could play in the future, a significant role in the changes in wind erosion for such surface types. Althgough the simulations of vegetation and surface characteristics (Z0) proved to be in good agreement with field observations, there is room for improvement especially for the period when most of wind erosion occurs, i.e. the late dry season and the early rainy season (May-June). Banizoumbou site: It is not the case in 2006, mainly because of the early start of millet growth, while the rangeland underwent a false-start and thus a delayed growth. In other words, the simulated horizontal flux from the millet field might be underestimated in 2006 because of an earlier start of millet growth in the simulations compared to the in situ measurements (Figure 5). Overall, phenology, especially in the early stages, as well as field management practices (including grazing) are important factors for wind erosion simulations in the Sahel, possibly more critical for erosion than for rangeland and cropland productivity modeling. Table 3: Simulated horizontal fluxes (kg.m-1.yr-1) for the rangeland and the millet field at Banizoumbou from 2006 to 2008. 2006 2007 2008 Total Millet field (measurements) 269 576 619 1463 Millet field (simulations) 154 527 885 1565 Rangeland (simulations) 173 440 455 1069 : Simulated horizontal fluxes (kg.m-1.yr-1) for the rangeland and the millet field at mbou from 2006 to 2008. 22 4. Concluding remarks Indeed, dry vegetation then is critical but vegetation growth models where not originally designed to specifically simulate vegetation during this period. Modeling of straws, litter and crop residues disapearance rates in these models could be improved, particularly if specific measurements are carried out. Parameterizations of Z0 could also be improved using more complete datasets, describing the contributions of litter and standing vegetation more precisely. Remote sensing might be helpful to quantify dry vegetation for regional studies (Kergoat et al., 2015). Overall, there are reasons to think that the difference between millet fields and rangelands or fallows that are found in this study is on the lower edge of what can be found in the Sahel. First, accounting for woody plants, which is not done in these case studies, would probably reduce the simulated horizontal fluxes, particularly from rangelands and old fallows, where woody plants are more numerous. Indeed, several studies measured an almost negligible horizontal flux over fallows (Rajot, 2001; Bielders et al., 2004): the discrepancy with the present simulations might partly be due to the woody population. Second, the millet stems, in the present case studies, were not collected at harvest, in agreement with the local observations. However, there is a Sahel-wide tendency to use millet stems increasingly either for building or as fodder for livestock. Fields without residues are exposed to wind erosion much earlier during the dry season. Third, the Agoufou site supports a 23 relatively high number of grazing animals, since it is close to a permanent pond. A significant part of the pastoral Sahel is probably less intensely grazed in the dry season, due to distance from water supply. Alltogether, these statements imply that the difference between millet fields and rangelands may be higher in many places. There is a need to address these different issues, especially at a larger scale. The simulations are also sensitive to several physical parameters that are difficult to constrain, e.g. in the drag partition scheme (Pierre et al., 2014b). Thus, further studies focusing on the effects of Sahelian land use on wind erosion represent an interesting perspective to the present work. Acknowledgments This work has been supported by the research program CAVIARS (ANR-12-SENV-0007-01) from the French Agence Nationale de la Recherche. The authors thank the AMMA-CATCH observatory (African Monsoon Multidisciplinary Analysis/Couplage de l’Atmosphère Tropicale et du Cycle Hydrologique: www.amma-catch.org) for supporting the Agoufou field measurements. Solar radiation data at Banizoumbou were obtained from the Atmospheric Radiation Measurement (ARM) Program sponsored by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Climate and Environmental Sciences Division. We also thank Manuela Grippa for providing updates of the STEP model, Mamadou Diawara for providing the Agoufou livestock observations and Nogmana Soumaguel for providing the vegetation measurements at Agoufou. Appendix For stem litter, K2 is the sum of a degradation coefficient set to 0.011 (d-1) and the same trampling coefficient as for standing stem. For leaves litter, K4 is the sum of a degradation coefficient also set to 0 011 (d-1) th t li ffi i t f t di l d th f d i t k For stem litter, K2 is the sum of a degradation coefficient set to 0.011 (d-1) and the same trampling coefficient as for standing stem. For leaves litter, K4 is the sum of a degradation coefficient also set to 0.011 (d-1), the same trampling coefficient as for standing leaves, and the same feed intake coefficient as for standing leaves. The coefficients are derived to be consistent with field studies (Schlecht et al. 2001; Hiernaux and Ayantunde, 2004; Kergoat et al., 2015) and with the STEP model (Mougin et al., in prep.; Delon et al., 2015). The litter degradation coefficients are directly taken from Abdourahmane Toure et al. (2011). Given the scarcity of field data, the difficulty to infer experimental conditions like stocking rates from literature, the number of prescribed values was kept low, and taken as close as possible to the STEP values and Abdourahamane Toure et al. (2011) values. The STEP model has a more detailed representation of processes, since it includes the effect of soil moisture on degradation, and the effect of different livestock species. Parameters were ajusted to match Agoufou dry season data, which benefits from livestock census and dry season mass measurements (Mougin et al., in prep; Delon et al., 2015; Diawara, 2015). Appendix Dry-season submodels have been developped for the STEP (Delon et al., 2015) and SarraH models (this study) in order to simulate the evolution over time of dry vegetation masses in response to biotic and abiotic factors. For SarraH, the additional state variables are the masses of standing stems, standing leaves, stem litter and leaves litter. At harvest, standing stems and leaves are initialised from their 'green' equivalent (stems and leaves), which in turn are set to zero. According to a prescribed crop management scenario, all or parts of the stems and leaves can be taken out of the field. This corresponds to the increasing trend of collecting most of the crop residues, either stored as livestock fodder or used as construction material (fences, huts, thatch …). In this study, all stems and leaves remain in the field as residues after harvest. During the dry season, the standing masses decay because of herbivory, decomposition, and abrasion. In addition, they are converted to litter (of stem and of leaves), mostly by trampling but also because of wind. Litter is also subject to herbivory (including by livestock and termites), decomposition, and burrying by livestock trampling. Field preparation for the next crop season usually occurs in the late dry season. It consists in turning all standing residues into litter. Here the date of field clearing is set to January 1st. A set of differential equations is used to simulate these different processes: 24 (A1) (A2) (A3) (A4) (A1) (A2) (A3) (A4) (A1) (A2) (A3) (A4) The stem degradation coefficient is set to 0.001 (d-1) and the trampling coefficient is set to 0.003 x livestock (d-1). Livestock density is expressed in Tropical Livestock Unit per km². Similarly, for leaves, the degradation coefficient is set to 0.001 (d-1), the trampling coefficient is set to 0.003 x livestok (d-1), and there is an additional feed intake coefficient set to 0.005 x livestock (d-1). The stem degradation coefficient is set to 0.001 (d-1) and the trampling coefficient is set to 0.003 x livestock (d-1). Livestock density is expressed in Tropical Livestock Unit per km². Similarly, for leaves, the degradation coefficient is set to 0.001 (d-1), the trampling coefficient is set to 0.003 x livestok (d-1), and there is an additional feed intake coefficient set to 0.005 x livestock (d-1). Baron, C., B. Sultan, M. Balme, B. Sarr, and S. B. Traoré, 2005: From GCM grid cell to agricultural plot: Scale issues affecting modelling of climate impact. Philos. Trans. R. Soc. B: Biol. Sci., 360, 2095–2108. References Abdourhamane Touré, A., J. L. Rajot, Z. Garba, B. Marticorena, C. Petit, and D. Sebag, 2011: Impact of very low crop residues cover on wind erosion in the Sahel, Catena, 85, 205–214, doi: 10.1016/j.catena.2011.01.002. Abdourhamane Touré, A., J. L. Rajot, Z. Garba, B. Marticorena, C. Petit, and D. Sebag, 2011: Impact of very low crop residues cover on wind erosion in the Sahel, Catena, 85, 205–214, doi: 10.1016/j.catena.2011.01.002. Bagnold, R.A., 1941: The physics of blown sand and desert dunes, Methuen, London, 265pp. Bagnold, R.A., 1941: The physics of blown sand and desert dunes, Methuen, London, 265pp. Balme, M., T. Vischel, T. Lebel, C. Peugeot and S. Galle, 2006: Assessing the water balance in the Sahel: Impact of small scale rainfall variability on ruoff. Part 1: Rainfall variability analysis, J. Hydrol., 331, 336-348. Baron, C., B. Sultan, M. Balme, B. Sarr, and S. B. Traoré, 2005: From GCM grid cell to agricultural plot: Scale issues affecting modelling of climate impact. Philos. Trans. R. Soc. B: Biol. Sci., 360, 2095–2108. 25 Bielders, C.L., J.P.A Lamers, and K. Michels, 2001a: Wind erosion technologies in the West African Sahel: The effectiveness of windbreaks, mulching and soil tillage, and the perspective of farmers, Ann. Arid Zone, 40, 369-394. Bielders, C.L., A. Vrieling, J.-L. Rajot, and E. Skidmore, 2001b: On-farm evaluation of field-scale soil losses by wind erosion under traditional management in the Sahel. In J.C. Ascough II and D.C. Flanagan (Eds). Soil erosion research for the 21st century, p. 494-497. Bielders, C. L., J.-L. Rajot, and M. Amadou, 2002: Transport of soil and nutrients by wind in bush fallow land and traditionally managed cultivated ﬁelds in the Sahel, Geoderma 109, 19–39. Bielders, C. L., J.-L. Rajot, and K. Michels, 2004: L’érosion éolienne dans le Sahel nigérien: influence des pratiques culturales actuelles et méthodes de lutte, Sécheresse, 15(1), 1-14. Boucher, O., et al., 2013: Clouds and Aerosols, In: Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the 5th Assessment Report of the IPCC, Cambridge Univ., Cambridge (UK), and New York (USA). Boulain, N., B. Cappelaere, D. Ramier, H. B. A. Issoufou, O. Halilou, J. Seghieri, F. Guillemin, J. Gignoux, and F. Timouk, 2009: Towards an understanding of coupled physical and biological processes in the cultivated Sahel - 2. Vegetation and carbon dynamics, J. Hydrol. 375 (1–2), 190–203. Breuning-Madsen, H., T.W. Awadzi, and G. References Lyngsie, 2015: Deposition of Nutrients from Harmattan Dust in Ghana, West Africa, Pedosphere, 25(4), 613-621. Chin, M., T. Diehl, Q. Tan, J. M. Prospero, R. A. Kahn, L. A. Remer, H. Yu, A. M. Sayer, H. Bian, I. V. Geogdzhayev, B. N. Holben, S. G. Howell, B. J. Huebert, N. C. Hsu, D. Kim, T. L. Kucsera, R. C. Levy, M. I. Mishchenko, X. Pan, P. K. Quinn, G. L. Schuster, D. G. Streets, S. A. Strode, O. Torres, and X.-P. Zhao, 2014: Multi-decadal aerosol variations from 1980 to 2009: a perspective from observations and a global model, Atmos. Chem. Phys., 14, 3657–3690, doi: 10.5194/acp-14-3657-2014. Cowie, S., P. Knippertz, and J. Marsham, 2013: Are vegetation-related roughness changes the cause of the recent decrease in dust emission from Sahel?, Geophys. Res. Lett., 40, 1868–1872, doi:10.1002/grl.50273. Dardel, C., L. Kergoat, P. Hiernaux, E. Mougin, M. Grippa, and C. J. Tucker, 2014: Re-greening Sahel: 30-years of remote sensing data and field observations (Mali, Niger), Remote Sens. Environ., 140, 350-364. Deacon, E. L., 1953: Vertical profiles of the mean wind in the surface layers of the atmosphere, Geophys., Mem. 91, Meteorol. Off., H. M. Stationery Off., U.K. Delon, C., E. Mougin, D. Serça, M. Grippa, P. Hiernaux, M. Diawara, C. Galy-Lacaux, and L. Kergoat, 2015: Modelling the effect of soil moisture and organic matter degradation on biogenic NO emissions from soils in Sahel rangeland (Mali), Biogeosciences, 12, 3253-3272. De Rosnay, P., C. Gruhier, F. Timouk, F. Baup, E. Mougin, P. Hiernaux, L. Kergoat, and V. Le Dantec, 2009: Multi-scale soil moisture measurements at the Gourma meso-scale site in Mali, J. Hydrol., 375, 241–252. 26 Diawara, M., 2015: Impact de la variabilité climatique au nord Sahel (Gourma) sur la dynamique des ressources pastorales et leurs conséquences sur les productions animales, 150 p., PhD thesis, Toulouse Univ. Diawara, M., 2015: Impact de la variabilité climatique au nord Sahel (Gourma) sur la dynamique des ressources pastorales et leurs conséquences sur les productions animales, 150 p., PhD thesis, Toulouse Univ. Dingkuhn, M., C. Baron, V. Bonnal, F. Maraux, and B. Sarr, 2003: Decision Support Tools for Rainfed Crops in the Sahel at the Plot and Regional Scales. In A Practical Guide to Decision-Support Tools for Agricultural Productivity and Soil Fertility Enhancement in Sub-Saharan Africa, IFDC, CTA; Struif- Bontkes, T.E., Wopereis, M.C.S., Eds.; International Fertilizer Development Center: Muscle Shoals, AL, USA, pp. 127–139. Doherty O.M., N. Riemer, and S. References Hameed, 2014: Role of the convergence zone over West Africa in controlling Saharan mineral dust load and transport in the boreal summer, Tellus, B 2014, 66, 23191, doi: 10.3402/tellusb.v66.23191. Fécan, F., B. Marticorena, and G. Bergametti, 1999: Parameterization of the increase of the aeolian erosion threshold wind friction velocity due to soil moisture for semiarid areas, Ann. Geophys., 17, 149–157. Frappart F., P. Hiernaux, F. Guichard, E. Mougin, L. Kergoat, M. Arjounin, F. Lavenu, M. Koite, J.E. Paturel, and T. Lebel, 2009: Rainfall regime across the Sahel band in the Gourma region, Mali, J. Hydrol., 375 (1-2), 128-142, doi:10.1016/j.jhydrol.2009.03.007. Fryrear, D. W., 1986: A field dust sampler, J. Soil Water Conserv., 41, 117-120. Gillette, D. A., 1979: Environmental factors affecting dust emission by wind erosion, Saharan Dust, Ed. C. Morales, 71-94, John Winley, New York. Guichard, F., L. Kergoat, E. Mougin, F. Baup, P. Hiernaux, and F. Lavenu, 2009: Surface thermodynamics and radiative budget in the Sahelian Gourma: Seasonal and diurnal cycles, J. Hydrol., 375(1–2), 161–177, doi:10.1016/j.jhydrol.2008.09.007. Haywood, J. M. and O. Boucher, 2000: Estimates of the direct and indirect radiative forcing due to tropospheric aerosols: A review, Rev. Geophys., 38, 513–543. Hermann L., R. Jahn, T. Maurer, 2010: Mineral dust around the Sahara, from source to sink. A review with emphasis on contributions of the German Soil Science Community in the last twenty years. J. Plant. Nutr. Soil Sci., 173, 811-821. Hiernaux P., and A. Ayantunde, 2004: The Fakara: a semi-arid agro-ecosystem under stress. First phase (July 2002 - June 2004) of the DMP-GEF Program (GEF/2711-02-4516) ILRI, www.jircas.affrc.go.jp/project/africa_dojo/Metadata/grad_research/15.pdf Hiernaux, P., and M. D. Turner, 2002: The influence of farmer and pastoralist management practices on desertification processes in the Sahel, in Global Desertification: Do Humans cause deserts?, Ed. J.F. Reynolds and D. M. Stafford Smith, Dahlem Univ. Press. , 135:148. Ikazaki, K, H. Shinjo, U. Tanaka, S. Tobita, S. Funakawa and T. Kosaki, 2011: “Fallow Band system”, a land management practice for controlling desertification and improving crop production in the Sahel, 27 West Africa. 1. Effectiveness in desertification control and soil fertility improvement, Soil Sci. Plant Nutr., 57 (4), 573-586. IPCC (Intergovernmental Panel on Climate Change), 2007: Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the IPCC. Jarlan L., E. Mougin, P. L. Frison, P. Mazzega, and P. Mahowald, N.M., S. Kloster, S. Engelstaedter, J. K. Moore, S. Mukhopadhyay, J. R. McConnell, S. Albani, S. C. Doney, A. Bhattacharya, M. A. J. Curran, M. G. Flanner, F. M. Hoffman, D. M. Lawrence, References Hiernaux, 2002 : Analysis of ERS wind scatterometer time series over Sahel (Mali), Remote Sens. Environ., 81(2-3), 404-415. Jickells, T. D., et al. (2005), Global iron connections between desert dust, ocean biogeochemistry, and climate, Science, 38(5718), 67–71, doi: 10.1126/science.1105959. Kawamura, R., 1951: Study of sand movement by wind, Univ. Tokyo, Institute for Science and Technology, Report 5. Kergoat, L., P. Hiernaux, C. Dardel, C. Pierre, F. Guichard, and A. Kalilou, 2015: Dry-season vegetation mass and cover fraction from SWIR1.6 and SWIR2.1 band ratio: Ground-radiometer and MODIS data in the Sahel, Int. J. Appl. Earth Obs. Geoinf., 39, 56–64, doi 10.1016/j.jag.2015.02.01 King, J., W. C. Nikling and J. A. Gillies, 2005: Representation of vegetation and other non erodible elements in Aeolian shear stress partitioning models for predicting transport threshold, J. Geophys. Res., 110, doi: 10.1029/2004JF000281. Kouressy, M., M. Dingkuhn, M. Vaksmann, and A.B. Heinemann, 2008: Adaptation to diverse semi- arid environments of sorghum genotypes having different plant type and sensitivity to photoperiod. Agric. For. Meteorol., 148, 357–371. Lebel, T., and A. Ali, 2009: Recent trends in the Central and Western Sahel rainfall regime (1990- 2007), J. Hydrol., 375, 52-64. Levy, P.E., and P. G. Jarvis, 1999: Direct and indirect measurements of LAI in millet and fallow vegetation in HAPEX-Sahel, Agric. For. Meteorol., 97, 199-212. Li, J., G. S. Okin, J. Tatarko, N. P. Webb, and J. E. Herrick, 2014: Consistency of wind erosion assessments across land use and land cover types: a critical analysis, Aeolian Res., 15, 253-260. Lo Seen D., E. Mougin, S. Rambal, A. Gaston, and P. Hiernaux, 1995: A regional Sahelian grassland model to be coupled with multispectral satellite data. II. Towards the control of its simulations by remotely sensed indices, Remote Sens. Environ., 52, 194-206. Lyles, L., and B. E. Allison, 1976: Wind erosion: the protective role of simulated standing stubble. Trans. ASAE, 19(l), 61-64. Mahowald, N. M., G. D. Rivera Rivera, and C. Luo, 2004: Comment on “Relative importance of climate and landuse in determining present and future global soil dust emission” by I. Tegen et al., Geophys. Res. Lett., 31, L24105, doi: 10.1029/2004GL021272. Mahowald, N.M., S. Kloster, S. Engelstaedter, J. K. Moore, S. Mukhopadhyay, J. R. McConnell, S. Albani, S. C. Doney, A. Bhattacharya, M. A. J. Curran, M. G. Flanner, F. M. Hoffman, D. M. Lawrence, 28 K. Lindsay, P. A. Mayewski, J. Neff, D. Rothenberg, E. Thomas, P. E. Thornton, and C. S. References Zender, 2010: Observed 20th century desert dust variability: impact on climate and biogeochemistry, Atmos. Chem. Phys., 10, 10875–10893, doi: 10.5194/acp-10-10875-2010. Marteau, R., B. Sultan, V. Moron, A. Alhassane, and C. Baron, 2011: The onset of the rainy season and farmers’ sowing strategy for pearl millet cultivation in Southwest Niger. Agric. For. Meteorol., 151, 1356–1369. Marticorena, B., and G. Bergametti, 1995: Modeling the atmospheric dust cycle: 1. Design of a soil derived dust production scheme, J. Geophys. Res., 100, 16415–16430, doi: 10.1029/95JD00690. Marticorena, B., M. Kardous, G. Bergametti, Y. Callot, P. Chazette, H. Khatteli, S. Le Hégarat-Mascle, M. Maillé, J-L. Rajot, D. Vidal-Madjar, and M. Zribi, 2006: Surface and aerodynamic roughness in arid and semi arid areas and their relation to radar backscatter coefficient, J. Geophys. Res., 111, F03017, doi: 10.1029/2006JF000462. Mougin, E., D. Lo Seen, S. Rambal, A. Gaston and P. Hiernaux, 1995: A regional Sahelian grassland model to be coupled with multispectral satellite data. I: Model description and validation, Remote Sens. Environ., 52, 181-193. Mougin, E., P. Hiernaux, L. Kergoat, M. Grippa, P. de Rosnay, F. Timouk, V. Le Dantec, V. Demarez, F. Lavenu, M. Arjounin, T. Lebel, N. Soumaguel, E. Ceschia, B. Mougenot, F. Baup, F. Frappart, P.L. Frison, J. Gardelle, C. Gruhier, L. Jarlan, S. Mangiarotti, B. Sanou, Y. Tracol, F. Guichard, V. Trichon, L. Diarra, A. Soumaré, M. Koité, F. Dembélé, C. Lloyd, N.P. Hanan, C. Damesin, C. Delon, D. Serça, C. Galy-Lacaux, J. Seghieri, S. Becerra, H. Dia, F. Gangneron, and P. Mazzega, 2009: The AMMA Gourma observatory site in Mali: Relating climatic variations to changes in vegetation, surface hydrology, fluxes and natural resources, J. Hydrol., 375(1–2), 14–33, doi: 10.1016/ j.jhydrol.2009.06.045. Mougin, E., P. Hiernaux, L. Kergoat, M. Grippa, P. de Rosnay, F. Timouk, V. Le Dantec, V. Dem Lavenu, M. Arjounin, T. Lebel, N. Soumaguel, E. Ceschia, B. Mougenot, F. Baup, F. Frapp Mougin E., V. Demarez, M. Diawara, P. Hiernaux, N. Soumaguel, and A. Berg, 2014 : Estimation of LAI, fCover and fAPAR of Sahel rangelands (Gourma, Mali), Agric. For. Meteorol., 198-199, 155-167. Mougin E., P. Hiernaux, M. Grippa, C. Delon, L. Kergoat, and V. Le Dantec, in prep.: Modelling seasonal and interannual vegetation dynamics in Sahel: a reconsideration of the STEP model. Moulin, C., and I. Chiapello, 2006: Impact of human-induced desertification on the intensification of Sahel dust emission and export over the last decades, Geophys. Res. Lett., 33, L18808, doi: 10.1029/2006GL025923. Pasquill, F., 1950 : The aerodynamic drag of grassland, Proc. R. Soc., Ser. A., 202, 143-153. References Mulitza, S., D. Heslop, D. Pittauerova, H.W. Fisher, I. Meyer, J-B. Stuut, M. Zabel, G. Mollenhauer, J.A. Collins, H. Juhnert and M. Schulz, 2010: Increase in African dust flux at the onset of commercial agriculture in the Sahel region, Nature, 466, doi: 10.1038/nature09213. Nandinstetseg, B., and M. Shinoda, 2015: Land surface memory effects on dust emission in a Mongolian temperate grassland, J. Geophys. Res., doi: 10.1002/2014/JG002708. Pasquill, F., 1950 : The aerodynamic drag of grassland, Proc. R. Soc., Ser. A., 202, 143-153. 29 Pierre, C., G. Bergametti, B. Marticorena, E. Mougin, T. Lebel, and A. Ali, 2011: Pluriannual comparisons of satellite-based rainfall products over the Sahelian belt for seasonal vegetation modeling, J. Geophys. Res., 116, D18201, doi: 10.1029/2011JD016115. Pierre, C., G. Bergametti, B. Marticorena, E. Mougin, T. Lebel, and A. Ali, 2011: Pluriannual comparisons of satellite-based rainfall products over the Sahelian belt for seasonal vegetation modeling, J. Geophys. Res., 116, D18201, doi: 10.1029/2011JD016115. Pierre, C., G. Bergametti, B. Marticorena, E. Mougin, C. Bouet, and C. Schmechtig, 2012: Impact of vegetation and soil moisture seasonal dynamics on dust emissions over the Sahel, J. Geophys. Res., 117 (D6), doi: 10.1029/2011JD016950. Pierre, C., G. Bergametti, B. Marticorena, A. Abdourhamane Touré, J-L. Rajot, and L. Kergoat, 2014a: Modeling wind erosion flux and its seasonality from a cultivated Sahelian surface: A case study in Niger, Catena, 122, 61-71, doi: 10.1016/j.catena.2014.06.006. Pierre, C., G. Bergametti, B. Marticorena, L. Kergoat, E. Mougin, and P. Hiernaux, 2014b: Description of a heterogeneous vegetation cover and its drag partition for the estimation of dust emission, J. Geophys. Res., 119 (10), 2291-2313, doi: 10.1002/2014JF003177. Priestley, C.H.B., 1959: Turbulent Transfer in the lower atmosphere, 130 pp., Univ. Chicago Press, Chicago. Rajot, J.-L., 2001: Wind blown sediment mass budget of Sahelian village land units in Niger, Bull. Soc. Geol. Fr., 172, 523–531, doi: 10.2113/ 172.5.523. Ridley D., C. Heald, and J. Prospero, 2014: What controls the recent changes in African mineral dust aerosol across the Atlantic? Atmos. Chem. Phys. Discus. 14(3), 3583–3627. Rockstrom, J., and A. de Rouw, 1997: Water, nutrients, and slope position in on-farm pearl millet cultivation in the Sahel, Plant Soil, 195, 311-327. Sabre, M., 1997: Etude dynamique du processus d’émission de poussières désertiques: impact sur le fractionnement physico-chimique entre sol et aérosol, PhD thesis, Paris VII Univ. Saugier, B., and E. A. Ripley, 1978: Evaluation of the aerodynamic method of determining fluxes over natural grassland, Q. J. R. Meteorol. References Soc., 104, 257-270. Schlecht E., I. Kadaouré, F. Graef, C. Hülsebusch, F. Mahler and K. Becker, 2001: Agro-pastoral Farming Systems of Western Niger, Die Erde, 132: 399-418. Schlecht, E., P. Hiernaux, F. Achard, and M. Turner, 2004: Livestock related nutrient budgets within village territories in western Niger, Nutr. Cycl. Agroecosys., 68, 199–211. Siddoway, F.H., W.S. Chepil, and D.V. Armbrust, 1965: Effect of kind, amount, and placement of residue on wind erosion control, Trans. ASAE, 8(3), 327-331. Stanelle, T., I. Bey, T. Raddatz, C. Reick, and I. Tegen, 2014: Anthropogenically induced changes in twentieth century mineral dust burden and the associated impact on radiative forcing, J. Geophys. Res. Atmos., 119, doi: 10.1002/2014JD022062. Sterk, G., 2003: Causes, consequences and control of wind erosion in Sahelian Africa: a review, Land Degrad. Develop., 14, 95-108, doi: 10.1002/ldr.526. 30 Swap, R., M. Garstang, S. Greco, R. Talbot and P. Källberg, 1992: Saharan dust in the Amazon Basin, Tellus, 44B, 133-149. Swap, R., M. Garstang, S. Greco, R. Talbot and P. Källberg, 1992: Saharan dust in the Amazon Basin, Tellus, 44B, 133-149. Tegen, I., and I. Fung, 1995: Contribution to the atmospheric mineral load from land surface modification, J. Geophys. Res., 100(D9), 18707–18726, doi: 10.1029/95JD02051. Tegen, I., P. Hollrig, M. Chin, I. Fung, D., Jacob, and J. Penner, 1997: Contribution of different aerosol species to the global aerosol extinction optical thickness: Estimates from model results, J. Geophys. Res., 102, 23895-23915, DOI: 10.1029/97JD01864. Tegen, I., M. Werner, S. P. Harrison, and K. E. Kohfeld, 2004: Relative importance of climate and landuse in determining present and future global soil dust emission, Geophys. Res. Lett., 31, L05105, doi: 10.1029/ 2003GL019216. Tidjani, A. D., C. L. Bielders, and K. J-M. Ambouta, 2009: Dynamique saisonnière des paramètres déterminant l’érosion éolienne sur les pâturages dunaires du Niger oriental, Geo-Eco-Trop., 33, 39- 56. Tracol, Y., E. Mougin, P. Hiernaux, and L. Jarlan, 2006: Testing a Sahelian grassland functioning model against herbage mass measurements, Ecol. Model., 193, 437–446. Traoré, S.B., A. Alhassane, B. Muller, M. Kouressy, and L. Somé, 2011: Characterizing and modeling the diversity of cropping situations under climatic constraints in west africa. Atmos. Sci. Lett., 12, 89– 95. Velluet, C., J. Demarty, B. Cappelaere, I. Braud, H. B. A. Issoufou, N. Boulain, D. Ramier, I. Mainassara, G. Charvet, M. Boucher, J. P. Chazarin, M. Oï, H. Yahou, B. Maidaji, F. Arpin-Pont, N. Benarrosh, A. Mahamane, Y. Nazoumou, G. Favreau, and J. White, B.R., 1979: Soil transport by winds on Mars, J. Geophys. Res., 84, 4643- 4651. References Seghieri, 2014: Building a field- and model-based climatology of local water and energy cycles in the cultivated Sahel – annual budgets and seasonality, Hydrol. Earth syst. Sci., 18, 5001-5024, doi: 10.5194/hess-18-5001-2014. Walker, I. J., 2005: Physical and logistical considerations of using ultrasonic anemometers in aeolian sediment transport research, Geomorphology, 68(1), 57-76. White, B.R., 1979: Soil transport by winds on Mars, J. Geophys. Res., 84, 4643- 4651. 31 31
https://openalex.org/W3011249203	https://www.frontiersin.org/articles/10.3389/fbioe.2020.00200/pdf	English	null	Antioxidant Nanotherapies for the Treatment of Inflammatory Diseases	Frontiers in bioengineering and biotechnology	2,020	cc-by	16,091	REVIEW REVIEW published: 18 March 2020 doi: 10.3389/fbioe.2020.00200 published: 18 March 2020 doi: 10.3389/fbioe.2020.00200 Antioxidant Nanotherapies for the Treatment of Inﬂammatory Diseases Chen-Wen Li 1†, Lan-Lan Li 1,2†, Sheng Chen 3, Jian-Xiang Zhang 1 and Wan-Liang Lu 4* 1 Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, Chongqing, China, 2 Department of Chemistry, College of Basic Medicine, Third Military Medical University, Chongqing, China, 3 Department of Pediatrics, Southwest Hospital, Third Military Medical University, Chongqing, China, 4 Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing, China Edited by: Carlotta Pucci, Istituto Italiano di Tecnologia, Italy Reviewed by: Ruibing Wang, University of Macau, China Anne Vejux, Université de Bourgogne, France Correspondence: Sheng Chen chenshengerk@163.com Jian-Xiang Zhang jxzhang1980@gmail.com Wan-Liang Lu luwl@bjmu.edu.cn Reviewed by: Ruibing Wang, University of Macau, China Anne Vejux, Université de Bourgogne, France Correspondence: Sheng Chen chenshengerk@163.com Jian-Xiang Zhang jxzhang1980@gmail.com Wan-Liang Lu luwl@bjmu.edu.cn Keywords: oxidative stress, nanoparticles, antioxidant nanotherapies, ROS, inﬂammatory diseases †These authors have contributed equally to this work †These authors have contributed equally to this work †These authors have contributed equally to this work †These authors have contributed equally to this work Antioxidant Nanotherapies for the Treatment of Inﬂammatory Diseases Chen-Wen Li 1†, Lan-Lan Li 1,2†, Sheng Chen 3, Jian-Xiang Zhang 1 and Wan-Liang Lu 4* 1 Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, Chongqing, China, 2 Department of Chemistry, College of Basic Medicine, Third Military Medical University, Chongqing, China, 3 Department of Pediatrics, Southwest Hospital, Third Military Medical University, Chongqing, China, 4 Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing, China Reactive oxygen species (ROS) are essential in regulating various physiological functions. However, overproduction of ROS is implicated in the pathogenesis of various inﬂammatory diseases. Antioxidant therapy has thus represented an effective strategy for the treatment of oxidative stress relevant inﬂammatory diseases. Conventional anti-oxidative agents showed limited in vivo effects owing to their non-speciﬁc distribution and low retention in disease sites. Over the past decades, signiﬁcant achievements have been made in the development of antioxidant nanotherapies that exhibit multiple advantages such as excellent pharmacokinetics, stable anti-oxidative activity, and intrinsic ROS-scavenging properties. This review provides a comprehensive overview on recent advances in antioxidant nanotherapies, including ROS-scavenging inorganic nanoparticles, organic nanoparticles with intrinsic antioxidant activity, and drug-loaded anti-oxidant nanoparticles. We highlight the biomedical applications of antioxidant nanotherapies in the treatment of different inﬂammatory diseases, with an emphasis on inﬂammatory bowel disease, cardiovascular disease, and brain diseases. Current challenges and future perspectives to promote clinical translation of antioxidant nanotherapies are also brieﬂy discussed. INTRODUCTION Specialty section: This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology Received: 10 January 2020 Accepted: 28 February 2020 Published: 18 March 2020 Specialty section: This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology Reactive oxygen species (ROS) are chemical species containing unpaired electrons of oxygen and oxidizing agents that are readily turned to free radicals, such as hydroxyl radical (•OH), superoxide anion radical (•O− 2 ), singlet oxygen (1O2), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) (Bayir, 2006; D’Autréaux and Toledano, 2007; Nosaka and Nosaka, 2017). Living organisms systematically keep a balance between ROS and antioxidant defenses including exogenous and endogenous antioxidants, such as vitamin C, vitamin E, glutathione, superoxide dismutase (SOD), catalase (CAT), and peroxiredoxins (PRXs). Basic levels of ROS play an essential role in the cellular signal transduction processes and maintain oxygen homeostasis in the physiological environment. However, excessive ROS can lead to oxidative stress, cause DNA fragmentation, protein oxidation, and lipid peroxidation, which are associated with various inﬂammatory diseases, such as cardiovascular diseases, inﬂammatory bowel disease (IBD), neurodegenerative diseases, asthma, diabetes, and arthritis (Andersen, 2004; Fraisl et al., 2009). Accordingly, antioxidant therapy has been recognized as a potentially plausible approach for treating ROS-related inﬂammatory diseases (Apostolova and Victor, 2015). Received: 10 January 2020 Accepted: 28 February 2020 Published: 18 March 2020 Keywords: oxidative stress, nanoparticles, antioxidant nanotherapies, ROS, inﬂammatory diseases Ceria Oxide Nanoparticles p Ceria oxide nanoparticles have been demonstrated to possess SOD-mimetic activity, resulting from the mixed valence states of Ce3+ and Ce4+ (Celardo et al., 2011; Bryant et al., 2016). It has become the most widely used inorganic nanomedicine for antioxidant therapy. At the nanoscale, Ce3+ and Ce4+ coexist on the surface of the Ce oxide lattice, and Ce4+ can be reduced to Ce3+ due to the presence of surface oxygen vacancies, therefore eﬀectively decreasing ROS levels. Previous studies suggested that the Ce3+/Ce4+ ratio has an important eﬀect on the antioxidant activity of ceria oxide nanoparticles, which is dominated by the microenvironment (Das et al., 2013). Li et al. reported a novel strategy to signiﬁcantly improve the superoxide-scavenging activity of ceria oxide nanoparticles (> 5 nm) with diﬀerent shapes and a negligible Ce3+/Ce4+ ratio through electron transfer (Li et al., 2015). Cerium oxide nanoparticles exhibited tissue protective actions in inﬂammatory diseases (Hirst et al., 2009), wound healing (Davan et al., 2012), photoreceptor degeneration (Cai et al., 2012), and neurodegenerative diseases (Kwon et al., 2016). Also, cerium oxide nanoparticles can accumulate in the liver and spleen following intravenous (i.v.) injection, showing Herein we aim to provide a comprehensive review on the recent progress in antioxidant nanoparticles for the treatment of inﬂammatory diseases. Diﬀerent antioxidant nanoparticles based on inorganic and/or organic materials as well as drug-loaded nanoparticles are ﬁrst introduced. Then we highlight biomedical applications of these nanoparticles in anti-inﬂammatory therapy, with emphasis on the treatment of IBD, cardiovascular disease, brain diseases, and other inﬂammatory diseases. Finally, major challenges regarding the clinical translation of these antioxidant nanotherapies are discussed. Fullerenes Fullerenes are the ﬁrst reported SOD-mimicking inorganic nanomaterials, representing the landmark in the development of ROS-scavenging inorganic nanoparticles. Fullerenes are three- dimensional carbon networks, and the most common form of fullerenes is buckminsterfullerene (C60) with 60 carbon atoms arranged in a sphere. C60 fullerene can behave as a free radical “sponge,” with each C60 molecule capable of quenching multiple radical species (Krusic et al., 1991; Markovic and Trajkovic, 2008). The molecular mechanism underlying antioxidant reactions of C60 compounds is that electron- deﬁcient regions of C60 cooperates with the attached malonyl groups to electrostatically guide and stabilize superoxide, thereby promoting its decomposition (Markovic and Trajkovic, 2008). C60(C(COOH)2)2 nanoparticles can be selectively internalized by oxidation-damaged cerebral endothelial cells, and greatly inhibit their apoptosis induced by ROS, which is related to the modulation of the c-Jun NH2-terminal kinase (JNK) signaling pathway (Lao et al., 2009). Several studies demonstrated that fullerenes can signiﬁcantly alleviate ROS-dependent neuronal injury induced by N-methyl-D-aspartate or K+ deﬁciency in vitro (Dugan et al., 1997; Ali et al., 2008). The neuroprotective eﬀects of fullerenes were also validated in cortical infarction induced by ischemia-reperfusion (Lin et al., 2002), amyotrophic lateral sclerosis in SOD mutant mice (Dugan et al., 1997), and Alzheimer’s disease in rats induced by amyloid-β25−35 peptide (Gordon et al., 2017). Treatment with fullerenes also inhibited mast cell function in vitro, showing potent therapeutic eﬀects on allergic asthma (Norton et al., 2012) and CCl4-mediated acute liver toxicity (Gharbi et al., 2005). Citation: Li C-W, Li L-L, Chen S, Zhang J-X and Lu W-L (2020) Antioxidant Nanotherapies for the Treatment of Inﬂammatory Diseases. Front. Bioeng. Biotechnol. 8:200. doi: 10.3389/fbioe.2020.00200 March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 1 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. Over the past decades, a number of natural and synthetic anti-oxidative compounds have been reported. For example, Edaravone is the only clinically approved low molecular weight radical scavenger used for the treatment of cerebral ischemic stroke, which has been proved to exert its therapeutic eﬀects through scavenging free radicals and upregulating endothelial NOS expression in the cerebral tissue, thus preventing lipid oxidation and reducing neuronal damage (Yamamoto et al., 1997; Edaravone Acute Infarction Study Group, 2003). Nevertheless, the clinical use of Edaravone has been limited due to its multiple adverse eﬀects such as hepatic and renal toxicity (Watanabe et al., 2008). Various adverse eﬀects of low molecular weight ROS scavengers are mainly due to their non-speciﬁc distribution, high renal clearance, and low delivery eﬃciency. The emergence of nanotechnology has considerably overcome these limitations and inspires the next wave of technological innovation in antioxidant therapy. Nanomedicines can remarkably improve the pharmacokinetics properties of antioxidative compounds and simultaneously decrease their side eﬀects. More excitingly, some types of nanomaterials, such as inorganic nanoparticles, possess inherent antioxidant activities, by directly reacting with ROS and/or mimicking the natural antioxidant enzymes, thereby showing powerful ROS-scavenging capability. Various ROS- scavenging and/or ROS-responsive antioxidative nanotherapies derived from organic materials, or inorganic/organic hybrid materials have also attracted extensive attention recently (Lu et al., 2016; Saravanakumar et al., 2017), yielding promising results to decrease oxidative damages in various animal models of diverse diseases. In addition, gas-generating (e.g., hydrogen) nanomaterials showed beneﬁcial therapeutic eﬀects in inﬂammatory diseases, due to their anti-inﬂammatory and anti- oxidative properties (He et al., 2017; Zhang B. et al., 2019). In this context, diﬀerent types of antioxidant nanotherapies have been constructed based on various materials, such as carbon, metal oxides, nanocrystals, lipids, and polymers, as well as a variety of newly developed materials (Petros and DeSimone, 2010). nanozymes by Yan in 2007 (Gao et al., 2007). ROS-scavenging inorganic nanoparticles show outstanding biological stability, versatile functionality, and regulatory activity. DIFFERENT ANTIOXIDANT NANOPARTICLES ROS-Scavenging Inorganic Nanoparticles Over the past decades, a variety of inorganic nanoparticles based on carbon, metal, and metal-organic frameworks (MOFs) with natural enzyme-like activities have been developed to decrease the damage induced by ROS in biomedical areas (Table 1). These enzyme-like antioxidative nanomaterials have been deﬁned as March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 2 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. TABLE 1 \| Representative ROS-scavenging inorganic nanoparticles. Nanoparticles Applications Evaluation models Administration routes References Fullerenes Cortical infarction In vivo in rats Intravenous or intracerebroventricular injection Lin et al., 2002 Amyotrophic lateral sclerosis In vivo in mice Loaded into mini-osmotic pumps and implanted into the peritoneum Dugan et al., 1997 Alzheimer’s disease In vivo in rats Intrahippocampal injection Gordon et al., 2017 Asthma In vitro in human lung mast cells; in vivo in mice Intranasal administration Norton et al., 2012 Ceria oxide nanoparticles Liver ﬁbrosis In vivo in rats Intravenous injection Oró et al., 2016 Photoreceptor degeneration In vivo in mice/rats Intracardial injection; Intravitreal injection Kong et al., 2011; Cai et al., 2012; Wong et al., 2013, 2015 Alzheimer’s disease In vivo in mice Unilateral subicular injection Kwon et al., 2016 Multiple sclerosis In vivo in mice Intravenous injection Heckman et al., 2013 Platinum nanoparticles Cerebral cavernous malformation disease In vitro in HeLa, MCF-7, and Caco-2 cells N/A Moglianetti et al., 2016 Cerebral ischemia In vivo in mice Intravenous injection Takamiya et al., 2012 Manganese-based nanoparticles Parkinson’s Disease In vitro in SH-SY5Y cells N/A Singh et al., 2017 Ear-inﬂammation In vitro in HeLa cells; in vivo in mice Subcutaneous injection Yao et al., 2018 Prussian blue nanoparticles Liver inﬂammation In vitro in HUVECs, RAW264.7, HBZY-1, NIH-3T3, and HT cells; in vivo in mice Intravenous injection Zhang W. DIFFERENT ANTIOXIDANT NANOPARTICLES et al., 2016 Molybdenum nanoparticles Acute kidney injury In vitro in HEK293 cells; in vivo in mice Intravenous injection Ni et al., 2018 V2O5@pDA@ MnO2 nanoparticles Ear inﬂammation In vitro in HEK293T cells; in vivo in mice Intravenous injection Huang et al., 2016 Ceria-zirconia nanoparticles Sepsis In vitro in RAW264.7 cells; in vivo in mice/rats Intravenous injection; intraperitoneal administration Soh et al., 2017 human cerebral cavernous malformation disease (Moglianetti et al., 2016), exhibited signiﬁcant neuroprotective eﬀects on ischemic mouse brains (Takamiya et al., 2012), eﬀectively protected keratinocytes against UV-induced inﬂammation (Yoshihisa et al., 2010), and inhibited pulmonary inﬂammation induced by acute cigarette smoking (Onizawa et al., 2009). Kim et al. conjugated platinum nanoparticles with a fusion protein based on a platinum binding peptide and a HIV-1 TAT-derived peptide. Of note, the TAT peptide is a cell-penetrating peptide capable of enhancing translocation and internalization of platinum nanoparticles into the cytoplasm. Compared with unconjugated platinum nanoparticles, the fusion protein- conjugated platinum nanoparticles drastically improved the bioavailability of platinum nanoparticles in Caenorhabditis elegans, leading to similar antioxidant eﬀects at only one hundredth dose (Kim et al., 2010). eﬀectiveness in therapy of liver ﬁbrosis in rats (Oró et al., 2016). Mechanistically, cerium oxide nanoparticles are able to reduce ROS levels in HepG2 cells and protect CCl4-treated rats against chronic liver injury by inhibiting liver steatosis, reducing portal hypertension, and decreasing overexpression of inﬂammatory genes (Figure 1). Platinum Nanoparticles Platinum is mainly used as a chemotherapeutic agent in clinical practice. Platinum nanoparticles were proven to have multiple activities mimicking SOD, catalase (CAT), and NADPH oxidoreductase (Hikosaka et al., 2008; Zhang et al., 2010; Oró et al., 2016; Pedone et al., 2017; Francesca et al., 2018), and they can catalyze the reduction of H2O2 to water and molecular oxygen, suggesting that platinum nanoparticles are potential medicinal candidates for oxidative stress diseases. Platinum nanoparticles possess strong and broad antioxidant properties, and they could signiﬁcantly decrease the lipopolysaccharide (LPS)-induced production of intracellular ROS and inﬂammatory cytokines in vitro (Rehman et al., 2012). As radical scavenging materials, platinum nanoparticles showed potent activity in a cellular model of Manganese-Based Nanoparticles g p A previous study demonstrated that natural MnSOD is superior to FeSOD and Cu/Zn SOD for the treatment of oxidative stress-related chronic diseases (Miriyala et al., 2012). This implicated that manganese-based nanoparticles may possess March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 3 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. FIGURE 1 \| Schematic, characterization, and in vivo evaluation of CeO2 nanoparticles (NPs). (A) Schematic illustration of therapeutic mechanisms of CeO2NPs. (B) High-resolution transmission electron microscopy (HR-TEM) photomicrograph at low (43,000×) magniﬁcation revealing loose CeO2NPs agglomerates. (C) High magniﬁcation image (400,000×) of individual 4 nm CeO2NPs with spherical shape. (D) (Left) The HR-TEM image of single CeO2 NP showing the spherical morphology; (middle) atomistic simulation of CeO2NP with a characteristic ﬂuorite-like electronic structure; (right) FFT of the selected nanoparticle with indicated atomic planes. (E) TEM image of liver tissue obtained from a CCl4-treated rat receiving vehicle and a CCl4-treated rat receiving CeO2NPs. Hepatocyte intracellular space containing mitochondria (M), organelles, fat droplets (asterisk), glycogen inclusions (arrow head), and aggregates of CeO2NPs (arrows). (F) Effect of CeO2NPs on hepatic steatosis, ﬁbrogenic and inﬁltrating cells. Reproduced with permission from Oró et al. (2016). FIGURE 1 \| Schematic, characterization, and in vivo evaluation of CeO2 nanoparticles (NPs). (A) Schematic illustration of therapeutic mechanisms of CeO2NPs. (B) High-resolution transmission electron microscopy (HR-TEM) photomicrograph at low (43,000×) magniﬁcation revealing loose CeO2NPs agglomerates. (C) High magniﬁcation image (400,000×) of individual 4 nm CeO2NPs with spherical shape. (D) (Left) The HR-TEM image of single CeO2 NP showing the spherical morphology; (middle) atomistic simulation of CeO2NP with a characteristic ﬂuorite-like electronic structure; (right) FFT of the selected nanoparticle with indicated atomic planes. (E) TEM image of liver tissue obtained from a CCl4-treated rat receiving vehicle and a CCl4-treated rat receiving CeO2NPs. Hepatocyte intracellular space containing mitochondria (M), organelles, fat droplets (asterisk), glycogen inclusions (arrow head), and aggregates of CeO2NPs (arrows). (F) Effect of CeO2NPs on hepatic steatosis, ﬁbrogenic and inﬁltrating cells. Reproduced with permission from Oró et al. (2016). disease-like cells from cytotoxicity induced by a neurotoxin 1-methyl-4-phenylpyridinium (MPP+) in vitro (Singh et al., 2017) (Figures 2A–E). In another study, Mn3O4 nanoparticles were synthesized via a hydrothermal method (Sun et al., 2019). Manganese-Based Nanoparticles These nanoparticles scavenged nearly 75% •O− 2 due to the two oxidation states of Mn2+ and Mn3+, which is superior excellent ROS-scavenging activities (Singh et al., 2018; Singh N. et al., 2019). A recent study reported the ﬂower-like Mn3O4 nanoparticles that functionally mimic three major antioxidant enzymes including SOD, CAT, and glutathione peroxidase (GPx). Mn3O4 nanoparticles exhibited excellent antioxidant activities and played a crucial role in protecting Parkinson March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 4 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. FIGURE 2 \| Characterization and antioxidative activity of manganese-based nanoparticles. (A,B) Scanning electron microscopy (SEM) (A) and TEM (B) images of Mn3O4 nanoparticles with ﬂower-like morphology (Mnf). (C–E) A comparison of CAT (C), glutathione peroxidase (D), and SOD (E)-like activity of Mnf with other metal oxide nanoparticles. (F) TEM image of Mn3O4 nanoparticles synthesized via a hydrothermal method. (G–I) Dependence between the elimination of •O2−(G), •OH (H), H2O2 (I) and concentrations of Mn3O4 and CeO2 nanoparticles. (J) In vivo ﬂuorescence imaging of mice with PMA-induced ear inﬂammation after treatment with Mn3O4 nanoparticles. Images in (A–E) are reproduced with permission from Singh et al. (2017). Images in (F–J) are reproduced with permission from Yao et al. (2018). FIGURE 2 \| Characterization and antioxidative activity of manganese-based nanoparticles. (A,B) Scanning electron microscopy (SEM) (A) and TEM (B) images of Mn3O4 nanoparticles with ﬂower-like morphology (Mnf). (C–E) A comparison of CAT (C), glutathione peroxidase (D), and SOD (E)-like activity of Mnf with other metal oxide nanoparticles. (F) TEM image of Mn3O4 nanoparticles synthesized via a hydrothermal method. (G–I) Dependence between the elimination of •O2−(G), •OH (H), H2O2 (I) and concentrations of Mn3O4 and CeO2 nanoparticles. (J) In vivo ﬂuorescence imaging of mice with PMA-induced ear inﬂammation after treatment with Mn3O4 nanoparticles. Images in (A–E) are reproduced with permission from Singh et al. (2017). Images in (F–J) are reproduced with permission from Yao et al. (2018). 13-acetate (PMA), UV irradiation, high-level glucose, oxidized low density lipoprotein (OxLDL), and hypoxia/hypoglycemia and reoxygenation in vitro (Zhang W. et al., 2016). Prussian blue nanoparticles also exerted desirable anti-inﬂammatory eﬀects in LPS-induced liver inﬂammation in mice. The peroxidase-like properties of Prussian blue nanoparticles are mainly ascribed to the FeNx units (Chen et al., 2018). Manganese-Based Nanoparticles Ruthenium nanoparticles could also decompose H2O2 and scavenge •OH, •O− 2 , 1O2, 2,2′-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid-derived free radicals (ABTS•+), and 1,1- diphenyl-2-picrylhydrazyl radical (•DPPH), and therefore exert cytoprotective eﬀects against H2O2-induced oxidative stress in vitro (Cao et al., 2017). However, in vivo eﬃcacies of ruthenium nanoparticles remain to be explored. In another study, Mugesh et al. found that V2O5 nanowires displayed notable GPx-like antioxidant activity to protect cells from oxidative damage (Vernekar et al., 2014). Ceria-zirconia composite nanozymes (Soh et al., 2017), Gd@C82-(ethylenediamine)8 nanoparticles to CeO2 nanoparticles. In addition, Mn3O4 nanoparticles can eliminate H2O2 and •OH. They demonstrated intracellular ROS-scavenging activities in HeLa cells in vitro and eﬀectively protected mice from ROS-induced ear-inﬂammation in vivo (Figures 2F–J). Other ROS-Scavenging Inorganic Nanoparticles p In addition to the above mentioned nanoparticles, some other inorganic nanoparticles have been examined for ROS- scavenging, such as nanoparticles derived from Prussian blue (Zhang W. et al., 2016; Chen et al., 2018), gold/platinum and diamond (Martín et al., 2010), ruthenium (Cao et al., 2017), molybdenum (Ni et al., 2018), and vanadium (Vernekar et al., 2014). In particular, Zhang et al. discovered that Prussian blue nanoparticles possess CAT- and SOD-like activities, thereby eﬀectively scavenging ROS, protecting cells from oxidative stress induced by cisplatin, diallyl trisulﬁde, LPS, phorbol 12-myristate March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 5 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. characteristics, such as free radical quenching, photoprotection, and photosensitization (Ju et al., 2011; Solano, 2017; Wang X. et al., 2019). However, melanins can only be dissolved in strongly alkaline aqueous solutions, which largely limits their biomedical applications (Wang X. et al., 2019). Bioinspired polymerization of dopamine can generate melanin-like nanoparticles with similar properties to natural melanins. These melanin-like nanoparticles, often known as polydopamine (PDA) nanoparticles, contain a population of antioxidant groups, thereby exhibiting strong free radical-scavenging ability. PDA nanoparticles have been widely studied in recent years (Chu et al., 2016; Liu et al., 2017; Zhu and Su, 2017; Bao et al., 2018; Zhao et al., 2018; Sun et al., 2019). Cai et al. designed a melanin-based antioxidant defense nanosystem for acute kidney injury (Sun et al., 2019). The ultrasmall Mn2+- chelated and PEG-decorated melanin (MMPP) nanoparticles exhibited good physiological stability and excellent anti-oxidative activities toward various toxic ROS. In vivo results indicated that MMPP nanoparticles can serve as a multifunctional nanotheranostic system for the treatment of acute kidney injury. Notably, Shi’s proved that bioinspired melanin nanoparticles (MeNPs) have excellent scavenging activity for multiple reactive oxygen and nitrogen species (RONS), such as •O− 2 , H2O2, •OH, •NO, and ONOO−(Liu et al., 2017). (Li et al., 2016), and V2O5@pDA@MnO2 nanocomposites (Huang et al., 2016) with multiple antioxidant activities were also investigated. Bilirubin-Derived Nanoparticles p Bilirubin, a natural metabolite of hemoglobin, has been suggested as a primary physiological antioxidant for scavenging various ROS and protecting cells/tissues from oxidative damage. However, clinical applications of bilirubin have been restricted due to its poor water-solubility and toxicity. To overcome the drawbacks of natural bilirubin, Jon and coworkers developed polyethylene glycol (PEG)-conjugated bilirubin nanoparticles through a combined covalent conjugation and self-assembly method, which were deﬁned as BRNPs (Lee et al., 2016a; Kim D. E. et al., 2017; Kim J. Y. et al., 2017). Compared to pristine bilirubin, BRNPs have more excellent water dispersibility, better pharmacokinetic properties, and higher accumulation in oxidative stress-induced inﬂammatory lesions. More importantly, BRNPs inherited the intrinsic powerful antioxidant capability of bilirubin, as evidenced by their eﬃcacies in the treatment of a variety of oxidative stress-associated diseases, such as hepatic ischemia-reperfusion injury (Kim J. Y. et al., 2017), asthma (Kim D. E. et al., 2017), IBD (Lee et al., 2016a), and pancreatic islet xenotransplantation (Kim M. J. et al., 2017). In addition, BRNPs protected diﬀerent cells against cytotoxicity caused by H2O2 and decreased inﬂammatory responses resulting from activated macrophages. Moreover, other bilirubin-derived nanoparticles can be used as a companion medicine for eﬀective treatment of cancers and colitis (Lee et al., 2017, 2018, 2020). As a typical paradigm, Lee et al. constructed a hyaluronic acid-bilirubin nanomedicine (HABN) for inhibition of acute colitis (Lee et al., 2020). In vivo experiments demonstrated that HABN could target the inﬂamed colonic epithelium, restore the dysregulated intestinal barriers, and simultaneously modulate the gut microbiota and immune responses in mice bearing colitis. Organic Nanoparticles With Intrinsic Antioxidant Activities Besides ROS-scavenging inorganic nanoparticles, organic nanoparticles with intrinsic antioxidant activities have been emerging as a potential platform for the treatment of numerous diseases associated with inﬂammatory disorders (Kim K. S. et al., 2015; Zhu and Su, 2017; Kwon et al., 2019). Several types of organic nanoparticles have shown higher antioxidative advantages over conventional antioxidants and radical scavengers, owing to their predominant properties in terms of pharmacokinetics, biodistribution, delivery eﬃciency, and robust scavenging capabilities of multiple radicals (Lu et al., 2016; Ferreira et al., 2018; Ye et al., 2019; Zafar et al., 2019). In this section, these organic nanoparticles will be brieﬂy introduced (Table 2). Free Radical-Containing Organic Nanoparticles Spurred by the development of enzymology, a series of ROS-scavenging organic nanoparticles with enzyme-mimicking functions have been engineered as new antioxidant candidates for the biomedical applications. As a representative example, antioxidative nanotherapies self-assembled by integrating a stable nitroxide radical-containing ROS trapper, 4-hydroxy-2,2,6,6- tetramethylpiperidin-1-oxyl (Tempol) into the side chain of amphiphilic copolymers have been intensively investigated by Nagasaki’s group (Yoshitomi and Nagasaki, 2014; Ikeda and Nagasaki, 2018). Two types of redox nanoparticles (RNPs), pH- sensitive RNPN and pH-insensitive RNPO have been synthesized and demonstrated remarkable protective and therapeutic eﬀects in diverse diseases, such as breast or colon cancers (Vong and Nagasaki, 2016; Shashni and Nagasaki, 2018), IBD (Vong et al., 2012, 2014, 2015), radiation-induced organ dysfunctions (Feliciano et al., 2017), ischemia reperfusion injury (Yoshitomi et al., 2011), non-alcoholic steatohepatitis (Eguchi et al., 2015), and neurological deﬁcit (Chonpathompikunlert et al., 2012). These studies have unambiguously substantiated the antioxidative eﬀects of RNPN and RNPO without discernible adverse side eﬀects. Phenolic Nanoparticles Phenolic compounds, such as hydroxybenzyl alcohol (HBA) and vanillyl alcohol have long been widely used for treating ischemic brain injury and coronary heart disease, because of their antioxidant, anti-inﬂammatory, and anti-nociceptive activities (Berwin Singh et al., 2018). Recently, HBA-containing copolyoxalate (HPOX) was designed and synthesized, which can be degraded completely to release therapeutic HBA and 1,4- cyclohexendimethanol in the presence of H2O2 (Yoo et al., Melanin-Like Nanoparticles Melanins are well-known endogenous biopolymers that are widely distributed in most living organisms. They have attracted much attention because of their fascinating biological March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 6 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. TABLE 2 \| Different organic nanoparticles with intrinsic antioxidant activities. Types of organic nanoparticles Applications Evaluation models Administration routes References Bilirubin-derived nanoparticles BRNPs Hepatic ischemia-reperfusion injury In vitro in primary mouse hepatocytes; in vivo in mice Intravenous injection Kim J. Y. et al., 2017 Asthma In vivo in mice Intravenous injection; intraperitoneal administration Kim D. E. et al., 2017 Inﬂammatory bowel disease In vivo in mice Intravenous injection Lee et al., 2016a Pancreatic islet xenotransplantation In vivo in mice Intraperitoneal administration Kim M. J. Melanin-Like Nanoparticles et al., 2017 DOX@BRNPs Dox@bt-BRNPs cisPt@BRNPs Tumor In vivo in mice Intravenous injection Lee et al., 2016b, 2017, 2018 HABN Colitis In vivo in mice Oral administration Lee et al., 2020 Melanin-like nanoparticles MMPP Acute kidney injury In vitro in HEK293 cells; in vivo in mice Intravenous injection Sun et al., 2019 PEG-MeNPs Ischemic stroke In vitro in Neuro 2A cells; in vivo in mice Intravenous injection Liu et al., 2017 PDA-PEG/CP Tumor In vitro in HeLa cells N/A Zhu and Su, 2017 Liposome-BSM Tumor In vivo in mice Subcutaneous injection Chu et al., 2016 PDA NPs Periodontal disease In vivo in mice Subgingival administration Bao et al., 2018 PDA Acute peritonitis and acute lung injury In vivo in mice Intravenous injection; intraperitoneal administration Zhao et al., 2018 Free radical-containing organic nanoparticles RNPN and RNPO Breast cancer In vitro in MDA-MB-231 cells; in vivo in mice Intravenous injection Shashni and Nagasaki, 2018 RNPO Inﬂammatory bowel disease In vivo in mice Oral administration Vong et al., 2012, 2014, 2015 RNPN and RNPO Colon cancer In vitro in C-26 cells; in vivo in mice Intravenous injection Vong and Nagasaki, 2016 RNPN and RNPO Epidermoid cancers In vitro in KB-3-1 and KB/MRP cells N/A Shashni et al., 2017 RNPN Radiation-induced organ dysfunctions In vitro in RAW264.7 cells; in vivo in mice Subcutaneous injection Feliciano et al., 2017 RNPN Ischemia reperfusion injury In vivo in mice Intravenous injection Yoshitomi et al., 2011 RNPs Nonalcoholic steatohepatitis In vivo in mice Intravenous injection Eguchi et al., 2015 RNPs Neurological deﬁcits In vivo in mice Intravenous injection Chonpathompikunlert et al., 2012 Phenolic nanoparticles HPOX Asthma In vitro in RAW264.7 cells; In vivo in mice Intratracheal administration Yoo et al., 2013 Hindlimb ischemia In vivo in mice Intramuscular injection Cho et al., 2015 H2O2-associated inﬂammatory diseases In vitro in RAW264.7 cells N/A Cho et al., 2012 PVAX Acute liver failure In vitro in RAW264.7 cells; in vivo in mice Intravenous injection Ko et al., 2014 PVO Hepatic ischemia-reperfusion injure In vivo in mice Intravenous injection Kang et al., 2016 Nanoparticles derived from functional cyclodextrin materials Nanoparticles based on PBAP-conjugated cyclodextrin Tumor In vitro in B16F10 and RAW264.7 cells; in vivo in mice Intravenous injection Zhang et al., 2015 Arterial restenosis In vitro in rat VSMCs; in vivo in rats Intravenous injection Feng et al., 2016 Inﬂammatory bowel disease In vivo in mice Oral administration Zhang Q. Melanin-Like Nanoparticles et al., 2016; Alaarg et al., 2017; Kotla et al., 2019). Interestingly, nanoparticles based on PBAP-conjugated cyclodextrin materials can eﬀectively eliminate H2O2, thereby inhibiting inﬂammatory responses and oxidative stress in stimulated macrophages (Zhang et al., 2017). These H2O2-eliminating cyclodextrin nanoparticles eﬃcaciously alleviated the symptoms of peritonitis and colitis in mice, by reducing the counts of neutrophils and macrophages as well as inhibiting the secretion of pro-inﬂammatory cytokines, chemokines, and oxidative mediators (Zhang et al., 2017; Zhang Q. et al., 2019). Also, we found that nanoparticles prepared from a luminol-conjugated β-CD material (LCD) can inhibit inﬂammatory response, oxidative stress, and recruitment of neutrophils and macrophages (Guo et al., 2019). In addition, LCD nanoparticles eﬀectively reduced the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, myeloperoxidase (MPO), and ROS in neutrophils stimulated with PMA. Nevertheless, almost all available organic nanoparticles can only eliminate limited oxygen species in ROS. To circumvent this shortcoming, our group synthesized a broad-spectrum ROS-eliminating material (i.e., TPCD), by simultaneously conjugating Tempol and phenylboronic acid pinacol ester onto β-CD (Li et al., 2018; Wang et al., 2018). TPCD can be easily produced into SOD/CAT-mimetic and size-controlled nanoparticles. The resulting TPCD nanoparticles can eﬀectively scavenge multiple reactive species, including •O− 2 , H2O2, radicals, and hypochlorite (Figures 3A,B). Compared with the corresponding nanotherapies with relative narrow-spectrum ROS-eliminating capability, TPCD nanoparticles more eﬀectively protected macrophages from H2O2-induced apoptosis and decreased inﬂammatory responses in vitro. Consistently, TPCD nanoparticles showed superior eﬃcacies in the animal models of acute and chronic inﬂammation, such as peritonitis, acute lung injury, drug-induced organ toxicity, and atherosclerosis. Importantly, preliminary in vitro and in vivo tests demonstrated the good safety proﬁle of TPCD nanoparticles. Melanin-Like Nanoparticles et al., 2016 March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. TABLE 2 \| Continued Types of organic nanoparticles Applications Evaluation models Administration routes References Atherosclerosis In vitro in RAW264.7 cells; in vivo in mice Intravenous injection Dou et al., 2017 Abdominal aortic aneurysm In vivo in mice Intravenous injection Cheng et al., 2018 Peritonitis In vitro in RAW264.7 cells; in vivo in mice Intraperitoneal administration Zhang et al., 2017 Colitis-associated colon cancer In vitro in C26 murine colon carcinoma cells; in vivo in mice Oral administration Zhang Q. et al., 2019 LCD nanoparticles Peritonitis, acute lung injury, and atherosclerosis In vitro in neutrophils and macrophages; in vivo in mice Intraperitoneal administration; intravenous injection Guo et al., 2019 TPCD nanoparticles Peritonitis, acute lung injury, drug-induced organ toxicity, and atherosclerosis In vivo in mice Intraperitoneal administration; intravenous injection Li et al., 2018; Wang et al., 2018 Other organic nanoparticles PVA-C Air pouch model In vitro in BMSCs; in vivo in rats Subcutaneous injection Wu et al., 2019 2013). The biocompatible HPOX nanoparticles exerted highly potent antioxidant and anti-inﬂammatory eﬀects by reducing the generation of ROS and suppressing the expression of pro-inﬂammatory mediators in stimulated macrophages. Given their beneﬁcial eﬀects in a mouse model of asthma by suppressing the recruitment of eosinophils and neutrophils as well as the expression of iNOS, HPOX nanoparticles exhibited tremendous potential as an anti-asthmatic agent. Likewise, HPOX nanoparticles also signiﬁcantly promoted angiogenesis and blood ﬂow perfusion in a mouse mode of hindlimb ischemia (Cho et al., 2015). Through similar procedures, vanillyl alcohol-containing copolyoxalate (PVAX) and poly (vanillin oxalate) (PVO) were synthesized by a simple one- step polymerization, and both of them are able to scavenge H2O2 (Ko et al., 2014; Jeong et al., 2016; Jung et al., 2019). PVAX nanoparticles and manganese porphyrin showed synergistic antioxidant and anti-inﬂammatory activities in mice with acetaminophen-induced acute liver failure (Ko et al., 2014). Kang et al. developed H2O2-triggered bubble-generating antioxidant PVO polymeric nanomaterials that can signiﬁcantly suppress liver damages due to ischemia/reperfusion injury by inhibiting inﬂammation and apoptosis (Kang et al., 2016). In view of the advantages of excellent biodegradability, antioxidant activity, anti-inﬂammatory property, and low cytotoxicity, these phenolic nanoparticles have great potential as therapeutics for inﬂammatory diseases. et al., 2013; Lamprecht, 2015; Zhang Q. Nanoparticles Derived From Functional Cyclodextrin Materials Most recently, our group has developed a series of ROS- responsive materials by conjugating phenylboronic acid pinacol ester (PBAP) onto β-cyclodextrin (β-CD), a cyclic oligosaccharide with excellent in vivo safety (Zhang and Ma, 2013). The obtained ROS-responsive materials can be used to construct nanoparticles for site-speciﬁc delivery and inﬂammation-responsive release of diﬀerent therapeutics (Juni March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 8 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. FIGURE 3 \| Design and preparation broad-spectrum ROS-scavenging nanoparticles for treatment of acute lung injury (ALI). (A) Schematic illustration of engineering of a broad-spectrum ROS-scavenging material and its nanoparticles based on functionalized β-CD. (B) The synthetic route of β-CD conjugated with Tempol (Tpl) and PBAP units (TPCD). CDI, 1,1-carbonyldiimidazole; DMAP, 4-dimethylaminopyridine; TCD, Tpl-conjugated β-CD; PBAP, 4-(hydroxymethyl) phenylboronic acid pinacol ester. (C–H) Treatment of ALI with TPCD nanoparticles (i.e., TPCD NP) in mice. (C) The lung wet-to-dry weight ratios after different treatments. (D–G) The expression levels of TNF-α (D), IL-1β (E), and H2O2 (F) in bronchoalveolar lavage ﬂuid from mice with LPS-induced ALI and subjected to different treatments. (G) Quantiﬁed neutrophil counts in pulmonary tissues of ALI mice. (H) H&E-stained pathological sections of lung tissues. Data are mean ± standard error (n = 6). P < 0.05, P < 0.01, *P < 0.001. Reproduced with permission from Li et al. (2018). FIGURE 3 \| Design and preparation broad-spectrum ROS-scavenging nanoparticles for treatment of acute lung injury (ALI). (A) Schematic illustration of engineering of a broad-spectrum ROS-scavenging material and its nanoparticles based on functionalized β-CD. (B) The synthetic route of β-CD conjugated with Tempol (Tpl) and PBAP units (TPCD). CDI, 1,1-carbonyldiimidazole; DMAP, 4-dimethylaminopyridine; TCD, Tpl-conjugated β-CD; PBAP, 4-(hydroxymethyl) phenylboronic acid pinacol ester. (C–H) Treatment of ALI with TPCD nanoparticles (i.e., TPCD NP) in mice. (C) The lung wet-to-dry weight ratios after different treatments. (D–G) The expression levels of TNF-α (D), IL-1β (E), and H2O2 (F) in bronchoalveolar lavage ﬂuid from mice with LPS-induced ALI and subjected to different treatments. (G) Quantiﬁed neutrophil counts in pulmonary tissues of ALI mice. (H) H&E-stained pathological sections of lung tissues. Data are mean ± standard error (n = 6). P < 0.05, P < 0.01, P < 0.001. Reproduced with permission from Li et al. (2018). FIGURE 3 \| Design and preparation broad-spectrum ROS-scavenging nanoparticles for treatment of acute lung injury (ALI). Nanoparticles Derived From Functional Cyclodextrin Materials (A) Schematic illustration of engineering of a broad-spectrum ROS-scavenging material and its nanoparticles based on functionalized β-CD. (B) The synthetic route of β-CD conjugated with Tempol (Tpl) and PBAP units (TPCD). CDI, 1,1-carbonyldiimidazole; DMAP, 4-dimethylaminopyridine; TCD, Tpl-conjugated β-CD; PBAP, 4-(hydroxymethyl) phenylboronic acid pinacol ester. (C–H) Treatment of ALI with TPCD nanoparticles (i.e., TPCD NP) in mice. (C) The lung wet-to-dry weight ratios after different treatments. (D–G) The expression levels of TNF-α (D), IL-1β (E), and H2O2 (F) in bronchoalveolar lavage ﬂuid from mice with LPS-induced ALI and subjected to different treatments. (G) Quantiﬁed neutrophil counts in pulmonary tissues of ALI mice. (H) H&E-stained pathological sections of lung tissues. Data are mean ± standard error (n = 6). P < 0.05, P < 0.01, P < 0.001. Reproduced with permission from Li et al. (2018). Other Organic Nanoparticles Other nanoparticles derived from small molecules such as ascorbic acid (Bhatia et al., 2019), citric acid (Wu et al., 2019), and trolox (Wattamwar et al., 2010), in combination with polymeric scaﬀolds, have also provided us with a powerful arsenal for antioxidant and anti-inﬂammatory treatment. For Drug-Loaded Nanoparticles With Antioxidant Activity In a thromboembolic rat model, sequential administration of CAT/PLGA and SOD/PLGA nanoparticles eﬀectively attenuated inﬂammatory responses, inhibited neuronal cell apoptosis, and suppressed edema formation, by protecting the blood-brain barrier (BBB) from ROS-mediated reperfusion injury. In a recent study, Kojima et al. constructed designer exosomes that can deliver CAT mRNA into target cells with high eﬃciency, thereby attenuating neurotoxicity and neuroinﬂammation in both in vitro and in vivo models of Parkinson’s disease (Figure 4) (Kojima et al., 2018). Both natural and synthetic antioxidants of small molecules have been broadly used or studied for anti-inﬂammatory therapies of numerous diseases associated with oxidative stress. The generally used natural antioxidants include vitamin C, vitamin E, glutathione, β-carotene, ﬂavonoids, and curcumin, while Edaravone, lipoic acid, N-acetylcysteine, and Tempol are frequently examined synthetic compounds. The main limitations of small molecule antioxidants lie in their systemic distribution, rapid metabolism, and low retention at the diseased site, thereby resulting in low bioavailability in target tissues/cells. In this aspect, nanoparticles can protect the loaded small molecule antioxidants from hydrolysis, achieve site-speciﬁc delivery and controlled release of antioxidants at disease sites, and therefore improve their bioavailability (Table 3) (Wang et al., 2015; Kang et al., 2016; Jung et al., 2018; Larrañaga et al., 2018; Gou et al., 2019). For example, our group fabricated a SOD/CAT- mimetic nanomedicine comprising H2O2-eliminating nanoparticles derived from a PBAP-conjugated β-CD material and a free radical scavenger Tempol (Zhang Q. et al., 2016). After oral administration, this nanotherapy was able to eﬃciently target the inﬂamed colon in colitic mice, and remarkably decrease non-speciﬁc distribution, thus notably alleviating manifestations relevant to colitis, with the eﬃcacy superior over free Tempol. Other Organic Nanoparticles instance, a chemically stable antioxidant molecule, citric acid was grafted with polyvinyl alcohol (Wu et al., 2019). The obtained polymer PVA-C showed anti-apoptotic ability in stem cells, and it also upregulated the nuclear peroxisome proliferator-activated receptor γ and MnSOD by releasing citric acid. March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 9 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. TABLE 3 \| Typical drug-loaded nanoparticles with antioxidant activity. Carrier materials Loaded drugs Applications Evaluation models Administration routes References OxbCD Tempol Inﬂammatory bowel disease In vivo in mice Oral administration Zhang Q. et al., 2016 Lipids Curcumin Sepsis In vitro in RAW264.7 cells; in vivo in mice Intraperitoneal injection Wang et al., 2015 Silk ﬁbroin Curcumin Inﬂammatory bowel disease In vitro in RAW264.7 cells; in vivo in mice Oral or intravenous administration Gou et al., 2019 Vanillyl alcohol-incorporated copolyoxalate Curcumin Peripheral artery disease In vitro in RAW264.7 cells, NIH3T3 ﬁbroblasts, and vascular endothelial cells; in vivo in mice Intramuscular injection Jung et al., 2018 PVO Vanillin Ischemia/reperfusion injury In vitro in RAW264.7 cells; in vivo in mice Intravenous injection Kang et al., 2016 β-Galactosidase- conjugated with anti-PECAM CAT Acute lung transplantation injury In vitro in HUVECs; in vivo in rats Intravenous injection Kozower et al., 2003 Polyketal SOD Myocardial ischemia-reperfusion injury In vitro in RAW264.7 cells; in vivo in rats Myocardial injection Seshadri et al., 2010 Engineered exosomes CAT mRNA Parkinson’s Disease In vitro in Neuro2A cells; in vivo in mice Subcutaneous implantation with Matrigel Kojima et al., 2018 TABLE 3 \| Typical drug-loaded nanoparticles with antioxidant activity. THERAPEUTIC APPLICATIONS OF ANTIOXIDANT NANOTHERAPIES IN INFLAMMATORY DISEASES Inﬂammation is a protective response of the innate immune system against pathogens or irritants (Karin and Clevers, 2016). However, uncontrolled inﬂammation plays a fundamental role in the progress of a large number of human diseases, such as asthma, pneumonia, rheumatoid arthritis, IBD, as well as cardiovascular and neurodegenerative diseases. It has been well- documented that excessive accumulation of ROS contributes to the pathogenesis of both acute and chronic inﬂammation (Zhang and Kaufman, 2008). In this section, we will provide an overview of the applications of antioxidant nanomedicines in the treatment of diﬀerent inﬂammatory diseases. In addition to the conventional small-molecule ROS scavengers, other biological agents, such as natural antioxidant enzymes SOD, CAT, and nucleic acids have been loaded into nanoparticles for anti-oxidative treatment (Kozower et al., 2003; Seshadri et al., 2010; Chen et al., 2013). Petro et al. constructed poly (D,L-lactide co-glycolide) (PLGA) nanoparticles separately loaded with SOD, CAT, or their combination (Petro et al., 2016). Inﬂammatory Bowel Disease y Inﬂammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis (UC), is a chronic inﬂammatory disorder in the gastrointestinal tract. The intestinal mucosa of patients suﬀering from IBD is characterized by overproduction of ROS and an imbalance of antioxidants, resulting in oxidative damage. It has March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 10 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. FIGURE 4 \| Applications of the engineered exosomes loaded with CAT mRNA in Parkinson’s disease. (A) Schematic illustration of protection against neurotoxicity in an in vitro experimental model of Parkinson’s disease by CAT mRNA delivery. (B) Engineered exosome producer cells were subcutaneously implanted with Matrigel in living mice. (C) Immunostaining result of tyrosine hydroxylase (TH)-positive neurons. Scale bars, 500 µm. (D) Attenuation of neuroinﬂammation caused by systemic LPS injection with catalase mRNA delivery in vivo. Reproduced with permission from Kojima et al. (2018). p < 0.05, *p < 0.01; n.s., no signiﬁcance. FIGURE 4 \| Applications of the engineered exosomes loaded with CAT mRNA in Parkinson’s disease. (A) Schematic illustration of protection against neurotoxicity in an in vitro experimental model of Parkinson’s disease by CAT mRNA delivery. (B) Engineered exosome producer cells were subcutaneously implanted with Matrigel in living mice. (C) Immunostaining result of tyrosine hydroxylase (TH)-positive neurons. Scale bars, 500 µm. (D) Attenuation of neuroinﬂammation caused by systemic LPS injection with catalase mRNA delivery in vivo. Reproduced with permission from Kojima et al. (2018). p < 0.05, *p < 0.01; n.s., no signiﬁcance. FIGURE 4 \| Applications of the engineered exosomes loaded with CAT mRNA in Parkinson’s disease. (A) Schematic illustration of protection against neurotoxicity in an in vitro experimental model of Parkinson’s disease by CAT mRNA delivery. (B) Engineered exosome producer cells were subcutaneously implanted with Matrigel in living mice. (C) Immunostaining result of tyrosine hydroxylase (TH)-positive neurons. Scale bars, 500 µm. (D) Attenuation of neuroinﬂammation caused by systemic LPS injection with catalase mRNA delivery in vivo. Reproduced with permission from Kojima et al. (2018). p < 0.05, p < 0.01; n.s., no signiﬁcance. This host-guest nanotherapy exhibited desirable antioxidant and anti-inﬂammatory eﬀects in macrophages and in mice with DSS- induced colitis (Xue et al., 2018). Most recently, we packaged a proresolving peptide Ac2-26 into nanoparticles derived from the aforementioned ROS-responsive material (i.e., PBAP-conjugated β-CD) to obtain a multifunctional nanotherapy, deﬁned as AON (Figure 5) (Li C. Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Inﬂammatory Bowel Disease et al., 2019). AON eﬀectively protected Ac2-26 from degradation under gastrointestinal conditions. By oral delivery of this nanotherapy to mice with DSS-induced colitis, site-speciﬁc accumulation of AON and triggerable release of Ac2-26 in response to high levels of ROS at the inﬂammatory colons were successfully achieved. In mice with DSS-induced acute and chronic colitis as well as in IL-10-deﬁcient mice that spontaneously develop colitis, oral administration of AON notably attenuated manifestations related to colitis, and signiﬁcantly decreased expression of proinﬂammatory cytokines and oxidative stress-related molecular mediators, which was more potent than free Ac2-26 or a non-responsive control nanotherapy based on PLGA. Mechanistically, AON can eﬀectively inhibit inﬂammatory responses and oxidative stress in stimulated macrophages, notably attenuate neutrophil traﬃcking, promote eﬀerocytosis of apoptotic neutrophils, as well as regulate phenotypic switching of macrophages in vitro. been found that the mucosal ROS concentrations are 10–100 times higher in patients with IBD (Simmonds et al., 1992; Sedghi et al., 1993). Treatment with antioxidant agents can mitigate IBD in both animal models and patients (Moura et al., 2015). Unfortunately, non-speciﬁc distribution and low retention of these compounds often results in multiple side eﬀects. So far, a number of studies have reported the construction of orally available antioxidant nanoparticles for IBD therapy with high inﬂamed colon retention and therapeutic eﬀects (Vong et al., 2012; Lamprecht, 2015; Alaarg et al., 2017; Bak et al., 2018; Gou et al., 2019; Kotla et al., 2019; Li S. et al., 2019; Schilrreﬀet al., 2019). For IBD treatment by site-speciﬁc delivery of a small-molecule antioxidant Tempol, our group fabricated a ROS-responsive nanoplatform based on a PBAP-conjugated β-CD material. The resulting SOD/CAT-mimetic nanotherapy was able to eﬃciently accumulate in the inﬂamed colon in mice and notably mitigated manifestations relevant to colitis, showing remarkable eﬃcacies in three mouse colitis models including DSS-induced acute and chronic colitis as well as TNBS- induced colitis (Zhang Q. et al., 2016). Also, we constructed aﬃnity nanoparticles via host-guest interactions between cyclodextrin-containing hydrophilic copolymers and Tempol. March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 11 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. FIGURE 5 \| A proresolving peptide nanotherapy for site-speciﬁc treatment of inﬂammatory bowel disease. (A,B) Schematic illustration of engineering of a ROS-responsive peptide nanotherapy (AON) (A) and targeted treatment of colitis (B). (C,D) TEM (C) and SEM (D) images of AON. Scale bars, 200 nm. Inﬂammatory Bowel Disease (E) Selective accumulation of AON in the inﬂamed colons of mice with acute colitis. (F) Representative mini-endoscopic images of colons from colitic mice at day 7 after different treatments. ON, blank nanoparticles based on the ROS-responsive material of PBAP-conjugated β-CD (i.e., OxbCD); APN, Ac2-26-containing nanoparticles based on a non-responsive polymer PLGA. Reproduced with permission from Li C. et al. (2019). FIGURE 5 \| A proresolving peptide nanotherapy for site-speciﬁc treatment of inﬂammatory bowel disease. (A,B) Schematic illustration of engineering of a ROS-responsive peptide nanotherapy (AON) (A) and targeted treatment of colitis (B). (C,D) TEM (C) and SEM (D) images of AON. Scale bars, 200 nm. (E) Selective accumulation of AON in the inﬂamed colons of mice with acute colitis. (F) Representative mini-endoscopic images of colons from colitic mice at day 7 after different treatments. ON, blank nanoparticles based on the ROS-responsive material of PBAP-conjugated β-CD (i.e., OxbCD); APN, Ac2-26-containing nanoparticles based on a non-responsive polymer PLGA. Reproduced with permission from Li C. et al. (2019). In vivo eﬃcacies of AON are most likely achieved by accelerating intestinal mucosal wound healing, expediting the resolution of inﬂammation, reshaping gut microbiota, and increasing the production of short-chain fatty acids. Moreover, oral delivery of AON showed excellent safety proﬁle in mice. myocardial hypertrophy, myocardial infarction, myocardial ischemia-reperfusion injury, and heart failure (Harrison et al., 2003). ROS in the cardiovascular system are predominantly produced by activated NADPH oxidase (NOX) which is the primary oxidase system underlying oxidative stress. NOX- derived ROS production induces activation of endothelial nitric oxide synthase (eNOS), leading to eNOS uncoupling and mitochondrial dysfunction, eventually causing sustained oxidative damage and the development of cardiovascular diseases (Zhang Y. et al., 2020). Brain Diseases Brain is an extremely metabolically active organ with low levels of antioxidant enzymes and high levels of redox-active substrates (e.g., Cu and Fe), and therefore it is especially vulnerable to oxidative stress. A growing body of evidence has substantiated that the excessive production of ROS plays a critical role in a common pathophysiology of brain diseases, such as cerebral infarction, Alzheimer’s disease (AD), and Parkinson’s disease (PD) (Barnham et al., 2004; Chen et al., 2011; Kim G. H. et al., 2015). Conventional antioxidant therapies cannot eﬀectively inhibit ROS-ampliﬁed brain injury for their limited ability to cross the BBB and target the disease sites. Antioxidant nanotherapies have been emerging as an eﬀective strategy to overcome the BBB and to provide targeted release of diﬀerent therapeutics in the impaired brain (Hu et al., 2015; Kwon et al., 2016, 2018; Liu et al., 2017). For example, Liu et al. fabricated PEG-coated melanin nanoparticles MeNPs (Liu et al., 2017). Neuroprotective and anti-inﬂammatory activities of MeNPs were evaluated by in vitro studies in Neuro 2A cells. It was found that MeNPs could decrease oxidative stress damage and inhibit CoCl2-induced ischemic injury, without notable eﬀects on mitochondrial function. Further in vivo studies in a rat model of ischemic stroke indicated that rats pretreated with MeNPs showed less infarct area in the ischemic brain (∼14%) compared with the control group (∼32%). Also, MeNPs eﬃciently suppressed the generation of •O− 2 in brains. Consequently, MeNPs could attenuate RONS-induced inﬂammatory responses through suppressing the expression of typical mediators related to oxidative stress and inﬂammation in vitro and in vivo. On the other hand, ROS-scavenging nanoparticles can serve as an eﬀective nanoplatform for site-speciﬁc delivery of therapeutics to the sites of vascular inﬂammation (Seshadri et al., 2010; Wu et al., 2018; Zhang R. et al., 2020). As well- documented, substantially increased ROS levels are positively related to endothelial dysfunction and pathogenesis of restenosis after percutaneous coronary interventions (Juni et al., 2013). To develop targeting nanotherapies for restenosis, our group engineered ROS-responsive nanotherapies by loading rapamycin (RAP) into nanoparticles derived from a PBAP-conjugated β- CD material (Figure 6) (Feng et al., 2016). Thus, obtained RAP-containing nanoparticles exhibited ROS-triggerable drug release, showing signiﬁcantly enhanced anti-migration and anti- proliferative eﬀects as compared to free RAP and a non- responsive RAP nanotherapy. In addition, the ROS-responsive nanoparticles can accumulate at the injured site in the carotid artery of rats subjected to balloon angioplasty injury. Cardiovascular Diseases Cardiovascular disease is a leading cause of mortality worldwide. Increasing evidence has demonstrated that excessive ROS production is closely linked to the mitochondrial dysfunction, cell apoptosis, endoplasmic reticulum stress, and autophagy, which facilitates the occurrence and development of cardiovascular disease (Brown and Griendling, 2015). The intracellular ROS have a critical role in the pathogenesis of various cardiovascular diseases, such as atherosclerosis, ROS-scavenging nanotherapies have demonstrated great potential in the prevention and treatment of cardiovascular disease. They can protect cardiac progenitor cells from oxidative stress (Pagliari et al., 2012), prevent ROS-induced death of implanted mesenchymal stem cells for myocardial March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 12 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. infarction treatment (Park et al., 2015), and eliminate excessive ROS to mitigate atherosclerosis (Chmielowski et al., 2017; Wang et al., 2018). As a typical example, our previously developed TPCD nanoparticles signiﬁcantly attenuated ROS- induced inﬂammation and cell apoptosis in macrophages, by eliminating broad-spectrum ROS in cells (Li et al., 2018; Wang et al., 2018). In addition, TPCD nanoparticles could eﬀectively inhibit foam cell formation in macrophages and vascular smooth muscle cells (VSMCs), by decreasing cellular internalization of oxLDL. After i.v. delivery, TPCD nanoparticles accumulated in atherosclerotic lesions of apolipoprotein E-deﬁcient (ApoE−/−) mice, which was mainly achieved by passive targeting through the dysfunctional endothelium and translocation via inﬂammatory cells. In ApoE−/−mice subjected to high-fat diets, treatment with TPCD nanoparticles by i.v. administration eﬃcaciously inhibited the development of atherosclerosis and stabilized atherosclerotic plaques, resulting less cholesterol crystals, smaller necrotic cores, thicker ﬁbrous caps, and lower levels of macrophages and matrix metalloproteinase-9, as compared to control drugs previously developed for antiatherosclerosis. These beneﬁcial eﬀects of TPCD nanoparticles were considered to be relevant to reduced systemic and local oxidative stress and inﬂammation. In view of the good safety proﬁle of TPCD nanoparticles in preliminary in vivo tests based on diﬀerent animal models (Li et al., 2018; Wang et al., 2018), this type of nanoparticles deserve further development as a potential antiatherosclerotic nanotherapy. In another study, Hao et al. developed injectable fullerenol nanoparticle-loaded alginate hydrogel, which showed excellent ROS-scavenging activity (Hao et al., 2017). This fullerenol/alginate hydrogel could serve as a cell delivery vehicle and suppress oxidative stress-mediated cell apoptosis in brown adipose-derived stem cells (BADSCs) in vitro, by attenuating JNK signaling pathways and activating p38 MAPK signaling pathways. Cardiovascular Diseases Moreover, this functional hydrogel remarkably enhanced the retention and survival of implanted BADSCs in myocardial infarction zone via regulating the ROS microenvironment, thereby reinforcing therapeutic eﬃcacy for cardiac repair. neointimal hyperplasia than free RAP and a non-responsive nanotherapy. Further studies demonstrated that in vivo eﬃcacy of the ROS-responsive RAP nanotherapy can be additionally improved by integrating with a pH-responsive β-CD material to aﬀord a pH/ROS dual-responsive nanotherapy (Zhang R. et al., 2020). Of note, surface engineering of the dual-responsive nanoparticles via a peptide (KLWVLPKGGGC) targeting type IV collagen can notably increase their accumulation at injured carotid arteries, thereby potentiating in vivo eﬃcacy of the dual- responsive RAP nanotherapy. Similarly, therapeutic advantages of the ROS-responsive RAP nanotherapy was demonstrated in a mouse model of atherosclerosis in ApoE−/−mice (Dou et al., 2017) and an animal model of abdominal aortic aneurysm in rats (Cheng et al., 2018). In both cases, treatment with ROS- responsive nanotherapies can signiﬁcantly reduce oxidative stress in diseased aortas. Consequently, ROS-responsive nanotherapies hold great potential for precision therapy of diﬀerent vascular inﬂammatory diseases. Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Brain Diseases In a rat model of arterial restenosis, treatment with the ROS-responsive RAP nanotherapy by i.v. injection more eﬀectively attenuated In addition, ROS-scavenging inorganic nanoparticles were used for treatment of neurodegenerative diseases. In this aspect, Kwon and coworkers constructed triphenylphosphonium (TPP)-conjugated CeO2 nanoparticles, for targeting the mitochondria and potential therapy of AD (Kwon et al., 2016). Such nanoparticles signiﬁcantly mitigated reactive gliosis and mitochondrial morphological damage in an AD model of 5XFAD transgenic mice. In a recent study, the same group designed three March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 13 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. FIGURE 6 \| Engineering of different inﬂammation-responsive nanotherapies for targeted treatment of restenosis. (A) Design of inﬂammation-triggerable nanoparticles. (B) Evans Blue staining indicates successful establishment of injury in the carotid artery. (C) DHE-stained cryosections showing oxidative stress in injured carotid arteries. The image at day 0 was acquired from the sample immediately collected after injury. (D) The levels of hydrogen peroxide in the carotid artery with or without injury. (E) H&E stained histological sections of carotid arteries isolated from rats subjected to various treatments. Scale bars, 200 µm (the upper panel), 50 µm (the lower panel). PLGA NP, Ac-bCD NP, and Ox-bCD NP represents nanoparticles based on a non-responsive polymer PLGA, a pH-responsive material of acetelated β-CD, and a ROS-responsive material of PBAP-conjugated β-CD, respectively. Data are mean ± standard deviation (n = 3). P < 0.001. Reproduced with permission from Feng et al. (2016). FIGURE 6 \| Engineering of different inﬂammation-responsive nanotherapies for targeted treatment of restenosis. (A) Design of inﬂammation-triggerable nanoparticles. (B) Evans Blue staining indicates successful establishment of injury in the carotid artery. (C) DHE-stained cryosections showing oxidative stress in injured carotid arteries. The image at day 0 was acquired from the sample immediately collected after injury. (D) The levels of hydrogen peroxide in the carotid artery with or without injury. (E) H&E stained histological sections of carotid arteries isolated from rats subjected to various treatments. Scale bars, 200 µm (the upper panel), 50 µm (the lower panel). PLGA NP, Ac-bCD NP, and Ox-bCD NP represents nanoparticles based on a non-responsive polymer PLGA, a pH-responsive material of acetelated β-CD, and a ROS-responsive material of PBAP-conjugated β-CD, respectively. Data are mean ± standard deviation (n = 3). P < 0.001. Brain Diseases Reproduced with permission from Feng et al (2016) FIGURE 6 \| Engineering of different inﬂammation-responsive nanotherapies for targeted treatment of restenosis. (A) Design of inﬂammation-triggerable nanoparticles. (B) Evans Blue staining indicates successful establishment of injury in the carotid artery. (C) DHE-stained cryosections showing oxidative stress in injured carotid arteries. The image at day 0 was acquired from the sample immediately collected after injury. (D) The levels of hydrogen peroxide in the carotid artery with or without injury. (E) H&E stained histological sections of carotid arteries isolated from rats subjected to various treatments. Scale bars, 200 µm (the upper panel), 50 µm (the lower panel). PLGA NP, Ac-bCD NP, and Ox-bCD NP represents nanoparticles based on a non-responsive polymer PLGA, a pH-responsive material of acetelated β-CD, and a ROS-responsive material of PBAP-conjugated β-CD, respectively. Data are mean ± standard deviation (n = 3). *P < 0.001. Reproduced with permission from Feng et al. (2016). CONCLUDING REMARKS Oxidative stress is at the basis of a variety of inﬂammatory pathologies, and therefore antioxidant nanotherapies have been extensively investigated as a new therapeutic strategy. Signiﬁcant advances have been achieved in the ﬁeld of antioxidant nanotherapies for diﬀerent inﬂammatory diseases, in which ROS-scavenging inorganic nanoparticles, organic nanoparticles with intrinsic antioxidant activity, and drug-loaded nanoparticles with antioxidant activity are generally used. Indeed, extensive preclinical studies have demonstrated desirable performances of ROS-scavenging nanotherapies in diﬀerent in vitro and in vivo inﬂammatory models. However, for the currently developed antioxidant nanotherapies, only a few of them have been intensively and systemically examined in diverse animal models. Even few translation studies have been conducted for antioxidant nanotherapies thus far. y g Acute lung injury (ALI), a heterogeneous pulmonary disease with the severe manifestation of acute respiratory distress syndrome, continues to cause high morbidity and mortality in critically ill patients (Rubenfeld et al., 2005; Sadikot et al., 2017). ALI is closely related to the systemic inﬂammatory response and the increased cellular ROS (Chabot et al., 1998). Cerium oxide nanoparticles were found able to reduce oxidative stress in vitro and in vivo (Arvapalli et al., 2015; Xu et al., 2016). They also exhibited protective eﬀects against sepsis-induced ALI and radiation-induced lung injury. In mice with paraquat-induced ALI, the levels of ROS, malondialdehyde, NF-κB, phosphorylated NF-κB, TNF-α, and IL-1β were signiﬁcantly reduced by porous Se@SiO2 nanospheres that contain Se to scavenge intracellular free radicals (Zhu et al., 2017). Also, resveratrol-loaded lipid- core nanocapsules (RSV-LNCs) were studied to ameliorate ALI via the ERK and PI3K/Akt pathways (de Oliveira et al., 2019). In addition, intrinsically bioactive nanoparticles derived from functional cyclodextrin materials TPCD and LCD that were developed by our group, were able to accumulate in the injured lungs, suppress oxidative stress, and signiﬁcantly reduce the inﬁltration of inﬂammatory cells in a mouse model of ALI (Figures 3C–H) (Li et al., 2018; Guo et al., 2019). Consequently, both inorganic and organic ROS-scavenging nanoparticles can serve as a potent remedy for the treatment of ALI. There are still some critical challenges need to be addressed for further clinical translation of these anti-inﬂammatory nanotherapies. First, synthesis processes for currently developed antioxidant nanoparticles need to be optimized to produce nanotherapies with good quality control, such as highly deﬁned structures and physicochemical characters as well as good batch-to-batch reproducibility, from the view point of preparation. Other Inﬂammatory Diseases diﬀerent types of ceria nanoparticles, i.e., ceria, TPP-ceria, and cluster-ceria nanoparticles, to selectively scavenge intracellular, mitochondrial, and extracellular ROS, respectively (Kwon et al., 2018). Therapeutic eﬀects of three ceria nanoparticles were compared in mice with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-induced PD. Mice treated with either ceria nanoparticles or TPP-ceria nanoparticles exhibited signiﬁcantly higher levels of tyrosine hydroxylase (a hallmark of PD) and lower levels of lipid peroxidation, compared to those treated with cluster-ceria nanoparticles. This ﬁnding indicated that decreasing mitochondrial and/or intracellular oxidative stress is critical to treat PD, while elimination of extracellular ROS is not an eﬀective strategy to prevent neurodegeneration. Other Inﬂammatory Diseases Asthma is a chronic pulmonary disease, characterized by recurrent airﬂow limitation, airway remodeling, and airway hyperreactivity. Inhaling corticosteroids is the most common treatment for asthma. However, adverse eﬀects associated with the frequent steroid administration precipitate the need for alternative therapeutics or novel delivery routes (Vij, 2011; Lim et al., 2016; Wang L. et al., 2019). Oxidative stress and inﬂammation play an important role in the pathogenesis of asthma. Growing evidence has indicated that nanoparticle-based anti-inﬂammation and antioxidant strategies are promising for the treatment of allergic airway inﬂammation and asthma (Fatani, 2014; Sahiner et al., 2018; Alexescu et al., 2019; March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 14 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. Singh A. P. et al., 2019). The anti-asthmatic eﬀects of two organic nanoparticles, i.e., BRNPs and HPOX nanoparticles with intrinsic antioxidant activity were evaluated using a murine model of asthma (Yoo et al., 2013; Kim D. E. et al., 2017). Both of them could reduce the recruitment of inﬂammatory cells and expression of inﬂammatory cytokines, thus may be used as potential nanotherapies for asthma. On the other hand, corticosteroids encapsulated in organic nanoparticles aﬀorded more sustained therapeutic eﬀects than free drugs. For example, a biodegradable polymer PVAX was modiﬁed to prepare dexamethasone (DEX)-loaded porous PVAX microparticles by a double emulsion method (Jeong et al., 2016). PVAX microparticles themselves remarkably reduced oxidative stress and down-regulated the expression of proinﬂammatory mediators such as TNF-α and iNOS. Notably, therapeutic eﬀects of DEX-loaded porous PVAX microparticles were much better than PVAX microparticles alone, indicating a signiﬁcant synergistic eﬀect. peritonitis, by reducing ROS production, inhibiting neutrophil inﬁltration and neutrophil-induced macrophage recruitment, as well as down-regulating the expression of inﬂammatory cytokines and chemokines (Zhang et al., 2017). REFERENCES Chemiluminescent and antioxidant micelles as theranostic agents for hydrogen peroxide associated-inﬂammatory diseases. Adv. Funct. Mater. 22, 4038–4043. doi: 10.1002/adfm.201200773 Bao, X., Zhao, J., Sun, J., Hu, M., and Yang, X. (2018). Polydopamine nanoparticles as eﬃcient scavengers for reactive oxygen species in periodontal disease. ACS Nano 12, 8882–8892. doi: 10.1021/acsnano.8b04022 Barnham, K. J., Masters, C. L., and Bush, A. I. (2004). Neurodegenerative diseases and oxidative stress. Nat. Rev. Drug Discov. 3, 205–214. doi: 10.1038/nrd1330 Chonpathompikunlert, P., Fan, C.-H., Ozaki, Y., Yoshitomi, T., Yeh, C.-K., and Nagasaki, Y. (2012). Redox nanoparticle treatment protects against neurological deﬁcit in focused ultrasound-induced intracerebral hemorrhage. Nanomedicine 7, 1029–1043. doi: 10.2217/nnm.12.2 Bayir, H. (2006). Reactive oxygen species. Crit. Care Med. 33, S498–S501. doi: 10.1097/01.CCM.0000186787.64500.12 Chu, M., Hai, W., Zhang, Z., Wo, F., Wu, Q., Zhang, Z., et al. (2016). Melanin nanoparticles derived from a homology of medicine and food for sentinel lymph node mapping and photothermal in vivo cancer therapy. Biomaterials 91, 182–199. doi: 10.1016/j.biomaterials.2016.03.018 Berwin Singh, S. V., Adam, A. G., Tripathy, N., Lee, D., and Khang, G. (2018). Reactive oxygen species responsive naturally occurring phenolic-based polymeric prodrug. Adv. Exp. Med. Biol. 1078, 291–301. doi: 10.1007/978-981-13-0950-2_15 Bhatia, S. K., Wadhwa, P., Hong, J. W., Hong, Y. G., Jeon, J. M., Lee, E. S., et al. (2019). Lipase mediated functionalization of poly(3-hydroxybutyrate-co- 3-hydroxyvalerate) with ascorbic acid into an antioxidant active biomaterial. Int. J. Biol. Macromol. 123, 117–123. doi: 10.1016/j.ijbiomac.2018.11.052 Das, S., Dowding, J. M., Klump, K. E., McGinnis, J. F., Self, W., and Seal, S. (2013). Cerium oxide nanoparticles: applications and prospects in nanomedicine. Nanomedicine 8, 1483–1508. doi: 10.2217/nnm.13.133 D’Autréaux, B., and Toledano, M. B. (2007). ROS as signalling molecules: mechanisms that generate speciﬁcity in ROS homeostasis. Nat. Rev. Mol. Cell Biol. 8, 813–824. doi: 10.1038/nrm2256 Brown, D. I., and Griendling, K. K. (2015). Regulation of signal transduction by reactive oxygen species in the cardiovascular system. Circ. Res. 116, 531–549. doi: 10.1161/CIRCRESAHA.116.303584 Davan, R., Prasad, R. G. S. V., Jakka, V. S., Aparna, R. S. L., Phani, A. R., Jacob, B., et al. (2012). Cerium oxide nanoparticles promotes wound healing activity in in-vivo animal model. J. Bionanosci. 6, 78–83. doi: 10.1166/jbns.2012.1074 Bryant, N., Johnson, M., Walker, M., Riley, K., and Sims, C. (2016). Antioxidant cerium oxide nanoparticles in biology and medicine. Antioxidants 5:E15. doi: 10.3390/antiox5020015 de Oliveira, M. T. P., de Sa Coutinho, D., Tenorio de Souza, E., Staniscuaski Guterres, S., Pohlmann, A. R., Silva, P. M. REFERENCES and potential molecular targets for neuroprotection. Antioxid. Redox Signal. 14, 1505–1517. doi: 10.1089/ars.2010.3576 Alaarg, A., Perez-Medina, C., Metselaar, J. M., Nahrendorf, M., Fayad, Z. A., Storm, G., et al. (2017). Applying nanomedicine in maladaptive inﬂammation and angiogenesis. Adv. Drug Deliv. Rev. 119, 143–158. doi: 10.1016/j.addr.2017.05.009 Chen, J., Wang, Q., Huang, L., Zhang, H., Rong, K., Zhang, H., et al. (2018). Prussian blue with intrinsic heme-like structure as peroxidase mimic. Nano Res. 11, 4905–4913. doi: 10.1007/s12274-018-2079-8 Chen, Y.-P., Chen, C.-T., Hung, Y., Chou, C.-M., Liu, T.-P., Liang, M.-R., et al. (2013). A new strategy for intracellular delivery of enzyme using mesoporous silica nanoparticles: Superoxide dismutase. J. Am. Chem. Soc. 135, 1516–1523. doi: 10.1021/ja3105208 Alexescu, T., Tarmure, S., Negrean, V., Cosnarovici, M., Ruta, V., Popovici, I., et al. (2019). Nanoparticles in the treatment of chronic lung diseases. J. Mind Med. Sci. 6, 224–231. doi: 10.22543/7674.62.P224231 Ali, S. S., Hardt, J. I., and Dugan, L. L. (2008). Sod activity of carboxyfullerenes predicts their neuroprotective eﬃcacy: a structure-activity study. Nanomed. Nanotechnol. 4, 283–294. doi: 10.1016/j.nano.2008.05.003 Cheng, J., Zhang, R., Li, C., Tao, H., Dou, Y., Wang, Y., et al. (2018). A targeting nanotherapy for abdominal aortic aneurysms. J. Am. Coll. Cardiol. 72, 2591–2605. doi: 10.1016/j.jacc.2018.08.2188 Andersen, J. K. (2004). Oxidative stress in neurodegeneration: Cause or consequence? Nat. Med. 10, S18–S25. doi: 10.1038/nrn1434 Chmielowski, R. A., Abdelhamid, D. S., Faig, J. J., Petersen, L. K., Gardner, C. R., Uhrich, K. E., et al. (2017). Athero-inﬂammatory nanotherapeutics: Ferulic acid-based poly(anhydride-ester) nanoparticles attenuate foam cell formation by regulating macrophage lipogenesis and reactive oxygen species generation. Acta Biomater. 57, 85–94. doi: 10.1016/j.actbio.2017.05.029 Apostolova, N., and Victor, V. M. (2015). Molecular strategies for targeting antioxidants to mitochondria: therapeutic implications. Antioxid. Redox Signal. 22, 686–729. doi: 10.1089/ars.2014.5952 Arvapalli, R., Manne, N., Rice, K., and Blough, E. (2015). Therapeutic eﬃcacy of cerium oxide nanoparticles against sepsis induced acute lung injury. FASEB J. 29, 620–614. Cho, B. R., Ryu, D. R., Lee, K. S., Lee, D. K., Bae, S., Kang, D. G., et al. (2015). P-hydroxybenzyl alcohol-containing biodegradable nanoparticle improves functional blood ﬂow through angiogenesis in a mouse model of hindlimb ischemia. Biomaterials 53, 679–687. doi: 10.1016/j.biomaterials.2015. 02.107 Bak, A., Ashford, M., and Brayden, D. J. (2018). Local delivery of macromolecules to treat diseases associated with the colon. Adv. Drug Deliv. Rev. 136–137, 2–27. doi: 10.1016/j.addr.2018.10.009 Cho, S., Hwang, O., Lee, I., Lee, G., Yoo, D., Khang, G., et al. (2012). CONCLUDING REMARKS The cost-eﬃcient mass production is also a critical factor that should be carefully considered according to the beneﬁts of diﬀerent antioxidant nanoparticles and their potential applications in clinical practice. Second, both acute and long-term chronic toxicities of various antioxidant nanotherapies must be comprehensively tested, particularly for ROS-scavenging inorganic nanoparticles. The physicochemical and biological properties of nanoparticles greatly inﬂuence their interactions with the living organisms. The developed antioxidant nanotherapies or nanocarriers should be biocompatible and easy to clear by the body. Thirdly, the ROS level varies among diﬀerent inﬂammatory disorders as well as throughout the diﬀerent stages of the same inﬂammatory disease, which largely decide the dose and dosing frequency of antioxidant nanotherapies. The appropriate dose should control the intracellular ROS toward the beneﬁcial therapeutic eﬀects without causing pathological ones. These issues unfortunately remain elusive. Also, a thorough assessment of risks and beneﬁts of antioxidative nanoparticles is a main ethical issue. The ideal antioxidative nanoparticles should maximize the well-being of patients and reduce or avoid therapy-induced side eﬀects, thereby aﬀording patients with the beneﬁts outweighing the risks. With the aforementioned challenges to be resolved, we are conﬁdent that the clinical applications of antioxidant nanotherapies for the treatment of inﬂammatory diseases will be realize in the foreseeable future, resulting in improved safety and individualized healthcare. Recently, diﬀerent antioxidant nanotherapies have been applied in the treatment of peritonitis, an inﬂammation of the peritoneum. Porﬁre and coauthors evaluated eﬃcacy of three diﬀerent Cu/Zn-SOD formulations in LPS-induced peritonitis (Porﬁre et al., 2014). PEGylated liposomes showed the most signiﬁcant antioxidant and anti-inﬂammatory properties in this case. Ac2-26-containing nanoparticles, developed using an anti-inﬂammatory peptide Ac2-26 as well as diblock copolymers PLGA-PEG and collagen IV-targeting PLGA-PEG, could signiﬁcantly inhibit the recruitment of polymononuclear neutrophils in a zymosan-induced peritonitis model (Kamaly et al., 2013). Our group developed H2O2- eliminating nanoparticles, based on PBAP-conjugated β-CD materials, also displayed desirable therapeutic eﬀects in mice with March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 15 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. Li et al. FUNDING C-WL and L-LL drafted the manuscript. C-WL, L-LL, and SC created the tables and ﬁgures and performed literature searches. J-XZ and W-LL revised the manuscript and edited the ﬁnal draft. This study was supported by the National Natural Science Foundation of China (Nos. 81971727 and 81901866) and the Program for Distinguished Young Scholars of TMMU. C-WL and L-LL drafted the manuscript. C-WL, L-LL, and SC created the tables and ﬁgures and performed literature searches. J-XZ and W-LL revised the manuscript and edited the ﬁnal draft. This study was supported by the National Natural Science Foundation of China (Nos. 81971727 and 81901866) and the Program for Distinguished Young Scholars of TMMU. This study was supported by the National Natural Science Foundation of China (Nos. 81971727 and 81901866) and the Program for Distinguished Young Scholars of TMMU. g p J-XZ and W-LL revised the manuscript and edited the ﬁnal draft. REFERENCES [60]fullerene is a powerful antioxidant in vivo with no acute or subacute toxicity. Nano Lett. 5, 2578–2585. doi: 10.1021/nl051866b Kang, C., Cho, W., Park, M., Kim, J., Park, S., Shin, D., et al. (2016). H2O2-triggered bubble generating antioxidant polymeric nanoparticles as ischemia/reperfusion targeted nanotheranostics. Biomaterials 85, 195–203. doi: 10.1016/j.biomaterials.2016.01.070 Gordon, R., Podolski, I., Makarova, E., Deev, A., Mugantseva, E., Khutsyan, S., et al. (2017). Intrahippocampal pathways involved in learning/memory mechanisms are aﬀected by intracerebral infusions of amyloid-β25-35 peptide and hydrated fullerene c60 in rats. J. Alzheimers. Dis. 58, 711–724. doi: 10.3233/JAD-161182 Karin, M., and Clevers, H. (2016). Reparative inﬂammation takes charge of tissue regeneration. Nature 529, 307–315. doi: 10.1038/nature17039 Gou, S., Huang, Y., Wan, Y., Ma, Y., Zhou, X., Tong, X., et al. (2019). Multi- bioresponsive silk ﬁbroin-based nanoparticles with on-demand cytoplasmic drug release capacity for CD44-targeted alleviation of ulcerative colitis. Biomaterials 212, 39–54. doi: 10.1016/j.biomaterials.2019.05.012 Kim, D. E., Lee, Y., Kim, M., Lee, S., Jon, S., and Lee, S. H. (2017). Bilirubin nanoparticles ameliorate allergic lung inﬂammation in a mouse model of asthma. Biomaterials 140, 37–44. doi: 10.1016/j.biomaterials.2017.06.014 Guo, J., Li, D., Tao, H., Li, G., Liu, R., Dou, Y., et al. (2019). Cyclodextrin– derived intrinsically bioactive nanoparticles for treatment of acute and chronic inﬂammatory diseases. Adv. Mater. 31:e1904607. doi: 10.1002/adma.201904607 Kim, G. H., Kim, J. E., Rhie, S. J., and Yoon, S. (2015). The role of oxidative stress in neurodegenerative diseases. Exp. Neurobiol. 24, 325–340. doi: 10.5607/en.2015.24.4.325 Kim, J., Takuji, S., and Yusei, M. (2010). The eﬀect of tat conjugated platinum nanoparticles on lifespan in a nematode caenorhabditis elegans model. Biomaterials 31, 5849–5854. doi: 10.1016/j.biomaterials.2010.03.077 Hao, T., Li, J., Yao, F., Dong, D., Wang, Y., Yang, B., et al. (2017). Injectable fullerenol/alginate hydrogel for suppression of oxidative stress damage in brown adipose-derived stem cells and cardiac repair. ACS Nano 11, 5474–5488. doi: 10.1021/acsnano.7b00221 Kim, J. Y., Lee, D. Y., Kang, S., Miao, W., Kim, H., Lee, Y., et al. (2017). Bilirubin nanoparticle preconditioning protects against hepatic ischemia-reperfusion injury. Biomaterials 133, 1–10. doi: 10.1016/j.biomaterials.2017.04.011 Harrison, D., Griendling, K. K., Landmesser, U., Hornig, B., and Drexler, H. (2003). Role of oxidative stress in atherosclerosis. Am. J. Cardiol. 91, 7–11. doi: 10.1016/S0002-9149(02)03144-2 Kim, K. S., Lee, D., Song, C. G., and Kang, P. M. (2015). Reactive oxygen species- activated nanomaterials as theranostic agents. Nanomedicine 10, 2709–2723. REFERENCES R., et al. (2019). Orally delivered resveratrol-loaded lipid-core nanocapsules ameliorate lps-induced acute lung injury via the erk and pi3k/akt pathways. Int. J. Nanomed. 14, 5215–5228. doi: 10.2147/IJN.S200666 Cai, X., Sezate, S. A., Seal, S., and McGinnis, J. F. (2012). Sustained protection against photoreceptor degeneration in tubby mice by intravitreal injection of nanoceria. Biomaterials 33, 8771–8781. doi: 10.1016/j.biomaterials.2012. 08.030 Cao, G.-J., Jiang, X., Zhang, H., Zheng, J., Croley, T. R., and Yin, J.-J. (2017). Exploring the activities of ruthenium nanomaterials as reactive oxygen species scavengers. J. Environ. Sci. Health C 35, 223–238. doi: 10.1080/10590501.2017.1391516 Dou, Y., Chen, Y., Zhang, X., Xu, X., Chen, Y., Guo, J., et al. (2017). Non-proinﬂammatory and responsive nanoplatforms for targeted treatment of atherosclerosis. Biomaterials 143, 93–108. doi: 10.1016/j.biomaterials.2017.07.035 Celardo, I., Pedersen, J. Z., Traversa, E., and Ghibelli, L. (2011). Pharmacological potential of cerium oxide nanoparticles. Nanoscale 3, 1411–1420. doi: 10.1039/c0nr00875c Dugan, L. L., Turetsky, D. M., Du, C., Lobner, D., Wheeler, M., Almli, C. R., et al. (1997). Carboxyfullerenes as neuroprotective agents. Proc. Natl. Acad. Sci. U.S.A. 94, 9434–9439. doi: 10.1073/pnas.94.17.9434 Chabot, F., Mitchell, J. A., Gutteridge, J. M., and Evans, T. W. (1998). Reactive oxygen species in acute lung injury. Eur. Respir. J. 11, 745–757. Edaravone Acute Infarction Study Group (2003). Eﬀect of a novel free radical scavenger, edaravone (mci-186), on acute brain infarction. Randomized, placebo-controlled, double-blind study at multicenters. Cerebrovasc. Dis. 15, 222–229. doi: 10.1159/000069318 Chen, H., Yoshioka, H., Kim, G. S., Jung, J. E., Okami, N., Sakata, H., et al. (2011). Oxidative stress in ischemic brain damage: Mechanisms of cell death March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 16 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. Eguchi, A., Yoshitomi, T., Lazic, M., Johnson, C. D., Vong, L. B., Wree, A., et al. (2015). Redox nanoparticles as a novel treatment approach for inﬂammation and ﬁbrosis associated with nonalcoholic steatohepatitis. Nanomedicine 10, 2697–2708. doi: 10.2217/nnm.15.87 Huang, Y., Liu, Z., Liu, C., Ju, E., Zhang, Y., Ren, J., et al. (2016). Self-assembly of multi-nanozymes to mimic an intracellular antioxidant defense system. Angew. Chem. Int. Ed. 55, 6646–6650. doi: 10.1002/anie.201600868 Ikeda, Y., and Nagasaki, Y. (2018). Antioxidative biointerface: Biocompatible materials scavenging reactive oxygen species. Biomed. Mater. 13:044103. doi: 10.1088/1748-605X/aab720 Fatani, S. H. (2014). Biomarkers of oxidative stress in acute and chronic bronchial asthma. J. Asthma 51, 578–584. REFERENCES doi: 10.3109/02770903.2014.892965 Jeong, D., Kang, C., Jung, E., Yoo, D., Wu, D., and Lee, D. (2016). Porous antioxidant polymer microparticles as therapeutic systems for the airway inﬂammatory diseases. J. Control. Release 233, 72–80. doi: 10.1016/j.jconrel.2016.04.039 Feliciano, C. P., Tsuboi, K., Suzuki, K., Kimura, H., and Nagasaki, Y. (2017). Long-term bioavailability of redox nanoparticles eﬀectively reduces organ dysfunctions and death in whole-body irradiated mice. Biomaterials 129, 68–82. doi: 10.1016/j.biomaterials.2017.03.011 Feng, S., Hu, Y., Peng, S., Han, S., Tao, H., Zhang, Q., et al. (2016). Nanoparticles responsive to the inﬂammatory microenvironment for targeted treatment of arterial restenosis. Biomaterials 105, 167–184. doi: 10.1016/j.biomaterials.2016.08.003 Ju, K. Y., Lee, Y., Lee, S., Park, S. B., and Lee, J. K. (2011). Bioinspired polymerization of dopamine to generate melanin-like nanoparticles having an excellent free-radical-scavenging property. Biomacromolecules 12, 625–632. doi: 10.1021/bm101281b Jung, E., Lee, J., Jeong, L., Park, S., Lee, M., Song, C., et al. (2019). Stimulus- activatable echogenic maltodextrin nanoparticles as nanotheranostic agents for peripheral arterial disease. Biomaterials 192, 282–291. doi: 10.1016/j.biomaterials.2018.11.022 Ferreira, C. A., Ni, D., Rosenkrans, Z. T., and Cai, W. (2018). Scavenging of reactive oxygen and nitrogen species with nanomaterials. Nano Res. 11, 4955–4984. doi: 10.1007/s12274-018-2092-y Fraisl, P., Aragonés, J., and Carmeliet, P. (2009). Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inﬂammatory disease. Nat. Rev. Drug Discov. 8, 139–152. doi: 10.1038/nrd2761 Jung, E., Noh, J., Kang, C., Yoo, D., Song, C., and Lee, D. (2018). Ultrasound imaging and on-demand therapy of peripheral arterial diseases using H2O2- activated bubble generating anti-inﬂammatory polymer particles. Biomaterials 179, 175–185. doi: 10.1016/j.biomaterials.2018.07.003 Francesca, G., Moglianetti, M., Pompa, P., and Bardi, G. (2018). Platinum nanoparticles decrease reactive oxygen species and modulate gene expression without alteration of immune responses in thp-1 monocytes. Nanomaterials 8:392. doi: 10.3390/nano8060392 Juni, R. P., Duckers, H. J., Vanhoutte, P. M., Virmani, R., and Moens, A. L. (2013). Oxidative stress and pathological changes after coronary artery interventions. J. Am. Coll. Cardiol. 61, 1471–1481. doi: 10.1016/j.jacc.2012.11.068 Gao, L., Zhuang, J., Nie, L., Zhang, J., Zhang, Y., Gu, N., et al. (2007). Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2, 577–583. doi: 10.1038/nnano.2007.260 Kamaly, N., Fredman, G., Subramanian, M., Gadde, S., Pesic, A., Cheung, L., et al. (2013). Development and in vivo eﬃcacy of targeted polymeric inﬂammation-resolving nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 110, 6506–6511. doi: 10.1073/pnas.1303377110 Gharbi, N., Pressac, M., Hadchouel, M., Szwarc, H., Wilson, S. R., and Moussa, F. (2005). REFERENCES Q., Dos Santos, J. C. F., Araujo, O. R. P., and Goulart, M. O. F. (2015). Antioxidant therapy for treatment of inﬂammatory bowel disease: does it work? Redox Biol. 6, 617–639. doi: 10.1016/j.redox.2015.10.006 Lao, F., Chen, L., Li, W., Ge, C., Qu, Y., Sun, Q., et al. (2009). Fullerene nanoparticles selectively enter oxidation-damaged cerebral microvessel endothelial cells and inhibit jnk-related apoptosis. ACS Nano 3, 3358–3368. doi: 10.1021/nn900912n Ni, D., Jiang, D., Kutyreﬀ, C. J., Lai, J., Yan, Y., Barnhart, T. E., et al. (2018). Molybdenum-based nanoclusters act as antioxidants and ameliorate acute kidney injury in mice. Nat. Commun. 9:5421. doi: 10.1038/s41467-018-07890-8 Larrañaga, A., Isa, I. L. M., Patil, V., Thamboo, S., Lomora, M., Fernández-Yague, M. A., et al. (2018). Antioxidant functionalized polymer capsules to prevent oxidative stress. Acta Biomater. 67, 21–31. doi: 10.1016/j.actbio.2017.12.014 Norton, S. K., Wijesinghe, D. S., Dellinger, A., Sturgill, J., Zhou, Z., Barbour, S., et al. (2012). Epoxyeicosatrienoic acids are involved in the C70 fullerene derivative– induced control of allergic asthma. J. Allergy Clin. Immunol. 130, 761–769. doi: 10.1016/j.jaci.2012.04.023 Lee, D. Y., Kim, J. Y., Lee, Y., Lee, S., Miao, W., Kim, H. S., et al. (2017). Black pigment gallstone inspired platinum-chelated bilirubin nanoparticles for combined photoacoustic imaging and photothermal therapy of cancers. Angew. Chem. Int. Ed. 56, 13684–13688. doi: 10.1002/anie.201707137 Nosaka, Y., and Nosaka, A. Y. (2017). Generation and detection of reactive oxygen species in photocatalysis. Chem. Rev. 117, 11302–11336. doi: 10.1021/acs.chemrev.7b00161 Lee, Y., Kim, H., Kang, S., Lee, J., Park, J., and Jon, S. (2016a). Bilirubin nanoparticles as a nanomedicine for anti-inﬂammation therapy. Angew. Chem. Int. Ed. 55, 7460–7463. doi: 10.1002/anie.201602525 Onizawa, S., Aoshiba, K., Kajita, M., Miyamoto, Y., and Nagai, A. (2009). Platinum nanoparticle antioxidants inhibit pulmonary inﬂammation in mice exposed to cigarette smoke. Pulm. Pharmacol. Ther. 22, 340–349. doi: 10.1016/j.pupt.2008.12.015 Lee, Y., Lee, S., and Jon, S. (2018). Biotinylated bilirubin nanoparticles as a tumor microenvironment–responsive drug delivery system for targeted cancer therapy. Adv. Sci. 5:1800017. doi: 10.1002/advs.201800017 Oró, D., Yudina, T., Fernández-Varo, G., Casals, E., Reichenbach, V., Casals, G., et al. (2016). Cerium oxide nanoparticles reduce steatosis, portal hypertension and display anti-inﬂammatory properties in rats with liver ﬁbrosis. J. Hepatol. 64, 691–698. doi: 10.1016/j.jhep.2015.10.020 Lee, Y., Lee, S., Lee, D. Y., Yu, B., Miao, W., and Jon, S. (2016b). Multistimuli- responsive bilirubin nanoparticles for anticancer therapy. Angew. Chem. Int. Ed. 55, 10676–10680. REFERENCES Local carboxyfullerene protects cortical infarction in rat brain. Neurosci. Res. 43, 317–321. doi: 10.1016/S0168-0102(02)00056-1 Kozower, B. D., Christoﬁdou-Solomidou, M., Sweitzer, T. D., Muro, S., Buerk, D. G., Solomides, C. C., et al. (2003). Immunotargeting of catalase to the pulmonary endothelium alleviates oxidative stress and reduces acute lung transplantation injury. Nat. Biotechnol. 21, 392–398. doi: 10.1038/n bt806 Liu, Y., Ai, K., Ji, X., Askhatova, D., Du, R., Lu, L., et al. (2017). Comprehensive insights into the multi-antioxidative mechanisms of melanin nanoparticles and their application to protect brain from injury in ischemic stroke. J. Am. Chem. Soc. 139, 856–862. doi: 10.1021/jacs.6b11013 Krusic, P. J., Wasserman, E., Keizer, P. N., Morton, J. R., and Preston, K. F. (1991). Radical reactions of C60. Science 254, 1183–1185. doi: 10.1126/science.254.5035.1183 Lu, Y., Aimetti, A. A., Langer, R., and Gu, Z. (2016). Bioresponsive materials. Nat. Rev. Mater. 2, 1–17. doi: 10.1038/natrevmats.2016.75 Markovic, Z., and Trajkovic, V. (2008). Biomedical potential of the reactive oxygen species generation and quenching by fullerenes (C60). Biomaterials 29, 3561–3573. doi: 10.1016/j.biomaterials.2008.05.005 Kwon, H. J., Cha, M.-Y., Kim, D., Kim, D. K., Soh, M., Shin, K., et al. (2016). Mitochondria-targeting ceria nanoparticles as antioxidants for alzheimer’s disease. ACS Nano 10, 2860–2870. doi: 10.1021/acsnano.5b08045 Martín, R., Menchon, C., Apostolova, N., Victor, V. M., Alvaro, M., Herance, J. R., et al. (2010). Nano-jewels in biology. Gold and platinum on diamond nanoparticles as antioxidant systems against cellular oxidative stress. ACS Nano 4, 6957–6965. doi: 10.1021/nn1019412 Kwon, H. J., Kim, D., Seo, K., Kim, Y. G., Han, S. I., Kang, T., et al. (2018). Ceria nanoparticle systems for selective scavenging of mitochondrial, intracellular, and extracellular reactive oxygen species in parkinson’s disease. Angew. Chem. Int. Ed. 130, 9552–9556. doi: 10.1002/ange.201805052 Miriyala, S., Spasojevic, I., Tovmasyan, A., Salvemini, D., Vujaskovic, Z., St. Clair, D., et al. (2012). Manganese superoxide dismutase, MnSOD and its mimics. Biochim. Biophys. Acta 1822, 794–814. doi: 10.1016/j.bbadis.2011.12.002 Kwon, S., Ko, H., You, D. G., Kataoka, K., and Park, J. H. (2019). Nanomedicines for reactive oxygen species mediated approach: An emerging paradigm for cancer treatment. Acc. Chem. Res. 52, 1771–1782. doi: 10.1021/acs.accounts.9b00136 Moglianetti, M., Luca, E. D., Pedone, D., Marotta, R., and Pompa, P. P. (2016). Platinum nanozymes recover cellular ros homeostasis in oxidative stress- mediated disease model. Nanoscale 8, 3739–3752. doi: 10.1039/C5NR08358C Lamprecht, A. (2015). Nanomedicines in gastroenterology and hepatology. Nat. Rev. Gastroenterol. Hepatol. 12, 195–204. doi: 10.1038/nrgastro.2015.37 Moura, F. A., de Andrade, K. REFERENCES doi: 10.1002/anie.201604858 Pagliari, F., Mandoli, C., Forte, G., Magnani, E., Pagliari, S., Nardone, G., et al. (2012). Cerium oxide nanoparticles protect cardiac progenitor cells from oxidative stress. ACS Nano 6, 3767–3775. doi: 10.1021/nn2048069 Lee, Y., Sugihara, K., Gillilland, M. G., Jon, S., Kamada, N., and Moon, J. J. (2020). Hyaluronic acid–bilirubin nanomedicine for targeted modulation of dysregulated intestinal barrier, microbiome and immune responses in colitis. Nat. Mater. 19, 118–126. doi: 10.1038/s41563-019-0462-9 Park, J., Kim, B., Han, J., Oh, J., Park, S., Ryu, S., et al. (2015). Graphene oxide ﬂakes as a cellular adhesive: Prevention of reactive oxygen species mediated death of implanted cells for cardiac repair. ACS Nano 9, 4987–4999. doi: 10.1021/nn507149w Li, C., Zhao, Y., Cheng, J., Guo, J., Zhang, Q., Zhang, X., et al. (2019). A proresolving peptide nanotherapy for site-speciﬁc treatment of inﬂammatory bowel disease by regulating proinﬂammatory microenvironment and gut microbiota. Adv. Sci. 6:1900610. doi: 10.1002/advs.201900610 Pedone, D., Moglianetti, M., Luca, E. D., Bardi, G., and Pompa, P. P. (2017). Platinum nanoparticles in nanobiomedicine. Chem. Soc. Rev. 46, 4951–4975. doi: 10.1039/C7CS00152E Li, J., Guan, M., Wang, T., Zhen, M., Zhao, F., Shu, C., et al. (2016). Gd@C82-(ethylenediamine) 8 nanoparticle: A new high-eﬃciency water- soluble ros scavenger. ACS Appl. Mater. Interfaces 8, 25770–25776. doi: 10.1021/acsami.6b08659 Petro, M., Jaﬀer, H., Yang, J., Kabu, S., Morris, V. B., and Labhasetwar, V. (2016). Tissue plasminogen activator followed by antioxidant-loaded nanoparticle delivery promotes activation/mobilization of progenitor cells in infarcted rat brain. Biomaterials 81, 169–180. doi: 10.1016/j.biomaterials.2015.12.009 Li, L., Guo, J., Wang, Y., Xiong, X., Tao, H., Li, J., et al. (2018). A broad-spectrum ros-eliminating material for prevention of inﬂammation and drug-induced organ toxicity. Adv. Sci. 5:1800781. doi: 10.1002/advs.201800781 Petros, R. A., and DeSimone, J. M. (2010). Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discov. 9, 615–627. doi: 10.1038/nrd2591 Li, S., Xie, A., Li, H., Zou, X., and Zhang, Q. (2019). A self-assembled, ros- responsive janus-prodrug for targeted therapy of inﬂammatory bowel disease. J. Control. Release 316, 66–78. doi: 10.1016/j.jconrel.2019.10.054 Porﬁre, A. S., Leucuta, S. E., Kiss, B., Loghin, F., and Parvu, A. E. (2014). Investigation into the role of Cu/Zn-SOD delivery system on its antioxidant and antiinﬂammatory activity in rat model of peritonitis. Pharmacol. Rep. 66, 670–676. doi: 10.1016/j.pharep.2014.03.011 Li, Y., He, X., Yin, J.-J., Ma, Y., Zhang, P., Li, J., et al. (2015). Acquired superoxide- scavenging ability of ceria nanoparticles. Angew. Chem. Int. Ed. REFERENCES doi: 10.2217/nnm.15.108 He, Y., Zhang, B., Chen, Y., Jin, Q., Wu, J., Yan, F., et al. (2017). Image-guided hydrogen gas delivery for protection from myocardial ischemia-reperfusion injury via microbubbles. ACS Appl. Mater. Interfaces 9, 21190–21199. doi: 10.1021/acsami.7b05346 Kim, M. J., Lee, Y., Jon, S., and Lee, D. Y. (2017). Pegylated bilirubin nanoparticle as an anti-oxidative and anti-inﬂammatory demulcent in pancreatic islet xenotransplantation. Biomaterials 133, 242–252. doi: 10.1016/j.biomaterials.2017.04.029 Heckman, K. L., DeCoteau, W., Estevez, A., Reed, K. J., Costanzo, W., Sanford, D., et al. (2013). Custom cerium oxide nanoparticles protect against a free radical mediated autoimmune degenerative disease in the brain. ACS Nano 7, 10582–10596. doi: 10.1021/nn403743b Ko, E., Jeong, D., Kim, J., Park, S., Khang, G., and Lee, D. (2014). Antioxidant polymeric prodrug microparticles as a therapeutic system for acute liver failure. Biomaterials 35, 3895–3902. doi: 10.1016/j.biomaterials.2014.01.048 Hikosaka, K., Kim, J., Kajita, M., Kanayama, A., and Miyamoto, Y. (2008). Platinum nanoparticles have an activity similar to mitochondrial nadh:Ubiquinone oxidoreductase. Colloids Surf. B Biointerfaces 66, 195–200. doi: 10.1016/j.colsurfb.2008.06.008 Kojima, R., Bojar, D., Rizzi, G., Hamri, G. C.-E., El-Baba, M. D., Saxena, P., et al. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for parkinson’s disease treatment. Nat. Commun. 9:1305. doi: 10.1038/s41467-018-03733-8 Hirst, S. M., Karakoti, A. S., Tyler, R. D., Sriranganathan, N., Seal, S., and Reilly, C. M. (2009). Anti-inﬂammatory properties of cerium oxide nanoparticles. Small 5, 2848–2856. doi: 10.1002/smll.200901048 Kong, L., Cai, X., Zhou, X., Wong, L. L., Karakoti, A. S., Seal, S., et al. (2011). Nanoceria extend photoreceptor cell lifespan in tubby mice by modulation of apoptosis/survival signaling pathways. Neurobiol. Dis. 42, 514–523. doi: 10.1016/j.nbd.2011.03.004 Hu, B., Dai, F., Fan, Z., Ma, G., Tang, Q., and Zhang, X. (2015). Nanotheranostics: Congo red/rutin-mnps with enhanced magnetic resonance imaging and h2o2- responsive therapy of alzheimer’s disease in appswe/ps1de9 transgenic mice. Adv. Mater. 27, 5499–5505. doi: 10.1002/adma.201502227 Kotla, N. G., Rana, S., Sivaraman, G., Sunnapu, O., Vemula, P. K., Pandit, A., et al. (2019). Bioresponsive drug delivery systems in intestinal inﬂammation: March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 17 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. State-of-the-art and future perspectives. Adv. Drug Deliv. Rev. 146, 248–266. doi: 10.1016/j.addr.2018.06.021 State-of-the-art and future perspectives. Adv. Drug Deliv. Rev. 146, 248–266. doi: 10.1016/j.addr.2018.06.021 Lin, M. Y., Su-Feng, F., Shin-Zong, L., Cheng-Kong, C., Tieng-Yau, L., and Low- Tone, H. (2002). REFERENCES 54, 1832–1835. doi: 10.1002/anie.201410398 Lim, Y. H., Tiemann, K. M., Hunstad, D. A., Elsabahy, M., and Wooley, K. L. (2016). Polymeric nanoparticles in development for treatment of pulmonary infectious diseases. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 8, 842–871. doi: 10.1002/wnan.1401 Rehman, M. U., Yoko, Y., Yusei, M., and Tadamichi, S. (2012). The anti- inﬂammatory eﬀects of platinum nanoparticles on the lipopolysaccharide- induced inﬂammatory response in raw 264.7 macrophages. Inﬂamm. Res. 61, 1177–1185. doi: 10.1007/s00011-012-0512-0 March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 18 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. the cytoprotective potential of vanadia nanowires. Nat. Commun. 5:5301. doi: 10.1038/ncomms6301 the cytoprotective potential of vanadia nanowires. Nat. Commun. 5:5301. doi: 10.1038/ncomms6301 Rubenfeld, G. D., Caldwell, E., Peabody, E., Weaver, J., Martin, D. P., Neﬀ, M., et al. (2005). Incidence and outcomes of acute lung injury. N. Engl. J. Med. 353, 1685–1693. doi: 10.1056/NEJMoa050333 Vij, N. (2011). Nano-based theranostics for chronic obstructive lung diseases: Challenges and therapeutic potential. Expert Opin. Drug Deliv. 8, 1105–1109. doi: 10.1517/17425247.2011.597381 Sadikot, R. T., Kolanjiyil, A. V., Kleinstreuer, C., and Rubinstein, I. (2017). Nanomedicine for treatment of acute lung injury and acute respiratory distress syndrome. Biomedicine Hub. 2, 1–12. doi: 10.1159/000477086 Vong, L. B., Mo, J., Abrahamsson, B., and Nagasaki, Y. (2015). Speciﬁc accumulation of orally administered redox nanotherapeutics in the inﬂamed colon reducing inﬂammation with dose-response eﬃcacy. J. Control. Release 210, 19–25. doi: 10.1016/j.jconrel.2015.05.275 syndrome. Biomedicine Hub. 2, 1–12. doi: 10.1159/000477086 Sahiner, U. M., Birben, E., Erzurum, S., Sackesen, C., and Kalayci, O. (2018). Oxidative stress in asthma: Part of the puzzle. Pediatr. Allergy Immunol. 29, 789–800. doi: 10.1111/pai.12965 Saravanakumar, G., Kim, J., and Kim, W. J. (2017). Reactive oxygen species- responsive drug delivery systems: Promises and challenges. Adv. Sci. 4:1600124. doi: 10.1002/advs.201600124 Vong, L. B., and Nagasaki, Y. (2016). Combination treatment of murine colon cancer with doxorubicin and redox nanoparticles. Mol. Pharm. 13, 449–455. doi: 10.1021/acs.molpharmaceut.5b00676 Schilrreﬀ, P., Simioni, Y. R., Jerez, H. E., Caimi, A. T., de Farias, M. A., Villares Portugal, R., et al. (2019). Superoxide dismutase in nanoarchaeosomes for targeted delivery to inﬂammatory macrophages. Colloids Surf. B Biointerfaces 179, 479–487. doi: 10.1016/j.colsurfb.2019.03.061 Vong, L. B., Tomita, T., Yoshitomi, T., Matsui, H., and Nagasaki, Y. (2012). An orally administered redox nanoparticle that accumulates in the colonic mucosa and reduces colitis in mice. Gastroenterology 143, 1027–1036.e3. doi: 10.1053/j.gastro.2012.06.043 Vong, L. REFERENCES B., Yoshitomi, T., Morikawa, K., Saito, S., Matsui, H., and Nagasaki, Y. (2014). Oral nanotherapeutics: Eﬀect of redox nanoparticle on microﬂora in mice with dextran sodium sulfate-induced colitis. J. Gastroenterol. 49, 806–813. doi: 10.1007/s00535-013-0836-8 Sedghi, S., Fields, J. Z., Klamut, M., Urban, G., Durkin, M., Winship, D., et al. (1993). Increased production of luminol enhanced chemiluminescence by the inﬂamed colonic mucosa in patients with ulcerative colitis. Gut 34, 1191–1197. doi: 10.1136/gut.34.9.1191 Seshadri, G., Sy, J. C., Brown, M., Dikalov, S., Yang, S. C., Murthy, N., et al. (2010). The delivery of superoxide dismutase encapsulated in polyketal microparticles to rat myocardium and protection from myocardial ischemia-reperfusion injury. Biomaterials 31, 1372–1379. doi: 10.1016/j.biomaterials.2009.10.045 Wang, J., Wang, H., Zhu, R., Liu, Q., Fei, J., and Wang, S. (2015). Anti- inﬂammatory activity of curcumin-loaded solid lipid nanoparticles in il- 1β transgenic mice subjected to the lipopolysaccharide-induced sepsis. Biomaterials 53, 475–483. doi: 10.1016/j.biomaterials.2015.02.116 Wang, L., Feng, M., Li, Q., Qiu, C., and Chen, R. (2019). Advances in nanotechnology and asthma. Ann. Transl. Med. 7:180. doi: 10.21037/atm.2019.04.62 Shashni, B., Alshwimi, A., Minami, K., Furukawa, T., and Nagasaki, Y. (2017). Nitroxide radical-containing nanoparticles as potential candidates for overcoming drug resistance in epidermoid cancers. Polymer. 116, 429–438. doi: 10.1016/j.polymer.2017.02.052 Wang, X., Sheng, J., and Yang, M. (2019). Melanin-based nanoparticles in biomedical applications: From molecular imaging to treatment of diseases. Chin. Chem. Lett. 30, 533–540. doi: 10.1016/j.cclet.2018.10.010 Shashni, B., and Nagasaki, Y. (2018). Nitroxide radical-containing nanoparticles attenuate tumorigenic potential of triple negative breast cancer. Biomaterials 178, 48–62. doi: 10.1016/j.biomaterials.2018.05.042 Wang, Y., Li, L., Zhao, W., Dou, Y., An, H., Tao, H., et al. (2018). Targeted therapy of atherosclerosis by a broad-spectrum reactive oxygen species scavenging nanoparticle with intrinsic anti-inﬂammatory activity. ACS Nano 12, 8943–8960. doi: 10.1021/acsnano.8b02037 Simmonds, N. J., Allen, R. E., Stevens, T. R. J., Niall, R., Van Someren, M., Blake, D. R., et al. (1992). Chemiluminescence assay of mucosal reactive oxygen metabolites in inﬂammatory bowel disease. Gastroenterology 103, 186–196. doi: 10.1016/0016-5085(92)91112-H Watanabe, T., Tahara, M., and Todo, S. (2008). The novel antioxidant edaravone: From bench to bedside. Cardiovas. Ther. 26, 101–114. doi: 10.1111/j.1527-3466.2008.00041.x Singh, A. P., Biswas, A., Shukla, A., and Maiti, P. (2019). Targeted therapy in chronic diseases using nanomaterial-based drug delivery vehicles. Signal Transduct. Target Ther. 4:33. doi: 10.1038/s41392-019-0068-3 Wattamwar, P. P., Mo, Y., Wan, R., Palli, R., Zhang, Q., and Dziubla, T. D. (2010). REFERENCES Antioxidant activity of degradable polymer poly (trolox ester) to suppress oxidative stress injury in the cells. Adv. Funct. Mater. 20, 147–154. doi: 10.1002/adfm.200900839 Singh, N., Geethika, M., Eswarappa, S. M., and Mugesh, G. (2018). Manganese- based nanozymes: Multienzyme redox activity and eﬀect on the nitric oxide produced by endothelial nitric oxide synthase. Chem. Eur. J. 24, 8393–8403. doi: 10.1002/chem.201800770 Wong, L. L., Hirst, S. M., Pye, Q. N., Reilly, C. M., Seal, S., and McGinnis, J. F. (2013). Catalytic nanoceria are preferentially retained in the rat retina and are not cytotoxic after intravitreal injection. PLoS ONE 8:e58431. doi: 10.1371/journal.pone.0058431 Singh, N., Savanur, M. A., Srivastava, S., D’Silva, P., and Mugesh, G. (2017). A redox modulatory Mn3 O4 nanozyme with multi-enzyme activity provides eﬃcient cytoprotection to human cells in a parkinson’s disease model. Angew. Chem. Int. Ed. 56, 14267–14271. doi: 10.1002/anie.201708573 Wong, L. L., Pye, Q. N., Chen, L., Seal, S., and McGinnis, J. F. (2015). Deﬁning the catalytic activity of nanoceria in the P23H-1 rat, a photoreceptor degeneration model. PLoS ONE 10:e0121977. doi: 10.1371/journal.pone.0121977 Singh, N., Savanur, M. A., Srivastava, S., D’Silva, P., and Mugesh, G. (2019). A manganese oxide nanozyme prevents the oxidative damage of biomolecules without aﬀecting the endogenous antioxidant system. Nanoscale 11, 3855–3863. doi: 10.1039/C8NR09397K Wu, T., Chen, X., Wang, Y., Xiao, H., Peng, Y., Lin, L., et al. (2018). Aortic Plaque- Targeted andrographolide delivery with Oxidation-Sensitive micelle eﬀectively treats atherosclerosis via simultaneous ROS capture and Anti-Inﬂammation. Nanomed. Nanotechnol. 14, 2215–2226. doi: 10.1016/j.nano.2018. 06.010 Soh, M., Kang, D. W., Jeong, H. G., Kim, D., Kim, D. Y., Yang, W., et al. (2017). Ceria-zirconia nanoparticles as an enhanced multi-antioxidant for sepsis treatment. Angew. Chem. Int. Ed Engl. 56, 11399–11403. doi: 10.1002/anie.201704904 Wu, X., Dai, H., Xu, C., Liu, L., and Li, S. (2019). Citric acid modiﬁcation of a polymer exhibits antioxidant and anti-inﬂammatory properties in stem cells and tissues. J. Biomed. Mater. Res. A 107, 2414–2424. doi: 10.1002/jbm.a.36748 Solano, F. (2017). Melanin and Melanin-Related polymers as materials with biomedical and biotechnological Applications-Cuttleﬁsh Ink and mussel foot proteins as inspired biomolecules. Int. J. Mol. Sci. 18:E1561. doi: 10.3390/ijms18071561 Xu, P. T., Maidment, B. W. III, Antonic, V., Jackson, I. L., Das, S., Zodda, A., et al. (2016). Cerium oxide nanoparticles: A potential medical countermeasure to mitigate radiation-induced lung injury in CBA/J mice. Radiat. Res. 185, 516–526. doi: 10.1667/RR14261.1 Sun, T., Jiang, D., Rosenkrans, Z. T., Ehlerding, E. REFERENCES B., Ni, D., Qi, C., et al. (2019). A Melanin-Based natural antioxidant defense nanosystem for theranostic application in acute kidney injury. Adv. Funct. Mater. 29:1904833. doi: 10.1002/adfm.201904833 Xue, F., Wang, Y., Zhang, Q., Han, S., Zhang, F., Jin, T., et al. (2018). Self- assembly of Aﬃnity-controlled nanoparticles via Host–guest interactions for drug delivery. Nanoscale 10, 12364–12377. doi: 10.1039/C8NR0 1518J Takamiya, M., Miyamoto, Y., Yamashita, T., Deguchi, K., Ohta, Y., and Abe, K. (2012). Strong neuroprotection with a novel platinum nanoparticle against ischemic Stroke- and tissue plasminogen activator-related brain damages in mice. Neuroscience 221, 47–55. doi: 10.1016/j.neuroscience.2012.06.060 Yamamoto, T., Yuki, S., Watanabe, T., Mitsuka, M., Saito, K.-I., and Kogure, K. (1997). Delayed neuronal death prevented by inhibition of increased hydroxyl radical formation in a transient cerebral ischemia. Brain Res. 762, 240–242. doi: 10.1016/S0006-8993(97)00490-3 Vernekar, A. A., Sinha, D., Srivastava, S., Paramasivam, P. U., D’Silva, P., and Mugesh, G. (2014). An antioxidant nanozyme that uncovers Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org March 2020 \| Volume 8 \| Article 200 19 Antioxidant Nanotherapies for Inﬂammatory Diseases Li et al. Yao, J., Cheng, Y., Zhou, M., Zhao, S., Lin, S., Wang, X., et al. (2018). ROS scavenging mn3o4 nanozymes for in vivo anti-inﬂammation. Chem. Sci. 9, 2927–2933. doi: 10.1039/C7SC05476A Zhang, Q., Zhang, F., Chen, Y., Dou, Y., Tao, H., Zhang, D., et al. (2017). Structure– property correlations of reactive oxygen species-responsive and hydrogen peroxide-eliminating materials with anti-oxidant and anti-inﬂammatory activities. Chem. Mater. 29, 8221–8238. doi: 10.1021/acs.chemmater.7b 02412 Ye, H., Zhou, Y., Liu, X., Chen, Y., Duan, S., Zhu, R., et al. (2019). Recent advances on reactive oxygen Species-responsive delivery and diagnosis system. Biomacromolecules 20, 2441–2463. doi: 10.1021/acs.biomac.9b00628 Zhang, Q., Zhang, F., Li, S., Liu, R., Jin, T., Dou, Y., et al. (2019). A multifunctional nanotherapy for targeted treatment of colon cancer by simultaneously regulating tumor microenvironment. Theranostics 9, 3732–3753. doi: 10.7150/thno.34377 Yoo, D., Guk, K., Kim, H., Khang, G., Wu, D., and Lee, D. (2013). Antioxidant polymeric nanoparticles as novel therapeutics for airway inﬂammatory diseases. Int. J. Pharm. 450, 87–94. doi: 10.1016/j.ijpharm.2013.04.028 Yoshihisa, Y., Honda, A., Zhao, Q.-L., Makino, T., Abe, R., Matsui, K., et al. (2010). Protective eﬀects of platinum nanoparticles against UV- light-induced epidermal inﬂammation. Exp. Dermatol. 19, 1000–1006. doi: 10.1111/j.1600-0625.2010.01128.x Zhang, R., Liu, R., Liu, C., Pan, L., Qi, Y., Cheng, J., et al. (2020). A pH/ROS Dual-responsive and targeting nanotherapy for vascular inﬂammatory diseases. Biomaterials 230, 119605. REFERENCES doi: 10.1016/j.biomaterials.2019.1 19605 Zhang, W., Hu, S., Yin, J.-J., He, W., Lu, W., Ma, M., et al. (2016). Prussian blue nanoparticles as multienzyme mimetics and reactive oxygen species scavengers. J. Am. Chem. Soc. 138, 5860–5865. doi: 10.1021/jacs.5b12070 Yoshitomi, T., Hirayama, A., and Nagasaki, Y. (2011). The ROS scavenging and renal protective eﬀects of pH-responsive nitroxide Radical-containing nanoparticles. Biomaterials 32, 8021–8028. doi: 10.1016/j.biomaterials.2011.07.014 Zhang, Y., Murugesan, P., Huang, K., and Cai, H. (2020). NADPH oxidases and oxidase crosstalk in cardiovascular diseases: Novel therapeutic targets. Nat. Rev. Cardiol. 17, 170–194. doi: 10.1038/s41569-019-0260-8 Yoshitomi, T., and Nagasaki, Y. (2014). Reactive oxygen species-scavenging nanomedicines for the treatment of oxidative stress injuries. Adv. Healthc. Mater. 3, 1149–1161. doi: 10.1002/adhm.201300576 Zhao, H., Zeng, Z., Liu, L., Chen, J., Zhou, H., Huang, L., et al. (2018). Polydopamine nanoparticles for the treatment of acute Inﬂammation-induced injury. Nanoscale 10, 6981–6991. doi: 10.1039/C8NR00838H Zafar, M. S., Quarta, A., Marradi, M., and Ragusa, A. (2019). Recent developments in the reduction of oxidative stress through antioxidant polymeric formulations. Pharmaceutics 11:505. doi: 10.3390/pharmaceutics11100505 Zhu, Y., Deng, G., Ji, A., Yao, J., Meng, X., Wang, J., et al. (2017). Porous Se@Sio2 nanospheres treated Paraquat-induced acute lung injury by resisting oxidative stress. Int. J. Nanomedicine 12, 7143–7152. doi: 10.2147/IJN.S1 43192 Zhang, B., Wang, F., Zhou, H., Gao, D., Yuan, Z., Wu, C., et al. (2019). Polymer dots compartmentalized in liposomes as a photocatalyst for in situ hydrogen therapy. Angew. Chem. Int. Ed. 58, 2744–2748. doi: 10.1002/anie.201813066 Zhang, D., Wei, Y., Chen, K., Zhang, X., Xu, X., Shi, Q., et al. (2015). Biocompatible reactive oxygen species (ROS)-responsive nanoparticles as superior drug delivery vehicles. Adv. Healthc. Mater. 4, 69–76. doi: 10.1002/adhm.201400299 Zhu, Z., and Su, M. (2017). Polydopamine nanoparticles for combined Chemo- and photothermal cancer therapy. Nanomaterials 7:160. doi: 10.3390/nano7070160 Zhang, J. X., and Ma, P. X. (2013). Cyclodextrin-based supramolecular systems for drug Delivery: Recent progress and future perspective. Adv. Drug Deliv. Rev. 65, 1215–1233. doi: 10.1016/j.addr.2013.05.001 Conﬂict of Interest: 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. Zhang, K., and Kaufman, R. J. (2008). From endoplasmic-reticulum stress to the inﬂammatory response. Nature 454, 455–462. doi: 10.1038/nature07203 Copyright © 2020 Li, Li, Chen, Zhang and Lu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org March 2020 \| Volume 8 \| Article 200 REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) 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. Zhang, L., Laug, L., Munchgesang, W., Pippel, E., Gosele, U., Brandsch, M., et al. (2010). Reducing stress on cells with Apoferritin-encapsulated platinum nanoparticles. Nano Lett. 10, 219–223. doi: 10.1021/nl903313r Zhang, Q., Tao, H., Lin, Y., Hu, Y., An, H., Zhang, D., et al. (2016). A superoxide dismutase/catalase mimetic nanomedicine for targeted therapy of inﬂammatory bowel disease. Biomaterials 105, 206–221. doi: 10.1016/j.biomaterials.2016.08.010 March 2020 \| Volume 8 \| Article 200 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 20
https://openalex.org/W3203937546	https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/85/e3sconf_icmed2021_01095.pdf	English	null	Finite element simulation of AISI 1025 and Al6061 specimen with coated and uncoated tools on turning process using deform-3D	E3S web of conferences	2,021	cc-by	3,287	Finite element simulation of AISI 1025 and Al6061 specimen with coated and uncoated tools on turning process using deform 3D K V Durga Rajesh1, Abdul Munaf Shaik1, A V S Ram Prasad1, Tanya Buddi2, and F M Mwema3 1Department of Mechanical Engineering, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Andhra Pradesh, India 2Department of Mechanical Engineering, GRIET, Bachupally, Hyderabad, Telangana, India 3Department of Mechanical Engineering, Dedan Kimathi University of Technology, Nyeri, Kenya K V Durga Rajesh1, Abdul Munaf Shaik1, A V S Ram Prasad1, Tanya Buddi2, and F M Mwema3 1Department of Mechanical Engineering, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Andhra Pradesh, India 2Department of Mechanical Engineering, GRIET, Bachupally, Hyderabad, Telangana, India 3Department of Mechanical Engineering, Dedan Kimathi University of Technology, Nyeri, Kenya Abstract. This paper describes how to use Deform-3D software to create a turning process model that can be used to simulate the turning on AISI 1025 carbon steel and Al6061 billets in industrial and automotive applications. The Deform-3D Software is used to build a 3D Finite Element turning model. Pre-processing, Simulation, and Post-processing are all modules that can be used to simulate. Tool and workpiece information, as well as appropriate necessary parameters, were taken into account in the software's Pre Processing module. Simulation was performed at two different rotational speeds for two different materials using with and without titanium nitride coated tungsten carbide tool. After 1000 steps of simulation, results such as damage, effective strain, effective stress, total velocity, total displacement, and Temperature are reported from the Post Processing module. From results, comparative analysis will be carried out on performance characteristics at different rotational speeds. During the turning process, to accurately predict metal removal Deform 3D software is used for finite element simulation. [1]. Chen Zhuo et al. carried out experiments on Titanium TC4 and curved model system with standard parameters to validate the accuracy of Deform-3D. Shraddha et al. [2] worked on machining of Ti-6Al-4V to reveal the thermal distribution using this 3D simulation software. A model for machining this alloy has been proposed for FEM of temperature distribution. Since laboratory experiments are expensive and time consuming, metal cutting simulation is a viable option in order to obtain a better distinguishing of fabricating processes under different cutting conditions. The effect of process parameters such as cutting speed and feed with a constant depth of cut of 0.25 mm was evaluated using a L9 orthogonal array based on configuration of experiments on Ti-6Al-4V as workpiece material by K. Satyanarayana et al. [3]. Finite element simulation of AISI 1025 and Al6061 specimen with coated and uncoated tools on turning process using deform 3D By using Deform-3D simulation, While machining AISI 1045 steel Tamizharasan et al. [4] attempted to minimize flank wear and performance quality characteristics were evaluated using statistical techniques for L9 orthogonal array. © 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/). * Corresponding author: kanchurajesh@kluniversity.in https://doi.org/10.1051/e3sconf/202130901095 https://doi.org/10.1051/e3sconf/202130901095 E3S Web of Conferences 309, 01095 (2021) ICMED 2021 DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 es/by/4 0/) 1 Introduction Process setup parameters for simulation Parameter Value Rotational Speed 1000 rpm & 2000 rpm Workpiece Diameter (D) 50 mm Depth of cut (d) 0.5 mm Feed rate (f) 0.3 mm/rev Environment Temperature 30 0C Convection coefficient 0.02 N/Sec/mm/C Shear friction factor 0.6 Heat Transfer coefficient 45 N/Sec/mm/C Table 2.1. Process setup parameters for simulation possibilities when using FEM. Analyzed various machining processes and projected cutting pressures, tool wear, temperatures and stresses, so that the cutting tool could be optimized. The use of the finite element method to work on metal cutting operations and chip forming has proven to be successful. Researchers, as well as computer and equipment manufacturers, may use the simulation findings to design new instruments and refine the cutting process. Orthogonal and oblique cutting operations were modelled by E. Ceretti et al. [8]. Libo Wu et al. [9] looked at cutting power, chip formation, chip morphology, and chip geometric parameters in relation to chip-breaking grooves in some studies. Rityuj Singh Parihar et al. [10] examined the reaction of key machining variables in ceramic tool turning on AISI H13 material. Interfacial temperature affects tool wear and can also influence the work piece and tool parameters in machining [11]. After considering the above mentioned parameters, tool setup details are to be entered. Where one can define new tool or existing tool from library. Here tool with and without coating options has been selected from existing library. Uncoated tungsten carbide tool details and parameters considered are mentioned in Table 2.2. Coated tungsten carbide tool with titanium nitride details and parameters considered are mentioned in Table 2.3. Tool holder setup of DTGNL was selected to perform ope. R. Rajesh et al. [12] has considered, titanium and aluminium alloys with coatings to study the tool wear characteristics. The base metal of the tool bit is tungsten carbide, which has been treated with three different materials: Ti, Al2O3, and TiCN. The turning mechanism of alloy steel AISI 4340 using a Nano Al2O3 tool is investigated in 3D numerical models at various turning speeds were studied by Hong-jun Hu &Wei-jiu Huang [13] to forecast the evolutions of tool turning powers, and wear rate in tools. L.R. Kilmetova & and P.V. Sletnev [14] compared the simulation of aluminium alloys with experimental process and analyzes the temperature distribution with respect to cutting speed. K. V. Durga Rajesh et al. 1 Introduction [15] was performed simulation process on Deform-3D software for forging operation which was considered as a basic to work on Deform-3D software for the present work. Table 2.2. Uncoated tool details Table 2.2. Uncoated tool details Tool type: TNMA332 (without coating) IC 9.53 mm B 13.494 mm T 4.76 mm H 3.81 mm R 0.8 mm Table 2.3. Coated tool details Table 2.2. Uncoated tool details Tool type: TNMA332 (without coating) IC 9.53 mm B 13.494 mm T 4.76 mm H 3.81 mm R 0.8 mm Table 2.3. Coated tool details Table 2.3. Coated tool details Tool type: TNMA332_KC90 (with titanium nitride coating) IC 9.53 mm B 13.494 mm T 4.76 mm H 3.81 mm R 0.8 mm Coated layer 1 Coated thickness 1 mm Coated material TiN Steel and Aluminium work pieces are taken for simulation under standard working parameters with change in rotational speeds. To obtain better results from finite element analysis, the turning set-up geometry, Tool setup data and workpiece setup details have been carefully described in this study. With and without tool coating, the effects of damage, effective stress, effective strain, total displacement, total velocity, and temperature were investigated. After considering all tool details, tool need to be meshed. With meshing the entire tool portion will be divided into finite number of nodes, elements and all required boundary conditions are constrained to perform simulation in later stage. Under Tool mesh generation option, size ratio of 4 and relative mesh size option was selected, where minimum half of the maximum elements to be entered to get better and quality mesh. Fig. 2.1. shows a solid mesh with 29804 components and 6888 nodes for simulation for a tungsten carbide uncoated tool. 1 Introduction Turning is a cutting technique in which a single point cutting tool, usually aided by cutting fluids, separates material from a rotating workpiece. The tool is usually mounted on a tool post, and the workpiece is kept in a chuck or other work holding unit. By extracting chips from a spinning workpiece that have been fed into a cutting tool and swept away by the work piece’s movement, the tool gradually creates a surface. Turning is often used as a secondary method to modify or refine features on parts that have already been produced. As a consequence, chip disposal in turning and cutting fluid effectiveness are crucial. Steel and aluminium are most commonly used materials in manufacturing industries. In the automotive and manufacturing industries, steel and aluminium are the most widely used materials. Since AISI 1025 carbon steel is used in machinery parts and trusses, and Al6061 is used in aircrafts and heat exchangers, these materials were chosen. Deform 3D is one of the Finite Element simulation software which performs several machining operations with variable parameters as constraints. Setting simulation controls plays an important role when analyzing. Many researchers indicated the various factors like selection of material, work piece, feed, depth of cut, convection coefficient etc. which influences the performance. Cutting forces and processes can perfectly simulated by Some researchers worked on different machining processes on Magnesium alloys, same has been simulated on Deform-3D. Using DEFORM 3D tools, Anirudh Muralidharan [5] investigated 6 different materials for 5 different depths of cut and different frequencies of feed. Amit Sharma et al. [6] examined and simulated the effects of variables on Ti-6Al-4V turning. Corina Constantin et al. [7] explains overview of Cutting forces and processes can perfectly simulated by Deform-3D Finite Element Modelling (FEM) software https://doi.org/10.1051/e3sconf/202130901095 E3S Web of Conferences 309, 01095 (2021) ICMED 2021 Table 2.1. Process setup parameters for simulation Parameter Value Rotational Speed 1000 rpm & 2000 rpm Workpiece Diameter (D) 50 mm Depth of cut (d) 0.5 mm Feed rate (f) 0.3 mm/rev Environment Temperature 30 0C Convection coefficient 0.02 N/Sec/mm/C Shear friction factor 0.6 Heat Transfer coefficient 45 N/Sec/mm/C Table 2.1. 2 Methodology The Design Environment for Forming (DEFORM 3D V6.1) software is used to simulate the turning process model in this paper. In this methodology, process to run simulation on turning process using DEFORM 3D Software is explained in detail. In Pre processor stage after opening the software, first project and operation names with unit system details are to be entered. In this work, System International (SI) has been selected. Machining type like turning, milling, drilling, boring need to be selected next depending upon type of operation performed on software, for this work turning operation has been identified. The following process setup details shown in table 2.1., are considered to perform simulation of turning operation. 2 https://doi.org/10.1051/e3sconf/202130901095 E3S Web of Conferences 309, 01095 (2021) ICMED 2021 Fig. 2.3. Simulation at step-1 Fig. 2.1. Tool meshed model Fig. 2.3. Simulation at step-1 Fig. 2.1. Tool meshed model In the present work, Simulation can be performed at two different rotational speeds like 1000 rpm and 2000 rpm, under optimal tool geometry. Two work piece materials AISI 1025 carbon steel and Al6061 were considered for the simulation process with titanium nitride coated and uncoated tungsten carbide tool. 8 datasets were considered to perform simulation. Data sets details are mentioned below in Table 2.5. After considering all tool required data, now workpiece related data need to be selected. To perform simulation on turning process both tool and workpiece plays key role. Workpiece type of plastic was opted with ambient temperature condition of 300C. Default workpiece shape was considered, which was available in the library of software to perform analysis. Like tool, workpiece also need to be meshed and constrained before go to simulation. With relative mesh size method, steel workpiece was meshed with 3821 elements and 967 nodes for simulation shown in Fig. 2.2. Table 2.5. Description of 8 datasets Data Set No. 2 Methodology Description 1 Simulation on AISI 1025 carbon steel workpiece with uncoated tungsten carbide tool at 1000 rpm rotational speed 2 Simulation on Al6061 workpiece with uncoated tungsten carbide tool at 1000 rpm rotational speed 3 Simulation on AISI 1025 carbon steel workpiece with titanium nitride coated tungsten carbide tool at 1000 rpm rotational speed 4 Simulation on Al6061 workpiece with titanium nitride coated tungsten carbide tool at 1000 rpm rotational speed 5 Simulation on AISI 1025 carbon steel workpiece with uncoated tungsten carbide tool at 2000 rpm rotational speed 6 Simulation on Al6061 workpiece with uncoated tungsten carbide tool at 2000 rpm rotational speed 7 Simulation on AISI 1025 carbon steel workpiece with titanium nitride coated tungsten carbide tool at 2000 rpm rotational speed 8 Simulation on Al6061 workpiece with titanium nitride coated tungsten carbide tool at 2000 rpm rotational speed Table 2.5. Description of 8 datasets Fig. 2.2. Workpiece meshed model Fig. 2.2. Workpiece meshed model Selection of workpiece material is an important task to perform simulation. In this work, two different materials like AISI 1025 carbon steel and Al6061 were considered for analysis. To run simulation, Simulation controls need to be selected and the same was shown in Table 2.4. Database need to be checked and generated before run the simulation. Then simulation stage will be started for 1000 steps. The process started at step-1 is selected for reference and shown in Fig. 2.3. After completion of 1000 steps simulation will be completed and results will be taken from post processor stage. After successful completion of the simulation process on 8 datasets, the results obtained are discussed in next section. Table 2.4. Process setup parameters for simulation Parameter Value Number of simulation steps 1000 Step increment to save 25 Arc length to cut 19.5 mm Table 2.4. Process setup parameters for simulation 3 Results and Discussion Simulation analysis is carried out with the defined optimal tool geometry, and performance characteristics like damage, total displacement, effective strain, effective 3 3 https://doi.org/10.1051/e3sconf/202130901095 E3S Web of Conferences 309, 01095 (2021) ICMED 2021 stress, temperature, and total velocity obtained dataset-1 are shown below. Fig. 3.1. Damage for Dataset-1 Fig. 3.2. Displacement for Dataset-1 Fig. 3.3. Strain Effective for Dataset-1 Fig. 3.4. Stress Effective for Dataset-1 stress, temperature, and total velocity obtained for dataset-1 are shown below. Fig. 3.1. Damage for Dataset-1 Fig. 3.2. Displacement for Dataset-1 Fig. 3.3. Strain Effective for Dataset-1 Fig. 3.5. Temperature for Dataset-1 Fig. 3.6. Velocity for Dataset-1 Fig. 3.7. Optimal load prediction in X-Load (N) for Dataset-1 From the Fig. 3.1. to 3.6., it was observed that the maximum damage, total displacement, effective strain, effective stress, temperature and total velocity are 47.4, 9.59 mm, 28.7 mm/mm, 1620 Mpa, 912 0C and 167000 mm/sec respectively. From the figure 3.7, the X-Load is maximum at 0.00222 sec. Similarly from the figure 3.8, the Y-Load is maximum at 0.00122 sec and from the figure 3.9, the Z-Load is maximum at 0.00174 sec. Same process is repeated for another 7 datasets. The results obtained from 7 datasets are shown in table 3.1 along with dataset-1 results. Table 3.1. Performance characteristics of 8 datasets Di l Effecti Effecti T Velocit Fig. 3.5. Temperature for Dataset-1 Fig. 3.6. Velocity for Dataset-1 Fig. 3.7. Optimal load prediction in X-Load (N) for Dataset-1 Fig. 3.5. Temperature for Dataset-1 stress, temperature, and total velocity obtained for dataset-1 are shown below. Fig. 3.1. Damage for Dataset-1 Fig. 3.5. Temperature for Dataset-1 Fig. 3.6. Velocity for Dataset-1 Fig. 3.1. Damage for Dataset-1 Fig. 3.1. Damage for Dataset-1 Fig. 3.2. Displacement for Dataset-1 Fig. 3.2. Displacement for Dataset-1 Fig. 3.3. Strain Effective for Dataset-1 Fig. 3.4. Stress Effective for Dataset-1 Fig. 3.6. Velocity for Dataset-1 Fig. 3.7. Optimal load prediction in X-Load (N) for Dataset-1 Fig. 3.7. Optimal load prediction in X-Load (N) for Dataset-1 Fig. 3.2. Displacement for Dataset-1 Fig. 3.3. Strain Effective for Dataset-1 Fig. 3.3. Strain Effective for Dataset-1 Fig. 3.7. Optimal load prediction in X-Load (N) for Dataset-1 From the Fig. 3.1. to 3.6., it was observed that the maximum damage, total displacement, effective strain, effective stress, temperature and total velocity are 47.4, 9.59 mm, 28.7 mm/mm, 1620 Mpa, 912 0C and 167000 mm/sec respectively. 3 Results and Discussion From the figure 3.7, the X-Load is maximum at 0.00222 sec. Similarly from the figure 3.8, the Y-Load is maximum at 0.00122 sec and from the figure 3.9, the Z-Load is maximum at 0.00174 sec. Same process is repeated for another 7 datasets. The results obtained from 7 datasets are shown in table 3.1 along with dataset-1 results. Fig. 3.3. Strain Effective for Dataset-1 Fig. 3.3. Strain Effective for Dataset-1 Fig. 3.4. Stress Effective for Dataset-1 Table 3.1. Performance characteristics of 8 datasets Data Set Dam age Displa cement (mm) Effecti ve Strain (mm/ mm) Effecti ve Stress (M pa) Tempe rature (o C) Velocit y (mm/s ec) 1 61.3 8.56 25.6 1310 1410 167000 2 53.7 8.46 44.3 1220 368 8810 Table 3.1. Performance characteristics of 8 datasets Fig. 3.4. Stress Effective for Dataset-1 4 https://doi.org/10.1051/e3sconf/202130901095 E3S Web of Conferences 309, 01095 (2021) ICMED 2021 3 47.4 9.59 28.7 1620 912 167000 4 83.7 8.21 29.5 1050 359 7520 5 73.2 8.87 28.6 1420 1687 214250 6 39 8.06 26.8 1430 417 29700 7 54.4 8.94 29.8 1590 878 223205 8 15.1 8.09 42.2 1890 467 47600 5. A. Muralidharan, Project report (SURGE 2015), IIT Kanpur. 6. A. Sharma, C.S. Kalra, R. Rohit., Int. J Engg. Res. Tech., 2, (2013). 7. C. Constantin, S.M. Croitoru, G. Constantin, C.F. Bisu., Adv. Vis., Imag. Sim., 41 (2010). From the above Table 3.1, the following points were observed. From the above Table 3.1, the following points were observed. 8. E. Ceretti, C. Lazzaroni, L. Menegardo, T. Altan, J Mat. Proc. Tech., 98, 99 (2000). • At 1000 rpm, except effective strain all the remaining performance characteritics are more for AISI 1025 carbon steel than Al6061 with uncoated tungsten carbide tool. 9. L. Wu., Int. J Adv. Mfg. Tech., 108, 1331. 10. R.S. Parihar, R.K. Sahu, G. Srinivasu., Mat. Tod. Proc., 4, 8432, (2017). • At 1000 rpm, using titanium nitride coated tungsten carbide tool the damage and effective strain values are less for AISI 1025 carbon steel than Al6061. 11. Y.R. Bhoyar, P.D. Kamble., Int. J Res Engg. Tech., 2, (2013). 12. R. Rajesh, J.L. Mercy, S.R. kumar, A. Singh., ARPN J Engg. App. Sci., 12, (2017). • At 2000 rpm, for Al6061 the value of damage is more while using the uncoated tungsten carbide tool. 13. H.J. Hu, W. J. Huang., Int. J Adv. Mfg. Tech., 67, 907 (2013). 4 Conclusion After performing turning operation at two different rotational speeds 1000 and 2000 rpm, from both coated and uncoated tungsten carbide tool it was observed that, with increase in rotational speed, damage rate is reduced and displacement remains unchanged. With decrease in rotational speed, effective stress, total velocity and temperature were decreased. With titanium nitride coated tool the effective stress, effective strain, velocity, and temperature were reduced than uncoated tool for the experimental datasets considered. So, it was concluded that the coated tools are preferable to produce optimal results in most cases. 3 Results and Discussion • For Al6061, at 2000 rpm all performance characteritics except damage are low for uncoated tool than titanium nitride coated tungsten carbide tool. 14. L.R. Kilmetova, P.V. Sletnev., J Engg. Sci. Tech Rev., 7, 29 (2014). • For AISI 1025 Carbon steel, at 1000 rpm the value of damage and temperature are more for uncoated tool than titanium nitride coated tungsten carbide tool. 15. K.V.D. Rajesh, T. Buddi, H. Mishra., Adv. Mat. Proc. Tech., (2021). (accepted and to be Published). At 1000 rpm, while machining with AISI 1025 carbon steel using uncoated tool displacement, temperature and velocity are more than machining Al6061 with coated tool. At 1000 rpm, while machining with AISI 1025 carbon steel using uncoated tool displacement, temperature and velocity are more than machining Al6061 with coated tool. References 1. C. Zhuo, Q.L. fang and Y.L. juan., Cutting Force Simulation of Titanium based on DEFORM-3D. Proceedings of the 3rd International Conference on Material Mechanical and Manufacturing Engineering, 1846 (2015). 2. S.S. Adewar, Int. Res. J Engg., 6, (2019) 3. K. Satyanarayana, A.V. Gopal, P.B. Babu., Proceedings of the ASME 2013 International Mechanical Engineering Congress and Exposition, San Diego, California, USA, (2013). 4. T. Tamizharasan, N.S. Kumar., Int. J Simul. Mod., 11, 65 (2012). 4. T. Tamizharasan, N.S. Kumar., Int. J Simul. Mod., 11, 65 (2012). 5
https://openalex.org/W4313328496	https://medkatjorn.ru/journal/nomera/2022/4/73-78.pdf	Russian	null	Need to Expand Official List of Conditions for which First Aid is Provided in the Russian Federation	Medicina katastrof	2,022	cc-by	5,926	НЕОБХОДИМОСТЬ РАСШИРЕНИЯ ДЕЙСТВУЮЩЕГО В РОССИЙСКОЙ ФЕДЕРАЦИИ ОФИЦИАЛЬНОГО ПЕРЕЧНЯ СОСТОЯНИЙ, ПРИ КОТОРЫХ ОКАЗЫВАЕТСЯ ПЕРВАЯ ПОМОЩЬ ф р р р р Ключевые слова: боль в груди, инсульт, инфаркт миокарда, острое нарушение мозгового кровообращения, острый коронарный синдром, официальный перечень состояний, первая помощь, Российская Федерация Конфликт интересов. Авторы статьи подтверждают отсутствие конфликта интересов р р др , ф ц р , р щ , Конфликт интересов. Авторы статьи подтверждают отсутствие конфликта интересов Для цитирования: Биркун А.А., Дежурный Л.И. Необходимость расширения действующего в Российской Федерации официального перечня состояний, при которых оказывается первая помощь // Медицина катастроф. 2022. №4. С. 73- 78. https://doi.org/10.33266/2070-1004-2022-4-73-78 Для цитирования: Биркун А.А., Дежурный Л.И. Необходимость расширения действующего в Российской Федерации официального перечня состояний, при которых оказывается первая помощь // Медицина катастроф. 2022. №4. С. 73- 78. https://doi.org/10.33266/2070-1004-2022-4-73-78 https://doi.org/10.33266/2070-1004-2022-4-73-78 UDC 616-083.98 Original article © Burnasyan FMBC FMBA Original article © Burnasyan FMBC FMBA НЕОБХОДИМОСТЬ РАСШИРЕНИЯ ДЕЙСТВУЮЩЕГО В РОССИЙСКОЙ ФЕДЕРАЦИИ ОФИЦИАЛЬНОГО ПЕРЕЧНЯ СОСТОЯНИЙ, ПРИ КОТОРЫХ ОКАЗЫВАЕТСЯ ПЕРВАЯ ПОМОЩЬ 1 Медицинская академия им. С.И. Георгиевского ФГАОУ ВО «Крымский федеральный университет им. В.И. Вернадского», Симферополь, Россия 2 ГБУЗ Республики Крым «Крымский республиканский центр медицины катастроф и скорой медицинской помощи», Симферополь, Россия 3 ФГБУ «Центральный научно-исследовательский институт организации и информатизации здравоохранения» Минздрава России, Москва, Россия 4 ФГБОУ ДПО «Российская медицинская академия непрерывного профессионального образования» Минздрава России, Москва, Россия 1 Медицинская академия им. С.И. Георгиевского ФГАОУ ВО «Крымский федеральный университет им. В.И. Вернадского», Симферополь, Россия 2 ГБУЗ Республики Крым «Крымский республиканский центр медицины катастроф и скорой медицинской помощи», Симферополь, Россия 3 ФГБУ «Центральный научно-исследовательский институт организации и информатизации здравоохранения» Минздрава России, Москва, Россия 4 ФГБОУ ДПО «Российская медицинская академия непрерывного профессионального образования» Минздрава России, Москва, Россия Резюме. Цели исследования – определить общий спектр критических состояний, при которых своевременно и правильно оказанная первая помощь (ПП) может способствовать сохранению жизни; оценить целесообразность расширения дей- ствующего в Российской Федерации официального перечня состояний, при которых оказывается первая помощь. Материалы и методы исследования. Материалы исследования – национальные и международные рекомендации по ока- занию первой помощи; данные официальной статистики; нормативные правовые акты Российской Федерации регули Резюме. Цели исследования – определить общий спектр критических состояний, при которых своевременно и правильно оказанная первая помощь (ПП) может способствовать сохранению жизни; оценить целесообразность расширения дей- ствующего в Российской Федерации официального перечня состояний, при которых оказывается первая помощь. Материалы и методы исследования. Материалы исследования – национальные и международные рекомендации по ока- занию первой помощи; данные официальной статистики; нормативные правовые акты Российской Федерации, регули- рующие оказание первой помощи; отечественные и зарубежные научные публикации. Для поиска научных публикаций использовались библиографические базы данных Google Scholar, Pubmed/Medline и Российский индекс научного цитиро- вания (РИНЦ). Поиск и анализ материалов выполнен в июле–августе 2022 г. р у Результаты исследования и их анализ. Представлены результаты анализа международных и национальных рекомендаций и соответствующих научных публикаций по оказанию первой помощи, предпринятого с целью определения общего спек- тра угрожающих жизни состояний, при которых своевременно и правильно оказанная первая помощь может определить благоприятный исход для больного или пострадавшего. Показано, что утверждённый в 2012 г. официальный перечень состояний, при которых оказывается ПП, не включает ряд широко распространённых и опасных для жизни нарушений здо- ровья, в их числе острый коронарный синдром и инсульт – состояния, в совокупности обусловливающие наибольшее коли- чество случаев смерти среди взрослого населения страны. Обоснована целесообразность расширения действующего в России официального перечня состояний, при которых оказывается первая помощь. ps://doi.org/10.33266/2070-1004-2022-4-73-78 Оригинальная статья ДК 616-083.98 © ФМБЦ им.А.И.Бурназяна НЕОБХОДИМОСТЬ РАСШИРЕНИЯ ДЕЙСТВУЮЩЕГО В РОССИЙСКОЙ ФЕДЕРАЦИИ ОФИЦИАЛЬНОГО ПЕРЕЧНЯ СОСТОЯНИЙ, ПРИ КОТОРЫХ ОКАЗЫВАЕТСЯ ПЕРВАЯ ПОМОЩЬ А.А.Биркун1,2, Л.И.Дежурный3,4 1 Медицинская академия им. С.И. Георгиевского ФГАОУ ВО «Крымский федеральный университет им. В.И. Вернадского», Симферополь, Россия 2 ГБУЗ Республики Крым «Крымский республиканский центр медицины катастроф и скорой медицинской помощи», Симферополь, Россия 3 ФГБУ «Центральный научно-исследовательский институт организации и информатизации здравоохранения» Минздрава России, Москва, Россия 4 ФГБОУ ДПО «Российская медицинская академия непрерывного профессионального образования» Минздрава России, Москва, Россия ps://doi.org/10.33266/2070-1004-2022-4-73-78 Оригинальная статья ДК 616-083.98 © ФМБЦ им.А.И.Бурназяна НЕОБХОДИМОСТЬ РАСШИРЕНИЯ ДЕЙСТВУЮЩЕГО В РОССИЙСКОЙ ФЕДЕРАЦИИ ОФИЦИАЛЬНОГО ПЕРЕЧНЯ СОСТОЯНИЙ, ПРИ КОТОРЫХ ОКАЗЫВАЕТСЯ ПЕРВАЯ ПОМОЩЬ А.А.Биркун1,2, Л.И.Дежурный3,4 1 Медицинская академия им. С.И. Георгиевского ФГАОУ ВО «Крымский федеральный университет им. В.И. Вернадского», Симферополь, Россия 2 ГБУЗ Республики Крым «Крымский республиканский центр медицины катастроф и скорой медицинской помощи», Симферополь, Россия 3 ФГБУ «Центральный научно-исследовательский институт организации и информатизации здравоохранения» Минздрава России, Москва, Россия 4 ФГБОУ ДПО «Российская медицинская академия непрерывного профессионального образования» Минздрава России, Москва, Россия ttps://doi.org/10.33266/2070-1004-2022-4-73-78 Оригинальная статья ДК 616-083.98 © ФМБЦ им.А.И.Бурназяна НЕОБХОДИМОСТЬ РАСШИРЕНИЯ ДЕЙСТВУЮЩЕГО В РОССИЙСКОЙ ФЕДЕРАЦИИ ОФИЦИАЛЬНОГО ПЕРЕЧНЯ СОСТОЯНИЙ, ПРИ КОТОРЫХ ОКАЗЫВАЕТСЯ ПЕРВАЯ ПОМОЩЬ А.А.Биркун1,2, Л.И.Дежурный3,4 1 Медицинская академия им. С.И. Георгиевского ФГАОУ ВО «Крымский федеральный университет им. В.И. Вернадского», Симферополь, Россия 2 ГБУЗ Республики Крым «Крымский республиканский центр медицины катастроф и скорой медицинской помощи», Симферополь, Россия 3 ФГБУ «Центральный научно-исследовательский институт организации и информатизации здравоохранения» Минздрава России, Москва, Россия 4 ФГБОУ ДПО «Российская медицинская академия непрерывного профессионального образования» Минздрава России, Москва, Россия https://doi.org/10.33266/2070-1004-2022-4-73-78 УДК 616-083.98 https://doi.org/10.33266/2070-1004-2022-4-73-78 УДК 616-083.98 A.A.Birkun1,2, L.I.Dezhurnyy3,4 A.A.Birkun1,2, L.I.Dezhurnyy3,4 A.A.Birkun1,2, L.I.Dezhurnyy3,4 1 Medical Academy named after S.I. Georgievskiy of V.I. Vernadskiy Crimean Federal University, Simferopol, Russian F d ti Federation 2 Crimean Republican Center for Disaster Medicine and Emergency Medical Care, Simferopol, Russian Federation 3 Federal Research Institute for Health Organization and Informatics of Ministry of Health of the Russian Federation, Moscow, Russian Federation Moscow, Russian Federation 4 Russian Medical Academy of Continuous Professional Education of the Ministry of Health of the Russian Federation, M R i F d ti Moscow, Russian Federation 4 Russian Medical Academy of Continuous Professional Education of the Ministry of Health of the Russian Federation, Moscow, Russian Federation * 1 International First Aid Resuscitation and Education Guidelines. Inter- national Federation of Red Cross and Red Crescent Societies. Red Cross Red Crescent Networks. 2020. URL: https://www.globalfirstaidcentre.org/wp-content/up- loads/2021/02/EN_GFARC_GUIDELINES_2020.pdf [Дата обра- щения: 07.09.2022] oscow, Russian Federation Russian Medical Academy of Continuous Professional Education of the Ministry of Health of the Russian Federation, oscow, Russian Federation Summary. The objectives of the study were to determine the general spectrum of critical conditions in which timely and correctly rendered first aid can contribute to the preservation of life; to assess the feasibility of expanding the official list of conditions in which first aid is rendered in the Russian Federation. Research materials and methods. The study materials are national and international recommendations for first aid; data of official statistics; legal acts of the Russian Federation regulating first aid; domestic and foreign scientific publications. Bibliographic data- bases Google Scholar, Pubmed/Medline and Russian Science Citation Index (RSCI) were used to search for scientific publica- tions. The materials were searched and analyzed in July-August 2022. y y g Study results and their analysis. The results of the analysis of international and national recommendations and relevant scientific publications on first aid, undertaken to identify the general spectrum of life-threatening conditions in which timely and properly provided first aid can determine a favorable outcome for the patient or victim are presented. The official list of conditions for 73 Медицина катастроф №4•2022 Медицина катастроф №4•2022 which first aid is provided, approved in 2012, does not include a number of widespread and life-threatening health disorders, including acute coronary syndrome and stroke — conditions that cumulatively cause the largest number of deaths among adult population of the country. The article substantiates the expediency of expanding the official list of conditions for which first aid is provided in Russia. which first aid is provided, approved in 2012, does not include a number of widespread and life-threatening health disorders, including acute coronary syndrome and stroke — conditions that cumulatively cause the largest number of deaths among adult population of the country. The article substantiates the expediency of expanding the official list of conditions for which first aid is provided in Russia. ords: acute coronary syndrome, acute impairment of cerebral circulation, chest pain, first aid, myocardial infarction, ist of conditions, Russian Federation, stroke Conflict of interest. The authors declare no conflict of interest For citation: Birkun A.A., Dezhurnyy L.I. Need to Expand Official List of Conditions for which First Aid is Provided in the Russian Federation. Meditsina Katastrof = Disaster Medicine. 2022;4:73-78 (In Russ.). https://doi.org/10.33266/2070-1004-2022- 4 73 78 Контактная информация: Contact information: Aleksey A. Birkun — Dr. Sc. (Med.), Associate Prof. of the Department of General Surgery, Anaesthesiology, Resuscitation and Emergency Medicine of Medical Academy named after S.I. Georgievskiy of V.I. Vernadskiy Crimean Federal University Address: 5/7, Lenin Blvd, Simferopol, 295051, Russia Phone: +7 (3652) 554-999 E-mail: birkunalexei@gmail.com Биркун Алексей Алексеевич — докт. мед. наук, доцент; доцент кафедры общей хирургии, анестезиологии-реаниматологии и скорой медицинской помощи Медицинской академии им. С.И. Георгиевского Крымского федерального университета им. В.И. Вернадского Адрес: Россия, 295051, Симферополь, бул. Ленина, д. 5/7 Биркун Алексей Алексеевич — докт. мед. наук, доцент; доцент кафедры общей хирургии, анестезиологии-реаниматологии и скорой медицинской помощи Медицинской академии им. С.И. Георгиевского Крымского федерального университета им. В.И. Вернадского Адрес: Россия, 295051, Симферополь, бул. Ленина, д. 5/7 д / Тел.: +7 (3652) 554-999 E-mail: birkunalexei@gmail.com / Тел.: +7 (3652) 554-999 Тел.: +7 (3652) 554-999 E-mail: birkunalexei@gmail.com E-mail: birkunalexei@gmail.com E-mail: birkunalexei@gmail.com оказанная первая помощь может способствовать сохра- нению жизни, и оценить целесообразность расширения дей- ствующего в Российской Федерации официального перечня состояний, при которых оказывается ПП2. Следует отметить, что, насколько известно авторам, со- ответствующий анализ до настоящего времени не пред- принимался. 2 Об утверждении перечня состояний, при которых оказывается первая помощь, и перечня мероприятий по оказанию первой по- мощи: приказ Минздравсоцразвития России от 04.05.2012 г. №477н. URL: http://www.consultant.ru/document/cons_doc_LAW_129862 [Дата обращения: 07.09.2022 3 Об основах охраны здоровья граждан в Российской Федера- ции: Федеральный закон от 21.11.2011 №323-ФЗ (ред. от 11.06.2022, с изм. от 13.07.2022). URL: http://www.consultant.ru/document/cons_doc_LAW_121895 [Дата обращения: 07.09.2022] Введение оказанная первая помощь может способствовать сохра- нению жизни, и оценить целесообразность расширения дей- ствующего в Российской Федерации официального перечня состояний, при которых оказывается ПП2. оказанная первая помощь может способствовать сохра- нению жизни, и оценить целесообразность расширения дей- ствующего в Российской Федерации официального перечня состояний, при которых оказывается ПП2. Следует отметить, что, насколько известно авторам, со- ответствующий анализ до настоящего времени не пред- принимался. Первая помощь (ПП) представляет собой комплекс ме- роприятий, выполняемых при различных нарушениях здо- ровья до оказания медицинской помощи с целью устра- нения угрозы для жизни и предупреждения возможных осложнений [1]1. Следует отметить, что, насколько известно авторам, со- ответствующий анализ до настоящего времени не пред- принимался. Раннее оказание ПП свидетелями внезапно возникших критических состояний, при которых исход в значитель- ной мере зависит от быстроты вмешательства, имеет до- казанный эффект в виде существенного снижения ле- тальности [2, 3]. Материалы и методы исследования. Материалы ис- следования – национальные и международные реко- мендации по первой помощи; данные официальной ста- тистики; нормативные правовые акты Российской Фе- дерации, регулирующие оказание ПП; отечественные и зарубежные научные публикации. Для поиска научных публикаций использовались библиографические базы дан- ных Google Scholar, Pubmed/Medline и Российский ин- декс научного цитирования (РИНЦ). Поиск и анализ ма- териалов выполнен в июле–августе 2022 г. Так, например, первая помощь имеет решающее значение в случае внегоспитальной остановки сердца, ког- да вследствие прогрессирующей гипоксии вероятность восстановления жизни с каждой минутой задержки ока- зания помощи сокращается приблизительно на 10%, а на немедленное прибытие бригады скорой медицинской помощи (СМП) к пострадавшему в большинстве случа- ев рассчитывать трудно [4]. При этом выполнение базовой сердечно-лёгочной реанимации (БСЛР) свидетелями остановки сердца до прибытия бригады СМП увеличи- вает шансы на выживание в 2–3 раза [5, 6]. Учитывая ис- ходно высокую инцидентность внегоспитальной остановки сердца, в масштабах Российской Федерации такой эф- фект снижения летальности может способствовать со- хранению десятков или сотен тысяч жизней ежегодно [7]. Результаты исследования и их анализ. Согласно ст. 31 «Первая помощь» Федерального закона от 21 ноября 2011 г. №323-ФЗ (ред. от 11.06.2022, с изм. от 13.07.2022)3 перечень состояний, при которых оказы- вается ПП, и перечень мероприятий по оказанию ПП утверждаются уполномоченным федеральным органом исполнительной власти, если иное не предусмотрено фе- деральными законами. Действующий перечень состояний, при которых ока- зывается первая помощь, был утверждён Минздравсоц- развития России в 2012 г. 2 Об утверждении перечня состояний, при которых оказывается первая помощь, и перечня мероприятий по оказанию первой по- мощи: приказ Минздравсоцразвития России от 04.05.2012 г. №477н. URL: http://www.consultant.ru/document/cons_doc_LAW_129862 [Дата обращения: 07.09.2022 3 Об Р й й Ф р 3 Об основах охраны здоровья граждан в Российской Федера- ции: Федеральный закон от 21.11.2011 №323-ФЗ (ред. от 11.06.2022, с изм. от 13.07.2022). URL: http://www.consultant.ru/document/cons_doc_LAW_121895 [Дата обращения: 07.09.2022] Введение Сравнение полученного списка опасных для жизни со- стояний с утверждённым в России официальным переч- нем состояний, при которых оказывается ПП, показало, 75 Медицина катастроф №4•2022 тельный вклад ишемической болезни сердца и инсультов в смертность и результирующий социально-экономиче- ский ущерб для государства, сокращение количества слу- чаев предотвратимой смерти, вызванных этими заболе- ваниями, составляет важнейшую задачу, стоящую перед отечественной системой здравоохранения [19–21]. На- ряду с интенсификацией первичной и вторичной профи- лактики, повышением эффективности оказания медицин- ской помощи и информированием населения о симптомах и правилах действий при развитии инсульта и ОКС, что предусмотрено федеральным проектом «Борьба с сердечно- сосудистыми заболеваниями»7, для снижения летальности и инвалидизации, обусловленных этой патологией, не- обходимо обеспечить максимально эффективное ис- пользование ресурса отечественной системы ПП, что предполагает включение боли в груди как типичного про- явления ОКС и инсульта в официальный перечень состояний, при которых оказывается первая помощь. Согласно современным международным рекоменда- циям, пострадавшего следует успокоить, исключить его физическую активность, помочь ему принять удобное по- ложение, обеспечить приём пострадавшим лекарственных препаратов, назначенных врачом — антиангинального средства и ацетилсалициловой кислоты (при возникно- вении боли в груди ранний приём последней более чем в 2 раза снижает летальность при инфаркте миокарда) и оставаться с пострадавшим до прибытия бригады СМП, внимательно наблюдая за его состоянием [15–17]4. Другим широко распространенным критическим со- стоянием, которое требует максимально быстрого ока- зания ПП, но отсутствует в утверждённом в России перечне состояний, при которых оказывается первая помощь, яв- ляется острое нарушение мозгового кровообращения (ОНМК) – инсульт. Как и в случае ОКС, при инсульте со- кращение времени от появления симптомов до оказания медицинской помощи определяет исход, однако даже в странах с высоким уровнем развития здравоохранения только 20–36% пострадавших с инсультом госпитали- зируют своевременно [18]. В связи с этим в междуна- родных рекомендациях по ПП подчёркивается важность раннего распознавания признаков инсульта с помощью таких простых оценочных систем, как FAST (англ. – Face, Arm, Speech, Time) или CPSS (англ. – Cincinnati Pre- hospital Stroke Scale), и немедленного вызова бригады СМП при подозрении на инсульт [17]5. Острый коронарный синдром и инсульт, будучи самыми частыми причинами смерти взрослых людей во всём мире8, очевидно, являются приоритетом для организации действенного массового оказания ПП с целью снижения смертности населения. Вместе с тем, при многих других неотложных состоя- ниях (при анафилактических реакциях, гипо- и гипер- гликемии, судорогах, состояниях, сопровождающихся за- труднённым дыханием и др.; см. Введение Он включает в себя 8 состояний: 1 – отсутствие сознания; 2 – остановка дыхания и кро- вообращения; 3 – наружные кровотечения; 4 – инородные тела верхних дыхательных путей; 5 – травмы различных В целом высокий уровень смертности населения Рос- сии определяет необходимость максимально эффек- тивного использования ПП как стратегического ресурса для предупреждения преждевременной смерти и со- кращения соответствующих социально-экономических по- терь государства. Цель исследования – определить общий спектр кри- тических состояний, при которых своевременно и правильно 74 Disaster Medicine No. 4•2022 областей тела; 6 – ожоги, эффекты воздействия высо- ких температур, теплового излучения; 7 – отморожение и другие эффекты воздействия низких температур; 8 – отравления. что последний не включает многие критические нарушения здоровья, при которых раннее и правильное оказание ПП может определять исход у пострадавшего и для которых сформулированы научно-обоснованные рекомендации по ПП (см. таблицу). Для того, чтобы определить общий спектр угрожающих жизни состояний, для которых мировым научно-меди- цинским сообществом рекомендовано оказание ПП, нами выполнен анализ международных и национальных ре- комендаций по первой помощи – 21 документ. Содер- жание каждого из них было изучено с целью определе- ния максимально полного списка состояний, для которых даны рекомендации по оказанию ПП. Массив данных с результатами анализа опубликован в свободном доступе в онлайн-репозитории Mendeley Data [8]. Количество со- стояний, представленных в одном документе, варьиро- вало от 1 до 52. Общее количество уникальных состоя- ний (без дублирования) – 57. Из общего количества со- стояний были выделены состояния (n=21), представляю- щие непосредственную угрозу для жизни (таблица). В числе таких нарушений — боль в груди — одна из са- мых частых причин обращения за медицинской помощью [8]. У 12–32% пациентов отделений СМП, предъявляющих жалобы на боль в груди, диагностируют острый коро- нарный синдром – ОКС – угрожающее жизни ослож- нение ишемической болезни сердца [9–11]. Риск смер- ти при ОКС и его разновидности — инфаркте миокар- да значительно повышается по мере увеличения про- межутка времени от возникновения боли в груди до на- чала лечения [12]. Следовательно, как можно более ран- нее выявление боли в груди как потенциального про- явления ишемии миокарда, своевременное обращение за медицинской помощью и оказание ПП до прибытия специалистов СМП к пострадавшему играют ключевую роль в предупреждении летального исхода [13, 14]. При этом комплекс мероприятий по оказанию ПП постра- давшим с болью в груди прост в освоении и исполнении. 6 Федеральная служба государственной статистики. Официаль- ная статистика. Население. Демография. Естественное движение населения. Число умерших по причинам смерти. 2022. URL: https://rosstat.gov.ru/storage/mediabank/demo24-2_2021.xlsx [Дата обращения: 07.09.2022] 7 Министерство здравоохранения Российской Федерации. Феде- ральный проект «Борьба с сердечно-сосудистыми заболеваниями». Паспорт федерального проекта. 2019. URL: https://minzdrav.gov.ru/poleznye-resursy/natsproektzdravoohrane- nie/bssz [Дата обращения: 07.09.2022] 8 World Health Organization. Fact sheets. Detail. The top 10 causes of death. 2020. URL: https://www.who.int/news-room/fact-sheets/de- tail/the-top-10-causes-of-death [Дата обращения: 07.09.2022 4 ANZCOR Guideline 9.2.1 – Recognition and First Aid Management of Suspected Heart Attack. The Australian and New Zealand Committee on Resuscitation. 2021. URL: https://resus.org.au/download/9_2_med- ical/anzcor-guideline-9-2-1-suspected-heart-attack-apr-2021.pdf [Дата обращения: 07.09.2022] 5 р 5 ANZCOR Guideline 9.2.2 – Stroke. The Australian and New Zealand Committee on Resuscitation. 2021. URL: https://resus.org.au/down- load/9_2_medical/anzcor-guideline-9-2-2-stroke-apr-2021.pdf [Дата обращения: 07.09.2022] Введение таблицу), , для которых в настоящее время в России отсутствуют формальные ос- нования оказывать ПП, также возможно развитие ско- ропостижной смерти, которую можно предотвратить при условии своевременного оказания первой помощи [22–25]. Включение этих состояний в официальный пе- речень состояний, при которых оказывается ПП, создаст базисные условия для повышения частоты, оперативно- сти и результативности оказания медицинской помощи в догоспитальном периоде в Российской Федерации, что должно в перспективе способствовать существенному сни- жению летальности при угрожающих жизни нарушениях здоровья и смертности населения страны. З р р у До прибытия бригады СМП необходимо придать по- страдавшему удобное и безопасное, учитывая риск его падения вследствие дисфункции нервной системы, по- ложение, оставаться рядом с пострадавшим, наблюдать за его состоянием, обеспечить ему эмоциональную под- держку. Так как некоторые симптомы инсульта совпадают с проявлениями гипо- или гипергликемии, при наличии глю- кометра и соответствующего навыка оказывающий пер- вую помощь может измерить уровень глюкозы в крови по- страдавшего с целью уточнения причины патологического состояния. Как при инсульте, так и при ОКС в случае усу- губления состояния пострадавшего – потеря сознания и прекращение нормального дыхания – требуется немед- ленно начать и непрерывно выполнять сердечно-лёгоч- ную реанимацию до прибытия специалистов бригады СМП; в случае потери сознания с сохранённым нор- мальным дыханием пострадавшему следует придать по- ложение на боку, запрокинуть голову для поддержания проходимости дыхательных путей и контролировать его состояние до прибытия медицинских специалистов. 4 ANZCOR Guideline 9.2.1 – Recognition and First Aid Management of Suspected Heart Attack. The Australian and New Zealand Committee on Resuscitation. 2021. URL: https://resus.org.au/download/9_2_med- ical/anzcor-guideline-9-2-1-suspected-heart-attack-apr-2021.pdf [Дата обращения: 07.09.2022] 5 ANZCOR Guideline 9.2.2 – Stroke. The Australian and New Zealand Committee on Resuscitation. 2021. URL: https://resus.org.au/down- load/9_2_medical/anzcor-guideline-9-2-2-stroke-apr-2021.pdf [Дата обращения: 07.09.2022] 6 Федеральная служба государственной статистики. Официаль- ная статистика. Население. Демография. Естественное движение населения. Число умерших по причинам смерти. 2022. URL: https://rosstat.gov.ru/storage/mediabank/demo24-2_2021.xlsx [Дата обращения: 07.09.2022] 7 REFERENCES Hasselqvist-Ax I., Riva G., Herlitz J., Rosenqvist M., Hollenberg J., Nordberg P., et al. Early Cardiopulmonary Resuscitation in out-of-Hospital Cardiac Arrest. N. Engl. J. Med. 2015;372;24:2307-2315. doi: 10.1056/NEJMoa1405796. 7. Биркун А.А., Фролова Л.П., Буглак Г.Н., Олефиренко С.С. Вне- госпитальная остановка кровообращения в Республике Крым: ана- лиз эпидемиологии и практики оказания помощи // Журнал им. Н В.Склифосовского Неотложная медицинская помощь. 2020. Т. 9. № 3. С. 338-347. doi: 10.23934/2223-9022-2020-9-3-338-347. 7. Birkun A.A., Frolova L.P., Buglak G.N., Olefirenko S.S. Out-of-Hospital Cardiac Arrest in the Republic of Crimea: Analysis of Epidemiology and Practice of Care. Zhurnal im. N.V.Sklifosovskogo Neotlozhnaya Meditsin- skaya Pomoshch = Russian Sklifosovsky Journal of Emergency Medical Care. 2020;9;3:338-347 (In Russ.). doi: 10.23934/2223-9022-2020- 9-3-338-347. 8. Birkun A. Coverage of Disorders / Conditions where First Aid is Recommended in Guidelines / Consensuses on First Aid // Mendeley Data. 2022. No. V1. doi: 10.17632/jjh9nkz6pm.1. 8. Birkun A. Coverage of Disorders / Conditions Where First Aid is Rec- ommended in Guidelines / Consensuses on First Aid. Mendeley Data. 2022;V1. doi: 10.17632/jjh9nkz6pm.1. 9. Bjørnsen L.P., Naess-Pleym L.E., Dale J., Grenne B., Wiseth R. De- scription of Chest Pain Patients in a Norwegian Emergency Department. Scand. Cardiovasc // J. 2019. V.53, No. 1. P. 28-34. doi: 10.1080/14017431.2019.1583362. 9. Bjørnsen L.P., Naess-Pleym L.E., Dale J., Grenne B., Wiseth R. De- scription of Chest Pain Patients in a Norwegian Emergency Department. Scand. Cardiovasc. J. 2019;53;1:28-34. doi: 10.1080/14017431.2019.1583362. 10. Knockaert D.C., Buntinx F., Stoens N., Bruyninckx R., Delooz H. Chest Pain in the Emergency Department: the Broad Spectrum of Causes // Eur. J. Emerg. Med. 2002. V.9, No. 1. P. 25-30. doi: 10.1097/00063110-200203000-00007. 10. Knockaert D.C., Buntinx F., Stoens N., Bruyninckx R., Delooz H. Chest Pain in the Emergency Department: the Broad Spectrum of Causes. Eur. J. Emerg. Med. 2002;9;1:25-30. doi: 10.1097/00063110- 200203000-00007. 11. George T., Ashover S., Cullen L., Larsen P., Gibson J., Bilesky J., et al. Introduction of an Accelerated Diagnostic Protocol in the Assess- ment of Emergency Department Patients with Possible Acute Coronary Syndrome: the Nambour Short Low-Intermediate Chest Pain Project // Emerg. Med. Australas. 2013. V.25, No. 4. P. 340-344. doi: 10.1111/1742-6723.12091. 11. George T., Ashover S., Cullen L., Larsen P., Gibson J., Bilesky J., et al. Introduction of an Accelerated Diagnostic Protocol in the Assessment of Emergency Department Patients with Possible Acute Coronary Syndrome: the Nambour Short Low-Intermediate Chest Pain Project. Emerg. Med. Aus- tralas. 2013;25;4:340-344. doi: 10.1111/1742-6723.12091. 12. REFERENCES 1. Дежурный Л.И., Шойгу Ю.С., Гуменюк С.А., Неудахин Г.В., За- курдаева А.Ю., Колодкин А.А. и др. Первая помощь: Учебное посо- бие для лиц, обязанных и (или) имеющих право оказывать первую помощь. М.: ФГБУ ЦНИИОИЗ Минздрава России. 2018. 68 с. 1. Dezhurnyy L.I., Shoygu Yu.S., Gumenyuk S.A., Neudakhin G.V., Za- kurdayeva A.Yu., Kolodkin A.A., et al. Pervaya Pomoshch = First Aid. A Training Manual for Persons Obliged and (or) Entitled to Provide First Aid. Moscow Publ., 2018. 68 p. (In Russ.). 2. Couper K., Abu Hassan A., Ohri V., Patterson E., Tang H.T., Bing- ham R., et al. Removal of Foreign Body Airway Obstruction: A System- atic Review of Interventions // Resuscitation. 2020. No. 156. P. 174-181. doi: 10.1016/j.resuscitation.2020.09.007. 2. Couper K., Abu Hassan A., Ohri V., Patterson E., Tang H.T., Bingham R., et al. Removal of Foreign Body Airway Obstruction: a Systematic Review of Interventions. Resuscitation. 2020;156:174-181. doi: 10.1016/j.re- suscitation.2020.09.007. 3. Eberhard K.E., Linderoth G., Gregers M.C.T., Lippert F., Folke F. Im- pact of Dispatcher-Assisted Cardiopulmonary Resuscitation on Neuro- logically Intact Survival in out-of-Hospital Cardiac Arrest: a Systematic Review // Scand. J. Trauma Resusc. Emerg. Med. 2021. V.29, No. 1. P. 70. doi: 10.1186/s13049-021-00875-5. 3. Eberhard K.E., Linderoth G., Gregers M.C.T., Lippert F., Folke F. Impact of Dispatcher-Assisted Cardiopulmonary Resuscitation on Neurologically Intact Survival in out-of-Hospital Cardiac Arrest: a Systematic Review. Scand. J. Trauma Resusc. Emerg. Med. 2021;29;1:70. doi: 10.1186/s13049-021-00875-5. 4. Böttiger B.W., Van Aken H. Kids Save lives - Training School Chil- dren in Cardiopulmonary Resuscitation Worldwide is Now Endorsed by the World Health Organization (WHO) // Resuscitation. 2015. No. 94. P. A5-7. doi: 10.1016/j.resuscitation.2015.07.005. 4. Böttiger B.W., Van Aken H. Kids Save lives - Training School Children in Cardiopulmonary Resuscitation Worldwide is Now Endorsed by the World Health Organization (WHO). Resuscitation. 2015;94:A5-7. doi: 10.1016/j.resuscitation.2015.07.005. 5. Holmberg M., Holmberg S., Herlitz J. Effect of Bystander Car- diopulmonary Resuscitation in out-of-Hospital Cardiac Arrest Patients in Sweden // Resuscitation. 2000. No. 47. P. 59-70. doi: 10.1016/s0300-9572(00)00199-4. 5. Holmberg M., Holmberg S., Herlitz J. Effect of Bystander Cardiopul- monary Resuscitation in out-of-Hospital Cardiac Arrest Patients in Sweden. Resuscitation. 2000;47:59-70. doi: 10.1016/s0300-9572(00)00199- 4. 6. Hasselqvist-Ax I., Riva G., Herlitz J., Rosenqvist M., Hollenberg J., Nordberg P., et al. Early Cardiopulmonary Resuscitation in Out-of-Hos- pital Cardiac Arrest // N. Engl. J. Med. 2015. V.372, No. 24. P. 2307- 2315. doi: 10.1056/NEJMoa1405796. 6. Заключение Выполненный анализ выявил ряд нарушений здоровья, которые требуют своевременного оказания ПП для со- хранения жизни пострадавших, но не входят в дей- ствующий в РФ официальный перечень состояний, при ко- торых должна оказываться первая помощь. Среди них – боль в груди как характерное проявление ОКС и инсульт, при том, что ОКС и инсульт в совокупности обусловли- вают наибольшее количество случаев смерти взрослых в России. Учитывая высокий потенциал ПП как ресурса, способного значительно снизить летальность при этих со- стояниях и ряде других, угрожающих жизни расстройств, не входящих в официальный перечень, имеется потреб- ность в расширении действующего перечня состояний, при которых оказывается ПП, и модификации перечня ме- роприятий по оказанию ПП. Это станет возможным Болезни системы кровообращения неизменно занимают лидирующую позицию в структуре смертности населения России. В 2021 г. эта патология привела к смерти 934 тыс. чел. – 38% от общего количества летальных исходов6. При этом в общем количестве случаев смерти населения от болезней системы кровообращения доля умерших от ишемической болезни сердца (ИБС) и её осложнений (508 тыс. случаев) и острых нарушений мозгового кровооб- ращения (132 тыс. случаев) составила 54 и 14% соот- ветственно. Учитывая высокую инцидентность и значи- Болезни системы кровообращения неизменно занимают лидирующую позицию в структуре смертности населения России. В 2021 г. эта патология привела к смерти 934 тыс. чел. – 38% от общего количества летальных исходов6. При этом в общем количестве случаев смерти населения от болезней системы кровообращения доля умерших от ишемической болезни сердца (ИБС) и её осложнений (508 тыс. случаев) и острых нарушений мозгового кровооб- ращения (132 тыс. случаев) составила 54 и 14% соот- ветственно. Учитывая высокую инцидентность и значи- 76 Disaster Medicine No. 4•2022 после внесения поправок в текст ст. 31 «Первая помощь» федерального закона от 21.11.2011 № 323-ФЗ9, ко- торыми предусмотрена возможность оказания ПП при до- полнительных состояниях и выполнения дополнительных мероприятий ПП. из расширенного перечня, включая: организацию мас- сового обучения населения принципам и навыкам ока- зания ПП; оснащение участников оказания ПП; орга- низацию учёта и анализа эффективности оказания ПП; создание условий для популяризации ПП и мотивации к оказанию ПП, а также внедрение в практику дистан- ционного консультирования свидетелей события и по- страдавших по вопросам оказания первой помощи, осуществляемого диспетчерами экстренных служб [26–30]. 9 Законопроект № 466977-7. О внесении изменения в статью 31 Федерального закона «Об основах охраны здоровья граждан в Рос- сийской Федерации». URL: https://sozd.duma.gov.ru/bill/466977-7 [Дата обращения: 07.09.2022] Заключение Решение такой комплексной задачи потребу- ет координированного взаимодействия специалистов в сфере организации здравоохранения и общественного здоровья, первой помощи и медицины неотложных со- стояний и, по мнению авторов, может быть наиболее эф- фективно реализовано в рамках работы экспертных со- ветов при профильной комиссии Минздрава России по направлению «Первая помощь». Кроме того, поправки предусматривают утверждение как регламентирующего документа правил оказания ПП, которые будут включать в себя необходимые меро- приятия первой помощи. Наряду с этим для обеспечения действенности оказания ПП потребуется реализация в отечественной системе первой помощи комплекса до- полнительных взаимосвязанных организационных пре- образований с их распространением на все состояния 9 Законопроект № 466977-7. О внесении изменения в статью 31 Федерального закона «Об основах охраны здоровья граждан в Рос- сийской Федерации». URL: https://sozd.duma.gov.ru/bill/466977-7 [Дата обращения: 07.09.2022] REFERENCES Zhuo L., Zhang Y., Zielke H.R., et al. Sudden unexpected death in epilepsy: Evaluation of forensic autopsy cases. Forensic Sci. Int. 2012;223;1-3:171-175. doi: 10.1016/j.forsciint.2012.08.024. 23. Zhuo L., Zhang Y., Zielke H.R., et al. Sudden Unexpected Death in Epilepsy: Evaluation of Forensic Autopsy Cases // Forensic Sci. Int. 2012. V.223, No. 1-3. P. 171-175. doi: 10.1016/j.forsciint.2012.08.024. 24. Kumar J.G., Abhilash K.P., Saya R.P., Tadipaneni N., Bose J.M. A Retrospective Study on Epidemiology of Hypoglycemia in Emergency De- partment. Indian J. Endocrinol. Metab. 2017;21;1:119-124. doi: 10.4103/2230-8210.195993. 24. Kumar J.G., Abhilash K.P., Saya R.P., Tadipaneni N., Bose J.M. A Retrospective Study on Epidemiology of Hypoglycemia in Emergency Department // Indian J. Endocrinol. Metab. 2017. V.21, No. 1. P. 119- 124. doi: 10.4103/2230-8210.195993. 25. Sessa F., Esposito M., Messina G., Di Mizio G., Di Nunno N., Salerno M. Sudden Death in Adults: a Practical Flow Chart for Pathologist Guidance. Healthcare (Basel). 2021;9;7:870. doi: 10.3390/health- care9070870. 25. Sessa F., Esposito M., Messina G., Di Mizio G., Di Nunno N., Salerno M. Sudden Death in Adults: a Practical Flow Chart for Patholo- gist Guidance // Healthcare (Basel). 2021. V.9, No. 7. P. 870. doi: 10.3390/healthcare9070870. 26. Dezhurnyy L.I., Boyarintsev V.V., Neudakhin G.V. The System of First Aid in the Russian Federation and its Interaction with the Emergency Serv- ices. Skoraya Meditsinskaya Pomoshch' = Emergency Medical Care. 2013;14;2:44-50 (In Russ.). 26. Дежурный Л.И., Бояринцев В.В., Неудахин Г.В. Система пер- вой помощи в России и ее взаимодействие со службой скорой меди- цинской помощи // Скорая медицинская помощь. 2013. Т.14, № 2. С. 44-50. 27. Birkun A.A., Dezhurnyy L.I. Dispatcher Assistance in out-of-Hospital Cardiac Arrest: Approaches for Diagnosing Cardiac Arrest by Telephone. Zhurnal im. N.V.Sklifosovskogo Neotlozhnaya Meditsinskaya Pomoshch = Russian Sklifosovsky Journal of Emergency Medical Care. 2019;8;1:60- 67 (In Russ.). doi: 10.23934/2223-9022-2019-8-1-60-67. 27. Биркун А.А., Дежурный Л.И. Диспетчерское сопровождение при угрозе внегоспитальной остановки кровообращения // Журнал им. Н.В.Склифосовского Неотложная медицинская помощь. 2019. Т.8, № 1. С. 60-67. doi: 10.23934/2223-9022-2019-8-1-60-67. 28. Dezhurnyy L.I., Gumenyuk S.A., Zakirov R.R., Maksimov D.A., Trofi- menko A.V. First Aid in the Russian Federation. Latest Changes and Imme- diate Prospects. Kremlevskaya Meditsina. Klinicheskiy Vestnik = Kremlin Medicine Journal. 2019;3:15-22 (In Russ.). 28. Дежурный Л.И., Гуменюк С.А., Закиров Р.Р., Максимов Д.А., Трофименко А.В. Первая помощь в Российской Федерации. По- следние изменения и ближайшие перспективы // Кремлевская ме- дицина. Клинический вестник. 2019. № 3. С. 15-22. 29. REFERENCES Hanifi N., Rezaee E., Rohani M. Time-to-Treatment and Its Asso- ciation with Complications and Mortality Rate in Patients with Acute My- ocardial Infarction: a Prospective Cohort Study // J. Emerg. Nurs. 2021. V.47, No. 2. P. 288-298.e4. doi: 10.1016/j.jen.2020.05.013. 12. Hanifi N., Rezaee E., Rohani M. Time-to-Treatment and Its Associ- ation with Complications and Mortality Rate in Patients with Acute My- ocardial Infarction: a Prospective Cohort Study. J. Emerg. Nurs. 2021;47;2:288-298.e4. doi: 10.1016/j.jen.2020.05.013. 13. Erhardt L., Herlitz J., Bossaert L., Halinen M., Keltai M., Koster R., et al. Task Force on the Management of Chest Pain // Eur. Heart. J. 2002. V.23, No. 15. P. 1153-1176. doi: 10.1053/euhj.2002.3194. 13. Erhardt L., Herlitz J., Bossaert L., Halinen M., Keltai M., Koster R., et al. Task Force on the Management of Chest Pain. Eur. Heart. J. 2002;23;15:1153-1176. doi: 10.1053/euhj.2002.3194. 77 Медицина катастроф №4•2022 14. Perkins G.D., Handley A.J., Koster R.W., Castrén M., Smyth M.A., Olasveengen T., et al. European Resuscitation Council Guidelines for Re- suscitation 2015: Section 2. Adult Basic Life Support and Automated Ex- ternal Defibrillation // Resuscitation. 2015. No. 95. P. 81-99. doi: 10.1016/j.resuscitation.2015.07.015. 14. Perkins G.D., Handley A.J., Koster R.W., Castrén M., Smyth M.A., Olasveengen T., et al. European Resuscitation Council Guidelines for Re- suscitation 2015: Section 2. Adult Basic Life Support and Automated Ex- ternal Defibrillation. Resuscitation. 2015;95:81-99. doi: 10.1016/j.resuscitation.2015.07.015. 15. Barbash I., Freimark D., Gottlieb S., Hod H., Hasin Y., Battler A., et al. Outcome of Myocardial Infarction in Patients Treated with Aspirin is En- hanced by Pre-Hospital Administration. Cardiology. 2002;98;3:141-147. doi: 10.1159/000066324. 15. Barbash I., Freimark D., Gottlieb S., Hod H., Hasin Y., Battler A., et al. Outcome of Myocardial Infarction in Patients Treated with Aspirin is Enhanced by Pre-Hospital Administration // Cardiology. 2002. V.98, No. 3. P. 141-147. doi: 10.1159/000066324. 16. Freimark D., Matetzky S., Leor J., Boyko V., Barbash I.M., Behar S., et al. Timing of Aspirin Administration as a Determinant of Survival of Pa- tients with Acute Myocardial Infarction Treated with Thrombolysis. Am. J. Cardiol. 2002;89;4:381-385. doi: 10.1016/s0002-9149(01)02256-1. 16. Freimark D., Matetzky S., Leor J., Boyko V., Barbash I.M., Behar S., et al. Timing of Aspirin Administration as a Determinant of Survival of Patients with Acute Myocardial Infarction Treated with Thrombolysis // Am. J. Cardiol. 2002. V.89, No. 4. P. 381-385. doi: 10.1016/s0002- 9149(01)02256-1. 17. Zideman D.A., Singletary E.M., Borra V., Cassan P., Cimpoesu C.D., De Buck E., et al. REFERENCES European Resuscitation Council Guidelines 2021: First Aid. Resuscitation. 2021;161:270-290. doi: 10.1016/j.resuscita- tion.2021.02.013. 17. Zideman D.A., Singletary E.M., Borra V., Cassan P., Cimpoesu C.D., De Buck E., et al. European Resuscitation Council Guidelines 2021: First Aid // Resuscitation. 2021. No. 161. P. 270-290. doi: 10.1016/j.resuscitation.2021.02.013. 18. Yang H., Huang X., Yang C., Zhu S., Chen X., Zhang M., et al. Time Window for Acute Stroke Management: a Cross-Sectional Study among Community Healthcare Practitioners in Primary Care. Int. J. Gen. Med. 2022;15:4483-4493. doi: 10.2147/IJGM.S361189. j 18. Yang H., Huang X., Yang C., Zhu S., Chen X., Zhang M., et al. Time Window for Acute Stroke Management: a Cross-Sectional Study among Community Healthcare Practitioners in Primary Care // Int. J. Gen. Med. 2022. No. 15. P. 4483-4493. doi: 10.2147/IJGM.S361189. 19. Kontsevaya A.V., Balanova YU.A., Imayeva A.E., KHudyakov M.B., Karpov O.I., Drapkina O.M. Economic Burden of Hypercholesterolemia in the Russian Federation. Ratsionalnaya Farmakoterapiya v Kardiologii = Rational Pharmacotherapy in Cardiology. 2018;14;3:393-401 (In Russ.). doi: 10.20996/1819-6446-2018-14-3-393-401. 19. Концевая А.В., Баланова Ю.А., Имаева А.Э., Худяков М.Б., Карпов О.И., Драпкина О.М. Экономический ущерб от гиперхоле- стеринемии на популяционном уровне в Российской Федерации // Рациональная фармакотерапия в кардиологии. 2018. Т.14, № 3. С. 393-401. doi: 10.20996/1819-6446-2018-14-3-393-401. 20. Kontsevaya A.V., Drapkina O.M., Balanova Yu.A., Imayeva A.E., Suvorova Ye.I., Khudyakov M.B. Economic Burden of Cardiovascular Dis- eases in the Russian Federation in 2016. Ratsionalnaya Farmakoterapiya v Kardiologii = Rational Pharmacotherapy in Cardiology. 2018;14;2:156- 166 (In Russ.). doi: 10.20996/1819-6446-2018-14-2-156-166. 20. Концевая А.В., Драпкина О.М., Баланова Ю.А., Имаева А.Э., Суворова Е.И., Худяков М.Б. Экономический ущерб от сердечно-со- судистых заболеваний в Российской Федерации в 2016 году // Ра- циональная фармакотерапия в кардиологии. 2018. Т.14, № 2. С. 156-166. doi: 10.20996/1819-6446-2018-14-2-156-166. 21. Samorodskaya I.V., Semenov V.Yu. Years of Potential Life Lost from Cardiovascular Diseases of the Economically Active Russian Population in 2013-2019. Rossiyskiy Kardiologicheskiy Zhurnal = Russian Journal of Cardiology. 2021;26;5:4161 (In Russ.). doi: 10.15829/1560-4071- 2021-4161. 21. Самородская И.В., Семёнов В.Ю. Потерянные годы потенци- альной жизни от болезней системы кровообращения экономически активного населения Российской Федерации в 2013-2019 годах // Российский кардиологический журнал. 2021. Т.26, № 5. С. 82-87. doi: 10.15829/1560-4071-2021-4161. 22. Moneret-Vautrin D.A., Morisset M., Flabbee J., Beaudouin E., Kanny G. Epidemiology of Life-Threatening and Lethal Anaphylaxis: a Review. Al- lergy. 2005;60;4:443-451. doi: 10.1111/j.1398-9995.2005.00785.x. 22. Moneret-Vautrin D.A., Morisset M., Flabbee J., Beaudouin E., Kanny G. Epidemiology of Life-Threatening and Lethal Anaphylaxis: a Review // Allergy. 2005. V.60, No. 4. P. 443-451. doi: 10.1111/j.1398-9995.2005.00785.x. 23. Материал поступил в редакцию 08.09.22; статья принята после рецензирования 24.10.22; статья принята к публикации 23.12.22 The material was received 08.09.22; the article after peer review procedure 24.10.22; the Editorial Board accepted the article for publication 23.12.22 REFERENCES Zhuravlev S.V., Kolodkin A.A., Maksimov D.A., Trofimenko A.V., Dezhurnyy L.I., Boyarintsev V.V. The Organization of Registration of Rate, Capacity and Effectiveness of First Aid Measures. Problemy Sotsialnoy Gigiyeny, Zdravookhraneniya i Istorii Meditsiny = Problems of Social Hy- giene, Public Health and History of Medicine. 2020;28;4:616-620 (In Russ.). doi: 10.32687/0869-866X-2020-28-4-616-620. 29. Журавлев С.В., Колодкин А.А., Максимов Д.А., Трофименко А.В., Дежурный Л.И., Бояринцев В.В. Организация учета частоты, объема и результативности мероприятий первой помощи // Про- блемы социальной гигиены, здравоохранения и истории медицины. 2020. Т.28, № 4. С. 616-620. doi: 10.32687/0869-866X-2020-28- 4-616-620. 30. Birkun A.A., Dezhurnyy L.I. Legal and Regulatory Framework for Provision of First Aid and Education in First Aid in out-of-Hospital Cardiac Arrest. Zhurnal im. N.V.Sklifosovskogo Neotlozhnaya Meditsinskaya Po- moshch = Russian Sklifosovsky Journal of Emergency Medical Care. 2021;10;1:141-152 (In Russ.). doi: 10.23934/2223-9022-2021-10- 1-141-152. 30. Биркун А.А., Дежурный Л.И. Нормативно-правовое регули- рование оказания первой помощи и обучения оказанию первой по- мощи при внегоспитальной остановке сердца // Журнал им. Н.В.Склифосовского Неотложная медицинская помощь. 2021. Т.10, № 1. С. 141-152. doi: 10.23934/2223-9022-2021-10-1-141-152. Материал поступил в редакцию 08.09.22; статья принята после рецензирования 24.10.22; статья принята к публикации 23.12.22 The material was received 08.09.22; the article after peer review procedure 24.10.22; the Editorial Board accepted the article for publication 23.12.22 оступил в редакцию 08.09.22; статья принята после рецензирования 24.10.22; статья принята к публикации 23.12.22 s received 08.09.22; the article after peer review procedure 24.10.22; the Editorial Board accepted the article for publication 23.12.22 78 Disaster Medicine No. 4•2022
https://openalex.org/W1965617583	https://cdr.lib.unc.edu/downloads/0z709361j	English	null	Accelerating the Inbreeding of Multi-Parental Recombinant Inbred Lines Generated By Sibling Matings	G3	2,012	cc-by	7,625	Accelerating the Inbreeding of Multi-Parental Recombinant Inbred Lines Generated By Sibling Matings Catherine E. Welsh and Leonard McMillan1 Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Catherine E. Welsh and Leonard McMillan1 Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 ABSTRACT Inbred model organisms are powerful tools for genetic studies because they provide reproducible genomes for use in mapping and genetic manipulation. Generating inbred lines via sibling matings, however, is a costly undertaking that requires many successive generations of breeding, during which time many lines fail. We evaluated several approaches for accelerating inbreeding, including the systematic use of back-crosses and marker-assisted breeder selection, which we contrasted with randomized sib-matings. Using simulations, we explored several alternative breeder-selection methods and monitored the gain and loss of genetic diversity, measured by the number of recombination-induced founder intervals, as a function of generation. For each approach we simulated 100,000 independent lines to estimate distributions of generations to achieve full-ﬁxation as well as to achieve a mean heterozygosity level equal to 20 generations of randomized sib-mating. Our analyses suggest that the number of generations to fully inbred status can be substantially reduced with minimal impact on genetic diversity through combinations of parental backcrossing and marker-assisted inbreeding. Although simulations do not consider all confounding factors underlying the inbreeding process, such as a loss of fecundity, our models suggest many viable alternatives for accelerating the inbreeding process. KEYWORDS marker-assisted breeding mouse simulation recombinant inbred lines Mouse Genetic Resource Recombinant inbred lines (RILs), ﬁrst developed in 1971 (Taylor et al. 1971; Bailey 1971), have long been an important resource for genetics. Typically, RILs are derived by crossing two inbred strains followed by repeated generations of selﬁng or sibling mating to produce an inbred line whose genome is a mosaic of its parental lines. More recently, panels of multiway RILs have been developed that combine the ge- nomes of multiple founder lines via an initial mixing stage followed by successive generations of inbreeding. Examples include mouse (Threadgill et al. 2002; Churchill et al. 2004; Chesler et al. 2008; Collaborative Cross Consortium 2012), maize (Buckler et al. 2009), Drosophila melanogaster (Gibson and Mackay 2002), and Arabidopsis thaliana (Paulo et al. 2008; Kover et al. 2009; Gan et al. 2011). For all species, inbreeding via either selﬁng or sibling mating is the primary process used for ﬁxing the genetic background. Copyright © 2012 Welsh and McMillan doi: 10.1534/g3.111.001784 Manuscript received July 11, 2011; accepted for publication November 6, 2011 This is an open-access article distributed under the terms of the Creative Commons Attribution Unported 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. Supporting information is available online at http://www.g3journal.org/lookup/ suppl/doi:10.1534/g3.111.001784/-/DC1 1Corresponding author: Department of Computer Science, 311 Sitterson Hall, University of North Carolina Chapel Hill, Campus Box 3175, Chapel Hill, NC 27599. E-mail: mcmillan@cs.unc.edu KEYWORDS marker-assisted breeding mouse simulation recombinant inbred lines Mouse Genetic Resource MOUSE GENETIC RESOURCES MOUSE GENETIC RESOURCES MOUSE GENETIC RESOURCES Accelerating the Inbreeding of Multi-Parental Recombinant Inbred Lines Generated By Sibling Matings The interval represen- tation has the advantage of implicitly representing every base pair in the genome while explicitly tracking every recombination. This ap- proach provides a conservative estimate of homozygosity because it treats every founder sequence as a separate genotype without taking into account regions of sequence identity among founders. Moreover, our interval model can be trivially converted to a string of alleles representation if given the founder sequences or markers from any platform. Despite the differences in the underlying representation, our sim- ulator produces results nearly indistinguishable from those presented by Broman (2005). Figure 2 shows the distribution of the number of generations to complete ﬁxation and number of segments for both the two-way and eight-way sib-mating RILs based on the simulation of 100,000 RILs. For a randomized eight-way RIL our simulations show that it takes an average of 38.21 6 7.1 (SD) generations of sib-matings to reach complete ﬁxation. The genomes of the resultant inbred lines have an average of 145.1 6 12.48 segments in their mosaic structure. Furthermore, 25.72 6 3.16 generations of sib-mating on average are needed to reach 99% ﬁxation. These baseline metrics are used for comparison against our accelerated inbreeding simulations. Marker-assisted breeding techniques have been used to ﬁx a sel- ected haplotype interval against a ﬁxed background in congenic strains (Markel et al. 1997). In mouse, marker-assisted “speed con- genics” have demonstrated a reduction in the number of generations of backcrossing from 10 generations to ﬁve. This reduction was achieved by selecting mice that retained the lowest proportion of heterozygous donor to recipient genome. These selection criteria have evolved overtime, as technology has allowed for more rapid genotyp- ing (Flaherty and Bolivar 2007). Just as marker-assisted techniques have been used to improve mapping resolution in self-pollinated spe- cies (Boddhireddy et al. 2009) and have been adapted for consomics (Armstrong et al. 2006), we adapt them for multiparental RILs. Rather than attempt to ﬁx one speciﬁc genomic region or one complete chromosome, our goal is to achieve complete ﬁxation of the genome in fewer generations than random sib-matings, without substantially impacting the overall genetic architecture of the inbred lines. In this article, we address accelerating the inbreeding process of outcrossing species by using a combination of alternative breeding strategies and marker-assisted inbreeding (MAI) techniques. Accelerating the Inbreeding of Multi-Parental Recombinant Inbred Lines Generated By Sibling Matings RILs derived by sibling matings from two parental backgrounds require multiple generations to ﬁx their genome as homozygous, and the number of generations depends on the diploid number. In mice, this requires at least 20 generations (Eisen 2005) and assuming an average of four generations per year, it takes a minimum of 5 years to create a new RIL. Moreover, a large fraction of the started RILs fail, presumably as the result of genetic incompatibilities affecting survival and reproduction (Silver 1995). Many recent efforts to generate RILs have focused on multiway crosses where more than two parental lines are initially mixed before inbreeding. Broman (2005) showed through simulation that eight-way RILs take on average 26.7 generations of sib-mating to reach 99% ﬁxation, and 38.9 generations, on average, to reach complete ﬁxation. Although a major source of genetic variation in a RIL is derived from the choice of founder strains, we focus on the additional genetic variations introduced by mixing of allele combinations via recombi- nations between founder genomes. This is the primary source of genetic variation between RILs. Therefore, the number of distinct founder segments, deﬁned as the regions between recombination breakpoints on the RIL chromosomes, can be used as a measure of Volume 2 \| February 2012 \| 191 Volume 2 \| February 2012 \| 191 genetic diversity. From now on, we refer to these distinct founder segments simply as segments. Using simulations, Broman (2005) tracked the number of segments generated through recombination in inbred lines and used it as a comparison between the genetic diversity of two-way and eight-way sib-mating RILs. Recombinations in early generations increase diversity, but eventually diversity peaks and the process of inbreeding leads to a loss of segments. In an eight-way cross, the peak in diversity is reached at the seventh generation of inbreeding on average and before 10 generations of inbreeding for 75% of line starts (Figure 1). Therefore, we will consider 10 generations of inbreed- ing as past the point of peak diversity. If inbreeding acceleration is started before this peak is reached, the resulting inbred lines are likely to see a reduction in the number of segments. Therefore, unless other- wise speciﬁed, we assume a randomized sib-pair mating scheme is used for the ﬁrst 10 generations, after which we apply various nonmarker- assisted and marker-assisted acceleration techniques. simulators (Broman 2005; Valdar et al. 2006). Accelerating the Inbreeding of Multi-Parental Recombinant Inbred Lines Generated By Sibling Matings Our analysis is based on an initial funnel-breeding scheme like that used in the eight-way Collaborative Cross (CC) (Churchill et al. 2004; Collaborative Cross Consortium 2012), where the mixing of eight inbred lines occurs in two initial crossing stages (G1 and G2), followed by successive generations of sib-matings (G2:F1, G2:F2, etc) until the line becomes fully inbred. The eight founder strains are represented by letters A-H, and these labels are used to track the descent of genomic segments (see Figure 1) (Collaborative Cross Consortium 2012). We introduce a notion of joint heterozygosity (JH) to express four possible states between the homologous alleles of a potential breeding pair. Figure 3 shows two homologous chromosomes from each parent of a potential breeding pair and depicts each of the JH states. The inbred state is achieved when both male and female samples are ho- mozygous for the same founder state. We call this state same-same (SS). Another possible state involves a breeding pair that is heterozy- gous with alleles from two founders while the mate is homozygous. We call this different-same (DS). This state occurs in two forms, DS2 when the heterozygous gene shares a founder allele with the homozygous allele of its mate, and DS3, when the heterozygous gene shares no Figure 1 The average number of founder segments in eight-way RILs at various gen- erations of inbreeding. This ﬁgure is based on 100,000 simulations, and the number of seg- ments was tracked until they reached com- plete ﬁxation. The average peak in the number of segments occurs at generation 7 and before generation 10 for 75% of all lines. Therefore, we consider generation 10 to be past the point of peak diversity. MATERIALS AND METHODS founder alleles with its mate. The third state is opposite-same (Ss), where the male is homozygous for one founder and the female is homozygous for another. The ﬁnal state is different-different (DD), where both male and female are heterozygous. This state comes in three variations, involving, two, three, and four founders, respectively. The two-founder state, called DD2, occurs when both male and female are heterozygous between the same founder alleles. DD3 refers to when both male and female are heterozygous but share one common founder allele. DD4 occurs when the male and female are heterozygous and do not share any founder alleles. Figure 4 shows a state diagram with these four states and their forms depicting all possible transitions between them in a single generation. The directed edge weights repre- sent the probability of transitioning between JH states. A similar tran- sition matrix, which uses 13 states instead of our seven, has also been derived by Broman (2012). It is a simple matter to extend our JH model to two generations by ﬁnding every path of length two within the graph and inserting an edge with weight equal to the product of the two edges along its path. The weights of edges from a common source to a common destination, but passing through different intermediate states, can be added and combined into a single edge. This approach can be extended to n generations, and as n increases all of the heaviest edges eventually lead to the inbred (SS) state. For analytical expressions for extending our JH model for n generations, see (Broman 2012; Collaborative Cross Consortium 2012). In early generations the CC lines include genomic intervals in JH states involving three or more founders (DD3, DD4, DS3), but in later generations these intervals eventually transition to states with two or less founders (DD2, DS2, SS, and Ss; Figure 5). We can see in Figure 4 that DD4, DD3, and DS3 are transient states, meaning that once this group of three states is left, there are no returning edges. In two-way RILs, the three transient states do not occur because there are at most two founders present. When selﬁng, the model further reduces to only two JH states, DD2 and SS. The transition probabilities to reach the inbred state are incorporated into our metric for selecting the best mating pair at each generation, which is discussed later in this section. MATERIALS AND METHODS We developed a simulator that represents a genome as a collection of intervals whose boundaries can be resolved at the resolution of a base pair rather than a string of alleles as is common in many breeding Figure 1 The average number of founder segments in eight-way RILs at various gen- erations of inbreeding. This ﬁgure is based on 100,000 simulations, and the number of seg- ments was tracked until they reached com- plete ﬁxation. The average peak in the number of segments occurs at generation 7 and before generation 10 for 75% of all lines. Therefore, we consider generation 10 to be past the point of peak diversity. 192 \| C. E Welsh and L. McMillan Figure 1 The average number of founder segments in eight-way RILs at various gen- erations of inbreeding. This ﬁgure is based on 100,000 simulations, and the number of seg- ments was tracked until they reached com- plete ﬁxation. The average peak in the number of segments occurs at generation 7 and before generation 10 for 75% of all lines. Therefore, we consider generation 10 to be past the point of peak diversity. Figure 1 The average number of founder segments in eight-way RILs at various gen- erations of inbreeding. This ﬁgure is based on 100,000 simulations, and the number of seg- ments was tracked until they reached com- plete ﬁxation. The average peak in the number of segments occurs at generation 7 and before generation 10 for 75% of all lines. Therefore, we consider generation 10 to be past the point of peak diversity. 192 \| C. E Welsh and L. McMillan Figure 2 The number of generations to complete ﬁxation (A) and the number of resulting founder segments (B) in two-way and eight-way RILs. On average, two-way RILs take 35.92 generations to reach complete ﬁxation and have 91.95 segments. Eight-way RILs take 38.21 generations and have 145.12 segments on average. These ﬁgures are based on 100,000 simulations and are consistent with previous simulations (Broman 2005). Figure 2 The number of generations to complete ﬁxation (A) and the number of resulting founder segments (B) in two-way and eight-way RILs. On average, two-way RILs take 35.92 generations to reach complete ﬁxation and have 91.95 segments. Eight-way RILs take 38.21 generations and have 145.12 segments on average. These ﬁgures are based on 100,000 simulations and are consistent with previous simulations (Broman 2005). MATERIALS AND METHODS Using the notion of JH state, we split the genome into intervals according to state and track the genomic fraction of each type. We combine these fractions to arrive at several useful measures. Adding the genomic fraction of all regions in the same-same state (SS) gives Figure 3 This image shows all possible JH states between a potential mating-pair and illustrates our notion of a genomic segment. DD stands for different-different and occurs in three variations. DD4 occurs when both breeders are heterozygous and do not share any founder alleles among them. DD3 occurs when both breeders are heterozygous and share one founder allele, whereas DD2 refers to both breeders being heterozygous for the same two founder alleles. DS stands for different-same and occurs in two variations. DS3 occurs when the heterozygous gene shares no founder alleles with the homozygous allele of its mate. DS2 refers to when the heterozygous gene shares one founder allele with its mate. Ss is opposite same, where the male is homozygous for one founder allele and the female is homozygous for another allele. The ﬁnal state, SS (same- same), is achieved when both male and female are homozygous for the same founder allele. All JH segments are depicted with a chromosome fraction of 0.15, except for Ss, with 0.10. CC lines begin inbreeding in one of the states, DD4, DD3,, and DD2. The desired inbred state for all intervals is SS. DS2 is the most likely to become SS. DD2 is the next most likely state to become ﬁxed. It takes at least two gener- ations to transit from Ss to SS, as there is no direct path between these two states. the ﬁxed genomic fraction (FGF). We call the complement of this, or 1-FGF, the mating pair’s combined heterozygous fraction (CHF). FGF and CHF can be used to assess how inbred a line is, such that FGF ¼ 1 refers to fully inbred. state in a breeding pair because it has no chance of becoming inbred in the next generation, as shown in Figure 4. In contrast, of the noninbred states, DS2 has the greatest probably of becoming ﬁxed in the next generation, and DD2 is the next most likely. In simulation, we tested a number of modiﬁed breeding schemes in an attempt to accelerate the inbreeding process. These nonmarker- assisted breeding schemes minimally impact the traditional RIL generation process. We considered several variations of backcrosses. The use of backcrosses was motivated by the analysis of Broman (2005), which identiﬁed a substantial advantage for selﬁng when com- pared to sib-mating. Selﬁng in two-way plant RILs takes on average 10.5 generations to reach complete ﬁxation, which is a substantial reduction from the 35 generations needed when two-way sib-mating. The steadily decreasing cost of high-density genotyping combined with the advantages of considering each sample’s individual full genetic makeup motivated us to also explore MAI techniques. The ability to compare potential breeding pairs based on their high density genotypes allows us to choose breeding pairs with the greatest likelihood of pro- ducing inbred offspring. The Ss (opposite same) is the least-preferred In simulation, we tested a number of modiﬁed breeding schemes in an attempt to accelerate the inbreeding process. These nonmarker- assisted breeding schemes minimally impact the traditional RIL generation process. We considered several variations of backcrosses. The use of backcrosses was motivated by the analysis of Broman (2005), which identiﬁed a substantial advantage for selﬁng when com- pared to sib-mating. Selﬁng in two-way plant RILs takes on average 10.5 generations to reach complete ﬁxation, which is a substantial reduction from the 35 generations needed when two-way sib-mating. Figure 3 This image shows all possible JH states between a potential mating-pair and illustrates our notion of a genomic segment. DD stands for different-different and occurs in three variations. DD4 occurs when both breeders are heterozygous and do not share any founder alleles among them. DD3 occurs when both breeders are heterozygous and share one founder allele, whereas DD2 refers to both breeders being heterozygous for the same two founder alleles. DS stands for different-same and occurs in two variations. DS3 occurs when the heterozygous gene shares no founder alleles with the homozygous allele of its mate. DS2 refers to when the heterozygous gene shares one founder allele with its mate. Ss is opposite same, where the male is homozygous for one founder allele and the female is homozygous for another allele. The ﬁnal state, SS (same- same), is achieved when both male and female are homozygous for the same founder allele. All JH segments are depicted with a chromosome fraction of 0.15, except for Ss, with 0.10. Volume 2 February 2012 \| Accelerating Inbreeding of RILs \| 193 193 Figure 4 A state diagram showing the tran- sitions between all JH states in a single generation. The directed edges are labeled with the transition probability. The grayed- out nodes represent transient states; once a segment moves away from these three states, there are no returning edges. Tran- sient states tend to go away after a few generations and are rarely seen past the point of peak diversity (as shown in Figure 5). CC lines begin inbreeding in one of the states, DD4, DD3,, and DD2. The desired inbred state for all intervals is SS. DS2 is the most likely to become SS. DD2 is the next most likely state to become ﬁxed. It takes at least two gener- ations to transit from Ss to SS, as there is no direct path between these two states. Figure 4 A state diagram showing the tran- sitions between all JH states in a single generation. The directed edges are labeled with the transition probability. The grayed- out nodes represent transient states; once a segment moves away from these three states, there are no returning edges. Tran- sient states tend to go away after a few generations and are rarely seen past the point of peak diversity (as shown in Figure 5). We choose the “best” breeding pair, by considering a weighted genomic mix of the JH types of all candidate mating pairs. The best pair is selected as the maximum of a weighted combination of transi- tion probabilities for all JH segments of a given mating pair considering all chromosomes. For each distinct JH segment of a chromosome the probability that it will become inbred in the next generation (i.e., the weight of the edge from the current JH state to the SS state) is mul- tiplied by the chromosome fraction of the segment, and the sum is accumulated over all segments on the chromosome. This calculation results in a chromosome score ranging from 0, when the entire chro- mosome is Ss, DD3, DD4, or DS3, to 1 when the entire chromosome is SS. This approximation ignores the relative ordering of segments, and, therefore, does not consider linkage. The individual chromosome scores are then multiplied together, modeling their independent Figure 5 A histogram of segments colored according to their JH state as a function of generation. In early generations, most segments have contributions from three or more founders, but by generation 10 (after the point of peak diversity), segments have contributions from two or fewer founders. This plot was created by tracking the JH states between breeder pairs and ﬁnding the average contribution of each state over 100,000 simulations. 194 \| C. E Welsh and L. McMillan Figure 5 A histogram of segments colored according to their JH state as a function of generation. In early generations, most segments have contributions from three or more founders, but by generation 10 (after the point of peak diversity), segments have contributions from two or fewer founders. This plot was created by tracking the JH states between breeder pairs and ﬁnding the average contribution of each state over 100,000 simulations. Figure 5 A histogram of segments colored according to their JH state as a function of generation. In early generations, most segments have contributions from three or more founders, but by generation 10 (after the point of peak diversity), segments have contributions from two or fewer founders. This plot was created by tracking the JH states between breeder pairs and ﬁnding the average contribution of each state over 100,000 simulations. Figure 5 A histogram of segments colored according to their JH state as a function of generation. Figure 5 A histogram of segments colored according to their JH state as a function of generation. In early generations, most segments have contributions from three or more founders, but by generation 10 (after the point of peak diversity), segments have contributions from two or fewer founders. This plot was created by tracking the JH states between breeder pairs and ﬁnding the average contribution of each state over 100,000 simulations. Nonmarker-assisted breeding schemes The ﬁrst breeding scheme examined was alternating back-crosses in successive generations, father-to-daughter in one generation followed by mother-to-son in the next (supporting information, Figure S1). This scheme has many practical advantages in that it leverages known-fertile samples. Furthermore, this strategy also serves as a use- ful fallback for preserving lines. We simulated this approach starting after the point of peak diversity, with a backcross between a father and daughter followed by a backcross between a mother and son in the next generation (each breeder is used in two successive generations, alternating dam and sire). This process was repeated for each sub- sequent generation until complete ﬁxation was achieved. Alternating backcrosses achieves a reduction in the number of generations to com- plete ﬁxation with an average number of generations of 33.45 6 5.88 (Figure 6). This represents a reduction of nearly ﬁve generations over randomized mating and a substantial reduction in variance. It decreases the number of segments in the resulting inbred lines to 141.21, a loss of about four segments on average. The alternating backcross also reduces the number of generations to 99% ﬁxation to 23.45 6 3.11, a reduction of two generations. We explored two marker-assisted breeding schemes. The ﬁrst of these is MAI, which modiﬁes the breeding scheme only after the point of peak diversity is reached. Once the peak is reached, the WSM discussed previously is applied to choose the best breeding pairs. The second is a marker-assisted advanced intercross, which modiﬁes the breeding scheme to choose sib-pairs to increase segments until either a speciﬁed generation or a desired number of segments is reached; it then reverts to choosing sib-pairs to accelerate inbreeding. Through simulations, we track the average number of generations to fully inbred and to 99% inbred as well as the average number of segments present in the inbred lines to compare the different breeding schemes. The simulator is written in Python and runs on a Dell Studio XPS with 8GB RAM, with dual-threaded quad-core processors. It takes approximately 5.5 hr to complete 100,000 simulations of eight-way RILs. For the purposes of this analysis, the eight-way CC funnel breeding scheme was used, but our simulator also supports the input of any breeding scheme using pedigree ﬁles. It has also been used to simulate two-way RILs, F2 crosses, and outbred populations. There are several practical limitations to the alternating back- crossing approach. In early generations, most segments have contributions from three or more founders, but by generation 10 (after the point of peak diversity), segments have contributions from two or fewer founders. This plot was created by tracking the JH states between breeder pairs and ﬁnding the average contribution of each state over 100,000 simulations. 194 \| C. E Welsh and L. McMillan 194 segregation, to arrive at the total pair score. Therefore, we assign a score for a given mating pair as: Array (MUGA). The SNPs on MUGA are evenly distributed with an average spacing of 325 Kb and a standard deviation of 191 Kb. In an eight-way cross, the genotypes at multiple markers (at a mini- mum three) are needed to distinguish among the founders. The founder assignments and recombination breakpoints are inferred from the genotypes using a hidden Markov model similar to the ones described by Mott et al. (2000), Zhang et al. (2009), and Liu et al. (2010). Because multiple markers are needed to distinguish each founder, the effective founder-ascertainment resolution of MUGA is approximately 1 Mb. Array (MUGA). The SNPs on MUGA are evenly distributed with an average spacing of 325 Kb and a standard deviation of 191 Kb. In an eight-way cross, the genotypes at multiple markers (at a mini- mum three) are needed to distinguish among the founders. The founder assignments and recombination breakpoints are inferred from the genotypes using a hidden Markov model similar to the ones described by Mott et al. (2000), Zhang et al. (2009), and Liu et al. (2010). Because multiple markers are needed to distinguish each founder, the effective founder-ascertainment resolution of MUGA is approximately 1 Mb. Scoreðn; mÞ 5 Y N i51 X JHSegn;m2Chri p JHSegn;m/SS JHSegn;m kChrik This score is an approximation of the actual likelihood that the entire genome will become inbred in the next generation. We refer to this score as the weighted state metric (WSM). JHSegn,m represents a JH segment on the speciﬁed chromosome i induced by the pairing n, m, and the best pair is the maximum of this score over all possible pairs n,m. In self-pollinated species, our score simpliﬁes to a scaled version of the FGF because the only relevant states are DD2 and SS, which has been described previously (Boddhireddy et al. 2009). Selected advanced intercrosses Although MAI achieves a substantial reduction in the number of generations required to ﬁx an RIL, it does so with an average loss of approximately seven segments per line. This result is unfortunate because the number of segments determines the resolution of a RIL panel for quantitative trait mapping (Aylor et al. 2011). Therefore, we attempted to overcome this loss by using marker-assisted techniques in the ﬁrst 10 generations of inbreeding to select mating pairs most apt to increase the number of recombination segments. We refer to these lines as selected advanced intercrosses (Darvasi and Soller 1995) in that they Using the WSM, we selected the best pair from sib-pairs, parent2child backcrosses, or a combination of both. To see what other pair relationships were worth considering, we simulated 100,000 lines such that ran- dom sib-matings were used for 15 generations, at which time three mating pairs were generated, producing two male and two female offspring each. The best breeding pair was then chosen by comparing every female to every male (both parents and offspring). The pair with the lowest CHF was selected. Sib-pairs were selected 63% of the time, whereas backcrosses were chosen 23% of the time. Cousin-pairs (off- spring from different mating pairs of the same generation) were the next most likely, being selected 6.9%. The remaining 7.1% included mating combinations such as aunt2nephew, uncle2niece, or grand- parent2grandchild. We concluded that non-sib, non-backcross matings should be used sparingly, except in the case of preserving a line. Figure 7 CHF as a function of number of generations. This plot shows that MAI reduces the CHF among breeding pairs much faster than random sib-matings. We can see the effect as soon as the breeding scheme is modiﬁed (at the point of peak diversity). Because sib-pairs were most often the best option, we limited subsequent simulations to selecting the best sib-pair and report those statistics in Figure 6 and Table 1. For the MAI sib-pairs breeding scheme, random sib-matings were simulated until the point of peak diversity was reached. After this point, four female and four male offspring were simulated (4-4), all pairs were considered, and the best pair was chosen as the breeders. This process was continued until the line reached complete ﬁxation. Our model is based on generation number and may require multiple litters to achieve the four females and four males assumed in simulation. Nonmarker-assisted breeding schemes For instance, female fertility often spans a limited window that might not allow for mother-son backcrossing. Therefore, we also explored, through simulation, a modiﬁed breeding scheme To test our MAI methods, we used the developing CC (Collab- orative Cross Consortium 2012) and a low-density genotyping plat- form we designed, referred to as the Mouse Universal Genotyping Figure 6 A comparison of ﬁve breeder selection alternatives for generating an eight-way RIL, showing the number of generations to reach complete ﬁxation (A) and the total number of segments (B) found in the ﬁnal inbred lines. Random sib-pair mating is used as our baseline. The alternating backcross swaps between father2daughter and mother2son matings in successive generations. The father2daughter scheme alter- nates between father2daughter and random sibling matings in successive generations. MAI uses our weighted state metric to choose between 16 breeding pairs after the point of peak diversity. The selected advanced intercross modiﬁes early stages of the breeding scheme to choose sib- pairs that maximize diversity, and then at a pre-established generation (10), it reverted to choosing sib-pairs to accelerate the inbreeding process. Figure 6 A comparison of ﬁve breeder selection alternatives for generating an eight-way RIL, showing the number of generations to reach complete ﬁxation (A) and the total number of segments (B) found in the ﬁnal inbred lines. Random sib-pair mating is used as our baseline. The alternating backcross swaps between father2daughter and mother2son matings in successive generations. The father2daughter scheme alter- nates between father2daughter and random sibling matings in successive generations. MAI uses our weighted state metric to choose between 16 breeding pairs after the point of peak diversity. The selected advanced intercross modiﬁes early stages of the breeding scheme to choose sib- pairs that maximize diversity, and then at a pre-established generation (10), it reverted to choosing sib-pairs to accelerate the inbreeding process. Volume 2 February 2012 \| Accelerating Inbreeding of RILs \| 195 195 n Table 1 Number of generations to 100% ﬁxation (fully inbred), 99% ﬁxation, and number of segments for different breeding schemes Breeding Scheme Average Generations to Fixation SD Gens to Fixation Average Generations to 99% Fixation SD Gens to 99% Fixation Average No. Segments SD, No. Nonmarker-assisted breeding schemes Segments Two-way 35.92 7.13 23.47 3.19 91.95 10.21 Eight-way random sib-pairs 38.21 7.10 25.72 3.16 145.12 12.48 Alternating backcross 33.45 5.88 23.45 3.11 141.21 12.07 Father2daughter backcross 37.06 7.55 24.70 3.54 142.39 12.24 MAI 22.10 4.41 16.44 1.00 138.83 11.83 Marker-selected advanced intercross 23.54 3.82 18.45 0.88 155.63 12.53 SD, standard deviation; MAI, marker-assisted inbreeding. ons to 100% ﬁxation (fully inbred), 99% ﬁxation, and number of segments for different breeding schemes n Table 1 Number of generations to 100% ﬁxation (fully inbred), 99% ﬁxation, and number of segme and number of segments for different breeding sche of setting up breeders as soon as any sibling mating pairs are available. Any form of MAI was always able to considerably reduce the number of generations to achieve inbred status regardless of sex balance or litter size. Moreover, waiting for a sufﬁciently large breeder-candidate set always outperformed the greedy approach of setting up matings as soon as any pair was available. More details on this analysis appear in the supplementary documentation (Figure S2). involving only father-daughter backcrosses. Starting after the point of peak diversity, a father-daughter backcross is followed in the next generation by a random sib-mating. This breeding scheme (Figure S1) is repeated for each subsequent generation until complete ﬁxation is achieved. The father-daughter backcross takes 37.06 6 7.55 genera- tions to reach complete ﬁxation, and the inbred lines contain on average 142.39 6 12.24 segments (Figure 6). This breeding scheme also takes 24.70 6 3.54 generations to 99% ﬁxation. Although the beneﬁts of father-daughter mating appear modest relative to random sib-mating, in practice they are compensated for by a reduction in generation time resulting from a mature and known fertile sire. Using this MAI breeding scheme, it was found that 99% ﬁxation can be reached in an average of 16.44 6 1.00 generations, whereas complete ﬁxation can be reached in 22.10 6 4.41 generations on average. These inbred lines have an average of 138.83 6 11.83 seg- ments. Figure 7 shows that MAI reduces the CHF among mating pairs much faster than random sib-matings. As soon as the breeding scheme is altered at the point of peak diversity, the effect is apparent. Marker-assisted inbreeding For all MAI techniques, random sib-matings were simulated until the point of peak diversity was passed. This was followed by subsequent generations of selecting the best breeding pair, until the line reached complete ﬁxation. ACKNOWLEDGMENTS We thank Fernando Pardo-Manuel de Villena and David Threadgill for their advice and comments on improving the simulator and the manuscript. In addition, we also want to thank the UNC Computa- tional Genetics research group. We modeled this reduced resolution by sampling the JH state at 1-Mb intervals. We simulated the breeding using the MAI breeding strategy discussed earlier, but modiﬁed the WSM to consider the JH state only at sample points. Furthermore, we declared lines inbred on the basis of the 1-Mb sampling (when all sample points were SS). We then inspected each declared “inbred” mouse to see if, at a base-pair resolution, all intervals were truly ﬁxed, and found them to be actually inbred only 38.3% of the time. On average we missed three nonﬁxed segments per line, and these segments were on average 3276 234 Kb. We also found that the lines were considered inbred approximately 2.5 generations earlier than MAI with complete observability. This ﬁnding implies that the inability to detect small recombinants might require additional inbreeding generations to attain the desired level of ﬁxation. Selected advanced intercrosses At the point of peak diversity, the lines had an average of 196.1 6 15.44 segments, compared with 167 segments in randomized sib-pair matings. The average number of segments in the ﬁnal inbred animals was 155.6 6 12.53. On the basis of our analysis, if genotyping is done at every generation, the lines will become inbred in approximately the same number of generations as the MAI breeding strategy but will have approximately 17 more segments per animal. This could lead to in- creased mapping resolution in the ﬁnal population. attempt to increase the number of segments on every chromosome by maximizing diversity until a designated generation is reached. This is similar to work done in self-pollinating populations to maximize map- ping resolution (Boddhireddy et al. 2009). After the designated gener- ation, the same MAI techniques as discussed previously are used to select the breeding-pairs until the line is ﬁxed. We found that it took on average 23.5 6 3.82 generations to become inbred. At the point of peak diversity, the lines had an average of 196.1 6 15.44 segments, compared with 167 segments in randomized sib-pair matings. The average number of segments in the ﬁnal inbred animals was 155.6 6 12.53. On the basis of our analysis, if genotyping is done at every generation, the lines will become inbred in approximately the same number of generations as the MAI breeding strategy but will have approximately 17 more segments per animal. This could lead to in- creased mapping resolution in the ﬁnal population. Low-resolution sampling The simulation software used in this analysis is available for download from http://sourceforge.net/p/breedingsim/. It has been adapted for many uses other than marker assisted inbreeding such as estimating the signiﬁcance of measured statistics in the developing CC (Collaborative Cross Consortium 2012). In our MAI analysis, we assumed that one is able to accurately assign genomic regions to founders at single base-pair resolution. In reality, genotyping platforms have a limited resolution with which they can ascertain a founder’s genomic sequence. This limited resolution cre- ates two main obstacles to the use of MAI methods: the possibility that small recombination intervals might escape detection, and the impre- cision with which the cross-over points of recombination can be detected. The impact of both of these limitations can, however, be modeled in a simulation. LITERATURE CITED Armstrong, N., T. C. Brodnicki, and T. P. Speed, 2006 Mind the gap: analysis of marker-assisted breeding strategies for inbred mouse strains. Mamm. Genome 17: 273–287. Aylor, D. L., W. Valdar, W. Foulds-Mathes, R. J. Buus, R. A. Verdugo et al., 2011 Genetic analysis of complex traits in the emerging Collaborative Cross. Genome Res. 21: 1213–1222. Bailey, D. W., 1971 Recombinant-inbred strains. An aid to ﬁnding identity, linkage, and function of histocompatibility and other genes. Transplan- tation 11: 325–327. Boddhireddy, P., J. L. Jannink, and J. C. Nelson, 2009 Selective Advance for Accelerated Development of Recombinant Inbred QTL Mapping Popu- lations. Crop Sci. 49: 1294. Broman, K. W., 2005 The genomes of recombinant inbred lines. Genetics 169: 1133–1146. Selected advanced intercrosses A potential shortcoming of our model is that we report the time to inbred as a function of generations, not the number of litters or calendar time required to produce enough viable offspring. However, we did perform additional simulations as- suming smaller litter sizes (two females, two males), and unbalanced sex-ratio (eight total offspring with one to seven females), and com- pared all three sets of assumptions (4-4, 2-2, 8) to the greedy approach Figure 7 CHF as a function of number of generations. This plot shows that MAI reduces the CHF among breeding pairs much faster than random sib-matings. We can see the effect as soon as the breeding scheme is modiﬁed (at the point of peak diversity). 196 \| C. E Welsh and L. McMillan 196 Taking fertility into account and prioritizing for the preservation of the lines, how do we select the ﬁnal breeders? WSM optimizes for becoming inbred in one generation, but it might be more advantageous in the early MAI generations to select for animals whose probability to become inbred in two or more generations is maximized. However, in simulations, the two-generation metric generally chooses the same breeding pairs as the single-generation model, leading to the same number of generations to achieve ﬁxation. Once lines reach small levels of residual heterozygosity, it might also be advantageous to maintain multiple breeding pairs selected to produce compatible offspring, which are more like sib-pairs than cousin-pairs. This provides more pair options, as well as a chance to compensate for uneven sex ratios or small litter sizes. Although it seems best to choose the optimal breeding pairs early on, ﬁnding good pairs near the end-game (ﬁxing the last 1%–2% of the genome) is a harder problem. The last few heterozygous regions can take several generations to ﬁx if compatible breeding pairs do not exist. attempt to increase the number of segments on every chromosome by maximizing diversity until a designated generation is reached. This is similar to work done in self-pollinating populations to maximize map- ping resolution (Boddhireddy et al. 2009). After the designated gener- ation, the same MAI techniques as discussed previously are used to select the breeding-pairs until the line is ﬁxed. We found that it took on average 23.5 6 3.82 generations to become inbred. DISCUSSION Broman, K. W., 2012 Genotype probabilities at intermediate generations in the construction of recombinant inbred lines. Genetics 190: 403–412. Through simulations, we have developed several alternatives to random sib-matings to dramatically accelerate the creation of RILs by as much as 16 generations. These include the judicious use of parental backcrossing and the selection of mating pairs based on genotypes from genome-wide SNPs. Both of these techniques, when applied after the point of peak diversity is reached, result in a negligible reduction in the number of segments. We also propose an advanced intercross variant in which MAI is applied during the early gen- erations to increase the number of haplotype segments for better mapping resolution. Buckler, E. S., J. B. Holland, P. J. Bradbury, C. B. Acharya, P. J. Brown et al., 2009 The genetic architecture of maize ﬂowering time. Science 325: 714–718. Chesler, E. J., D. R. Miller, L. R. Branstetter, L. D. Galloway, B. L. Jackson et al., 2008 The Collaborative Cross at Oak Ridge National Laboratory: develop- ing a powerful resource for systems genetics. Mamm. Genome 19: 382–389. Churchill, G. A., D. C. Airey, H. Allayee, J. M. Angel, A. D. Attie et al., 2004 The Collaborative Cross, a community resource for the genetics analysis of complex traits. Nat. Genet. 36: 1133–1137. Collaborative Cross Consortium, 2012 The genome architecture of the Collaborative Cross Mouse Genetic Reference Population. Genetics 190: 389–401. In simulation we also have the luxury of assuming uniform litter sizes and equal sex ratios, but in reality the fecundity of a RIL and the sex-balance of litters are complicating issues. As lines become more inbred, fertility generally decreases (Silver 1995). One way to address this is to use backcrosses as discussed previously. However fertility issues might override the choice of “best breeding pair.” To address this problem we calculate backups that, when used, may extend the number of generations required to achieve ﬁxation. Darvasi, A., and M. Soller, 1995 Advanced intercross lines, an experimental population for ﬁne genetic mapping. Genetics 141: 1199–1207. Eisen, E. J., 2005 The Mouse in Animal Genetics and Breeding Research. Imperial College Press, London. Flaherty, L., and V. Bolivar, 2007 Congenic and consomic strains, chapter 7 in Neurobehavioral Genetics: Methods and Applications, edited by B. Jones and P. Mormede. CRC Press, Boca Raton, FL. Edited by Lauren M. McIntyre, Dirk-Jan de Koning, and 4 dedicated Associate Editors DISCUSSION Volume 2 February 2012 \| Accelerating Inbreeding of RILs \| 197 consisting of a half diallel of two-way hybrids in Arabidopsis. Euphytica 161: 107–114. Gan, X., O. Stegle, J. Behr, J. G. Steffen, P. Drewe et al., 2011 Multiple reference genomes and transcriptomes for Arabidopsis thaliana. Nature 477: 419–423. Gibson, G., and T. F. C. Mackay, 2002 Enabling population and quantita- tive genomics. Genet. Res. 80: 1–6. Silver, L. M., 1995 Mouse Genetics. Oxford University Press, Oxford, UK. Taylor, B. A., H. Meier, and D. D. Myers, 1971 Host-gene control of C- type RNA tumor virus: inheritance of the group-speciﬁc antigen of murine leukemia virus. Proc. Natl. Acad. Sci. USA 68: 3190–3194. Kover, P. X., W. Valdar, J. Trakalo, N. Scarcelli, I. M. Ehrenreich et al., 2009 A multiparent advanced generation inter-cross to ﬁne-map quantitative traits in Arabidopsis thaliana. PLoS Genet. 5: e1000551. Threadgill, D. W., K. W. Hunter, and R. W. Williams, 2002 Genetic dis- section of complex and quantitative traits: from fantasy to reality via a community effort. Mamm. Genome 13: 175–178. Liu, E. Y., Q. Zhang, L. McMillan, F. P. de Villena, and W. Wang, 2010 Efﬁcient genome ancestry inference in complex pedigrees with inbreeding. Bioinformatics 26: i199–i207. Valdar, W., J. Flint, and R. Mott, 2006 Simulating the Collaborative Cross: power of quantitative trait loci detection and mapping resolution in large sets of recombinant inbred strains of mice. Genetics 172: 1783–1797. Markel, P., P. Shu, C. Ebeling, G. A. Carlson, D. L. Nagle et al., 1997 Theoretical and empirical issues for marker-assisted breeding of congenic mouse strains. Nat. Genet. 17: 280–284. Zhang, Q., W. Wang, L. McMillan, F. Pardo-Manuel de Villena, and D. Threadgill, 2009 Inferring genome-wide mosaic structure. Pac. Symp. Biocomput. 150–161. Mott, R., C. J. Talbot, M. G. Turri, A. C. Collins, and J. Flint, 2000 A new method for ﬁne-mapping quantitative trait loci in outbred animal stocks. Proc. Natl. Acad. Sci. USA 97: 12649–12654. Paulo, M. J., M. Boer, X. Huang, M. Koornneef, and F. van Eeuwijk, 2008 A mixed model QTL analysis for a complex cross population 198 \| C. E Welsh and L. McMillan
https://openalex.org/W1997972137	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0106453&type=printable	English	null	Monte-Carlo Modeling of the Central Carbon Metabolism of Lactococcus lactis: Insights into Metabolic Regulation	PloS one	2,014	cc-by	14,276	Abstract Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models. Citation: Murabito E, Verma M, Bekker M, Bellomo D, Westerhoff HV, et al. (2014) Monte-Carlo Modeling of the Central Carbon Metabolism of Lactococcus lactis : Insights into Metabolic Regulation. PLoS ONE 9(9): e106453. doi:10.1371/journal.pone.0106453 Editor: Ying Xu, University of Georgia, United States of America Received February 10, 2014; Accepted August 7, 2014; Published September 30, 2014 Received February 10, 2014; Accepted August 7, 2014; Published September 30, 2014 Copyright: 2014 Murabito 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. Ettore Murabito1, Malkhey Verma1, Martijn Bekker2, Domenico Bellomo3, Hans V. Westerhoff1,4,5, Bas Teusink3, Ralf Steuer6,7 1 Manchester Institute of Biotechnology, School of Chemical Engineering and Analytical Sciences (CEAS), Manchester Centre for Integrative Systems Biology (MCISB), The University of Manchester, Manchester, United Kingdom, 2 Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands, 3 Systems Bioinformatics IBIVU and Netherlands Institute for Systems Biology (NISB), VU University Amsterdam, Amsterdam, The Netherlands, 4 Synthetic Systems Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands, 5 Molecular Cell Physiology, FALW, VU University Amsterdam, Amsterdam, The Netherlands, 6 CzechGlobe - Global Change Research Center, Academy of Sciences of the Czech Republic, Brno, Czech Republic, 7 Humboldt-University Berlin, Institute for Theoretical Biology, Berlin, Germany Abstract Funding: HVW acknowledges support by the transnational program Systems Biology of MicroOrganisms (SysMO) and ERASysBio program and its funders EPSRC/ BBSRC for the following projects: R111828, MOSES project, ERASysBio, BB/F003528/1, BB/C008219/1, BB/F003544/1, BB/I004696/1, BB/I00470X/1, BB/I017186/1, EP/D508053/1, BB/I017186/1. RS acknowledges funding by the OPVK project "Local Team and International Consortium for Computational Modelling of a Cyanobacterial Cell, Reg. No. CZ.1.07/2.3.00/20.0256". As of 1.1.2013, RS is supported by the framework e:Bio of the BMBF (Fo¨rderkennzeichen 0316192). 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. * Email: ettore.murabito@manchester.ac.uk (EM); ralf.steuer@hu-berlin.de (RS) September 2014 \| Volume 9 \| Issue 9 \| e106453 The Central Metabolism of Lactococcus lactis The starting point of our analysis is a stoichiometric represen- tation of the central metabolism of Lactococcus lactis, defined here as the carbon and energy metabolism of this organism that generates most of its free-energy and C3 carbon precursors during fermentative growth. Drawing upon earlier kinetic models [3,4,7,9,15] and several available genome-scale reconstructions [5,31], a set of enzymes involved in fermentative metabolism of L. lactis was selected. A graphical overview is shown in Figure 1. The metabolic network was chosen so as to describe the main glycolytic intermediates, the ATP regeneration cycle, and the dynamics of inorganic phosphate (Pi) and redox carriers (NAD/NADH). We neglect flux through the pentose phosphate pathway, since it accounts for less than 2% of glycolytic flux [32]. Main fermentation products are lactate (LAC), ethanol (EtOH), acetate and butanediol. Stoichiometric analysis reveals that the systems has three conserved moieties, ATP/ADP, NAD/NADH, as well as conservation of a phosphate group involving 11 metabolites. The concentrations of formate (FMT) and coenzyme A (CoA) are considered constant. The stoichiometry of the network allows for either LAC as the only fermentation product, or for equimolar amounts of butanediol and EtOH or acetate and EtOH as end products. The latter branch, fermentation to acetate and EtOH, results in the highest yield of ATP per glucose consumed. Building upon these methods, we aim to elucidate and understand the regulatory properties of the central metabolism of the lactic acid bacterium Lactococcus lactis. In particular, we seek to understand how allosteric regulation, together with its associated feedback and feedforward loops, influence the control exerted by the various enzymatic steps. Making only minimal assumptions about rate equations and kinetic parameters, and data from direct experimentation, we show that it is possible to study and elucidate the control properties of a metabolic pathway. In a second step, we investigate the dynamics of a corresponding kinetic pathway model in periods of starvation and show that allosteric control and regulatory interactions are crucial to maintaining metabolic viability in times of nutrient scarcity. Our probabilistic approach directly builds upon measured properties, such as the concentrations of metabolic intermediates and flux distributions, rather than enzyme-kinetic parameters, to constrain the possible dynamics of a metabolic pathway. Introduction but also via redox (NADH) and energy carriers (ATP). Under- standing the dynamic response arising from such a complex network of interactions is often not possible through heuristic reasoning alone, but requires the construction of detailed computational models. Such kinetic models of biochemical pathways are typically built in a bottom-up approach. First, the detailed enzyme-kinetic properties of each individual step are collected, having been sourced from either dedicated experiments or from literature research and databases. Subsequently, the model is constructed and revised until a faithful in-silico representation of the pathway is obtained. Despite considerable efforts, however, the construction of such models is still hampered by the extensive data requirements necessary to parametrize each enzymatic step. Several approaches have been suggested to alleviate this problem, including the use of approximative kinetics [18,19] and Monte-Carlo sampling of reaction parameters Lactic acid bacteria (LAB) are industrially important microor- ganisms used in the fermentation of milk, meat, and certain vegetables [1,2]. LAB produce lactic acid as their main catabolic byproduct and are frequently used as starter cultures in various food-fermentation processes. LAB have a long tradition of safe use and, because the central metabolism of LAB has high activity, is an interesting target for metabolic engineering. A variety of metabolic models, detailed kinetic models as well as large-scale stoichiometric reconstructions, have been proposed, in particular for the best- studied LAB Lactococcus lactis [3–10]. Despite a relative wealth of data, however, many aspects of the fermentative metabolism of LAB are still insufficiently understood [2,11–17]. Even compar- atively small metabolic pathways, such as the glycolytic pathway of LAB, are highly interconnected not only via carbon metabolites, September 2014 \| Volume 9 \| Issue 9 \| e106453 1 PLOS ONE \| www.plosone.org Monte-Carlo Modeling of L. lactis Central Metabolism Figure 1. A pathway map of L. lactis central metabolism. The pathway involves 21 metabolic interconversions between 24 metabolites and includes three conserved moieties and two internal metabolites whose concentrations are assumed to be constant (FMT, CoA). External metabolites are indicated by light blue boxes. The inset provides an overview of the regulatory interactions. Abbreviations are defined in Text S2. doi:10.1371/journal.pone.0106453.g001 Figure 1. A pathway map of L. lactis central metabolism. The pathway involves 21 metabolic interconversions between 24 metabolites and includes three conserved moieties and two internal metabolites whose concentrations are assumed to be constant (FMT, CoA). External metabolites are indicated by light blue boxes. Introduction The inset provides an overview of the regulatory interactions. Abbreviations are defined in Text S2. doi:10.1371/journal.pone.0106453.g001 regulatory properties that enable large-scale metabolic networks to function reliably in uncertain environments. [20,21]. As one of the first applications of a Monte-Carlo sampling procedure accounting for the uncertainty in kinetic parameters, Wang et al. [22] employed statistical tools for the identification of the rate-controlling steps in a model of yeast glycolysis. Shortly afterwards, the formalism of generalized modeling [23] was extended to study the dynamic properties of models of metabolic pathways by using a general parametric representation of their Jacobian matrix [24,25]. Later, these methods were adopted and modified by several other groups [26–30]. September 2014 \| Volume 9 \| Issue 9 \| e106453 Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism The resulting distribution of flux control coefficients allows us to assess, qualitative and fairly independent of the precise values of the enzyme kinetic parameters, the potential effects of perturba- tions in enzyme levels on the system flux at the metabolic state. Most importantly, the distributions of the control coefficients were not arbitrarily. Rather, the results shown in Figure 3 exhibit strong patterns and, in accordance with earlier studies [37], indicate that qualitative knowledge of model properties can already result in well-constrained predictions, even when individual parameters are only poorly constrained. With respect to the interpretation of the distributions of scaled flux control coefficients, three properties are of particular interest: (i) The width of the distribution. Narrow distributions indicate control coefficients that do not change appreciably due to parameter sampling, whereas broad distribu- tions indicate that the precise value of the coefficient is highly dependent on parameter values. (ii) The median of the distribu- tion. For several reactions, the respective distributions are appreciably shifted away from zero towards either negative or positive values. (iii) The dominant sign of the control coefficients. Often, it is only of interest whether an increase in enzyme amount will result in an increase or decrease of a specific flux in the network, irrespective of the exact magnitude. To this end, Figure 4 provides the probabilistic sign distribution of flux control coefficients, grey-scale coded to indicate the percentage of sampled control coefficients that lie on the positive semi-axis. therefore the concentration of free inorganic phosphate (Pi) is an (inverse) measure of the amount of the glycolytic intermediates plus ATP. Further reactions that are under regulatory control include the pyruvate dehydrogenase (PDH), as well as the alcohol dehydrogenases (ADH). A complete list of all regulatory interac- tions is summarized in Figure 1 and given in Text S1. Following the workflow outlined above, a kinetic rate equation was assigned to each reaction step. In good agreement with earlier models [3], reaction rates were assumed to follow conventional Michaelis- Menten kinetics. Regulatory interactions were implemented as multiplicative factors to the rate equations, using Hill-type kinetics [33]. Detailed reaction equations are provided in Text S2. Subsequently, equilibrium constants were assigned to all steps, using values obtained by the group contribution method [34]. Probabilistic Analysis of Control Properties To quantify the extent to which a system property, such as a specific flux or metabolite concentration, is influenced by a specific enzymatic step, we make use of the concepts of Metabolic Control Analysis (MCA). The control coefficient CP i is defined as the relative change of a steady-state property P (the response of the system) divided by the small relative modulation of the activity of an enzyme i that caused that change [35,36]. In the following, we mostly focus on the flux control coefficients CJ, where the system properties P represent the fluxes within central metabolism. These control coefficients can only be evaluated for a fully characterized system, that is, their evaluation requires knowledge of the stoichiometry, as well as of all kinetic parameters or elasticity coefficients. Because neither all kinetic parameters nor all elasticity coefficients were known, we employed a Monte-Carlo scheme to iteratively sample enzyme-kinetic parameters, in particular Michaelis-Menten constants, to obtain insight into what we shall call the probabilistic distribution of control coefficients. To determine the respective intervals for the Monte-Carlo sampling of parameters, we make use of the fact that the control coefficients are essentially determined by the scaled elasticities, the logarithmic partial derivatives of the rate equations with respect to metabolic compounds. In a first approximation, these values are determined by the ratio of the Michaelis-Menten constants to their respective concentration [24,28]. Motivated by the saturation properties of enzymatic rate equations, and making use of knowledge of the metabolic phenotype, we assume each Michaelis-Menten constant to be distributed around the concentration value of its associated metabolic compound. The width of the sampling interval is varied to test for robustness of the results; in the following all results are reported for a sampling interval spanning both, one order of magnitude above and below the respective concentration. After choosing all Michaelis constants, the forward velocity Vmax was adjusted so as to ensure that the reaction rate was not affected by the chosen constant. Applying this Monte-Carlo procedure, we obtain a distribution, rather than a single value, for each flux control coefficient. We emphasize that our approach ensures that each set of parameters is sampled such that it is consistent with the experimentally observed state. That is, each sampled set of paremeters indeed gives rise to the experimentally observed state. All subsequent evaluations are specific to this state. Probabilistic Analysis of Control Properties Prior to the calculation of control coefficients, the stability of the model is tested for each sampled set of parameters and only sets corresponding to a stable phenotype are retained (<92% of all sampled instances). Our workflow is outlined in Figure 2. Monte-Carlo Modeling of L. lactis Central Metabolism The metabolic state was based on values for extracellular fluxes and metabolites specifically measured for this study, supplemented with values from the literature. We consider a state with high external glucose (20 mM) and high-flux activity, see also Text S1. The Central Metabolism of Lactococcus lactis We demonstrate that (i) the control coefficients of biochemical network models show intelligible patterns and trends that are accessible without detailed knowledge of enzyme-kinetic parameters; (ii) the regula- tory structure of a biochemical network models has profound effects on the possible dynamics that are largely independent of specific kinetic parameters; and (iii) more specifically, that the topology of the regulation network is instrumental to ensure the stability of an observed state and to enable the patwhay to survive periods of starvation. We expect that our methodology will be of high utility to elucidate and understand the dynamic and Beyond the reaction stoichiometries, our model incorporates the currently known regulatory features found in L. lactis central metabolism. Fructose 1,6-bisphosphate (FBP) activates the forma- tion of pyruvate (PYR) by the pyruvate kinase (PYK), activates the conversion from PYR to LAC by the lactate dehydrogenase (LDH), and inhibits the phosphotransferase system (PTS). A potentially important regulator is the concentration of free phosphate. In our model, the total pool of phosphate is constant, September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 2 September 2014 \| Volume 9 \| Issue 9 \| e106453 The Control of Flux Figures 3 and Figure 4 show the resultant distributions of control coefficient based on the sampling procedure described above. All sampled sets of parameters share the common property that they are consistent with the experimentally observed metabolic state of the pathway. However, other than that, we have no knowledge as to whether an actual control coefficient is close to the median value of the respective distribution, or whether evolutionary pressure has selected for a value at the extreme fringe of this distribution. Clearly, for each control coefficient, both scenarios are possible. To provide a meaningful interpretion of these results, we therefore proceed in two steps. First, we asses to what extend the observed distributions correspond to our intuitive assumptions about control within the pathway. Overall, the pattern of flux control coefficients shown in Figures 3 and 4 are indeed in good agreement with conventional assumptions about the distribution of control within the pathway. For example, the glycolytic enzymes (PGI to ENO) predominantly show positive control over the glycolytic flux, with more than 80% of the sampled control coefficients having positive values. Reactions close to equilibrium, such as PGM or ENO, typically also have narrow distributions around zero, indicating that they typically exert little control on the fluxes through the system, irrespective of precise parameter values. Indeed, the average standard deviation of the scaled control coefficients correlates with the distance from equilibrium of the respective reaction, as shown in Figure 5, and in accordance with theoretical considerations [38]. Other straightforward results include the predominantly negative control the LDH exerts upon the competing branches involved in the production of ethanol, acetate and butanediol. However, other properties of the distributions shown in Figure 3 and Figure 4 are less straightforward to explain. For example, the predominantely negative control that the enzymes involved in acetate production (PTA and ACK) exert upon the glycolytic flux. Although increasing activity of PTA and ACK might be expected to pull more flux through the upper part of glycolysis, the flux control coefficients indicate that this is, for the vast majority of parameters, not the case. Rather, increasing the activity of the acetate branch September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 3 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 2. Probabilistic Modeling of L. lactis. The topological properties of the pathway, including the stoichiometry and all known regulatory interactions, are assembled. The Control of Flux The analysis is then based on knowledge of a metabolic state of the system, as defined by a steady-state flux distribution and a set of associated thermodynamically consistent metabolite concentrations. Based on this information, the state-specific dynamic properties of the corresponding pathway model are evaluated. Of particular interest are control coefficients, the role of regulatory interactions, as well as the dynamic response to periods of starvation. doi:10.1371/journal.pone.0106453.g002 Figure 2. Probabilistic Modeling of L. lactis. The topological properties of the pathway, including the stoichiometry and all known regulatory interactions, are assembled. The analysis is then based on knowledge of a metabolic state of the system, as defined by a steady-state flux distribution and a set of associated thermodynamically consistent metabolite concentrations. Based on this information, the state-specific dynamic properties of the corresponding pathway model are evaluated. Of particular interest are control coefficients, the role of regulatory interactions, as well as the dynamic response to periods of starvation. doi:10.1371/journal.pone.0106453.g002 Figure 3. Probabilistic distribution of flux control coefficients. Shown is the distribution of the scaled flux control coefficients corresponding to the pathway model of L. lactis central metabolism given in Figure 1. Each plot corresponds to the interval [21,1] on the abscissa. The diagram in the i th column and on the j th row gives the distribution of the control coefficient quantifying the extent to which enzyme i controls the flux through the reaction j. Each distribution provides information about the magnitude and uncertainty of one control coefficient. Narrow distributions indicate control coefficients that do not change appreciably due to parameter sampling, whereas broad distributions indicate that the precise value of the coefficient is more strongly dependent on parameter values. The corresponding sign distribution is shown in Figure 4. doi:10.1371/journal.pone.0106453.g003 September 2014 \| Volume 9 \| Issue 9 \| e106453 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 4 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 4. Probabilistic sign distribution of flux control coefficients. Grey-scale representation of the sign distribution of the flux control coefficient shown in Figure 3. The shade of the entry represents the percentage of the calculated control coefficients that are positive. Dark colors correspond to a distribution of flux control coefficients that lies predominantly on the negative semiaxis, whereas light colors indicate that the sampled control coefficients are predominantely positive. For example, for this metabolic phenotype, an increase of the enzyme PYK will for almost all sampled parameter values result in decreased flux through the LDH reaction as indicated by the dark circle in the row for LDH and the column for PFK. doi:10 1371/journal pone 0106453 g004 Figure 4. Probabilistic sign distribution of flux control coefficients. Grey-scale representation of the sign distribution of the flux control coefficient shown in Figure 3. The shade of the entry represents the percentage of the calculated control coefficients that are positive. Dark colors correspond to a distribution of flux control coefficients that lies predominantly on the negative semiaxis, whereas light colors indicate that the sampled control coefficients are predominantely positive. For example, for this metabolic phenotype, an increase of the enzyme PYK will for almost all sampled parameter values result in decreased flux through the LDH reaction as indicated by the dark circle in the row for LDH and the column for PFK. doi:10.1371/journal.pone.0106453.g004 Figure 5. The width of the distribution of control coefficients correlates with distance from equilibrium. Shown is the average standard deviation of the sampled flux control distribution as a function of displacement C from equilibrium of the respective enzyme. Reactions close to equilibrium (C close to unity) typically have narrow distributions of flux control coefficients, centered at zero, indicating they can only exert little control over the flux through the system. Contrary, reactions far from equilibrium (C%1) exhibit broad distribu- tions, indicating a potential, but no necessity, for high control coefficients. For definitions see Materials and Methods. doi:10.1371/journal.pone.0106453.g005 prevents the regeneration of NAD required by the glyceraldehyde- 3-phosphate-dehydrogenase (GAPDH), consequently slowing down the glycolytic pathway. We emphasize that the probabilistic distributions shown in Figures 3 and 4 depict control properties of the pathway model for values of kinetic parameters that are consistent with the experimentally observed state, but are otherwise random. Clearly, the peaks of these distributions do not necessarily correspond to the actual control profile of the organism. Selection pressure during evolution might have easily led to parameters on the fringes or even outside of the range in which we sampled, precisely because the organism’s requirements were different from what arises as ‘typical’ from random sampling. We therefore must compare the distributions shown in Figures 3 and Figure 4 with known and empirically obtained control coefficients in L. lactis [39–42]. In particular, control coefficients that take values far from the median value of the respective distribution might point to additional evolutionary pressure, or errors in the topology of the pathway, and therefore provide valuable information for further analysis. Figure 5. The width of the distribution of control coefficients correlates with distance from equilibrium. Shown is the average standard deviation of the sampled flux control distribution as a function of displacement C from equilibrium of the respective enzyme. Reactions close to equilibrium (C close to unity) typically have narrow distributions of flux control coefficients, centered at zero, indicating they can only exert little control over the flux through the system. Contrary, reactions far from equilibrium (C%1) exhibit broad distribu- tions, indicating a potential, but no necessity, for high control coefficients. For definitions see Materials and Methods. doi:10.1371/journal.pone.0106453.g005 Using a series of mutants with altered GAPDH activity, Solem et al. (2003) [40] determined that changes in GAPDH activity had virtually no effect on glycolytic flux in growing as well as nongrowing cells. Control of Glycolytic Flux by PTS As a rather counter-intuitive finding, the distribution of control coefficients indicates that for most sets of parameters the PTS would exert a negative control on flux through the pathway. This finding is in contrast to our expectation that an increase in PTS activity results in higher glucose uptake, hence an increased flux through glycolysis. These results emphasize that the control coefficients are indeed systemic properties of the pathway and may exhibit non-intuitive patterns, depending on the specific metabolic phenotype and details of the pathway stoichiometry and regula- tion. To verify that the non-intuitive distribution of the influence of the glucose transport upon the glycolytic flux is indeed a true feature of the pathway model, we varied the maximal velocity (Vmax) of the PTS using an explicit kinetic model. The resulting flux is shown in Figure 6 for a specific set of reference parameters. Repeating the analysis for a large number of sampled parameters yielded similar results. At the specified metabolic state, the slope of the curve representing glucose uptake as a function of Vmax is almost always negative. The universality of the negative slope indicates that the control profile of the network model is indeed strongly dependent on the specific metabolic phenotype at which the probabilistic control profile is evaluated. That is, the experimentally observed metabolic state itself shapes the resultant distribution of control coefficients. To verify this assertion, we evaluated the control profile of a second metabolic state, corresponding to a situation with low external glucose. The results are provided and discussed in Text S2. In this case, the control of the PTS system on glycolytic flux is predominantly positive. Repeating the Monte-Carlo sampling of kinetic parameters in the absence of regulatory interactions, we first note that the percentage of stable models, as evaluated by an analysis of the largest real part of the eigenvalues of the Jacobian, drops to about 83% of sampled instances, as compared to 92% for the regulated system considered above. The analysis was repeated for the second metabolic state, characterised by low external glucose, in the Text S2, showing that a similar drop in average stability is again associated with the absence of regulatory interactions. Figure 7 shows the probabilistic distribution of the scaled flux control coefficients in the absence of metabolic regulation, the corre- sponding sign distribution is shown in Figure 8. Likewise, in a series of studies, the enzymes PFK, PYK, and LDH, encoded together on the las operon, were shown to have no significant control on the glycolytic flux in PLOS ONE \| www.plosone.org September 2014 \| Volume 9 \| Issue 9 \| e106453 5 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 6. Dependency of the glycolytic flux on the maximal activity of the glucose transporter (PTS). Shown is the glucose uptake as a function of Vmax, evaluated using a kinetic model with a representative set of the sampled parameters. The black dot indicates the reference state. doi:10.1371/journal.pone.0106453.g006 exponentially growing cells [39,41]. These findings are in agreement with the rather narrow distributions, centered at zero, of the respective probabilistic control coefficients. Furthermore, Koebmann et al. (2005) [41] showed that the enzyme LDH has a strong negative control over the flux to mixed acids and formate formation, whereas PYK has a strong negative control over these fluxes – again in agreement with the respective distributions. PFK was found to have no control on either acetate or lactate flux, again corresponding to the respective distribution. An interesting case is the control of ATP consuming-processes outside of the pathway (ATPase) on the glycolytic flux. The respective probabi- listic distribution indicates a large potential for control, as manifested by the very broad distribution of the corresponding flux control coefficients. Such a high level of control by ATP consuming reactions outside of the pathway was indeed reported recently [17], and a similar high control of demand for ATP on glycolytic flux has been observed for E. coli [43]. From the introduction of an uncoupled ATPase activity, however, Koeb- mann et al. (2002) [39] concluded that the flux control by ATP demanding processes was close to zero over a range of ATP/ADP ratios. Both scenarios are consistent with the broad distribution shown in Figure 3. We emphasize that in the following, despite the good agreement with experimental data, we do not interpret the probabilistic distributions as a likelihood for the actual control coefficients. Rather the distributions indicate ranges of control coefficients that are typical for the metabolic state, given our sampling procedure, whereas deviations provide a highly valuable starting point for further analysis. We note, however, that as yet most empirically determined control coefficients available for L. lactis do seem to be consistent with the bulk of the respective distribution. Figure 6. Control of Glycolytic Flux by PTS The absence of regulation affects the control profile in terms of the dominant sign of the control coefficients, as well as the amplitude and width of their distribution. A comparison between Figures 3 and 7 shows, for example, that the control of PGI and PFK over the glycolytic flux is predominantly positive in the regulated system, but almost always negative in the unregulated systems. Differences are also Dependency of the glycolytic flux on the maximal activity of the glucose transporter (PTS). Shown is the glucose uptake as a function of Vmax, evaluated using a kinetic model with a representative set of the sampled parameters. The black dot indicates the reference state. doi:10.1371/journal.pone.0106453.g006 Each instance of sampled parameters included randomly selected values for all regulatory interactions. To delineate the impact of metabolic regulation, we subsequently removed all regulatory interactions and repeated the analysis to pinpoint specific differences between the regulated and unregulated network model. In particular, our re-scaling and sampling procedure allows us to analyze both systems at an identical steady state and therefore enables a direct comparison between both scenarios. We note that this approach is different to a simple change of parameters within a kinetic model, for example by decreasing the value of a certain feedback parameter. While such a change would indeed modify the feedback properties, it would also alter the metabolic state of the pathway and thereby complicate a direct comparison of systemic properties between both scenarios. In contrast, here, we assume that the metabolic phenotype, the set of concentrations and fluxes, has evolved to satisfy the particular functional requirements of the cell. By removing regulatory interactions while keeping the metabolic state unchanged, we ask the question how this evolved state would typically behave in the absence of metabolic regulation, whilst fulfilling the same metabolic function. As is demonstrated below, this approach allows us to perform a detailed assessment of the role of individual regulatory interac- tions. September 2014 \| Volume 9 \| Issue 9 \| e106453 Monte-Carlo Modeling of L. lactis Central Metabolism The probability of the system to have recovered from starvation also depends on the duration of the Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism recorded for the regulation of the glycolytic flux by AS, ACLACD and BDH, which is negative in the regulated case and evenly distributed between negative and positive values in the absence of regulation. A strong difference between the probabilistic control profiles is also observed for the ATPase, representing general ATP demand outside of the pathway. In the presence of regulation, the distribution of the control coefficients of the ATPase on the glycolytic flux is rather broad, indicating a high potential for control, and symmetrically distributed around zero. In contrast, in the absence of regulation, the distribution is shifted to higher values, with a median value close to unity. Consequently, in the absence of metabolic regulation, the pathway model exhibits a strong sensitivity with respect to ATP demand for almost all possible sets of parameters. Similar differences are observed for the possible control of PYK on the glycolytic flux. The respective distribution is significantly narrower in the presence of metabolic regulation, indicating less potential for control but an increased robustness of the flux with respect to perturbations in enzyme levels. Similar differences in widths and signs were observed for the second metabolic state as discussed in Text S2. numeric simulations for 103 instances of sampled parameters in the presence and absence of regulatory interactions. After a period of t~1 min, the external glucose was lowered from its original value of [GLCx] = 20 mM to a value of [GLCx] = 0:1 mM. The pathway model then adapted to the new conditions and converged to a new metabolic state. At a simulation time of t~10 min, the external glucose was restored to its original value [GLCx] = 20 mM. As expected, and shown in Figure 9, withdrawal of external glucose resulted in a quick drop of intracellular metabolites. Typical time-courses of selected model instances are provided in the Text S2. Due to the autocatalytic nature of the glycolytic pathway, the concentration of ATP typically first exhibits a slight increase, corresponding to the cessation of ATP utilization in the upper part of glycolysis. Subsequently, the concentration of ATP, and likewise of other metabolites, drops to a new steady-state, characterized by a significantly decreased ATP concentration. The average value of ATP concentration during starvation is 0:5+0:5 mM for regu- lated systems versus 0:11+0:07 mM in the absence of regulation. Monte-Carlo Modeling of L. lactis Central Metabolism Both scenarios exhibit drastically different behavior after external glucose is restored to its original value. For the regulated system, the distribution of ATP concentration after a suitable recovery time is clearly bimodal (Figure 9A). Approximately 54% of all models recover to the original metabolic state (542 of 1000 instances tested) and the ATP level reaches its original value (&4 mM). In the absence of regulation, the probability of the system to recover drops significantly. More than 95% of the simulations correspond to a failure in restoring the original concentration of ATP. The probability of the system to have recovered from starvation also depends on the duration of the numeric simulations for 103 instances of sampled parameters in the presence and absence of regulatory interactions. After a period of t~1 min, the external glucose was lowered from its original value of [GLCx] = 20 mM to a value of [GLCx] = 0:1 mM. The pathway model then adapted to the new conditions and converged to a new metabolic state. At a simulation time of t~10 min, the external glucose was restored to its original value [GLCx] = 20 mM. As expected, and shown in Figure 9, withdrawal of external glucose resulted in a quick drop of intracellular metabolites. Typical time-courses of selected model instances are provided in the Text S2. Due to the autocatalytic nature of the glycolytic pathway, the concentration of ATP typically first exhibits a slight increase, corresponding to the cessation of ATP utilization in the upper part of glycolysis. Subsequently, the concentration of ATP, and likewise of other metabolites, drops to a new steady-state, characterized by a significantly decreased ATP concentration. The average value of ATP concentration during starvation is 0:5+0:5 mM for regu- lated systems versus 0:11+0:07 mM in the absence of regulation. Both scenarios exhibit drastically different behavior after external glucose is restored to its original value. For the regulated system, the distribution of ATP concentration after a suitable recovery time is clearly bimodal (Figure 9A). Approximately 54% of all models recover to the original metabolic state (542 of 1000 instances tested) and the ATP level reaches its original value (&4 mM). In the absence of regulation, the probability of the system to recover drops significantly. More than 95% of the simulations correspond to a failure in restoring the original concentration of ATP. The Impact of Metabolic Regulation Up to this point, the system had been evaluated in the presence of metabolic regulation additional to substrate/product effects. September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 6 Starvation and Dynamic Recovery To investigate the impact of metabolic regulation on the dynamic properties of the system, we have to go beyond the steady-state control coefficients and consider explicit time-courses of the system. In particular, we are interested in the dynamic response to periods of starvation and in the subsequent recovery when external nutrients are replenished. To this end, we set up an ensemble of models, each at the steady-state defined by the metabolic state considered above. Subsequently, we ran explicit Figure 7. Probabilistic distribution of flux control coefficients in the absence of metabolic regulation. Same as Figure 3 except for the absence of metabolic regulation. Any diagram refers to the control of one flux (i.e. through the step indicated to the left of the row) by one enzyme (i.e. the enzyme indicated above each diagram corresponds to the interval [-1,1] on the abscissa. doi:10.1371/journal.pone.0106453.g007 Figure 7. Probabilistic distribution of flux control coefficients in the absence of metabolic regulation. Same as Figure 3 except for the absence of metabolic regulation. Any diagram refers to the control of one flux (i.e. through the step indicated to the left of the row) by one enzyme (i.e. the enzyme indicated above each diagram corresponds to the interval [-1,1] on the abscissa. doi:10.1371/journal.pone.0106453.g007 September 2014 \| Volume 9 \| Issue 9 \| e106453 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 7 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 8. Probabilistic sign distribution of flux control coefficients in the absence of regulation. The shade of the entry represents the percentage of the calculated control coefficients that are positive. Dark colors correspond to a distribution of flux control coefficients that lies predominantly on the negative semiaxis, whereas light colors indicate that the sampled control coefficients are predominantly positive. doi:10.1371/journal.pone.0106453.g008 Figure 8. Probabilistic sign distribution of flux control coefficients in the absence of regulation. The shade of the entry represents the percentage of the calculated control coefficients that are positive. Dark colors correspond to a distribution of flux control coefficients that lies predominantly on the negative semiaxis, whereas light colors indicate that the sampled control coefficients are predominantly positive. doi:10.1371/journal.pone.0106453.g008 starvation (Ts) and recovery time (Tr). Figure 10 shows the percentage of recovered systems as a function of Ts and Tr for both scenarios. Starvation and Dynamic Recovery In the presence of regulation, the probability to recover does not seem to depend appreciably on the duration of the starvation and recovery time. In the absence of regulation, however, a longer starvation time strongly decreases the proba- bility of recovery. In addition, unregulated systems tend to recover more slowly, as also observed in the individual time-courses provided in the Text S2. These results are in agreement with the previous observation that regulatory interaction can accelerate response times of simple biological network motifs [44,45]. FBP as an activator of PYK, followed by free inorganic phosphate (Pi) as an inhibitor of the same reaction. A slight, but significant, increase is also observed for FBP as an inhibitor of PTS. The results are in good agreement with previous work on metabolic stability using minimal pathway models [2], such as the study of Voit et al. [15] who investigate the feedforward activation system in L. lactis using a six variable model with generalized mass action kinetics. Likewise, recent work on regulation of PEP utilization in E. coli also emphasized the importance of FBP as a regulator allowing rapid adaptation to changing environmental conditions [46]. September 2014 \| Volume 9 \| Issue 9 \| e106453 FBP as a regulator of PYK and PTS The importance of allosteric regulation of PYK by FBP for metabolic functioning, in particular for the levels of ATP and glycolytic intermediates, is widely recognized and the regulatory mechanisms is present in almost all glycolytic pathways [2,46]. Our results suggest that for L. lactis at the metabolic state studied here, a major role of the activation of PYK by FBP is to prevent an irreversible metabolic collapse during brief times of starvation. Indeed, as argued previously [2,15], the mechanism introduces a safety valve for the utilization of PEP. In the absence of regulation, and after the withdrawal of glucose, PEP is continually converted to pyruvate and short-lived ATP until all resources are depleted. However, in the presence of regulation, a decrease of FBP concomitantly decreases the utilization of PEP by PYK. Since no external glucose is present, utilization of PEP by PTS is likewise diminished. Hence a substantial amount of PEP is retained in the To investigate the role of regulation in the response to periods of starvation and the subsequent metabolic recovery in more detail, we tested the response of the system in the presence of individual regulation mechanisms. To do so, we performed the aforemen- tioned parameter sampling on models of the pathway endowed with only a single regulatory interaction. The percentage of models that recover after a period of starvation strongly depends on the specific regulatory interaction. Table 1 lists the percentage of recovering models for each individual regulation mechanism, along with the percentage of stable Jacobians for the respective system. Most of the regulatory mechanisms do not have any appreciable effect on the ability of the system to recover from starvation. Three of the ten tested regulation mechanisms, however, result in a significantly increased probability to recover after a period of starvation. The strongest increase is observed for September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 8 Monte-Carlo Modeling of L. lactis Central Metabolism Figure 9. Metabolic recovery after periods of starvation. Starting at the defined metabolic state, external glucose is lowered from 20 mM to 0.1 mM at time t = 1 min, mimicking a brief period of starvation. At time t = 10 min external glucose is restored to its orginal value. The upper panel shows a histogram of intracellular ATP after a recovery period at time t = 100 min. FBP as a regulator of PYK and PTS For the regulated system (A), approximately 54% of all models recover to the initial metabolic state (542 of 1000 instances tested), whereas in the absence of regulation the probability of recovery is below 5% (39 of 1000 instances tested). The lower panel shows the median of the time-course, with .25 and 0.75 quantiles included as dashed lines. For the regulated system, the time-course is split between recovering and non-recovering instances. doi:10.1371/journal.pone.0106453.g009 Figure 9. Metabolic recovery after periods of starvation. Starting at the defined metabolic state, external glucose is lowered from 20 mM to 0.1 mM at time t = 1 min, mimicking a brief period of starvation. At time t = 10 min external glucose is restored to its orginal value. The upper panel shows a histogram of intracellular ATP after a recovery period at time t = 100 min. For the regulated system (A), approximately 54% of all models recover to the initial metabolic state (542 of 1000 instances tested), whereas in the absence of regulation the probability of recovery is below 5% (39 of 1000 instances tested). The lower panel shows the median of the time-course, with .25 and 0.75 quantiles included as dashed lines. For the regulated system, the time-course is split between recovering and non-recovering instances. doi:10.1371/journal.pone.0106453.g009 system, which allows glucose uptake and regeneration of ATP as soon as external glucose is restored to its pre-starvation levels. Figure 11 shows the time-course of the median of the concentra- tions of FBP, ATP, and PEP following the withdrawal of external glucose at t~1 min for models that include the activation of PYK by FBP. As expected, after withdrawal of glucose, the concentra- tion of PEP quickly rises and attains a new steady state – system, which allows glucose uptake and regeneration of ATP as soon as external glucose is restored to its pre-starvation levels. Figure 11 shows the time-course of the median of the concentra- tions of FBP, ATP, and PEP following the withdrawal of external glucose at t~1 min for models that include the activation of PYK by FBP. As expected, after withdrawal of glucose, the concentra- tion of PEP quickly rises and attains a new steady state – interestingly with a median concentration similar to the value before starvation. With the restoration of external glucose at t~10 min, PEP undergoes a quick drop, fuelling the uptake of glucose, and the subsequent production of ATP. FBP as a regulator of PYK and PTS The percentage of the recovering systems is always relative to the instances of stable systems tested. doi:10 1371/journal pone 0106453 t001 The table gives the percentage of stable instances of the Jacobian in the presence of a single regulation mechanisms (83% in the absence of regulation) and the probability of metabolic recovery (4% in the absence of any regulation). The percentage of the recovering systems is always relative to the instances of stable systems tested. d i 10 1371/j l 0106453 t001 stable instances of the Jacobian in the presence of a single regulation mechanisms (83% in the absence of regulation) and the 4% in the absence of any regulation). The percentage of the recovering systems is always relative to the instances of stable systems systems that fail to recover after the external glucose has been restored to its original value must at least possess one additional stable state for the respective concentration of external glucose, that is, the systems must be at least bistable. To test for this hypothesis, we varied the concentration of external glucose and progressively decreased the concentration from the initial 20 mM to 0:1 mM. For each level of external glucose, the respective systems were allowed to relax to a new steady state. Subsequently, the external glucose was progressively increased again, back to the original value of 20 mM. Two typical examples are shown in Figure 12. Indeed, bistability and hysteresis was observed for all non-recovering systems. For recovering systems, in almost all cases (approximately 99%) no hysteresis was observed. A small subset of recovering systems (approximately 1%), however, also exhibited hysteresis. In this case the hysteresis loop is usually fully contained within the considered interval of external glucose and the system is monostable for an external glucose concentration of 20 mM. In corresponding plot and its discussion is provided the Text S2. We note that a similar reasoning might also hold in the absence of a PTS. In this case, the substrate ATP is required for the hexokinase and PFK in upper glycolysis, which is provided by the PYK reaction. The functional role of the FBP-mediated negative feedback on the PTS is more difficult to delineate and has received only little attention so far [2]. While the feedback also contributes to metabolic stability, its quantitative effect is rather small. Functionally, the regulation constitutes a traditional negative feedback loop, providing a mechanism for pathway homeostasis. FBP as a regulator of PYK and PTS The time-course shown in Figure 11 is in strong contrast to the situation in the absence of feedforward regulation, even when selecting for parameters that likewise allow for metabolic recovery. The Figure 10. Recovery as a function of starvation time. Shown is the percentage of model instances that recovered to the original metabolic state after a starvation time Ts and a recovery time Tr in the presence (left panel) and absence (right panel) of regulation. We emphasise the different scales on the z-axis on both panels. In the presence of regulation, the probability to recover does not appreciably depend on length of starvation and recovery time. In the absence of regulation a longer starvation time decreases the probability to recover. doi:10.1371/journal.pone.0106453.g010 Figure 10. Recovery as a function of starvation time. Shown is the percentage of model instances that recovered to the original metabolic state after a starvation time Ts and a recovery time Tr in the presence (left panel) and absence (right panel) of regulation. We emphasise the different scales on the z-axis on both panels. In the presence of regulation, the probability to recover does not appreciably depend on length of starvation and recovery time. In the absence of regulation a longer starvation time decreases the probability to recover. doi:10.1371/journal.pone.0106453.g010 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 9 Monte-Carlo Modeling of L. lactis Central Metabolism Table 1. Percentage of recovering systems in the presence of individual regulation mechanisms. REACTION REGULATOR TYPE PERCENTAGE STABLE STATES PERCENTAGE RECOVERING SYSTEMS PTS FBP Inhibitor 87% 11% GAPDH NADH Inhibitor 81% 4% PYK Pi Inhibitor 97% 26% PYK FBP Activator 87% 51% PDH DHAP Inhibitor 83% 4% PDH GAP Inhibitor 83% 3% ADH ATP Inhibitor 82% 3% LDH Pi Inhibitor 84% 2% LDH FBP Activator 83% 2% LDH NADH/NAD Inhibitor 83% 3% The table gives the percentage of stable instances of the Jacobian in the presence of a single regulation mechanisms (83% in the absence of regulation) and the probability of metabolic recovery (4% in the absence of any regulation). The percentage of the recovering systems is always relative to the instances of stable systems tested. doi:10.1371/journal.pone.0106453.t001 The table gives the percentage of stable instances of the Jacobian in the presence of a single regulation mechanisms (83% in the absence of regulation) and the probability of metabolic recovery (4% in the absence of any regulation). Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism very rare cases, the system also returned to its original state, despite hysteresis and the existence of a second stable state with low metabolic activity. In these cases, the sudden withdrawal and reestablishment of external glucose may induce dynamic transients that allow the system to leave the lower state. Figure 12 shows a non-recovering system in the absence of regulation (panels A and B), as well as a recovering system in the presence of regulation (panels C and D). We note that hysteresis was tested numerically and therefore the existence of further metabolic states cannot be excluded. However, the numeric results obtained from the tested 103 instances clearly showed that recovery was predominantely due to the monostability of the steady state for the respective concentration of the external glucose. Furthermore, the absence of bistability was clearly linked to the regulatory structure of the system, almost irrespective of the precise paramater values. Our results therefore imply that the regulatory structure indeed has a structural influence on the possible bifurcations of the pathway independent of a specific fine-tuned set of parameters. dynamic properties of a corresponding pathway model. To this end, our first aim was to demonstrate that the distributions of control coefficients obtained from conditional Monte-Carlo sampling is highly structured. Based on a sampling scheme, such that all considered sets of parameters are in accordance with the experimentally observed state, the resulting patterns of control coefficients were consistent with many well-known control control principles of the pathway. Given these findings, two aspects of our study must be emphasized: First, we did not conduct a straightforward Monte- Carlo study such that the kinetic parameters were drawn from a random distribution. Such an approach would likely result in a plethora of different metabolic states – most of which are totally irrelevant for the functioning of the system under any condition. Rather, our approach is based on the assertion that evolution and selective pressure can easily restrict the metabolic state, and hence the parameters, to values that are beneficial for the organism’s survivial, even if these parameter sets represent only an exceedingly small portion within parameters. Therefore, we restrict the sampling to an evolved metabolic state, such that all sampled parameters are consistent with this experimentally observed state. Monte-Carlo Modeling of L. lactis Central Metabolism The underlying assumption is that the primary function of a metabolic pathway is embodied in its flux and, to a lesser extend, in the set of metabolite concentrations. It is these values that are the targets of selective pressure, not the kinetic parameters per se. We therefore seek to study how the dynamic properties of the pathway differ for different potential sets of kinetic parameters that are equally suited to support the observed steady-state flux. Such an approach allows us to straightforwardly Metabolic Collapse and Hysteresis As demonstrated, the regulatory structure of the glycolytic pathway is crucial to allow for metabolic recovery after periods of starvation. This raises the question about the detailed dynamic mechanisms through which recovery is achieved. By definition, Figure 11. Time-courses of intermediate metabolites. The median of the concentration of FBP, ATP, and PEP following a withdrawal of external glucose at t~1 min in models that include activation of PYK by FBP is shown. After withdrawal of glucose, the concentration of PEP quickly rises and attains a new steady state. With the restoration of external glucose at t~10 min, PEP undergoes a quick drop, fuelling glucose uptake and subsequent production of ATP. The corresponding figure for systems that lack regulatory interactions but are nonetheless able to recover from periods of starvation is discussed in the Text S2. doi:10.1371/journal.pone.0106453.g011 Figure 11. Time-courses of intermediate metabolites. The median of the concentration of FBP, ATP, and PEP following a withdrawal of external glucose at t~1 min in models that include activation of PYK by FBP is shown. After withdrawal of glucose, the concentration of PEP quickly rises and attains a new steady state. With the restoration of external glucose at t~10 min, PEP undergoes a quick drop, fuelling glucose uptake and subsequent production of ATP. The corresponding figure for systems that lack regulatory interactions but are nonetheless able to recover from periods of starvation is discussed in the Text S2. doi:10.1371/journal.pone.0106453.g011 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 10 From Stoichiometry to Dynamics y y Our approach consists of a series of well-defined steps and is based upon related strategies that utilize Monte-Carlo sampling in the study of metabolic networks [22,24,26,28,30,48]. The starting point of our analysis is a stoichiometric representation of a metabolic system, as obtained either from textbook knowledge or extracted from genome-scale models of the respective organism. The stoichiometric representation is tested to allow for meaningful flux patterns, for example by an analysis of metabolic flux modes [49] or flux-balance analysis [50]. In addition to stoichiometric dependencies, we assume that the basic regulatory interactions are known. That is, for each enzymatic interconversion there may be a set of metabolites that either inhibits or activates the respective step, albeit with unknown strength. Differing from conventional bottom-up modelling, and following the definitions given in Grimbs et al. [51] and Murabito et al. [28], the subsequent analysis is then based on knowledge of a specific metabolic phenotype of the system. That phenotype is defined by a steady- state flux value for each metabolic reaction, as well as by a set of concentration values for all metabolic intermediates, and has to fulfill two prerequisites. First, its flux distribution must be consistent with the mass-balance constraint. Second, the set of concentration values must be thermodynamically consistent with the directions of the fluxes, namely that given a set of concentration values and the set of equilibrium constants, the Gibbs free energy of all reactions must be negative in the direction indicated by the phenotype’s flux distribution. To obtain insights into kinetic properties, each reaction rate is then assigned a rate equation that specifies the dependence of the reaction rate with respect to its substrates and products, as well as with respect to possible allosteric or competitive effectors. In case the actual rate equation of the respective step is unknown, a generic Michaelis- Menten equation is employed [33,52]. Once these data are assembled, the system of differential equations that determines the dynamic properties of the network has been fully specified except for lacking numerical values for most enzyme-kinetic parameters. As shown previously [28], however, with these definitions and the known metabolic phenotype, it is possible to systematically sample the parametric space, such that the resulting set of enzyme-kinetic parameters is consistent with the known metabolic state. Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism compare the distributions of dynamic properties in the presence and absence of a specific metabolic feedback. In summary, sampling unknown kinetic parameters for a kinetic model of a metabolic pathway, such that all sampled parameters give rise to the same metabolic state, can lead to fundamental insights into the control properties of the underlying system. In particular, the emergent control profile exhibits a structure that helps us to draw conclusions about the possible behavior of the system. The probabilistic control profile represents what dynamics we should expect as typical, given no further knowledge about specific constraints the system is subject to – a tremendously useful information when interpreting experimentally observed data. In this respect, some of the probabilistic distributions are in line with what we expect given the topology of the network, while other properties are counter-intuitive and therefore point the direction for further investigation. Our approach is particularly suited to investigate the qualitative effects that result from changes in pathway topology, in particular from the presence or absence of regulatory interactions. Our computational approach is straight- forward to implement and numerically efficient even for large systems. We therefore expect it to be of high utility also in other studies of metabolic, gene-expression and signal transduction systems. p The second aspect of our study relates to the question: How relevant are such distributions of control properties, given that any actual system only attains a single set of parameters. To what extent is, therefore, average behavior an indicator for actual behavior, given that the actual set of parameters may as well be located on the extreme fringes of the distribution. While we reject any interpretation of average behavior as a likelihood for actual behavior, we claim that the distributions obtained using our computational procedure indeed have functional, as well as explanatory, relevance. The explanatory relevance lies in the fact that probabilistic control profiles allow us to evaluate to what extent experimental findings are rooted in the topological structure of the pathway, rather than in fine-tuning of Michaelis-Menten parameters. Monte-Carlo Modeling of L. lactis Central Metabolism Specifically, if almost all sampled parameters attain a value within a certain narrow range, and the empirically observed value is in good agreement with these values, then we face no further explanatory challenge: The empirical value simply corresponds to what we expect as typical behavior, given no further knowledge about additional contraints the system is subject to. On the other hand, an empirically obtained control coefficient that is located at the extreme fringe of the respective distribution provides valuable information for further analysis and points to additional constraints or requirements the pathway is subject to. The functional relevance of our probabilistic evaluation is then given by the fact that control properties that are already inherent in the topological structure allow for an evolutionary adaptation of parameters with respect to other objectives. Specifically, it seems favorable, within the course of evolution, to adopt a network topology that exhibits a certain desired behavior, such as recovery after periods of starvation, for a broad range of parameter values. These parameters can then be further fine-tuned according to other objectives, without impeding the core functionality of the pathway. September 2014 \| Volume 9 \| Issue 9 \| e106453 Conclusions Metabolic regulation is a crucial aspect of cellular function. While significant progress has been made on the stoichiometric properties of large-scale metabolic systems, the organizing principles of metabolic regulation that enable stability and rapid adaptation are still insufficiently understood [15,29,38,47]. In this work, we have presented a Monte-Carlo approach to study the regulatory feedback topology of the homo-fermentative LAB strain Lactococcus lactis. In particular, we have used knowledge of an experimentally acquired metabolic state to evaluate typical Figure 12. Bistability and hysteresis with respect to external glucose. Shown is a non-recovering system in the absence of regulation (A,B) and a recovering system in the presence of regulation (C,D). In the upper panels (A,C), the concentration of external glucose was varied between the initial level of 20 mM to a lower value of 0:1 mM and back. The lower panels (B,D) show a corresponding time-course of the rate of glucose uptake. The original level of external glucose, 20 mM, was lowered to 0:1 mM within the time interval t = 1 min to 10 min. doi:10.1371/journal.pone.0106453.g012 Figure 12. Bistability and hysteresis with respect to external glucose. Shown is a non-recovering system in the absence of regulation (A,B) and a recovering system in the presence of regulation (C,D). In the upper panels (A,C), the concentration of external glucose was varied between the initial level of 20 mM to a lower value of 0:1 mM and back. The lower panels (B,D) show a corresponding time-course of the rate of glucose uptake. The original level of external glucose, 20 mM, was lowered to 0:1 mM within the time interval t = 1 min to 10 min. doi:10.1371/journal.pone.0106453.g012 September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 11 Monte-Carlo Modeling of L. lactis Central Metabolism Defining the system and its evaluation All code is provided in File S1. J’ : ~N’:Lv LSDS0 :L: ð4Þ ð4Þ The control coefficients are only evaluated for stable metabolic states, characterized by an invertible Jacobian with all real parts of the eigenvalues below zero. The information required to evaluate the control coefficients therefore consists of: (i) The stoichiometry of the system, as encoded by N’ and L; (ii) the metabolic state, as encoded in the matrices Dv 0 and DS0; and (iii) the kinetic properties of the reactions, as encoded in the partial derivatives. The partial derivatives are also known as the unscaled elasticity coefficients. Our probabilistic evaluation of the system is then based on the fact that the metabolic state is often directly experimentally accessible, whereas information about kinetic parameters, and hence the elasticities, is generally lacking. We therefore evaluate the possible values of the unscaled elasticities by drawing random instances of parameters and evaluating the equations for the control coefficients. In particular, we proceed along the following steps: First, each reaction is associated with a kinetic reaction equation. We adopt general Michaelis-Menten kinetics of the form From Stoichiometry to Dynamics In this In this sense, we argue that our approach has led to increased understanding of principles of metabolic regulation in Lactococcus lactis: We compared the typical response of the pathway with respect to short periods of starvation in the presence and absence of metabolic regulation. It was shown that the regulatory interactions, irrespective of the particular parameter values, result in qualitative differences in the dynamics. Our results therefore shows that the topology of the regulation alone is, to a large extent, already sufficient to ensure dynamic stability and recovery of the pathway. While fine-tuning of parameters may achieve a similar increase in stability, an appropriate regulatory structure dramat- ically increases the set of accessible parameters space and therefore opens the possibility to optimize Michaelis-Menten parameters for other secondary objectives, such as the trade-off between affinity and catalytic rate which are not considered here. The main result presented in this study is therefore that the regulatory architecture of the Lactococcus lactis central metabolism induces qualitative changes in the probabilistic control profile, as well as in the dynamic behavior after a brief period of starvation. Further, we were able to delineate the role of individual regulation terms: Three of the known regulatory mechanisms play a major role in the recovery ability of the system. These are (i) the inhibition of PYK by inorganic phosphate, (ii) the activation of PYK by FBP and (iii) the inhibition of PTS by FBP. We showed that in cases of non-recovery the zero-flux metabolic state is an attractor for the system. In the presence of regulation the ability of the system to escape metabolic death is, in the overwhelming number of cases, mirrored by the absence of such attractor. September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 12 Monte-Carlo Modeling of L. lactis Central Metabolism way, insight into the typical dynamic properties of a specific metabolic phenotype can be obtained. way, insight into the typical dynamic properties of a specific metabolic phenotype can be obtained. u~Vmax:f (S,K,Keq), ð5Þ ð5Þ Defining the system and its evaluation where K denotes a vector of unknown Michaelis-Menten parameters and Keq denotes an equilibrium constant. The function f includes terms for possible inhibition and activation. For example, for an unregulated uni-uni reaction A?B, the equation reads The probabilistic approach used within this work has been described previously [28]. In the following, we briefly outline our workflow. We assume that a metabolic system of interest consisting of m metabolites and r reactions is described by a system of ordinary differential equations of the form, u~Vmax: A{ B Keq K1 1z A K1 z B K2 ð6Þ dS dt ~N:v(S), ð1Þ ð1Þ ð6Þ where N denotes the m\|r stoichiometric matrix and S the m{dimensional vector of metabolite concentrations. The r{dimensional vector v specifies the nonlinear dependencies of the reaction rates as a function of the associated metabolite concentrations and kinetic parameters. To evaluate the dynamics, we assume the existence of a feasible metabolic state, defined by a concentration vector S0 and its associated flux values v(S0), such that Nv(S0)~0. We note that the metabolic state does not necessarily has to be asymptotically stable. The matrices of flux control coefficients CJ and concentration control coefficients CS can be expressed as, The number of unknown reaction parameters typically equals the number of associated substrates, products and modifiers (plus Vmax and the equlibrium constant). A full list of rate equations is provided in the Text S2. Second, the kinetic parameters K are sampled from intervals chosen according to the associated metabolite concentration, such that Ki[ 10{a1S ,10za2S : ð7Þ i0 i0 Ki[ 10{a1S ,10za2S : ð7Þ i0 i0 ð7Þ CS~{D{1 S0 :L:J’{1:N’:Dv0 ð2Þ For each simulation, the set of Michaelis-Menten parameters is chosen at random. All results are reported for a1~a2~1, but the results are highly robust for different choices of a and b. Sampling was linear in log space, i.e., the logarithm of Ki=S i0 is equidistributed in the interval. Once the parameters are specified, the values of Vmax are adjusted so as to deliver the known steady- state flux, ð2Þ and and CJ~1zD{1 v0 :Lv LSDS0 :DS0:CS ð3Þ ð3Þ where Dv0 and DS0 denote diagonal matrices with elements v0 and S0 on the diagonal, respectively, N’ denotes the reduced stoichiometric matrix and L the link matrix. See [28] for details. Defining the system and its evaluation The Jacobian J’ accounts for possible mass conservation and is defined as Vmax~ u0 f (S0,K,Keq) , ð8Þ ð8Þ and the derivatives of the reaction equation can be computed. Parameter sampling and the evaluation of control coefficients were repeated 2:104 times for each regulatory scenario. The values obtained for the control coefficients were largely robust with respect to different choices of the rate equations. Specifically, the evaluation of the control coefficient only depends on the values attained by the partial derivative, which are primarily determined by the ratio of substrate concentrations with respect to their corresponding Michaelis-Menten constants, as well as by param- eter-independent thermodynamic contributions. Using a similar sampling scheme with slightly modified reaction equations is therefore unlikely to result in major deviations with respect to overall control properties [38]. We note that our approach is straightforward to implement and its robustness with respect to different sampling schemes has been tested previously [28]. In particular, the evaluation of the control coefficients does not require explicit kinetic simulation of nonlinear differential equations, making it applicable also for medium- and large-scale systems. The key requirements are knowledge of the network topology, including its regulatory interactions, the metabolic state, as well as the respective thermodynamic equilibrium constants. All code is provided in File S1. and the derivatives of the reaction equation can be computed. Parameter sampling and the evaluation of control coefficients were repeated 2:104 times for each regulatory scenario. The values obtained for the control coefficients were largely robust with respect to different choices of the rate equations. Specifically, the evaluation of the control coefficient only depends on the values attained by the partial derivative, which are primarily determined by the ratio of substrate concentrations with respect to their corresponding Michaelis-Menten constants, as well as by param- eter-independent thermodynamic contributions. Using a similar sampling scheme with slightly modified reaction equations is therefore unlikely to result in major deviations with respect to overall control properties [38]. We note that our approach is straightforward to implement and its robustness with respect to different sampling schemes has been tested previously [28]. In particular, the evaluation of the control coefficients does not require explicit kinetic simulation of nonlinear differential equations, making it applicable also for medium- and large-scale systems. The key requirements are knowledge of the network topology, including its regulatory interactions, the metabolic state, as well as the respective thermodynamic equilibrium constants. Fermentation experiments KH2PO4, 0.6 g ammonium citrate, 1 g acetate, 0.25 g tyrosine, 0.24 g alanine, 0.125 g arginine, 0.42 g aspartic acid, 0.13 g cysteine, 0.5 g glutamic acid, 0.15 g histidine, 0.21 g isoleucine, 0.475 g leucine, 0.44 g lysine, 0.275 phenylalanine, 0.675 g proline, 0.34 g serine, 0.225 g threonine, 0.05 g tryptophan, 0.325 g valine, 0.175 g glycine, 0.125 g methionine, 0.1 g asparagine, 0.2 g glutamine, 10 g glucose, 0.5 g L-ascorbic acid, 35 mg adenine sulfate, 27 mg guanine, 22 mg uracil, 50 mg cystine, 50 mg xanthine, 2.5 mg D-biotin, 1 mg vitamin B12, 1 mg riboflavin, 5 mg pyridoxamine-HCl, 10 mg p-aminobenzoe¨ic acid, 1 mg pantothenate, 5 mg inosine, 1 mg nicotinic acid, 5 mg orotic acid, 2 mg pyridoxine, 1 mg thiamine, 2.5 mg lipoic acid, 5 mg thymidine, 200 mg MgCl2, 50 mg CaCl2, 16 mg MnCl2, 3 mg FeCl3, 5 mg FeCl2, 5 mg ZnSO4, 2.5 mg CoSO4, 2.5 mg CuSO4, (NH4)6Mo7O24. Mid-exponentially grown cells were harvested by centrifugation at 5000 RPM for 10 minutes at room temperature, washed twice with 50 mM MES buffer (pH = 6.5), and finally suspended in the indicated buffer solution. Anaerobic conditions were established by flushing with nitrogen for 10 min. Glucose (20 mM for L. lactis) was added and samples were taken at regular time intervals. 400 ml samples were taken and mixed immediately with 200 ml of a cold perchloric acid (3.5 M) solution. The extracts were kept on ice for maximal 60 minutes. The pH was neutralized with 160 ml 2 M KOH. The pH-adjusted samples were centrifuged and the supernatants were stored at 280uC for subsequent analysis. All metabolites were quantified by enzymatic methods coupled to the spectrophotometric determination of NAD(P)H. The strain NZ9000 was used [54]. where DRG0 denotes the change in standard Gibbs free energy occurring in the corresponding reaction and RT the gas constant multiplied by the absolute temperature. The value of DRG0 was estimated using, DRG0~ X products Df G0{ X substrates Df G0, ð10Þ ð10Þ where Df G0 denotes the Gibbs free energy of formation of the different metabolites. The values were adopted from Feist et al. [34], who use the group contribution method developed by Mavrovouniotis [56,57]. The full list of equilibrium constants is provided in the Text S1. The metabolic state was checked for thermodynamics feasibility. Acknowledgments We would like to thank Adam Wilkins for his kind help and contribution to revise linguistic aspects of the manuscript. Determination of the metabolic state Fluxes for lactate, acetate, formate, pyruvate and ethanol were calculated using their fermentation broth concentration, dilution rate (0:5h{1) and steady state bacterial cell dry weight. Fluxes are given in Text S1. Steady state intracellular metabolites concen- trations were gathered from previously published articles in various journals. The steady state data and its sources are provided in Text S1. Fermentation experiments In particular for any reaction S1zS2?P1zP2 with positive flux, the following relationship must hold at steady state, S 1 0S 2 0{P 1 0P 2 0=Keq w0: ð11Þ 2 0{P 1 0P 2 0=Keq w0: ð11Þ ð11Þ In Figure 5, we consider the distance C from equilibrium, defined as the ratio C : ~ 1 Keq Pj P j0 Pi S i0 , ð12Þ ð12Þ for a reaction that converts a set of substrates S into a set of products P. Only considering the positive direction of flux, the value of C is zero for irreversible reactions and approaches unity for reactions close to equilibrium. Determination of the equilibrium constants Determination of the equilibrium constants The equilibrium constants Keq were derived using the following equation, Conceived and designed the experiments: EM MV MB BT HVW RS. Performed the experiments: EM MV MB. Analyzed the data: EM MV MB DB HVW RS. Contributed reagents/materials/analysis tools: MB DB BT. Wrote the paper: RS MV EM. DRG0~{RT ln Keq, ð9Þ ð9Þ Analysis of carbon fluxes Bacterial dry weight was measured as described previously [55]. External glucose, pyruvate, lactate, formate, acetate, succinate, and ethanol were determined by high-pressure liquid chromatog- raphy (HPLC; LKB) with a Rezex organic acid analysis column (Phenomenex) at a temperature of 45uC with 7.2 mM H2SO4 as the eluent, using a RI 1530 refractive index detector ( Jasco) and AZUR chromatography software for data integration. Discrimi- nation between d- and l-lactate was performed using a d-/l-lactate assay kit (Megazyme). Fermentation experiments L. lactis cells were grown anaerobically at 37uC in CDM-LAB medium [53]. The medium contained per liter: 1 g K2HPO4, 5 g September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 13 Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism KH2PO4, 0.6 g ammonium citrate, 1 g acetate, 0.25 g tyrosine, 0.24 g alanine, 0.125 g arginine, 0.42 g aspartic acid, 0.13 g cysteine, 0.5 g glutamic acid, 0.15 g histidine, 0.21 g isoleucine, 0.475 g leucine, 0.44 g lysine, 0.275 phenylalanine, 0.675 g proline, 0.34 g serine, 0.225 g threonine, 0.05 g tryptophan, 0.325 g valine, 0.175 g glycine, 0.125 g methionine, 0.1 g asparagine, 0.2 g glutamine, 10 g glucose, 0.5 g L-ascorbic acid, 35 mg adenine sulfate, 27 mg guanine, 22 mg uracil, 50 mg cystine, 50 mg xanthine, 2.5 mg D-biotin, 1 mg vitamin B12, 1 mg riboflavin, 5 mg pyridoxamine-HCl, 10 mg p-aminobenzoe¨ic acid, 1 mg pantothenate, 5 mg inosine, 1 mg nicotinic acid, 5 mg orotic acid, 2 mg pyridoxine, 1 mg thiamine, 2.5 mg lipoic acid, 5 mg thymidine, 200 mg MgCl2, 50 mg CaCl2, 16 mg MnCl2, 3 mg FeCl3, 5 mg FeCl2, 5 mg ZnSO4, 2.5 mg CoSO4, 2.5 mg CuSO4, (NH4)6Mo7O24. Mid-exponentially grown cells were harvested by centrifugation at 5000 RPM for 10 minutes at room temperature, washed twice with 50 mM MES buffer (pH = 6.5), and finally suspended in the indicated buffer solution. Anaerobic conditions were established by flushing with nitrogen for 10 min. Glucose (20 mM for L. lactis) was added and samples were taken at regular time intervals. 400 ml samples were taken and mixed immediately with 200 ml of a cold perchloric acid (3.5 M) solution. The extracts were kept on ice for maximal 60 minutes. The pH was neutralized with 160 ml 2 M KOH. The pH-adjusted samples were centrifuged and the supernatants were stored at 280uC for subsequent analysis. All metabolites were quantified by enzymatic methods coupled to the spectrophotometric determination of NAD(P)H. The strain NZ9000 was used [54]. Supporting Information Text S1 A pdf document detailing the experimental procedure and the metabolic state. (PDF) Text S2 A pdf document providing additional informa- tion about computational methods and results. (PDF) 4. Hoefnagel MHN, van der Burgt A, Martens DE, Hugenholtz J, Snoep JL (2002) Time dependent responses of glycolytic intermediates in a detailed glycolytic model of Lactococcus lactis during glucose run-out experiments. Mol Biol Rep 29: 157–161. 5. Oliveira AP, Nielsen J, Fo¨rster J (2005) Modeling Lactococcus lactis using a genome-scale flux model. BMC Microbiol 5: 39. g 6. Oddone GM, Mills DA, Block DE (2009) A dynamic, genome-scale flux model of Lactococcus lactis to increase specific recombinant protein expression. Metab Eng 11: 367–381. Monte-Carlo Modeling of L. lactis Central Metabolism Monte-Carlo Modeling of L. lactis Central Metabolism 33. Liebermeister W, Uhlendorf J, Klipp E (2010) Modular rate laws for enzymatic reactions: thermodynamics, elasticities and implementation. Bioinformatics 26: 1528–1534. 7. Andersen AZ, Carvalho AL, Neves AR, Santos H, Kummer U, et al. (2009) The metabolic pH response in Lactococcus lactis: an integrative experimental and modelling approach. Comput Biol Chem 33: 71–83. 8. Lall R, Donohue TJ, Marino S, Mitchell JC (2011) Optimizing ethanol production selectivity. Mathematical and Computer Modelling 53: 13631373. 34. Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, et al. (2007) A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. Mol Syst Biol 3: 121. p y p g 9. Oh E, Lu M, Park C, Park C, Oh HB, et al. (2011) Dynamic modeling of lactic acid fermentation metabolism with Lactococcus lactis. J Microbiol Biotechnol 21: 162–169. 35. Burns J, Cornish-Bowden A, Groen A, Heinrich R, Kacser H, et al. (1985) Control analysis of metabolic systems. Trends Biochem Sci 10: 16. Control analysis of metabolic systems. Trends Biochem Sci 10: 1 10. Levering J, Musters MWJM, Bekker M, Bellomo D, Fiedler T, et al. (2012) Role of phosphate in the central metabolism of two lactic acid bacteria–a comparative systems biology approach. FEBS J 279: 1274–1290. 36. Heinrich R, Schuster S (1996) The Regulation Of Cellular Systems. Chapman & Hall, New York. 37. Gutenkunst RN, Waterfall JJ, Casey FP, Brown KS, Myers CR, et al. (2007) Universally sloppy parameter sensitivities in systems biology models. PLoS Comput Biol 3: 1871–1878. 11. Garrigues C, Loubiere P, Lindley ND, Cocaign-Bousquet M (1997) Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio. J Bacteriol 179: 5282–5287. p 38. Steuer R, Junker BH (2009) Computational models of metabolism: Stability and regulation in metabolic networks. Advances in Chemical Physics (ed Stuart A Rice).John Wiley & Sons 142. 12. Neves AR, Ramos A, Shearman C, Gasson MJ, Almeida JS, et al. (2000) Metabolic characterization of Lactococcus lactis deficient in lactate dehydroge- nase using in vivo 13C-NMR. Eur J Biochem 267: 3859–3868. 39. Koebmann BJ, Andersen HW, Solem C, Jensen PR (2002) Experimental determination of control of glycolysis in Lactococcus lactis. Antonie Van Leeuwenhoek 82: 237–248. g J 13. Neves AR, Ventura R, Mansour N, Shearman C, Gasson MJ, et al. Monte-Carlo Modeling of L. lactis Central Metabolism I: framework development. Metab Eng 8: 133–141. 47. van Heerden J, Wortel M, Bruggeman F, Heijnen J, Bollen Y, et al. (2014) Lost in transition: Startup of glycolysis yields subpopulations of nongrowing cells. Science 343: 1245114ff. p g 21. Wang L, Hatzimanikatis V (2006) Metabolic engineering under uncertainty–II: analysis of yeast metabolism. Metab Eng 8: 142–159. y y g 22. Wang L, Birol I, Hatzimanikatis V (2004) Metabolic control analysis under uncertainty: framework development and case studies. Biophys J 87: 3750–3763. 48. Soh KC, Miskovic L, Hatzimanikatis V (2012) From network models to network responses: integration of thermodynamic and kinetic properties of yeast genome- scale metabolic networks. FEMS Yeast Res 12: 129–143. 23. Gross T, Feudel U (2006) Generalized models as a universal approach to the analysis of nonlinear dynamical systems. Phys Rev E Stat Nonlin Soft Matter Phys 73: 016205. 49. Schuster S, Fell DA, Dandekar T (2000) A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic networks. Nat Biotechnol 18: 326–332. y 24. Steuer R, Gross T, Selbig J, Blasius B (2006) Structural kinetic modeling of metabolic networks. Proc Natl Acad Sci U S A 103: 11868–11873. 50. Orth JD, Thiele I, Palsson B (2010) What is flux balance analysis? Nat Biotechnol 28: 245–248. 25. Steuer R, Nesi AN, Fernie AR, Gross T, Blasius B, et al. (2007) From structure to dynamics of metabolic pathways: application to the plant mitochondrial TCA cycle. Bioinformatics 23: 1378–1385. 51. Grimbs S, Selbig J, Bulik S, Holzhuetter HG, Steuer R (2007) The stability and robustness of metabolic states: identifying stabilizing sites in metabolic networks. Mol Syst Biol 3: 146. y 26. Tran LM, Rizk ML, Liao JC (2008) Ensemble modeling of metabolic networks. Biophys J 95: 5606–5617. 52. Rohwer JM, Hanekom AJ, Crous C, Snoep JL, Hofmeyr JHS (2006) Evaluation of a simplified generic bi-substrate rate equation for computational systems biology. Syst Biol (Stevenage) 153: 338–341. 27. Tan Y, Rivera JGL, Contador CA, Asenjo JA, Liao JC (2011) Reducing the allowable kinetic space by constructing ensemble of dynamic models with the same steady-state flux. Metab Eng 13: 60–75. 53. Jo¨nsson M, Saleihan Z, Nes IF, Holo H (2009) Construction and characteriza- tion of three lactate dehydrogenase-negative Enterococcus faecalis V583 mutants. Appl Environ Microbiol 75: 4901–4903. y g 28. Monte-Carlo Modeling of L. lactis Central Metabolism (2002) Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? kinetics of NAD(+) and NADH pools determined in vivo by 13C NMR. J Biol Chem 277: 28088–28098. 40. Solem C, Koebmann BJ, Jensen PR (2003) Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis MG1363. J Bacteriol 185: 1564–1571. 14. Neves AR, Pool WA, Kok J, Kuipers OP, Santos H (2005) Overview on sugar metabolism and its control in Lactococcus lactis - the input from in vivo NMR. FEMS Microbiol Rev 29: 531–554. 41. Koebmann B, Solem C, Jensen PR (2005) Control analysis as a tool to understand the formation of the las operon in Lactococcus lactis. FEBS J 272: 2292–2303. 15. Voit E, Neves AR, Santos H (2006) The intricate side of systems biology. Proc Natl Acad Sci U S A 103: 9452–9457. 42. Solem C, Koebmann B, Yang F, Jensen PR (2007) The las enzymes control pyruvate metabolism in Lactococcus lactis during growth on maltose. J Bacteriol 189: 6727–6730. 16. Voit EO, Almeida J, Marino S, Lall R, Goel G, et al. (2006) Regulation of glycolysis in Lactococcus lactis: an unfinished systems biological case study. Syst Biol (Stevenage) 153: 286–298. 43. Koebmann BJ, Westerhoff HV, Snoep JL, Nilsson D, Jensen PR (2002) The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J Bacteriol 184: 3909–3916. ( g ) 17. Papagianni M, Avramidis N, Filiousis G (2007) Glycolysis and the regulation of glucose transport in Lactococcus lactis spp. lactis in batch and fed-batch culture. Microb Cell Fact 6: 16. J 44. Rosenfeld N, Elowitz MB, Alon U (2002) Negative autoregulation speeds the response times of transcription networks. J Mol Biol 323: 785–793. 18. Heijnen JJ (2005) Approximative kinetic formats used in metabolic network modeling. Biotechnol Bioeng 91: 534–545. 45. Bluethgen N (2010) Transcriptional feedbacks in mammalian signal transduction pathways facilitate rapid and reliable protein induction. Mol Biosyst 6: 1277– 1284. 19. Bulik S, Grimbs S, Huthmacher C, Selbig J, Holzhuetter HG (2009) Kinetic hybrid models composed of mechanistic and simplified enzymatic rate laws–a promising method for speeding up the kinetic modelling of complex metabolic networks. FEBS J 276: 410–424. 46. Xu YF, Amador-Noguez D, Reaves ML, Feng XJ, Rabinowitz JD (2012) Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase. Nat Chem Biol 8: 562–568. J 20. Wang L, Hatzimanikatis V (2006) Metabolic engineering under uncertainty. References 1. Teusink B, Smid EJ (2006) Modelling strategies for the industrial exploitation of lactic acid bacteria. Nat Rev Microbiol 4: 46–56. 1. Teusink B, Smid EJ (2006) Modelling strategies for the industrial exploitation of lactic acid bacteria. Nat Rev Microbiol 4: 46–56. 4. Hoefnagel MHN, van der Burgt A, Martens DE, Hugenholtz J, Snoep JL (2002) Time dependent responses of glycolytic intermediates in a detailed glycolytic model of Lactococcus lactis during glucose run-out experiments. Mol Biol Rep 29: 157–161. 2. Teusink B, Bachmann H, Molenaar D (2011) Systems biology of lactic acid bacteria: a critical review. Microb Cell Fact 10 Suppl 1: S11. bacteria: a critical review. Microb Cell Fact 10 Suppl 1: S11. 3. Hoefnagel MHN, Starrenburg MJC, Martens DE, Hugenholtz J, Kleerebezem M, et al. (2002) Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis. Microbiology 148: 1003–1013. 6. Oddone GM, Mills DA, Block DE (2009) A dynamic, genome-scale flux model of Lactococcus lactis to increase specific recombinant protein expression. Metab Eng 11: 367–381. September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 14 Monte-Carlo Modeling of L. lactis Central Metabolism Murabito E, Smallbone K, Swinton J, Westerhoff HV, Steuer R (2011) A probabilistic approach to identify putative drug targets in biochemical networks. J R Soc Interface 8: 880–895. 54. Wouters JA, Frenkiel H, de Vos WM, Kuipers OP, Abee T (2011) Cold shock proteins of lactococcus lactis MG1363 are involved in cryoprotection and in the production of cold-induced proteins. Appl Environ Microbiol 67: 5171–5178. 29. Girbig D, Grimbs S, Selbig J (2012) Systematic analysis of stability patterns in plant primary metabolism. PLoS One 7: e34686. p p y 30. Murabito E (2013) Targeting breast cancer metabolism: A metabolic control analysis approach. Curr Synthetic Sys Biol 1: 104. 55. Alexeeva S, de Kort B, Sawers G, Hellingwerf KJ, de Mattos MJ (2000) Effects of limited aeration and of the arcAB system on intermediary pyruvate catabolism in escherichia coli. J Bacteriol 182: 4934–4940. 31. Notebaart RA, van Enckevort FHJ, Francke C, Siezen RJ, Teusink B (2006) Accelerating the reconstruction of genome-scale metabolic networks. BMC Bioinformatics 7: 296. 56. Mavrovouniotis ML, Stephanopoulos G, Stephanopoulos G (1990) Computer- aided synthesis of biochemical pathways. Biotechnol Bioeng 36: 1119–1132. 32. Nova´k L, Loubiere P (2000) The metabolic network of Lactococcus lactis: distribution of (14)Clabeled substrates between catabolic and anabolic pathways. J Bacteriol 182: 1136–1143. 57. Mavrovouniotis ML (1991) Estimation of standard Gibbs energy changes of biotransformations. J Biol Chem 266: 14440–14445. September 2014 \| Volume 9 \| Issue 9 \| e106453 PLOS ONE \| www.plosone.org 15
https://openalex.org/W4312190728	https://link.springer.com/content/pdf/10.1140/epjc/s10052-022-11128-9.pdf	English	null	Constraints on light leptophilic dark matter mediators from decay experiments	European physical journal. C, Particles and fields	2,022	cc-by	9,505	Received: 15 September 2022 / Accepted: 13 December 2022 / Published online: 22 December 2022 © The Author(s) 2022 Abstract We study the inﬂuence of leptophilic dark matter interactions on decays of muons and ground state mesons in existing experiments. We consider a secluded dark sector exclusively interacting with leptons via either a (leptophilic) scalar or vector mediator. These interactions will therefore inﬂuence leptonic decays and deform the energy spectra. We ﬁrst study the Michel decay of muons, μ+ →e+νe ¯νμ, which allow us to constrain the parameter space reasonably well. Secondly, the rare π±, K ±, D± and D± s decays to eν will be considered. Scalar mediators would remove the Standard Model helicity suppression, so that strong constraints can be derived. The resulting bounds on the couplings of the light mediators to electrons and muons still turn out to be some- what weaker than those from searches at low-energy e+e− colliders and the magnetic moment of the muon, respectively. Finally, we show that kaon and pion decays basically exclude a “Co-SIMP” scenario where a scalar dark matter particle has a dimension-5 coupling to electrons. accurately, DMh2 = 0.120 ± 0.001 [2]; here DM is the scaled DM mass density and h is the rescaled Hubble param- eter. In the absence of data pinning down the properties of DM, a wide range of models has been proposed. In spite of inten- sive efforts no clear signal for DM particles has yet been found in either direct or indirect detection experiments [1], leading to severe constraints on many models [3]. This is true in particular for models with weakly interacting mas- sive particles (WIMPs), with masses very roughly at the weak scale. This has led to increased interest in sub-GeV masses [4], which for purely kinematic reasons are much less con- strained. Here we consider models that couple the potential dark sector to the Standard Model with light leptophilic media- tors, i.e. mediators that couple directly only to leptons. One class of models assumes a gauged lepton-family number; after spontaneous symmetry breaking these models contain a massive vector mediator, sometimes called a “dark photon”, that couples to some leptons of the Standard Model and in the dark sector to Dirac dark matter [5,6]. This lepton coupling introduces a kinetic mixing term with the ordinary photon, resulting in a small coupling between the dark photon and all electrically charged particles which can also be used to con- strain the model. a e-mail: bickendorf@th.physik.uni-bonn.de (corresponding author) b e-mail: drees@th.physik.uni-bonn.de Received: 15 September 2022 / Accepted: 13 December 2022 / Published online: 22 December 2022 © The Author(s) 2022 Another class of models assumes a scalar mediator, which again only couples directly to some or all charged leptons. Since this breaks the SU(2) gauge symme- try, models of this kind can at most be an effective theory. Finally, we consider the so-called “Co-SIMP” model [7] con- taining a light scalar DM particle with a non-renormalizable coupling to electrons. Eur. Phys. J. C (2022) 82:1163 https://doi.org/10.1140/epjc/s10052-022-11128-9 Regular Article - Theoretical Physics Constraints on light leptophilic dark matter mediators from decay experiments Gerrit Bickendorfa, Manuel Dreesb Bethe Center for Theoretical Physics and Physikalisches Institut, Universität Bonn, Nussallee 12, 53115 Bonn, Germany Gerrit Bickendorfa, Manuel Dreesb Bethe Center for Theoretical Physics and Physikalisches Institut, Universität Bonn, Nussallee 12, 53115 Bonn, Germany , Bethe Center for Theoretical Physics and Physikalisches Institut, Universität Bonn, Nussallee 12, 53115 Bonn, Germany Received: 15 September 2022 / Accepted: 13 December 2022 / Published online: 22 December 2022 © The Author(s) 2022 1 Introduction Evidence for the existence of substantial non-baryonic mass in the universe, in addition to the baryonic contribution from the known Standard Model (SM) particles, has been piling up for decades [1]. Cosmological observations, from the CMB at the largest scales, the structure of galaxy clusters and grav- itational lensing on intermediate scales, down to the rotation of single galaxies, all essentially only probe the gravitational interactions of this dark matter (DM), leaving the properties of the constituents of DM largely obscure. We do know that DM should be “cold”, i.e. non-relativistic well before the CMB decoupled. Moreover, within the minimal cosmolog- ical framework, the overall DM density can be determined We present a novel approach to constraining the parameter space using measurements of the decays of muons or ground- state ﬂavored mesons into ﬁnal states containing an electron. The spectrum of electrons produced in muon decays has been measured accurately; it agrees with SM predictions, which The spectrum of electrons produced in muon decays has been measured accurately; it agrees with SM predictions, which 12 3 1163 Page 2 of 11 Eur. Phys. J. C (2022) 82 :1163 Fig. 1 Kinetic mixing of A′ with the SM photon allows us to put upper bounds on the coupling of spin-1 medi- ators to electrons or muons. However, the resulting bounds turn out to be more than one order of magnitude weaker than the best constraint from e+e−colliders. New spin-0 particles coupling to electrons would remove the helicity suppression in charged meson decays into eνe ﬁnal states; the result- ing bounds on the renormalizable couplings of light scalar mediators are tighter than those from muon decay, but still somewhat weaker than those from e+e−colliders. However, bounds from pion and kaon decays sufﬁce to exclude a ther- mal “Co-SIMP” for masses below 0.8 MeV; in the allowed mass range χ does not behave like a SIMP any more. Fig. 1 Kinetic mixing of A′ with the SM photon Fig. 1 Kinetic mixing of A′ with the SM photon as L = LSM −1 4 F′ μν F′μν + m2 A′ 2 A′ μA′μ + ϵ 2 F′ μν Fμν − l=e,μ,τ e′ l ¯lγ μA′ μl + ¯νlγ μA′ μνl + ¯χ(i /∂−mχ)χ −gD ¯χγ μA′ μχ . (1) The remainder of this article is structured as follows: in Sect. 2 we present the leptophilic models considered here. In Sects. 1 Introduction 3 and 4 we describe the method of obtaining limits on the parameter space from the Michel decays of muons and pseudoscalar meson decays respectively. Section 5 presents our results and compares them to existing bounds. Section 6 ﬁnishes with some concluding remarks. (1) Here Fμν and F′ μν are the ﬁeld strength tensors of QED and of U(1)D, respectively. The new coupling constants gD and e′ l are free parameters of the model, and the term ∝ϵ describes kinetic mixing between the new gauge boson and the photon. In order to avoid anomalies, one may chose to gauge any combination X = yB − xi Li of baryon number B and lepton family number Li, with constraint 3y = xe + xμ + xτ [9]. Popular choices include gauged B −L or Lμ −Lτ [10,11]. While the former is highly constrained by direct collider searches and other 5th force experiments, the latter still exhibits rather weak constraints with a region that is even favored by the gμ −2 anomaly [12–14]. Here Fμν and F′ μν are the ﬁeld strength tensors of QED and of U(1)D, respectively. The new coupling constants gD and e′ l are free parameters of the model, and the term ∝ϵ describes kinetic mixing between the new gauge boson and the photon. In order to avoid anomalies, one may chose to gauge any combination X = yB − xi Li of baryon number B and lepton family number Li, with constraint 3y = xe + xμ + xτ [9]. Popular choices include gauged B −L or Lμ −Lτ [10,11]. While the former is highly constrained by direct collider searches and other 5th force experiments, the latter still exhibits rather weak constraints with a region that is even favored by the gμ −2 anomaly [12–14]. 2 Models The strongest bound on many DM models comes from “direct” search experiments, which look for elastic scatter- ing of ambient DM particles off the nuclei in a detector [1]. Leptophilic dark matter models, where the dark matter par- ticles primarily couple to the Standard Model leptons, either directly or via another “mediator” particle, avoid most of these bounds. In such models the DM particles can interact with nucleons only via loop diagrams. The term proportional to ϵ has been added to work with the most general renormalizable gauge invariant Lagrangian. Even though this term might be absent at tree level, as is the case for some GUT theories, it can be generated by loop contributions such as the diagram shown in Fig. 1, where the fermion running in the loop is charged both under the Standard Model and dark U(1)-group. In the case at hand this leads to One way of incorporating these ideas is a hidden sec- tor that contains only singlets under the Standard Model gauge group. However, the simplest (thermal) DM produc- tion mechanism requires some coupling to SM particles. To this end one may introduce additional ﬁelds which mediate interactions between both sectors. These are then ﬁttingly called portals. ϵ = l ee′ l 12π2 ln m2 l μ2 (2) (2) 2.1 Vector mediator The extension of the Standard Model by a new vector boson is well motivated both from a bottom up as well as from a top down perspective, e.g. from grand uniﬁed theories [8]. Here we consider scenarios where the gauge group is extended by another U(1)D gauge group which is spontaneously broken such that the associated particles become massive. The new vector boson A′ is often called a dark photon. By assumption the Dark Matter particles χ are charged under U(1)D. In this article we are concerned with the production of a light A′ which decays invisibly. The exact nature of χ therefore is not relevant for us. Assuming it to be a Dirac fermion for simplicity’s sake, the resulting Lagrangian can the be written where the dependence on the renormalization scale μ can- cels in the sum. The term ∝ϵ in Eq. (1) leads to additional effective interactions of the form L ⊃eϵ A′ μJ μ em , (3) (3) L ⊃eϵ A′ μJ μ em , where J μ em = ψ Qψ ¯ψγ μψ is the electromagnetic current; hence every electrically charged particle interacts with the dark photon as it is now millicharged under U(1)D [15]. This can be used to put strong constraints on the parameter space. where J μ em = ψ Qψ ¯ψγ μψ is the electromagnetic current; hence every electrically charged particle interacts with the dark photon as it is now millicharged under U(1)D [15]. This can be used to put strong constraints on the parameter space. 123 123 Eur. Phys. J. C (2022) 82 :1163 Eur. Phys. J. C (2022) 82 :1163 Page 3 of 11 1163 If m A′ < 2mχ the new gauge boson will mostly decay to the leptons to which it couples directly: from gauge invariant (but non-renormalizable) dimension 5 operators [21]: (A′ →l¯l) = e ′2 l 12π dlm A′ 1 + 2m2 l m2 A′ 1 −4m2 l m2 A′ , (4) cl φ ¯Liei R + h.c. , (8) (8) (4) where is the Standard Model Higgs ﬁeld. Once obtains a vacuum expectation value v, the interactions in (8) lead to the scalar couplings in Eq. (7), with where dl = 1 for charged leptons while dl = 1/2 for left-handed neutrinos. If m A′ > 2ml± one may search for visible A′ →l+l−decays. 2.1 Vector mediator Here we are instead inter- ested in scenarios with mostly invisible A′ decays, either because m A′ < 2ml± for the relevant charged lepton, or because m A′ > 2mχ and gD ≫e′ l. A′ →χ ¯χ decays are also described by Eq. (4), with the obvious replacements el →gD, ml →mχ and dl →1. e′ l = clv √ 2 . (9) e′ l = clv √ 2 . (9) Another possibility is to have the light scalar φ mix with the neutral component of a (second) scalar doublet, which can in principle have renormalizable O(1) couplings to leptons.1 Most lepton-speciﬁc scalar mediator models considered in the literature assume the effective scalar couplings cl to be proportional to the charged lepton masses, in which case the e′ l follow the lepton mass hierarchy. Within a given cosmological scenario the χ relic density imposes one constraint on the parameters of the model [4, 14]; even the case e′ l = 0, in which case A′ couples to SM particles only via Eq. (3), can lead to the correct relic density in minimal cosmology [16]. Here we implicitly assume that this constraint is used to determine the DM mass mχ, which allows us to vary the mass and couplings of A′ freely. A nonvanishing e′ l leads at 1-loop to an effective coupling of the scalar to two photons, through the diagram shown in Fig. 2. If mφ < 2ml, this decay can be used to search for φ in the diphoton invariant mass distribution. The decay width to photons is2 [24] Additional motivation for direct interactions with the muon speciﬁcally come from the anomalous magnetic moment of the muon, aμ = (g−2)μ/2. The Standard Model prediction [17] differs from the experimental result [18]: aμ = aμ(Exp) −aμ(SM) = (251 ± 59) × 10−11. The additional 1-loop contribution from a vector boson coupling to muons is [19,20]: (φ →γ γ ) = α2m3 φ 256π3 l=e,μ,τ e′ l ml F1/2(xl) 2 , (10) (10) a A′ μ = e′2 μ 4π2 1 0 dz m2 μz(1 −z)2 m2μ(1 −z)2 + m2 A′z . 2.1 Vector mediator 2 One loop contribution to the φ-photon coupling The main avenue for collider experiments to constrain this model is then missing energy searches. On the other hand, for 2mχ > mφ > 2ml the mediator decays mostly visibly to leptons it directly couples to, with decay width Fig. 3 Effective operator of the Co-SIMP model we consider (φ →¯ll) = e′2 l mφ 8π 1 −4m2 l mφ 3/2 . (13) ∝¯eeχ3 + h.c.. Observation of cosmological structures imply mχ ≳5 keV, while mχ ≲me is required in order to avoid a WIMP-like freeze-out [7]. Note that this model does not contain additional free parameters that allow to tune the relic density independent of the laboratory limits which are the main topic of this work. In order to compute the cosmo- logically preferred value of we therefore solve numerically the Boltzmann equation describing the freeze-out of χ [7]: (13) We’ll be interested in scenarios where \|e′ l\| ≳10−3 for l = e or μ. The decay width (13) then corresponds to a lifetime τφ ≲1.5 · 10−14(1 MeV)/mφ seconds; even accounting for a Lorentz boost, φ →l+l−decays will then usually be “prompt” if the corresponding decay is kinematically allowed. sH(T )x dYχ dx = −s3⟨σ32v2⟩ Y 2 χ −YχY eq χ Y eq e . (17) (17) A light scalar coupling to muons can also explain the (g − 2)μ results. Its contribution is given by [19,24]: Here x = mχ/T , T being the temperature, s is the total entropy density, and Yψ = nψ/s where nψ is the number density of ψ particles, with ψ ∈{χ, e} in our case; the super- script eq denotes the equilibrium value of the corresponding quantity. The thermally averaged cross section [25] is also obtained numerically from the integral aφ μ = e′2 μ 8π2 1 0 dz m2 μ(1 + z)(1 −z)2 m2μ(1 −z)2 + m2 φz . (14) (14) For m2 φ ≪m2 μ this simpliﬁes to For m2 φ ≪m2 μ this simpliﬁes to aφ μ ≈ 3e′2 μ 16π2 ≈1.9 × 10−10 e′ μ 10−4 2 . (15) ⟨σ32v2⟩= 1 2neq e neq χ neq χ 5 i=1 gid3 pi (2π)32Ei (2π)4 ·δ4(p1 + p2 + p3 −p4 −p5) f1 f2 f3\|M\|2. (15) 2.3 Co-SIMP (18) (18) (18) Finally, we consider the Co-SIMP mechanism proposed by Smirnov et al. [7]. 2.1 Vector mediator where xl = 4m2 l m2 φ and the loop function F1/2 reads where xl = 4m2 l m2 φ and the loop function F1/2 reads (5) F1/2(xl) = ⎧ ⎪⎪⎨ ⎪⎪⎩ −2xl 1 + (1 −xl) arcsin2(x−1/2 l ) xl ≥1 −2xl 1 −1−xl 4 −iπ + log 1+√1−xl 1−√1−xl 2 xl < 1 . (11) For m A′ ≪mμ this simpliﬁes to For m A′ ≪mμ this simpliﬁes to a A′ μ ≈ e′2 μ 8π2 ≈1.3 × 10−10 e′ μ 10−4 2 . (6) (6) (11) 2.2 Scalar mediator 2.2 Scalar mediator For xl ≫1, the loop function F1/2 →−4/3. For xl ≫1, the loop function F1/2 →−4/3. For gD > e′ l and 2mχ < mφ the scalar mediator decays mostly invisibly to the dark sector. The corresponding decay width is Another possibility is that a scalar particle mediates inter- actions between the dark matter and the SM particles. Here we consider a real scalar ﬁeld φ with mass mφ that is a sin- glet under the Standard Model gauge group, and again a dark matter Dirac fermion χ. The Lagrangian is: (φ →¯χχ) = g2 D mφ 8π 1 − 4m2 χ mφ 3/2 . (12) (12) L = LSM + 1 2∂μφ∂μφ − m2 φ 2 φ2 − l=e,μ,τ e′ l ¯llφ + ¯χ(i /∂−mχ)χ −gD ¯χχφ. (7) 1 Such a doublet would have to be quite heavy, with masses well beyond the range we consider here; φ can therefore not itself be part of such a doublet. In principle φ can also mix with the SM Higgs ; however, the resulting (renormalizable) couplings to electrons and muons would be uninterestingly small. (7) This Lagrangian respects QED gauge invariance, but the interactions of the scalar with the leptons break electroweak gauge invariance explicitly. These couplings might originate This Lagrangian respects QED gauge invariance, but the interactions of the scalar with the leptons break electroweak gauge invariance explicitly. These couplings might originate 2 The corresponding loop diagram for the SM Higgs was ﬁrst computed numerically in [22] and analytically in [23]. 12 3 1163 Page 4 of 11 Eur. Phys. J. C (2022) 82 :1163 Fig. 2 One loop contribution to the φ-photon coupling Fig. 3 Effective operator of the Co-SIMP model we consider Fig. 2.1 Vector mediator A real scalar particle χ with strong self- interactions is assumed as dark matter. An interaction with the Standard Model of the form χχe →χe is introduced in order to dissipate entropy from the dark sector whilst a Z3 symmetry stabilizes χ (Fig. 3). Here gi denotes the number of internal degrees of freedom of particle i (1 for χ and 4 for e), fi = 1/(exp Ei/T ± 1) is the phase space distribution function for the i-th particle in the initial state, and \|M\|2 is the averaged squared matrix element for the relevant process χ + χ + e →χ + e. We consider an electrophilic version of this model, in which case the relevant interaction is described by the effec- tive operator 3 Bounds from µ−decays Our ﬁnal estimate for the sensitivity of these measurements to the new coupling is obtained by comparing these ﬁtted Michel parameters to the measured values [1]: FIS = x(1 −x) + 2 9ρ(4x2 −3x −x2 0) +ηx0(1 −x); FAS = 1 3ξ x2 −x2 0 · 1 −x + 2 3δ 4x −4 + 1 −x2 0 . (20) (20) ρ = 0.74979 ± 0.00026; η = 0.057 ± 0.034; δ = 0.75047 ± 0.00034; \|Pμξ\| = 1.0009+0.0016 −0.0007. Here the Michel parameters ρ, η, ξ and δ have Standard Model values 3/4, 0, 1 and 3/4 respectively. Measurements of these parameters have been used to put constraints on the effective parameters of additional four fermions interactions. Here the Michel parameters ρ, η, ξ and δ have Standard Model values 3/4, 0, 1 and 3/4 respectively. Measurements of these parameters have been used to put constraints on the effective parameters of additional four fermions interactions. Here we use precise measurements of the muon decay spec- trum to put constraints on four-body decays μ−→e−νμ¯νeX where X is a light leptophilic mediator. The contributing Feynman diagrams for the case of a vector mediator coupling to electron number are shown in Fig. 4. We assume that X is either long lived or decays invisibly, so that the four-body ﬁnal state has the same basic signature as the three-body ﬁnal state. However, the observable electron spectrum of this four- body mode differs from the spectrum predicted by the SM. Whenever a best-ﬁt Michel parameter deviates further than 1.645σ from the experimental one, the parameter point is rejected at more than 95% C.L. This conservative approach is taken to justify the simplistic modeling of the experimental setup. We assume muon polarization Pμ = 1, as predicted by the SM for the relevant case of muons produced in meson decays. Here we use precise measurements of the muon decay spec- trum to put constraints on four-body decays μ−→e−νμ¯νeX where X is a light leptophilic mediator. The contributing Feynman diagrams for the case of a vector mediator coupling to electron number are shown in Fig. 4. We assume that X is either long lived or decays invisibly, so that the four-body ﬁnal state has the same basic signature as the three-body ﬁnal state. However, the observable electron spectrum of this four- body mode differs from the spectrum predicted by the SM. 3 Bounds from µ−decays Oe = ¯ee χ3 2 . (16) Having introduced the models we will consider, we turn to a discussion of the decays which we use to derive bounds on the parameters of these models. We begin with a discussion of the muon decay spectrum. Since this has widely been used as a high precision test of the electroweak theory [26], it might provide a good chance to constrain our models. The double (16) Once again this does not respect the electroweak gauge sym- metry; as before gauge invariance can be restored by replac- ing the dim-5 operator of Eq. (16) by a dim-6 operator 123 Eur. Phys. J. C (2022) 82 :1163 Page 5 of 11 1163 differential width for μ−→e−νμ¯νe decays at rest can be written as [1]: by the numerical integration over all kinematic parameters except x and cos θ. The resulting two dimensional distribu- tion is then added to the pure Standard Model spectrum. The Michel parameters that best describe this new distribution were extracted from Eqs. (19) and (20) by means of a χ2 ﬁt, using the same binning as in Ref. [26]. This serves as a ﬁlter that condenses the spectrum to the parameters that have been measured. More exactly, we minimized d2 dxd cos θ = mμ 2π3 W 4 eμG2 F x2 −x2 0 · FIS(x) −Pμ cos θ FAS(x) . (19) (19) Here Weμ = (m2 μ + m2 e)/2mμ is the maximum electron energy (neglecting possible neutrino masses), x = Ee/Weμ is the rescaled electron energy, x0 = me/Weμ is its minimum value, Pμ is the degree of muon polarization and θ is the angle between the polarization vector of the muon and the outgoing electron. The functions for the isotropic part FIS(x) and the anisotropic part FAS(x) are given by [1]: χ2 = i i(Michel) −i(Z′) 2 i(Z′) . (22) (22) Here i is the muon decay width in the i−th bin; i(Michel) is computed from Eqs. (19) and (20) and depends on the values of the Michel parameters, while i(Z′) is computed from Eq. (21) and depends on the mass and coupling of the new Z′ boson. 3 Our result agrees with that of Ref. [31], up to an overall factor of 2 which arises because they consider a spin-0 particle coupling only to νL. There is also a contribution ∝e′2 e m2 e, which is IR divergent for mφ →0 [32]. As predicted by the KLN theorem, these terms are canceled by loop diagrams [33]. Our simple expression (25) therefore accurately captures the most important contribution due to φ emission. 4 Bounds from leptonic decays of charged pseudoscalar mesons In the limit of vanishing electron and neutrino masses, the total width for P →eνeφ decays can easily be computed analytically:3 At tree level the total width for the decay of a charged pseu- doscalar meson P± to a lepton pair is given by [30] (P →eνeφ) = e′2 e G2 F F2 P\|Vq1q2\|2m3 P 384π3 · 1 − mφ m P 6 + mφ m P 2 9 + 6 ln m2 φ m2 P − mφ m P 4 9 −6 ln m2 φ m2 P . (25) (P−→l−¯νl) = G2 F\|Vq1q2\|2 4π F2 Pm Pm2 l · 1 −m2 l m2 P 2 . (23) (23) (25) Here G F is the Fermi constant, m P and ml are the meson and lepton mass, respectively, FP is the P decay constant, and Vq1q2 is a CKM matrix element, P+ being a (q1 ¯q2) bound state. Owing to the V −A nature of charged current weak interactions, both the charged lepton and the neutrino “like” to be left-handed, which however is forbidden by angular momentum conservation. This leads to the well known helic- ity suppression represented by the factor m2 l in Eq. (23). Evi- dently this factor strongly suppresses the decay to an electron and neutrino. This is often exploited for tests of lepton uni- versality in the ratio of decays to electrons and muons. The SM prediction for the ratio of decay widths is Here G F is the Fermi constant, m P and ml are the meson and lepton mass, respectively, FP is the P decay constant, and Vq1q2 is a CKM matrix element, P+ being a (q1 ¯q2) bound state. Owing to the V −A nature of charged current weak interactions, both the charged lepton and the neutrino “like” to be left-handed, which however is forbidden by angular momentum conservation. This leads to the well known helic- ity suppression represented by the factor m2 l in Eq. (23). Evi- dently this factor strongly suppresses the decay to an electron and neutrino. This is often exploited for tests of lepton uni- versality in the ratio of decays to electrons and muons. The SM prediction for the ratio of decay widths is This expression manifestly avoids the m2 e suppression. 3 Bounds from µ−decays er, the emission of collinear mediators can give rise to m X) enhanced terms in the decay distribution, which n the total muon decay width by the Kinoshita–Lee– erg (KLN) theorem [28,29] once loop diagrams are d. We therefore expect that a comparison of different ments of G F has much poorer sensitivity to the light rs we consider than the measurement of the muon pectrum discussed above. where δRQED describes the effect of QED corrections, including real photon emission. It is important to note that the emission of a spin-1 boson does not change the helic- ity structure of the amplitude, and therefore does not lift the helicity suppression. In contrast, if a scalar or pseudoscalar particle couples to the electron, the helicity suppression is removed, which can enhance the electronic decay mode sig- niﬁcantly. Since measurements are in agreement with the SM prediction (24), this lifting of the helicity suppression can be used to derive bounds on the couplings of new light spin-0 particles. Fig. 4 Diagrams contributing to μ−→e−A′νμ ¯νe decays for the case that A′ couples to electron number Fig. 4 Diagrams contributing to μ−→e−A′νμ ¯νe decays for the case that A′ couples to electron number surement of G F – with, however, much poorer precision [1]. Moreover, the emission of collinear mediators can give rise to ln(mμ/m X) enhanced terms in the decay distribution, which cancel in the total muon decay width by the Kinoshita–Lee– Nauenberg (KLN) theorem [28,29] once loop diagrams are included. We therefore expect that a comparison of different measurements of G F has much poorer sensitivity to the light mediators we consider than the measurement of the muon decay spectrum discussed above. 3 Bounds from µ−decays A technical subtlety arises because experiments do not ﬁt to the whole spectrum: cuts have to be applied in order to cover detector inefﬁciencies and blind spots. We modeled the effects of these cuts by restricting our kinematical ﬁt to the ﬁducial region covered by the TWIST detector [26]: approxi- mately x ∈[0.45, 0.98] and \|cos θ\| ∈[0.54, 0.96]. We found that these cuts affect the sensitivity limit on the coupling only by an O(1) factor, for the mediator masses considered here. Our sensitivity limits should not be confused with experi- mental bounds; we did not use real data, nor did we include QED corrections when modeling the SM prediction for the decay spectrum. Our procedure should nevertheless give a reasonable estimate of the sensitivity of the measurements of muon decays to the new mediators. The obvious Standard Model background is the radiative muon decay, μ−→e−γ νμ¯νe, where the photon escapes detection but nevertheless carries some energy, thereby also altering the electron spectrum. Since our signal involves a four particle ﬁnal state, this case cannot be mapped directly onto Eqs. (19) and (20). How- ever, when the dark mediator remains invisible the observ- able ﬁnal state has the same topology as in the SM, i.e. the combination d(μ →e−¯νeνμ) dxd cos θ + e′2 l d(μ →e−¯νeνμX) dxd cos θ e′ l=1 (21) (21) We ﬁnally note that the measurement of the muon lifetime cannotbeuseddirectlytoconstrainourmodels.IntheSMthis measurement is used to determine the experimental value of G F, which is a free parameter of the theory. A deviation from the SM could therefore only be detected by comparing this measurement with a second, independent determination of G F. Assuming unitarity of the quark mixing (CKM) matrix, the experimental “CKM unitarity test” can be recast as a mea- will be observed. In order to derive estimates of possible constraints we studied the sensitivity of ﬁtting procedures similar to those employed in the experimental determination of the spectral parameters. The four body matrix elements were derived with help of the Mathematica package FeynCalc [27] followed 12 3 Eur. Phys. J. C (2022) 82 :1163 1163 Page 6 of 11 iagrams contributing e−A′νμ ¯νe decays for hat A′ couples to umber t of G F – with, however, much poorer precision [1]. 4 Bounds from leptonic decays of charged pseudoscalar mesons The acceptance is then approximated by the efﬁciency after selecting events with Ee ≥52 MeV (i.e. the fraction of events passing the cut). The resulting acceptance correction is shown in Fig. 5. • PIENU: Search for the three body decay π+ →l+νX where l is an electron or muon and X an invisible neutral boson. Pions were stopped in a detector and the spectrum of the charged lepton was measured. A search for the smooth signal spectrum was then carried out below the energy of the two body decay. This sets limits on the branching ratio π+ →e+νX / π+ →μ+ν for X−masses in the range 0 < m X < 120 MeV [43]. For the Co-SIMP model the acceptance is approximately constant in the allowed mass range, with ϵ ≈11%. This lim- itation does not apply to K ±, D± and D± s decays, which are studied in ﬂight, so that even the two-body decay mode has a broad energy spectrum. The most precise measurement of RK comes from the NA62 Collaboration [36]. Here a Kaon beam decays in ﬂight inside the detector. Since the accepted range of electron energies has a width of several 10 GeV we simply assume that all decay modes have the same accep- tance. Our estimated sensitivity of existing muon decay data to the couplings of a new vector mediator are shown in Fig. 6. Unfortunately these estimated sensitivities are considerably weaker than the best existing bounds; the electron coupling is constrained by BaBar, while the muon coupling is con- strainedbytheCCFRtridentdata.Weﬁndaconsiderablybet- ter sensitivity to the electron coupling, since near-collinear A′ emission off the electron, which is enhanced by a large logarithm, reduces the energy of the electron and thus leads to an observable effect. In contrast, near-collinear A′ emission off the muon neutrino leaves the electron energy essentially unchanged. Below a mediator mass of O(10 MeV) the bound on the Michel parameter δ determines the sensitivity limit. For larger mediator masses the sensitivity limit is set by the ξ parameter. Of course, the sensitivity is worse for larger mediator masses due to the closing phase space. The decay of D± or D± s mesons to eν has not yet been observed, but 90% c.l. upper bounds on the corresponding branching fractions have been set by the CLEO and Belle Collaboration respectively [37,38]. 4 Bounds from leptonic decays of charged pseudoscalar mesons We use these to derive limits on the couplings of our light spin-0 mediators. Ourprojectedboundsonthecouplingsofthescalarmedia- tor φ are depicted in Fig. 7. Muon decay is much less sensitive to these couplings than to those of a spin-1 mediator shown in Fig. 6, since collinear emission of a soft spin-0 boson off a fermion is suppressed. This also explains why the bounds on e′ e and e′ μ from muon decay are now quite similar. 4 Bounds from leptonic decays of charged pseudoscalar mesons (26) Here X stands for either a single spin-0 mediator φ or for the three Co-SIMP scalars χ, and ϵ is an acceptance cor- rection factor due to the softer electron spectrum. For the most precise measurement of Rπ, by the PIENU Collabo- ration [34], this factor is computed as follows. This exper- iment analyses decays of a stopped π+ beam. In order to discriminate between direct π+ →e+ decays and the dom- inant background from π+ →μ+ →e+, an energy cut Ee ≥52 MeV was used in the experimental deﬁnition of π+ →e+νe decays, which have a nominal positron energy of Ee = 69.8 MeV. If we want to apply this analysis to our π+ →e+νφ decay, the same cut on the positron energy should be applied. 105 Monte Carlo pion decays weregeneratedusingMadGraph5_aMC@NLO 3.2.0[35]. The acceptance is then approximated by the efﬁciency after selecting events with Ee ≥52 MeV (i.e. the fraction of events passing the cut). The resulting acceptance correction is shown in Fig. 5. Here X stands for either a single spin-0 mediator φ or for the three Co-SIMP scalars χ, and ϵ is an acceptance cor- rection factor due to the softer electron spectrum. For the most precise measurement of Rπ, by the PIENU Collabo- ration [34], this factor is computed as follows. This exper- iment analyses decays of a stopped π+ beam. In order to discriminate between direct π+ →e+ decays and the dom- inant background from π+ →μ+ →e+, an energy cut • Belle II: Search for e+e−→μ+μ−Z′ with beam ener- gies of 4 and 7 GeV where the Z′ is radiated off of one of the muons and decays invisibly with m Z′ < 6 GeV [41]. The limits can then be recast to muonphilic scalar mediators [42]. • Belle II: Search for e+e−→μ+μ−Z′ with beam ener- gies of 4 and 7 GeV where the Z′ is radiated off of one of the muons and decays invisibly with m Z′ < 6 GeV [41]. The limits can then be recast to muonphilic scalar mediators [42]. Ee ≥52 MeV was used in the experimental deﬁnition of π+ →e+νe decays, which have a nominal positron energy of Ee = 69.8 MeV. If we want to apply this analysis to our π+ →e+νφ decay, the same cut on the positron energy should be applied. 105 Monte Carlo pion decays weregeneratedusingMadGraph5_aMC@NLO 3.2.0[35]. 4 Bounds from leptonic decays of charged pseudoscalar mesons In the Co-SIMP model one instead has to emit three scalar χ particles, leading to a considerably more complicated phase space integral; however, since the new vertex again violates chirality, also in this case the helicity suppression is lifted. To obtain the decay width numerically the phase space integral is carried out using the Monte Carlo integration rou- tines provided by the GNU Scientiﬁc Library. Whenever the new scalars remain invisible the event will have the same topology as a rare decay into e + νe, albeit with a softer electron energy spectrum. This change of the RSM P ≡(P →eν(γ )) (P →μν(γ )) = me mμ 2 m2 P −m2 e m2 P −m2μ 2 (1 + δRQED), (24) RSM P ≡(P →eν(γ )) (P →μν(γ )) = me mμ 2 m2 P −m2 e m2 P −m2μ 2 (1 + δRQED), (24) 123 123 Page 7 of 11 1163 Eur. Phys. J. C (2022) 82 :1163 electron spectrum can reduce the sensitivity due to kinematic cuts employed by the experiments. Bounds are then extracted by saturating the maximal allowed difference P between the theory prediction RSM p and the experimental result Rexp p , i.e. • BaBar: Dedicated search for the dark photon in e+e−→ γ A′; A′ →invisible with CM energies near the ϒ reso- nances. 90% c.l. limits were derived on the dark photon coupling constant ε2 for m A′ ≤8 GeV [39]. This can directly be applied to our model with a vector mediator, with the replacement e′ e = eε [6]. i.e. P ≥ϵ(e+νe + X) (μνμ(γ )) . (26) e • CCFR: Measurement of neutrino trident production events νN →νNμ−μ+ using a muon-neutrino beam with average energy ⟨Eν⟩= 160 GeV. The observed NCCFR = 37.0 ± 12.4 events agree with the Standard Model prediction NSM = 45.3 ± 2.3 [40]. This is used to set limits on additional contributions from a vector mediator coupling to the muon-neutrino and to the muon [9]. • CCFR: Measurement of neutrino trident production events νN →νNμ−μ+ using a muon-neutrino beam with average energy ⟨Eν⟩= 160 GeV. The observed NCCFR = 37.0 ± 12.4 events agree with the Standard Model prediction NSM = 45.3 ± 2.3 [40]. This is used to set limits on additional contributions from a vector mediator coupling to the muon-neutrino and to the muon [9]. 5 Results In the left frame, the dashed black, and solid yellow, red and orange lines indicate limits from additional contributions to P+ →e+νe decays (dashed black: P = π; yellow: P = K; orange: P = D; red: P = Ds). The dot-dashed black line represents the limits derived from the result of the search of PIENU for π+ →eνX; the drop of sensitivity at mφ ∼55 MeV is due to the signal being similar to π+ →μ+ →e+ [43]. The region shaded in gray in the left frame is excluded by an older BaBar limit recast to scalar mediators [44]. In the right frame the orange shaded region is excluded by a recast of a Belle II search for e+e−→μ+μ−A′, (A′ →invisible) [42], and the green band indicates parameters that bring the experimen- tal and theoretical values of (g −2)μ within 2σ Acceptance correction for π+ →e+νeφ and π+ →e+νeχχχ decays due to the cut on Ee by the PIENU experiment Fig. 6 The blue curves indicate our estimated 90% c.l. sensitivity limit on the coupling constant between electron or muon and the spin-1 mediator A′ from existing measurements of muon decay. The grey shaded region in the left frame is excluded by the BaBar search for e+e−→γ A′(A′ →invisible) [39], rescaled to e′ e = εe, while the red shaded region in the right frame is excluded by an analysis [9] of CCFR data on νN →νNμ−μ+. The green band in the right frame indicates parameters that bring the experimental and theoretical values of (g −2)μ within 2σ Fig. 7 90% c.l. upper bounds on the coupling constant between elec- tron or muon and the spin 0 mediator φ. The blue curves in both frames indicate the sensitivity derived from muon decay. In the left frame, the dashed black, and solid yellow, red and orange lines indicate limits from additional contributions to P+ →e+νe decays (dashed black: P = π; yellow: P = K; orange: P = D; red: P = Ds). The dot-dashed black line represents the limits derived from the result of the search of PIENU for π+ →eνX; the drop of sensitivity at mφ ∼55 MeV is due to the signal being similar to π+ →μ+ →e+ [43]. The region shaded in gray in the left frame is excluded by an older BaBar limit recast to scalar mediators [44]. 5 Results Here we show our estimated sensitivity to the new coupling as function of the mass of the postulated new boson, derived frommuonandchargedmesondecays. Wealsoshowexisting limits found in the literature, where we focus on the strongest bounds for the case at hand. μ However, bounds on P+ →e+νe decays, where P+ is a pseudoscalar ground state meson, lead to quite strin- gent bounds on new scalar couplings of the electron. For The relevant current bounds come from: 12 3 Eur. Phys. J. C (2022) 82 :1163 1163 Page 8 of 11 Fig. 5 Acceptance correction for π+ →e+νeφ and π+ →e+νeχχχ decays due to the cut on Ee by the PIENU experiment Fig. 6 The blue curves indicate our estimated 90% c.l. sensitivity limit on the coupling constant between electron or muon and the spin-1 mediator A′ from existing measurements of muon decay. The grey shaded region in the left frame is excluded by the BaBar search for e+e−→γ A′(A′ →invisible) [39], rescaled to e′ e = εe, while the red shaded region in the right frame is excluded by an analysis [9] of CCFR data on νN →νNμ−μ+. The green band in the right frame indicates parameters that bring the experimental and theoretical values of (g −2)μ within 2σ Fig. 5 Acceptance correction for π+ →e+νeφ and π+ →e+νeχχχ decays due to the cut on Ee by the PIENU experiment Fig. 5 Acceptance correction for π+ →e+νeφ and π+ →e+νeχχχ decays due to the cut on Ee by the PIENU experiment Fig. 6 The blue curves indicate our estimated 90% c.l. sensitivity limit on the coupling constant between electron or muon and the spin-1 mediator A′ from existing measurements of muon decay. The grey shaded region in the left frame is excluded by the BaBar search for e+e−→γ A′(A′ →invisible) [39], rescaled to e′ e = εe, while the red shaded region in the right frame is excluded by an analysis [9] of CCFR data on νN →νNμ−μ+. The green band in the right frame indicates parameters that bring the experimental and theoretical values of (g −2)μ within 2σ Fig. 7 90% c.l. upper bounds on the coupling constant between elec- tron or muon and the spin 0 mediator φ. The blue curves in both frames indicate the sensitivity derived from muon decay. 6 Conclusion Above this mass the DM particle χ doesn’t behave like a SIMP any more, since its relic density is greatly affected by annihilation into SM particles (namely e+e−pairs). Our analysis therefore excludes the electrophilic Co-SIMP scenario. In Figs. 6 and 7 we assumed that the mediator couples only to electrons or only to muons. The constraints from gμ −2 and from the BaBar and CCFR experiments are sensitive to only one of these couplings, and thus hold in full general- ity. Since meson decays into scalar mediators constrain the electron coupling much more than the muon coupling, they will remain essentially unchanged in models where both cou- plings are comparable. Data Availability Statement This manuscript has no associated data or the data will not be deposited. [Authors’ comment: There is no useful data to be deposited since the information is contained in the graphs. The custom code immediately generated the coupling-mass parameter pairs that are shown by the blue lines in Figs. 6 and 7.] Finally, constraints on the leptonic Co-SIMP scenario are summarized in Fig. 8. The blue curve indicates the value of required to obtain the correct relic density via χχe →χe reactions, which change the number of χ parti- cles without changing the number of SM particles; this is the essence of the Co-SIMP scenario of Ref. [7]. The constraint from K + →e+νe decays excludes this value of unless mχ > 0.8 MeV. Note that for mχ > 2me/3 another reac- tion becomes possible, χχχ →e+e−; however, this is not a SIMP scenario any more. Meson decays therefore exclude the whole region of parameter space where the correct χ relic density is determined by the Co-SIMP reaction.4 Open Access This article is licensed under a Creative Commons Attri- bution 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, pro- vide 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 indi- cated otherwise in a credit line to the material. 6 Conclusion In this paper we investigated the inﬂuence of leptophilic dark matter models on two well measured decays of Standard Model particles: deviations from the muon decay spectrum due to additional undetected particles and the removal of the helicity suppression in the leptonic decays of charged ground state pseudoscalar mesons. The measured muon decay spec- trum proved to be less sensitive to the new couplings than published searches at e+e−colliders and from neutrino trident production. Limits on the electrophilic scalar cou- pling derived from meson decays are an order of magni- tude stronger than those from muon decays but still weaker than existing limits by a factor of ∼2. Hence a moder- ate precision improvement in leptonic kaon decays could probe new regions of parameter space. On the other hand, the measurement of the muon decay spectrum would have to become a lot more accurate to yield competitive limits, which currently looks unlikely. The muonphilic scalar limit at mφ < 1 MeV is competitive with the recast search from Belle II at eμ ∼5·10−2, but lies well above the upper bound from the measurement of the magnetic dipole moment of the muon. Fig. 8 90% c.l. lower bounds on the scale parameter of the Co-SIMP model. Again the black dashed and dot-dashed lines indicate limits from pion and kaon decays respectively. The solid blue line indicate param- eter pairs that result in χh2 ≈0.12 via the freeze-out mechanism in minimal cosmology mφ ≤400 MeV the strongest bound originates from Kaon decays; decays of D+ and Ds mesons are considerably weaker, but extend to larger mediator masses. However, even the constraint from K + decays is somewhat weaker than that from an older BaBar search for e+e−→φγ with invisible φ. We do not show bounds from charged meson decays on the muon coupling. Since the helicity suppression of P+ →μ+νμ is much weaker than for the electron mode, the resulting bounds on e′ μ are considerably less stringent than those on e′ e, and are thus not competitive with the con- straint from the measurement of gμ −2. For the purely electrophilic Co-SIMP we showed that the model is ruled out for mχ < 800keV by the kaon decay branching ratio because at allowed couplings χ is overpro- duced by freeze-out. 4 In our calculation we neglected the chemical potential of the elec- trons. Since the electron asymmetry is, like the baryon asymmetry, only O(10−9), this will affect the required value of only at very small χ masses, where is already much below the bound from meson decays. 5 Results In the right frame the orange shaded region is excluded by a recast of a Belle II search for e+e−→μ+μ−A′, (A′ →invisible) [42], and the green band indicates parameters that bring the experimen- tal and theoretical values of (g −2)μ within 2σ Fig. 7 90% c.l. upper bounds on the coupling constant between elec- tron or muon and the spin 0 mediator φ. The blue curves in both frames indicate the sensitivity derived from muon decay. In the left frame, the dashed black, and solid yellow, red and orange lines indicate limits from additional contributions to P+ →e+νe decays (dashed black: P = π; yellow: P = K; orange: P = D; red: P = Ds). The dot-dashed black line represents the limits derived from the result of the search of PIENU for π+ →eνX; the drop of sensitivity at mφ ∼55 MeV is due to the signal being similar to π+ →μ+ →e+ [43]. The region shaded in gray in the left frame is excluded by an older BaBar limit recast to scalar mediators [44]. In the right frame the orange shaded region is excluded by a recast of a Belle II search for e+e−→μ+μ−A′, (A′ →invisible) [42], and the green band indicates parameters that bring the experimen- tal and theoretical values of (g −2)μ within 2σ 12 123 Page 9 of 11 1163 Eur. Phys. J. C (2022) 82 :1163 Fig. 8 90% c.l. lower bounds on the scale parameter of the Co-SIMP model. Again the black dashed and dot-dashed lines indicate limits from pion and kaon decays respectively. The solid blue line indicate param- eter pairs that result in χh2 ≈0.12 via the freeze-out mechanism in minimal cosmology References 19. J.P. Leveille, The second order weak correction to (G-2) of the muon in arbitrary gauge models. Nucl. Phys. B 137, 63–76 (1978). https://doi.org/10.1016/0550-3213(78)90051-2 1. P.A. Zyla et al., Review of particle physics. PTEP 2020(8), 083–01 (2020). https://doi.org/10.1093/ptep/ptaa104 20. Y. Kahn, G. Krnjaic, N. Tran, A. Whitbeck, M3: a new muon missing momentum experiment to probe (g −2)μ and dark mat- ter at Fermilab. JHEP 09, 153 (2018). https://doi.org/10.1007/ JHEP09(2018)153. arXiv:1804.03144 [hep-ph] 2. Aghanim, N., et al., Planck 2018 results. VI. Cosmological param- eters. Astron. Astrophys. 641, 6 (2020). https://doi.org/10.1051/ 0004-6361/201833910. arXiv:1807.06209 [astro-ph.CO]. [Erra- tum: Astron.Astrophys. 652, C4 (2021)] 21. B. Batell, N. Lange, D. McKeen, M. Pospelov, A. Ritz, Muon anomalous magnetic moment through the leptonic Higgs por- tal. Phys. Rev. D 95(7), 075003 (2017). https://doi.org/10.1103/ PhysRevD.95.075003. arXiv:1606.04943 [hep-ph] 3. L. Roszkowski, E.M. Sessolo, S. Trojanowski, WIMP dark mat- ter candidates and searches—current status and future prospects. Rep. Prog. Phys. 81(6), 066201 (2018). https://doi.org/10.1088/ 1361-6633/aab913. arXiv:1707.06277 [hep-ph] 22. J.R. Ellis, M.K. Gaillard, D.V. Nanopoulos, A phenomenological proﬁle of the Higgs boson. Nucl. Phys. B 106, 292 (1976). https:// doi.org/10.1016/0550-3213(76)90382-5 4. S. Knapen, T. Lin, K.M. Zurek, Light dark matter: models and constraints. Phys. Rev. D 96(11), 115021 (2017). https://doi.org/ 10.1103/PhysRevD.96.115021. (rXiv:1709.07882 [hep-ph]) 23. M.A. Shifman, A.I. Vainshtein, M.B. Voloshin, V.I. Zakharov, Low-energy theorems for Higgs boson couplings to photons. Sov. J. Nucl. Phys. 30, 711–716 (1979) 5. W. Altmannshofer, S. Gori, S. Profumo, F.S. Queiroz, Explain- ing dark matter and B decay anomalies with an Lμ −Lτ model. JHEP 12, 106 (2016). https://doi.org/10.1007/JHEP12(2016)106. arXiv:1609.04026 [hep-ph] 24. C.-Y. Chen, J. Kozaczuk, Y.-M. Zhong, Exploring leptophilic dark matter with NA64-μ. JHEP 10, 154 (2018). https://doi.org/10. 1007/JHEP10(2018)154. arXiv:1807.03790 [hep-ph] 6. Essig, R., et al.: Working group report: new light weakly coupled particles (2013) 25. J.M. Cline, H. Liu, T. Slatyer, W. Xue, Enabling forbidden dark matter. Phys. Rev. D 96(8), 083521 (2017). https://doi.org/10. 1103/PhysRevD.96.083521. arXiv:1702.07716 [hep-ph] 7. J. Smirnov, J.F. Beacom, New freezeout mechanism for strongly interacting dark matter. Phys. Rev. Lett. 125(13), 131301 (2020). https://doi.org/10.1103/PhysRevLett.125.131301. arXiv:2002.04038 [hep-ph] 26. A. Hillairet et al., Precision muon decay measurements and improved constraints on the weak interaction. Phys. Rev. D 85, 092013 (2012). https://doi.org/10.1103/PhysRevD.85.092013. arXiv:1112.3606 [hep-ex] 8. F. del Aguila, G.D. Coughlan, M. Quiros, Gauge coupling renor- malizationwithseveralU(1)factors.Nucl.Phys.B307,633(1988). https://doi.org/10.1016/0550-3213(88)90266-0. [Erratum: Nucl. Phys. B 312, 751 (1989)] 27. V. Shtabovenko, R. Mertig, F. Orellana, FeynCalc 9.3: new features and improvements. Comput. Phys. Commun. 256, 107478 (2020). References https://doi.org/10.1016/j.cpc.2020.107478. arXiv:2001.04407 [hep-ph] 9. W. Altmannshofer, S. Gori, M. Pospelov, I. Yavin, Neutrino tri- dent production: a powerful probe of new physics with neutrino beams. Phys. Rev. Lett. 113, 091801 (2014). https://doi.org/10. 1103/PhysRevLett.113.091801. arXiv:1406.2332 [hep-ph] 28. T. Kinoshita, Mass singularities of Feynman amplitudes. J. Math. Phys. 3, 650–677 (1962). https://doi.org/10.1063/1.1724268 29. T.D. Lee, M. Nauenberg, Degenerate systems and mass singular- ities. Phys. Rev. 133, 1549–1562 (1964). https://doi.org/10.1103/ PhysRev.133.B1549 10. P.S.B. Dev, B. Dutta, K.J. Kelly, R.N. Mohapatra, Y. Zhang, Light, long-lived B −L gauge and Higgs bosons at the DUNE near detector. JHEP 07, 166 (2021). https://doi.org/10.1007/ JHEP07(2021)166. arXiv:2104.07681 [hep-ph] 30. S. Scherer, Introduction to chiral perturbation theory. Adv. Nucl. Phys. 27, 277 (2003). arXiv:hep-ph/0210398 11. M. Escudero, D. Hooper, G. Krnjaic, M. Pierre, Cosmology with a very light Lμ −Lτ gauge boson. JHEP 03, 071 (2019). https:// doi.org/10.1007/JHEP03(2019)071. arXiv:1901.02010 [hep-ph] 31. J.M. Berryman, A. De Gouvêa, K.J. Kelly, Y. Zhang, Lepton- number-charged scalars and neutrino beam strahlung. Phys. Rev. D 97(7), 075030 (2018). https://doi.org/10.1103/PhysRevD.97. 075030. arXiv:1802.00009 [hep-ph] 12. P. Fayet, U-boson production in e+ e- annihilations, psi and Upsilon decays, and light dark matter. Phys. Rev. D 75, 115017 (2007). https://doi.org/10.1103/PhysRevD.75.115017. arXiv:hep-ph/0702176 32. V.D. Barger, W.-Y. Keung, S. Pakvasa, Majoron emission by neutrinos. Phys. Rev. D 25, 907 (1982). https://doi.org/10.1103/ PhysRevD.25.907 13. M. Pospelov, Secluded U(1) below the weak scale. Phys. Rev. D 80, 095002 (2009). https://doi.org/10.1103/PhysRevD.80.095002. arXiv:0811.1030 [hep-ph] y 33. P.S. Pasquini, O.L.G. Peres, Bounds on neutrino-scalar yukawa coupling. Phys. Rev. D 93(5), 053007 (2016). https://doi.org/10. 1103/PhysRevD.93.053007. arXiv:1511.01811 [hep-ph]. [Erra- tum: Phys.Rev.D 93, 079902 (2016)] 14. P. Foldenauer, Light dark matter in a gauged U(1)Lμ−Lτ model. Phys. Rev. D 99(3), 035007 (2019). https://doi.org/10.1103/ PhysRevD.99.035007. arXiv:1808.03647 [hep-ph] 34. A. Aguilar-Arevalo et al., Improved search for heavy neutrinos in the decay π →eν. Phys. Rev. D 97(7), 072012 (2018). https://doi. org/10.1103/PhysRevD.97.072012. arXiv:1712.03275 [hep-ex] 15. T.G. Rizzo, Kinetic mixing and portal matter phenomenology. Phys. Rev. D 99(11), 115024 (2019). https://doi.org/10.1103/ PhysRevD.99.115024. arXiv:1810.07531 [hep-ph] 35. J. Alwall, R. Frederix, S. Frixione, V. Hirschi, F. Maltoni, O. Mattelaer, H.-S. Shao, T. Stelzer, P. Torrielli, M. Zaro, The auto- mated computation of tree-level and next-to-leading order dif- ferential cross sections, and their matching to parton shower simulations. J High Energy Phys (2014). https://doi.org/10.1007/ jhep07(2014)079 16. E. Izaguirre, G. Krnjaic, P. Schuster, N. Toro, Testing GeV- scale dark matter with ﬁxed-target missing momentum experi- ments. Phys. Rev. D 91(9), 094026 (2015). https://doi.org/10.1103/ PhysRevD.91.094026. 6 Conclusion 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 permit- ted use, you will need to obtain permission directly from the copy- right holder. To view a copy of this licence, visit http://creativecomm ons.org/licenses/by/4.0/. g y Funded by SCOAP3. SCOAP3 supports the goals of the International Year of Basic Sciences for Sustainable Development. 12 123 1163 Page 10 of 11 Eur. Phys. J. C (2022) 82 :1163 42. B. Zhu, X. Liu, Probing the ﬂavor-speciﬁc scalar media- tor for the muon (g - 2) deviation, the proton radius puz- zle and the light dark matter production. Sci. China Phys. Mech. Astron. 65(3), 231011 (2022). https://doi.org/10.1007/ s11433-021-1819-5. arXiv:2104.03238 [hep-ph] References arXiv:1411.1404 [hep-ph] 17. T. Aoyama et al., The anomalous magnetic moment of the muon in the Standard Model. Phys. Rept. 887, 1–166 (2020). https://doi. org/10.1016/j.physrep.2020.07.006. arXiv:2006.04822 [hep-ph] 36. C. Lazzeroni et al., Precision measurement of the ratio of the charged kaon leptonic decay rates. Phys. Lett. B 719, 326–336 (2013). https://doi.org/10.1016/j.physletb.2013.01.037. arXiv:1212.4012 [hep-ex] 18. B. Abi et al., Measurement of the positive muon anoma- lous magnetic moment to 0.46 ppm. Phys. Rev. Lett. 126(14), 141801 (2021). https://doi.org/10.1103/PhysRevLett.126.141801. arXiv:2104.03281 [hep-ex] 37. B.I. Eisenstein et al., Precision measurement of B(D+ –> mu+ nu) and the pseudoscalar decay constant f(D+). Phys. Rev. D 78, 052003 (2008). https://doi.org/10.1103/PhysRevD.78.052003. arXiv:0806.2112 [hep-ex] 12 3 Eur. Phys. J. C (2022) 82 :1163 Page 11 of 11 1163 42. B. Zhu, X. Liu, Probing the ﬂavor-speciﬁc scalar media- tor for the muon (g - 2) deviation, the proton radius puz- zle and the light dark matter production. Sci. China Phys. Mech. Astron. 65(3), 231011 (2022). https://doi.org/10.1007/ s11433-021-1819-5. arXiv:2104.03238 [hep-ph] 38. A. Zupanc et al., Measurements of branching fractions of lep- tonic and hadronic D+ s meson decays and extraction of the D+ s meson decay constant. JHEP 09, 139 (2013). https://doi.org/10. 1007/JHEP09(2013)139. arXiv:1307.6240 [hep-ex] 39. J.P. Lees et al., Search for invisible decays of a dark photon produced in e+e−collisions at BaBar. Phys. Rev. Lett. 119(13), 131804 (2017). https://doi.org/10.1103/PhysRevLett.119.131804. arXiv:1702.03327 [hep-ex] 43. A. Aguilar-Arevalo et al., Search for three body pion decays π+→l+νX. Phys. Rev. D 103(5), 052006 (2021). https://doi.org/ 10.1103/PhysRevD.103.052006. arXiv:2101.07381 [hep-ex] 44. R. Essig, J. Mardon, M. Papucci, T. Volansky, Y.-M. Zhong, Constraining light dark matter with low-energy e+e−colliders. JHEP 11, 167 (2013). https://doi.org/10.1007/JHEP11(2013)167. arXiv:1309.5084 [hep-ph] 40. S.R. Mishra et al., Neutrino tridents and W Z interference. Phys. Rev. Lett. 66, 3117–3120 (1991). https://doi.org/10.1103/ PhysRevLett.66.3117 41. I. Adachi et al., Search for an invisibly decaying Z′ boson at Belle II in e+e−→μ+μ−(e±μ∓) plus missing energy ﬁnal states. Phys. Rev. Lett. 124(14), 141801 (2020). https://doi.org/10.1103/ PhysRevLett.124.141801. arXiv:1912.11276 [hep-ex] 123 123 123
https://openalex.org/W3092760518	https://www.nature.com/articles/s41598-021-98434-6.pdf	English	null	Evaluation of Reference Genes for qRT-PCR Normalization in Angelica decursiva under Various Experimental Conditions	null	2,020	cc-by	7,932	Evaluation of Angelica decursiva reference genes under various stimuli for RT‑qPCR data normalization Yuedong He1,2,4, Yuan Zhong1,4, Zhenzhen Bao1, Weiqi Wang1, Xiaoqing Xu1, Yanan Gai3* & Jie Wu1* Angelica decursiva is one of the lending traditional Chinese medicinal plants producing coumarins. Notably, several studies have focused on the biosynthesis and not the RT-qPCR (quantitative real- time reverse transcription polymerase chain reaction) study of coumarins. This RT-qPCR technique has been extensively used to investigate gene expression levels in plants and the selection of reference genes which plays a crucial role in standardizing the data form the RT-qPCR analysis. In our study, 11 candidate reference genes were selected from the existing transcriptome data of Angelica decursiva. Here, four different types of statistical algorithms (geNorm, NormFinder, BestKeeper, and Delta Ct) were used to calculate and evaluate the stability of gene expression under different external treatments. Subsequently, RefFinder analysis was used to determine the geometric average of each candidate gene ranking, and to perform comprehensive index ranking. The obtained results showed that among all the 11 candidate reference genes, SAND family protein (SAND), protein phosphatase 2A gene (PP2A), and polypyrimidine tract-binding protein (PTBP) were the most stable reference genes, where Nuclear cap binding protein 2 (NCBP2), TIP41-like protein (TIP41), and Beta-6-tubulin (TUBA) were the least stable genes. To the best of our knowledge, this work is the first to evaluate the stability of reference genes in the Angelica decursiva which has provided an important foundation on the use of RT-qPCR for an accurate and far-reaching gene expression analysis in this medicinal plant. Angelica decursiva (Miq.) Franch. et Sav. is an important traditional medicinal plant form the genus Angelica, family Umbelliferae. The major bioactive components of this herbal drug are coumarins and this drug has been listed as one of the main special coumarin sources by the Chinese Pharmacopoeia. Coumarin has a core struc- ture (2H-1-benzopyran-2-one core), that widely exists in higher plant species like the Rutaceae, Leguminosae, and Umbelliferae. Besides, they are slightly distributed in both animals and microorganisms1. Recent pharma- cological studies have proved that the 70% ethanol extract of A. decursiva has an anti-inflammatory activity2,3, antioxidant4,5, inhibitory effect on C6 rat glioma cells6 and inhibits the proliferation of osteogenic sarcoma cells7. Hence, certifying the supply of coumarin compounds from the drug has been a long-term practice8. Conversely, there is a huge shortage of coumarin raw materials, which has been attributed to low abundance, season, or region-dependent sourcing. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Evaluation of Angelica decursiva reference genes under various stimuli for RT‑qPCR data normalization Also, because of its complicated structure and multiple chiral centers9, there lacks a feasible method that has the capacity of mass chemical synthesis. Therefore, genetic and metabolic engineering strategies used in the production of coumarin compounds have become the main developing research direction. Subsequently, the study of functional genes that are present in their biosynthetic pathways is a study of interest. q y y g p y p y y In recent years, the rapid development of next-generation sequencing (NGS) technologies such as Roche / 454 and Illumina HiSeq platforms, has made it possible to study the distribution of mRNA and their expres- sion levels in different tissues and cells. Currently, due to its high sensitivity, accurate quantification, and strong specificity, the RT-qPCR technology has become one of the most powerful tools for studying gene transcription levels. Besides, it quantifies the relative abundance and improves the quantitative accuracy of target genes10–12. Nonetheless, the accuracy of this technique is affected by several factors13, like the amplification efficiency of the 1School of Pharmacy, Jiangsu Health Vocational College, Nanjing, Jiangsu, China. 2College of Horticulture, Hunan Agricultural University, Changsha, Hunan, China. 3Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, Jiangsu, China. 4These authors contributed equally: Yuedong He and Yuan Zhong. *email: gaiyanan@cnbg.net; wujie1760@126.com \| https://doi.org/10.1038/s41598-021-98434-6 Scientific Reports \| (2021) 11:18993 www.nature.com/scientificreports/ Table 1. Details of primers used in this study. Gene abbreviation Gene name Arabidopsis homolog locus Primer sequences (5′–3′) (forward/ reverse) length Tm (°C) PCR efficiency R2 CYP2 Cyclophilin 2 AT4G33060 CGCCACTTTTTTTGTTCTCT/ TTGCGGATTATATTCCGACA 108 82.9 1.711 0.971 EXP-1 S-adenosyl-l-methionine-dependent methyltransferases superfamily protein AT2G32170 CCAAGTAGGAGCTTGGGATG/ CACACCACCGTCCTTTAGAA 109 81.7 1.789 0.958 GAPDH Glyceraldehyde 3-phosphate dehy- drogenase AT1G42970 TGGTTCCCTTAACGATACCA/ CTTACGTTGTTGGCGTGAAT 138 82.1 1.713 0.999 NCBP2 Nuclear cap binding protein 2 AT5G44200 TAGATGGCATGACAGGTGGA/ CAGGTACGCGATGAATATCG 145 85.6 1.333 0.993 PP2A protein phosphatase 2A subunit A3 AT1G13320 GGTTGCCAATCAGCTCTATG/ AACTTGGTGACTTTGCCAGC 141 85 1.880 1.000 PTBP Polypyrimidine tract-binding protein AT3G01150 AATGTAAAGGCCTTCAGCGA/ GGAGCAGCATGAGGATTCTG 110 85.2 1.847 1.000 SAND SAND family protein AT2G28390 GAGCCTCATGAATCCCTCAG/ CCCAAGCAAAGGTGTCATAT 126 82.1 1.845 0.972 TIP41 TIP41-like family protein AT4G34270 CACTTGCATCAAAAGAGCCT/ GAAGAAACCAACAGCTTGGC 126 82.7 1.838 1.000 UBQ10 Polyubiquitin 10 AT4G05320 TTAAGCAGGGATCAAAACCC/ TTTGCTGGAAAGCAGCTAGA 124 84.6 1.721 0.888 ACT Actin 2 AT3G18780 TGCTGGTCGTGATCTCACTG/ GGTTTCAAGCTCTTGCTCGT 148 85.2 1.850 1.000 TUBA Beta-6-tubulin AT5G12250 CACCCAGCTTTGGTGATCTC/ AAGCCTAGGGAAGGGGATTA 134 86 1.685 0.999 Table 1. Details of primers used in this study. target gene, the RNA yield, quality, and the reverse transcription efficiency that is present in different samples. Evaluation of Angelica decursiva reference genes under various stimuli for RT‑qPCR data normalization Therefore, to overcome experimental errors and assure accuracy of experimental results, a certain number of internal reference genes, also called housekeeping genes, are often selected for calibration and standardization14,15. There genes are usually used as a reference for either tissues or cells when they are stably expressed in a diversity of experimental conditions, and when changes are detected in the expression level of a target gene. Among the most commonly used internal reference genes for RT-qPCR are Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Elongation factor-1α (EF-1α), Tubulin (TUB), β-Actin (ACT), 18S ribosomal RNA protein (18S), Polyubiquitin 10 (UBQ10), etc. These housekeeping genes play a significant role in the cell structure maintenance and during primary metabolic activities. Moreover, more internal reference genes have been identified from the gene expression chip data. They include protein phosphatase 2A (PP2A), SAND family protein (SAND), Tap42- interacting protein of 41 kDa (TIP41), etc. Consequently, the reference gene screening and evaluation have been accomplished for most species, such as rice16, Lycoris aurea17, radish18, potato19, Peucedanum praeruptorum20, etc. However, no study has demonstrated a systematic selection of reference genes in A. decursiva under exter- nal challenges (abiotic stress and hormone treatments). The study of the coumarin biosynthesis pathway is one of our primary research interests relates. Here, previous studies have characterized some candidate genes that associate with A. decursiva8. However, it is imperative to find suitable reference genes, that enhance the detection of differential gene expression levels under various experimental conditions in A. decursiva using the RT-qPCR application. pp In our study, 11 candidate reference genes (SAND, PP2A, PTBP, ACT, CYP2, EXP-1, GAPDH, TUBA, NCBP2, UBQ10, and TIP41) were selected about the transcriptome sequencing datasets of A. decursiva. Besides, 7 dif- ferent forms of treatments such as cold (4 °C), drought (20% PEG 6000), methyl jasmonate (25 mM MeJA), salt (600 mM NaCl), oxidative (50 mM H2O2), ultraviolet (UV) induction, and metal (500 mM CuSO4) were set. Next, the application of four statistical algorithms (geNorm, NormFinder, BestKeeper, and Delta Ct) to evaluate their expression stability for normalization, and comprehensive stability ranking was also performed by RefFinder. In conclusion, our work provides a basis for further studies on gene expression profiling and the regulation mechanisms of coumarin biosynthesis in A. decursiva under diverse experimental conditions. Results l i Selection of candidate reference genes, evaluation of amplification specificity and PCR effi‑ ciency. Here, 11 candidate reference genes were selected based on the transcriptome data of A. decursiva (unpublished). Table 1 lists all the candidate reference gene names and abbreviations, homologous genes from Arabidopsis, primer sequences, amplification length, annealing temperature (°C), PCR efficiency (E), and corre- lation coefficient (R2). Next, conventional PCR and RT-qPCR were used to verify the primer-specific amplifica- tion of all candidate reference genes. As illustrated in Fig. S1, based on agarose gel electrophoresis and melting curve analysis which showed a peak, the 11 primer pairs were amplified by a single specific amplicon. Here, the amplification efficiency range of CYP2 and PP2A was 1.711 and 1.880 respectively whereas the correlation coef- ficient range of UBQ10 was 0.888, and that of PP2A, PTBP, TIP41, and ACT was 1.000. Scientific Reports \| (2021) 11:18993 \| https://doi.org/10.1038/s41598-021-98434-6 www.nature.com/scientificreports/ Figure 1. The RT-qPCR Cp values for 11 candidate reference genes in all samples. The expression data is shown as the Cp value of each reference gene in the samples of A. decursiva. Boxes indicate the 25th/75th percentiles, the lines represent the median, squares represent the means and whiskers represent the maximum and minimum values. Figure 1. The RT-qPCR Cp values for 11 candidate reference genes in all samples. The expression data is shown as the Cp value of each reference gene in the samples of A. decursiva. Boxes indicate the 25th/75th percentiles, the lines represent the median, squares represent the means and whiskers represent the maximum and minimum values. Expression profile of candidate reference genes. Here, the cycle threshold values (Cp) showed the number of cycles when the generated fluorescent signal reached a level that could be detected. Therefore, in this study, the expression profile of the candidate reference genes were directly determined using the obtained Cp values. As shown in Fig. 1, the average Cp values of these 11 reference genes are distributed between 10 and 30, and a majority of them are distributed between 23 and 27. Among all the candidate reference genes, NCBP2 had the lowest average Cp values, whereas CYP2, PTBP, and TUBA had the highest average Cp values. Moreover, each reference gene had a different coefficient of variation under different conditions. Also, it was observed that SAND and PTBP had the lowest variability, whereas TUBA, which had the highest Cp value, had the highest variability. Results l i This value ranged between 25 and 32. Therefore, it may not be qualified as a reference gene. Through the Cp value combined with box-plot not only display the expression profile of the reference gene, but also give us a glimpse in their stability (Table S2). However, considering the complexity of their surroundings, we need to check the proper use of the references in every particular experimental condition. Thus, more statistical tools and further analyses are required. Expression stability of candidate reference genes. Based on the relative expression levels of the 11 candidate reference genes, four algorithms such as BestKeeper, geNorm, NormFinder, and Delta Ct were used to examine their expression stability. Subsequently, the RefFinder tool was employed to sequence the expression stability of all these candidate reference genes and select the most suitable ones.h y g geNorm analysis: The geNorm analysis evaluated the stability of all the 11 candidate reference genes using the M value (reference expression stability measure). These M values were calculated from the average variation of the gene relative against other candidate reference genes, and the lower M values indicated a higher gene expression stability21. As illustrated in Fig. 2, all the candidate reference genes had different levels of stability under different treatments. Here, the M value of PP2A and SAND was the lowest in most treatments and was deliberated as the most stable reference genes. Besides, ACT showed good stability under H2O2, MeJA, NaCl, and UV treatments, whereas CYP2 was one of the most stable reference genes in the CuSO4 and UV treatment groups. On the other hand, the stability of NCBP2 seemed to be less satisfactory than other candidate reference genes. Subsequently, the geNorm algorithm can also determine the optimal number of normalized reference genes21 by calculating pairwise mutations (Vn/n+1). Normally, the ideal paired variation (V) score is less than 0.15, which means that the addition of any other gene will not have a substantial impact on standardization. In our study, in the NaCl, Cold, UV, H2O2, CuSO4, and WT subsets, their pairwise variation values (V 2/3) were all less than 0.15. This showed that the addition of a third reference gene lacked significant effects on the normalization of the target gene, thus, the number of the optimal reference genes that were determined was two. In contrast, as shown in Fig. Results l i 3 and Table S3, the pairwise variation values (V 3/4) of the PEG and MeJA subsets, was also less than 0.15. This indicated that the number of reference genes that were most suitable for these two treatments was three. h g NormFinder analysis: To normalize raw data and measure the expression stability of candidate reference genes through intra- and inter-group variation, the NormFinder analysis uses the 2−ΔCt method. Like the geNorm analysis, its lower values show higher stability. Table 2 shows the ranking of all candidate genes as calculated using the NormFinder algorithm. Among the PEG, Cold, and ’total’ treatment subsets, it was observed that SAND was the most stable candidate gene, and also ranked higher in other subsets. After MeJA treatment (0.078) on all sample subsets, TUBA was deliberated as the most stable candidate gene. Besides, among the other five groups (NaCl, UV, H2O2, CuSO4, and WT), PTBP (0.670), TIP41 (0.307), GAPDH (0.495), CYP2 (0.483), and UBQ10 (0.451) were observed to be the most stable genes, respectively. Similar to the geNorm analysis, our study found that the NCBP2 was the most unstable candidate gene. Scientific Reports \| (2021) 11:18993 \| https://doi.org/10.1038/s41598-021-98434-6 www.nature.com/scientificreports/ p / Figure 2. Average expression stability values (M) of the 11 candidate reference genes using geNorm software Expression stability was evaluated in samples from A. decursiva was submitted to cold stress, drought stress (20% PEG), Methyl jasmonate (MeJA) stress, salt stress (0.5 M NaCl), oxidative stress (H2O2), ultraviolet (UV induction, Metal stress (CuSO4), untreated (WT) and ’total’ (all treated samples). The least stable genes are on the left with higher M-value and the most stable genes on the right with lower M-value. Figure 2. Average expression stability values (M) of the 11 candidate reference genes using geNorm software. Expression stability was evaluated in samples from A. decursiva was submitted to cold stress, drought stress (20% PEG), Methyl jasmonate (MeJA) stress, salt stress (0.5 M NaCl), oxidative stress (H2O2), ultraviolet (UV) induction, Metal stress (CuSO4), untreated (WT) and ’total’ (all treated samples). The least stable genes are on the left with higher M-value and the most stable genes on the right with lower M-value. Figure 2. Average expression stability values (M) of the 11 candidate reference genes using geNorm software. Expression stability was evaluated in samples from A. Results l i decursiva was submitted to cold stress, drought stress (20% PEG), Methyl jasmonate (MeJA) stress, salt stress (0.5 M NaCl), oxidative stress (H2O2), ultraviolet (UV) induction, Metal stress (CuSO4), untreated (WT) and ’total’ (all treated samples). The least stable genes are on the left with higher M-value and the most stable genes on the right with lower M-value. BestKeeper analysis: Here, the raw data of the Ct value was directly calculated and the more stable candidate reference genes was evaluated by calculating the standard deviation (SD) and the Ct value22. Notably, lower SD and Ct values indicated higher gene expression stability, particularly when the SD was greater than 1 which indicated that the reference gene was unstable23 and could not be utilized for normalization. Table 3 shows that in the NaCl, MeJA, UV, WT, and ’total’ treatment subsets, the PTBP gene was considered the most stable. How- ever, the MeJA and ’total’ treatment subsets were postulated to be expression-insensitive since their SD values were exceed 1 and could not be used for normalization. For this reason, such values should be excluded. Besides, Scientific Reports \| (2021) 11:18993 \| https://doi.org/10.1038/s41598-021-98434-6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Determination of the optimal numbers of reference genes for normalization by pairwise variation (Vn/n+1). Different treatments are marked as square frame with different colors. The cut-off value is 0.15 and used to determine the optimal number of candidate reference genes for qRT-PCR normalization. Figure 3. Determination of the optimal numbers of reference genes for normalization by pairwise variation (Vn/n+1). Different treatments are marked as square frame with different colors. The cut-off value is 0.15 and used to determine the optimal number of candidate reference genes for qRT-PCR normalization. Results l i Table 2 Stability rank of 11 candidate genes by NormFinder Rank NaCl PEG Cold MeJA UV H2O2 CuSO4 WT Total 1 PTBP SAND SAND TUBA TIP41 GAPDH CYP2 UBQ10 SAND (0.670) (0.319) (0.146) (0.078) (0.307) (0.495) (0.483) (0.451) (0.568) 2 PP2A PP2A ACT PTBP) PTBP PTBP SAND SAND PP2A (0.672) (0.399) (0.313) (0.452 (0.606) (0.528) (0.500) (0.656) (0.624) 3 ACT UBQ10 TUBA SAND CYP2 TIP41 UBQ10 GAPDH PTBP (0.676) (0.560) (0.407) (0.472) (0.615) (0.594) (0.535) (0.672) (0.696) 4 SAND ACT PP2A GAPDH PP2A PP2A GAPDH TIP41 ACT (0.714) (0.616) (0.418) (0.502) (0.744) (0.706) (0.571) (0.692) (0.772) 5 TIP41 EXP-1 PTBP UBQ10 SAND SAND ACT PTBP GAPDH (0.719) (0.739) (0.493) (0.502) (0.776) (0.766) (0.597) (0.699) (0.918) 6 UBQ10 PTBP CYP2 PP2A ACT CYP2 PP2A PP2A UBQ10 (0.730) (0.869) (0.667) (0.641) (0.851) (0.855) (0.604) (0.728) (1.198) 7 GAPDH GAPDH GAPDH ACT GAPDH UBQ10 TIP41 CYP2 CYP2 (0.999) (0.957) (0.998) (0.675) (0.954) (0.960) (0.773) (0.815) (1.275) 8 CYP2 TIP41 UBQ10 TIP41 UBQ10 ACT PTBP ACT EXP-1 (1.014) (1.086) (1.032) (0.699) (1.176) (1.044) (1.075) (1.011) (1.373) 9 EXP-1 TUBA EXP-1 CYP2 EXP-1 TUBA EXP-1 TUBA TIP41 (1.075) (1.398) (1.505) (1.085) (1.210) (1.284) (1.222) (1.089) (1.422) 10 TUBA CYP2 NCBP2 EXP-1 NCBP2 EXP-1 TUBA EXP-1 TUBA (1.528) (2.677) (2.711) (1.324) (3.510) (1.291) (2.200) (2.013) (2.635) 11 NCBP2 NCBP2 TIP41 NCBP2 TUBA NCBP2 NCBP2 NCBP2 NCBP2 (2.918) (3.037) (3.636) (3.498) (6.852) (3.698) (2.350) (2.764) (3.449) Table 2. Stability rank of 11 candidate genes by NormFinder. under H2O2, CuSO4, Cold, and PEG treatments, the most stable genes were observed to be CYP2, SAND, TUBA, and PP2A. Generally, as illustrated in Table 3, it was observed that the NCBP2 gene was still the most unstable under all treatments.h Delta Ct analysis: This method evaluated gene expression stability by calculating the mean standard devia- tion (SD) of each gene. Here, the smaller the value, the higher the stability24. As shown in Table S4, the results of this analysis are consistent with the geNorm analysis. The only difference is that in the Cold and H2O2 subsets, TUBA and PP2A candidate genes are the most stable respectively. According to the geNorm analysis, the two most stable candidate genes are SAND and ACT. Hence, SAND and PP2A are the most qualified reference genes. i RefFinder analysis: As shown in Fig. 4, we further calculated the geometric mean of the ranking of each candi- date gene using the RefFinder algorithm (http://150.216.56.64/referencegene.php#). Results l i This was based on the results obtained from the three statistical algorithms such as geNorm, NormFinder, and BestKeeper. Tables S5 and S6, show the comprehensive index ranking, whereby, the smaller the index, the more stable the gene expression19. Results l i Scientific Reports \| (2021) 11:18993 \| https://doi.org/10.1038/s41598-021-98434-6 www.nature.com/scientificreports/ k f d d b Rank NaCl PEG Cold MeJA UV H2O2 CuSO4 WT Total 1 PTBP PP2A TUBA PTBP PTBP CYP2 SAND PTBP PTBP 0.97 ± 0.29 2.73 ± 0.65 2.50 ± 0.68 4.22 ± 1.23 0.54 ± 0.15 3.45 ± 1.07 1.04 ± 0.26 1.10 ± 0.30 4.63 ± 1.32 2 PP2A PTBP CYP2 SAND SAND SAND CYP2 SAND SAND 1.76 ± 0.46 2.89 ± 0.82 2.76 ± 0.80 4.92 ± 1.30 0.87 ± 0.22 3.70 ± 1.03 1.13 ± 0.31 1.17 ± 0.28 5.40 ± 1.40 3 SAND SAND PTBP TUBA PP2A TIP41 UBQ10 ACT EXP-1 2.23 ± 0.63 3.17 ± 0.81 3.44 ± 0.94 6.08 ± 1.70 0.89 ± 0.20 3.98 ± 1.13 1.83 ± 0.42 1.19 ± 0.23 6.33 ± 1.70 4 ACT EXP-1 GAPDH TIP41 CYP2 PP2A PP2A PP2A PP2A 2.32 ± 0.56 3.88 ± 1.04 3.92 ± 0.98 6.30 ± 1.69 1.21 ± 0.33 4.27 ± 1.11 2.03 ± 0.47 1.34 ± 0.30 6.34 ± 1.52 5 CYP2 UBQ10 ACT UBQ10 ACT PTBP ACT CYP2 CYP2 2.48 ± 0.77 4.52 ± 1.05 4.07 ± 0.86 6.35 ± 1.65 1.49 ± 0.32 4.27 ± 1.30 2.51 ± 0.51 2.73 ± 0.73 6.35 ± 1.84 6 UBQ10 GAPDH SAND GAPDH TIP41 GAPDH PTBP UBQ10 GAPDH 2.91 ± 0.74 4.52 ± 1.19 4.31 ± 1.08 6.39 ± 1.71 3.90 ± 0.98 4.44 ± 1.29 2.54 ± 0.71 3.20 ± 0.68 6.88 ± 1.82 7 EXP-1 ACT PP2A CYP2 UBQ10 ACT TIP41 TUBA TIP41 3.16 ± 0.88 4.81 ± 1.05 5.04 ± 1.17 6.46 ± 1.94 3.94 ± 0.87 4.87 ± 1.18 3.09 ± 0.74 3.63 ± 0.92 7.27 ± 1.90 8 TIP41 TIP41 EXP-1 PP2A GAPDH UBQ10 GAPDH TIP41 ACT 3.96 ± 1.12 5.19 ± 1.35 5.15 ± 1.39 6.80 ± 1.70 4.52 ± 1.15 5.05 ± 1.34 3.30 ± 0.83 4.05 ± 0.99 7.30 ± 1.59 9 TUBA TUBA UBQ10 ACT EXP-1 TUBA EXP-1 GAPDH UBQ10 4.32 ± 1.28 5.87 ± 1.64 5.91 ± 1.47 7.24 ± 1.62 5.09 ± 1.36 6.37 ± 1.93 4.10 ± 1.03 4.15 ± 1.01 7.30 ± 1.76 10 GAPDH CYP2 TIP41 EXP-1 TUBA EXP-1 TUBA EXP-1 TUBA 4.36 ± 1.27 6.49 ± 1.93 9.03 ± 2.34 7.96 ± 2.20 15.44 ± 3.98 6.71 ± 1.93 5.34 ± 1.44 7.75 ± 1.94 7.30 ± 2.02 11 NCBP2 NCBP2 NCBP2 NCBP2 NCBP2 NCBP2 NCBP2 NCBP2 NCBP2 23.24 ± 2.56 21.11 ± 2.17 17.41 ± 1.52 21.69 ± 2.06 27.51 ± 3.12 24.04 ± 4.27 19.57 ± 1.98 22.34 ± 2.55 8.19 ± 0.82 Figure 4. Table 3. Rank of 11 candidate genes by BestKeeper. Discussion l Currently, a majority of medicinal plants enhance the bioactivity of their medicinal components by inducing and regulating functional genes present in their biosynthetic pathway25. Several reports have shown that in plants, there is a significant correlation between the synthesis and accumulation of secondary metabolites and the expression levels found in the functional pathways of their biosynthetic pathways26,27. Coumarins are one of the main active components extracted from A. decursiva, which is one of the main sources of coumarin found in China and is listed as a special coumarin resource by the Chinese Pharmacopoeia. However, there exist no previous reports on RT-qPCR studies, which confines the research on the biosynthetic pathway of coumarin compounds from A. decursiva. p Here, we screened a total of 11 potential reference genes from the transcriptome data of A. decursiva (unpub- lished). First, as shown in Table 1 and S1, their primer specificity and PCR experimental conditions were con- firmed, whereby the single peak of the dissolution curve exposed the specificity of these 11 pairs of primers (Fig. S1). As illustrated in Fig. 1 and Table S2, the average Ct-values for the 11 candidate reference genes is between 8.04 and 32.65, and the majority of these values were found to be between 23 and 27, which means that most of the 11 candidate reference genes were likely to afford accurate normalization20. The SAND and PTBP genes have the lowest variability, which suggests that they could have stable expression levels under different treatments. Conversely, considering the complexity of the ambient environment, the expression stability of these reference genes under different conditions must be investigated further28. Therefore, there is a need to use more statistical tools and conduct further analyses. y To ensure the accuracy and reliability of experimental data, four commonly used statistical algorithms (geNorm, NormFinder, BestKeeper, and Delta Ct) were combined for reference gene selection. Subsequently, RefFinder analysis was used to calculate the geometric ranking mean of each candidate reference gene, and then a comprehensive index ranking was done. Here, the results from this analysis showed that the ranking results acquired using different statistical algorithms were not similar, and the candidate reference genes also showed different levels of stability under different treatments. Discussion l For example, Table 2 shows that TUBA is the best reference gene in the MeJA subset, and this is consistent with the results obtained when Atropa belladonna29 was exposed to different hormone treatments which showed that it had the most stable expression. On the other hand, when this gene was exposed to other treatments, its stability was unsatisfactory. This is most likely because some genes belonging to the tubulin family and that express isomers in specific developmental stages or tissues are regulated by different developmental processes30. Similar to the results from tomato31 and Arabidopsis thaliana32, UBQ10 is the most stable gene in the WT group (Table 2). Also, the PTBP gene performed well in the UV subset, unlike in the Cold, PEG, and CuSO4 which is illustrated on Table 2 and S5). This result contrasted with the most stable performance of the PTBP gene in Cold, PEG, and CuSO4 subsets in Peucedanum praeruptorum20. Table 3 shows that under cold stress (4 °C) and the ’total’ group, the TUBA and PTBP genes were the most stable reference genes, respectively, but this was different from the results obtained from geNorm and NormFinder analyses which showed that SAND was the gene with the best stability. Since various calculation principles are inconsist- ent, even under the same conditions, the ranking of these genes could be different. Therefore, the single software analysis has certain disadvantages when evaluating the stability of the reference gene. Our study recommends that a comprehensive evaluation and analysis need to be used to ensure the reliability and accuracy of the results. Besides, it is necessary to flexibly select the most appropriate reference gene for different experimental treatments. yl y pp p gf p Also, bearing in mind that a single reference gene could cause errors in the expression level of the target gene33,34, the geNorm statistical algorithm calculates the pairwise variation (Vn/n+1) to attain the optimal number of reference genes35. Notably, the ideal pairwise variation value must be lower than the critical value of 0.1533,36. For instance, as illustrated in Fig. 3 and Table S3, the pairwise variation V 2/3 in the Cold, NaCl, CuSO4, H2O2, UV, WT, and the ’total’ treatment subsets, is less than 0.15. This demonstrated that only two genes are required to complete the gene expression normalization under these conditions. Discussion l Consequently, it was observed that the PP2A and ACT are relatively stable reference genes under the treatment of MeJA, their pairing value V 3/4 was < 0.15, and their optimal number of reference genes was three. g Taken together, the analysis results from the three algorithms (geNorm, NormFinder, and Delta Ct) seemed to be consistent, whereas those of BestKeeper are different. Figure 4 and Table S5, confirm that under most exter- nal conditions, the stability of SAND and PP2A genes are significantly better than that of other reference genes, whereas NCBP2 and TUBA are nearly always defined as the most unstable reference genes. Of note, the SAND and PP2A are new reference genes that were identified through screening the data from the Arabidopsis gene chip. To date, many studies choose the SAND and PP2A genes for their reference gene standardization studies. For instance, studies that used Arabidopsis thaliana32, tomato37, Petunia hyrbrida38, Peucedanum praeruptorum20, etc. Conventionally, reference genes, such as TUBA, GAPDH, UBQ10, and ACT play a housekeeping role in the maintenance of cell structure or primary metabolic activities. Nevertheless, increasing research indicates that the expression levels in most of these traditional reference genes can somehow vary greatly and in many species, they are unsuitable for gene normalization under specific conditions39,40. Therefore, given a subset of experimental conditions, the housekeeping genes used as reference genes in each different species should be handled carefully41,42. Results l i Comprehensive ranking of BestKeeper, NormFinder, and geNorm. Using BestKeeper, NormFinder, and geNorm algorithms, the 11 candidate reference genes were ranked comprehensively, and the geometric mean of each reference gene was calculated. Figure 4. Comprehensive ranking of BestKeeper, NormFinder, and geNorm. Using BestKeeper, NormFinder, and geNorm algorithms, the 11 candidate reference genes were ranked comprehensively, and the geometric mean of each reference gene was calculated. This study showed that SAND and PP2A ranked the highest in most subsets, whereas NCBP2 and TUBA ranked the lowest, making them the most unstable reference genes. In contrast, in the MeJA and Cold subsets, TUBA seemed to be a relatively stable reference gene. Despite the different assessment methods, this resulting difference is reasonable and acceptable. In summary, the stability of these 11 candidate reference genes from the highest to the lowest is: SAND, PP2A, PTBP, ACT, CYP2, EXP-1, GAPDH, UBQ10, TIP41, TUBA, and NCBP2. These results were similar to those obtained from the geNorm and NormFinder analysis, but slightly different from those of the BestKeeper analysis. https://doi.org/10.1038/s41598-021-98434-6 Scientific Reports \| (2021) 11:18993 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ tion of plant material is in full compliance with relevant institutions, national and international guidelines and legislation. Then, these accessions were transplanted into plastic pots that contained a mixture of vermiculite, perlite, and peat moss (1:1:1 v/v). Next, the plants were grown in a greenhouse at a temperature of 25 °C, a long photoperiod of 16 h light and 8 h darkness, 40–65% relative humidity, and 3000 lx light intensity until treated. For drought treatments, a 200 mL solution of 25% PEG 6000 (w / v, polyethylene glycol, Sangon, China) was used to treat the plants for one week. For salt stress treatment, an approximate amount of 200 mL (600 mM) of NaCl was used to water the plants for 7 days. For cold stimulation treatments, the plants were incubated at 4 °C for 48 h. To study hormone therapy, 25 mM MeJA was applied for 6 h. To assess heavy metal stress, 500 mM of Copper sulfate (CuSO4) was applied to the plants for 24 h. In the case of oxidative stress, 50 mM H2O2 was used for 24 h. To induce ultraviolet (UV) light, these plants were irrigated using distilled water (100 mL) and then exposed to ultraviolet light for 24 h. Notably, all the above treatments had three biological replicates. The remain- ing plants that received no treatment served as controls. Lastly, all samples from each treatment were washed with MINIQ- filtered water, quickly frozen in liquid nitrogen, and stored at − 80 °C. Total RNA isolation and cDNA synthesis. About 100 mg of the different frozen tissue samples were used to extract total RNA using the Spectrum Plant Total RNA kit (Sigma, USA). Next, the quality and purity of the extracted total RNA was determined using the NanoDrop spectrophotometer 2000 (Thermo Scientific, USA), and its integrity confirmed on a 1.5% agarose gel. Here, only RNA with A260/280 ratios between 1.8 and 2.2 and an A260/230 above 2.0 were used for cDNA synthesis. Subsequently, RNA samples were pre-treated with RNase-free DNase I (Takara Biotechnology, Dalian, China) to remove contaminating traces of genomic DNA and then used for reverse transcription. Lastly, following the instructions from the HiScript Q RT SuperMix for qPCR (Vazyme, China) was used for first-strand cDNA synthesis with oligo (dT) as the primer. Primer design and RT‑qPCR conditions. Here, a total of 11 candidate reference genes were selected from the transcriptome data of A. www.nature.com/scientificreports/ decursiva (unpublished). Next, the built-in program in the Bioedit Sequence Alignment Editor (v7.0.9) was used to screen and select potential single genes using local blast (TBLASTN). Sub- sequently, the corresponding homologs of these reference genes were selected from the database of The Arabi- dopsis Information Resource (TAIR) (http://www.arabidopsis.org), and only single genes with lower E-values and higher scores were selected for subsequent analysis. Table S1, shows the reference gene IDs, homologous loci, gene sequences, and the different expression levels (FPKM, Fragments Per Kilobase per Million) of all the 11 candidate reference genes.i g To avoid amplification of any contaminating genomic DNA, primers were designed to cross at least one intron/exon border that contained both donor and acceptor sites, and then exon analysis was performed using the AlignX program in the vector NTI advance 11.5 package. Subsequently, the Primer3Plus (http://prime r3plus.com/cgi-bin/dev/primer3plus.cgi) was used to design primers with the following characteristics: ampli- con length was 100 to 150 bp, GC content was between 40 and 60%, primer length was 18 to 24 bp, temperature difference of each primer pair was less than 1 °C, and the melting temperature (Tm) was between 55 and 60 °C. Consequently, all the primer pairs were tested using conventional PCR to determine the combination of forward and reverse primers that performed optimally and the resulting products were examined using 1.0% agarose gel electrophoresis. Besides, from a series of standard curves of five different cDNA dilutions, the amplification efficiency (E) and correlation coefficient (R2) were calculated. Table 1 lists all the gene-specific primer pairs that were designed and used in the RT-qPCR analysis. Stability evaluation of candidate reference genes. To obtain the RT-qPCR data, three biological and technical replicates were done for each sample, and all data presented as mean ± standard error of the mean (SEM). Consequently, statistical analyses were performed using the Student’s t-test. Next, representative graphs were generated using OriginPro 9.1 (OriginLab Corporation, Northampton, MA, USA). Subsequently, data analysis was performed using GeNorm (ver.3.5), NormFinder (ver.0.953), BestKeeper (ver.1.0), and the Delta Ct method following the instructions from the manufacturer. Lastly, a comprehensive stability ranking analysis was performed using RefFinder (http://150.216.56.64/referencegene.php). Conclusions Selecting appropriate reference genes is a significant prerequisite for quantifying gene expression using RT-qPCR. This is a conducive research on the key enzyme genes that are involved in the biosynthesis of coumarins and other interesting secondary metabolites found in A. decursiva. So far, this is the first systematic screening experiment of the most suitable reference genes for A. decursiva under various external treatments, like cold stress (4 °C), drought stress (20% PEG), Methyl jasmonate (MeJA), salt stress (0.5 M NaCl), oxidative stress (H2O2), ultraviolet (UV) induction, metal stress (CuSO4), untreated (WT), and ’total’ (all treated samples). Our results have exposed that the 11 candidate genes have different stability in A. decursiva when exposed to different experimental treat- ments. From the overall stability ranking, SAND is the most stable candidate reference gene, followed by PP2A and then PTBP. On the other hand, the NCBP2 gene has the lowest stability making it unsuitable for further research. In summary, the reference genes evaluated in this study can be helpful for accurate normalization of the RT-qPCR data and any other future work on the gene expression of coumarin synthesis present in A. decursiva. Materials and methods Samples preparation and treatments. Here, one-year-old plants of A. decursiva were collected from Ningguo City, Anhui Province, China (longitude: 118.95E, latitude: 30.62 N), and was identified as Angelica decursiva (Miq.) Franch. et Sav. by Zhang Ning, school of pharmacy, Jiangsu Health Vocational College, and deposited in the herbarium of the medicinal botanical garden (ID: JSJK-AD-021). The planting field of A. decur- siva was a private land and the landowner has allowed us to collect plant materials for further study. The collec- https://doi.org/10.1038/s41598-021-98434-6 Scientific Reports \| (2021) 11:18993 \| www.nature.com/scientificreports/ References 8, 1605 (2017). p y 9. Tang, X., Zhang, N., Si, H. & Calderón-Urrea, A. Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress. Plant Methods 13, 85 (2017). 0. Zhao, Y. C. et al. Selection of reference genes for gene expression normalization in Peucedanum praeruptorum dunn under abiotic stresses, hormone treatments and different tissues. PLoS ONE 11, e0152356 (2016). f 1. Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple interna control genes. Genome Biol. 3, 1–11. https://doi.org/10.1186/gb-2002-3-7-research0034 (2002).flf 22. Pfaffl, M. W., Tichopad, A., Prgomet, C. & Neuvians, T. P. Determination of stable housekeeping genes, differentially regu 22. Pfaffl, M. W., Tichopad, A., Prgomet, C. & Neuvians, T. P. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–515 (2004). 23. Xiao, X. et al. Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR. Front. Plant Sci. 5, 788 (2014). g g p g y p g p , ( ) 23. Xiao, X. et al. Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR. Front. Plant Sci. 5, 788 (2014). q 4. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method Methods 25, 402–408. https://doi.org/10.1006/meth.2001 (2001). Methods 25, 402–408. https://doi.org/10.1006/meth.2001 (2001). 25. Kumar, P. et al. Expression analysis of biosynthetic pathway genes vis-à-vis podophyllotoxin content in Podophyllum hexandrum Royle Protoplasma 252 1253–1262 (2015) g 5. Kumar, P. et al. Expression analysis of biosynthetic pathway genes vis-à-vis podophyllotoxin content in Podophyllum hexandrum Royle. Protoplasma 252, 1253–1262 (2015).f y p 6. Olofsson, L., Engström, A., Lundgren, A. & Brodelius, P. E. Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L. BMC Plant Biol. 11, 45 (2011). 7. Lau, W. & Sattely, E. S. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science 34 1224–1228 (2015).h 28. Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622 (2009). 29. Li, J. D. et al. Reference gene selection for gene expression studies using quantitative real-time PCR normalization in Atropa bel- ladonna. Plant Mol. Biol. Rep. References References 1. Ojala, T. et al. Antimicrobial activity of some coumarin containing herbal plants growing in Finland. J. Ethnopharmacol. 73, 299–305 (2000). 1. Ojala, T. et al. Antimicrobial activity of some coumarin containing herbal plants growing in Finland. J. Ethnopharmacol. 73, 299–305 (2000).l ( ) 2. Lim, H. J. et al. Inhibition of airway inflammation by the roots of Angelica decursiva and its constituent, columbianadin. J. Ethnop- harmacol. 155, 1353–1361 (2014).l ( ) 2. Lim, H. J. et al. Inhibition of airway inflammation by the roots of Angelica decursiva and its constituent, columbianadin. J. Ethnop- harmacol. 155, 1353–1361 (2014).l ( ) 3. Islam, M. N. et al. Mechanism of anti-inflammatory activity of umbelliferone 6-carboxylic acid isolated from Angelica decursiva J. Ethnopharmacol. 144, 175–181 (2012).l p 4. Zhao, D. et al. In vitro antioxidant and anti-inflammatory activities of Angelica decursiva. Arch. Pharm. Res. 35, 179–192 (2012). 5 Ali M Y et al Coumarins from Angelica decursiva inhibit α glucosidase activity and protein tyrosine phosphatase 1B Chem Biol p hao, D. et al. In vitro antioxidant and anti-inflammatory activities of Angelica decursiva. Arch. Pharm. Res. 35, 179–192 (2012). Ali, M. Y. et al. Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B. Chem. Biol. nteract. 252, 93–103 (2016). p 4. Zhao, D. et al. In vitro antioxidant and anti-inflammatory activities of Angelica decursiva. Arch. Pharm. Res. 35, 179–192 (2012). 4. Zhao, D. et al. In vitro antioxidant and anti inflammatory activities of Angelica decursiva. Arch. Pharm. Res. 35, 179 192 (2012). 5. Ali, M. Y. et al. Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B. Chem. Biol Interact. 252, 93–103 (2016). 6. Cho, S. H. et al. Induction of apoptosis by Angelica decursiva extract is associated with the activation of caspase in glioma cells. J Korean Soc. Appl. Bi. 52, 241–246 (2009).hf 6. Cho, S. H. et al. Induction of apoptosis by Angelica decursiva extract is associated with the activation of caspase in glioma cells. J. Korean Soc. Appl. Bi. 52, 241–246 (2009).hf pp 7. Lee, S. W. et al. The effects of Angelica decursiva extract in the inhibition of cell proliferation and in the induction of apoptosis in osteogenic sarcoma cells. J. Med. Plants Res. 3, 241–245 (2009). hf osteogenic sarcoma cells. J. Med. Plants Res. 3, 241–245 (2009 g 8. Zhao, Y. C. et al. References Elucidation of the biosynthesis pathway and heterologous construction of a sustainable route for producing umbelliferone. J. Biol. Eng. 13, 44 (2019).f g 9. Feher, M. & Schmidt, J. M. Property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry. J. Cheminform. 34, 218 (2003).i y f 0. Dekkers, B. J. et al. Identification of reference genes for RT–qPCR expression analysis in Arabidopsis and tomato seeds. Plant Cel Physiol. 53, 28–37 (2012).i y 1. Maltseva, D. V. et al. High-throughput identification of reference genes for research and clinical RT-qPCR analysis of breast cancer samples. J. Clin. Bioinform. 3, 13 (2013). p f 2. Sun, H. P., Jiang, X. F., Sun, M. L., Cong, H. Q. & Qiao, F. Evaluation of reference genes for normalizing RT-qPCR in leaves and suspension cells of Cephalotaxus hainanensis under various stimuli. Plant Methods 15, 31 (2019).h 13. Dheda, K. et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normaliza- tion. Anal. Biochem. 344, 141–143 (2005). ( ) 14. Suzuki, T., Higgins, P. J. & Crawford, D. Control selection for RNA quantitation. Biotechniques 29, 332–337 (2000). fi f l ( ) d d bl l 15. Bustin, S. A. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–39. https://doi.org/10.1677/jme.0.0290023 (2002).f i 29, 23–39. https://doi.org/10.1677/jme.0.0290023 (2002). g j 6. Pabuayon, I. M. et al. Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance. Rice 9, 32 (2016). genotypes in rice for drought tolerance. Rice 9, 32 (2016). 7. Ma, R., Xu, S., Zhao, Y. C., Xia, B. & Wang, R. Selection and validation of appropriate reference genes for quantitative real-time 17. Ma, R., Xu, S., Zhao, Y. C., Xia, B. & Wang, R. Selection and validation of appropriate referen PCR analysis of gene expression in Lycoris aurea. Front. Plant Sci. 7, 536 (2016).i 17. Ma, R., Xu, S., Zhao, Y. C., Xia, B. & Wang, R. Selection and validation of appropriate reference genes for quantitative real-time PCR analysis of gene expression in Lycoris aurea. Front. Plant Sci. 7, 536 (2016).i 18. Duan, M. M. et al. Identification of optimal reference genes for expression analysis in radish (Raphanus sativus L.) and its relatives based on expression stability. Front. Plant Sci. 8, 1605 (2017). i based on expression stability. Front. Plant Sci. Data availability Th d d y The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. https://doi.org/10.1038/s41598-021-98434-6 Scientific Reports \| (2021) 11:18993 \| www.nature.com/scientificreports/ Received: 12 November 2020; Accepted: 17 August 2021 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 0. Zhu, J. et al. Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions. PLoS ONE 8, e53196 (2013).f 40. Zhu, J. et al. Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions. PLoS ONE 8, e53196 (2013). l b l f d d f f d d ff d b 40. Zhu, J. et al. Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions. PLoS ONE 8, e53196 (2013).f 1. Ma, S. et al. Expression stabilities of candidate reference genes for RT-qPCR under different stress conditions in soybean. PLoS ONE 8, e75271 (2013). ( ) 42. Lin, Y. et al. Validation of potential reference genes for qPCR in Maize across abiotic stresses, hormone treatments, and tissue types. PLoS ONE 9, e95445 (2014). 42. Lin, Y. et al. Validation of potential reference genes for qPCR in Maize across abiotic stresses, hormone treatments, and tissue types. PLoS ONE 9, e95445 (2014). Acknowledgementsh g This project is funded by Practical Training Program for Natural Science Foundation of the Jiangsu Higher Education Institutions of China (16KJB150042, 19KJD430005). This Project was also funded by Qinglan project of excellent teaching team in Jiangsu and teaching and research project of Jiangsu Health Vocational College (JKKYTD201701, JKA201706, JKA201812, JKB201839, JKA201902, JKB201911)and the Jiangsu "Six one" Project of Health top talent program (LGY2018089). Author contributions Y.H., Y.G., and J.W. conceived and designed the experiments. Y.H. and Y.Z. performed the experiments. Y.Z., Z.B., and W.W. analyzing the data. Z.B., W.W., and X.X. contributed reagents, materials, and analysis tools. Y.H., Y.G., and J.W. wrote and revised the paper. All authors read and approved the manuscript. References 32, 1002–1014 (2014). p 0. Montoliu, L., Rigau, J. & Puigdomènech, P. A tandem of α-tubulin genes preferentially expressed in radicular tissues from Zea mays. Plant Mol. Biol. 14, 1–15 (1990). y 31. Løvdal, T. & Lillo, C. Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal. Biochem. 387, 238–242 (2009).i g 32. Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K. & Scheible, W. R. Genome-wide identification and testing of superior refer- ence genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5–17. https://doi.org/10.2307/4281837 (2005). g p p y p g 3. Gu, C. et al. Reference gene selection for quantitative real-time PCR in Chrysanthemum subjected to biotic and abiotic stress. Mol Biotechnol. 49, 192–197 (2011). 34. Veazey, K. J. & Golding, M. C. Selection of stable reference genes for quantitative rtPCR comparisons of mouse embryonic and extra-embryonic stem cells. PLoS ONE 6, e27592 (2011). 35. Sgamma, T., Pape, J., Massiah, A. & Jackson, S. Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce. Plant Methods 12, 21. https://doi.org/10.1186/s13007-016-0121-y (2016). p y p g y 36. Huang, Y. X. et al. Stable internal reference genes for normalizing real-time quantitative PCR in Baphicacanthus cusia u hormonal Stimuli and UV irradiation, and in different plant organs. Front. Plant Sci. 8, 668 (2017). f p g 37. Expósito-Rodríguez, M., Borges, A, A., Borges-Pérez, A. & Pérez, J.A. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol. 8, 131–142 (2008). f 37. Expósito-Rodríguez, M., Borges, A, A., Borges-Pérez, A. & Pérez, J.A. Selection of internal con RT-PCR studies during tomato development process. BMC Plant Biol. 8, 131–142 (2008). 37. Expósito-Rodríguez, M., Borges, A, A., Borges-Pérez, A. & Pérez, J.A. Selection of internal control genes for quantitativ RT-PCR studies during tomato development process. BMC Plant Biol. 8, 131–142 (2008). 38. Mallona, I., Lischewski, S., Weiss, J., Hause, B. & Egea-Cortines, M. Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol. 10, 4 (2010). gl p y 9. Die, J. V., Román, B., Nadal, S. & González-Verdejo, C. I. Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232, 145–153. https://doi.org/10.2307/23391626 (2010). https://doi.org/10.1038/s41598-021-98434-6 Scientific Reports \| (2021) 11:18993 \| Competing interests h p g The authors declare no competing interests. p g The authors declare no competing interests. © The Author(s) 2021 Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-98434-6. Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-98434-6. Correspondence and requests for materials should be addressed to Y.G. or J.W. Correspondence and requests for materials should be addressed to Y.G. or J.W. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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https://openalex.org/W2950341929	https://zenodo.org/records/3971603/files/1736-Article%20Text-6061-1-10-20190608.pdf	English	null	Integration of Knowledge Management in E-CRM: A Collaborative Study	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	6,388	1. Introduction This paper discusses the relationship between knowledge management concept and e-CRM efforts. The academic nature of this paper explores how eCRM, manifest itself in the form of a knowledge management system (KMS henceforth), to enhance its efforts within the organization. Many SMEs progress in the globalized business environment portrayed greater efficiency and effectiveness. The battle gain competitive advantage is rapidly increasing day by day due to the dynamic nature of business environment. This has produced increase pressure on management of SMEs to become world-class enterprises (Lu & Beamish, 2001), In attempt to reduce their manufacturing cost, improve product quality, respond to their customers and other business partner, its highly needed to concentrate on their customer’s needs and expectations are in a better situation to gain long term success when compared to others(Louis et al., 2016) With this view, many SMEs firms have made substantial investment in information technology and especially in Internet/Web-based technology (Grandon and Pearson, 2004; Harrigan et al., 2010-2011;Solaymani et al., 2012) to invest huge amount of money to implement Customer Relationship Management (CRM) to sustain in this turbulent environment. CRM can improve business intelligence by making better decisions with cordial customer relations, along with good quality of services and product offering (Tae, Cheol, and Ingoo, 2005) and hence there is a heave in the CRM solution. Abstract The paper aims to present a collaborative study on Electronic Customer Relationship Management (eCRM) and Knowledge Management (KM) within four dimensions; technology, knowledge, customer and business based on Resource-based View Theory (RBV). With comprehensive knowledge about customer, SMEs organisations with web presence can provide more customised products and services to individual customers. Hence, the proposition of this paper is the success of e-CRM performance can be improved by interweaving knowledge management concept. This approach is based on four models of CRM, eCRM and KM which are within four dimensions which are technology, knowledge, customer and business based on Resource-based View Theory (RBV). The study identified that CRM Development Stages Model and Balance Scorecard Perspective of eCRM Model provides better fit to the four resource dimensions which in turn can develop a KM-based eCRM model. This paper concludes that eCRM system designed to support KM must be linked closely to the ideas that within the domain of eCRM which can be decentralized and contextualized.This research had only focused exclusively on conceptual and models of past researches; a comprehensive user study, extensive performance and scalability testing is left for future work.A key contribution of this paper is the four models of CRM, eCRM and KM which are analyzed with the four dimensions i.e. technology, knowledge, customer and business based on Resource-based View Theory (RBV) Keywords: CRM; eCRM; customer; knowledge management; performance; resource-based view; integration; collaborative. Integration of Knowledge Management in E-CRM: A Collaborative Study Farida Bhanu Bt. Mohd Yousoof 1 & Kalaivani Jayaraman2 1 Department of Global Economics, Faculty of Accountancy and Management, University Tunku Abdul Rahman, 2 Department of International Businesses, Faculty of Accountancy and Management, University Tunku Abdul Rahman Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Volume 9, Issue 1 Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 688 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Gartner has estimated that CRM software market had exceeded $7.4 billion (£3.6 billions) in 2007, up by 14 per cent from 2006.The rapid growth in the Internet technology is the key enabler for eCRM functions to be delivered in a more effective manner. The Internet technology provides an incomparable opportunity for better relationship building by means of strong interactivity and personalizability (Tae, Cheol, and Ingoo, 2005). Hence, web-based CRM software is increasingly popular option for many organizations and it’s becoming an important issue for marketing strategies and their subsequent implementation. eCRM encompasses all aspects needed to acquire, maintain and build customer relationship all the way through e-business operations ( Clark, Yunxuan, Han, Cheong, and Billy, 2005, Yan, Chang, and Ying, 2006) eCRM aims at attracting and retaining customers who are economically valuable simultaneously eliminating the economically invaluable ones (Romano, and Fjermedestas, 2003). Though eCRM promises improved customer service and loyalty, Gartner claims that 65% of all eCRM projects have failed and this is expected to increase to an even higher rate of 85% (Kimiloglu, and Zarali, 2009). Anderson Consultants also found that 64% of the variation in return on sales between high and average performing companies is mainly due to eCRM capability. These evidences indicate that a good implementation of an eCRM can make the organizations more successful. According to (Coltman, and Dolnicar, 2004), while the payoff from eCRM programs is a growing issue in marketing and IT research, at present there is no robust relationship between eCRM investment and performance. Review of the literature about CRM shows that performance measurement is one of the less popular areas of research in this domain (Kimiloglu and Zarali, 2009). In support, (Hasan, and Tibbits, 2000). Hassan and Tibbits (2000), noted that measuring the performance of the implementation of IT applications and IS projects has grown to be considerably important in order to assess whether the investments proposed to these areas are meaningful. Review of CRM by (Ngai, 2005) which encompasses the period 1992-2002, categorizes the major areas in CRM research, claimed that studies relating to performance management are scarce. Noticeably the focus seems to be on why and how to engage in eCRM applications rather than on evaluating the performance of CRM (Kimiloglu, and Zarali, 2009). Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss The current climate of eCRM shows that high rate of failure and the rate is expected to increase in future (Kimiloglu, and Zarali, 2009). Hence, it is important to measure eCRM performance to ensure whether the investment directed to this area is fruitful. Additionally, at present there is lack of studies related to eCRM performance. Many researchers in this domain stress that there is growing interest on knowledge management (KM) in transferring the customer data to meaningful information. This is evident in the table 1 below which shows the KM as one of the lowest number of research. This paper therefore, seeks to explore the literature review to ascertain the relationship between knowledge management and eCRM performance. This paper proceeds as follows. First section describes literature review on CRM, eCRM, and knowledge management. Next, the paper discusses the methodology of this paper. The section continues with the highlights on findings of the research. The paper concludes with the researchers’ discussion on the KM concept in improving the E-CRM performance. 2.1 Customer Relationship Management (CRM) Traditional marketing had focus on how to win customers but CRM centers around both wining and retaining customers. “Retaining customers is more profitable than building new relationships” it is very true in the internet marketing (Jeong, Serk, and Kyung, 2003). The CRM paradigm has brought a tremendous change in traditional marketing. CRM had evolved from business processes, like relationship marketing and improved customer retention through valuable and efficient management of customer relationship. Few authors had explained that CRM is an approach which is based on maintaining positive relationship with customers, by increasing their loyalty and expanding their customer lifetime value. CRM primarily revolves around marketing and starts with an in-depth analysis of customer behavior. A 3 stage model of CRM shows how customer relationships can be managed. According to the model, customers are first acquired via clear communications and an influential value proposition and are retained through good quality services; and finally the relationship is extended using proper delivery of tailored products/services to a clearly defined customer segments. This indicates that CRM makes use of information and communication technology (ICT) to collect data, and analyze it to provide all needed information in order to have a more personal and cordial interaction with the customer (Lopez, Nicholas, & Castillo, 2008, Goldenberg, 2000) CRM, if properly implemented can be a multifunctional, customer-driven technology which integrates business process management strategy which tends to maximize the relationship throughout the organization (Goldenberg, 2000). Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 689 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Despite the benefits derived from the system, the CRM failure rate is still high (Zablah. Bellenger, and Johnston, 2004). In 2001, the failure rate of CRM projects had increased from 55% to 75% (Kotorov, 2003). CRM can be classified into three levels namely strategic, operational, and analytical (Buttle, 2004). Strategic type deals with creating the customer centric business culture which in turn creates a better value over competitors by making them to take better investment decisions. The operational type is concerned about customers’ processes automation, and service automation. Operational CRM is concerned with automation and streamlining the workflow especially at the front office collecting data, processing transaction, at the sales, marketing and services (Lun, Jinlin, and Yingying, 2008, Zhang, Chen, and Fu, 2006) The analytical CRM roots from operational CRM and scrutinize customer data in order to create information regarding the segmentation of customer and their behavior, as well as their value to the organization through data mining (Gefen, and Ridings, 2002, Dyche, 2002). 2.2 eCRM eCRM has been defined by (Lee-Kelley, Gilbert, and Mannicom, 2003) as “the marketing activities, tools and techniques delivered over the with a specific aim to locate, build and improve long term customer relationships to enhance their individual potential”. Internet is highly essential to understand the unique distinction in the customer behavior, their choice and opinion about the company products and services. eCRM is not only about technology but also about alignment of business processes with customer strategies (Rigby, Richheld and Scheffer, 2002). According to (Anton, 1996), emphasis must be placed on the management of customer relation with “continuous improvement” or reengineering for better services and competitive positioning. From previous researches, five major eCRM research areas were identified namely, eCRM markets, eCRM knowledge management, eCRM business models, e-CRM technology, and eCRM human factors out of which the least popular area was knowledge management (103,28%) still an emerging field which certainly need more impetus and attention from academicians(Romano and Fjermedestas, 2003) Management of customer information is a prime success factor in eCRM. Proactive and practical use of internet technologies smoothen the process of collection of customer information from all possible customer contact points by combining other relevant data in order to develop customer insight(Payne and Frow, 2006) which in turn will enable the organization to predict the future behavior and personalize offerings towards more valuable customers. eCRM process is classified into the front-end and back-end process. eCRM starts with the front-end tool when customers visit or browse the company’s website. Customers browsing activities will be captured by a cookie file and logged in a server for initial profiling purposes. This will allow the company to understand customer’s surfing pattern which would be very useful for future marketing strategy eCRM is not able to function effectively without the support of a well-built back end system that utilizes the power of intelligent agents and database engines. (Noor Raihan, 2005). A very powerful retention tool in Ecrm is personalizing the experience of valuable customers especially those 20% who contribute to the 80% of the company business that will provide noteworthy long term rewards. Customer support aspect is yet another crucial tool which will build trust. “Contact Us” or “Help” button promotes the company brand and build trust. 2.3 Knowledge Management (KM) Knowledge management is a concept of systematically collect, share, organize, store, disseminate and apply knowledge in an organizational context to improve the visibility and usability of knowledge. Knowledge management encompasses a wide range of disciplines. Groupware, decision support systems, expert systems and other forms of collaborative systems are examples of technology related to knowledge management (Gupta and Sharma, 2004) Knowledge can be viewed from several perspectives for example “as a state of the mind, as an object, as a process, a situation of having access to information or even as a capability” (p.109). A single knowledge can be tacit or explicit (Nonaka and Takeuchi, 1995). KM facilitates the selective application of knowledge from past experiences during critical moments of decision making currently as well as in future leading to a well efficient organizational practices. In addition, the dynamic nature of eCRM, coupled with different inputs and requirements from various eCRM stakeholders, creates the information overload to the managers which can prevent timely and accurate decision making. A well tested KMS in this context can help to decide which areas to look at, and what sort of decisions to focus on, well in advance. 690 Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 2.4 Theoretical Background The resource-based view theory (RBV) stipulate and recommend that the main driver of firms resources are the main driver of performance enabling them to achieve competitive advantage which leads to superior long-term performance. Empirical studies pertaining to firm performance using the RBV have found vast differences between firms in the same industry, as well as groups within industries suggesting the significant effect of individual firm- specific resources on performance. Resources that are attributing to the core competencies will lead to gain competitive advantage which can sustain over a longer period of time. In general, empirical studies have strongly favored the resource-based view. Researchers interested in the RBV have used a variety of terminology to talk about a firm’s resources, including competencies (Garg and Prahaled, 1990), skills (Rajiv and Sarv 2003), strategic assets (Garg and Prahaled, 1990), assets (Raphael and Paul 1993), and stocks (Ross, Beath, C.M. and Goodhue, 1996) and these have been problematic for research as it is unclear about the key terminology and its meaning. Information system researchers have highlighted the potential of the RBV and related theories to provide explanations as to how and why firms can derive strategic value from IT investments (Guoquan, 2005). Proponents of the RBV suggest that the competitive advantage of the firm could be the organizational resources, capabilities, and competences that are valuable, unique, rare, difficult to imitate and difficult to substitute. This topic had been debated for almost two decades by Information Systems (IS) researchers and IT business value research, investigate the impact of IT investment on firm performance. Though the proposition on computers does not improve the productivity (Jason, Vijay, & Kenneth, 2003), researchers are still trying to find how IT improves firm performance (Bharadwaj, 2000). Meta-analytic studies on IT business value came up with two directions for future research viz., application of RBV of the firm which explains IT business value, and further research which helps to identify management practices which might complement IT in establishing the business value. RBV believes that competitive advantage and better firm performance can be achieved by effectively combining the resources which are unique, valuable, rare, and hard to imitate (Barney, 1991). Studies based on IT business value reveal that RBV theory explains how IT investments can be changed into firm performance (Michael, and John, 2004) Researchers have identified tangible and intangible resources that are complementary to IT. Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 KM is an action discipline and knowledge should be used in order to have an impact. CRM relies on the use of knowledge from both historical and current transactions in order to generate current and future action plan and procedures. Lessons learned practically enable eCRM managers to prepare a well-structured strategic plan. 3. Integrating eCRM and Knowledge Management 3. KM emphasises the practice of applying the knowledge selectively from previous experiences too make good decisions (Jennex, 2005). Interweaving KM into eCRM tends to improve the CRM’s effectiveness and helps to understand the customer to provide superior services. In addition, we further add that given the dynamic nature of market, coupled with different customer expectations, a CRM or sales manager and related departments therein, are subjected to information overload, which can prevent timely and accurate decision making. A well-integrated. CRM with KM characteristics in this context can help to decide what to look at, what decisions to focus on, and what decisions can be made automatically and/or in advance. CRM with KM characteristics in this context can help to decide what to look at, what decisions to focu what decisions can be made automatically and/or in advance. According to (Bose, and Sugumaran, 2003) in order to integrate marketing, sales and service activities, CRM requires integration of all the business processes involving customers. Customer knowledge constitutes the following: They should understand what customers really want, which is usually called as knowledge about customers (Garcia, Munillo and Annabi, 2002). Customers know well about the products and services they use as well as about how they view the offerings that led to the purchase. This knowledge from customers is valuable as it helps to improve the products and services. Efforts needs to be made to channel this knowledge back into the enterprise (Garcia, Munillo and Annabi, 2002) 2.4 Theoretical Background Firms can gain competitive advantage by effectively identifying and assembling resources that work together in order to create organizational core competencies (Santhanam, and Hartono, 2003) and they tend to measure the firm’s ability to combine many resources effectively and efficiently to achieve the preset objective (Coltman, 2007, Dutta, Narasimhan, and Rajiv, 2005) Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 4. Methodology The research approach for this study is based on secondary data. Two recent customer knowledge management models and two eCRM models that are closely fit to the topic were selected. The papers were selected from the result of search on keywords – knowledge management and CRM for KM-CRM models and eCRM for eCRM Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 5. Four Models on KM-CRM and eCRM Four Models on KM-CRM and eCRM Two models that discusses on KM concept for implementation of CRM are as follows: Figure 1. CRM Development Stages Model - Stefanou and Sarmaniotis (2003) Figure 2. Knowledge CRM Model – Lin, Su and Chien (2006) Two models that discusses on KM concept for implementation of CRM are as follows: Figure 1. CRM Development Stages Model - Stefanou and Sarmaniotis (2003) Figure 1. CRM Development Stages Model - Stefanou and Sarmaniotis (2003) Figure 1. CRM Development Stages Model - Stefanou and Sarmaniotis (2003) Figure 2. Knowledge CRM Model – Lin, Su and Chien (2006) Figure 2. Knowledge CRM Model – Lin, Su and Chien (2006) Figure 2. Knowledge CRM Model – Lin, Su and Chien (2006) Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 691 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 models. The search was launched in five major online databases – Emerald, Science Direct, IEEE Xplore, and MIS Quarterly. These models were then analyzed in terms on its characteristics and elements that contribute to an effective integrated KM-based eCRM. The findings were presented in the proceeding section. 5. Four Models on KM-CRM and eCRM Two models that discusses on KM concept for implementation of CRM are as follows: Figure 1. CRM Development Stages Model - Stefanou and Sarmaniotis (2003) Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 692 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Figure 3. CKM Model for e-Commerce - Nicholas and Castillo (2008) Figure 3. CKM Model for e-Commerce - Nicholas and Castillo (2008) Figure 4. Balance Scorecard Perspective of e-CRM –Kımılog˘lu and Zarali (2009) . Outcome Based on the RBV theory, any firm should focus on their key resources and evaluate the resources based on four main criteria which are valuable, rare, in-imitable and non-substitutable. Firms should care and protect their resources in order to improve organizational performance (Barney, 1991) and achieve competitive advantage. In line with this theory, the four models were analyzed based on four major competitive dimensions such as technology, customer, knowledge and business. The resulting findings are presented here. Figure 4. Balance Scorecard Perspective of e-CRM –Kımılog˘lu and Zarali (2009) 6. Outcome Based on the RBV theory, any firm should focus on their key resources and evaluate the resources based on four main criteria which are valuable, rare, in-imitable and non-substitutable. Firms should care and protect their resources in order to improve organizational performance (Barney, 1991) and achieve competitive advantage. In line with this theory, the four models were analyzed based on four major competitive dimensions such as technology, customer, knowledge and business. The resulting findings are presented here. Figure 4. Balance Scorecard Perspective of e-CRM –Kımılog˘lu and Zarali (2009) Figure 4. Balance Scorecard Perspective of e-CRM –Kımılog˘lu and Zarali (2009) . Outcome Based on the RBV theory, any firm should focus on their key resources and evaluate the resources based on four main criteria which are valuable, rare, in-imitable and non-substitutable. Firms should care and protect their resources in order to improve organizational performance (Barney, 1991) and achieve competitive advantage. In line with this theory, the four models were analyzed based on four major competitive dimensions such as technology, customer, knowledge and business. The resulting findings are presented here. Figure 4. Balance Scorecard Perspective of e-CRM –Kımılog˘lu and Zarali (2009) 6. Outcome Based on the RBV theory, any firm should focus on their key resources and evaluate the resources based on four main criteria which are valuable, rare, in-imitable and non-substitutable. Firms should care and protect their resources in order to improve organizational performance (Barney, 1991) and achieve competitive advantage. In line with this theory, the four models were analyzed based on four major competitive dimensions such as technology, customer, knowledge and business. The resulting findings are presented here. 693 Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss This gives a better view of the dimensions that can be focused on in developing a KM- based e-CRM model. In comparison to all the four models, (Kimiloglu and Zarali, 2009) and Gebert, Geib, Kolbe, and Brenner, 2003) seems to be covering all of the dimensions mentioned. Kımılog˘lu and Zarali’s model (2009) is from the E- CRM category and Stefanou and Sarmaniotis (2003) is in KM-CRM category. Figure 2 and 3 have unclear elements for business dimensions. This gives a better view of the dimensions that can be focused on in developing a KM- based e-CRM model. 694 Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 ISSN 2395-6283 Table 1- KM-CRM Models and E-CRM Models in Four Resource Dimensions. Dimensio ns KM- based e- CRM Figure 1 - Stefanou andSarmanioti s (2003) Figure 2 – Lin et al (2006) Figure 3 – Nicholas & Castillo (2008) Figure 4 – Kımılog˘lu and Zaralı (2009) Technology IT Level Unclear tie Perceived risk associated to each CKM tool Increasing usage of technology Customer  Custo mer Satisfaction- Complain Management  Custo mer Accounts- Orders Management Unclear tie Perceived risk Internet preference  Improved customer awareness and perceptions  Increased amount, satisfaction, transaction, and loyalty Knowledge Customer Personalizatio n Management  Customer- knowledge sources  Customer knowledge managemen t Internet knowledge  Learning  Development of new services Business Defensive relationship marketing Unclear tie Unclear tie  Faster, more effective, high-quality and more reliable business processes  Continuous improvement of current services Net Benefit Effective KM- CRM Performanc e – hard & soft Purchase Intention e-CRM Performance In comparison to all the four models, (Kimiloglu and Zarali, 2009) and Gebert, Geib, Kolbe, and Brenner, 2003) seems to be covering all of the dimensions mentioned. Kımılog˘lu and Zarali’s model (2009) is from the E- CRM category and Stefanou and Sarmaniotis (2003) is in KM-CRM category. Figure 2 and 3 have unclear elements for business dimensions. This gives a better view of the dimensions that can be focused on in developing a KM- Table 1- KM-CRM Models and E-CRM Models in Four Resource Dimensions. In comparison to all the four models, (Kimiloglu and Zarali, 2009) and Gebert, Geib, Kolbe, and Brenner, 2003) seems to be covering all of the dimensions mentioned. Kımılog˘lu and Zarali’s model (2009) is from the E- CRM category and Stefanou and Sarmaniotis (2003) is in KM-CRM category. Figure 2 and 3 have unclear elements for business dimensions. This gives a better view of the dimensions that can be focused on in developing a KM- based e-CRM model. In comparison to all the four models, (Kimiloglu and Zarali, 2009) and Gebert, Geib, Kolbe, and Brenner, 2003) seems to be covering all of the dimensions mentioned. Kımılog˘lu and Zarali’s model (2009) is from the E- CRM category and Stefanou and Sarmaniotis (2003) is in KM-CRM category. Figure 2 and 3 have unclear elements for business dimensions. Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 7. Discussion Based on the above outcome, we agree with Jennex’s (Jennex, 2008) claim that we need KM to help organisation to make sense of what they know, to know what they know, and to effectively use what they know based on the following reasons: i. to help organisations identify, capture, store, and retrieve critical knowledge. ii. to help us deal with the transience of knowledge workers. iii. to help organisations manage a glut of knowledge We therefore reinstate that importance of KM for eCRM is evident. Implementing knowledge management systems in organizations can help to increase competitiveness (Krogh, 1998) and lead to greater innovation and responsiveness (Hackbarth, 1998). An organization’s ability to survive given dynamic changes within its environment is contingent upon its ability to quickly respond to change (Burnell, Priest, and Durrett, 2004). This includes among others the ability to effectively manage its customer knowledge resources. Assert that “an effective knowledge-based organization is one that correctly captures, shares, applies and maintains it knowledge resources to achieve its goals” (p.203). This echoes the view of (March, and Simon 1958) who states that successful organizations are able to adapt to any dynamic environment. The information processing theory states that the role of having accurate and up to date information is vital particularly when organizations deal with large pool of customers (Burnell, Priest, and Durrett, 2004). Implementation of a knowledge management system that can eCRM managers to proactively respond to ever-changing customer needs will benefit an organization (Burnell, Priest, and Durrett, 2004). eCRM implementation by interweaving KM concept promises 3 benefits to organizations: (i) enhanced visibility of customer related knowledge in organizations through the use of maps, hypertexts, yellow pages; directories etc. (ii) building knowledge sharing culture i.e. create avenues for employees and customers to share knowledge and (iii) a knowledge infrastructure, not confined to technology solely, rather create an environment that permits collaborative work (Lee-Kelley, Gilbert, and Mannicom, 2003). This implies that knowledge management systems can support an organization in planning for and dealing with customers effectively. It could be inferred and ascertained that any eCRM designed to support KM, must be linked very closely to ideas that are within the domain of eCRM. A specific technology that was selected to support eCRM should lead to knowledge that can be decentralized, and contextualized. Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 8. Conclusion As remarks, this paper is timely especially in a situation where there is lack of research pertaining to KM and eCRM. The researchers have extra miles by integrating four models, two each from KM and eCRM models to develop a KM-based e-CRM model based on Resource-based Theory. The findings of this paper contributes to the existing knowledge of eCRM and RBV in the domain of KM. Future researchers may expand this findings by conducting an extensive search on models in the domain of KM and develop more integrated and comprehensive model for business firms to have a clear vision on how to improve performance and competitive advantages through an effective KM-based eCRM. As remarks, this paper is timely especially in a situation where there is lack of research pertaining to KM and eCRM. The researchers have extra miles by integrating four models, two each from KM and eCRM models to develop a KM-based e-CRM model based on Resource-based Theory. The findings of this paper contributes to the existing knowledge of eCRM and RBV in the domain of KM. Future researchers may expand this findings by conducting an extensive search on models in the domain of KM and develop more integrated and comprehensive model for business firms to have a clear vision on how to improve performance and competitive advantages through an effective KM-based eCRM. 7. Discussion Open source systems viz., Wiki technology might be a choice for organizations that tend to use and design any information system to manage customers. Open source systems will be ideal for knowledge that is instable, dynamic and decentralized (Raphael and Paul, 1993). However, technology alone cannot be sufficient to promote effective eCRM-KM but need an effective design and usage considerations. The following are essential when we think about integrating KM into eCRM: • A “fit” between the knowledge management system and CRM policies should be there. In other words, technology should support CRM initiatives and should not act as hurdles. • A “fit” between the knowledge management system and CRM policies should be there. In other words, technology should support CRM initiatives and should not act as hurdles. • eCRM organizations should cultivate acknowledge sharing culture between entities that are involved in the process. • eCRM organizations should cultivate acknowledge sharing culture between entities that are involved in the process. Adoption of eCRM with KM integration being widely practised in various sectors. Healthcare organisation has adopted eCRM which is widely recognised as electronic health record. eCRM help healthcare professionals store and process various types of clinical, administrative, and financial data about their patients, and they often interface with systems, such as remote medical monitoring devices, billing, health insurance, public health surveillance, and patient-centered web portals . Past researches show that accurate and complete interoperable electronic clinical records can lead to more efficient healthcare processes, improved clinical operations, better governance, improved research data, increased adherence to guidelines, decreased medication errors, and facilitation of effective management and communication (Ronnie, Nava, and Lior, 2014). Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss References [1] Anton, J, (1996). ‘Customer relationship management: Making hard decision with soft numbers”, Englewood Cliffs, N.J: Prentice Hall. [2] Barney, J., (1991). Firm Resources and Sustained Competitive Advantage, Journal of Management, 1799- 120. [3] Bharadwaj, A.S. (2000). A Resource Based Perspective on Information Technology Capability and Firm Performance: An Empirical Investigation, MIS Quarterly, 24(1), 169-196. [4] Bose, R, & Sugumaran, V., (2003). Application of Knowledge Management Technology in Customer Relationship Management, Knowledge and Process Management, 10(1), 3-17. [5] Burnell, L., Priest, J., & Durrett, (2004). Developing and Maintaining Knowledge Management System for Dynamic, Complex Domains. In J. Gupta and S. Sharma (Eds.), Creating Knowledge Based Organizations. London: IGP. [6] Buttle, F. (2004). Customer relationship Management: Concepts and Tools: Oxford:Elsevier. [7] Clark, H, Yunxuan, H. Han, S., Cheong, J. & Billy, B., (2005). E-relational Characteristics on Hospitality and Tourism Program Web Sites, Journal of Hospitality & Tourism Research, 29. [8] Coltman, T. & Dolnicar, S., (2004). eCRM success and the value of managerial discretion, ANZMAC (CD), Conferance Proceedings of the Australian and New Zealand Marketing Academy, , [9] Coltman, T.R. (2007). “Why build a customer relationship management capability”?Journal of Strategic Information Systems, 16(3), 301-320. [10] Dutta. S., Narasimhan, O., & Rajiv.,S, (2005). “Conceptulizing and measuring capabilities: Methodology and empirical application. Strategic Management Journal 26(3), 277-285. [11] Dyche, J, (2003). The CRM Handbook: A Business Guide to Customer Relationship Manageme Wesley, 2002. [12] Garg, H., & Prahaled C.K., (1990). Strategic Intent, McKinsey Quartely, 36-61. [13] Garcia, M., Munillo & Annabi, H., (2002). Customer Knowledge Management, Journal of Operational Research Society, 53(8), 875-888. [14] Gefen, D. & Ridings, C.M, (2002). Implementation Team Responsiveness and User Evaluation of Customer Relationship Management: A Quasi –Experimental Design Study of Social Exchange Theory, Journal of Management Information System, 119, 47-69. [15] Gebert, H., Geib, M., Kolbe, L & Brenner, W., (2003). Knowledge enabled customer relationship management and knowledge management concepts, Journal of Knowledge Management, 7(5), 107-123. [16] Goldenberg, B. (2000). What is CRM? What is an e-customer? Why you need them now, in Proceedings of DCI Customer Relationship Management Conference, Boston, MA, 27-29 June. [17] Grandon E.E. & Pearson J.M. (2004). Electronic commerce adoption: an empirical study of small and medium US Business. Information and management 42: 197-216. [18] Guoquan, C., (2005). An organizational learning model based on Western and Chinese management thoughts and practise, Management Decision, 43(4). [19] Gupta, J. N. D. & Sharma, S. K. Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 695 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 References (2004). “Creating knowledge based organizations”. Hershey, USA: IDEA Group Publishing. [20] Hackbarth, G., (1998). The Impact of Organizational Memory on IT Systems, Paper presented at the Fourth Americas Conference on Information Systems, [21] Harrigan, P. (2011). Conceptualising social CRM in SMEs. In Australian and New Zealand Marketing Association Conference. Australian and New Zealand Marketing Association Conference. Retrieved from http://eprints. soton.ac.uk/185425/1/ANZMAC_Paper_2011.doc [22] Harrigan,P., Ramsey, E., & Ibbotson, P., (2010). Critical factor underpinning the e-CRM activities of SMEs. Journal of Marketing Management. 27(5-6), 503-529. [23] Hasan, H & Tibbits, H.R, (2000). Strategic management of electronic commerce: an adaptation of the balance scorecard, Internet Research: Electronic Networking Applications and Policy, 10(5), 439-50. Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 696 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 [24] Jason, D., Vijay G., & Kenneth, L.K. (2003), Information Technology and Economic Performance: A Critical Review of the Empirical Evidence. ACM Computing Surveys. 35(1), 1. [25] Jeong, Y.A, Serk, K.K & Kyung, S.H., (2003). On the Design Concepts for CRM Syste Management & Data System, 103(5). [26] Jennex, M.E, (2005). Productivity Impacts from Knowledge Management, Proc American Conference on Information System (AMCIS), Association for Information Systems. [27] Jennex, M.E., (2008). Current issues in knowledge management. Information Science Reference, Hershey: New York, [28] Kimiloglu, H. & Zarali, H., (2009). What signifies success in e-CRM, Marketing Intelligence and Planning, 27(2). [29] Kotorov, R. (2003). Customer Relationship Management Strategic Lessons and Future Directions, Business Process Management Journal, 9,566-571. 30] Krogh, V.G. (1998). Care in Knowledge Creation. California Management Review, 40(3), 133-153. [31] Lee-Kelley, L. Gilbert, D, & Mannicom, R, (2003). How e-CRM can enhance customer loyalty, Marketing Intelligence & Planning, 21(4), 239-48. [32] Lin, Y., Su, H., & Chien, S. (2006). A knowledge-enabled procedure for customer Relationship Management. Industrial Marketing Management Journal, 35. [33] Louis, D. N., Perry, A., Reifenberger, G., von Deimling, A., Figarella-Branger, D., & Cavenee, W. K., (2016). The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol. 131, 803–820. doi: 10.1007/s00401-016-1545-1 [34] Lopez-Nicolas, C. & Molina-Castillo, F.J. (2008) Customer Knowledge Management and E- commerce: The Role of Customer Perceived Risk. International Journal of Information Management, 28, 102-113. [35] Lun, Z, Jinlin, L. & Yingying, W, (2008). References Customer relationship management system framework design of Beijing Rural Commercial Bank, in IEEE International Conference on Service Operations and Logistics, and Informatics (SOLI), 97-101. [36] Lu, Jane W., Beamish & Paul W. (2001). “The Internationalization and Performance of SMEs,” Strategic Management Journal, 22, 565-586. [37] March, J. G., & Simon, H. A. (1958). Organizations. New York: John Wiley & Sons. [38] Michael, W., & John, H., (2004). The Resource Based View and Information System Research: Review, Extension and Suggestion for Future Research, MIS Quarterly, 28(1), 107-142. [39] Ngai, E.W.T, (2005). Customer relationship management research (1992-2002): An academic literature review and classification, Marketing Intelligence & Planning, 23(6), 582-605. [40] Noor Raihan Ab Hamid, (2005). E-CRM: Are we there yet?, Journal of American Academy of Business, 6. 41] Nonaka, I., & Takeuchi, H. (1995). The Knowledge-Creating Company. New York: Oxford Univers [42] Payne, A & Frow, P. (2006). Customer relationship management: from strategy to implementation, Journal of Marketing Management, 22 Nos ½, , 135-68 [43] Rajiv, K., & Sarv, D., (2003). Performance Impacts of Information Technology: Is Actual Usage the Missing Link, Management Science, 49(3), 273-289. [44] Raphael, A., & Paul S.J.H, (1993). Strategic Assets and Organizational Rent, Strategic Management Journal, 14(1), 33-46. [45] Rigby, D.Richheld, F, & Scheffer P, (2002). “Avoid the four perils of CRM” Harvard Business Review, February 101-110. [46] Romano, N.C., & Fjermedestas, J., (2003). Electronic commerce customer relationship management: A research agenda, Information Technology and Management, 2/3, 233-258. [47] Ross, J.W.Beath, C.M. & Goodhue, D.L, (1996). Developing long term competitiveness through IT assets, Sloan Management Review, 38(1), 31-42. Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss 697 Journal of Progressive Research in Social Sciences (JPRSS) ISSN 2395-6283 [48] Ronnie Ben Zion, Nava Pliskin & Lior Fink., (2014). Critical Success Factors For Adoption of Electronic Health Record Systems: Literature Review and Prescriptive Analysis, Information Systems Management, 31,296-312. [49] Santhanam, R, & Hartono, (2003). E”Issues in linking information technology capability to firm performance”MIS Quarterly, 27(1), 125-153. [50] Solaymani, S., Sohaili, K., & Yazdinejad, E. A. (2012). Adoption and use of e-commerce in SMEs. A case study. Electron. Commer. Res. 1, 249–263. doi: 10.1007/s10660-012-9096-6 [51] Stefanou, C., Sarmaniotis, C., & Stafyla, A., (2003). CRM and customer centric-knowledge management: an empirical research, Business Process Management Journal, 9(5), 617-634. [52] Tae, H.P, Cheol, K.A. & Ingoo, H., (2005). Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss References The priority factor model for customer relationship management system success, Expert Systems with Application, 28(4), 641-654. [53] Yan L, Chang–F.Z & Ying, W. C, (2006). Determinants of E-CRM in influencing Customer Satisfaction, 4099, Springer Berlin / Heidelberg. [54] Zablah. A. R. Bellenger, D.N. & Johnston, W. J., (2004). Customer Relationship Management Implementation Gaps, Journal of Personal Selling & Sales Management, 24, 279-295. [55] Zhang, G, Chen, Y. & Fu. C. (2006). A study on the relation between enterprise competitive advantage and CRM based on data mining in International Technology and Innovation Conference (ITIC 2006), 1710-1714. 698 Volume 9, Issue 1 available at www.scitecresearch.com/journals/index.php/jprss
https://openalex.org/W2140137811	https://europepmc.org/articles/pmc2981565?pdf=render	English	null	Analysis of Mice Lacking DNaseI Hypersensitive Sites at the 5′ End of the IgH Locus	PloS one	2,010	cc-by	9,381	Abstract The 59 end of the IgH locus contains a cluster of DNaseI hypersensitive sites, one of which (HS1) was shown to be pro-B cell specific and to contain binding sites for the transcription factors PU.1, E2A, and Pax5. These data as well as the location of the hypersensitive sites at the 59 border of the IgH locus suggested a possible regulatory function for these elements with respect to the IgH locus. To test this notion, we generated mice carrying targeted deletions of either the pro-B cell specific site HS1 or the whole cluster of DNaseI hypersensitive sites. Lymphocytes carrying these deletions appear to undergo normal development, and mutant B cells do not exhibit any obvious defects in V(D)J recombination, allelic exclusion, or class switch recombination. We conclude that deletion of these DNaseI hypersensitive sites does not have an obvious impact on the IgH locus or B cell development. Citation: Perlot T, Pawlitzky I, Manis JP, Zarrin AA, Brodeur PH, et al. (2010) Analysis of Mice Lacking DNaseI Hypersensitive Sites at the 59 End of the IgH Locus. PLoS ONE 5(11): e13992. doi:10.1371/journal.pone.0013992 Editor: Sebastian D. Fugmann, National Institute on Aging, United States of America Editor: Sebastian D. Fugmann, National Institute on Aging, United States of America Received June 28, 2010; Accepted October 26, 2010; Published November 15, 2010 Received June 28, 2010; Accepted October 26, 2010; Published November 15, 2010 Received June 28, 2010; Accepted October 26, 2010; Published November 15, 2010 Copyright: 2010 Perlot 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. nding: This work was funded by National Institutes of Health R01 AI020047 (to FWA) and GM36064 (to PHB). FWA is an Investigator dical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manu Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alt@enders.tch.harvard.edu . These authors contributed equally to this work. Thomas Perlot1.¤a, Inka Pawlitzky2.¤b, John P. Manis3, Ali A. Zarrin1¤c, Peter H. Brodeur2, Frederick W. Alt1* Thomas Perlot1.¤a, Inka Pawlitzky2.¤b, John P. Manis3, Ali A. Zarrin1¤c, Peter H. Bro Alt1* nka Pawlitzky2.¤b, John P. Manis3, Ali A. Zarrin1¤c, Peter H. Brodeur2, Frederick W 1 The Howard Hughes Medical Institute, The Children’s Hospital, Immune Disease Institute, and Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Immunology Program, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, United States of America, 3 Children’s Hospital Boston and Joint Program in Transfusion Medicine, Harvard Medical School, Boston, Massachusetts, United States of America Abstract ¤a Current address: IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria ¤b Current address: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands ¤c Current address: Department of Immunology, Genentech, San Francisco, California, United States of America A large effort has been made to elucidate mechanisms of IgH locus regulation and a number of cis-regulatory elements have been described and characterized. The IgH intronic enhancer (Em) resides in the JH – CH intron and was shown to be necessary for efficient V(D)J recombination by promoting both D to JH and VH to DJH recombination [6,7]. Downstream of the CH genes at the very 39 end of the IgH locus a cluster of DNaseI hypersensitive sites was described, termed 39 IgH regulatory region (39IgH RR). So far two main functions have been assigned to this regulatory region: the 39IgH RR plays an important role in promoting CSR to most IgH isotypes, and the 39IgH RR was shown to be necessary for high level expression of the functionally assembled HC gene from the promoter 59 of the VHDJH exon [8]. PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 Introduction The variable region of an immunoglobulin heavy chain (IgH) is assembled from V (variable), D (diversity), and J (joining) gene segments that lie upstream of several IgH constant (C) region exons in a process called V(D)J recombination [1]. The mouse IgH locus contains large numbers of VH segments and multiple D and JH segments but an individual IgH V(D)J exon is assembled from only one VH, one D, and one JH segment. V(D)J recombination of the IgH locus takes place in pro-B cells in an ordered way such that D to JH recombination precedes VH to DJH recombination [2]. In this regard, activation of the IgH locus is thought to progress in a stepwise manner [3]. D to JH rearrangement efficiently occurs on both alleles, however, allelic exclusion ensures that VH to DJH recombination results in expression of a functional heavy chain (HC) from only one of the two alleles [4]. An additional potential regulatory region was identified at the 59 end of the IgH locus, consisting of four DNaseI hypersensitive sites [9]. One of these sites, HS1, was shown to be pro-B cell specific, the stage during which IgH V(D)J recombination takes place, and was suggested to include binding sites for the transcription factors PU.1, Pax5 and E2A [9]. These observations led to the suggestion that this region might represent a new regulatory region for IgH rearrangements. In this regard, the 59 end of the IgH locus is an attractive location for a regulatory element because it would not be deleted during the course of V(D)J recombination, and it might explain control of several unresolved phenomena in the IgH locus. Among these is the regulation of VH germline transcripts as so far Mature B-cells can undergo further alterations of their HCs. IgH class switch recombination (CSR) causes expression of different immunoglobulin isotypes which confer different effector functions. During this recombination process one of several sets of downstream CH exons replaces the Cm exons and the intervening sequence is deleted from the chromosome, which results in expression of a new C region without changing the specificity of the IgH variable region [5]. PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 1 59IgH Enhancer no cis-regulatory element has been identified that controls activity of the bulk of unrearranged VH promoters. IgH class switch recombination assay CD43- splenocytes were isolated by MACS, cultured with LPS or IL4/aCD40, and analyzed by flow cytometry as described previously [13]. Introduction Furthermore, it is not known how it is achieved that proximal and distal VH segments are activated independently or why usage of distal versus proximal VH gene families varies significantly. PCR assay for V(D)J rearrangements hi Pro-B cells (IgM- B220+ CD43hi), pre B-cells (IgM- B220+ CD43lo), and double positive T-cells (B220- CD4+ CD8+) were isolated by FACS on a FACSAria (BD Biosciences) and genomic DNA was extracted. 50 ng DNA or 5-fold dilutions were analyzed by PCR for DH–JH, VH-DJH, Vk–Jk, and Vl–Jl rearrangements with primers listed in Table S1. Input DNA amounts were normalized upon PCR amplification within DLG5. PCR was performed at 95uC for 49, 30 cycles of 95uC for 300, 60uC for 900, and 72uC for 29, followed by 72uC for 59. PCR products were transferred from ethidium bromide gels to nylon membranes and visualized with end labeled oligonucleotide probes (Table S1). CDR 3 lengths were generated from IgH VDH rearrangements from mature B cells using oligonucleotides for V558 and JH4 rearrangements. PCR fragments were amplified using iProof (Bio- Rad) polymerase and cloned into Zero Blunt Topo vectors (Invitrogen), and sequenced. Here we report the targeted deletion of the pro-B cell specific 59IgH HS1 as well as combined deletion of HS1, HS2, HS3a,b in mice. We analyzed potential implications on B cell development, V(D)J recombination, and IgH CSR. Flow cytometry and cell sorting Single cell suspensions from spleen, thymus, or bone marrow were stained in PBS 2% FBS with various antibodies: FITC-aIgM, PE-Cy5-aB220, PE-aCD8a, PE-aCD43, FITC-aLy9.1, APC- aIgM, APC-Cy7-aB220 (BD Pharmingen), PE-aAA4.1, FITC- aCD4 (eBioscience). FACS analysis was performed on a FACSCalibur (BD Biosciences) and a FACSAria (BD Biosciences) apparatus. Cell sorts were performed on a FACSAria (BD Biosciences) apparatus. EF1 heterozygous IgHa/b embryonic stem (ES) cells, generated in the Alt laboratory, were transfected with PvuI linearized RHS1 targeting vector to obtain RHS1/+ ES cells. To obtain DHS1/+ ES cells, the PGK-NeoR cassette was deleted by applying a Cre – expressing adenovirus vector. DHS1/+ ES cells were transfected with PvuI linearized R39HSs targeting vector to obtain R39HSs/+ ES cells. R39HSs/+ ES cells were selected for homozygocity of the targeted allele through increasing concentration of G418 to obtain R39HSs/R39HSs ES cells. Cre – loxP mediated deletion of the PGK-NeoR cassette resulted in DHSs/DHSs ES cells. Targeted ES cells were injected into Rag2-/- blastocysts to obtain RDBC chimeras [11] or into C57BL/6 blastocysts to obtain chimeras that could be crossed to 129Sv mice to achieve germline transmission of the targeted allele. Generation of mice with targeted deletion of 59IgH DNaseI hypersensitive sites To determine the in vivo function of the cluster of DNaseI hypersensitive sites described at the 59 end of the IgH locus [9] we first replaced a ,340 bp BccI – AatII fragment, harboring HS1, with a loxP flanked PGK-NeoR cassette. All targeting experiments were performed in heterozygous IgHa/b EF1 ES cells which have the advantage that IgHa (129 strain) and IgHb (C57BL/6 strain) alleles can be distinguished by antibodies against the different allotypes or by detection of restriction fragment length polymor- phisms (RFLP). Targeting vector homology arms were cloned from 129 strain genomic DNA, resulting in correct targeting events only on the IgHa allele. In heterozygous targeted ES cells, the IgHb allele always remained in the untargeted wildtype configuration. RT-PCR analysis RNA was extracted using TriPure Isolation Reagent (Roche). 200 ng–1 mg of total RNA was reverse transcribed for one hour at 50uC using random hexamers (Roche) and Superscript III (Invitrogen) reverse transcriptase. PCR was performed at 94uC for 49, 30–39 cycles of 94uC for 300, annealing temperature (Table S1) for 300, 72uC for 300, followed by 72uC for 59. cDNA input amount was normalized upon PCR amplification of b-actin cDNA. PCR products were visualized on ethidium bromide gels and/or subsequently transferred to nylon membranes and visualized with end labeled oligonucleotide probes (Table S1). Targeted deletion of 59IgH DNaseI hypersensitive sites in ES cells and generation of mutant mice All mouse were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by Animal Research of Children’s Hospital Boston (Protocol # 08 11 1253R). The RHS1 targeting vector was assembled in pLNTK [10]. As a 59 homology arm a 2.2 kb PCR product was generated with primers 59 GTCGACGGATTTAGGAGGATACACAAC 39 and 59 GTCGACCTTGGATAACACAGAACTCTG 39 containing a SalI site at their 59 ends, which facilitate cloning of the PCR product into the SalI site of pLNTK. As a 39 homology arm a 7.3 kb AatII – ApaI fragment was blunt end cloned into the XhoI site of pLNTK. The R39HSs targeting vector was generated by blunt end cloning a 4.4 kb EcoRI fragment into the SalI site of pLNTK as a 59 homology arm, and a 7.0 kb KpnI fragment into the XhoI site as the 39 homology arm. Correct targeting events and cre – loxP deletion events were confirmed by Southern blotting (Fig 1). Probe 1 is a 830 bp PCR product amplified with primers 59 GCTCATGTACCAATCTGCACTCAC 39 and 59 CACTGT- GACCTCCATCTTATGTCTG 39. Probe 2 is a 1.2 kb PstI – EcoRI fragment 59 of HS2. Probe 3 is a 0.8 kb PstI – XbaI fragment about 11 kb 39 of HS3b. To confirm single integration of the targeting vectors a 525 bp NeoR probe was used, amplified with primers 59 GCAGCCATATGGGATCGGC 39 and 59 GTTCGGCTGGCGCGAGCCCC 39. November 2010 \| Volume 5 \| Issue 11 \| e13992 B cell hybridomas DHS1 ES cells exhibit a 15.7 kb DHS1 band and a 26.3 kb band for the wildtype IgHb allele (lane 9), R39HSs ES cells show a 11.1 kb R39H and a 26.3 kb band for the wildtype IgHb allele (lane 11), DHSs ES cells are identified by the presence of a 14.1 kb DHSs band in additio 26.3 kb wildtype IgHb allele (lane 10). All targeting events occured on the IgHa allele, whereas the IgHb allele remained in wildtype config Drawings not to scale. B - BglII; S - SacI; P - SphI. Figure 1. Targeting strategy for the generation of the RHS1, DHS1, and DHSs alleles. (A) the wildtype (wt) IgH locus and its 59 flanking region are shown. VH, DH, JH indicate representative IgH V, D, and J segments. Exons 1, 2, and 3 of Zfp386 are shown as grey rectangles, DNaseI hypersensitive sites HS1, HS2, HS3a, and HS3b are shown as black ovals. Correct targeting events of the RHS1 targeting vector were identified by Southern blotting on BglII digested ES cell DNA using probe 1, which results in a 15.8 kb band (lane 1) in addition to the 14.2 kb wildtype band (lane 2). Cre – loxP (black triangles) mediated deletion of the PGK-NeoR cassette (NeoR) from the RHS1 allele results in the DHS1 allele. Deletions were identified by Southern analysis of SacI digested DNA utilizing probe 2. A targeted clone before Cre – mediated deletion exhibits a 6.7 kb RHS1 band and a 5.1 kb wildtype band (lane 3). Upon deletion of the PGK-NeoR cassette, a 4.8 kb DHS1 band and a 5.1 kb wildtype band are visible (lane 4). Lane 5 shows untargeted wildtype DNA. (B) The DHS1 allele was targeted with the R39HSs targeting vector to introduce a PGK-NeoR cassette flanked by loxP sites. Correct targeting events were confirmed by Southern blotting on SphI digested ES cell DNA with probe 1, resulting in a 2.6 kb band for the wildtype IgHb allele and a 7.2 kb band for R39HSs, the targeted IgHa allele (lane 6). Cre – mediated recombination between the first and the third loxP site generates the DHSs allele (14.1 kb, lane 8). Homozygous R39HSs ES cells were generated under increasing concentrations of G418, resulting in a single 7.2 kb R39HSs band (lane 7). Southern analysis on SphI digested DNA with probe 3 confirms correct targeting events of the R39HSs targeting vector. B cell hybridomas CD43- splenocytes were isolated by MACS, stimulated with LPS (20 mg/ml), and fused to NS-1 plasmacytoma cells (TIB-18, ATCC) as described previously [12]. IgH V(D)J rearrangement status was analyzed by Southern blotting of EcoRI digested genomic DNA of clonal hybridomas with three different probes, a 1.6 kb HindIII - EcoRI fragment 39 of JH4, a 0.38 kb SacI - ApaI fragment 39 of DHQ52, and a 0.75 kb PCR product 59 of DHFL16.1 generated with oligonucleotides 59 GAACAG- CAACCCTTGACTGACTCTG 39 and 59 GATTGGTTCT- TATGGAATGGGTGG 39. Targetings were performed with the RHS1 targeting vector (Fig. 1A) to obtain the RHS1 allele and, upon cre/loxP deletion, PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 November 2010 \| Volume 5 \| Issue 11 \| e13992 2 59IgH Enhancer Figure 1. Targeting strategy for the generation of the RHS1, DHS1, and DHSs alleles. (A) the wildtype (wt) IgH locus and region are shown. VH, DH, JH indicate representative IgH V, D, and J segments. Exons 1, 2, and 3 of Zfp386 are shown as grey recta hypersensitive sites HS1, HS2, HS3a, and HS3b are shown as black ovals. Correct targeting events of the RHS1 targeting vector were Southern blotting on BglII digested ES cell DNA using probe 1, which results in a 15.8 kb band (lane 1) in addition to the 14.2 kb wildty 2). Cre – loxP (black triangles) mediated deletion of the PGK-NeoR cassette (NeoR) from the RHS1 allele results in the DHS1 allele. D identified by Southern analysis of SacI digested DNA utilizing probe 2. A targeted clone before Cre – mediated deletion exhibits a 6.7 and a 5.1 kb wildtype band (lane 3). Upon deletion of the PGK-NeoR cassette, a 4.8 kb DHS1 band and a 5.1 kb wildtype band are visible 5 shows untargeted wildtype DNA. (B) The DHS1 allele was targeted with the R39HSs targeting vector to introduce a PGK-NeoR casse loxP sites. Correct targeting events were confirmed by Southern blotting on SphI digested ES cell DNA with probe 1, resulting in a 2.6 k wildtype IgHb allele and a 7.2 kb band for R39HSs, the targeted IgHa allele (lane 6). Cre – mediated recombination between the first and site generates the DHSs allele (14.1 kb, lane 8). Homozygous R39HSs ES cells were generated under increasing concentrations of G418 single 7.2 kb R39HSs band (lane 7). B cell hybridomas Southern analysis on SphI digested DNA with probe 3 confirms correct targeting events of the R3 vector. DHS1 ES cells exhibit a 15.7 kb DHS1 band and a 26.3 kb band for the wildtype IgHb allele (lane 9), R39HSs ES cells show a 11.1 k and a 26.3 kb band for the wildtype IgHb allele (lane 11), DHSs ES cells are identified by the presence of a 14.1 kb DHSs band in a 26 3 kb wildtype IgHb allele (lane 10) All targeting events occured on the IgHa allele whereas the IgHb allele remained in wildtype 5 Figure 1. Targeting strategy for the generation of the RHS1, DHS1, and DHSs alleles. (A) the wildtype (wt) IgH locus and its 59 region are shown. VH, DH, JH indicate representative IgH V, D, and J segments. Exons 1, 2, and 3 of Zfp386 are shown as grey rectangles hypersensitive sites HS1, HS2, HS3a, and HS3b are shown as black ovals. Correct targeting events of the RHS1 targeting vector were iden Southern blotting on BglII digested ES cell DNA using probe 1, which results in a 15.8 kb band (lane 1) in addition to the 14.2 kb wildtype ba 2). Cre – loxP (black triangles) mediated deletion of the PGK-NeoR cassette (NeoR) from the RHS1 allele results in the DHS1 allele. Deletio identified by Southern analysis of SacI digested DNA utilizing probe 2. A targeted clone before Cre – mediated deletion exhibits a 6.7 kb RH and a 5.1 kb wildtype band (lane 3). Upon deletion of the PGK-NeoR cassette, a 4.8 kb DHS1 band and a 5.1 kb wildtype band are visible (lane 5 shows untargeted wildtype DNA. (B) The DHS1 allele was targeted with the R39HSs targeting vector to introduce a PGK-NeoR cassette fla loxP sites. Correct targeting events were confirmed by Southern blotting on SphI digested ES cell DNA with probe 1, resulting in a 2.6 kb band wildtype IgHb allele and a 7.2 kb band for R39HSs, the targeted IgHa allele (lane 6). Cre – mediated recombination between the first and the th site generates the DHSs allele (14.1 kb, lane 8). Homozygous R39HSs ES cells were generated under increasing concentrations of G418, resul single 7.2 kb R39HSs band (lane 7). Southern analysis on SphI digested DNA with probe 3 confirms correct targeting events of the R39HSs t vector. Development of homozygous RHS1, DHS1, and DHSs lymphocytes Development of homozygous RHS1, DHS1, and DHSs lymphocytes y p y Lymphocytes of different developmental stages can be identified by FACS analysis of cells from lymphoid tissues such as bone marrow, thymus, or spleen. We analyzed 8 week old wildtype mice, homozygous RHS1, and homozygous DHS1 mice that carry the mutant alleles in their germline, as well as lymphocytes from RDBC chimeras generated from homozygous DHSs ES cells (Fig. 2). In wildtype bone marrow, pro-B cells can be identified as IgM- B220+ CD43hi and pre-B cells as IgM- B220+ CD43lo cells, respectively. Defects in B-cell development can be revealed by the increase or decrease of certain lymphocyte populations. In this regard, impaired IgH V(D)J recombination leads to an accumu- lation of pro-B cells and to reduced numbers of pre-B cells [7]. We performed FACS analyses of bone marrow from three mice of each genotype to measure the percentage of pro- and pre-B cells in the lymphocyte gate. These analyses revealed the average percentage (6 standard deviation) of pro-B and pre-B cells, respectively of B220+/CD43+ events in the total lymphocyte gate were 1462 and 50620 for wildtype, 963 and 5666 for DHS1, and 963 and 42611 for RHS1 mice (Fig. 2A). Thus, there were no obvious differences in early B-cell development in wildtype and mutant mice. However we cannot exclude minor developmental defects not readily detectable by such analyses. Homozygous mutant DHSs bone marrow cells were analyzed in a similar fashion, but only Ly9.1+ cells were included in the analysis. Ly9.1 is exclusively expressed on cells derived from the DHSs ES cells but not on cells derived from the Rag2-/- blastocyst. The presence of a large compartment of blastocyst derived Rag-deficient pro-B cells in the bone marrow can interfere with development of ES cell It was speculated that HS1 might regulate the differential activation of distal versus proximal VH families [9]; therefore, we analyzed the rearrangement efficiencies of the proximal VH7183 family (Fig. 3C), the distal VHJ558 familiy (Fig. 3D), and the distal most VH segment VHJ558.55 (Fig. 3E). We found that pro-B cells and pre-B cells from wildtype, homozygous RHS1, and homozy- gous DHS1 mice rearrange the proximal VH7183 family at similar levels (Fig. 3C). Also, the distal family VHJ558 (Fig. 3D) as well as the distal most VH segment VHJ558.55 (Fig. 3E) rearranged at comparable efficiencies in pro-B cells and pre-B cells from the three different genotypes. B cell hybridomas Subsequently, targeted ES cells were injected into Rag2-/- blastocysts to obtain Rag-deficient blastocyst complementation (RDBC) chimeras, and into wildtype blastocyts to generate mice that carry the RHS1 or DHS1 allele in their germline. In order to delete all four hypersensitivity sites (HS1, HS2, HS3a, and HS3b), ES cells containing the DHS1 allele were targeted with the R39HSs targeting vector to obtain the R39HSs allele (Fig. 1B). Cre/loxP recombination between the loxP site originating from the DHS1 allele and the loxP site 39 of the PGK- NeoR cassette results in the replacement of a 8.9 kb region, harboring all described 59IgH DNaseI hypersensitive sites, with a single loxP site, referred to as the DHSs allele. Germline transmission could not be achieved for either of the R39HSs or DHSs heterozygous ES cell lines. Therefore, we placed ES cells containing the R39HSs allele under increasing concentrations of G418 to select for homozygous mutant ES cells. The homozygous mutant ES cells were subsequently subjected to cre/loxP recombination to delete the Neor gene and generate ES cells homozygous for the DHSs allele. The homozygous mutant DHSs ES cells were injected into Rag2-/- blastocysts, and chimeras generated by RDBC and lymphocytes were analyzed. and did not suggest a block in B-cell development (Fig. 2A). Next we analyzed spleens for IgM+ B220+ AA4.1+ transitional B-cells and IgM+ B220+ AA4.1- mature B-cells (Fig. 2B). In homozygous RHS1, and homozygous DHS1 mice transitional (19.7%–27.5%) and mature (65.3%–72.7%) B-cell compartments similar to wildtype were identified; whereas, in spleens from RDBC chimeras generated from homozygous DHSs ES cells strongly reduced numbers of transitional B-cells were observed (6.44%). This reduction in the transitional B-cell compartment compared to the mature B-cell compartment (75%) might be due to overall reduced numbers of developing B cells in the obtained RDBC chimeras and to the accumulation of mature B-cells in the periphery of these mice and not to a defect in B cell development. Finally, we observed normal development of T-lymphocytes in the thymi of wildtype, homozygous RHS1, and homozygous DHS1 mice as well as RDBC chimeras generated from homozygous DHSs ES cells (Fig. 2C). The DHS1, RHS1, and DHSs alleles show no significant defect in V(D)J recombination The data indicating that HS1 is pro-B cell specific and contains binding sites for the transcription factors PU.1, Pax5, and E2A led to the suggestion that HS1 could be involved in regulation of V(D)J recombination at the IgH locus [9]. We utilized a PCR based assay to assess V(D)J recombination efficiencies in developing lymphocytes from mice with homozygous deletion of HS1. FACS-sorted pro-B cells (IgM- B220+ CD43hi) and pre-B cells (IgM- B220+ CD43lo) from bone marrow and double positive (DP) T-cells (B220- CD4+ CD8+) from thymus were analyzed for D to JH, VH to DJH, Vk to Jk and Vl to Jl rearrangements. Intensities of PCR bands for DHQ52 to JH (Fig. 3A) and DSP to JH rearrangements (Fig. 3B) were comparable in pro-B cells, pre-B cells, and DP T-cells from wildtype, homozygous RHS1, and homozygous DHS1 mice indicating that deletion of the pro-B cell specific HS1 site does not detectably affect the D to JH recombination step. DNA input amounts were normalized to the presence of a genomic sequence within the murine DLG5 gene (Fig. 3H). B cell hybridomas DHS1 ES cells exhibit a 15.7 kb DHS1 band and a 26.3 kb band for the wildtype IgHb allele (lane 9), R39HSs ES cells show a 11.1 kb R39HSs band and a 26.3 kb band for the wildtype IgHb allele (lane 11), DHSs ES cells are identified by the presence of a 14.1 kb DHSs band in addition to the 26.3 kb wildtype IgHb allele (lane 10). All targeting events occured on the IgHa allele, whereas the IgHb allele remained in wildtype configuration. Drawings not to scale. B - BglII; S - SacI; P - SphI. doi:10.1371/journal.pone.0013992.g001 PLo November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 3 59IgH Enhancer 59IgH Enhancer g PLoS ONE \| www.plosone.org 4 November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 59IgH Enhancer Figure 2. Development of homozygous DHS1, RHS1, and DHSs lymphocytes. (A) Bone marrow from wildtype (wt), homozygous DHS1, homozygous RHS1 mice, and RDBC chimeras generated from homozygous DHSs ES cells was subjected to FACS analysis. Gates were set on the lymphocyte population, Ly9.1 positive population, and on IgM negative population (upper three blots, left to right) to analyze pro-B cell (IgM- B220+ CD43hi) and pre-B cell (IgM- B220+ CD43lo) populations (lower blots). (B) FACS analysis of splenocytes from wildtype (wt), homozygous DHS1, homozygous RHS1 mice, and RDBC chimeras generated from homozygous DHSs ES cells. Gates were set on the lymphocyte population and on the IgM positive population (upper two blots, left to right) to analyze transitional B-cell (IgM+ B220+ AA4.1+) and mature B-cell (IgM+ B220+ AA4.1-) populations (lower blots). (C) FACS analysis of thymocytes gated on the lymphocyte population (upper blot) from wildtype (wt), homozygous DHS1, homozygous RHS1 mice, and RDBC chimeras generated from homozygous DHSs ES cells (lower blots). doi:10.1371/journal.pone.0013992.g002 derived B-lymphocytes. However, FACS analysis of DHSs bone marrow B cells indicated the presence of both pro- and pre-B cells and did not suggest a block in B-cell development (Fig. 2A). Next we analyzed spleens for IgM+ B220+ AA4.1+ transitional B-cells and IgM+ B220+ AA4.1- mature B-cells (Fig. 2B). B cell hybridomas In homozygous RHS1, and homozygous DHS1 mice transitional (19.7%–27.5%) and mature (65.3%–72.7%) B-cell compartments similar to wildtype were identified; whereas, in spleens from RDBC chimeras generated from homozygous DHSs ES cells strongly reduced numbers of transitional B-cells were observed (6.44%). This reduction in the transitional B-cell compartment compared to the mature B-cell compartment (75%) might be due to overall reduced numbers of developing B cells in the obtained RDBC chimeras and to the accumulation of mature B-cells in the periphery of these mice and not to a defect in B cell development. Finally, we observed normal development of T-lymphocytes in the thymi of wildtype, homozygous RHS1, and homozygous DHS1 mice as well as RDBC chimeras generated from homozygous DHSs ES cells (Fig. 2C). the DHS1 allele. Correct targeting events (Fig1) and single integration of the targeting vector (Figure S1) were confirmed by Southern blotting. Subsequently, targeted ES cells were injected into Rag2-/- blastocysts to obtain Rag-deficient blastocyst complementation (RDBC) chimeras, and into wildtype blastocyts to generate mice that carry the RHS1 or DHS1 allele in their germline. In order to delete all four hypersensitivity sites (HS1, HS2, HS3a, and HS3b), ES cells containing the DHS1 allele were targeted with the R39HSs targeting vector to obtain the R39HSs allele (Fig. 1B). Cre/loxP recombination between the loxP site originating from the DHS1 allele and the loxP site 39 of the PGK- NeoR cassette results in the replacement of a 8.9 kb region, harboring all described 59IgH DNaseI hypersensitive sites, with a single loxP site, referred to as the DHSs allele. Germline transmission could not be achieved for either of the R39HSs or DHSs heterozygous ES cell lines. Therefore, we placed ES cells containing the R39HSs allele under increasing concentrations of G418 to select for homozygous mutant ES cells. The homozygous mutant ES cells were subsequently subjected to cre/loxP recombination to delete the Neor gene and generate ES cells homozygous for the DHSs allele. The homozygous mutant DHSs ES cells were injected into Rag2-/- blastocysts, and chimeras generated by RDBC and lymphocytes were analyzed. derived B-lymphocytes. However, FACS analysis of DHSs bone marrow B cells indicated the presence of both pro- and pre-B cells and did not suggest a block in B-cell development (Fig. 2A). the DHS1 allele. Correct targeting events (Fig1) and single integration of the targeting vector (Figure S1) were confirmed by Southern blotting. Development of homozygous RHS1, DHS1, and DHSs lymphocytes VH to DJH recombination was absent in DP T-cells from wildtype, homozygous RHS1, and homozygous DHS1 mice as the VH to DJH recombination step is restricted to the B-lineage (Fig 3C, D, E). These data show that HS1 is not PLoS ONE \| www.plosone.org PLoS November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 5 59IgH Enhancer Figure 3. V(D)J recombination in DHS1 and RHS1 mice. Pro-B cells, pre-B cells and double positive (DP) T-cells from wildtype 129 mice, from homozygous DHS1, and homozygous RHS1 mice were sorted by FACS. 5-fold dilutions of genomic DNA were subjected to PCR analysis. IgH V(D)J recombination efficiencies were assessed using a reverse primer downstream of JH4 and a forward primer recognizing DQ52 (A), DSPs (B), the VH7183 family (C), the VHJ558 family (D), or the VHJ558.55 segment (E). Rearrangements can occur to JH1, JH2, JH3, or JH4 as indicated. GL indicates PCR product from germline configuration. Igk rearrangements were quantified (F), rearrangements can occur to Jk1, Jk2, Jk4, or Jk5 as indicated. Igl rearrangement efficiency was analyzed (G). Bands correspond to Vl2 - Jl2, Vl1 - Jl3, or Vl1 - Jl1 rearrangements as indicated. DNA input was normalized to DLG5 PCR products (H). doi:10.1371/journal.pone.0013992.g003 Figure 3. V(D)J recombination in DHS1 and RHS1 mice. Pro-B cells, pre-B cells and double positive (DP) T-cells from wildtype 129 mice, from homozygous DHS1, and homozygous RHS1 mice were sorted by FACS. 5-fold dilutions of genomic DNA were subjected to PCR analysis. IgH V(D)J recombination efficiencies were assessed using a reverse primer downstream of JH4 and a forward primer recognizing DQ52 (A), DSPs (B), the VH7183 family (C), the VHJ558 family (D), or the VHJ558.55 segment (E). Rearrangements can occur to JH1, JH2, JH3, or JH4 as indicated. GL indicates PCR product from germline configuration. Igk rearrangements were quantified (F), rearrangements can occur to Jk1, Jk2, Jk4, or Jk5 as indicated. Igl rearrangement efficiency was analyzed (G). Bands correspond to Vl2 - Jl2, Vl1 - Jl3, or Vl1 - Jl1 rearrangements as indicated. DNA input was normalized to DLG5 PCR products (H). Development of homozygous RHS1, DHS1, and DHSs lymphocytes Numbers for D to JH rearranged alleles (DJ) and nonproductive VH to DJH rearranged alleles (VDJ-) are shown. doi:10.1371/journal.pone.0013992.t001 Wildtype B cells undergo D to JH rearrangements on both alleles; but still, consistent with earlier studies, about 5% of hybridomas harbor an IgH allele in germline configuration which presumably originates from tripartite fusions involving non B-cells [12] (not shown). The number of mutant alleles in germline configuration was not increased compared to wildtype indicating that RHS1, DHS1, and DHSs alleles can undergo efficient D to JH recombination (not shown). In 50–60% of wildtype B-cells the nonproductive allele is in DJH configuration; whereas in 40–50% the nonproductive allele is in VHDJH configuration [15]. An increased percentage of DJH alleles could indicate less efficient VH to DJH recombination: in contrast, an increased percentage of VHDJH alleles might indicate a break in allelic exclusion. IgMa expressing hybridomas generated from B-cells heterozygous for RHS1, DHS1, and DHSs were analyzed for their rearrangement status of the wildtype IgMb allele and show ratios of DJH (56%– 61%), and VHDJH alleles (39%–44%) in the expected range (Table 1). IgMb expressing hybridomas were analyzed for the necessary for rendering the distal part of the VH cluster accessible and, therefore, suggest that HS1 does not play a major role in regulation of usage or accessibility of distal versus proximal VH families. Recently, it has been shown that IgH and Igk loci can colocalize during B-cell development, mainly at the pre-B cell stage, and it was suggested that this colocalization induces decontraction of the IgH locus [14]. We therefore performed an assay to evaluate Igk (Fig. 3F) and Igl (Fig. 3G) V(D)J recombination efficiencies. Both Igk and Igl loci show similar V(D)J recombination levels in the analyzed developing B cells from wildtype, homozygous RHS1, and homozygous DHS1 mice, while light chain rearrangements were absent in DP T-cells from the three different genotypes. Therefore, we conclude that deletion of HS1 does not markedly affect Ig light chain gene rearrangements. Figure 4. IgMa versus IgMb expression in DHS1, RHS1, and DHSs B cells. Heterozygous IgMa/b B-cells from spleen (A) and bone marrow (B) of 129 wildtype (wt) or DHS1, RHS1, and DHSs RDBC chimeras were analyzed IgMa and IgMb expression. In DHS1, RHS1, and DHSs B-cells the IgMb allele is in wildtype configuration whereas the IgMa allele is the mutant allele. doi:10.1371/journal.pone.0013992.g004 Figure 4. Development of homozygous RHS1, DHS1, and DHSs lymphocytes doi:10.1371/journal.pone.0013992.g003 November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 6 59IgH Enhancer As an independent method to evaluate D to JH and VH to DJH recombination efficiencies, we generated clonal hybridoma lines from splenic B-cells of IgHa/b heterozygous RHS1, DHS1 mice carrying the mutant allele in their germline and of RDBC chimeras generated from heterozygous DHSs ES cells (Table 1). In each case the IgHa allele was the mutant allele while the IgHb allele was the wildtype allele. In splenic B-cells, one allele exists as a functional VHDJH rearrangement, while the second allele can either be in germline configuration, or it exists as a DJH or an nonproductive VHDJH rearrangement. The rearrangement status of the second IgH allele was assessed by Southern blot analysis. Consequently, hybridomas expressing the mutant IgHa allele can be analyzed for rearrangement efficiency of the wildtype IgHb allele, and vice versa, in hybridomas expressing the wildtype IgHb allele, the rearrangement status of the mutant IgHa allele can be assessed. Table 1. DHS1, RHS1, and DHSs hybridoma analysis. DJ VDJ- DHS1 IgMa+ 66 (61%) 43 (39%) DHS1 IgMb+ 52 (57%) 39 (43%) RHS1 IgMa+ 62 (56%) 49 (44%) RHS1 IgMb+ 45 (52%) 41 (48%) DHSs IgMa+ 51 (61%) 32 (39%) DHSs IgMb+ 55 (69%) 25 (31%) Hybridomas were generated from heterozygous IgMa/b DHS1, RHS1, and DHSs splenic B-cells. In each case, IgMb is the wildtype allele and IgMa is the mutant allele. IgMa expressing hybridomas (IgMa+) and IgMb expressing hybridomas (IgMb+) of each genotype were analyzed for the rearrangement status of their nonproductive allele. Numbers for D to JH rearranged alleles (DJ) and nonproductive VH to DJH rearranged alleles (VDJ-) are shown. doi:10.1371/journal.pone.0013992.t001 Hybridomas were generated from heterozygous IgMa/b DHS1, RHS1, and DHSs splenic B-cells. In each case, IgMb is the wildtype allele and IgMa is the mutant allele. IgMa expressing hybridomas (IgMa+) and IgMb expressing hybridomas (IgMb+) of each genotype were analyzed for the rearrangement status of their nonproductive allele. Numbers for D to JH rearranged alleles (DJ) and nonproductive VH to DJH rearranged alleles (VDJ-) are shown. doi:10.1371/journal.pone.0013992.t001 Hybridomas were generated from heterozygous IgMa/b DHS1, RHS1, and DHSs splenic B-cells. In each case, IgMb is the wildtype allele and IgMa is the mutant allele. IgMa expressing hybridomas (IgMa+) and IgMb expressing hybridomas (IgMb+) of each genotype were analyzed for the rearrangement status of their nonproductive allele. The DHS1, RHS1, and DHSs alleles do not affect allelic exclusion FACS analysis of wt B cells from spleen (Fig. 4A) and bone marrow (Fig. 4B) shows distinct populations of similar size for B cells that are single positive for either IgHa or IgHb, but intact allelic exclusion prevents the appearance of an obvious IgHa, IgHb double producing population. Similarly, RDBC chimeras gener- ated from heterozygous RHS1, DHS1, and DHSs ES cells exhibited IgHa or IgHb single positive B-cell populations of similar size in spleen (Fig. 4A) and bone marrow (Fig. 4B) but no IgHa, IgHb double producing population. These data indicate that the deleted sequences of the targeted alleles do not contain a regulatory element that is necessary for implementation of allelic exclusion. Development of homozygous RHS1, DHS1, and DHSs lymphocytes IgMa versus IgMb expression in DHS1, RHS1, and DHSs B cells. Heterozygous IgMa/b B-cells from spleen (A) and bone marrow (B) of 129 wildtype (wt) or DHS1, RHS1, and DHSs RDBC chimeras were analyzed IgMa and IgMb expression. In DHS1, RHS1, and DHSs B-cells the IgMb allele is in wildtype configuration whereas the IgMa allele is the mutant allele. doi:10.1371/journal.pone.0013992.g004 November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 7 59IgH Enhancer rearrangement status of their mutant IgMa allele. RHS1, DHS1, and DHSs alleles do not show significantly increased or decreased (Fisher’s exact test) rearrangement ratios compared to wt alleles, as 52%–69% of mutant alleles were in DJH configuration while 31%–48% were in VHDJH configuration. Furthermore, data from hybridoma analysis (Tab. 1) support this notion as in the case of a break in allelic exclusion increased numbers of hybridomas with VH to DJH rearrangements on both alleles would be expected. Such an increase compared to wildtype alleles could not be observed (Tab. 1), which indicates intact allelic exclusion of RHS1, DHS1, and DHSs alleles. FACS analysis was performed on B-cells from spleens (Fig. 4A) and bone marrow (Fig. 4B) of RDBC chimeras generated from heterozygous RHS1, DHS1, and DHSs ES cells. IgMa expressing populations, representing the targeted allele, and IgMb expressing populations, representing the wildtype allele, were of similar size both in bone marrow and in spleen from RHS1, DHS1, and DHSs chimeras, suggesting that the RHS1, DHS1, and DHSs alleles can undergo V(D)J recombination at the IgH locus at similar efficiencies as wildtype alleles. The 59IgH DNaseI hypersensitive sites are not required for efficient class switch recombination To assess a potential effect of the 59IgH DNaseI hypersensitive sites on CSR, B-cells were stimulated to undergo CSR and analyzed by FACS (Fig. 5). Stimulation with LPS induces IgH isotype switching to c3, while stimulation with IL4+ aCD40 promotes switching to c1. B-cells from AID-/- mice served as negative controls, while wildtype B-cells represented a positive control and therefore switched to the appropriate isotypes under LPS or IL4+ aCD40 stimulation. CSR in homozygous DHSs B- cells occurs at similar levels as in wildtype B-cells implying that the cluster of 59IgH DNaseI hypersensitive sites is not required for efficient CSR to c1 (Fig. 5A) and c3 (Fig. 5B). The DHS1, RHS1, and DHSs alleles do not affect allelic exclusion Complex phenotypes without an obvious relation to the IgH locus in DHS1 mice Currently, we do not know what causes these phenotypes, but we exclude that this phenotype is caused by a second integration of the targeting vector at an undefined site in the genome (Figure S1). The deletion in the DHS1 allele deletes 340 bp within intron 1 of Zfp386. Therefore, age, likely due to a lack of food intake. These mice exhibit an abnormal limp grasping phenotype, i.e. mice clasp their front and hind feet almost immediately upon being lifted by their tail (Fig. 6A, B). Furthermore these mice develop a hydrocephalus, which is already visible at about one week of age and is enlarged over the following weeks (Fig. 6C, D). Histological analysis confirmed the presence of a hydrocephalus, revealed abnormal hindbrain development, and revealed retinal abnormalities (Fig. 6E, F, G). The wildtype retina is organized in a delicate layer system (Fig. 6E): stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). In the DHS1 mutant mice, the organization of retinal layers is impaired in such a way that nuclei from the outer nuclear layer are aberrantly located in the layer of rods and cones (Fig. 6F). In some more severe cases rosette formation in the outer nuclear layer is evident (Fig. 6G). Currently, we do not know what causes these phenotypes, but we exclude that this phenotype is caused by a second integration of the targeting vector at an undefined site in the genome (Figure S1). The deletion in the DHS1 allele deletes 340 bp within intron 1 of Zfp386. Therefore, Complex phenotypes without an obvious relation to the IgH locus in DHS1 mice We performed targeted deletion experiments of the 59IgH DNaseI hypersensitive sites to test their suggested function in IgH locus regulation. So far no major IgH related phenotype was identified. However, about 20% of homozygous DHS1 mice develop a complex neurological phenotype and die at 3–5 weeks of Figure 5. Ig class switch recombination in absence of the 59IgH DNaseI hypersensitive sites. MACS purified splenic B-cells were stimulated in culture with LPS or IL4+ aCD40 as indicated. FACS analysis shows B-cells that underwent CSR as B220+ IgG1+ or B220+ IgG3+ cells, respectively. AID-/- B-cells served as negative controls, wildtype (wt) 129 B-cells as positive controls. Homozygous DHSs B-cells were isolated from RDBC chimeras. doi:10.1371/journal.pone.0013992.g005 Figure 5. Ig class switch recombination in absence of the 59IgH DNaseI hypersensitive sites. MACS purified splenic B-cells were stimulated in culture with LPS or IL4+ aCD40 as indicated. FACS analysis shows B-cells that underwent CSR as B220+ IgG1+ or B220+ IgG3+ cells, respectively. AID-/- B-cells served as negative controls, wildtype (wt) 129 B-cells as positive controls. Homozygous DHSs B-cells were isolated from RDBC chimeras. doi:10.1371/journal.pone.0013992.g005 PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 8 59IgH Enhancer 59IgH Enhancer misregulation of that poorly described gene might cause the described phenotypes although other possibilities are conceivable. age, likely due to a lack of food intake. These mice exhibit an abnormal limp grasping phenotype, i.e. mice clasp their front and hind feet almost immediately upon being lifted by their tail (Fig. 6A, B). Furthermore these mice develop a hydrocephalus, which is already visible at about one week of age and is enlarged over the following weeks (Fig. 6C, D). Histological analysis confirmed the presence of a hydrocephalus, revealed abnormal hindbrain development, and revealed retinal abnormalities (Fig. 6E, F, G). The wildtype retina is organized in a delicate layer system (Fig. 6E): stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). In the DHS1 mutant mice, the organization of retinal layers is impaired in such a way that nuclei from the outer nuclear layer are aberrantly located in the layer of rods and cones (Fig. 6F). In some more severe cases rosette formation in the outer nuclear layer is evident (Fig. 6G). Discussion This study aimed for elucidating the potential regulatory functions of a cluster of recently described DNaseI hypersensitive sites at the 59 end of the IgH locus [9]. We performed targeted deletion of either the pro-B cell specific site HS1 (DHS1) or deletion of the entire cluster of hypersensitive sites (DHSs) in mice or in their lymphocytes, respectively. A potential regulatory element at the 59end of the IgH locus was speculated to regulate processes such as IgH allelic exclusion, VH germline transcription, differential accessibility or usage of distal versus proximal VH gene families. Furthermore, it was suggested that the 59end of the IgH locus might play a role in positioning the IgH locus in distinct subnuclear compartments [16,17,18], and it was suggested to harbor insulator or boundary capacity [19]. B- and T-lymphocytes homozygous for the DHS1, RHS1, and DHSs alleles appear to proceed through lymphocyte development in an unimpaired way. Data from RDBC chimeras generated from Figure 6. Complex phenotypes of homozygous DHS1 mice. Homozygous DHS1 mice exhibit an abnormal limp grasping phenotype (B) whereas wildtype (wt) mice do not (A). DHS1 mice can develop severe hydrocephalus as indicated by arrows in (C) and (D). A wildtype mouse without hydrocephalus is shown in (C). The wildtype retina is organized in distinct layers (E): Stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). The retina of homozygous DHS1 mice shows external nuclei from the outer nuclear layer (5) in the layer of rods and cones (6) indicated by arrows in (F), or rosette formation of the outer nuclear layer (5) indicated by arrows in (G). doi:10.1371/journal.pone.0013992.g006 Figure 6. Complex phenotypes of homozygous DHS1 mice. Homozygous DHS1 mice exhibit an abnormal limp grasping phenotype (B) whereas wildtype (wt) mice do not (A). DHS1 mice can develop severe hydrocephalus as indicated by arrows in (C) and (D). A wildtype mouse without hydrocephalus is shown in (C). The wildtype retina is organized in distinct layers (E): Stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). Acknowledgments We thank Drs. Meinrad Busslinger, Thomas Decker, Andrew Franklin, Cosmas Giallourakis, Gang Li, Raul Mostoslavsky, Sheila Ranganath, Jing Wang, and Shan Zha for helpful discussions, Lisa Acquaviva and Aimee Williams for mouse work, and Natasha Barteneva for cell sorting. Overall, our analysis of the deletion of the pro-B cell specific site HS1 or the whole cluster of 59IgH DNaseI hypersensitive sites did not support the existence of a cis-regulatory function of these elements regarding the IgH locus. References 11. Chen J, Lansford R, Stewart V, Young F, Alt FW (1993) RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc Natl Acad Sci U S A 90: 4528–4532. 1. Perlot T, Alt FW (2008) Cis-regulatory elements and epigenetic changes control genomic rearrangements of the IgH locus. Adv Immunol 99: 1–32. 1. Perlot T, Alt FW (2008) Cis-regulatory elements and epigenetic changes control genomic rearrangements of the IgH locus. Adv Immunol 99: 1–32. 2. Alt FW, Yancopoulos GD, Blackwell TK, Wood C, Thomas E, et al. (1984) Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J 3: 1209–1219. 12. Dudley DD, Sekiguchi J, Zhu C, Sadofsky MJ, Whitlow S, et al. (2003) Impaired V(D)J recombination and lymphocyte development in core RAG1-expressing mice. J Exp Med 198: 1439–1450. 3. Chowdhury D, Sen R (2001) Stepwise activation of the immunoglobulin mu heavy chain gene locus. EMBO J 20: 6394–6403. 13. Franco S, Gostissa M, Zha S, Lombard DB, Murphy MM, et al. (2006) H2AX prevents DNA breaks from progressing to chromosome breaks and transloca- tions. Mol Cell 21: 201–214. 4. Jung D, Giallourakis C, Mostoslavsky R, Alt FW (2006) Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu Rev Immunol 24: 541–570. 14. Hewitt SL, Farmer D, Marszalek K, Cadera E, Liang HE, et al. (2008) Association between the Igk and Igh immunoglobulin loci mediated by the 39 Igk enhancer induces ‘decontraction’ of the Igh locus in pre-B cells. Nat Immunol 9: 396–404. 5. Chaudhuri J, Basu U, Zarrin A, Yan C, Franco S, et al. (2007) Evolution of the immunoglobulin heavy chain class switch recombination mechanism. Adv Immunol 94: 157–214. 6. Sakai E, Bottaro A, Davidson L, Sleckman BP, Alt FW (1999) Recombination and transcription of the endogenous Ig heavy chain locus is effected by the Ig heavy chain intronic enhancer core region in the absence of the matrix attachment regions. Proc Natl Acad Sci U S A 96: 1526–1531. 15. Mostoslavsky R, Alt FW, Rajewsky K (2004) The lingering enigma of the allelic exclusion mechanism. Cell 118: 539–544. 16. Kosak ST, Skok JA, Medina KL, Riblet R, Le Beau MM, et al. (2002) Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296: 158–162. 7. Perlot T, Alt FW, Bassing CH, Suh H, Pinaud E (2005) Elucidation of IgH intronic enhancer functions via germ-line deletion. Table S1 Found at: doi:10.1371/journal.pone.0013992.s003 (0.08 MB DOCX) The only observed phenotypes so far occurred in homozygous DHS1 mice and seem to be independent of the IgH locus. DHS1 mice show abnormal limp grasping indicating a neurological abnormality, DHS1 mice can develop severe hydrocephalus and exhibit retinal impairments. A possible explanation for these phenotypes is a potential defect in regulation of the zinc finger protein Zfp386. DHS1 deletes a 340 bp region from intron 1 of Zfp386 which might result in different splice forms, impaired expression levels, or expression patterns of this gene. Discussion heterozygous DHS1, RHS1, and DHSs ES cells indicated that allelic exclusion is not affected in mutant B-cells and that mutant IgH alleles can undergo efficient V(D)J recombination of their IgH locus. Furthermore, data from PCR assays to analyze V(D)J recombination efficiency in mice with HS1 deleted on both alleles supports the notion that HS1 is not necessary for neither the D to JH nor the VH to DJH recombination step. Both proximal and distal VH families as well as the distal most VH segment VHJ558.55 rearrange as efficiently as on wildtype alleles. Similarly, IgL loci in HS1 deleted B-cells rearrange at the same efficiency as wildtype IgL loci. Analysis of IgH V(D)J rearrangement status in hybridomas generated from heterozygous DHS1, RHS1, and DHSs B-cells also strengthens the idea that the deleted DNAseI hypersensitive sites would not regulate IgH V(D)J recombination. We tested for potential alterations associated with DNA end processing during V(D)J recombination by examining the CDR3 sequence obtained from homozygous DHS1 B cells and found a distribution in length that was similar to wildtype B cells [20] (Figure S2). Author Contributions Conceived and designed the experiments: TP IP PB FWA. Performed the experiments: TP IP. Analyzed the data: TP IP JPM AZ PB. Wrote the paper: TP IP FWA. Conceived and designed the experiments: TP IP PB FWA. Performed the experiments: TP IP. Analyzed the data: TP IP JPM AZ PB. Wrote the paper: TP IP FWA. Supporting Information Figure S1 Single integration of the RHS1 targeting vector. The targeting vector (targeting vector RHS1), the targeted locus (RHS1), and the wildtype (wt) IgH locus with its 59 flanking region are shown. VH, DH, JH indicate representative IgH V, D, and J segments. Exons 1, 2, and 3 of Zfp386 are shown as grey rectangles, DNaseI hypersensitive sites HS1, HS2, HS3a, and HS3b are shown as black ovals, the NeoR specific Southern probe as a black rectangle. X - XbaI. Southern analysis of XbaI digested genomic DNA from the targeted RHS1 clones 5 (lane 1) and 23 (lane 2) utilizing the NeoR specific probe shows a single 16.0 kb band. No bands are visible from untargeted wildtype ES cell DNA (lane 3). M - Fermentas 1 kb ladder. Found at: doi:10.1371/journal.pone.0013992.s001 (0.17 MB TIF) Figure S2 CDR3 length in DHS1 B cells. Peripheral B cells were isolated from one DHS1 mouse and a wildtype mouse and amplified for V558-JH4 rearrangements. Heavy Chain CDR3 lengths were calculated as the number of nucleotides between the consensus Cys residue and the Trp residue. 23 individual sequences were analyzed from DHS1 B cells and 10 from wildtype. Found at: doi:10.1371/journal.pone.0013992.s002 (0.05 MB PDF) Figure S2 CDR3 length in DHS1 B cells. Peripheral B cells were isolated from one DHS1 mouse and a wildtype mouse and amplified for V558-JH4 rearrangements. Heavy Chain CDR3 lengths were calculated as the number of nucleotides between the consensus Cys residue and the Trp residue. 23 individual sequences were analyzed from DHS1 B cells and 10 from wildtype. Found at: doi:10.1371/journal.pone.0013992.s002 (0.05 MB PDF) We tested a potential effect of the cluster of DNaseI hypersensitive site on the process of IgH CSR. Assaying class switching upon different in vitro stimulations in wildtype and homozygous DHSs B-cells let us conclude that the cluster of 59IgH DNaseI hypersensitive sites does not play a crucial role in CSR. Discussion The retina of homozygous DHS1 mice shows external nuclei from the outer nuclear layer (5) in the layer of rods and cones (6) indicated by arrows in (F), or rosette formation of the outer nuclear layer (5) indicated by arrows in (G). doi:10.1371/journal.pone.0013992.g006 Figure 6. Complex phenotypes of homozygous DHS1 mice. Homozygous DHS1 mice exhibit an abnormal limp grasping phenotype (B) whereas wildtype (wt) mice do not (A). DHS1 mice can develop severe hydrocephalus as indicated by arrows in (C) and (D). A wildtype mouse without hydrocephalus is shown in (C). The wildtype retina is organized in distinct layers (E): Stratum opticum and ganglionic layer (1), inner plexiform layer (2), inner nuclear layer (3), outer plexiform layer (4), outer nuclear layer (5), layer of rods and cones (6), pigment layer (7). The retina of homozygous DHS1 mice shows external nuclei from the outer nuclear layer (5) in the layer of rods and cones (6) indicated by arrows in (F), or rosette formation of the outer nuclear layer (5) indicated by arrows in (G). doi:10.1371/journal.pone.0013992.g006 November 2010 \| Volume 5 \| Issue 11 \| e13992 PLoS ONE \| www.plosone.org 9 59IgH Enhancer heterozygous DHS1, RHS1, and DHSs ES cells indicated that allelic exclusion is not affected in mutant B-cells and that mutant IgH alleles can undergo efficient V(D)J recombination of their IgH locus. Furthermore, data from PCR assays to analyze V(D)J recombination efficiency in mice with HS1 deleted on both alleles supports the notion that HS1 is not necessary for neither the D to JH nor the VH to DJH recombination step. Both proximal and distal VH families as well as the distal most VH segment VHJ558.55 rearrange as efficiently as on wildtype alleles. Similarly, IgL loci in HS1 deleted B-cells rearrange at the same efficiency as wildtype IgL loci. Analysis of IgH V(D)J rearrangement status in hybridomas generated from heterozygous DHS1, RHS1, and DHSs B-cells also strengthens the idea that the deleted DNAseI hypersensitive sites would not regulate IgH V(D)J recombination. We tested for potential alterations associated with DNA end processing during V(D)J recombination by examining the CDR3 sequence obtained from homozygous DHS1 B cells and found a distribution in length that was similar to wildtype B cells [20] (Figure S2). References Proc Natl Acad Sci U S A 102: 14362–14367. 17. Fuxa M, Skok J, Souabni A, Salvagiotto G, Roldan E, et al. (2004) Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy- chain gene. Genes Dev 18: 411–422. 8. Khamlichi AA, Pinaud E, Decourt C, Chauveau C, Cogne M (2000) The 39 IgH regulatory region: a complex structure in a search for a function. Adv Immunol 75: 317–345. 18. Yang Q, Riblet R, Schildkraut CL (2005) Sites that direct nuclear compartmentalization are near the 59 end of the mouse immunoglobulin heavy-chain locus. Mol Cell Biol 25: 6021–6030. 9. Pawlitzky I, Angeles CV, Siegel AM, Stanton ML, Riblet R, et al. (2006) Identification of a candidate regulatory element within the 59 flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A. J Immunol 176: 6839–6851. 19. Reddy KL, Zullo JM, Bertolino E, Singh H (2008) Transcriptional repression mediated by repositioning of genes to the nuclear lamina. Nature 452: 243–247. 20. Jolly CJ, Klix N, Neuberger MS (1997) Rapid methods for the analysis of immunoglobulin gene hypermutation: application to transgenic and gene targeted mice. Nucleic Acids Res 25(10): 1913–9. 10. Sleckman BP, Bardon CG, Ferrini R, Davidson L, Alt FW (1997) Function of the TCR alpha enhancer in alphabeta and gammadelta T cells. Immunity 7: 505–515. PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e13992 10
W2178611454.txt	https://zenodo.org/record/2444762/files/article.pdf	en		THE TREATMENT OF UNUNITED FRACTURES	Medical journal of Australia	1,934	public-domain	0
W2069818243.txt	https://downloads.hindawi.com/journals/jfs/2005/203281.pdf	en		Traces of multipliers in pairs of weighted Sobolev spaces	Journal of function spaces and applications/Journal of Function Spaces and Applications	2,005	cc-by	8,947	()+ , +- . / 0 1.2.. !!"""#$%&'# '& " &3 -4 5'& % "3& '') 3'# ! " $ % ' Ê # % % % % & ( ) , # % # (+) & % % % . () * & 0 / / . Ê Ê 1 Ê Ê ½ Ê " % % Ê ½ $ , Ê # 2 Ê * 2 Ê 3 456 " # ' ! Ê & * ** ( ) 7 ( ) ' Ê Ê # 8 (7) Ê ( ) Ê * . " 2 Ê Ê Ê . ! 2 Ê " 2 Ê 5 2 Ê 2 Ê . (7) 2 Ê Ê * # Ê ! 2 # # 2 Ê 2 Ê # # # 2 Ê Ê " . . # 9 " # Ê Ê $ # (7) . : " 2 Ê 2 2 Ê ; Ê (7) Ê * 2 2 Ê Ê . 2 Ê 2 # # ! * " 2 Ê 2 Ê * 2 Ê " 2 Ê Ê " 2 Ê # Ê * 2 # ! 2 2 Ê 2 Ê 3 456 " # ' Ê · $ Ê Ê % . % & 2 Ê & ' Ê # 7 * Ê Ê ( ) * $ % Ê %& 2 Ê # + (+) Ê Ê Ê Ê Ê () * < = " # " . Ê " # & 2 Ê # Ê & Ê & ' # (!) # > >! ' , Ê Ê Ê %,2 # # # , 9 Ê %,2 # # " , 8 + , , ? " " / 1 # ( . # 8 (9) # 79! , ! , ! , % ) * * , ? $ Ê & ) * ) ( . , " * 2 Ê . 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L7? % % # >+ ' % (, " G >+ - " " % (, - 3E < 3 @> # 3 : ' " 4 (7) 3 456 " # ' (9) (+) & D < # ' ?. ? !>!L7.7 M 2 E 4 # " >@ @. !L9+ 4 : #E+> >7 $ 'F $ 'F # . / 01 . / 1 2 / & 344* Advances in Operations Research Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Advances in Decision Sciences Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Applied Mathematics Algebra Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Probability and Statistics Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Differential Equations Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 Submit your manuscripts at http://www.hindawi.com International Journal of Advances in Combinatorics Hindawi Publishing Corporation http://www.hindawi.com Mathematical Physics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Complex Analysis Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Mathematics and Mathematical Sciences Mathematical Problems in Engineering Journal of Mathematics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Discrete Mathematics Journal of Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Discrete Dynamics in Nature and Society Journal of Function Spaces Hindawi Publishing Corporation http://www.hindawi.com Abstract and Applied Analysis Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Journal of Stochastic Analysis Optimization Hindawi Publishing Corporation http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Volume 2014
https://openalex.org/W2263956376	https://europepmc.org/articles/pmc4646364?pdf=render	English	null	High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner	Journal of biological chemistry/The Journal of biological chemistry	2,015	cc-by	10,748	3 The abbreviations used are: GT, glycosyltransferase; GTA, -(133)-N-acetyl- galactosaminyltransferase; GTB, -(133)-galactosyltransferase; HA, H antigen acceptor; UDP-C-Gal, UDP-phosphono-galactose; MPD, 2-methyl- 2,4-pentanediol; DI, 3-deoxy inhibitor. High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner* Susannah M. L. Gagnon‡1, Peter J. Meloncelli§, Ruixiang B. Zheng§, Omid Haji-Ghassemi‡, Asha R. Johal‡, Svetlana N. Borisova‡, Todd L. Lowary§, and Stephen V. Evans‡2 From the ‡Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada and the §Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada Svetlana N. Borisova‡, Todd L. Lowary§, and Stephen V. Evans‡2 From the ‡Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada and the §Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada From the ‡Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada and the §Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada Background: Substrate hydrolysis has impeded structural investigation of human ABO(H) glycosyltransferase specificity. Results: Complexes with natural and isosteric non-hydrolyzable donor analogs show multiple stable intermediate donor bind- ing conformations. Conclusion: Subtle stereochemical differences from natural donor prevent isosteric donor analog from displaying full mimicry. Significance: High resolution structural analysis provides insight into inhibitor development and the multistage process of substrate binding. Homologous glycosyltransferases -(133)-N-acetylgalacto- saminyltransferase (GTA) and -(133)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investi- gation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chime- ras have been determined to between 1.55 and 1.39 A˚ resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a con- stant location, the sugar moieties lie in four distinct conforma- tions, varying from extended to the “tucked under” conforma- tion associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disor- dered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. * This work was supported in whole or part by a grant from the Natural Sci- ences and Engineering Research Council of Canada, Alberta Innovates Technology Futures, and the Canadian Institutes of Health Research. The authors declare that they have no conflicts of interest with the contents of this article. The atomic coordinates and structure factors (codes 5C36, 5C38, 5C3A, 5C3B, 5C3D, 5C47, 5C48, 5C49, 5BXC, 5C1G, 5C1H, 5C1L, 5C4B, 5C4C, 5C4D, 5C4E, 5C4F, and 5C8R) have been deposited in the Protein Data Bank (http://wwpdb.org/). 2 Recipient of a Senior Scholarship from the Michael Smith Foundation for Health Research. To whom correspondence should be addressed: Dept. of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055 STN CSC, Victoria, British Columbia V8W 3P6, Canada. E-mail: svevans@uvic.ca. crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 45, pp. 27040–27052, November 6, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 45, pp. 27040–27052, November 6, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. ssmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 45, pp. 27040–27052, November 6, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 45, pp. 27040–27052, November 6, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. 1 Recipient of a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada. Experimental Procedures Ligands—UDP-C-Gal, l-Fuc-(132)--d-Galp-O(CH2)7CH3 (HA), and -l-Fuc-(132)--d-(3-deoxy)-Galp-O(CH2)7CH3 (DI) were synthesized as reported previously (29, 39, 41). Donor substrates UDP-Gal and UDP-Glc were purchased from Sigma-Aldrich. Crystallization—Chimeric AABB, ABBB, and ABBA pro- teins were produced as described by Alfaro et al. (12) and crys- tallized in protein stock solution. Native GTA/GTB proteins were grown at 4 °C from a much higher concentration of pro- tein (30–40 mg ml1 for GTB and 16–20 mg ml1 for GTA) along with 1% polyethylene glycol (PEG) 4000, 4.5–5% 2-meth- yl-2,4-pentanediol (MPD), 100 mM ammonium sulfate, 70 mM sodium chloride, 50 mM N-[2-acetamido]-2-iminodiacetic acid buffer, pH 7.5, 30 mM sodium acetate buffer, pH 4.6, and 5 mM manganese chloride (MnCl2) for GTB crystallization and 5–8 mM CoCl2 for GTA crystallization. Drops of 10–15 l were placed against a reservoir containing 3.7% PEG 4000, 7% MPD, 0.3 M ammonium sulfate, 0.25 M sodium chloride, 0.2 M N-[2- acetamido]-2-iminodiacetic acid buffer, and 0.1 M sodium ace- tate. Crystals grew for 5–10 days at 4 °C. The challenges associated with soaking or co-crystallizing these enzymes with natural donors and donor analogs (12, 30–33), such as substrate hydrolysis, oxidation, and concomi- tant conformational changes that lead to destruction of the crystal lattice, have been obstacles to furthering understanding of enzyme action. Although several electrospray ionization-MS and NMR studies have examined GTA and GTB interactions with their respective donors in the presence of Mn2 (33–35), it remains difficult to understand the influence of substrate bind- ing on conformational changes in the absence of fully liganded structures. Protein crystals were washed with artificial mother liquor-2 containing 3.5% PEG 4000, 50 mM ammonium sulfate, 40 mM sodium chloride, 35 mM N-[2-acetamido]-2-iminodiacetic acid buffer, and 15% MPD prior to substrate addition. Crystals of AAAA, AABB, ABBB, ABBA, and BBBB in complex with UDP- Gal, UDP-C-Gal, UDP-Glc, DI, and HA were obtained by soak- ing them in mother liquor-2 with 15% MPD, 60–70 mM UDP- Gal, 60–80 mM UDP-Glc, 40–60 mM UDP-C-Gal, 12–20 mM DI, 20 mM HA, and 10 mM MnCl2 for 2–5 days at 4 °C. Short soaking times and reduced concentrations of UDP-C-Gal and DI were also used for ABBA and ABBB chimeras. Donors, acceptors, and analogs thereof were added incrementally over a period of a few minutes to hours to avoid crystal fracture. The data sets and structures presented were from those crystals that diffracted to highest resolution. Donor Recognition in ABO(H) Blood Group Glycosyltransferases Co-crystallization with other chemically related but inactive donors, such as natural donor sugar epimers, can also yield significant structural insight into donor binding as well as the basis for enzyme stereoselectivity. UDP-glucose (UDP-Glc) is a C4 epimer of UDP-Gal that binds GTB with comparable affin- ity (31), but whereas STD NMR experiments have shown the bound conformations of these nucleotide sugars to be compa- rable (32), UDP-Glc yields much lower enzymatic activity, 0% compared with UDP-GalNAc for GTA, which has a kcat of 17.5 s1, and 0.02% compared with UDP-Gal for GTB, which has a kcat of 5.1 s1 (26, 40). forms of GTA and GTB (16, 25, 26), so biosynthesis terminates at the H antigen. The similarity of the GTA and GTB enzyme donor substrates results in the blood group A and B individuals differing only in the replacement of an acetamido with a hydroxyl group on the terminal galactose residue. GTA and GTB are homologous, dif- fering in just four critical amino acid residues: Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268 (16, 25). Prior kinetic and structural studies using GTA/GTB chimeras have provided insight into the roles of these key amino acids. GTA, GTB, and their chimeras can be described by four-letter codes, where each letter corresponds to a critical residue. For example, AAAA refers to GTA, BBBB refers to GTB, and AABB refers to the chimera with the first two critical residues of GTA and the last two critical residues of GTB. Previous findings implicate the first amino acid in enzyme turnover (12, 26), the second and third in acceptor recognition (14, 27), and the third and fourth in donor selection (14, 17, 25, 28, 29). Here we report the structures to high resolution of GTA, GTB, AABB, ABBA, and ABBB grown in complex with UDP- C-Gal, UDP-Gal, and UDP-Glc with either synthetic H antigen disaccharide -l-Fuc-(132)--D-Galp-O(CH2)7CH3 (HA) or the 3-deoxy inhibitor (DI), -l-Fuc-(132)--D-(3-deoxy)-Galp- O(CH2)7CH3, providing fresh insight into the glycosyl transfer reaction. High resolution structures have revealed two flexible regions near the active site (Fig. 1B): an internal loop (amino acids 176– 188) and C-terminal -helix (amino acids 346–354) (12, 14). In the absence of substrate, these enzymes tend to adopt the “open” conformation, where both regions have higher disorder and/or the internal loop faces away from the active site (12). High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner* Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C- Gal was designed to be “isosteric” with natural donor, the small differences in structure imposed by changing the epimeric oxy- gen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB. Glycosyltransferases (GTs)3 mediate glycoside synthesis, a ubiquitous enzymatic process, by catalyzing the transfer of monosaccharides from activated donor to acceptor. GTs gen- erate glycan and glycoconjugate products that are critical for cell signaling, immunity, plant cell wall formation, and molec- ular recognition. These enzymes are essential for normal cell development, and aberrant GT function can result in a number of infections and inflammatory disease states (1). Despite their functional and physiological diversity, GTs exhibit structural phenotype conservation even in the absence of sequence homo- logy (1–12). There are currently 97 GT families based on sequence identity (2, 13), with almost all of the enzymes falling into two major fold types: GT-A and GT-B. Two family six GTs with the GT-A fold type (14), -(133)- N-acetylgalactosaminyltransferase (GTA) and -(133)-galac- tosyltransferase (GTB) perform the final step in human A and B blood group antigen synthesis through monosaccharide trans- fer to the H antigen acceptor (HA) terminal sequence -l-Fuc- (132)--d-Gal-O-R (14–21). As shown in Fig. 1A, GTA cata- lyzes the transfer of an N-acetylgalactosaminyl (GalNAc) residue from UDP-GalNAc to HA to form the A antigen, and GTB catalyzes the transfer of a galactosyl (Gal) residue from UDP-Gal to HA to form the B antigen (14, 15, 20, 22–25). Blood group O individuals generally express truncated or mutated VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 27040 JOURNAL OF BIOLOGICAL CHEMISTRY Donor Recognition in ABO(H) Blood Group Glycosyltransferases When UDP and manganese ion (Mn2) bind, there is a shift to the “semi-closed” conformation, where the internal loop is more ordered and oriented toward UDP so as to occlude the active site entrance, whereas the C-terminal loop remains dis- ordered (12). With the addition of UDP or UDP-Gal in combi- nation with acceptor or acceptor analog, GTA and GTB are observed to assume the “closed” state associated with catalytic activity (Fig. 1B), where the internal loop and C-terminal region are both ordered and interacting with donor and acceptor (12). Experimental Procedures Before freezing crystals for data collection, the cryoprotectant concentration was adjusted to 20% MPD. The synthesis of non-hydrolyzable nucleotide donors at- tempts to remedy one of these problems, and previous studies have shown that similar sugar nucleotide analogs can act as competitive, mechanism-based GT inhibitors (36–39), but there is a dearth of corresponding complex enzyme structures. Replacement of the anomeric oxygen of UDP-Gal with a meth- ylene group (Fig. 1A) generates UDP-C-Gal, a non-hydrolyz- able substrate designed to be isosteric to UDP-Gal, which could be used as a probe of the GTA/GTB glycosyl transfer reaction. The substrate analog was previously used in structural studies to probe the UDP galactopyranose mutase reaction but was found to bind the enzyme in an alternate conformation; the -phosphate and galactosyl moiety of the analog were shifted relative to the “U-shaped” conformation of natural donor (39). NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 27041 27041 NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 27042 JOURNAL OF BIOLOGICAL CHEMISTRY TABLE 2 Data Collection and Reduction—X-ray diffraction data were collected at 160 °C for all crystals using a CryoStream 700 crystal cooler. Each crystal was incubated with a cryoprotectant solution consisting of mother liquor with 20% (v/v) MPD pro- portionally replacing the volume of water in the initial mother liquor. Data were collected on a Rigaku R-AXIS IV area detector at distances of 72 mm and exposure times between 4.0 and 7.0 min for 0.5° oscillations. X-rays were produced by an MM-002 generator (Rigaku/MSC, College Station, TX) cou- pled to Osmic 9 “Blue” confocal x-ray mirrors (Osmic, Auburn Hills, MI). Data were scaled, averaged, and integrated using d*trek and CrystalClear (42). Data collection and refinement statistics for GTA, GTB, and their chi- mera in complex with UDP-Gal and DI Data collection and refinement statistics for GTA, GTB, and their chi- mera in complex with UDP-Gal and DI All structures are in space group C2221 with unit-cell dimensions a 52.5–52.7, b 148.9–149.4, and c 79.2–80.0 Å. AAAA ABBA ABBB BBBB Data collection Resolution (Å) 20-1.40 20-1.46 20-1.55 20-1.40 Rmerge (%)a 3.3 (30.7)b 3.1 (24.0) 3.4 (27.2) 3.2 (31.1) Completeness (%) 95.1 (81.4) 96.5 (97.0) 91.8 (91.4) 96.1 (81.7) Unique reflections 59,210 52,594 42,208 59,299 Refinement Rwork (%)c 18.0 17.5 17.8 17.2 Rfree (%)c,d 19.9 19.2 21.4 19.6 No. of waters 308 224 238 274 r.m.s. bond (Å)e 0.0100 0.0097 0.0096 0.0095 r.m.s. angle (degrees) 1.507 1.506 1.555 1.561 Protein Data Bank ID 5BXC 5C1G 5C1H 5C1L a Rmerge Iobs Iavg/Iavg. b Values in parentheses are for the highest resolution shell. c Rwork Fo Fc/Fo. d 10% of reflections were omitted for Rfree calculations. e r.m.s., root mean square. Structure Determination—Although the structures were nearly isomorphous, for completeness, they were solved by molecular replacement using the CCP4 module Phaser (43) with the structures of wild type GTA and GTB as starting models (Protein Data Bank accession codes 1LZ0 and 1LZ7, respectively). Structures were then refined using the CCP4 module REFMAC5 (44, 45), and all figures were produced with SetoRibbon. internal loop (residues 176–188) and the C-terminal loop (res- idues 346–354) for all structures is summarized in Table 4. With the exception of these sometimes ordered and sometimes disordered regions, the entire length of the polypeptide chains had excellent electron density. FIGURE 1. GTA/GTB glycosyl transfer reaction scheme, with key donor bond angles and lengths shown, and overview of flexible loop organization. A, GTA and GTB catalyze GalNAc and Gal transfer from uridine diphosphate (UDP)-GalNAc (1) and UDP-Gal (2) to the H antigen acceptor (3), forming the human blood group A (4) and B antigens (5). Donor analog UDP-C-Gal (6) only differs from UDP-Gal in replacement of the anomeric oxygen with a methylene group, whereas UDP-Glc (7) differs in the equatorial position of Gal-C4-OH. Labeled UDP-C-Gal carbon-carbon (6A) and carbon-phosphorus (6B) bonds are longer than UDP-Gal carbon-oxygen (2A) and oxygen-phosphorus (2B) bonds, and the UDP-C-Gal C-C-P bond angle (6) is much smaller than the UDP-Gal C-O-P bond angle (2). B, superimposition of unliganded ABBB (Protein Data Bank code 2RIZ) in the open state with AABB (Protein Data Bank code 2RJ7) in the closed state. The closed internal loop (residues 176–188) and C-terminal domain (residues 346–354) are shown in red, whereas the open loops of ABBB are shown in yellow. The location of Arg-176 is indicated, and the C-terminal loop is labeled C. The dashed arrow indicates the movement of the internal loop toward the donor in going from the open state to both the semi-closed and closed states. Mn2 is shown as a magenta sphere, donor is shown in blue, and acceptor is shown in orange. TABLE 2 Table 4 notes which residues in the loops have ordered atoms, which have interpretable density for the main chain atoms only, and which residues are com- pletely disordered. Generally, when only main chain atoms are observed, the relative temperature factors are higher than the proximal protein atoms, indicating that there is still consider- able lability in such a region of polypeptide. TABLE 1 TABLE 1 Data collection and refinement statistics for GTA GTB and their chimera in complex with UDP C Gal TABLE 1 Data collection and refinement statistics for GTA, GTB, and their chimera in complex with UDP-C-Gal All crystal structures are in space group C2221 with unit-cell dimensions a 52.5–52.7, b 149.0–149.9, and c 79.2–79.6 Å. Acceptor analogs and enzymes are shown at the top. DI with AAAA DI with AABB DI with ABBA (short soak) DI with ABBA (long soak) DI with ABBB (short soak) DI with ABBB (long soak) DI with BBBB HA with BBBB Data collection Resolution (Å) 20-1.55 20-1.45 20-1.40 20-1.40 20-1.39 20-1.39 20-1.46 20-1.49 Rmerge (%)a 4.2 (28.7)b 4.3 (30.8) 3.2 (30.2) 3.3 (29.3) 3.0 (28.6) 3.2 (26.9) 4.1 (26.9) 3.7 (27.6) Completeness (%) 98.8 (100.0) 99.5 (100.0) 96.9 (85.0) 96.6 (82.4) 96.0 (87.0) 98.0 (91.1) 99.1 (98.0) 95.3 (91.1) Unique reflections 45,201 55,396 60,216 59,599 60,710 61,958 54,000 48,905 Refinement Rwork (%)c 18.2 18.1 20.0 18.4 19.7 17.8 18.2 17.8 Rfree (%)c,d 21.7 19.5 21.8 20.4 21.3 19.9 19.0 19.1 No. of waters 231 262 217 259 248 243 223 215 r.m.s. bond (Å)e 0.0105 0.0098 0.0097 0.0094 0.0092 0.0091 0.0097 0.0102 r.m.s. angle (degrees) 1.516 1.504 1.532 1.553 1.437 1.528 1.540 1.542 Protein Data Bank ID 5C36 5C38 5C3A 5C3B 5C3D 5C47 5C48 5C49 a Rmerge Iobs Iavg/Iavg. b Values in parentheses are for the highest resolution shell. c Rwork Fo Fc/Fo. d 10% of reflections were omitted for Rfree calculations. e r.m.s., root mean square. 1 lection and refinement statistics for GTA, GTB, and their chimera in complex with UDP-C-Gal Data collection and refinement statistics for GTA, GTB, and their chimera in complex with UDP-C-Gal All crystal structures are in space group C2221 with unit-cell dimensions a 52.5–52.7, b 149.0–149.9, and c 79.2–7 at the top. Donor Recognition in ABO(H) Blood Group Glycosyltransferases Donor Recognition in ABO(H) Blood Group Glycosyltransferases VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 Donor Recognition in ABO(H) Blood Group Glycosyltransferases TABLE 1 Results Loop Ordering—Data collection and refinement statistics are given in Table 1 for the structures of enzymes in complex with UDP-C-Gal, Table 2 for UDP-Gal, and Table 3 for UDP-Glc. Overall, the resolution limit ranges from 1.55 to 1.39 Å, Rwork ranges from 17.2 to 20.0%, and Rfree ranges from 18.6 to 21.8%. In the space group in which all of the enzymes crystallized, C2221, there is a solvent channel that allows considerable mobility to the two loops of mobile polypeptide that surround the active site. Observed electron density surrounding the Loop Ordering—Data collection and refinement statistics are given in Table 1 for the structures of enzymes in complex with UDP-C-Gal, Table 2 for UDP-Gal, and Table 3 for UDP-Glc. Overall, the resolution limit ranges from 1.55 to 1.39 Å, Rwork ranges from 17.2 to 20.0%, and Rfree ranges from 18.6 to 21.8%. In the space group in which all of the enzymes crystallized, C2221, there is a solvent channel that allows considerable mobility to the two loops of mobile polypeptide that surround the active site. Observed electron density surrounding the When in complex with UDP-C-Gal, GTA, GTB, and all chi- meras have disordered C termini, even with acceptor or accep- NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 27043 27043 NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 TABLE 3 Data collection and refinement statistics for GTA, GTB, and their chimera in complex with UDP-Glc All crystal structures are in space group C2221 with unit-cell dimensions a 52.5–52.6, b 149.3–149.7, and c 79.2–79.5 Å. Acceptor analogs and enzymes are shown at the top. DI with AAAA DI with AABB DI with ABBA DI with ABBB DI with BBBB HA with BBBB Data collection Resolution (Å) 20-1.54 20-1.41 20-1.45 20-1.43 20-1.40 20-1.55 Rmerge (%)a 4.2 (35.1)b 3.6 (32.8) 3.0 (22.1) 3.2 (26.3) 3.3 (27.8) 4.6 (27.3) Completeness (%) 94.4 (89.1) 97.2 (82.0) 97.9 (98.8) 97.7 (96.3) 98.1 (87.1) 98.5 (99.7) Unique reflections 44,020 58,865 54,336 56,554 60,723 45,267 Refinement Rwork (%)c 18.2 18.8 17.6 18.5 17.5 17.3 Rfree (%)c,d 21.0 20.5 18.6 20.9 18.9 19.8 No. of waters 235 252 217 220 272 275 r.m.s. bond (Å)e 0.0101 0.0095 0.0091 0.0097 0.0095 0.0114 r.m.s. angle (degrees) 1.552 1.503 1.509 1.501 1.532 1.612 Protein Data Bank ID 5C4B 5C4F 5C4R 5C4C 5C4D 5C4E a Rmerge Iobs Iavg/Iavg. b Values in parentheses are for the highest resolution shell. Loop ordering in AAAA, BBBB, and chimeric enzyme complexes Loop ordering in AAAA, BBBB, and chimeric enzyme complexes Black single-letter amino acid codes correspond to electron density for main and side chain atoms; blue letters correspond to electron density for main chain atoms only; red letters correspond to weak electron density for both main and side chain atoms; lowercase letters represent amino acids omitted from refined models. The “open” state of the enzymes is defined by the internal and C-terminal loops disordered, the “semi-closed” state is defined by the internal loop becoming ordered so as to occlude the active site while the C-terminal loop remains disordered, and the “closed” state is defined by the ordering of both loops over the active site. p g , , y p Black single-letter amino acid codes correspond to electron density for main and side chain atoms; blue letters correspond to electron density for main chain atoms only; red letters correspond to weak electron density for both main and side chain atoms; lowercase letters represent amino acids omitted from refined models. The “open” state of the enzymes is defined by the internal and C-terminal loops disordered, the “semi-closed” state is defined by the internal loop becoming ordered so as to occlude the active site while the C-terminal loop remains disordered, and the “closed” state is defined by the ordering of both loops over the active site. a Value in parentheses indicates a lower occupancy alternate conformation. b As reported by Alfaro et al. (12); Protein Data Bank code 2RJ7. a Value in parentheses indicates a lower occupancy alternate conformation. b As reported by Alfaro et al. (12); Protein Data Bank code 2RJ7. when in complex with UDP-Gal and DI, similar to the previ- ously published AABB structure (12). ABBB is in the semi- closed state with a flexible C terminus, and GTB is in the open state with both domains disordered. tor analog bound. In this set of structures, only GTA is in the semi-closed state with a structured internal loop. The remain- ing complexes are in the open state, although ABBB becomes more ordered in the long soak condition. Due to weak or ambig- uous density, the UDP-C-Gal sugar moiety was not modeled for GTA and GTB (with HA) complexes. Structures observed in complex with UDP-Glc generally have more disordered C-terminal and internal loops except for GTA and GTB (the latter in complex with HA instead of DI). TABLE 3 LE 3 collection and refinement statistics for GTA, GTB, and their chimera in complex with UDP-Glc Results c Rwork Fo Fc/Fo. d 10% of reflections were omitted for Rfree calculations. e r.m.s., root mean square. Donor Recognition in ABO(H) Blood Group Glycosyltransferases Donor Recognition in ABO(H) Blood Group Glycosyltransferases Donor Recognition in ABO(H) Blood Group Glycosyltransferases all complexes except BBBB, where electron density was ambiguous. Donor Sugar Occupancy—In four of the structures, unex- plained electron density was observed in the active sites sur- rounding the donor sugar moiety (Fig. 5, left). Inspection showed that the density was explained by the donor sugar (and sometimes the donor phosphate groups) being disordered over two conformations, with the second conformation lying in the difference density (Fig. 5, right). The occupancy of the primary conformation in all four of these structures was adjusted to yield temperature factors comparable with adjacent protein atoms. The difference electron densities in Fig. 5 are unbiased because they do not include refinement of the alter- nate conformation. Substrate Binding and Donor Conformation—Among the structures with an ordered donor sugar, a total of four distinct donor conformations are observed, each with examples that show excellent electron density (Fig. 2) and each with a differ- ent set of hydrogen-binding partners with the protein (Fig. 3). In all cases, the donor sugar moiety is observed to shift with respect to the enzyme, with some movement about the -phos- phate, whereas the uracil maintains its position. The so-called “tucked under” conformation that has been associated with catalysis (conformation 1 in Figs. 2A and 3A) has the epimeric carbon positioned under the phosphate groups, was first reported by Alfaro et al. (12) for UDP-Gal in complex with AABB, and is also observed here for UDP-Glc in complex with ABBA. Specifically, in each of the ABBA (short and long soak) struc- tures, UDP-C-Gal was found to be disordered over conforma- tions 2 and 3 (Fig. 5). In the short soak structure, the extended conformation predominates in a ratio of 80:20, whereas in the long soak structure, conformation 2 predominates in a ratio of 70:30. In contrast, UDP-C-Gal was observed to be 100% occu- pied when in complex with AABB, ABBB (short soak), ABBB (long soak), and GTB (with DI) and completely disordered when in complex with GTA and GTB (with HA). Conformation 2 is observed for GTB (Figs. 2B and 3B) in complex with DI and UDP-Gal and with DI and UDP-C-Gal. Over longer soaking times, UDP-C-Gal adopts this conforma- tion in ABBB in the presence of DI. Discussion Four distinct donor conformations were observed in total using three different donors or donor analogs (Figs. 2 and 3 and Table 4). GTA, GTB, and their chimeras and mutants have only been observed in complex with intact donor in two previous structures, most notably AABBUDP-GalDI (12), where UDP-Gal is observed in the classic tucked under conformation (Table 4, conformation 1); a second UDP-Gal conformation (conformation 2) was observed in GTB Cys/Ser mutant struc- tures (48). The observation of several new structures with intact bound donor represents a significant advance in understanding donor recognition in this class of enzymes. A third conformation (number 3) is observed in the ABBB (short soak) structure, where two water molecules are intro- duced into the active site (Figs. 2C and 3C). DI is absent as the C-galactosyl moiety extends into the acceptor-binding site such that there are contacts between Glu303 and Gal-O-2 and Gal- O-3 as well as between His-233 and Gal-O-3 and Gal-O-4. Active site residues Arg-188, Asp-211, Asp-302, and His-301 are no longer involved in binding the C-Gal group. UDP-C-Gal Cannot Fully Mimic Bound Donor—There is a paucity of glycosyltransferase structures determined in com- plex with donor substrates, which has been attributed partly to the enzymes’ ability to hydrolyze donor in the absence of accep- tor and partly to the enzymes’ tendency to undergo conforma- tional rearrangement upon binding substrate and so shatter the crystal lattice (12, 30–33). UDP-Glc occupies the donor-binding sites of GTA, AABB, ABBB, and GTB in a fourth distinct conformation, where the glucose moiety is rotated so that each hydroxyl group changes binding partners relative to the first UDP-Gal conformation (Figs. 2D and 3D). Glc-O-2 no longer contacts with Asp-211 and instead interacts with DI; Glu-303 interacts with Glc-O-3; Arg-188 and Asp-211 make contacts to Glc-O-4; and Asp-302 forms a hydrogen bond to Glc-O-6. When in complex with ABBA, Glc is in an orientation similar to the first UDP-Gal conformation but with equatorial Glc-O-4 (Figs. 2E and 3E). The position of Glc-O-4 permits an additional interaction with Arg-188 and causes a shift in Asp-302 to maintain the hydrogen bond to Glc-O-4. The introduction of non-hydrolyzable donors like UDP-C- Gal provides an opportunity to negate the first barrier but, because the analog necessarily contains structural differences from the natural donors, does not allow complete mimicry (Fig. 1A and Table 4). Donor Recognition in ABO(H) Blood Group Glycosyltransferases In conformation 1, the galactosyl moiety is oriented such that Arg-188 and Asp-211 form hydrogen bonds to Gal-O-3, Asp-302 interacts with Gal- O-4, His-301 interacts with Gal-O-6, the -phosphate oxygen interacts with Asp-211, and His-233 and Glu-303 make con- tacts to DI (Figs. 2A and 3A). In this conformation, the internal loop residue Trp-181 lies far from Gal-O-6 (Fig. 4). Also, natural donor UDP-Gal was found to be disordered over conformations 1 and 2 in an approximate ratio of 70:30 in the active site of ABBA and with an approximate ratio of 40:60 in the active site of ABBB (Fig. 5). Again in contrast, UDP-Gal was 100% occupied when in complex with GTB and completely disordered when in complex with GTA. All enzymes were soaked with the same batch of donor, so the disorder observed in the GTAUDP-Gal structure was probably not due to hydrolyzed donor. UDP-Glc was ordered when in complex with GTA, AABB, ABBA, ABBB, and GTB (DI) and disordered when in complex with GTB (with HA). In conformation 2 (Figs. 2B and 3B), the galactosyl group shifts so that Arg-188 and Asp-211 no longer contact Gal-O-3 directly and instead interact with Gal-O-2 through a water mol- ecule. In this conformation, Asp-302 binds Gal-O-3, and Glu- 303 interacts with Gal-O-2. His-301 still forms a hydrogen bond to Gal-O-6, and Glu-303 still contacts DI. The new posi- tion of Gal-O-6 prevents internal loop residue Trp-181 from approaching the active site to form the closed conformation (Fig. 4). With the galactosyl moiety in this conformation, GTB is in the semi-closed state with a partially disordered internal loop and a highly unstructured C terminus (Table 4). Loop ordering in AAAA, BBBB, and chimeric enzyme complexes GTA is in the semi-closed state with UDP-Glc and DI bound, whereas AABB, ABBA, ABBB, and BBBB are in the open state. The UDP-Glc sugar moiety could be modeled for Structures in complex with the native donor UDP-Gal gen- erally show much greater order than those in complex with UDP-C-Gal or UDP-Glc. GTA and ABBA adopt the closed state with structured internal and C-terminal loops (Table 4) 27044 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 Discussion For example, the anomeric C–O bond length of 1.4 Å changes to 1.55 Å for the C–C bond. More significantly, NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 27045 JOURNAL OF BIOLOGICAL CHEMISTRY NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 Donor Recognition in ABO(H) Blood Group Glycosyltransferases whereas the C-O-P bond angle is close to a trigonal 123 11° (averaged from UDP-sugar structures in the RCSD Protein Data Bank) because of the ability of the anomeric oxygen to assume partial sp2 character, the purely sp3 methylene group in UDP-C-Gal would be expected to be closer to a tetrahedral 109°. These small changes lead to significant differences in binding. all wild type and chimera, and whereas it is observed in a closed form in complex with UDP-Gal, it can only achieve the semi- closed form in complex with UDP-C-Gal. All of the remaining UDP-C-Gal structures are observed to lie in the open confor- mation. This can be directly attributed to the inability of UDP- C-Gal to assume the tucked under conformation, which leads to a steric clash with Trp-181 of the internal loop and with the acceptor (evidenced by weaker electron density), which itself impacts the ability of the C-terminal tail to adopt the closed state. Trp-181 was noted in a previous study to form a stabiliz- ing interaction with Arg-352 of the terminal loop (12), explain- ing why internal loop order is a prerequisite to C-terminal loop order. UDP-C-Gal is observed in two distinct conformations in the active sites of six of the eight structures in which it was soaked. The analog displays the greatest order in the chimera (Table 4) because the galactosyl residue is completely disordered in GTA and GTB. In four of the chimeras, it is observed in a single conformation (either conformation 2 or 3), whereas in two of the structures, there is clear evidence that it is disordered over these two distinct conformations. In none of the structures does UDP-C-Gal assume the tucked under conformation asso- ciated with catalysis. The inability of UDP-C-Gal to assume the catalytically competent conformation is probably due to the small differences in bond angles and lengths compared with the native donor. UDP-Gal Is Observed Ordered in the Active Site of Wild Type GTB—The search for GTA, GTB, and other GT structures in complex with UDP-Gal has been ongoing, involving various preparations of donor and acceptor and different soaking con- ditions, to yield finally this report of the crystal structure of GTB in which unambiguous electron density is seen for its nat- ural donor UDP-Gal. When soaking times are short and substrate concentrations are low, the substrate primarily assumes an extended confor- mation (conformation 3 in Fig. Donor Recognition in ABO(H) Blood Group Glycosyltransferases 2C). In this conformation, UDP- C-Gal utilizes almost none of the stabilizing donor-enzyme interactions of the tucked under conformation, and the galac- tosyl moiety makes enzyme contacts normally reserved for acceptor, including His-233 and Glu-303 hydrogen bonds to C-Gal hydroxyl groups O-2, O-3, and O-4, which explains the poor or absent acceptor electron density in the two structures where the donor has assumed conformation 3. The rotated donor sugar makes space for two new water molecules in the active site to form bridging hydrogen bonds with residues Asp- 302, Arg-188, and Asp-211 normally used to form direct hydro- gen bonds to the donor sugar (Fig. 2A). Interestingly, the substrate adopts the same conformation (number 2) in the wild-type GTB structure that was observed in the crystal structure of GTB Cys/Ser mutants (48) (Fig. 2C) but without achieving the closed state. Although their conforma- tions may appear to be similar (Fig. 2), the donor-enzyme con- tacts in conformations 1 and 2 are significantly different. Rela- tive to the tucked under donor (Figs. 2A and 3A), the enzyme no longer interacts with Gal-O-6, and Arg-188 and Asp-211 no longer interact with the galactosyl moiety directly but make bridging contacts through a newly introduced water molecule. Both differ from the conformation for bound donor first pre- dicted by Heissigerova et al. (49). UDP-Glc Can Adopt the Tucked under Conformation in ABBA—The structures reported here show UDP-Glc in two positions. The fourth distinct conformation (Figs. 2D and 3D) is observed only for UDP-Glc. As seen for other conformations with UDP-Gal, five hydrogen bonds stabilize the glucosyl moi- ety (Figs. 2D and 3D) with an additional contact between DI and Glc-O-2. UDP-C-Gal Biases Loop Structures toward the Open Confor- mation—Previous studies have documented that the GTA/ GTB internal loop generally becomes increasingly ordered in the presence of both donor and acceptor (12), with a tendency for enzymes with greater GTA character to order more readily than enzymes with greater GTB character (12, 46, 47). The UDP-Gal-bound structures presented in this study obey this trend (Table 4, middle). GTAUDP-Gal is in the closed form, ABBAUDP-Gal is nearly closed, ABBBUDP-Gal is semi- closed, and BBBBUDP-Gal is open. UDP-Glc is observed to assume the tucked under conforma- tion (conformation 1; Figs. 2E and 3E) in ABBA, which was not expected, given that UDP-Glc is known to be a poor donor for these enzymes. FIGURE 2. Four donor conformations and key enzyme-substrate interactions. UDP-Gal is tucked under in ABBAUDP-GalDI (A) and is in an alternate conformation in GTBUDP-GalDI (B). In B, relative to A, the Gal hydroxyl groups have changed binding partners, but DI-enzyme interactions are the same. Donor analog UDP-C-Gal is in an extended conformation in ABBBUDP-C-GalDI short soak (C). Here acceptor is absent, and donor extends into the acceptor binding site. Glu-303 and His-233 interact with C-Gal-O-2, -O-3, and -O-4 instead of DI, and Arg-188, Asp-211, and Asp-302 contact Gal via two water molecules. UDP-Glc is in a fourth conformation in D, AABBUDP-GlcDI. DI makes a new contact to His-233, Glu-303 interacts with Glc-O-3, Arg-188 and Asp-211 interact with Glc-O-5, and Asp-302 interacts with Glc-O-6. In E, ABBAUDP-GlcDI, Glc is in the same conformation as in A but with equatorial Glc-O-4. Here, Asp-302 contacts Glc-O-4, and there is an additional interaction between Arg-188. In both D and E, the contact between Glc and His-301 is lost, and in A–E Glu-303 consistently makes two contacts to DI. Donor substrate has excellent electron density in all five structures. F, stereoview of overlapped donor conformations shows movement of the saccharide moiety and the -phosphate. ABBA UDP-Gal is in green, GTB UDP-Gal is in yellow, ABBB short UDP-C-Gal is in orange, and AABB UDP-Glc is in magenta, with conformation numbers indicated in the corresponding color. Electron density diagrams are 2Fo Fc maps contoured at 1.0. Protein atoms are colored by element with red for oxygen, yellow for phosphorus, white for carbon, and blue for nitrogen. Waters are shown as red spheres, and Mn2 is shown as a magenta sphere. Green dotted spheres represent hydrogen bonds. Donor Recognition in ABO(H) Blood Group Glycosyltransferases Donor Recognition in ABO(H) Blood Group Glycosyltransferases Donor Recognition in ABO(H) Blood Group Glycosyltransferases 7046 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 45•NOVEMBER 6, VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 27046 JOURNAL OF BIOLOGICAL CHEMISTRY 27046 JOURNAL OF BIOLOGICAL CHEMISTRY Donor Recognition in ABO(H) Blood Group Glycosyltransferases unproductive because transfer to the H antigen acceptor does FIGURE 3. Hydrogen bond schematic for donor- and acceptor-enzyme intera A is tucked under UDP-Gal bound to ABBA, B is UDP-Gal in conformation 2 bou UDP-Glc in conformation 4 bound to AABB, and E is UDP-Glc in conformation 1 donor and acceptor are indicated. Hydrogen bonds are shown as dashed lines. unproductive because transfer to the H antigen acceptor does mechanism where Asp-302 and Glu-303 lock Gal in the tucked FIGURE 3. Hydrogen bond schematic for donor- and acceptor-enzyme interactions in the four observed conformations. A–E correspond to Fig. 2, where A is tucked under UDP-Gal bound to ABBA, B is UDP-Gal in conformation 2 bound to GTB, C is UDP-C-Gal in conformation 3 bound to ABBB (short soak), D is UDP-Glc in conformation 4 bound to AABB, and E is UDP-Glc in conformation 1 bound to ABBA. Enzyme residues that participate in hydrogen bonding with donor and acceptor are indicated. Hydrogen bonds are shown as dashed lines. FIGURE 3. Hydrogen bond schematic for donor- and acceptor-enzyme interactions in the four observed conformations. A–E correspond to Fig. 2, where A is tucked under UDP-Gal bound to ABBA, B is UDP-Gal in conformation 2 bound to GTB, C is UDP-C-Gal in conformation 3 bound to ABBB (short soak), D is UDP-Glc in conformation 4 bound to AABB, and E is UDP-Glc in conformation 1 bound to ABBA. Enzyme residues that participate in hydrogen bonding with donor and acceptor are indicated. Hydrogen bonds are shown as dashed lines. mechanism where Asp-302 and Glu-303 lock Gal in the tucked under conformation to facilitate formation of the transition state. They suggest that, unlike the natural donor, UDP-Glc unproductive, because transfer to the H antigen acceptor does not readily occur (31, 32). On the basis of NMR data for UDP- Glc bound to GTB, Angulo et al. (32) proposed a “tweezers” 27048 JOURNAL OF BIOLOGICAL CHEMISTRY Donor Recognition in ABO(H) Blood Group Glycosyltransferases The observed kcat for GTA is too low to mea- sure; however, the small transfer for GTB of kcat 0.0010 s1 (50) is well above background. In contrast, enzymes in complex with UDP-C-Gal and DI do not obey this trend but are consistently biased toward the open form (Table 4, top). GTA is well established as the least labile of g NMR studies have shown that UDP-Gal and UDP-Glc bind GTB with the same affinity, but in the latter case, binding is FIGURE 2. Four donor conformations and key enzyme-substrate interactions. UDP-Gal is tucked under in ABBAUDP-GalDI (A) and is in an alternate conformation in GTBUDP-GalDI (B). In B, relative to A, the Gal hydroxyl groups have changed binding partners, but DI-enzyme interactions are the same. Donor analog UDP-C-Gal is in an extended conformation in ABBBUDP-C-GalDI short soak (C). Here acceptor is absent, and donor extends into the acceptor binding site. Glu-303 and His-233 interact with C-Gal-O-2, -O-3, and -O-4 instead of DI, and Arg-188, Asp-211, and Asp-302 contact Gal via two water molecules. UDP-Glc is in a fourth conformation in D, AABBUDP-GlcDI. DI makes a new contact to His-233, Glu-303 interacts with Glc-O-3, Arg-188 and Asp-211 interact with Glc-O-5, and Asp-302 interacts with Glc-O-6. In E, ABBAUDP-GlcDI, Glc is in the same conformation as in A but with equatorial Glc-O-4. Here, Asp-302 contacts Glc-O-4, and there is an additional interaction between Arg-188. In both D and E, the contact between Glc and His-301 is lost, and in A–E Glu-303 consistently makes two contacts to DI. Donor substrate has excellent electron density in all five structures. F, stereoview of overlapped donor conformations shows movement of the saccharide moiety and the -phosphate. ABBA UDP-Gal is in green, GTB UDP-Gal is in yellow, ABBB short UDP-C-Gal is in orange, and AABB UDP-Glc is in magenta, with conformation numbers indicated in the corresponding color. Electron density diagrams are 2Fo Fc maps contoured at 1.0. Protein atoms are colored by element with red for oxygen, yellow for phosphorus, white for carbon, and blue for nitrogen. Waters are shown as red spheres, and Mn2 is shown as a magenta sphere. Green dotted spheres represent hydrogen bonds. NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 27047 27047 NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 Donor Recognition in ABO(H) Blood Group Glycosyltransferases Donor Recognition in ABO(H) Blood Group Glycosyltransferases UDP-Gal is Donor Selectivity and Acceptor Influence in the Wild Type Enzymes—GTA in complex with UDP-Gal and UDP-C-Gal shows unambiguous density for UDP bound to the donor-bind- ing site but weak or absent density for the sugar moiety, indi- cating, certainly for the non-hydrolyzable UDP-C-Gal, that the moiety is disordered in the active site. This is interesting in the case of GTAUDP-GalDI because the enzyme achieves the fully closed state although GTA displays a very low transfer rate of UDP-Gal to the natural acceptor (HA). However, the corresponding complexes with GTB show the sugar to be fully ordered. These observations speak directly to the specificity of these enzymes toward their cognate donor substrates. cannot undergo this conformational transition because Glc- O-4 is unable to interact with Asp-302. However, the structure of ABBA shows clearly that Glc-O-4 can and does form a strong hydrogen bond (2.62 Å) to Asp-302 (Figs. 2E and 3E). These structures show that the ability of the enzymes to dis- tinguish between UDP-Gal and UDP-Glc more likely depends on their internal and C-terminal loop organization. The struc- ture of ABBA in complex with UDP-Glc (conformation 1) dis- plays considerably more disorder than the corresponding com- plex with UDP-Gal (Table 4). Further, ABBA in complex with UDP-Glc shows that residues Met-186 and Ser-185 are offset significantly compared with their position in the ordered Alfaro et al. (12) AABBUDP-Gal structure. The resulting disorder and displacement of the internal loop and C-terminal tail mean that key stabilizing residues, including Trp-181, Lys-346, and Arg-352 are no longer positioned to stabilize substrate for transfer. Although the orientation of C4-OH seems compatible with conformation 1, it has a significant effect on mobile loop organization and the enzymes’ ability to adopt the catalytically active closed state. Whereas the selectivity of the smaller GTB active site against the larger blood group A-donor (UDP-GalNAc) could always be explained on steric grounds, the reverse was not true because the GTA active site could easily accommodate the smaller UDP-Gal donor. These structures show that the selectivity of GTA against the B-donor (UDP-Gal) may simply depend on the inability of the GTA enzyme to stabilize the B-donor in a cata- lytically active conformation. Donor Recognition in ABO(H) Blood Group Glycosyltransferases FIGURE 4. UDP-Gal conformations influence enzyme ordering. UDP-Gal is in conformation 1 (yellow), and the enzyme is in the closed state in ABBAUDP-GalDI. Here Trp-181 is distant from Gal-O-6, and Lys-346 inter- actswiththe-phosphateoxygen.WhenUDP-Galisinconformation2(cyan), asseenincomplexwithGTB,theenzymeisinthesemi-closedstatewithaless ordered internal loop and a disordered C-terminal tail. Here Trp-181 is near Gal-O-6, and Asp-211 interacts with the -phosphate oxygen. Mn2 is shown as a magenta sphere. Green dotted spheres represent hydrogen bonds, and black dashed lines indicate measured distances that are not hydrogen bond interactions. donor in conformation 3 clearly show that this conformation sterically impedes the binding of acceptor (29). Observed Donor Sugar Disorder Supports the Mechanism of Substrate Binding—Four of the structures show clear evidence that the donor sugar is disordered over two conformations (Fig. 5), indicating that the enzymes do indeed bind the substrates in the predominant extended conformation 3 and shift it in a step- wise fashion to the tucked under conformation 1. The most significant evidence comes from the two structures of ABBA soaked in UDP-C-Gal for a shorter and longer time, respectively. The short soak structure shows the donor analog predominantly in the extended conformation (number 3), with a small proportion in conformation 2. The longer soaking time shows that a greater proportion is observed in conformation 2. Further evidence of the stepwise insertion of donor comes in the structures of ABBA and ABBB in complex with UDP-Gal, which show disorder over conformations 1 and 2, with ABBA being predominantly conformation 1 and ABBB being predom- inantly conformation 2. Together, these structures show how the enzyme facilitates the rotation of the donor into the cata- lytically active tucked under conformation 1. FIGURE 4. UDP-Gal conformations influence enzyme ordering. UDP-Gal is in conformation 1 (yellow), and the enzyme is in the closed state in ABBAUDP-GalDI. Here Trp-181 is distant from Gal-O-6, and Lys-346 inter- actswiththe-phosphateoxygen.WhenUDP-Galisinconformation2(cyan), asseenincomplexwithGTB,theenzymeisinthesemi-closedstatewithaless ordered internal loop and a disordered C-terminal tail. Here Trp-181 is near Gal-O-6, and Asp-211 interacts with the -phosphate oxygen. Mn2 is shown as a magenta sphere. Green dotted spheres represent hydrogen bonds, and black dashed lines indicate measured distances that are not hydrogen bond interactions. FIGURE 4. UDP-Gal conformations influence enzyme ordering. 27048 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 27048 Donor Recognition in ABO(H) Blood Group Glycosyltransferases Finally, the UDP-C-Gal donor was designed in an effort to allow the visualization of the donor sugar residue by preventing donor hydrolysis, so it is somewhat paradoxical that the sugar moiety is not observed in two of the structures (Table 4). In both wild type enzymes, GTA and GTB, soaked with donor analog and intact HA, there is unambiguous density for UDP bound to the donor-binding site but weak or absent density for the C-Gal moiety, indicating that the moiety is disordered in the active site. This is understandable for GTA, because its active site accommodates the much larger N-acetylgala- tosamine residue, and the UDP-Gal substrate is also observed to be disordered. Insight into Substrate Binding—Angulo et al. (32) observed conformation 1 in the active site of GTB using NMR methods, prompting the hypothesis that the enzymes selected this cata- lytically competent conformation despite its sparse population in solution. However, the second UDP-Gal orientation is actu- ally similar to the dominant low energy conformer observed in solution (32), and these data suggest that the enzyme binds the abundant low energy UDP-Gal conformer and shifts it to the catalytically competent conformation. The wild type GTB is also interesting, because the complex with the DI shows an ordered active site with the donor analog UDP-C-Gal in conformation 2, whereas the presence of the 3-OH group on the natural acceptor (HA) galactosyl residue causes the donor analog sugar group to become completely Indeed, the selection of the extended conformer (i.e. approx- imately conformation 3) from solution offers an explanation of the kinetically observed phenomenon that donor binding pre- cedes acceptor binding because the structures with the bound NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 27049 NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY Donor Recognition in ABO(H) Blood Group Glycosyltransferases FIGURE5.StereoviewoftheelectrondensityintheactivesiteofthestructuresdisplayingUDP-C-GalandUDP-Galdisorderedovertwoconformations. The left column shows the dominant conformation, colored by element with gray for carbon, red for oxygen, and yellow for phosphorus, along with the supporting 2Fo Fc map, contoured at 1.0 (green) and the Fo Fc electron density maps contoured at 3.0 (red), 2.00 (magenta), and 4.00 (blue). The right column shows just the unbiased difference map (i.e. without the inclusion of the alternate conformation in the refinement) with the dominant conformation in gray and with the alternate conformation modeled into the difference density and colored by element with green for carbon, red for oxygen, and yellow for phosphorus. Donor Recognition in ABO(H) Blood Group Glycosyltransferases Alternate conformations are modeled into the difference maps and were not included during structure refinement for map generation. A, ABBAUDP-C-GalDI short soak where the donor is primarily in conformation 3 (0.8 occupancy, left) and in conformation 2 (right). B, ABBAUDP-C-GalDI long soak where the donor is in conformations 2 (0.7 occupancy, left) and 3 (right). C, ABBAUDP-GalDI the donor is in conformation 1 (0.7 occupancy, left) and conformation 2 (right). D, ABBBUDP-GalDI the donor is in conformations 1 (0.6 occupancy, left) and 2 (right). Donor Recognition in ABO(H) Blood Group Glycosyltransferases FIGURE5.Stereoviewoftheelectrondensityintheactivesiteofthestructures The left column shows the dominant conformation, colored by element with g supporting 2Fo Fc map, contoured at 1.0 (green) and the Fo Fc electron dens FIGURE5.StereoviewoftheelectrondensityintheactivesiteofthestructuresdisplayingUDP-C-GalandUDP-Galdisorderedovertwoconformations. The left column shows the dominant conformation, colored by element with gray for carbon, red for oxygen, and yellow for phosphorus, along with the supporting 2Fo Fc map, contoured at 1.0 (green) and the Fo Fc electron density maps contoured at 3.0 (red), 2.00 (magenta), and 4.00 (blue). The right column shows just the unbiased difference map (i.e. without the inclusion of the alternate conformation in the refinement) with the dominant conformation in gray and with the alternate conformation modeled into the difference density and colored by element with green for carbon, red for oxygen, and yellow for phosphorus. Alternate conformations are modeled into the difference maps and were not included during structure refinement for map generation. A, ABBAUDP-C-GalDI short soak where the donor is primarily in conformation 3 (0.8 occupancy, left) and in conformation 2 (right). B, ABBAUDP-C-GalDI long soak where the donor is in conformations 2 (0.7 occupancy, left) and 3 (right). C, ABBAUDP-GalDI the donor is in conformation 1 (0.7 occupancy, left) and conformation 2 (right). D, ABBBUDP-GalDI the donor is in conformations 1 (0.6 occupancy, left) and 2 (right). disordered. This missing hydroxyl group clearly results in large steric clashes, because a superposition of the two structures would put the 3-OH group only 2.5 Å away from C-1 of the ring, 3.3 Å from the methylene carbon, and 2.89 Å from one of the phosphate oxygen atoms. GTB transfer mechanism may be limited. The exquisite speci- ficity of these enzymes is remarkable because even a substrate analog designed to be isosteric can encounter major structural barriers. References Hu, Y., and Walker, S. (2002) Remarkable structural similarities between diverse glycosyltransferases. Chem. Biol. 9, 1287–1296 9. Qasba, P. K., Ramakrishnan, B., and Boeggeman, E. (2005) Substrate-in- duced conformational changes in glycosyltransferases. Trends Biochem. Sci. 30, 53–62 30. Boix, E., Swaminathan, G. J., Zhang, Y., Natesh, R., Brew, K., and Acharya, K. R. (2001) Structure of UDP complex of UDP-galactose:-galactoside- -1,3-galactosyltransferase at 1.53-Å resolution reveals a conformational change in the catalytically important C terminus. J. Biol. Chem. 276, 48608–48614 10. Breton, C., Snajdrova´, L., Jeanneau, C., Koca, J., and Imberty, A. (2006) Structures and mechanisms of glycosyltransferases. Glycobiology 16, 29R–37R 31. Blume, A., Angulo, J., Biet, T., Peters, H., Benie, A. J., Palcic, M., and Peters, T. (2006) Fragment-based screening of the donor substrate specificity of human blood group B galactosyltransferase using saturation transfer dif- ference NMR. J. Biol. Chem. 281, 32728–32740 11. Letts, J. A., Rose, N. L., Fang, Y. R., Barry, C. H., Borisova, S. N., Seto, N. O. L., Palcic, M. M., and Evans, S. V. (2006) Differential recognition of the type I and IIH antigen acceptors by the human ABO(H) blood group A and B glycosyltransferases. J. Biol. Chem. 281, 3625–3632 32. Angulo, J., Langpap, B., Blume, A., Biet, T., Meyer, B., Krishna, N. R., Peters, H., Palcic, M. M., and Peters, T. (2006) Blood group B galactosyl- transferase: insights into substrate binding from NMR experiments. J. Am. Chem. Soc. 128, 13529–13538 12. Alfaro, J. A., Zheng, R. B., Persson, M., Letts, J. A., Polakowski, R., Bai, Y., Borisova, S. N., Seto, N. O. L., Lowary, T. L., Palcic, M. M., and Evans, S. V. (2008) ABO(H) blood group A and B glycosyltransferases recognize substrate via specific conformational changes. J. Biol. Chem. 283, 10097–10108 33. Soya, N., Shoemaker, G. K., Palcic, M. M., and Klassen, J. S. (2009) Com- parative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases. Glycobiology 19, 1224–1234 13. Coutinho, P. M., and Henrissat, B. (1999) in Recent Advances in Carbohy- drate Bioengineering (Gilbert, H. J., Davies, G., Henrissat, B., and Svensson, B., eds) pp. 3–12, Royal Society of Chemistry, Cambridge, UK 34. Sindhuwinata, N., Munoz, E., Munoz, F. J., Palcic, M. M., Peters, H., and Peters, T. (2010) Binding of an acceptor substrate analog enhances the enzymatic activity of human blood group B galactosyltransferase. Glyco- biology 20, 718–723 14. Patenaude, S. I., Seto, N. O. Donor Recognition in ABO(H) Blood Group Glycosyltransferases question the potential utility of C-phosphonate analogs as biologically relevant mimics of sugar nucleotides. In addition, the multiple observed conformers suggest that donor binding to GTA and GTB is a multistage process that implements sig- nificant stepwise shifts to achieve the catalytically active conformation. for blood type B in humans. Biochem. Biophys. Res. Commun. 32, 272–277 16. Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990) Molecular genetic basis of the histo-blood group ABO system. Nature 345, 229–233 17. Seto, N. O. L., Palcic, M. M., Compston, C. A., Li, H., Bundle, D. R., and Narang, S. A. (1997) Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activ- ity and progressively modifies substrate recognition in human recombi- nant enzymes. J. Biol. Chem. 272, 14133–14138 Author Contributions—S. M. L. G. performed some of the experi- ments, processed and analyzed the data, made the figures, and wrote a major part of the paper. P. J. M., R. B. Z., and T. L. L. performed the synthesis of the non-hydrolyzable donor analog and contributed to writing the paper. O. H. G. processed some of the data and contrib- uted to Fig. 1 and Fig. 3A. A. R. J. processed and analyzed data and contributed to the paper. S. N. B. performed many of the experi- ments. S. V. E. conceived many of the experiments and wrote a major part of the paper. All authors reviewed the results and approved the final version of the manuscript. 18. Hamasaki, N., and Yamamoto, M. (2000) Red blood cell function and blood storage. Vox Sang. 79, 191–197 19. Yamamoto, M., Lin, X. H., Kominato, Y., Hata, Y., Noda, R., Saitou, N., and Yamamoto, F. (2001) Murine equivalent of the human histo-blood group ABO gene is a cis-AB gene and encodes a glycosyltransferase with both A and B transferase activity. J. Biol. Chem. 276, 13701–13708 20. Hearn, V. M., Smith, Z. G., and Watkins, W. M. (1968) An -N-acetyl-D- galactosaminyltransferase associated with human blood-group A charac- ter. Biochem. J. 109, 315–317 21. Landsteiner, K. (2001) [Agglutination phenomena of normal human blood]. Wien. Klin. Wochenschr. 113, 768–769 22. Morgan, W. T. J., and Watkins, W. M. (1959) Some aspects of the bio- chemistry of the human blood-group substances. Brit. Med. Bull. 15, 109–113 23. Kabat, E. A. (1949) Immunochemical studies on blood group substances. Bacteriol. Rev. References 24. Watkins, W. M., Greenwell, P., and Yates, A. D. (1980) Blood group A and B transferase levels in serum and red cells of human chimeras. Rev. Fr. Transfus. Immunohematol. 23, 531–544 1. Sinnott, M. L. (1990) Catalytic mechanism of enzymic glycosyl transfer. Chem. Rev. 90, 1171–1202 2. Coutinho, P. M., Deleury, E., Davies, G. J., and Henrissat, B. (2003) An evolving hierarchical family classification for glycosyltransferases. J. Mol. Biol. 328, 307–317 25. Yamamoto, F., and Hakomori, S. (1990) Sugar-nucleotide donor specific- ity of histo-blood group A and group B transferases is based on amino acid Substitutions. J. Biol. Chem. 265, 19257–19262 3. Paulson, J. C., and Colley, K. J. (1989) Glycosyltransferases: structure, lo- calization, and control of cell type-specific glycosylation. J. Biol. Chem. 264, 17615–17618 26. Lee, H. J., Barry, C. H., Borisova, S. N., Seto, N. O. L., Zheng, R. X. B., Blancher, A., Evans, S. V., and Palcic, M. M. (2005) Structural basis for the inactivity of human blood group O-2 glycosyltransferase. J. Biol. Chem. 280, 525–529 4. Campbell, J. A., Davies, G. J., Bulone, V., and Henrissat, B. (1997) A clas- sification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem. J. 326, 929–939 27. Letts, J. A., Persson, M., Schuman, B., Borisova, S. N., Palcic, M. M., and Evans, S. V. (2007) The effect of heavy atoms on the conformation of the active-site polypeptide loop in human ABO(H) blood-group glycosyl- transferase B. Acta Crystallogr. D Biol. Crystallogr. 63, 860–865 5. Unligil, U. M., and Rini, J. M. (2000) Glycosyltransferase structure and mechanism. Curr. Opin. Struct. Biol. 10, 510–517 6. Bourne, Y., and Henrissat, B. (2001) Glycoside hydrolases and glycosyl- transferases: families and functional modules. Curr. Opin. Struct. Biol. 11, 593–600 28. Seto, N. O. L., Compston, C. A., Evans, S. V., Bundle, D. R., Narang, S. A., and Palcic, M. M. (1999) Donor substrate specificity of recombinant hu- man blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli. Eur. J. Biochem. 259, 770–775 7. Breton, C., Mucha, J., and Jeanneau, C. (2001) Structural and functional features of glycosyltransferases. Biochimie 83, 713–718 29. Kamath, V. P., Seto, N. O. L., Compston, C. A., Hindsgaul, O., and Palcic, M. M. (1999) Synthesis of the acceptor analog Fuc(1 3 2) Gal- O(CH2)(7) CH3: a probe for the kinetic mechanism of recombinant hu- man blood group B glycosyltransferase. Glycoconj. J. 16, 599–606 8. Donor Recognition in ABO(H) Blood Group Glycosyltransferases It was also observed that the donors and donor analogs bind these enzymes in more than one conformation and, further, that these multiple conformations have similar free energies of binding in the active site. UDP-Glc binds in an orientation dis- tinct from those previously observed with significantly different enzyme-donor sugar hydrogen bond interactions. It also binds in the tucked under conformation associated with catalysis, but the slight differences in binding induced by the epimeric posi- tion of the 4-OH group prevent loop closure and so allow the enzymes to distinguish between epimers. Conclusions—Despite being soaked under identical condi- tions, the UDP-C-Gal donor analog in the active site of GTA, GTB, or their chimera approaches but cannot achieve the tucked under conformation associated with catalysis. This is probably due to the differences in chemical geometries imposed when substituting the glycosidic oxygen atom for car- bon to make a non-hydrolyzable donor analog. Although the enzymes do bind the UDP moiety of UDP-C-Gal in the same manner as the native donors, the different conformation of the altered sugar moiety does not permit formation of the semi- closed or closed states, and its utility as a probe of the GTA/ Together, these structures provide insight into the devel- opment of specific GT inhibitors and, in particular, calls into VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 27050 JOURNAL OF BIOLOGICAL CHEMISTRY Donor Recognition in ABO(H) Blood Group Glycosyltransferases 13, 189–202 Acknowledgment—We gratefully acknowledge Professor Monica Pal- cic for helpful discussions. NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 Acknowledgment—We gratefully acknowledge Professor Monica Pal- cic for helpful discussions. Donor Recognition in ABO(H) Blood Group Glycosyltransferases malm, G., and Peters, T. (2010) NMR-based exploration of the acceptor binding site of human blood group B galactosyltransferase with molecular fragments. Glycoconj. J. 27, 349–358 Donor Recognition in ABO(H) Blood Group Glycosylt roni, L. C., and Read, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 malm, G., and Peters, T. (2010) NMR-based exploration of the acceptor binding site of human blood group B galactosyltransferase with molecular fragments. Glycoconj. J. 27, 349–358 44. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 36. Beaton, S. A., Huestis, M. P., Sadeghi-Khomami, A., Thomas, N. R., and Jakeman, D. L. (2009) Enzyme-catalyzed synthesis of isosteric phosphono- analogues of sugar nucleotides. Chem. Commun. 10.1039/b808078j 45. Collaborative Computational Project, Number 4 (1994) The CCP4 Suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 37. Gordon, R. D., Sivarajah, P., Satkunarajah, M., Ma, D., Tarling, C. A., Vizitiu, D., Withers, S. G., and Rini, J. M. (2006) X-ray crystal structures of rabbit N-acetylglucosaminyltransferase I (GnT I) in complex with donor substrate analogues. J. Mol. Biol. 360, 67–79 46. Johal, A. R., Blackler, R. J., Alfaro, J. A., Schuman, B., Borisova, S., and Evans, S. V. (2014) pH-induced conformational changes in human ABO(H) blood group glycosyltransferases confirm the importance of elec- trostatic interactions in the formation of the semi-closed state. Glycobiol- ogy 24, 237–246 38. Clarke, A. J., Hurtado-Guerrero, R., Pathak, S., Schu¨ttelkopf, A. W., Borodkin, V., Shepherd, S. M., Ibrahim, A. F. M., and van Aalten, D. M. F. (2008) Structural insights into mechanism and specificity of O-GlcNAc transferase. EMBO J. 27, 2780–2788 47. Johal, A. R., Schuman, B., Alfaro, J. A., Borisova, S., Seto, N. O. L., and Evans, S. V. (2012) Sequence-dependent effects of cryoprotectants on the active sites of the human ABO(H) blood group A and B glycosyltrans- ferases. Acta Crystallogr. D Biol. Crystallogr. 68, 268–276 39. Partha, S. K., Sadeghi-Khomami, A., Slowski, K., Kotake, T., Thomas, N. R., Jakeman, D. L., and Sanders, D. A. R. (2010) Chemoenzymatic syn- thesis, inhibition studies, and x-ray crystallographic analysis of the phos- phono analog of UDP-Galp as an inhibitor and mechanistic probe for UDP-galactopyranose mutase. J. Mol. Biol. 403, 578–590 48. Schuman, B., Persson, M., Landry, R. C., Polakowski, R., Weadge, J. T., Seto, N. O. L., Borisova, S. References L., Borisova, S. N., Szpacenko, A., Marcus, S. L., Palcic, M. M., and Evans, S. V. (2002) The structural basis for specificity in human ABO(H) blood group biosynthesis. Nat. Struct. Biol. 9, 685–690 35. Rademacher, C., Landstro¨m, J., Sindhuwinata, N., Palcic, M. M., Wid- 15. Kobata, A., Grollman, E. F., and Ginsburg, V. (1968) An enzymatic basis NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 NOVEMBER 6, 2015•VOLUME 290•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 27051 JOURNAL OF BIOLOGICAL CHEMISTRY 27051 Donor Recognition in ABO(H) Blood Group Glycosyltransferases N., Palcic, M. M., and Evans, S. V. (2010) Cysteine-to-serine mutants dramatically reorder the active site of human ABO(H) blood group B glycosyltransferase without affecting activity: structural insights into cooperative substrate binding. J. Mol. Biol. 402, 399–411 40. Marcus, S. L., Polakowski, R., Seto, N. O. L., Leinala, E., Borisova, S., Blancher, A., Roubinet, F., Evans, S. V., and Palcic, M. M. (2003) A single point mutation reverses the donor specificity of human blood group B- synthesizing galactosyltransferase. J. Biol. Chem. 278, 12403–12405 41. Lowary, T. L., and Hindsgaul, O. (1993) Recognition of synthetic deoxy and deoxyfluoro analogs of the acceptor -L-Fuc p-(132)--D-Gal p-OR by the blood-group A and B gene-specified glycosyltransferases. Carbo- hydr. Res. 249, 163–195 49. Heissigerova, H., Breton, C., Moravcova, J., and Imberty, A. (2003) Molec- ular modeling of glycosyltransferases involved in the biosynthesis of blood group A, blood group B, Forssman, and iGb(3) antigens and their interac- tion with substrates. Glycobiology 13, 377–386 50. Seto, N. O. L., Compston, C. A., Szpacenko, A., and Palcic, M. M. (2000) Enzymatic synthesis of blood group A and B trisaccharide analogues. Car- bohydr. Res. 324, 161–169 42. Pflugrath, J. W. (1999) The finer things in x-ray diffraction data collection. Acta Crystallogr. D Biol. Crystallogr. 55, 1718–1725 y g y g 43. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Sto- 3. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Wi VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 VOLUME 290•NUMBER 45•NOVEMBER 6, 2015 27052 JOURNAL OF BIOLOGICAL CHEMISTRY
W4293431499.txt	https://www.scielo.br/j/csp/a/gNnz4VX7VmvYRRb5hSpd7fR/?lang=en&format=pdf	en		Contributions from research on the Health Economic-Industrial Complex to the teaching project in Public Health	Cadernos de Saúde Pública	2,022	cc-by	2,480	PERSPECTIVAS PERSPECTIVES Contributions from research on the Health Economic-Industrial Complex to the teaching project in Public Health Contribuições da pesquisa sobre o Complexo Econômico-Industrial da Saúde ao projeto político-pedagógico da Saúde Coletiva Contribuciones de la investigación sobre el Complejo Económico-Industrial de la Salud al proyecto político-pedagógico de la Salud Colectiva Eduardo Levcovitz 1 doi: 10.1590/0102-311XEN224720 A critical reflection on recent research production in Brazil’s Public Health field After 16 years working away from Brazil, I attempted to update my knowledge on research production in Health Policy, Planning, and Administration, urged by recent analyses 1,2,3,4, especially Schraiber’s seminal work 5. I revisited the pioneering research methodology on the theme from 1974 to 2000 6 and reviewed the articles published from 2001 to 2018 in the journals Cadernos de Saúde Pública, Ciência & Saúde Coletiva, Revista de Saúde Pública, and Saúde em Debate, as well as books, anthologies, and theses, selected intuitively. There has been a major reduction in this production on the themes of the national State and its role in guaranteeing universal social protection, as well as in macro policies in Social Security and the Brazilian Unified National Health System (SUS) 2,3,4,5,7, steadily replaced by case studies, reports of local experiences, program evaluations 2,4, and exaltations of identity particularisms, in addition to a virtually total abandonment of the theoretical and methodological approach of Political Economy 8. Intellectual interaction between professors, researchers, and students with different academic and professional backgrounds, whether Marxist structuralists or post-structuralists, post-Modernists or neo-Institutionalists, has been present since the pioneering writings in Public Health 6,9,10. This plurality has not been limited to the organic linkage between original research production and policy, services, and the development of capacities for understanding, interpretation, and transformative intervention in the sector’s reality, based on the “ideology-knowledge-practice triad” 11. This organic linkage was established over the course of the 1970s, oriented by a shared understanding in the “field” concerning the Brazilian health sector: a “Grand Narrative” on the population’s living and health conditions; the evolution of health and corporate-social security policies; the social organization of medical work and the “hospital-centered” healthcare model vis-à-vis the capitalist mode of production; segmented access to medical and hospital care; the “commodification” of health and the power relations spearheaded by the medical profession and the nascent entrepreneurial class, all under the influence of Historical Materialism and Political Economy 3,8,10, which was “The” reference in the subsequent decades for the politically engaged and innovative production of the field’s objects, methods, and products 6,7,12. This article is published in Open Access under the Creative Commons Attribution license, which allows use, distribution, and reproduction in any medium, without restrictions, as long as the original work is correctly cited. Instituto de Medicina Social, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil. 1 Correspondence E. Levcovitz Instituto de Medicina Social, Universidade do Estado do Rio de Janeiro. Rua São Francisco Xavier 524, 7o andar, Rio de Janeiro, RJ 20550-013, Brasil. eduardo.levcovitz@gmail.com Cad. Saúde Pública 2022; 38 Sup 2:e00224720 2 Levcovitz E The foundations for the theoretical and methodological density and complexity of this research output were the seminars held by the Pan American Health Organization (PAHO) from 1969 to 1978, the teachings of Juan Cezar Garcia 13, with Marxist structuralist orientation, reinforced in the case of various authors by their activism in the Brazilian Communist Party (PCB) 12,14, and Latin American critical thinking. “There is an immeasurable wealth of academic production that has come to light in the recently inaugurated field of Collective Health, in which studies have been developed taking the issue of the State, democracy, and social policies as the line for updating the Marxist debate (...), exposing the forms of commodification of health, the dynamics of capitalist accumulation in the sector, and the different organizational configurations and linkages between the public and private sectors” 3 (p. 17-8). “In the late 1980s, with the new liberal hegemony, critical thinking had clearly lost its vitality, while many Marxist structuralists had adhered to the liberal-conservative project in one way or another” 15 (p. 33). The 1990s witnessed a profound change in the political and ideological references, accompanying the impacts on the Socialist movements from the electoral defeats of the Social Democratic parties and the dissolution of the USSR, aggravated in Brazil by the self-dissolution of the PCB, the exhaustion of Keynesian and ECLAC (Economic Commission for Latin America)-inspired developmentalism, and intellectuals’ increasing disinterest in studying the Brazilian State, thereby favoring the hegemony of utilitarian views of the State and the narrow limits of public policies. The Public Health field in Brazil initially resisted this change 2,4,6,7, but since the transition into the 21st century we have lost our reference to the Grand Narrative and thus the depth, complexity, intelligence, and relevance of our production 4,7,12, which “is becoming increasingly fragmented and fragmentary” 3 (p. 22). We are collectively responsible for the abandonment of Marxism’s central methodological category, namely Dialetical Totality, a tendency which adds to the growing depoliticization and “theoretical rarefaction of our production” 5 (p. 36) and has resulted in the impoverishment of critical reflection in articles, books, and theses, now peopled by an inductive empiricism with limited explanatory power, constituting the reason for frustration for many of us. “Pragmaticism, opportunism, and post-Modernism serve as a pretext to block reflection on projects and the future” 11 (p. 302). Notwithstanding its disciplinary and epistemological diversity, our original research production expressed the authors’ reflections on their political, professional, and institutional praxis 3, and even having concentrated the output on academic studies 2,6, the field kept a critical eye on the dynamics of the State, the market, society, and politics 4,6,7. In the 21st century, our production has swayed increasingly away from praxis 3, thus leading to an important theoretical-empirical dissociation, expressed in the incapacity to construct a dialogue between the theoretical elements and the research results 5, which leads to the risk of contributing to the dissemination of speculations, alternative facts, and post-truths. This process is aggravated by the multiplication of research groups that are the “property” of a few researchers and expressions of their personal interests (or disinterests), backed by a “laissez-faire” understanding of department-chair freedom, by “a certain eclecticism in the definition of approaches and methods” 16 (p. 116), and by the growing transformation of our courses/debates into spaces for collective training in manifestations of selfaffirmation of identity groups, in which “places of speech” override reflections on praxis. The results of this production largely fail to expand or update knowledge, nor do they promote an understanding of reality. They do not contribute to critical reflection and rarely propose innovative research agendas or recommend transformative interventions. They are mostly limited to the description of sociopolitical/health processes. It is never too much to recall Marx’s warning: “All science would be superfluous if the outward appearance and the essence of things directly coincided” 17 (p. 939). Cad. Saúde Pública 2022; 38 Sup 2:e00224720 CONTRIBUTIONS FROM RESEARCH ON THE HEALTH ECONOMIC-INDUSTRIAL COMPLEX Contribution of research on the Health Economic-Industrial Complex to the explanation/understanding/interpretation of the Brazilian health sector in the 21st century Studies on the consolidation/expansion of the Health Economic-Industrial Complex (CEIS, in Portuguese), acknowledging it as the productive base of health and simultaneously as the space for capital accumulation and innovation, competition, and financialization, are critical for reconstructing the Grand Narrative. A prime example is the study on the Health Economic-Industrial Complex, Innovation, and Capitalist Dynamic: Structural Challenges for Building the Universal System in Brazil, led by the Research and Documentation Group in Health Entrepreneurship, Institute for Studies in Collective Health, Federal University of Rio de Janeiro (GPDES/IESC/UFRJ). The group has moved beyond theoretical reflections 8 to study the health market’s current reality and its impacts on social protection policies, adopting the Political Economy approach to explain/understand/interpret Brazilian capitalism from a theoretical and historical perspective, analyzing companies that develop direct activities in the provision of individual care or indirectly in training human resources, marketing of health plans/insurance, or production/marketing of materials, equipment, and medicines, further assessing the activity in political-institutional spaces. In my view, it is in Marxist Political Economy that we find the theoretical and methodological basis for a renewed teaching project in Public Health in Brazil, since it approaches the reality simultaneously from a historical, economic, political, and ideological perspective. The analysis of the evolution and challenges posed by the Health Industrial Complex necessitate the introduction of a dynamic economic focus in the health field, concentrating on the processes of development, capital accumulation, and innovations, reclaiming approaches from Political Economy 18. In addition to analyzing “the contradictory dynamic of capital, whose essence is given by the processes of accumulation, distribution [and] innovation” 19 (p. 107) and relations of power and ownership, Marxist Political Economy establishes “a highly complex link between the process of knowledge and the process of political organization of social forces” 20, by moving towards transformative praxis, as proposed by Marx in the 11th Thesis on Feuerbach. This approach also induces the recovery of the political engagement at the origins of Public Health and the methodological orientation from the category of Dialectical Totality, understood as “the perception of social reality as an organic, structured whole in which one cannot understand an element, an aspect, a dimension without losing its relationship to the totality” 21 (p. 23-4), rejecting both causal linearity in the explanation of complex phenomena and positivist pseudo-neutrality in the interpretation of such phenomena, so much in vogue in the age of evidenced-based and value-free policies. The approach understands such phenomena as permeated by social worldviews, as “structured sets of values, representations, ideas, and cognitive orientations” 21 (p. 21). Based on Marxist Political Economy, the renewal of the teaching project in Brazil’s Public Health field will require rebuilding the reflection on the Brazilian State and its irreplaceable role in strategic leadership in the development, reduction of inequalities, and universalization of social protection. For this to happen, our debates and intellectual efforts should be oriented by a collective set (agreed upon with solidarity) of political, institutional, and academic challenges and objectives: (1) Priority for cooperative research and collective production of knowledge based on theoreticalconceptual density in reclaiming the complexity, reconstructing the theoretical-empirical dialogue, critical reflection, technical and scientific innovation, and ethical and political engagement; (2) Shared construction of a Grand Narrative on the Brazilian health sector in the 21st century, based on reflections on the State, market, social classes, distributive conflict, policy, development projects, and expansion of the CEIS; (3) Sharing of knowledge and analyses, based on the ideology-knowledge-practice triad; (4) Formation of a new generation of professors, researchers, administrators, and technicians with capacity for description/explanation – understanding/interpretation – and technical-political-institutional intervention/transformative action in the State, economy, society, and health policy. Cad. Saúde Pública 2022; 38 Sup 2:e00224720 3 4 Levcovitz E Additional information References ORCID: Eduardo Levcovitz (0000-0003-39238481). 1. Acknowledgments The authors wish to thank the teams from Research and Documentation Group in Health Entrepreneurship, Institute for Studies in Public Health, Federal University of Rio de Janeiro, the Health Policy Analysis Group, Institute of Social Medicine, University of the State of Rio de Janeiro, and Pan American Health Organization/World Health Organization, who shared in the debates used in the manuscript’s production. Cad. Saúde Pública 2022; 38 Sup 2:e00224720 Baptista TWF, Azevedo CS, Machado CV, editors. Políticas, planejamento e gestão em saúde: abordagens e métodos de pesquisa. Rio de Janeiro: Editora Fiocruz; 2015. 2. Santos JS, Teixeira CF. Produção científica sobre política de saúde no Brasil (1988-2014). Contribuição ao debate sobre a Reforma Sanitária Brasileira. In: Teixeira CF, editor. Observatório de análise de política em saúde. Abordagens, objetos e investigações. Salvador: EdUFBA; 2016. p. 42-72. 3. Fleury S. Reforma Sanitária: múltiplas leituras, diálogos e controvérsias. In: Fleury S, editor. Teoria da reforma sanitária. Diálogos críticos. Rio de Janeiro: Editora Fiocruz; 2018. p. 15-30. 4. Teixeira CF. Produção científica na área de política, planejamento e gestão em saúde 1975-2010: temas e teorias. In: Baptista TWF, Azevedo C, Machado CV, editors. Políticas, planejamento e gestão em saúde: abordagens e métodos de pesquisa. Rio de Janeiro: Editora Fiocruz; 2015. p. 81-111. 5. Schraiber LB. Engajamento ético-político e construção teórica na produção científica do conhecimento em Saúde Coletiva. In: Baptista TWF, Azevedo C, Machado CV, editors. Políticas, planejamento e gestão em saúde: abordagens e métodos de pesquisa. Rio de Janeiro: Editora Fiocruz; 2015. p. 33-57. 6. Levcovitz E, Baptista TWF, Uchoa SAC, Nespoli G, Mariani M. Produção do conhecimento em política, planejamento e gestão em saúde e políticas de saúde no Brasil (1974-2000). Brasília: Organização Pan-Americana da Saúde; 2003. 7. Paim J, Teixeira CF. Política, planejamento e gestão em saúde: balanço do estado da arte. Rev Saúde Pública 2006; 40(spe):73-8. 8. Mendes A, Ianni AMZ, Marques MCC, Ferreira MJ, Silva THS. A contribuição do pensamento da saúde coletiva à economia política da saúde. Saúde Soc 2017; 26:841-60. 9. Donnangelo MCF. A pesquisa na área de Saúde Coletiva no Brasil: a década de 70. In: Associação Brasileira de Pós-Graduação em Saúde Coletiva, editor. Ensino da Saúde Pública, Medicina Preventiva e Social no Brasil. Rio de Janeiro: Associação Brasileira de Pós-graduação em Saúde Coletiva; 1983. p. 17-35. 10. Canesqui AM. Ciências sociais, a saúde e a saúde coletiva. In: Canesqui AM, editor. Dilemas e desafios das ciências sociais na saúde coletiva. São Paulo: Hucitec Editora; 1995. p. 19-35. 11. Paim J. Reforma sanitária brasileira: contribuição para a compreensão e crítica. Salvador: EdUFBA/Rio de Janeiro: Editora Fiocruz; 2008. 12. Sophia DC. Saúde & utopia. O Cebes e a Reforma Sanitária Brasileira (1976-1986). São Paulo: Hucitec Editora/Sobravime; 2014. CONTRIBUTIONS FROM RESEARCH ON THE HEALTH ECONOMIC-INDUSTRIAL COMPLEX 13. Garcia JC. Juan Cezar Garcia entrevista Juan Cezar Garcia. In: Nunes ED, editor. As Ciências Sociais em saúde na América Latina, tendências e perspectivas. Brasília: Organização Pan-Americana da Saúde; 1985. p. 21-8. 14. Arouca S. Crise brasileira e reforma sanitária. Saúde Debate 1991; 4:15-20. 15. Fiori JL. As trajetórias intelectuais do debate sobre desenvolvimento na América Latina. In: Brandão CA, editor. Teorias e políticas de desenvolvimento latino-americano. Rio de Janeiro: Contraponto; 2018. p. 17-46. 16. Machado CV, Lima, LD. Perspectivas históricas na análise de políticas de saúde. In: Baptista TWF, Azevedo C, Machado CV, editors. Políticas, planejamento e gestão em saúde: abordagens e métodos de pesquisa. Rio de Janeiro: Editora Fiocruz; 2015. p. 115-45. 17. Marx K. O Capital – crítica da economia política. Rio de Janeiro: Civilização Brasileira; 2008. 18. Gadelha CAG. O complexo industrial da saúde e a necessidade de um enfoque dinâmico na economia da saúde. Ciênc Saúde Colet 2003; 8:521-35. 19. Teixeira A. Marx e a economia política: a crítica como conceito. Econômica 2000; II:85-109. 20. Dos Santos T. Economia política Marxista: um balanço. https://www.nodo50.org/cubasiglo XXI/taller/dossantos_290204.pdf (accessed on 18/Dec/2018). 21. Löwy M. Ideologias e ciência social. Elementos para uma análise marxista. São Paulo: Cortez Editora; 2015. Submitted on 28/Jul/2020 Final version resubmitted on 26/Aug/2020 Approved on 31/Aug/2020 Cad. Saúde Pública 2022; 38 Sup 2:e00224720 5
https://openalex.org/W2966921342	https://periodicos.utfpr.edu.br/rbect/article/download/7296/pdf	Portuguese	null	O uso de mídias digitais, associados ao ambiente virtual de ensino e de aprendizagem, no ensino de química: explorando a radioatividade por meio da educação a distância	Revista brasileira de ensino de ciência e tecnologia	2,019	cc-by	6,625	ERROR: type should be string, got "https://periodicos.utfpr.edu.br/rbect R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. RESUMO Maurício Severo da Silva \nmss@universo.univates.br \n0000-0003-1271-6981 \nUniversidade do Vale do Taquari - \nUnivates, Lajeado, Rio Grande do \nSul, Brasil. Maurício Severo da Silva \nmss@universo.univates.br \n0000-0003-1271-6981 \nUniversidade do Vale do Taquari - \nUnivates, Lajeado, Rio Grande do \nSul, Brasil. O presente texto é um relato de experiência que teve como objetivo possibilitar aos \nestudantes de uma turma do 1º ano do Ensino Médio o estudo do tema radiação por meio \ndo Ambiente Virtual de Ensino e de Aprendizagem. Os professores organizaram sua prática \npedagógica em seis encontros, um presencial e os seguintes a distância. Os docentes \nutilizaram mídias digitais, tais como: animações, vídeos, textos, infográficos e o jogo \ncomercial SimCity, bem como uma ferramenta de comunicação e interação, o fórum de \ndiscussão, presente no AVEA. Este recurso possibilitou que os estudantes interagissem \nentre si, proporcionando ricas e acaloradas discussões. No entanto, o jogo SimCity foi o que \nprovocou maior engajamento entre o grupo de alunos. Como forma de avaliação, os \ndocentes solicitaram que os estudantes se organizassem em grupos para gravar um vídeo, \nno qual os discentes deveriam demonstrar o que aprenderam sobre os assuntos abordados. A qualidade do conteúdo e da produção apresentada pelos estudantes surpreendeu os \ndocentes e evidenciou a preferência dos alunos por este tipo de atividade. Os docentes \nacreditam que o uso de forma estratégica do AVEA e das mídias digitais, em conjunto com \natividades que possibilitaram aos discentes serem autores de suas próprias discussões, \nreflexões e vídeos, auxiliou no desenvolvimento de uma postura mais proativa dos alunos \ne de uma maior autonomia nos seus estudos. Katiele Stefani Zotti \nkatyszotti@gmail.com \n0000-0001-9446-927X \nUniversidade do Vale do Taquari - \nUnivates, Lajeado, Rio Grande do \nSul, Brasil. Marcia Jussara Hepp Rehfeldt \nmrehfeld@univates.br \n0000-0002-0007-8639 \nUniversidade do Vale do Taquari - \nUnivates, Lajeado, Rio Grande do \nSul, Brasil. Miriam Ines Marchi \nmimarchi@univates.br \n0000-0003-2546-7072 \nUniversidade do Vale do Taquari - \nUnivates, Lajeado, Rio Grande do \nSul, Brasil. INTRODUÇÃO A inserção de mídias digitais no ensino vem sendo discutida e estudada por \ndiversos autores, tais como Saeed (2010) e Leite (2016). Contudo, estes mesmos \nautores concordam que é difícil para um docente que não possui uma formação \nvoltada ao uso de tecnologias educacionais - ou não as utilizou durante sua \nformação - inseri-las em suas práticas pedagógicas. Isso ocorre por diversos \nfatores, como a falta de habilidade com o uso de tecnologias digitais, \ndesconhecimento quanto aos recursos disponíveis, a falta de tempo para planejar \no uso de mídias digitais em sala de aula, entre outros. Além destes fatores, Prensky (2001) apresenta uma reflexão que agrega outro \nmotivo às dificuldades dos docentes em adicionar mídias digitais em suas práticas \npedagógicas. O autor argumenta que o distanciamento geracional existente entre \nos professores que nasceram em uma época onde pouca ou quase nenhuma \ntecnologia digital era existente amplia as dificuldades de diálogo com as novas \ngerações, que já nasceram imersas em um mundo onde a comunicação e a \ninteração são cada vez mais digitais. Para Prensky (2001), os docentes nascidos e \nformados na era pré-digital foram ensinados que o professor é o meio de \ninformação mais substancial que deve haver na formação de um indivíduo, sendo \no detentor do conhecimento, condição esta que não é mais válida em tempos \natuais. Diante disso, o autor destaca que o abismo geracional entre os que \nnasceram antes e após as tecnologias digitais é tão grande que parecem conversar \nem diferentes idiomas. Com base nisso, é possível perceber que um docente que não acompanha a \nevolução das tecnologias e não atualiza suas práticas pedagógicas está \ndistanciando-se ainda mais da realidade dos atuais estudantes. Um estudo recente \npublicado pelo Cetic.br1 (2016) demonstra que o número de dispositivos capazes \nde acessar a Internet disponíveis aos estudantes cresce a cada dia. Segundo o \nrelatório, 89% dos estudantes entre 15 e 17 anos, de escolas públicas, utilizam \nmeios de acesso à Internet como instrumentos para efetuarem pesquisas \nescolares, número este que chega a 97% no ensino privado. Vale destacar que o \nrelatório não deixa claro se os estudantes utilizam a Internet a pedido dos docentes \nou se a buscam de forma espontânea. Já, ao contrário, docentes que buscam inserir as tecnologias, por meio do uso \nde mídias digitais, em suas práticas tendem a tornar as atividades de ensino e de \naprendizagem mais atraentes aos estudantes. Nesse sentido, Santos et al. Miriam Ines Marchi PALAVRAS-CHAVE: Relato de experiência. Prática pedagógica. Ensino à distância. Ambiente \nvirtual de ensino e de aprendizagem. Mídias digitais. Página \| 37 Página \| 37 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. INTRODUÇÃO (2016) \nafirmam que os Ambientes Virtuais de Aprendizagem (AVAs) constituem-se em \nespaços fecundos que podem ser utilizados para promover a comunicação e \ninteratividade, além de serem capazes de potencializar os processos de ensino e \nde aprendizagem. Neste trabalho, os docentes optaram pela utilização do \nAmbiente Virtual de Ensino e Aprendizagem (AVEA) como instrumento de \ncomunicação, interação e disponibilização de mídias digitais coletadas na Internet \ne, também, produzidas pelos professores. É importante destacar que tanto o uso de AVEAs quanto de mídias digitais no \nensino não resolve os problemas de aprendizagem dos estudantes. Sendo assim, \nmesmo as atividades que utilizam tais recursos necessitam de planejamento e de \nobjetivos pedagógicos claramente definidos. Isso porque, sem estas etapas, o \ndocente corre o risco de reduzir o potencial destes recursos a meros artifícios para \ndeixar a aula mais divertida, conforme argumenta Demo (2016). Quanto a isto, Lames (2011) afirma que o docente precisa refletir sobre sua \nprática pedagógica de modo a superar o uso inadequado das tecnologias. Os \ndocentes precisam compreender o que é aprender e o que é ensinar para que, \nassim, possam criar e oportunizar aos discentes novas metodologias de ensino, “de \nproduzir, comunicar e representar o conhecimento” (LAMES, 2011, p. 23), o que, \nsegundo a autora, é possibilitado pelas novas mídias. Foi com base na reflexão sugerida por Demo (2016) e Lames (2011) que os \ndocentes e autores deste trabalho chegaram a seguinte questão de pesquisa: quais \nos efeitos pedagógicos do uso de um Ambiente Virtual de Ensino e de \nAprendizagem, associado a conteúdos que explorem diferentes mídias digitais, e \nda educação a distância, no ensino do tema radioatividade em uma turma de 1º \nano do Ensino Médio? Tendo em vista a questão norteadora, os docentes efetuaram o planejamento \nde suas atividades, de modo a propiciar a seus discentes uma experiência no uso \nde diversos tipos de mídias digitais, que puderam ser acessadas por meio de um \nAmbiente Virtual de Ensino e de Aprendizagem. Posto isso, a pesquisa teve como \nobjetivo desvelar os efeitos de diferentes mídias digitais por meio de um Ambiente \nVirtual de Ensino e Aprendizagem, com discentes do 1º ano do Ensino Médio, na \ndisciplina de Química. Como forma de fundamentar a prática realizada, a seção a \nseguir apresenta um breve resumo da abordagem teórica que guiou os docentes \ndurante a escolha das ferramentas e tipos de mídias a serem utilizadas. INTRODUÇÃO Na \nsequência, a seção 3 apresenta as particularidades da metodologia adotada pelos \ndocentes, bem como o detalhamento da prática efetivada. Por fim, são \napresentados os resultados obtidos e as considerações finais. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. ABORDAGEM TEÓRICA De acordo com Messa (2010), os AVEAs estão sendo cada vez mais utilizados \nno âmbito educacional, uma vez que permitem o uso de mídias para veicular \nconteúdos e possibilitam a interação por meio de suas ferramentas, fornecendo \nsuporte a diversos tipos de atividades propostas aos alunos. Diante disso, é preciso \nelucidar que a prática pedagógica apresentada neste relato foi planejada de modo \na aproveitar os recursos que o AVEA Moodle disponibiliza. Sendo assim, neste \ntrabalho, a abordagem teórica objetiva situar o leitor quanto a alguns dos \nconceitos utilizados pelos docentes durante o planejamento e efetivação da \nprática pedagógica, que usou as mídias digitais com o apoio do Ambiente Virtual. Como forma de elucidar todos os recursos utilizados pelos docentes, cabe, \nneste momento, apresentar o Quadro 1, onde estão listados os recursos do AVEA, \nos tipos de mídias digitais de que os docentes se beneficiaram, bem como o \nobjetivo de cada um destes instrumentos. Página \| 39 Página \| 39 Quadro 1 - Recursos do AVEA e mídias digitais utilizadas \nRecurso \nObjetivos \nFerrament\nas do \nAVEA \nFórum \n‐ Promover a comunicação; \n‐ Realizar postagem de atividades; \n‐ Proporcionar discussões acerca dos temas estudados. E-mail \n‐ Promover a comunicação. Livro \n‐ Elaborar roteiros de estudos. Rótulo \n‐ Orientar os estudantes quanto às atividades a serem \ndesenvolvidas. Mídias \nDigitais \nHipertexto \n(Roteiros de \nEstudo) \n‐ Guiar o estudante por meio dos diversos tipos de \nmídia previamente selecionados pelos docentes. Textos \ninformativos \n‐ Servir de material base para os estudos individuais. Infográficos \n‐ Apresentar informações e dados de forma visual, com \no objetivo de ampliar a compreensão do tema em \nestudo. Vídeos \n‐ Servir de material base para os estudos individuais; \n‐ Estimular a reflexão sobre o tema em estudo; \n‐ Buscar indícios do aprendizado dos estudantes. Animação \ninterativa \n‐ Aproximar o estudante de uma realidade fictícia, \npossibilitando-lhe interagir e observar os efeitos de \nsuas ações no ambiente. Jogo (SimCity) \n‐ Aproximar o estudante de uma realidade fictícia, \npossibilitando-lhe interagir e observar os efeitos de \nsuas ações no ambiente; \n‐ Estimular a reflexão sobre as verdades, mentiras e \nadaptações da realidade promovida nos jogos digitais. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. ABORDAGEM TEÓRICA (Fonte: Elaborado pelos autores com base em (MORAN, 1995; MACIEL et al, 2012; \nCUNHA; PAIVA, 2003; TRINDADE; REHFELDT; MARCHI, 2016; MÓDOLO, 2007; ALVES, \n2008; FIALHO, 2008)) Quadro 1 - Recursos do AVEA e mídias digitais utilizadas (Fonte: Elaborado pelos autores com base em (MORAN, 1995; MACIEL et al, 2012; \nCUNHA; PAIVA, 2003; TRINDADE; REHFELDT; MARCHI, 2016; MÓDOLO, 2007; ALVES, \n2008; FIALHO, 2008)) (Fonte: Elaborado pelos autores com base em (MORAN, 1995; MACIEL et al, 2012; \nCUNHA; PAIVA, 2003; TRINDADE; REHFELDT; MARCHI, 2016; MÓDOLO, 2007; ALVES, \n2008; FIALHO, 2008)) Dentre as ferramentas expostas, a mais utilizada pelos docentes e discentes \nfoi o fórum. Segundo Cunha e Paiva (2003), os fóruns facilitam a integração dos \nparticipantes e o hábito do trabalho aberto e exposto, oportunizando ao professor \numa visão geral dos interesses e empenho dos alunos, bem como a evolução da \naprendizagem. Maciel et al. (2012) corroboram destacando que os fóruns são \nespaços que possibilitam a discussão e a troca de ideias, favorecendo, assim, a \nconstrução coletiva do conhecimento. Outro recurso amplamente utilizado pelos docentes, tanto em contextos de \nensino quanto de aprendizagem, foi o vídeo. Isso se deve ao fato de que os vídeos \nsão úteis para despertar o interesse e a curiosidade dos alunos, uma vez que, de acordo com Moran (1995), utilizá-los é partir do visível, tornando o assunto \nabordado mais próximo dos discentes. Trindade, Rehfeldt e Marchi (2016) \ncomplementam esta ideia, destacando que os vídeos “normalmente resumem os \nconteúdos de forma prática e divertida, auxiliando a motivação acerca do tema” \n(Ibid, 2016, p. 4). Além dos vídeos, os docentes utilizaram animações para romper com a \nlinearidade com que os conteúdos foram abordados e instigar nos alunos uma \nmaior predisposição para a aprendizagem. Para Kenski (2015), a imagem, o som e \no movimento oferecem informações mais realistas ao sujeito que está sendo \nensinado, contribuindo para o aprendizado e aprofundamento do conteúdo \nestudado. Outra mídia digital inserida nos materiais disponíveis no AVEA foi o infográfico, \nque, de acordo com Módolo (2007), é um termo derivado da expressão em inglês \ninformational graphics, que significa gráficos informativos. Segundo a autora, esta \né uma mídia que alia texto e imagem com o intuito de passar uma mensagem \nvisualmente atrativa para o leitor. ABORDAGEM TEÓRICA A autora destaca, ainda, que os infográficos são \nrecursos de comunicação que transmitem sua mensagem por meio de elementos \nvisuais juntamente com o texto verbal reduzido, além de serem a porta de entrada \npara outros textos, já que estão no primeiro nível de leitura. Em complemento aos recursos e mídias digitais disponibilizados no AVEA, os \ndocentes utilizaram o jogo comercial SimCity, um simulador em que o jogador \nconstrói e administra uma cidade. Os pesquisadores decidiram por utilizar este \njogo em virtude da possibilidade de os estudantes poderem construir uma cidade \nonde houvesse uma usina nuclear. Adiciona-se a isso o fato de o jogo ser atrativo \ne familiar aos estudantes inseridos na turma em que a prática foi realizada. Além \nde ratificar esta afirmação, Alves (2008) defende que, quando explorado de \nmaneira correta, o jogo comercial pode ser uma ferramenta pedagógica que \nproduz resultados significativos. Outro autor que defende a utilização de jogos comerciais em contextos de \nensino e de aprendizagem é Fialho (2008), cujo trabalho destaca que os jogos são \nimportantes porque “promovem situações de ensino-aprendizagem e aumentam \na construção do conhecimento, introduzindo atividades lúdicas e prazerosas, \ndesenvolvendo a capacidade de iniciação e ação ativa e motivadora” (Ibid, 2008, \np. 12299). Para Alves (2008), os jogos possibilitam ao jogador “criar caminhos e \ncenários inexistentes, utilizando os conteúdos que permeiam o seu imaginário \npara construir narrativas que não estão definidas, imergindo em um universo de \nhistórias” (ALVES, 2008, p. 5). Neste momento, com base no referencial teórico exposto, cabe apresentar o \ndetalhamento da metodologia utilizada pelos docentes para o desenvolvimento \ndas atividades. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. METODOLOGIA UTILIZADA Como forma de fundamentar a metodologia utilizada no presente trabalho, os \npesquisadores utilizaram como referencial terórico a obra de Silveira e Córdova \n(2009), na qual realizam uma revisão de literatura quanto às metodologias de \npesquisa existentes. No entendimento dos pesquisadores, estes autores fornecem \nsubsídios o suficiente para que este trabalho seja caracterizado como uma pesquisa de cunho qualitativo. Para Silveira e Córdova (2009), em uma pesquisa \nqualitativa o foco não é a representatividade numérica, e sim a análise dos \naspectos da realidade, buscando a compreensão e a explicação dos fatos. Além \ndisso, os pesquisadores optaram por trazer, neste trabalho, os resultados e \ndiscussões como um relato de experiência. Como instrumentos de coletas de dados, os pesquisadores utilizaram um \ndiário de bordo compartilhado entre os docentes e os dados gerados pelos \nestudantes por meio da iteração com os conteúdos e atividades disponibilizados \nno Ambiente Virtual de Ensino e de Aprendizagem. A pesquisa foi realizada na \ndisciplina de Química, com os alunos do 1º ano do Ensino Médio (EM), no Colégio \nCenecista Mário Quintana, localizado no município de Encantado - RS. A turma é \nformada por 17 alunos, com idades entre 15 e 16 anos. As atividades desenvolvidas com a utilização do AVEA foram extraclasse e os \nconteúdos abordados ocorreram a distância. O tema explorado no decorrer da \natividade foi Radioatividade, sendo disponibilizado aos alunos um tópico semanal, \no que totalizou cinco encontros. Antes de iniciar as atividades semanais, os \ndocentes reuniram os alunos no auditório da escola para uma breve explanação \nquanto aos objetivos e atividades propostas. Aproveitou-se o momento para \ndemonstrar como acessar o AVEA e realizar estas atividades. Já o primeiro encontro a distância, “Papo inicial sobre Radioatividade”, teve \ncomo objetivo compreender porque alguns átomos são radioativos e diferenciar \nos tipos de radiação. Para tanto, disponibilizou-se o roteiro de estudos “Átomos, \nisótopos e radioatividade”, elaborado por Kare Kullerud, da Universidade de \nTronso, Noruega. Em seguida, foram solicitadas aos alunos a realização de uma \npesquisa sobre meia vida e decaimento radioativo e a postagem de um resumo \nsobre o que compreenderam em um fórum de perguntas e respostas. Ainda neste \ntópico, foi disponibilizada, como material complementar, uma animação do \nLaboratório Didático Virtual2 (LabVir) sobre Raio X, que tem por finalidade explicar \no funcionamento do Raio X e, de maneira geral, dos demais tipos de radiação. Ao \nfinal da animação, os alunos deveriam responder a cinco questões sobre o assunto \nestudado. METODOLOGIA UTILIZADA A Figura 1 apresenta a organização do AVEA utilizada pelos docentes no \nprimeiro encontro. nol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. Figura 1 - Organização dos materiais do encontro 1 no AVEA \n \n(Fonte: Elaborado pelos autores) Figura 1 - Organização dos materiais do encontro 1 no AVEA Página \| 42 (Fonte: Elaborado pelos autores) (Fonte: Elaborado pelos autores) R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. O segundo encontro, “Níveis de radiação e seus efeitos”, teve como objetivo \nanalisar os níveis de radiação aos quais estamos submetidos e seus efeitos no \norganismo. Para atender a este objetivo, os docentes criaram dentro do Ambiente \nVirtual um roteiro de estudos formado por mídias digitais extraídas da internet e \num vídeo elaborado pelos autores. Na primeira seção do roteiro, foi \ndisponibilizado aos alunos o vídeo “Os locais mais radioativos da Terra”, que \nexplana sobre os níveis de radiação a que estamos expostos diariamente, assim \ncomo a radiação em diversos lugares da Terra. Na sequência, a seção 2 apresentou \num infográfico3 que busca quantificar os níveis de radiação a que estamos \nsubmetidos. Na seção 3, outro infográfico4 foi utilizado para demonstrar os efeitos \ncausados por cada nível de radiação no organismo. Para finalizar o roteiro de \nestudos, os docentes disponibilizaram um vídeo por eles gravado que abria a \ndiscussão sobre uma reportagem5 realizada na cidade de Encantado, pela RBS TV. A notícia abordava medições, aferidas no município, de radioatividade, cujos \nvalores encontrados seriam considerados anormais. Neste segundo encontro, após realizar o roteiro de estudos, os alunos foram \nconvidados a discutir, em um fórum geral, os níveis de radiação encontrados na \ncidade de Encantado, elaborando uma síntese comparativa entre os dados \ndisponibilizados no infográfico com as informações apresentadas na reportagem. Foi solicitado, também, que os discentes elencassem os tipos de radiação aos quais \nestão submetidos, seus efeitos no organismo e quais as prováveis fontes de \nradiação encontradas na cidade. Já o terceiro encontro, “Simcity e um desastre nuclear”, teve por objetivo \nobservar os impactos causados por um desastre nuclear com o auxílio do jogo \nSimcity 6, que é um simulador de cidades no qual os estudantes desempenharam a \nfunção de prefeito. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. METODOLOGIA UTILIZADA A atividade deste encontro consistia em construir uma cidade \ncom uma usina nuclear, conduzindo-a a entrar em colapso para, então, observar \nos efeitos da radiação emitidos na cidade em diferentes situações, como na \nenergia, água, saúde da população, ambiente. Para os discentes que não possuíam \no jogo, foi disponibilizado um gameplay7 do jogo com uma situação semelhante a \nque deveriam ocasionar. Na sequência, os alunos elaboraram individualmente um \nrelato dos impactos observados, procurando refletir se, em uma situação real, os \nefeitos seriam os mesmos. O relatório foi postado na “Tarefa”, ferramenta \ndisponível no AVEA. A Figura 2 apresenta duas capturas de tela do gameplay \ndisponibilizado aos alunos, sendo, ao lado esquerdo, a cidade em modo normal de \njogo e, à direita, no modo medidor de poluição radioativa. Figura 2 - Cidade do SimCity cujo reator nuclear entrou em colapso \n \n(Fonte: Elaborado pelos autores por meio de capturas de tela do vídeo \nhttps://youtu.be/PHh2EmMTYvw) Figura 2 - Cidade do SimCity cujo reator nuclear entrou em colapso (Fonte: Elaborado pelos autores por meio de capturas de tela do vídeo \nhttps://youtu.be/PHh2EmMTYvw) Página \| 43 Página \| 43 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. Dando continuidade às atividades, o quarto encontro, denominado “Energia \nNuclear”, teve como objetivo demonstrar a importância e os possíveis riscos da \nenergia nuclear. Para tanto, foi disponibilizado aos alunos um roteiro de estudos \ncontendo três vídeos e um infográfico. O primeiro vídeo, intitulado “Ciclo do \nUrânio”, produzido pela Indústria Nuclear do Brasil (INB), relata o ciclo do \ncombustível nuclear, ou seja, como o urânio contido em um minério transforma-\nse em energia. Trata-se de uma animação de aproximadamente cinco minutos no \nqual a INB percorre todo o processo de tratamento do urânio, desde sua extração \naté a produção de energia elétrica. Os outros dois vídeos desta unidade se intitulam “A vida no entorno da mina \nde Urânio”, mina está localizada na cidade de Caetité, na Bahia. Os vídeos \nmencionados demonstram os diferentes pontos de vista sobre a exploração da \nmina: a dos empreendedores, que ressaltam as melhorias realizadas na \ncomunidade e o crescimento econômico da cidade, e a dos moradores do entorno \nda mina, que destacam os impactos ambientais aos quais foram submetidos com \na extração do Urânio. Em complemento aos vídeos, o infográfico apresentado neste encontro \ndestaca o caminho dos resíduos radioativos, desde a sua mineração até a produção \nde energia. METODOLOGIA UTILIZADA Ao final do roteiro de estudos, os alunos elaboraram um resumo \ndiscutindo a importância da energia nuclear, os desastres que já ocorreram e quais \nseriam as maneiras para evitar futuras catástrofes. A fim de finalizar as atividades propostas para o estudo sobre radioatividade, \nno quinto encontro, os alunos formaram pequenos grupos e tiveram como \natividade elaborar um vídeo em que foram convidados a demonstrar o que \naprenderam sobre a temática em estudo. O estilo de vídeo ficou à critério de cada \ngrupo. Posto isso, cabe apresentar os resultados e discussões resultantes da prática \npedagógica exposta. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. RESULTADOS E DISCUSSÕES Os ambientes virtuais de aprendizagem são espaços fecundos e estratégicos, \nnos quais tem-se a possibilidade de desenvolver a postura autônoma do aluno. No \nentanto, Messa (2010) destaca que a qualidade do processo educativo depende \ndo envolvimento do aprendiz. Nesse sentido, procurou-se observar como os \ndiscentes se envolveram e participaram das atividades propostas. Além disso, cabe \nesclarecer que, nesta pesquisa, não se buscou desvelar a contribuição individual \nde cada mídia digital utilizada e, sim, a forma como os estudantes interagiram com \no conjunto de mídias e como reagiram quando expostos ao processo de ensino a \ndistância. Posto isso, quanto à participação dos alunos nos fóruns, os docentes \nobservaram diferentes tipos de interação. Inicialmente, por não estarem \nhabituados com propostas de trabalho deste cunho, a participação e o cuidado \ncom as datas estabelecidas ficou a desejar, porém, no decorrer das semanas, o \nenvolvimento e empenho aumentou. No fórum de perguntas e respostas, no qual \nos estudantes precisavam fazer uma postagem antes de ter acesso aos \ncomentários dos colegas, os alunos não interagiram entre si. No entanto, todos \npostaram o resumo solicitado e complementaram com gráficos e outras sugestões de leituras para o aprofundamento da temática. Já nos fóruns gerais, que são \nespaços onde é possível trocar mensagens livremente, os alunos demonstraram \nmaior envolvimento e interagiram com mais frequência, contribuindo com \ncolocações, sugestões e opiniões. Vale ressaltar que os fóruns estavam sendo \nmonitorados, e, sempre que era pertinente, os docentes realizaram \nquestionamentos, convidando-os, assim, para debaterem novas ideias. Ainda quanto aos fóruns, um, em particular, chamou a atenção dos docentes. O fórum relativo à discussão sobre os níveis de radiação encontrados na cidade \nonde vivem os estudantes foi o que mais recebeu postagens, 28 no total. Este \nnúmero representa quase 50% das postagens realizadas durante o período no qual \na prática foi realizada. Para os docentes, existem dois fatores que podem ter \ninfluenciado para que houvesse este número expressivo de mensagens. Primeiro, \no vídeo que provocou a discussão foi o único produzido pelos docentes abordando \no conteúdo em estudo. Segundo, o fato de os professores terem utilizado como \ncenário de discussão a cidade na qual tanto docentes quanto discentes residem. Além disso, neste fórum em específico, alguns estudantes reagiram a uma \npostagem na qual uma aluna utilizou erroneamente um conceito. Em suas \npostagens, os colegas a auxiliaram a compreender o porquê de sua colocação ser \nproblemática, contribuindo para o aprendizado da aluna. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. RESULTADOS E DISCUSSÕES Para os docentes \nenvolvidos, estas mensagens evidenciam quão ricas são as interações realizadas \nentre os estudantes por meio da ferramenta Fórum do Ambiente Virtual e \nconfirmam o pensamento de Maciel et al. (2012) e Cunha e Paiva (2003) sobre este \nrecurso. Mesmo com o sucesso apresentado durante as discussões nos fóruns, foi a \natividade envolvendo o jogo que mais mobilizou e envolveu os alunos. Como a \natividade foi extraclasse e a distância, a docente titular da turma utilizava as aulas \npresenciais para abordar outros conteúdos, ainda assim, foi perceptível o \nenvolvimento e discussões dos alunos. No decorrer da semana, observou-se uma \natenção especial a esta atividade, uma vez que o assunto em sala de aula girou em \ntorno do nível em que se encontravam e dos avanços que apresentavam. Este fato \nestá em consonância com a colocação de Fialho (2008), na qual destaca que, ao \njogar, o indivíduo se depara com a vontade de vencer, provocando assim uma \nsensação agradável, visto que os desafios são situações que mexem com o \nemocional das pessoas. No entanto, a tarefa na qual deveriam elaborar um \nrelatório sobre os impactos observados durante a interação com o jogo teve baixa \nadesão, dado que poucos alunos postaram a atividade solicitada. Quando \nindagados sobre o motivo da não postagem, a resposta foi o acúmulo de atividades \nescolares próximas ao período de entrega do relatório. Ainda assim, mesmo com \na prorrogação do prazo, poucos estudantes finalizaram a tarefa. Já a elaboração do vídeo, no qual deveriam explanar alguns dos assuntos \nabordados durante o período de estudos, evidenciou a preferência dos alunos por \natividades envolvendo a produção de vídeos. A criação deste material possibilitou \naos discentes trabalharem de forma autônoma, uma vez que, de acordo com Demo \n(2016, texto digital), o que interessa “é como o estudante faz o vídeo, tornando-se \nautor do conteúdo, não seu consumidor”. Além disso, os docentes surpreenderam-\nse com a qualidade do material elaborado pelos estudantes e com suas habilidades \nde interpretação e intimidade com a tecnologia e formato de mídia utilizado. Este \nresultado corrobora com a tese defendida por Prensky (2001, p. 1) de que as novas \ngerações são fluentes na “linguagem digital dos computadores, vídeo games e internet”. Posto isso, os professores sugerem aos leitores deste trabalho que \nassistam a um dos vídeos produzidos pelos estudantes, cujo link está disponível na \nFigura 3. RESULTADOS E DISCUSSÕES Figura 3- Estudante interpretando um aluno que está coletando informações para seu \ntrabalho de conclusão de curso \n(Fonte: Elaborado pelos autores por meio de captura de tela do vídeo \nhttps://youtu.be/OYJ2-6--qB8) Figura 3- Estudante interpretando um aluno que está coletando informações para seu \ntrabalho de conclusão de curso Figura 3- Estudante interpretando um aluno que está coletando informações para seu \ntrabalho de conclusão de curso (Fonte: Elaborado pelos autores por meio de captura de tela do vídeo \nhttps://youtu.be/OYJ2-6--qB8) R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. CONSIDERAÇÕES FINAIS Diante dos resultados obtidos, os docentes possuem indícios para acreditar \nque a prática realizada atingiu seu objetivo, que foi desvelar os efeitos de \ndiferentes mídias digitais por meio de um Ambiente Virtual de Ensino e \nAprendizagem, com discentes do 1º ano do Ensino Médio, na disciplina de \nQuímica. Quanto à expectativa dos docentes, esta foi superada. O uso da educação \na distância, com o suporte do Ambiente Virtual de Ensino e Aprendizagem, \nassociado ao uso de um leque variado de mídias digitais, possibilitou aos \nestudantes experienciarem uma prática diferente da qual estão acostumados. Ainda assim, adequaram-se ao formato proposto e responderam bem à grande \nmaioria das atividades propostas. Existem dois pontos a serem considerados que contribuíram para o sucesso \nobtido nesta empreitada. O primeiro deles é o fato de que os docentes, além de \ntrabalharem de forma colaborativa, tiveram tempo para planejar, discutir e refletir \nsobre todas as etapas que a prática pedagógica seguiria. O segundo é que as \nbarreiras quanto ao uso das tecnologias digitais descritas por Prensky (2001) \npuderam ser superadas em virtude de ambos os docentes estarem desenvolvendo \npesquisas relativas ao uso de tecnologias em âmbito educacional, sendo um deles \nprofissional da área tecnológica como experiência em educação a distância e o \noutro, uma professora que constantemente atualiza suas práticas, buscando \ninserir as mídias digitais em contextos de ensino e de aprendizagem. Como nem tudo é perfeito, surgiram também alguns pontos para reflexão dos \ndocentes. O primeiro está relacionado ao uso da ferramenta fórum configurada \npara perguntas e respostas. Este modelo de fórum produziu diversas respostas semelhantes dos alunos e pouca interação entre eles. Com isso, seu \nfuncionamento aproximou-se muito mais de um questionário do que o de um \nfórum, uma vez que cada estudante apenas depositou suas respostas, sem \nreflexão, discussão ou questionamentos quanto às respostas dos demais colegas. O segundo ponto que cabe reflexão é o fato de que, mesmo havendo um grande \nenvolvimento dos alunos na atividade relacionada ao jogo SimCity, poucos alunos \nconcluíram o relatório solicitado ao final da proposta. Em relação a isso, ficam \nalguns questionamentos: mesmo que os alunos não tenham escrito o relatório \nsolicitado, eles fizeram a reflexão proposta pela atividade ou apenas utilizaram o \njogo como forma de entretenimento? Será que a escolha do relatório como \nresultado final da atividade foi a mais adequada? R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. CONSIDERAÇÕES FINAIS Um terceiro ponto para reflexão \nsurgiu em virtude de a atividade relativa ao jogo ter sido planejada de modo que \nos pais/responsáveis dos alunos pudessem participar e envolverem-se na \nelaboração do relatório, o que, de fato, não ocorreu. Quanto a isso, cabe salientar \nque os docentes entraram em contato com os familiares dos estudantes e os \nconvidaram para participar da atividade, tendo, alguns, acenado positivamente. Além disso, os docentes gravaram um vídeo explicando como a atividade ocorreria \ne encaminharam aos pais que, inicialmente, haviam aceitado participar. Diante \ndisso, adiciona-se uma nova questão para reflexão quanto ao ponto anterior: será \nque o não envolvimento dos pais fez com que os estudantes desistissem de \nentregar o relatório? Como contribuição para os estudos na área de ensino, os autores destacam \nque existem indicativos nesta investigação para afirmar que a educação a \ndistância, associada a um Ambiente Virtual de Ensino e de Aprendizagem e às \nmídias digitais, pode ser utilizada como um recurso que permite ao docente \nexperimentar práticas pedagógicas que extrapolem o espaço físico da sala de aula \nou da escola. Neste sentido, é possível citar como indícios a adesão dos estudantes \nao fórum de discussão relacionado ao vídeo produzido pelos docentes e a \nprodução do vídeo autoral por parte dos alunos. Neste último, em especial, fica \nevidente que os estudantes se apropriaram dos conceitos trabalhados durante os \nencontros a distância. Por fim, os autores gostariam de destacar que o uso de mídias digitais e dos \ndiversos tipos de tecnologias em sala de aula pode colaborar para que o ensino de \nalgumas matérias, como o da química, seja menos tedioso, além de contribuir para \nque o aprendizado seja efetivamente significativo. Assim, cabe aos docentes que \nutilizam estes recursos o papel de incentivar e estimular outros professores a \nusufruírem de tecnologias e mídias digitais em sala de aula, divulgando suas \nexperiências, compartilhando resultados e, se necessário, auxiliando no \nplanejamento e execução de práticas empíricas, como a relatada neste trabalho. Página \| 47 Página \| 47 KEYWORDS: Experience report. Pedagogical practice. Distance learning. Virtual learning \nenvironment. Digital media. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. ABSTRACT The present text is a experience report witch had the objective of making possible to high \nschool students approach the study of radiation using an Learning Management System. The teachers organized the pedagogical practice in six meetings, one presential and the \nfollowings remotely. The teachers made use of digital medias as: animations, videos, text, \ninfographics and the comercial game SimCity, as well as a internal comunication and \nintegration tool and the discussion forum present in the LMS. This resource made possible \nthe integration of the students, providing rich and heated discussions. However, SimCity \ncaused the most engagement in the group. As the evaluation method, the teachers \nrequested the students to form groups and produce a video, presenting what they learned \nabout the subject. The quality of the content and production presented by the students \nsurprised the teachers and made evident the preference on this kind of activity. The \nteachers belive that the strategic use of LMS and digital medias together with activities that \nmake possible the students to be authors of their own discussions, reflections and videos \nhelped in the development of a more proactive position and a bigger autonomy on their \nstudies. Página \| 48 Página \| 48 NOTAS 1 Centro Regional de Estudos sobre o Desenvolvimento da Sociedade da \nInformação (Cetic.br) do Núcleo de Informação e Coordenação do Ponto BR \n(NIC.br) - http://cetic.br/ 2 Segundo Leite (2016), no LabVir ou Laboratório Didático Virtual, encontram-se \nvárias páginas com material didático, bem como questões elaboradas por \nespecialistas da área. Um dos objetivos do laboratório é incentivar o pensamento \ne uso do método científico, além de estimular o gosto pela ciência. O LabVir pode \nser acessado pelo link: <www.labvirt.fe.usp.br> 3 \nO \ninfográfico \nutilizado \nfoi \nextraído \ndo \nsite \n<http://n.i.uol.com.br/ultnot/1103/radioativo.jpg> \n4 \nO \ninfográfico \nutilizado \nfoi \nextraído \ndo \nsite \n<https://i2.wp.com/diariodebiologia.com/wp-\ncontent/uploads/sites/4/2015/09/radiac3a7c3a3o-no-\norganismo.jpg?resize=635%2C478&ssl=1> 3 \nO \ninfográfico \nutilizado \nfoi \nextraído \ndo \nsite \n<http://n.i.uol.com.br/ultnot/1103/radioativo.jpg> 4 \nO \ninfográfico \nutilizado \nfoi \nextraído \ndo \nsite \n<https://i2.wp.com/diariodebiologia.com/wp-\ncontent/uploads/sites/4/2015/09/radiac3a7c3a3o-no-\norganismo.jpg?resize=635%2C478&ssl=1> 5 A reportagem da RBS TV sobre a radiação encontrada na cidade de Encantado – \nRS está disponível em: <https://www.youtube.com/watch?v=mNVLoiyXgXI> 6 O Simcity é um jogo no qual o objetivo é que o jogador crie e gerencie recursos \nde uma cidade virtual. Uma de suas características é não possuir um fim, podendo \nser jogado por tempo indeterminado. Trata-se de um jogo comercial produzido \npela empresa Eletronic Arts e está disponível para a compra no site dos \ndesenvolvedores. ABSTRACT 7 Gameplay é um formato de vídeo no qual os jogadores gravam e comentam suas \nações enquanto jogam e, posteriormente, os publicam na Internet. O objetivo \ndestes vídeos é aproximar o público que compartilha o gosto por jogos digitais. Além disso, uma estratégia dos desenvolvedores de jogos é liberar seus últimos \nlançamentos para os produtores deste tipo de conteúdo, fazendo com que outras \npessoas conheçam seus jogos e os comprem. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. REFERÊNCIAS ALVES, Lynn. Relações entre os jogos digitais e aprendizagem: delineando \npercurso. Educação, Formação & Tecnologias-ISSN 1646-933X, v. 1, n. 2, p. [3-\n10], 2008. Disponível em: ALVES, Lynn. Relações entre os jogos digitais e aprendizagem: delineando \npercurso. Educação, Formação & Tecnologias-ISSN 1646-933X, v. 1, n. 2, p. [3-\n10], 2008. Disponível em: <http://eft.educom.pt/index.php/eft/article/view/58/38>. Acesso em: 20 mai. 2017. CETIC.BR (Brasil). TIC Educação. 2016. Disponível em: CETIC.BR (Brasil). TIC Educação. 2016. Disponível em: <http://cetic.br/pesquisa/educacao/>. Acesso em: 02 jan. 2017. <http://cetic.br/pesquisa/educacao/>. Acesso em: 02 jan. 2017. CUNHA, Fernando; PAIVA, João. A utilização de fóruns em contexto de \nEnsino/aprendizagem. In: Actas da III Conferência Internacional sobre \nTecnologias de Informação e Comunicação na Educação. Braga: Portuga Página \| 49 Página \| 49 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. FIALHO, Neusa Nogueira. Os jogos pedagógicos como ferramentas de ensino. In: \nCongresso Nacional de Educação. 2008. p. 12298-12306. Disponível em: \nhttp://www.pucpr.br/eventos/educere/educere2008/anais/pdf/293_114.pdf> \nAcesso em: 24 mai. 2017. ______. CAETITE/BA 2014 - INB. Disponível em: \n<https://www.youtube.com/watch?v=zCofeK_zS64> . Acesso em: 25 nov. 2016. KENSKI, Vani Moreira. Educação e tecnologias: o novo ritmo da informática. Campinas, SP: Papirus, 2015. E-book. KENSKI, Vani Moreira. Educação e tecnologias: o novo ritmo da informática. Campinas, SP: Papirus, 2015. E-book. ULLERUD,Kare. Átomos, isótopos e radioatividade. Universidade do Tromso, \noruega. Disponível em: LAMES, Liliane da Costa Jacobs. Docência no ensino superior: o uso das mídias \ndigitais como estratégia pedagógica. 2011. 159 f. Dissertação (Mestrado) - Curso \nde Mestrado em Ciências Contábeis, 1 Fundação Escola de Comércio Álvares \nPenteado – Fecap, São Paulo, 2011. Disponível em: <http://tede.fecap.br:8080/jspui/bitstream/tede/494/1/Liliane_da_Costa_Jacobs\n_Lames.pdf>. Acesso em: 15 mai. 2017. LEITE, Bruno Silva. Discussões sobre Ambientes Pessoais de Aprendizagem. Educaonline, Rio de Janeiro, v. 10, n. 1, p.37-54, jan./abr. 2016. Quadrimestral. Disponível em: <http://www.latec.ufrj.br/revistas/index.php?journal=educaonline&page=article\n&op=view&path;[]=809&path;[]=738>. Acesso em: 05 mai. 2017. MACIEL, Cristiano et al (Org.). Ambientes Virtuais de Aprendizagem. Cuiabá: \nEdufmt, 2012. 262 p. Disponível em: \n<https://edisciplinas.usp.br/pluginfile.php/129865/mod_resource/content/1/Am\nbientes%20Virtuais.pdf>. Acesso em: 18 mai. 2017. MESSA, Wilmara Cruz. Utilização de ambientes virtuais de aprendizagem-AVAs: a \nbusca por uma aprendizagem significativa. Revista brasileira de aprendizagem \naberta e a distância, v. 9, p. 8, 2010. Disponível em: \n<http://www.abed.org.br/revistacientifica/Revista_PDF_Doc/2010/2010_246201\n0174147.pdf>. Acesso em: 25 mai. 2017. MACIEL, Cristiano et al (Org.). Ambientes Virtuais de Aprendizagem. Cuiabá: \nEdufmt, 2012. 262 p. Disponível em: Página \| 50 Página \| 50 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. MESSA, Wilmara Cruz. Utilização de ambientes virtuais de aprendizagem-AVAs: a \nbusca por uma aprendizagem significativa. Revista brasileira de aprendizagem \naberta e a distância, v. 9, p. 8, 2010. Disponível em: \n<http://www.abed.org.br/revistacientifica/Revista_PDF_Doc/2010/2010_246201\n0174147.pdf>. Acesso em: 25 mai. 2017. MESSA, Wilmara Cruz. Utilização de ambientes virtuais de aprendizagem-AVAs: a \nbusca por uma aprendizagem significativa. Revista brasileira de aprendizagem \naberta e a distância, v. 9, p. 8, 2010. Disponível em: MÓDOLO, Cristiane Machado. Infográficos: características, conceitos e princípios \nbásicos. In: XII Congresso Brasileiro de Ciências da Comunicação da Região \nSudeste. 2007. Disponível em: MORAN, José Manuel. O vídeo na sala de aula. Comunicação & Educação, n. 2, p. 27-35, 1995. Disponível em: PRENSKY, Marc. Digital Natives, Digital Immigrants. On the Horizon, v. 9, n. 5, p. 1-6, 2001. Disponível em: <http://www.marcprensky.com/writing/Prensky%20-\n%20Digital%20Natives,%20Digital%20Immigrants%20-%20Part1.pdf>. Acesso em: \n18 mai. 2017. SAEED, Nauman. Integration and acceptance of Web 2.0 technologies in higher \neducation. 2010. 200 f. Tese (Doctor of Philosophy) - Melbourne, Swinburne \nUniversity of Technology, 2010. Disponível em: \n<http://researchbank.swinburne.edu.au/vital/access/manager/Repository/swin:1\n8535>. Acesso em: 17 out. 2016. SANTOS, Simone Andrade et al. Uso pedagógico do ambiente virtual de \naprendizagem Moodle como apoio a aula presencial. Revista EDaPECI, [S.l.], v. 16, n. 1, p. 78-94, abr. 2016. ISSN 2176-171X. Disponível em: \n<https://seer.ufs.br/index.php/edapeci/article/view/3897>. Acesso em: 20 mai. 2017. SILVEIRA, Denise Tolfo. CÓRDOVA, Fernanda Peixoto. Unidade 2 – A pesquisa \ncientífica. In: GERHARDT, Tatiana Engel. SILVEIRA, Denise Tolfo. Métodos de \nPesquisa. POA: Editora da UFRGS, 2009. Disponível em: <http://www.ufrgs.br/cursopgdr/downloadsSerie/derad005.pdf>. Acesso em: 23 \nmai. 2017. TRINDADE, Kelli Cristina Freitas; REHFELDT, Marcia Jussara Hepp; MARCHI, \nMiriam. Ambiente Virtual de Aprendizagem: repaginando antigas práticas \npedagógicas. Disponível em: <http://tecedu.pro.br/wp-\ncontent/uploads/2016/09/REL3-ano8-vol17-dez2016.pdf>. Acesso em: 20 mai\n2017. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019. TV Zabelê. INB: a vida no entorno da mina de urânio. Disponível em: \n<https://www.youtube.com/watch?v=69KhzmY-DY0>. Acesso em: 25 nov. 2016. Veritasium. Os lugares mais radioativos da Terra. Disponível em: \n<https://www.youtube.com/watch?v=TRL7o2kPqw0>. Acesso em: 25 nov. 2016. Recebido: 2017-11-06 \nAprovado: 2018-08-29 \nDOI: 10.3895/rbect.v12n2.7296 \nComo citar: SILVA, M. S.; ZOTTI, K. S.; REHFELDT, M. J. H.; MARCHI, M. I. TV Zabelê. INB: a vida no entorno da mina de urânio. Disponível em: \n<https://www.youtube.com/watch?v=69KhzmY-DY0>. Acesso em: 25 nov. 2016. \nVeritasium. Os lugares mais radioativos da Terra. Disponível em: \n<https://www.youtube.com/watch?v=TRL7o2kPqw0>. Acesso em: 25 nov. 2016. TV Zabelê. INB: a vida no entorno da mina de urânio. Disponível em: \nhtt\n//\nt b\n/\nt h?\n69Kh\nY DY0\nA\n25\n2016 MACIEL, Cristiano et al (Org.). Ambientes Virtuais de Aprendizagem. Cuiabá: \nEdufmt, 2012. 262 p. Disponível em: O uso de \nmídias digitais, associados ao ambiente virtual de ensino e de aprendizagem, no ensino \nde química: explorando a radioatividade por meio da educação a distância. Revista \nBrasileira de Ensino de Ciência e Tecnologia, v. 12, n. 2, 2019. Disponível em: \n<https://periodicos.utfpr.edu.br/rbect/article/view/7296>. Acesso em: xxx. Correspondência: Maurício Severo da Silva - mss@universo.univates.br \nDireito autoral: Este artigo está licenciado sob os termos da Licença Creative \nCommons-Atribuição 4.0 Internacional. Página \| 52 Página \| 52 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 37-52, mai./ago. 2019."
https://openalex.org/W2117215559	https://europepmc.org/articles/pmc3997970?pdf=render	English	null	Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases	Biotechnology Research International	2,014	cc-by	25,166	Research Article Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases Christian Bach,1 William Sherman,2 Jani Pallis,3 Prabir Patra,1 and Hassan Bajw 1 University of Bridgeport, Biomedical Engineering, 221 University Avenue, Bridgeport, CT 06604, USA 2 Physics Faculty, BHSEC Queens, 30-20 Thomson Avenue, Long Island City, NY 11101, USA 3 University of Bridgeport, Mechanical Engineering, 221 University Avenue, Bridgeport, CT 06604, USA 4University of Bridgeport, Electrical Engineering, 221 University Avenue, Bridgeport, CT 06604, USA Correspondence should be addressed to Christian Bach; cbach@bridgeport.edu Received 13 September 2013; Revised 2 January 2014; Accepted 2 January 2014; Published 6 April 2014 Academic Editor: Michael Hust Academic Editor: Michael Hust Copyright © 2014 Christian Bach 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. Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable tools to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger. Hindawi Publishing Corporation Biotechnology Research International Volume 2014, Article ID 970595, 27 pages http://dx.doi.org/10.1155/2014/970595 Hindawi Publishing Corporation Biotechnology Research International Volume 2014, Article ID 970595, 27 pages http://dx.doi.org/10.1155/2014/970595 Hindawi Publishing Corporation Biotechnology Research International Volume 2014, Article ID 970595, 27 pages http://dx.doi.org/10.1155/2014/970595 2. The Translational Case for Using SP1 to Design a ZFN with Low Side Effects for Sickle-Cell Anemia Zinc fingers describe a class of DNA binding proteins with a modular design [5, 14, 15] in which single fingers can be assembled to form multifinger arrangements and recognize any desired target sequence on the genome. Each individual finger binds preferentially to a specific DNA triplet “with defined three-base-specificity” [3, page 1]. Naturally occur- ring protein-binding domains typically contain three fingers that bind to a DNA-binding site of a 9-base pair long DNA sequence (9-mers). The modularity of the fingers lends itself naturally to a broad variety of bioengineering applications. Protein/DNA hybrid structures have applications, for exam- ple, in the fabrication of nanoscale functional assemblies [16]. Of course, their primary application is as a versatile tool for designing DNA binding proteins for any target sequence on the human genome [5, 14] for the purpose of gene regulation and genome modification.i g g g g This is due to the unprecedented advances in the biomed- ical sciences which provide the capability to induce the creation of stem cells from our own ordinary skin cells and then grow them in numbers to replace burned skin or entire organs. In the case of sickle-cell anemia [17], induced pluripo- tent stem (iPS) cells carrying the disease have been repaired by introducing a healthy HBB gene (Hb A) near the mutated location of the diseased gene (Hb S) (Figures 1 and 2). Cutting the HBB gene at the specific location GTGGAG (Figure 1) using a nuclease and introducing a healthy donor gene completes the correction. Nucleases are proteins with the enzymatic capability to cut the genome at any location. In order to introduce one specific cut at one location, the nuclease is guided by a bespoken zinc finger protein designed to bind to the specific DNA sequence next to the HbS mutation (Figure 2). gi Such designer zinc fingers have been successfully used in curing genetic diseases, for example, for curing sickle-cell anemia [17], in disrupting the HIV CCR5 gene, for example, [18–20], in advanced stem cell therapies, for example, [21, 22], cancer [23], and in other potential applications, for example, [24–26], as well as modifying plant and animal genomes [27, 28]. In addition, a fast growing number of translational applications and test assays in biotechnology are reported in, for example, [20, 29, 30]. 1. Introduction Exchangeability of small molecules, protein structures, DNA sequences, and entire functional units and systems denotes that the modularity principle is fundamentally used by nature to manage life in an uncomplicated manner. An example reported by [1] on gene regulatory regions shows exchange of four TFs and binding sequences to control activation and repression of genes in the same and between yeast species, in which nature did not change nucleotides and amino acids to develop new units and functions but con- served the TF structures and binding sites to exchange entire functional units [1, page 69, Figure 2]. Nature, therefore, operates via relocating functional units in the same organism and between species beyond the need of changing amino acids and nucleotides to adapt to evolutionary pressure. A strong argument for universal modularity is that it is a tool through which nature is primed to efficiently and effectively manage instant changes. This might lead to the assumption that nature had reasons to create and conserve the frameworks of zinc finger domains and use and reuse them over long evolutionary distances of time. To utilize particular inherent evolutionary traits may turn out to be Indications of intense complexity [1] of DNA recognition are manifested in many forms including observed diversity and equal functionality of secondary binding motifs [2], degeneracy, modularity and “overlap problem” [3–5], cyto- toxicity [6], high failure rate [7] and dependencies of context [8, 9], and condition [10] and DNA sequence deformability [9, 11]. Widespread use of zinc finger proteins in nature [2, 12], however, suggests that natural zinc finger domains have nature-given advantages and that those evolutionary traits should be replicated or reused to produce molecular tools such as zinc finger nucleases (ZFNs). Also to be considered is that single zinc fingers which contain 28–30 amino acids are simple structures with an unusual high degree of functional flexibility and structural malleability to bind distinctively any triplet depending on certain tissue condition, protein context, and form of DNA sequence. In addition, they are used in a naturally occurring setting by nature to fulfill a variety of dissimilar and exchangeable functions in the same or between organisms in a modular fashion [1]. 2. The Translational Case for Using SP1 to Design a ZFN with Low Side Effects for Sickle-Cell Anemia However in all these cases “off- target” binding is a problem with unacceptable side effects [14, 31] for which the goal of this study is to show potentially novel ways to significantly improve these emerging technologies by increasing accuracy of binding to a single target site and thus reducing side effects. Two nuclease domains are required at the same location but on opposite strands of the genome’s DNA sequence to form a dimer (Fok I nuclease domains in orange in Figure 2) that can induce a cut at both strands [6]. To get the two nuclease domains to the one desired location each domain is tethered to the binding domain of a zinc finger protein that specifically recognizes and attaches to its binding site, which is a nine-base pair long DNA string, for example, TCCTCAGTC in Figure 2. The hope of this strategy is that, through modular assembly of individual fingers, zinc finger nucleases can be created that specifically bind to one desired DNA sequence [3]. In the HBB example symbolized in Figure 2, the upper three-finger binding domain should recognize exclusively the binding site TCCTCAGTC (lower: GGCAGACTT) where each finger binds to one nucleotide triplet. The two three-finger DNA binding domains com- bined should have the unique quality of bringing the two nuclease domains together at only the one specific target site GGCAGACTT - - - - - - TCCTCAGTC.h gf From the literature it is clear that practical application of engineered zinc fingers in humans is severely limited due to cytotoxic side effects caused by “off-target” binding site activities leading to cell death and apoptosis [32]. To add to the challenge, recent findings indicate discrepancies and inconsistencies of results produced by various in vitro and in vivo assays [3, 7, 33]. This may be caused by evolutionary plasticity [34] in which the binding capabilities of single fingers vary significantly due to the high malleability of their 3-dimensional structure, which leads to changes in their binding preferences in various tissue conditions [35]. Because of “our limited understanding of even simple DNA This technique, called gene targeting, has been success- fully applied to cure sickle-cell anemia in a mouse model [17]. It has been suggested that statistically the two three- finger binding domains should enable the formation of the nuclease dimer only at the one desired location. 1. Introduction Biotechnology Research International 2 2 critical in the design of zinc finger domains to deal with the overwhelming complexity of DNA binding.f proteininteraction” [36, page 2500], limited knowledge of transcription factors (TF) functions [35, page 253], and lack of precise data to accurately predict binding recognition [37, 38], page 144, progress is slow to systematically translate brilliant therapies from, for instance, animal models [17] into clinical practice.h In an effort to reduce complexity and to develop solutions in a timely fashion, it might be realistic to use a natural zinc finger binding domain and exchange amino acids in the alpha helical region of one of the fingers to change the domains’ binding preferences [8, 13]. To test the feasibility of changing the rank-ordered binding preferences, the three- finger binding domain of SP1 is used to reduce complexity by focusing on exchanging amino acids in the alpha helical region of the 2nd finger. Therefore, to progress the science, it is critical to investi- gate the nature of “off-target” binding, to identify and elimi- nate the potential factors which prevent medical implemen- tation, and to gain insights from diverse sources for directing further research efforts and technological advances. These efforts will provide the means to create critical knowledge and technological breakthroughs with broad research and societal impact. This is especially true since today molecular biology enables us to modify the human genome to cure inherited genetic diseases and in the foreseeable future has the potential to replace damaged and aging tissues and organs.h 2. The Translational Case for Using SP1 to Design a ZFN with Low Side Effects for Sickle-Cell Anemia An exact match search on the NCBI-HuRef genome (National Center for Biotechnology Information) revealed that the 3 Biotechnology Research International 3 Figure 1: Normal HBB gene retrieved from NCBI website. Figure 1: Normal HBB gene retrieved from NCBI website. “off-target” binding is tied into the observation that zinc fingers typically bind degenerated motifs of hundreds of similar sequences [2] connoting that three-base specificity [3, page 1] does not signify that a single zinc finger only binds to one or few best triplets. Diseased HBB sequence in human hemoglobin (HbS): Fok I nuclease domain 9-mer binding site Normal HBB sequence in human hemoglobin (HbA): GTG GAG TCC TCA GTC GAG GAG TCC TCA GTC GGC AGA CTT CAC CTC GGC AGA CTT CTC CTC Three finger binding domain 2-finger 3-finger 1-finger 2-finger 3-finger 1-finger - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Figure 2: Mutated HBB diseased gene. Normal HbA target sequence versus single point mutation of diseased HbS gene and target sequence of a three-finger binding domain. In the last two decades, the binding specificity of hun- dreds of artificial and natural zinc fingers has been character- ized. Yet despite fast progress, little is known about even sim- ple DNA-protein interactions [36] and computational tools to design proteins and predict binding sites lack accuracy [37, 38, 42]. Accompanying large scale studies have shown an unmanageable diversity of DNA recognition [2] where the massive amounts of data on transcription factor domains and binding sites increased complexity to a point where more data contribute little to gain vital understanding of DNA-protein interactions. Figure 2: Mutated HBB diseased gene. Normal HbA target sequence versus single point mutation of diseased HbS gene and target sequence of a three-finger binding domain. At this point it might be rational to reduce complexity and bring it onto a manageable level by using an exemplary case that focuses on generating data about one finger to gain insight before further proceeding. 3. Material and Methods Expression of three-finger domain using plasmid pPacSpl- 516c is provided by Tjian’s lab and purified by FPLC Mono S chromatography [13]. The DNA binding capability of the 2nd finger of SP1 and mutants has been assessed by incu- bating the 64 (Tables 2 and 3) P32-labeled double-stranded oligonucleotides (Figure 4(a)) by performing electrophoresis mobility shift assays (EMSA). P32 counts of band shifts have been produced by Phosphor Imager Screening (Molecular Dynamics) [13, 43, 44]. 3.4. Electrophoretic Mobility Shift Assay (EMSA). Oligonu- cleotides for electrophoretic mobility shift assays (EMSA) were synthesized on 380A Applied Biosystems DNA Syn- thesizer. Proteins CB1, MR14, MQ91, MQ135, and MQ151 were incubated (15 ng) with 10 𝜇L labeled double-stranded oligonucleotide in a 30 𝜇L standard electrophoretic mobility shift assay (EMSA). The reaction mixture consisted of 10 𝜇L of 3x band shift buffer (15 mM NaCl, 150 mM KCl, 36 mM HEPES pH 7.9, 36% glycerol, and 5 mM MgCl, 300 𝜇M ZnCl), 6 𝜇L H20, 3 𝜇L DTT (10 mM), 10 𝜇L labeled oligonucleotides (10000–20000 Cerenkov cpm), and 1 𝜇L protein (15 ng). Proteins were diluted by addition of H2O. The band shift reactions were incubated for 30 min at R.T. and loaded onto a 6% polyacrylamide band shift gel (acrylamide/bisacrylamide 30% : 0.8%) containing 100 𝜇M ZnCl and 0.25x Tris-borate electrophoresis buffer (TBE). By performing EMSA analysis, the fragments of mutants present in plasmids were identified to bind to the majority of 64 possible triplets. The binding sites in the mutant plasmids were determined by dideoxy sequence analysis [13]. 3.1. Oligonucleotides. Oligonucleotides for site-directed mu- tagenesis and for electrophoretic mobility shift assays (EMSA) were synthesized on 380A Applied Biosystems DNA Synthesizer. The oligonucleotide 1892 used in EMSA contains one SP1 binding site TTGGGGCGGGGCTT sur- rounded by cassette sequences, which contain the appropriate primer annealing sites for primer A and primer B. For the EMSA analysis cassette, oligonucleotide 3028 was gener- ated (Table 2) resulting in 5󸀠GTCGGATCCTGTCTGAG- GTGAGTTGGGNNNGGGCTTGTCTTCCGACGTCGA- ATTCGCG3󸀠. Site-directed mutagenesis oligonucleotide 2744 (AAGTCGTCTGCCCTAATTAGTCACAAACGT- ACACACACAGGTGAGAAG) and oligonucleotide 2745 (GTGACTAATTAGGGCAGACGACTTTGTGAAGCG- TTTCCCACAGTATGA) were synthesized encoding lysine (K) at zinc finger position 15, serine (S) at position 17, alanine (A) at position 18, isoleucine (I) at position 20, and serine at position 21. The oligonucleotides 393 (GTAAAACGACGG- CCAGTG) and 392 (AAACAGCTA TGACCATG), which are universal primers of Bluescript plasmid (Stratagene), have been used together with the oligonucleotides 2744 and 2745 in PCR mutagenesis. 2. The Translational Case for Using SP1 to Design a ZFN with Low Side Effects for Sickle-Cell Anemia SP1, one of the most ubiquitous transcription factors, has been chosen with the intent to test which of the 64 putative triplets (Table 3) for its 2nd finger still allows the entire three-finger domain to form a DNA-protein complex. The focus on one finger and 64 triplets as a first step appears to be reasonably manageable and more productive than testing the 262,144 putative binding sites of the entire three-finger domain. TCCTCAGTC (AGGAGTCAG) sequence occurs 18,279 times and the GGCAGACTT sequence 8,676 times, whereas the GGCAGACTTGTGGAGAGGAGTCAG sequence was found exactly one time at the proper location in the HBB gene, which provides some rational that this approach might produce clinically feasible products. However, despite the fact that the target sequence occurs just one time, cytotoxicity is observed and attributed to the zinc finger nuclease’s ability to bind not only to the one desired target site but also to numerous “off-target” sites that induce deleterious genetic changes preventing cells from functioning properly and causing cell death and apoptosis. In addition, the lack of technologies to precisely control genome modifications hampers human application [6, 17, 32, 39–41]. Concomitant TCCTCAGTC (AGGAGTCAG) sequence occurs 18,279 times and the GGCAGACTT sequence 8,676 times, whereas the GGCAGACTTGTGGAGAGGAGTCAG sequence was found exactly one time at the proper location in the HBB gene, which provides some rational that this approach might produce clinically feasible products. However, despite the fact that the target sequence occurs just one time, cytotoxicity is observed and attributed to the zinc finger nuclease’s ability to bind not only to the one desired target site but also to numerous “off-target” sites that induce deleterious genetic changes preventing cells from functioning properly and causing cell death and apoptosis. In addition, the lack of technologies to precisely control genome modifications hampers human application [6, 17, 32, 39–41]. Concomitant Referring to Lam et al.’s report on general degeneracy, it can be realistically expected that the outcome should be fairly degenerated 64 three-base-specificity codes [3] that could provide guidance to develop concomitant core and sup- porting technologies to focus on further investigations and generate precise data on the mechanisms ruling the reversible 4 Biotechnology Research International Biotechnology Research International Biotechnology Research International mutants. Standard PCR conditions were applied. Introduced mutations were verified by dideoxy sequence analysis. mutants. Standard PCR conditions were applied. Introduced mutations were verified by dideoxy sequence analysis. formation and dissolution processes of a model DNA-protein complex. 3. Material and Methods Oligonucleotide 1956 (CAGCCCGGG- AGATCTGCCACCTGCA TGAC) introduces a BglII site at the 3󸀠end of the SP 1 fragment in pB-516c. 2. The Translational Case for Using SP1 to Design a ZFN with Low Side Effects for Sickle-Cell Anemia Among the many known and unknown factors we focus in this paper on selected factors with the highest proba- bility of having practical relevance to advancing translational research. 3.3. E. coli Expression. Mutated SPl protein was expressed and purified according to the procedure described for the analogous wild type SP1 protein. Mutated SP1 protein was diluted 1 : 10 in buffer A (8 M urea, 20 mM MES pH 5.0, and 2 mM EDTA), subjected to FPLC Mono S chromatog- raphy, and eluted with an increasing salt gradient of buffer B (1 M NaCl, 8 M urea, 20 mM MES pH 5.0, and 2 mM EDTA). Peak fractions were collected and analyzed together with recently purified SP1 on 15% polyacrylamide-SDS gel. Fractions containing the mutated SP1 protein were pooled. Protein concentrations were determined by the method of Bradford to be 0.5 mg/mL [43]. 4. Results and Discussion Remarkably, six binding patterns in Figure 4(a) with significant differences Biotechnology Research International 5 C C G I Q M K C C C C H G G G G G K K K K K K V Y K T T T T T T T I S S S H H H H H H H Zn L L L R R R R R R R Y S A A W E E E P P P W F F F F D Zn Zn M SP1-binding domain (three fingers) R R H K S D E L Q Figure 3: Amino acid sequence and structure of the SP1 binding domain. SP1-binding domain (three fingers) C C K K K K T I S S H H H Zn L R R A E P P F F D M Figure 3: Amino acid sequence and structure of the SP1 binding domain. Table 1: List of exchanged amino acids in 2nd finger of SP1. Table 1: List of exchanged amino acids in 2nd finger of SP1. Table 1: List of exchanged amino acids in 2nd finger of SP1. 2nd finger Amino acids in alpha helical region SP1 (wild type) R S D E L K R H K Exchanged Amino Acids CB1 H S S R L I R H E MR14 R S S T L I Q H K MQ91 Q S S Y L I K H K MQ135 Q S S H L I Q H K MQ151 Q S S Y L T Q H K genome. The rank denotes the sensitivity of the protein to bind a specific DNA sequence in which the binding is sensitized to the contextual influences the fingers exert on each other, the environmental condition of tissues and organisms, and the shape of the DNA [2, 3, 11]. The lower the rank (higher number) in Table 4 is, the less time a complex has to form, which is extremely important for zinc finger nucleases because the time factor is a crucial indicator to reduce cytotoxic behavior at off-target sites. 4. Results and Discussion The exchange of amino acids in the alpha helical region of the 2nd finger of SP1 (Figure 3, colored in blue and underlined) produced the five mutants CB1, MR14, MQ91, MQ135, and MQ151 as displayed in Table 1. The exchanged amino acids are double underlined.h 3.2. Site-Directed Mutagenesis. The BamHI-BglII fragment derived from pPacSpl-516c, encoding 3 zinc fingers of the human transcription factor SP1, was cloned into the BamHI site of Bluescript (Stratagene) to yield pB-516c. Two poly- merase chain reactions (PCR) were performed using oligonu- cleotide pairs 393/2745 and 2744/392 together with pB-516c generating SP1 fragments A and B. Each fragment harbors the introduced mutations at either the 3󸀠or 5󸀠site. They were isolated from a 6070 polyacrylamide gel. The complete SP1 fragment encoding the desired mutations and a restored BglII site was generated by performing a second PCR using primers 393 and 1956 on SP1 fragments A and B. The PCR product was extracted with phenol/chloroform, digested with BamHI and BglII, gel-purified, and cloned into pAR3039 to yield pAR-SP1 The EMSA assay results in Figures 4(a) and 4(b) show significant changes in the binding preferences of the 64 triplets for the 2nd finger of SP1, CB1, MR14, MQ91, MQ135, and MQ151. 4.1. Malleability of Binding Preferences. Variations of the SP1 binding domain have been created via site-directed mutagenesis of nonconserved positions in the alpha helical region of the 2nd finger of which CB1, MR14, MQ91, MQ135, and MQ151 are listed in Table 1 and of which the binding capability has been tested using electrophoretic mobility shift assay (EMSA) with P32 labeled oligonucleotides. Remarkably, six binding patterns in Figure 4(a) with significant differences 4.1. Malleability of Binding Preferences. Variations of the SP1 binding domain have been created via site-directed mutagenesis of nonconserved positions in the alpha helical region of the 2nd finger of which CB1, MR14, MQ91, MQ135, and MQ151 are listed in Table 1 and of which the binding capability has been tested using electrophoretic mobility shift assay (EMSA) with P32 labeled oligonucleotides. 4. Results and Discussion Instead of assembling finger arrays from modified Zif268 and SP1 fingers [3], our findings suggest the viability of a strategy to adjust the natural framework of a zinc finger domain by exchanging amino acids of one finger at a time to alter binding preferences of the entire domain. In addition, two three- finger domains in a 2 × 3 strategy [5] can be combined to form a six-finger domain binding an 18-base pair long DNA sequence that is unlikely to occur twice in the human genome. This could be a way to sensitize the domain to a point that allows producing clinical viable molecular tools to influence the human genome. have been obtained that show extraordinary diversity of bind- ing occurrences with distinct dissimilar binding preferences, which supports the notion of context dependency among the three-domain fingers and beyond degeneracy; the paper by [3] noted “unanticipated specificity” [3, page 4683] and that by [2] noted “rank-ordered listing of the (DNA binding site) preferences” amid millions of measurements, of which one can derive that the patterns in Figure 4(b) and the systematic 1 to 64 ranking in Table 4 are specific rank-ordered listings of binding preferences [2], in which the altered 2nd finger changes the rank of binding preferences of the entire domain. Instead of assembling finger arrays from modified Zif268 and SP1 fingers [3], our findings suggest the viability of a strategy to adjust the natural framework of a zinc finger domain by exchanging amino acids of one finger at a time to alter binding preferences of the entire domain. In addition, two three- finger domains in a 2 × 3 strategy [5] can be combined to form a six-finger domain binding an 18-base pair long DNA sequence that is unlikely to occur twice in the human genome. This could be a way to sensitize the domain to a point that allows producing clinical viable molecular tools to influence the human genome. 4.2. Rank-Specific Recognition of the 2nd Finger of SP1 and Mutants. The rank-specific recognition (RSR) code in Table 4 signifies the rank ordered stability of the DNA- protein complex in a certain condition, in which complex stability denotes the degree of binding reversibility or in other words the time a zinc finger protein sticks to the 4.2. Rank-Specific Recognition of the 2nd Finger of SP1 and Mutants. 4. Results and Discussion Depending on the assay and measurement technique, degeneracy of rank-specific recognition can be defined as (1) time period a DNA/protein complex holds together (visible spectroscopy), (2) complex reversibility (binding energy of formation and dissolution, change induced by physical parameter—thermal, ph, UV, etc.), (3) complex stability (delta of binding energies of formation and dis- solution process), (4) binding sensibility (binding energy of initiation before formation), (5) influence on biological functionality, and (6) condition-dependent shift of rank- specific recognition and functionality. Following this notion, the rank order from 1 to 64 represents the (1) decrease of the time period a DNA/protein complex holds together, (2) increase of complex reversibility, (3) decrease of complex stability, (4) increase of binding sensibility/sensitivity, (5) control of biological functionality (e.g., gene expression and double-strand cleavage of ZFN), and (6) shift of rank-specific recognition and functionality of the same zinc finger in a different environmental condition (tissue, organism). The observation of Badis in which secondary binding motifs (2nd–64th rank) potentially execute biological functionality (gene expression) to the full extent and “independent of the primary motif” (1st rank) [2, page 1723] denotes that the rank of the “DNA binding capability of zinc finger domains” does not influence the quality of the biological functionality (gene expression) but that the rank represents the control to which extent the biological functionality is executed by limiting the time period a DNA-protein complex’s activity is active at a specific location on the genome in a specific condition. In other words, nature is limiting the time period a DNA/protein complex is functional by choosing “alternate recognition interfaces” [2, page 1723] which in this case means a sequence of secondary binding preference. In regard have been obtained that show extraordinary diversity of bind- ing occurrences with distinct dissimilar binding preferences, which supports the notion of context dependency among the three-domain fingers and beyond degeneracy; the paper by [3] noted “unanticipated specificity” [3, page 4683] and that by [2] noted “rank-ordered listing of the (DNA binding site) preferences” amid millions of measurements, of which one can derive that the patterns in Figure 4(b) and the systematic 1 to 64 ranking in Table 4 are specific rank-ordered listings of binding preferences [2], in which the altered 2nd finger changes the rank of binding preferences of the entire domain. 4. Results and Discussion The rank-specific recognition (RSR) code in Table 4 signifies the rank ordered stability of the DNA- protein complex in a certain condition, in which complex stability denotes the degree of binding reversibility or in other words the time a zinc finger protein sticks to the 6 Biotechnology Research International 6 A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T SP1-ANN SP1-CNN SP1-GNN SP1-TNN CB1-ANN CB1-CNN CB1-GNN CB1-TNN MQ91-ANN MQ91-CNN MQ91-GNN MQ91-TNN MQ135-ANN MQ135-CNN MQ135-GNN MQ135-TNN MR14-ANN MR14-CNN MR14-GNN MR14-TNN MQ151-ANN MQ151-CNN MQ151-GNN MQ135-TNN 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T SP1-ANN 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T CB1-ANN 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T CB1-TNN 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T CB1-ANN 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MR14-TNN 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MR14-ANN 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MR14-CNN 2󳰀A 3󳰀A MQ135-TNN A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MQ135-TNN 2󳰀A 3󳰀A A A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MQ151-CNN 2󳰀A 3󳰀A A A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MQ151-ANN 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MQ151-GNN 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MQ151-GNN 2󳰀A 3󳰀A MQ151-GNN MR14-CNN A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MQ91-TNN 2󳰀A 3󳰀A MQ135-GNN A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MQ135-TNN 2󳰀A 3󳰀A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MQ135-CNN 2󳰀A 3󳰀A MQ91-GNN MQ91-ANN A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T MQ91-GNN 2󳰀A 3󳰀A MQ135-GNN MQ151-CNN A A A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T 2󳰀A 3󳰀A (a) Figure 4: Continued. MQ151-CNN 7 Biotechnology Research International 0 1000 2000 3000 4000 5000 CGG GCG CGT GGC TTG TGG GAG CCC TAG CCG AGG GGT GAC GTG GGA AGT TAA CAG GTC GCT TAT GTA GAT GCA AAT CGC TCT TCG TAC CCT CAA GTT GGG CAT TGT TCA GCC TGA TTT CGA ATG AAG ACT CTG ATA AGA TTA TCC CCA TGC TTC AGC ACA ACG CAC ATT CTA ATC CTT GAA CTC ACC AAC AAA S1 0 10000 20000 30000 CCC GAG GGT AGG GGA TAA CAG GTG GGG TGG TGT TAT CAT ATG GTT CGT CGG GTC TAG TCG CAC GAA GAC GTA GCG AGA CGC GGC AAG ACA CCG AAT GAT AGT CAA GCT TGA TCC CCT CGA AAA TTG CCA CTG TCA TAC CTA ATT CTT AGC ACT ATA TTA GCC TTT CTC TCT AAC ACC ACG ATC GCA TGC TTC 0 10000 20000 30000 40000 GAG GGA GTG GAA GGG GGT GTA AGG GGC GTT TGG CCC AGT CGG GAT TGA AAG GAC GCG TGC TTG TAG GCA TTT CGC CGT GTC ATG AGA CAG CCT GCT ATA CAA CTG TGT CGA CAT CTA AAT CCG CTT TAA AGC TAT ATT ACA CAC ACT CTC ACG TTA CCA TCA ACC GCC AAA AAC TAC TCG TCT ATC TCC TTC Rank-specific recognition code SP1 Rank-specific recognition code CB1 Rank-specific recognition code MR14 Rank-specific recognition code MQ91 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 AGG CGG GGA GAG AAG GGC GTG AGA ATG AAT AAA GGT CAG GGG GAC GAA AGT ACA ATT ACC GCG GCT TAG ATA GTA CAA CGT TAA GTC TGG GCA GCC CCC CGA GAT ACT TGA CAT CCG ACG GTT AGC CCT CAC CGC CTA TAC AAC TCG TAT CCA TGC CTT TCT CTG TCA TCC CTC TGT TTG ATC TTT TTA TTC (b) Rank-specific recognition code SP1 0 1000 2000 3000 4000 5000 CGG GCG CGT GGC TTG TGG GAG CCC TAG CCG AGG GGT GAC GTG GGA AGT TAA CAG GTC GCT TAT GTA GAT GCA AAT CGC TCT TCG TAC CCT CAA GTT GGG CAT TGT TCA GCC TGA TTT CGA ATG AAG ACT CTG ATA AGA TTA TCC CCA TGC TTC AGC ACA ACG CAC ATT CTA ATC CTT GAA CTC ACC AAC AAA S1 30000 Rank-specific recognition code SP1 Rank-specific recognition code CB1 0 10000 20000 30000 CCC GAG GGT AGG GGA TAA CAG GTG GGG TGG TGT TAT CAT ATG GTT CGT CGG GTC TAG TCG CAC GAA GAC GTA GCG AGA CGC GGC AAG ACA CCG AAT GAT AGT CAA GCT TGA TCC CCT CGA AAA TTG CCA CTG TCA TAC CTA ATT CTT AGC ACT ATA TTA GCC TTT CTC TCT AAC ACC ACG ATC GCA TGC TTC Rank specific recognition code CB1 Rank-specific recognition code MR14 0 10000 20000 30000 40000 GAG GGA GTG GAA GGG GGT GTA AGG GGC GTT TGG CCC AGT CGG GAT TGA AAG GAC GCG TGC TTG TAG GCA TTT CGC CGT GTC ATG AGA CAG CCT GCT ATA CAA CTG TGT CGA CAT CTA AAT CCG CTT TAA AGC TAT ATT ACA CAC ACT CTC ACG TTA CCA TCA ACC GCC AAA AAC TAC TCG TCT ATC TCC TTC Rank specific recognition code MR14 Rank-specific recognition code MQ91 Rank specific recognition code MQ91 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 AGG CGG GGA GAG AAG GGC GTG AGA ATG AAT AAA GGT CAG GGG GAC GAA AGT ACA ATT ACC GCG GCT TAG ATA GTA CAA CGT TAA GTC TGG GCA GCC CCC CGA GAT ACT TGA CAT CCG ACG GTT AGC CCT CAC CGC CTA TAC AAC TCG TAT CCA TGC CTT TCT CTG TCA TCC CTC TGT TTG ATC TTT TTA TTC (b) Figure 4: Continued. MQ151-CNN Results of complete recognition code of the 64 binding sites of the 2nd finger of SP1 (nonstandardized P32 count). 5󸀠GTCGGATCCTGTCTGAGGTGAGTTGGGNNNGGGCTTGTCTTCCGACGTCGAATTCGCG3󸀠 to [45] observation of a relatively poor relationship between sequence specificity in vitro and nuclease targeting capacity in vivo might indicate that degeneracy can be defined as a “loss of functionality” [33] or “loss of pioneer factors” [45, page 289]. However, considering the dependencies on context, condition, and DNA shape together with rank- specific recognition rather denotes that degeneracy can be defined as the “shift of functionality” to dissimilar binding sites in a different condition. does not denote the quality of gene expression but rather the duration of gene expression. Following this notion, certain sequences in the rank in Table 4 might be associated with a certain biological functionality. However, a higher rank in Table 4 does not indicate improved functionality and the rank does not determine the type and strength of functionality in the notion that weaker affinity does not result in less functionality but rather retained functionality independent of affinity. Rank-specific recognition then means that dependencies of context, condition, and DNA shape are consistent with the general concept of modularity [3] and are applicable to single fingers as well as an entire multifinger domain. Because of context dependency in which each finger influences the binding behavior of adjacent fingers and the entire binding domain [8], the modularity and binding character of the entire domain can be altered and adjusted to recognize any DNA sequence. This delivers a significant advantage over randomly altering single fingers of Zif268 and SP1 and assembling them to arrays with high affinity of uncontrollable 4.3. Rank-Specific Recognition of Altered SP1 Zinc Finger: CB1, MR14, MQ91, MQ135, and MQ151. The exchange of amino acids in the 2nd finger of Sp1 induces a change in the domain context of the entire three-finger binding domain and a shift to a distinctively different rank order of binding preferences, in which a zinc finger is able to execute biological functions at dissimilar target sequences. Rank-specific recognition then denotes a ranking of locations on the genome where a zinc finger potentially induces a biological function rather than a gradual loss of a function’s quality. In other words, the rank 4.3. Rank-Specific Recognition of Altered SP1 Zinc Finger: CB1, MR14, MQ91, MQ135, and MQ151. MQ151-CNN Figure 4: Continued. Biotechnology Research International 8 8 0 10000 20000 30000 40000 50000 60000 70000 80000 GGA GAC GAG TAA GGG GGT AGA GAT GTG AGT GGC GTA AGG TGA CGA GCT GTT GCG ATG GCA AGC TGT CGT GAA ATT ACT TCA ACC CGG ACA TGG CAA CCT GCC AAT AAG ATA TTT CGC ACG AAA TTA AAC TAG TTC GTC CAT CCC TAC TGC CAG CAC TTG TAT CTT TCG CCA CCG CTG TCT ATC TCC CTC CTA 0 10000 20000 30000 40000 50000 AGG GGA AAG GAG GTA AGT TGA GTG TTG GCA GGG GGT CGT TTT GCG GCT GAT TGG GTC AAT ATT TCT CGG TGT GGC TAG TAT ACT AGA TTC CCC GTT AGC ATA TGC TTA TCC AAA ACA CGA GAC AAC ATG ACC CGC GCC TCA CTT CTG CCG GAA TAA TAC CAA CAG CTA ACG CAT CTC CCA CCT CAC TCG ATC Rank-specific recognition code MQ151 (b) Rank-specific recognition code MQ135 ure 4: (a) Band shifts (SP1 protein-DNA P32 oligonucleotide complex). (b) Results from Phosphor Imager Screening (Molecular namics). Results of complete recognition code of the 64 binding sites of the 2nd finger of SP1 (nonstandardized P32 count). T 2 P32 l b l d d bl t d d li l tid 58 b i Rank-specific recognition code MQ135 0 10000 20000 30000 40000 50000 60000 70000 80000 GGA GAC GAG TAA GGG GGT AGA GAT GTG AGT GGC GTA AGG TGA CGA GCT GTT GCG ATG GCA AGC TGT CGT GAA ATT ACT TCA ACC CGG ACA TGG CAA CCT GCC AAT AAG ATA TTT CGC ACG AAA TTA AAC TAG TTC GTC CAT CCC TAC TGC CAG CAC TTG TAT CTT TCG CCA CCG CTG TCT ATC TCC CTC CTA Rank-specific recognition code MQ135 0 10000 20000 30000 40000 50000 AGG GGA AAG GAG GTA AGT TGA GTG TTG GCA GGG GGT CGT TTT GCG GCT GAT TGG GTC AAT ATT TCT CGG TGT GGC TAG TAT ACT AGA TTC CCC GTT AGC ATA TGC TTA TCC AAA ACA CGA GAC AAC ATG ACC CGC GCC TCA CTT CTG CCG GAA TAA TAC CAA CAG CTA ACG CAT CTC CCA CCT CAC TCG ATC Rank-specific recognition code MQ151 (b) Rank-specific recognition code MQ151 (b) Figure 4: (a) Band shifts (SP1 protein-DNA P32 oligonucleotide complex). (b) Results from Phosphor Imager Screening (Molecular Dynamics). Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. context dependency. The electrophoretic mobility shift assay (EMSA) bandshifts show context dependency in that he 2nd finger influence the binding ability of the 1st and 3rd finger via three-dimensional-malleability of the domain structure which results in the six distinctly different binding patterns shown in Figures 3 and 4. The binding ability of the 1st and 3rd fingers via three-dimensional malleability of the domain structure. This might be interpreted as degeneracy in that the domain binds a significant number of related individual sequences [3, page 1]. However, the pattern does not indicate that the domain either binds or not (on/off binding) but rather shows subtle differences of specific binding represent- ing a decreasing gradient of complex stability.t binding capability. Following the notion of functionality, the inference is that binding specificity is not degenerated, which means no loss or degradation of functional activity, but is rank-ordered degenerated time sensitivity at multiple target sequences in which a module shifts its DNA binding capability to dissimilar DNA sequences and furthermore retains the same or has new function in dissimilar context and conditions. 4.4. DNA-Protein Interactions. Because of condition depen- dency, results derived from a single assay are tentative and are disallowing generalizability, but substantial inferences about the influence of evolutionary traits on the malleability of binding preferences can be drawn that can lead research to a pragmatic direction to produce clinical viable molecular approaches and tools. Reportedly, the binding domain of Sp1 in its natural conditions within a large number of cellular and viral promoters, for example, [8] binds GC-rich boxes and especially the second finger of the triplet GCG. Looking at the RSR in Figure 4(a) and Table 4 under the unique (unnatural) EMSA conditions, SP1 recognizes AT-rich triplets at ranks 5 and 9 as well as AT-boxes at ranks 17, 21, and 25. It can be inferred that in the same condition the SP1 zinc finger domains potentially bind any triplets and that patterns of shifting preferences of certain nucleotide positions in the triplet emerge when comparing the six patterns. The finding that the 2nd finger’s best binding site is CGG might be due to the specific condition in EMSA; however, it has to be con- sidered that in vivo the observed preference to GCG is likely. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. For MQ91 the ranks 60, 61, 62, and 64 (TTG, TTT, TTA, and TTC) might show that at the third position G, T, A, and C do not play a role and that the 2nd finger binds GTT in which the overlap mechanism that stabilizes the DNA-protein complex is disabled and cannot initiate complex formation. In Figure 5, the band shifts in the upper portion of the pictures represent the stable DNA-protein complexes of each of the 64 assays. Relative comparison of the band shifts with the unbound P32-labeled DNA oligonucleotides in the lower portion of the pictures using a Phosphor Imager infers that the complex stabilities in a specific condition systematically decrease. In Table 5, the columns list the 64 (9-mer) GGGNNN- GGG (#P32) are the Phosphor Imager Screening counts and (#loc) is the number of locations the 9-mer string occurs as exact matches in the human genome using the NCBI-HuRef database. Table 5 and Figure 6 contains the exact number of loca- tions of the 64 NNN nucleotide combinations (Table 3) of the 9-mer DNA strings GGGNNNGGG in the human genome (NCBI-HuRef) which might represent potential “off-target” locations. Of the 26 highest P32counts the SP1 binding domain recognizes around 70% of (18/26) GC-rich triplets of which 27% (6/26) are GC-triplets. In addition, of noticeable impor- tance is the observation that 30% (8/26) are AT-rich triplets of which 10% (3/26) are AT-triplets, which in turn signifies that the 2nd finger sufficiently influences the formation of a DNA-protein complex to create a distinguished recognition pattern. The sorting of the Phosphor Imager readings from the highest to the lowest P32count shows gradually decreasing formation of 26 DNA-protein complexes (band shifts) with P32 counts above 500 and 35 below 500. The three triplets AAA, AAC, and ACC did not yield detectable measures; however, the binding ability of a transcription factor can change with conditions [3]; thus it can be assumed that complex formation is possible under altered circumstances. In general the outcome confirms that the SP1 domain not only preferably binds GC-rich triplets but also has the ability to bind AT-rich sequences. Despite the attempt to reduce the quantity of information to one altered finger and six proteins, the complexity of results already exceeds full analysis and understanding. Biotechnology Research International Biotechnology Research International 9 5󸀠GTCGGATCCTGTCTGAGGTGAGTTGGGNNNGGGCTTGTCTTCCGACGTCGAATTCGCG3󸀠 The exchange of amino acids in the 2nd finger of Sp1 induces a change in the domain context of the entire three-finger binding domain and a shift to a distinctively different rank order of binding preferences, in which a zinc finger is able to execute biological functions at dissimilar target sequences. Rank-specific recognition then denotes a ranking of locations on the genome where a zinc finger potentially induces a biological function rather than a gradual loss of a function’s quality. In other words, the rank Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. SP1-ANN SP1-CNN SP1-GNN SP1-TNN A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A Figure 5: Band shifts of the SP1 protein-DNA P32 oligonucleotide complex. Figure 5: Band shifts of the SP1 protein-DNA P32 oligonucleotide complex. Figure 5: Band shifts of the SP1 protein-DNA P32 oligonucleotide complex. 0 1000 2000 3000 4000 5000 (1)16697 (2)19345 (3) (4) (5) (6) (7) (8) (9) (10) (11)4 7 (12) (13) (14) (15) (16) 26799 (17) (18) 34437 (19) (20) (21) (22) (23) (24) (25) 17749 (26) (27) (28) (29) (30) (31) 15503 (32) (33) (34) 14458 (35) (36) (37) (38) (39) (40) (41) 58 (42) 36175 (43) 16928 (44) (45) 7850 (46) 38448 (47) (48) (49) (50) (51) (52) 6704 (53) 26050 (54) 6423 (55) 3769 (56) 2 (57) (58) 1919 (59) (60) (61) (62) 3170 (63) 2925 (64) 29730 Figure 6: Results from Phosphor Imager Screening (Molecular Dynamics) of the complete binding spectrum of the 64 binding triplets of the 2nd finger of SP1 and number of exact GGGNNNGGG (Table 3) matches in the human genome. Figure 6: Results from Phosphor Imager Screening (Molecular Dynamics) of the complete binding spectrum of the 64 binding triplets of the 2nd finger of SP1 and number of exact GGGNNNGGG (Table 3) matches in the human genome. modularity and that one finger binds to one triplet thus cast- ing doubt on the feasibility of producing zinc finger domains that allow precise modifications of the human genome [3]. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. However, it shows the possibilities from a full data set of 262,144 DNA sequences to which a three-finger protein can bind; important inferences can be drawn regarding the clinical viability of a domain. With microarrays there is the capability to produce data sets of the entire range of 262,144 nine-base pair binding sites. It remains open if in vitro data can be triangulated with in vivo data to generate a clearer picture of specific DNA- protein interactions. A more pragmatic approach is to mea- sure the formation and dissolution of DNA-protein complex. 4.5. Electrophoretic Mobility Shift Assay (EMSA). The elec- trophoretic mobility shift assay (EMSA) band shifts in Figure 4(a) and computational results in Figure 4(b) show Biotechnology Research International 10 SP1-ANN SP1-CNN SP1-GNN SP1-TNN A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T A A A C C C C C G G G G G T T A C G T A C G T A C G T T T T 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A 2󳰀A 3󳰀A Figure 5: Band shifts of the SP1 protein-DNA P32 oligonucleotide complex. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. Nonetheless, with the complexity and doubts at hand, zinc fingers are the right candidates primarily because nature uses them extensively, because they are the most important for gene regulation, have a reasonably small structure (binding domain), and seem to have evolutionary traits that might be of practical importance in the design and function of molecular tools to safely influence genomes.i The findings are consistent with evidence that emerged over the last few years and in particular highlights the challenges to produce clinical viable molecular zinc finger tools. Research on transcription factors has advanced rapidly and data and knowledge have created a multifaceted pic- ture with an overwhelming abundance of aspects. Extensive reviews, for example, [1, 3, 5, 11], and detailed discoveries, for example, [2, 3, 7, 35, 46], paint a picture of an increasingly complex situation regarding the DNA binding properties of transcription factors.h The goal addressed here in particular is to investigate the feasibility to produce clinical viable tools to securely modify the human genome with the current state of knowledge and technical capabilities. Zinc finger proteins seem to be interesting candidates despite the correct assessment of [3] presenting a complex collection of challenges to the notion of l From the start there was the hope that a single finger that consists of 28–30 amino acids is a simple enough structure that can easily be studied in detail and assembled into bespoken multifinger domains for any desired DNA sequence thus specifically reaching any location in the human genome. However, the efforts of the last two decades resulted Biotechnology Research International 11 Table 4: Listed rank-specific recognition codes for SP1, CB1, MR14, MQ91, MQ135, and MQ151. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. SP1 CB1 MR14 MQ91 MQ135 MQ151 (1) CGG CCC GAG AGG GGA AGG (2) GCG GAG GGA CGG GAC GGA (3) CGT GGT GTG GGA GAG AAG (4) GGC AGG GAA GAG TAA GAG (5) TTG GGA GGG AAG GGG GTA (6) TGG TAA GGT GGC GGT AGT (7) GAG CAG GTA GTG AGA TGA (8) CCC GTG AGG AGA GAT GTG (9) TAG GGG GGC ATG GTG TTG (10) CCG TGG GTT AAT AGT GCA (11) AGG TGT TGG AAA GGC GGG (12) GGT TAT CCC GGT GTA GGT (13) GAC CAT AGT CAG AGG CGT (14) GTG ATG CGG GGG TGA TTT (15) GGA GTT GAT GAC CGA GCG (16) AGT CGT TGA GAA GCT GCT (17) TAA CGG AAG AGT GTT GAT (18) CAG GTC GAC ACA GCG TGG (19) GTC TAG GCG ATT ATG GTC (20) GCT TCG TGC ACC GCA AAT (21) TAT CAC TTG GCG AGC ATT (22) GTA GAA TAG GCT TGT TCT (23) GAT GAC GCA TAG CGT CGG (24) GCA GTA TTT ATA GAA TGT (25) AAT GCG CGC GTA ATT GGC (26) CGC AGA CGT CAA ACT TAG (27) TCT CGC GTC CGT TCA TAT (28) TCG GGC ATG TAA ACC ACT (29) TAC AAG AGA GTC CGG AGA (30) CCT ACA CAG TGG ACA TTC (31) CAA CCG CCT GCA TGG CCC (32) GTT AAT GCT GCC CAA GTT (33) GGG GAT ATA CCC CCT AGC (34) CAT AGT CAA CGA GCC ATA (35) TGT CAA CTG GAT AAT TGC (36) TCA GCT TGT ACT AAG TTA (37) GCC TGA CGA TGA ATA TCC (38) TGA TCC CAT CAT TTT AAA (39) TTT CCT CTA CCG CGC ACA (40) CGA CGA AAT ACG ACG CGA (41) ATG AAA CCG GTT AAA GAC (42) AAG TTG CTT AGC TTA AAC (43) ACT CCA TAA CCT AAC ATG (44) CTG CTG AGC CAC TAG ACC (45) ATA TCA TAT CGC TTC CGC (46) AGA TAC ATT CTA GTC GCC (47) TTA CTA ACA TAC CAT TCA (48) TCC ATT CAC AAC CCC CTT Table 4: Continued. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. SP1 CB1 MR14 MQ91 MQ135 MQ151 (49) CCA CTT ACT TCG TAC CTG (50) TGC AGC CTC TAT TGC CCG (51) TTC ACT ACG CCA CAG GAA (52) AGC ATA TTA TGC CAC TAA (53) ACA TTA CCA CTT TTG TAC (54) ACG GCC TCA TCT TAT CAA (55) CAC TTT ACC CTG CTT CAG (56) ATT CTC GCC TCA TCG CTA (57) CTA TCT AAA TCC CCA ACG (58) ATC AAC AAC CTC CCG CAT (59) CTT ACC TAC TGT CTG CTC (60) GAA ACG TCG TTG TCT CCA (61) CTC ATC TCT ATC ATC CCT (62) ACC GCA ATC TTT TCC CAC (63) AAC TGC TCC TTA CTC TCG (64) AAA TTC TTC TTC CTA ATC in high failure rates of modular assembled zinc finger arrays [7, page 374] and cytotoxicity which is thought to be caused by cleavage at “off-target” sites [6, 39, 40] when used in zinc finger nucleases. In addition, despite the fact that several quantitative methods have been developed to model DNA-protein interactions with specific focus on the C2H2 zinc finger proteins, the overall predictive accuracy of current computational tools is still limited [37]. Tompa et al. concluded earlier that sequence variability among the binding sites of a given transcription factor and the nature of variability itself are not well understood (page 137) and that the accuracy of prediction of computational tools cannot be accomplished because “we do not understand the full truth about transcription factor binding sites [38, page 144].” In a more recent study, [36] uncovered some surprising results highlighting “our limited understanding of even simple protein-DNA interactions [36, page 2500].” When looking at the number of 1,261,301 exact locations for the 64 considered 9-mers in Table 5, which are just 64 combinations out of 262,144 (64 × 64 × 64 or 49) possible combinations of 9- mers (a multiplier of 4,100), the following question arises: how nature ensures evolutionary success and functionality of natural three-finger domains. One answer might be that transcription factors are part of a regulatory network system and are controlled by factors that are absent using artificially created zinc finger arrays. However, this would not explain why nature would create and extensively use three-finger domains that can interfere with millions of exact locations without any evolutionary purpose and sustainable biological functionality. 4.6. Observations Relevant to Understand Cytotoxicity. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. The extraordinary evolutionary success of C2H2 binding proteins has been attributed to the modularity and three-base speci- ficity of single zinc fingers (Figure 3) and the ability to chain them together to form a multifinger domain that possesses Table 4: Listed rank-specific recognition codes for SP1, CB1, MR14, MQ91, MQ135, and MQ151. Table 4: Continued. 4.6. Observations Relevant to Understand Cytotoxicity. The extraordinary evolutionary success of C2H2 binding proteins has been attributed to the modularity and three-base speci- ficity of single zinc fingers (Figure 3) and the ability to chain them together to form a multifinger domain that possesses Biotechnology Research International 12 Table 5: (a) Visible band shifts with value ranging from 4570 to 500 counts: 1–26 (40%). (b) Less/no visible band shifts with value <500 counts: 27–64 (38, 60%). Table 5: (a) Visible band shifts with value ranging from 4570 to 500 counts: 1–26 (40%). (b) Less/no visible band shifts with value <500 counts: 27–64 (38, 60%). Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. (a) 9-mers #P32 #loc 9-mers #P32 #loc 9-mers #P32 #loc 9-mers #P32 #loc 9-mers #P32 #loc (1) CGG 4570 16697 (2) GCG 3450 22741 (4) GGC 2850 16087 (8) CCC 1900 6153 (19) GTC 950 6246 (3) CGT 3125 6508 (5) TTG 2320 28372 (9) TAG 1850 14922 (20) GCT 800 42489 (6) TGG 2300 55636 (10) CCG 1850 8662 (21) TAT 660 7319 (7) GAG 2180 58721 (11) AGG 1750 41257 (22) GTA 630 15656 (12) GGT 1440 38721 (23) GAT 560 23908 (13) GAC 1215 5109 (24) GCA 555 40076 (14) GTG 1200 69427 (25) AAT 550 17749 (15) GGA 1175 50364 (26) CGC 500 4098 (16) AGT 1150 26799 (17) TAA 1100 9037 (18) CAG 1010 34437 (b) 9-mers #P32 #loc 9-mers #P32 #loc 9-mers #P32 #loc (27) TCT 475 16364 (40) CGA 225 5420 (53) ACA 63 26050 (28) TCG 450 7161 (41) ATG 188 28958 (54) ACG 62 6423 (29) TAC 380 1172 (42) AAG 188 36175 (55) CAC 50 3769 (30) CCT 300 27004 (43) ACT 187 16928 (56) ATT 50 11132 (31) CAA 300 15503 (44) CTG 150 38542 (57) CTA 40 9049 (32) GTT 280 24314 (45) ATA 124 7850 (58) ATC 30 1919 (33) GGG 260 18460 (46) AGA 123 38448 (59) CTT 20 15023 (34) CAT 250 14458 (47) TTA 110 10717 (60) GAA 10 36693 (35) TGT 245 20901 (48) TCC 110 2733 (61) CTC 10 4500 (36) TCA 238 23485 (49) CCA 80 22765 (62) ACC 0 3170 (37) GCC 235 12890 (50) TGC 75 4411 (63) AAC 0 2925 (38) TGA 230 24010 (51) TTC 73 1854 (64) AAA 0 29730 (39) TTT 225 16500 (52) AGC 65 6704 Total: 1,261,301 the binding specificity to only recognize one primary DNA target sequence at which it exerts biological activity [5]. This is an indispensable requirement to ensure genome modifications occur at only one desired location to prevent damaging changes in the human genome that could interfere with cell functions and lead to cell death and apoptosis [6]. However, reported degeneracy and the overlap problem [3, page 2] as well as supporting observations in Tables 6 and 7 have complicated the straightforward approach of one finger binding to one primary triplet.h 4.7. Genetic and Functional Conservation and Universality of TFs. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. Degeneracy is the most recognizable challenge since the precise clinical use of zinc finger nucleases requires three- finger C2H2 domains having a binding preference to only a single 9-base DNA sequence on the entire human genome [3, page 7]. Consequently, this requirement should be applicable to a single finger as well and the observed recognition pattern in Figures 4(a) and 5 that at first glance seems to be a serious threat for its clinical use and, first of all, would certainly explain the abundant binding occurrences at “off-target” sites as observed with engineered zinc finger domains.i This section selectively discusses observations that might most evidently determine and regulate the reversible nature of the DNA-protein complex, in particular, its stability and formation and dissolution mechanisms. Particularly consid- ered are the genetic and functional conservation on one hand and universality on the other hand that defines evolutionary success of TFs, the DNA-protein complex stability, and the role of single fingers. Finally evolutionary issues are considered. These observations together seem to provide the pivotal insights of nature’s success that may lead to a distinguished research strategy and clinical success. i Similarly, the natural zinc finger SP1 should to some extent bind at undesired locations as well; however, there is no evidence that SP1 introduces deleterious genome modifications or displays other side effects, which in turn indicates that the observations in Figure 3 do not just show degenerated binding at multiple triplets but that the more accurate interpretation would be what [3] specified as “unanticipated specificity” [3, page 7]. Furthermore, it has been well documented that degeneracy is common among transcription factors and it is discussed that the flexibility Biotechnology Research International 13 Table 6: Evolutionary success of C2H2 binding proteins. Relevant observations concerning evolutionary success of C2H2 binding proteins. Observations References and comments Degeneracy (i) Engineered ZFAs typically yielded degenerate motifs, binding dozens to hundreds of related individual sequences [3]. (ii) Observed clear secondary DNA binding preferences and the secondary motifs were bound nearly as well as the primary motifs [2]. (iii) The secondary motif can recruit genomic loci independently of the primary motif [2]. (iv) Beyond simply providing a DNA binding site motif, these data provide rank-ordered listing of the preference of a protein [2]. (v) Observed “secondary motif” phenomenon had not been described before, and it has important implications for understanding how proteins interact with their DNA binding sites [2]. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. High failure rates The modular assembly method of engineering zinc finger arrays has an unexpectedly higher failure rate [7]. Evolutionary plasticity (i) The dramatic expansion of the number of C2H2-ZFs in mammals appears to be a recent evolutionary event [3]. (ii) Evolutionary plasticity [34, 47]. (iii) Conserved expression without conserved regulatory sequence: the more things change, the more they stay the same [1]. Complexity (i) Half of the proteins: each recognized multiple distinctly different sequence motifs [2]. (ii) 10605 combinations for a 1000 bp long gene [48]. (iii) The dramatic expansion of the number of C2H2-ZFs in mammals appears to be a recent evolutionary event [3]. Simplicity (i) Origami structure: [49–53]. (ii) Fractal organization: [54–56]. Directional evolution (i) Expression of ftz changed at least three times during arthropod evolution: [47]. (ii) The complexity, robustness, and evolvability of regulatory systems [1]. Evolutionary traits (i) “The contribution of finger 1 to the DNA binding affinity of SP1 is smaller than that of fingers 2 and 3, but the presence of finger 1 is still essential for the high DNA binding affinity. These unique features have never been detected in other zinc fingers [8]. Cytotoxicity Cell death and apoptosis associated with ZFN expression are most likely the result of excessive cleavage at off-target sites, which, in turn, suggests imperfect target-site recognition by the ZF DNA-binding domains. [6, 39, 40] to bind dissimilar sequences and the capacity of functioning at different binding regulatory sequences could be beneficial in the evolutionary process for establishing new regulatory systems [2, 59]. Especially, the interesting finding of [1] demonstrates that fully conserved promoter sequences can be replaced in a gene and fully conserved proteins take over the functionality in the new regulatory system. For this, nature does not rely on single-base pair mutations alone but can rearrange DNA sequences of any length on the human genome while at the same time preserving them. With this in mind, the observation by [2] of “rank-ordered listing of DNA binding site preferences” for a wide range of transcription factors might help to explain the significantly high number of DNA-triplets with which the 64 triplets of the 2nd finger of SP1 form a noticeable complex [2, page 1720]. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. Carrying the rank-ordered thought forward, Figure 3 shows that the binding capability of the 2nd finger of SP1 is not reduced to one or a few triplets but that the DNA-protein complex can possess any degree of stability in which the binding site specificity and affinity primarily determine the stability of the complex. Lam noted that degeneracy actually is specific binding, leading to the conclusion that the pattern (RSR) in Figure 3 is actually a rank-specific recognition (RSR) code.ifi More importantly, beyond specificity and affinity ranks the well-documented condition dependency ultimately con- notes that the RSR code primarily depends on the condition of a specific environment (tissue, organism) that determines specificity and affinity. Condition dependency has been observed by [60] who reported a relatively poor relationship between sequence specificity in vitro and nuclease targeting capacity in vivo [60] and [2] who reported that secondary binding motifs do bind in vivo and that the secondary motifs are used independently of the primary motif [2]. Condition dependency also is likely to be responsible for the high failure rate of zinc finger arrays because the intended target binding site is not the preferred binding site in a specific test condition [7, 33]. This might be of importance because it could indicate that degeneracy and condition dependency are vital evolutionary traits that allow TFs to conserve the amino acid sequence but do not exclude its use for executing different functionalities, which explains the widespread use of TFs in nature [47]. This allows conjecture that the 2nd finger has inherited the potential of binding any triplet Biotechnology Research International 14 Table 7: Evolutionary traits to be considered or reused in the design of zinc finger domains. Evolutionary trait References and comments Binding spectra A single zinc finger binds many triplets[2, 3] related to Lam’s degeneracy Condition dependency Binding of dissimilar sequence in various conditions [1–3, 10, 11] and high failure rates of intended target site [7] Context dependency The interdependence between all fingers in a domain through the overlap loci [3, 8] Sequence dependency Form of DNA determines the binding sequence a domain can recognize [11] p. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. 242 Dynamic biological 3D malleability Of the three-dimensional structure of single fingers and entire domains and the form and deformability of DNA structure [8, 11, 57] Reversibility Formation and dissolution of DNA-protein complex is an indispensable property of a functional regulatory network system Evolutionary dualisms Duality of conserving and changing gene and protein sequences [1] and reversibility of biological processes and functions [1, 2, 11, 57] 4th base overlap loci Complex stabilization and dissolution loci to control the reversibility process [3], Binding initiation: 1st finger of SP1 The 1st finger has unique evolutionary traits never detected in other fingers as well as relaxed binding specificity and affinity and therefore is likely to initiate complex formation [8]. The 1st finger’s condition dependency is unlike other fingers [58] Table 7: Evolutionary traits to be considered or reused in the design of zinc finger domains. Of the three-dimensional structure of single fingers and entire domains and the form and deformability of DNA structure [8, 11, 57] Formation and dissolution of DNA-protein complex is an indispensable property of a functional regulatory network system Duality of conserving and changing gene and protein sequences [1] and reversibility of biological processes and functions [1, 2, 11, 57] Complex stabilization and dissolution loci to control the reversibility process [3], The 1st finger has unique evolutionary traits never detected in other fingers as well as relaxed binding specificity and affinity and therefore is likely to initiate complex formation [8].hii The 1st finger’s condition dependency is unlike other fingers [58] The 1st finger’s condition dependency is unlike other fingers [58] AT-triplets seem to indicate that the 2nd finger adjusted the structure of the entire domain to fit AT-triplets by also utilizing DNA deformability in specific conditions in which AT-rich sequences can take forms that allow the formation of a complex. The RSR thus supports [57] observation that both the DNA and protein have structural malleability that provides an evolutionary advantage, which is more efficient than building new biological systems, components, and function from scratch via Darwinian randomness to adapt to evolutionary demands. The three-dimensional malleability (3D malleability), however, significantly raises the complexity for designing simple zinc finger based tools for clinical applications. In particular, both three-dimensional structures (2 × 3D malleability) can change in many ways and quite inconsistently under various conditions, which severely chal- lenges our ability for predicting recognition and biological functionality. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. under certain circumstances and that when circumstances change so does the order of the rank-specific recognition code. Considering the number of exact matches (1,261,301) found in the human genome of the 64 possible 9-mers in Table 5, to be clinically useful only one of them should be recognized, leading to the conclusion that nature must have the ability to make small incremental changes in the protein structure that might be induced by changes in the condition, which among other factors make TFs only bind at one or a few very specific locations. The RSR code together with condition dependency demonstrates the challenge to cope with potentially millions of putative “off-target” binding loca- tions and highlights an increased complexity in coping with cytotoxicity. 4.8. DNA-Protein Complex Stability: Role of Single Fingers. To find ways to better investigate the molecular mechanisms through which nature might use rank-specific recognition and condition dependency, for example, [10], [11] persua- sively argue that the three-dimensional structures of both the DNA and the protein change when forming a DNA- protein complex and subsequently both the DNA and the protein are able to morph their three-dimensional structure to adapt to altering conditions [11, 57]. Because of the fact that the nucleotide and amino acid sequences do adapt their structures to each other, the rank-specific recognition (RSR) code in Figures 4(a) and 4(b) shows a coordinated analog pattern of decreasing recognition, where the stability of the DNA-protein complex decreases in small incremental degrees. It has to be noted that the recognition pattern is highly complex in that even the 2nd finger prefers GC-rich triplets (18/26 in Table 5); the remaining 5 AT-rich and 3 4.9. Evolutionary Dualism and Reversibility of DNA-Protein Complex. One of the fundamental underpinning principles is that evolution is a process in which nature needs to accomplish the duality of conserving and changing gene and protein sequences as well as structures and biological func- tionalities [1, 2, 9, 11, 12, 34, 47, 57]. The evolutionary dualism significantly increases the chances of having straightforward ways of dealing with complexity, change and conservation, and time. Nature after all needs to have pragmatic ways to cope with the extraordinary complexity to adapt in a timely manner to required modifications. In addition, evolutionary dualism has not received particular attention regarding what traits zinc fingers need to make a multi-finger domain viable for clinical application. 4.9. Biotechnology Research International For TFs in general, evolutionary dualism entails “reversibility” of the DNA-protein complex formation. It involves a fundamental mechanism that nature employs to control biological functionality and prevent undesired activities. Nature thus has to create the means through which it can control stability to achieve balanced reversibility in which specificity and affinity are important to arrange binding at the right location but in a way to allow reversibility of binding. High affinity in this regard would result in a highly stable complex with a low ability of controlling reversibility. High specificity, however, does not necessarily result in diminished reversibility, which then would lead to the conclusion that zinc finger domains with high specificity and low affinity are preferable and could be designed with the ability to avoid cytotoxicity. Testing zinc finger arrays for high affinity sequences then may result in arrays with less favorable binding occurrences for the intended target site, especially, because a substantial number of binding occurrences could occur at undesired locations. For clinical tools that are employed to fight genetic diseases it is, however, desirable to have extensive affinity to form irreversible or covalent binding to deter the growth of microorganisms, such as, for example, HIV, by disabling the CCR5 gene with a high affinity zinc finger array. It is relatively evident that binding to a fourth nucleotide increases stability of the DNA-protein complex without necessarily increasing affinity of a zinc finger domain [3, page 2]. With this feature, nature added the capability to delicately adjust the reversibility apparatus to form and dissolve a complex in small degrees. Behind the term “overlap” therefore seems to be the larger issue of a “complex stabilization and dissolution” mechanism that is part of the reversibility apparatus in which the 4th base converse nucleotides assist as complex dissolution points for potential factors in a regulatory network system (see Figure 7).h g y y g The overlap with the two loci connecting 1st/2nd and 2nd/3rd fingers (see Figure 7) strongly indicates general context dependency of the entire three-finger domain that allows transcription factors to have the capacity to recognize “secondary binding sites” [11, page 235] or secondary motifs [2]. There is a complex blending between general modularity of single fingers and overall context dependency of an entire domain. Biotechnology Research International Especially, in regard to condition dependency (tissues, organisms, and genomes), the recognition of a single finger and the whole domain can shift to a dissimilar binding site. In other words, the specific binding capability seems to be influenced by complex relations in the context of adjacent fingers (context dependency) as well as to specific environmental conditions (condition dependency) allowing a finger to change binding preferences at any incremental degree to recognize secondary binding sites [2, 3]. This interconnection between the three fingers might indicate that 3D malleability could affect the three fingers simultaneously, which makes it a powerful tool for effective and sensitive reversibility. However, in static conditions 3D malleability may not occur [8, page 16034]. These evolutionary traits integral to the reversibility apparatus significantly increase the complexity of specific modularity in the sense that subtle changes in the environment can lead to instant subtle changes in the context of the whole domain. This may considerably complicate design of single fingers and the predictability of which the triplet might be recognized in various conditions. 4.10. DNA-Protein Complex Stability and Overlap. To regu- late reversibility, nature may have ways beyond specificity and affinity to influence the formation and dissolution of a DNA-protein complex. An indication for this may be the ability of the 2nd finger to distinguish specific DNA triplets. This demonstrates that three-base modularity in general is plausible, but specifically to further use the modular character for designing zinc finger arrays it has to be taken into account that the binding domains of the 2nd and 3rd fingers reach the 4th converse nucleotide of the binding triplet of the adjacent finger (see Figure 7) (usually referred to as “target site overlap problem” [3, 61]). However, the overlap should not be seen as a problem but rather as an evolutionary trait exerting a certain biological function. The specific binding preferences seen in the RSR code indicate that the overlap has no adverse effect on general three-base modularity and it might be in the range of possibility that nature uses the overlap as part of a reversibility apparatus. Table 3: The 64 triplets of the 2nd finger are divided into four 16-triplet series starting with A, C, G, and T. Evolutionary Dualism and Reversibility of DNA-Protein Complex. One of the fundamental underpinning principles is that evolution is a process in which nature needs to accomplish the duality of conserving and changing gene and protein sequences as well as structures and biological func- tionalities [1, 2, 9, 11, 12, 34, 47, 57]. The evolutionary dualism significantly increases the chances of having straightforward ways of dealing with complexity, change and conservation, and time. Nature after all needs to have pragmatic ways to cope with the extraordinary complexity to adapt in a timely manner to required modifications. In addition, evolutionary dualism has not received particular attention regarding what traits zinc fingers need to make a multi-finger domain viable for clinical application. 15 Biotechnology Research International Biotechnology Research International Biotechnology Research International Biotechnology Research International The RSR code for that provides strong indications that, in order to distinguish between potentially millions of target binding sites in Table 5 by means of inducing incremental differences in cell conditions, the reversibility apparatus needs to include highly sophisticated and delicate mechanisms of which one of them is the ability of a finger to bind to the 4th converse nucleotide. That it is the converse nucleotide might be a purposeful feature in that the location of the converse position is accessible to potential factors that can incept a mechanism to form and dissolve a complex. Following this notion, natural zinc fingers with high affinity that are part of a regulatory network system can be regulated through factors that can initiate a dissolution process at the 4th converse nucleotide. This is not possible for artificial zinc finger arrays that are not part of a regulatory network system. 4.11. Binding Initiation and the Role of the 1st Finger of SP1. Oka reported on previous studies which found unique features in the DNA recognition mode of the 1st finger that “have never been detected in other zinc fingers” [8, page 16027]. According to those accounts the 1st finger has a more relaxed sequence and site specificity than other Cys2 His2 zinc fingers in general. Because of this relaxed base recognition of finger 1, Sp1-(530-623) can bind more various sequences than other multi-C2H2-type zinc fingers, and such a property may be required for the ubiquitous transcription factor Sp1, which activates transcription of many genes [8, page 16034]. The 1st finger contributes less to the DNA binding affinity of SP1, but “the presence of finger 1 is still essential for the high DNA binding affinity” of the entire domain [8, page 16027] [62]. This is a strong attestation for the pivotal context influence the 1st finger exerts instantly on the entire three-finger domain. Eventually, nature has created a delicate system of context dependencies among the three fingers in which the 1st finger was given a key role for establishing and maintaining 16 Biotechnology Research International G G G G G G G G C C C 1 2 3 9 3 - - - - - - - Figure 7: Evolutionary traits of SP1. Figure 7: Evolutionary traits of SP1. might be measurable via a change in the binding energies of both. Biotechnology Research International The inference that can be drawn is that each finger in a domain may have different evolutionary traits and exercise distinct functions at its defined location, which might limit modularity in a way that a finger’s functional traits have to be considered at its original location. Thenceforth, the uniqueness of features is the evolutionary trait of the 1st finger that needs to be replicated or preserved in the design of a clinical viable zinc finger domain. The evolutionary traits are of crucial clinical relevance to utilize the evolutionary mechanisms that control the formation and dissolution of a DNA-protein complex. Because of the potential millions of putative binding sites a three-finger domain can recognize, with a number that is actually significantly greater than that for the 2nd finger in Table 5, one of the unique evolutionary functions of the 1st finger is to initiate the binding process. This is mainly due to its relaxed specificity and affinity and the fact that it does not engage in 4th base overlap binding [8]. The relaxed nature of the 1st finger emphasizes the importance of this feature in that it allows the 1st finger to touch at many positions on the human genome without initiating binding in which specificity and affinity are not the vital features in a “binding initiation mechanism.” The type of measures that can be employed for testing clinical viability might come from [9] who reported that “the sequence context of a binding site significantly influences binding energetics” and that the binding energy provides the “full contextual information” about a complex [9, page 4544]. The evolutionary advantage however demands that the process must be reversible, for which again the relaxed nature and the overlap locus between the 1st and 2nd finger might play key roles in that the protein and the DNA sequence can change binding energies by deforming the 1st finger slightly to trigger the dissolution process. The deformation could be initiated either at the overlap locus between the 1st and 2nd fingers by a factor docking at the overlap locus at the converse nucleotide or by changing the conformation of the DNA via a change in the condition [11]. The overlap mechanism has the evolutionary trait of stabilizing and destabilizing the DNA- protein complex of the regulatory binding mechanism that importantly does not interfere with general modularity of zinc finger design. 4.12. Biotechnology Research International Now with three data sets of the binding energies of the protein domain, the DNA sequence and the DNA- protein complex, we might be able to understand and predict the 3D malleability of protein and DNA in various clinical conditions. Accurate measurement of binding energy might be a fast and efficient way to design and test clinical viable zinc finger proteins and improve their binding recognition capability to the point that only one location has the condition for forming a complex. The changes in binding energies in the binding initiation phase are probably the most delicate and important and together with the observation that “flanking sequence influences binding properties to an unexpected degree” [9], thus influencing binding energy as well, they are a further property that can be used to pinpoint the location a DNA-protein complex can form via compatibility of binding energies of the protein and the DNA sequence. In this way, when the 1st finger touches a DNA sequence at the right position, both 3D structures change and so do their respective binding energies. Complex formation then is only initiated if there is compatibility of structure and binding energy of both DNA and protein. Designing zinc finger domains by measuring binding energies that can be confirmed with struc- tural insight at a later stage might be a more pragmatic and manageable way for fast success of producing clinical viable proteins. In conclusion, if the binding energy of the entire three-finger domains is compatible with the binding energy of the DNA sequence, including the influence of the flanking sequences, the 1st finger will initiate the binding process and will utilize the two overlap loci to stabilize the complex.h a functional domain. The main functions of the 1st finger are binding initiation and the timing of the formation and dissolution of a DNA-protein complex to correctly maneuver reversibility. Notably, [58] found that the 1st finger peptide of SP1 is not stable in acidic solution as are other finger peptides [58]. This may demonstrate that the 1st finger does not function in the same way as other fingers and is able to exert quite different functions in the same condition that can be distinctively different from the functions of the other fingers in the same domain. 5. Evolutionary Issues 5.1. Evolutionary Traits. The evolutionary traits listed in Table 7 seem to be planned and purposeful products of nature which provide vital mechanisms that might be utilized in the design of zinc finger domains to cope with the pervasive complexity.h These evolutionary traits that are part of the various reversibility processes engaged in regulating the formation and dissolution processes of a DNA-protein complex deter- mine the functionality of engineered zinc finger domains. For this, high affinity makes a complex less reversible to the point where tools like zinc finger nucleases stick for an extended period of time on the genome interfering with cell function or causing damaging effects. Looking at Table 7 with potentially millions of putative exact locations for a three-finger domain (which represents just a small sample of 64 out of the 262,144 combinations of 9-mers), it becomes clear that there is an abundance of possibilities to form a stable and enduring complex on the genome. 5.3. Simplicity. The evolutionary success of TFs has to do with adapting quickly to environmental changes and to do so necessitates flexibility to change the components of a regulatory network system on a genetic and functional level. Going through an unsystematic process of trial and error to find simultaneously the one amino acid sequence out of 2.736 and the one nucleotide sequence out of 10605 possible com- binations strongly indicates that nature utilizes underlying simplistic rules to produce modular structures with a high degree of flexible malleability that can be turned into different functional units via minute structural and genetic changes. Existing concepts that might serve as examples for producing complexity out of underlying simplicity can be found in a simple fractal equation’s ability to grow structures that are ever more complex and origami where one plain plane sheet of paper can be folded in unlimited ways to form endless forms. 5.2. Complexity. The immense complexity resulting from previous findings listed in Table 7 still appears to have deeper roots. Observations describe SP1 as regulating transcription “through synergistic effects with other transcription factors” [12, page 36] [63, 64] and supporting cofactors [47] in gene regulatory systems [2]. The role of transcription factors therefore is part of a delicate network which has to emerge entirely and simultaneously in an already existing organism in order to survive the evolutionary selection process. Biotechnology Research International Binding Energy as a Key for Binding Initiation and Complex Formation. With the full contextual information “binding energy” [9, page 4547] might provide a complex and considering the context dependency [8] of a finger domain as well as sequence dependency of DNA structure [11, pages 246- 247], [9], there might be the possibility to assign “potential binding energy” to a protein domain and a DNA sequence and consider them in various conditions in computational models. Because both the DNA and protein can change their conformation to initiate, stabilize, and/or enhance protein binding [11, page 247], this change in the 3D structure i Reversibility of the DNA-protein complex appears to be of essential significance in the design of viable clinical zinc finger proteins. Employing different technologies to measure the complex formation and dissolution properties in various 17 Biotechnology Research International 17 Inferences from the probabilities of a hypothetical sim- plistic gene regulatory network that might contain (1) one target binding site of 9-mers in the promoter region, (2) one 28-amino-acid long zinc finger, and (3) a small 1000-base pair long gene that would deliver the following numbers: (a) 9-mers randomly appear every 700,000 years [46], (b) there are 2.736 different amino acid combinations for one finger, and (c) 10605 possible combinations are to arrange one thousand nucleotides [65], which represents “a complexity for which we have no imagination” especially in comparison to the fact that “only 10108 hydrogen atoms would fit into the whole universe with a radius of 1010light-years” [48]. Notably, this setting still would require a functional organism, which is not considered. This might lead to the conclusion that the practical success of nature to establish ad hoc such an oversimplified regulatory network is so remote in any evolutionary distance of time, that nature more likely employs a strategy of underlying simplicity and modularity where complexity results from a reductionist scientific approach that produces detailed, but fragmented, pieces of data from which the whole of biological functionalities cannot be deduced. conditions might be a manageable way to create a critical mass of data and knowledge to build cytotoxicity-free zinc finger domains. With accurate and clinical relevant data sets it is possible to establish a reversibility index for each DNA-protein complex that can assist in ensuring the clinical feasibility of the zinc finger domain. 5. Evolutionary Issues In this way it is not the amino acid or nucleotide sequence that determines what information is contained, but the underlying intrinsic information of a whole system defines what sequence is needed. In this way, a zinc finger protein as a whole evolves like a landscape out of a simple information fractal or a repeated elementary fold in origami whence building an infinite manifold of things in which the amino acid sequence is the resulting representation of the underlying information. Important in this notion is that the 3D structure and function of the protein are not only determined by the amino acid sequence but also by yet unknown information-related properties that lie outside the observable scope of science. Following the thought of simplicity, the human genome then seems to have an underly- ing information fabric from which nature forms appropriate configurations. entire domain (context dependency), which has significant implications for strategies to produce clinically viable zinc finger domains in which each finger can be gradually adjusted to find a sensible complex for a specific DNA sequence which might produce better molecular tools to achieve successful clinical outcomes. From a historical perspective, since we know that amino acid alteration of natural fingers results in bondage to new DNA target sites [13], it should have become feasible very early on to pursue the creation of libraries of altered domains instead of focusing on single fingers. i Producing precise measures of DNA-protein interactions under one condition does not provide relevant clinical knowledge. Thus to further reduce complexity, there should be a focus on the one condition of iPS cells. A more realistic way to go about this is to think along the lines of comparison of rank-specific recognition codes within large data sets in one condition. Rules can then be deduced that govern certain evolutionary traits that are simple enough to be directly used and modified to designer domains. If one reduces complexity to a point where new discoveries have the most clinical relevance, it is reasonable to argue that the condition of iPS cells among individuals is identical and thus genome modifications are accomplished under standard and repeatable conditions before being differentiated into dissimilar cell types. Most importantly, proper technologies need to be developed that allow continuous measurement of gene expression. 5. Evolutionary Issues The ability alone to create and place all components entirely and simultaneously seems to have to overcome profound complexity that requires consideration of more fundamental issues. Perhaps the most striking findings are the staggering complexity and diversity of DNA binding observed in [2], the fact that transcription factors encode a significant portion of the genome, for example, [5, 35], and that nature has devel- oped gene regulatory networks in a rather short evolutionary period of time [2]. Considering the binding to secondary motifs, which bind equivalently and independently to the primary motif [2], and the observations presented here of rank-specific recognition (Figure 4) together with the number of locations in Table 5 might lead to the serious question of how nature manages to produce viable regulatory systems and what possible ways nature might have taken to produce them. Considerations of these fundamental issues might help to exclude ways that cannot succeed in handling complexity and prepare for taking into account that new and unconventional ideas and approaches from a broad inter- disciplinary perspective are needed for producing clinically relevant outcomes. It has been considered that the human genome has an underlying fractal structure that repeats itself in a modular fashion, for example, [54–56], and that with repeated folding and unfolding processes in origami, for example, [49–53], limitless information might be reversibly used, archived, and revitalized in dynamic information processing cycles, which are the tools of evolution to directly produce and change biological functionalities. Such directional evolution is capable of directly fabricating a selection of modules, in which minute structural differences in the modules can be produced via changes in the microconditions for executing dissimilar functions. The evolutionary selection process then determines the success of the closely related modules from which the capability arises to adapt to evolutionary pressure from changes in the environmental macrocondition. Modu- larity thus is an evolutionary trait that is extensively used by nature to cope with complexity. In terms of building protein and DNA structures, modularity is the repeated use of simple Biotechnology Research International 18 elementary information processing modules that determine the functionality of the protein and the specific amino acid sequence. In other words, the underlying simplicity consists of information processing modules that pinpoint out of the 2.736 and 10605 combinations the exact amino acid and nucleotide sequence. 5. Evolutionary Issues Because of lack of evolutionary traits that could control the biological activity of artificial zinc finger nucleases, it is indeed chal- lenging to build cytotoxicity-free ZFNs in a straightforward way by assembling high specificity and affinity fingers into multifinger domains. More needs are to be understood about the reversible nature of the DNA-protein complex beyond 6.1. Cytotoxicity and Proposition for a Solution. Considering the above findings, the inference that can be made on the nature of cytotoxicity is that engineered zinc finger nucleases bind specifically to an unpredictably high number of locations that are determined by the rank-specific recog- nition of each of the fingers and the binding domain as a whole. In particular, the problem is compounded because zinc finger domains usually have been selected for high affinity and specificity. High affinity causes the complex to remain too long at undesirable locations which cause uncontrollable genome breaches and cell death. Because of lack of evolutionary traits that could control the biological activity of artificial zinc finger nucleases, it is indeed chal- lenging to build cytotoxicity-free ZFNs in a straightforward way by assembling high specificity and affinity fingers into multifinger domains. More needs are to be understood about the reversible nature of the DNA-protein complex beyond 5.4. Evolutionary Plasticity. The dynamic Gestalt form of the human genome then explains the high evolvability and extraordinary evolutionary plasticity needed to react to changes while minimizing the risk of failure as well as having the flexibility to allow minor variation of a sequence and structure that drives expression in a given tissue without otherwise altering the regulatory properties of a gene [1, page 71]. With the extraordinary evolutionary plasticity, nature is able to address the evolutionary dualism of conserving and changing life in an organized fashion. 5.4. Evolutionary Plasticity. The dynamic Gestalt form of the human genome then explains the high evolvability and extraordinary evolutionary plasticity needed to react to changes while minimizing the risk of failure as well as having the flexibility to allow minor variation of a sequence and structure that drives expression in a given tissue without otherwise altering the regulatory properties of a gene [1, page 71]. With the extraordinary evolutionary plasticity, nature is able to address the evolutionary dualism of conserving and changing life in an organized fashion. 5. Evolutionary Issues Such functional nanobiology would provide extremely valuable insight about clinical behavior of zinc finger based molecular tools.h Nature is able to fold and unfold information in the human genome in limitless ways which provides the ability to create endless forms and expresses them via the gene expression path to timely adapt to environmental changes without the need of going through an unmethodical evo- lutionary selection process. In this way, positive selective pressures guide the information unfolding and component forming mechanisms. Reflecting on what is said, it is clear that beyond complexity rules simplicity, and it might be reasonable to see the human genome not merely in a reduced view as a string of three billion nucleotides with a rather fixed static structure encoding only the information for building proteins, but holistically as a dynamic Gestalt that is not the sum of its parts, but always in its totality is an information singularity that has no parts that would encode less than an infinite amount of inseparable information [66]. In such a Gestalt form, the human genome rather functions like an organism with the ability of expressing an interminable variability of forms and systems thus being capable of unde- viating dynamic formability under purposeful evolutionary pressures of directional evolution. i Thus, to guarantee clinical success, it is crucial to focus the development of technologies on delivering the two main ingredients: producing precise data in one condition and modifying the human genome at one location. In a first step, assays need to be developed to make data comparable among the different zinc finger domains. The most practical way to produce precise and repeatable measures is the formation and dissolution process of the DNA-protein complex in various conditions, which at a later stage can be complimented with precise data regarding DNA-protein interaction. 6.1. Cytotoxicity and Proposition for a Solution. Considering the above findings, the inference that can be made on the nature of cytotoxicity is that engineered zinc finger nucleases bind specifically to an unpredictably high number of locations that are determined by the rank-specific recog- nition of each of the fingers and the binding domain as a whole. In particular, the problem is compounded because zinc finger domains usually have been selected for high affinity and specificity. High affinity causes the complex to remain too long at undesirable locations which cause uncontrollable genome breaches and cell death. 6. Discussion Managing Cytotoxicity via Mutated and Sensitized SP1 Domains. The SP1 binding domain has unique evolutionary traits which are not found in other fingers and which are quite clearly responsible for its universal employment throughout nature; see, for instance, [12]. In particular, the widespread appearance of C2H2 zinc fingers in mammals as a recent evo- lutionary event [3] indicates that simplistic underlying rules and procedures keep the observed complexity manageable through inherent evolutionary traits of which 3D malleability allows in general the targeted adjustment of each finger and the context of a particular domain to fulfill a distinct function in various conditions. Of particular interest are both the complex stabilization and dissolution points and the binding initiating capacity of the 1st finger that allows the design of either high or low stabilization (affinity) or high and low dissolution properties as part of the reversibility apparatus. The complexity of the possible combinations that cannot rationally be tested in reasonable evolutionary time suggests 6.3. Managing Cytotoxicity via Mutated and Sensitized SP1 Domains. The SP1 binding domain has unique evolutionary traits which are not found in other fingers and which are quite clearly responsible for its universal employment throughout nature; see, for instance, [12]. In particular, the widespread appearance of C2H2 zinc fingers in mammals as a recent evo- lutionary event [3] indicates that simplistic underlying rules and procedures keep the observed complexity manageable through inherent evolutionary traits of which 3D malleability allows in general the targeted adjustment of each finger and the context of a particular domain to fulfill a distinct function in various conditions. Of particular interest are both the complex stabilization and dissolution points and the binding initiating capacity of the 1st finger that allows the design of either high or low stabilization (affinity) or high and low dissolution properties as part of the reversibility apparatus. The complexity of the possible combinations that cannot rationally be tested in reasonable evolutionary time suggests p The 1st finger of SP1 has the unique evolutionary trait of initiating binding, which makes it the first and foremost tool for controlling the formation of a DNA-protein complex. Of practical importance then is the sensibility of the 1st finger to contact many locations without initiating complex formation through which control of cleavages at off-target sites can be implemented. 6. Discussion The most important finding is that the exchange of amino acids in one finger alters the binding preference of the 19 Biotechnology Research International to allow the nuclease dimer to make one cleavage, which requires high specificity and particularly low affinity of the three-finger domain. This is in particular significant to avoid inducing cleavage at off-target locations where the domain might bind but with such low intensity that the initiation of complex formation is diverted by the sensitized 1st finger. In order to avert off target binding, both the 1st finger and the three-finger domain should have high specificity and low affinity in which ideally the complex should only be held in place at the overlap loci in order to easily release the contact but just long enough to induce cleavage at one location. In addition, high specificity of the domain can be achieved with the influence of flanking sequences [9] next to the binding site that might deter or encourage the formation of a complex. Specifically, the careful design of the 1st finger will improve binding accuracy of a sensitized domain by determining the three-dimensional fit to the target sequence in many ways that influence the 3D malleability of both the protein domain and DNA sequence. The three- dimensional fit between a protein domain and a DNA sequence can be determined when producing measures of the complex formation and dissolution by detecting changes, for example, in the binding energies, thermal differences, and optical absorption. In particular, the potential behavior of a complex can be drawn by characterizing structural changes associated with on- and off-target zinc finger binding as well as their thermal and pH dependence via circular dichro- ism spectroscopy, ultraviolet/visible absorption spectroscopy, dynamic light scattering, and colocalization confocal fluo- rescence microscopy. In combination, the resulting accurate data sets will eventually provide the much needed clinical relevant information to select and verify constructs in various combinations. To ensure single location modification (SLM), further supporting technologies are essential to fully control insertion of genetic material at a single location. For this DNA tagging technologies can be considered to tag the genome at a single location for controlling site-directed modification in which for verification microscopy might be used to detect and verify modifications at the right location. specificity and affinity. 6. Discussion To cope with cytotoxicity, a reasonable approach however would be that the 1st finger would have high specificity without having any affinity to a triplet until the 1st overlap locus supports complex formation, in which event the affinity of the 1st finger should switch to a balanced affinity to stabilize the complex. The life span of the complex should be just long enough to regulate a gene and short enough to get dislodged before inducing any irregularities. This precise balance is nature’s key for achieving evolutionary success which needs to be replicated to build clinically viable binding domains. 6.2. Evolutionary Traits and Aspects of SP1. A practical way to achieve clinical solutions is to modify the natural framework of SP1 by leaving each finger at its evolutionary location. In this way, there might be the opportunity to retain known and unknown evolutionary traits of SP1 and utilize them for binding new target sequences that might give enough control for successfully using them in clinical applications. Strong support for SP1 as a candidate is the finding that SP1 is both highly conserved throughout evolution and used in many organisms, tissues, and stages during development [12, page 39] [1, page 70]. The key question is how nature can use the highly conserved SP1 binding domain for fulfilling a variety of different functions in different conditions, and the most meaningful answer is via the malleability of its 3D structure of the binding domain without changing the amino acid sequence. The flexibility that provides SP1 with the universality to be used throughout nature is a result of its inherent evolutionary traits of which two are illustrated in Figure 7.i Considering the narrowly defined purpose of this study to produce clinically viable tools in an ethically meaningful time frame and manageable way, the discussed observa- tions (listed in Table 7) indicate a potential way to suc- ceed without gaining full understanding of all components. Of practical importance for a manageable approach are reversibility, the rank-specific recognition code, the 1st fin- ger, and the overlap loci which can be influenced and designed in a way to create a clinical viable domain. The complexity following most evolutionary traits in Table 7 might be beyond the practical capabilities of direct mea- surement and influence; however, they are indirectly being accounted for when studying the reversibility mechanisms of the formation and dissolution processes of the DNA-protein complex.hi 6.3. 6. Discussion When using zinc finger nuclease, however, the sensibility needs to be particularly refined and the 1st finger particularly sensitized because of a lack of a regulatory network system that controls binding initiation and reversibility of the complex. To avoid cytotoxicity, the complex should contact the target location just briefly enough Biotechnology Research International 20 Table 8: Sensitizing the SP1 framework. Potential design strategies for sensitizing versions of the SP1 framework. 1 × 1st finger 321 2 × 1st finger 3211 2 × 3 design 321321 3f + 4f design 3213211 2 × 4f design 32113211 (to lower affinity and higher specificity) Table 8: Sensitizing the SP1 framework. Potential design strategies for sensitizing versions of the SP1 framework. the 1󸀠󸀠󸀠-Finger initiates binding because if any of the other fingers binds before the domain as whole cannot be sensitized and the frequency of off-target binding occurrences would be uncontrollable. The binding sequence should follow a zipper pattern: starting with the 1󸀠󸀠󸀠-Finger and concluding with the 3󸀠-Finger. After forming a DNA-protein complex with the first subdomain, the second crucial point to sensitize the domain is the 1󸀠-Finger in the second subdomain to prevent the complex formation of the entire domain if the complex of the first subdomain is at an off-target location. It is notable that the affinities of all the fingers are the lowest possible just at the point to form a DNA-protein complex (lower rank in the RSR code). The first finger might be able to be designed by substituting amino acids to be sensible to certain triplets in the sense of having low affinity and high specificity to a triplet. Considering the 1,261,301 exact 9-mer locations in the human genome it is of importance to eliminate as many of those 9-mer locations as possible by making the 3-mer initiation binding occurrence as sensible as possible. To design the most sensitive binding, the 1st finger needs to be adjusted to the cell type environment and context to the other fingers and the nucleotide sequence of the target site that is highly flexible due to deformability, a feature that is used by proteins to recognize specific DNA sequences rephrase [11, page 242]. that simple underlying rules do let the right combination emerge at a particular time and such rules might be revealed by studying in depth a natural zinc finger domain and its modifications. 6. Discussion Thus it is a prudent approach to take advantage of inherited evolutionary traits to improve binding accuracy. It is reasonable to assume that each SP1 finger can be modified by substituting amino acids in zinc fingers that result in altered DNA binding recognition [8, 12, 13, 67] and it might be possible to utilize some of nature’s evolutionary traits. Depending on the form of the DNA [11, page 242], amino acids can be replaced in the fingers of SP1 to recognize AT- rich boxes. Indication for this can be seen in the RSR code as the occurrence of AT-rich boxes with high P32 counts: TTC (the 5th highest), TAG (9th) and AT-boxes TAA (17th), TAT (21st), and AAT (25th). For the HBB example several strategies might improve accuracy of binding to significantly reduce cytotoxicity. The kernel of several potential strategies listed in Table 8 is the use of the 1st finger and the SP1 framework as a whole to create combinations out of the two components to increase sensibility and specificity in order to obtain clinical viable domains. (1) Strategy 1 incorporates the exchange of amino acids in the alpha helical region of SP1 to create mutants with a different rank-specific recognition code; (2) in strategy 2 it might be of use to add a second 1st finger to increase the sensibility and specificity of the initial contact; (3) strategy 3 follows the Klug reviewed approach to thread together two three-finger domains to obtain a six-finger domain with higher domain specificity; (4) strategy 4 adds a second 1st finger to create a seven-finger domain; and (5) strategy 5 is an eight-finger domain which includes four 1st fingers. 6.4. A Practical Approach: Interdisciplinary Innovation and New Technologies. The complexity of the matter at hand seems to coerce a clinical solution consisting of an alliance of scientific and managerial skills and the concerted effort of genuine collaborators. For medical and social purposes, genuine collaborative environments must be formed to create an ethical value which cannot be created by individuals or institutions alone. It is of ethical urgency to make therapies that have been successfully developed in animal models available to cure patients. In the case of sickle-cell anemia this requires a full understanding of the nature and mechanisms of “off-target” binding. 6. Discussion It is crucial that only Biotechnology Research International 21 - - - - - - - - - - - - - - - - - - C A C C T G T G G A C G T C T C A T C T A C T G A A A C C C T G G A C - - C G G C G First subdomain Second subdomain Complex stabilization/ dissolution overlap loci Binding-initiation domain Fok I nuclease domain 3-finger 3-finger 2-finger 1-finger 1󳰀-finger 1󳰀-finger 2󳰀-finger 3󳰀-finger 1󳰀󳰀󳰀-finger 1󳰀󳰀󳰀-finger 1󳰀󳰀-finger Binding initiation site 2 × 12-mer binding sites SP1 sensitized designer domain Figure 8: Sensitizing the SP1 framework.Potential design strategies for sensitizing versions of the SP1 framework for HBB gene target. Figure 8: Sensitizing the SP1 framework.Potential design strategies for sensitizing versions of the SP1 framework for HBB gene target. (1) in vitro and in vivo cell-based assays, (2) customized high precision detection instruments, (3) functional nanobiology, for example, to measure continuous gene expression, and (4) computational tools to capture, process, analyze, and reuse data. In this, focusing on the 64 binding sites for each finger of SP1 reduces complexity to a point where it might be man- ageable to generate precise and repeatable data with a variety of instruments and assays that can be used to develop accurate computational tools to predict complex formation in various conditions. (1) in vitro and in vivo cell-based assays, (2) customized high precision detection instruments, (3) functional nanobiology, for example, to measure continuous gene expression, and (4) computational tools to capture, process, analyze, and reuse data. In this, focusing on the 64 binding sites for each finger of SP1 reduces complexity to a point where it might be man- ageable to generate precise and repeatable data with a variety of instruments and assays that can be used to develop accurate computational tools to predict complex formation in various conditions. conditions. Furthermore, integration of data sets from exist- ing assays such as DNA affinity precipitation assay, dual- luciferase promoter activity assay, SP1-knockout mice [12], microarrays [2], and a variety of other methods [6] might complement the overall effort. The core technologies also provide the ability to study DNA-binding properties of transcription activator-like effectors (TALEs) that can be developed into robust tools for controlling the introduction of genetic material, for instance [68]. 6. Discussion The purpose of ethical research is to enable concerted collaborative efforts to reduce suffering by developing end-point therapies in an accelerated and manageable way. Because of the complexity at hand, the goal of understanding protein-DNA interactions remains elusive until the underlying simplistic rules can be determined. To manage technical progress in the short term, complexity needs to be reduced to a point where accurate and repeatable data can be produced and fully understood in the exemplary case of the three-finger SP1 domain and each of its fingers. Also, technologies which can be applied on a broad scale must be developed. While most of the research efforts are dedicated to detect binding sites and identifying TFs on genomes, little has been done to understand the biological functions [35]. The general lack of understanding of TFs [36] promotes the idea to reduce complexity and develop core technologies that delve into the very details of DNA- protein interactions, complex formation and dissolution, and evolutionary fundamentals [35]. To bridge this gap which sig- nificantly hinders scientific progress of gene regulation and genome modification, research needs to address issues about the fundamental aspects here. This should include three parts: ii It remains to be seen which strategy is more practical and manageable to produce viable outcomes. To discuss the various features, the eight-finger domain of strategy 5 has been drawn in Figure 8 and might have an enriched sensibility to the point of clinical relevance.h The strategy illustrated in Figure 8 is to use the SP1 frame- work as a whole to fully utilize the different evolutionary traits and functions of each finger. The entire binding domain is composed of two SP1 subdomains each enhanced with an additional 1st finger. The 1st finger of SP1 that initiates the binding process is of significant importance for preventing the two domains from binding at off-target sites and having two 1st fingers in each subdomain allows successively placing the fingers resulting in the first subdomain to complete half of the complex formation starting with 1󸀠󸀠󸀠-Finger which has the function of initiating the binding process. It requires that the 1󸀠󸀠󸀠-Finger needs to have a slightly higher affinity than all the other fingers in the domain. 7. Limitations The reduction of complexity brought about the valuable insight of rank-specific recognition. However, many aspects remain to be discovered. For example, of interest is to determine the number of exact matches in Table 5 that occur in promoter regions of genes to define more precisely how many matches should be regarded as “off-target.” In particular, because of the strong influence of condition dependency, single assay results remain tentative. For each clinical condition a rank-specific recognition code needs to be established together with more precise assays that make cytotoxicity, reversibility, and genotoxicity precisely quantifiable. 6. Discussion In order to escape cytotoxicity, however, the core chal- lenge is to fully control the introduction of genetic material at a single location in the human genome, which is for sickle- cell anemia the cleavage of the genome and introduction of the healthy donor via homologous recombination at the exact HBB location signified in Figures 1 and 2. Most importantly, these technologies are applicable to introducing the factors for creating induced pluripotent stem (iPS) cells at the proper locations in the human genome. To further ensure single location modification (SLM), supporting technologies such as DNA tagging at a single location are essential to fully control and verify insertion of genetic material at a clinically relevant single location. Especially clinically relevant are technologies that measure the formation and dissolution of a DNA-protein complex which can provide feedback on the sensitivity and reversible behavior of a binding domain. With this in mind, on the technical side we have supplemented our experimental capabilities by taking advantage of the broad selection of tools available in the Soft and Biological Nanomaterials Section of the Center for Functional Nano- materials in Brookhaven National Laboratory. We will be characterizing structural changes associated with on- and off-target zinc finger binding, as well as their thermal and pH dependence, via circular dichroism spectroscopy, ultra- violet/visible absorption spectroscopy, dynamic light scatter- ing, and colocalization confocal fluorescence microscopy. In combination, the resulting accurate data sets will eventually provide the much needed understanding of the functional biology of the binding mechanisms. 8. Contributions 8.1. Overall Contribution. The overall contribution of this study is that we persuasively argue that there are no general rules for affinity and specificity of DNA binding of zinc finger domains because of condition dependency of binding. Refinements of existing as well as additional definitions are provided. 8.1.1. The Existing Literature Appears to Describe Affinity in Consensus as the Strength of Noncovalent Temporary Binding of a Zinc Finger Domain to a DNA Sequence. However, strength of binding to DNA is not the only translational important and clinical relevant measure of affinity. Refined definitions of affinity should include the circumstance that preferably one zinc finger domain should bind to only one single location in the human genome. This would make it safe 8.1.1. The Existing Literature Appears to Describe Affinity in Consensus as the Strength of Noncovalent Temporary Binding of a Zinc Finger Domain to a DNA Sequence. However, strength of binding to DNA is not the only translational important and clinical relevant measure of affinity. Refined definitions of affinity should include the circumstance that preferably one zinc finger domain should bind to only one single location in the human genome. This would make it safe When those data sets and constructs are available, two major technological and scientific achievements have been accomplished: (1) a scientific base for clinical viable constructs and (2) the technological base to examine the actual DNA-protein interactions and behavior in various Biotechnology Research International 22 for clinical application to modify one diseased location [17] in the human genome.i and adherence that takes condition dependency into account should result in identifying and consequently developing novel strategies for reducing side effects in which the “goal for optimal zinc finger design is to generate high affinity to the intended target, with low affinity to additional sites in the genome [14, page 3] [70]” and that this might be accomplishable by using evolutionary traits to sensitize a three-finger domain (making a domain sensible to only bind to one location) to the point that a zinc finger nuclease (ZFN) only induces a functional effect at the intended target site but not at additional locations it binds to in the genome.i We contend that the three-finger domain of the zinc finger protein (ZFP) SP1 significantly increases its affinity to a specific DNA 9-mer sequence by “locking in” binding by means of a 4th base overlap mechanism of its 2nd and 3rd fingers. Comments (i) Unregulated binding of artificial zinc finger domains to locations on the human genome causes side effects. Consequently, we argue for an additional definition of location specificity (in contrast to sequence specificity) in which natural zinc finger proteins form a DNA/protein complex at particular locations in the human genome. The DNA sequences can be dissimilar at the different locations because of the condition dependency of forming a biological active complex. (ii) Unregulated binding of natural zinc finger domains does not induce biological activity or functional effect. (i) Regulated binding by a largely unknown reversibility apparatus, (ii) 1st finger initiating binding, (ii) 1st finger initiating binding, (iii) 4th base overlap loci “locking in” to form the DNA/protein complex that allows the protein being active to perform its function. 8.1.2. Sequence Specificity Is the Selective Binding of a Zinc Finger Domain to Preferably Only One Specific DNA Sequence. Our own as well as other previous findings show that a 9-mer DNA sequence to which a three finger zinc finger domain binds occurs thousands of times in the human genome. This degeneration of sequence specificity, for example, [2, 3] where there is more than one DNA sequence that a zinc finger domain binds to, requires further refinement and additional definitions of specificity. ZFP/DNA Binding Definition. Binding of natural and artificial zinc finger proteins (ZFPs) or their binding domains to many locations in the human genome without inducing a biologicalf ZFP/DNA Binding Definition. Binding of natural and artificial zinc finger proteins (ZFPs) or their binding domains to many locations in the human genome without inducing a biological activity or having a functional effect (no formation of a DNA/protein complex): in contrast, artificial zinc finger domains with high unregulated affinity can establish bindings that allow unregulated functional effects (e.g., the nuclease of a ZFN tool that induces side effects). In refinement, we contend that there is no general sequence specificity of a zinc finger domain to specific DNA target sites but that targeted specificity is accomplished by a cell-type specific reversibility apparatus of which the 4th base overlap mechanism is an important factor to accomplish targeted specificity at specific locations. 8.3. Difference of Complex Formation and Zinc Finger Binding We may be able to replicate or preserve nature’s reversibil- ity apparatus by carefully modifying natural domains to bind novel intended target sites as has been previously demonstrated [13, 69]. DNA/Protein Complex Definition. Active DNA/protein com- plex that has the authority to induce a functional effect or biological activity with regulated affinity (adherence) by a condition-dependent reversibility apparatus. To distinguish between the two affinities we define reg- ulated affinity as adherence of zinc finger domains to a single location in the human genome. The zinc finger domain forms a complex only at particular locations in the human genome and because of condition dependency of binding the DNA target site at the different locations can be dissimilar. Comments (i) Regulated binding by a largely unknown reversibility apparatus, 8. Contributions This mechanism locks and stabilizes the DNA/protein complex and enables the complex to induce a functional effect or biological activity. Consequently, we contend that there are two types of affinities: regulated and unregulated affinity; for regulated affinity nature employs a reversibility apparatus to regulate affinity of three-finger domains where it controls the formation and dissolution of the DNA/protein complex but not for unregulated affinity.h In summary, our own as well as other previous findings indicate that there are three translational factors that regulate biological activity of natural C2H2 zinc finger domains: reversibility, adherence and specificity [40], and to a lesser extend unregulated affinity. We suspect that high unregulated affinity is associated with elevated toxicity and side effects.i fi This makes DNA binding well planned and reversible. A zinc finger domain has to be “locked in” in order to induce an effect. In contrast, unregulated affinity allows uncontrolled binding at many locations in the human genome which may induce severe clinical side effects. Natural zinc finger proteins do not display side effects because unregulated binding at a location does not induce a functional effect or biological activity. fif Based on our own and previous findings, we conclude with the following definitions that have the potential of fostering advancements of translational research. 8.3. Difference of Complex Formation and Zinc Finger Binding Comments (i) Artificial designer zinc finger domains are not regu- lated by a reversibility apparatus. (ii) Artificial designer zinc finger domains are able to form a functionally active binding. In zinc finger nucleases (ZFNs), the nuclease can execute its func- tion of cutting a single strand of DNA at many locations on the human genome which results in toxic side effects. (ii) Artificial designer zinc finger domains are able to form a functionally active binding. In zinc finger nucleases (ZFNs), the nuclease can execute its func- tion of cutting a single strand of DNA at many locations on the human genome which results in toxic side effects. (4) Functional Reversibility of DNA-Binding Complex Defini- tion. Functional reversibility is the regulatory mechanism that governs attachment of an active DNA-binding complex at a specific location in the human genome. It is the time frame of activity during which a DNA/protein complex can exert a functional effect or biological activity at specific locations in the human genome. (iii) Modifying natural zinc finger’s specificity without changing its framework [13, 69] might still be regu- lated by a specific cell’s reversibility apparatus. (iii) Modifying natural zinc finger’s specificity without changing its framework [13, 69] might still be regu- lated by a specific cell’s reversibility apparatus. (iii) Modifying natural zinc finger’s specificity without changing its framework [13, 69] might still be regu- lated by a specific cell’s reversibility apparatus. (i) Atomic forces are condition dependent.i (ii) General rules for zinc finger domains for binding the same target site for all conditions cannot be established. 9. Definitions Arrived at and Used in This Paper 8.2. Translational Research: Reversibility and Adherence . We persuasively argue that translational research on reversibility (1) Functional Adherence (Regulated Affinity) Definition. Functional adherence is regulated affinity that is defined as Biotechnology Research International 23 adhesion or binding (attachment) that lasts for a specifically controlled time frame; a DNA/protein complex is functionally active to induce a functional effect or biological activity. The attachment is regulated by a cell-specific reversibility apparatus. Part of a reversibility apparatus is the 4th base overlap mechanism that increases the strength of noncovalent bonds. Comments (i) The longer the time the higher the specificity, (i) The longer the time the higher the specificity, (ii) if the time is too short, there is no formation of a DNA/protein complex, so (iii) the longer the time the higher the probability of forming a DNA/protein complex, (iii) the longer the time the higher the probability of forming a DNA/protein complex, (iv) the time a ZFP is attached at a specific location in the human genome where induces a clinically relevant activity. Comments (i) Binding regulated by reversibility apparatus. (2) Unregulated Affinity Definition. Unregulated affinity is defined as noncovalent temporary and uncontrolled adhe- sion or binding (attachment) that lasts for a random time frame. Unregulated bindings of natural DNA-binding proteins do not induce a functional effect or biological activity.i (2) Unregulated Affinity Definition. Unregulated affinity is defined as noncovalent temporary and uncontrolled adhe- sion or binding (attachment) that lasts for a random time frame. Unregulated bindings of natural DNA-binding proteins do not induce a functional effect or biological activity.i (ii) Induced and timed biological activity and artificial functional effect. (iii) Regulation of binding accomplished using the 4th base overlap loci that lie at the opposite site of the DNA/protein binding grooves. Adhesion or binding of artificial DNA-binding proteins and, especially, zinc finger nucleases (ZFNs) with high affinity to a condition-dependent thus unspecifiable number of DNA sequences lasts longer than a certain nonfunctional time frame with the ability to induce a functional effect that can lead to clinical side effects. (iv) Binding initiated by the 1st finger enhances selectivity and decreases affinity. Binding sites that would have high affinity but low specificity to a domain do not undergo binding-initiation by the 1st finger. (5) Nonfunctional Reversibility of DNA Binding Definition. Nonfunctional reversibility of DNA binding of, for example, unregulated zinc finger protein (ZFP) binding: an engineered zinc finger nuclease (ZFN) tool affects and changes the genome uncontrollably producing clinical side effects. Box 1 Box 1 High location specificity + high adherence →high reversibility → manageable side effects/low–very low toxicity Box 2 natural biological activity or artificial functional effect that is reversible and well planned. research effort needs to be organized to uncover the “biologi- cal truth” [38, page 141] and “underlying biology of regulatory mechanisms (which) is very incomplete understood” [38, page 140]. The rank-specific recognition code of a single finger sheds light on the nature and scope of “off-target” binding and associated cell death and apoptosis [6]. A simple table of all triplets as has been deemed “extremely useful” [3, page 9] for each finger of SP1 would be particularly helpful in estimating the level of cytotoxicity that might be associated with a three-finger domain. The known and utilizable evolutionary traits of overlap, specificity, condi- tion dependency, and context dependency together might be a viable way to produce cytotoxicity-free zinc finger domains. Combined with data from RSR, various in vitro and in vivo assays with computational analytic tools, the binding accuracy of a binding domain can be significantly increased. Our recommendation is that a three-finger domain with high location specificity, high adherence and high reversibil- ity, and low unregulated affinity will show the lowest toxicity and clinical side effects. We contend that unregulated affinity of artificial zinc finger domains is the problem while trans- lational researchers tend to consider that adherence induced by the 4th base overlap mechanism of the 2nd and 3rd fingers of SP1 stabilizes the DNA/protein complex. Adherence occurs when the 4th base overlap of the 2nd and 3rd fingers of SP1 “locks in.” The consequence is that the “lock-in” of the DNA/protein complex allows the protein to fulfill its unique function. The “lock in” function is associated with a “lock-out” function. It allows nature to control DNA/protein complex binding at a single location in the genome with the same or different target DNA sequences at different locations by changing the conditions. Dealing with three rank-specific recognition codes of the three fingers of the SP1 domain and considering the interdependency among the adjacent C2H2 fingers while distinguishing between relevant and nonrelevant 9-mers under certain conditions are an immense computational task that needs to be done in order to use the technology in clinical settings. This can help to identify the biological active 9-mers out of a pool of 262,144 putative 9-mers. Box 1 This number of combinations cannot be lab-tested even with high throughput testing. In addition, data sets from one assay alone will not supply sufficient information to build accurate computational tools to design novel proteins for any location on the human genome and predict target- binding sites. To bring research onto a manageable level the focus on the three fingers and the framework of SP1 as an exemplary case to gain full understanding should supply knowledge on how to approach other venues of research. For this, standards and reproducible methods need to be established. Such a task needs an unprecedented concerted collaborative interdisciplinary effort as well as organizational and managerial tasks. Clinical endpoints, so to speak, might be pursued by an interdisciplinary approach including the specific disciplines of biology, biomedical engineering, nan- otechnology, bioinformatics, computational protein folding, fractal, and origami to generate accurate data sets to yield 10. Assessments Assessment of toxicity of artificially created three-finger domains with unregulated binding affinity is, according to our and previous findings, displayed in Box 1.ii i Natural three-finger frameworks of natural zinc finger domains that are carefully modified to alter their binding specificity that keeps their reversible regulated binding affin- ity intact would presumably have low or no toxicity that might prove successful in personalized therapies; see Box 2. Comments (iii) ZFP might bind to a specific DNA sequence in one condition (cell type) but to another DNA sequence in another condition (cell type). (iii) ZFP might bind to a specific DNA sequence in one condition (cell type) but to another DNA sequence in another condition (cell type). (i) Binding of natural zinc finger proteins does not induce a functional effect or biological activity. Bind- ing of artificial zinc finger domains with high affinity is not released in a timely manner causing side effects. (iv) Nuclease of artificial zinc finger nucleases (ZFNs) seems to be causing damage at casual ZFP/DNA binding locations on the human genome. (iv) Nuclease of artificial zinc finger nucleases (ZFNs) seems to be causing damage at casual ZFP/DNA binding locations on the human genome. (ii) Artificial zinc finger domains can form an unregulated DNA/protein complex of ZFNs causing clinical side effects because the “lock-in” situation initiated by the 4th base overlap remains intact unregulated. (v) If no time sensitive regulation occurs via the 4th base mechanism, a zinc finger domain binds unregulated to many locations inducing a functional effect causing side-effects. Our own and previous findings support the idea that it is necessary to shift the research focus of translational research from specificity and affinity to reversibility, adherence, and specificity of a DNA/protein complex and to a lesser extent to unregulated affinity of a zinc finger domain. We see the 4th base overlap of the 2nd and 3rd fingers of SP1 as a “lock- in” mechanism that stabilizes a DNA/protein complex that allows a natural zinc finger protein to induce its intended (3) Specificity Definitions. (1) Sequence specificity is the binding of zinc finger domain and DNA-binding factors to preferably only one specific DNA sequence. (2) Location specificity is the binding to preferably only one location in the human genome. 24 Biotechnology Research International Biotechnology Re High affinity + high specificity →medium reversibility →medium toxicity high affinity + low specificity →low reversibility →high toxicity low affinity + medium specificity →medium reversibility →medium toxicity Box 1 High location specificity + high adherence →high reversibility → manageable side effects/low–very low toxicity Box 2 References [18] J. Wang, G. Friedman, Y. Doyon et al., “Targeted gene addition to a predetermined site in the human genome using a ZFN- based nicking enzyme,” Genome Research, vol. 22, no. 7, pp. 1316–1326, 2012. [1] M. T. Weirauch and T. R. Hughes, “Conserved expression with- out conserved regulatory sequence: the more things change, the more they stay the same,” Trends in Genetics, vol. 26, no. 2, pp. 66–74, 2010. [19] R. van Rensburg, I. Beyer, X.-Y. Yao et al., “Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells,” Gene Therapy, vol. 20, no. 2, pp. 201–214, 2012. [2] G. Badis, M. F. Berger, A. A. Philippakis et al., “Diversity and complexity in DNA recognition by transcription factors,” Science, vol. 324, no. 5935, pp. 1720–1723, 2009. [20] F. Buchholz and J. Hauber, “Engineered DNA modifying enzymes: components of a future strategy to cure HIV/AIDS,” Antiviral Research, vol. 97, no. 2, pp. 211–217, 2013. [3] K. N. Lam, H. van Bakel, A. G. Cote, A. van der Ven, and T. R. Hughes, “Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays,” Nucleic Acids Research, vol. 39, no. 11, pp. 4680–4690, 2011. [21] A. Trounson, R. G. Thakar, G. Lomax, and D. Gibbons, “Clinical trials for stem cell therapies,” BMC Medicine, vol. 9, no. 1, article 52, 2011. [4] S. Myers, R. Bowden, A. Tumian et al., “Drive against hotspot motifs in primates implicates the PRDM9 gene in meiotic recombination,” Science, vol. 327, no. 5967, pp. 876–879, 2010. [22] L.-T. Cheng, L.-T. Sun, and T. Tada, “Genome editing in induced pluripotent stem cells,” Genes to Cells, vol. 17, no. 6, pp. 431–438, 2012. [5] A. Klug, “The discovery of zinc fingers and their applications in gene regulation and genome manipulation,” Annual Review of Biochemistry, vol. 79, pp. 213–231, 2010. [23] C. Yan and P. J. Higgins, “Drugging the undruggable: transcrip- tion therapy for cancer,” Biochimica et Biophysica Acta, vol. 1835, no. 1, pp. 76–85, 2013. [6] T. Cathomen and J. Keith Joung, “Zinc-finger nucleases: the next generation emerges,” Molecular Therapy, vol. 16, no. 7, pp. 1200–1207, 2008. [24] N.-H. Zschemisch, S. Glage, D. Wedekind et al., “Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat,” BMC Immunology, vol. 13, no. 1, article 60, 2012. [7] C. L. Ramirez, J. E. Foley, D. A. Acknowledgments The authors would like to thank R. Tjian for supplying the plasmid pPacSP1-516c as well as IBM and Intel for graciously supplying computational infrastructure. They are especially thankful for the support of Paul Kontogiorgis at IBM and Brian Lenaghan at Intel and H.-J. Thiesen for his continuous support. They also thank members of the Center of Functional Nanotechnology at BNL for providing scientific insights and technological support. Research was carried out in part at the Center for Functional Nanomaterials, Proposal no. 30097, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract no. DE-AC02-98CH10886. Special thanks are due to M. Bishop and C. Gajdusek for reviewing the work. [13] H.-J. Thiesen and C. Bach, “Determination of DNA binding specificities of mutated zinc finger domains,” FEBS Letters, vol. 283, no. 1, pp. 23–26, 1991. [14] D. Davis and D. Stokoe, “Zinc Finger Nucleases as tools to understand and treat human diseases,” BMC Medicine, vol. 8, no. 42, pp. 1–11, 2010. [15] P. Perez-Pinera, D. G. Ousterout, and C. A. Gersbach, “Advances in targeted genome editing,” Current Opinion in Chemical Biology, vol. 16, no. 3-4, pp. 268–277, 2012. [16] C. M. Niemeyer, “Semisynthetic DNA-protein conjugates for biosensing and nanofabrication,” Angewandte Chemie, vol. 49, no. 7, pp. 1200–1216, 2010. [17] J. Hanna, M. Wernig, S. Markoulaki et al., “Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin,” Science, vol. 318, no. 5858, pp. 1920–1923, 2007. 11. Conclusion Cytotoxicity is the outcome of deleterious genetic changes in the human genome which are not well understood and beyond the control of present technology. Observation of cell death and apoptosis is widely associated with excessive cleavage at “off-target” sites, which has been attributed to imperfect target site recognition by a zinc finger binding domain [6, 39, 40]. In order to meet the ethical requirements of bringing cures to patients in an uncompromised safe as well as morally fastest way, a concerted interdisciplinary 25 Biotechnology Research International function,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 10, pp. 4544–4549, 2010. molecular tools, comprehensive knowledge, and collabora- tion that forms the basis for a branch of ethical research to cure unprofitable diseases. [10] M.-S. Kim, G. Stybayeva, J. Y. Lee, A. Revzin, and D. J. Segal, “A zinc finger protein array for the visual detection of specific DNA sequences for diagnostic applications,” Nucleic Acids Research, vol. 39, no. 5, article e29, 2011. Conflict of Interests [11] R. Rohs, X. Jin, S. M. West, R. Joshi, B. Honig, and R. S. Mann, “Origins of specificity in protein-DNA recognition,” Annual Review of Biochemistry, vol. 79, pp. 233–269, 2010. The authors declare that there is no conflict of interests regarding the publication of this paper. [12] C.-J. Lin, T.-H. Hsiao, Y.-S. Chung et al., “Zebrafish Sp1-like protein is structurally and functionally comparable to human Sp1,” Protein Expression and Purification, vol. 76, no. 1, pp. 36– 43, 2011. Biotechnology Research International 7243, pp. 73–76, 2009. [33] J.-S. Kim, H. J. Lee, and D. Carroll, “Genome editing with modularly assembled zinc-finger nucleases,” Nature Methods, vol. 7, no. 2, p. 91, 2010. [51] C. E. Castro, F. Kilchherr, D.-N. Kim et al., “A primer to scaffolded DNA origami,” Nature Methods, vol. 8, no. 3, pp. 221– 229, 2011. [34] S. J. Maerkl and S. R. Quake, “Experimental determination of the evolvability of a transcription factor,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 44, pp. 18650–18655, 2009. [52] P. W. K. Rothemund, “Folding DNA to create nanoscale shapes and patterns,” Nature, vol. 440, no. 7082, pp. 297–302, 2006. [35] J. M. Vaquerizas, S. K. Kummerfeld, S. A. Teichmann, and N. M. Luscombe, “A census of human transcription factors: function, expression and evolution,” Nature Reviews Genetics, vol. 10, no. 4, pp. 252–263, 2009. [53] Y. He, T. Ye, M. Su et al., “Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra,” Nature, vol. 452, no. 7184, pp. 198–201, 2008. [54] B. V. S. Iyer, M. Kenward, and G. Arya, “Hierarchies in eukaryotic genome organization: insights from polymer theory and simulations,” BMC Biophysics, vol. 4, no. 1, article 8, 2011. [36] T. A. Desai, D. A. Rodionov, M. S. Gelfand, E. J. Alm, and C. V. Rao, “Engineering transcription factors with novel DNA- binding specificity using comparative genomics,” Nucleic Acids Research, vol. 37, no. 8, pp. 2493–2503, 2009. [55] E. Lander, “Initial sequencing and analysis of the human genome,” Nature, vol. 409, no. 6822, pp. 860–921, 2001. [37] J. Liu and G. D. Stormo, “Context-dependent DNA recognition code for C2H2 zinc-finger transcription factors,” Bioinformatics, vol. 24, no. 17, pp. 1850–1857, 2008. [56] E. S. Lander, “Initial impact of the sequencing of the human genome,” Nature, vol. 470, no. 7333, pp. 187–197, 2011. [57] R. Rohs, S. M. West, P. Liu, and B. Honig, “Nuance in the double- helix and its role in protein-DNA recognition,” Current Opinion in Structural Biology, vol. 19, no. 2, pp. 171–177, 2009. [38] M. Tompa, N. Li, T. L. Bailey et al., “Assessing computational tools for the discovery of transcription factor binding sites,” Nature Biotechnology, vol. 23, no. 1, pp. 137–144, 2005. [58] V. A. Narayan, R. W. Kriwacki, and J. P. Caradonna, “Structures of zinc finger domains from transcription factor Sp1. Insights into sequence-specific protein-DNA recognition,” Journal of Biological Chemistry, vol. 272, no. Biotechnology Research International Biotechnology Research International Biotechnology Research International 26 [44] H.-J. Thiesen and C. Bach, “DNA recognition of C2H2 zinc- finger proteins. Evidence for a zinc-finger-specific DNA recog- nition code,” Annals of the New York Academy of Sciences, vol. 684, pp. 246–249, 1993. [27] V. K. Shukla, Y. Doyon, J. C. Miller et al., “Precise genome modification in the crop species Zea mays using zinc-finger nucleases,” Nature, vol. 459, no. 7245, pp. 437–441, 2009. [28] J. A. Townsend, D. A. Wright, R. J. Winfrey et al., “High- frequency modification of plant genes using engineered zinc- finger nucleases,” Nature, vol. 459, no. 7245, pp. 442–445, 2009. [45] K. Kim, A. Doi, B. Wen et al., “Epigenetic memory in induced pluripotent stem cells,” Nature, vol. 467, no. 7313, pp. 285–290, 2010. [29] D. Mittelman, C. Moye, J. Morton et al., “Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 24, pp. 9607–9612, 2009. [46] S. Behrens and M. Vingron, “Studying the evolution of pro- moter sequences: a waiting time problem,” Journal of Compu- tational Biology, vol. 17, no. 12, pp. 1591–1606, 2010. [47] A. Heffer, J. W. Shultz, and L. Pick, “Surprising flexibility in a conserved Hox transcription factor over 550 million years of evolution,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 42, pp. 18040–18045, 2010. [30] P. Li, Y. Xiao, Z. Liu, and P. Liu, “Using mouse models to study function of transcriptional factors in T cell development,” Cell Regeneration, vol. 1, no. 1, article 8, 2012. [31] A. Gupta, R. G. Christensen, A. L. Rayla, A. Lakshmanan, G. D. Stormo, and S. A. Wolfe, “An optimized two-finger archive for ZFN-mediated gene targeting,” Nature Methods, vol. 9, pp. 588–590, 2012. [48] M. Eigen, “The origin of genetic information: viruses as mod- els,” Gene, vol. 135, no. 1-2, pp. 37–47, 1993. [49] P. Yin, H. M. T. Choi, C. R. Calvert, and N. A. Pierce, “Programming biomolecular self-assembly pathways,” Nature, vol. 451, no. 7176, pp. 318–322, 2008. [32] T. Cathomen, “Zinc-finger nucleases: finding the balance between activity and toxicity,” Human Gene Therapy, vol. 20, no. 11, p. 1356, 2009. [50] E. S. Andersen, M. Dong, M. M. Nielsen et al., “Self-assembly of a nanoscale DNA box with a controllable lid,” Nature, vol. 459, no. References Wright et al., “Unexpected failure rates for modular assembly of engineered zinc fingers,” Nature Methods, vol. 5, no. 5, pp. 374–375, 2008. [25] M. Lusser, C. Parisi, D. Plan, and E. Rodr´ıguez-Cerezo, “Deploy- ment of new biotechnologies in plant breeding,” Nature Biotech- nology, vol. 30, no. 3, pp. 231–239, 2012. [8] S. Oka, Y. Shiraishi, T. Yoshida, T. Ohkubo, Y. Sugiura, and Y. Kobayashi, “NMR structure of transcription factor Sp1 DNA binding domain,” Biochemistry, vol. 43, no. 51, pp. 16027–16035, 2004. [26] M. J. Ortu˜no, A. R. Susperregui, N. Artigas, J. L. Rosa, and F. Ventura, “Osterix induces Col1a1 gene expression through binding to Sp1 sites in the bone enhancer and proximal promoter regions,” Bone, vol. 52, no. 2, pp. 548–556, 2013. [9] C. D. Carlson, C. L. Warren, K. E. Hauschild et al., “Specificity landscapes of DNA binding molecules elucidate biological Biotechnology Research International 12, pp. 7801–7809, 1997. [39] D. Carroll, “Progress and prospects: zinc-finger nucleases as gene therapy agents,” Gene Therapy, vol. 15, no. 22, pp. 1463– 1468, 2008. [40] T. I. Cornu, S. Thibodeau-Beganny, E. Guhl et al., “DNA- binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases,” Molecular Therapy, vol. 16, no. 2, pp. 352–358, 2008. [59] X. Meng, S. Thibodeau-Beganny, T. Jiang, J. K. Joung, and S. A. Wolfe, “Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method,” Nucleic Acids Research, vol. 35, no. 11, article e81, 2007. [41] J. C. Miller, M. C. Holmes, J. Wang et al., “An improved zinc- finger nuclease architecture for highly specific genome editing,” Nature Biotechnology, vol. 25, no. 7, pp. 778–785, 2007. [60] H. J. Kim, H. J. Lee, H. Kim, S. W. Cho, and J.-S. Kim, “Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly,” Genome Research, vol. 19, no. 7, pp. 1279–1288, 2009. [42] A. V. Persikov, R. Osada, and M. Singh, “Predicting DNA recognition by Cys2His2 zinc finger proteins,” Bioinformatics, vol. 25, no. 1, pp. 22–29, 2009. [61] R. R. Beerli, D. J. Segal, B. Dreier, and C. F. Barbas III, “Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger pro- teins constructed from modular building blocks,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 25, pp. 14628–14633, 1998. [43] H.-J. Thiesen and C. Bach, “Target detection assay (TDA): a versatile procedure to determine DNA binding sites as demonstrated on SP1 protein,” Nucleic Acids Research, vol. 18, no. 11, pp. 3203–3209, 1990. 27 Biotechnology Research International Biotechnology Research International [62] M. Yokono, N. Saegusa, K. Matsushita, and Y. Sugiura, “Unique DNA binding mode of the N-terminal zinc finger of transcrip- tion factor Sp1,” Biochemistry, vol. 37, no. 19, pp. 6824–6832, 1998. [63] E. Pascal and R. Tjian, “Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism,” Genes and Development, vol. 5, no. 9, pp. 1646–1656, 1991. [64] K. Thomas, J. Wu, D. Y. Sung et al., “SP1 transcription factors in male germ cell development and differentiation,” Molecular and Cellular Endocrinology, vol. 270, no. 1-2, pp. 1–7, 2007. [65] M. Eigen and P. Schuster, “Stages of emerging life—five princi- ples of early organization,” Journal of Molecular Evolution, vol. 19, no. 1, pp. 47–61, 1982. [66] C. Bach and S. Belardo, The General Theory of Information: Origin of Truth and Hope, Amazon, North Charleston, SC, USA, 2012. [67] P. Bouwman and S. Philipsen, “Regulation of the activity of Sp1-related transcription factors,” Molecular and Cellular Endocrinology, vol. 195, no. 1-2, pp. 27–38, 2002. [68] F. Zhang, L. Cong, S. Lodato, S. Kosuri, G. M. Church, and P. Arlotta, “Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription,” Nature Biotechnology, vol. 29, no. 2, pp. 149–154, 2011. [69] J. R. Desjarlais and J. M. Berg, “Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins,” Proceedings of the National Academy of Sciences of the United States of America, vol. 90, no. 6, pp. 2256– 2260, 1993. [70] S. M. Pruett-Miller, J. P. Connelly, M. L. Maeder, J. K. Joung, and M. H. Porteus, “Comparison of zinc finger nucleases for use in gene targeting in Mammalian Cells,” Molecular Therapy, vol. 16, no. 4, pp. 707–717, 2008.
https://openalex.org/W2231746824	https://europepmc.org/articles/pmc4700569?pdf=render	English	null	Brief report on ecological momentary assessment: everyday states predict HIV prevention behaviors	BMC research notes	2,016	cc-by	5,493	© 2016 Cook 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 Background: Prevention behaviors help persons living with HIV (PLWH) to avoid transmitting HIV, and psychological variables have been found to predict HIV prevention behaviors. These variables have typically been measured using retrospective questionnaires about average psychological states over a period of time, which are likely to be biased by selective recall and interpretation. Measuring the same variables as momentary states, in the day-to-day context where they actually occur, may reveal different relationships to behavior. Findings: 21 PLWH completed daily surveys about momentary states and prevention behaviors. Brief, validated measures were used to assess control beliefs, mood, stress, coping, social support, stigma, knowledge, and motiva‑ tion. We used multilevel models to predict prevention behaviors from momentary states the previous day, while controlling for the effect of multiple observations from the same person over time. Participants reported a moderate overall level of HIV prevention behaviors during the 6-month study. Although lapses in prevention were infrequent, there was room for improvement. Control beliefs, mood, and motivation had significant prospective effects on HIV prevention behaviors, rs = 0.07−0.21. Stress and coping had effects approaching significance. Conclusions: Some momentary states predicted prevention behaviors, providing partial support for the motiva‑ tional model. This finding supports past research showing effects of momentary states on behavior, and advances the science by testing multiple predictors. High within-sample diversity strengthened generalizability, but the overall sample size was small and the findings require replication. Future research should continue to examine the everyday experiences of PLWH as influences on their behavior. Keywords: Ecological momentary assessment, HIV, Motivation, Prevention, Theory with men (MSM) are unprotected even when their part- ner is HIV-seronegative or of unknown serostatus [2]. In addition to reducing new infection rates, prevention using condoms benefits PLWH by reducing their chance of acquiring other sexually transmitted diseases or treat- ment-resistant strains of HIV. Paul F. Cook1, Catherine J. McElwain1 and Lucy A. Bradley‑Springer2 Paul F. Cook1, Catherine J. McElwain1 and Lucy A. Bradley‑Springer2 Correspondence: paul.cook@ucdenver.edu 1 University of Colorado College of Nursing, Campus Box C288‑04, Aurora, CO 80045, USA Full list of author information is available at the end of the article BMC Research Notes BMC Research Notes Cook et al. BMC Res Notes (2016) 9:9 DOI 10.1186/s13104-015-1814-4 Momentary states as predictors of HIV prevention behavior Given that a single failure of HIV prevention behav- iors can lead to transmission, and that these behaviors occur relatively infrequently in the context of everyday life, the future development of both theory and inter- ventions depends on a better understanding of state- level predictors of behavior. Overall patterns tend to be easier to discern than the variables that predict specific incidents, but they are less useful when attempting to intervene [19]. Although many of the predictor variables included in this pilot study are well supported by prior research, the element of time in state-level analyses intro- duces additional within-person variability and limits the applicability of trait-level theories in predicting state- level behaviors [20]. Our goal in the current study was therefore to screen multiple predictors suggested by the motivational model of momentary state influences on behavior [15], in order to guide further theory develop- ment and research. Momentary states as predictors of HIV prevention behavior Many health phenomena including HIV prevention behaviors can be meaningfully understood as momen- tary states that have high levels of within-person vari- ability and fluctuate during everyday life [5–7]. However, most research [4] has measured variables retrospectively and in aggregate as traits, which are long-term and sta- ble patterns of interpreting and responding to events. For example, researchers might ask PLWH about their typical mood or usual motivation over a period of days or weeks, then correlate these reports with similar aggregate measures of behavior. Studies have shown only moderate correlations between state and trait metrics of the same construct [8], and state- versus trait-level measures of the same construct may predict behavior in different ways. This discrepancy results from cognitive biases in recall and interpretation, which link trait-level aggregate meas- ures more closely to people’s ideas about themselves than to their actual experiences in the moment [9]. Ecological momentary assessment (EMA) methods evaluate momentary states and behaviors close to the time they actually occur [10], and can reveal new insights about the predictors of HIV prevention behaviors. A few studies using EMA suggest that HIV prevention behav- iors depend on momentary mood [11, 12] and contextual factors including alcohol use [13, 14]. Momentary states as predictors of HIV prevention behavior Mustanski’s study [12] included a specific comparison of the same variables measured as momentary states and as stable traits, and found that state-level emotion measures—in particular high anxiety and low positive affect—had stronger effects than trait-level emotion measures on HIV prevention behaviors. Theoretical model and goals of the current studyh This pilot study examined potential momentary state predictors of HIV prevention behaviors based on a moti- vational model in which five momentary state variables predict motivation for HIV prevention, which in turn predicts HIV prevention behaviors [15]. The model was derived from leading theories and research on trait-level predictor variables: control beliefs and social support as in the Theory of Planned Behavior [TPB] [16]; motiva- tion as in the Information-Motivation-Behavioral Skills [IMB] Model [17]; stress, coping, and mood as in Lev- enthal’s dual-process model of health behavior [18]. We also tested HIV stigma as an additional variable inversely related to social support, and information as a variable Participantsh This study was conducted in accordance with the Dec- laration of Helsinki and approved by the Colorado Mul- tiple Institutional Review Board (protocol #06-0948). Participants were 21 PLWH recruited from an outpatient infectious disease practice in Denver, CO. Another 15 PLWH invited to the study declined to participate, pri- marily because of the time commitment required. Quota sampling was used to recruit a diverse sample in terms of race/ethnicity and gender, approximating the cur- rent demographics of the U. S. HIV epidemic [15]. Par- ticipants’ demographic characteristics are summarized in Table 1. Attrition was 33 % over 6 months, with some evidence that minority PLWH were more likely to refuse participation and also to leave the study early. Detailed recruitment, participation, and attrition data are pub- lished elsewhere [15]. Methods Data were collected in a pilot study using daily electronic surveys with PLWH. The study’s methods are published elsewhere [15], but summarized below. Findings Background Preventing transmission of human immunodeficiency virus (HIV) is a major public health issue. Behaviors like abstinence, condom use, and/or using clean needles help persons living with HIV (PLWH) avoid transmitting HIV to others [1]. Although many PLWH take precautions to avoid transmitting HIV, a significant number engage in behaviors that place others at risk: for instance, 26 % of anal sex occurrences for HIV-positive men who have sex Although prevention programs for PLWH have been developed based on various theoretical models and many of these are efficacious [3], the predictors of PLWH’s HIV prevention behaviors are still not well understood. One meta-analysis found that the strongest predictors of prevention behaviors among PLWH were belief in one’s control over situations and behaviors, positive mood, having information about HIV, being concerned about Correspondence: paul.cook@ucdenver.edu 1 University of Colorado College of Nursing, Campus Box C288‑04, Aurora, CO 80045, USA Full list of author information is available at the end of the article Correspondence: paul.cook@ucdenver.edu 1 University of Colorado College of Nursing, Campus Box C288‑04, Aurora, CO 80045, USA Full list of author information is available at the end of the article Page 2 of 6 Page 2 of 6 Cook et al. BMC Res Notes (2016) 9:9 HIV, social support, ability to communicate about HIV, motivation to have safer sex, lack of perceived barriers to condom use, absence of avoidant coping strategies, and absence of psychotic symptoms or sexual compulsiveness [4]. that some authors have considered important (as in the IMB model) but that others have found to be unrelated to behavior at the trait level (as in the TPB). Crepaz and Marks’s meta-analysis [4] also found trait-level effects for information, control beliefs, mood, social support, cop- ing and motivation on prevention. Procedure Each participant was given a personal digital assistant (PDA) handheld computer and asked to complete a 5-minute questionnaire (26–46 questions, depending on skip logic) once daily at a time they had pre-selected. Page 3 of 6 Cook et al. BMC Res Notes (2016) 9:9 Table 1 Participant demographics (N = 21) MSM men who have sex with men, IDU injection drug user Characteristic M (SD) or frequency (%) Age 42.0 years (8.8 years) Gender 15 men (71 %) 6 women (29 %) Race/ethnicity 9 African–American (43 %) 7 White non-Hispanic (33 %) 5 White Hispanic (24 %) Sexual orientation 14 MSM (67 %) 1 heterosexual man (5 %) 6 heterosexual women (28 %) Injection drug use 3 injection drug users (14 %) 18 non-IDU (86 %) Table 1 Participant demographics (N = 21) comprehensiveness of the list of potential predictors, we asked participants at the end of the study to suggest any other important predictors of their HIV prevention behaviors that we did not consider. For the dependent variable in the analysis, participants answered questions about their HIV prevention behav- iors (e.g., carrying condoms, avoiding risky situations). Items were taken from the Behavioral Risk Factor Screen- ing Survey, a measure of prevention behaviors devel- oped by the Centers for Disease Control and Prevention (CDC) and used for population-level epidemiological data collection [21]. To improve reliability of the CDC’s original yes/no items, our 7-item measure substituted response choices from a well-validated tool for daily sur- vey data collection, the Diary of Ambulatory Behavioral States [22], that asked participants to rate their perfor- mance of each behavior on a 4-point scale (1 = NO!!, 2 = no??, 3 = yes??, 4 = YES!!). In a prior study, partici- pants said that these response choices were easy to use and to understand, with the lower-case font and ques- tion marks indicating less certainty than the capital font and exclamation points, and items using this response scale showed good reliability [15]. Because multiple pre- vention strategies may be used, any prevention is better than none, and using more strategies may be more effec- tive, an average of the seven prevention items was used for analysis. Other items asked about HIV risk behav- iors (e.g., unprotected sex, sharing needles), but these had too little variability for analysis because most PLWH reported no risk behaviors. 1 Although this variable was labeled “negative thoughts” in our initial meth- ods paper, it is more accurately described as “control beliefs” based on item content. This label is also more useful for placing current findings in the context of prior research. Procedure The risk items also appeared to be more vulnerable to social desirability bias than the prevention items. Most measures were non-reactive with repeated use and all had adequate reliability. Additional measurement detail is provided in [15]. MSM men who have sex with men, IDU injection drug user Participants completed the questionnaire once at the start of the study with a research assistant, to ensure that they understood the questions and the response choices. PDAs were password-protected. Daily responses were date- and time-stamped. Participants completed questionnaires on 72 % of study days (range 37–92 %) for an average of 4 months (range 1–8). A total of 2319 daily questionnaires were completed by 21 PLWH dur- ing the 6-month study. The original study was designed to identify the optimal duration of daily monitoring, so variability in the number of data points per individual was expected. PLWH returned to the clinic in person to download data from their PDA every 2 months. At these visits, PLWH also answered open-ended ques- tions to identify any additional factors that they believed impacted their HIV prevention behaviors. Participants were paid $25 per visit, and were allowed to keep their PDAs if they participated for at least 2 months. Additional possible predictors identified by participants In addition to the momentary state variables included in daily surveys, participants said that health context vari- ables were important for their HIV prevention behaviors. For instance, participants said they were more likely to take steps for prevention when they were feeling well, but that they also might be more likely to encounter risky situations. Participants suggested that the researchers should ask further questions about medication use, side effects, and stigma experiences with health care provid- ers, especially new providers. Despite a lack of significant findings on the coping scale, participants’ feedback indi- cated that this set of items was interesting and relevant to their daily lives: some participants stated that the ques- tionnaire made them more aware of how stress impacted their daily moods and reactions, while others said it made them more aware of their own coping strategies. Data analysis BMC Res Notes (2016) 9:9 Page 4 of 6 Table 2 Predictors of PLWH’s self-reported HIV prevention behaviors p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001 a Variable names clarified from Cook, et al., 2010: Control Beliefs were originally labeled “Negative Thoughts” which sounds misleadingly like Mood; and in the current study Motivation is a proxy variable as described in the text, labeled “Evaluation of Source” in the 2010 paper Construct Effect on next-day prevention scale score Control beliefsa T (1630) = 3.95, p < 0.001, r = 0.10* Mood T (1639) = 2.96, p = 0.004, r = 0.07 Stigma HIV stigma scale: T (1618) = −0.84, p = 0.40, r = 0.02 Stress Acute stress item: T (1622) = 0.08, p = 0.94, r < 0.01 Chronic stress item: T (466) = −1.52, p = 0.13, r = 0.07 Coping Assessment of daily coping: T (231) = 0.86, p = 0.39, r = 0.06 Successful resolution item: T (466) = −1.30, p = 0.19, r = 0.06 Social support T (1631) = −0.57, p = 0.57, r = 0.01 HIV stigma T (1631) = 0.06, p = 0.95, r < 0.01 Information source T (85) = 0.80, p = 0.43, r = 0.09 Motivationa T (85) = 1.97, p = 0.05, r = 0.21* Risk behaviors T (1612) = 0.55, p = 0.58, r = 0.01 Table 2 Predictors of PLWH’s self-reported HIV prevention behaviors * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001 a Variable names clarified from Cook, et al., 2010: Control Beliefs were originally labeled “Negative Thoughts” which sounds misleadingly like Mood; and in the current study Motivation is a proxy variable as described in the text, labeled “Evaluation of Source” in the 2010 paper Construct Effect on next-day prevention scale score Control beliefsa T (1630) = 3.95, p < 0.001, r = 0.10* Mood T (1639) = 2.96, p = 0.004, r = 0.07 Stigma HIV stigma scale: T (1618) = −0.84, p = 0.40, r = 0.02 Stress Acute stress item: T (1622) = 0.08, p = 0.94, r < 0.01 Chronic stress item: T (466) = −1.52, p = 0.13, r = 0.07 Coping Assessment of daily coping: T (231) = 0.86, p = 0.39, r = 0.06 Successful resolution item: T (466) = −1.30, p = 0.19, r = 0.06 Social support T (1631) = −0.57, p = 0.57, r = 0.01 HIV stigma T (1631) = 0.06, p = 0.95, r < 0.01 Information source T (85) = 0.80, p = 0.43, r = 0.09 Motivationa T (85) = 1.97, p = 0.05, r = 0.21* Risk behaviors T (1612) = 0.55, p = 0.58, r = 0.01 predictor variable individually. Data analysis Alpha was not corrected across models in this preliminary study. Table 2 Predictors of PLWH’s self-reported HIV prevention behaviors Prediction of HIV prevention behaviors from momentary states Table 2 shows the relationship of each momentary state variable to PLWH’s self-reported HIV prevention behav- iors at the next daily data collection point. Scores on the prevention behaviors scale were significantly related to participants’ prior-day report of higher control beliefs, r = 0.10, p < 0.001, better mood, r = 0.07, p = 0.004, and higher motivation based on a positive evaluation of new information received, r = 0.21, p = 0.05. Chronic stress, r = 0.07, p = 0.13, and successful coping, r = 0.06, p = 0.19, did not reach conventional levels for statistical significance as predictors of prevention behaviors, and their effect sizes were small. Furthermore, neither acute HIV prevention behaviors p Participants reported an overall moderate level of HIV prevention behaviors during the study, M = 2.52 out of 4 possible points (SD = 0.45). The range of participant responses was slightly restricted, 1.22–3.11 on a 1–4 scale, meaning that no participant ever said they used no prevention strategies, and no participant ever endorsed all prevention items over a 6-month monitoring period. There was some negative skew in the prevention meas- ure, skew/SEskew = −2.63, meaning that participants gave higher scores on the prevention scale more often than they gave lower ones. We corrected for skew using non- parametric analyses. A multilevel model with time as a level-1 predictor and data clustered within individuals showed that individual participants’ prevention behav- iors were relatively stable based on a flat linear trend, t(8) = −0.25, p = 0.81, β = −0.0001. Although relatively high ICCs for all momentary state variables mean that these states fluctuated only slightly over time, the ICC for prevention behavior was particularly stable at .93. This suggests that gaps in HIV prevention behaviors are rela- tively infrequent and therefore potentially hard to predict. k d d h b l * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001 a Variable names clarified from Cook, et al., 2010: Control Beliefs were originally labeled “Negative Thoughts” which sounds misleadingly like Mood; and in the current study Motivation is a proxy variable as described in the text, labeled “Evaluation of Source” in the 2010 paper stress nor the number of coping strategies used predicted HIV prevention behaviors. Additionally, HIV-related stigma, social support, and information each failed to prospectively predict HIV prevention behaviors. In an interview question asked during the baseline assessment, 100 % of participants said they had adequate knowledge of how to prevent HIV transmission. Interest- ingly, 70 % (16/21) said they also had adequate knowledge of how to prevent HIV infection at the time they were originally infected with HIV. Neither current nor past self-reported knowledge of HIV prevention had any rela- tionship to current HIV prevention behaviors as reported on the daily questionnaires, ps > 0.58. Furthermore, there were no significant relationships between HIV preven- tion behaviors and any of PLWH’s momentary states measured on the same day, all ps > 0.05. Data analysis Daily PDA questionnaires were used to evaluate partici- pants’ state-level control beliefs,1 mood, perceived social support, experience of stigma related to HIV, information received about HIV, stress experienced, coping strategies used, and motivation for HIV prevention. The stress scale had separate items for acute and chronic stressors. The coping scale included a multi-item scale on specific cop- ing strategies used, plus a separate single item about whether the selected coping strategies successfully resolved the problem. The information scale was coded yes/no to correct for significant skew. Motivation was measured using a proxy variable, based on participants’ positive or negative evaluation of any new information received about HIV prevention. As a check on the Data were analyzed using a prospective prediction strat- egy in which each participant’s scores on each momen- tary state predictor variable were compared with their self-reported behavior at the subsequent data collection point, which was usually the next day. Multilevel models were used to correct for the intra-class correlation (ICC) of data points from the same participant, which in this study was high for most variables, ICCs = 0.64–0.93 [15]. Nonparametric multilevel models were used to cor- rect for skew in the dependent variable [23]. In tests of within-person relationships, power for multilevel models is based on the number of observations adjusted for the ICC, rather than on the number of participants. With an average of 110 data points from each of 21 participants, power was .80 to detect moderate effects of r = 0.48 or larger at α = 0.05 [24]. We first tested for the effect of time on prevention behaviors, then tested each potential Cook et al. Discussion Based on these considerations, the relevance of stress and coping at the momentary state level remains unclear.hii The current study confirms previous findings about the state-level effects of mood [12], supports other state-level predictors that have previously been tested only as traits, and advances the science of momentary state influences on behavior by testing multiple predictors in an inte- grated theory of state-level influences on behavior. Prior studies have examined only one or a few state-level pre- dictors and had limited theoretical grounding. Although not all variables in the theoretical model underlying this study were confirmed to predict HIV prevention behav- iors, the model received partial support.h y To partially address the limitations of significance test- ing with a small sample, we reported effect sizes (correla- tion coefficients) for all analyses to quantify the relative importance of each studied predictor variable. Addi- tionally, we asked participants open-ended questions to identify other variables that might be important to study. These questions revealed that PLWH saw healthcare variables including adherence, side effects, and provider relationships as potentially important for future research of this type. In addition, PLWH emphasized the impor- tance of coping as an important factor in HIV preven- tion behaviors; even though this study showed small and nonsignificant effects of stress and coping on preven- tion behaviors, based on the findings of other studies [4] and participants’ suggestions in the current study these may still be potentially important variables for future research. Because the receipt of new information showed no relationship to prevention, but motivation for preven- tion after receiving new information was a better predic- tor, future studies should examine motivation separately from whether participants received any new information about HIV. The current study tested whether momentary states prospectively predicted HIV prevention behaviors on the next day, which is a slightly different strategy from many EMA studies that look at relationships between momentary states and behaviors measured concur- rently—e.g., [26]. Even though the next day’s prevention behaviors were more distant in time from the assess- ment of momentary states, our primary objective was to establish potential causation between predictor variables and prevention behaviors when both were measured as state-level constructs, a goal that was supported by the prospective daily data analysis strategy employed. Discussion PLWH reported relatively high and consistent preven- tion behaviors over the course of this study, and these did not correlate with momentary states measured on the same day. However, momentary states did prospectively Cook et al. BMC Res Notes (2016) 9:9 Page 5 of 6 U.S. HIV epidemic as a whole, results still may generalize primarily to White PLWH because of the higher attrition among minority PLWH. All data provided by minority participants were included in the analysis, but the total number of data points from this group was smaller. Sta- tistical power was adequate to detect moderate effects based on over 2300 daily questionnaires completed by participants, but high between-participant variability in a small sample might have contributed to weaker than expected effects. The number of participants is compara- ble to the small N in some prior EMA studies of PLWH [27], although at least two larger-scale studies have been conducted on individual risk factors: alcohol [13] and mood [12]. Statistical models with multiple simultane- ous predictors were not used in the current analysis because of the high risk of sample-dependent results and a potential lack of generalizability when data-driven deci- sion rules are used with a small sample. Future research should (a) include a larger number of participants; (b) collect many daily data points per participant to capture state-level variability; (c) include multiple predictors of prevention behaviors in a single multiple-variable statis- tical model to test their relative importance; and (d) test predictors such as alcohol and mood that are well sup- ported by prior research as well as those identified as potentially meaningful in small-scale pilot work such as the current study.i predict HIV prevention behaviors the next day. Consist- ent with research on trait-level predictors of HIV preven- tion behaviors [25], this included significant effects for control beliefs, mood, and motivation. Consistent with theory [15] and prior research [25], new information did not predict prevention behaviors. Contrary to theory [15], neither social support nor stigma predicted preven- tion behaviors; this was contrary to past research at the trait level showing a significant effect of social support [25]. Stress and coping were each measured with two subscales, which showed nonsignificant effects. These predictor variables have each been found significant at the trait level [25], and their failure to predict behaviors in the current study may have been due to sample size or measurement limitations. Discussion Addi- tionally, most prevention behaviors are actions taken prospectively (e.g., carrying condoms), so it may be most useful to focus on variables that predict next-day preven- tion behaviors as early warning signs that can be used to direct needed interventions before lapses in prevention occur. Limitations and directions for future researchi Because the current findings are based on a small sample, they may not generalize to all PLWH, and even predictors with weak effects might still be appropriate for further research. Although this study used quota sampling to recruit PLWH who were demographically similar to the Abbreviations EMA l i l EMA: ecological momentary assessment; HIV: human immunodeficiency virus; ICC: intra-class correlation coefficient; IDU: injection drug user; IMB: information-motivation-behavioral skills model; MSM: men who have sex with men; PDA: personal digital assistant device; PLWH: persons living with HIV; TPB: theory of planned behavior. 11. Barta WD, Tennen H, Kiene SM. Alcohol-involved sexual risk behavior among heavy drinkers living with HIV/AIDS: negative affect, self-efficay, and sexual craving. Psychol Addict Behav. 2010;24:563–70. 12. Mustanski B. The influence of state and trait affect on HIV risk b daily diary study of MSM. Health Psychol. 2007;26:618–26. 13. Barta WD, Portnoy DB, Kiene SM, Tennen H, Abu-Hasaballah KS, Ferrer R. A daily process investigation of alcohol-involved sexual risk behavior among economically disadvantaged problem drinkers living with HIV/ AIDS. AIDS Behav. 2008;12:729–40. Received: 2 July 2015 Accepted: 17 December 2015 Received: 2 July 2015 Accepted: 17 December 2015 25. Crepaz N, Hart TA, Marks G. Highly active antiretroviral therapy and sexual risk behavior: a meta-analytic review. J Am Med Assoc. 2004;292:224–36. 26. Reis HT. Why researchers should think “real-world”: A conceptual rationale. In: Mehl MR, Conner TS, editors. Handbook of research methods for studying daily life. New York: Guilford; 2012. p. 3–21. 27. Janda LH, Markowski E, Derlega VJ, Nezlek J, McCain N. Daily events and mood state among individuals living with HIV: examination of the within- persons approach to data collection using daily diary methodology. J Nurs Meas. 2006;14:116–28. Acknowledgements This research was supported by a developmental award from the Colorado Center for AIDS Research, NIH Grant #P30 AI054907-02, with additional sup‑ port from the UCDHSC General Clinical Research Center, NIH Grant #M01 RR00051. The authors also wish to thank Dr. Elizabeth Connick, Dr. Steven Johnson, and Abigail Lord for consultation on research and clinical topics, the staff and patients at the University of Colorado Infectious Disease Group Prac‑ tice for their support of the study, and Dr. Samantha MaWhinney for statistical consultation during study design. 18. Teel CS, Meek P, McNamara AM, Watson L. Perspectives unifying symptom interpretation. Image J Nurs Sch. 1997;29(2):175–81. 19. Silver N. The signal and the noise. 2013. 20. Riley WT, Rivera DE, Atienza AA, Nilsen W, Allison SM, Mermelstein R. Health behavior models in the age of mobile interventions: are our theories up to the task? Transl Behav Med. 2011;1(1):53–71. 21. Centers for Disease Control and Prevention (CDC). Behavioral risk factor surveillance system. Retrieved 8 Aug 2006 from http://www.cdcgov/ brfss/2006. Author details 1 1 University of Colorado College of Nursing, Campus Box C288‑04, Aurora, CO 80045, USA. 2 University of Colorado School of Medicine, 12631 E. 17th Ave., Mail Stop 8204, Aurora, CO 80045, USA. 16. Ajzen I, Fishbein M. Questions raised by a reasoned action approach: comment on Ogden (2003). Health Psychol. 2004;23:431–4. 17. Fisher JD, Fisher WA, Amico KR, Harman JJ. An information-motivation- behavioral skills model of adherence to antiretroviral therapy. Health Psychol. 2006;25:462–73. Authors’ contributions PFC and LBA designed the study. PFC and CJM carried out the data collection and completed the statistical analyses. CJM and LBA contributed clinical interpretation during data analysis and writing. PFC drafted the manuscript, with editorial assistance from LBA. All authors read and approved the final manuscript. 14. Mckirnan DJ, Ostrow DG, Hope B. Sex, drugs and escape: a psychological model of HIV-risk sexual behaviors. AIDS Care. 1996;8:655–70. 15. Cook PF, McElwain CJ, Bradley-Springer L. Feasibility of a PDA diary method to study daily experiences in persons living with HIV. Res Nurs Health. 2010;33:221–34. Conclusions Momentary states are under-studied, proximal vari- ables that appear to predict HIV prevention behaviors Cook et al. BMC Res Notes (2016) 9:9 Page 6 of 6 among PLWH, and that may have different effects from trait-level predictors because they are less affected by biases associated with recall and interpretation. Despite a small sample size, our study gathered a large amount of data from individual participants and examined a range of predictor variables in order to enhance the lim- ited literature on momentary state predictors of PLWH’s prevention behaviors. Further, we used a prospective analysis strategy examining the effects of momentary states on behavior at the next daily data collection point, a novel method that strengthens causal interpretations. Additional research using EMA is needed to clarify the momentary states that are most conducive to HIV pre- vention behaviors. 3. Crepaz N, Lyles CM, Wolitski RJ, Passin WF, Rama SM, Herbst JH, et al. Do prevention interventions reduce HIV risk behaviours among peo‑ ple living with HIV? A meta-analytic review of controlled trials. AIDS. 2006;20:143–57. 4. Crepaz N, Marks G. Towards an understanding of sexual risk behavior in people living with HIV: a review of social, psychological, and medical find‑ ings. AIDS. 2002;25:135–49. 5. Barta WD, Kiene SM, Tennen H, Abu-Hasaballah KS, Ferrer R. The idi‑ ographic study of inconsistent condom use behavior of persons living with HIV. AIDS Care. 2007;19:1058–64. 6. Courvoisier DS, Eid M, Nussbeck FW. Mixture distribution latent state-trait analysis: basic ideas and applications. Psychol Methods. 2007;12:80–104. 6. Courvoisier DS, Eid M, Nussbeck FW. Mixture distribution latent state-trait analysis: basic ideas and applications. Psychol Methods. 2007;12:80–104. 7. Fridhandler BM. Conceptual note on state, trait, and the state–trait distinction. J Pers Social Psychol. 1986;50:169–74. 7. Fridhandler BM. Conceptual note on state, trait, and the state–trait distinction. J Pers Social Psychol. 1986;50:169–74. 8. Ptacek JT, Pierce GR, Thompson EL. Finding evidence of dispositional cop‑ ing. J Res Personal. 2006;40:1137–51. 9. Schwarz N. Why researchers should think “real-time”: a cognitive rationale. In: Mehl MR, Conner TS, editors. Handbook of research methods for studying daily life. New York: Guilford; 2012. p. 22–42. 10. Tennen H, Affleck G, Coyne JC, Larsen RJ, DeLongis A. Paper and plastic in daily diary research: comment on Green, Rafaeli, Bolger, Shrout, and Reis (2006). Psychol Methods. 2006;11:112–8. 1. Centers for Disease Control and Prevention (CDC). Advancing HIV preven‑ tion: new strategies for a changing epidemic-United States, 2003. Morbid Mortal Wkly Rep. 2003;52(15):1–4. y p 2. Crepaz N, Marks G, Liau A, Mullins MM, Aupont LW, Marshall KJ, et al. Prevalence of unprotected anal intercourse among HIV-diagnosed MSM in the United States: a meta-analysis. AIDS. 2009;23:1617–29. Competing interests 22. Kamarck TW. The diary of ambulatory behavioral states: a new approach to the assessment of psychosocial influences on ambulatory cardiovas‑ cular activity. In: Krantz DS, Baum A, editors. Technology and methods in behavioral medicine. Mahwah: Erlbaum; 1998. p. 163–85. In the past 3 years, author PFC has received grant support from Merck & Co. Inc. and from the Colorado Health Foundation, and has served as a consultant for Takeda Inc., the Optometric Glaucoma Society, Medical Simulation Corpo‑ ration, Competency and Credentialing Institute Inc., and Academic Impres‑ sions Inc. In addition, authors PFC and LBS have each received grant support from several U.S. Federal agencies: HRSA, CDC, SAMHSA, and/or NIH. behavioral medicine. Mahwah: Erlbaum; 1998. p. 163–85. 23. HLM 6: Hierarchical linear and nonlinear modeling [computer program]. Lincolnwood: Scientific Software International; 2006. 24. Maas CJM, Hox JJ. Sufficient sample sizes for multilevel modeling. Meth‑ odology. 2007;1:86–92.
https://openalex.org/W1982293214	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0004823&type=printable	English	null	Cage Matching: Head to Head Competition Experiments of an Invasive Plant Species from Different Regions as a Means to Test for Differentiation	PloS one	2,009	cc-by	5,209	Abstract 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. Funding: An NSERC Discovery Grant to Dr. Lortie funded this 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. * E-mail: lortie@yorku.ca * E-mail: lortie@yorku.ca Abstract Many hypotheses are prevalent in the literature predicting why some plant species can become invasive. However, in some respects, we lack a standard approach to compare the breadth of various studies and differentiate between alternative explanations. Furthermore, most of these hypotheses rely on ‘changes in density’ of an introduced species to infer invasiveness. Here, we propose a simple method to screen invasive plant species for potential differences in density effects between novel regions. Studies of plant competition using density series are a fundamental tool applied to virtually every aspect of plant population ecology to better understand evolution. Hence, we use a simple density series with substitution contrasting the performance of Centaurea solstitialis in monoculture (from one region) to mixtures (seeds from two regions). All else being equal, if there is no difference between the introduced species in the two novel regions compared, Argentina and California, then there should be no competitive differences between intra and inter-regional competition series. Using a replicated regression design, seeds of each species were sown in the greenhouse at 5 densities in monoculture and mixed and grown till onset of flowering. Centaurea seeds from California had higher germination while seedlings had significantly greater survival than Argentina. There was no evidence for density dependence in any measure for the California region but negative density dependence was detected in the germination of seeds from Argentina. The relative differences in competition also differed between regions with no evidence of differential competitive effects of seeds from Argentina in mixture versus monoculture while seeds from California expressed a relative cost in germination and relative growth rate in mixtures with Argentina. In the former instance, lack of difference does not mean ‘no ecological differences’ but does suggest that local adaptation in competitive abilities has not occurred. Importantly, this method successfully detected differences in the response of an invasive species to changes in density between novel regions which suggests that it is a useful preliminary means to explore invasiveness. Citation: Lortie CJ, Munshaw M, Zikovitz A, Hierro J (2009) Cage Matching: Head to Head Competition Experiments of an Invasive Plant Species from Different Regions as a Means to Test for Differentiation. PLoS ONE 4(3): e4823. doi:10.1371/journal.pone.0004823 Editor: Andrew Wilby, University of Lancaster, United Kingdom Received December 16, 2008; Accepted February 11, 2009; Published March 13, 2009 Received December 16, 2008; Accepted February 11, 2009; Pub Copyright: 2009 Lortie et al. Cage Matching: Head to Head Competition Experiments of an Invasive Plant Species from Different Regions as a Means to Test for Differentiation Christopher J. Lortie1, Michael Munshaw2, Andrea Zikovitz3, Jose Hierro4 1 Department of Biology, York University, Toronto, Ontario, Canada, 2 Department of Biology, York University, Toronto, Ontario, Canada, 3 Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario, Canada, 4 Conicet, University of La Pampa, Santa Rosa, La Pampa, Argentina University, Toronto, Ontario, Canada, 2 Department of Biology, York University, Toronto, Ontario, Canada, 3 Ecology and Evolutionar oronto, Ontario, Canada, 4 Conicet, University of La Pampa, Santa Rosa, La Pampa, Argentina Department of Biology, York University, Toronto, Ontario, Canada, 2 Department of Biology, York University, Toronto, Ontario, Can iology, University of Toronto, Toronto, Ontario, Canada, 4 Conicet, University of La Pampa, Santa Rosa, La Pampa, Argentina Citation: Lortie CJ, Munshaw M, Zikovitz A, Hierro J (2009) Cage Matching: Head to Head Competition Experiments of an Invasive Plant Species from Different Regions as a Means to Test for Differentiation. PLoS ONE 4(3): e4823. doi:10.1371/journal.pone.0004823 Introduction process within each novel range wherein an introduced species is increasing in density is not tested. Hence, differential responses to density could be an important first step in the identification of invasiveness or in determining traits associated with spread. Here, we propose a simple experimental method using the biogeograph- ical filter as a first approximation to test whether there is evidence for differences in an invasive species in any response characters in competition when introduced to more than one novel region. Understanding the success of invasive plants is not necessarily simple [1,2]. Invasion is primarily a biogeographical issue as it involves the movement (either intentionally or accidentally) of a species from one region to another [3,4]. The application of this filter as a means to infer differences is powerful, and there are a variety of broad applications such as (i) population-level experi- ments, i.e. comparison of success in home versus away regions [5,6], assessment of variation in dominance in novel ranges [7], or gradient studies [8,9] and (ii) individual-based tests such as evidence for differences in plasticity [10], genetics [11,12], fitness [13], or ecotypic differentiation in morphology such as size [14,15]. Equally fundamental to the biogeographical approach to studying invasions is the use of density following movement to a region to infer invasiveness. Relative changes in the population density of an introduced species (i.e. increases) in a novel region is arguably the primary, yet informal means, to infer that a plant species is invasive. Yet, the importance of density as a regulating Competition in plants is a fundamental concept tested and used extensively as a tool to understand population dynamics, patterns of diversity, and community composition with literally over 6000 papers published within the last 10 years on the topic (Web of Science V.4.3, query ‘plant competition’). As such, it is reasonable to propose that at some level use of simple competition experiments can also potentially elucidate mechanisms associated with an introduced species becoming invasive. Introduction Certainly, the success of plant species when introduced is not necessarily attributable solely to changes in competitive effects or responses, but we propose that, regardless of the reason for the success of PLoS ONE \| www.plosone.org March 2009 \| Volume 4 \| Issue 3 \| e4823 1 March 2009 \| Volume 4 \| Issue 3 \| e4823 PLoS ONE \| www.plosone.org Invasive Plant Competition some invasives, simple competition experiments within a single invasive plant species from different regions (i.e. intra-specific but inter-regional) can test whether there is evidence for differences in competitiveness associated with density when introduced (provided seeds are collected widely from each region and sample more than one region). If the reason for success is not related to density when introduced, then there is no reason to expect that competition between individuals from the same region should differ from competition between individuals from different regions under controlled conditions. In summary, we predict that competition between individuals of invasive plant species from different novel regions is a useful first step in screening invasive plant species. We use a highly successfully invader, Centaurea solstitalis or yellow starthistle, to test the prediction that differentiation (either due to sampling effects or local adaptation) leads to relative differences in competition within and between novel regions using standard pot- based competition experiments in the greenhouse. general differences between regions [23,24]. Firstly, broad-scale patterns were identified using generalised linear models [25], and factors identified as significant (alpha set at p,0.01 to control for table-wide errors [26]) were further tested for density dependence via simple regressions of the mean summary data per level. Additional analysis is necessary since a direct relationship between density and a response variable does not necessarily imply that there is density dependence, i.e. more seeds should equal more plants. Only when there is a disproportionate (i.e. curvilinear) increase or decrease in the response with density do we infer density dependent regulation. Non-linearity for population level measures such as proportionate germination or survival thus indicates density dependence (with an increase of r2 of at least 10%), and for individual plant measures such as rgr, a slope significantly different from 0 indicates density effects [27,28]. Secondly, to test for differences in the mean competitive effects of individuals [29] from the same region versus individuals mixed with different regions, the ‘relative interaction index’ or Rii was calculated for each of the response measures recorded [30]. Experimental Design Seeds of C. solstitalis were collected widely from seed heads in 2005 from 10 populations in each of the two introduced regions, California and Argentina [4], sorted into pappus and non-pappus, and thoroughly mixed within region by each seed type. General differences between regions in seed ecology and the importance of competitive effects intra and inter-regionally were compared using density series in a greenhouse at York University, Canada. These two levels of contrast were tested by sowing seeds in 15 cm diameter pots with standard potting mix in the following density series: 1, 2, 5, 10, and 20 seeds per pot using seeds from only one region and both regions mixed at a 50:50 ratio (modified replacement series, i.e. proportion varied, 100% or 50%, with density held constant but more than one density tested, [22,23]). Ten replicates per density per region per seed type were tested (pappus and non-pappus seeds were tested independently). A standardized grid-based planting was used to ensure identity of each seed and subsequent seedling, and initial application of water was done carefully to ensure that seeds did not move prior to germination. Germination, relative growth rate (rgr) of individuals (total number of leaves recorded weekly from emergence date for each individual and biomass at the end of the growing season by total number of days since emergence), and survival were recorded for the span of the experiment (4 months total, ended when the first individuals flowered in the greenhouse September 15th 2007). Water (added to saturation every 2–3 days), nutrients (20:20:20 NPK added at onset), and light were not limiting in this experiment. All plants were harvested, dried for 48 h at 60uC, and weighed. Table 1. A summary of the generalised linear models used to test the importance of density, region, and density by region on the four responses measured in this greenhouse experiment of C. solstitialis. Introduction This index is a direct measure of effect size and is calculated as following: Experimental Design Measure Factor DF Chi-square Prob.Chi-square Germination Density 3,78 6.55 0.01 Region 1,78 16.77 0.0001 Density6Region 3,78 12.77 0.0004 RGR leaves Density 3,196 2.98 0.08 Region 1,196 1.1 0.3 Density6Region 3,196 4.11 0.04 RGR mass Density 3,152 3.73 0.05 Region 1,152 2.37 0.12 Density6Region 3,152 5.55 0.018 Survival Density 3,78 70.75 0.0001 Region 1,78 4.97 0.0001 Density6Region 3,78 4.49 0.03 Seeds collected from two invaded regions were tested (California and Argentina), densities included 1, 2, 5, 10, and 20 seeds per pot, and details for the responses are reported in the text. Proportionate germination and survival were tested with logistic models and relative growth rates (rgr) with linear models. Bold denotes significant effects. doi:10.1371/journal.pone.0004823.t001 Measure Factor DF Chi-square Prob.Chi-square Seeds collected from two invaded regions were tested (California and Argentina), densities included 1, 2, 5, 10, and 20 seeds per pot, and details for the responses are reported in the text. Proportionate germination and survival were tested with logistic models and relative growth rates (rgr) with linear models. Bold denotes significant effects. doi:10.1371/journal.pone.0004823.t001 Study species Centaurea solstitalis is a highly invasive weed from Eurasia [16]. It is a prolific seed producer with up to 1000 s of seeds produced per plant [17], 125–250 millions seeds per hectare reported in an invaded region [18], produces two types of seeds – pappus and non-pappus each possessing unique dormancy attributes [19,20] - all making it a perfect candidate to explore the importance of density and competition. In California, it is reported in 56 of 58 counties [21], and in Argentina, it is also highly invasive and widespread [6]. Rii~ C{T ð Þ= CzT ð Þ Controls are designated as performance in monocultures (i.e. seeds from same region) and treatments as the individuals grown in mixture with seeds from the second region at every density. The metric ranges from +1 to 21 with negative values indicating competition and positive indicates facilitation, and two-tailed t- tests are used to test for differences from 0 (i.e. no relative difference in the effect of neighbours in mix to mono, alpha also set at 0.01). The net differences in actual density between mixture and monocultures was also tested directly as a predictor of effect size (i.e. 10 seeds sown but 5 germinate in monoculture and 8 in PLoS ONE \| www.plosone.org Statistics The replicated regression design used here (10 reps per density per region per seed type) permits two set of analyses to test for March 2009 \| Volume 4 \| Issue 3 \| e4823 2 Invasive Plant Competition PLoS ONE \| www.plosone.org Figure 1. The importance of density on measures of the invasive weed C. solstitialis. See text for generalized linear model statistics. Data is summarized by plotting the mean response per seed densities tested (1, 2, 5, 10, & 20). doi:10.1371/journal.pone.0004823.g001 mixture and the difference between the two treatments in actual density changes the competitive environment experienced by individuals therein). Density dependence and broad-scale patterns of seeds from the two invaded regions In monocultures, seeds from California expressed significantly greater germination relative to Argentina under these controlled conditions (Table 1, CA: 65%+/23%, AR: 41%+/24%). Survival to final census also significantly differed between regions in favour of California (Table 1, CA: 70%+/23%, AR: 60%+/24%). There were no differences in any measure by seed type (pappus/non- pappus, all GLMs p.0.05) nor an effect of census on any measure, i.e. timing of germination did not differ (GLMs p.0.05). Germination and survival of C. solstitialis significantly responded to changing seed densities (Table 1). Negative density dependence was detected in the germination of seeds from Argentina (Table 1, significant density6region effect, Fig. 1, best fit curvilinear r2 = 0.97 on summarized data) while survival of plants responded positively at first to increasing seed densities but then began to decrease – particularly for the California populations (Fig. 1, best fit curvilinear r2 = 0.77 on summarized data). Intra versus inter-regional differences in competi- tion. There was no evidence for differential competitive effects of C. solstitialis collected from Argentina when grown in mixtures with seeds from California (Table 2, Fig. 2). However, seeds from California had significantly greater germination when grown in monocultures than with seeds from Argentina, and also expressed a relatively higher rate of rgr leaves in the absence of intra-regional competition (Table 2, Fig. 2). In the former instance, the difference in the density of germinated seedlings between treatments positively predicted the strength of the relative interaction indices for the germinants from California (Fig. 3). Table 2. Tests of the relative interaction index (Rii) contrasting performance in mixtures to that in monocultures. Region Measure DF t p sign AR Germination 96 20.21 0.83 0 RGR leaves 96 2.27 0.03 0 RGR mass 57 1.45 0.15 0 Survival 96 20.7 0.5 0 CA Germination 100 2.75 0.007 + RGR leaves 100 3.24 0.001 + RGR mass 58 0.8 0.42 0 Survival 100 0.42 0.68 0 Mixtures refers to the performance of individuals of C. solstitialis in competition with seeds sown from two regions, Argentina (AR) and California (CA), at 2, 10, and 20 seeds per pot or in monocultures, i.e. seeds from only one invaded region. Two-tailed t-tests were used to determine whether the mean Rii values were significantly different from 0 (at p,0.01). doi:10.1371/journal.pone.0004823.t002 Table 2. Tests of the relative interaction index (Rii) contrasting performance in mixtures to that in monocultures. Discussion Hypotheses explaining the relative success of invasive species span the entire spectrum of population and community ecology including evolutionary arguments such as local adaptation, i.e. evolution of increased competitive ability, EICA [31,32] to more stochastically driven processes such as disturbance [33,34] or pure sampling effects such as propagule pressure [35]. Nonetheless, these hypotheses need not be mutually exclusive, but it would be useful to be able to sort invasive species with simple, standardized experimentation into at least the most broad set of potentially applicable hypotheses [2], i.e. is there evidence for differences in the competitiveness of the species or is opportunity/and or the local environment likely causal. Here, we successfully tested whether competition between individuals of an invasive species from different regions is sensitive to mixed versus monocultures by region. Interestingly, greater differences in the actual densities of seedlings recorded in the paired monoculture versus mixture pots positively predicted the effect size estimates for germination in California. These relative increases suggest that germination in the field is likely not regulated at all by potential increases in the density of other C. solstitialis in California within the local neighbourhood (i.e. within a 15 cm range). Admittedly this is a simplistic first approximation to understanding the dynamics of invasive plant species and the myriad of causal factors, but it did clearly demonstrate that differentiation within an invasive species is detectable using competition experiments. Interestingly, greater differences in the actual densities of seedlings recorded in the paired monoculture versus mixture pots positively predicted the effect size estimates for germination in California. These relative increases suggest that germination in the field is likely not regulated at all by potential increases in the density of other C. solstitialis in California within the local neighbourhood (i.e. within a 15 cm range). Admittedly this is a simplistic first approximation to understanding the dynamics of invasive plant species and the myriad of causal factors, but it did clearly demonstrate that differentiation within an invasive species is detectable using competition experiments. Conceptually, this experimental approach is highly novel as competition between invasive and native species has been tested [36–38] but not within the invasive species directly [39]. Importantly, even if no difference is detected, this experimental design (i.e. Density dependence and broad-scale patterns of seeds from the two invaded regions PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org March 2009 \| Volume 4 \| Issue 3 \| e4823 3 Invasive Plant Competition Figure 3. Regression of the relative differences in density of germinants of C. solstitialis from California when planted with only seeds from the same region or mixed with C. solstitialis seeds from Argentina and the relative interaction indices for germinants from this region. Control (C) refers to individuals grown in monoculture (California only) and the paired treatment (T) to the mixture of two regions. The fit curve is described by y = 20.03+0.06x20.004(x23.8)2 (r2 = 0.37 and p = 0.0001). doi:10.1371/journal.pone.0004823.g003 Figure 2. The relative effect of growth in competition with C. solstitialis plants from another invaded region (California and Argentina) to performance in monocultures, i.e. seeds from the same region. The mean relative interaction indices are plotted (Rii)+/ 21 s.e. and were calculated for each paired density (2, 10, & 20 seeds per pot). doi:10.1371/journal.pone.0004823.g002 Figure 3. Regression of the relative differences in density of germinants of C. solstitialis from California when planted with only seeds from the same region or mixed with C. solstitialis seeds from Argentina and the relative interaction indices for germinants from this region. Control (C) refers to individuals grown in monoculture (California only) and the paired treatment (T) to the mixture of two regions. The fit curve is described by y = 20.03+0.06x20.004(x23.8)2 (r2 = 0.37 and p = 0.0001). Figure 2. The relative effect of growth in competition with C. solstitialis plants from another invaded region (California and Argentina) to performance in monocultures, i.e. seeds from the same region. The mean relative interaction indices are plotted (Rii)+/ 21 s.e. and were calculated for each paired density (2, 10, & 20 seeds per pot). doi:10.1371/journal.pone.0004823.g002 Figure 2. The relative effect of growth in competition with C. solstitialis plants from another invaded region (California and Argentina) to performance in monocultures, i.e. seeds from the same region. The mean relative interaction indices are plotted (Rii)+/ 21 s.e. and were calculated for each paired density (2, 10, & 20 seeds per pot). doi:10.1371/journal.pone.0004823.g002 Figure 3. Regression of the relative differences in density of germinants of C. solstitialis from California when planted with only seeds from the same region or mixed with C. solstitialis seeds from Argentina and the relative interaction indices for germinants from this region. Density dependence and broad-scale patterns of seeds from the two invaded regions Control (C) refers to individuals grown in monoculture (California only) and the paired treatment (T) to the mixture of two regions. The fit curve is described by y = 20.03+0.06x20.004*(x23.8)2 (r2 = 0.37 and p = 0.0001). doi:10.1371/journal.pone.0004823.g003 PLoS ONE \| www.plosone.org Discussion cage matching an invasive species) has biological relevance in that it points towards explanations that focus more on disturbance or environmental drivers such as climate matching [40] rather than explanations that necessarily invoke change such as EICA or enemy release [41]. The outcome of the test does not thus determine the usefulness of the experiment, and these approaches are of course the best types of studies – even if preliminary. Furthermore, it challenges a dogma which seems to be common in the general perception of invasives in that if an introduced species is numerically dominant, i.e. increases in density, it must also always be a good competitor and free from regulation [35]. If the relative abundances of the invasive in different regions or locally are documented, this design can also be used more finely to assess whether there is evidence for differential competitive effects as related to dominance or density (sensu Goldberg 1996). Centaurae solstitialis from one the two invaded regions, Argentina, did not differ in performance in the intra versus inter-regional comparisons which cursorily suggests that competitive ability has not changed in this region. Hence, hypotheses related to stochastic processes such as disturbance might be more powerful explanatory avenues of research, and evidence to date indicates this is likely the case [6]. However, C. solstitialis from California performed relatively better in monocultures for some key responses which suggests that either local adaptation has occurred or founder effects sampled individuals, and now populations (10 sampled throughout region), with divergent competitive abilities (i.e. increased germination and leaf growth rates in California comes at a cost when in competition with plants from Argentina which continue to adopt a more conservative strategy). This is not to say that disturbance is necessarily unimportant in California [6], but that it is clear that individuals of C. solstitialis differ in this region in the expression of traits in the context of plant competition. PLoS ONE \| www.plosone.org March 2009 \| Volume 4 \| Issue 3 \| e4823 March 2009 \| Volume 4 \| Issue 3 \| e4823 4 Invasive Plant Competition Another strength of coupling standard plant population ecology experiments with biogeography is that the relative importance of density dependence can be inferred, seed biology described, and broad differences in the relative importance of life-stage screened. References Lortie CJ, Cushman JH (2007) Effects of a directional abiotic gradient on plant community dynamics and invasion in a coastal dune system. Journal of Ecology 95: 468–491. 29. Goldberg DE (1996) Competitive ability: definitions, contingency, and correlated traits. Philosophical Transactions of the Royal Society of London B 351: 1377–1385. 9. MacDougall AS, Boucher J, Turkington R, Bradfield GE (2006) Patterns of plant invasion along an environmental stress gradient. Journal of Vegetation Science 17: 47–56. 30. Armas C, Ordiales R, Pugnaire F (2004) Measuring plant interactions: a new comparative index. Ecology 85: 2682–2686. 31. Joshi J, Schmid B, Caldeira MC, Dimitrakopoulos PG, Good J, et al. (2001) Local adaptation enhances performance of common plant species. Ecology Letters 4: 536–544. 10. Alpert P, Bone E, Holzapfel C (2000) Invasiveness, invasibility and the role of environmental stress in the spread of non-native plants. Perspectives in Plant Ecology, Evolution and Systematics 3: 52–66. 32. Blossey B, Notzold R (1995) Evolution if increased competitive ability in invasive nonindiginenous plants: a hypothesis. Journal of Ecology 83: 887–889. 11. Figueroa ME, Castillo JM, Redondo S, Luque T, Castellanos EM, et al. (2003) Facilitated invasion by hybridization of Sarcocornia species in a salt-marsh. Journal of Ecology 91: 616–626. 33. D’Antonio CM, Dudley TL, Mack M (1999) Disturbance and biological invasions: Direct effects and feedbacks. In: Walker LR, ed. Ecosystems of disturbed ground. 16 ed. New York: Elsevier. pp 413–452. 12. Meirmans PG, Vlot EC, Den Nijs JCM, Menken SBJ (2003) Spatial ecological and genetic structure of a mixed population of sexual diploid and apomictic triploid dandelions. Journal of Evolutionary Biology 16: 343–352. g pp 34. Mack MC, D’Antonio CM (1998) Impacts of biological invasions on disturbance regimes. Trends in Ecology and Evolution 13: 68–75. 13. Gerlach JD, Rice KJ (2003) Testing life history correlates of invasiveness using congeneric plant species. Ecological Applications 13. 13. Gerlach JD, Rice KJ (2003) Testing life history corre g gy 35. Stohlgren TJ, Barnett DT, Jarnevich CS, Flather CH, Kartesz JT (2008) The myth of plant species saturation. Ecology Letters 11: 313–322. congeneric plant species. Ecological Applications 13. 14. Willis AJ, Memmott J, Forrester RI (2000) Is there evidence for the post-invasion evolution of increased size among invasive plant species? Ecology Letters 3: 275–283. 36. Bakker JP, Wilson SD (2001) Competitive abilities of introduced and native grasses. Plant Ecology 157: 117–125. 37. References 21. Pitcairn MJ, O’Connell RA, Gendron JM (1998) Yellow starthistle: survey of statewide distribution. In: Agriculture CDoFa, ed. Biological control program and annual summary. Sacramento. 1. Sax DF, Brown JH (2000) The paradox of invasion. Global Ecology and Biogeography 9: 363–371. 2. Rejmanek M (2000) Invasive plants: approaches and predictions. Austral Ecology 25: 497–506. 22. Gibson DJ, Connolly J, Hartnett DC, Weidenhamers JD (1999) Designs for greenhouse studies of interactions between plants. Journal of Ecology 87: 1–16. 23. Jolliffe PA (2000) The replacement series. Journal of Ecology 88: 371–385. 22. Gibson DJ, Connolly J, Hartnett DC, Weidenhamers JD (1999) Designs for greenhouse studies of interactions between plants. Journal of Ecology 87: 1–16. 3. Hierro JL, Maron JL, Callaway RM (2005) A biogeographical approach to plant invasions: the importance of studying exotics in their introduced and native range. Journal of Ecology 93: 5–15. 23. Jolliffe PA (2000) The replacement series. Journal of Ecology 88: 371–385. 24. Cottingham KL, Lennon JT, Brown BL (2005) Knowing when to draw the line: designing more informative ecological experiments. Frontiers in Ecology and the Environment 3: 145–152. 4. Pauchard A, Cavieres L, Bustamante RO (2004) Comparing alien plant invasions among regions with similar climates: where to from here? Diversity and Distributions 10: 371–375. 25. Carrete M, Tella JL, Sanchez-Zapata JA, Moleon M, Gil-Sanchez JM (2008) Current caveats and further directions in the analysis of density-dependent population regulation. Oikos 117: 1115–1119. 5. Maron JL, Vila` M, Arnason J (2004) Loss of natural enemy resistance among introduced populations of St. John’s Wort, Hypericum perforatum. Ecology 85: 3243–3253. 26. Moran MD (2003) Arguments for rejecting the sequential Bonferroni in ecological studies. Oikos 100: 403–405. 6. Hierro JL, Villarreal D, Eren O, Graham JM, Callaway R, M (2006) Disturbance facilitates invasion: the effects are stronger abroad than at home. American Naturalist 168: 144–156. 27. Goldberg DE, Turkington R, Olsvig-Whittaker L, Dyer AR (2001) Density dependence in an annual plant community: variation among life history stages. Ecological Monographs 71: 423–446. 7. Von Holle B (2005) Biotic resistance to invader establishment of a southern Appalachian plant community is determined by environmental conditions. Journal of Ecology 93: 16–26. g g p 28. Lortie CJ, Turkington R (2002) The effect of initial seed density on the structure of a desert annual plant community. Journal of Ecology 90: 435–445. J gy 8. Author Contributions Conceived and designed the experiments: CJL JH. Performed the experiments: MM AZ. Analyzed the data: CJL. Wrote the paper: CJL. Conceived and designed the experiments: CJL JH. Performed the experiments: MM AZ. Analyzed the data: CJL. Wrote the paper: CJL. Discussion In this particular invasive species, the evidence concurs with field studies describing the importance of life-history traits and the seed biology of C. solstitialis in California in that a large proportion of seeds can germinate and frequently do so very quickly [18]. While not measured directly here, it is likely that C. solstitialis can express the phenomenon described as adaptive acceleration in competitive contexts [42]. In this study, increased germination by seeds from California was detected, and acceleration was potentially expressed via an increased relative growth rate similar to field studies [13]. Biogeographically, the differences in density depen- dence between Argentina, negative effects on germination, and California, higher germination and survival and even positive effects of initial increases in density, clearly suggest that in Argentina prolific seed production and subsequent seed and seedling densities do not benefit this species in an intra-specific competitive context here while in California it does not come at a cost. This strongly suggests that C. solstitialis in California is either able to capitalize on opportunity via high seed densities or is positively influenced by increasing local abundances of its seed. Few studies of density dependence fail to detect negative effects on germination [27,28], and this is thus a unique finding suggestive of an important trait related to invasiveness. Hence, experimentally pairing an invasive plant species from different regions not only facilitated the detection of evidence for potential differentiation by region but provided a clear signal of the relative importance of seed and seedling densities within each invaded region. References D’Antonio CM, Hughes RF, Vitousek PM (2001) Factors influencing dynamics of two invasive C4 grasses in seasonally dry Hawaiian woodlands. Ecology 82: 89–104. 15. Genton BJ, Kotanen PM, Cheptou PO, Adolphe C, Shykoff JA (2005) Enemy release but no evolutionary loss of defence in a plant invasion: an inter- continental reciprocal transplant experiment. Oecologia 146: 404–414. 38. MacDougall AS, Turkington R (2004) Relative importance of suppression-based and tolerance-based competition in an invaded oak savanna. Journal of Ecology 92: 422–434. 16. Maddox DM, Mayfield A, Poritz NH (1985) Distribution of yellow starthistle (Centaurea solstitialis) and Russin knapweed (Centaurea repens). Weed Science 33: 315–327. 39. White EM, Wilson JC, Clarke AR (2006) Biotic indirect effects: a neglected concept in invasion biology. Diversity and Distributions 12: 445–455. 17. Sheley RL, Larson LL (1994) Observation: comparative life-history of cheat- grass and yellow starthistle. Journal of Range Management 47: 450–456. 40. Richardson DM, Thuiller W (2007) Home and away from home - objective mapping of high-risk source areas for plant introductions. Diversity and Distributions 13: 299–312. 18. Benefield CB, DiTomaso JM, Kyer GB, Tshohl A (2001) Reproductive biology of yellow starthistle: maximizing later-season control. Weed Science 49: 83–90. 19. Joley DB, Maddox DM, Schoenig SE, Mackey BE (2003) Parameters affecting germinability and seed bank dynamics in dimorphic achenes of Centaurea solstitialis in California. Canadian Journal of Botany 81: 993–1007. 41. Ewel JJ (1986) Invasibility: some lessons from South Florida. In: Drake JA, ed. Ecology of biological invasions of North America and Hawaii. New York: Springer-Verlag. pp 214–230. J y 20. Joley DB, Maddox DM, Supkoff DM, Mayfield A (1992) Dynamics of Yellow Starthistle (Centaurea solstitialis) achenes in field and laboratory. Weed Science 40: 190–194. 42. Dyer AR, Fenech A, Rice KJ (2000) Accelerated seedling emergence in interspecific competitive neighbourhoods. Ecology Letters 3: 523–529. PLoS ONE \| www.plosone.org March 2009 \| Volume 4 \| Issue 3 \| e4823 March 2009 \| Volume 4 \| Issue 3 \| e4823 5
https://openalex.org/W2541677156	http://liu.diva-portal.org/smash/get/diva2:913071/FULLTEXT02	English	null	Conclusions: Glocal pharma and the Swedish Viagra man	DiVA (Linkoping University)	2,016	cc-by	63,410	Glocal Pharma Glocal Pharma An exploration of how global pharmaceutical products are localized – of what happens when they become ‘glocal’ – this book examines the tensions that exist between a global pharmaceutical market and the locally bounded discourses and regulations encountered as markets are created for new drugs in particular con- texts. Employing the case study of the emergence, representation and regulation of Viagra in the Swedish market, Glocal Pharma offers analyses of commer- cial material, medical discourses and legal documents to show how a Swedish, Viagra-consuming subject has been constructed in relation to the drug and how Viagra is imagined in relation to the Swedish man. Engaging with debates about pharmaceuticalization, the authors consider the ways in which new identities are created around drugs, the redefinition of health problems as sits of pharmaceutical treatment and changes in practices of gov- ernance to reflect the entrance of pharmaceuticals to the market. With attention to ‘local’ contexts, it reveals elements in the nexus of pharmaceutcalization that are receptive to cultural elements as new products become embedded in local markets. An empirically informed study of the ways in which the presence of a drug can alter the concept of a disease and its treatment, understandings of who suf- fers from it and how to cure it – both locally and internationally – this book will appeal to scholars of sociology and science and technology studies with interests in globalization, pharmaceuticals, gender and the sociology of medicine. Ericka Johnson is Assistant Director of the Technology and Social Change Divi- sion at Linköping University, Sweden and co-editor of Technology and Medical Practice: Blood, Guts and Machines. Ebba Sjögren is Associate Professor in the Department of Accounting at Stock- holm School of Economics, Sweden. Cecilia Åsberg is Associate Professor and Head of Gender Studies at Linköping University, Sweden. Global Connections Series Editor: Robert Holton, Trinity College, Dublin Global Connections builds on the multidimensional and continuously expanding interest in globalization. The main objective of the series is to focus on ‘connect- edness’ and provide readable case studies across a broad range of areas such as social and cultural life and economic, political and technological activities. The series aims to move beyond abstract generalities and stereotypes: ‘Global’ is considered in the broadest sense of the word, embracing connections between different nations, regions and localities, including activities that are transnational and trans-local in scope; ‘Connections’ refers to movements of people, ideas, resources and all forms of communication as well as the opportunities and con- straints faced in making, engaging with and sometimes resisting globalization. The series is interdisciplinary in focus and publishes monographs and collections of essays Ey new and estaElished scholars. ,t fills a niche in the market for Eooks that make the study of globalization more concrete and accessible. For a full list of titles in this series, please visit www.routledge.com/series/ASHS ER1306 Also published in this series: Violence and Gender in the Globalized World: The Intimate and the Extimate Edited by Sanja Bahun and V. G. Julie Rajan Global Exposure in East Asia: A Comparative Study of Microglobalization Ming-Chang Tsai Ericka Johnson, Ebba Sjögren and Cecilia Åsberg Ericka Johnson, Ebba Sjögren and Cecilia Åsberg First published 2016 by Routledge 2 Park Square, Milton Park, Abingdon, Oxon OX14 4RN First published 2016 by Routledge 2 Park Square, Milton Park, Abingdon, Oxon OX14 4RN and by Routledge 711 Third Avenue, New York, NY 10017 and by Routledge 711 Third Avenue, New York, NY 10017 Routledge is an imprint of the Taylor & Francis Group, an informa business Routledge is an imprint of the Taylor & Francis Group, an informa business © 2016 Ericka Johnson, Ebba Sjögren and Cecilia Åsberg © 2016 Ericka Johnson, Ebba Sjögren and Cecilia Åsberg The right of Ericka Johnson, Ebba Sjögren and Cecilia Åsberg to Ee identified as authors of this work has Eeen asserted Ey them in accordance with sections 77 and 78 of the Copyright, Designs and Patents Act 1988. The Open Access version of this book, available at www.tandfebooks.com, has been made available under a Creative Commons Attribution-Non Commercial-No Derivatives 3.0 license. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and e[planation without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data The LOC data has been applied for. Library of Congress Cataloging-in-Publication Data The LOC data has been applied for. ISBN: 9781472481634 (hbk) ISBN: 9781315585185 (ebk) ISBN: 9781315585185 (ebk) Typeset in Times New Roman by Apex CoVantage, LLC Contents Notes on contributors Acknowledgements Notes on contributors Acknowledgements 1 Introduction: Glocal pharmaceuticalization ERICKA JOHNSON PART 1 Changes in forms of governance 13 2 Governing by drugs: Conniving patients, beguiled doctors and disciplining drugs 15 EBBA SJÖGREN AND ERICKA JOHNSON 3 ‘A few good men’ are not enough: Upsetting general categories with specific knowledge when making reimbursement decisions 29 EBBA SJÖGREN PART 2 Changes in the medical discourse 49 4 The Swedish medical discourse: Impotence, erectile dysfunction and Viagra in Läkartidningen 51 ERICKA JOHNSON 5 Alpha-blockers and a weaker pharmaceutical influence on medical discourse 63 ERICKA JOHNSON PART 1 PART 1 Changes in forms of governance 2 Governing by drugs: Conniving patients, beguiled doctors and disciplining drugs 15 EBBA SJÖGREN AND ERICKA JOHNSON 3 ‘A few good men’ are not enough: Upsetting general categories with specific knowledge when making reimbursement decisions 29 EBBA SJÖGREN PART 2 Changes in the medical discourse 49 4 The Swedish medical discourse: Impotence, erectile dysfunction and Viagra in Läkartidningen 51 ERICKA JOHNSON PART 2 Changes in the medical discourse 4 The Swedish medical discourse: Impotence, erectile dysfunction and Viagra in Läkartidningen 51 ERICKA JOHNSON 5 Alpha-blockers and a weaker pharmaceutical influence on medical discourse 63 ERICKA JOHNSON 63 vi Contents PART 3 Techno-social relationships and identities 73 6 Enrolling men, their doctors and partners: Individual and collective responses to erectile dysfunction 75 ERICKA JOHNSON AND CECILIA ÅSBERG 7 Viagra selfhood: Pharmaceutical advertising and the visual formations of Swedish masculinity 88 CECILIA ÅSBERG AND ERICKA JOHNSON 8 Conclusions: Glocal pharma and the Swedish Viagra man 99 ERICKA JOHNSON References 108 Index 121 vi Contents PART 3 73 Techno-social relationships and identities Notes on contributors Ericka Johnson is a Senior Lecturer at the Department of Thematic Studies, Tech- nology and Social Change. Her research has looked at medical technologies and their relationship to the patient body and gender, with a particular focus on pharmaceuticals. Currently she is working on discursive constructions of the ageing prostate. Her publications include the edited volume Technology and Medical Practice: Blood, Guts and Machines (with Boel Berner 2010), Ash- gate, and Dreaming of a Mail Order Husband (2007), Duke University Press. Ebba Sjögren is Associate Professor at the Department of Accounting, Stock- holm School of Economics. Her research centres on the role of knowledge- based management tools in organizational control and decision-making, notably in the health care sector, and the practical undertaking of valuation work in the construction of markets. She combines these two interests in an ongoing cross-country comparative study of price formation and assessment of inpatient pharmaceuticals. Her work has previously been published in journals including European Accounting Review, Journal of Common Market Studies, The Sociological Review, Valuation Studies and Governance. Cecilia Åsberg is Professor in Gender Studies at the Department of Thematic Studies at Linköping University. Focusing on reciprocity and vulnerability as a more-than-human condition, she has published on the natures and cultures of genetics, Alzheimer’s disease, pharmaceuticals, laboratory life and environed bodies. Currently working on toxic embodiment, she heads a Swedish research programme in environmental humanities. Her most recent book is A Feminist Companion to the Posthumanities (with Rosi Braidotti 2015), Springer. Acknowledgements This book is a result of interdisciplinary discussions and collaborative writing practices between the three authors and our colleagues in many different academic fields and constellations. We each came to the material from our own disciplines and have also brought with us critics and conversation partners from different places and perspectives. In addition, we have been invited to put chapter drafts forward in many different seminar groups, which has been invaluable. Thank you to all of you who haYe helped us find our way through this proMect ,n particular, we would like to thank Boel Berner, Elin Björk, Maria Björkman, Anna Bredström, Jelmer Brüggemann, Oscar Javier Maldonado Casteneda, Isabelle Dussauge, Mark Elam, Kathrin Friedrich, Andreas Gunnarsson, Lisa Guntram, Ali Hanbury, Kath- erine Harrison, Marie-Louise Holm, Klasien Horstman, Ine van Hoyweghen, Sari ,rni, 6onMa -erak=uiderent, 0arianne Winther -\|rgensen, &orinna .ruse, %engt Larsson, Francis Lee, Marin Letell, Lisa Lindén, Sebastian Linke, Nina Lykke, Erik Malmqvist, Tara Mehrabi, Ulf Mellström, Shai Mulinari, Alma Persson, Anders Persson, Jesper Petersson, Celia Roberts, Kerstin Sandell, Göran Sundqvist, Håkan 7h\|rn, 6teYe Woolgar, .ristin =eiler and 7eun =uiderent-erak. We would also like to thank the funders who haYe made large parts of this work possiEle the 6wedish 6cience &ouncil, which financed the proMect µ&ulture, Eio- medicalization and changing masculinities: Viagra™ as symbol and technology’, the European Research Council under the European Union’s Seventh Frame- work Programme (FP7/2007–2013)/ERC grant agreement no. 263657, PPPHS, and the -an Wallander and 7om +edelius )oundation. ,n addition, the proMect has Eenefitted from institutional support at 7he 6wedish &ollegium for $dYanced Study, Gothenburg University, Stockholm School of Economics and, primarily, Linköping University. 1 Introduction Glocal pharmaceuticalization Ericka Johnson The pharmaceutical nexus is large, international and successful. It is also very complex. Heterogeneous in its components, stretching both upstream to research, clinical trials, product development and disease construction, and downstream through doctors and experts, it enables and enrols regulatory bodies, lawmakers, lobbyists, judicial systems, marketing professionals, producers, medical practi- tioners and consumers. And it does this on a global scale, dominating the medical approach in adYanced Western countries and spreading its territory to domains in the developing world. Pharmaceuticals are colonizing and creating new mar- kets in geographically and socially diverse parts of the world and throughout all aspects of the industry. The pharmaceutical industry is trying, and succeeding, to work and profit in Yery different conte[ts, with Yery different regulatory frame- works, marketing needs and consumer bases. And while the industry has had an international approach since Eefore World War ,,, the gloEal market for pharma- ceuticals and the profit margins, which large, multinational companies are chas- ing, have grown exponentially in recent decades. These themes are prevalent in critical studies of global pharmaceuticals from within the social sciences (see Elliot 2003; Moynihan and Cassels 2005; Petryna and .leinman Williams et al. 2011a). In this book, we present a close look at the glocal of gloEal pharma in 6weden. %y attending to the specificities of the local in Sweden within a conceptual framework of global pharmaceuticals, we will Ee showing gloEal trends and local responses in a Western1orthern, highly developed and regulated state. To do so, we employ the term glocal to signify that the local specificities of a cultural conte[t, including its regulatory Eodies, do something to the global pharmaceuticals that are integrated into it, and, likewise, global pharmaceuticals impact the local context. Critical studies of pharmaceuticals have developed out of academic work on medicalization, a concept often traced back to work inspired by Parsons’s analysis of the sick role in the 1950s. Medicalization became a sociological tool with which to think about the interplay between medicine, individuals and society. Parsons’s proposition, that the sick role allows the individual to avoid blame for his/her ill- ness while simultaneously legitimating and excusing his/her shortcomings in the workplace or family as long as the individual seeks medical help (Parsons 1951), has resonated throughout medical sociology and influenced the deYelopment of Ericka Johnson 2 the field since. 1 Introduction Glocal pharmaceuticalization Within sociology, studies of medicali]ation initially focused on the hospital as a professional institution within which the patient figured, Eut where doctors (and to a lesser extent, nurses) were particularly interesting to study as they assumed professional roles, made decisions, directed practice and policy and almost peripherally attended the ill see (aton and Weil %alint )o[ 1959; Becker et al. &oser )reidson . 2ne finds echoes of the sick role in Illich (1976), Fox refers to it in her work on medicalization in America (Fox 1977, 15), and current interests in the process of pathologizing emotions (Healy 2004) and behaviour (Hart et al. 2006; Conrad 2007) touch on the inter- play between illness and society. p y y How exactly medicalization occurs is, of course, up for debate, as is what should be included in the term. Illich famously called it ‘iatrogenesis’, combining the Greek iatros, ‘physician’, and genesis, ‘origin’ (Illich 1976, 3), yet it is used to convey a very broad set of processes, sites and actors beyond the physician and his/her workplace. The concept of medicalization has expanded beyond the idea of the sick role, to include ideas of how a patient’s complaint becomes a medi- cal diagnosis (Balint 1957), and how social deviance becomes medicalized (cf. Fox 1977). It now applies to ‘a process by which nonmedical problems become defined and treated as medical proElems, usually in terms of illness and disorders¶ (Conrad 2007, 4), including, and worryingly, the transformation of difference into pathology (Conrad 2007, 148). Forty years ago, Illich pointed to the way ill health is created by what he termed the medical bureaucracy, which defined the need for medical care ± defined nonnormatiYe ways of Eeing as diseased and in need of medical treatment – and discursively limited the ability or opportunity for other forms of care, be that social, familial, spiritual or self-care (Illich 1976, 40). Critical studies of medicalization in the social sciences today look beyond the immediate medical context to explore how commercial, state and media inter- ests also produce illness. 1 Introduction Glocal pharmaceuticalization And while, within medical sociology, medicalization is still largely used to direct attention to issues of how illness is understood and used in social contexts, work by Mol (2002) and other science and technology studies (STS) researchers broaches and questions the illness/disease divide, and has begun to approach biomedical technologies with a critical lens (Berg and Mol 1998; Johnson and Berner 2010). These examine how illness and disease are enacted, but also how their particular formations shape medical practice and governance. Medicalization conceptually sets the stage for the idea of pharmaceuticaliza- tion, understood to mean the introduction and acceptance of drug-based responses to (and creation of) health issues, as illustrated in Dumit’s (2012) book Drugs for Life. Williams, 0artin and aEe use the term pharmaceuticalization to interrogate how many different aspects of life are becoming opportunities for pharmaceuti- cal intervention. In their usage, pharmaceuticalization is a broad term, and can be applied to processes of ‘discovery, development, commercialization, use and governance of pharmaceutical products centred around chemistry-based technol- ogy¶ Williams et al. 2011a, 711). This is opposed to Abraham’s more medically focused use of the term $Eraham , . ,n its Eroader definition, which 3 Introduction we embrace in this book, pharmaceuticalization can also be applied to the use of pharmaceuticals to address issues currently outside of medical practice, like some lifestyle drugs or the use of nicotine replacement therapies in chewing gum or e-cigarettes (see Elam 2012). This broader stance is not as new or controversial as the $Eraham Yersus Williams and colleagues E e[change would suggest. As Illich pointed out long before our current obsession with lifestyle drugs, pharmaceuticals do not need doctors and hospitals to pervade society, nor are most ‘poisons’, ‘remedies’ and ‘placebos’ necessarily destined for the sick (Illich 1976, 61). Reminding us of pharma’s ambiguity, Illich noted: ‘The Greek’s only word for “drug” – pharmakon – did not distinguish between the power to cure and the power to kill¶ ,llich , . While not as radical in their take on pharmaceu- ticals as ,llich, Williams and colleagues point out that Eoth medicalization and pharmaceuticalization are ostensiEly Yalueneutral terms Williams et al. 2011a, 711), and medicalization and pharmaceuticalization both describe processes that may imply Eenefits or drawEacks to society and indiYiduals. 1 Introduction Glocal pharmaceuticalization It is positioned in social science and cultural studies approaches to pharmaceuticals, and employs theories and terms that attend to the fle[iEility of pharmaceuticals as medical technologies, especially when they become mobile across countries, regulatory frameworks and value systems (cf. Dugdale 2000; Kruse 2016). Our book can be read as a study of pharmaceuticals at an intersection of political, economic and ethical dimensions (cf. Petryna and Kleinman 2006; Brody 2007). Approaching such a multi-scaled and complex nexus demands an analytical toolbox which is heterogeneous and broad, so we have mixed liber- ally from our disciplinary backgrounds to create an approach drawing from post- humanities studies, STS and medicine and management and organizational studies. hi h i i fl d E h h ¶ E d i i d i hi The theoretical framework of pharmaceuticalization employs analysis of hetero- geneous aspects of pharmaceuticals in society, and can productively be approached from within different disciplines. Because of this, the work in this book is multi- disciplinary. It is positioned in social science and cultural studies approaches to pharmaceuticals, and employs theories and terms that attend to the fle[iEility of pharmaceuticals as medical technologies, especially when they become mobile across countries, regulatory frameworks and value systems (cf. Dugdale 2000; Kruse 2016). Our book can be read as a study of pharmaceuticals at an intersection of political, economic and ethical dimensions (cf. Petryna and Kleinman 2006; Brody 2007). Approaching such a multi-scaled and complex nexus demands an analytical toolbox which is heterogeneous and broad, so we have mixed liber- ally from our disciplinary backgrounds to create an approach drawing from post- humanities studies, STS and medicine and management and organizational studies. 7his approach is influenced Ey the authors¶ own Eoundary crossings into and within interdisciplinary fields gender studies 676 and medicine and social studies of accounting. We Ering with us theoretical and methodological Eaggage from our respectiYe fields, including a shared interest in the materialities of pharmaceuticali- zation. Methodologically, our research, like much of that we draw inspiration from, is qualitative, and relies on close readings of visual and written discourses. 1 Introduction Glocal pharmaceuticalization %ut, as has Eeen the case with medicalization, in social science studies of pharmaceuticalization there seems to be a tendency to see these processes as negative, or at least suspi- cious. Abraham echoes this reticence towards pharmaceuticals when he writes µthat increased pharmaceuticali]ation can sometimes Ee suEoptimal for signifi- cant therapeutic advances in the interests of public health’ (Abraham 2010, 603). The relationship between pharmaceuticalization and medicalization is some- times very intertwined, and many critical studies of pharmaceuticals have shown how drugs are being used to manufacture diseases which can then be treated with them. But the process of pharmaceuticalization can imply more than just increased medicalization. Many examples exist where pharmaceuticalization changes the method of treating an already existing medical condition. As Abraham notes, ‘pharmaceuticalization can grow without expansion of medicalization, because some drugs are increasingly used to treat an established medical condition involv- ing no transformation of a non-medical problem into a medical one’ (Abraham , emphasis in the original. 7his Yolume discusses the influence of phar- maceuticalization on the treatment of established medical conditions, as well as on marketing communication and the governance of access to such treatment, using the example of Viagra and the treatment of impotence. What is the suEMect of the process of pharmaceuticali]ation" Williams and col- leagues use the term pharmaceutical regime to cover the networks of institutions, organizations, actors, artefacts and cultural values one can identify in studies of pharmaceuticalization. Others have used the term pharmaceutical nexus (cf. Pet- ryna and Kleinman 2006), and Abraham talks in terms of processes (Abraham . What we take from these discussions is the idea that pharmaceuticals are one actor within a complex and heterogeneous collectif (Callon and Law 1995) of actors, institutions and ideas, including clinicians, patients, consumers, regulators, sales reps and marketing departments. This collectif of actors notably includes the drugs themselYes in Yery specific technological forms ± pills, patches, sticks and shots. We treat these material artefacts as a useful prism to see through and with, Ericka Johnson 4 to bring into focus and refract various values, ideas and desires that are manifested in and through the drugs we are studying. The theoretical framework of pharmaceuticalization employs analysis of hetero- geneous aspects of pharmaceuticals in society, and can productively be approached from within different disciplines. Because of this, the work in this book is multi- disciplinary. 1 Introduction Glocal pharmaceuticalization These discourses are taken from regulatory contexts (legal and court documents), profes- sional debates (medical journals and testimonials from medical experts, court wit- nesses and committee members) and commercial material (advertisements for the drugs, often on ‘informational websites’ and other Internet forums, to circumvent the Swedish prohibition on direct-to-consumer (DTC) advertising of prescription pharmaceuticals). The different discourses are then analyzed to trace the glocal contours of Swedish Viagra and the Swedish Viagra man. g g 'espite our disciplinary promiscuity, or µtheoretical eclecticism¶ cf. Williams et al. 2011a, 722), and the diversity of material we analyze, the overarching theo- retical framework in all three sections of this book can be related to the concept of pharmaceuticali]ation $Eraham Williams et al. 2011a), from which we garner specific Tuestions to Tuery the shapes and forms that gloEal pharmaceuti- cals assume as they are integrated into local discourses, and how the discourses and the pharmaceuticals change in the process. Pharmaceuticalization has many aspects. Abraham, for example, argues that it involves dimensions from biomedi- calism, medicalization, industry drug promotion, consumerism and the ideology or policy of the regulatory state $Eraham , . Williams and colleagues (2011a) identify several more aspects, including the role of the media and the use of drugs outside of the medical domain. ,n this Eook, we specifically attend to three aspects of pharmaceuticalization that we think are particularly tangible and visible in the case of Swedish Viagra, yet also relevant to a discussion of glocal pharmaceuticalization. These are: the way pharmaceuticals change forms of gov- ernance the redefinition of health proElems as issues with a pharmaceutical solu- tion; and the creation of new techno-social identities around drugs and the way 5 Introduction pharmaceuticals become essential actors in relationships between subjects. Our analysis of these aspects in Sweden shows how the local context is an important influence on the process of pharmaceuticali]ation.i 7he first aspect of pharmaceuticali]ation, which we will discuss in this Eook, is the way pharmaceuticals reshape forms of governance. In the global debate, examples of pharma-governance are often related to questions about emerging markets, equitable access, cost and patent protection, with political decisions at the nation state level contravening international decisions and regulations – and sometimes even forcing these regulations to change (see Biehl 2006). 1 Introduction Glocal pharmaceuticalization However, the presence and influence of international pharmaceuticals is also Yery tangiEle in governance decisions and policy responses in established and well-regulated nation states, like Sweden, that have a reputation for being obedient to interna- tional treaties and understandings. While our material does not proYide e[amples of patent infringement and black or grey market infringement, it does show a nation state finding new ways of regulating and goYerning pharmaceutical access and subsidies, changing the role of medical doctors and regulatory decision mak- ers at the point of clinical diagnosis.i Critical studies of pharmaceuticals also discuss the redefinition of health problems as issues with a pharmaceutical solution (see Healy 2004; Kassirer 2005; Moyni- han and &assels /aw 0oynihan and 0int]es Williams et al. 2009, 712). As Moynihan notes, this process involves, among other things, exam- ples of how pharmaceutical advertising and regulation turn ordinary ailments into medical problems, present mild symptoms as serious, and treat inter-personal problems as medical (Moynihan and Cassels 2005). Our study of the medical discourses around Viagra show this happening very clearly in the Swedish con- text. Predictably, impotence became erectile dysfunction, and urology replaced relationship counselling and sex therapy as the site of treatment for erectile prob- lems. Yet because of the ideological framework of the Swedish health care sys- tem, Viagra was also discursively connected to diabetes, multiple sclerosis and spinal injuries, which tints the identity of the drug and the Swedish Viagra man in the medical discourse. The creation of new techno-social identities around drugs In our analysis of the Swedish patient information pages on pharmaceutical web- pages, men, doctors and the partners of men with erectile dysfunction are enrolled into a 9iagra discourse which presents them with specific tasks and functions in the recognition of, need for and enabling of Viagra. As Chapters 6 and 7 describe, these roles are similar to patient, doctor and partner identities in North American 9iagra adYertising yet also tweaked to fit perceiYed 6wedish Yalues and norms. $s the concept of pharmaceuticalization would predict, Viagra is positioned as a nec- essary component in these identities and, important, in the relationships between the subject positions that Viagra facilitates. But the shape and shades of these identities and relationships reflect Yery traditional understandings of the local cul- ture, an interesting example of glocal identities. 6 Ericka Johnson 6 Much of the work on global pharmaceuticals explores encounters between deYeloping, nonWestern1orthern countries and a commercial pharmaceutical industry. We see the Yalue of this work, and laud its approach to Tuestions of social justice, equitable access to health care and global commercial forces as they discoYer and create new markets see /akoff . We draw theoretical lessons from these studies. For our work, the most useful studies of this global process examine local manifestations of pharmaceuticals with close, qualitative and eth- nographically inspired approaches i.e. 3etryna and .leinman Wailoo et al. 2010) For example, analysis of knowledge about, access to and use of antiret- roviral therapies in Uganda articulates how social relations and distinctions are embedded in the social meanings of medicines, as well as how the medicines enaEle and articulate the doing of family relationships Whyte et al. 2006, 260), articulations we see resonances of in our material on Viagra. Just as Applbaum (2006) shows in his study of antidepressants in the Japanese market, and with Lakoff’s work on antidepressant use in Argentina (Lakoff 2006, 133), our work shows how the market, medical profession and regulatory actors all struggle to define disease and patient groups, and to influence each other in the process. $s Lakoff writes, ‘the mutual imbrication of science, regulation and business in the circulation of pharmaceuticals is best seen not as a contamination of pure science but rather as part of a distinctive and emergent regime for authorizing knowledge claims and e[pert action¶ /akoff , ±. We draw inspiration from this approach in our analysis of relationships enabled by pharmaceuticals, as well, and with an eye to our own culture, but we challenge the tendency in this body of research to see global as a euphemism for West Yersus the 5est /\|wy 6tockel and /indpn are e[ceptions to this. We assert that it is also interesting and valuable to explore relationships between the local and the global in a deYeloped, Western1orthern, non86 conte[t. Pharmaceuticals in Sweden Medical care available in Sweden can be characterized as technically advanced, µscientifically grounded¶, eYidenceEased medicine soundly positioned in the Western1orthern medical knowledge paradigm. 7his knowledge paradigm is based on the idea that universal medical facts about the body and health exist, and that these should not be dependent on cultural context. Medical facts should travel unhindered across geographical borders. It is this assumption that makes the global reach of pharmaceuticals (and other medical responses to health and illness) possible at the theoretical level. It is also this assumption that allows the gloEal analysis of pharmaceuticals to assume that the West1orth is one unit of analysis. y In our close reading of pharmaceuticals in Sweden, however, we problematize this assumption by showing the uniqueness of the Swedish case, and demonstrat- ing that Western1orthern medicine is not a useful analytical category. Western Northern is actually a trope that hides a great deal of diversity, even beyond the oEYious difficulties of placing countries like -apan and $ustralia in this nominally 7 Introduction geographical category see $Eraham , . 7he nation states that would fit into it are a heterogeneous group which responds to global pharma in unique ways. This is an important point to remember in social science and cultural stud- ies work on the pharmaceutical industry and the medical practices it engenders. Living in an oft-cited example of socialist medicine, Swedish citizens have access to state-subsidized and state-delivered medical care at very little out-of- pocket cost. Funded by the tax system, the Swedish health care structures are based on the idea that everyone should have equal access to the same high quality of care. 7his is a concept and a system which is firmly ideological and politically linked to the history of socialist rule in Sweden during most of the twentieth cen- tury 6henkin . While the 6wedish health care system has not Eeen immune to privatization and neo-liberal impulses during the past decades (see Johans- son Krafve 2015), it still remains a system which attempts to provide close-to- free health care for close-to-everyone. This understanding of health care includes heavily subsidized pharmaceutical treatments. y p Because the Swedish state subsidizes pharmaceutical costs for all drugs approved within the scheme with a very minimal individual co-pay, our study presents a context in which the individual’s theoretical ability to pay is of little interest. Pharmaceuticals in Sweden 7he first has allowed us to analy]e µ3atient ,nformation¶ websites about erectile dysfunction and the localization elements they incorpo- rate. The second has created a very interesting economic context within which doctors debate the use of pharmaceuticals in relation to other costs, the state takes interest in limiting pharmaceutical use, and the judicial system tests these deci- sions, at the same time as the state has developed industry regulations in close collaboration with commercial interests. Abraham has pointed out that pharmaceuticalization is occurring even in coun- tries which have a DTC advertising ban on prescription pharmaceuticals, sug- gesting that we need to look at shifts in regulatory ideology or policy to explain pharmaceuticali]ation, not Must commercial forces $Eraham , . While this is certainly true, we would assert that analysis of DTC advertising may also need to be expanded to the actual practices pharmaceutical companies employ, not only the legal frameworks they work within. Sweden has a ban on DTC advertising, like the rest of Europe, but there may be reason to suspect that this ban is only partially functional. The industry-supported information pages about diseases sometimes shamelessly propose the use of pharmaceutical solutions. At times, media reports aEout new drugs read like industry press releases. When Viagra arrived it was advertised on full-sized posters at bus stops in southern Sweden, despite the ban. And, as research has shown, in Sweden (as in the United Kingdom) there seems to be widespread disregard for the regulations restricting DTC advertisement, and little bite to the regulatory bodies which are supposed to police it (Zetterqvist and Mulinari 2013; Zetterqvist et al. 2015). Thus, in the (uropean conte[t, the legality of '7& adYertising is not necessarily a sufficiently well-formulated research parameter. Pharmaceuticalization and the interplay of pharmaceuticals and state regula- tory bodies has a long tradition in Sweden, which began regulating approval and marketing of pharmaceuticals in 1934 (Abraham and Lewis 2000, 55), but its form, and that of regulatory systems in the West1orth in general, has changed significantly oYer the past decades. 7he current state of today¶s regulatory frame- work for pharmaceuticals, developed under neo-liberalism, shows how regulatory bodies have been refashioned to facilitate drug development, rather than protect the public, and in particular how these regulatory bodies are co-opted to serve the industry’s will rather than the public’s need (Brody 2007; Abraham and Ballinger 2012). Pharmaceuticals in Sweden We argue that in 6weden, gloEal pharma is confronted with a Yery regu- lated market populated by hyper-individualized patients who are also comfortable with a paternalistic welfare state that has long been responsible for giving them their drugs for almost free, as opposed to the privatized and decentralized health care systems in which states are losing (or have never really had) control of phar- maceutical use (see Petryna and Kleinman 2006). Individuals in Sweden are not familiar with the idea that they should pay for their pharmaceuticals at the point of purchase, but are concerned about the collective costs of pharmaceuticals to their tax bill, even as they demand access to treatment. This setting problematizes discussions in the extant literature which centre on individuals’ willingness (and ability) to pay. The development of some pharma- ceutical therapies (for erectile dysfunction, male pattern baldness or wrinkles, for example) and not others (malaria is a commonly cited one), and the growth of pharmaceutical treatments labelled lifestyle drugs, is testament to the underly- ing market mechanisms which encourage drug companies to target markets (con- ceived of as groups of individuals with similar medical and/or pharmaceutical needs) which are capable and willing to pay for their products. ‘As drug costs escalate and access becomes hyperindividualized, pharmaceuticals markets gen- erate new social distinctions based on the individual’s ability to pay’ (Petryna and .leinman , . 7his common assertion is to some e[tent an oYersimplifica- tion. 2ur Eook shows with specific empirical studies how this is a more compli- cated process of market subdivision and creation within well-regulated markets and distriEution channels. 6pecifically, our work proYides an e[ample of what happens in pharmaceutical marketing and distribution practices when the tension between cost to the individual meets the Swedish solidarity principle and idea that health care is a general puElic good. We highlight the tensions in this con- cept of ‘hyperindividualized’ pharmaceutical consumption, teasing out how state Ericka Johnson 8 structures and local and temporal medical consensus can influence access to and use of brand name pharmaceuticals. Two aspects of the Swedish case are particularly relevant to the glocal relation- ships of pharmaceuticals: 1) there is a ban on DTC advertising on prescription medicine (with the exception of vaccines), yet the industry seems to sidestep this ban with little reprimand; and 2) the state pays for the majority of the prescription costs for the population. Pharmaceuticals in Sweden In Sweden, this has seen the creation of the Medical Products Agency in 1990, which was funded entirely by industry fees and was supposed to accelerate drug approval times (Abraham and Ballinger 2012, 448). As Abraham and Ball- inger note, the pattern of establishing a regulatory body to improve approval pro- cesses for industry also occurred in the United Kingdom and in Germany during 9 Introduction the s and s, which then influenced the (8 supranational drug regulatory system with their neo-liberal approach (Abraham and Ballinger 2012, 449; Junker 2014). Much academic critique of the regulatory system looks at the regulatory bod- ies that approve pharmaceuticals before they are made available to the public. The work we present in Part 1, however, particularly in Chapter 2, examines a less commonly discussed regulatory actor – the legal system and court decisions on the appropriate use and funding of drugs. Here the Swedish case is very dif- ferent from that of the United States, as the state pays for prescription drugs and the use and cost of drugs are thereby regulated by a separate administrative body the 'ental and 3harmaceutical %enefits $gency, preYiously the 3harmaceutical %enefits %oard, whose decisions can Ee appealed through the court system see Sjögren 2006). This has traditionally not been a course of action (Abraham and Lewis 2000, 71), but the legal battles over subsidizing Viagra marked a change in the regulatory praxis. Outline of this book The Swedish case shows how a highly developed nation, which provides univer- sal health care, including universal access to prescription medicine, responds to and shapes the pharmaceutical solutions that a global market presents through the health care system. The existence of a single, tax-based payer for pharmaceuticals can change the faces (in the STS spirit, we would argue this changes the very products themselves) of the pharmaceutical options available to the public. As the following chapters will show, a global blockbuster drug like Viagra becomes a different product in Sweden than Viagra in the United States, and the users of Viagra are glocalized, too. This book is divided into three parts. In Part 1, we consider policy and bureau- cratic responses to the introduction of new pharmaceuticals, in particular Viagra and the possiEility of pharmacogenomics. 7he work here identifies certain argu- ments which appear to direct the response: solidarity; provision to those in need; how need is determined; and how the pharmaceutical technologies can actually assist or even replace medical doctors in the determination of need. Close reading of the debates surrounding these products shows how a system founded on the principles of solidarity and equal access to health care, including pharmaceuticals, can respond to the profitdriYen industry which is proYiding these products to the state. Part 1 begs questions about how solidarity, citizen rights and biological rights are related, and how they are created in relationship with each other and with pharmaceuticals. In Part 2, we analyze the medical discourses that surround the introduction of new pharmaceuticals. 7he first chapter looks at 9iagra and how it has influenced the medical understandings of impotence and erectile dysfunction in Sweden. It shows how the presence of an international drug can have implications for medi- cal terminology and the colloquial use of words to describe a social and medical problem, even in a small, linguistically bounded community like Sweden (with Ericka Johnson 10 approximately nine million speakers). Chapter 5 traces debates around the intro- duction of alpha-blockers for benign prostate hyperplasia to the Swedish health care market and the shift in treatment methods they implied for an already med- icalized condition. Together, these chapters present local examples of how the pharmaceutical products redefine e[isting health proElems as issues with phar- maceutical solutions and how their presence frames the medical community’s responses. Outline of this book Impotence shifts from being a condition treated with sex and couple’s therapy to a condition for which one takes a pill. Lower urinary tract symptoms for older men move from being treated with surgery to being treated with a medi- cation. ,nterestingly, this shift is nearly complete in the 9iagra case, Eut is, fifteen years later, still being contested in the case of alpha-blockers. Part 3 articulates subject positions for pharmaceutical consumers which are created in commercial discourses in 6weden. 6pecifically, we e[amine the adYer- tising, disguised as web-based disease informational pages, for Viagra. By think- ing through our cases within a theoretical framework of pharmaceuticalization, we see that pharmaceuticals are creating new subject positions around the drugs. Chapter 6 examines the construction and enrolment of (consumer) subjects which are not directly consuming the pill. Here we see how the commercial Viagra dis- course produces and enrols the man’s partner and his doctor in the production of the Swedish Viagra man. This discourse also directs the relationships the man develops and maintains with his partner and his doctor, and tries to approach the partner and doctor to help them maintain Viagra-mediated relationships with the consuming Swedish Viagra man. Chapter 7 then discusses the creation of the Swedish Viagra man and the particular characteristics assigned to him. It pre- sents an analysis of the culturally specific aspects of 6wedish masculinity which 3fi]er or at least its marketing departments and agencies deemed strong and stable enough to be associated with a product which addresses an image of failed masculinity. Chapter 8 concludes this volume by returning to the multiplicity of aspects that the term pharmaceuticalization contains. Using the literary trope of the Swed- ish Viagra man, we discuss how the local and the global interact in the Swedish context. Glocal pharma 7he changes in goYernance practices to reflect pharmaceuticals¶ presence, the redefinition of health proElems as sites of pharmaceutical treatment and the crea- tion of new techno-social identities around drugs are general aspects of pharma- ceuticalization apparent in the Swedish case material we present in this book. +oweYer, they are also aspects of the local, specific to the 6wedish conte[t, which are important to analyze and integrate into a concept of pharmaceuticalization because they show nodes in the pharmaceutical network which are receptive to influence Ey local specificities. 7he results of these local influences are what we refer to as the µglocal of pharmaceuticali]ation¶, the local specificities which appear in the discourses embedding global pharmaceutical products and practices 11 Introduction Introduction Introduction in local markets. When local responses to gloEal pharma are enacted in 6weden, the local pushes back. Persistent traces of the local can be interpreted in a couple of different ways. One could see the process of pharmaceuticalization as an irresistible, all-consuming process, and these local specificities can point to weaknesses in the process. 2ur observations could be interpreted as aspects of pharmaceuticals where they are not quite strong enough to resist and redirect the local context to their will. Or, these nodes of difference could be seen as examples of where the pharmaceutical dis- course is fle[iEle enough to consume and adapt to the local conte[t, where it shows its strength through a colonizing, but not neutralizing practice of meeting local cultures. $s Williams and colleagues note, µthere are important sources of resist- ance to the expansion of pharmaceutical markets from the media, government, medicine, patients and diverse publics thereby making de-pharmaceuticalization a possiEility in principle, if infreTuent in practice¶ Williams et al. 2011a, 722). Perhaps our examples of the glocal response to subsidizing Viagra in Sweden in Chapter 2 can indicate that this is more than mere optimism. Resistance is possible. But our analysis of the information sites for Viagra treatments and the medical discourse around Viagra and alpha-blockers in Sweden are harder to read as support for this optimism. In drawing conclusions about the glocal of pharmaceuticalization, it is useful to ask how a drug’s presence has altered the concept of a disease and its treatment, who suffers from it, and how to cure it, in the local context and internationally. Glocal pharma This book shows how drugs interact with stereotypical imaginaries of a patient, and include markers of class, race and se[uality. 'rugs can influence laws and policies to regulate the practices of both doctors and patients. Yet it is not the chemical compound that prescribes behaviours or identities, it is the network of decision makers, commercial actors, medical experts and consumers who attach the drug to specific demands, images and e[pectations to influence the EehaYiours of groups they are trying to govern, cajole or cure. And because actors in different countries have different cultural starting points and are working within different institutional frameworks, how they use a pharmaceutical varies. By looking at the pharmaceutical nexus and its actors, we can see that a global drug does more than fi[ the Eiomedical Eody¶s proElems. $s the following chap- ters will show, the mere existence of a pharmaceutical product impacts medi- cal knowledge and discourses, reinforces and even constructs cultural ideas and identities, changes the practices of experts and laypeople, and changes policy. But local circumstances also influence how drugs are presented and prescriEed and what they are allowed to be. Together, the global and local aspects of the pharma- ceutical nexus create glocal pharma. This page intentionally left blank Part 1 Changes in forms of governance This page intentionally left blank 2 Governing by drugs Conniving patients, beguiled doctors and disciplining drugs1 Ebba Sjögren and Ericka Johnson The Viagra cases marked a new way of using the courts in the regulation of pharmaceutical policy, but did so within a changed bureaucratic Ebba Sjögren and Ericka Johnson 16 landscape that had a new regulatory Eody and was grappling with how to define disease, with questions of trust, and with the involvement of patient groups and commercial actors. 0any Western welfare states haYe, in recent decades, faced the Tuestion of how to set Eoundaries for puElic fiscal responsiEility for health care serYices. 2ne recurrent solution has been to create central health care assessment organizations tasked with evaluating the appropriateness of undertaking and publicly funding particular treat- ment methods. 7he importance of using scientific facts as a decisionmaking tool in these organizations is typically emphasized (Jost 2005) and has been explored and criticized by social scientists (Pope 2003; Timmermans and Berg 2003, Gray and Harrison 2004; May et al. 2006). But whatever the shortcomings of, for example, evidence-based medicine and clinical trials, the organized collection, evaluation, codification and distriEution of eYidence aEout treatment effects is still conceiYed of as a means to achieve a more fair and effective prioritization of limited resources (cf. Cuyler and Newhouse 2000; Mitton and Donaldson 2004). In response to the conceptualization of an evidence-based prioritization imperative, state bodies have Eeen constructed and tasked with the MoE of turning scientific facts into puElic policy. The TLV, which was formally launched in October 2002, is an example of such an organization. By law, the decision-making board of the TLV is instructed to approve subsidy for a drug when: ‘the cost of using the pharmaceutical . . . is reasonable from med- ical, humanitarian and socio-economic perspectives’ (Swedish Book of Statutes 2002, 160, Section 15; authors’ emphasis added). The TLV is further instructed to take into consideration the three principles of priority-setting that Parliament approved in 1997, namely: equal human value, meaning all people have an equal right to life and health, need solidarity, that those with greatest need of treatment have priority over those with lesser need and cost-effectiveness, that the Eenefit of treatment must be reasonable in relation to its cost (Socialutskottet 1997).4i ( ) 7he suEsidi]ation status of 9iagra was one of the first cases the 7/9 decided. After a period of deliberation, the TLV decided to exclude Viagra entirely from the 6wedish pharmaceutical Eenefits scheme on 0arch . 2 Governing by drugs Conniving patients, beguiled doctors and disciplining drugs1 Ebba Sjögren and Ericka Johnson Ebba Sjögren and Ericka Johnson In April 2001, the Swedish government decided to immediately remove Viagra from the public pharmaceutical reimbursement system (Swedish Book of Statutes 2001, 140). All patients who still wanted their use of these pharmaceuticals to be subsidized were henceforth required to submit individual applications for subsidy to the government itself. This highly publicized and much-debated decision marked a clear break with the established practice whereby prescription drugs that were granted marketing approYal were consistently included in the puElic pharmaceutical Eenefits scheme. Under this scheme, patients prescribed subsidized drugs paid only a portion of their direct cost. Following the government’s decision, Viagra was still available for purchase in pharmacies by those holding prescriptions, but the patients were now to assume the cost.i 7he decision to e[clude 9iagra from the Eenefits scheme was made in a setting of long debate concerning the rising costs for health care in general, the growing public expenditure on pharmaceuticals in particular, and a concern that a block- buster drug like Viagra would put undue pressure on the public purse.2 Yet having the government decide on the subsidization of Viagra on a case-by-case basis was as extraordinary as it sounds. Prior to this, decisions were made on an aggregate leYel, and this change is an e[ample of the first aspect of pharmaceuticali]ation we will Ee discussing, when drugs change the form of goYernance Williams et al. 2011a). Because of Viagra (and concerns about pharmaceutical costs in general at the time, caseEycase decision practice was first applied and then a new goY- ernmental agency was instituted with the responsibility for deciding which phar- maceuticals to suEsidi]e the 3harmaceutical %enefits %oard here called the 7/9 to reflect its current name, the 'ental and 3harmaceutical %enefits $gency.3 This new goYernment agency changed the configuration of commercial, regulatory and public interests at the pharmaceutical nexus in Sweden, and led to a series of court cases questioning the TLV’s decisions, which we will examine in this chapter. 7hat the decision to deny 9iagra from the puElic pharmaceutical Eenefits scheme was appealed through the courts is particularly interesting because, while this option had existed in Sweden for some time, it was rarely employed (Abraham and Lewis 2000, 71). 2 Governing by drugs Conniving patients, beguiled doctors and disciplining drugs1 Ebba Sjögren and Ericka Johnson 7he marketing company, 3fi]er $%, soon appealed the 7/9¶s decision. $ series of court Eattles ensued, ending with a decision by the Swedish Supreme Administrative Court on 14 March 2008. In its ruling, the court upheld the TLV’s decision to exclude Via- gra from the pharmaceutical Eenefits scheme in 6weden. 3rior to this, two lower courts had overturned the TLV’s decision and approved restricted subsidy for 9iagra when treating specific categories of patients, which were deemed to fulfil the legal criteria for subsidy. Still today, Viagra is not eligible for use with public subsidy. As the Swedish Supreme Administrative Court took this decision, there is no further legal recourse for appeal of the case. However, the option remains for the pharmaceutical marketing company to submit a new application for subsidy if and when substantively new developments open for the possibility of a different outcome.i ,n this chapter, we will place the argumentation in the 7/9¶s decision Mustifica- tion documents and the various court rulings in a discussion of trust in regulatory bodies and expert practices by looking at how the TLV and the courts conceive 17 Governing by drugs 1 of patients, doctors and subsidized pharmaceuticals. In particular, we will focus on how patients, doctors and drugs are framed within the subsidy discourse sur- rounding Viagra and how drugs themselves are used as diagnostic tools and to enforce suEsidy decisions. We will argue that, notwithstanding the legal criteria for approving subsidy, the ultimate denial of subsidy in the Viagra case relied on a construction of imagined behaviours and roles for patients, doctors and drugs as recipients and enforcers of subsidy decisions. A comparison of the various deci- sion Mustifications points to a concern for patient misuse of the suEsidy, difficulties for doctors to deny subsidy and an organizational concern for bracket creep, all of which were thought to potentially lead to unreasonably high public spending on Viagra. In our discussion we problematize some of the assumptions on which the framing of actors is based.i While the particular circumstances of the 9iagra case are specific to 6weden, we see the TLV and its work as an example of a strengthened emphasis on mechanical objectivity and bureaucratic compliance in the management and control of health care services, issues which are relevant in other contexts, as well. 2 Governing by drugs Conniving patients, beguiled doctors and disciplining drugs1 Ebba Sjögren and Ericka Johnson This develop- ment – while by no means unidirectional – is nonetheless relevant to consider in light of the many rules, standards and decisions which target the contents of medi- cal practice from a distance (Timmermans and Berg 2003; Lagrelius and Sjögren . We see that the e[ample of 9iagra raises a numEer of pertinent practical and theoretical concerns linked to the emergence of technosocial identities. We also want to emphasize the way the pharmaceuticals themselves, especially the materiality of their delivery technologies, were enrolled in the regulatory deci- sions and that these actors shaped the local regulatory practices. The Pharmaceutical Benefits Board, the law and Viagra With the creation of the 7/9, Ey law, the decision aEout whether to suEsidi]e a pharmaceutical was to be determined by characteristics of its use.5 Regardless of which conclusion the TLV reaches as regards a pharmaceutical’s subsidization status, it must puElicly Mustify this outcome in a socalled decision Mustification document. This document has an important legal status. It is the basis for any appeal of the TLV’s decision (Swedish Book of Statutes 2002, Section 26, 160). Various procedural requirements are set out for the appeals process. Notably, the law restricts the right of appealing the decision concerning a particular drug to the company marketing that pharmaceutical. The appeal must be submitted within a strict time frame. The legislation also sets out that it is the Stockholm County Administrative Court (henceforth ‘the Lower Court’) which will rule on an appeal, followed by the Administrative Court of Appeal in Stockholm (henceforth ‘the Appellant Court’) and the Supreme Administrative Court (‘the Supreme Court’). In comparison to the fairly detailed procedural regulation of the appeals process, the suEstantiYe Easis for Mudicial reYiew of the 3harmaceutical %enefits %oard¶s decision-making is less clear-cut. The legislation does not specify in detail what it means for a pharmaceutical to haYe a µreasonaEle¶ cost of use. 6ome clarification is proYided in the legislatiYe Eill 1ew 3harmaceutical %enefits %ill . +ow- ever, this document also includes repeated references to the need for imprecise instructions for the 7/9 due to the difficulty of specifying practice for an organi- zation with a new and complicated task. The onus is therefore on the TLV to make decisions that can Ee Mustified and Mudged as Eeing in accordance with the 7/9¶s goYerning legislation. ,t is to a consideration of these Mustifications, and the court¶s Mudgements, to which we now turn. 7hese can Ee Eriefly summari]ed as follows With the creation of the 7/9, Ey law, the decision aEout whether to suEsidi]e a pharmaceutical was to be determined by characteristics of its use.5 Regardless of which conclusion the TLV reaches as regards a pharmaceutical’s subsidization status, it must puElicly Mustify this outcome in a socalled decision Mustification document. This document has an important legal status. It is the basis for any appeal of the TLV’s decision (Swedish Book of Statutes 2002, Section 26, 160). Various procedural requirements are set out for the appeals process. 2003: The TLV denies subsidy for Viagra ,n its decision Mustification document from 0arch , the 7/9 acknowledges that Viagra may be a reasonable drug to subsidize for patients with severe ED. However, the judgement is that the agency does not have the means for ensur- ing that suEsidy is restricted only to those patients whose treatment needs fulfil the legal criteria for publicly funded pharmaceutical use (Pharmaceutical Ben- efits %oard c, the risk that nonworthy patients would receiYe suEsidi]ed medicine and opening the door for diagnostic bracket creep is such that all use is denied subsidy. Empirical material 7he following account focuses on how the 7/9 Mustified its decision to deny suEsidy for 9iagra use, and how these Mustifications were Mudged. 2ur material deriYes from the 7/9¶s original decision Mustification document, 0arch 3harmaceutical %enefits %oard a the argumentation set out in court rulings made by the Stockholm Lower Administrative Court in June 2004 (Länsrätten 2004a); the argumentation set out in court rulings made by the Stockholm Admin- istrative Appellant Court in April 2005 (Kammarrätten in Stockholm 2005a); the argumentation set out in court rulings made by the Supreme Administrative Court in March 2008 (Regeringsrätten 2008 a, b, c).i We will also consider the contents of the 3harmaceutical %enefit %oard¶s deci- sion Mustification documents concerning two other pharmaceuticalEased treat- ments of erectile dysfunction for which the TLV approved subsidy: Caverject and Bondil. The TLV’s decisions in these two cases were referenced in several court proceedings, thereby making them empirically relevant for our analysis. Of note is that the decisions these documents present were made by collec- tives, and they were not unanimous decisions. Dissenting members of the TLV’s decision-making board and the Supreme Administrative Court have stated their grounds for disagreeing with the final outcome of particular decisionmaking pro- cesses in writing. Ebba Sjögren and Ericka Johnson 18 The Pharmaceutical Benefits Board, the law and Viagra Notably, the law restricts the right of appealing the decision concerning a particular drug to the company marketing that pharmaceutical. The appeal must be submitted within a strict time frame. The legislation also sets out that it is the Stockholm County Administrative Court (henceforth ‘the Lower Court’) which will rule on an appeal, followed by the Administrative Court of Appeal in Stockholm (henceforth ‘the Appellant Court’) and the Supreme Administrative Court (‘the Supreme Court’). In comparison to the fairly detailed procedural regulation of the appeals process, the suEstantiYe Easis for Mudicial reYiew of the 3harmaceutical %enefits %oard¶s decision-making is less clear-cut. The legislation does not specify in detail what it means for a pharmaceutical to haYe a µreasonaEle¶ cost of use. 6ome clarification is proYided in the legislatiYe Eill 1ew 3harmaceutical %enefits %ill . +ow- ever, this document also includes repeated references to the need for imprecise instructions for the 7/9 due to the difficulty of specifying practice for an organi- zation with a new and complicated task. The onus is therefore on the TLV to make decisions that can Ee Mustified and Mudged as Eeing in accordance with the 7/9¶s goYerning legislation. ,t is to a consideration of these Mustifications, and the court¶s Mudgements, to which we now turn. 7hese can Ee Eriefly summari]ed as follows 2004: The Lower Court approves restricted subsidy to ‘reasonable’ patients with certain medical conditions or a treating specialist In 2004, the Lower Court rules to reverse the TLV’s denial of subsidy, and approves subsidy with restrictions to patients with certain medical conditions such as diabetes or cardiovascular diseases, or who were prescribed the drugs by a urologist. Unlike the 7/9, the /ower &ourt concludes that the scientific studies the pharmaceuti- cal companies submitted show the pharmaceutical to be generally cost-effective, 19 Governing by drugs and therefore reasonable. In contrast to the TLV, the Lower Court also judges that the two other pharmaceutical-based treatments of male impotence – Caverject and Bondil – are relevant comparisons. Viagra (and Cialis and Levitra) was deemed cost-effective in comparison to Caverject and Bondil, which had been approved for subsidy in the intervening year between the TLV’s decision on Viagra and the Lower Court’s decision (see also Länsrätten in Stockholm 2004b; 2004c, 13, 25). ( ) 7he 7/9 immediately appealed this decision. While the appeal was pending, Viagra (and Cialis and Levitra) was not subsidized. 2005: The Appellant Court approves restricted subsidy on different grounds: specialists to make appropriate diagnosis of ‘reasonable’ patients The Appellant Court did not uphold the TLV’s subsequent appeal of the Lower Court’s ruling. However, the Appellant Court did change the definition of the restrictions for approved subsidy. In the Appellant Court’s ruling, Viagra was granted restricted subsidy for: ‘patients with the [medical condition] severe erec- tile dysfunction, regardless of underlying illness. Initial prescription [is] to be made by a physician with specialist competence in urology’ (Kammarrätten in Stockholm 2005, 18). In contrast to the Lower Court’s conclusion, the Appellant Court agreed with the TLV’s initial conclusion that the cited clinical studies showed that only patients with severe male impotence were reasonable to treat. However, the Appellant Court agreed with the Lower Court’s argument that medical practitioners could depend- ably identify patients who were ‘reasonable’ to subsidize. Yet the diagnosis-based restriction the Lower Court set was not deemed to correctly identify those patients who should receive subsidized treatments. Rather, the Appellant Court argued that restricting subsidy to initial prescription by a medical specialist on male reproductive organs (urologist) was a (more) dependable way of determining ‘reasonable’ patients. The TLV also immediately appealed this decision. It would take three years for the Supreme Court to make its decision in the case. 2008: The Supreme Court denies all subsidy Based on the existence of Bondil and Caverject, the uncertainty of a ‘severe ED’ diagnosis, the imagined behaviours of patients and doctors and the risk for diag- nostic bracket creep, the Supreme Court denied all subsidy of Viagra (and Cialis and /eYitra in . We will now discuss this decision. Diagnostic dilemmas: identifying the right patients to treat with subsidized drugs ,n the final 6upreme &ourt decision that 9iagra would not Ee suEsidi]ed, the 7/9 argued it would be impossible to limit the subsidization of Viagra to appropriately Ebba Sjögren and Ericka Johnson 20 needy and deserving patients, and therefore the drug should not be subsidized at all. As the TLV puts forward in its argument: Doctors will rely upon the patient’s responses and understandings [to ques- tions about their ED], which in practice means that it is impossible for the individual doctor to deny the patient a subsidized prescription if the patient demands it. Likewise, the patient can nearly always be expected to want to have the medication subsidized. (Regeringsrätten 2008a, 3) 7he implication is that all, or at least a significant numEer of, patients with mild ED – those not considered deserving of the subsidy – could be expected to con- nive and wile their way into a diagnosis of severe ED, beguiling their doctors into making this diagnosis and thereby receiving subsidized Viagra instead of paying for it themselves. Why are patients and doctors represented this way" Why does the 7/9 and apparently the Supreme Court, which agreed to its arguments) distrust patients and declare doctors incapable of withstanding a patient’s wishes in the clini- cal setting, implying that the laypersone[pert relationship has Eroken down"6 Other research has shown this dynamic does occur in the patient/doctor meet- ing (cf. Hirschauer 1998, 16; Timmermans and Berg 2003, 121; Friberg 2006). And empowered, knowledgeable patients who make demands on their health care providers exist, and are cultivated by both patient groups and care providers. But one would assume the belief still exists that doctors are capable of objectively diagnosing disease. Yet, actually, the (in)ability of doctors to assess the severity of ED was already questioned and debated in the original TLV decision document, although three board members disagreed with this, stating: The only ones who can determine if a patient has severe ED are the patient, his partner, and his doctor. This type of decision occurs daily and repeat- edly for all sorts of illnesses in a doctor’s day-to-day practice, with varying degrees of certainty. Diagnostic dilemmas: identifying the right patients to treat with subsidized drugs 7o determine (' can hardly Ee more difficult than to determine if a patient is suffering from depression, pain or any other state for which the doctor must primarily rely on information from the patient.i 3harmaceutical %enefits %oard c, 3harmaceutical %enefits %oard c, But they were in the minority. The idea of conniving patients and beguiled doctors continued to appear and became one of the important framing arguments in the 6upreme &ourt decision. We suggest that when it appears in the arguments put forward to the Supreme Court, the construction of conniving patients and beguiled doctors is relating and responding to earlier arguments about the reasonableness of Viagra and prior decisions to subsidize other medical treatments of ED. It is important to keep in mind that throughout the debate about subsidies, it was generally agreed that Viagra was a ‘reasonable’ drug to subsidize for severe ED.7 The Supreme Court, for example, stated that it was ‘uncontested that medical 21 Governing by drugs treatment of severe ED is to be considered urgent and that treatment with Viagra in these cases is cost-effective’ (Regeringsrätten 2008a, 9). And in the original decision Mustification document, the 7/9 formulated its Yiews on the µreasonaEle ness’ of treating patients with severe ED as follows: [T]he diagnosis [of] ED is so wide that one can include everything from com- plete inability [to achieve an erection] to the occasional inability [to achieve an erection]. The Board can therefore determine that the degree of suffering for all individuals who are affected by ED is not so great that it can dictate that the group as a whole has a large need of care in relationship to many other patient groups. In addition, a gradual reduction in erectile ability is a natural part of aging. According to the Board’s understanding, it can hardly be the general public’s (allmännas) task to ensure that a person can live with the same functionality for the entire life. . . . The Board has considered if it would Ee possiEle to adopt a Tualified decision which would allow 9iagra to be subsidized only for those who suffer from severe ED, which can be more appropriate to treat medically and where one can assume that the medicine would Ee costeffectiYe. Diagnostic dilemmas: identifying the right patients to treat with subsidized drugs 7he %oard cannot, howeYer, find appropriate restric- tions which would make it possible in this case to limit the subsidy to only those who have a socioeconomically reasonable need of subsidized treatment. 3harmaceutical %enefits %oard c, Thus, already in the original decision document the TLV admits it may be reason- able to subsidize Viagra for patients with severe ED, but that existing medical practice is not capable of or trusted enough to provide diagnoses that meet the level of certainty that the TLV as a bureaucratic institution demands, that is to distinguish without a doubt which patients have mild ED and which have severe ED. The problem was, for the TLV and the Supreme Court, there was no reliable way to determine if a patient suffered from severe or merely mild ED. Debates over diagnostic tools: questionnaires, urologists and patients’ accounts A tool exists for measuring ED: the International Index of Erectile Function (IIEF). However, in the various decision-making processes involving the subsidization status of Viagra disagreement recurred about its ability to actually determine the severity of ED. According to the TLV in the court documents, this questionnaire was developed for other (research) purposes and is only really used as a support or crutch in the patient meeting, and not as a diagnostic instrument (Regeringsrät- ten 2008a, 3). The TLV’s position, as described in the Supreme Court ruling, is that: ‘the questionnaire provides no guarantee that the information provided by the questions is a mirror of reality’ (Regeringsrätten 2008a, 3). Furthermore, the TLV does not see the IIEF as a necessary diagnostic tool because doctors can diagnose their patients’ ED without it. Yet when making this diagnosis, doctors will haYe to rely on patients¶ information and e[periences, rather than a scientific 22 Ebba Sjögren and Ericka Johnson tool. Therefore, the TLV claims that doctors are powerless to deny the truth of patients’ stories, thus making it impossible to deny the patients subsidized Viagra if the patients demand it (presumably by telling doctors they have severe ED) (Regeringsrätten 2008a, 3). Expert testimony, particularly in the Lower Court, supported the claim that the IIEF was primarily used as a conversational aid, not a diagnostic tool, and that doctors did have to rely on conversations with patients to determine the sever- ity of the ED. Like in the dissenting opinion of the original decision document, arguments were put forth through the entire debate that this type of diagnosis is something doctors are Tualified to do, and do in their daily practice with many other diseases. +oweYer, these arguments did not proYide sufficient reassurance to the TLV or the Supreme Court that the diagnosis of severe ED was being made on certain, scientific grounds.i 7he difficulty of diagnosing seYere (' also carried with it another proElem for the TLV. According to the Supreme Court documents, because it was impossible to identify oEMectiYely, scientifically and with certainty the group of patients who should be covered, it would also be impossible to develop bureaucratic structures which could check that the subsidy was correctly applied. Debates over diagnostic tools: questionnaires, urologists and patients’ accounts Following on from this was a series of arguments against the use of specialist doctors to regulate the pre- scription of Viagra to those with severe ED, which the Appellant Court decision had dictated. However, one of the biggest problems with relying on specialists to diagnose severe ED was an uneven distribution of specialists in the country, which meant many people would not have access to a specialist. If patients were forced to rely on visiting a specialist for the prescription of the drug, those patients liv- ing in areas without access to them would be discriminated against by the health care system, which would go against the principle of equal access to health care guaranteed by law (Swedish Book of Statutes 1982, 763). Additionally, the TLV argued that restricting prescription rights to specialist doctors could only be moti- vated for control reasons, not medical reasons, which would not lead to an optimal use of resources within the health care system (Regeringsrätten 2008a, 3–4). Likewise, when discussing the use of specialist doctors the TLV asserted that Eecause the disease of (' is fle[iEle and patients¶ e[perience of it YariaEle, Eoth oYer time and from patient to patient, it is not a sufficiently staEle condition to war- rant one initial truth-determining moment with a specialist. An initial prescription by a specialist would, therefore, not guarantee that the medicine would only be used for the most severe cases over an extended period of time (Regeringsrätten 2008a, 4). A man with severe ED at one appointment may get better and have mild ED later. Thus there was no trusting that even a specialist could apply the criteria for subsidizing the medication appropriately. However, we feel that the most interest- ing element in the debate is the existence of and subsidy for Bondil and Caverject. Pharmaceuticals as alternative tools for resolving problems of diagnosis The TLV granted subsidy for Bondil and Caverject a few months after the agency denied suEsidy for 9iagra 3harmaceutical %enefits %oard d e. %ondil 23 Governing by drugs is a medication in ‘dissolvable stick’ form inserted into the urethra. It can be administered by the man himself, after initial training by a medical professional, and produces an erection after about ten minutes. Caverject is injected directly into the erectile tissue of the penis. It is also possible for the man to inject him- self with Caverject, although training with a medical professional is also rec- ommended. Both Caverject and Bondil were, at the time, more expensive than 9iagra, although they now cost aEout the same per dose. 6o, as 3fi]er argued in the court cases (and as was also noted in the dissenting opinions in both the orig- inal &aYerMect and %ondil decision documents 3harmaceutical %enefits %oard 2003d; 2003e), Viagra was more cost-effective than Caverject and Bondil – all else being equal (Länsrätten in Stockholm 2004, 9; Kammarrätten in Stockholm 2005, 16). As previously noted, whether all else was equal was very hotly con- tested. For both the TLV and the Supreme Court, it was important for the decision to deny subsidy for Viagra that neither Caverject nor Bondil was particularly easy or pleasant to use. 1otaEly, the 6upreme &ourt Mustified its decision to uphold the TLV’s decision to deny subsidy for Viagra since: It is true that the drugs Caverject and Bondil are subsidized. In the decision regarding these two substances, however, it was assumed that because of the methods of use, they will primarily be used to treat patients who suf- fer from the most severe forms of ED. The general subvention decision is, therefore, in practice limited to only those cases of ED for which would also be reasonable to subsidize Viagra. The subsidy for Caverject and Bondil can therefore not be used as an argument that Viagra should also be granted general subsidy. ( i 2008 8) (Regeringsrätten 2008a, 8) Also in the Lower and Appellant Court rulings it was implied, and sometimes stated outright, that a patient with mild ED might be tempted to try Viagra, but he would not be tempted to try injecting or inserting medicine directly into his penis. Pharmaceuticals as alternative tools for resolving problems of diagnosis Repeatedly in the documents it is claimed that Bondil and Caverject have built- in mechanisms that prevent their ‘misuse’ or at least their ‘mis-subsidization’ by patients suffering from only mild ED, and the TLV can thus claim that it is provid- ing a subsidized treatment for patients who suffer from severe ED, which will not be misused (Länsrätten in Stockholm 2004, 9). Thus, one could assert that when the TLV argues before the Supreme Court that Viagra should not be subsidized, it is really saying that there is no sufficiently reliaEle medical tool in clinical prac- tice to determine the seYerity of (' which would fulfil the oEMectiYity demands of the bureaucratic subsidy system. According to the TLV’s arguments, patients can be expected to exaggerate the severity of their problem in order to receive (desirable) subsidized drugs. Doctors will probably be beguiled or bullied into agreeing with the patients. And the diagnostic questionnaire the industry and cer- tain medical practitioners suggest for determining severity is not a reliable tool, either. Instead, in the case of ED, the bureaucracy that regulates subsidies relies on the physical characteristics of two other medicines to discipline the patients and determine the severity of disease. Bondil and Caverject become ‘bitter pills’ that Ebba Sjögren and Ericka Johnson 24 only a sufficiently ill person could reasonaEly Ee e[pected to swallow. 7hus, their use Ey a giYen patient reifies a diagnosis of seYere ('. Reliance on the physical characteristics of a technology (pharmaceutical) to prevent misdiagnosis and mis-subsidizing of drugs against ED is important because it is thought to reduce the risk of diagnostic bracket creep, that is to limit the risk that the drug will be prescribed for a larger group of symptoms or for less severe symptoms that were previously untreated. There are other examples where the TLV pinned its hopes on a technology that might serve to remove ambiguity related to the correct diagnosis of ‘treatable’ patients, such as the organization’s review of pharmaceuticals for the treatment of stomach acid–related disorders 3harmaceutical %enefits %oard E see also 6M\|gren . Conclusion The TLV was purposefully created to decide the subsidization status of prescrip- tion pharmaceuticals and to take part in shaping the use of public funds for phar- maceutical spending. In this chapter, we have focused on how the TLV and the courts conceived of patients, doctors and subsidized pharmaceuticals when deter- mining the subsidization status of Viagra. Notwithstanding the legal criteria for approving subsidy, we have argued in the previous section that the ultimate denial of subsidy in the Viagra case relied on a construction of imagined behaviours and roles for patients, doctors and drugs as recipients and enforcers of subsidy decisions. $ comparison of the Yarious decision Mustifications points to a suspi- cion of patient misuse of the suEsidy, difficulties for doctors to deny suEsidy and an organizational concern for bracket creep, all of which could potentially lead to unreasonaEly high puElic spending on 9iagra. ,n this section, we will reflect on the consequences of these observations, against the organizational remit of the 7/9 and the role of pharmaceuticals in fulfilling the regulatory amEitions of the organization. In particular, we will touch on the use of pharmaceuticals as disciplining technology and a possible differentiation between bureaucratic and treatment compliance. Pharmaceuticals as alternative tools for resolving problems of diagnosis The assertion of a risk of diagnostic bracket creep goes back to the argument that a patient is always going to want to have his medication subsidized, even if he knows that he really only has mild ED (or a stomach ache), and will therefore pressure his doctor into agreeing that he actually has severe ED. Since, in this scenario, the patient’s description of ED is not to be trusted, alternative methods for creating unambiguous knowledge about the condition must be used. Doctors, in the 7/9¶s ideal world, must Ee aEle to rely on scientific diagnostic tools to help them make correct decisions in their clinical practice. But since the available tool (the IIEF) is deemed unreliable, instead the TLV relies on the drugs themselves to do the disciplining work. Use of pharmaceuticals as disciplining technology The term disease mongering is often used for the process by which the existence of a drug (or other form of treatment) is used to create and/or promote the notion Governing by drugs 25 of a disease meriting treatment. Marshall (2002; 2006) and Tiefer (2000; 2006) have shown this development using the case of erectile dysfunction and the mar- keting of Viagra, but other drug–disease pairs have also been connected to dis- ease mongering: the development of SSRI inhibitors and the increased diagnosing of depression (cf. Healy 2004), and lifestyle drugs and diseases like hair loss (Rogaine), wrinkles (Botox) and shyness (beta-blockers) (Elliott 2003; Moynihan and Cassels 2005). In these cases, the existence of a treatment serves as a motiva- tion Eehind the medicali]ation of a particular state of Eeing and the definition of a disease. Studies within science, technology and medicine have examined how medica- tions can be ascribed even stronger ontological roles. For example, in his explo- ration of Eronchodilators, Willems demonstrates how these drugs work within a network of researchers, laboratory assistants and measurement devices to create disease classifications. ,n this e[ample, the drugs µdefine diseases and reorgani]e the Eody Ey creating new identities for it¶ Willems , . 6imilar eYents haYe been detailed for arteriosclerosis (Mol 2002), liver disease (Law and Singleton 2005) and IUD contraception practices (Dugdale 2000). Medical technologies can Ee silent and unrecogni]ed actors that take part in producing particular configura- tions of diseases or patients, as an outcome of the technologies’ use in a network of actors. In the present case, however, we understand the TLV’s use of Caverject and Bondil as a conscious and explicit deployment of technologies to this purpose. p p y g p p As described earlier, the TLV deemed Viagra reasonable to subsidize for patients with severe ED. However, the tools necessary to identify and sort patients Eased on the seYerity of (' were deemed insufficiently reliaEle. 7his highlights that the clinical practice involved in prescribing Viagra is messy. That clinical practice is messy makes it difficult to regulate, difficult to define and certainly difficult to standardi]e %erg and 0ol 7immermans and %erg . Use of pharmaceuticals as disciplining technology 6o when a bureaucratic body such as the TLV is confronted with obvious examples of how that messy clinical practice is going to Ee difficult to regulate and difficult to enforce any regulation within), the organization looks for other means of dis- ciplining it. By granting Bondil and Caverject subsidy while denying subsidy for Viagra, the TLV is relying on particular physical characteristics of these two drugs to replace the diagnostic responsibilities and perceived shortcomings of medical doctors in clinical practice.ii We suggest that the way that the 7/9 searches for and finally finds in the case of &aYerMect and %ondil an µoEMectiYe¶ tool to define and diagnose seYere (' can be related to the two different types of objectivity explicated by Porter (1995). Porter differentiates between disciplinary objectivity and mechanical objectivity. The former is found in specialist communities and can be characterized by con- sensus between equals, trust, tacit knowledge and the artful application of insight. It is also associated with a disdain for standards. In many ways, disciplinary objectivity is what the expert witnesses relied on when they claimed throughout the various decision documents analyzed in this chapter that doctors are entirely capable of diagnosing severe ED when meeting patients, with or without the assis- tance of the IIEF. It is also what the Appellant Court decided to rely on when it ruled that subsidized Viagra should initially be prescribed by urologists. 26 Ebba Sjögren and Ericka Johnson Mechanical objectivity, on the other hand, replaces trust in experts with mechanical rules, procedures and numEers, and is what the 7/9 is trying to find when relying on &aYerMect and %ondil to define seYere ('. 3orter argues that mechanical oEMectiYity through independently YerifiaEle rules and procedures can be used by an expert community (like the TLV) to create legitimacy. It ‘is a way of making decisions without seeming to decide >it@ lends authority to officials who have very little of their own’ (Porter 1995, 8). Perhaps it is no surprise, then, that the TLV, which was created only in 2002, would rely on this type of mechanical objectivity. Not only does its young age mean that it needs to create legitimacy for its decision, but also the very fact that the agency is being taken to court over its decisions indicates that the TLV’s authority is being questioned. Use of pharmaceuticals as disciplining technology Furthermore, the TLV is looking to intervene in what has historically been an activity (treat- ment choice) that has constituted an important part of the mandate of a strong professional group (physicians) (cf. Porter 1995, 228). Taken together, this would arguably encourage reliance on mechanical objectivity, rather than the trust and legitimacy of an expert community, and the Supreme Court decision supports the employment of this kind of mechanical objectivity. Bureaucratic versus treatment compliance Given how the process of determining Viagra’s subsidization status unfolded, there was no moYe to adopt any kind of more fle[iEle Yersion of oEMectiYity. $s a parallel to Porter’s differentiation between disciplinary and mechanical objectiv- ity, we would posit that the TLV’s deployment of Caverject and Bondil is intended to take part in creating circumstances that minimize the risk of non-compliance. But it is non-compliance in a particular manner and mode. p p As a consequence of the TLV’s formal and delimited task to make knowledge- based decisions about the subsidization status of prescription pharmaceuticals, we see a concern with the bureaucratic compliance to these decisions. This is in contrast with the general compliance to the agency’s individual decisions within medical practice. This contrasts with what we would loosely term treatment com- pliance, by which we refer to compliance that involves consideration of multiple rules, decisions and principles in an individual case. The latter form of compli- ance is typically inferred in relation to treatment guidelines, for example, where it is common practice to state that the guideline is a standard, which is discretion- ary to follow, rather than a directive, which must be adhered to (cf. Brunsson and Jacobsson 2000). These two ideas of compliance imply very different ver- sions of what constitutes ‘good practice’, where the latter is more malleable and ambiguous. It is worth reminding the reader here that the decisions the TLV takes, and its desire for objective diagnostics and disciplining tools, come at a point in time when the 6wedish health care and social insurance agencies are under fire for excessively permissive long-term sick leave in what the media presented as unreasonably large segments of the populations, thereby draining the coffers of the social welfare state. 27 Governing by drugs The more ‘singular’ version of bureaucratic compliance constructs a differ- ent scope and means for balancing judgement and distributing responsibility between patient/doctor/TLV and the state than does the idea of treatment compli- ance. Notably, scholars have argued that regulation, which maintains ambiguity over the conditions for appropriate use, contributes to moving responsibility away from those with the formal power to regulate use (cf. Rappert 2001). p g ( pp ) We therefore see certain parallels Eetween how the 7/9 itself is regulated and evaluated, and how the agency seeks to regulate medical professionals. 1 An earlier version of this chapter has been published as Sjögren, E. and Johnson, E. (2012). Conniving Patients, Beguiled Doctors and Disciplining Drugs: Justifying the Denial of Subsidy for Viagra Use in Sweden. In Martin Letell, Bengt Larsson and Bureaucratic versus treatment compliance In the case of the TLV, there are precise procedural requirements for the organization’s decision-making process and comparatively loose substantive criteria. Due to the manner and means of oversight – primarily through the requirement for pub- lic accounting of the grounds for every decision outcome, and further through appeals – it is the mechanical objectivity of the TLV that is emphasized rather than the expert judgement that the agency is encouraged to use.8 That the TLV is evalu- ated for how it makes and Mustifies decisions could arguaEly serYe to emphasi]e the organization’s procedural, bureaucratic compliance (cf. Power 1997). This could, in turn, shift the responsibility for perceived shortcomings in how bounda- ries for puElic fiscal responsiEility for pharmaceutical use are drawn to the 7/9 (and, by extension, individual medical practitioners) – and away from the national policymakers who have given the organization this challenging task. How the TLV was tasked with regulatory responsibility and how it perceived its role as a regulating Eody are e[amples of local, nation state±specific responses to the integration of international pharma within an allegedly culturally neutral, oEMectiYe and scientific medical knowledge paradigm. While of course, the 7/9 exists within an international pharmaceutical regulatory framework, especially the EU one, as Abraham and Lewis (2000) explore, it and its decisions are none- theless indelibly tinted by the cultural aspects of the Swedish medical system. The introduction of a drug like Viagra refracts uniquely within it as the drug and framework encounter each other. A new drug and a new regulatory body led to new regulatory tools and, because of local structures (like the uneven distribution of urologists throughout the country and the culturally specific ideology of the health care system’s framework (with the principle of equal access to health care guaranteed by law), the drug precipitated a debate and decision about subsidy based on local structural and ideological aspects of the Swedish health care sys- tem. The glocal of Viagra is inseparable from the local of the TLV and erectile dysfunction. This chapter articulates the construction of a Swedish Viagra col- oured by the TLV and value judgements about what ED is within the ideology of the Swedish health care system. This glocal Viagra becomes a drug with uniquely Swedish diagnostic and prescription values, practices and concerns. Notes 28 Ebba Sjögren and Ericka Johnson Håkan Thorn, Re-engineering the Social? Transformations of the Swedish Welfare State. Basingstoke: Palgrave, 181–96. Håkan Thorn, Re-engineering the Social? Transformations of the Swedish Welfare State. Basingstoke: Palgrave, 181–96. Håkan Thorn, Re-engineering the Social? Transformations of the Swedish Welfare State. Basingstoke: Palgrave, 181–96. 2 For a discussion of this process, see Christensen and Lægreid (2002); Lindbom (2002); Premfors and colleagues (2003). 2 For a discussion of this process, see Christensen and Lægreid (2002); Lindbom (2002); Premfors and colleagues (2003). 2 For a discussion of this process, see Christensen and Lægreid (2002); Lindbom (2002); Premfors and colleagues (2003). 3 As of 1 September 2008, the TLV broadened its scope of responsibility, which now also includes the evaluation of dental treatments. 3 As of 1 September 2008, the TLV broadened its scope of responsibility, which now also includes the evaluation of dental treatments. 3 As of 1 September 2008, the TLV broadened its scope of responsibility, which now also includes the evaluation of dental treatments. 4 From the TLV’s webpage, www.tlv.se, accessed 5 June 2008. This and all other transla- tions are the authors’ own.ii 4 From the TLV’s webpage, www.tlv.se, accessed 5 June 2008. This and all other transla- tions are the authors’ own.ii 4 From the TLV’s webpage, www.tlv.se, accessed 5 June 2008. This and all other transla- tions are the authors’ own.ii When the pharmaceutical Eenefits scheme was first launched, the inclusion of a drug in the scheme was determined by the medical condition(s) it was approved to treat. 0edicinalstyrelsen, a precursor to the 1ational %oard of +ealth and Welfare, was responsible for deciding which medical conditions were granted subsidized pharma- ceutical treatment. Whether an indiYidual patient¶s pharmaceutical use was suEsidi]ed then depended on the diagnosis set by the treating medical professional. The diagnosis- based system for deciding subsidy was abandoned a few decades later in favour of a product-based system, which was in place at the time of the TLV’s creation. The overarching principle of a product-based system is that subsidy is decided by product: either a pharmaceutical is approved for subsidy or it is not. And since the inception of the TLV, a pharmaceutical is not subsidized unless the TLV has reached the decision to approve subsidy. See Sjögren (2006). 6 Our analysis builds in part on the assumption that doctors/experts are not asking for this steering from above. Notes Studies of the emergence of evidence-based medicine (EBM) note that it has its ideological and practical foundations in intra-professional qual- ity improvement efforts (see, for example, Claridge and Fabian 2005; Hult 2006). However, attempts to exercise control of medical treatment choice decisions based on standards of best treatment have since been appropriated by actors and agendas beyond the medical professions, such as administrators, politicians and third party spe- cial interests (Pope 2003; Timmermans and Berg 2003). 7 The documents we have seen all seem to agree that at least severe forms of ED are a medical condition worthy of treatment. There were no references to the morality debates that surrounded Viagra when it was introduced, probably because the docu- ments we haYe analy]ed were from and later, at least fiYe years after the initial introduction of Viagra. 8 Notably, the legislative bill that was submitted to Parliament explicitly states that the precise interpretation of the law is left to the TLV as it ‘develops practice’ (New Phar- maceutical %enefits %ill , . ‘A few good men’ are not enough A recurrent conclusion is that ‘[t]he translation of this sci- ence into new technology is far more difficult, costly and timeconsuming than many policy-makers believe’ (Nightingale and Martin 2004, 564).i While pharmacogenomics has many definitions, a recurrent core claim is that increased knowledge about the human genome and the genetic precursors of dis- ease can contribute to greater certainty of diagnosis and improved effectiveness of treatment. Thus, in addition to generically promoting the continued possibility of drug-based treatment for a growing number of health problems, the pharmacog- enomics proMect seeks Eoth to reconfigure how patients, diseases and treatments are characterized and categorized, and how categories of patients, diseases and treatments are linked. 2ne oYerarching e[planation for the difficulty of deYel- oping and implementing pharmacogenomic technologies is that they – like all pharmaceutical-based treatments – must be embedded in existing organizational, professional and financial arrangements. 1ew sociotechnological networks emerge as incumEent structures that are reconfigured Ey the introduction of new technologies and attendant material and discursive practices (Callon 1987; Bijker 1995; Jasanoff 2004). This chapter seeks to contribute to an understanding of how pharmaceuticals shape governance by foregrounding one setting in which such technologies must be situated: decision-making about health care coverage, specifically pharmaceutical reimEursement. Determining the scope of coverage is a generic concern for both private insur- ers and health care systems, which operate with public funding. Unsurprisingly, the matter of health care provision, and especially the evaluation of products’ costeffectiYeness, has Eeen identified as an important contriEuting factor to the integration of any new pharmacogenomics product into local health care systems (Hedgecoe 2004, 179–80; Hopkins et al. 2006; Gurwitz et al. 2009). The use of evidence-based evaluation mechanisms has been seen as an important means of resolving the economic challenges expected to arise from the treatment pos- sibilities more personalized therapies afford (Phillips et al. 2004). A dominant research theme has been the development of techniques for analyzing the eco- nomic impact of pharmacogenomic technologies. To date these studies have been largely exploratory (for example Flowers and Veenstra 2004; Dervieux and Bala 2006; Payne and Shabaruddin 2010). Relatively few pharmacogenomic technolo- gies have reached the development stage where the matter of coverage is raised. ‘A few good men’ are not enough 3 Ebba Sjögren Two overarching arguments in this volume are that situating pharmaceuticals in any particular locality recasts the identities and actions of various actors and forges new relationships between health problems and pharmaceutical-based solutions. The other chapters trace various avenues through which ostensibly globally stand- ardized drugs become enmeshed in local discourses and socio-technical arrange- ments. However, the localization of pharmaceuticals does not automatically give rise to increased pharmaceuticalization. The cases of Viagra and alpha-blockers discussed in Chapters 4 and 5, respectively, highlight differences in the trajec- tory of pharmaceuticalization when drugs are introduced into areas with varying degrees of medicalization. In the previous chapter, the protracted legal battle over the reimbursement status of Viagra was ultimately resolved by a broadly exclud- ing court ruling which denied all public funding. This outcome was argued on the basis of structural and ideological aspects of the Swedish health care system, notably including the principle of equal access to health care. Here, I extend the analysis of how pharmaceutical treatment changes forms of governance and I argue that the possibility for making broadly exclusive or inclusive decisions may be strained by the widespread ambition to make pharmaceutical treatment more individualized, notably through technical advancements in the targeting of pharmaceutical-based treatment. To date, such commercially available products are rare. However, their introduction will argu- ably add to existing challenges with addressing subcategories of users and uses in the regulation of access to pharmaceutical treatment. I use the examples of man- agement of reimbursement for Viagra and other conventional drugs to suggest that a greater precision in delimiting and linking categories of treatable patients and reimEursaEle drugs can make e[isting arrangements for defining the Eoundaries of fiscal responsiEility for pharmaceutical use less roEust. 7his nuances the promis- sory discourse around widespread access to individualized pharmaceutical-based treatment in particular, and the sustained momentum of pharmaceuticalization in general.i ,n the past decade, detractors haYe aimed intensified critiTue against the reYo- lutionary model of biotechnology previously espoused by many researchers, poli- cymakers and representatives of the pharmaceutical industry. Notwithstanding Ebba Sjögren 30 the formatiYe influence of such e[pectations on the deYelopment traMectories of the field +edgecoe and 0artin , a growing Eody of research Erings into question the linear model of innovation based on observations of an incremen- tal spread of so-called pharmacogenomic technologies ‘from bench to bedside’ (Hopkins et al. 2007). ‘A few good men’ are not enough Thus, the actual practices of deciding the scope of health care provision have garnered comparatively little attention from scholars, as compared to the impact of pharmacogenomics on drug deYelopment /esko and Woodcock ins- burg et al. 2005; Phillips and Van Bebber 2006; Pendergast 2008) and clinical use (Hedgecoe 2004, 2008; Kirchheiner et al. 2005; Lakoff 2006). However, scholars have previously posited challenges with incorporating the results of tools such as costEenefit analysis into e[isting regulatory systems. )or e[ample, 5aM 31 ‘A few good men’ are not enough notes that many existing systems for deciding coverage for so-called orphan drugs generally fail to acknowledge the problem of scarce resources. With the adYent of pharmacogenomics technologies, he argues, these arrange- ments will haYe difficulties addressing the likely emergence of a larger numEer of ‘new orphan groups’, that is, categories of patients for which it is commercially unattractive to develop tailored therapies. In anticipation of a growing number of pharmacogenomic products that allow for more individually targeted drug-based treatment, a purpose of this chapter is to inquire into the workings of one critical regulatory setting where evaluations of such technologies’ cost-effectiveness will be used as part of determining coverage. This is done through a detailed inquiry into the activities of one of the many governmental health care assessment bodies that haYe Eeen created in Western welfare states oYer the past decades -ost . In studying the Swedish governmental agency tasked with deciding the scope of the puElic pharmaceutical Eenefits scheme, , engage with an issue that preYious research has identified as piYotal to the deYelopment and implementation traMecto- ries of biotechnology in general and pharmacogenomics in particular: the politics of classification %owker and 6tar 0iller and 5ose . ,t is well known that the delineation and management of populations as objects of knowledge and interYention is a political proMect with significant material impact +acking Foucault 2007, 2008). A growing number of studies highlight the considerable commercial, clinical and ethical stakes that a genetici]ation of disease classifica- tions +edgecoe , and the possiEility and practice of patient stratification (Hedgecoe and Martin 2003; Hedgecoe 2004; Reardon 2004; Egalite and Godard 2007; Prainsack 2007), bring to the fore. This chapter focuses on how categories of ‘treatable patients’ and ‘reimburs- able pharmaceutical use’ are constructed. ‘A few good men’ are not enough Crucially, these categories are inter- dependent: patients are treatable if they can be linked to a functioning treatment regime. Similarly, a pharmaceutical use is only reimbursable if it treats patients in a medical and economically viable manner. An evaluation of viability takes into consideration various forms of knowledge about pharmaceuticals’ medical and economic effects, derived from clinical studies and clinical practice. I will illus- trate how this knowledge is articulated at different levels of aggregation and with varying degrees of accuracy, which gives rise to recurrent ambiguity as to how patients and interventions should be characterized and categorized. This ambi- guity must be addressed in order to construct stable categories of patients, dis- eases and drugs that can serve as objects for regulatory intervention. I suggest that knowledge at lower levels of aggregation and the increased degree of accuracy that pharmacogenomic technologies may provide could strain the means by which decisions about pharmaceutical coverage are currently made. That more precise and accurate knowledge poses a challenge to an existing regulatory setting is somewhat contrary to the findings of /akoff. +is study of efforts to identify genetic markers for bipolar disease in Argentina makes vis- ible the close intertwining of fundamental notions of disease causality with the regulation and production of knowledge about psychiatric treatment regimens and the organization and undertaking of clinical care. Notably, he highlights the Ebba Sjögren 32 perYasiYe influence of µpharmaceutical reason¶ /akoff , ± on a wide range of actors. This strategic logic operates with the underlying rationale of link- ing drugs directly to diagnosis. Citing Rosenberg (2002), Lakoff characterizes this rationale as µdisease specificity¶ , ±, according to which illnesses are understood as stable entities which can be explained through universal causal mechanisms that are identifiaEle in the Eodies of sufferers. 2f particular releYance is my understanding of disease specificity as a tool of administratiYe management that makes it possible to rationalize health practices through the production of standardi]ed eYidence of treatment efficacy a deYelopment discussed Ey %erg 1997; Pope 2003; Timmermans and Berg 2003). Lakoff concludes that pharma- cogenomic technologies support the norm of specificity as eYoked in regulatory arrangements since µgenomic technology seeks to make the >specificity@ model more accurate. Pharmacogenomics serve as a mechanism of adjustment between drug and disease entity – a way of calibrating intervention more closely to illness’ (Lakoff 2006, 174). ‘A few good men’ are not enough This characterization of pharmacogenomics suggests that the momentum of pharmaceuticalization could be further enhanced by a more wide- spread aYailaEility of indiYiduali]ed drugEased treatments. While /akoff goes on to illustrate how more precise calibrations of drugs to diseases may clash with other configurations of functionality ± notaEly those found in clinical practices see also Hedgecoe (2008) on the usefulness of pharmacogenomic technologies as a situated achieYement ± , argue that disease specificity is also a difficult norm to adhere to in a regulatory setting. A widespread commercial availability of individ- ualized pharmaceutical treatments is therefore likely to have more diverse impli- cations for the manner in which pharmaceuticals are situated and the momentum of pharmaceuticalization. The following empirical account will inquire into how the Swedish Dental and 3harmaceutical %enefits $gency henceforth µ7/9¶, the same agency discussed in the previous chapter) makes decisions about conventional drugs. The organi- ]ation¶s classification of pharmaceuticals as included or e[cluded from the puE- lic pharmaceutical Eenefits scheme moEili]es particular modes of characteri]ing and categorizing patients, diseases and drug-based therapies. Foregrounding the means by which the TLV characterizes and categorizes ‘treatable’ patients and µreimEursaEle¶ pharmaceutical use contriEutes to the final purpose of this chapter to articulate the versions of solidarity that are produced. Previous studies illustrate how the regulatory setting can haYe a consideraEle influence on how legitimate uses and aEuses of Eiotechnology are defined and materiali]ed see, for e[ample, Jasanoff 2005; Prainsack and Firestine 2006). As previously mentioned, the delin- eation of coverage is a practical concern in both publicly and privately funded local health care systems. This is because a balance between individual and aggre- gate needs for treatment is a fundamental problem for any attempt to undertake a Must distriEution of limited resources ± irrespectiYe of how this Mustice is defined. Nevertheless, this matter arguably has particular salience in welfare systems with espoused ideals of µuniYersal coYerage¶. +ere the Eoundary for collectiYe fiscal responsibility2 for individuals’ health care needs must be drawn in a manner that is perceived as just and equitable. This is part of the construction and maintenance 33 ‘A few good men’ are not enough of a systemic solidarity, on which any such health care system depends. ‘A few good men’ are not enough It is therefore important to understand the mechanisms whereby patients, diseases and pharmaceuticals are categorized in the process of determining coverage, as the scope of the puElic pharmaceutical Eenefits scheme has direct conseTuences for what manner of solidarity is achieved. In the forthcoming empirical account, I follow how the TLV evaluates the reimbursement status of various non-pharmacogenomic pharmaceuticals. The decision to study conventional pharmaceuticals is partly premised on the limited experience of evaluating pharmacogenomic technologies’ coverage. However, the evaluation uncertainties related to pharmacogenomic technologies are similar to other innovations (Ling and Raven 2006). Furthermore, previous sociologically informed studies of pharmacogenomics in practice lend support to the supposition that such technologies become ‘ordinary’ when embedded in existing organiza- tional arrangements +edgecoe , . 7hus, the difficulties faced when eYalu- ating conventional pharmaceuticals can be used to indicate potential challenges to ambitions of a more widespread introduction of pharmacogenomic drugs. The remainder of this chapter is organized as follows. The next section will introduce the empirical setting of the study and then account for four controversies centred on the evaluation of pharmaceutical-based treatments for stomach acid– related disorders, migraines and erectile dysfunction. In each case, the focus is on incoherence between the level of aggregation and the degree of accuracy with which patients, diseases and pharmaceuticals are characterized, categorized and linked. The following section will discuss how these incoherencies are addressed and what forms of solidarity this produces. Different categories or not? Dealing with incoherent characterizations of patients and pharmaceutical use The Dental and Pharmaceutical Benefits Board: an organization to set boundaries for publicly funded pharmaceutical spending in Sweden The Dental and Pharmaceutical Benefits Board: an organization to set boundaries for publicly funded pharmaceutical spending in Sweden Whether patients must pay for their prescription pharmaceutical use can haYe potentially far-reaching effects on usage patterns. This is perhaps most obviously the case in welfare states, where patients’ out-of-pocket expenditure for drugs has historically been low and the reimbursement of pharmaceutical use relates to an overarching concern with fair and equitable resource allocation. How to prioritize resources is an enduring concern in the field of health care. ,n recent decades, the debate over resource allocation within health care has tended to take its point of departure in the perception of a widening gap between the supply and demand of health care intervention. This has brought the question of prioritization to the fore, whether between different forms of medical interventions or between different therapy areas and patient groups. 2ne way many Western welfare states haYe sought to address the Tuestion of which treatments to provide and reimburse is through the creation of central health Ebba Sjögren 34 care technology assessment (HTA) organizations. These organizations typically haYe instructions which emphasi]e the use of scientific facts in decisionmaking (Jost 2005). The organized collection and evaluation of evidence about treatment effects is conceived as a means to achieve a more fair and effective prioritization of limited resources (Cuyler and Newhouse 2000; Mitton and Donaldson 2004). The creation of HTA organizations as such is therefore in line with the pervasive idea that the evidence-based standardization of treatment decisions and medical clinical practice can secure Eroadly defined Eetter outcomes 'rori et al. 2003; Pope 2003; Timmermans and Berg 2003). In Sweden, a governmental agency was created in 2002 with the task of deter- mining the reimbursement status of all prescription pharmaceuticals.3 Prior to the creation of the TLV, all use of prescription pharmaceuticals in Sweden was µautomatically suEsidi]ed¶ 1ew 3harmaceutical %enefits %ill , .4 Accord- ing to the TLV’s governing legislation, pharmaceuticals should now be granted reimbursement when: the cost for using the pharmaceutical . . . is reasonable from medical, humani- tarian and socio-economic perspectives.i the cost for using the pharmaceutical . . . The Dental and Pharmaceutical Benefits Board: an organization to set boundaries for publicly funded pharmaceutical spending in Sweden is reasonable from medical, humani- tarian and socio-economic perspectives.i $ct on 3harmaceutical %enefits, 6ection $ct on 3harmaceutical %enefits, 6ection When a pharmaceutical is e[cluded from the pharmaceutical Eenefits scheme, patients must pay for all outpatient use of the product.5 In contrast, the cost of a pharmaceutical that has been approved for reimbursement is carried by the patients’ counties of residence.6 The legislation gives the TLV the option to approve reimbursement for restricted uses of a drug. However, this alternative is to be used sparingly in order to uphold the principle of a product-based reimburse- ment system $ct on 3harmaceutical %enefits, 6ection . The work of the TLV to decide pharmaceuticals’ reimbursement status is divided between the Board and the Bureau. The Board has the formal decision- making power. 7he goYernment appoints its eleYen memEers to reflect different interest groups within the health care sector (Ordinance with Instructions for the 3harmaceutical %enefits %oard, 6ection . 3resent and preYious %oard mem- bers include practising general physicians, health economists, medical special- ists, medical ethicists, individuals with experience from patient organizations and county health administrators. The role of the Bureau is to undertake evaluations to support the Board in its decision-making. The Bureau employs approximately fifty indiYiduals, many of whom hold doctorates in pharmacy or health economics. By law, the TLV must publicly justify its decision outcomes in so-called deci- sion Mustification documents. +oweYer, as it is not specified what it means for a pharmaceutical to have a reasonable cost of use, the onus is on the TLV to make decisions that can Ee puElicly Mustified as Eeing in accordance with the agency¶s governing legislation. The legislative bill submitted to Parliament provides some guidance for interpreting the legal framework. As mentioned in the previous chapter, the bill instructs the TLV to adopt ‘a broad approach’ (Pharmaceutical 35 ‘A few good men’ are not enough %enefits %ill , and to take into consideration the three principles of pri- ority setting that Parliament approved in 1997. These are: equal human value, meaning all people have an equal right to life and health; need solidarity, that those with greatest need of treatment have priority over those with lesser need; and cost-effectiveness, that the Eenefit of treatment must Ee reasonaEle in relation to its cost (Socialutskottet 1997). The Dental and Pharmaceutical Benefits Board: an organization to set boundaries for publicly funded pharmaceutical spending in Sweden There are no detailed instructions about what it means in practice to take these principles into consideration. However, the agency is explicitly encouraged to use health economic techniques for evaluat- ing drugs 1ew 3harmaceutical %enefits %ill . 1otaEly, the 7/9 must not take into consideration the budgetary impact of implementing its decisions. Nor does the agency haYe any targets to fulfil as regards the leYel of pharmaceutical spending.l 7he following section descriEes four instances of conflicting characteri]a- tions and categorizations of treatable patients and reimbursable pharmaceutical use. Three of these accounts are based on a detailed study of the evaluation of pharmaceuticals for the treatment of migraine and two stomach acid–related dis- orders. These decision-making processes were followed using a combination of fiftyseYen semistructured interYiews with memEers of the 7/9¶s two proMect groups and members of the organization’s decision-making Board, analysis of working documents and decision Mustification documents and participant oEser- vation (described in greater detail in Sjögren 2006, 59–67). Following the com- pletion of this study, the resulting controversy concerning one decision outcome was opportunistically identified and followed Yia articles selected through key word searches in a proprietary media database (Mediearkivet). The fourth account looks at the legal appeal of the TLV’s denial of reimbursement for Viagra. The empirical material deriYes from the 7/9¶s decision Mustification documents and the various court rulings in the case (for a more detailed discussion of methodol- ogy, see Chapter 2). In each account, the focus is on how the TLV deals with incoherencies which emerge between different levels of aggregation and degrees of accuracy when characteri]ing and categori]ing patients, diseases and drug use. 7he first account is from the stomachacid group, where the 7/9 had to address the significant prescription of certain pharmaceuticals to ‘untreatable’ patients. Identifying ‘untreatable’ patients: sorting similar symptoms into different diagnosis categories? The stomach-acid group was one of two pilot projects for the TLV’s review of the existing pharmaceutical assortment. It was launched in October 2003. At an early stage, members of the stomach-acid group described a ‘well-known’ problem of offlaEel prescription. 7he specific suspicion was that physicians were prescriE- ing a class of drugs called proton pump inhibitors (PPIs) to patients who had stomach-ache rather than gastro-oesophageal reflux disease (GERD). These two conditions could have similar symptoms. However, numerous sources agreed that Ebba Sjögren 36 only patients with GERD could be successfully treated with PPIs. Unfortunately, it was often difficult for clinicians to determine whether a patient had (5' Everything else is clear-cut . . . you have a bacterial infection; you have an ulcerous sore, and so on. With (5', there is so much Yariation. . . . <ou have symptoms with sores, sores but no symptoms, symptoms with no sores.7 It was considered problematic that the means of identifying ‘treatable’ patients was so ambiguous.8 In an attempt to resolve this ambiguity, the stomach-acid group proposed that the Board restrict reimbursement of PPIs to patients with a confirmed diagnosis of (5'. ,n order to ensure a confirmed diagnosis, the suggestion was to make reimbursement of treatment for patients with GERD con- tingent on a gastroscopic examination. However, the Board ultimately discarded this and other suggestions to delimit a smaller category of treatable patients linked to reimbursed drug use. Instead, all of the PPIs were included in the pharma- ceutical Eenefits scheme. ,nformants e[plained that this outcome was largely a consequence of the Board’s recognition that it was not possible for the TLV to interYene in diagnostic practices in the manner needed to make a more specific category of ‘reimbursed pharmaceutical use’ stable and feasible to implement in clinical practice: The Board took a pragmatic view and said that we can’t tell the doctors how they should make a diagnosis . . . we don’t know enough about what it would mean, what kind of equipment they have access to today.9 Had the Board chosen to include a restriction in line with the Bureau’s original proposal, this could have become a ‘hard strike in the air’.10 This would have been potentially problematic for the perceived legitimacy of the TLV’s work. Identifying ‘untreatable’ patients: sorting similar symptoms into different diagnosis categories? Had the Board chosen to include a restriction in line with the Bureau’s original proposal, this could have become a ‘hard strike in the air’.10 This would have been potentially problematic for the perceived legitimacy of the TLV’s work. Furthermore, informants noted that if the Board were to take differences in diagnosis-related treatment regimens into consideration for the case of PPIs there was a strong likelihood that more reimEursement decisions would haYe specific inclusion criteria. The widespread use of diagnosis-based reimbursement restric- tions was not in line with the principle of a product-based system for pharma- ceutical reimbursement, which the TLV’s governing legislation instructed the organi]ation to uphold 1ew 3harmaceutical %enefits %ill , ±. 2ne informant noted that it was a principal problem that a product-based system inferred that all pharmaceuticals had only one cost and effect, which could be clearly measured and systematically evaluated.11 That a pharmaceutical in prac- tice might have multiple effects (and costs) related to the treatment of different patient groups was not in line with the legislation’s basic premise.i ,n summary, the 7/9 discarded the possiEility of formulating more specific inclusion criteria for patients with GERD in the face of epistemological prob- lems with delineating a correct, stable and clinically feasible category of patients with this diagnosis. )ormulating a more specific inclusion criterion was also at ‘A few good men’ are not enough 37 odds with the principle of a product-based reimbursement system. By approving reimbursement for all the PPIs, the TLV constructed a broadly inclusive category of treatable patients. The ambiguity concerning who belonged to this category and should have their pharmaceutical use reimbursed was delegated to medical practitioners. A similar strategy of delegating ambiguity was used to resolve incoherency in the comparison of treatment effects for products in the migraine group. If one took cost-effectiveness into consideration. But there are other things that need to be taken into account as well, of course. If one took cost-effectiveness into consideration. But there are other things that need to be taken into account as well, of course. (Interview project manager migraine group, 22 February 2005) group, 22 February 2005) The preceding example from the stomach-acid group revealed problems with defining a staEle category of µtreataEle¶ patients using a diagnosisspecific inclu- sion criterion. In the present case, the migraine group struggled with how to estab- lish a stable and coherent characterization of the evaluated pharmaceuticals. An intended comparison of treatment effects was thwarted due to a failure to achieve comparable data. This failure resulted from the lack of a stable object of knowl- edge: there was no aggregate of patients for whom the treatment effects of dif- ferent drugs could Ee compared. ,nstead of identifiaEle patient groups, there were individual patients with different and unforeseeable responses to treatment. The 7/9¶s solution to this amEiguity was to forego any specific inclusion criteria that might risk the exclusion of pharmaceuticals that were potentially the only treat- ment alternative for certain patients. Instead, a broad category of reimbursable drug use was established. The work to characterize and categorize a particular tripane as appropriate to reimEurse when used Ey a specific patient was delegated to medical practitioners. In summary, both of the preceding cases saw the delegation of ambiguity over the characterization and categorization of treatable patients and reimbursable drug use to medical practitioners. In the following example, concerns over the feasi- bility of implementing reimbursement restrictions in clinical practice meant that such delegation was not considered appropriate. From one comparison to none: the failure to match the treatment effect of migraine drugs to stable patient groups ,n the preceding account, the identification of specific µtreataEle¶ patients was del- egated to medical practitioners. The challenge in the migraine group was some- what different, since there were no perceived problems for clinicians to diagnose patients with migraine. Therefore, the TLV’s migraine group initially intended to evaluate the treatment effects of different pharmaceuticals using data from vari- ous clinical trials. However, it soon became apparent that this data was incoher- ent. Notably, the studies used different measurements of treatment effect and also defined different categories of µcomparaEle¶ drugs. 7his made it impossiEle to make a general comparison of all the drugs using a common metric. To resolve this problem, the Bureau’s project group decided to use the golden standard for measuring treatment effect as defined Ey the ,nternational +eadache 6ociety. $s most clinical trials included this metric, it could be used to compare some – if not all – results from the studies.12i 2nce the comparaEle effect metric had Eeen defined, the suEseTuent eYaluation was initially couched as a matter of deciding how many pharmaceuticals were needed to ensure adequate product diversity. One project member likened this to an analysis of µmarginal rate of return¶, where the first product might treat a certain percentage of patients and subsequent pharmaceuticals could treat an increasingly smaller group of new patients.13 However, the project group’s seconded medical experts and various other parties soon highlighted a critical problem: it was not possible to foresee which patient would respond to which pharmaceutical within a chemically similar group of drugs called triptanes. This meant that ‘[i]n practice, doctor and patient must test until a triptane with good effect and acceptable toler- ance is identified¶ 7/9 a, . 6ince the triptanes were not interchangeaEle treatment alternatiYes for identifiaEle groups of patients, it was not possiEle to compare their treatment effect using the chosen metric.14 The failure to compare treatment effects, in combination with the high-calculated cost of not treating migraine, was described by informants as a strong contributing factor behind the TLV’s subsequent approval of reimbursement for all the triptanes. Yet, while the 7/9 approYed reimEursement for all of these drugs, the agency¶s final report for the migraine group also included a ranking of the evaluated pharmaceuticals in order of calculated cost-effectiveness. From one comparison to none: the failure to match the treatment effect of migraine drugs to stable patient groups It was stressed that the ranking was not a general treatment recommendation and the TLV would not monitor compliance Ebba Sjögren 38 to the ranking, However, the intention was that the ranking could guide medical practitioners in choosing an order in which to test the triptanes. ‘A few good men’ are not enough: the exclusion of Viagra from the pharmaceutical benefits scheme As detailed in the previous chapter, in March 2008, the Supreme Administrative Court of Sweden ruled to exclude Viagra (and two other pharmaceuticals used for the treatment of erectile dysfunction from the puElic pharmaceutical Eenefits scheme. This decision ended a process that had started seven years earlier, when the Swedish government decided to immediately cease general reimbursement of Viagra. In the aftermath of a heated public debate, the government took steps to reverse its decision. Less than six months later, the TLV began its operations. The reimEursement status of 9iagra was one of the first cases the new agency decided. In March 2003, Viagra was once again excluded from coverage. The decision Mustification document issued Ey the agency gaYe two reasons for this outcome.i 7he first argument was that 9iagra was not generally reasonaEle to reimEurse for treatment of all forms of erectile dysfunction. Notably, the TLV argued that the medical studies cited in the application for reimbursement showed neither that the 39 ‘A few good men’ are not enough product was generally cost-effective nor for which particular patient groups the drug was cost-effective (TLV 2003, 4). The second argument was that no depend- aEle way e[ists to identify specific groups of treataEle patients that could Ee deemed societally reasonable to provide with reimbursed treatment. Had this been possible, the TLV might have approved reimbursement for Viagra to patients with severe erectile dysfunction. However, the perceived inability of medical prac- titioners to determine the medically treatable and societally reasonable patients made it necessary to deny reimbursement to all patients. As described in Chapter 2, the company marketing Viagra appealed the TLV’s decision and a series of court battles ensued. Prior to the Supreme Court’s rul- ing, two lower courts overturned the TLV’s decision and approved restricted reimEursement for 9iagra for a specific category of patients. 7he /ower &ourt defined this category as patients with certain medical conditions such as diaEetes or cardiovascular diseases, or patients who were prescribed the drug by a urolo- gist 6tockholm &ounty $dministratiYe &ourt . While the 7/9¶s suEse- quent appeal of the Lower Court’s ruling was not upheld, the Appellant Court did change the category of patients to be included in coverage. Instead of a primarily diagnosis-based categorization, the Appellant Court restricted reimbursement to initial prescription by a medical specialist on male reproductive organs. Ensuring reimbursed pharmaceutical use for a deserving few: implementing the decision to exclude all Losec use from coverage 6ince its inception, the 7/9 has eYaluated hundreds of products. While most deci- sions have met with few public reactions, this account concerns one case which did become a matter of some public attention: the general exclusion of Losec from coverage. Losec, a brand-name pharmaceutical in the stomach-acid group, was deemed unreasonable to subsidize on the grounds that other products with the same actiYe suEstance had a significantly lower price. 7hese drugs were inter- changeable with Losec, according to the list maintained by the Swedish Medical Products Agency. Thus, the TLV argued that patients could receive another prod- uct when filling their /osec prescriptions. 7he result would Ee significant cost savings, without a loss of treatment quality. However, in the aftermath of the TLV’s decision in February 2006, reports sur- faced about an unforeseen consequence of excluding Losec from coverage. The TLV’s decision was based on the fact that patients using Losec could remain in treatment with another brand of drug. This premise was derived in part from an analysis of data from clinical trials and sales statistics, which suggested that a number of products were interchangeable for the patient population using Losec. However, information now surfaced about a group of patients for which Losec was the only treatment option. This patient group was young children with GERD. 7hey had difficulties swallowing capsules and therefore needed to take the actiYe ingredient in a form that could be broken apart and softened. The exclusion of this group of patients from reimbursed treatment was not considered a reasonaEle outcome. 6eYeral counties¶ solution was to finance this particular use of /osec through an alternatiYe route. 6pecifically, the /osec these patients used was re-categorized as inpatient use. By doing so, the counties man- aged to put in place a number of extra routines so that the National Corporation of Pharmacies could directly bill the cost to the counties.i ,n summary, this final case makes YisiEle the incoherence Eetween different levels of aggregation and degrees of accuracy in both the characterization and categorization of patients, and the matching of patient categories with pharma- ceuticals. The data the TLV used crafted a large category of patients for whom the active substance – not a particular brand or dosage form – was important for treataEility. %ut this category was not staEle in other settings. ‘A few good men’ are not enough: the exclusion of Viagra from the pharmaceutical benefits scheme The court described this as a more dependable way of identifying the medically treatable and societally reasonable patients (Stockholm Administrative Court of Appeals 2005).i 7hree years later, the 6upreme $dministratiYe &ourt Mustified its denial of all reimbursement on three grounds (Supreme Administrative Court of Sweden 2008): 1 The existence of two alternative treatments for patients with severe erectile dysfunction [i.e. those patients who were both treatable and reasonable to provide with reimbursed treatment; author’s note] 2 The uncertainty of a diagnosis for severe erectile dysfunction, and 3 The imagined behaviours of patients and doctors that created a clear risk for off-label prescription of Viagra to patients other than those whom it was ‘rea- sonable’ to treat with reimbursed pharmaceuticals. 3 The imagined behaviours of patients and doctors that created a clear risk for off-label prescription of Viagra to patients other than those whom it was ‘rea- sonable’ to treat with reimbursed pharmaceuticals. The Supreme Court’s second and third arguments are similar to those the TLV made in its original decision Mustification document. 7he perceiYed risk, that med- ical practitioners would be too inclusive when categorizing patients as eligible for reimEursed 9iagra, Mustified the e[clusion of all 9iagra use from coYerage.i ,n summary, the first two cases centred on Yarious proElems that the 7/9 had with creating correct and stable categories of treatable patients and reimbursable drug use that were feasible to implement in clinical practice. In both the migraine and stomach-acid groups, the epistemological problem of characterizing, cat- egorizing and matching patients, diseases and drugs was delegated to medical practitioners through the delineation of broad, inclusive categories. As argued in Chapter 2, the case of Viagra further illustrates how the basis for characterization Ebba Sjögren 40 and categorization needed to be stable and feasible to implement in clinical prac- tice. 7he fourth and final e[ample elaEorates further on the issue of clinical fea- siEility Ey looking at the implementation of a 7/9 decision for a specific patient group. Discussion 7he four preceding accounts illustrate Yarious detailed and casespecific proE- lems related to the evaluation of reimbursement status for pharmaceutical-based treatments of stomach acid–related disorders, migraine and erectile dysfunction. However, the examples also highlight common challenges in the TLV’s work to appropriately and reliably create and maintain stable categories of ‘treatable’ patients, and link these patients with ‘societally reasonable’ pharmaceutical use in line with the legal criteria for reimbursement. I will now discuss how perceived epistemological problems in this categorization work were addressed. The sec- ond part of the section will elaEorate on the difficulty of achieYing high disease specificity when making decisions aEout coYerage. 7he final part of the section will relate the observed processes of decision-making about pharmaceutical reim- bursement to the production of different modes of solidarity. Ensuring reimbursed pharmaceutical use for a deserving few: implementing the decision to exclude all Losec use from coverage 6pecifically, it clashed with a more finegrained category of patients in medical practice for whom dosage form was a key component of treatability. To ensure that the treat- ment of these patients remained reimbursed, a new organizational arrangement was put in place. This involved the re-categorization of Losec from outpatient to inpatient use. 7hus, the inclusion of the specific category of patients in coYerage ‘A few good men’ are not enough 41 was achieYed outside the regulatory arrangements of the pharmaceutical Eenefits scheme. The following section will compare and contrast the four accounts of how inco- herent and unstable categorizations of treatable patients and reimbursable drugs were addressed. The focus is on the mechanisms for resolving ambiguities, and on the consequences for inclusion and exclusion that these solutions provide. Making decisions with ambiguous knowledge by delegating ambiguity to others ,n the first case, the 7/9 struggled with how to differentiate Eetween µtreataEle¶ and ‘untreatable’ patients in its reimbursement decisions for drugs in the stomach- acid group. The Bureau project group made various proposals to delimit a more specific category of patients with the µtreataEle¶ disease (5'. 2ne e[ample was the suggestion to make reimEursement contingent on a µconfirmed diagnosis¶. 7his was defined as a diagnosis made after a gastroscopic e[am. +oweYer, the Board discarded these suggestions. In part, this behaviour can be attributed to the Board’s recognition of a lack of supporting technologies. Notably, the Board was aware that lack of access to gastroscopes could impede the implementation of such a restriction. ,n foregoing more specific inclusion criteria in its decision outcome, the 7/9 delegated amEiguity oYer which specific indiYiduals were to Ee categorized as treatable patients to medical practitioners. The second account of the migraine group described work to characterize and categorize pharmaceuticals using a comparison of treatment effect. By deciding to use the ,nternational +eadache 6ociety¶s metric, the 7/9 defined an ostensi- bly common means of measuring treatment effect. This opened up the possibil- ity of formulating more narrowly defined criteria for what could Ee categori]ed as ‘reimbursable’ drug use. Yet this means for comparing drugs broke down in later stages of the decision-making process, when the TLV was unable to sustain- aEly link the measurements of treatment effect to a staEle and identifiaEle patient Ebba Sjögren 42 population. The subsequent decision to approve reimbursement for all the trip- tanes can also be described as delegation to medical practitioners: this time of the work to determine whether a given pharmaceutical made a patient treatable, and thus made the drug reasonable to prescribe with reimbursement.ii 7he first two cases illustrate how attempts to craft more specific categories of treatable patients and reasonable drugs were abandoned in favour of broadly inclu- sive decision outcomes that delegated the ambiguity of categorization to medical practitioners. The story of Viagra reads differently. Despite a widespread agree- ment aEout the e[istence of a specific group of patients that merited reimEursed use of Viagra (individuals with severe erectile dysfunction), this characterization of patients was not deemed stable and feasible in clinical practice. The agency doubted the ability of medical practitioners to appropriately limit the prescription of Viagra to these deserving few. ‘Specific’ concerns in crafting categories of included and excluded patients and pharmaceuticals The preceding section discussed the TLV’s responses to ambiguity in the charac- terization and categorization of patients, diseases and pharmaceuticals. Arguably, one foundation for the aforementioned epistemological proElems is the codified norm of disease specificity 5osenEerg , cited in /akoff . 7his is most clearly made visible in the description of the legally sanctioned product-based reimbursement system as a model which assumes each drug has one set of treat- ment effects which can Ee attriEuted to a clearly defined patient population. 7his assumption is recurrently challenged in the preceding four accounts. Nevertheless, the option of formulating more specific inclusion criteria for coYerage appears to be a comparatively weak one. This might be the case for several reasons. One obvious reason is that the legislation governing the agency’s work instructs it to strive to uphold a product-based reimbursement system. However, a further e[planation is that the agency lacks the means of influencing the work practices of medical practitioners. The TLV has no mechanisms to ensure compliance to any more specific restrictions it might set out. $ third reason, highlighted Ey the proE- lem of diagnosing GERD and prescribing migraine drugs with treatment effect, is that adhering to the underlying assumption of disease specificity when making decisions about coverage makes it problematic for the TLV to both justify deci- sions based on evidence, as is required by law, and reach outcomes that can be appropriately implemented in clinical practice, in line with its intended task of setting priorities for public pharmaceutical spending. This dual imperative makes it difficult to disregard knowledge from either clinical trials or clinical practice. This creates a basis for clashes between the level of aggregation and degree of accuracy with which patients and pharmaceuticals are possible to characterize and categorize. It is against this backdrop that the advent of pharmacogenomic technologies should be considered. What this chapter suggests is that a greater accuracy at lower leYels of aggrega- tion than pharmacogenomic technologies could provide would further strain the construction of staEle oEMects of regulatory interYention. 7he difficulties of ren- dering stable representations of patients that can encompass variation – notably as regards diagnosis practices and treatment response – contributes to blurring the category of medically treatable, societally reasonable and therefore regulate-able patients. Making decisions with ambiguous knowledge by delegating ambiguity to others 7hese e[amples thus illustrate how more precise and detailed knowledge about patient subgroups and their treatment responses did not 43 ‘A few good men’ are not enough immediately and automatically resolve the challenge of justifying decisions based on correct, stable and clinically feasible knowledge. Making decisions with ambiguous knowledge by delegating ambiguity to others Rather than the broadly inclusive category in the previous two cases, the outcome in the Viagra case was a broadly exclusive category that made it necessary for all patients to take priYate fiscal responsiEility for their Viagra use. While this chapter does not consider what responses the denial of 9iagra may haYe triggered, the final case of /osec offers insight into how an ostensiEly staEle category of treatable patients was upset after the TLV had made its reimbursement decision. The agency’s decision to exclude Losec from the pharmaceutical ben- efits scheme was premised on the comparison of treatment effects within a patient population for whom the actiYe suEstance defined treataEility. 7he suEseTuent breakdown of this category of patients – which had been sourced from clinical trials – came in the intersection with a particular clinical practice. Suddenly there was a specific group of patients whose treataEility was closely entwined with /osec as a specific product. 7his group of patients had not Eeen rendered YisiEle for the TLV. Following the TLV’s decision, efforts were made by other parties to de facto include the specific patient group in coYerage. The four accounts provide examples of how the TLV dealt with ambiguity to reach a decision outcome. 7o make a decision that could Ee Mustified, there was a need for the characterization and categorization of patients, diseases and drugs to Ee correct, staEle and feasiEle to implement in clinical practice. ,f this was diffi- cult to achieve, then an alternative recourse was for the TLV to remove ambiguity about who was a treatable patient and what was reimbursable drug use through delegation to others, notably medical practitioners (see also Rappert 2001). In the first case from the stomachacid group, memEers of the 7/9¶s %ureau and %oard expressed a practical understanding of the socio-technical network of clinical work. 7hus, the reTuirement to haYe a confirmed diagnosis in order to reimEurse pharmaceutical treatment of GERD was discarded since it was dependant on a sup- porting network of elements, such as a gastroscope, which the agency could not moEili]e. 7he Mustification that certain 9iagra use could not Ee granted restricted reimEursement due to concerns oYer compliance similarly reflects an understand- ing of diagnosis as a situated accomplishment, which can be malleable to the influence of different actors. ‘Specific’ concerns in crafting categories of included and excluded patients and pharmaceuticals Thus, in contrast to earlier conclusions about the capacity for pharma- cogenomic technologies to support a further calibration of health care rationali- zation (Lakoff 2006, 174) and provide momentum to pharmaceuticalization, the present study suggests a potentially more destaEili]ing effect. 'isease specificity is a difficult norm to adhere to in the current regulatory practice. $ more precise knowledge about how to characterize and categorize treatable patients and rea- sonable drugs is not obviously more useable and useful when reaching a decision Ebba Sjögren 44 outcome. Rather, one could argue that the possibility of articulating a greater form of disease specificity in certain forms of knowledge could further contribute to the need for delegation of ambiguity and the attendant distribution of responsibility that this entails. However, the viability of this practice can be questioned. First, the possibility for ad hoc tinkering in those situations where unreasonable out- comes result from decisions aEout coYerage, as e[emplified Ey the case of /osec, does not provide – much less guarantee – a systematic treatment of such prob- lems. Second, an increased ambition to standardize, control and direct the content of medical practice using eYidenceEased policy would make tinkering difficult to maintain (Berg 1997; Berg and Mol 1998; Timmermans and Berg 2003). The present inquiry into the construction and workings of a particular ‘redistributive machinery¶ thus proYides a Easis for reflecting on Eoth the e[isting production of solidarity, and what the advent of more individualized treatment and diagnosis could portend. Inclusive forms of evidence-based solidarity? This chapter highlights a possible underlying ‘dysfunctional norm of disease specificity¶ in decisionmaking concerning resource allocation in the 6wedish health care system. 7hese findings suggest that pharmaceuticals which allow for more individually targeted therapy could make it even more complicated and potentially controversial to make such a local ‘redistributive machinery’ work. However, it would appear to be a difference of degree, rather than kind. The four accounts discussed earlier concern the evaluation of reimbursement status for conventional drugs. By analogy, it could be argued that technological advances – in particular when paired with more knowledge claims about matters such as individual patients’ drug responses – might exacerbate the prevalence of upset- ting precision in the Swedish context. This argument is supported, in part, by the results of a comparative study of how Germany, the Netherlands and the United Kingdom have incorporated three kinds of genetic technologies (Aarden 2009). Based on a comparison of the local regulatory and medical practices by which medical technologies become available to individuals, Germany has a reticent pattern of provision for such technologies due to the relative importance of policy decision-making about health care provision. In these processes, cost-effectiveness and efficacy play an important role, such that the reTuirement for inclusion is tangible proof of disease or risk (Aarden 2009, 145). In a broader setting in which evidence-based decision-making is emphasized, this would tend to exclude groups of patients or pharmaceuticals for which evidence is unavailable or incoherent or where diagnosis and treatment in clinical practice remains complex and variable. This contrasts with the case of the Netherlands and the United Kingdom, where other forms of solidarity are achieved through practices that lead to the emphasis of alternatiYe leYels of specificity, for e[ample of niche communities that match patients and technologies in the case of the Netherlands (ibid., 146). The current study in Sweden suggests a tendency to forego the incorporation of more specific knowledge and inclusion criteria into decision outcomes in faYour ‘A few good men’ are not enough 45 of broadly inclusive or exclusive categorizations of patients and pharmaceutical use. This could be taken to suggest that the solidarity such arrangements produce remains true to a principle of universal coverage. However, such a judgement must also take into consideration the settings into which decisions are implemented. Inclusive forms of evidence-based solidarity? As previously mentioned, at the time of the study the TLV had no responsibility for considering the financial impact of its decisions. $ changed reTuirement that eYal- uations of public coverage should consider not only cost-effectiveness but also economic feasibility would arguably also limit the possibility of making broadly inclusive decisions at the policy level (see also discussion in Rai 2002). Similarly, the linking of remuneration to health care service providers to compliance with treatment guidelines and reimbursement decisions would arguably contribute to limiting the room for interpretation when implementing decisions in clinical prac- tice. Such developments could become topical in a setting where the widespread expectation and fear is that conventional and pharmacogenomic technologies will continue to drive the direct cost of pharmaceutical-based treatment. Conclusions This chapter has inquired into a national governmental agency’s efforts to include or e[clude prescription drugs from a national pharmaceutical Eenefits scheme. I draw attention to the challenge ambiguous knowledge poses in the work of craft- ing stable categories of treatable patients and reimbursable drug use with which to justify local coverage decisions. The effort on the part of the studied agency to craft correct, stable and clinically feasible categories within a regulatory frame- work premised on disease specificity resulted in decisions with Eroadly inclusiYe or exclusive categories. The former type of decisions involves a delegation of ambiguity regarding the characterization and categorization of treatable patients and reimbursable drug use to medical practitioners. I have argued that different levels of aggregation and degrees of accuracy in knowledge claims about patients, diseases and pharmaceuticals are a potential source of ‘upset’ in categorization work. The observed delegation of ambiguity balances the dual need to make and justify evidence-based decisions which are feasible to implement in clinical prac- tice. However, such outcomes are not obviously in line with the legislative ambi- tions to centrally prioritize resource allocation. This study illustrates how a governmental agency’s efforts to include or exclude drugs from a national pharmaceuticals Eenefits scheme is normatiYe work that takes part in a wider politics of distribution for health care resources. A critical issue is how the dynamic tensions which emerge in the work of constructing locally rea- sonable and legitimate coverage decisions are addressed. This arguably requires an understanding of the mutual constitution of medical technologies and the dis- tributive practices embedded within any local health care system. In particular, it implies sensitivity to the broader range of mechanisms and arenas through which new technologies in health care practices are situated, beyond the clinical setting. A related concern is how to make visible the normative work of organizations such as the TLV. Little visible public debate concerning the TLV’s work exists in Ebba Sjögren 46 Sweden. This contrasts with the experience in many other countries, where the work of HTA organizations has prompted controversy. For example, several deci- sions by the British National Institute for Clinical Excellence (NICE) have been the target of public outcry and the visible involvement of various advocacy groups 0oreira . g y, ( ), 2 Public funding of health care can be supplied in various ways. One such method, which is in place in 6weden, is the direct proYision or third party financing of health care services using taxes. An alternative model, which is used in Germany and Holland, is Conclusions 7he lack of conflicts and YisiEle interest group inYolYement in the Swedish case is partly a methodological artefact of the study, which focuses on the internal workings of the 7/9. +oweYer, it is also an empirical finding that has the- matic antecedents linked to the localization of technologies. In a comparative study of regulatory responses to agricultural biotechnology and GM food in Germany, the United States and the United Kingdom, Jasanoff (2005) she observed differ- ences in the topic and manner of controversy within each country. He explained the observed differences in part by differences of civic epistemology, the ‘cultur- ally specific, historically and politically grounded, puElic knowledgeways¶ that encompass shared understandings of how knowledge should be ‘presented, tested, Yerified, and put to use in puElic arenas¶ -asanoff , , . ,n a similar vein, Prainsack and Firestine (2006) have argued that the absence of public contro- versies concerning biobanks in Israel is due to a series of narratives that construct biotechnology as crucial for the continuity of Jewish existence in the region. 7he lack of YisiEle conflict aEout 7/9 decisions in 6weden can also Ee attriE- uted to the workings of a ‘machinery’ of social technologies and devices, which renders the TLV’s decision-making processes opaque (Casula and Sjögren 2011) and formats the participation of interest groups (Sjögren and Fernler 2010). A publicly funded health care system is ultimately dependant on the boundaries for puElic fiscal responsiEility Eeing set in a manner that is perceiYed as legiti- mate. 7he present chapter highlights the comple[ interplay Eetween scientific and clinical knowledge claims with different levels of aggregation and with varying degrees of accuracy, which gives rise to recurrent ambiguity as to how patients and interventions should be characterized, categorized and linked. This ambiguity must be addressed in order to construct stable categories of patients, diseases and drugs that can serve as objects for regulatory intervention. Building on previous chapters, the examples of Viagra and other conventional drugs highlight that the future of pharmaceutical access is not limited to enrolling participants in clinical practice but enrolling society to subsidize access to situated understandings of ‘legitimate’ drug use. Thus, even with a more incremental introduction of indi- vidualized technologies for diagnosis and treatment, there is an ongoing need to follow the avenues whereby they become embedded in existing local systems for deciding the scope and content of health care provision. 1 Substantive parts of this chapter have been previously published as Sjögren, Ebba . 8psetting &ategories" 7he &onseTuences of 3harmacogenomics for 0aking Knowledge-based Decisions in Sweden. New Genetics and Society, 29 (4), 389–411. 1 Substantive parts of this chapter have been previously published as Sjögren, Ebba . 8psetting &ategories" 7he &onseTuences of 3harmacogenomics for 0aking Knowledge-based Decisions in Sweden. New Genetics and Society, 29 (4), 389–411. 2 Public funding of health care can be supplied in various ways. One such method, which is in place in 6weden, is the direct proYision or third party financing of health care services using taxes. An alternative model, which is used in Germany and Holland, is Notes ‘A few good men’ are not enough 47 Eased around statutory health insurance schemes see Wagstaff et al. 1999 for a brief overview). Eased around statutory health insurance schemes see Wagstaff et al. 1999 for a brief overview). ) 3 The term pharmaceutical reimbursement is commonly used in the literature. How- ever, the term subsidization status more accurately captures the structure of the Swed- ish pharmaceutical Eenefits scheme. 7his scheme is designed to minimi]e priYate expenses in connection with illness. In the current set-up, a patient pays the full cost of prescription pharmaceuticals up to 1,100 SEK (approximately 112 Euro). A graduated subsidy then reduces the patient’s direct cost for prescription medication so that (s)he never pays more than 2,200 SEK (approximately 225 Euro) in a twelve-month period. 4 The term was used to describe how a drug was included in the public pharmaceutical Eenefits scheme once the 6wedish 0edical 3roducts $gency or its (uropean eTuiYa- lent, EMEA) had approved it for use. ) pp 5 ‘Outpatient use’ refers to pharmaceutical use, which takes place outside of monitored hospital care. This can include pharmaceuticals which have been prescribed during inpatient care, Eut which are intended for use after discharge from hospital. When a pharmaceutical is e[cluded from the pharmaceutical Eenefits scheme, this in no way limits the right of physicians to prescribe the drug. The use of pharmaceuticals for inpatient treatment is not subject to centralized regulation by the TLV. This coverage is decided by the individual counties.i y 7he twentyone counties finance the maMority of health care serYices directly through an income tax levied on all county residents who are in paid employment. The cost of the pharmaceutical reimbursement is billed to patients’ resident counties by the state- owned National Corporation of Swedish Pharmacies, which held a legally sanctioned monopoly on the distribution of pharmaceuticals in Sweden until 1 July 2009. p y p 7 Interview project manager stomach-acid group, 17 May 2005. 8 Interview project manager stomach-acid group, 2 September 2005; interview health economist, 16 November 2005. 9 Interview project manager stomach-acid group, 23 November 2005. 10 Interview project manager stomach-acid group, 23 November 2005. 11 Interview health economist stomach-acid group, 28 September 2004. g p p 12 Interview project manager migraine group, 10 June 2004. See also discussion about the lack of ‘ideal effect metric’ (TLV 2006, 41–2). Notes ( , ) 13 Interview project manager migraine group, 19 April 2004. nterview project manager migraine group, 19 A 14 Interview health economist migraine group, 22 February 2005. This page intentionally left blank Part 2 Changes in the medical discourse This page intentionally left blank 4 The Swedish medical discourse Impotence, erectile dysfunction and Viagra in Läkartidningen1 Ericka Johnson Ericka Johnson 52 Ericka Johnson Sexuality is not the only area of life to be medicalized and later pharmaceutical- ized with the development of pharmaceutical solutions to health ‘problems’. The processes of medicalization and pharmaceuticalization depend on the social and technical networks within which people and diseases are placed. As Oudshoorn states, µhealth proElems can only Ee classified as illness and Ee medicali]ed if there exists a cultural climate and a medical infrastructure that actively transforms health complaints into diseases’ (Oudshoorn 1997, 143). Thus, solutions to prob- lems like depression, anxiety, obesity, hair loss and ageing can become medical solutions when both the medical community and general population recognize them as such. This process is not uncontroversial, however, and the pharmaceuti- cal solutions to obesity, hair loss and ageing can be grouped together with the treatment for erectile dysfunction in the category of drugs sometimes called life- style drugs (Mamo and Fishman 2001, 16; Elliott 2003; Loe 2004a; Moynihan and &assels Williams et al. a. While some of these cures address proE- lems that most people would at least nowadays call diseases (like depression), the disease status of others is more contested and their development has spawned the term disease mongering to denote the process of medicalization that uses medical practice and medical technologies to promote a concept of improvement unto per- fection and the idea of medication for instant, scientific solutions for physiologi- cal and psychological distress (see Fishman 2004, 193; Tiefer 2006, 274). Disease mongering not only serves the purposes of pharmaceutical companies looking for conditions that can fit their pharmaceutical cures, it also e[pands the areas of life that doctors can claim as their territory – and for which they can charge consulta- tion fees and sell drugs (Elliott 2003). Some of these lifestyle drugs and diseases are related to a change in attitudes towards activity, sexual and otherwise, in older age. The concept of successful ageing allows the medical community to offer medical solutions to health prob- lems traditionally associated with ageing, problems like hair loss, menopause and, as will be discussed in this chapter, erectile dysfunction. As the medical com- munity claims these areas as its domain (rather than the domain of diet, witch- craft, antisocial behaviour or psychoanalysis, for example), the problems become diseases and the solutions are often decidedly medical, in the form of surgeries, drugs and physiological treatments. Medicalizing the ageing male The inability to achieve and maintain an erection has had many causes and many cures. Ancient Greeks attributed it to a diet of dry, cooling foods. During the European Middle Ages, impotency could be the result of a curse – dealt out by one’s enemies, a witch or a slighted lover who tied knots in a string. Later, sexual excess, youthful indulgences and masturbation were blamed. During the nine- teenth century both a wife’s aversion to sex and her desire for it could cause impotence, as well as a glimpse of her ‘unattractive’ female genitals. The stress of modern, urban living was (and still is) made a culprit. Then, of course, comes the litany of 2edipal urges, domineering mothers, incestuous fi[ations and the Freudian analysts needed to cure these. In the mid twentieth century, especially in the United States, therapists looked to relationship issues for a cause and the point of treatment, while in a wider arena impotence was variously attributed to feminism, the sexual revolution and the contraceptive pill. And most recently, impotence has become a vascular issue (for a cultural history of impotence, see McLaren 2007). Tracing the changing expectations of men’s potency and vitality in Europe and North America shows how male sexuality has, with time, become more and more medicalized. This is true of sexuality in general. It has slowly gained recogni- tion as a field in medicine conferences are Eeing held aEout Yarious aspects of sexuality; medical journals on the subject are sprouting up; medical schools are offering courses in sexuality and some are even opening entire departments in the field doctors are Eeing encouraged to speak with their patients aEout se[ual histories during routine medical exams; and pharmaceutical solutions to sexual µproElems¶ help define these proElems as medical )ishman and 0amo , 7iefer , f. $t the same time, nonmedical e[perts are flourishing, offer- ing sexual advice on the Internet, radio talk shows and TV, in magazines and newspapers (Tiefer 2006, 275). But while female reproductive health has tradi- tionally been a focus of medical intervention (see Martin 1992; Oudshoorn 1994; Dugdale 2000), for men it has been virility, with strength and vitality instead of reproductive capacity, that has long attracted medical attention (Sengoopta 2006; McLaren 2007). Ericka Johnson Because these solutions to ageing are creating diseases out of changes during the life span, the idea of getting older successfully has been critiqued for promoting an idea of ageing that really means not ageing at all (Marshall 2006, 350). Viagra and its promise of returning male sexual per- formance to a youthful, erection-on-demand state so that anyone can have sexual intercourse at any time and any age is currently one of the most talked about treat- ments for successful ageing. This development should be placed in the history of medicalized male vital- ity. In the early part of the twentieth century, it was thought that the secret to masculine vitality was found in the sex glands, and much research about these glands, in both people and animals, was conducted (see Oudshoorn 1994; Mar- shall 2006; Sengoopta 2006). One of the medical treatments developed during the 1920s (sometimes called the decade of the testicle because of the intensity of 53 Swedish medical discourse research into testicles during this time) to treat a loss of vitality in ageing men was the Steinach operation. Developed by Austrian Eugen Steinach, and said to have Eeen performed on Eoth )reud and <eats, the operation redirected fluid from the testicles to Ee reaEsorEed into the Eody rather than released outside. 7his fluid was then thought to help revitalize the patients (Marshall 2006, 347; Sengoopta 2006; McLaren 2007). In the 1930s and 1940s, male rejuvenation treatments moved into more mainstream medical practices and Eegan to reflect new knowledge aEout testosterone. Hormone therapy, that is testosterone treatment, was developed as a treatment for the ‘male climacteric’. But again, the goal of treatment was gen- eral male vitality as displayed through physical and mental, but not necessarily sexual, prowess. In fact, increased sexual function was sometimes seen almost as an undesired and slightly embarrassing side effect of treatments (Marshall 2006, 347–8). In the middle of the twentieth century, new research into sexuality and sexual behaviour changed the way sex and sexual dysfunction was perceived and treated. No longer was sexual decline in the male thought to be a natural part of ageing. Experts asserted that sexual activity and sexual intercourse were important parts of healthy ageing (Marshall 2006, 349). Impotence was thought to be caused by a fear of impotence. Ericka Johnson It was perceived as something that could be avoided and treated through therapy, often involving the partner, rather than through biomedi- cal interventions (Tiefer 2006, 283). During the middle of the century and up into at least the 1980s, it was generally agreed that in 80% of impotence cases the problem was psychological and therapy was the best treatment. p p y g py According to feminist sex therapist Tiefer (2006), the psychology-based approach to sexuality in general and sexual dysfunction in particular changed during the 1980s. She notes that in the United States, several different factors contributed to a shift. For one thing, the American Psychiatric Association (APA) decided to define se[ual proElems as disorders in performing a seTuence of geni- tal functions, which coincided with a broader acceptance of a biomedical and psy- chopharmacological model of mental health. At the same time, the health industry began to use the Diagnostic and Statistical Manual of Mental Disorders and its definition of se[ual dysfunction when determining which diseases would Ee eli- gible for reimbursement (Tiefer 2006, 283). Shortly thereafter, urology specialists began to take on more sexual dysfunction cases, which served to cement the idea that impotence was a biophysical issue of the penis, and led to examinations and treatments for sexual dysfunction that did not include the involvement of wives or partners (Tiefer 2006, 285). Thus, impotence, which only a decade before had been called a psychological problem with physiological results, began to be seen as a physiological problem that could lead to psychological suffering (Marshall 2006, 350). The 1990s also saw other changes in the view of sexual dysfunction. Impotence became known as erectile dysfunction (', specifically located in the penis. %y the late s, largely thanks to research and adYertising funded Ey 9iagra¶s maker, 3fi]er, the new reigning explanation for impotence stated that 70–80% of cases stemmed from physical causes, a direct reversal of the earlier ideas and one which supported Ericka Johnson 54 medical consultations and prescription-based solutions rather than behavioural therapy or couples counselling (Plante 2006, 379). Sexual function was no longer seen as a controversial side effect of anti-ageing treatments; it was now the main goal. g The narrative of impotence and ED within the US context is also relevant to what has happened in Sweden, although with a bit of delay on some points. Ericka Johnson To examine the pharmaceuticalization of erectile dysfunction in this discourse in Sweden, I have analyzed articles dealing with impotence and erectile dysfunction in the generalist medical journal Läkartidningen.2 I have looked at the articles published between 1990 and 2015, starting eight years before Viagra’s introduc- tion in 1998. Doing so shows that the construction of impotence, erectile dysfunc- tion and male sexuality in Läkartidningen in some ways follows very closely with how these ideas have been framed in the international medical community, despite the framework of socialized medicine within which Läkartidningen pub- lishes and the debates about subsidies of Viagra which have surrounded its intro- duction to the Swedish market. For example, in Läkartidningen the discourse has moved from social causes of impotence to a focus on mechanical and molecular aspects of ED, as has also happened in English-language journals. Reports in Läkartidningen from studies about impotence are also frequently linked to phar- maceutical funding after the introduction of Viagra, and there seems to be more column space granted to these discussions than there was before Viagra. In 2006, a study was published in Läkartidningen which reaffirmed that social aspects of (' contriEute to indiYiduals¶ treatment options. 7his suggests that a Eroader defi- nition and re-evaluation of male sexuality could have appeared in the Swedish discourse. But it is rather unique. The local structures of health care provision also influenced the 9iagra discourse in 6weden, framing it in deEates aEout chronic medical conditions that produce ‘legitimate’ and severe ED. These debates are particularly Swedish in that they stem from questions about subsidizing Viagra with tax money. Later I will present my analysis drawn from a careful reading of the Läkartidnin- gen articles. In examining the articles, I have looked at the construction of patients and symptoms associated with impotence, as well as how the authors define and propose impotence and its cure(s). I have then contextualized this against results of similar analysis of the international discourse on impotence and ED. The arti- cles studied have been found using the search words: impotence, impotence treat- ment, penis erection, erectile dysfunction, erectile difficulties, potency treatment, 9iagra and 6ildenafil.3 $ total of fiftythree articles was sourced for the years ±. )ortyfiYe of these were puElished after 9iagra was introduced. Impotent patients before and after Viagra Prior to Viagra, impotence was presented in Läkartidningen as a combination of psychological and physiological conditions. It was also something ‘natural’. 7his understanding of the condition is reflected in a Eook reYiew which asserts µ$ppro[imately ten percent of western men suffer from se[ual difficulties 55 Swedish medical discourse associated with impotence, and if men can stay healthy and live long enough, they all become old-age-impotent’4 (Mellgren 1993, 984). Old-age impotence, my translation for åldersimpotent, can Ee read as a specific type of impotence, and one that seems to disappear from the discourse after the arrival of Viagra and the presentation of the term erectile dysfunction. Also of note is that this number, , is Yery low compared to 3fi]er statistics that Eegan to appear with the intro- duction of Viagra. (On the Viagra website,5 (' is said to afflict more than half of men oYer fifty. Impotence, if natural, was, however, already a medicalized condition before Viagra arrived, although for fewer people. Pre-Viagra, impotence was also very complex in Läkartidningen. In an article about the causes of impotence from 1990, the authors spend an entire section speaking about feelings. They discuss the way men, particularly after not engaging in sex for a longer period of time, can want to have sex, but still not have sexual urges. They explain this by stating that ‘many people interpret desire as the same thing as libido. But desire and libido are not the same thing’ (Olsson et al. 1990, 4456). Five years later, in an article written by two of the same authors, the pre9iagra discourse denied the feasiEility and Eenefit of distinguishing between physical and emotional causes of impotence, something Viagra relies on. ‘To create a distinction between somatic and psychological cases of impotence has been shown to match poorly with reality. Instead, one must for every patient evaluate biological factors and his feelings, his relationship to his partner, his family and work’ (Olsson et al. 1995, 313). Thus, pre-Viagra, impo- tence is a result of a combination of (mostly social and emotional) factors. It is not just the number of impotent patients and the emotional causes of impo- tence that change through the 1990s in Läkartidningen the definition of an impo- tent patient also proYes fle[iEle. Ericka Johnson After the appearance of Viagra, types or groups of patients are still discussed in association with ED in Läkartidningen, but usually these are connected to indi- viduals who have diseases whose symptoms can include, or whose treatments can induce, impotence, that is diabetes, multiple sclerosis, anxiety attacks and heart disease. Thus patients are distinguished into categories based on medical diseases rather than social factors. This later discussion of the types of ED patients in Läkartidningen was probably triggered by the debates raging after the introduc- tion of Viagra over who should receive subsidies for the prescriptions of Viagra (see Landtblom and Ertzgaard 2000; Örn 2001). Using Viagra in these cases is presented as a solution to ED for patients with a legitimate need for the drug. I use the word legitimate because it is in these cases that the Swedish courts have heard arguments for and against the subsidized use of Viagra as a treatment for severe ED (see Chapter 2). Impotent patients before and after Viagra ,n the pre9iagra article aEout impotence (Olsson et al.), impotent patients are constructed as a heterogeneous group, within which one finds some patients easier to treat than others. 7hose most likely to respond successfully to treatments are men in long-term relationships, and when discussing them, this ‘patient’ is frequently spoken about as the couple. More dif- ficult to treat are single men, of which the authors identify three types the young, shy man; the older man who has been sexually inactive for a while; and the loner carrying a secret. Of these, the older, sexually inactive man is seen as the easiest to treat (Olsson et al. 1995, 314). This typology of patients is based on social fac- tors, and focuses on the men’s relationships with others, not on the biological or mechanical causes of impotence. Respect for the social aspects of impotence also appears when, in the same article, the authors discuss reasons it can Ee difficult to treat impotent patients: because some impotent men can have rigid stereotypes aEout normal se[uality they can find intimacy threatening they think se[ is aEout performance and thus develop performance anxiety; they are unable to recognize their own emotional signals; they see the ability to have intercourse as a sign of power; and/or because some of them experience impotence as shameful (Olsson et al. , . $nd, as the authors later go on to say, the most difficult thing to deal with as a doctor is the rage that some patients feel and can at times project upon the doctor when treating impotence. 56 Sexual problems and pharmaceuticals From their studies of US cases, sociologists Mamo and Fishman note that pre- scribing drugs like Viagra can, in some cases, lead to and justify polypharmica (Mamo and Fishman 2001, 27). Läkartidningen takes up the occurrence of poly- pharmica as well. Three years before the introduction of Viagra, the discussion of se[ual difficulties and pharmaceuticals was raised in Läkartidningen in an article called ‘Sex life and pharmaceuticals’ (Lundberg 1995). In it, Lundberg discusses both how pharmaceuticals have been developed to treat sexual problems, and how pharmaceuticals taken for other reasons can influence one¶s se[ life. 7hus, Ey 1995, pharmacological sources of, and solutions to, sexual problems were gain- ing acceptance within the Swedish medical community. But it also shows that the understanding of the concept of sexual problems was very broad before Viagra. In this article, a great deal of time is spent discussing the influence of other drugs i.e. dopamine and serotonin blockers) on desire rather than on the mechanical ability to have sex, for both men and women. Most interesting, however, is the way sex is defined more widely than it often is in post9iagra discussions. )or e[ample, se[ is presented as involving not just intercourse but also orgasms, even for women. $nd after noting that serotonin Elockers can make orgasm difficult for women, the author states, µ7here are, howeYer, few reports of orgasm difficulties in men using this type of antidepressant. We do not know if there is a se[specific difference or if the proElem is hidden in men Eecause of the difficulties in differentiating between ejaculation and orgasm’ (Lundberg 1995, 2745). The idea that men could experience ejaculation without orgasm is completely absent from any later discus- sions of Viagra, as is the possibility of having an erection without ejaculation. But in the 1995 article, a nuanced way of discussing pharmaceuticals and sexual health is presented, one that involves desire, ability and pleasure. Much of this nuance is lost with the arrival of Viagra, and discussions about libido and desire also tend to disappear in the literature about ED, in Läkartidningen and other medical journals, despite the fact that the makers of Viagra insist that it will not produce an erection without sexual stimulation. Sexual problems and pharmaceuticals But, as many social scientists and critics have 57 Swedish medical discourse noted about Viagra in general, it relies on the traditional understanding of male sexuality, that men always want sex (see Fishman and Mamo 2001, 183; Mamo and Fishman 2001, 23; Marshall 2002, 2006; Loe 2004b; Tiefer 2006). Returning to the discourse in Läkartidningen, libido appears only in connection with impotence post-Viagra in two articles. One is the 2004 column ‘Pharmaceuti- cal questions’ (Kimland and Ståhle 2004), which is a brief compilation of topics discussed at regional pharmaceutical information centres. There, in 2004, the terms libido and impotence appear together when reporting a study that examined these in connection with the use of lithium (as the 1995 Lundberg article did when talking about sexual problems and pharmaceuticals). The other is a 2008 article that dis- cusses the Eenefits of offlaEel use of 6ildenafil for women suffering from se[ual dysfunction as a result of antidepressants (Bodlund 2008). But these two articles can be read as special cases dealing with the effects of psycho-pharmaceuticals rather than the use of Viagra for a wider population. Ericka Johnson Ericka Johnson To better contextualize his use of the term erectile dysfunction, it is important to note that the discursiYe sliding Eetween, and in some cases conflation of, impo- tence and (' has a history outside of the 6wedish conte[t, largely in the field of urology and closely connected to the development of pharmaceutical therapies. Social scientist Barbara Marshall, in her work on Viagra, relays the story of how Dr Giles Brindley in 1983, in front of an audience of medical peers, injected his penis with phenoxybenzamine and obtained an erection, essentially removing the connection between emotional or tactile stimulation and erection. Ten years later, in 1993, the US National Institute of Health created consensus around the use of the term erectile dysfunction (Marshall 2002, 136). In Sweden, in the 1998 Hedelin article, erectile dysfunction becomes equated with impotence in Läkartidningen.l 6usan %ordo claims that impotence as a term reflects a characteristic of the per- son, not a disease – one says of a man, ‘he is impotent’ while one would not say ‘he is a headache’ (Bordo 1998, 87). And, as Loe (2004a) has noted, the shift in Eng- lish from impotence to ED can be quite comfortable for the individual. ED allows the man to maintain his identity and self untainted, and treat only the penis. Potts notes this as well: ‘This term [impotence] infers that a man loses power through his “failure” to achieve an erection, and demonstrates how important a notion of “potency” is in constructions of conventional masculine sexuality. Consequently, an inability to produce erections may be perceived as tantamount to a destruction of the male self¶ 3otts , . With the introduction of (' and 9iagra, a medi- cal term and a pill exist that can prevent damage to the individual and erase blame for the failure to produce an erection on demand. These comments on terminology are also applicable to the Swedish usages of impotence and erectile dysfunction. Another interesting observation in connection with the use of the term erectile dysfunction is that, in the 1998 Läkartidningen article, Hedelin asserts that ED is ‘the most common form of sexual function problems’ around the world. This statement was very common in 1998, internationally. Pharmaceuticals and erectile dysfunction Pharmaceutical solutions for impotence existed prior to Viagra’s arrival in 1998, although most of these involved needles or pellets inserted directly into the penis and were therefore not as easy to administer as a pill. However, in a Läkartidnin- gen article from 1997 (Hedelin and Abramsson), the use of orally ingested medi- cines for erectile dysfunction is discussed and their pending introduction to the market predicted. In this article, the term erectile dysfunction,6 rather than impo- tence, is first used in Läkartidningen.7 With this term, the discussion is shifted to the mechanical aspects of Elood flow, Yascular systems and muscle cells. 0en haYe µerectile difficulties¶ and these can Ee treated. 7he authors start the article with the statement, ‘The ability for a man to have an erection that facilitates inter- course and insemination is a prerequisite for the continuation of the human race’ (Hedelin and Abramsson 1997, 2548), and then go on to discuss various possibili- ties for treatment to be subsidized by the Swedish state, a discussion that later takes on enormous proportions in Läkartidningen (Hedelin 1998; Sjöstrand 1998; Beerman 2000; Byström 2000a, 2000b; Landtblom and Ertzgaard 2000; Landtb- lom 2004; Ströberg et al. 2006). In 1998, Viagra appears in Läkartidningen, with articles about the drug itself and about the way it is being received in the United States (Bergström 1998; %ranke , its introduction to 6weden, specifics of its use, Tuestions of its costs to the individual and society (Hedelin 1998) and warnings that it is being sold illegally through the mail (Aldstedt 1998). It is at this point that impotence, which in the and articles was Eroadly defined, often with social causes, and which occurred in many different types of patients, including couples, is now directly equated with ED. In the introduction to his 1998 article ‘New treatment for impotence’, aforementioned urologist Hans Hedelin articulates this discursive coupling: ‘Erectile dysfunction (impotence), that is the inability to achieve and maintain an erection for a sufficiently long period for se[ual actiYity, is the most common form of sexual functioning problems’ (Hedelin 1998, 4558). 58 Ericka Johnson 1995, 316) (Olsson et al. 1995, 316) However, this is exactly what Viagra promises, as noted in a 1998 Läkartidningen article, which argues for subsidies for Viagra because its use cures two patients, not just one, that is also the partner of the man suffering from ED (Sjöstrand 1998). Potts and colleagues (2006) have shown how this idea is also prevalent in English-language commercials for Viagra, and the partner in the Swedish com- mercial discourse is discussed in Chapter 6. However, this is exactly what Viagra promises, as noted in a 1998 Läkartidningen article, which argues for subsidies for Viagra because its use cures two patients, not just one, that is also the partner of the man suffering from ED (Sjöstrand 1998). Potts and colleagues (2006) have shown how this idea is also prevalent in English-language commercials for Viagra, and the partner in the Swedish com- mercial discourse is discussed in Chapter 6. The shift in the use of the term impotence to erectile dysfunction occurs in Läkartidningen and the post-Viagra articles written largely by urologists, and is not unique to the Swedish discourse. It is in line with what has occurred in other journals and media outlets internationally. As Tiefer notes in her critique of the English-language use of the term, ‘erectile dysfunction, a condition in the man’s genitalia, has become the only acknowledged focus of interest, focus of evalu- ation, and focus of treatment. This represents a substantial narrowing from sex therapy – erasing the partner, erasing subjective meaning, and, ironically, perpetu- ating the obsession with penile hardness, which many sex therapists have argued is itself a primary cause of sexual unhappiness’ (Tiefer 2000, 278). Examples of the same refocusing of the discourse in Sweden can be seen in Läkartidningen. Prior to Viagra, mention is made of another closely related sexual problem: premature ejaculation (Olsson et al. 1990). But after Viagra, this prob- lem is not discussed again in connection with se[ual difficulties until , when a notice about a new drug treatment is presented (Hansen 2006) which mentions that 20–30% of men suffer from premature ejaculation. (Compare with the pre- Viagra book review that states an estimated 10% of men suffer from impotence 0ellgren . Ericka Johnson That ED became the most common form of sexual function problem just when a medicine to cure it was introduced has been discussed elsewhere in relation to medicalization, biomedi- calization and pharmaceuticalization (Mamo and Fishman 2001, 16; Elliott 2003; /oe a 0oynihan and &assels Williams et al. 2011a). In Läkartidnin- gen, articles after 1998 spend a great deal of time talking about the physiology of erections and their molecular and biological aspects, and very little, if any time discussing counselling, couples therapy and the social or relationship issues related to impotence. p Perhaps most indicative of the direction articles about impotence and ED in Läkartidningen took after the introduction of Viagra is the 2000 article ‘Viagra is the first option for treating erectile dysfunction,¶ coauthored Ey urologist +ans +edelin who defined erectile dysfunction as impotence and 3fi]er employee Lena Jacobsson. In this article they discuss a study which compared treatments for ED in Sweden. Gone from this study are all questions about the emotional or social aspects of impotence, or even the fact that sexual problems can take other forms than ED. Instead the study only focuses on how ED can be treated pharmacologically. The authors start their article by stating, ‘In the last few years different methods to successfully treat erectile dysfunction (ED) have appeared, 59 Swedish medical discourse methods which work largely unrelated to the cause of the erectile problems and which demand a minimum of evaluation before the treatment can be initiated’ (Hedelin and Jacobsson 2000, 2616). Thus, the treatment for ED (which impo- tence had become) is suddenly a relatively simple procedure, rather than one which, as suggested in a 1995 article, demands an empathetic doctor who can giYe hope and understanding, and who can spend sufficient time with the patient to discuss his/their problems, often over a period of several consultations (Olsson et al. 1995, 313). Likewise, in articles appearing prior to Viagra, references to alternative treatments like self-injections to the penis and the use of penis pumps and surgical implants appear, often as later-stage complements to couples therapy (see Olsson et al. 1995). This is particularly true with the penis implant, which is discussed with the warning: Those patients who expect that an implant will not only create erections but also improve their relationship with their partner and bring them closer to a harmonic life are often disappointed. (Olsson et al. The impotent man, the partner patient and a woman’s responsibility Before Viagra, in several of the Läkartidningen articles about impotence, the patient is presented as the partner unit. For example, in an article from 1995, the roles of each partner in dealing with impotence are narrowly defined. µ&onYersa- tion with the couple is the most important diagnostic and therapeutic instrument. One should strive to work with the couple rather than the man alone, though one should never try to force the partner’s cooperation. Men and women have differ- ent ways of expressing themselves and therefore misunderstandings can easily arise. Women must learn to Ee clearer and men to Ee more receptiYe¶ 2lsson et al. , . ,n addition to charging each partner with a specific way of com- municating and the responsibility to change this, the article notes: ‘The best help for a man with disappointing erections is, besides his own courage to speak about it, an understanding and sensual partner who is sexually keen but not demanding’ (Olsson et al. 1995, 314). Although, as the authors go on to say, ‘of course, this isn’t always enough’ (Olsson et al. 1995, 314). The couple-patient is also present when talking about other sexual problems pre-Viagra, as here, when discussing early ejaculation: ‘Naturally, the sexual act can easily be a failure in these situations unless the female partner is wise and possibly experienced, and can focus primary attention on physical contact and intimacy, and reduce the importance of genital contact’ (Olsson et al. 1990, 4456). The woman is charged with responsibility for ensuring that the sexual act is a success, despite the man’s sexual problems. This is slightly different than when speaking about impotence as a partner issue or presenting the impotent patient as a partner constellation. Instead, the solution to the sexual problem is in the hands of the female partner. This same shift of responsibility for curing the patient occurs in the discussion aEout male liEido. When e[panding on the difference between desire and libido, and their relationship to impotence, the authors state that, ‘Naturally, even here the female partner’s behaviour is very important’ (Ols- son et al. 1990, 4456). Also of note is the distinct sense that impotence occurs only in heterosexual relations. Ericka Johnson 7his seems to confirm 0arshall¶s assertion, drawn from her analysis of English-language articles about Viagra, that ‘even though premature ejaculation (an “orgasmic disorder”) has higher prevalence rates than “erectile dysfunction” in many studies, we do not hear of an “epidemic” of premature ejac- ulation’ (Marshall 2002, 137). Viagra has shifted the focus onto ED, and redirected Ericka Johnson 60 attention away from other se[ual difficulties, including early eMaculation and a lack of libido, internationally and also in the Swedish medical discourse. Reopening the debate Not until 2006 does the partner-patient unit of ED appear again in the post-Viagra Läkartidningen. In this year the partner becomes one of the people who should be asked about evaluation of the treatment and one of the reasons patients chose to discontinue treatment (Ströberg et al. , . While the integration of the partner in the discourse can be related to his/her presence in the pre-Viagra articles, this is a somewhat new role for the partner. Rather than being part of the cure, as in the articles from the early 1990s, now the partner is part of the wider conte[t that influences a patient¶s decision to follow a medical cure. ,t is also in this article that the social factors Eehind (' are finally reintroduced to the discourse, after having been absent for eight years. In the discussion about the dis- continuation of pharmaceutical treatments for (', the results of the 3fi]erfunded study showed that more than half of the patients prescriEed 6ildenafil stopped using it within two years. To explain this, the authors report that ‘Often the rea- sons are multi-factoral and factors like increasing age, diminished libido, rela- tionship problems, health problems, social and cultural background all together can influence the decision to stop treatment¶ 6tr\|Eerg et al. 2006, 2866). Issues concerning the physiology of ED were not the only, or even primary, answers they received from patients. This article in Läkartidningen shows that when a study is conducted which actually asks Viagra patients about their experiences and the reasons for their use or disuse of the drug, a disjuncture of the drug’s patients and their medically prescribed sexual identities and practices begins to (re)appear. That international medical research about the use of pharmaceutical treatments for ED is focused on biological, mechanical or molecular aspects of erections has been noted. As Tiefer wrote in 2000, ‘There’s little attention to the person or couple attached to the penis, or recognition that relational factors might modify the meaning or importance of penile rigidity or sexual intercourse in a couple’s sexual script. It would appear that industry-sponsored research wishes simply to wave away the complexities introduced by the psychosocial context of sexuality’ (Tiefer 2000, 278). As an example of pharmaceuticalization, the case of Viagra in Sweden before the 2006 study complicates the medicalization hypothesis that general medicine is trying to constantly expand the domain over which it reigns. The impotent man, the partner patient and a woman’s responsibility And, as the earlier discussion about men and women’s communication issues implies, not only is the patient a heterosexual couple, it is a couple with Yery stereotypical, genderspecific interaction patterns.i ,n , after the introduction of 9iagra, the definition of the patient with (' shifts from the couple to the man in Läkartidningen. The only articles which sug- gest the presence of a female Viagra patient is the aforementioned 1998 article which argues that the debate about whether to subsidize Viagra should take into consideration that the pill helps two patients, not one (Sjöstrand 1998) and the 2008 article about using Viagra for women on antidepressants (Bodlund 2008).8 Other than these, however, the post-Viagra ED patient is primarily the man pre- scribed the pills, and often only the genitals of that man. 61 Swedish medical discourse The assumption of heterosexual patients in the Swedish case is not unique and mirrors a wider heteronormativity in the English-language discussions about Via- gra and impotence, this despite the widespread use of Viagra within homosexual communities, and despite the use of the gender-neutral term partner in 3fi]er advertising (see Chapter 6). For further discussion, see McLaren (2007) and Vares and Braun (2006). Reopening the debate The examples of medical intervention for impotence in the pre-Viagra articles in Läkartidningen suggest that doctors were willing to intervene in the biomedical and social aspects of their patients’ sexuality, but that after Viagra’s appearance, medical intervention is narrowed to the biomechanical functions of a man’s penis. This narrowing runs counter to some expected processes of medicalization, but Ericka Johnson 62 shows how pharmaceuticali]ation tries to confine the condition of impotence to one disease (ED) with a universal, pharmaceutical treatment.i ( ) , p 7his tendency makes the 3fi]erfunded 6tr\|Eerg, +edelin and %ergstr\|m (2006) study in Läkartidningen even more noteworthy, as it perhaps suggests that Viagra has not successfully reduced impotence to ED. Their article reopens the discussion of factors that can influence se[ual health and simultaneously remedi- calizes the larger context of patients’ sexual health. One could have hoped that this signalled a return to a more nuanced discussion of sexual problems and their treat- ments within Läkartidningen and that acknowledging that there may be diverse reasons for patients to discontinue treatment with Viagra and similar medications may lead the medical discourse to include aspects from the early 1990s, that is recognition that there are different types of patients who have different reasons for and understandings of their impotence, along with the existence of a pill. By comparison, it is relatively uncomplicated to assert that women’s sexualities are comple[ and conte[t dependent, and that they are influenced Ey feelings and emo- tions, even with older women (see, for example, Loe’s (2004b) study). One could have hoped that the 2006 article in Läkartidningen was a sign that soon Swed- ish men, too, would be granted the right to (once again) own a complicated and conte[tdependent se[uality influenced Ey feelings, emotions and social situa- tions, not just kicked into action with a drug. But in the years that followed, little seemed to change. Viagra continued to be discussed in articles about subsidies and in relation to other medical conditions, like cardiovascular and prostate problems. Thus, in the Swedish medical discourse, the local structures doctors and health care providers worked in combined with Viagra’s global traits to create a glocal Viagra in Läkartidningen, one which presented and maintained a pharmaceutical solution to erectile dysfunction. Notes 1 An earlier version of this chapter has been published as Johnson, E. (2008). Chemis- tries of Love: Impotence, Erectile Dysfunction and Viagra in Läkartidningen, NORMA, 3 (1), 31–47. 1 An earlier version of this chapter has been published as Johnson, E. (2008). Chemis- tries of Love: Impotence, Erectile Dysfunction and Viagra in Läkartidningen, NORMA, 3 (1), 31–47. ( ) 2 Läkartidningen is the trade journal of the Swedish Medical Association (Läkarför- bund). It is published about once a week and covers international and Swedish devel- opments in medicine and medical care. 2 Läkartidningen is the trade journal of the Swedish Medical Association (Läkarför- bund). It is published about once a week and covers international and Swedish devel- opments in medicine and medical care. 3 In Swedish: Impotens, impotensmedel, peniserektion, erektil dysfunktion, erektil svårigheter, potensmedel, Viagra, Sildenafil. 3 In Swedish: Impotens, impotensmedel, peniserektion, erektil dysfunktion, erektil svårigheter, potensmedel, Viagra, Sildenafil. 4 All translations are the author’s. 5 www.viagra.se (October 2015). 6 Much later, in 2005, an interesting shift is made when erectile dysfunction, which had been a side effect of some diseases, also becomes a symptom. Läkartidningen reported that erectile dysfunction may be a symptom of undiagnosed heart disease and encour- aged doctors who haYe a patient with (' to find out if that patient actually has heart disease (Gunnarsdottar 2005). 7 Masters and Johnson used the term erectile dysfunction in the 1950s (McLaren 2007, 221). However, it was generally not taken up by the medical community until adopted Ey urologists and populari]ed Ey 3fi]er see 0arshall /oe a. 7 Masters and Johnson used the term erectile dysfunction in the 1950s (McLaren 2007, 221). However, it was generally not taken up by the medical community until adopted Ey urologists and populari]ed Ey 3fi]er see 0arshall /oe a. y g p p yi 8 See Loe (2004b) for an analysis of senior women in the United States and the drug. 8 See Loe (2004b) for an analysis of senior women in the United States and the drug. 5 Alpha-blockers and a weaker pharmaceutical influence on medical discourse Ericka Johnson As Illich pointed out many years ago, a drug’s commercial and medical success is not terribly dependent on its chemical effects on the body (Illich 1976, 72). This observation has given impetus to critical work on pharmaceuticals that explores factors which make drugs as varied as Viagra, antidepressants and Botox so suc- cessful (Fishman and Mamo 2001; Elliot 2003; Moynihan and Cassels 2005; Marshall 2006; Petryna and Kleinman 2006), and spawned studies of the way pharmaceuticali]ation influences medical and social practices Williams et al. 2011a). In this body of work it becomes clear that one important factor, among many, that can impact a drug’s success is the way it is constructed and situated in the professional medical discourse and the medical infrastructure of a specific country, a trait pharmaceuticals share with many other medical technologies (see Healy 2000; Löwy 2015). This section of this book explores local examples of a pharmaceutical’s ability to influence the treatment of estaElished medical conditions and redefine health problems as issues with a pharmaceutical solution. The previous chapter explored the influence 9iagra had on the medical discourse in 6weden. ,n this chapter, , am again relying on an analysis of the medical discourse in the Swedish-language medical journal Läkartidningen. From it, I have retrieved research articles and debate pages that appear when the journal’s online archive is searched for the Swedish words associated with lower urinary tract symptoms secondary to benign prostate hyperplasia (LUTS/BPH): BPH, prostatahyperplasi and prostatahyper- trofi.1 I have conducted a search for the years between 1990 and 2015, since alpha-blockers started to become a more common treatment method in Europe for LUTS/BPH in the 1990s (EUA 2006, 35) and were registered as a treatment for BPH in Sweden in the early 1990s (Carlsson and Spångberg 1996a, 4549; Hallin 1999, 3520). This time frame mirrors that used for Viagra in the previous chapter. Alpha-blockers are an interesting case for a book about glocal pharmaceuti- calization because they show an example of incomplete change in the medical discourse and clinical practice for the treatment of LUTS/BPH, as opposed to Viagra, which completely changed the face of a disease and its treatment. Alpha- blockers are also related to an analysis of Viagra because one of their side effects is impotence and/or erectile dysfunction. 5 Alpha-blockers and a weaker pharmaceutical influence on medical discourse Medical guidelines suggest that men tak- ing alpha-blockers may experience decreased desire and erectile and ejaculatory Ericka Johnson 64 difficulties ($8 $8$ 6%8 . 0any men who receiYe a pre- scription for alpha-blockers will also receive a prescription for Viagra, although probably to very limited success, given that Viagra does not address desire. Alpha-blockers are being prescribed to treat LUTS/BPH in Sweden as in other deYeloped, Western countries. ,n fact, some 6wedish e[perts assert that they are being overprescribed. Yet, despite their presence in the Swedish cadre of treat- ments for close to twentyfiYe years, and despite their place as an early course of action for /876%3+, according to an official 6wedish diagnostic and treat- ment report (SBU 2011), they have not managed to replace surgical treatments as completely as Viagra managed to replace sex therapists in the Läkartidningen discourse or out in the urology clinics. By the early 2000s, 40,000 men were being treated pharmaceutically for BPH in Sweden (Dahlstrand 2003, 2678), and by 2009, this number had risen to approximately 115,000 (Spångberg and Dahl- gren , , a significant increase, eYen if one considers the possiEility that pharmaceutical treatments also increased the gross number of patients diagnosed with LUTS/BPH. At the same time, surgical procedures had declined, but were still relatively high. In 1987, before alpha-blockers, 12,000 surgeries for enlarged prostates were performed in Sweden. In 2009, just under 5,000 were performed (Spångberg and Dahlgren 2013, 685). This is a decrease, but shows that surgery is still the treatment option for thousands of Swedish men each year and is still presented as an option in the discourse in Läkartidningen. LUTS/BPH and alpha-blockers Around the turn of the twentieth century, surgeries were performed for prostate issues, but with very high risks primarily due to infection. These surgeries would access the prostate from the abdomen or, more commonly, through a Y-shaped incision behind the testicles. These days, the LUTS/BPH prostate is usually surgically accessed through the urethra and the most common method is by transurethral resection of the prostate (TURP), which sends a tool in through the urethra to scrape away and cauterize the prostate tissue from the inside (AUA 2010, 70). There are other mechanical ways of removing or destroying a prostate diagnosed with LUTS/BPH, including microwave thermo- therapy, radiofrequency needle ablation and laser therapies, but besides pharma- ceuticals, surgery is the most common method in Sweden (SBU 2011). Pharmaceutical treatment can be traced to the introduction of alpha-blockers in the 1970s (Heyns and de Klerk 1989, 226) and their eventual position as a stand- ard treatment starting in the 1990s. However, even before alpha-blockers, LUTS/ BPH had been treated by suppositories, herbal treatment, chemicals and hor- mones. Chemical preparations doctors historically prescribed for patients include opium, silver nitrate and belladonna suppositories, potassium iodine, potassium bromide, ergot (a type of fungi that grows on rye) and large quantities of dis- tilled water (Marting 1903, 52; O’Shea 2012, 14). Many of these treatments were directed at the symptoms the man presented with, primarily urination problems, and some of the elixirs irrigated the bladder, but others, like ergot, were actually thought to shrink the prostate gland (O’Shea 2012, 14). At the end of the nineteenth century, it was generally thought that ‘orchidec- tomy’ (surgically removing one or both of the testicles) and vasectomy could be two treatments for the enlarged prostate that may haYe Eeneficial effects +eyns and de Klerk 1989, 204; Shackley 1999, 777). This theory was in part developed because the then-current surgical methods (primarily suprapubic prostatectomy) had a mortality rate of about 20%, so other treatment methods were very welcome by patients and doctors alike. But the ideas about using castration and vasectomy could also be traced to the observation that eunuchs and those with non-developed testicles never presented with ‘hypertrophy’ of the prostate (Ciechanowski 1903, 91), indicating that the testes were somehow involved in the development of pros- tate growth later in life. LUTS/BPH and alpha-blockers The search terms I used for this study are the Swedish words which are or have recently been applied to what is today known as LUTS/BPH, lower urinary tract symptoms secondary to benign prostate hyperplasia in English. This is a condition in which the prostate becomes enlarged but without being cancerous. The prostate can start to grow again in older men, gradually increasing in size with age, and this is thought to happen in the majority of men. For some, it happens around age fifty, for others, not until they are seYenty or older. ,t is a normal deYelopment, but this normal growth can cause problems with urination and is thus considered pathological. By the age of seventy, 75% of men will have LUTS/BPH symptoms, that is problems urinating (Parsons 2007, 395), and by age eighty, nearly all men will have an enlarged prostate (Fall 1999, 2227), 80% of whom will have prob- lems with urination (Dahlstrand 2003, 2678). These statistics tend to vary a bit, but the general understanding is that the prostate gets larger as men get older, and that causes problems urinating. Urologists like to point out that urination issues for men have existed for thou- sands of years, referring to drawings on (gyptian papyrus from the fifteenth century BC and to writings by Hippocrates (Shackley 1999, 776). Medically, however, the prostate as a separate organ was not represented in European anat- omy diagrams until 1536, and not named until 1611 (Marx and Karenberg 2009, 209). Successful methods of surgically addressing the prostate for urination dif- ficulties did not really eYolYe until Must more than years ago 6hackley , 65 Alpha-blockers 776). Today, a connection is often made between male urinary problems and an enlarged prostate. It is thought that when the prostate gets larger it may also start to block the urethra, the tube that lets the urine pass from the bladder and which the prostate surrounds, like a straw stuck through a Eall. When the prostate Eegins to Elock the flow of urine through the urethra, its enlargement Eecomes a proElem.i l g g p 7he first step of treatment for /876%3+ is currently pharmaceutical. 7his is a relatively recent change and it has complemented but not entirely replaced the traditionally more common surgery. LUTS/BPH and alpha-blockers While clinical practice did not really support the use of castration for treatment – many doctors reported that it was not successful and that patients were not pleased with the results (Marting 1903) – the possibility that it could work eventually led to theories about the role of hormones in prostate enlargement. However, it took until sometime into the twentieth century before hormone therapies were widely developed and used for prostate issues (O’Shea Ericka Johnson 66 2012, 17; see also Oudshoorn 1994, 2003 and Sengoopta 2006 for general histo- ries of hormone treatments and their relationship to masculinity). In the 1930s and 1940s, androgens, especially testosterone, were used to treat patients with what was then called ‘prostatism’, but without much success. The next step was to try oestrogen and combinations of oestrogen and testosterone. This did not really work to reduce the prostate, either, although it did seem to give some relief of symptoms and increase urine flow +eyns and de .lerk , . ,n the s and 1980s a series of trials were done on antiandrogens and progestins. Some of these showed symptom improvement and others did not, and many of the trials had quite a few side effects, like vertigo, shivering, tiredness, loss of libido and impotence (Heyns and de Klerk 1989, 221–3). Cholesterol-lowering drugs were also tested, Eut with no significant Eenefit +eyns and de .lerk , ±. 2012, 17; see also Oudshoorn 1994, 2003 and Sengoopta 2006 for general histo- ries of hormone treatments and their relationship to masculinity). In the 1930s and 1940s, androgens, especially testosterone, were used to treat patients with what was then called ‘prostatism’, but without much success. The next step was to try oestrogen and combinations of oestrogen and testosterone. This did not really work to reduce the prostate, either, although it did seem to give some relief of symptoms and increase urine flow +eyns and de .lerk , . ,n the s and 1980s a series of trials were done on antiandrogens and progestins. Some of these showed symptom improvement and others did not, and many of the trials had quite a few side effects, like vertigo, shivering, tiredness, loss of libido and impotence (Heyns and de Klerk 1989, 221–3). Cholesterol-lowering drugs were also tested, Eut with no significant Eenefit +eyns and de .lerk , ±. LUTS/BPH and alpha-blockers At the same time, in the 1970s and 1980s, doctors also began to try alpha- blockers, with more success. They seemed better than placebos at treating peak and aYerage flow rates of urination, the amounts of residual urine and eYen the prostatic urethral pressure (Heyns and de Klerk 1989, 226), so much so that it has been suggested that their relative success may also have increased the interest in diagnosing LUTS/BPH (Ekman 1999, 3504). It is this category of drugs, alpha- blockers, that I will explore in the Swedish medical literature. Alpha-blockers Alpha-blockers’ main purpose as a treatment for LUTS/BPH is to reduce the symptoms and bother of urination problems (SBU 2011, 317), and their use has steadily increased since the 1990s, probably in part because patients (and their doctors) see them as a way of avoiding surgery and in part because of increased marketing from pharmaceutical companies (EUA 2006, 35). Alpha-blockers work on the smooth muscle tissue of the prostate. One the- ory about the cause of LUTS/BPH is that, as the prostate increases in size, the increase in prostatic smooth muscle tissue interferes with urethral constriction and impairs the flow of urine $8$ , . 5educing this process and changing the behaviour of the smooth muscle cells by ingesting alpha-blockers can then help to relieve the blockage and enable better urination. However, alpha-blockers, which are ingested orally, do not only work on the muscle cells of the prostate; they can affect smooth muscle cells all over the body, which leads to side effects like head- aches, dizziness, hypertension, retrograde ejaculation and, as mentioned earlier, sometimes erectile dysfunction. Newer versions of alpha-blockers, which are bet- ter at targeting the prostate specifically, are Eeing deYeloped and marketed, Eut for the most part, treatment with alpha-blockers is followed by various side effects. If alpha-blockers are going to work for a man, he should notice a difference relatively quickly, some within forty-eight hours, and no longer than a month after beginning treatment (EUA 2006, 36). Different ways of measuring the results of treatment are commonly used to determine if alpha-blockers are effective, includ- ing reiterations of the symptom scale Tuestionnaire, pressure, flow and Yolume measures and by measuring the volume of the prostate, to see if it has shrunk at 67 Alpha-blockers all. A third of men will not notice any symptom improvement at all (EUA 2006, 36) and, according to Swedish pharmaceutical registry statistics, two-thirds of the men will stop taking alpha-blockers within three years (Spångberg and Dahlgren 2013, 685). If they do work, the patient can continue taking the alpha-blockers for the rest of his life. Thus, the actual success of treating LUTS/BPH with alpha-blockers is con- tested. The American Urology Association (AUA) says that alpha-blockers ‘pro- duce significant symptom improYement compared to placeEo that the aYerage patient will appreciate as a moderate improvement from baseline’ (AUA 2010, . %ut what is significant is a matter of deEate. Alpha-blockers ,n the national 6wedish report for treatment of LUTS/BPH done by the Swedish Agency for Health Technology Assessment, alpha-blockers are said to reduce the symptom bother slightly and increase the flow rate slightly Eetter than placeEos 6%8 , . 7he results are statistically significant Eut rather small 6%8 , . $s the (uropean Association of Urology (EAU) guidelines suggest, there are very real placebo effects to take into consideration (EAU 2006, 35) and, as the SBU overview noted, there is always a publication bias to statistical evaluations based on published studies; unsuccessful studies do not tend to be published. Of all the published studies that the 6%8 found to eYaluate, none had Eeen financed Ey independent sources; all were funded by the pharmaceutical industry (SBU 2011, 317–18). 7hey suggest that it is important to rememEer that scientific and commercial inter- ests haYe influenced the design, conduct and eYaluation of the studies on which conclusions about alpha-blockers are based (SBU 2011, 325). My analysis of the medical discourse in Läkartidningen would suggest that resource distribution and professional hierarchies implicit in the structural organization of professional health care in Sweden are also relevant factors in the moderate success of alpha- blockers within the process of pharmaceuticalization of LUTS/BPH. They are not the only factors, but they are involved. Alpha-blockers for LUTS/BPH in Läkartidningen Treatment for LUTS/BPH in Sweden can take one of three paths – and sometimes all three through the course of the disease. One can engage in ‘watchful wait- ing’, which means the patient is sent home and told to keep an eye on things and come back if his urination problems become more serious. If the patient is already so bothered by his LUTS/BPH that watchful waiting is not an option, the treat- ment can either be lifelong medication, beginning with the use of alpha-blockers, potentially combined with 5-Alpha-reductase inhibitors (5-ARIs), or the problem can be addressed surgically or through other means of removing or destroying the prostate. (There are natural dietary supplements, herbal medicines and exercise programmes which are also said to help, but these are not covered by the urology- dominated discourse in Läkartidningen.) Alpha-blockers could have been a game changer for the treatment of LUTS/ BPH when they were introduced in the 1990s. And to some extent, they have Eeen, Eut not entirely. When they started Eeing used, the main treatment for seYere Ericka Johnson 68 BPH was surgery. The surgical removal of parts of the prostate through the urethra has been in general use in Sweden since the end of the 1960s (Hedelin et al. 2003, 2441). However, the introduction of alpha-blockers has not removed surgery as an option for treatment, eYen if the official diagnostic and treatment oYerYiew 6%8 suggests that alphaElockers are a firststep treatment, to Ee tried Eefore surgical removal or reduction of the prostate. Yet surgery for an enlarged prostate and the urination problems it is thought to cause is still a very widely used method in Sweden (and in other countries), and the surgical techniques are continually being advanced technically, with hospitals purchasing expensive robotic surgery systems, partly as recruitment tools to attract urology surgeons (Lindgren 1999), and TURP simulators for training purposes are being developed and used (Käll- ström 2010). The most recent articles in Läkartidningen mention both surgery and pharmaceutical treatments on equal grounds (Degerblad et al. 2014). So, as an example of pharmaceuticals changing the treatment practices of already medical- ized diseases, alpha-blockers are only partially successful. When they arriYed on the scene, alphaElockers did not haYe to do any con- vincing about the ‘realness’ of LUTS/BPH or its presence in the enlarged prostate diagnosis. Alpha-blockers for LUTS/BPH in Läkartidningen Problems with urination had already been accepted, both the legiti- macy of patient suffering and the legitimacy of paying for treatment with tax money in Sweden. LUTS/BPH was an established disease with established treat- ments that were costly and that cost was not controYersial. What was controYersial in the Swedish medical discourse was shifting that cost from surgery provision to pharmaceutical distribution. In 1996, an article about new, expensive pharma- ceuticals for urological treatments, written by a leading urologist, compared their increasing costs (and potential share of a limited health care budget) with the cost of more traditional urology care, claiming that the increased costs for medicines for prostate cancer, BPH and erectile dysfunction ‘would equal the annual costs of running Eetween fiYe and si[ medium si]ed urology clinics¶2 (Carlsson and Spångberg 1996a, 4552). In this argument the cost of medication is directly com- pared to the cost of funding clinical treatments in urology. Resources for drugs are rhetorically placed in direct competition with urologists’ salaries and clinical costs. The concern about cost is particularly relevant to the local context of alpha- Elockers in 6weden, reflecting Eoth the ta[Eased structure of health care funding in Sweden and the fact that LUTS/BPH treatments are accessed within a health care system that has designated specialists – urologists – who treat LUTS/BPH and gatekeepers to those specialists, general practitioners. With alphaElockers, there was debate and concern about cost in general, although the concern seems to be more related to the fact that use of alpha-blockers for the treatment of LUTS/ BPH is a lifelong medication, whereas the (arguably expensive) surgery it is some- times thought to replace is a one-off cost (Hedelin et al. 2001, 2441). However, in the debate about whether to treat with alpha-blockers or to treat with surgery also lurked an implication of which type of doctor was allowed to treat LUTS/ BPH and where the state resources for it would then go. In Sweden, a man with suspected LUTS/BPH cannot make an appointment with an urologist on his own; 69 Alpha-blockers he must receive a referral to the urology specialist from his general practitioner. General practitioners are not allowed to conduct TURP surgeries, but they can – at least theoretically – prescribe alpha-blockers (Hassler 2002, 2174). Alpha-blockers for LUTS/BPH in Läkartidningen By shifting the first line of treatment to alphaElockers, there is the potential that /876%3+ patients will become a case for general practitioners but not urologists. To some extent this has happened, but not entirely, and here, too the medical discourse has focused on how alpha-blockers can potentially shift cost and resource distribution within the health care system (Carlsson and Spångberg 1996b, 4557). In the early 2000s, it was estimated that one in four patients was beginning treatment with alpha-blockers or other pharmaceuticals before visiting a urologist (Hedelin et al. 2003, 1435). This does not mean that urologists have lost 25% of their LUTS/ BPH patients – on the contrary. There has been a dramatic rise in the number of patients presenting with LUTS/BPH since the early 1990s, probably due to the existence of alpha-blockers and their use as a treatment at an earlier stage in the disease. But nonetheless, the discourse in Läkartidningen shows a concern about patients being treated outside of, and prior to, consultation with a urology spe- cialist, despite that the 6wedish urology field is currently understaffed and there are long waiting lists for meeting a urologist, as the field is Yery occupied with prostate cancer. p For the treatment of LUTS/BPH, a shift from being a disease treated by special- ists to one managed by general practitioners can also carry with it a step down the prestige ladder see Wit] /indgren , and one which could mean a transfer of resources from the more prestigious speciality (urology) to the less prestigious world of general practitioners, as the comparison of increasing costs of pharmaceutical treatments to clinic costs could suggest. This competition between GPs and urologists has been articulated elsewhere, like in the debate aEout whose finger is Eetter at conducting the digital rectal e[am of the prostate, the general practitioner’s or the urologist’s (see Kirby et al. 1995). It may have had an influence on the e[tent to which alphaElockers replaced surgery in the Swedish context.i 7he final words in the deEate Eetween alphaElockers and surgical treatments for LUTS/BPH may yet remain to be written. One of the urologists I interviewed suggested that the new framing of BPH as primarily LUTS in the 2013 European Association of Urology clinical guidelines (Oelke et al. Strong and weak pharmaceuticalization: alpha-blockers and Viagra in Läkartidningen As discussed in Chapter 4, Viagra changed the concept of ED and impotence, its treatment and even who its patient was in the Swedish medical discourse, as it did in the international discourse. Yet particularly local Swedish aspects appeared in the discourse around Viagra, related to local (nation state) health care provision specificities, particularly the diYision of laEour Eetween 3s and specialists, and the funding structures for clinical care and pharmaceutical provision. There are some similarities here to what happened with alpha-blockers in the Läkartidnin- gen discourse, Eut also some significant differences. One similarity between the Viagra discourse and that surrounding the use of alphaElockers is the concern aEout cost, yet there are some significant differences between them, too. This was a very heated debate about Viagra, and dealt primar- ily with the potential costs a state subsidy of the pharmaceutical would entail, an argument that spilled over into the court system, as Chapter 2 detailed. The Viagra cost debate focused on the sheer (potential) cost of the drug for a desiring public and its legitimacy within a tax-funded system, based on the question of whether oldage impotence was a legitimate disease to spend ta[ resources on. Within the alpha-blocker debate, the question of cost was framed as one of where within the health care system the disease would be treated, and whether resources should be used to support clinics or pharmaceuticals. The legitimacy of spending tax money to treat LUTS/BPH was never questioned. Viagra also changed the way impotence (now ED) was treated, which alpha- blockers only partially managed to do. Here the most interesting difference with alpha-blockers can be found. Perhaps the incompleteness of the shift from surgery to pharmaceutical treatment is because the representatives of the other method of treatment surgery are not from a less prestigious and weaker field of medicine. I h Vi h Vi i d d i hif d h f i Viagra also changed the way impotence (now ED) was treated, which alpha- blockers only partially managed to do. Here the most interesting difference with alpha-blockers can be found. Perhaps the incompleteness of the shift from surgery to pharmaceutical treatment is because the representatives of the other method of treatment surgery are not from a less prestigious and weaker field of medicine. Alpha-blockers for LUTS/BPH in Läkartidningen 2013) is a result of alpha- blockers – or lobbying by their producers and supporters – to change the focus from the enlarged prostate to the lower urinary tract symptoms and thereby shift the focus from potentially removing an enlarged gland and instead treating the muscles in and around it with pharmaceuticals. More recently, there has been little debate about the use of alpha-blockers in Läkartidningen, which would suggest that the usage has stabilized. Alpha-blockers’ place in treatment practice has also been formalized in the 2011 overview pub- lished by the Swedish Agency for Health Technology Assessment. The chair of the committee who authored this report is the same urologist who raised the com- parison of costs in relation to urology clinics in 1996, and who wrote a 2013 article in Läkartidningen reviewing the overview. His voice, together with the Ericka Johnson 70 health care policy analysts he co-authored with, raised concerns about the (over) use of alpha-blockers, while simultaneously noting their relatively small success rate (Spångberg and Dahlgren 2013, 683). Strong and weak pharmaceuticalization: alpha-blockers and Viagra in Läkartidningen In the Viagra case, when Viagra was introduced it shifted the event of impotence from a problem for sexual and couples therapists to a disease (ED) under the remit of urologists. The urologists were able to claim this territory from the sex thera- pists without much visible resistance. However, LUTS/BPH was already a disease of urology when the pharmaceuticals arrived on the scene and despite indica- tions that general practitioners may be beginning to prescribe alpha-blockers for it, urologists are trying to keep LUTS/BPH in their remit. Rather than facilitating a shift from one medical field to another, alphaElockers merely created two dif- ferent factions within urology: those who were still using surgery, and those who were willing to treat LUTS/BPH with pharmaceuticals. Given that the introduction of alpha-blockers could have replaced an expen- sive, invasive surgical procedure, one could have expected it to cause a change in treatment practices more readily and thoroughly, as Viagra did. But this was not Alpha-blockers 71 the case. 6urgical procedures are down significantly from their occurrence rate Eefore alphaElockers were officially part of the treatment cadre, Eut there are still many thousands conducted each year in Sweden. Perhaps one clue to why can be found in the fact that with Viagra there was no actor who/which had traditionally received large resources for the treatment of impotence. Sexual therapists existed (and still exist) in Sweden, but their branch of medicine was peripheral compared to urology, and not nearly as well funded. The occurrence of impotence was medi- calized, but its treatment costs were minimal and it was somewhat considered a natural part of getting older that men should just accept (see McLaren 2007). This was not the case with urination problems. The shift of ownership of impotence from sex therapists to urologists that Viagra facilitated was from a low-prestige field to a higherprestige one. %ut for alphaElockers, the potential shift was from a surgical branch of urology to a non-surgical branch of urology. This can either Ee considered two eTual Eranches in the same field, or enYisioned as a shift from a highprestige to a lowerprestige Eranch in the same field of medicine, depending on how one sees it (cf. Lindgren 1999). Alpha-blockers were not a tool that could be used to capture the right to diagnose and treat a problem from one specialty to another. 7here has Eeen a more recent deEate aEout the dangers of using an antiinflammatory drug for BPH because of its negative effect on heart patients already taking glucosa- mine, a deEate that has primarily Eeen related to 3fi]er¶s heaYy marketing of $rtro[, its alleged influence on 6wedish safety and sideeffect recommendations to doctors, 3fi]er¶s alleged attempts to deEunk studies which critici]e its effectiYeness and safety, and its increased cost, compared to other drugs (see Beermann 2003; Fuberg 2003a, 2003b; Järhult and Lindahl 2003a, 2003b, 2003c; Lohm and Lindh 2003;; Nilsson et al. 2003; Lohm et al. 2003; Järhult 2005). This is not an alpha-blocker and is therefore not analyzed in this chapter. Strong and weak pharmaceuticalization: alpha-blockers and Viagra in Läkartidningen Urologists were already conducting surgeries for LUTS/BPH, and it is primarily urologists who are prescribing alpha-blockers. Increasing their use could potentially allow GPs more space in the game, but the urologists seem to be a more powerful voice in the discourse. In fact, no GPs were authoring arti- cles about LUTS/BPH and alpha-blockers in Läkartidningen during the period studied, unlike the Viagra discourse, which saw a shift in who was the author of most of these texts. In the alpha-blockers for LUTS/BPH discourse, nearly all of the articles are written by or giving the opinions of urologists – eleven of them – throughout the entire time period searched, with the exception of a few pharma- cists and pharmaceutical policy analysts. This is different than the authors, who were given space in the Viagra discussion, where sex therapists and sexologists were writing about impotence before Viagra, and urologists were writing about erectile dysfunction after Viagra’s arrival. The case of alpha-blockers in Sweden can nuance our understanding of phar- maceuticalization by showing that the existence of the drug and its acceptance into the official retinue of treatment options is not enough to predict the success of a new pharmaceutical treatment. Success is also related to which actors (individu- als, commercial interests and medical practitioners, a heterogeneous category) use the drugs to make the shifts that occur. Locally, with the case of alpha-blockers in 6weden, the specific concerns with cost and resource distriEution related to the goYernmentfinanced organi]ation of speciality clinics may haYe impacted the amount of success the alpha-blocker treatment has had in replacing existing therapies, and seems to certainly haYe influenced the way it was presented in the medical discourse. Alpha-blockers have become an earlier step in the disease trajectory, a sort of middle stage before eventual treatment with surgery, even though many men are prescribed alpha-blockers with the idea that they will have to take them for the rest of their lives, and may avoid surgery by doing so. The ability of alpha-blockers to Ericka Johnson 72 shift the treatment paradigm of an existing medical condition from surgery to phar- maceutical has been weaker than that of Viagra. But they have still managed to take over some of the patients and, perhaps more important, they have managed to increase the numEer of patients identified as haYing /876%3+. Strong and weak pharmaceuticalization: alpha-blockers and Viagra in Läkartidningen $lphaElockers have been part of a process of pharmaceuticalization of an existing medical condi- tion, but this process has been only partially successful. 2 All translations are the author’s. Notes 7here has Eeen a more recent deEate aEout the dangers of using an antiinflammatory drug for BPH because of its negative effect on heart patients already taking glucosa- mine, a deEate that has primarily Eeen related to 3fi]er¶s heaYy marketing of $rtro[, its alleged influence on 6wedish safety and sideeffect recommendations to doctors, 3fi]er¶s alleged attempts to deEunk studies which critici]e its effectiYeness and safety, and its increased cost, compared to other drugs (see Beermann 2003; Fuberg 2003a, 2003b; Järhult and Lindahl 2003a, 2003b, 2003c; Lohm and Lindh 2003;; Nilsson et al. 2003; Lohm et al. 2003; Järhult 2005). This is not an alpha-blocker and is therefore not analyzed in this chapter. 6 Enrolling men, their doctors and partners Individual and collective responses to erectile dysfunction1 Ericka Johnson and Cecilia Åsberg This chapter examines how men, their doctors and their partners are enrolled by the 3fi]ersponsored weEsite for potential 6wedish 9iagra customers. We read this enrolment as an example of how new techno-social identities are created by a drug, in this case, Viagra. The Swedish-language site www.potenslinjen.se2 (in English, ‘potency hotline’) is framed as a source of information for laypeople concerned about erectile dysfunction.3 We haYe e[amined how the site¶s te[t and imagery address different audiences in the construction of the Swedish Viagra man. Our analysis builds on existing literature about the promotion of Viagra which addresses the construction of erectile dysfunction (ED) and masculinity in other national contexts, and we therefore make mention of alternative images and readings in other contexts throughout our analysis. Like previous critical stud- ies of Viagra (Fishman and Mamo 2001; Marshall 2006; Tiefer 2006; Vares and Braun 2006), we are examining the construction of an ideal user of Viagra, but we also discuss the way the enrolment of doctors and partners serves to position ED in the man and define its treatment as a solitary act of taking a pill while simulta- neously involving the other actors to help the medicine function.i 2ur contriEution delineates the specific roles the Yarious suEMects are granted in the commercial discourse, in particular by looking at the invisible work the ‘pas- sive’ female partner is tasked with as she is told to actively guide and support her partner in the 9iagra traMectory. 7o think through the creation of the 9iagraspecific technosocial identities within the pharmaceuticali]ation framework Williams et al. 2011a), we use the Actor Network Theory (ANT) concept of enrolment, which articulates the roles given to various actors as they are enrolled into a network that discursively constructs ED as an illness and Viagra as a cure. The global and the local become relevant in this chapter when the empirical material is read against the framework of regulations regarding DTC marketing of pharmaceuticals. The legality of DTC advertising of prescription pharmaceuticals in the United States and New Zealand has spawned much of the critical research about Viagra and its role in the media climate (see Mamo and Fishman 2001; Elliot 2003; Loe 2004b; Moynihan and Cassels 2005; Potts and Tiefer 2006). Part 3 Techno-social relationships and identities This page intentionally left blank 6 Enrolling men, their doctors and partners Individual and collective responses to erectile dysfunction1 And the illegal advertising with near impunity of pharmaceuticals in countries which do ban DTC advertising has been detailed by Zetterqvist and Mulinari (2013) and Zetterqvist and colleagues (2015). Our study, looking at Viagra in the Swedish Ericka Johnson and Cecilia Åsberg 76 conte[t, which Eans '7& adYertising for prescription drugs, confirms the results of much of this work Eut contriEutes an important insight to the strength and fle[- ibility of Viagra marketing in a globalized pharmaceutical market. There is a good deal of harmonization between the Swedish site and other Viagra sites, yet also local adaptation (see also Chapter 7). And as we show here, the local adaptation of Viagra marketing that is not considered DTC advertising relies on the construc- tion of subjectivities for the man, his doctor and his partner. The Swedish site is a ‘purely informational’ site about erectile dysfunction (although it is obviously advertising Viagra) and therefore has information explaining what sex therapy can offer men suffering from impotence in addition to pharmaceutical solutions to ED. The site has previously (2009) listed the telephone number to a sexual medicine centre at a large hospital in Stockholm that receives undirected funds from 3fi]er and recently suggests that men use se[ual or psychological therapy for cases when erectile problems have psychological or social causes. In these cases, the site suggests men turn to The Yellow Pages. Despite the DTC ban, 9iagra figures largely on the weEsite in its colour schemes and 3fi]er Eranding, even if the word Viagra is generally absent.i 9iagra has figured largely in the µcollectiYe psyche¶ in 6weden Eoth as a suE- ject of newspaper articles (some reading as if they have been taken directly from drug company PR sheets) and of public debate, thanks in large part to the drawn- out discussions and court cases about whether the Swedish health care system should or would subsidize Viagra (see Chapters 2 and 4). Despite the ban on DTC advertising, a wide range of commercially produced informational material about Viagra is available in Sweden: free pamphlets and booklets which men or their partners can order from 3fi]er literature for doctors and other medical profession- als; press coverage in local and national newspapers; informational material on puElicly funded weEpages aEout men¶s health. 6 Enrolling men, their doctors and partners Individual and collective responses to erectile dysfunction1 We haYe, for this chapter, focused on the website www.potenslinjen.se for a number of reasons: it is produced by 3fi]er for a 6wedish audience in the 6wedish language it is easily accessiEle to anyone with an Internet connection and does not require interfacing with a medical practitioner; it focuses on impotence and erectile dysfunction rather than men’s health in general; it can be accessed by and addresses individuals not fac- ing impotence personally, like partners and friends. Additionally, the website is a good e[ample of how 3fi]er tweaks its material to locali]e a gloEal message for its global product. Viagra is available in Sweden, but falls outside of the state- subsidized pharmaceutical scheme, which means patients must pay for the drug themselves, creating challenges for marketing the drug. This is addressed by a short film on the weEsite, encouraging men to aYoid dangerous, Elack market pur- chases and explaining how easy it is to get a prescription and buy the ‘real thing’ at a drugstore. By analyzing the discourse on www.potenslinjen.se, we argue that the challenge of encouraging men to buy Viagra has been met on the website in part by enrolling men, their doctors and their partners in the ways we detail later in this chapter. In analyzing the text and images on this site, we have been inspired by the criti- cal studies of Viagra mentioned earlier in this book as well as Foucault’s idea of an Responses to erectile dysfunction 77 economy of discourses aEout se[uality. We are looking at the material presented on the website as an example of an economy of discourses in an attempt to articu- late ‘the necessities of their operation, the tactics they employ [and] the effects of power which underlie them and which they transmit’ (Foucault 1987 [1976], f. We also draw inspiration from studies of scientific discourse and naturali]ed embodiment that feminist scholars have produced since the late 1970s on power- ful ideological processes (cf. Merchant 1980; Haraway 1989; Butler 1990; Martin 1991; Fox Keller 1992; McClintock 1995; Bryld and Lykke 2000; Franklin et al. 2000; Braidotti 2006). 6 Enrolling men, their doctors and partners Individual and collective responses to erectile dysfunction1 These researchers have used feminist critiques to inves- tigate how science as a discourse and notions of the natural are used to support dominant ideologies.i ,n our final discussion, we ask what this enrolment says aEout social, rather than individual, aspects of ED, drawing inspiration from early medical sociology work on community responses to mental health (aton and Weil and recent qualitative studies of men’s responses to ED (Oliffe 2005; 2006). By exploring alternative narratives of illness, we suggest, an alternative reading of erectile dys- function and its subjects could be told. Background Sexual response models within sex therapy have taken foremost physiol- ogy, Eut also EehaYiour penetration into account in defining healthy or non healthy sex, which coloured the term impotence pejoratively, and later replaced it with the term erectile dysfunction. However, the medical community did not generally take up the term until it was adopted by urologists and popularized by 3fi]er see %ordo 0arshall and .at] /oe E see &hapter for the 6wedish e[ample. 7his rhetorical shift narrows the definition of what impotence is from a condition of the psyche, the emotions or the relationship, to a biomedi- cal complaint (Marshall and Katz 2002; Tiefer 2006; McLaren 2007; Johnson . 3fi]er marketing has also introduced the concept of erectile Tuality (4 to expand the market to include younger men (Fishman and Mamo 2001, 181; Mar- shall 2002, 139), playing on ‘erectile insecurity’ (Tiefer 2006, 279). And, impor- tantly, Viagra has been a phenomenon around which multiple, different, vested interests have gathered to sell it and construct the disease of ED, as Loe articu- lately shows in her examination of the US case (Loe 2004b).i 1950s (McLaren 2007, 221). In psychiatric discourse, erectile dysfunction has Eeen articulated as a proElem of arousal since the s, when (' is defined as a proElem of attaining and maintaining an erection sufficient for intercourse $3$ 2000). Sexual response models within sex therapy have taken foremost physiol- ogy, Eut also EehaYiour penetration into account in defining healthy or non healthy sex, which coloured the term impotence pejoratively, and later replaced it with the term erectile dysfunction. However, the medical community did not generally take up the term until it was adopted by urologists and popularized by 3fi]er see %ordo 0arshall and .at] /oe E see &hapter for the 6wedish e[ample. 7his rhetorical shift narrows the definition of what impotence is from a condition of the psyche, the emotions or the relationship, to a biomedi- cal complaint (Marshall and Katz 2002; Tiefer 2006; McLaren 2007; Johnson . 3fi]er marketing has also introduced the concept of erectile Tuality (4 to expand the market to include younger men (Fishman and Mamo 2001, 181; Mar- shall 2002, 139), playing on ‘erectile insecurity’ (Tiefer 2006, 279). Background Science and technology studies use the term enrolment within ANT to denote how human and non-human agents are called on and woven into complicated networks (Latour 1993; 1998). Although it has been rightly criticized for implying a heroic, entrepreneurial actor in the process of enrolment (Star 1991), the concept is use- ful for our study Eecause it articulates the sense that there are actors with specific interests here, the pharmaceutical company 3fi]er and its marketing e[perts who use specific methods those descriEed in this chapter are discursiYe strategies to involve heterogeneous constellations of human and non-human actors in the con- struction of a 6wedish 9iagra man. We will Ee using the concept of enrolment to e[amine how a specific weE of actors ± potential users, their medical doctors and their partners – is woven together by a discrete discourse to construct an identity and agenda for the Swedish Viagra man. In Sweden, Viagra has been available by prescription since its approval in 1998, but, like the similar drugs Cialis and Levitra, it is not covered by the national sub- sidy programme for medicines. Thus, men can get a prescription for the drug, but they must pay for it out of their own pockets, which is unusual for the Swed- ish consumer. 3roduced and sold Ey the pharmaceutical company 3fi]er, 9iagra works physically in some men and in some situations by blocking the return of Elood flowing out of the penis. 7hus, if a man Eecomes aroused and Elood flow to the penis increases, Viagra will help keep it there and produce an erection. Discursively, however, Viagra does many other things. Viagra has, for example, changed our language about impotence in both English and Swedish. The market- ing around Viagra has helped to introduce the term erectile dysfunction (ED) to the general public, replacing the more negative term impotence (Bordo 1998, 90; Potts 2004, 23). Masters and Johnson used the term erectile dysfunction in the Ericka Johnson and Cecilia Åsberg 78 1950s (McLaren 2007, 221). In psychiatric discourse, erectile dysfunction has Eeen articulated as a proElem of arousal since the s, when (' is defined as a proElem of attaining and maintaining an erection sufficient for intercourse $3$ 2000). Enrolling men Men are enrolled through the information on the pages that constructs them as potential patients with ED. They are welcomed in what could be considered a respectful and tactful manner: ‘Potency problems can be a sensitive issue in spite of the fact that many men – and their partners – are affected,’5 they are told. The picture attached to the welcoming sentence, a photo of a tanned, grey-haired man in his fifties on his Eack with hands Eehind his head, smiling as he looks up into a blue sky that matches his blue t-shirt, suggests a relaxed, laid-back attitude. The blue colours of clothing and sky seem to allude to the iconic colour of Viagra and to 3fi]er¶s logotype, and are a common feature of 9iagra ads, as /oe E and Baglia (2005) have discussed. In the image and text, the cultural stigma of ED is mitigated by such a respectful yet relaxed approach. Further, the mode of address to the large number of men and their partners who suffer from ED normalizes the prevalence of the problem and works to alleviate the concern the visitor to this site may be experiencing. The accessibility of an easy, safe and comforting solution to erectile proElems, in the form of the drug, is also highlighted Ey the film on the first page, complete with dancing, erect penises and reassuring statistics aEout how common ED is. Aside from the overwhelming discourse of disease associated with erectile dif- ficulties including mention of cardioYascular disease, which will Ee discussed later), one of the primary tools used to enrol the men is an interactive quiz in which they are first asked to rate their se[ual health Ey answering a series of fiYe questions) then encouraged to talk to their doctor. Unlike much of the other text on this site, the quiz is a short version of the International Index of Erectile Func- tion (IIEF), and is directly translated from the US Viagra site.6 As Marshall has discussed, this process of inviting quizzes, generous medical advice and sexual education effectively creates an ostensibly benevolent regime of self-surveillance on the website for the individual through assisted self-monitoring and remedial action (Marshall 2006, 356; see also Mamo and Fishman 2001; Baglia 2005). Background And, impor- tantly, Viagra has been a phenomenon around which multiple, different, vested interests have gathered to sell it and construct the disease of ED, as Loe articu- lately shows in her examination of the US case (Loe 2004b).i $s has Eeen noted widely, 9iagra has reinforced the definition of se[ as penetra- tion, and masculinity as the ability to achieve penetration, relying on a reworked version of the notion Sigmund Freud previously had reserved for the female mind: the ‘anatomy is destiny’ determinism of the naturalized body in the construc- tion of the late modern male identity. The physicality of male embodiment boils down to the sexual (and not necessarily reproductive) performativity4 of the vis- ible sexual organ. Thus, within the Viagra discourse, as Baglia’s (2005) study of 3fi]er promotional material in the 8nited 6tates has shown, se[ual performance is defined Ey a narrow se[ual function model starting with arousal and progressing through erection, penetration and ejaculation. As long as a man can perform this penetrative sex, his masculinity is intact. The idea that penetration produces (or at least proves) masculinity reinforces the importance of penetration for both the sex act itself and the concept of sex-life expectancy. As sociologist Barbara Marshall notes, this concept of sex-life expectancy, with its calls to vigilant self-monitoring of healthy practices and appropriate sexual behaviour, relies on the disciplined individual taking responsibility for managing the risks of lost masculinity (i.e. lost ability to penetrate) even before ‘old age’ (Marshall 2006, 335). Viagra connects this individual responsibility to the medical and pharmaceutical networks within which Viagra is active, which also reinforces the scientism of sex and the natural- ized body, attaching both to systems of expertise while simultaneously assigning responsibility for functionality to the man. The Viagra discourse of sex relies on a three-step paradigm of arousal, pen- etration and ejaculation, and then demands Viagra as a solution to (age, stress or illness related) declining sexual performance (Plante 2006, 380). According to this discourse, a person (or couple) can maintain a successful sex life, as long as penetration is possible, that is with the help of Viagra. This idea ignores and tends to silence suggestions of alternative sexual practices and a sex life that is not 79 Responses to erectile dysfunction dependent on penetration (Tiefer 2006). In this narrative, emotions all but disap- pear. Background And where they do play a role, responsibility for them is given to the partner, as we will discuss later. Enrolling participants in the Viagra discourse ([amining the 6wedish weEsite it Eecomes apparent that 3fi]er enrols three dif- ferent groups of human participants to assist in constructing a subject position for men as potential consumers of Viagra: the men themselves; medical doctors; and the men’s partners. Throughout the discourse, the Viagra pill is also enrolled as a non-human actor, nearly given a hero’s identity. Enrolling men Such a mode of address, and ways of enticing and enrolling potential consum- ers of Viagra, can be read as part of a larger discourse, a sexual regimen of the individual. This sexual self-governance and monitoring, to borrow ideas from Ericka Johnson and Cecilia Åsberg 80 )oucault, is centred on male penetration at the 9iagra weEsite. iYen the flour- ishing market for self-health guides, books and websites and so forth, this mode of address is hardly surprising. This website merges the commercial aims of a product-selling site with sexual education and health advice in a manner charac- teristic of the rather recent new media genre of ‘edutainment’ where entertaining features such as quizzes and educational imagery blend for accessible, online dis- play (Åsberg 2005).i +ealth matters are, in such genres, firmly placed within the world of consumer- ism, as detailed in Stacey’s description of self-help literature in her cultural study of cancer (Stacey 1997). The Viagra consumer is enrolled into a mode of being sustained by the ideal of the self-caring subject position of a health consumer. In line with Stacey’s work on health consumerism, this also resonates with what Rose and Novas termed ‘biological citizenship’ (Rose and Novas 2005, 14). The biological citizen invests heavily in self-education on health matters and develops the medical literacy needed to pursue a highTuality, selfsufficient, personally and socially ‘responsible’ lifestyle (Rose and Novas 2005, 14). Through the quiz on se[ual health, the selfsurYeillance discourse redefines se[ual health in a Yery specific, determinist way se[ual health for men is the achieYement and mainte- nance of an erection and ability to complete intercourse. Their anatomy (particu- larly the functioning of certain parts of their anatomy) becomes their destiny. The website not only encourages self-surveillance and individual responsibil- ity, it also provides the tools for individuals to take on the task of monitoring and disciplining their erections: ,f you are Eeing treated with potency medicine from 3fi]er, you can receiYe support and encouragement for your treatment through the web.7 Do you lack the time, desire or opportunity to pick up your medicine from the drugstore" 1ow you can haYe your impotence medicine deliYered to your home by mail. Enrolling doctors 0edical doctors are also enrolled through 3fi]er¶s efforts on the 6wedish www. potenslinjen.se website (beyond the infamous drug rep sales techniques [see 5eidy @. 3art way down on the Yery first page, an anatomical sketch of a heart accompanies a text that reads ‘Potency problems – an important warning sign¶ and delineates how potency proElems might Ee the first µuseful¶ sign of car- dioYascular diseases. ,t Eecomes clear that 9iagra is not merely a matter of fleshy pleasures and an improved sex life, and that the ED Viagra is supposed to allevi- ate relates to serious health issues and eYen has a function as a first warning sign. Such medical appeals to cardiovascular health issues as linked to ED both play to the scientism of the naturalized body and work to medically legitimize Via- gra. Swedish men seeking medical attention for ED are addressed as upstanding citizens taking responsibility for their personal overall health, and doctors are encouraged to help them with that. This use of medical complaints other than ED to legitimate Viagra can be read against the efforts in Sweden to associate Viagra with specific diseases rather than lifestyle choices as part of the deEate oYer state subsidies (see Chapter 2).i 7he medical dimension of 9iagra is further enhanced with a figure of author- ity that confirms Eoth the rela[ed personal tone and the urgency of the matter, namely a headshot of a physician in scruEs with a hint of a smile on the first page. The photo of the doctor creates a close proximity between medical authority and the potential Viagra consumer in another sense: the ambivalence of the picture in this setting suggests that even a medical doctor can have a use for Viagra. Most importantly, howeYer, this small photo, emitting medical confidence and trust- worthiness, serves a particular function within the website: to illustrate a search engine for finding a local, 9iagrafriendly doctor. ,n addition to encouraging doc- tors to be ‘proactive’ in asking their patients about sexual function during routine exams and when taking medical histories,8 3fi]er has included a national dataEase of µ('aware¶ doctors, or µaffiliated e[perts¶ as /oe E calls them, which lets visitors to the website submit a query and generates a list of doctors near them who can be consulted for information about erectile dysfunction (and, presum- ably, for prescriptions of Viagra). Enrolling men Similarly, in a special section called the Potency Coach, illustrated by an animated cartoon figure with a megaphone, one finds that The Potency Coach is an easy to use, interactive patient support that will help you achieve the expected and pre-determined results with your treat- ment. +ere you can also find information aEout the underlying causes of potency problems and about other patients’ experiences. Working within a EeneYolent discursiYe frame to help him help himself, the weE- site also reveals the assumption of a shy, Swedish man of few words implied by such a mode of address. 7he targeted suEMect is one who does not easily confide in his physician, especially not regarding sexually related matters, and must be reassured and coaxed to bring up the topic during a health care visit: Unfortunately, it is common that men hesitate before seeking help. This is a shame, since the vast majority of those who seek help can be successfully treated for their problems. 81 Responses to erectile dysfunction Perhaps surprisingly, it is not a sexually liberated or outspoken subject who is addressed, but someone rather inhibited when it comes to articulating sexual problems. From this arises a Swedish man who is non-articulate with respect to his own malleable body and sexual health, a man who might need encouragement when asking for a 9iagra prescription at the doctor¶s office since doing so could be interpreted as a defeat in the masculinized struggle to control the body. Impor- tantly, this can Ee read as a remarkaEle handson approach of 3fi]er in facilitating the individual and his care of the self. Enrolling doctors A clear example of local manifestations and adaptations of global market- ing methods, the same type of database can be found on other national Viagra sites, also paired with suggested phrases that men can use when speaking to their Ericka Johnson and Cecilia Åsberg 82 doctors, addressing the fact that some men may find it difficult to Ering up the subject of sexual dysfunction during an exam. On the Swedish site, men are told, µWhen you meet your doctor, she or he will proEaEly interYiew you and ask you questions about previous illnesses and if you are currently taking any medica- tions. Try to provide as accurate information as possible, including if you still have early morning erections or if your erectile ability has disappeared suddenly or gradually. ,t may feel difficult to speak aEout these issues, Eut it is completely OK to be embarrassed. Remember that doctors are used to speaking about these things and their job is to help you.’ In this way the men and doctors are also positioned to enrol each other and maintain each other’s investment in the Viagra discourse. We suggest that this part of the 9iagra weEsite seems to connect Eio- logical citi]enship with medical literacy and affiliated e[perts, to thus secure the commercial success of the drug. Enrolling partners On the Swedish site partners are also enrolled in the process of positioning men as subjects for whom Viagra is the solution to a waning sex life and/or issues of sexual dysfunction. Potency issues are continuously addressed as a joint problem, for the female partner as well as for the man. In a special section of the Swedish webpages, partners are told about the ways ED can affect a relationship, above all by letting coldness, distance and worry creep in and replace the sensitivity, near- ness and trust that had been in the relationship before. On the connecting pages, partners are encouraged to be supportive, and then to let their partners know that treatments are available for the problem: Today there are many different treatment methods. There are medicines that are prescribed in connection with a doctor’s visit. Apart from medical treat- ment, sometimes sexual therapy can be the most appropriate approach. It is good for you as a relative to know about this and to be able to support and encourage your partner to seek help. The partners are also encouraged to order the free brochure ‘A man’s best support is Ey his side¶, puElished Ey 3fi]er with a smiling, heterose[ual couple on its front page. The way partners are enrolled to support the men experiencing ED plays strongly on the assumption that the partner is steadfastly unreflectiYely" con- senting to reproduce certain practices and maintain a supportive position within a relationship with the man (cf. Potts et al. 2003). There is also information about how ED makes a man feel and what sorts of ‘normal’ behaviour it can provoke in one’s partner. ‘Many [men with ED] distance themselves from their partners sim- ply to aYoid conflict and to aYoid situations which can lead to se[. 0any deYelop a new hobby, immerse themselves in their work, or make sure they don’t go to bed at the same time as their partner in the evening. Many consciously or subcon- sciously eYen create conflicts to aYoid Eeing close to their partner.¶ $s this Tuote implies, sexual intercourse seems to be an active achievement, where ‘success’ Responses to erectile dysfunction 83 needs to be granted. Enrolling partners Moreover, the female partner has a supportive rather than a leading role in this sexual achievement which combines the traditional, passive recipient of penetration (waiting in the bed for her partner with the new hobby) with an active subject tasked with leading and directing the man to Viagra and/ or sex therapy. She can guide him on his way, yet he is the doer behind the deed. This reverberates with the traditional assumption about heterosexual femininity as sexual passivity and masculinity as sexual activity, but more importantly it also points to the enormous effort by the woman that in reality lies behind achieving the µpassiYity¶, which can confirm his actiYe and YaluaEle status as a heterose[ual male. There are many subtle manoeuvres, enticing practices and persuasive, yet necessarily non-direct rhetorical moves a woman must master in order to achieve the right amount of sex-inspiring passivity. A lot of hard work lies behind her µpassiYe¶ affirmation of his masculinity.i $ specific section of the 6wedish weEsite is dedicated to the prescriptiYe dis- cursive patterns available to the partner. As the main heading on the partners’ page suggests, she should ask herself how µcan , help"¶ to receiYe the answer, µ6peak to and encourage: It is best to speak openly with your partner; support each other’.i ,n this section of the 6wedish weEpage one finds a discussion aEout how (' and the normal behaviour it provokes in men can make the partner feel. These partner responses build on feelings of guilt and inadequacy. However, the partners are encouraged to perseYere and help their men seek help Eecause µWhen one has received help and solved the problem, many discover that their relationship has in fact become stronger.’ Again, the partner’s discursive work is one of maintain- ing a seemingly effortless and natural attitude that avoids putting pressure on her partner and is achieved by another rhetorical strategy here provided by the Viagra site text, namely the ‘we’. She can address her sexual needs and his sexual proElems if they are addressed as a µwe¶ issue, enrolling a se[ual dyad, a figure of heterosexual complementarity. Within the 6wedish conte[t, it is worth noting that pre9iagra prior to medical advice about impotence underlined how important it was for doctors to warn their patients that merely solving a man’s inability to produce an erection would not necessarily solve relationship problems (Olsson et al. Enrolling partners 1995), something the 3fi]er information seems to Eelie. ,nternationally, this assertion can Ee read in light of a Japanese study where a survey on the level of satisfaction derived from using 9iagra indicated that while the male patient was e[tremely satisfied, his part- ner was not satisfied at all. Women reported their husEands¶ erections as trouEle- some, that they had to use supplements to increase vaginal lubrication and in some cases even take hormones (Castro-Vázquez 2006, 123). Loe (2004a) and Potts and colleagues (2003) also provide examples of women’s responses to and concerns about Viagra use in the United States, demonstrating a wide diversity in opinions and practices. Additional studies on Swedish women’s accounts of Viagra would here be needed, but looking at the website it is clear that the female partner, since heterosexuality becomes further implied in the illustrative photos of both older and younger heterosexual couples that frame the text, is enrolled as responsible for the man’s health and for their relationship; she can help him help himself to become the 84 Ericka Johnson and Cecilia Åsberg Viagra-empowered, potent man. At the website, her task becomes one of ensuring that penetrative intercourse can occur, since sexual intercourse is what consolidates the relationship and makes it strong. 7hrough the figure of the sympathetic partner conjured up on the website, the responsibility not only for the general health and well-being of the man but also for the emotional health of the couple is presented as a feminized task. It is here, in the partner section, that emotions and feelings are mentioned on the website with the references to coldness, distance, worry, sensitivity, nearness, trust, guilt and encouragement. Physiologically, Viagra only works if a man is se[ually aroused in the first place, so partners are encouraged to help achieYe 9ia- gra’s success by ensuring the necessary feelings are in place. Thus responsibility for the emotional aspects of sex, not just the relationship, is also effectively given to the partner. Partners are reminded that ED is ‘the man’s symptom, the couple’s shared problem’, so the partner is directed to ‘speak to and encourage’ the man. 6he is the one who in practice can confirm his potency. +er assignment within the Viagra discourse is to manage this talk as she takes the emotional responsibility for discussing and reflecting oYer the role of se[ for their relationship. Enrolling partners +ere, too, emotions come into the discourse but so do the co-constitutive agencies of medi- cal expertise, female partners and Viagra as embodied, chemical effect as well as an expectation on virile manhood giving shape to the ideal Viagra man. Partners are encouraged to learn about ED because, ‘with knowledge in hand, you will find it easier to speak with your partner. 7ogether you can discuss your feelings and thoughts, and give each other support, and in the end, solve your relationship problems’. We suggest that this assignment of emotions and responsiEility for the relation- ship’s well-being to the female partner enables her to legitimately address the proElem of (' as a shared issue. Within the discourse on the 9iagra pages, the tool she is often given to solve the problem is the little blue pill, but because of the particularly Swedish, legally dictated, ‘informational’ role of the website, the partner is also provided with information about alternative treatments like sexual therapy (even if this information is sparse and even as the site is branded in a very Viagra blue). But as we will discuss later, enrolling the partner in the ED discourse this way could also open alternatiYe solutions and alternatiYe definitions of the problem. Discussion ,n our analysis, we haYe identified three enrolled participants the weEsite addresses to help create a suEMect position for the consuming 9iagra man. 7he first of these is the male patient, for whom anatomy and age become his destiny, but who can con- sume Viagra to control that destiny and discipline it in line with youthful expecta- tions. The second is the doctor, both enrolled to help ensure the male patient can access Viagra and used to represent scientism, which legitimates the use of Viagra Ey associating it with networks of scientific e[pertise. $nd thirdly, the partner of the patient is also enrolled in the process of creating a subject position for the 85 Responses to erectile dysfunction Viagra-consuming man. Responsibility for his emotions is given to his partner, who simultaneously consents to supporting a pharmaceutical solution for the man and the relationship. Viagra-consuming man. Responsibility for his emotions is given to his partner, who simultaneously consents to supporting a pharmaceutical solution for the man and the relationship. The enrolment of these three participants in the commercial discourse creates a network of actors who can perform the desire for, distribution of and context to contribute to successful use of a pharmaceutical as a solution to impotence. Their presence in the commercial Viagra discourse is particularly striking when one considers that they all but disappeared from the medical discourse around Viagra when it was introduced in 1998. As Chapter 4 discusses, the Swedish medical journal Läkartidningen supported a Yery heterogeneous definition of impotence and impotent patients in the early 1990s, one which recognized many different types of men with different reasons behind their impotence, and which encour- aged the involvement of partners during treatment, enrolling the partner in much the same way as the Viagra website does (Olsson et al. 1995, 313). This approach was not necessarily benign; the imagined partner was a woman in this (also) very heterosexual discourse, and she was ascribed a narrow position in the discourse: ‘Men and women have different ways of expressing themselves and therefore misunderstandings can easily arise. Women must learn to Ee clearer and men to be more receptive’ (Olsson et al. 1995, 313). Her ideal sexuality was also lim- ited: unthreatening, dependent on and receptive to her male partner’s desires. Discussion But we would like to suggest that the type of ‘enrolment’ that the men are displaying is also dif- ferent in another way. Their enrolment is an activity which creates a community of people, all of whom can help to define the medical proElem as medical or not and as a problem or not. And, importantly, it is also a community that seems to at least tacitly recognize that the solution, when there is one, is one that needs to be acted on and participated in by more than just the man. In particular, these interYiew studies would seem to highlight the partner¶s need to Ee actiYe in defin- ing the problem, and also the solution, as co-produced and as something that both parts of the couple are actively participating in. This is in contrast to perceiving ED as a disease of the penis and the penis alone, and for which responsibility to enact a solution (take a pill which will maintain an erection) is the man’s. As we have shown, in the Viagra discourse, the partner is enrolled to help the man see how important it is for him to take 9iagra. 7his demands a significant amount of work on her part, actively enabling the man to recognize the problem as ED and the solution as Viagra. An alternative would be for partners to be enrolled as par- ticipants who can also define alternatiYe se[ual practices and solutions. Thinking about the enrolment of not only new, male patients, but also their doc- tors and their partners, we were reminded of an early study of mental health by (aton and Weil , which found that relatiYely isolated, $naEaptist communi- ties’ responses to patients who developed mental illness were very different than the response to mental illness found in the wider American society at the time. Rather than isolating the individual, institutionalizing him or her, and stigmatiz- ing the patient, the Hutterite communities tried to help the individual continue to play a role in the community, contributing and working as best they could, and Eeing cared for Ey their family during the course of the illness (aton and Weil 1955, 212). Reading this study today, it is obvious that it was written before the pharmaceutical industry had colonized the discourse of mental illness, and illness in general. Discussion ‘The best help for a man with disappointing erections is, besides his own courage to speak about it, an understanding and sensual partner who is sexually keen but not demanding’ (Olsson et al. 1995, 314). However, with the 1998 advent of Viagra, the medical discourse in Läkartidningen narrowed the definition of an impotent patient to the male penis and removed the varied social and sexual backgrounds, and actors, which had previously been present. Yet, in the commercial discourse, the partner and factors like stress and tiredness are present alongside Viagra. Despite the stigma attached to impotence and the common assumption that men would not want to talk about ED (as the nudging encouragement provided by www.potenslinjen.se implies), qualitative research (primarily interview studies) on men who are dealing with erectile dysfunction shows that not all men deal with their ED problems alone and in silence. Many men are already enrolling medical professionals and partners in their quest for a solution (Oliffe 2005; 2006; Grace et al. 2006). These men turn to medical professionals to procure treatments for their impotence (Viagra and similar drugs, but also injections and vacuum pump treatments) and some men engage their partners both in treatment therapies and as discussion partners with whom they can talk aEout their difficulties and e[plore alternative sexual practices (Oliffe 2005). As much current research within mas- culinities studies supports, men’s experience of illness, especially a condition as related to masculinity as erectile dysfunction, is influenced Ey how the men and those around them, that is the network of actors enrolled in definition and solution work, think about and practise masculinity (cf. Marshall and Katz 2002; Aucoin and Wessersug 6andEerg . 7hese studies also suggest that some men who experience sexual dysfunction are already comfortable using a network of actors to help them Eoth define their proElem and seek treatment options. 86 Ericka Johnson and Cecilia Åsberg We ask, then, how this practice and these enrolled actors patients, doctors and partners) differ from the enrolment we have observed on the Swedish webpage. We ask, then, how this practice and these enrolled actors patients, doctors and partners) differ from the enrolment we have observed on the Swedish webpage. The obvious answer, of course, is that in the conversations detailed in Oliffe (2005) the partner pair can explore non-pharmaceutical solutions. Discussion Rather than talking about patient-centred, individualized cures to ill- ness, the study relied on concepts of social cohesion, social structures and group expectations as explanatory models and as treatment options. It pays special atten- tion to sociological variables, the cultural and social dimensions of health (Eaton and Weil , . We are not suggesting that a theory of social cohesion and mental health from 1955 may be a good way to reinterpret erectile dysfunction. But as a reminder that our research material, our oEserYations and our interpretations are influenced by the paradigm within which we are working, it is very useful. Going back to the material we have discussed in this chapter, and looking at the way patients, doctors, partners and pills are enrolled in the production of Viagra consumers, we see first that these actors are enrolled to produce pharmaceutical consumption as a treatment option. Secondly, the men and their partners are not discussed as explanatory factors. Although it may seem unnecessary to reintroduce the part- ner as the source of impotence (for a discussion of historical, cultural and social Responses to erectile dysfunction 87 explanations of impotence, see McLaren (2007)), this enrolment can explain what other critical research on Viagra has shown; that its existence and doctors’ partici- pation in its prescription practices have created ED. Social structures (the phar- maceuticalized framework) and group expectations (of lifelong sexual activity and successful ageing) have contributed to the ‘epidemic’ of erectile dysfunction. The illness, itself, is constructed by the enrolled actors. Only then can they be engaged as a network to help the man find a solution. 6tarting from this insight, we ask: how might these same human actors be enrolled into creating a different solution" ,f 9iagra was not aYailaEle, what solutions could this cast of characters work together to find" Whowhat else could possiEly Ee enrolled" $nd how would the concept of (' change" Like mental illness, impotence has traditionally been a situation that is not gen- erally flouted or discussed puElicly. 7herefore we find it interesting that the 9iagra solution suddenly enrols a wider group of actors to help the man find a solu- tion. Discussion The Viagra solution demands these other actors; the regulatory framework in Sweden means that doctors are a gatekeeper to the drug; and Viagra’s reliance on sexual desire means that the sexual partner can be important to initiate, develop or maintain arousal. What we are asking is if these three groups of actors men, their doctors and their partners) can be enrolled to address ED through Viagra, how could they Ee enrolled to address (' without 9iagra" +ow would a dis- tributed response to ED place responsibility for dealing with the problem at the relationships and community leYel rather than only Ey indiYidual" We suggest that these e[amples, Eoth 2liffe¶s TualitatiYe research on men with impotence (Oliffe 2005; 2006) and the pre-Viagra treatment advice for doctors, show that enrolling a wider community to respond to a health issue is possible. (nrolling a larger network of actors can inYolYe finding and supporting alterna- tive behaviours, alternative demands and alternative expectations, both by and of the ‘individual’ with a condition, such as erectile dysfunction, and by the people around him who are also affected by it. Notes 1 An earlier version of this chapter has been published as Johnson, E. and Åsberg, C. (2012). Enrolling the Swedish Viagra Man. Science and Technology Studies, 25 (2), 46–60. 2 Accessed in October 2007, February and April 2008, November 2009 and October 2015 7his is in contrast to another 3fi]ersponsored weEsite, www.Yiagra.se, which is framed as an informational site for medical professionals. The different readership is constructed to legally avoid DTC advertising of Viagra. g y g g We would like to point out that this performatiYity is more directly connected to spe- cific physical actions than the discursiYe performatiYity often found in gender studies, that is Butler’s (1990) work. We would like to point out that this performatiYity is more directly connected to spe- cific physical actions than the discursiYe performatiYity often found in gender studies, that is Butler’s (1990) work. ( ) 5 www.potenslinjen.se, accessed 4 November 2009. 6 www.viagra.com 7 The quotes cited later are (unless otherwise mentioned) taken from www.potenslinjen. se, accessed 5 November 2009. 8 This encouragement and advice in how to meet and speak with patients with ED is pre- sented on the website http://viagra.se, which is directed solely to health professionals (Accessed 28 October 2007). 7 Viagra selfhood Pharmaceutical advertising and the visual formations of Swedish masculinity1 Cecilia Åsberg and Ericka Johnson ,n this chapter, we will inYestigate the Yisual configuration of what we term a Swedish Viagra imaginary, a cultural phantasy landscape that produces and repro- duces certain subject positions of great interest for feminists and other scholars invested in social change. More precisely, we interrogate a set of key images pre- sented Ey the 3fi]ersponsored weEsite for potential 6wedish 9iagra customers with erectile dysfunction in order to explore how this particular Viagra imaginary proYides reference points for shared and collectiYe identities. We e[plore here the visual formation, and the naturalization, of the nationally shaped masculinity of the potential consumers of Viagra at a Swedish-language site, www.potenslinjen.se/,2 the same site discussed in Chapter 6. This site is produced by the pharmaceutical company 3fi]er for the e[plicit purpose of proYiding the 6wedish puElic with health information on erectile problems. Drawing on feminist science studies, cultural studies and medical technology studies, we argue that Viagra is a cultural phenomenon, or rather a material-semiotic node for discursiYe production and identity formation ± one inflected Ey indiYidual- ized health concerns as well as by the social authority of medicine and the compel- ling appeal of so-called lifestyle pharmaceuticals. Following masculinity theorist and feminist cultural scholar Graham Dawson, a cultural imaginary, as a kind of cultural phantasy landscape, is not monolithic and not hegemonic, Eut fluid and mul- tiple. It consists of ‘those vast networks of interlinking discursive themes, images, motifs, and narrative forms that are publicly available within a culture at any one time, and articulate its psychic and social dimensions’ (Dawson 1994, 48). Cultural communities not only mirror but also continually reinvent themselves through cul- tural imaginaries, as these are sites of identity negotiation and formation. The reality-producing and identity-constituting effect of such discursive activ- ities is why it is of the essence to study emerging subjectivities, such as those appearing publicly at Viagra websites. Moreover, in line with other critical stud- ies of pharmaceutical use and promotion,3 we argue that Viagra is a feminist issue. 7he tendency to diYide the psychic, the social and the technoscientific into separate domains of knowledge has been resisted in a rich body of work, including that of feminist cultural studies of techno-science, and is an interdisci- plinary mode in which we feel largely at home; see Åsberg and Johnson (2009) 89 Viagra selfhood and -ohnson . 7 Viagra selfhood Pharmaceutical advertising and the visual formations of Swedish masculinity1 We attempt to align and estaElish crossdisciplinary dia- logue between cultural theory and gender studies, media ethnography and stud- ies of science, medicine and technology in society. Our work implies that we see sexual difference within a cultural theory setting, understood as the effect of the human suEMect¶s entry into the symEolic systems of its culture. While subjectivity is neither universal nor ahistorical, it is the product of structuring social relations, shaped by materialities and physical embodiment as much as by social phantasy. Shared and collective, these phantasies organize a screen through which the material world appears and social relations are experienced. As cultural imaginaries, they furnish public forms, organize knowledge of the social world and give shape to phantasies that are constitutive of, in this case, masculine identities. Our method is materialist in that it tries to stay clear of both cultural and biological determinisms while approaching material-semiotic phe- nomena discursively (cf. Haraway 1989). The target of our study here is not the lived, sensuous body-self, but the hermeneutic, cultural embodiment of Viagra masculinity in discourse. Our analysis builds on discussions of the promotion of Viagra (a drug mar- keted Ey 3fi]er since for men and couples as a cure for erectile dysfunction and the construction of masculinities and sexuality as a lifelong duty and health concern of the individual also in other comparative national contexts, such as the United States, New Zealand and Japan (see Bordo 1998; Fishman and Mamo 2001; Marshall 2002; Potts 2004; Marshall 2006; Tiefer 2006; Johnson 2008). Like other critical readings of Viagra (Fishman and Mamo 2001; Marshall 2006; Tiefer 2006; Vares and Braun 2006), we are examining the construction of ideal users of Viagra and especially the nationalized, classed, racialized and sexual- ized identities they are assigned. This, however, does not imply that we naively aim to reveal any kind of Viagra ideology as a singular site of reality distortion produced by the discursive operations of Big Pharma (ideology critique). The political driving forces of social change are today much more complex and highly individualized, which makes us interested in the creation of cultural appeal rather than the creation of (hegemonic) consent. 7 Viagra selfhood Pharmaceutical advertising and the visual formations of Swedish masculinity1 In order to investigate in local detail, for instance, how retiring sexually became a sign of pathology through the new Via- gra discourse, we are aligning slightly different insights from feminist approaches to intersectionality4 (identity as a crossroad of intersecting social categories such as gender, age, ethnicity and embodiment) with the notions of the social imaginary and suEMect positioning the cultural encouragement for one to take up a specific sense of self) from visual cultural studies, and further, with previous work within medical sociology and feminist science studies on the ideological processes of medicalization and naturalization. Interrogating the many cultural dimensions of ‘the natural’ as a rhetorical device at work in the Viagra imaginary, we consult the work that a range of femi- nist scholars working on (or in) science, medicine and technology have produced since the late 1970s on the powerful, ideological processes of naturalization (cf. Merchant 1980; Haraway 1989; Butler 1990; Fox Keller 1992; McClintock 90 Cecilia Åsberg and Ericka Johnson 1995; Bryld and Lykke 2000; Franklin et al. 2000). It has, through such research, become clear that authoritative speech of the natural has maintained stereotypical definitions of se[ and gender throughout late modernity. In the case we present, Viagra seems – somewhat oxymoronically – to be natu- ralizing a hegemonic form of masculinity as closely associated with virility, and as determined by erectile capacity for penile penetration of the female body. On one hand, such masculine subjectivity is about successful ageing and the maintain- ing of an autonomous and androcentric, welldefined and impenetraEle selfhood, one disassociated from health problems and embodiment in general. On the other hand, Viagra discourse invites interrogations of volatile masculinity and male ageing, pharmaceutical incorporation and prosthetic virility, and of commerce in liaison with medical science. These paradoxical Viagra connections function in direct opposition to the teleological ethos of science imagined to rationally and purposefully promote social development. 7 Viagra selfhood Pharmaceutical advertising and the visual formations of Swedish masculinity1 This is evidenced by the narrative of how 9iagra, with the actiYe suEstance sildenafil citrate, came into commercial existence as the accidental result of heart medicine trials in which the male test subjects reported regained capacities for erectile functioning, and is an example of how we see Viagra performing ontologically as a curious material-semiotic node, an entangled case of meaning and materiality with some unexpected outcomes, especially regarding the choreography of selfhood, which we see as inflected Ey the formative powers of gender, nationality and sexuality. Delineating the glocal of the creation of new techno-social identities around drugs, we discuss what we see as the performance of ‘Swedish’ elements of the Viagra man constructed through the text and images on the site, as this collective identity is imagined as slightly different from that presented in the advertisements that have helped to market Viagra to the US and New Zealand customer base (see www.viagra.com and www.viagra.co.nz). US advertising has seen a conceptual shift from impotence to erectile dysfunction (ED), and later to erectile quality (4 ± as a much more inclusiYe category of erectile insecurity. 7hese shifts, closely associated to the use of Viagra to maintain masculinity against the threats of ageing and other stresses in life, have meant that the potential market for the drug has expanded from older men with serious medical problems (as represented by early Bob Dole endorsements) to ageing but active, sporty types (presented through endorsements by football stars like Pele and baseball stars in the United States), to ‘every man’ as in the ads with non-famous, regular Joes (called ‘Sven- ssons’ in Swedish) in their thirties. Much of the critical research about Viagra has so far come from the United States and New Zealand, two countries which allow DTC advertising of prescription medicine (see Mamo and Fishman 2001; Elliott 2003; Loe 2004a; Moynihan and Cassels 2005). DTC advertising of pharmaceu- ticals creates a media climate that allows drug companies like 3fi]er to deliYer information about their products to the general population through billboards, tel- evision, infomercials, magazines, newspapers, radio and so forth, pumping out a Viagra message and producing a Viagra-primed, ED-aware audience (Potts and Tiefer 2006, 268). The messages about Viagra are also mirrored in jokes, televi- sion programmes and moYies, documentaries and news reports, reflecting the fact 91 Viagra selfhood that the line between paid advertising and media coverage is often blurry. 7 Viagra selfhood Pharmaceutical advertising and the visual formations of Swedish masculinity1 This has bearing on our case study as well. Medical and popular imagery (with an almost global scope) take part in co-shaping a Viagra imaginary that provides a backdrop onto which virility can be measured and masculine subjectivities posi- tioned. As discussed in Chapter 6, because of the ban on DTC advertising of pre- scription medicine, the Swedish situation is slightly different (although this ban is not always easy to enforce. 3fi]er ran an ad campaign for 9iagra on Eusstop billboards in 2004, and satellite television broadcasts from abroad have created a loophole for television ads as well; see Zetterqvist et al. (2015).) As mentioned earlier, 9iagra has also figured largely in the collectiYe psyche in 6weden as a suE- ject both of newspaper articles and of debate, thanks in large part to the drawn-out discussions and court cases about whether the Swedish health care system would subsidize Viagra. g Concerned with the co-constitutive work of national and global imagina- tion $nderson , and with what has Eeen theori]ed as the unfinished and open-ended ‘new universalisms of global culture’ (Stacey 2000), we study how digital imagery employed within pharmaceutical marketing positions embodied subjects through appeals to both the national and the natural. Appeals to the national and the natural are highly potent cultural tropes through which identity formation can take place Williams )ranklin et al. 2000). National dif- ferences, understood in line with Benedict Anderson’s (1983) anthropological notion of nationhood as non-essential imagined communities, are here com- paratiYe indicators of the glocal dimensions of the 9iagra imaginary. While Anderson assumed that national media, like everyday print press or museums, maintained such a collective imaginary, we propose to investigate how such national imagery today also is co-constituted by commercial, sometimes con- flicting, gloEal flows of imagery in general, and Ey the pharmaceutical industry on the Internet in particular. It is in that vein we closely read the cultural pro- cesses that produce the suEMect position of a specific type of 6wedish man as a potential consumer of Viagra. This ideal consumer, as he is addressed in the web te[t and imagined in the selected photos, is a cultural figuration we refer to as the Swedish Viagra man. Naturalizing the Swedish Viagra man $n e[ample of how 3fi]er¶s 6wedish erectile dysfunction site for the general puE- lic takes an active part in the public negotiation of the meaning of masculinity as related to penetrative sex is a list published on the site of the top ten characteristics of ‘a manly man’. This list is based on a public poll and can be read against the backdrop of a rather large interest in issues of gender, in what constitutes ‘manli- ness’ and ‘womanliness’, in Swedish public debate. Gender is a positive notion in Sweden, yet the content of such national awareness, informed as it is by decades of gender equality policy work, sometimes amounts to no more than an uncritical celebration of bipolar difference (Elvin-Nowak and Thomsson 2001, 409). How- eYer, in the summer of , 3fi]er hired a nationally renowned polling company Cecilia Åsberg and Ericka Johnson 92 to create a top ten list of what Swedish people associated with manliness, and the result reads as follows:5 to create a top ten list of what Swedish people associated with manliness, and the result reads as follows:5 1 To be practical and handy (a handyman of sorts) 1 To be practical and handy (a handyman of sorts) 2 To have good potency (presumed sexual) 3 To have a well-paying job 4 To have an attractive partner 7o haYe a physically fit, muscular Eody 6 To have an attractive outer appearance 7 To be interested in sports 8 To be always ready for sex 9 To own technical gadgets 10 To have a cool car 2 To have good potency (presumed sexual) 9 To own technical gadgets The desire to perform an all-around able-bodied, and even enhanced (with cars, gadgets or other statusraising attriEutes form of masculinity is clearly a defin- ing feature of the potential Viagra consumer. As an underlying and unarticulated assumption, such a notion is, in a commercial, yet serious health informational setting like this website, spoken of in terms of ‘good potency’ and visualized through imagery of active men appearing in non-erotic, everyday life settings as to not risk ‘indecent’, or even homoerotic, associations. This might be regarded as somewhat surprisingly prudish in a national setting famous for its high level of sexual education. Naturalizing the Swedish Viagra man Viagra has, however, been promoted in accordance with a class-related sense of ‘respectability’, and such a legitimating mode of address is achieved at this site through the staging of health information and references to medical professionals. Next to the list, the visitor is given the opportunity to vote online for which of the top four points she finds most manly. 7he results from that direct online poll name, unsurprisingly, potency as the number one characteristic and handi- ness as number two. This poll also indicates how Viagra, while in a limited and framed manner on this site, actually is very effective in inviting the addressed web visitor to contemplate the meaning of masculinity as strongly linked to, or even framed by, erectile capacity. Possible anxieties around potency are also in such a benevolent manner (as an open poll) simultaneously alleviated (in how it becomes a collective issue) and enforced (by the underlining of potency as the most important manly feature). The solution to any private feelings of inadequacy is placed in the hands of the individual, as he (or a female partner) on the website is encouraged to ask a medical professional for a Viagra prescription. This echoes the recurring signature of US television commercials for pharmaceutical products where the viewer, at the end of the ad, is always prompted to ask his or her physi- cian aEout the new drug. We read this as part of a larger transnational discourse of ‘biological citizenship’ (Rose and Novas 2005), where the multinational company is positioned as a benevolent assistant to the good and healthy sex life that, in the website rhetoric, becomes the duty of the individual consumer. 93 Viagra selfhood This able-bodied and individualized ideal masculinity, as it emerges in the list, is to a large part construed in antagonist relation to common associations to lived femininity (soft embodiment, a low-paid job, dependency, not always ready for sex and less interested in male sports, cars and technical gadgets). In turn, this points to a phantasy masculinity inflected Ey class, se[ual orientation and age, as some of the gendered ideals (owning a cool car) seem formatted already within teenage boyhood, although usually only attainable in adult life. Of note is that this list is framed and presented at the blue-tinted Viagra website along with a picture of a man in a Viagra-blue overall changing a tyre. Naturalizing the Swedish Viagra man By analogue, Viagra seems an easy solution, as a technical Tuick fi[, to any kind of common feelings of insufficient manhood related to Eeing aEleEodied oYerall. 7he image of the man changing the tyre manages to displace a commonplace cultural fetish of abil- ity where almost everything mundane and less than erotic (like changing tyres) that defines such masculinity seems hinged on erectile capacities. While clean in design, the website imagery is perhaps not completely devoid of any hints or visual reference to intercourse as a forceful male accomplishment, as the man is prosthetically enhanced by his phallic jack and tyre iron, screwing wheel lugs on an equally Viagra-blue car. Many of the other images on the site, however, portray romantic couples. The Swedish Viagra man seems to predominately appear, whether alone and able-bodied or in a relationship, through the discourses of heteronormativity (Butler 1990). Nonetheless, there is an ambiguity to the imagery that opens up for slightly differ- ent interpretations. We found one photo on the 6wedish weEsite where a couple¶s sunlit feet are flirtatiously entangled on a Eed with white Eed sheets, where the gender of the couple is hard to decipher. It could be the feet of two men, or even two women, just as well as the feet of a heterosexual couple. As an example of erectile desires not Eeing confined to heterose[ual men, this picture could in fact lend itself to the possibility of portraying a male homosexual couple. Aligning the erectile function of the drug with sensuous imagery of couples or active men sustains, however, a reductive idea where penetration becomes the sole means through which relationships can Ee confirmed and a se[ual act manifested. $lso, it becomes clear that Viagra is not intended as a drug for the single man wishing to enjoy masturbatory sex on his own. The masculine identity on the Swedish website is marked with nationalized signifiers of socioeconomic class in seYeral ways. )or e[ample, on one of the information pages about facts and myths of impotence, there is the myth that ‘wet dreams give early morning erections’. Nocturnal erections are then explained by the assertion that µWhen a man is sleeping his penis e[ercises to keep in shape. Naturalizing the Swedish Viagra man A healthy man will have an erection approximately every 70th to 100th min- ute when he is sleeping, regardless of whether he is thinking about sex or tax deductiEles.¶ 7his same message appears in the short film on the weEsite¶s first page. The reference to tax deductibles, in a country of progressive income taxes and, until recently, high capital gains taxes, could be interpreted as an appeal to the interests of the middle and upper-middle classes, or, at least, to the desire to Cecilia Åsberg and Ericka Johnson 94 Ee associated with financially saYYy methods of aYoiding ta[es. 7hus, it Eecomes part of respectable upper-middle-class life to consume Viagra. Perhaps most clearly indicative of Swedishness and its association with healthy, physical activity in the outdoors is the dominant imagery of people (men and cou- ples) in wilderness settings. As a collective phantasy of national belonging and camaraderie, the overall use of this imagery can be seen in many commercials for a wide range of products in 6weden. We feel that one image on the site in particular poignantly plays on a charged version of Swedish nature. This image has a blurred, male body jumping into the water in what appears to be a summer day in the Swedish archipelago. It draws on a widely viewed beer commercial from the 1990s. This commercial, with a distinguishable aesthetic reproduced in several versions over the past decades, has come to represent the much-sought- after, idealized Swedish summer. It is a commercial for Pripp’s Blue Beer which shows mixed-sex groups of white Swedes in their late twenties and early thirties gathering on the cliffs of the Swedish coast on what is obviously a crisp, yet warm and salty afternoon during the summer holidays. This group of Swedes, with their well-built, muscular bodies and attractive and slender partners, all wearing swim- wear and seemingly without make-up, brings with them a large case of Pripp’s Blue Beer and spends the duration of the commercial barbequing, sailing or jumping in and out of the water while laughing to the background sounds of a then-popular Swedish pop-song, ‘Blue, blue winds and water’.6 After more than a decade, this advertisement was still running on Swedish television, and the imagery from the commercial has fastened in the cultural imaginary of what constitutes Swedish- ness; namely, naturalness and freedom merged with an aestheticized form of sum- mer night melancholia. Naturalizing the Swedish Viagra man Serendipitously for Viagra, ‘Blue’ is the iconic brand name of a particular Pripp’s beer and, for millions of Swedes tuned into popular culture, it represents blue water, blue skies and a laid-back, yet youthful attitude of sexual yearning (blue as in nostalgic, yet not unhappy, feelings) – all staged within a coastal setting very much like that in an image on the Viagra website. The www.potenslinjen.se site taps into this Swedish imagery of the outdoor life, where several other pictures feature men and couples by the seaside. A rather simple yet highly effective aesthetic works thus to naturalize Swedish masculinity and connect it to 9iagra. We would like to emphasi]e the central role of scenic landscapes and visualized Swedish nature as the photographic backdrop in this process of naturalizing the Swedish Viagra man. At the same time, class plays in the background. Access to the seaside, to a private sailboat that enables access to unpopulated islands, cliffs and bathing areas during the short summer, is a privi- lege that is somewhat restricted to members of the capital-owning classes. Such an upper- and middle-class summer lifestyle is thus the iconic ideal and unique selling point for both this rather widely accessible beer and the slightly less acces- sible pharmaceutical. It is a national phantasy of Swedish nature and the yearning nature of 6wedes as they long for that µElue¶ summer feeling. We contend that the Viagra illustrations on www.potenslinjen.se effectively make use of such a blue and youthfully sexual, class-coded and nature-loving, national imaginary of white Swedish masculinity. 95 Viagra selfhood This nationalized construction of a middle-class Viagra man in Sweden should be seen in light of the prolonged court deliberations over subsidizing Viagra for the general puElic, which was finally decided in 0arch . $s 9iagra is not subsidized in Sweden (see Chapter 2), individuals have to cover the full cost of prescriptions. In a country in which all people are covered by state health care which substantially subsidizes most pharmaceutical products, the idea of paying for a drug, particularly a lifestyle drug, is relatively foreign. Many people would probably think twice before purchasing Viagra with their own money when they are accustomed to receiving medication much cheaper. Naturalizing the Swedish Viagra man Thus, the fact that Viagra is not subsidized means that the market for the drug is probably smaller than it would otherwise be, at least for Viagra received through a doctor’s prescription, which is the method of procurement www.potenslinMen.se promotes. 7he film on the first weEpage claims that , 6wedes purchase 9iagra illegally oYer the ,nternet and warns strongly against this. 3fi]er has proEaEly therefore consciously chosen to market the drug to those social groups with more expendable income. The image construction of Viagra on the site is also involved in positioning men as sufferers of legitimate potency problems related to underlying health concerns, thus avoiding connotations to Viagra as a recreational sex drug for party-happy youngsters. The target group is instead respectable yet mundane, middle-aged, Swedish, middle-class men, perhaps residing in the northern countryside. The images on www.potenslinjen.se all depict a very traditional and a very white Scandinavian man. This despite the fact that Sweden is, today, a multi-ethnic country with a significant urEan population. y gi p p Among the photos illustrating the claims about potency, health and Viagra, there are, besides the medical expert (a white, slightly balding, middle-aged yet vital-looking man in glasses and blue scrubs), relatively few portraits of men. One exception to this is an image which shows the face of the naturalized, Swedish Viagra man in a winter coat in front of an unpopulated and cold, coastal land- scape. Most photographed people are otherwise depicted with their backs to the camera from afar so their faces cannot be discerned, or metonymically repre- sented through parts of their (tanned, yet white-skinned) bodies, like the entan- gled feet belonging to the couple in bed. Conversely, the somewhat older man in a winter coat looks confidently Eack at the audience with cheeks Elushed Ey the cold. The untamed shoreline in a frosty landscape associates him (and Viagra) to the wilderness rather than a cosmopolitan life. It links directly to notions of nature and the natural. Ideas of the natural are often working through a cultural imperative of preser- vation, of safeguarding the natural as if God-given, and it is probably the most powerful trope through which subject positioning and identity formation works Williams )ranklin et al. 2000). Naturalizing the Swedish Viagra man The drug Viagra can be conceptualized as natural, as it is presented in 3fi]er marketing material, since it enaEles men an assumed ‘normal’ control over their bodily functions. It makes naturalized masculinity possible. Taking the pill merely enables ‘nature to take its course’ 0amo and )ishman , . 6o at the 3fi]er weEsite, the task of the Eiomedical drug industry seems, somewhat oxymoronically, to be one of ‘seconding nature’ Cecilia Åsberg and Ericka Johnson 96 (Franklin et al. 2000, 21f). In other words, it is one of preserving naturalized mas- culinity through the prosthetic virility Viagra can provide. The imagery online conceptualizes both Swedish nature and Viagra as a drug seconding such nature, as a quality of both the inner, personal and outer, physical landscapes of the people depicted. From landscapes by the sea in the southern coastal areas or winters up north, images of Swedish nature sustain notions of Viagra as a normal and natural ingredient through the idea of ‘letting nature take its course’. The Swedish man becomes one with the enhanced natural surround- ings, as Viagra becomes one with the ‘natural’ Swedish man. It is with surpris- ing ease that the marketing of a pharmacological product can associate ageing Northern manhood to harsh and untamed landscapes, as it seems sustained by gendered national demographics where women move south to work in the urban areas and men stay in the north for the fishing and hunting. 6uch a phantasy of seconded nature remains by implication unsubdued by civilization (gender equal- ity discourse or multiculturalism) as it takes on the oppositional characteristic of feminized and multicultural cosmopolitanism. Interestingly, Swedish masculin- ity works here not in opposition to nature and wildlife, but through Viagra, as realigned with natural virility. It seems almost as if the defeminization of the north requires the enforcement of heterosexual masculinity. Viagra virility, also as the commodification of men¶s se[ual health, presents itself as a striking and counterintuitive account of white masculinity coupled with a national phantasy of uncompromised, and uncompromising, nature. Concluding remarks Likewise, a television ad in New Zealand has featured a couple riding a motor- bike on a sheep farm with the woman driving (Vares and Braun 2006, 318). How this advertising has been localized for the Swedish context says much about how and to whom Viagra is marketing itself in Sweden, and about what stereotypical images of masculinity and gender relations it feels are legitimate to base such a campaign on. Perhaps our results are surprising for a nation that prides itself on gender equality. These images can also be read as suggestions of what concepts of masculinity are so stable and unassailable that they can withstand association with a drug that is, in essence, an acknowledgement of ‘failed’ masculinity and ‘dysfunctional’ sexuality. Or, as Vares and Braun call it, ‘a pill not only to repair, but also to enhance or improve, both erectile functioning and masculinity’ (Vares and Braun 2006, 325). As such, the online imagery of the Swedish Viagra man provides a telling account of naturalized Swedish masculinity as intersected by sexual orientation, ethnicity, age and class. And, like the work of Mamo and Fish- man, our analysis has shown that Viagra is a gendered drug, ‘transmitting cultural scripts which serve as enforcers of normatively gendered expressions of sex and sexuality’ (Mamo and Fishman 2001, 20). According to the international Viagra script, male sexuality is always active and desirous, the on-demand erected penis is central in its penetrative function (Potts and Tiefer 2006, 268–9), and accord- ing to the Swedish site, the Viagra man can easily be transposed into a different national context, sustaining its hegemonic, naturalized and nationalized image of the common (or, rather common-ized) ‘Svensson’ who in effect is not so anony- mous or unmarked but visibly heterosexual and white, while haunted by blue yearnings of se[ual unfulfilment. +e is associated with a wide, middleclass socioeconomic group, one which has the capital to access the seaside and take holidays in the Swedish archipelago, perhaps using the peaceful nights there to dream about tax deductions.i ,n spite of the mythical figure created online of the ordinary 6wedish 9iagra man, users of Viagra come in many shapes, sizes, ages, classes and genders. In addition, internationally, there are less than positive stories about using Viagra (Grace et al. Concluding remarks In this investigation of the Viagra imaginary and the techno-social identities it enacts, we dealt predominately with identity-forming intersections such as a nationalized (Swedish), middle-class sexuality and naturalized, white masculin- ity. This is a masculinity which nonetheless is conspicuously malleable, vulner- able and a target for a virility-enhancing drug like Viagra. Importantly, we dealt with a visual and spectacular kind of embodied subjectivation to both public and medical scrutiny, a subjectivity creating processes otherwise historically pre- serYed for the female Eody -ordanoYa 0c&lintock . ,n fact, 3fi]er has positioned the drug as important to ‘every’ man through discourses that pre- sent masculinity as the opposite of sexual impotence. Masculinity is equated with µsuccessful¶ se[, a Yery specific, narrowly defined act of penetration in usually heterosexual) intercourse. Being able to perform with an erect penis in this spe- cific way is eTuated with manhood. %y aligning masculinity with physiological sexual performance and promising reliable performance with the assistance of a pill, this discourse asserts that a man can maintain his masculinity regardless of age. ,nflected Ey a strong sense of constant Yigilance or disciplining of the male body, Viagra discourse demands individual responsibility so to manage the risk of losing such a virile masculinity even before one enters ‘old age’ (Marshall 2006, 355). As the consumption of Viagra promises a youthful sexuality long into ‘old age’, it also becomes another building block in the construction of an ideology 355). As the consumption of Viagra promises a youthful sexuality long into ‘old age’, it also becomes another building block in the construction of an ideology 97 Viagra selfhood about ‘successful ageing’ as the responsibility of the individual as s/he is framed within a liberal discourse of individualized self-care and biological citizenship (cf. Stacey 1997, 2000; Rose and Novas 2005; Åsberg and Johnson 2009). ( y g ) Our observations about the glocal images of the Swedish Viagra man can be read in line with preYious research, which has already shown that 3fi]er adMusts its marketing strategy to appeal to local consumers. For example, whereas in the US advertisements for Viagra have used baseball stars to promote Viagra, in New =ealand, 3fi]er enrolled sports heroes from rugEy 9ares and %raun , . Concluding remarks , reflected in the fact that at least half of 9iagra customers do not refill their prescriptions ± which is a statistic also found in 6wedish studies of Viagra users (Ströberg et al. 2006). Further, an interview study by Potts and col- leagues (2004) suggests that the importance of bodily experience of both erection and impotence is far more complex. Interviewees in that study challenged medi- cal definitions of the meaning of se[ual function as well as the straightforward link between Viagra and a satisfying sexual life. Instead, reports were given of Cecilia Åsberg and Ericka Johnson 98 how erectile difficulties led to more gratifying intimate relations Ey e[panding the sexual repertoire. Some even reported how the use of Viagra, contrary to being a Tuick fi[ for relationships, affected se[ual relationships for the worse. At the same time, alternative uses of Viagra, not least of all by women, have come to light, suggesting that as Viagra is integrated into our understandings of se[ual practice, it Eecomes a fle[iEle medical technology that means differ- ent things to different people. Viagra discourse is symptomatic of sociocultural imaginaries that are glocally situated, as these involve branding techniques and marketing strategies that breach distinctions between public and private (even intimate, health and commerce. While widely promoted as a pharmaceutical suc- cess and a scientific Ereakthrough, 9iagra is also a symEolic materiali]ation of the commodification of se[uality and gender, health and old age. ,t is in this sense that the Viagra imaginary is important to study (and by implication then take part in and intervene into) as it provides an indicator of embodied subjectivities as they take shape. 1 An earlier version of this chapter has been published as Åsberg, C. and Johnson, E. (2009). Viagra Selfhood: Pharmaceutical Advertising and the Visual Formation of Swedish Masculinity. Health Care Analysis, 17 (2), 144–59. y y ( ) 2 Accessed in October 2007, February and April 2008, November 2009 and October 2015 3 See the special issue of Sexualities. Sexualities, 9 (3), 2006. 8 Conclusions Glocal pharma and the Swedish Viagra man Ericka Johnson In this book we have been looking at how pharmaceuticals are localized in a specific conte[t in 6weden, with its welldeYeloped, 1orthernWestern medical system and the welfare policies that provide this medical system at very little point-of-contact cost to the majority of people living there.il 6pecifically, we haYe analy]ed the presence and influence of 9iagra through the theoretical lens of pharmaceuticali]ation, as Williams and colleagues a described. Following their lead, we look at pharmaceuticals even ‘outside the medical domain and explore the broader way in which pharmaceutical futures are shaping how we think about innovation, policy and the very meaning of health and illness, therapy and enhancement¶ Williams et al. 2011b; 730 emphasis in the original. We haYe paid particular attention to three aspects the way pharmaceu- ticals change forms of governance and are changed themselves by local policies; the redefinition of health proElems as issues with a pharmaceutical solution and the creation of new techno-social identities around drugs and the way pharma- ceuticals become essential actors in relationships between subjects. Doing so, we see quite clearly that Viagra and its perceived threat of eventually emptying the state coffers by over-demand has been part of a discussion that changed the way pharmaceuticals are subsidized and governed (and govern) in Sweden. Viagra has redefined impotence in the 6wedish conte[t as erectile dysfunction, and Eeen presented as the solution aYailaEle through medical treatment. While this is not a process that has only happened in Sweden, the medical discourse around it has incorporated aspects of pharmaceutical treatment that are particularly relevant in a context which provides subsidized medicine. Finally, as the last two chapters showed, Viagra has created a ‘Swedish Viagra man’ drawing on unique – if ste- reotypical – Swedish masculinities. Viagra has also been placed as an essential element in this man’s relationships with sexual partners and doctors. Our work in this book has focused on discourses, but the carrier or medium of those discourses is sometimes text, visual images and the materialities of the pills themselves. Our approach to discourse, and the subjects one can read from it, is framed by science and technology studies (STS) understandings of relational agency and non-human actors. Notes y y , ( ), 2 Accessed in October 2007, February and April 2008, November 2009 and October 2015. 3 S h i l i f S l S l 9 (3) 2006 4 Intersectionality accounts for how identities are never just sexed and gendered, but also conditioned by nationhood, ethnicity and racialization, class and so forth. An ana- lytical insistence on intersectionality emerged from within the social sciences and anti- racist feminist theory (Haraway 1989; McClintock 1995; Davis 2008). In that vein, we argue that the educational advertising of Viagra in Swedish in fact does more than merely provide information. It generates cultural negotiations over gender and sexual- ity as these are intersected by issues of, for instance, age and ability, nationhood and ethnicity. 5 All translations are the authors’. 6 Lyrics and melody by Thomas Ledin. 8 Conclusions Glocal pharma and the Swedish Viagra man These understandings allow us to articulate the role of Viagra as an actor in processes of pharmaceuticalization, but we are also keen to show – as the previous chapters have done – that other (human) actors Ericka Johnson 100 and (policy and commercial) interests and structures are implicated in pharma- ceuticalization, too. Articulating pharmaceuticals in the local context with these theoretical concerns helps us show specific aspects of gloEal pharmaceuticals as they are refracted in 6weden. +ere, in the final chapter, we would like to present the reader with an analysis of glocal pharma through the trope of the Swedish Viagra man, to give form to these glocal manifestations of pharmaceuticalization. The Swedish Viagra man We haYe used 9iagra as a prism through which to oEserYe the glocal aspects of pharmaceuticalization. Cultural and social studies of Viagra in other contexts haYe shown how it influences more than Must Elood flow in the penis. 9iagra has introduced the term erectile dysfunction (ED) to the general public, changing how impotence is perceived and treated (Chapter 4; Bordo 1998; Marshall 2002; Loe 2004a; Tiefer 2006). It has reinforced a coital imperative – the idea that all sex and intimacy must involve penetrative intercourse – stressing quick, hard (youthful) erections and constant male desire (Tiefer 2000, 2006; Fishman and Mamo 2001; Mamo and Fishman 2001; Marshall 2002, 2006; Potts 2004; Loe 2004b), and it has connected successful ageing with successful sex and successfully taking one’s medications (Marshall and Katz 2002; Moynihan and Cassels 2005; Marshall 2006). Yet much of the critical work about Viagra comes from the North American context, where DTC advertising has been widespread and overpowering (Elliot 2003; Moynihan and Cassels 2005). In our work, we have asked how Viagra has influenced ideas aEout disease, se[ and pharmaceutical use in this small, periph- eral country, Sweden, with its laws against DTC advertising of prescription drugs, with state-funded, universal health care and a history of, or at least a reputation for, sexual freedom. Taking a look at ‘downstream’ effects of pharmaceutical sci- ence, we show that the specific structural characteristics of the health care system and the cultural landscape influence how 9iagra acts and is receiYed in 6weden. We use the figuratiYe 6wedish 9iagra man in our analysis to represent a suEMect position which is facilitated, described and prescribed by the multitude of local and global responses to Viagra as it is called into being in the Swedish context and discourse. 7his figure sits at the Muncture of the 6wedish state, 6wedish cultural identity (as it is imagined, not necessarily as it is), the internationally acclaimed pharmaceutical product and the consumer(s) of it (see Johnson et al. 2011). State subsidies and ED Who should pay for the 9iagra man¶s 9iagra" 7his Tuestion has generated heated debate, both in the United States (should insurance companies pay for Viagra but not Eirth control pills" and in 6weden what if a huge puElic demand drains the state coffers". +istorically, goYernment regulation has often Eeen seen as part of the process to bolster medical expertise (Starr 1982; Petryna and Kleinman 2006). As medical practices were regulated by the state, they were simultaneously granted legitimacy and positioned against traditional medicine and unscientific 101 Conclusions cures. Likewise, the development of regulated medical schools and the licensing practices they facilitate creates legitimate experts out of medical doctors. ‘The increased standardization of the therapeutic process was believed to promote sci- entific progress in medicine while protecting the puElic against inflated claims about the effects and uses of substances claimed as remedies to restore health. One unintended effect of such regulation has been that it works as a barrier to mar- ket entry for prescription drugs ± thus ensuring profits for those who are allowed to enter’ (Petryna and Kleinman 2006, 10). In the discussion about Viagra’s subsidy debate in Sweden (Chapter 2), however, we detailed an example of this happening, but in a nuanced way that responded to local concerns and structures. In 2003, the newly formed Swedish 3harmaceutical %enefits %oard decided that 9iagra would not Ee suEsidi]ed. 'oc- tors could prescribe Viagra, but the Swedish Viagra man would have to pay for it himself. The decision was controversial because people in Sweden had, until then, been accustomed to receiving prescription medication free, past a basic co-pay leYel, and 3fi]er promptly appealed the decision. 'uring the course of the ne[t few years, the question of Viagra subsidies made its way through a series of court cases and appeals until, in 2008, the Supreme Court upheld the initial decision. Today, Viagra is still not subsidized in Sweden.i 7he 3harmaceutical %enefits %oard was conYinced suEsidi]ing 9iagra would be a legitimate use of tax money for severe ED but not for mild ED. However, it was also convinced that patients would claim to suffer from the severe form of the disease to beguile doctors into prescribing subsidized Viagra, which would both give subsidies to men who really did not deserve them, and lead to diagnostic bracket creep, that is an expansion of the diagnostic categories to match medica- tion. Ericka Johnson Chapters 2 and 3 of this book show that the regulatory changes we noted were a response to pharmaceuticals, especially Viagra. Analyzing the court documents that followed Viagra through the long and arduous series of court cases surround- ing the decision finally upheld to not suEsidi]e it in 6weden, we saw that the regulatory body tasked with deciding about pharmaceutical subsidies in Sweden was producing Yery local, nation state±specific responses to the integration of international pharma within an allegedly culturally neutral, oEMectiYe, scientific, medical knowledge paradigm. This regulatory board was, of course, working within an international (European) regulatory framework, but its decisions were influenced Ey cultural aspects of the 6wedish medical system. $ new drug ± 9ia- gra – and a new regulatory body led to new regulatory tools. Because of local peculiarities like the uneven distribution of urologists throughout the country and the political ideology of the health care system’s framework with the principle of equal access to health care guaranteed by law, the drug precipitated a debate and decision about subsidy based on structural and ideological aspects of the Swedish health care system. The glocal of Viagra provision became inseparable from the local context of the regulatory body. Chapter 3 then discussed how this Swedish government agency, the Pharma- ceutical %enefits %oard, has tried to include or e[clude certain other prescrip- tion drugs from the national pharmaceutical Eenefits scheme. %y looking at cases which involve ambiguous knowledge, the chapter showed how regulatory bodies appreciate stable objects and stable categories, both of patients and diseases, and of pharmaceutical treatments. When technology destaEili]es these things, as new pharmaceuticals do, and as pharmacogenomics technologies threaten to do even more, the regulatory Eodies haYe to find ways of reacting to unstaEle categories. How this is done can vary from nation state to nation state, even within a collec- tive framework like the EU, which is striving towards regulatory harmonization. ,n 6weden, we haYe seen a tendency to disregard specific knowledge and inclu- sion criteria and instead use broadly inclusive or exclusive categories for treatable patients and reasonable drugs. State subsidies and ED 7hus, rationali]ed Eoth the %enefits %oard and the court, it would Ee Eetter not to suEsidi]e the drug at all. 7he decision reflected concerns aEout patient and doctor compliance with government policies. The clincher in their argument was that two other drugs against severe ED, one injected by syringe into the penis and one inserted as a stick into the urethra, were already subsidized in Sweden. $ccording to the %enefits %oard and the court, these two drugs are so unpleasant to administer, compared to taking a pill, that men with mild ED would not reason- ably be expected to use them, de facto limiting their subsidized use to ‘legitimate’ patients with severe ED. In the discourse the state (represented by the regulatory agency) presented in court documents, medical doctors were presented as invalid experts, as easily beguiled individuals who could not diagnose a patient correctly. And the result- ing regulation removed the responsibility to diagnose severe erectile dysfunc- tion from the medical expert, placing it instead with the pharmaceutical and its delivery method, a clear example of what Biehl terms pharmaceutical forms of governance %iehl , . $s predicted, this regulation had significant effects on which pharmaceuticals were sold and subsidized, working as a market entry barrier (although probably not a particularly effective one; see Chapters 4 and . 7he unsuEsidi]ed 6wedish 9iagra man was influenced Ey the particular local structural framework of the Swedish welfare state. 102 Ericka Johnson The impotent Swedish man and his dysfunctional penis In our analysis of impotence and ED in the Swedish medical literature, using the weekly trade journal Läkartidningen, we noted a distinct change before and after Viagra’s 1998 introduction. Prior to Viagra, impotent men were written about as a heterogeneous group: some had partners, others were older, single men; some were shy, young men with problems relating to women, others were men ‘with a secret’, although what that secret could be was never clearly articulated. The treatment options for these various patients differed, but the doctor was always supposed to Ee a trusting confidant who saw the man on seYeral occasions, lis- tened to his feelings and discussed his concerns. Furthermore, the patient’s partner was encouraged to be involved in these discussions because she (the partner was always imagined to be a woman in this literature) could play an important role in the man’s treatment. After the introduction of Viagra to Sweden, the partner almost 103 Conclusions disappeared from the medical discourse. So did the term impotence. Instead, erec- tile dysfunction was discussed, an affliction of the man¶s penis, a disease of Elood flow and tissues rather than an illness related to relationships, feelings, e[pecta- tions and disappointments (which has also been seen in other national contexts; see Tiefer 2006). Since 1998, Viagra has dominated the Swedish medical dis- course so completely that its availability determined the concept of the patient (reduced to a penis) and the disease (a biomechanical shortcoming) in the medical discourse. Not until 2006, when statistics showed more than half of the men pre- scriEed 9iagra in 6weden chose not to refill their prescriptions, was this discourse undermined. Reporting on interviews with men who stopped taking Viagra, an article in Läkartidningen suggested that Viagra failed because of social, cultural and relationship issues, unwittingly bringing the discussion back to the relation- ship and lifestyle causes of impotence that had been prominent in the early 1990s. But this article is the exception to the rule. Viagra today still dominates the way impotence(' is defined and treated in the 6wedish Läkartidningen: impotence has become erectile dysfunction and is a condition to be treated pharmaceutically. Addressing this aspect of pharmaceuticalization in Chapters 4 and 5, we demon- strate the way health problems which already had a medical solution, that is were already medicalized, became issues with a pharmaceutical solution at a glocal level in the 6wedish medical discourse. The impotent Swedish man and his dysfunctional penis We analy]ed how 9iagra and alphaElockers for BPH (Viagra is often prescribed together with alpha-blockers because of side effects of the alpha-blockers) were discussed in the Swedish medical journal Läkartidningen. We also paid close attention to who was allowed to giYe Yoice to concerns about the use of Viagra and alpha-blockers. Distinct global trends were visible in the Swedish material, like in the construction of impotence and erectile dysfunction as a condition related solely to Elood flow after the introduction of Viagra. Discussions shifted from social and relationship causes of impotence to mechanical and Eiomedical e[planations in 6weden as in other Western1orthern countries. 6pecifically 6wedish aspects of the 9iagra and alphaElocker medi- cal discussion were also prominent, many related to the solidarity principle in Swedish health care. These aspects included debates about the (un)availability of urologists in different parts of the country, the right a patient has to the best care regardless of his geographical location, the extent to which erections were an aspect of health that should be provided by state-funded medicine (and who was the patient in such cases, the man or the partner), and the connection between erectile dysfunction and other serious medical conditions which had already been deemed Mustified to treat from the puElic purse. Viagra has (largely) replaced sexual and relationship therapy for the Swed- ish Viagra man, and it far outsells other, earlier, more mechanical solutions like pumps, implants and insertable sticks. The alpha-blockers discussed in Chapter 5, on the other hand, have not had this same effect on treatment options. To some extent, alpha-blockers have become a pharmaceutical solution to a medical prob- lem – enlarged prostates – that previously relied on surgery to correct, but not nearly as completely as Viagra took over erectile dysfunction. In Sweden today, surgeons still perform large numbers of surgeries on benign prostate hyperplasia. 104 Ericka Johnson So while alpha-blockers are being prescribed to shrink the prostate more than they were twenty years ago, they have not completely pharmaceuticalized this already medicalized problem in the same way Viagra did. Analyzing the discourse in Läkartidningen and comparing it to that of Viagra, one thing that strikes us is that the authorship of articles about impotence/erectile dysfunction shifted dra- matically with the introduction of Viagra, from being dominated by sex therapists to being dominated by urologists. The impotent Swedish man and his dysfunctional penis But within the discourse on enlarged prostate treatments, urologists were always the main authors of articles in Läkartidningen. The introduction of alpha-blockers to the enlarged prostate treatment regime did not imply a change in which medical specialty claimed the disease. Perhaps this can in part explain why the original treatment, surgery, is still performed. Rather than shifting care site, the pharmaceuticals merely added another weapon to the arsenal at the urology clinic. The relative success of Viagra compared to alpha- blockers may have more to do with the relative strengths and weaknesses of urol- ogy surgeons and sexologists as professional groups than the drugs themselves. Viagra has wrestled customers away from the sexologists, but alpha-blockers are haYing a more difficult time taking prostate operation patients away from the urol- ogy surgeons. Commercial images of the Swedish Viagra man The discursive contours of the Swedish Viagra man’s subject position become very clear in the commercial marketing material for Viagra in Sweden. Pharmaceutical marketing has received a good deal of academic attention, in both its pure form as advertisements (see Moynihan and Cassels 2005) and its more subtle forms, like clinical trials, ghostwritten scientific articles, medical actiYism Ey supported patient groups and disease awareness campaigns (Healy 2006, 62). The marketing of pharmaceuticals is international in its scope, and our Swedish material featured many characteristics which were similar to, for example, marketing produced for the 8nited 6tates. +oweYer, there were also distinctions. While these similari- ties and differences are interesting in detail (and are discussed in Chapters 6 and , what we find more releYant to an analysis of pharmaceuticali]ation is how the marketing discourses created both diseases and subjects which and who are then both actively produced and produce action through their relationship to the drugs. As Healy succinctly notes, ‘companies now sell diseases rather than just drugs’ (Healy 2006, 82). Our material supports this and shows that the concept of µconsumer¶ must also Ee Yiewed as fle[iEle. 3harmaceuticals enrol and enaEle relationships to sell their diseases and drugs. Such advertisements target not only the person injecting, ingesting or applying the product, but also the person’s fam- ily, doctor, school, job or prison facility suggesting, encouraging, prescribing or mandating consumption. g p Most DTC advertising of prescription medicine is forbidden in Sweden, so instead of using television commercials, pharmaceutical companies provide infor- mation about drugs and medical conditions in pamphlets distributed by doctors and nurses, through supporting patient advocacy groups and on informational 105 Conclusions websites – a useful way to circumvent DTC rules, according to industry insiders, and seen in other contexts, too (Applbaum 2006, 103–4). This sort of advertising is a clear example of the selling of sickness which can occur; the construction of an illness with a pharmaceutical solution that Moynihan and Mintzes (2010) and Williams and colleagues a discuss. 3harmaceuticals are often presented to the patient together with online quizzes that can be used for self- diagnosis or for diagnosis in the clinical setting. Commercial images of the Swedish Viagra man These quizzes are frequently translated and appear on websites in many different languages and for many dif- ferent diseases (erectile dysfunction, depression, benign prostate hyperplasia, female sexual dysfunction, to name a few).i When we analy]ed the images of men that populate the 3fi]erfunded, 6wedish language webpage about ED, we saw examples of both global harmonization and local adaptation. Much of the information about ED is similar to that found in US commercial material, but the Swedish Viagra man as a collective trope is a slightly different man than the one(s) found in the United States. He is, for starters, very white – which mirrors a traditional image of the Swedish man even though 6weden is, at this point, a country which has a significant non&aucasian popula- tion – and the Swedish Viagra man is slightly older than the middle-aged men and sports stars found as Viagra representatives in the United States. The Swedish Viagra man is also very connected to nature; he is presented in wilderness scenes, toughing the winter cold or jumping into pristine water from rugged, stony out- croppings. The Swedish Viagra man is comfortable in the uncivilized wilderness, which by association naturalizes both his condition and its cure, Viagra.l y g We also notice that the 6wedish 9iagra man is not alone in his affliction he is accompanied by his partner. Images of smiling women next to their men, couples walking along the seaside and two sets of feet sticking out from under a blanket pepper the weEsites and informational literature. While the partner all Eut disap- peared from the medical discourse when Viagra appeared in Sweden, she (there is little to suggest homosexual relationships in the material, even if the language is gender neutral) is actively enrolled and present in the commercial material. We suspect this is because Viagra needs sexual stimulation to function properly. For some men and in some cases, Viagra will ensure the maintenance of an erection, but initial sexual stimulation has to come from somewhere or someone else, and the partner is a conYenient ally for 3fi]er. Commercial images of the Swedish Viagra man The Swedish Viagra man does not act in solitude, and in Chapter 6 we have shown how Viagra is used to create three subject positions in the commercial/ informational discourse: a shy and reluctant Swedish Viagra man; his helpful doctor who represents science and knowledge and who is concerned about his patient’s impotence in part because of what it says about his patient’s heart and general health status; and a supportive partner who can facilitate and produce the nearness and intimacy that a loving, sexual relationship needs. Chapter 7 paid closer attention to the representations of masculine traits that are giYen to the 6wedish 9iagra man within the pages of the 3fi]ersponsored weEsite providing health information on erectile dysfunction to potential Swedish Viagra customers. We haYe articulated the images of a potential 6wedish 9iagra man in Ericka Johnson 106 relation to se[ual health and cultural cues of masculinity. We argue that the mas- culine traits highlighted in the webpages are indicative of Swedish cultural values of male virility that are strong enough to withstand discursive coupling with a pharmaceutical used to address ‘failed’ virility. In both of these chapters, the global and the local are relevant as the websites and informational pamphlets we have analyzed are local adaptations of interna- tional marketing approaches (including the self-help quiz and the database of local, Viagra-friendly doctors) and are produced and distributed within a regula- tory framework that (more or less successfully) forbids DTC advertising of pre- scription pharmaceuticals. They therefore provide a glocalized cultural imaginary of Viagra, the subject positions it engenders and the particular relationships it facilitates. Glocal pharma 3harmaceuticals and the commercial forces Eehind them are incrediEly fle[iEle and determined in their drive to conquer new markets and ensure a global reach (Petryna and Kleinman 2006, 7). However, we also see in our material that the local can push Eack. &ulturalspecific discourses can Ee and are incorporated into ideas about the consumer subject, and perhaps this is testimony to their obduracy. Medical professionals and opinion makers address the integration of pharmaceuti- cal solutions into clinical practice and treatment, and while many of them may be enthusiastic supporters and/or be receiving pharmaceutical industry support, not all are. Their participation in this discourse is also testimony to a belief that their locally generated opinions are useful, valid and objective. Likewise, the industry supports their role as independent opinion makers with valuable expert advice to be considered by policymakers and other political and administrative bodies. Both industry’s use of local doctors and the doctors’ willingness to participate attend to a belief that the local is relevant. And that regulatory bodies in a small nation state like Sweden still feel that they have a duty to the citizen (the citizen patient and the citizen taxpayer), which should be considered before the industry’s demands, and a court system which facilitates this, attests to the relational agency that develops as global drugs meet local structures. As we mentioned in the introduction, in drawing conclusions about the glocal of pharmaceuticalization, it is useful to ask how a drug’s presence has altered the concept of a disease and its treatment, who suffers from it and how to cure it, in the local context and internationally. Drugs can both create new and refract with existing stereotypical images of a patient, images which carry markers of class, race and sexuality. And they lead to new laws and policies to regulate the practices of both doctors and patients. But of course it is not only the drug that prescribes behaviours or identities, it is decision makers, commercial actors and medical experts who attach the drug to specific demands, images and e[pectations to influence the EehaYiours of groups they are trying to govern, cajole or cure. These actors are located in different coun- tries, have different cultural starting points and work within different institutional 107 Conclusions frameworks, so how they construct and use a pharmaceutical varies. Glocal pharma Thus, the series of 6wedish court cases and suEsidy deEates aEout 9iagra reflect the specif- ics of Swedish law, its national health care programme and its basis in the con- cept of solidarity. 7his Eecomes a framework which creates a specific, local legal conte[t within which 9iagra must Ee goYerned, and which also influences the discursiYe contours of 9iagra in 6weden. While 9iagra¶s influence on the medical discourse in Sweden was similar to the international one, it also contained a great deal of debate about the connection of ED to other established medical conditions like diaEetes and 06, reflecting the suEsidy controYersy and concern that 9iagra would be unavailable to ‘legitimate’ patients. Likewise, the commercial construc- tion of 9iagra on 3fi]er¶s 6wedish weEpage in many ways parallels that on the US pages, with a self-help diagnostic quiz, a database of Viagra-friendly doctors and information for partners. <et there are also specific elements manipulated to reflect and resonate with 6wedish sensiEilities, like the imagined race of the user, the connection to Swedish forests and coastlines and the Swedish survey of mas- culine personality traits on the 3fi]ersponsored 6wedish potency weEsite. 7hese details show a global pharmaceutical being localized. When an analysis of pharmaceuticali]ation teases out the actors Eehind a drug, one can see that a drug is much more than the pill that is claimed to cure a disease or alleYiate a symptom. ,t can Eecome a discursiYe conflation of Yalues, actors, structures, biomedical understandings, social identities and personal desires. The mere e[istence of a pharmaceutical product can influence the medical discourse, reinforce and even construct cultural ideas and identities, change the practices of experts and lay people and reimagine ways relationships are performed between patients and doctors and Eetween patients and their loYed ones see Whyte et al. 2006). Pharmaceuticals are global in their reach and regulated by international institutions, Eut our work here articulates drugs as fle[iEle artefacts as they encounter local social and institutional frameworks. While there are Yery 6wedish aspects of Viagra in Sweden, the drug has carried with it previously established ideas about disease, medical treatments for ageing and appropriate intimacy prac- tices. 7hese are strong, disciplining discourses that influence eYen as they Eecome embedded in the cultures that encounter them. Glocal pharma The globalization of the pharma- ceutical market not only makes medicines available to international consumers, it also spreads ideas about the healthy subjectivities those medicines are prescrib- ing. This, we feel, calls for further consideration, to articulate the prescribed sub- jectivities that prescription medicines carry when they are sold on a globalized pharmaceutical market. We want to consider the images of healthy identities, relationships and practices they claim to facilitate. We also call for careful atten- tion to the local particularities they challenge and are challenged by: the medical structures that provide access to them; the ideological basis of local health care provision; and the regulatory frameworks that govern them. These are the local aspects we have examined which, when combined with a global pharmaceutical, relationally construct glocal pharma. A question this awakes is if, and how, these local productions of pharmaceutical artefacts and subject positions can be seen to inform backwards, from the peripheral local to the industry’s centre. References Läkartidningnen, 97 (8), 87 %eerman, %. . /ls rekommendationerna Läkartidningen, 100 (21), 1918. Berg, M. (1997). Rationalizing Medical Work: Decision Support Techniques and Medical Practices. Cambridge: MIT Press. g Bergstörm, L. (1998). Potensmedel tar USA med storm. Läkartidningen, 95 (25), 2927–9. Berner, B., and Johnson, E. (2010). Technologies and Medical Practices: Blood, Guts and Machines. Surrey: Ashgate. Biehl, J. (2006). Pharmaceutical Governance. In A. Lakoff, A. Petryna and A. Kleinman, Global Pharmaceuticals: Ethics, Markets, Practices (pp. 206–39). Durham, NC: Duke University Press. %iMker, W. (. . Of Bicycles, Bakelites and Bulbs: Towards a Theory of Sociotechnical Change. Cambridge: MIT Press. Bodlund, O. (2008). Sildenadil hjälper även kvinnor med sexuella biverkningar av SSRI. Läkartidningen, 105 (43), 3005. Bordo, S. (1998). Pills and Power Tools. Men and Masculinities, 1 (1), 87–90. Braidotti, R. (2006). Transpositions: On Nomadic Ethics. Cambridge: Cambridge Polity Press.l %ranke, .. . $llehanda om 3arMehanda. (n personlig refleksMon lYer 86$¶s nya potensdebatt. Läkartidningen, 95 (25), 3016. Brody, H. (2007). Hooked: Ethics, the Medical Professional and the Pharmaceutical Industry. /anham, 0' 5owman /ittlefield. Brunsson, N., and Jacobsson, B. (2000). A World of Standards. Oxford: Oxford University Press. Bryld, M., and Lykke, N. (2000). Cosmodolphins: Feminist Cultural Studies of Technol- ogy, Animals and the Sacred. London: Zed Books. Butler, J. (1990). Gender Trouble: Feminism and the Subversion of Identity. New York: Routledge. %ystr\|m, -. a. ,mpotensmedicin pn µn\|Meskontot¶" Läkartidningen, 97 (3), 214. Byström, J. (2000b). Visst rekommenderar Läkarmedelsverket. Läkartidningen, 97 (8), 870. Callon, M. (1987). Society in the Making: The Study of Technology as a Tool for Socio- logical $nalysis. ,n W. (. %iMker, 7. 3. +ughes and 7. -. 3inch, The Construction of Technological Systems (pp. 83–103). Cambridge: MIT Press. Callon, M., and Law, J. (1995). Agency and the Hybrid Collectif. South Atlantic Quartely, 94, 481–507. &arlsson, 3., and 6pnngEerg, $. a. 'yra, nya llkemedel Eatter ln kirugi" Läkartid- ningen, 93 (49), 4549–55. Carlsson, P., and Spångberg, A. (1996b). Har Patienten Rätt att Välja. Läkartidingen, 93 (49), 4556–8. Castor-Vázquez, G. (2006). The Politics of Viagra: Gender, Dysfunction and Reproduction in Japan. Body and Society, 2 (2), 109–29. &asula, c. 9., and 6M\|gren, (. . 0ore .nowledge, %etter oYernment" &onse- Tuences of .nowledgeEased 'ecision 0aking in 6wedish 3harmaceutical %enefits. Governance, 24 (1), 85–110. Ciechanowski, S. (1903). References Aarden, E. (2009). Politics of Provision: The Co-production of Genetic Technologies and Health Care Arrangements in Germany,the Netherlands and the United Kingdom. Maas- tricht: Maastricht University. Abraham, J. (2010). Pharmaceuticalization of Society in Context: Theoretical, Empirical and Health Dimensions. Sociology 44 (4), 602–22. Abraham, J. (2011). Evolving Sociological Analyses of Pharmaceuticalization: A Response to William, 0artin and aEe. Sociology of Health and Illness, 33 (5), 726–8. Abraham, J., and Ballinger, R. (2012). The Neoliberal Regulatory State, Industry Interests and the ,deological 3enetration of 6cientific .nowledge 'econstructing the 5edefini- tion of Carningens in Pharmaceuticals. Science and Technology & Human Values, 37 (5), 442–77. Abraham, J., and Lewis, G. (2000). Regulating Medicines in Europe: Competition, Exper- tise and Public Health. London, New York: Routledge. g Aldstedt, E. (1998). Viagra sälgs olagligt via postorder. Läkartidningen, 95 (51–2), 5859 American Psychiatric Association (1980) Diagnostic and Statistical Manual of Mental Disorders rd edn. Washington, '& $merican 3sychiatric $ssociation. American Urological Association (AUA). (2010). American Urological Association Guideline: Management of Benign Prostate Hyperplasia(BPH). Linthicum, MD: AUA Education and Research. Anderson, B. (1983). Imagined Communities: Reflections on the Origin and Spread of Nationalism. London and New York: Verso. Applbaum, K. (2006). Educating for Global Mental Health: The Adoption of SSRIs in Japan. In A. Lakoff, A. Petryna and A. Kleinman, Global Pharmaceutical: Ethics, Mar- kets, Practices (pp. 85–110). Durham, NC: Duke University Press. Application for Subsidy: Levitra, 8369–04 (Kammerräten I Stockholm December 2004). Åsberg, C. (2005). Genetiska Föreställingar: Mellan genus och gener i populär/vetenska- pens visuella kulturer. Linkjöping: Linkjöping University Press. Åsberg, C. (2005). Genetiska Föreställingar: Mellan genus och gener i populär/vetenska pens visuella kulturer. Linkjöping: Linkjöping University Press. Åsberg, C., and Johnson, E. (2009). Viagra Selfhood: Pharmaceutical Advertising and the Visual Formation of Swedish Masculinity. Health Care Analysis, 17 (2), 144–59. $ucoin, 0., and Wassersug, 5. . 7he 6e[uality and 6ocial 3erformance of Androgen-deprived (Castrated) Men Throughout History: Implications for Modern Day Cancer Patients. Social Science and Medicine, 63, 3162–73. Baglia, J. (2005). The Viagra Adventure: Masculinity, Media and the Performance of Sex- ual Health. New York: Peter Lang. Balint, M. (1957). The Doctor, His Patient and the Illness. London: Pitman. Balint, M. (1957). The Doctor, His Patient and the Illness. London: Pitman. 109 References Becker, H., Geer, B. and Hughes, E. (1961). Boys in White: Student Culture in Medical School. Chicago: Chicago University Press. Beerman, B. (2000). Rabattering är en politisk fråga. References Prostatic Hypertrophy: Anatomical Researches on the So-called ‘Prostatic Hypertrophy’ and Allied Processes in the Bladder and Kidneys. Translated by E. R. Pelton. Krakow: University of Krakow. 110 References Christensen, T., and Lægreid, P. (2002). New Public Management: The Transformation of Ideas and Practice. Aldershot: Ashgate. Claridge, J. A., and Fabian, T. C. (2005). History and Development of Evidence-based Medicine. World Journal of Surgery, 29 (5), 547–53. Clarke, A., Mamo, L., Fishman, J. and Shim, J. (2010). Biomedicalization, Technoscience, Health and Illness in the U.S. Durham, NC and London: Duke University Press. Conrad, P. (2007). The Medicalization of Society. Baltimore, MD: Johns Hopkins Univer- sity Press. Coser, R. L. (1963). Alienation and the Social Structure: Case Analysis of a Hospital. In Eliot Freidson, The Hospital in Modern Society (pp. 231–265). London: The Free Press. Cuyler, A., and Newhouse, J. (2000). Handbook of Health Economics. Amsterdam: Elsevier. Dahlstrand, C. (2003) Ett bra och säkert terapival – för både patienten och sjukvården. Läkartidningen, 100 (35), 2678. Davis, K. (2008). Intersectionality as Buzzword. A Sociology of Science Perspective on What 0akes a )eminist 7heory 6uccessful. Feminist Theory 9 (1), 67–85. Dawson, G. (1994). Soldier Heroes: British Adventure, Empire and the Imagining of Mas- culinities. London and New York: Routledge. Degerblad, M., Elmér, C., Falconer, C., Jonasson, A. F., Lauritzen, M. and Lökk, J. (2014). Nokturi-en underdiagnostiserad och ofta obehandlad folksjukdom. Läkartidningen, 1111: CUWD. Dervieux, T., and Bala, M. V. (2006). Overview of the Pharmacoeconomics of Pharmaco- genetics. Pharmacogenomics, 7 (8), 1175–84. 'rori, ., 0eyer, -. W., 5amire], 2. ). and 6chofer, (. . Science in the Modern World Polity: Institutionalization and Globalization. Stanford, CA: Stanford University Press. Dugdale, A. (2000). Intrauterine Contraceptive Devices, Situated Knowledges and the 0aking of Women¶s %odies. Australian Feminist Studies, 15 (32), 165–76.i Dumit, J. (2012). Drugs for Life: How Pharmaceutical Companies Define Our Health. Durham, NC: Duke University Publishing. (aton, -., and Weil, 5. . Culture and Mental Disorders. Glencoe, IL: The Free Press. Dumit, J. (2012). Drugs for Life: How Pharmaceutical Companies Define Our Health Durham, NC: Duke University Publishing. Durham, NC: Duke University Publishing. (aton, -., and Weil, 5. . Culture and Mental Disorders. Glencoe, IL: The Free Press. y g (aton, -., and Weil, 5. . Culture and Mental Disorders. Glencoe, IL: The Free Press. Egalite, O. N., and Godard, B. (2007). References Pharmacogenomics Research Involving Racial &lassification 4ualitatiYe 5esearch )indings on 5esearchers¶ 9iews, 3erceptions and Attitudes Towards Socioethical Responsibilities. Pharmacogenomics, 8 (9), 1115–26. p g ( ) Ekman, P. (1999). Många behandlingsalternatiiv vid prostathyperplasi. Läkartidningen, 96 (34), 3504–5. Elam, M. (2012). Pharmaceutical Incursion on Cigarette Smoking at the Birth of the Brain Disease Model of Addiction. In J. Netherland, Critical Perspectives on Addiction: Advances in Medical Sociology (Vol. 14, pp. 53–75). London: Emerald Press. Elliott, C. (2003). Better Than Well. American Medicine Meets the American Dream. New <ork W.W. 1orton and &ompany. Elvin-Nowak, Y. and Thomsson, H. (2001). Motherhood Is Idea and Practice. A Discursive Understanding of Employed Mothers in Sweden. Gender & Society, 15 (3), 407–28. European Association of Urology (EAU). (2006). Guidelines on Benign Prostate Hyper- plasia. Arnhem, NL: European Association of Urology. )all, 0. . 8rininkontinens hos mannenett f\|rsummat proElem" Läkartidningen, 96 (18), 2227–31.i )ishman, -. . 0anufacturing 'esire 7he &ommodification of )emale 6e[ual 'ys- function. Social Studies of Science, 34 (2), 187–218. )ishman, -., and 0amo, /. . What¶s in a 'isorder $ &ultural $nalysis of 0edical and Pharmaceutical Constructions of Male and Female Sexual Dysfunction. Women & Therapy, 24 (1/2), 179–93. 111 References 1 References Flowers, C. R., and Veenstra, D. (2004). The Role of Cost Effectiveness Analysis in the Era of Pharmagenomics. PharmacoEconomics, 22 (8), 481–93. Foucault, M. (1987) [1976]. The History of Sexuality. An introduction. Translated by Rob- ert Hurley. New York: Penguin Books. Foucault, M. (2007). Security, Territory, Population: Lectures at the College de France, 1977–1978. Basingstoke: Palgrave Macmillan. Foucault, M. (2008). The Birth of Biopolitics: Lectures at the College de France, 1978– 1979. Basingstoke: Palgrave Macmillan. Fox, R. (1959). Experiment Perilous: Physicians and Patients Facing the Unknown. Glen- coe, IL: The Free Press. Fox, R. (1977). The Medicalization and Demedicalization of American Society. Daedalus, 106 (1), 9–22. Fox Keller, E. (1992). Secrets of Life, Secrets of Death: Essays on Language, Gender and Science. New York: Routledge. Franklin, S., Lury, C. and Stacey, J. (2000). Global Nature, Global Culture. London, Thou- sand Oaks, and New Delhi: Sage Press. g Freidson, E. (1963). The Hospital in the Modern Society. London: The Free Press. Friberg, T. (2006). Diagnosing Burn Out: An Anthropological Study of a Social Concept in Sweden. Lund: Lund University. Fuberg, C. (2003a). Minska förskrivningen av dyra läkemedel utan mervärde. Läkartidnin- gen, 100 (49), 4113. Fuberg, C. (2003b). References 9em 6kall %ehandla (rektil 'ysfunktion" Läka- retidningen 94 (28–9), 2548–52. Hedelin, H., Grenabo, L. and Holmäng, S. (2001). Både stora genombrott och stagnation inom urologin. Läkartidningen, 98 (20), 2440–4. Hedelin, H., and Jacobsson, L. (2000). Viagra förstahandsmedel mot erektil dysfunktion. Läkartidningen, 97 (21), 2616–17. Hedelin, H., Johansson, N. and Ströberg, P. (2003). Ojämn kvalitet på remisser för blåstom- ningensproblem hos män. Läkartidningen, 100 (16), 1435.i +edgecoe, $. . 5einYenting 'iaEetes &lassification, 'iYision and the eneti]ation of Disease. New Genetics and Society, 21 (1), 7–27. Hedgecoe, A. (2004). The Politics of Personalized Medicine-pharmacogenetics in the Clinic. Cambridge: Cambridge University Press. Hedgecoe, A. (2008). From Resistance to Usefulness: Sociology and the Clinical Use of Genetic Tests. BioSocieties, 3 (2), 183–94. +edgecoe, $., and 0artin, 3. . 7he 'rugs 'on¶t Work ([pectations and the 6haping of Pharmacogenetics. Social Studies of Science, 33 (3), 327–64. Heyns, C. F., and de Klerk, D. P. (1989). Pharmaceutical Management of Benign Prostatic Hyperplasia. In D. Paulson, Prostatic Disorders. London: Lea & Febiger. Hirschauer, S. (1998). Performing Sexes and Genders in Medical Practices. In A. Mol and M. Berg, Differences in Medicine: Unraveling Practices, Techniques and Bodies (pp. 13–27). Durham, NC: Duke University Press. Hopkins, M. M., Ibarreta, D., Gaisser, S., Enzing, C., Ryan, J., Martin, P., Lewis, G., Det- mar, S., Elske van den Akker-van Marle, M., Hedgecoe, A., Nightingale, P., Dreiling, 0., +artig, .., 9ullings, W. and )orde, 7. . 3utting 3harmacogenetics into 3rac- tice. Nature and Biotechnology, 24 (4), 403–10. Hopkins, M. M., Martin, P. A., Nightingale, P., Kraft, A. and Mahdi, S. (2007). The Myth of the Biotech Revolution: An Assesment of Technological, Clinical and Organizational Change. Research Policy, 36 (4), 566–89. Hult, E. (2006). Vetenskap i paketerad form: Framväxten av evidensbaserad medicine. In &. Waks, and &. /eYay, Sträven efter transparens: Gransking, styring och organisering i sjukvårdens nätverk (pp. 77–100). Stockholm: SNS Förlag. Illich, I. (1976). Limits to Medicine/Medical Nemesis: The Expropriation of Health. Lon- don: Marion Boyars.i -lrhult, %. . 3fi]er uppmanas sYara pn kritik och delta i det offentliga samtalet. Läkartidningen, 100.i -lrhult, %., and /indahl, 6.2. . µ%lodtrycksmaffians¶ agerande har kostat skatteEeta- larna milijarder. Läkartidningen, 100 (22), 1917–18. Jasanoff, S. (2004). States of Knowledge: The Co-production of Science and Social Order. London: Routledge. Jasanoff, S. (2005). Designs on Nature: Science and Democracy in Europe and the United States. Princeton, NJ: Princeton University. Johansson Krafve, L. References Alfadil-positiv dokumentantation för säkerhet saknas. Läkartidningen, 51–2, 4329. Ginsburg, G., Konstance, R., Allsbrook J. and Schulman, K. (2005). Implications of Pharmacogenomics for Drug Development and Clinical Practice. Archives of Internal Medicine, 165 (20), 2331–6. Grace, V., Potts, A. Gavey, N. and Vares, T. (2006). The Discursive Condition of Viagra. Sexualities, 9 (3), 295–314. Gray, A., and Harrison, S. (2004). Governing Medicine: Theory and Practice. London: Open University Press. Gunnarsdotter, S. (2005). Erektil dysfunktion kan tyda på hjärt-kärlsjukdom. Läkartidnin- gen, 102 (36), 2458. Gurwitz, D. Z., Hopkins, M. M., Gaisser, S. and Ibarreta, D. (2009). Pharmacogenetics in Europe: Barriers and Opportunities. Public Health Genomics, 12 (3), 134–41. Hacking, I. (1999). The Social Construction of What? Boston, MA: Harvard University Press. Hallin, A. (1999). Produktutvekling efterlyses för termoterapi vid prostataförstoring. Läk- d 96 (34) 3 20 2 Hacking, I. (1999). The Social Construction of What? Boston, MA: Harvard University Press. Hallin, A. (1999). Produktutvekling efterlyses för termoterapi vid prostataförstoring. Läk- ertidningen, 96 (34), 3520–2. H A (2006) N fö idi lö i Läk d 103 (40) 2943 Hallin, A. (1999). Produktutvekling efterlyses för termoterapi vid prostataförstoring. Läk- ertidningen, 96 (34), 3520–2. Hansen, A. (2006). Nytt preparat mott för tidig utlösning. Läkartidningen, 103 (40), 2943. Hansen, A. (2006). Nytt preparat mott för tidig utlösning. Läkartidningen, 103 (40), 2943. Haraway, D. (1989). Primate Visions: Gender, Race and Nature in the World of Modern Science. New York: Routledge Haraway, D. (1989). Primate Visions: Gender, Race and Nature in the World of Modern Science. New York: Routledge Hart, N., Grand, N. and Riley, K. (2006). Making the Grade: The Gender Gap, ADHD, and the Medicalization of Boyhood. In D. Rosenfeld and C. A. Faircloth, Medicalized Masculinities (pp. 132–64). Philadelphia, PA: Temple University Press. Hassler, E. (2002). Att behandla prostatiker i primärvård inte enkelt-trots frågeformulär. Läkartidningen, 99 (19), 2174. Healy, D. (2004). Let Them Eat Prozac. The Unhealthy Relationship between the Pharma- ceutical Industry and Depression. New York: New York University Press. Healy, D. (2006). The New Medical Oikumene. In A. Lakoff, A. Petryna and A. Kleinman, Global Pharmaceuticals: Ethics, Markets, Practices (pp. 61–84). Durham, NC: Duke University Press. 112 References Healy, K. (2000). Embedded Altruism: Blood Collection Regimes and the European Union’s Donor Population. American Journal of Sociology, 105 (6), 1633–57. Hedelin, H. (1998). Nytt medel mot impotens. Läkartidningen, 95 (42), 4558 +edelin, +. and $Eramsson, /. . References (2015). Valuation in Welfare Markets: The Rulebooks, Whiteboards and Swivel Chairs of Care Choice Reforms. Linköping: Linköping University. Johnson, E. (2008). Chemistries of Love: Impotence, Erectile Dysfunction and Viagra in the Läkartidningen. Läkartidningen, NORMA 3 (1), 31–47. 113 References 113 References Johnson, E., and Åsberg, C. (2012). Enrolling the Swedish Viagra Man. Science and Tech- nology Studies, 25 (2), 46–60. Johnson, E., and Sjögren, E. (2009). Conniving Patients, Beguiled Doctors and Dis- ciplining Drugs: Justifying the Denial of Reimbursment for Viagra Use in Sweden. Paper Presented at the 6th Nordic Medical Anthropology Conference. Gothenburg, Sweden. Johnson, E., Sjögren, E. and Åsberg, C. (2011). Prescribing the Swedish Viagra Man. Clin- ical Pharmacology and Therapeutics, A Nature Publication. January. Jordanova, L. (1999). Nature Displayed: Gender, Science and Medicine 1760-1820. Lon- don: Longman. Jost, T. (2005). Health Care Coverage Determinations: An International Comparative Study. Maidenhead: Open University Press. Junker, S.-O. (2014). Att Skapa gemenskap: hur beslut fattas i en EU myndighet. PhD Dis- sertation. Stockholm: Stockholm School of Economics. Källström, R. (2010). Construction, Validation and Application. Linkjöping: Linkjöping University Press. Kammarrätten in Stockholm (Stockholm Administrative Court of Appeals). (2005). Appli- cation for Subsidy: Viagra. Case no. 5014-04. Issued 15 April 2005. Kassirer, J. (2005). On the Take: How Medicine’s Complicity with Big Business Can Endanger Your Health. Oxford: Oxford University Press. g y Kimland, E., and Ståhle, L. (2004). Läkemedelsfrågan. Läkartidningen, 101 (35), 2666–7. Kimland, E., and Ståhle, L. (2004). Läkemedelsfrågan. Läkartidningen, 101 (35), 2666–7. Ki b R Fit t i k J Ki b M d Fit t i k A (1995) D l t A fö t t Kimland, E., and Ståhle, L. (2004). Läkemedelsfrågan. Läkartidningen, 101 (35), 2666–7. Kirby, R., Fitzpatrick, J., Kirby, M. and Fitzpatrick, A. (1995). Delat Ansvar för prostata kd S kh l M k Sh Kirby, R., Fitzpatrick, J., Kirby, M. and Fitzpatrick, A. (1995). Delat Ansvar för prostata sjukdomar. Stockholm: Merck Sharp. Kirchheiner, J., Fuhr, U. and Brockmoller, J. (2005). Pharmacogenetics-based Therapeutic 5ecommendations ± 5eady for &linical 3ractice" Nature Reviews Drug Discovery, 4 (8), 639–47. Kruse, C. (2016). The Social Life of Forensic Evidence. Oakland: University of California Press. Lagrelius, A. M., and Sjögren, E. (2004). Reglers mångfold: Läkemedelsreglering i Sver- ige. In G. Ahrne and B. Nils, Regelexplosionen (pp. 93–125). Stockholm: EFI. Lakoff, A. (2005). Pharmaceutical Reason: Knowledge and Value in Global Psychiatry. Cambridge: Cambridge University Press. Lakoff, A. (2006). References High Contracts: Gifts and Surveillance in Argentina. In A. Lakoff, A. Petryna and A. Kleinman, Global Pharmaceuticals: Ethics, Markets, Practices (pp. 111–35). Durham, NC: Duke University Press. /andtElom, $.0. . 6t\|d permanent raEatt aY 9iagra och &ialis Yid 06 Läkartid- ningen, 101 (35), 2666–7. Landtblom, A.-M. and Ertzgaard. P. (2000). Tillstånded – inte läkemedlet – ska avgöra frågan om subventionering. Läkartidningen, 97 (21), 2612–14. Länsrätten in Stockholm (Stockholm County Administrative Court). (2004a) Application for Subsidy: Viagra. Case no. 8032-03. Issued 30 June 2004. Länsrätten in Stockholm (Stockholm County Administrative Court). (2004b) Application for Subsidy: Cialis. Case no. 11092-03. Issued 30 June 2004. Latour, B. (1993). The Pasteurization of France. Translated by Alan Sheridan. Cambridge: Harvard University Press. Latour, B. (1998). On Recalling ANT. Published by the Department of Sociology, Lancaster University, at www.comp.lancs.ac.uk/sociology/papers/Latour-Recalling-ANT.pdf (accessed 4 April 2008). 114 References Law, J. (2006). Big Pharma: How the World’s Biggest Drug Companies Control Illness. London: Constable and Robinson. Law, J., and Singleton, V. (2005). Object Lessons. Organization, 12 (3), 331–5. /esko, /. -., and Woodcock, -. . 7ranslation of 3harmacogenomics and 3harmacoge- netics: A Regulatory Perspective. Nature Reviews Drug Discovery, 3 (9), 763–9. /indEom, $. . 'ismantling the 6ocial 'emocratic Welfare 0odel" +as the 6ocial Wel- fare 6tate /ost ,ts 'efining &haracteristics" Scandinavian Political Studies, 24 (3), 171–93. /indpn, /. . µWhat do (Ya and $nna haYe to 'o with &erYical &ancer"¶ &onstruct- ing Adolescent Girl Subjectivities in Swedish Gardasil Advertisements. Girlhood Stud- ies, 6 (2), 83–100. Lindgren, G. (1999). Klass, kön och kirurgi: relationer bland vårdpersonal i organisa- tionsförändringarnas spår. Malmö: Liber. Ling, T., and Raven, A. (2006). Pharmacogenetics and Uncertainty: Implications for Policy Makers. Studies in History and Philosophy of Science, 37 (3), 533–49. Loe, M. (2004a). The Rise of Viagra: How the Little Blue Pill Changed Sex in America. New York: New York University Press. /oe, 0. E. 6e[ and the 6enior Woman 3leasure and 'anger in the 9iagra (ra. Sexualities, 7 (3), 303–26. Lohm, L., and Lindh, E. (2003). Älder och nyare hypertonimedel har likvärdig effekt. Läkartidningen, 100 (21), 1920. /\|wy, ,. . +39 9accination in &onte[t $ 9iew from )rance. ,n .. Wailoo, -. /iY- ingston, S. Epstein and R. Aronoqitz, Three Shots at Prevention: The HPV Vaccine and the Politics of Medicine’s Simple Solution (pp. 270–91). Baltimore, MD: John Hopkins University Press. Löwy, I. (2015). References Norms, Values and Constraints: The Case of Prenatal Diagnosis. In I. Dus- sauge, C.-F. Helgesson and F. Lee, Values Practices in the Life Sciences & Medicines (pp. 186–205). Oxford: Oxford University Press. (pp ) y Lundberg, P. O. (1995). Sexualliv och Läkemedel. Läkartidningen, 92 (28–9), 2742–7. Marshall, B. (2002). ‘Hard Science’: Gender Constructions of Sexual Dysfunction in the ‘Viagra Age’. Sexualities, 5 (2), 131–58. Marshall, B. (2006). The New Virility: Viagra, Male Aging and Sexual Function. Sexuali- ties, 9 (3), 345–62. Marshall, B., and Katz, S. (2002). ‘Forever Functional’: Sexual Fitness and the Ageing Male Body. Body & Society, 8 (4), 3–70. Martin, E. (1991). The Egg and the Sperm: How Science Constructed a Romance Based on Stereotypical Male-Female Roles. Signs, 16 (3), 485–501. Martin, E. (1992). The Woman in the Body: A Cultural Analysis of Reproduction. Boston, MA: Beacon Press. Marting, S. J. (1903). Prostatic Hypertrophy from Every Surgical Standpoint by George M Philips, M.D. and Forty Distinguished Authorities. St. Louis, MO: The Ajod Company Medical Publishers. Marx, F. J., and Karenberg, A. (2009). History of the Term Prostate. The Prostate, 69, 208–13. May, C, Rapley, T., Moreira, T, Finch, T. and Heaven, B. (2006). Technogovernance: Evi- dence, subjectivity, and the clinical encounter in primary care medicine. Social Science and Medicine, 62 (4):1022–30. McClintock, A. (1995). Imperial Leather: Race, Gender and Sexualities in the Colonial Context. London, New York: Routledge. McClintock, A. (1995). Imperial Leather: Race, Gender and Sexualities in the Colonial Context. London, New York: Routledge. Mclaren, A. (2007). Impotence: A Cultural History. Chicago: University of Chicago Press. g Mclaren, A. (2007). Impotence: A Cultural History. Chicago: University of Chicago Press. 115 References Mellgren, A. (1993). Ny Behandling of Impotens Review of the Book Pyschomastik der Impotenz by D Langer, U Hartmann. Läkartidningen, 90 (10), 984.i y g g Merchant, C. (1980). The Death of Nature: Women, Ecology and the Scientific Revolution. San Francisco, CA: Harper and Row. Miller, P., and Rose, N. (2008). Governing the Present: Administering Economic, Social and Personal Life. Cambridge: Polity Press. Mitton, C., and Donaldson, C. (2004). Priority Setting Toolkit: A Guide to the Use of Eco- nomics in Healthcare. London: BMJ Publishing Group. Mol, A. (2002). The Body Multiple: Ontology in Medical Practice. Durham, NC: Duke University Press. Mol, A., and Berg, M. (1998). Differences in Medicine: Unraveling Practices, Techniques and Bodies. Durham, NC: Duke University Press. 0oreira, 7. . References When are 7rials 1ot (nough" &linical 9ersus &osteffectiYeness in the &ontroYersy 2Yer $ccess to 'ementia 'rugs in the 1+6. ,n &atherine Will and T. Moreira, Medical Proofs, Social Experiments: Clinical Trials in Shifting Contexts (pp. 85–102). Farnham: Ashgate. Moynihan, R., and Cassels, A. (2005). Selling Sickness: How Drug Companies are Turning Us All into Patients. Crows Nest, Australia: Allen & Unwin. Moynihan, R., and Mintzes, B. (2010). Sex, Lies and Pharmaceuticals: How Drug Com- panies Plan to Profit from Female Sexual Dysfunction. Crows Nest, Australia: Allen & Unwin. 1ew 3harmaceutical %enefits %ill . 6uEmitted to 3arliament on 'ecemEer Available from the Swedish Government, www.regeringen.se/sb/d/108/a/1435. Nightingale, J., and Martin, J. (2004). The Myth of the Biotech Revolution. Trends in Bio- technology, 22 (11), 564–9. Nilsson, P., Hedner, T., Himmelmann, A., Kahan, T. and Menhem, K. (2003). Samarbete med industrin önskvart och nödvandigt-med bevarad integritet. Läkartidningen, 100 (21), 1919. 2elke, 0., %achmann, $., 'esc]euad, $., (mEerton, 0., raYas, 6., 0ichel, 0. &., 1ૃdow, J., Nordling, J. and de la Rosette, J. J. (2013). EAU Guidelines on the Treatment and the Follow-up of Non-neurogenic Male Lower Urinary Tract Symptoms Including Benign Prostatic Obstruction. European Urology, 65 (1), 118–40. Oliffe, J. (2005). Constructions of Masculinity Following Prostatectomy-Induced Impo- tence. Social Science & Medicine, 60 (2005), 2249–59. Oliffe, J. (2006). Embodied Masculinity and Androgen Deprivation Therapy. Sociology of Health & Illness, 28 (4), 410–32. Olsson, A. M., Abramsson, L., Höjerback, T., Malmberg, L. and Pedersen, J. (1995). Är det 0\|Mligt att Eota impotens" Läkartidningen, 92 (4), 313–16. Olsson, A. M., Forsberg, L., Höjerback, T. and Nilsson, A. (1990). Impotens på grund av arteriell insufficiens kan ha funktionella orsaker. Läkartidningen, 87 (51–2), 4456. Örn, P. (2001). Fler får dispens för lakemedel mot fetma. Läkartidningen, 98 (47), 5294. O’Shea, C. (2012). ‘A Plea for the Prostate’: Doctors, Prostate Dysfunction, and Male Sexuality in Late 19th- and Early 20th-Century Canada. Canadian Bulletin of Medical History CBMH/BCHM, 29 (1), 7–27. Oudshoorn, N. (1994). Beyond the Natural Body: An Archeology of Sex Hormones. Lon- don: Routledge. 2udshoorn, 1. . 0enopause 2nly for Women" 7he 6ocial &onstruction of 0eno- pause as an Exclusively Female Condition. Journal of Psychosomatic Obstretrics and Gynecology, 18, 137–44. 116 References Oudshoorn, N. (2003). The Male Pill: A Biography of a Technology in the Making. Dur- ham, NC: Duke University Press. Parsons, T. (1951). References Social Structure and Dynamic Process: The Case of Modern Medical Practice. London: Routledge. Payne, K., and Shabaruddin, F. H. (2010). Cost-effectiveness Analysis in Pharmacogenom- ics. Pharmacogenomics, 11 (5), 643–6. Pendergast, M. K. (2008). Regulatory Agency Consideration of Pharmacogenomics. Experimental Biology and Medicine, 233 (12), 1498–503. Petryna, A. (2006). Globalizing Human Subjects Research. In A. Lakoff, A. Petryna and A. Kleinman, Global Pharmaceuticals: Ethics, Markets, Practices (pp. 1–32). Durham, NC: Duke University Press. 3harmaceutical and 'ental %enefits $gency. a, 'ecemEer . $pplication for 6uE- sidy: Levitra (online) 2004. Retrieved from www.tlv.se.i 3harmaceutical and 'ental %enefits $gency. E, 'ecemEer . $pplication for 6uE- sidy: Viagra (online) 2003.i 3harmaceutical and 'ental %enefits $gency. c, 'ecemEer . $pplication for 6uE- sidy: Viagra (online). Stockholm County Administrative Court 2004.i 3harmaceutical %enefits %oard. a, -anuary . Autonativ. Stockholm, Sweden. Retrieved from www.tvl.se.i 3harmaceutical %enefits %oard. E, -anuary . Robinul. Stockholm, Sweden. Retrieved from www.tvl.se.i 3harmaceutical %enefits %oard. c, 0arch . Viagra. Stockholm, Sweden. Retrieved from www.tvl.se.i 3harmaceutical %enefits %oard. d. Caverjet. Stockholm, Sweden. Retrieved from www. tvl.se.i 3harmaceutical %enefits %oard. e. Bondil. Stockholm, Sweden. Retrieved from www. tvl.se. Phillips, K. A., Veenstra, D., Ramsey, S., van Bebber, S. and Sakowski, J. (2004). Genetic Testing and Pharmacogenomics: Issues for Determining the Impact to Healthcare Deliv- ery and Costs. American Journal of Managed Care, 10 (7), 425–32. Phillips, K. A., and Van Bebber, S. L. (2006). Regulatory Perspectives on Pharmacogenom- ics: A Review of the Literature on Key Issues Faced by the United States Food and Drug Administration. Medical Care Research and Review, 63 (3), 301–26. Plante, R. (2006). Review of Loe, Meika, The Rise of Viagra: How the Little Blue Pill Changed Sex in America. Sexualities, 9 (3), 379–80. Pope, C. (2003). Resisting Evidence: The Study of Evidence-based Medicine as a Contem- porary Social Movement. Health, 7 (276), 267–82. Porter, T. M. (1995). Trust in Numbers: Objectivity in Science and Public Life. Princeton, NJ: Princeton University Press. 3otts, $. . 'eleu]e on 9iagra 2r, What can a µ9iagra%ody¶ 'o". Body & Society, 10 (1), 17–36. 3otts, $., aYey, 1., race, 9. and 9ares, 7. . 7he 'ownside of 9iagra Women¶s Experiences and Concerns. Sociology of Health and Illness, 25 (7), 697–719. 3otts, $., race, 9. and aYey, 1. . 6e[ for /ife" 0en¶s &ounterstories on µ(rectile Dysfunction’, Male Sexuality and Ageing. Potts, A., and Tiefer, L. (2006). Introduction. Sexualities, 9 (3), 267–72. References Sociology of Health & Illness, 28 (3), 306–29. Potts, A., Grace, V., Gavey, N. and Vares, T. (2004). Viagra Stories: Challenging ‘Erectile Dysfunction’. Social Science & Medicine, 59, 489–99. Potts, A., Grace, V., Gavey, N. and Vares, T. (2004). Viagra Stories: Challenging ‘Erectile Dysfunction’. Social Science & Medicine, 59, 489–99. Potts, A., and Tiefer, L. (2006). Introduction. Sexualities, 9 (3), 267–72. Potts, A., and Tiefer, L. (2006). Introduction. Sexualities, 9 (3), 267–72. 117 References 117 References Power, M. (1997). The Audit Society: Rituals of Verification. Oxford: Oxford University Press. Power, M. (1997). The Audit Society: Rituals of Verification. Oxford: Oxford University Press. Prainsack B (2007) Research Populations: Biobanks in Israel New Genetics and Society Power, M. (1997). The Audit Society: Rituals of Verification. Oxford: Oxford University Press. Prainsack, B. (2007). Research Populations: Biobanks in Israel. New Genetics and Society, 26 (1), 85–103. Power, M. (1997). The Audit Society: Rituals of Verification. Oxford: Oxford University Press. Prainsack, B. (2007). Research Populations: Biobanks in Israel. New Genetics and Society, 26 (1), 85–103. Prainsack, B., and Firestine, O. (2006). Science for Survival: Biotechnology Regulation in Israel. Science and Public Policy, 33 (1), 33–46. Premfors, R., Ehn, P., Haldén, E. and Sundstöm, G. (2003). Demokrati och byråkrati (Democracy and Bureaucracy). Lund: Studentlitteratur. Raj, A. K. (2002). Pharmacogenetic Interventions, Orphan Drugs and Distributive Justice: 7he 5ole of &ost %enefit $nalysis. Social Philosophy & Policy, 19 (2), 246–70. Rappert, B. (2001). The Distribution and Resolution of the Ambiguities of Technologies or Why %oEEy &an¶t 6pray %oEEy &an. Social Studies of Science, 31 (4), 557–91. Reardon, J. (2004). Race to the Finish: Identity and Governance in an Age of Genomics. Princeton, NJ: Princeton University Press. Regeringsrätten. (2008a, March 14). Judgement in the Case 2713–05 Concerning the Sub- sidization of Viagra. Regeringsrätten. (2008b, March 14). Judgement in the Case 2716–05 Concerning the Sub- sidization of Cialis. 5. Regeringsrätten. (2008c, March 14). Judgement in the Case 2719–05 Concerning the Sub- sidization of Levitra. Reidy, J. (2005) Hard Sell: The Evolution of a Viagra Salesman. Riverside, NJ: Andrews McMeel Publishing. Rose, N. and Novas, C. (2005). Biological Citizenship. In A. Ong and S. Collier, Global Assemblages: Technology, Politics and Ethics as Anthropological Problems (pp. 439–463). Oxford: Blackwell. Sandberg, L. (2011). Getting Intimate: A Feminist Analysis of Old Age, Masculinity & Sexuality. Linköping: LiU Tryck. SBU Swedish Council on Health Technology Assessment (Statens Beredning for Medicinsk utYlrdering . References odartad 3rostataf\|rstoring med aYfl\|deshinder %enign 3rostate Hyperplasia with Flow Restrictions). Rapport 209. Sengoopta, C. (2006). The Most Secret Quintessence of Life. Chicago and London: The University of Chicago. Shackley, D. (1999). A Century of Prostatic Surgery. BJU International, 83, 776–82. Shenkin, B. (1973). Politics and Medical Care in Sweden: The Seven Crowns Reform. New England Journal of Medicine, 288, 555–9. Sjögren, E. (2006). Reasonable Drugs: Making Decisions with Ambiguous Knowledge. Dissertation. Stockholm: Stockholm School of Economics. 6M\|gren, (., and )ernler, .. . 'emocratic 3rioritysetting" 2rgani]ing 0ultiple 6take- holders to Make Decisions in the Healthcare Sector. In L. Sonderyd, G. Sundström and S. Furusten, Organizing Democracy: The Construction of Agency in Practice (pp. 94–112). Cheltenham: Edward Elgar. 6M\|strand, 0. . )\|rEattring aY erektionssYikt oftast Era f\|r tYn personer Läkartid- ningen, 95 (46), 5139. Socialutskottet. (1997). Socialutskottets betänkende 1996/97: SoU14. Prioritetingar inom hälso och sjukvården. Swedish Goverment.l 6pnngEerg, $., and 'ahlgren, +. . odartad prostataf\|rstoring med aYfl\|deshinder. Läkertidningen, 110 (13–14), 682–5. Stacey, J. (1997). Teratologies. A Cultural Study of Cancer. London and New York: Routledge. 118 References 6tacey, -. . 7he loEal Within &onsuming 1ature, (mEodying +ealth. ,n 6. )ranklin, C. Lury and J. Stacey, Global Nature, Global Culture (pp. 97–145). London, Thousand Oaks and New Delhi: Sage Press. Star, S. (1991) Power, Technology, and the Phenomenology of Conventions: On Being Allergic to Onions. In J. Law, A Sociology of Monsters: Essays on Power, Technology and Domination (pp. 22–56). London: Routledge. Star, S. L., and Bowker, G. C. (1999). Sorting Things Out: Classification and Its Conse- quences. Cambridge: MIT Press. Starr, P. (1982). The Social Transformation of American Medicine. Cambridge: Basic Books. Stockholm County Administrative Court (2004). Application for Subsidy: Viagra. Avail- able from: www.tlv.se (Accessed 12 December 2008), www.tlv.se/Upload/Ovrigt/ OVR_050418_dom_KR_viagra.pdf (Issued 15 April 2005). Stöckl, A. (2010). Public Discourses and Policy Making. The HPV Vaccination from the (uropean 3erspectiYe. ,n .. Wailoo, -. /iYingston and 6. (pstein, Three Shots at Pre- vention: The HPV Vaccine and the Politics of Medicine’s Simple Solutions (pp. 254–69). Baltimore, MD: John Hopkins University Press. 6tr\|Eerg, 3., +edelin, +. and %ergstr\|m, $. . 6e[Eara f\|r rika och friska" Läkartid- ningen, 103 (39), 2865–6. Ströberg, P., Hedelin, H. and Ljunggren, C. (2006). Behandling av erektil dysfunktion med PDE-5 hämmare. Läkartidningen, 103 (14), 1107–11. Swedish Book of Statutes (SFS). (1982:763). The Health and Medical Service Act. References Retrieved from www.regeringen.se/sb/d/108/a/23172.i 6wedish %ook of 6tatutes 6)6. , $pril . $ct on 3harmaceutical %enefits. 5etrieYed from www.notisum.se/rnp/sls/lag/20020160.HTM. Swedish Book of Statutes (SFS). (2001/140, April 2). Förordning om ändring i förordnin- gen(1996:1294) om högkostnadsskydd vid köp av läkemedel m.m. Retrieved from www. notisum.se/rnp/sls/fakta/a9961294.htm.i Swedish Parliament. (2001, December 2001). New Pharmaceutical Benefits Bill. Retrieved December 12, 2008, from www.regeringen.se: www.regeringen.se/sb/d/108/a/1435. Tiefer, L. (2000). Sexology and the Pharmaceutical Industry. The Journal of Sex Research, 37 (3), 273–94. Tiefer, L. (2006). The Viagra Phenomenon. Sexualities, 9 (3), 273–94. TLV (2003). Application for Subsidy: Viagra. Available from: www.tlv.se (Accessed 12 December 2008), www.tlv.se/Upload/Beslut/BES_030326_Viagra.pdf (Issued 26 March 2003). TLV (2005). Genomgången av läkemedel mot migrän. Available from: www.tlv.se, www. tlv.se/Upload/Genomgangen/rapport-migran.pdf (Accessed 12 December 2008). TLV (2006). Genomgången av läkemedel mot sjukdomar orsakade av magsyra. Avail- able from: www.tlv.se, http://www.tlv.se/Upload/Genomgangen/rapport-magsyra.pdf (Accessed 12 December 2008). Timmermans, S., and Berg, M. (2003). The Gold Standard: The Challenge of Evidence- based Medicine and Standardization in Health Care. Philadelphia. PA: Temple Univer- sity Press. 9ares, 7., and %raun, 9. . 6preading the Word, %ut What Word ,s 7hat" 9iagra and Male Sexuality in Popular Culture. Sexualities, 9 (3), 315–32. Wagstaff, $., 9an 'oorslaer, (., 9an der %urg, +., &alonge, 6., &hristiansen, 7., &itoni ., erdtham, 8, erfin, 0., ross, /. and +akinnen, 8. . (Tuity in the )inance 119 References of Health Care: Some Further International Comparisons. Journal of Health Economics, 18, 263–90. of Health Care: Some Further International Comparisons. Journal of Health Economics, 18, 263–90. Wailoo, .., /iYingston, -. and (pstein, 6. . Three Shots at Prevention: The HPV Vaccine and the Politics of Medicine’s Simple Solutions. Baltimore, MD: Johns Hopkins University Press. Whyte, 6. 5., Whyte, 0. $., /otte, 0. and .yaddondo, %. . 7reating $,'6 'ilem- mas of Unequal Access in Uganda. In A. Petryna, A. Lakoff and A. Kleinman, Global Pharmaceuticals: Ethics, Markets, Practices (pp. 240–62). Durham, NC: Duke Univer- sity Press. Willems, '. . ,nhaling 'rugs and 0aking Worlds $ 3roliferation of /ungs and Asthmas. In M. Berg and A. Mol, Differences in Medicine: Unraveling Practices, Tech- niques and Bodies (pp. 105–18). Durham, NC: Duke University Press. Williams, 5. . Culture and Materialism. Selected Essays. London and New York: Radical Thinkers, Verso. Williams, 6. -., aEe, -. and 'aYis, 3. . Pharmaceuticals and Society: Critical Dis- courses and Debates. West 6usse[ Wiley%lackwell. Williams, 6. References -., 0artin, 3. and aEe, -. a. 7he 3harmaceuticalisation of 6ociety" A Framework for Analysis. Sociology of Health and Illness, 33 (5), 710–25. Williams, 6. -., 0artin, 3. and aEe, -. E. (YolYing 6ociological $nalysis of µ3har- maceuticalisation’: A Reply to Abraham. Sociology of Health & Illness, 33 (5), 729–30. Wit], $. . Professions and Partriarchy. London: Routledge. Zetterqvist, A., Merlo, J. and Mulinari, S. (2015). Complaints, Complainants, and Rulings 5egarding 'rug 3romotion in the 8nited .ingdom and 6weden ± $ 4uantita- tiYe and 4ualitatiYe 6tudy of 3harmaceutical ,ndustry 6elf5egulation. 12 (2), e1001785 doi:10.1371. Zetterqvist, A., and Mulinari, S. (2013). Misleading Advertising for Antidepressants in Sweden: A Failure of Pharmaceutical Industry Self Regulation. PLOS one, 8 (5), 1–12. This page intentionally left blank Index $Eraham, -. ௘±௘, , $Eraham, -. ௘±௘, , commercial discourse ௘±௘ compliance ௘±௘ couples therapy , ௘±௘, cultural imaginary ௘±௘, cultural phantasy 88, 94, 96 commercial discourse ௘±௘ compliance ௘±௘ couples therapy , ௘±௘, cultural imaginary ௘±௘, cultural phantasy 88, 94, 96 Actor Network Theory (ANT) 75, 77 Administrative Court of Appeal in 6tockholm ௘±௘, , , ageing: medicalization of 52; old-age impotence 55; prostate enlargement se[uality and ௘±௘, ௘±௘ Dawson, Graham 88 decision Mustification documents ௘±௘, decisionmaking amEiguous ௘±௘ Eureaucratic compliance ௘±௘ classification disciplinary oEMectiYity 25; general compliance 26; mechanical oEMectiYity ௘±௘ depression 20, 25, 52, 105 Diagnostic and Statistical Manual of Mental Disorders 53 diagnostic Eracket creep ௘±௘, , direct-to-consumer (DTC) advertising 4, 8, ௘±௘, , ௘±௘ disciplinary objectivity 25 disease mongering ௘±௘, disease specificity , ௘±௘ doctors: disease mongering 52; general practitioners ௘±௘ imagined behaviours and roles 17, 19, 24; patient pressure on ௘±௘, specialist work practices of 43; see also urologists drug-disease pairing 25 drugs see pharmaceuticals Dawson, Graham 88 decision Mustification documents ௘±௘, decisionmaking amEiguous ௘±௘ Dawson, Graham 88 decision Mustification documents ௘±௘, decisionmaking amEiguous ௘±௘ alphaElockers effectiYeness of ௘±௘, p , /876%3+ treatment ௘±௘, ௘±௘ side-effects of 66; urination problems 66 /876%3+ treatment ௘±௘, ௘±௘ side-effects of 66; urination problems 66 Eureaucratic compliance ௘±௘ classification disciplinary oEMectiYity 25; general compliance 26; mechanical American Psychiatric Association (APA) 53 American Psychiatric Association (APA) 53 25; general compliance 26; mechanical American Urology Association (AUA) 67 Anderson, Benedict 91 androgens 66 American Urology Association (AUA) 67 Anderson, Benedict 91 androgens 66 oEMectiYity ௘±௘ depression 20, 25, 52, 105 Diagnostic and Statistical Manual of Mental Disorders 53 diagnostic Eracket creep ௘±௘, , direct-to-consumer (DTC) advertising 4, 8, Mental Disorders 53 diagnostic Eracket creep ௘±௘, , direct-to-consumer (DTC) advertising 4, 8, ANT see Actor Network Theory (ANT) antidepressants 57, 63 Ballinger, R. 8 beta-blockers 25 Biehl, J. 101 Eiological citi]enship ௘±௘, , Eiotechnology ௘±௘ %ondil , , ௘±௘, ௘±௘ Bordo, Susan 58 Botox 25, 63 Ballinger, R. 8 beta-blockers 25 Biehl, J. Index ௘±௘, imagery glocal , 3fi]er weEsite , ௘±௘, 6wedish 9iagra man ௘±௘ i t b li f b t 51 53 55 identity: collective 90; formation of 88, 91; intersectionality and 89; masculine 58, 78, 93; national 77, 91, 96, 100; naturali]ation technosocial ௘±௘, , 17, 75, 90, 96, 99 identity: collective 90; formation of 88, 91; intersectionality and 89; masculine 58, 78, 93; national 77, 91, 96, 100; naturali]ation technosocial ௘±௘, , 17, 75, 90, 96, 99 Index 101 Eiological citi]enship ௘±௘, , Eiotechnology ௘±௘ %ondil , , ௘±௘, ௘±௘ Bordo, Susan 58 Botox 25, 63 ( ) g , , ௘±௘, , ௘±௘ disciplinary objectivity 25 disease mongering ௘±௘, disease specificity , ௘±௘ doctors: disease mongering 52; general practitioners ௘±௘ imagined behaviours and roles 17, 19, 24; patient pressure on ௘±௘, specialist work practices of 43; see also urologists drug-disease pairing 25 d h i l ( ) g , , ௘±௘, , ௘±௘ disciplinary objectivity 25 disease mongering ௘±௘, disease specificity , ௘±௘ doctors: disease mongering 52; general practitioners ௘±௘ imagined behaviours and roles 17, 19, 24; patient pressure on ௘±௘, specialist work practices of 43; see also urologists drug-disease pairing 25 d h ti l ௘±௘, , ௘±௘ disciplinary objectivity 25 disease mongering ௘±௘, disease specificity , ௘±௘ doctors: disease mongering 52; general practitioners ௘±௘ imagined behaviours and roles 17, 19, 24; patient pressure on ௘±௘, specialist work practices of 43; see also urologists Bordo, Susan 58 Botox 25, 63 , BPH see lower urinary tract symptoms secondary to benign prostate hyperplasia (LUTS/BPH) Braun, V. 97 Brindley, Giles 58 British National Institute for Clinical Excellence (NICE) 46 bronchodilators 25 Eureaucratic compliance ௘±௘ &aYerMect , , ௘±௘, ௘±௘ Cialis 19 classification, politics of ௘±௘ clinical practice ௘±௘ BPH see lower urinary tract symptoms secondary to benign prostate hyperplasia (LUTS/BPH) p ; g drug-disease pairing 25 drugs see pharmaceuticals Eaton, J. Index 86 enrolment: Actor Network Theory (ANT) , creation of community ௘±௘ doctors ௘±௘, , ௘±௘ men ௘±௘, , ௘±௘ partners ௘±௘ 9iagra discourse , ௘±௘ y ( ) , creation of community ௘±௘ doctors ௘±௘, , ௘±௘ men ௘±௘, , ௘±௘ partners ௘±௘ 9iagra discourse , ௘±௘ erectile dysfunction (ED): alpha- Elockers and ௘±௘ construction of ௘±௘, , ௘±௘, , defining , diagnosis of ௘±௘, ௘±௘ glocal 122 Index ideology of 27; management of 17, 32; pharmaceutical reimEursement ௘±௘ resource allocation , ௘±௘ scope of coYerage ௘±௘ selfeducation ௘±௘, 105; solidarity principle 103; universal coverage 32, 45; urological treatments 68 health care technology assessment (HTA) organizations 34 health issues: medicalization of 2, 103; pharmaceuticalization of 5, 10, 25, 99; redefinition of +edelin, +ans ௘±௘ heteronormativity 61, 83, 85, 93 pharmaceuticals 62; medicalization of ௘±௘, , , , partnerpatient unit 61, 86; pharmaceuticalization of , ௘±௘, ௘±௘, , , , pre Viagra discourse 56, 59, 87, 102, 104; psychiatric discourse 78; severity of ௘±௘, se[ therapy , , , , , ௘±௘, social causes of state suEsidies for , ௘±௘, , ௘±௘, , ௘±௘ treatments for ௘±௘, , ௘±௘, ௘±௘, , , , 9iagra discourse , ௘±௘, , ௘±௘, ௘±௘ see also impotence; Viagra subsidy erectile Tuality (4 European Association of Urology (EAU) 67 evidence-based evaluation mechanisms 30, 34 health care technology assessment (HTA) organizations 34 health issues: medicalization of 2, 103; pharmaceuticalization of 5, 10, 25, 99; redefinition of health issues: medicalization of 2, 103; pharmaceuticalization of 5, 10, 25, 99; redefinition of +edelin, +ans ௘±௘ heteronormativity 61, 83, 85, 93 hormone therapy , ௘±௘ +edelin, +ans ௘±௘ heteronormativity 61, 83, 85, 93 hormone therapy , ௘±௘ identity: collective 90; formation of 88, 91; intersectionality and 89; masculine 58, 78, 93; national 77, 91, 96, 100; naturali]ation technosocial ௘±௘, , 17, 75, 90, 96, 99 ,llich, ,. females see women , , , , ,llich, ,. ௘±௘, feminism cultural studies ௘±௘ naturali]ation and ௘±௘ science discourse 77; sex therapy and 53; Viagra and 88 Firestine, O. 46 Fishman, J. 56, 97 5-Alpha-reductase inhibitors (5-ARIs) 67 )oucault, 0. ௘±௘, Fox, R. 2 Freud, Sigmund 78 imagery glocal , 3fi]er weEsite , ௘±௘, 6wedish 9iagra man ௘±௘ impotence: beliefs about 51, 53, 55; construction of , , ௘±௘, couples therapy , ௘±௘ defining ௘±௘ liEido and medicali]ation of ௘±௘, ௘±௘ oldage impotence 55; pharmaceuticalization of 57; as a physiological proElem ௘±௘ pre9iagra discourse ௘±௘, , , se[ therapy , , , social causes of ௘±௘, , treatment for ௘±௘ 9iagra discourse 58, 76, 103; see also erectile dysfunction (ED) impotence: beliefs about 51, 53, 55; construction of , , ௘±௘, couples therapy , ௘±௘ defining ௘±௘ liEido and medicali]ation of ௘±௘, ௘±௘ oldage impotence 55; pharmaceuticalization of 57; as a physiological proElem ௘±௘ pre9iagra discourse ௘±௘, , , se[ therapy , , , social causes of ௘±௘, , treatment for ௘±௘ 9iagra discourse 58, 76, 103; see also erectile dysfunction (ED) Fox, R. females see women 2 Freud, Sigmund 78 gastrooesophageal reflu[ disease (5' ௘±௘, ௘±௘ gender issues 91 general compliance 26 general practitioners see doctors gloEali]ation local conte[t and ௘±௘ marketing methods 76, 81, 91; pharmaceutical industry , ௘±௘, , , 100, 107; pharmaceutical nexus 1 glocal, defined glocal identities 5 glocal pharmaceuticals , ௘±௘ alpha Elockers local conte[t and ௘±௘, , ௘±௘ 6wedish 9iagra man techno-social identities 90; Viagra 27, 97 governance: medicalization and 2; pharmaceuticals and ௘±௘, , , ௘±௘, , gastrooesophageal reflu[ disease (5' ௘±௘, ௘±௘ gender issues 91 general compliance 26 general practitioners see doctors gloEali]ation local conte[t and ௘±௘ marketing methods 76, 81, 91; pharmaceutical industry , ௘±௘, , , 100, 107; pharmaceutical nexus 1 glocal, defined glocal identities 5 glocal pharmaceuticals , ௘±௘ alpha Elockers local conte[t and ௘±௘, , ௘±௘ 6wedish 9iagra man techno-social identities 90; Viagra 27, 97 governance: medicalization and 2; pharmaceuticals and ௘±௘, , , ௘±௘, , gastrooesophageal reflu[ disease *(5' ௘±௘, ௘±௘ gender issues 91 general compliance 26 general practitioners see doctors gloEali]ation local conte[t and ௘±௘ marketing methods 76, 81, 91; pharmaceutical industry , ௘±௘, , , 100, 107; pharmaceutical nexus 1 glocal, defined glocal identities 5 glocal pharmaceuticals , ௘±௘ alpha Elockers local conte[t and ௘±௘, , ௘±௘ 6wedish 9iagra man techno-social identities 90; Viagra 27, 97 governance: medicalization and 2; pharmaceuticals and ௘±௘, , , ௘±௘, , general compliance 26 general compliance 26 general practitioners see doctors International Headache Society 37, 41 International Index of Erectile Function gloEali]ation local conte[t and ௘±௘ marketing methods 76, 81, 91; pharmaceutical industry , ௘±௘, , , 100, 107; pharmaceutical nexus 1 ,,() ௘±௘, intersectionality 89 ,,() ௘±௘, intersectionality 89 Jacobsson, Lena 58 Jasanoff, S. 46 Johnson, V. E. 77 Jacobsson, Lena 58 Jasanoff, S. 46 Johnson, V. E. 77 glocal, defined glocal identities 5 Läkartidningen ௘±௘, ௘±௘, , ௘±௘ /akoff, $. ௘±௘ Levitra 19 Lewis, G. 27 liEido ௘±௘, lifestyle drugs 7, 25, 52, 95 Loe, M. 58 Losec 40, 42 lower urinary tract symptoms secondary to benign prostate hyperplasia Läkartidningen ௘±௘, ௘±௘, , ௘±௘ /akoff, $. ௘±௘ Levitra 19 Lewis, G. 27 liEido ௘±௘, lifestyle drugs 7, 25, 52, 95 Loe, M. females see women 58 Losec 40, 42 lower urinary tract symptoms secondary to benign prostate hyperplasia Levitra 19 governance: medicalization and 2; pharmaceuticals and ௘±௘, , , ௘±௘, , hair loss 25 health care: assessment organizations hair loss 25 health care: assessment organizations Losec 40, 42 ௘±௘, , cost management , ௘±௘, ௘±௘, ௘±௘, , , , ௘±௘ ௘±௘, , cost management , ௘±௘, ௘±௘, ௘±௘, , , , ௘±௘ lower urinary tract symptoms secondary to benign prostate hyperplasia Index 123 123 Index 123 Index (LUTS/BPH): alpha-blockers and ௘±௘, ௘±௘ pharmaceuticali]ation of ௘±௘, ௘±௘ surgical treatments and ௘±௘, , ௘±௘ treatments for ௘±௘ roles , , treataEle , , ௘±௘, ௘±௘, ௘±௘ untreataEle i roles , , treataEle , , ௘±௘, ௘±௘, ௘±௘ untreataEle i roles , , treataEle , , ௘±௘, roles , , treataEle , , ௘±௘, ௘±௘, ௘±௘ untreataEle i 3fi]er ,nc. , , , , , ௘±௘, ௘±௘, , i 3fi]er weEsite enrolment ௘±௘, ௘±௘ global harmonization 105; local context and national imagery ௘±௘, ௘±௘ social phantasy ௘±௘, , 6wedish masculinity and ௘±௘, male rejuvenation treatments 53 male se[uality construction of , ௘±௘, male rejuvenation treatments 53 male se[uality construction of , ௘±௘, y e[pectations aEout , ௘±௘, , medicali]ation of ௘±௘ Yitality and ௘±௘ see also masculinity pharmaceutical-based treatment: indiYiduali]ed ௘±௘ pharmacogenomics and ௘±௘ 3harmaceutical %enefit %oard see Swedishi pharmaceutical-based treatment: y Mamo, L. 56, 97 Mamo, L. 56, 97 pharmacogenomics and ௘±௘ 3harmaceutical %enefit %oard see Swedishi 0arshall, %arEara ௘±௘, ௘±௘ p g 3harmaceutical %enefit %oard see Swedishi masculinity: construction of 75, 78, , ௘±௘ identity and , , naturali]ed ௘±௘ partners and penetration and ௘±௘, , ௘±௘, phantasy ௘±௘ potency and social class and ௘±௘ stereotypical images of 6wedish concept of ௘±௘, ௘±௘ virility and 51, 90, 96; volatile 90; wilderness settings and ௘±௘, 'ental and 3harmaceutical %enefits Agency (TLV) pharmaceutical forms of governance 101 pharmaceutical industry: direct-to- consumer adYertising , , ௘±௘, ௘±௘ disease mongering gloEali]ation marketing ௘±௘ research funding 6weden ௘±௘i 0asters, W. +. mechanical oEMectiYity ௘±௘ medical discourse , , ௘±௘, i 0asters, W. +. females see women mechanical oEMectiYity ௘±௘ medical discourse , , ௘±௘, i pharmaceuticali]ation defined ௘±௘ gloEali]ation ௘±௘ glocal , , ௘±௘, goYernance and , , ௘±௘ local conte[t and , ௘±௘ medicali]ation and 3; medicine and 63; of sexuality ௘±௘ social practices and theoretical framework of 4 medicali]ation ௘±௘ defining pharmaceuticalization and 3, 25; of se[uality ௘±௘ Medical Products Agency 8 medical sociology ௘±௘ pharmaceutical marketing: direct-to- consumer '7& , , ௘±௘, ௘±௘ global imagination and 91; national imagination and 91i gy mental health: psychopharmacological model 53, 86; responses to 86 migraine drugs: pharmaceutical reimEursement ௘±௘ treatment effects ௘±௘, ௘±௘ Mintzes, B. 105 Moynihan, R. 105 mental health: psychopharmacological model 53, 86; responses to 86 migraine drugs: pharmaceutical model 53, 86; responses to 86 migraine drugs: pharmaceutical reimEursement ௘±௘ treatment effects ௘±௘, ௘±௘ migraine drugs: pharmaceutical reimEursement ௘±௘ treatment effects ௘±௘, ௘±௘ pharmaceutical ne[us defining gloEal impact 11; Sweden 15 pharmaceutical reason 32 pharmaceutical regime 3 p ; pharmaceutical reason 32 pharmaceutical regime 3 Mintzes, B. 105 Mintzes, B. 105 Moynihan, R. 105 Moynihan, R. 105 pharmaceutical reimEursement ௘±௘ assessment organi]ations ௘±௘, ௘±௘ classification of costeffectiYeness ௘±௘, ௘±௘, , , ௘±௘, ௘±௘, ௘±௘, , ௘±௘ cost management , ௘±௘ decision Mustification documents ௘±௘ diagnosisEased system erectile dysfunction ௘±௘ eYaluation of ௘±௘, ௘±௘ /osec , migraine drugs ௘±௘ organi]ational arrangements ௘±௘ productEased system ௘±௘, stomachacid patients ௘±௘ usage patterns and see also Viagra subsidy national identity 77, 91, 96, 100 national imagery ௘±௘ naturali]ation ௘±௘, ௘±௘ nonmedical problems: medicalization of ௘±௘, pharmaceuticali]ation of , , 25, 52 Novas, C. 80 old-age see ageing Oliffe, J. 86 orchidectomy 65 orphan drugs 31 Oudshoorn, N. 52 Parsons, T. 1 patients: characterization of 42; distrust of ௘±௘, imagined EehaYiours and nonmedical problems: medicalization of nonmedical problems: medicalization of ௘±௘, pharmaceuticali]ation of , , ௘±௘, pharmaceuticali]ation of , , 25, 52 25, 52 Novas, C. 80 old-age see ageing Oliffe, J. 86 orchidectomy 65 orphan drugs 31 Oudshoorn, N. 52 Parsons, T. 1 patients: characterization of 42; distrust of ௘±௘, imagined EehaYiours and old-age see ageing Oliffe, J. 86 orchidectomy 65 orphan drugs 31 Oudshoorn, N. 52 Parsons, T. females see women 46 premature eMaculation ௘±௘ prostate enlargement of ௘±௘, surgical treatments and , ௘±௘ urination proElems ௘±௘ see also lower urinary tract symptoms secondary to benign prostate hyperplasia (LUTS/BPH) proton pump inhiEitors 33,s ௘±௘ pharma-governance 5 p g polypharmica 56 3orter, 7. 0. ௘±௘ potency 92 3otts, $. ௘±௘, Prainsack, B. 46 premature eMaculation ௘±௘ testosterone treatment 53 Tiefer, L. 53, 59, 61 TLV see Swedish Dental and 3harmaceutical %enefits $gency 7/9 transurethral resection of the prostate 7853 , ௘±௘ treatment compliance 27 triptanes ௘±௘, TURP see transurethral resection of the prostate enlargement of ௘±௘, surgical treatments and , ௘±௘ urination proElems ௘±௘ see also lower urinary tract symptoms secondary to benign prostate hyperplasia (LUTS/BPH) prostate hyperplasia (LUTS/BPH) proton pump inhiEitors 33,s ௘±௘ 7853 , ௘±௘ treatment compliance 27 triptanes ௘±௘, 5aM, $. .. ௘±௘ refraction 4, 27, 100 regulatory system ௘±௘, , ௘±௘, ௘±௘ research funding, pharmaceutical industry 54 Rogaine 25 Rose, N. 80 TURP see transurethral resection of the prostate TURP see transurethral resection of the prostate universal coverage 32, 45 urination proElems ௘±௘, , urological treatments ௘±௘ urologists , , , ௘±௘, ௘±௘ Rose, N. 80 science and technology studies 99 se[ therapy , , , , ௘±௘, , , , ௘±௘ science and technology studies 99 se[ therapy , , , , ௘±௘, , , , ௘±௘ Vares, T. 97 Vares, T. females see women 1 patients: characterization of 42; distrust of ௘±௘, imagined EehaYiours and pharmaceuticals: ability to pay 7; diagnostic Eracket creep ௘±௘, futures 99; gendered 97; globalization ௘±௘, goYernance and lifestyle pharmaceuticals: ability to pay 7; diagnostic Eracket creep ௘±௘, futures 99; gendered 97; globalization ௘±௘, goYernance and lifestyle of ௘±௘, imagined EehaYiours and 124 Index 124 Index 124 Index Stacey, J. 80 Steinach, Eugen 53 Steinach operation 53 Stockholm County Administrative Court ௘±௘, ௘±௘, stomach-ache 35 stomachacid patients confirmed diagnosis 36, 41; pharmaceutical reimbursement drugs 7, 25; local context and 6, 8, 29, ௘±௘ misdiagnosis of ontological roles regulatory system ௘±௘ se[uality and state payment for ௘±௘, , state regulatory Eodies and ௘±௘ success of 63; techno-social identities ௘±௘, , , , , , stomach-ache 35 stomachacid patients confirmed diagnosis pi g 36, 41; pharmaceutical reimbursement ௘±௘ 36, 41; pharmaceutical reimbursement ௘±௘ pharmacogenomic technologies 9; classification ௘±௘ economic impact of 30; evidence-based evaluation mechanisms 44; improved treatment and indiYiduali]ation and ± pharmacogenomic technologies 9; classification ௘±௘ economic impact of 30; evidence-based evaluation mechanisms 44; improved treatment and 6upreme $dministratiYe &ourt ௘±௘, ௘±௘ 6upreme $dministratiYe &ourt ௘±௘, ௘±௘ ; mechanisms 44; improved treatment and , indiYiduali]ation and ௘±௘ pharma-governance 5 polypharmica 56 3orter, 7. 0. ௘±௘ potency 92 3otts, $. ௘±௘, Prainsack, B. 46 premature eMaculation ௘±௘ prostate enlargement of ௘±௘, surgical treatments and , ௘±௘ urination proElems ௘±௘ see also lower urinary tract symptoms secondary to benign prostate hyperplasia (LUTS/BPH) proton pump inhiEitors 33,s ௘±௘ p ௘±௘ mechanisms 44; improved treatment and , indiYiduali]ation and ௘±௘ Swedish Agency for Health Technology Assessment 67 Swedish Dental and Pharmaceutical %enefits $gency 7/9 ௘±௘, , ௘±௘, ௘±௘, ௘±௘ 6wedish 9iagra man ௘±௘, , , , ௘±௘, ௘±௘ , indiYiduali]ation and ௘±௘ pharma-governance 5 polypharmica 56 3orter, 7. 0. ௘±௘ potency 92 3otts, $. ௘±௘, Prainsack, B. 46 premature eMaculation ௘±௘ prostate enlargement of ௘±௘, surgical treatments and , ௘±௘ urination proElems ௘±௘ see also lower urinary tract symptoms secondary to benign prostate hyperplasia (LUTS/BPH) proton pump inhiEitors 33,s ௘±௘ , indiYiduali]ation and ௘±௘ pharma-governance 5 polypharmica 56 3orter, 7. 0. ௘±௘ potency 92 3otts, $. ௘±௘, Prainsack, B. females see women 97 vasectomy 65 se[ual dysfunction , ௘±௘ see also erectile dysfunction (ED) vasectomy 65 se[ual dysfunction , ௘±௘ see also erectile dysfunction (ED) y 9iagra adYertising , ௘±௘, , , ௘±௘, ௘±௘, ௘±௘, , cultural imaginary ௘±௘ as a feminist issue 88; high public spending on 17, 24; masculinity and 78, 89; national imagery ௘±௘ promotion of , satisfaction with , ௘±௘, success of 63; techno-social identities 17; use by women 60, 98; see also Swedish Viagra man 9iagra adYertising , ௘±௘, , , ௘±௘, ௘±௘, ௘±௘, , cultural imaginary ௘±௘ as a feminist issue 88; high public spending on 17, 24; masculinity and 78, 89; national imagery ௘±௘ promotion of , satisfaction with , ௘±௘, success of 63; techno-social identities 17; use by women 60, 98; see also Swedish Viagra man y ( ) se[uality attitudes towards ௘±௘, ௘±௘ couples therapy , ௘±௘, defining , emotional responsiEility female responsiEility ௘±௘ heteronormativity 61, 83, 85; libido ௘±௘, medicali]ation of ௘±௘, partner-patient unit 61; penetration and ௘±௘, pharmaceuticali]ation of ௘±௘, post9iagra discourse ௘±௘ pre9iagra discourse , psychology-based approach 53 sick role ௘±௘ 6ildenafil , social phantasy ௘±௘, , SSRI inhibitors 25 Viagra discourse: commercial discourse ௘±௘, cultural imaginary erectile dysfunction ௘±௘, ௘±௘, female responsiEility ௘±௘ glocal 4, 102, 107; heterosexuality 61, ௘±௘, local conte[t and ௘±௘, Index 125 125 Index 125 Index Viagra website see 3fi]er weEsite virility 51, 90, 96 volatile masculinity 90 Weil, 5. Western1orthern medicine ௘±௘ Willems, '. Williams, 6. -. ௘±௘, , , , women emotional responsiEility ௘±௘ partner support , , ௘±௘, responses to 9iagra use ௘±௘ se[ual dysfunction sexuality and 60, 62, 85; Viagra use 60, 98 sexual performance and 96; sexual selfgoYernance ௘±௘ ta[ suEsidies 54, 70, 106; techno-social identities 5, 79, 96 see also 3fi]er weEsite 9iagra imaginary ௘±௘ Viagra subsidy: appeals 19; court cases , ௘±௘, , ௘±௘, deEates aEout , , , ௘±௘ decision Mustification documents ௘±௘ erectile dysfunction diagnosis ௘±௘, glocal responses Mustification for denial ௘±௘, , , Viagra website see 3fi]er weEsite virility 51, 90, 96 volatile masculinity 90 Weil, 5. Western1orthern medicine ௘±௘ Willems, '. Williams, 6. -. females see women ௘±௘, , , , women emotional responsiEility ௘±௘ partner support , , ௘±௘, responses to 9iagra use ௘±௘ se[ual dysfunction sexuality and 60, 62, 85; Viagra use 60, 98 women emotional responsiEility ௘±௘ partner support , , ௘±௘, responses to 9iagra use ௘±௘ se[ual dysfunction sexuality and 60, 62, 85; Viagra use 60, 98
https://openalex.org/W4385546704	https://journal.formosapublisher.org/index.php/ajae/article/download/4189/5082	English	null	Academic Success in the Oral Presentation Course and Overall Self-Confidence	Asian Journal of Applied Education	2,023	cc-by	3,916	Asian Journal of Applied Education (AJAE) Vol. 2, No. 3, 2023: 293 - 302 Asian Journal of Applied Education (AJAE) Vol. 2, No. 3, 2023: 293 - 302 A R T I C L E I N F O A R T I C L E I N F O A B S T R A C T Key Words: Oral Performance, Academic Achievement, General Self-Confidence Received : 17 May Revised : 19 June Accepted: 19 July ©2023 Hidayad, Lestari, Laoli, Anam, Despita: This is an open- access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. The investigation of the relationship between overall self-confidence and academic success in the oral presentation course was a special focus of the study. 53 female undergraduate English majors from PGRI University of Palembang participated in the study. The General Self- Confidence Questionnaire (GSCQ) and final evaluation scores for the Oral Presentation course were used to gather data. Data analysis was done using SPSS. The findings showed a significant, positive association between general self- confidence and academic success. High GSCQ scorers also performed well on the oral achievement test. In order to improve their students' achievement in oral performance, language instructors were advised to increase the development of their students' self-confidence. The investigation of the relationship between overall self-confidence and academic success in the oral presentation course was a special focus of the study. 53 female undergraduate English majors from PGRI University of Palembang participated in the study. The General Self- Confidence Questionnaire (GSCQ) and final evaluation scores for the Oral Presentation course were used to gather data. Data analysis was done using SPSS. The findings showed a significant, positive association between general self- confidence and academic success. High GSCQ scorers also performed well on the oral achievement test. In order to improve their students' achievement in oral performance, language instructors were advised to increase the development of their students' self-confidence. Academic Success in the Oral Presentation Course and Overall Self-Confidence Ferri Hidayad1, Dewi Lestari2, Adieli Laoli3, Samsul Anam4, Despita5, 1Universitas PGRI Palembang, 2,4Universitas Baturaja, 3Universitas Nias, 5STIA Satya Negara Palembang Corresponding Author: Ferri Hidayad ferri6591@gmail.com INTRODUCTION The process of learning a language is intricate (Young, 1991). It is impacted by cognitive and affective elements, which, according to (Tallon, 2009), are the primary causes of individual variability in foreign language learning. The affective domain, which includes personality traits including emotion, motivation, attitude, anxiety, and personality, is the emotional side of human behavior, according to (Brown, 2003; Skehan, 1991). One of these factors that has the greatest impact on learning is self-confidence. It is one of the main motivations for people and can have a major impact on someone's life, either positively or negatively. (Dörnyei & Skehan, 2003, p. 211)stated that the concept of self- confidence is closely related to self-esteem, both share a common emphasis on the individual’s perception of his or her abilities as a person. The phrases "self-confidence," "self evaluation," "self worth," "self appraisal," and "self satisfaction" are frequently used interchangeably in studies, according to (Gregersen & Horwitz, 2002). In its most basic form, it can be described as a psychological and social phenomenon in which a person assesses himself or herself in accordance with certain values that may cause various emotional states, become developmentally stable, but are still subject to change depending on individual circumstances (Rubio, 2021). It is described as an individual's "overall evaluation or appraisal of themselves, whether they approve of themselves or disapprove of themselves, like themselves or dislike themselves" by (Craven & Marsh, 2004; Higgins, 1996, p. 178). According to (Chou, 2018), it is the way a person feels about themselves or the "sense of personal worth and competence that people associate with their self concept" (Brockner, 1988, p. 243). According to (Pierce et al., 1989), it is the extent to which a person believes that they are competent, relevant, and deserving (Chang & Read, 2008). It is "a set of attitudes, judgments that a person brings with him or herself when facing the world," (Chang & Read, 2006) noted. It also involves expectations for success or failure, according to (Harris, 2009, p. 9). It often refers to perceptions of oneself and attitudes about oneself. The development of general self-confidence occurs during childhood and results from the accumulation of interpersonal and intrapersonal experiences. The literature on how to increase general self-confidence points to a number of different origins from which it derives. ( DOI: https://doi.org/10.55927/ajae.v2i3.4189 ISSN-E: 2963 - 5241 https://journal.formosapublisher.org/index.php/ajae ( DOI: https://doi.org/10.55927/ajae.v2i3.4189 ISSN-E: 2963 - 5241 https://journal.formosapublisher.org/index.php/ajae 293 Hidayad, Lestari, Laoli, Anam, Despita INTRODUCTION The most crucial elements are: (1) personal experiences; positive events foster the growth of strong self- confidence, whereas negative experiences have the reverse impact; (2) Social cues from other people. Peers, peers, community, and home are all crucial for self-confidence development. Being exposed to negative signals lowers self- confidence levels, whereas sending good messages to others is regarded to be harmful to the development of strong self-confidence (Miraei, 2005). In order to successfully learn a foreign language, one individual characteristic that supports success is self-confidence. According to several studies, it is necessary for all language learning activities to be successful (Kimura, 2000). Academic success might be aided or hindered. Learners of foreign languages who are generally confident do well and probably think highly of their own abilities. 294 Asian Journal of Applied Education (AJAE) Vol. 2, No. 3, 2023: 293 - 302 Asian Journal of Applied Education (AJAE) Vol. 2, No. 3, 2023: 293 - 302 Conversely, "learners suffer from uncertainty, insecurity, fear, and social distance" when there is low self-confidence. (Yen et al., 2015, p. 9) Conversely, "learners suffer from uncertainty, insecurity, fear, and social distance" when there is low self-confidence. (Yen et al., 2015, p. 9) The development of oral performance, one of the four language skills, is regarded to be closely tied to self-assurance. Without some degree of it, FL learners cannot speak the language or express themselves freely and eloquently (H. D. Brown, 2000). The primary goal of this study was to investigate the relationship between learners' academic success on an oral presentation examination and their overall sense of confidence. LITERATURE REVIEW 295 Hidayad, Lestari, Laoli, Anam, Despita Speaking anxiety is linked to lack of confidence, which has a negative impact on performance. (Gregersen & Horwitz, 2002; Chen & Chang, 2004). g g Contrary to the results of the above studies, some more recent studies indicated a modest or low correlation between self-confidence and academic achievement such as (Brockner, 1988; Pullmann & Allik, 2008; Nagar et al., 2008), and the results of other studies revealed that no correlation was found between the two variables (Ahmed, 2011; Zubrick & Silburn, 2006; Gabuardi & Sánchez, 2015 ;Naderi et al., 2009; Yahaya & Ramli, 2009). In conclusion, it clearly appeared from the review of this survey that there is contradiction between the results of the previously mentioned studies. The possible explanation is that the above studies were conducted in different countries on different samples, gender, levels and used different scales. It was also obvious that there did not exist much research literature on the correlation between general self-confidence and academic achievement within a Saudi context. The studies conducted on Indonesia learners were very few such as (Ahmed, 2011; Purwanto, 2022). The current paper, therefore, aimed at investigating the correlation between general self-confidence and academic oral presentation achievements of female English majors, to find out if general self-confidence had an influence on learners’ oral performance. LITERATURE REVIEW The majority of teachers find that the hardest part of teaching a language is helping students improve their oral communication abilities. Competence in the L2 may not be sufficient, even though doing various communicative tasks is the greatest strategy to help learners speak the language fluently. Learners must be willing to communicate as well as able to do so, according to (Wright, 2011). A willingness to communicate is described as "the intention to initiate communication, given a choice" by (Molberg, 2010), as cited in (J. C. Brown & Golston, 2004). The most crucial element that influences whether or not students will be willing to participate in speaking activities in language classrooms is self-confidence (Yashima et al., 2004). In other words, we might assert that effective communication results from self-assurance. Conversely, it is believed that the most perilous obstacle to successful communication is a lack of confidence. The impact of learners' self-confidence on their preparedness to engage in expressive activities in the classroom has been the subject of several studies (Al-Sibai, 2005; Ahmed, 2011). This research showed that self-assured language learners frequently take risks when speaking a foreign language, even when they make mistakes. Regardless of the subject being covered or the number of pupils in the class, they participate in a variety of speech exercises. They put in a lot of effort, learn from their mistakes, and gradually they become more fluent in the language. On the other hand, low confident learners usually look away from instructor to avoid being called on. They feel uncomfortable when using the language orally because they are concerned about being criticized or disapproved of. As a result, they tend to perform less successfully (Al-Sibai, 2004). Literature has shown a connection between general self-confidence and academic success. It is a contentious relationship, and several investigations have produced contradictory findings. Numerous research has shown a beneficial relationship between self-confidence and language course grades. High self-confidence can also encourage communication among learners and advance language skills, which can lead to improved academic success. The relationship between self-assurance and academic success is dynamic. Academic achievement rises as self-confidence levels do. Academic achievement suffers as a result of low self-confidence in students (Yamini & TAHRIRI, 2006; Pullmann & Allik, 2008). Learners' speaking performance was particularly impacted by their level of confidence. It was correlated with the output they generated, which had an impact on L2 communicative proficiency. METHODOLOGY To determine the connection between overall self-confidence and academic success, a descriptive-correlation methodology was used. The current condition of the study's subjects was described using the descriptive design. The correlation study, on the other hand, was conducted to ascertain whether there was a relationship between the study's variables or not in order to make predictions or recommendations (Fook et al., 2011). The study's participants were 53 English-majoring undergraduate students from PGRI University of Palembang. Their ages ranged from 20 to 22 and they were female. They have studied English in school since the intermediate stage, just like other children their age. They didn't have much prior experience conversing in English with one other or native speakers before they joined the English Department. They also had little chance to use their English outside of the classroom. They were introduced to a few speaking- improvement courses by the English Department, the most crucial of which was Oral Presentation. Every participant registered for the oral presentation course during the 2021 academic year. The main objective of this course was to teach learners how to construct, compose, present and deliver information through oral interaction in more fluent and correct English. (El-Anzi, 2005) developed the General Self-Confidence Questionnaire, which was used to gather data relevant to the study's goal. Participants received it during the first semester of the 2021 school year. There were 25 assertions about GSC in it, and students were asked to rate how much they agreed or disagreed with each one. (Kesumawati et al., 2017) said the responses were graded using a five-point Likert scale, with no being the lowest score and too much being the highest. The questionnaire's rating scale included a minimum score of 25 points and a maximum score of 125 points. A high score 296 Asian Journal of Applied Education (AJAE) Vol. 2, No. 3, 2023: 293 - 302 on this questionnaire indicated a high level of GSC, whereas a low score suggested a low amount of GSC. Academic accomplishment was also evaluated for the study's purposes using the teachers' final evaluation "grades." The course instructor provided grades to students as a measure of their performance. Learners took the oral presentation test individually. Each student was given unlimited freedom to present anything they choose, along with some time to prepare. This assignment was to be completed in front of the teacher and other students. METHODOLOGY Ten minutes were allotted to each participant for her presentation. The following criteria were used by the instructor to evaluate students' performance: fluency, accuracy, organization, facts conveyed, delivery, nonverbal communication, and social skills. SPSS (version 18.0) was used to evaluate the information gathered from the learners' results on the oral test and self-confidence questionnaire. To better comprehend the results of the study instruments' scores, descriptive statistics were computed. ANOVA, T-test, and correlation analysis were used to assess the relationship between the study variables. The outcomes were shown in the table below. RESULTS AND DISCUSSION The results of the computed descriptive statistics of the general self- confidence questionnaire revealed that the mean score of the participants was (92.41), and participants‟ standard deviation was (16.786). See table (1). Table 1. Self-Confidence Questionnaire N Minimum Maximum Mean Std. Deviation Sum 53 59.00 155.00 92.4151 16.78648 It appeared from the results reported in table (2) that the mean score of the participants on oral presentation test was (81.84) and the standard deviation score was (4.89). Table 2. Score of the Participants on Oral Presentation N Minimum Maximum Mean Std. Deviation OPT 53 70.00 93.00 81.8491 4.89268 To examine the correlation between general self-confidence and academic achievement scores, correlation analysis was conducted. The Pearson correlation coefficient, presented in Table (3), was r (.707). The Spearman’ s rank correlation coefficient was also employed to determine the correlation between the two variable. The results presented in Table (4) revealed that the correlation coefficient was (.806). 297 Hidayad, Lestari, Laoli, Anam, Despita Table 3. The Pearson Correlation Coefficient GSC OPT GSC Pearson Correlation 1 . .707() Sig. (2-tailed) .000 N 53 53 OPT Pearson Correlation .707() 1 Sig. (2-tailed) .000 N 53 53 * Correlation is significant at the 0.01 level (2-tailed). Table 3. The Pearson Correlation Coefficient Table 4. The Spearman’ S Rank Correlation Coefficient GSC OPT Spearman's rho GSC Correlation Coefficient 1.000 .806() Sig. (2-tailed) . .000 N 53 53 OPT Correlation Coefficient .806() 1.000 Sig. (2-tailed) .000 . N 53 53 Correlation is significant at the 0.01 level (2-tailed). Table 4. The Spearman’ S Rank Correlation Coefficient The findings above indicated that the participants’ scores on the general self-confidence questionnaire correlated positively with oral test scores. It appeared that general self-confidence could affect the quality of oral performance. The participants were divided in two groups according to the scores they acquired on the general self-confidence questionnaire: the higher self-confidence group and the lower self-confidence group. The ANOVA analysis was run, using self-confidence as the independent variable (high & low) and oral performance as the dependent variable. The results of the analysis revealed that self-confidence had a significant effect on the participants’ oral performance F (20.82) = P<0.01. Further, correlation analysis was employed to examine if there was a significant correlation between possessing high levels of self-confidence and high scores in the oral presentation test. RESULTS AND DISCUSSION The results, reported in table (5), showed a statistically positive correlation at the level of P <0.01. The positive correlation showed that the higher the participants’ level of general self- confidence, the higher were their oral presentation scores and conversely participants with low self-confidence had lower scores. In other words, the participants’ level of general self-confidence was a significant predictor for their academic achievement. Table 5. Correlation Analysis Group_s N Meant Std. Deviation Std. Error Mean Spearman's rho Group_p 59-79 13 1.1538 .55470 .15385 749() 107-155 13 3.4615 .66023 .18311 ** Correlation is significant at the 0.01 level (2-tailed). 298 Asian Journal of Applied Education (AJAE) Vol. 2, No. 3, 2023: 293 - 302 Asian Journal of Applied Education (AJAE) Vol. 2, No. 3, 2023: 293 - 302 The findings mentioned above highlighted the importance of self- confidence in speaking a foreign language. Selfconfident learners are ready to speak in public. They work hard, perform well and accordingly, achieve academic progress. On the contrary, the issue of developing oral communication skills becomes problematic when learners suffer from a lack of self-confidence. Low confident learners feel uncomfortable, afraid and frustrated in the classroom. As a result, they tend to perform with less effectiveness and satisfaction, which is affecting their academic achievement in general. CONCLUSION The study looked at the relationship between overall self-confidence and performance on the oral performance test among female English major undergraduate students. The findings showed a substantial positive association between the two factors. The greater the learners' oral test scores, the more self- assured they were. The most self-assured students were willing to try speaking in front of others. On the other side, a lack of overall confidence led to a lack of motivation to try for a high-caliber oral performance. Less assured students had doubts about their prowess. They frequently made fewer attempts, which resulted in below-average achievement. The study's conclusions also emphasized the significance of encouraging all-around self-confidence in language learners in order to advance their oral skills. As a result, it is advised that language teachers concentrate on boosting their students' self-confidence by creating a safe learning atmosphere that inspires them to talk and take part in oral activities without inhibition. They can assist students in identifying their concerns and teaching them coping mechanisms. They can combat unfavorable opinions and attitudes while promoting optimistic thinking. They should maintain a laid-back and amusing mood during oral activities, create engaging activities that allow more time and opportunity, and focus on the positive. REFERENCES Ahmed, J. (2011). The Relationship between Self Concept and Response to Word Students’ Academic Achievement among Students Learners in University Putra Malaysia. International Journal of Instructio, 4(2), 23–38. Al-Sibai, D. (2004). Promoting oral fluency of second language learners. Retrieved on October, 13. Al-Sibai, D. (2005). L2 anxiety of Saudi female university students enrolled in speaking courses. Retrieved on July, 26, 2011. Brockner, J. (1988). Self-esteem at work: Theory, research, and practice. Lexington, MA: Lexington Books, 159–163. Brown, H. D. (2000). Principles of language learning and teaching (Vol. 4). Longman New York. Brown, H. D. (2003). Language assessment: Principles and classroom practices. 299 Hidayad, Lestari, Laoli, Anam, Despita Pearson Education. Brown, J. C., & Golston, C. (2004). The Evolution of Hierarchical Structure in Language. Annual Meeting of the Berkeley Linguistics Society, 30(1), 13. https://doi.org/10.3765/bls.v30i1.959 Chang, A. C.-S., & Read, J. (2008). Reducing Listening Test Anxiety through Various Forms of Listening Support. TESL-EJ, 12(1), n1. CHANG, A. C.-S., & READ, J. (2006). The Effects of Listening Support on the Listening Performance of EFL Learners. TESOL Quarterly, 40(2), 375–397. https://doi.org/https://doi.org/10.2307/40264527 Chen, T.-Y., & Chang, G. B. Y. (2004). The Relationship between Foreign Language Anxiety and Learning Difficulties. Foreign Language Annals, 37(2), 279–289. https://doi.org/https://doi.org/10.1111/j.1944- 9720.2004.tb02200.x Chou, M.-H. (2018). Speaking Anxiety and Strategy Use for Learning English as a Foreign Language in Full and Partial English-Medium Instruction Contexts. TESOL Quarterly, 52(3), 611–633. https://doi.org/https://doi.org/10.1002/tesq.455 Craven, R. G., & Marsh, H. W. (2004). The Challenge for Counsellors: Understanding and Addressing Indigenous Secondary Students’ Aspirations, Self-concepts and Barriers to Achieving their Aspirations. Journal of Psychologists and Counsellors in Schools, 14(1), 16–33. https://doi.org/DOI: 10.1017/S1037291100002648 Dörnyei, Z., & Skehan, P. (2003). Individual differences in second language learning. The Handbook of Second Language Acquisition, 589–630. El-Anzi, F. O. (2005). Academic achievement and its relationship with anxiety, self-esteem, optimism, and pessimism in Kuwaiti students. Social Behavior and Personality: An International Journal, 33(1), 95–104. https://doi.org/https://doi.org/10.2224/sbp.2005.33.1.95 Fook, C., Sidhu, G. K., Rani, N., & Aziz, N. A. (2011). Analyzing Factors Associated with Students‟ Oral Test Performance. The International Journal of Educational and Psychological Assessment, 9(1), 27–46. Gabuardi, V. F., & Sánchez, K. F. (2015). EFL Learners’ Preferences in the Implementation of Direct Learning Strategies in Learning Grammar. MEMORIA, 167. Gregersen, T., & Horwitz, E. K. (2002). Language Learning and Perfectionism: Anxious and Non-Anxious Language Learners’ Reactions to Their Own Oral Performance. The Modern Language Journal, 86(4), 562–570. https://doi.org/https://doi.org/10.1111/1540-4781.00161 Harris, S. L. (2009). The relationship between self-esteem and academic success among African American students in the minority engineering program at a research extensive university in the southern portion of the United States. Louisiana State University and Agricultural & Mechanical College. 300 Asian Journal of Applied Education (AJAE) Vol. 2, No. 3, 2023: 293 - 302 Asian Journal of Applied Education (AJAE) Vol. 2, No. 3, 2023: 293 - 302 Higgins, E. T. (1996). The" self digest": self-knowledge serving self-regulatory functions. Journal of Personality and Social Psychology, 71(6), 1062. Kesumawati, N., Retta, A. M., & Sari, N. (2017). Pengantar Statistika Penelitian. Depok: Rajawali Pers. Kimura, M. (2000). Pearson Education. Affective Factors of Japanese EFL Learners at Junior Coliege in the Oral Communication Tasks. Eibeibunka: Studies in English Language, Literature and Culture, 30, 5–19. Miraei, R. (2005). The relationship between self esteem, self concept and academic achievement among junior of high school’students. University of Tarbiat Moallem, Tehran, Iran. Molberg, H.-K. K. (2010). The consequence of motivation and linguistic self- confidence in relation to pupils’ oral interaction. Universitetet i Tromsø. Naderi, H., Abdullah, R., Aizan, H. T., Sharir, J., & Kumar, V. (2009). Self- esteem, gender and academic achievement of undergraduate students. American Journal of Scientific Research, 3(1), 26–37. Nagar, S., Sharma, S., & Chopra2, G. (2008). Self Esteem among Rural Adolescent Girls in Kangra District of Himachal Pradesh. The Anthropologist, 10(2), 151–154. https://doi.org/10.1080/09720073.2008.11891041 Pierce, J. L., Gardner, D. G., Cummings, L. L., & Dunham, R. B. (1989). Organization-Based Self-Esteem: Construct Definition, Measurement, And Validation. Academy of Management Journal, 32(3), 622–648. https://doi.org/10.5465/256437 Pullmann, H., & Allik, J. (2008). Relations of academic and general self-esteem to school achievement. Personality and Individual Differences, 45(6), 559–564. https://doi.org/https://doi.org/10.1016/j.paid.2008.06.017 Purwanto, M. B. (2022). Learning Community In Learning English Speaking Skills. Language and Education Journal, 7(2), 87–99. https://doi.org/https://doi.org/10.52237/lej.v7i2.383 Rubio, F. (2021). Self-esteem and foreign language learning. Cambridge Scholars Publishing. Skehan, P. (1991). Individual Differences in Second Language Learning. Studies in Second Language Acquisition, 13(2), 275–298. https://doi.org/DOI: 10.1017/S0272263100009979 Tallon, M. (2009). Foreign language anxiety and heritage students of Spanish: A quantitative study. Foreign Language Annals, 42(1), 112–137. https://doi.org/https://doi.org/10.1111/j.1944-9720.2009.01011.x Wright, G. B. (2011). Student-centered learning in higher education. International Journal of Teaching and Learning in Higher Education, 23(1), 92–97. Yahaya, A., & Ramli, J. (2009). The relationship between self-concept and 301 Hidayad, Lestari, Laoli, Anam, Despita communication skills towards academic achievement among secondary school students in Johor Bahru. International Journal of Psychological Studies, 1(2), 25. communication skills towards academic achievement among secondary school students in Johor Bahru. International Journal of Psychological Studies, 1(2), 25. Yamini, M., & TAHRIRI, A. R. (2006). On the relationship between foreign language classroom anxiety and global self-esteem among male and female students at different educational levels. Yashima, T., Zenuk‐Nishide, L., & Shimizu, K. (2004). The influence of attitudes and affect on willingness to communicate and second language communication. Language Learning, 54(1), 119–152. https://doi.org/https://doi.org/10.1111/j.1467-9922.2004.00250.x Yen, Y.-C., Hou, H.-T., & Chang, K. E. (2015). Pearson Education. Applying role-playing strategy to enhance learners’ writing and speaking skills in EFL courses using Facebook and Skype as learning tools: a case study in Taiwan. Computer Assisted Language Learning, 28(5), 383–406. https://doi.org/10.1080/09588221.2013.839568 Young, D. J. (1991). Creating a low-anxiety classroom environment: What does language anxiety research suggest? The Modern Language Journal, 75(4), 426– 439. https://doi.org/https://doi.org/10.2307/329492 Zubrick, S., & Silburn, S. (2006). Western Australian Aboriginal Child Health survey: improving the Educational experiences of Aboriginal children and Young people. Telethon Institute for Child Health Research and Curtin University of Technology. 302
https://openalex.org/W4221103879	https://edukatif.org/index.php/edukatif/article/download/2094/pdf	Indonesian	null	Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur	Edukatif	2,022	cc-by-sa	6,033	Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur Veronika Genua1, Alexander Bala2 Universitas Flores, Indonesia1,2 E-mail : nikaruing1971@gmail.com1, naradiring@gmail.com2 Abstrak Tujuan dari penulisan ini adalah untuk memaparkan khazanah teks ritual letu umn pada etnik Flores Timur. Ritual letu umne merupakan ritual menutup lubang tanah yang telah digali untuk membuat rumah tempat tinggal etnik tersebut. Ritual tersebut tetap dijalankan sebagai warisan dari pada leluhur secara turun temurun dan tetap dilaksanakan hingga saat ini. Rumusan masalah yang diangkat pada tulisan ini adalah;1) bagaimanakah bentuk khazanah teks ritual adat letu umne pada etnik Flores Timur NTT; 2) bagaimanakah fungsi khazanah teks ritual adat letu umne pada etnik Flores Timur NTT; 3) bagaimanakah makna khazanah teks ritual adat letu umne pada etnik Flores Timur NTT. Tujuannya adalah untuk menemukan dan mendeskripikan bentuk, fungsi dan makna khazanah teks ritual letu umne pada etnik Flores Timur. Metode yang digunakan adalah deskriptif kualitatif untuk memaparkan teks ritual letu umne Penelitian tersebut menunjukkan bahwa terdapat khazanah kebahasaan dalam kajian linguistik yakni bentuk morfologi yang meliputi nomina, verba, pronomina, adverbial, dan numeralia. Untuk fungsi bahasa meliputi fungsi interpersonal dan fungsi kekerabatan, sedangkan makna yag terkandung dalam tek meliputi makna religius, perlindungan dan makna simbol seperti tuak “moke” , dan braha “kapas”. Kata Kunci: Teks, letu umne, ritual, tradisi. Edukatif : Jurnal Ilmu Pendidikan Volume 4 Nomor 1 Tahun 2022 Halm 1263 - 1273 EDUKATIF: JURNAL ILMU PENDIDIKAN Research & Learning in Education https://edukatif.org/index.php/edukatif/index PENDAHULUAN Setiap etnik atau daerah tentunya memiliki tradisi atau adat istiadat setempat. Setiap tradisi tentu menggunakan bahasa untuk mengkominkasikan semua ide atau hal apa saja yang akan dilaksanakan. Bahasa tersebut memiliki peranan penting dalam kehidupan sehari-hari untuk dapat berinteraksi antar etnik yang satu dengan yang lainnya. Kenyataan yang dihadapi saat ini terlihat bahwa semua orang dalam kehidupan sehari-hari selalu mempraktekan bahasa baik dalam komunikasi secara lisan maupun tulisan. Orang menyadari bahwa interaksi dalam segala kegiatan dalam etnik akan lumpuh tanpa bahasa. Begitu pula melalui bahasa, kebudayaan suatu bangsa dapat dibentuk, dibina, dan dikembangkan serta dapat diturunkan kepada generasi-generasi mendatang (Fauzan 2021) Bahasa sebagai alat komunikasi verbal lisan hanya dimiliki manusia, karena hanya manusia yang memiliki bahasa. Tidak ada manusia yang hidup tanpa bahasa. Manusia dan bahasa bagai benang dan jarum. Jadi, dimana ada bahasa di situ ada manusia, dan atau sebaliknya dimana ada manusia, di situ ada bahasa. Bahasa merupakan alat komunikasi yang digunakan manusia dalam kehidupan sehari-hari untuk menyampaikan pesan dan maksud penutur kepada lawan tutur. Peran bahasa sangat penting bagi kehidupan manusia. Begitu pentingnya peran bahasa maka setiap orang dituntut untuk dapat menguasai atau memiliki bahasa dan memahami bahasa yang disampaikan. Bahasa merupakan instrument atau sarana berpikir dan bertindak yang digunakan manusia untuk saling berkumnikasi dan berinteraksi secaralisan maupun tulisan, maupun gerakan badan atau isyarat (Eiilerz 1995). Bahasa daerah adalah sebuah komponen kebudayaan. Bahasa daerah tidak hanya berfungsi sebagai salah satu unsur budaya lokal, jati diri etnik penutur, lambang kebanggaan daerah, fakta kebhinekaan dan keragaman budaya bangsa, serta dapat menambah khazanah budaya nasional. Selain itu juga bahasa digunakan sebagai sarana untuk memahami dan menginformasikan makna dan nilai berbagai fenomena sosial budaya etnik (Pampe 2009). Bahasa daerah tidak dapat dipisahkan dari kehidupan etnik itu sendiri, karena bahasa daerah disajikan dan dimanifestasikan untuk kepentingan etnik. Dengan demikian dapat dikatakan bahasa daerah merupakan milik etnik daerah. Bahasa memiliki peranan penting dalam kehidupan etnik Hal tersebut dikarenakan bahasa merupakan alat pemersatu dalam kehidupan etnik. Selain itu, bahasa digunakan sebagai alat untuk mnyampaikan maksud dan pikiran seseorang untuk dapat berkomunikasi dengan orang lain. Pada dasarnya, manusia adalah makluk sosial. Artinya manusia diciptakan untuk hidup bersama dan berdampingan dengan manisia yang lain. Manusia harus hidup bersama untuk memenuhi kebutuhan, antara yang satu dengan yang lainnya. Interaksi ini terlihat jelas melalui pemakaian bahasa. Bahasa daerah menjadi media utama etnik di pedesaan dalam berinteraksi. Bahasa daerah merupakan milik etnik secara kolektif. Abstract The purpose of this paper is to describe the treasures of the letu umn ritual text on the East Flores ethnicity. The letu umne ritual is a ritual to cover a hole in the ground that has been dug to make a house for the ethnic group to live in. The ritual is still carried out as a legacy from the ancestors from generation to generation and is still carried out today. The formulation of the problems raised in this paper are; 1) what is the form of the treasures of the traditional letu umne ritual text on the East Flores ethnicity of NTT; 2) what is the function of the traditional ritual text of letu umne in the East Flores ethnicity of NTT; 3) what is the meaning of the traditional letu umne ritual text for the East Flores ethnicity of NTT. The aim is to find and describe the form, function and meaning of the treasures of the letu umne ritual text on the East Flores ethnicity. The method used is descriptive qualitative to describe the ritual text of letu umne. The research shows that there are linguistic treasures in linguistic studies, namely morphological forms which include nouns, verbs, pronouns, adverbs, and numerals. The language function includes interpersonal functions and kinship functions, while the meaning contained in tek includes religious meaning, protection and the meaning of symbols such as tuak "moke", and braha “cotton”. d l l d Keywords: Text, letu umne, ritual, tradition Copyright (c) 2022 Veronika Genua, Alexander Bala Copyright (c) 2022 Veronika Genua, Alexander Bala Copyright (c) 2022 Veronika Genua, Alexander Bala Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Copyright (c) 2022 Veronika Genua, Alexander Bala  Corresponding author Email : nikaruing1971@gmail.com ISSN 2656-8063 (Media Cetak) DOI : https://doi.org/10.31004/edukatif.v4i1.2094 ISSN 2656-8071 (Media Online) ISSN 2656-8063 (Media Cetak) ISSN 2656-8071 (Media Online) ISSN 2656-8063 (Media Cetak) ISSN 2656-8071 (Media Online) Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 1264 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 1264 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 PENDAHULUAN Etnik mengggunakan bahasa daerah untuk bertindak, berpikir dan mengungkapkan apa yang ada dalam dirinya. Sebagai alat komunikasi bahasa merupakan saluran perumusan maksud seseorang melahirkan perasaan dan memungkinkan seseorang menciptakan kerja sama dengan sesama warga. Bahasa mengatur berbagai aktivitas keetnikan, merencanakan dan mengarahkan masa depan kita. Bahasa juga memunginkan manusia menganalisa masa lampaunya untuk memetik hasil- hasil yang berguna bagi masa kini dan masa yang akan datang (Keraf 2004). Menurut (Pampe 2009) bahasa daerah tidak dapat dipisahkan dari kehidupan etnik itu sendiri, karena bahasa daerah disajikan dan dimanifestasikan untuk kepentingan etnik. Dengan demikian dapat dikatakan bahasa daerah merupakan milik etnik daerah. Manusia pada dasarnya ialah makluk sosial. Artinya manusia diciptakan untuk hidup bersama dan berdampingan dengan manisia yang lain. Manusia harus hidup bersama untuk memenuhi kebutuhan, antara yang satu dengan yang lainnya. Interaksi ini terlihat jelas melalui pemakaian bahasa. Bahasa daerah menjadi media utama etnik di pedesaan dalam berinteraksi. Bahasa daerah merupakan milik etnik secara kolektif. Etnik mengggunakan bahasa daerah untuk bertindak, berpikir dan Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 1265 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 1265 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 mengungkapkan apa yang ada dalam dirinya. Pemberian pengetahuan yang berbudaya lokal (Nur, Fadhilah and Rahmawati 2020). mengungkapkan apa yang ada dalam dirinya. Pemberian pengetahuan yang berbudaya lokal (Nur, Fadhilah and Rahmawati 2020). Menurut Haugen (Ola 2013:133) meyatakan bahwa bahasa hadir dalam pikiran penuturnya dan bahasa hanya dapat berfungsi ketika penuturnya dapat menghubungkan para penutur satu sama lain dan dapat menghubungkan bahasa dengan alam. Sebagai alat komunikasi bahasa merupakan saluran perumusan maksud seseorang melahirkan perasaan dan memungkinkan seseorang menciptakan kerja sama dengan sesama warga. Bahasa mengatur berbagai aktivitas keetnikan, merencanakan dan mengarahkan masa depan kita. Bahasa juga memunginkan manusia menganalisa masa lampaunya untuk memetik hasil- hasil yang berguna bagi masa kini dan masa yang akan datang (Keraf 2004). Bahasa daerah dapat dikenali melalui suatu daerah dan kebudayaan tertentu. Di sisi lain, bahasa daerah dapat membantu seseorang memahami kehidupan kelompok etnik yang memakai bahasa daerah tersebut. Bahasa bersifat unik artinya tiap bahasa mempunyai sistem yang khas dan tidak harus ada dalam bahasa lain. PENDAHULUAN Selain sebagai sarana komunikasi sehari-hari bahasa daerah Lamaholot di Flores Timur merupakan sarana teks adat yang digunakan dalam ritual letu umne tradisi pembuatan rumah adat itu pada hakekatnya tidak dituturkan sebagai alat komunikasi biasa dalam kehidupan sehari-hari tetapi diungkapkan khusus dalam ritual-ritual adat. Apresiasi terhadap kebudayaan adalah penilaian atau penghargaan terhadap nilai-nilai yang terkandung dalam setiap kebudayaan yang merupakan hasil cipta, rasa, dan karsa manusia. Kebudayaan bukanlah sesuatu yang bersifat individual, tetapi merupakan milik bersama dalam suatu kelompok warganya milik bersama. Selanjutnya terdapat sejumlah pola pikir dan dapat dikembangkan melalui proses belajar. Hal tersebut wajib dimiliki bersama menjadi sesuatu yang khas dan unik, bagi setiap pemiliknya. Proses penciptaan kebudayaan merupakan proses pencerdasan yang hasilnya mampu meningkatkan harkat dan martabat bangsa dan negara. Jika anak bangsa dibiarkan menjadi user hasil karya budaya asing, maka yang terjadi adalah proses keterasingan atau modernisasi semu (Rahyono 2015: 59). Tuturan adat erat kaitannya dengan perkembangan budaya berpikir dan bertindak, etnik pendukung dan pelakunya. Di samping itu tuturan adat dapat pula digunakan sebagai sarana pendidikan bagi generasi muda melalui etnik setempat. Salah satu ritual yang tetap dilaksanakan oleh etnik Flores Timur yakni ritual Letu umne pada saat pembuatan rumah dengan menggunakan bahasa daerah Lamaholot. Sudah menjadi suatu tradisi bagi bahwa dalam teks ritual adat terkandung berbagai makna yang terkandung di dalamnya. Upacara rumah biasanya dilaksanakan setelah diadakan renovasi bangunan atau membuat rumah baru. Upacara ini merupakan suatu pesta besar yang harus dihadiri oleh seruruh kerabat dan keluarga yang berasal dari rumah adat tersebut (Isu 2021). Bahasa daerah dapat dikenali melalui suatu daerah dan kebudayaan tertentu. Memperkenalkan suatu budaya tradisi adat istiadat dengan tujuan untuk membangun kecintaan setiap masyarakat akan budaya yang dimilikinya. Selain itu setiap masyarakat berusaha untuk tetap melestarikanya seperti tarian atau pun pembuatan rumah adat (Riyanti, Irfani, and Prasetyo 2022). Di sisi lain mengenai bahasa daerah dapat membantu seseorang memahami kehidupan kelompok etnik serta pola tingkah laku yang dapat mengatur etnik tersebut. Selain sebagai sarana komunikasi sehari-hari bahasa merupakan sarana teks adat yang dapat digunakan dalam ritual letu umne ” pengerjaan rumah” sebagai tempat huni setiap warganya. Pengerjaan rumah berkaitan dengan kebudayaan yang merupakan aspek yang paling inti dalam kehidupan manusia. Segala hirarki kajian bahasa termasuk dalam bidang antropolinguistik yang membicarakan tentang bahasa dan budaya (Sibarani 2015). Di tengah peradaban yang begitu pesat, setiap etnik selalu berpegang teguh pada warisan dari para leluhur yang telah diwariskan secara turun temurun. PENDAHULUAN Rasa kekeluargaan dan kebersamaan selalu dipupuk oleh setiap etnik yang berada pada wilah tersebut (Hasanah and Sukmawan 2021). Berdasarkan pandangan yang disampaikan tersebut dapat dikatakan bahwa etnik Flores Timur tetap mewariskan tradisi yang diturunkan oleh leluhur pada zaman dahulu. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 1266 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 Kebudayaan yang diturunkan sangat erat kaitan dengan teks ritual yang disesuaikan dengan perkembangan budaya berpikir dan bertindak suatu etnik. Pengajaran budaya melalui suatu cerita memiliki pesan moral yang sudah dilakukan ribuan tahun lalu. Cerita yang sama tersebut dapat diteruskan dari generasi ke generasi dan mengalami distori sehingga tak lagi diketahui kebenarannya (Angeline 2015). Selain cerita, tuturan adat dapat pula digunakan. Hal tersebut sudah menjadi suatu tradisi dalam ritual adat digunakan. Setiap teks adat yang mengungkapkan perasaan, perwujudan sikap etnik dalam hidupnya, karena etnik Flores Timur yakin melalui ritual tersebut para leluhur mengabulkan keinginan dan harapan yang disampaikan. Dalam ritual letu umne pada saat pembuatan rumah terdapat makna tuturan adat yang dituturkan oleh tua adat. Setiap teks tersebut terkandung berbagai makan di dalamnya yang mempunyai dampak bagi etnik setempat. Ritual Letu umne pada saat pembuatan rumah terdapat bentuk tuturan adat yang dituturkan oleh Tua Adat atau juru bicara. Teks dalam pembuatan rumah dapat dijadikan sebagai unsur penting untuk pembinaan dan pengembangan bahasa dapat dilakukan dengan berbagai cara. Salah satunya adalah penelitian dan inventarisasi bahasa secara khusus bahasa daerah (Muhidin 2015). Selain sebagai sarana komunikasi sehari-hari, bahasa juga sebagai sarana tuturan adat yang dapat digunakan dalam ritual-ritual adat misalnya, ritual-ritual mohon berkat hasil panen, ritual perkawinan serta ritual-ritual resmi lainnya. Ritual letu umne pada saat pembuatan rumah merupakan salah satu ritual yang sering dilakukan etnik Flores Timur ketika sebuah rumah siap untuk dihuni. Tuturan dalam ritual letu umne pada saat pembuatan rumah merupakan sarana ekspresi dalam hubungan adat untuk bersyukur kepada sang pencipta dan kepada nenek moyang sekalian sebagai acara syukuran karena telah menyelesaikan pembangunan rumah dan sebagai tanda bahwa rumah tersebut telah layak untuk dihuni. PENDAHULUAN Ritual letu umne pada saat pembuatan rumah juga secara turun temurun dilakukan etnik Flores Timur sangat penting dan sebagai bukti untuk pemahaman generasi muda agar lebih mengenal dan mengetahui setiap makna teks yang terkandung di dalamnya. Perencanaan berbasis lokal untuk menjawan tantangan global yang dapat mengkikis pemahaman masyarak tentang budaya dan adat istiadat (Umri , Ulili and Hadiyanto 2021). Berikut ini salah satu teks adat letu umne dalam mengerjakan rumah adat pada etnik Flores Timur. Ina ratu rera wulan, Tuhan ratu yang maha bulan Tuhan yang Maha Kuasa ama nini tanah ekan, Bapak ibu tanah bumi Para leluhur di bumi teti modi lodo hau, di atas dengan turun datang Datanglah di tengah kami Lali modi gere haka, Berikut ini salah satu teks adat Ina ratu rera wulan, Tuhan ratu yang maha bulan Tuhan yang Maha Kuasa ama nini tanah ekan, Bapak ibu tanah bumi Para leluhur di bumi teti modi lodo hau, di atas dengan turun datang Datanglah di tengah kami Lali modi gere haka, Teks yang dipaparkan tersebut bermakna religius, yakni memohon kepada Tuhan Yang Maha Kuasa dan juga meneghadirkan leluhur untuk bersama merestui dan meyaksikan pengerjaan rumah tempat hunian dari etnik setempat. Teks yang dituturkan oleh tua adat setempat agar proses yang akan dilakukan dapat berjalan dengan lancar. Sikap mempertahankan budaya menggambarkan kepatuhan masyarakat terhadap warisan leluhur (Manek 2021). Hal tersebut juga sebagai bahan pembelajaran dari generasi penerus untuk dapat mengetahuinya agar budaya setempat tetap diwariskan agar tidak punah. Kenyataan yang terjadi saat ini, pada umumnya generasi penerus atau generasi muda kurang mempedulikan tentang budaya daerah setempat. Generasi muda saat ini, begitu sibuk dengan permainan game secara online, sibuk catting, tik tok, dan berbagai media sosial lainnya. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 1267 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 1267 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 Untuk itu penelitian ini perlu dipublikasikan untuk pengetahuan generasi selanjutnya. Selain itu dapat menjadi bahan pembelajaran dalam muatan lokal bagi para siswa di sekolah untuk mengenal dan memahami pesan dan makna yang terkandung dalam budaya daerah. METODE PENELITIAN Penelitian yang digunakan dalam kajian ini yakni penelitian deskriptif kualitatif untuk mendeksripsikan secara lengkap tentang teks adat yang dituturkan pada saat pengerjan rumah dan proses yang dilakukan. Penelitian ini dilaksanakan di Flores Timur Flores NTT tentang teks pengerjaan rumah yakni letu umne. Data yang digunakan yakni data lisan tentang teks adat yang dituturkan oleh tua adat setempat. Sumber data diperoleh dari tua adat setempat, budayawan, tokoh etnik dan orang yang paham tentang budaya dan adat istiadat setempat. Teknik dalam pengumpulan data yakni penelitian lapangan dengan dengann melakukan wawancara dengan narasumber untuk mendapatkan informasi tentang teks adat dan makna dibalik setiap teks letu umne dituturkan oleh narasumber. Setelah melakukan wawancara, data tersebut ditranskripkan ke dalam bahasa Indonesia untuk menentuk bentuk,dan makna yang terkandung didalamnya. Selain melakukan wawancara, penulis mencatat hal-hal penting yang berkaitan dengan teks adat dalam pengerjaan rumah. Selanjutnya menggunakan teknik rekam dan dokumentasi untuk untuk memperoleh data secara keseluruhan yang berkaiatan dengan tradisi setempat. Analisis data dengan menggunakan model analisis interaktif yang dikembangkan oleh (Miles, M., & Huberman 1992:16) ada empat komponen yang dilakukan dalam model ini, yakni pengumpulan data, reduksi data display data, menarik kesimpulan atau verifikasi. Komponen tersebut dilakukan untuk menganalisis data secara terperinci. PENDAHULUAN Permasalahan yang diangkat dalam tulisan ini adalah 1) bagaimanakah bentuk khazanah teks ritual letu umne pada etnik Flores Timur, 2) bagaimanakah fungsi khazanah teks ritual letu umne pada etnik Flores Timur. 3) bagaimanakah makna khazanah teks ritual letu umne pada etnik Flores Timur Tujuannya untuk menemukan dan mendeskripsikan bentuk dan makna khazanah teks ritual letu umne pada etnik Flores Timur. Metode yang digunakan dalam kajian tersebut yakni deskripsi kualitatif untuk memaparkan khazanah teks letu umne serta bentuk dan makna yang terkandung didalamnya. Teknik yang digunakan yakni catat, wawancara, dan dokumentasi. Teori yang digunakan dalam tulisan ini yakni teori linguistik kebudayaan. Linguistik kebudayaan/linguistik budaya membuka cakrawala baru untuk mencari tahu lebih lanjut, apa yang ada di dalamnya, jati diri, prinsip, metode, dan cara kerjanya. Linguistik kebudayaan sesungguhnya adalah bidang ilmu interdisipliner yang mengkaji hubungan kovariatif antara struktur bahasa dengan kebudayaan suatu etnik menurut Mbete dalam (Genua 2019). Berdasarkan teori tersebut terdapat kebaruan dalam penelitin yakni terdapat makna sosiologis yakni hubungan masyarakat dengan para leluhur yang diyakini ada namu tidak terlihat secara kasat mata. Penelitian terdahulu tentang teks dalam suatu tarian adat namun, tulisan ini berkaitan dengan teks yang berkaitan dengan pengerjan rumah yang dihuni masyarakat. Penelitian ini sangat penting dilakukan dalam kaitan dengan budaya agar masyarakat secara khusus generasi muda dapat memahami makna yang terkandung dalam teks adat letu umne tersebut. a. Nomina Gute mala witi sora pile mala wawe wajo Ambil ambil kambing itu, pilih ambil babi itu Ambillah kambing dan babi itu Penggunaan kata benda witi ‘kambing’ dan wawe ‘babi’ terlihat pada kalimat sebagai berikut: Data tersebut dikatakan bahwa nomina witi ‘kambing’ dan wawe ‘babi’ berkategori nomina karena merupakan hewan korban yang akan di ambil darah untuk dioles pada tiang rumah dan hatinya untuk disajikan kepada nenek moyang. HASIL DAN PEMBAHASAN PENELITIAN Teks letu umne merupakan ritual yang dilakukan menutup lubang tanah yang digali pada saat pembuatan rumah untuk dihuni oleh pemiliknya. Setiap teks yang disampaikan dalam ritual adat tersebut dianalisis dari bentuk, fungsi dan makna yang terkandung didalamnya yang dapat dijadikan sebagai bahan pembelajaran terhadap bahasa. Berikut ini akan dipaparkan bentuk, fungsi, dan makna dalam teks letu umne tradisi etnik Flores Timur. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 1268 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 1. Bentuk Teks adat Letu Umne pada Etnik Flores Timur Setiap teks memiliki bentuk yang terkandung di dalamnya. Bentuk teks letu umne dapat dikaji dari bentuk mikrolinguistik berikut ini. Ciri Morfologi dalam teks adat dalam Ritual Letu umne dapat dipaparkan berikut ini. a. Nomina Nomina merupakan kelas kata yang berfungsi sebagai subjek dan objek yang yang mengacu pada orang, benda dan hal lain yang dibendakan. Nomina merupakan kelas kata yang berfungsi sebagai subjek atau objek dari klausa. Nomina berpadanan dengan orang atau benda atau hal lain yang dibendakan (Kridalaksana, 2008:163). Data nomina dapat dilihat pada kata /frasa yang bercetak tebal berikut. Tahap Pertama Soro Nei “Pemberian sesajian kepada nenek moyang” D t (1) Tahap Pertama Soro Nei “Pemberian sesajian kepada nenek moyang” Nene moyang wahan kae, pi ume lamak mio Nenek moyang sudah semua, ini makanan kamu Para leluhur terimalah semua pemberian kami Arak, teluk, taha, no braha ume lamak mio Moke, telur, beras, dan kapas makanan kamu Moke, telur, beras, dan kapas kami sajikan Maja ge menu hama-hama Nene moyang wahan kae, pi ume lamak mio Nenek moyang sudah semua, ini makanan kamu Para leluhur terimalah semua pemberian kami Arak, teluk, taha, no braha ume lamak mio Moke, telur, beras, dan kapas makanan kamu Moke, telur, beras, dan kapas kami sajikan Maja ge menu hama-hama Nene moyang wahan kae, pi Berdasarkan data (1) nomina seperti frasa nenek moyang, ume lamak termasuk nomina majemuk. Frasa nomina nenek moyang merupakan nomina orang yang telah meninggal sedangkan dan ume lamak bentuk makanan yang telah disajikan peda para leluhur. Selanjutnya nomina Arak, teluk, taha, dan braha bahan sesajen yang akan dipersembahkan kepada para leluhur. Kelompok nomina selanjutnya terlihat pada kalimat berikut ini . Hone no’o lango kae, wani no’o uli kae Membuat dengan rumah, sudah membuat dengan tempat istirahat Sudah membangun rumah kediaman, sudah membuat tempat peristirahatan Lango puna dike kae, uli liput sare kae Rumah sudah jadi, tempat istirahat sudah dibuat dengan baik Rumah kediaman sudah dibangun Kelompok nomina selanjutnya terlihat pada kalimat berikut ini . Hone no’o lango kae, wani no’o uli kae Membuat dengan rumah, sudah membuat dengan tempat istirahat Sudah membangun rumah kediaman, sudah membuat tempat peristirahatan Lango puna dike kae, uli liput sare kae Rumah sudah jadi, tempat istirahat sudah dibuat dengan baik Rumah kediaman sudah dibangun Nomina dalam teks tersebut terlihat pada kata yang bercetak tebal yakni lango ‘’rumah’ lango ‘merupakan suatu tempat yang akan dihuni oleh pemilik rumah pada etnik setempat. Selanjutnya data lain yang menunjukkan nomina dapat dilihat pada kalimat berikut ini yang menunjukkan nomina dapat dilihat pada kalimat berikut ini. yang menunjukkan nomina dapat dilihat pada kalimat berikut ini. Pana nala dute waen, dei nala jadi ana Pergi mengambil istri, berdiri melahirkan anak Pergi mengambil istri, berdiri melahirkan anak Pergi mengambil istri, dan melahirkan anak-anak Pemakaian kata ‘dute’ ‘ambil’ nala ‘ambil’ dei nala ‘ berdiri melahirkan’ pada kalimat tersebut menggambarkan atau menyatakan untuk mengambil istri dan sudah melahirkan anak-anak. Verda pada kaliat tersebut merupakan kelompok verba berderet. c. Numeralia Numeralia merupakan kelas kata atau kategori yang dapat mendampingi nomina dalam konstruksi sintaksis. bilangan adalah kata yang menyatakan jumlah, urutan dan tingkatan suatu benda (Djajasudarma, 2010: 49). Numeralia pada teks letu umne dapat dilihat pada teks atau kalimat beriku ini. Kata bilangan terdapat terdapat pada kata yang bercetak tebal berikut ini. Numeralia puluh pito ‘tujuh puluh’ dan numeralia puluh lema ‘lima puluh’ merupakan numeralia utama atau disebut denan bilangan penuh. Secara lengkap numeralia dapat dilihat pada kalimat berikut ini. Numeralia puluh pito ‘tujuh puluh’ dan numeralia puluh lema ‘lima puluh’ merupakan numeralia utama atau disebut denan bilangan penuh. Secara lengkap numeralia dapat dilihat pada kalimat berikut ini. Teti wan puluh pito, lali wade puluh lema dan puluh lema ‘lima puluh’ dari tuturan Diatas tujuh puluh lapis, Dibawah lima puluh lapis Diatas tujuh puluh lapis, Dibawah lima puluh lapis Yang berada dilangit yang ketujuh puluh, dan dibawah lapisan bumi kelima puluh’. Penggunaan kata puluh pito ‘tujuh puluh’ tersebut mengandung arti bahwa lapisan keturunan yang sudah banyak. b. Verba Verba merupakan sebuah kata yang menyatakan keadaan, perbuatan atau tindakan yang dapat berubah bentuk dengan sistem pengkonjungsian dalam kalimat dan berfungsi sebagai predikator. Verba dalam teks letu umne menggambarkan perbuatan, keadaan, atau proses yang dilakukan pada saat ritual mbama berlangsung. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 1269 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 1269 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 Verba yang dimaksud yakni merupakan kata yang menjelaskan suatu kegiatan atau pekerjaan. Contoh verba dapat dilihat pada kata/ frasa yang bercetak tebal pada kalimat berikut ini. Maja ge menu hama-hama Maja ge menu hama-hama Panggil makan sama-sama Mengajak semua leluhur dimanapun berada supaya bisa makan bersama’ Mengajak semua leluhur dimanapun berada supaya bisa makan bersama’ Mengajak semua leluhur dimanapun berada supaya bisa makan bersama’ Verba pada teks tersebut terlihat pada kata yang bercetak tebal yakni maja ‘panggil’ Kata maja ‘mengajak para leluhur untuk datang berkumpul makan bersama. Verba pada teks tersebut terlihat pada kata yang bercetak tebal yakni maja ‘panggil’ Kata maja ‘mengajak para leluhur untuk datang berkumpul makan bersama. Verba selanjutnya terdapat pada kalimat berikut ini . Hone no’o lango kae, wani no’o uli kae Membuat sebuah rumah, membuat tempat istirahat membangun rumah kediaman sebagai tempat tinggal Verba dalam teks tersebut yakni hone ‘membuat’ dan wani ‘membuat’ merupakan verba berderet yang terdapat dalam satu kalimat. Pana nala dute waen, dei nala jadi ana e. Adverbial Adverbia merupakan kata yang memerikan verba, adjektiva, preposisi, atau adverbial lain (Kridalaksana, 2009: 2). Adverbia merupakan kata yang menunjukkan atau menjelaskan berlangsungnya suatu peristiwa dalam suatu peristiwa dalam suatu waktu. Data keterangan terlihat pada kata yang bercetak tebal berikut ini. Teti : di atas sana (keterangan tempat) Teti : di atas sana (keterangan tempat) Lali : di bawah sana (keterangan tempat) Lewo : kampung (keterangan tempat) Kata keterangan teti ‘di atas sana’ dan lali ‘di bawah sana’ terdapat dalam tuturan ritual Letu umne kalimat sebagai berikut: Kata keterangan teti ‘di atas’ dan lali ‘di sana’ terdapat dalam tuturan upacara letu tana umne kalimat sebagai berikut: Teti modi lodo hau, lali modi gere haka modi gere haka Diatas sana juga datang kesini, dibawah sana naik kesini Diatas sana juga datang kesini, dibawah sana naik kesini Turunlah ditengah-tengah kami, dan masuklah juga ditengah-tengah kami Turunlah ditengah-tengah kami, dan masuklah juga ditengah-tengah kami Turunlah ditengah-tengah kami, dan masuklah juga ditengah-tengah kami Pemakaian kata teti ‘di atas sana’ dan lali ‘di bawah sana’ menunjukkan suatu tempat yang menjadi ediaman nenek moyang. Data selanjutnya yang menunjukkan adverbial dapat dilihat pada kalimat berikut ini. Data selanjutnya yang menunjukkan adverbial dapat dilihat pada kalimat Kaka pia tobo lewo kame, bapa pia pae tana kame Kakak duduk dikampung kami, bapak juga mendiami tanah kami Kakak mendiami kampung kami, bapak juga mendiami tanah kami’ Dari tuturan tersebut yakni lewo ‘kampung’ merupakan tempat tinggal untuk semua orang yang mendiami wilayah tersebut yang merupakan pengelompokkan adverbial. d. Pronomina Pronomina atau kata ganti adalah jenis kelas kata yang menggantikan nomina atau frasa nomina. Teks letu umne juga memiliki beberapa pronominal baik dalam satu kalimat maupun pada kalimat lainnya. Data pronomina dalam teks ritual letu umne yakni kami “kami” dan miu “kamu” dapat dilihat pada kalimat berikut ini. Kaka pia tobo Kaka pia tobo lewo kame lewo kame lewo ka kaka sini duduk kampong 2TGL kaka duduk di kampung kami Pronomina atau kata ganti kami merupakan kata ganti orang pertama jamak. Kata ganti tersebut menunjukkan bahwa guyub kultur setempat menggunakan kata kami ‘kami”yakni etnik setempat untuk duduk di kampung untuk menempati rumah yang sudah disiapkan. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 1270 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 Berikut kata pia ‘sini” menunjukkan pronominal demontrativa yang digunakan untuk menunjuk suatu tempat tinggal yang baru yaitu di kampung asal. 3. Makna Teks Letu Umne pada Etnik Flores Timur Setiap teks tentunya terdapat berbagai makna yang terkandung di dalamnya. sama halnya dengan teks letu umne pada etnik Flores Timur. beberapa makna yang terkandung didalamnya dapat dipaparkan berikut ini. a. Makna Religius Makna religius merupakan penyampaian permohonan dan syukur kepada Tuhan sebelum melaksanakan kegiatan upacara letu umne tua adat memohon Tuhan dan kepada nenek moyang yang telah meninggal dunia untuk merestui kegiatan letu umne dan sebagai makluk yang lemah dan selalu berharap dan bersandar pada Tuhan seperti terlihat pada kalimat berikut ini. Ina ratu rera wulan, Ama nini tana ekan Penguasa matahari dan bulan, penguasa bumi Tuhan yang maha kuasa dan para nenek moyang Teti wan puluh pito, lali wade puluh lema Yang berada dilangit yang ketujuh puluh, dan dibawah lapisan bumi kelima puluh’ Tuturan dalam makna religius di atas mengandung pengertian bahwa Tuhan yang Maha Kuasa dan para leluhur dimana pun berada agar melindungi dan memberi keamanan pada rumah kami. Selain itu sebagai media untuk menjalin hubungan dengan sesuatu yang lebih tinggi yang diyakini sebagai sang pencipta. b. Fungsi Kekerabatan Fungsi kekerabatan secara khusus yang merupakan gambaran hubungan atau kedekatan antara warga masyarakat pewaris kebudayaan tersebut. Fungsi kekerabatan dengan maksud untuk mendorong dan menumbuhkan nilai positif yang sudah mulai merdup dalam masyarakat seperti suka menolong atau berbuat baik (Genua 2013). Kalimat-kalimat yang menyatakan fungsi kekerabatan dapat terlihat pada kalimat berikut ini. Nene moyang wahan kae, pi ume lamak mio Nene moyang wahan kae, pi ume lamak mio Nenek moyang semua, ini makanan kamu Para leluhur semua terimalah bagianmu ini Maja ge menu hama-hama Panggil makan sama-sama Meyapa luluhur untuk makan bersama Berdasarkan kalimat tersebut menggambarkan sebuah upacara pemberian makan kepada nenek moyang atau leluhur sebagai bentuk jalinan kekerabatan. Selain itu merupakan gambaran hubungan atau kedekatan antara warga masyarakat pewaris kebudayaan tersebut. b. Makna Perlindungan Makna perlindungan ini adalah Tuhan Penguasa Yang Maha Kuasa serta penguasa bumi lindungilah agar rumah kediaman dan tempat peristirahatan yang di bangun dapat memberi ketentraman dan kedamaian kepada kami seisi rumah. Dalam memilih kebiasaan hidup sehari-hari, bahkan dalam hal kecil sekalipun, masyarakat selalu memilih kebiasaan hidup sesuai sesuai dengan pemikirannya. Hal tersebut berkaitan dengan pembuatan rumah rempat tinggal yang dapat memberi perlindungan bagi penghuni didalammnya (Lokanawa 2013). Data tuturan tersebut dapat terlihat berikut ini. 2. Fungsi Tuturan dalam Upacara Letu Umne a. Fungsi Interpersonal Fungsi interpersonal merupakan fungsi bahasa yang digunakan untuk menjalin dan memelihara hubungan sosial. Hal ini terlihat pada tuturan tahap soro nei yaitu semua leluhur yang telah meninggal diberi arak, teluk, taha, serta braha untuk nenek moyang merupakan suatu bentuk penghargaan dalam menjalin suatu hubungan yang erat agar hubungan itu tidak hanya kepada yang masih hidup, karena nenek moyang sebagai media dalam menyampaikan permohonan kepada Maha Kuasa. Masyarakat atau etnik setempat yakin bahwa segala sesuatu harus bergapang teguh pada peninggalan leluhur yang telah diwariskan secara turun – temurun. dan tetap dipertahankan gar tidak punah.(Hasanah and Sukmawan 2021). Hal tersebut dapat dilihat pada fungsi interpersonal pada kutipan kalimat berikut ini. Nene moyang wahan kae, pi ume lamak mio Nenek moyang semua, ini makanan kamu Para leluhur semua terimalah bagianmu ini Maja ge menu hama-hama Panggil makan sama-sama Panggilah semua leluhur dimanapun berada supaya bisa makan bersama Kalimat tersebut mengandung pengertian bahwa suatu bentuk penghargaan dalam menjalin suatu hubungan yang erat agar hubungan itu tidak hanya kepada yang masih hidup, karena nenek moyang merupakan media dalam menyampaikan permohonan kepada Tuhan. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 c. Makna Simbolis Makna simbol yang dimaksudkan dalam tulisan ini merupakan sarana yang yang akan digunkan sebagai bahan sesajian kepada para leluhur seperti berikut ini moke, telur, beras, dan kapas makan kamu yang bisa kami sajikan (Khoiri 2009). Simbol-simbol dalam upacara yang dilakukan bertujuan sebagai sarana untuk menunjukkan secara semu maksud dan tujuan upacara yang dilakukan oleh masyarakat pendukungnya. Data tersebut yakni berupa arak ‘moke’, teluk ‘telur’, taha ‘beras’, dan braha ‘kapas’. Melambangkan kebersamaan. Kebersamaan dalam hal ini simbol dalam menjamu orang yang datang bertamu sekaligus menjalin relasi dengan sesama baik yang masih hihup maupun dengan para arwah agar hubungannya intim dan erat. Arak‘moke’, teluk ‘telur’, taha ‘beras’, dan braha ‘kapas’ sebagai persembahan pada upacara letu umne merupakan simbol dalam menjamu orang yang datang bertamu sekaligus menjalin relasi dengan sesama yang datang bertamu maupun para arwah nenek moyang Tradisi tentang pembuatan simbol litu umne menjadi bagian dalam suatu ritual adat. Terdapat kesesuaian hasil penelitian ini dengan peneitian lainnya yakni setiap ritual memiliki simbol terutama dalam memberikan sesajian atau pengormatan kepada para leluhur . Pemberian makna simbolis dalam kegiatan memiliki peran penting yakni leluhur sebagai perantara. Kaka pia tobo lewo, bapa pia pae tana Kakak duduk dikampung kami, bapak juga mendiami tanah kami Kakak mendiami kampung kami, bapak juga mendiami tanah kami Kakak mendiami kampung kami, bapak juga mendiami tanah kami Rumah sudah jadi, tempat istirahat sudah dibuat dengan baik. Rumah kediaman sudah dibangun dengan baik, tempat peristirahatan sudah dibentuk dengan baik. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 1272 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 Kalimat tersebut menyatakan mohon penyertaan pada Tuhan untuk penghuni rumah tempat tinggal yang nyaman dan bahagia. KESIMPULAN Teks letu umne merupakan ritual menutup lubang tanah pada saat membuat rumah baru. Setiap teks dalam ritual letu umne memiliki khazanah kebahasaan yang meliputi bentuk fungsi dan makna yang terkandung dalam setiap teks yang merupakan ciri khas pada etnik Flores Timur NTT. Bentuk teks letu umne memiliki beberapa kategori morfologi yakni kelompok nomina, verba, pronomna, adverbial dan numelaria. Setiap teks memiliki kata, frasa, dan kalimat yang memiliki ciri tersendiri. Selain bentuk, terdapat pula fungsi teks yakni fungsi interpersonal dan fungsi kekerabatan. Selanjutnya, setiap teks memiliki makna yang terkandung di dalam setiap kalimat yakni makna religius, makna, makna perlindungan dan juga makna simbol yang nerupakan sarana yang digunakan selama ritual berlangsung. Salah satu contoh makna kapas putih sebagai bagian dari alat pemersatu anara para lelehur dam masyarakat setempat. Hal tersebut merupakan bagian dari pembejaran agar masyarakat atau generasi penerus dapat memahami serta tetap mempertahankan budaya yang ada sebagai bagian dari budaya adat istiadat. DAFTAR PUSTAKA Angeline, Mia. 2015. “Mitos Dan Budaya.” Humaniora 6(2): 190. Angeline, Mia. 2015. “Mitos Dan Budaya.” Humaniora 6(2): 190. Eiilerz, Yosef-Frans. 1995. Berkomunikasi Antara Budaya. Ende: Nusa Indah. Fauzan. 2021. “Dwifungsi Tuturan Oposisi Dalam Masyarakat Bersosiokultur Jawa.” Kembara: Jurnal Keilmuan Bahsa, Sastra, Dan Pengajarannya 7(1): 17–29. Genua, Veronika. 2013. Nilai Kehidupan Dalam Legenda Rendo Rate Rua Sebagai Jati Diri Masyarakat Kabupaten Ende Ntt. —. 2019. “Teks Sole Oha Tradisi Budaya Guyub Kultur Lembata Flores Ntt.” Retorika 1: 30–41. Hasanah, Holifatul, And Sony Sukmawan. 2021. “Berbingkai Kemajemukan Budaya, Bersukma Desakalapatra: Selidik Etnografi Atas Tradisi Tengger.” Diglosia: Jurnal Kajian Bahasa, Sastra, Dan Pengajarannya 4(1): 79–90. Hasanah, Holifatul, And Sony Sukmawan. 2021. “Berbingkai Kemajemukan Budaya, Bersukma Desakalapatra: Selidik Etnografi Atas Tradisi Tengger.” Diglosia: Jurnal Kajian Bahasa, Sastra, Dan Pengajarannya 4(1): 79–90. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 1273 Teks Ritual Letu Umne Tradisi Pengerjaan Rumah pada Etnik Flores Timur Nusa Tenggara Timur – Veronika Genua, Alexander Bala DOI: https://doi.org/10.31004/edukatif.v4i1.2094 Isu, Rudolof Jibrael. 2021. “Pengaruh Upacara Rumah Adat Terhadap Ketertinggalan Pendidikan Anak Di Desa Oenbit.” Retorika 2: 16–33. Keraf, Goris. 2004. Komposisi Sebuah Pengantar Kemahiran Berbahasa. Ende: Nusa Indah. Khoiri, Madhan. 2009. “Makna Simbol Dan Pergeseran Nilai Tradisi Upacara Adat Rebo Pungkasan.” : 1–90. Http://Digilib.Uin-Suka.Ac.Id/3907/1/Bab I%2cv%2c Daftar Pustaka.Pdf. Lokanawa, Petrus. 2013. “Agama Dan Pembentukan Cara Pandang Serta Perilaku Hidup Manusia.” Humaniora 4(45): 790–99. Manek, Agnes Odiyanti. 2021. “Makna Budaya Topa Ma Ta Auba Pada Masyarakat Desa Lasi , Kecamatan Kuanfatu , Kabupaten Timor Tengah Selatan.” Retorika 2: 45–59. Miles, M., & Huberman, M. 1992. 5 Bmc Public Health Qualitative Data Analysis A Method Sourcebook Third Edition. California: Sage Publications. Https://Ejournal.Poltektegal.Ac.Id/Index.Php/Siklus/Article/View/298%0ahttp://Repositorio.Unan.Edu. Ni/2986/1/5624.Pdf%0ahttp://Dx.Doi.Org/10.1016/J.Jana.2015.10.005%0ahttp://Www.Biomedcentral.C om/1471-2458/12/58%0ahttp://Ovidsp.Ovid.Com/Ovidweb.Cgi?T=Js&P. Miles, M., & Huberman, M. 1992. 5 Bmc Public Health Qualitative Data Analysis A Method Sourcebook Third Edition. California: Sage Publications. Third Edition. California: Sage Publications. Https://Ejournal.Poltektegal.Ac.Id/Index.Php/Siklus/Article/View/298%0ahttp://Repositorio.Unan.Edu. Ni/2986/1/5624.Pdf%0ahttp://Dx.Doi.Org/10.1016/J.Jana.2015.10.005%0ahttp://Www.Biomedcentral.C om/1471-2458/12/58%0ahttp://Ovidsp.Ovid.Com/Ovidweb.Cgi?T=Js&P. Muhidin, Rahmat. 2015. “Kosakata Budaya Yang Berhubungan Dengan Adat Perkawinan Melayu Bangka Di Kota Pangkalpinang.” Widyaparwa 43(1): 79–88. Http://Www.Widyaparwa.Com/Index.Php/Widyaparwa/Article/View/95. Nur, Fadhilah, Aisyah, And Laili Etika Rahmawati. 2020. “Muatan Kearifan Lokal Pada Buku Bacaan Siswa Terbitan Kemdikbud.” Diglosia: Jurnal Kajian Bahasa, Sastra, Dan Pengajarannya 3(3): 317–30. Ola, Simon Sabon. 2013. Buku Ajar Sosiolinguistik. Kupang: Lembaga Penelitian Undana. Ola, Simon Sabon. 2013. Buku Ajar Sosiolinguistik. Kupang: Lembaga Penelitian Undana. Pampe, Pius. 2009. Alineansi Verbal Pada Umat Katolik Etnik Manggarai. Pampe, Pius. 2009. Alineansi Verbal Pada Umat Katolik Etnik Manggarai. Rahyono, F.X. 2015. Kearifan Budaya Dalam Kata. DAFTAR PUSTAKA Jakarta: Wedatama Wydia Sastra. 2015. Kearifan Budaya Dalam Kata. Jakarta: Wedatama Wydia Sastra. Riyanti, Dwi, Sabit Irfani, And Danang Prasetyo. 2022. “Pendidikan Berbasis Budaya Nasional Warisan Ki Hajar Dewantara.” Edukatif: Jurnal Ilmu Pendidikan 4(1): 345–54. Sibarani, Robert. 2015. “Pendekatan Antropolinguistik Terhadap Kajian Tradisi Lisan.” In Retorika: Jurnal Ilmu Bahasa, , 94–107. Umri , Ulili, Ganefri, And Hadiyanto. 2021. “Perencanaan Pengembangan Dan Pendidikan Berbasis Kearifan Lokal.” Edukatif: Jurnal Ilmu Pendidikan 3(5): 2025–31. Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071 Edukatif : Jurnal Ilmu Pendidikan Vol 4 No 1 Tahun 2022 p-ISSN 2656-8063 e-ISSN 2656-8071
https://openalex.org/W2118838194	https://journal.fi/afs/article/download/5855/5053	English	null	A dynamic programming model for optimising feeding and slaughter decisions regarding fattening pigs	Agricultural and food science	2,006	cc-by	73,029	A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Preface Several persons have contributed to this thesis and supported me. I take this opportunity to thank those people. First, I wish to express my gratitude to my two supervisors, Docent Kyösti Pietola (MTT Eco- nomic Research) and Professor Matti Ylätalo (University of Helsinki). Docent Pietola encouraged me to start post-graduate studies, supported me throughout, and assisted me in my efforts to deal with a variety of methodological and theoretical issues. Professor Ylätalo supported me during my studies in several ways, provided guidance during my research, and made constructive comments on this study. In addition, I would like to express my gratitude to Marja-Liisa Sévon-Aimonen (MTT Animal Production Research), who provided biological data and helped me to formalise the model. I thank Professor Ruud Huirne (Wageningen University, The Netherlands) and Professor Anders Ring- gaard Kristensen (Royal Veterinary and Agricultural University, Denmark) for their careful pre-examina- tion of the thesis and for their many valuable comments. I am also grateful to Professor Pekka Ilmakunnas (Helsinki School of Economics) and Professor Asko Mäki-Tanila (MTT Biotechnology and Food Re- search) who agreed to be my public examiner. I wish to acknowledge my colleagues at MTT Economic Research as well as several people who have participated in this research project at MTT Animal Production Research: Esa Mäntysaari (Ph.D.), Hilkka Siljander-Rasi (M.Sc.), Ismo Stranden (Ph.D.), Jarmo Valaja (Ph.D.) and Kaarina Vuori (M.Sc.). I also thank Saara Raulo (Ph.D.) (the National Veterinary and Food Research Institute) and Marie-Josée Mangen (Ph.D.) (Agricultural Economics Research Institute, The Netherlands) for suggestions concerning early versions of Chapter 7. I am also grateful to the supervisors of the research project “Economic comparison of feeding strategies in pigs” for their remarks. This study is based on research projects which were funded by Ministry of Agriculture and Forestry, MTT Agrifood Research Finland and Raisio Group Plc., with additional support provided by the Finnish Animal Breeding Association (FABA). In addition, the August Johannes and Aino Tiura Foundation of Agricultural Research, the Science and Research Foundation of the Finnish Association of Academic Agronomists as well as the Finnish Cultural Foundation provided ﬁnancial support for this thesis. I gratefully acknowledge the funding and information provided by these organisations. I am also grateful to MTT for giving me the op- portunity to work on this topic and for providing me with excellent facilities for this study. Kurikka, February 2006 A dynamic programming model for optimising feeding and slaughter decisions regarding fattening pigs Jarkko K. Niemi MTT Agrifood Research Finland, Economic Research, Luutnantintie 13, FI-00410 Helsinki, Finland e-mail: jarkko.niemi@mtt.ﬁ ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in Auditorium XII, Unioninkatu 34, Helsinki, on April 21st, 2006, at 12 o’clock. ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in Auditorium XII, Unioninkatu 34, Helsinki, on April 21st, 2006, at 12 o’clock. Supervisors: Docent Kyösti Pietola Economic Research MTT Agrifood Research Finland Professor Matti Ylätalo Department of Economics and Management University of Helsinki, Finland Reviewers: Professor Ruud Huirne Business Economics Group Wageningen University, The Netherlands Professor Anders Ringgaard Kristensen Department of Large Animal Sciences Royal Veterinary and Agricultural University, Denmark Opponents: Professor Pekka Ilmakunnas Department of Economics Helsinki School of Economics, Finland Professor Asko Mäki-Tanila Biotechnology and Food Research MTT Agrifood Research Finland A G R I C U L T U R A L A N D F O O D S C I E N C E Preface I thank Emeritus Professor Henry Fullenwider (Ph.D.) for reading the manuscript for English-language usage. I’m also grateful to the editorial board of Agricultural and Food Science for accepting this study for publication in their journal. Finally, I wish to express my gratitude to my friends and relatives for their support. Warm thanks go to my parents Erkki and Raila, who have encouraged and supported me throughout my life. My warmest thanks go to my wife Sari for her love, care and understanding, and to our sons, Ville and Jere, who have always brought sunshine into my life. Kurikka, February 2006 Jarkko Niemi Jarkko Niemi Jarkko Niemi A G R I C U L T U R A L A N D F O O D S C I E N C E Contents Contents Abstract ........................................................................................................................ 6 1 Introduction ............................................................................................................. 7 1.1 Background of the study .................................................................................. 7 1.2 Modelling feeding and the timing of slaughter decisions ................................ 8 1.3 Objectives and structure of the study ............................................................... 10 2 Previous studies on pig production management and optimisation ........................ 11 2.1 Production and pricing of pig meat in Finland ................................................. 11 2.2 Features of optimised pig management ............................................................ 12 2.2.1 The planning horizon ............................................................................. 12 2.2.2 Choosing the optimal feeding patterns .................................................. 12 2.2.3 The optimality of slaughter timing ........................................................ 16 2.2.4 Uncertainty of slaughter income ............................................................ 19 2.3 Methods for optimising pig fattening ............................................................... 20 2.3.1 The optimality of an allocation .............................................................. 20 2.3.2 Production and cost functions ................................................................ 21 2.3.3 Convex and concave sets and functions ................................................. 22 2.3.4 Dynamic programming .......................................................................... 24 2.4 Scope and method of the study ........................................................................ 26 3 A dynamic programming model for pig fattening ................................................... 29 3.1 Model speciﬁcation problems .......................................................................... 29 3.2 The optimisation problem ................................................................................ 31 3.3 Decomposing the control variables .................................................................. 32 3.4 The pig growth model ...................................................................................... 33 3.5 Restricting pig growth ...................................................................................... 35 3.6 One-period returns function ............................................................................. 36 4 Precision feeding – a benchmark scenario .............................................................. 37 4.1 Data and scenarios ............................................................................................ 37 4.2 Estimation method ............................................................................................ 41 4.3 Results .............................................................................................................. 44 4.4 Summary .......................................................................................................... 49 5 Adjusting pig production management to market conditions ................................. 50 5.1 Data and scenarios ............................................................................................ 50 5.1.1 Price movements .................................................................................... 50 5.1.2 Quality price premiums .......................................................................... 52 5.2 Results .............................................................................................................. 52 5.2.1 The effects of price changes .................................................................. 52 5.2.2 Elasticity estimates for price movements .............................................. 56 5.2.3 The effects of quality price premiums ................................................... 58 5.3 Summary .......................................................................................................... 61 4 A G R I C U L T U R A L A N D F O O D S C I E N C E 6 The value and management implications of animal genetics .................................. 63 6.1 Data and scenarios ............................................................................................ 63 6.2 Results .............................................................................................................. 66 6.2.1 The effects of animal genotype .............................................................. 66 6.2.2 Split-sex feeding .................................................................................... 69 6.3 Summary .......................................................................................................... 72 7 The importance of slaughter timing ........................................................................ 74 7.1 Data and scenarios ............................................................................................ 74 7.1.1 The case of exogenously delayed slaughter ........................................... 74 7.1.2 Modelling the value of slaughter timing ................................................ 76 7.2 Results .............................................................................................................. 78 7.2.1 Adjusted feeding patterns when the slaughter is delayed ...................... 78 7.2.2 Income losses in the event of a short slaughter delay ............................ Contents 79 7.2.3 Income losses in the event of a long slaughter delay ............................. 82 7.3 Summary .......................................................................................................... 83 8 Conclusions and discussion ..................................................................................... 85 8.1 Management implications ................................................................................ 85 8.2 Policy implications ........................................................................................... 89 8.3 Suggestions for further research ....................................................................... 91 References .................................................................................................................... 93 Selostus ........................................................................................................................ 98 Appendices Appendix A. Computer code of the basic model Appendix B. Sensitivity analysis of two-phase feeding Appendix C. Feeding patterns of female pigs and castrated male pigs Appendix D. Sensitivity analysis for genotype scenarios 5 A G R I C U L T U R A L A N D F O O D S C I E N C E A dynamic programming model for optimising feeding and slaughter decisions regarding fattening pigs Jarkko K. Niemi MTT Agrifood Research Finland, Economic Research, e-mail: jarkko.niemi@mtt.ﬁ Costs of purchasing new piglets and of feeding them until slaughter are the main variable expenditures in pig fattening. They both depend on slaughter intensity, the nature of feeding patterns and the technological constraints of pig fattening, such as genotype. Therefore, it is of interest to examine the effect of production technology and changes in input and output prices on feeding and slaughter decisions. This study examines the problem by using a dynamic programming model that links genetic characteristics of a pig to feeding decisions and the timing of slaughter and takes into account how these jointly affect the quality-adjusted value of a carcass. The state of nature and the genotype of a pig are known in the analysis. The results suggest that producer can beneﬁt from improvements in the pig’s genotype. Animals of im- proved genotype can reach optimal slaughter maturity quicker and produce leaner meat than animals of poor genotype. In order to fully utilise the beneﬁts of animal breeding, the producer must adjust feeding and slaughter patterns on the basis of genotype. The results also suggest that the producer can beneﬁt from ﬂexible feeding technology. Typically, such a technology provides incentives to feed piglets with protein-rich feed. When the pig approaches slaughter maturity, the share of protein-rich feed in the diet gradually decreases and the amount of energy-rich feed increases. Generally, the optimal slaughter weight is within the weight range that pays the highest price per kilogram of pig meat. The optimal feeding pattern and the optimal timing of slaughter depend on price ratios. Particularly, an increase in the price of pig meat provides incentives to increase the growth rates up to the pig’s biological maximum by increasing the amount of energy in the feed. Price changes and changes in slaughter premium can also have large income effects. Key words: barley, carcass composition, dynamic programming, feeding, genotypes, pig fattening, preci- sion agriculture, productivity, slaughter weight, soybeans 1.1 Background of the study weight and leanness (Kure 1997, Chapter 4). Stud- ies even suggest that providing an efﬁcient supply of nutrients according to the pig’s genetic growth potential requires continuous rather than discrete adjustment of feed ratios (Glen 1983). The beneﬁts of continuous adjustments are, however, quite un- known. Decisions related to feeding regimen and slaugh- ter timing in pig meat production have vital sig- niﬁcance because they affect economic perform- ance of pig farms and more broadly, the economic performance of the pork industry. Both decisions can signiﬁcantly affect the value of pigs sold for slaughter and the income ﬂows of a pig farm, es- pecially as the prices of feed and of piglets are the major variable production expenses. Tuppi (2004), for instance, estimates that on ProAgria Rural Advisory Centres’ sample farms specialised in pig fattening, the average feed cost in 2003 was 22% (€39 per pig) and the purchase cost of pig- lets was 32% (€57 per pig) of the total produc- tion cost of pig meat. Taken together, these two items account for 93% of the total material cost incurred in bringing a pig to slaughter. Therefore, changes in the allocation of feeds, the length of the fattening period, and factors that determine the optimality of these decisions, can have large impact on production cost and proﬁtability of pig fattening. Studies suggest that improvements in genotype can signiﬁcantly increase returns to pig farming. Since a pig’s genotype and carcass leanness are correlated and since carcass is priced according to its weight and leanness, increases in returns have been observed above all to improvements that in- crease the growth rate of lean meat (Boland et al. 1993). Full utilisation of production potential of improved genotypes can require the adjustment of slaughter and feeding patterns accordingly (cf. Boland et al. 1999). Therefore, it is important to examine how genotype affects pig production management, and how management and genotype jointly affect the value of a capacity unit. This can be important particularly when aiming at obtaining unbiased assessment of economic advantages of a particular animal breeding strategy. An example of a segregated feeding pattern is the case where the producer has an option to group pigs into batches according to their sex, and there- after to optimise feeding and slaughter decisions separately for both sexes (split-sex feeding). Since feeding and slaughter decisions frequently involve interactions (Chavas et al. A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs soy meal. Dros and Kriesch (2003, p. 10–11), for instance, have reported approximately a 10% price difference. The price gap is expected to increase over time. Regulations prohibiting the use of GMO soy meal therefore imply higher feed costs to do- mestic producers than to foreign competitors with less stringent regulations. Thus, estimates of the effects of regulations require taking into account to which extent producers can make adjustments to production through input substitution. timal to adjust the timing of slaughter of pigs ac- cording to their weight and leanness (cf. Kure 1997, Roemen and de Klein 2000). Measurement of carcass quality can also be inaccurate and affect the optimal timing of marketing (Jorgensen 1993, Boland et al. 1996). Furthermore, it may take time to observe changes in growth rates of pigs (White et al. 2004), and manager may not always reach desired carcass composition (Parsons et al. 2004). Studies such as Jorgensen (1993), Boland et al. (1996) and Kure (1997) have carefully examined the impacts of uncertainty and biological varia- tion in pig fattening. Less work has been done to analyse the connections between feeding and slaughter decisions and carcass quality. Hence, this study emphasises these linkages and shows how to estimate to the value of genetic improve- ments by taking their management effects into ac- count. Another important policy issue is decoupling subsidies from production. The slaughter premium paid for each slaughtered carcass affects the timing of slaughter, because the amount of premiums paid to the producer depends on slaughter intensity. The premium is also known to have an impact on pro- ducer income (Sipiläinen and Ryhänen 1996). De- coupling the slaughter premium from production so that producer has no obligation to produce meat is the equivalent of removing it from the optimisa- tion problem. Since the decoupled premium is ex- ogenous, decoupling can decrease the proﬁtability of high slaughter intensity. Decoupling can in- crease the value of existing production capacity but decrease producer incentives to invest in new production capacity. This is so because decoupled payment is independent of production capacity. Hence, more consideration could be given to giv- ing the producer incentives to invest in new pro- duction capacity. From an economic point of view, price ratios of inputs and outputs largely determine the opti- mality of feeding and slaughter decisions for a given production technology (Dent et al. 1970, Sonka et al. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 1976, Jolly et al. 1980, Glen 1983, Chavas et al. 1985, Broekmans 1992, Rydstedt and Andersson 1993, Kure 1997, Boland et al. 1999). The factors affecting the optimal pig production management patterns include price adjustments that depend on carcass weight and leanness (Boland et al. 1993). Although a producer can ben- eﬁt from adjusting feeding and/or slaughter pat- terns according to the markets, studies have gener- ally found that input substitution is inelastic with respect to input and output prices. Income, on the other hand, has responded very elastically to changes in input and/or output prices (Chavas et al. 1985, Boland et al. 1993, Sipiläinen and Ryhä- nen 1996, Kure 1997). 1.1 Background of the study 1985), it is important to take into account also the income that simultane- ous adjustment of feeding and slaughter decisions can contribute to the capacity unit through im- proved carcass quality as a result of the enhance- ment of the genetic characteristics of the animal. This aspect is often omitted and an inﬂexible feed- ing technology is used instead. The effects of prices and technological con- straints on production management and the value of a capacity unit are inextricably related to choice of feeding patterns and to slaughter decisions. The genetic production potential of an animal (i.e. gen- otype) can have a particularly large impact on the market value of a carcass and as well as on input efﬁciency of feeds. It is known that the pig’s re- quirements for protein (Fuller et al. 1989) and en- ergy (Whittemore 1983) depend on its maturity stage and genotype (Boland et al. 1999). Hence, producer can beneﬁt from using feeding schemes that have two or more phases, each of which has a different feed composition (two or three-phase feeding), as opposed to schemes that have the same feed composition (one-phase feeding) throughout the fattening period (Boland et al. 1999, Campos 2003, p. 80) and from adjusting the timing of slaughter of pigs in a batch according to their Successful pig management entails the ability to ﬁnd solutions to problems arising from uncer- tainty and biological variation. Pigs are often managed as groups where individuals may have different growth patterns. This causes variation in carcass value, with the results that it becomes op- 7 Vol. 15 (2006): Supplement 1. feeds and how feeding affects the market value of a carcass. The second aspect is that economically optimal production decisions take both allocation of inputs and the timing of replacement of an asset into account (Burt 1965, 1993, Dillon and Ander- son 1990, p. 87–88). No matter how the problem of the optimal timing of marketing of an animal is solved, the general principle is simple: It is opti- mal to market a pig when the marginal net revenue from fattening an additional day is equal to the op- portunity cost of replacement (Chavas et al. 1985, p. 639, Dillon and Anderson 1990, p. 87–94, Bo- land et al. 1993, p. 148). In other words, the re- placement pig can contribute higher proﬁt than the current pig (for analytical solution, see Chavas et al. (1985, p. 639)). and the pig reaches the speciﬁed live weight quick- er than before the increase. Therefore, the daily feed allowance signiﬁcantly affects carcass quality (Whittemore 1998, p. 48–52). The growth of a pig and constraints related to its growth can be characterised using a set of equa- tions (Emmans 1995, p. 113N). The idea is to split the pig’s live weight into energy, protein, water and ash and then to model the growth of the com- ponents separately. As Emmans and Kyriazakis (1999) summarise, this can be a very useful form of simplifying pig growth, because most important processes of pig growth can be reduced to a func- tion of either an energy or a protein component. The genotype of a pig plays an important part in this functional approach. In this study, the geno- type of a pig refers to the maximum rate at which the amount of lean and fatty tissue in the pig’s body can increase (i.e. the growth potential). Lean tissue refers to the protein component and meas- ures the amount of all fat-free components in the body, whereas fatty tissue refers to the energy component and measures all fat in the body. p In this study, pig fattening is examined with a structural-form optimisation model that is based on characterisation of the growth mechanism of pigs. An optional approach would be to estimate a reduced-form pig growth model. Vol. 15 (2006): Supplement 1. Discussion on how to feed fattening pigs includes solving wheth- er it is optimal to feed the pig according to its bio- logical growth potential (ad libitum feeding) (Dil- lon and Anderson 1990, p. 113). The problem is economically relevant because, as Boland et al. (1999) note, maximisation of live weight growth only does not recognise the impact of diminishing returns on inputs. Even when the growth rates are exogenously given, it is useful to solve feed ratios by maximising proﬁts rather than weight gain. When the Gompertz function characterises the maximum growth pattern of a pig, the parameter known as mature weight measures the weight that an animal can reach when it matures, and the pa- rameter known as maturing rate measures the maximum daily weight gain of a tissue component (Whittemore 1998, p. 59–68, Emmans and Kyria- zakis 1999; see also Black 1988, Boland et al. 1993, Sevón-Aimonen 2001). Clearly, these two parameters are of interest when estimating the val- ue of genetic improvements. Generally, an increase in the rate of daily weight gain is associated with an increase in maturing rate, whereas an increase in carcass leanness is associated with an increase in the mature weight of lean tissue. Both increases, however, result in a higher rate of daily weight gain (Whittemore 1998, p. 59–68, Emmans and Kyriazakis 1999). The beneﬁts of restricting the supply of feed below stomach capacity are related to a carcass merit based meat pricing scheme (Boland et al. 1993, Boland et al. 1996, Sipiläinen and Ryhänen 1996) and to the fact that restricting the feed sup- ply gives the producer the option to extract the quality price premium from the markets at the cost of decreased growth rate. Although pig growth it- self is a complex process, the principle of how to control carcass quality through feeding is quite simple. As the amount of protein in feed increases, carcass becomes leaner (i.e. the share of red meat in the carcass increases) and its value (€ per kg) increases. As the amount of energy in feed increas- es, carcass becomes fattier, and as the total amount of feed increases, the daily weight gain increases Mature weight is conceptually different from slaughter weight. Mature weight depends on the pig’s genotype, whereas the producer decides the slaughter weight. 1.2 Modelling feeding and the timing of slaughter decisions Economic optimisation of feeding and slaughter decisions requires simultaneous analysis of both decisions. This is mainly due to two aspects. The ﬁrst is that feeding decisions, conditional on the genotype of an animal, affect both carcass value and daily growth rates (Chavas et al. 1985, Kennedy 1986, p. 11, Burt 1993). Hence, making the optimal feeding decisions requires information on how a pig of a particular genotype responds to Feeding and slaughter decisions are also in- volved in several policy issues such as the subsi- dising of pig fattening and regulations affecting the choice of inputs, outputs and production tech- nology. An example of a regulatory problem in- volving the need to evaluate the impact of regula- tions on production is whether to prohibit the use of genetically modiﬁed (GMO) soy meal in feeds. GMO soy meal is less expensive than non-GMO 8 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs of inﬂexible two-phase feeding technology (Chapter 4) layed can provide a producer with incentives to minimise reductions in the value of a carcass by marketing the pigs prematurely. Yet, little attention has been paid on examination of the economic im- pacts of delayed timing of slaughter, and of the op- tions available to an individual producer to adjust pig farming to delayed slaughter. This problem is common, because slaughterhouses are generally responsible for the collection of animals for slaughter, and an individual producer cannot al- ways fully control the date of slaughter. Thus, failed co-ordination of the production chain can alter the value of a carcass and the quality of the industry’s product. • How the optimal feeding and slaughter policies and the value of a capacity unit change when carcass quality premiums, slaughter premiums or the prices of pig meat, feed and piglets change in a given production technology (Chapter 5) • How much the producer can beneﬁt from changes in production technology such that the genotype of an animal is improved, and how these technological improvements affect the optimal feeding and slaughter patterns (Chap- ter 6) • How much the producer can beneﬁt from opti- mising the timing of slaughter, and how he/she can minimise income losses when there is a possibility that the production technology al- lows timing of slaughter to be delayed (Chap- ter 7) Similar problem can occur also in special cir- cumstances, such as when animal disease regula- tions expose farms to the risk of a long exogenous delay in the timing of slaughter. Then, producer’s problem is to consider the options to minimise in- come losses through adjusting feeding patterns. The problem of premature slaughter is interesting also from society’s point of view because prema- ture slaughter can have externalities for a large number of farms in the event of an animal disease outbreak, and because it can increase society’s ex- posure to additional disease losses. These problems are studied by modelling the producer’s decision making in a ﬁnite horizon re- cursive dynamic programming (Bellman, 1957) framework. Dynamic programming provides an efﬁcient tool for linking feeding and feed compo- nents to carcass quality and for optimising feeding and the timing of slaughter simultaneously. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs The value of a capacity unit depends on both volume and quality of pig meat produced, which is condi- tional on quality price premiums paid according to the share of red meat in the carcass. The scope of this study is practical in the sense that the model characterises the growth mechanism of a pig (cf. Lucas’s critique of the validity of macroeconomic models (Sargent 1987, p. 40–41)). A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Vol. 15 (2006): Supplement 1. Since slaughter pigs are valued according to their weight and carcass leanness, in- creasing the probability for the slaughter to be de- 9 Vol. 15 (2006): Supplement 1. mate absolute feed levels and the timing of slaugh- ter or the parameters themselves. the producer receives for ﬁxed inputs and other variable inputs than feed cost and cost of replace- ment piglets. The results are based on the data from growth experiments on Finnish low-fat York- shire and Landrace crossbreed pigs, literature and meat market records in 2001–2003. The results are conditional on model formulation factors such as producer’s ability to control carcass quality of an individual pig and that states of nature (i.e. geno- type, carcass composition, prices) are known each period. Hence, the main goal of this thesis is to analyse how changes in model parameters, such as market information, animal genotype or feeding technology, affect feeding and slaughter patterns and the value of a capacity unit rather than to esti- This study is organised as follows. Chapter 2 discusses the optimisation of pig fattening in gen- eral, and results obtained in previous studies. Chapter 3 presents the modelling framework used in this study. Each of the four research problems given above is examined separately, one at a time, in Chapters 4–7. More detailed description of the problem in question is given in the beginning of each of these Chapters, followed by scenario and data description, the results and a summary at the end of the Chapter. Finally, Chapter 8 draws con- clusions and discusses the results. 1.3 Objectives and structure of the study This study focuses on modelling the production technology of pig fattening in cases where produc- er can observe the technology and state of nature of pig and control feeding and slaughter of a pig accordingly. The objective is to examine changes in pig producer’s production decisions and income when production technology and input and output prices change. In more detail, the goal of this study is to estimate: The main contribution of this study to the lit- erature of pig production management is the dy- namic approach that explicitly takes into account carcass quality while simultaneously optimising feeding and slaughter decisions. The model esti- mates the optimal feeding and slaughter patterns and corresponding value functions over time and over subsequent fattening periods. The term fat- tening period refers to the time that is used to feed an individual piglet until slaughter maturity. The model is normalised for a capacity unit. The value of a capacity unit refers to the compensation that • How much the producer can beneﬁt from using a ﬂexible feeding technology (‘precision feed- ing’), which allows him/her to continuously control feed ratios and carcass quality, instead 10 2.2.1 The planning horizon Farm management is often deﬁned as the alloca- tion of limited resources to maximise the farm’s satisfaction. The management functions include planning the allocation, implementing the plans, and ﬁnally controlling the activities. Agricultural producers can have different objectives in the short run and in the long run. Planning in the short run involves the operational planning and implemen- tation of a chosen strategy whereas strategic plan- ner expands decisions to long run planning and to fundamental issues of ﬁrm operations (Boehlje and Eidman 1984, p. 6–27 and 242–261). Interme- diate length horizon planning is known as tactical planning. Feeding and slaughter decisions, for in- stance, can be adjusted at relatively short intervals Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Strategic planning Tactical planning Operational planning Implementation Control Fig. 1. The management cycle (Huirne 1990). premiums or discounts may differ from slaughter- house to slaughterhouse. The producer price con- sists of the base price, where no quality adjust- ments apply, and quality premiums or discounts that depend on carcass weight and leanness. In ad- dition, the base price can be differentiated accord- ing to producer category. For instance, producers contracted to produce a certiﬁed quality generally receive a higher base price than producers without a contract. Often, the base price is paid for carcasses con- taining 59–59.9% lean meat and being within cer- tain weight range (such as 75–80 kg). Each addi- tional percentage point of red meat increases above (below) the reference receives an additional pre- mium (discount). Each kilogram of carcass weight that is below or above the target weight range, re- sults in a price discount. The discount increases as the carcass is lighter or heavier. For instance, when the discount is €0.02 per additional kg and the car- cass weight is 2 kg above the target, the price per kg is discounted €0.04 below the base price. Fig. 1. The management cycle (Huirne 1990). whereas investments in costly new production technology require planning for a period of several years, because the new technology can increase ef- ﬁciency of input use and thus contribute value added in the long run. Management decisions can be represented as a cyclical process (Figure 1) in which strategic, tac- tical and operational decisions combine to affect the implementation and control of management plans. After the plans have been developed, the managers are concerned with implementing the plans, controlling and monitoring the outcome over time, as well as with adjusting the plans if conditions change. The decisions examined in this study mainly relate to strategic and operational de- cision-making. 2.1 Production and pricing of pig meat in Finland many, the Netherlands or Spain, which produce large amounts of pig meat (Eurostat 2004). The producer price of pig meat in Finland is adjusted according to carcass weight and the share of red meat in the carcass. The advantage of car- cass component based pricing is that it sends pig meat producers an observable price signal reﬂect- ing the value of carcass quality at later stages of the marketing chain (Boland et al. 1996, p. 46). Carcass leanness upon slaughter is graded accord- ing to the SEUROP classiﬁcation scheme com- monly used in European Union. The measurement is carried out with a Hennessy grading probe 4 measure or a pork fat thickness analyser. In 2003, 40.6% of the carcasses qualiﬁed as class S (more than 60% red meat), 54.6% as class E (55–59% red meat), and 4.6% as class U (50–54% red meat) carcasses. The share of carcasses in class S was higher in 2003 than in any of the years from 1999– 2002 (Tike 2004a). Approximately 2.3 million fattening pigs are slaughtered in Finland per year. The average car- cass weight of a slaughtered pig in the past few years has been 82–84 kg. Thus, the annual amount of pig meat produced by fattening pigs in Finland has been 162–187 million kg (Tike 2005). Finnish pig farms house approximately 520,000 fattening pigs (>50 kg) and almost 300,000 young pigs at 20–50 kg live weight, of which most become fat- tening pigs (Tike 2004b). Since 1995, the price of pig meat has been less volatile in Finland than in most member countries of European Union (EU). Both increases and decreases in the EU average price seem to diffuse into Finnish meat markets with lags, and the difference between the highest and the lowest meat price is generally smaller in Finland than in such countries as Denmark, Ger- Major slaughterhouses in Finland use similar principles when pricing pig meat, but the quality 11 Vol. 15 (2006): Supplement 1. amount of feed an animal can eat is limited by its stomach capacity (a genetic constraint) (cf. Black 1995, Emmans 1995, Whittemore 1998, Sevón- Aimonen 2001). Therefore, it is important to know whether the actual growth meets the poten- tial. If not, then other factors, such as feed avail- ability, restrict growth (Emmans and Kyriazakis 1999). Finnish feeding recommendations advise that young piglets be fed with a protein-rich diet. As the recommended amount of energy in feed in- creases almost every week, pigs approaching slaughter maturity are fed with energy-rich feed. The daily amount of energy supplied to young pigs increases by 0.2 fodder units each week. At later stages of growth, an increase of 0.1 fodder units is typical, and eventually the amount supplied stabi- lises at a constant level. In addition, the recom- mendations suggest discretionary reduction of the share of protein in feed once or twice during the fattening period. The ﬁrst reduction is recom- mended at 55 kg live weight and the second at 80 kg live weight (MTT 2004). Both maximum rate of protein retention at dif- ferent stages of growth and mature weights of the animal can differ by sex (Whittemore et al. 1988, Emmans and Kyriazakis 1999, Sevón-Aimonen 2001). Similarly, changes in such factors as quality of feed, housing environment, animal health, diet composition, the number of pigs in a pen, and ﬂoor allowance can affect the growth rates obtained with a given amount of feed (Kornegay and Knot- ter 1984, Heikkonen 1997, Whittemore 1998). Animals having the freedom to choose their diet may prefer, for instance, large amounts of energy feed (such as barley) and dislike diets that contain a very high or very low share of protein (Arsenos et al. 2000). In addition, the producer can control feed intake causing the pig to eat less that its stom- ach capacity allows and hence, reducing growth rates below the genetic potential. Changes in recommended feeding patterns are linked to the fact that economically optimal feed- ing decisions change as growth rates change (Fig- ure 2). Figure 2 illustrates the fact that an increase in the daily feed allowance affects the three factors measured in the vertical axis, viz. the share of red meat in the carcass, the amount of daily weight gain and the feed cost per additional kilogram of meat for a given feed allowance. 2.2.2 Choosing the optimal feeding patterns The biological basis of examining the optimality of feeding patterns is usually the partitioning of the nutritional needs of the growing pig into additive parts, the maintenance needs being met ﬁrst, fol- lowed by growth needs. In addition, nutritional needs can be separated into protein (i.e. amino ac- ids), energy and minerals needs. If an animal con- sumes just enough nutrients to meet the mainte- nance requirements, then no growth occurs. Thus, nutritional information is critical in the dynamic modelling of animal growth. Furthermore, the 12 1 For further details on the method, see for instance, Kamien and Schwartz (1992, p. 109–243), Seierstad and Sydsaeter (1993, p. 69–410) or Bertsekas (1995a, p. 97– 112). A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Optimal region fodder units of feed per pig per day )Share of red meat )Daily weight gain )Feed consumption The amount of measured variables ) Optimal region fodder units of feed per pig per day )Share of red meat )Daily weight gain )Feed consumption The amount of measured variables ) Fig. 2. The impact of daily feed allowance (fodder units per pig per day) on feed consumption (fodder units per additional kilo- gram of meat), daily weight gain (kg), the share of red meat in car- cass at slaughter (%), and the re- gion where the economically op- timal daily feed allowance with respect to the combination of the three factors can be found (Source: Danske slagterier 2001, p. 1). The amount of measured variables ) Fig. 2. The impact of daily feed allowance (fodder units per pig per day) on feed consumption (fodder units per additional kilo- gram of meat), daily weight gain (kg), the share of red meat in car- cass at slaughter (%), and the re- gion where the economically op- timal daily feed allowance with respect to the combination of the three factors can be found (Source: Danske slagterier 2001, p. 1). Fig. 2. The impact of daily feed allowance (fodder units per pig per day) on feed consumption (fodder units per additional kilo- gram of meat), daily weight gain (kg), the share of red meat in car- cass at slaughter (%), and the re- gion where the economically op- timal daily feed allowance with respect to the combination of the three factors can be found (Source: Danske slagterier 2001, p. 1). fodder units of feed per pig per day )Share of red meat )Daily weight gain )Feed consumption )Share of red meat )Daily weight gain )Feed consumption Since interactions frequently occur between feed inputs, it is important to consider different in- put combinations over a feasible operational range. Both static and dynamic analyses of the optimal feeding patterns suggest that it is optimal to de- crease the share of protein in feed as the pig grows. Dent et al. (1970) examined the effect of feed in- take in different combinations on pig growth and economic outcomes using static regression analy- sis. They observed that response of lean meat and live weight gain to both lysine and protein intake was almost linear for young pigs. Vol. 15 (2006): Supplement 1. The daily weight gain increases when feed allowance increases, but the marginal weight gain is smaller when the al- lowance is large than when it is small. This can be due, for instance, to limits in stomach capacity and the fact that the daily weight gain starts to decrease when the animal becomes mature enough. Simul- taneously, the average feed cost per kilogram of marketed meat decreases. At a certain inﬂection point, and thereafter, the efﬁciency of converting feed into meat decreases and average feed cost in- creases. Furthermore, increasing a feed allowance causes trade-off between carcass leanness and dai- ly weight gain. The shaded box between the two vertical lines illustrates the region where an eco- nomically optimal feed allowance is likely to be found. The optimum is where the feed allowance and the three components are suitably balanced. In general, if the daily feed allowance is excessively high (low), then decreasing (increasing) it results in higher returns to producer. The exact location of the proﬁt-maximising optimum in the shaded re- gion, however, depends on such factors as feed and piglet prices. Studying potential and actual growth rates re- quires knowledge of growth as a function of weight and feed intake, and of growth potential. When carcass leanness needs to be taken into account, growth needs to be further separated into energy and protein needs (cf. Chavas et al. 1985, Glen 1983, Boland et al. 1993, Emmans 1995, Kure 1997, Sevón-Aimonen 2001). According to Whit- temore (1998, p 50), the ratio of fat growth to lean growth responds quite steadily to increased use of feeds when growth of fatty and lean tissue is re- stricted below the maximum. When lean growth reaches its maximum, the growth rate of fatty tis- sue increases with respect to the growth rate of lean tissue. Even if feeding is initially restricted so that tissue grows below the potential, the pig can have the capacity to partly compensate for reduced weight gain afterwards if the amount of feed is in- creased up to the level where the pig can utilise the nutrients up to its growth potential (Valaja et al. 1992, Kyriazakis and Emmans 1999). 13 Vol. 15 (2006): Supplement 1. Chavas et al. (1985) have characterised the op- timal feeding pattern as follows. The amount of soybean meal increases until the pig reaches ap- proximately 73 kg live weight. Thereafter, the amount of soybean meal in feed decreases. The amount of corn, on the other hand, increases dur- ing the fattening period. During the ﬁnishing stage, the amount of corn increases very rapidly and the share of protein in feed decreases linearly. In addi- tion, Chavas et al. (1985) computed positive cross- price elasticity estimates with soybean meal and corn. Thus, the feeds were substitutes for each other. When the price of one feed increased by one percent the amount of the substitute feed increased less than 0.15%. Increasing the price of a piglet by one percent, however, increased the demand for corn by 1.84% and the demand for soybean meal by 0.94%. stricted feeding yielded a higher surplus than unre- stricted feeding, whereas at price ratios for 1995, this was the case only for castrated male pigs. In general, these results suggest that castrated male pigs beneﬁted more, in terms of returns, from re- stricted feeding than female pigs. Kure (1997, p. 71–72) also showed substantial income effects of changes in input and output pric- es. An increase of 10% in the base price of pig meat increased annual net revenues by 81.7% and decreased the optimal slaughter weight by 2 kg. A similar increase in feed prices decreased annual net revenues by 40% and slaughter weight by 0.2 kg. Studies suggest that a producer can increase proﬁts by adjusting the composition and amount of feed according to the pig’s current needs. The ad- justment can be done continuously or, as Boland et al. (1999) and Campos (2003, p. 73–100) illus- trate, in phases. The advantage of allowing adjust- ment of feeding patterns in economic analysis is that it recognises the impact of diminishing returns on feed ratios (Boland et al. 1999). In other words, using two- or three-phase feeding instead of a sin- gle feed ration increases the cost of replacement (i.e. opportunity cost of feeding) (Campos 2003, p. 98). This is due to the fact that under two or three- phase feeding, the loss of nutrients and the cost of an additional day of fattening period are smaller than under one-phase feeding. A G R I C U L T U R A L A N D F O O D S C I E N C E Response of live weight gain to increased amount of energy was continuous though diminishing in the later stages of growth, and a connection between low response to protein and low intake of energy was observed. In early stages of growth, lean growth showed positive though a diminishing response to energy. Since interactions frequently occur between feed inputs, it is important to consider different in- put combinations over a feasible operational range. continuous growth curve. Sonka et al. (1976), ob- served a similar change in the feeding pattern as the pig grows. They estimated that the optimal share of protein in the diet varies between 12–18% depending on the growth stage of the animal as well as on input prices. Sonka et al. (1976, p. 473) also observed that when the price of protein is low in comparison with the price of corn, the cost ad- vantage of using low amounts of protein decreas- es. Both static and dynamic analyses of the optimal feeding patterns suggest that it is optimal to de- crease the share of protein in feed as the pig grows. Chavas et al. (1985) estimated the optimal feeding pattern of fattening pigs using optimal control theory1. They concluded that pig meat pro- ducers have incentives to feed high protein ration for young pigs, but to use lower protein ration in the ﬁnishing phase. The result was quite robust to changes in relative prices. They observed that the marginal product of each nutrient had a negative relationship with respect to the level of the other nutrient. They also observed a diminishing mar- ginal utility of growth with respect to feed intake. As the ratio of energy intake to dietary lysine decreases along the iso-growth line, the cost of producing a daily weight gain simultaneously de- creases. Thus, under conditions orientated purely to achieving live-weight gain at the minimum feed cost, nutrient combinations should be selected to- wards the high energy-low lysine end of the iso- growth curve (Dent et al. 1970, p. 202). These re- sults, however, do not permit the construction of a 14 2.2.3 The optimality of slaughter timing (cf. Dillon and Anderson 1990, p. 87–94, Boland et al. 1993, p. 148). (cf. Dillon and Anderson 1990, p. 87–94, Boland et al. 1993, p. 148). (cf. Dillon and Anderson 1990, p. 87–94, Boland et al. 1993, p. 148). Economically optimal production decisions take both allocation of inputs and the timing of replace- ment of an asset into account (Burt 1965, 1993, Dillon and Anderson 1990, p. 87–88). The prob- lem of timing is related to the fact that extracting an output from a resource system exchanges a fu- ture loss for an immediate gain, whereas allocating an input into a resource system exchanges an im- mediate loss for a future gain (Kennedy 1986, p. 11). Several studies have elaborated on the opti- mality of the timing of slaughter or on the timing of marketing pigs for slaughter (Jolly et al. 1980, Chavas et al. 1985, Giesen et al. 1988, Broekmans 1992, Boland et al. 1993, Burt 1993, Rydstedt and Andersson 1993, Lloyd et al. 1994, Kure 1997, Toft 2000, Roemen and de Klein 2000, Campos 2003). Chavas et al. (1985, p. 639, for further ap- plication, see Campos and Andersson 2003, p. 32– 35) derive a principle that it is optimal to keep the animal until the age at which the marginal net rev- enue is equal to the opportunity cost of replace- ment. Furthermore, Burt (1993) provides a solu- tion to the dynamic and simultaneous replacement and feeding problem under input and output price uncertainty by extending Hotelling’s replacement and depreciation theory to pig fattening. Another approach is to make feeding and slaughter deci- sions that maximise average proﬁt per unit of time The concept of retention pay-off, despite the fact that it is used for reproduction animals (sows), illustrates the basic idea of the optimal timing of replacement of an animal as a function of time (Figure 3). The optimal timing for replacement is at time t2, when average revenue equals marginal revenue (R2). Any deviation from the optimal tim- ing may result in a loss. Replacing (cf. slaughter- ing) the animals at the optimal time yields higher revenues than replacing them at a later time be- cause the replacement animal can provide higher income than the current animal would provide in the future if no replacement took place. Vol. 15 (2006): Supplement 1. Glen (1983), studied another dynamic pro- gramming application by optimising feeding pat- terns of fattening pigs for the entire fattening pe- riod. The pigs had a speciﬁed slaughter weight. Glen (1983) divided feeding and live weight into energy and protein (for development of the pig growth model see Whittemore and Fawcett (1976), and for use of the growth model to determine the least costly feed ratio see Fawcett et al. (1978)). Glen (1983) estimated that with a diet consisting of soy and barley it is optimal to increase the amount of feed throughout the fattening period. The optimal diet for 20 kg piglet contained 9.7% soy whereas the optimal diet for 25–50 kg pig con- tained 19.3–21.6% soy. Thereafter, on pigs heavier than 50 kg, the share of soy decreased approxi- mately to 2%. Also Boland et al. (1999) showed that it is economically optimal to decrease the share of protein in feed when the pig approaches the optimal slaughter maturity. Wet feeds, for instance, allow producers more ﬂexibility than dry feeds in adjusting feeding pat- terns. Use of wet feeds can also decrease the amount of wasted feed. Thus, using a ﬂexible feed- ing technology, such as wet feeds, can improve the efﬁciency of inputs use and both increase and sta- bilise income (Campos 2003, p. 61–66, Campos and Andersson 2003, p. 41–42). As adjustments include reducing the share of protein in feed in ac- cordance with the pig’s needs, the adjustments can also reduce environmental externalities such as nutrient leakages (Boland et al. 1998, Campos 2003, p. 20–21, 83–84 and 98–100). Hence, fewer nutrients are excreted and higher returns for pro- ducers are obtainable under ﬂexible feeding tech- nology than under inﬂexible feeding technology (Boland et al. 1999). Using a production and cost theoretical ap- proach, Sipiläinen and Ryhänen (1996, p.157–200) estimated that at the price ratios observed in 1993– 1996, and at the price ratios estimated for 2000, a producer could achieve a higher income per slaughtered pig by using unrestricted feeding in- stead of restricted feeding. When the analysis fo- cused on maximising the daily surplus, the result was more ambiguous. At price ratios for 1996, re- 15 Vol. 15 (2006): Supplement 1. slaughter weight would increase by 6.9 kg and producer’s income would decrease by 18%. In other cases, the effect of piglet price on slaughter weight was smaller. In the above cases, feeding was restricted and the carcass quality premiums and discounts applied for carcasses containing less than 59% or more than 61% red meat. When the base leanness (implies no discounts or premiums) was 59–60%, the optimal slaughter weight was generally 76.6 kg, whereas income effects were similar to the previous case. Finally, removing the slaughter premium increased the optimal slaughter weight (Sipiläinen and Ryhänen 1996, p. 191). iance of pig traits as well as by the ﬂexibility of piglet supply. The terminal weight of a batch in- creased a little when the variance between traits increased (Kure 1997, Chapter 4). Thus, when a producer has to market a group of pigs of different qualities, he/she beneﬁts from marketing fast- growing pigs when they are close to the upper limit of the target weight range and slowly grow- ing pigs at a lower weight. Similar result has been conﬁrmed in several studies (cf. Jorgensen 1993, Rantala 2004, Toft et al. 2005), which is due to the fact that producers can compensate for the value of lost growth potential of slowly growing pigs by increased rotation of animal stock and improved productivity of replacement pigs. Giesen et al. (1988) developed a model that op- timised delivery of pigs to the slaughter according to their weight in an all-in/all-out production sys- tem. Their results indicated that producer can ex- tract positive returns by using such an optimisation model. Ross (1980) analysed production function in order to maximise pig meat producer proﬁts. He showed that the optimal slaughter weight varies within relatively narrow range and is generally close to the maximum carcass index. Jolly et al. (1980, p. 809) also provided evidence that market weights of slaughter pigs are rather insensitive to changes in feed or meat prices and that net farm income re- sponds sluggishly to changes in slaughter weights. Roemen and de Klein (2000) used a Markov decision model to study the production problem of how to organise delivery of groups of pigs. They took into account that each group consists of sub- groups of pigs with different growth rates and that meat prices vary in time stochastically and interde- pendently. 2.2.3 The optimality of slaughter timing As early replacement leaves some of the current animal’s production potential unused, replacement prior to t2 is also non-optimal. When the replacement ani- mal is non-identical to the current animal, the opti- mum may shift away from t2 (Huirne et al. 1993, Huirne at al. 1997, p. 86). Sipiläinen and Ryhänen (1996) estimated that increasing the price of pig meat 10% above the price of 1996 would decrease the optimal slaugh- ter weight from 81.6 kg to 79.5 kg. Simultaneous- ly, producer’s daily surplus would increase by 32%. If feed price increased by 10%, the optimal slaughter weight would decrease by 2.1 kg and producer’s income would decrease by 12%. If the price of a piglet increased by 10%, the optimal Net revenue R1 R2 R3 Net revenue R1 16 Time Marginal revenue Average revenue Net revenue t1 t2 t3 R1 R2 R3 Fig. 3. The optimal time for re- placement as a function of time in a situation without an alternative opportunity (t3), and in situations of an identical (t2) and a non- identical (improved) (t1) replace- ment animal (Huirne et al. 1997, p. 86). Time Marginal revenue Average revenue Net revenue t1 t2 t3 R1 R2 R3 Fig. 3. The optimal time for re- placement as a function of time in a situation without an alternative opportunity (t3), and in situations of an identical (t2) and a non- identical (improved) (t1) replace- ment animal (Huirne et al. 1997, p. 86). R3 16 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs served that pigs of lean genotype had heavier opti- mal slaughter weight than pigs of fatty genotype. The heaviest weights were close to the limit above which weight price discounts applied. Further- more, pigs priced according to carcass merits were heavier upon slaughter and they required a longer fattening period, but also contributed higher in- come than pigs priced according to live weight. In addition, pricing based on six size components of hams and loins resulted in slightly heavier pigs than standard carcass merit pricing. The difference was larger for fatty pigs than for lean pigs. The explanation is largely in the efﬁciency of convert- ing a kilogram of feed into lean meat, because lean pigs and barrows have higher lean and feed efﬁ- ciency than fatty pigs and gilts. The interpretation of the feed efﬁciency ratio is, however, problem- atic because the choice of diet, length of fattening period and slaughter weight affect the ratio. De- spite improved feed efﬁciency per kilogram of pig meat, proﬁts can fall if slaughter weight simultane- ously falls (Campos and Andersson 2003, p. 43). Despite this drawback, the positive impact of car- cass merits on producer proﬁts increases when the genetic background of pigs is conducive to the production of leaner meat (Boland et al. 1993). weight of a pig upon marketing increased with in- put prices and decreased with output prices. The timing of marketing and the marketing weight re- sponded quite elastically to changes in piglet price, at least when compared to other input prices. Boland et al. (1999) conclude that under optimised feeding, an increase in feed prices has a larger im- pact on average net revenues than on the marginal cost of the timing of replacement. Chavas et al. (1985) and Boland et al. (1999) also observe that prices of inputs and the price of pig meat have a strong impact on producer income. In analyses such as those done by Boland et al. (1993) and Kure (1997), input allocation has responded less elastically to price changes, and income effects have been more elastic than in the analysis of Cha- vas et al. (1985). Boland et al. (1993), for instance, report elasticity estimates of decision variables with respect to price changes to be less than 0.25, whereas income elasticity estimates with respect to price changes are 2.35 or larger. Vol. 15 (2006): Supplement 1. They concluded that the optimal deliv- ery pattern depends on the combination of pig’s age and pig meat price. They concluded that pigs in a larger pig group should be delivered if the ani- mals are sufﬁciently close to the delivery weight and if the price is in the preferred price set. Other- wise it is optimal to postpone the delivery. Kure (1997) analysed the marketing manage- ment of a pig fattening farm. Speciﬁcally, he stud- ied how to select and when to market individual pigs from batches, and when to deliver the remain- ing pigs of a batch for slaughter (i.e. terminate the batch). Kure (1997) concluded that selection of the timing of slaughter based on carcass leanness and weight is only slightly superior to selection based on pig’s live weight only. Therefore, very little ﬁ- nancial leeway was left for on-farm leanness meas- urement. The leeway, however, may depend on the meat quality premiums and discounts. Chavas et al. (1985) estimated the optimal marketing time of fattening pigs using the optimal control theory. The results suggest that the optimal marketing time is very close to the maximum of the price premium function. Thus, they conclude that, in a continuous pig operation, the presence of a price premium or discount can be very effective in inﬂuencing the marketing weight of pigs. How- ever, they emphasise that further research is re- quired to study the relationship between quality- adjusted market signals and producer response in terms of carcass quality characteristics. The quali- ty price adjustment of Chavas et al. (1985) was based on a quadratic function of live weight, which may not reﬂect the Finnish policy of basing meat pricing on carcass merit. Kure (1997) noticed that pig meat producers can beneﬁt from marketing individual pigs of a batch according to their weight. In other words, fast-growing pigs should be marketed before they suffer from discounts in meat price due to excess weight, whereas slowly growing pigs should be kept until they reach sufﬁcient live weight. The optimal delivery patterns were affected by the var- In contrast to previous studies that neglect op- portunity cost of replacement, Chavas et al. (1985) and Boland et al. (1999) observed that the age and 17 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Vol. 15 (2006): Supplement 1. placement decisions. Mourits et al. (1999, 2000) optimised nutrition and timing of insemination of dairy heifers using stochastic dynamic program- ming and the hierarchic Markov process technique. Nielsen et al. (2004) optimised winter feed levels, grazing strategy and slaughter policy of organic steer production in Denmark, using a multi-level hierarchic Markov process. Pihamaa and Pietola (2002) used dynamic programming to optimise feeding and slaughter patterns of beef cattle in Fin- land. Vargas et al. (2001) also paid attention to feeding strategies in their dynamic programming model. However, while decisions on replacing the dairy herd were optimised, their model did not ex- plicitly optimise feeding decisions. Earlier work on dynamic optimisation of feeding and timing of marketing include Talpaz et al. (1988) and Kennedy et al. (1976) for broilers, and Kennedy (1972), Yager et al. (1980) and Feinerman and Siegel (1988) for beef cattle. The ﬁrst three studies have deterministic optimisation models, Yager et al. (1980) presents stochastic optimisation model for beef cows, and Feinerman and Siegel (1988) has a farm-level optimisation model for beef feedlots. placement decisions. Mourits et al. (1999, 2000) optimised nutrition and timing of insemination of dairy heifers using stochastic dynamic program- ming and the hierarchic Markov process technique. Nielsen et al. (2004) optimised winter feed levels, grazing strategy and slaughter policy of organic steer production in Denmark, using a multi-level hierarchic Markov process. Pihamaa and Pietola (2002) used dynamic programming to optimise feeding and slaughter patterns of beef cattle in Fin- land. Vargas et al. (2001) also paid attention to feeding strategies in their dynamic programming model. However, while decisions on replacing the dairy herd were optimised, their model did not ex- plicitly optimise feeding decisions. Earlier work on dynamic optimisation of feeding and timing of marketing include Talpaz et al. (1988) and Kennedy et al. (1976) for broilers, and Kennedy (1972), Yager et al. (1980) and Feinerman and Siegel (1988) for beef cattle. The ﬁrst three studies have deterministic optimisation models, Yager et al. (1980) presents stochastic optimisation model for beef cows, and Feinerman and Siegel (1988) has a farm-level optimisation model for beef feedlots. Another source of income uncertainty is that producers cannot always accurately assess current quality of pigs. Accuracy of carcass assessment can affect the optimal timing of marketing (Jor- gensen 1993, Boland et al. Vol. 15 (2006): Supplement 1. 1996) and incentives for preferring genotypes that conduce to carcass leanness at the farm level (Boland et al. 1996). When the number of pigs per pen increases, chanc- es to control animal quality decrease. Thus, ani- mals can be delivered at an inappropriate weight and producer can suffer income losses compared to the case of perfect information. Ignoring possi- ble sources of uncertainty can result in overestima- tion of the value of information. The effect of un- certainty over the pig’s actual weight may not be large in all the cases, however, and depends on the problem under investigation (Jorgensen 1993). Recent advances in precision farming have ﬁlled some of the information gaps in swine pro- duction. These include visual analysis of pigs and on-line weighing of animals that are ready for de- livery (see e.g. Schoﬁeld et al. 1999, Kristensen 2003) as well as individual-speciﬁc feeding tech- nology. Jorgensen (1993) recognised that the opti- mal delivery policy is relatively robust to varia- tions in weighing precision. Weighing precision was measured through variance of weighing. When the pen had more than 16 pigs, the variation was large enough so that improved weighing precision left only very little ﬁnancial leeway for invest- ments in weighing equipments. Nevertheless, in- creasing the weighing precision could increase slaughter weights at most by 4 kg. An increase in both the variance of pig quality and weighing pre- cision could decrease the net present value of pig fattening by almost 5% (Jorgensen 1993). Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs On gilts, in- come elasticity with respect to the price of pig meat is as high as 12.79. The impacts of quality adjustments are also quite large. Female and male pigs have different growth rates and thus require different time periods to reach slaughter maturity. Female pigs have higher yields of lean meat than male pigs. Thus, produc- ers may beneﬁt from marketing female and male pigs separately (Boland et al. 1993, Boland et al. 1996). This view is supported by Sipiläinen and Ryhänen (1996, p. 186), who estimated that, de- pending on the combination of feeding pattern (re- stricted/unrestricted), price ratios and meat quality premiums and discounts, the optimal slaughter weight of female pigs can be higher, lower or the same as the optimal slaughter weight of castrated male pigs. These results emphasise the importance of technological options, such as separate facilities for pig groups, separation of feeding patterns or genotype of pigs (cf. Chavas et al. 1985, p. 643, Boland et al. 1993, p. 161). Some studies (e.g. Jolly et al. 1980), however, estimate that penalties from simultaneous non-optimal marketing of gilts and barrows are small. Rydstedt and Andersson (1993) developed a dynamic optimisation model to estimate the batch slaughter pattern that maximises pig meat produc- er income over a given planning horizon. They showed that although slaughter weights differ sea- sonally and an increase in the price of pig meat increases incentives for adjusting the timing of marketing, pig meat producers have insufﬁcient incentives to keep capacity units idle in order to produce pigs speciﬁcally for Christmas markets. Only a large increase (+20%) in the price of pig meat over time resulted in seasonal synchronisa- tion of the timing of marketing. Thus, they con- cluded that producing at full capacity is among the most important factors affecting proﬁtability of pig fattening operations. Few of the studies mentioned above have si- multaneously optimised decisions on how to feed and slaughter fattening pigs. Recent studies con- ducted with other animals provide more examples of joint dynamic optimisation of feeding and re- Returns to management under alternative pric- ing systems vary widely. Boland et al. (1993) ob- 18 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs real-time control of weight gain and leanness of pig can be challenging. Parsons et al. (2004), com- pared pig groups that were fed in order to reach 50 kg or 60 kg liveweight and groups that were fed in order to reach 12 mm or 16 mm backfat thickness. They found that it is possible to control carcass quality to some extent. They also found that the proposed management could not reach the target leanness, particularly the 16 mm back fat target. and static optimisation is the time aspect: while dynamic optimisation takes into account the inter- temporal nature of the problem, static optimisation generally focuses on a single-period optimal solu- tion. Occasionally, a mixture of both methods is used. Linear programming, for instance, can solve least cost ratios within dynamic programming ap- plications (Glen 1983, Bertsekas 1995b, p. 51). Several ways to model production dynamics have been presented. One approach discussed by Dillon and Anderson (1990, p. 87–94) is to specify production response as a function of time and total input used during the response period. Another ap- proach is to characterise production as a dynamic process that follows differential or difference equa- tions. The latter approach allows us to examine a biological process in greater detail than the ﬁrst approach, but it also has high requirements for data accuracy. No matter which approach is cho- sen, it is important to pay attention to the charac- terisation of dynamic processes. Chavas et al. (1985, p. 640) have criticised some earlier analy- ses of pig growth because their ability to character- ise growth response continuously for all stages of pig growth is impaired due to static bias, and hence they do not facilitate determination of the optimal timing of slaughter and replacement. The ﬁnal example of income uncertainty is that by Toft et al. (2005), who show that the optimal delivery policy depends on such risk factors as the possibility of animal disease. When disease pres- sure increases, producer can beneﬁt from market- ing a larger share of animals at earlier growth stages. This is due to the fact that the producer can slow down the epidemic by early delivery, which reduces the potential shortfall in weight gain due to the disease. Another explanation may be in the expectation that the next batch will be more pro- ductive than the current (infected) batch. 2.2.4 Uncertainty of slaughter income It is known that volatility and uncertainty of in- come can affect optimal production decisions. Broekmans (1992) observed differences in the du- ration of the fattening period when evaluating the effect of price ﬂuctuations on delivery patterns for slaughter pigs. Broekmans (1992) concluded a producer has more incentives for a longer fatten- ing period under ﬁxed prices than under uncertain prices. An exception was the case where the prices were favourable (i.e. low feed prices and/or high pig meat price). When prices became uncertain, producer maximised the net present value of future income ﬂow by slaughtering the pigs as soon as possible, i.e. when prices were still favourable. This was earlier than when prices were ﬁxed and deterministic (Broekmans 1992). The result, how- ever, can depend on the current market conditions so that expectations on whether prices will fall or rise can affect the timing of slaughter. Among the potential beneﬁts of real-time per- formance measurement of pigs are characterisa- tion of growth response to nutrients in the context of speciﬁc pig and farm types, and optimisation of pig performance with reference to a given target (cf. Whittemore 2004). White et al. (2004) ob- served that visual image analysis was able to de- tect a change in pig state after 3–5 days fattening period with 80% conﬁdence, and after 8–10 days fattening period with 95% conﬁdence. The results of Parsons et al. (2004), however, suggest that 19 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs There- fore, the opportunity cost of replacement decreases when the number of infected pigs in the current batch increases, and the producer accepts a small immediate loss if he/she expects to gain rewards in the future. Thus, the optimal disease control policy includes delivery policy (Toft et al. 2005, p. 11– 12). This result emphasises the importance of con- sidering slaughter decisions simultaneously with most other pig herd management problems. The fundamental idea of both dynamic and static optimisation is to ﬁnd the resource allocation that maximises a given objective, such as maxim- ising the value of a capacity unit. A standard ap- proach to obtain the optimal resource allocation for a constrained optimisation problem is to for- mulate the maximisation problem into a La- grangean form and then to characterise the ﬁrst- order conditions. In general, the optimal resource allocation is such that increasing or decreasing the amount of a particular resource cannot yield addi- tional proﬁts. In other words, marginal revenue is zero (Gravelle and Rees 1992, p. 12–42, Mas- Colell et al. 1995, p. 956–964). Vol. 15 (2006): Supplement 1. in the dynamic framework. Chavas et al. (1985, p. 639) derive a result where the optimal resource allocation in pig fattening is at the point, where the marginal rate of technical substitution between two inputs with respect to growth function equals the ratio of net marginal value product of the two inputs. The ratio at time t is equal to the marginal value of the current product minus input cost. Therefore, both growth rate function and produc- tion function play a role in the optimality condi- tions. One of the fundamental ideas of production economics is that a relationship exists between in- puts z and outputs y. Generally, the relationship is written in a convenient mathematical form called the production function y=f(z). Thus, a corre- spondence between y and z exists (Chambers 1988, p. 7–9). When modelling pig growth, the corre- spondence generally means that growth potential and growth rate can be measured using factors such as the amounts of feed, composition of the diet, live weight and carcass composition as ex- planatory factors. Several models based on such detailed or simpliﬁed biological understanding of pig growth have been developed (e.g. Sonka et al. 1976, Whittemore and Fawcett 1976, Glen 1983, Chavas et al. 1985, Black et al. 1986, Emmans 1995, Sevón-Aimonen 2001, Lizardo et al. 2002). in the dynamic framework. Chavas et al. (1985, p. 639) derive a result where the optimal resource allocation in pig fattening is at the point, where the marginal rate of technical substitution between two inputs with respect to growth function equals the ratio of net marginal value product of the two inputs. The ratio at time t is equal to the marginal value of the current product minus input cost. Therefore, both growth rate function and produc- tion function play a role in the optimality condi- tions. The optimal solution holds only momentarily and can be altered by any deviation in any param- eter. On the other hand, when prices change but relative prices remain constant, the optimal solu- tion to the problem can remain. Thus, the optimal allocation of resources primarily depends on rela- tive prices whereas the ﬁnancial outcome (such as proﬁt) may depend also on the absolute prices (Mas-Colell et al. 1995, p. 50–57 and 129–143). In pig farming, the optimal feeding and slaughter de- cisions can change over time when markets change (Chavas et al. 1985). Vol. 15 (2006): Supplement 1. The aspect of shifting price ratios is particularly important in dynamic optimi- sation, because it takes into account how future decisions and dynamics of production affect cur- rent optimal decisions and vice versa. A set of assumptions often ensures that the pro- duction function will have the desired properties (Varian 1984, Chambers 1988, Gravelle and Rees 1992, Mas-Colell et al. 1995). The production function has the property of being (strictly) monot- onic. This implies that output does not decrease when the amount of inputs increases. In addition, the input requirement set V(y) is preferably con- cave, which implies that the production function is strictly quasi-concave. Convexity of the produc- tion possibilities set, on the other hand, is required for the law of the diminishing marginal rate of technical substitution. An alternative requirement is the law of diminishing marginal productivity. Further properties of the production function in- clude that a positive output requires the use of scarce inputs; the production possibilities set is closed and nonempty; that the production function is ﬁnite, nonnegative, real valued and single-val- ued for all ﬁnite and nonnegative inputs; and that the production function is everywhere continuous or twice-continuously differentiable. The last property rules out discontinuous jumps in the pro- duction technology (Chambers 1988, p. 9–18). 2.3.1 The optimality of an allocation In the past, the analysis of agricultural production response has heavily relied on static analysis (Heady and Dillon 1972, Kennedy 1986). Live- stock production processes, however, are never instantaneous or static, but occur in a dynamic set- ting in which time can have a decisive impact on outcomes. The major difference between dynamic In a static framework, the optimal allocation of inputs is such that the marginal rate of technical substitution equals the input price ratio (Dillon and Anderson 1990, p. 11–25, Mas-Colell et al. 1995, p. 129–143). Since the static criteria omit the op- portunity cost of replacement, the result may differ 20 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs feed, the pig cannot eat more feed and thus the growth rate no longer increases even if the amount of feed fed increases. The law of diminishing mar- ginal returns holds for a concave production func- tion, because the second derivative of such a func- tion is negative. The law implies that the amount of output gained by using an additional unit of in- put starts decreasing after the marginal product has reached its maximum, whereas the average prod- uct per unit of input reaches the maximum when the marginal product drops below the average product. The marginal product, however, is posi- tive until the total output reaches the maximum (Intriligator 2002, p. 179–189). es. Concavity is a direct consequence of the funda- mental inequality of cost minimisation, and sug- gests that the shape of the cost curve with respect to prices is similar to that of an upturned bowl (Varian 1984, p. 44–46, Chambers 1988, p. 50– 59). Fig. 4. Convex and non-convex sets (Mas-Colell et al. 1995, p. 947). 2.3.2 Production and cost functions In order to ﬁnd a global solution to an optimisation problem, the problem has to meet certain condi- tions. These necessary conditions are embodied in the Weirstrass Theorem. The conditions are that the objective function is continuous, and that the feasible set is non-empty, closed and bounded (Gravelle and Rees 1992, p. 25–26). These four conditions are important although in discrete time models the objective function is often a discrete approximation of a continuous function. Without boundedness, for instance, the solution can be inﬁ- nite (Bertsekas 1995b, p. 9–11). Because the con- ditions are closely related to the properties of the functions that characterise underlying processes, Chapter 2.3. brieﬂy examines the issues that fre- quently arise in production economics. As an example of the property of the law of diminishing marginal productivity, we may con- sider a producer who keeps on increasing the amount of feed fed to a pig. For small amounts of feed, the growth rate of a pig increases when the amount of feed increases. After having enough 21 A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. T z z y y* a) Convex technology b) Non-convex technology z y T Fig. 5. Illustration of a convex (a) and a non-convex (b) production technology (Chambers 1988, p. 253). Fig. 5. Illustration of a convex (a) and a non-convex (b) production technology (Chambers 1988, p. 253). b) Non-convex technology a convex set, is no smaller when using the total amount az1+(1–a)z2 of inputs to obtain an output than the expected value of outputs obtained when using inputs z1 and z2 separately: technology has no ‘holes’ in the boundary of the production possibilities frontier. The requirement is essentially a mathematical requirement that can- not always be contradicted by observable data (Chambers 1988, p. 252). Output y in Figure 5 il- lustrates, for instance, the daily weight gain of a pig when it is given amount z of feeds. Then, Fig- ure 5a can illustrate a ﬂexible feeding technology and Figure 5b two-phase feeding technology. The optimal resource allocation lies on the frontier of the production possibilities set, such as at the inter- section of z* and y* in Figure 5a. The kink in Figure 5b suggests that there is a region where increasing the use of input z yields no additional output de- spite the fact that higher output is available. (2) f(az1+(1–a)z2) ≥ a f(z1) +(1–a) f(z2), where 0<a<1. The inequality theorem implies that when a producer deviates from the optimal solution, he/ she suffers losses and cannot increase the value of a capacity unit. Thus, non-convexity (or linearity) of production technology can have the result that the optimal solution ﬂuctuates over time. This phe- nomenon is known as the bang-bang control (e.g. Intriligator 2002, p. 358). Figure 6 illustrates the case where the costs are either convex or non-con- vex. pyy is income from selling the output y at price py, whereas c(pz,y) is production cost as a function of output y and input prices pz. A producer who is a rational proﬁt maximiser never chooses the re- gion y< y< y1 if y and y1 are available. Rather, such an individual hops over this non-convex re- gion (broken line) (Chambers 1988, p. 142, Grav- elle and Rees 1992, p. 204–205). 2.3.3 Convex and concave sets and functions The properties of production and cost functions can have important implications for optimisation. The property of convexity is closely related to the concepts of half space and hyperplane. A half space is the set of all points lying on one side of a hyperplane in a real space. Graphically this means that drawing a line (hyperplane) across the real space creates half spaces both below and above the line. Since all half spaces and hyperplanes are con- vex sets, a closed convex set can be deﬁned using hyperplanes that support the set. For a convex set it holds that the line az+(1–a)z’, where 0<a<1, can connect any two points in the set so that all points lying on the connecting line belong to the convex set. For a non-convex set, the points at the con- necting line can lie outside the set (Figure 4) (Mas- Colell et al. 1995, p. 947–949). The cost of producing a certain amount of out- put is another issue that is of interest in the eco- nomic analysis. Hence, the cost function: (1) c(p,y) = min z≥0 {pz:z ∈ V(y)}, where p is a vector of strictly positive exogenous input prices, and pz is the inner product. As Equa- tion 1 illustrates, the cost function represents the minimum cost of producing a given level of output at a given time and given input and output prices (Varian 1984, p. 21, Chambers 1988, p. 50–59). According to Varian (1984, p. 37), the cost func- tion summarises all economically relevant infor- mation about the production technology of the ﬁrm. Cost functions also have desirable properties, such as concavity and non-decreasingness in pric- The production possibilities set should be a closed convex set in order to guarantee that the z z’ az+(1-a)z’ Fig. 4. Convex and non-convex sets (Mas-Colell et al. 1995, p. 947). b) Non-convex technology. 22 z z’ az+(1-a)z’ z z’ az+(1-a)z’ Fig. 4. Convex and non-convex sets (Mas-Colell et al. 1995, p. 947). a) Convex technology b) Non-convex technology. z z’ az+(1-a)z’ Fig. 4. Convex and non-convex sets (Mas-Colell et al. 1995, p. 947). b) Non-convex technology. z z’ az+(1-a)z’ a) Convex technology az+(1-a)z’ az+(1-a)z’ Fig. 4. Convex and non-convex sets (Mas-Colell et al. 1995, p. 947). b) Non-convex technology. b) Non-convex technology. a) Convex technology 22 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs y pyy c(pz,y) y* c(pz,y) pyy y1 Fig. 6. Proﬁt maximisation with non-convex costs (Cham- bers 1988, p. 142). y pyy c(pz,y) y* c(pz,y) pyy y1 fully applied to both discrete and continuous time problems in economics and management (for ex- amples, see Glen 1983, Kennedy 1986, Ljunqvist and Sargent 2000, Stokey and Lucas 1989). The fundamental principle of dynamic programming is Bellman’s principle of optimality (Bellman 1957, p. 83): ”An optimal policy has the property that, whatever the initial state and optimal ﬁrst decision may be, the remaining decisions constitute an op- timal policy with regards to the state resulting from the ﬁrst decision.” Elements deﬁning a dynamic programming problem are (Kennedy 1986): 1) A sequence of decisions ut, t=1,…,T 2) a set of states xt for t=1,…,T where a decision has to be made, Fig. 6. Proﬁt maximisation with non-convex costs (Cham- bers 1988, p. 142). 3) a transformation (or transition) function g(xt, ut), and 4) a stage return function Rt(xt, ut) that gives in- stantaneous returns for each decision at each stage and point of time. at both y* and y1. Although pigs can respond to changes in feeding by changing their eating behav- iour (Whittemore et al. 2002), large ﬂuctuations in daily feed allowance should be avoided. Optimisa- tion models often assume decreasing marginal en- ergetic efﬁciency above maintenance require- ments. However, this assumption has been criti- cised for its lack of empirical support (Emmans and Kyriazakis 1995). From the biological point of view the criticism can be justiﬁed, while from the economic point of view such assumptions can be also due to the way the problem is formulated, such as taking into account the amount of wasted feed (cf. Campos 2003). 5) A set of constraints that may restrict free choice of choosing the optimal sequence of decision variables. Modifying the notation of Kennedy (1986, p. 27–31) for the purposes of this study, the deci- sion problem of how to use resource xt (such as production facilities) can be considered as the problem of maximising recursively the value Vt(xt) of the resource with respect to the decision varia- ble ut: (3) Vt(xt) = max ut {Rt(xt, ut)+βVt+1(xt+g(xt, ut))}, for t=T,…,1, subject to VT(xT)=F(xT) and x1=∞x1, A G R I C U L T U R A L A N D F O O D S C I E N C E Observationally equivalent convex technology would, between y* and y1, follow the broken line instead of the solid line. If production function is concave, then the im- plicit cost function (i.e. the cost of deviating from the optimal bounded resource allocation (Gravelle and Rees 1992, p. 36–39)) is convex in y. Convex- ity of the implicit cost function for a twice-con- tinuously differentiable cost function is equivalent to the traditional neoclassical assumption of rising marginal cost, which is usually taken as a neces- sary condition for a stable equilibrium to exist (Chambers 1988, p. 138–140). Furthermore, if the objective function is quasi-concave and the feasi- ble set is convex, then the function has concave contours, and a local maximum is also a global maximum (Gravelle and Rees 1992, p. 22–28). Lack of sufﬁcient concavity or convexity is probably the most common reason for the conver- gence problems of optimisation models. An exam- ple of such a problem is that the optimal solution varies between y* and y1 when the proﬁts are equal The deﬁning property of a concave function is known as Jensen’s inequality (Mas-Colell p. 185– 186 and 931). The inequality theorem says that the expected value of a concave function f, deﬁned in 23 Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. planning horizon, and x1 is the given initial state of the resource. spond to changes in the state of nature over time, then he/she can beneﬁt from taking this option into account when the future development of the proc- ess is uncertain. The argument is based on an op- tions approach and recursivity of decision-making (for details, see Dixit and Pindyck 1994, Chapter 2). An applied example could be that if producer observes that pigs are growing poorly, then a re- cursive model allows producer to adjust feeding patterns or to replace pigs prematurely with pigs that are expected to be more productive than the current pigs. Under uncertainty this option yields higher returns than the case where feeding and slaughter decisions are chosen deterministically in the beginning according to the average case, and producer cannot respond to his/her observations of pig growth. Therefore, beneﬁts from ﬂexible feed- ing technology under uncertainty could be even larger in a dynamic adjustment model than in a de- terministic model. The rules that summarise the optimality condi- tions for control problems are, in general, referred to as the maximum principle. In dynamic program- ming, the principle of optimality implies the Mark- ovian property, which says that the future state of the resource depends only on the current state of the resource and the current decision (Ljunqvist and Sargent 2000, p. 1). Thus, past events do not affect the optimal future decisions that maximise the value function in Equation 3. This emphasises the importance of deﬁning the state so that it in- cludes all information that is relevant for choosing ut optimally at a given xt. The principle of optimal- ity holds also for other separable objective func- tions besides additive functions (Nemhauser 1966, p. 34–39). The Markovian property is also related to the recursivity of the decision problem. Recursivity refers to the fact that the decision problem is split into small pieces over time and the pieces have in- tertemporal connections. Recursive methods are a very powerful approach to economic dynamics as they focus on the trade-off between the current pe- riod’s utility and a continuation value for utility in all future periods (Ljunqvist and Sargent 2000, p. xxiv). Since determining Vt+1(xt+1) enables Vt(xt) to be determined, the value function is recursive (Kennedy 1986, p. 27). Due to the recursive nature of the optimal policy (i.e. 2.3.4 Dynamic programming where Vt(xt) is the maximised value of the resource at stage t=T,…,1, index t indicates the stage (such as time) under consideration, Rt(xt,ut) is the func- tion giving the instantaneous stage return, β is the discount factor, Vt+1 is the maximised value of the resource in the next stage, g(xt, ut) is the transition equation that deﬁnes both autonomous and con- trolled changes from the current stage xt of the re- source to the next-period stage xt+1 of the resource when the decision ut is taken, VT(xT)=F(xT) is the terminal value of the resource at the end of the The literature provides a number of techniques for solving the dynamic optimisation problems that were examined in the previous chapters. The tech- niques have differences as well as similarities. Of these techniques, dynamic programming (Glen 1983) and optimal control (Chavas et al. 1985) have been applied to pig fattening. Dynamic programming (Bellman 1957) was developed in the 1950’s by Richard Bellman for the planning of military logistics. It has been fruit- 24 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs One drawback of dynamic programming is that complex models having several state or decision variables frequently face excess computational burdens. This problem is known as “the curse of dimensionality” (Bellman and Dreyfus 1962, p. 323). Although advances in information tech- nology have facilitated the analysis of complex farm decision problems, most applications still re- quire considerable amounts of computational ca- pacity. Several approaches, such as the Hierarchi- cal Markov process (HMP) (Kristensen 1987), which omits the states of nature that are ‘useless’ in terms of the ﬂow of the underlying process, have been developed to solve the problem of di- mensionality. One of the largest HMP applications was reported by Houben et al. (1994), who consid- ered 6,821,724 states of nature. Kure (1997, p. 83– 98) also proposes an option to solve efﬁciently a recursive dynamic programming model in pig fat- tening. sibilities set, and then solve for the value of the resource under the chosen policy. The second step is to choose an alternative policy and to solve for the value of the resource under the alternative pol- icy. The third step is to compare alternative poli- cies and choose the policy with the highest value unless a policy with a higher value of the resource is available by repeating the second step (policy improvement) (Ljunqvist and Sargent 2000, p. 32– 33 and p. 633–635, Bertsekas 1995b, p. 35–49). Value function iteration includes policy itera- tion. Value function iteration is suitable for solving both ﬁnite-horizon and inﬁnite-horizon problems. It maximises the internal rate of return by solving the Bellmann equation explicitly and recursively. One of the differences between value iteration and policy iteration is how they handle time. Policy it- eration optimises the control variable over (gener- ally) inﬁnite time horizon whereas value function iteration requires a sufﬁciently long ﬁnite time ho- rizon in order to have stable solutions. Initiating the value function iteration requires giving a start- ing vector, such as VT(xT)=0 (Ljunqvist and Sar- gent 2000, p. 32). The idea is to iterate the solution to the Bellman equation over time for a sufﬁciently large number of successive iterations t* (note that the time can run virtually, from the future to the past). Ultimately, the optimal decision vector be- comes the same for iteration t* and for all subse- quent iterations (Ljunqvist and Sargent 2000, p. 32, Bertsekas 1995b, p. 19–34). Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs In other words, the optimal control policy converges so that in- creasing t* does not alter the decision vector (Stokey and Lucas 1989, p. 332). Ultimately Vt(xt) also converges to V(x) if t* is large enough (Kennedy 1986, p. 30). Dynamic programming has similarities with optimal control theory (for an application see Cha- vas et al. 1985). In optimal control problems, the variables are also divided into state variables and control variables. The control inﬂuences the objec- tive function directly through its own value and indirectly through its impact on the evolution of the state variable over time (Kamien and Schwartz 1992, p. 121–123, Seierstad and Sydsaeter 1993, p. 74–75). Despite the fact that the optimal solu- tion is a function of time, the method applies also to problems involving constraints on the state vari- ables or on the derivatives of the functions sought (cf. Kamien and Schwartz 1992, p. 121–123, Seierstad and Sydsaeter 1993, p. 73–73 and 194– 219, Bertsekas 1995a, p. 110–112). The discount factor is important in determining t, because a discrete change in the value function depends on how much additional value the optimal decision contributes to the asset in comparison with the depreciation of the asset over time. Thus, a large discount rate implies faster convergence than small discount rate (Bertsekas 1995b, p. 25–27). The contraction mapping theorem proves that the solu- tion to the optimisation problem is feasible only if the discount factor has a value that is between zero and one (Stokey and Lucas 1989, p. 49–55). Vol. 15 (2006): Supplement 1. a sequence of decisions over time), sequential decision problems can be solved by using backwards recursion. Thus, it is possible to obtain feedback from future decisions in order to optimise current decisions so that they depend only on the current state of nature. Dynamic programming can solve both analyti- cal and numerical decision problems. Analytically a dynamic programming problem can be solved by using the Hamiltonian or the Lagrange method. An analytical solution to the problem includes differ- entiating the objective function with respect to the decision variable ut and with respect to a change in state of nature over time (a time derivative) as a consequence of the current state and current deci- sion. The problem is to ﬁnd a solution where forth- coming returns and instantaneous returns are bal- anced so that no additional aggregate returns to the resource are available by adjusting sequence of de- cisions (for details, see Kennedy 1986 p. 10–23, Ljunqvist and Sargent 2000, p. 35–36, or Stokey and Lucas 1989, p. 66–102). Using the repeated substitution method, and by starting from the ter- minal period T, the optimal solution can be ob- tained stepwise for each moment of the time hori- zon. The solution for the ‘terminal’ period is known as the transversality condition (Intriligator 2002, p. 315–317). Recursivity can have important managerial im- plications. Uncertainty in decision making is known to decrease the utility of a risk-averse deci- sion maker (e.g. Mas-Colell et al. 1995, p. 193– 194). Such an uncertainty can arise, for instance, from inability to observe the current quality of a pig. On the other hand, Brealey and Myers (2003, p. 578) note that the option value of having the op- portunity to adjust decisions over time always ex- ceeds its minimum value (except when stock price is zero). Hence, if producer has an option to re- Common techniques used to solve numerical dynamic programming optimisation problems are value function iteration and policy iteration. In the policy improvement algorithm (Howard 1960), the ﬁrst step is to choose a policy from a decision pos- 25 Vol. 15 (2006): Supplement 1. designed to solve the problems identiﬁed in Chap- ter 1.3. Since the focus of this study is on econom- ics, which examines how scarce resources should be allocated (Black 1997, p. 137), a natural start- ing point is to maximise the utility of a pig meat producer with respect to decision variables. In this study, the measure of utility is the value Vt(xt) of a capacity unit at a given moment t and given state of nature xt of a capacity unit. The value of a ca- pacity unit is a suitable measure because it is the net present value of the most important variable income ﬂows that depend on feeding and slaughter decisions and genotype of a pig. sidy rates, animal welfare regulations, and biologi- cal constraints of animals, such as their genetic quality and the nature of their growth process (Fig- ure 7). The factors that producer can control can im- ply considerable adjustment costs. In other words, it may not be proﬁtable to change decisions once they are taken even if markets no longer provide incentives for choosing the action again. For in- stance, when a producer has invested in a capacity unit, it is costly for him/her to keep the unit idle as long as the slaughter income exceeds variable cost of production (cf. Ross 1980). Similarly, even if the prices are currently unfavourable, it can be proﬁtable to put effort into animal breeding in the long run. Since the value of a capacity unit gives information on a producer’s incentives to invest in new production capacity in the long run, this study indirectly examines also pig farm investment deci- sions. In conclusion, a producer can optimise feed- ing and slaughter patterns conditional on exoge- nous constraints and the dynamics of the produc- tion process. The goal of the study is to examine how a pro- ducer can maximise the value of a capacity unit by simultaneously optimising feeding and slaughter patterns so that the quality of a carcass is taken into account (White region inside the shaded box in Figure 7). Both exogenous and endogenous fac- tors determine how much utility producers can re- ceive from pig farming over time. Exogenous fac- tors set limits for pig fattening operations, whereas endogenous factors can be chosen by an individual producer himself/herself. 2.4 Scope and method of the study Until now, this study has focused on the result and research approaches of previous studies. This chapter proposes an approach and a method that is 26 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs explicitly on time, dynamic programming can be a more convenient method for solving discrete time models than optimal control method. Dynamic programming can have generality and simplicity for numerical solution of low dimension problems because concavity/convexity assumptions are not required and inequality and integer constraints can simplify computation (Burt 1982, p. 383–384). Lack of sufﬁcient concavity or convexity can, however, result in instability of the optimal control policy at certain points of the state space (e.g. In- triligator 2002, p. 358). Furthermore, the solution to the value function, the state variables and the control variables can freely choose any functional form. that economic efﬁciency enforces on the optimal feeding decisions. While being theoretically strong, the major disadvantage of static production function analysis is that it can hardly take into ac- count the timing of allocation of resources. Opti- mal control theory and dynamic programming, on the other hand, emphasise the time aspect. Agricultural resource management decisions frequently have irreversible effects on the resource itself or on the cost of reversal. This irreversibility and uncertainty may signiﬁcantly affect the value of a decision (Dixit and Pindyck 1994). Hence, one of the advantages of dynamic programming is the option to take into account information gained from the underlying process over time and to re- vise production decisions according to this infor- mation. In dynamic programming, the optimal se- quence of decisions takes into account the effects that current decisions have on future decisions. With rotating modiﬁcations, similar feedback is obtainable also from an optimal control problem (Chavas et al. 1985, p. 643). For analytical problems optimal control can be more useful than dynamic programming, as the maximum principle can solve the control variables and the co-state variables separately (Intriligator 2002, p. 355–357). Dynamic programming is, however, suitable for solving hardly tractable problems possibly containing nonlinear functions and sequential aspects (Kennedy 1986, p. 6, In- triligator 2002, p. 365). Pig fattening involves both nonlinear growth functions and sequential slaugh- ter decisions. Dynamic programming is also tech- nically attractive for solving the problem at hand numerically, because it requires placing fewer re- strictions on the state variables rather than using optimal control. Dynamic programming has prov- en to be useful in simultaneous optimisation of feeding and slaughter decisions in cattle (Feiner- man and Siegel 1988, Mourits et al. 1999, Pihamaa and Pietola 2002, Nielsen et al. 2004) and broilers (Kennedy et al. Vol. 15 (2006): Supplement 1. Endogenous factors in- clude such decisions as which feeds to use, how much to use, when to market pigs for slaughter and what kind of animals to buy. Nevertheless, exoge- nous factors largely determine which decisions are optimal for an individual producer. Exogenous factors include, for instance, prices of inputs and outputs, quality premiums paid for lean meat, sub- Studies (see Chapter 2.2) suggest that optimi- sation methods are powerful tools for analysing the optimality of feeding and slaughter decisions. As Chavas et al. (1985), Dillon and Anderson (1990, p. 113), Boland et al. (1999) and some oth- ers suggest, the method of study needs to be able to take into account the dynamic and sequential na- ture of pig fattening, as well as the requirements 27 Endogenous factors Objective: max V(xt) The choice of feeding pattern The timing of slaughter Long term objectives: - Technology - Breeding strategy Markets -Meat price -Input prices Agricultural policy & regulations Exogenous shocks to pig farming Biological limitations Farm-specific constraints Other exogenous factors Fig. 7. The scope of this study is to examine feeding and slaughter decisions that maximise the value of a capacity unit (V(xt)), condi- tional on exogenous and prede- termined factors such as market conditions and technology, and the effects that changes in these factors have on the decisions. Endogenous factors Objective: max V(xt) The choice of feeding pattern The timing of slaughter Long term objectives: - Technology - Breeding strategy Markets -Meat price -Input prices Agricultural policy & regulations Exogenous shocks to pig farming Biological limitations Farm-specific constraints Other exogenous factors Markets -Meat price -Input prices Agricultural policy & regulations Exogenous shocks to pig farming Biological limitations Fig. 7. The scope of this study is to examine feeding and slaughter decisions that maximise the value of a capacity unit (V(xt)), condi- tional on exogenous and prede- termined factors such as market conditions and technology, and the effects that changes in these factors have on the decisions. Fig. 7. The scope of this study is to examine feeding and slaughter decisions that maximise the value of a capacity unit (V(xt)), condi- tional on exogenous and prede- termined factors such as market conditions and technology, and the effects that changes in these factors have on the decisions. Objective: max V(xt) Farm-specific constraints Long term objectives: - Technology - Breeding strategy Other exogenous factors 27 3.1 Model speciﬁcation problems The objective of this study is to examine how pro- duction technology and input and output prices may affect pig production management and in- come in cases where the state of nature and pig’s growth pattern are known. Rather than to estimate feeding levels, the goal is to study how the value of a capacity unit changes and how the optimal feed- ing and slaughter patterns should be adjusted when pig genotype or market price changes. The analy- sis should therefore focus on comparing differ- ences between alternative scenarios, because the results arise from differences in model parameters rather than from absolute values of the parameters. The goal is also to study how much producer can beneﬁt from using a ﬂexible feeding technology, which allows him/her to control feed ratios and carcass quality continuously, instead of two-phase feeding technology. The main issue is whether it is proﬁtable to invest in technology where feeds can be supplied according to the current state of the pig. Dynamic optimisation of feeding and slaugh- ter decisions is suitable method for estimation of such a ﬂexible production technology. Studies (e.g. Glen 1983, Boland et al. 1993, Kure 1997, Sevón-Aimonen 2001) have used growth functions to characterise pig growth as a function of pig’s age. Growth functions are often used in a mechanical growth model to characterise pig genotype in terms of stomach capacity (cf. Glen 1983, Black et al. 1986, Emmans 1995). The functions can separate growth into components that can characterise carcass leanness, weight and market value. This characterisation can be inter- preted as a production technology, and input use can be optimised accordingly. In this study, growth of an individual pig under unrestricted feeding is based on stomach capacity. The problems are approached by modelling the pig management problem under a speciﬁc produc- tion technology. The studies reviewed in Chapter 2 suggest that such a modelling problem ought to take several biological and economic aspects into account. These include characterisation of pig growth, markets, and uncertainty related to deci- sions. Economic analyses traditionally separate feeding and slaughter decisions. Chavas et al. (1985), Kure (1997) and Boland et al. (1999) how- ever argue that feeding and slaughter decisions should be optimised simultaneously. This issue is of great importance because it is linked to carcass quality and value. 3 A dynamic programming model for pig fattening and it uses information obtained from literature and growth experiments. The advantage of the structural form model is that it can explicitly char- acterise the mechanism of how feeding and slaugh- ter decisions and genotype of a pig affect growth, carcass quality and carcass value, and how chang- es in meat price quality premiums, input and meat prices and genotype affect optimal decisions. Tak- ing into account simultaneity of decisions causes trade-offs, such that there is insufﬁcient computa- tional power to take into account stochastic aspects that have been elaborated in empirical models. The novelty of this study is therefore in that it explic- itly analyses the simultaneity of feeding decisions and slaughter decisions and their links to pig geno- type and carcass quality. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 1976, Talpaz et al. 1988). Dynamic programming can therefore provide a useful ap- proach for optimising feeding and slaughter deci- sions in pig fattening as well. One of the differences between the two meth- ods is that the optimal solution of dynamic pro- gramming depends only on the current state of nature, whereas the optimal solution of the optimal control is a function of the state of nature and time. Although a dynamic programming problem is more general than the classical calculus of varia- tions problem, the maximum principles of the two methods have much in common (Intriligator 2002, p. 330–334). Both principles can be deﬁned for discrete-time and continuous-time problems. A discrete-time formulation is appropriate for most practical resource problems in agriculture, because outputs are frequently obtained and inputs applied periodically (Kennedy 1986, p. 11). As the optimal solution in dynamic program- ming depends only on the state variable and not 28 A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs (e.g. Burt 1992, Broekmans 1992). Market uncer- tainty can be dealt with by modelling the impact of uncertainty or removing the uncertainty. The ap- proach taken in this study is to remove the uncer- tainty from the model by allowing producer to make ﬁxed-price meat marketing contracts such as futures contracts. The approach is justiﬁed because pig meat producer prices in Finland are quite sta- ble in the short run when compared to most other EU countries, and because slaughterhouses that buy slaughter pigs and trade piglets in Finland pro- vide also price lists that remain in effect until a speciﬁed delivery date. (cf. Chavas et al. 1985, Sipiläinen and Ryhänen 1996). This study therefore allows producer to control both feed supply and carcass quality through feeding decisions. This is in contrast with many previous studies that assume that growth fol- lows function based on the stomach capacity. p y Previous studies have related time to the dy- namics of the growth process of production ani- mals (Glen 1983, Chavas et al. 1985, Kure 1997, Sevón-Aimonen 2001, Nielsen et al. 2004) and to the timing of replacement and marketing decisions (Giesen et al. 1988, Huirne et al. 1993, Kure 1997, Mourits et al. 1999, Roemen and de Klein 2000, Toft et al. 2005). These relationships emphasise the irreversible nature of time. For instance, past feeding decisions cannot be undone. The analysis conducted in this study assumes that producer can control feeding and slaughter decisions daily ac- cording to the current quality of the pig. This is justiﬁed because this study focuses on characteris- ing production technology, where time has an im- portant role. Daily adjustment is justiﬁed also be- cause textbooks (cf. Dixit and Pindyck 1994, p. 335, Hayashi 2000, p. 121) suggest the use of high-frequency data when estimating variables that are of interest. Low-frequency data that has long time steps could alter slaughter decisions be- cause the value of marketing a pig now versus marketing it in the subsequent period is affected by the length of the time step, especially if it is large. Decisions in pig fattening may also include biological uncertainty and variation. Uncertainty arises, for instance, when a heterogeneous group of pigs needs to be fed and managed but their ge- netic characteristics are unknown. Variation can arise when growth rates of pigs in a group differ. Studies (e.g. 3.1 Model speciﬁcation problems Another important issue that needs to be taken into account in modelling pig growth is feed sup- ply. Feed supply should be adjusted according to the maturity stage of a pig because requirements for protein, energy and other nutrients change as the pig grows (MTT 2004). In addition, producer can minimise feed costs and nutrient leakages by controlling feed supply (Boland et al. 1998, Cam- pos 2003). Studies reviewed in Chapter 2 suggest that it is justiﬁed to separate feed supply into com- ponents. It is economically important to take into account options to control carcass quality through feed supply because producer can (in suitable con- ditions) beneﬁt from controlling carcass quality As opposed to studies that use predictive em- pirical models (such as Kure 1997), this study is mainly based on a characterisation of the pig’s growth mechanism (cf. studies such as Glen 1983) 29 3.2 The optimisation problem The dynamic programming model that is intro- duced in Chapter 3 is a recursive optimisation model. The model takes into account that producer can optimise current decisions according to the current state of nature and expected returns, and adjust his/her decisions in the forthcoming periods if necessary on the basis of additional information on production incentives. Hence, the model is also well suited to solve stochastic optimisation prob- lems such as the one presented in Chapter 7. As Dixit and Pindyck (1994, Chapter 2) and Brealey and Myers (2003, p. 578) illustrate, the ability to apply a model with a recursive structure gives an option to adjust decisions after observing the state of nature and thus guides to the optimal decisions and higher returns. The model is solved numeri- cally by iterating on the Bellman equation (for de- tails, see Chapters 2.3.4 and 4.2). Value function iteration is used because it maximises internal rate of return to the capacity unit and solves the opti- mal feeding and slaughter decisions recursively. This method it is suitable for solving inﬁnite-hori- zon problems. It can also be used effectively when the planning horizon is ﬁnite (as is often the case for individual producers). This chapter focuses on modelling the pig meat producer’s decision problem using dynamic pro- gramming (DP). The model developed here will be applied to solve speciﬁc decision problems in Chapters 4–7. The goal of the pig meat producer is to ﬁnd a management policy that maximises the expected net present value of a capacity unit. The objective includes controlling carcass quality so that the value of a capacity unit is also maximised when the timing of slaughter is exogenously deter- mined. The value to be maximised is the discount- ed net income from selling the animals for slaugh- ter plus related slaughter subsidy minus by the cost of producing ready-to-slaughter animals over a given planning horizon. In order to reach the ob- jective, producer controls the feeding and slaugh- ter decisions simultaneously. The economic model of the problem follows a Bellman equation of the form: (4) Vt(xt) = max ut {Rt (xt,ut) + βVt+1(xt+1)}, t = 0,1,…,T subject to: xt+1 = g(xt,ut) (transition equations) x0 given (initial state given) VT+1(xT+1)given (the terminal value), Decision and state variables of the model are evaluated discretely, at given nodes of the state and control space. Vol. 15 (2006): Supplement 1. growth of an individual pig accordingly. This ap- proach is chosen because models of the growth of group of heterogeneous pigs are very liable to the curse of dimensionality (Bellman and Dreyfus 1962, p. 323). In other words, computational bur- den of such a stochastic model becomes excessive (see Chapter 4.2). However, the theoretical model can be extended to cases where production tech- nology and growth rates are unknown. The impacts of the underlying assumptions on the results are discussed in Chapter 8. and the cost of replacement piglets are excluded from the analysis. The impact of these constant factors vanishes when alternative scenarios are compared (for details, see Robinson and Barry 1996, p. 66). Hence, even if these constant factors affect the value function, they do not affect the ranking of the scenarios. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Jorgensen 1993, Kure 1997) suggest that these factors decrease returns to pig fattening. Models should therefore take into account the fact that biological variation within a pig group can be large and that producer cannot fully observe geno- type of a pig or state of nature of a pig. Studies such as Jorgensen (1993), Boland et al. (1996) and Kure (1997) have explored the effects of biological uncertainty and variation in pig fat- tening in a detailed manner. Less work has been done to characterise simultaneous optimisation of feeding and slaughter timing of pigs, and their link to the carcass quality. Simultaneous analyses have been conducted on other animals (see e.g. Talpaz et al. 1988, Mourits et al. 1999, Pihamaa and Pie- tola 2002, Nielsen et al. 2004). Chavas et al. (1985), Kure (1997) and Boland et al. (1999) argue that feeding and slaughter decisions interact and should be optimised jointly. The contribution of this study to the literature of pig management lies in its ability to take into account simultaneity of feeding and slaughter decisions and link them to carcass quality. The issue is of great importance because carcass quality determines quality premi- ums paid for a carcass. Even if production decisions are made each day, it should be noted that in practice it can be very difﬁcult to control carcass quality with de- sired accuracy in a group of pigs (cf. Hansen 1992, Parsons et al. 2004). It can take time to observe how growth rates change after adjusting feeding (White et al. 2004) and producer may not reach very high lean percentages of carcass through feeding (Parsons et al. 2004). The model therefore imposes restrictions, such that the pig has limited ability to utilise very large amounts of feed or diets with very high protein content. When modelling pig production technology it is also important to take uncertainty into account. Market uncertainty related to future prices of in- puts and outputs affects production decisions be- cause it makes returns to a capacity unit uncertain The analysis conducted in this study assumes that producer can observe production technology (e.g. genotype) and state of a pig and control 30 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs ning of the planning horizon. The transition equa- tion determines the connection between the cur- rent state of nature and the next-period state of nature for a given control. The transition equation consists of the pig growth model, the constraints that genotype imposes on the growth model, and other constraints and controls that may affect evo- lution of the state of nature over time. Although the Bellman equation above is deterministic, the model can be extended to have stochastic features by attaching an expectations operator so that the next period’s value function is uncertain. producer produces two outputs: lean meat and by- products. The control vector ut decomposes into three generic control variables, viz. the decision to slaughter the animal ut cull, the amount of digestible protein supplied in the feeds ut prot, and the amount of digestible energy supplied in the feeds ut ener. The decision to sell the pig for slaughter is a binary choice variable. It takes the value of one when the animal is slaughtered instantaneously in period t, and the value of zero when feeding of the animal is continued. After slaughter, the animal is replaced by a newly weaned piglet whenever t<T. After the terminal period T, the value function equals the value of the slaughtered animal (i.e. the value of the carcass). The time unit of the model is one day. The state vector xt includes input and output prices and animal weight measures. The effects of price uncertainty are not considered in this study because prices have a ﬁxed value that is exoge- nously given to the producer in each scenario. Prices are nevertheless included as state variables in the model because they give information that is relevant for making optimal decisions in each sce- nario. The weight measures contain three compo- nents, viz. the weight of lean tissue xt lean, the weight of fatty tissue xt fat and the animal’s live weight xt weight. The live weight is not an independent state variable, because the two tissue components deter- mine the live weight component. Live weight vari- able is, however, used in Equations 6–12 as an auxiliary variable. Live weight is obtained as a function of lean and fatty tissues (cf. Glen 1983, Sevón-Aimonen 2001): 3.2 The optimisation problem The state and control spaces are piecewise linear because linear interpolation is carried out between the nodes (cf. Keane and Wolpin 1994). The model described in Chapter 3 could be solved for continuous space as well be- cause functions of the model are generally contin- uous. The model is normalised for a capacity unit, the results are estimated for a period of approxi- mately 5 years, and other costs than feeding cost where the optimal value function Vt is the function of the current state vector xt; t is the time index that measures time in days; ut is the control vector; Rt(.) is the one-period return function; β is the dis- count factor; Vt+1(xt+1) is the next-period value function; g(.) is the transition equation, VT+1(xT+1) is the value of a capacity unit after the terminal period T, and x0 is the state of nature in the begin- 31 3.3 Decomposing the control variables Pigs use protein and energy supplied in feed to grow lean and fatty tissue. The control variable ut ener measures the net energy (MJ) supplied in the feeds, whereas the control variable ut prot measures the amount of digestible protein supplied in the feeds in terms of lysine. Of the 22 commonly found amino acids, 9 are essential to the pig (Whit- temore 1998, p. 339). Because lysine is often the ﬁrst amino acid in the diet to limit the protein syn- thesis, the amount of protein in feed is measured by its lysine contents. Other amino acids besides lysine are supplied in amounts that do not restrict protein growth (cf. Glen 1983, p. 513–515). This approach helps to avoid the problem known as the curse of dimensionality. The mixture of amino ac- ids is balanced so that at least 7% of digestible pro- tein ut prot supplied in the feeds is lysine (Agricul- tural Research Council 1981). In addition, the pig can utilise 70% of supplied protein after absorp- tion (Fuller et al. 1989). (5) xt weight = (xt lean + xt fat + θ51(xt lean) θ53 + θ52(xt lean) θ54)/0.95, where θ5j, j=1,2,3,4, are parameters for the at- tached variables. The measure of live weight ﬁrst combines the amounts of lean tissue , fatty tissue , water and ash in the body to give empty weight. Dividing the empty weight by 0.95 then yields the live weight (see e.g. Whittemore 1998, p. 57). The weight of water in the body is estimated using a simple allometric function of live weight (param- eters θ51 and θ53). A similar function also estimates the weight of ash in the body (parameters θ52 and θ54). Therefore, lean tissue measures the amount of fat-free components (i.e. the amount of protein) in the body, and fatty tissue measures the amount of fat (i.e. the amount of energy) in the body. Hence, where θ5j, j=1,2,3,4, are parameters for the at- tached variables. The measure of live weight ﬁrst combines the amounts of lean tissue , fatty tissue , water and ash in the body to give empty weight. Dividing the empty weight by 0.95 then yields the live weight (see e.g. Whittemore 1998, p. 57). The weight of water in the body is estimated using a simple allometric function of live weight (param- eters θ51 and θ53). 3.4 The pig growth model where θ6j, j=1,2,3, are parameters (coefﬁcients) for the attached decision and state variables. Parame- ters θ61 and θ63 measure the amount of lysine re- quired for body maintenance as a function of live weight, and parameter θ62 refers to the amount of lean tissue that one unit of lysine given in the feeds can grow. The ileal digestible lysine required for body maintenance is at least 36 mg lysine per kg live weight per day (Fuller et al. 1989, p. 255). The parameter values are summarised in Table 1. When ut cull = 1, the pig is replaced with a newly weaned piglet, and Equation 6 reduces to xt+1 lean = x0 lean. The pig growth model measures how pig growth responds to the amount of nutrients supplied in the feeds. The growth model presented in this chapter is subject to the restriction that the stomach capac- ity of a pig does not restrict growth. This restric- tion is introduced into the optimisation model in Chapter 3.5. The model measures the amounts of protein and energy deposited into the body each day. This is also referred to below as the growth of lean and fatty tissue. The model takes into account interactions between the tissue components despite the fact that lean and fatty tissue growth are meas- ured using separate equations. The next-period states of nature given in Equations 6 and 7 are linked to each other such that xt fat affects xt+1 lean and xt lean affects xt+1 fat. Therefore, for a given control, the Markov property is satisﬁed for the system of growth equations and two state variables. The next problem is to deﬁne growth of fatty tissue. Partitioning of energy is affected by such factors as composition and amount of feed, hous- ing conditions, herd health, live weight and carcass composition and stocking density (for an over- view, see Black et al. 1986, Emmans 1999). Black (1995) has illustrated that metabolisable energy is used with an efﬁciency of 100% when providing heat to maintain body temperature below her lower critical temperature. It is therefore important for models to be capable of predicting which nutrients are to be used for particular purposes and to apply the appropriate energetic efﬁciencies. Vol. 15 (2006): Supplement 1. necessary amino acid is lacking, growth of protein tissue cannot increase unless the amount of that particular amino acid increases. The result is use- ful, because the mixture of amino acids can be de- ﬁned so that only one amino acid rations protein growth (Moughan 1995, p.75–76). The producer can partly substitute synthetic amino acid supple- mentation for amino acids required in feed (Valaja et al. 1993). For instance, adding lysine in solid or crystalline form when lysine restricts protein growth increases the daily weight gain and cost ef- ﬁciency of feed conversion and can also increase carcass leanness (Valaja 1992). Supplementation at later stages of growth may sometimes level off the beneﬁts of supplementation gained during the early stages of growth (Valaja et al. 1996). whereas the feeds used in farms generally contain both protein and energy. Therefore, producer con- trols the two generic control variables via the amounts of three feeds: barley ut barley, soy meal ut soy and synthetic amino acids ut amino. Producer can choose feeding and slaughter pat- terns simultaneously and separately for each day within the boundaries set by the pig’s natural growth and feed uptake characteristics. This means that the producer can apply unrestricted feeding, where the animal grows according to its growth potential. Alternatively, he/she may restrict the amount of protein feed, energy feed, or both so that the daily weight gain is below the animal’s growth potential. Hence, producer can control the value and quality of the carcass within limits set by the pig’s growth potential (Whittemore 1998, p. 48–52). The transition equation for lean tissue utilises the growth rate of protein in body: (6) xt+1 lean = xt lean – θ61xt weightθ63 + θ62ut prot, when ut cull = 0, (6) 3.3 Decomposing the control variables A similar function also estimates the weight of ash in the body (parameters θ52 and θ54). Therefore, lean tissue measures the amount of fat-free components (i.e. the amount of protein) in the body, and fatty tissue measures the amount of fat (i.e. the amount of energy) in the body. Hence, The two generic control variables ut prot and ut ener determine the growth of lean and fatty tissues, 32 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs (MTT 2004). This means that energy is supplied to the pig as metabolizable energy (ME). An alterna- tive would be to handle feeds in the form of digest- ible energy (DE) (Patience 1996). As noted above, the difference is that the net energy approach in- corporates the effects of feed quality, environmen- tal and animal speciﬁc factors in the transforma- tion process whereas in the DE approach, energy is supplied only to digestion process. in which the efﬁciency of energy use depends on diet composition, genotype and housing environ- ment of an animal as well as on the fat and lean tissue composition of the carcass. In this study, the utilisation of energy is sepa- rated into three parts, viz. energy required for maintenance, protein synthesis, and growth of fat- ty tissue. Because heavy animals require more maintenance energy than light animals, mainte- nance energy (mener(xt lean, xt fat, ut ener)) is a function of live weight. As one of the three state variables is redundant, maintenance energy is ultimately a function of the amount of protein and fatty tissue. Consumption of energy for maintenance is based on Collin et al. (2001). The estimate includes the energy required for fasting heat production and heat production due to physical activity. The energy required for lean growth is param- eterised as follows. First, let ∆xt lean and ∆xt fat denote the growth potential of lean and fatty tissues for a given current state of nature. These parameters re- ﬂect the maximum daily growth rate as a function of the state variable. They are derived in Equations 8, 9 and 10 by differentiating the Gompertz growth func- tion (Equation 8) with respect to time. Second, de- ﬁne a constant that measures the energy required for the growth of a given amount of protein tissue. Third, deﬁne a slope that increases the amount of energy required for lean growth when the growth of fatty tissue decreases and the growth of lean tissue re- mains constant. In order to optimise feed conversion to lean tis- sue, producers aim to achieve high lean growth rates without excessive fat deposition (Kanis 1988, Boland et al. 1993, Williams et al. 1994). Animals with reduced feed intake achieve lower lean growth rates, grow slower, and allocate a higher percentage of their energy intake to maintenance. 3.4 The pig growth model Then the model effectively becomes a “net energy model” Lean tissue growth is a function of the digesti- ble protein given in the feeds and the current live weight composition of the animal. Protein reten- tion is constrained by the fact that the pig needs amino acids in a balanced mixture. Thus, when a 33 Vol. 15 (2006): Supplement 1. The foregoing steps produce the transition equation for fatty tissue on the conditions that feeding is continued (for details, see appendix A, p. 108–109): such condition holds. In other words, unrestricted growth equals the animal’s growth potential that, due to limited stomach capacity, cannot be exceed- ed by increasing the amount of feeds given to the animal. Thus, feeding the pig in excess of its stom- ach capacity brings no monetary compensation. The growth terms could also be reversed as, in the event of unrestricted feeding, genotype restricts growth instead of feed supply. In addition to the upper growth limit, animal can have limits for the minimum growth of tissue for given time period (Whittemore 1998, p. 69). The minimum growth can be implicitly set, for instance, for animal wel- fare reasons. Like unrestricted growth, growth po- tential can also be separated into lean and fatty tis- sue. (7) xt+1 fat = xt fat + θ71ut ener – θ72(xt weight) θ73 – mprot(xt lean, xt fat, ut ener) where mprot(xt lean, xt fat, ut ener) = θ74∆xt lean + θ75∆xt lean (ut ener – θ79(xt weigth) θ73), θ76∆xt lean + θ77∆xt fat – θ78(xt weigth) θ73 where θ7j denote parameters for j=1,…,9, and ∆xt lean and ∆xt fat refer to the daily growth potential of xt lean and xt fat, respectively. Parameter θ71 in Equation 7 refers to how many megajoules of en- ergy are converted into fatty tissue, the term θ72(xt weight) θ73 refers to the energy used for mainte- nance as a function of live weight, whereas mprot(xt lean, xt fat, ut ener) measures the energy used in protein growth. It is a function of a constant θ74∆xt lean, which quantiﬁes the demand for energy for protein synthesis as a function of the amount of lean tissue in the basic situation and of a slope that adjusts demand for energy used in protein synthe- sis according to the current state of nature and the current growth rates. The slope is due to the fact that potential growth of lean tissue starts decreas- ing at a lower live weight that potential growth of fatty tissue. When ut cull = 1, the pig is replaced with a newly weaned piglet, and Equation 7 reduces to xt+1 fat = x0 fat. Under unrestricted feeding, the weight of both lean and fatty tissue follows the Gompertz func- tion. Vol. 15 (2006): Supplement 1. The literature often characterises the Gom- pertz function as the functional form that best ap- proximates the growth of pigs. The Gompertz function represents a sigmoidal (S-shaped) weight curve. The weight of a young animal grows slowly but the growth rate accelerates rapidly. After an in- ﬂection point, the growth decelerates (e.g. Whit- temore 1998, p. 59–76). Formally, the function is: (8) xτ i = α iexp(–kiexp(–ϕ iτ)) where i={lean, fat}. The subscript τ is the age of the animal measured in days, and αi, ki, and ϕi are parameters. Parameters αi and ϕi are usually re- ferred to as adult weight and maturing rate (Em- mans and Kyriazakis 1999). Equation 8 is not di- rectly applicable to the model, because the state variable is speciﬁed differently than in Equations 4–7. Utilising Equation 8 in the model requires transforming it so that is consistent with the equa- tions given in Chapter 3.3. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs When energy intake increases above that needed for maximum lean growth, the ratio of fat:lean deposition, backfat thickness, and efﬁciency of feed conversion into lean tissue increase (for a review, see Schinckel and de Lange 1996). To evaluate alternative manage- ment or genetic changes that affect energy intake, the relationship between energy intake and protein accretion must be characterised. Computing the slope of protein accretion on (energy) feed intake is a common way for describing the partitioning of energy between lean and fat growth (cf. Campbell et al. 1983). An optional way to express the rela- tionship between protein accretion and energy in- take is to use the ratio of lipid to protein deposition (cf. Whittemore and Fawcett 1976). The slope for the energy required for lean growth is computed by ﬁrst allowing ∆xt lean to de- crease by 60% below the growth potential, and thereafter measuring the change in energy required for lean growth when the growth of fatty tissue de- viates below the reference value ∆xt fat. The devia- tion is obtained from the amount of energy availa- ble for growth. Finally, the change in the growth of lean tissue is compared to the aggregate growth of both tissue components and weighted with the en- ergy that is available for growth after subtracting maintenance energy. The parameters are approxi- mated by converting the estimates of Quiniou (1999) to the net energy. The conversion is solved numerically so that the daily weight gain is the same in both the net energy approach and the di- gestible energy approach. Thereafter, the require- ment is linearly interpolated and extrapolated for animals having different values of the weight measures. The complexity of deriving the slope is due to the fact that the desired change of energy tissue growth in terms of net energy is observable only indirectly as a function of the weight meas- ures. The energy required for protein growth mprot(xt lean, xt fat, ut ener) is such that it maintains the protein level and is a function of the amount of fatty and lean tissue. Energy that is left after main- tenance and protein growth goes to the growth of fatty tissue. The model utilises the net energy ap- proach (NE) to growth of fatty tissue because the Finnish feed classiﬁcation is based on net energy 34 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Thus, the next-period state variable is the current state plus the growth rate of a tissue component during a time period. Finally, the terms are rear- ranged to obtain the next-period state for cases where the animal is not slaughtered in period t. Therefore, the transition equation under unrestrict- ed feeding is: when deriving the energy required for protein growth. For both lean and fatty tissue, the daily growth potential in Equation 7 follows: (10) ∆xi t+1 = ϕixi t(lnαi – lnxi t) when ut cull = 0. when ut cull = 0. In addition to having a maximum growth po- tential, the growth of fatty and lean tissue can also have minimum values (Whittemore 1998, p. 68– 75). In this study, no weight loss is allowed in ei- ther of the components. In addition, the ratio of the growth of fatty tissue with respect to the growth of lean tissue is restricted from below by factor λ. According to Whittemore (1998, p. 69), some fat is required in the body. The minimum target of fat: lean tissue ratio in a young pig is at least 1. The ratio, however, depends on the genotype of a pig. Physiologically, even ratio 0.3 may sufﬁce to sus- tain life. A similar target ratio holds for the ani- mal’s growth rates. The minimum ratio of fatty to lean tissue varies according to the animal’s matu- rity stage and genetic characteristics, and no gen- eral cut-off values are available for a minimum ratio of fatty to lean tissue. Whittemore (1998, p. 68–70 and p. 557–558), however, provides esti- mates of λ that can be used in modelling. This study utilises the estimates for commercial female and castrated male pigs. (9) xi τ+1 = xi t(1 + ∂lnxτ i/∂τ)  ≅ xi τ (1 + kiϕiexp(–ϕiτ))  = xi τ (1 + ϕilnαi – ϕilnαi +  when uτ cull = 0, ϕikiexp(–ϕiτ))  and = xi τ (γ i – ϕilnxi τ),  xi τ+1 = xi 0 when uτ cull =1, where γ i = 1 + ϕilnαi. The superscript i and parameters αi, ki and ϕi refer to the parameters as used in Equation 8. Since the next-period state no longer depends explicitly on age, Equation 9 can take the form of a general transition equation when τ is replaced with t. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Therefore, the transition equation for those cases where the pig utilises feeds up to its biological growth potential is xi t+1 = xi t(γ i – ϕ ilnxi t). When the animal is slaughtered at age τ (i.e. uτ cull = 1), Equa- tion 9 simpliﬁes to xi t+1 = xi 0 , because the next-pe- riod state then corresponds a newly weaned pig- let. The feed uptake function for Equation 9 fol- lows the Gompertz function. It should be noted that energy tissue of a pig grows according to Equation 9 only if energy supply from feeds is suf- ﬁciently large to produce a weight gain in Equa- tion 7 that is at least as high the growth potential in Equation 9. The same applies to the growth of lean tissue and supply of protein. Therefore, Equation 9 is subject to the condition that pig growth in Equa- tions 6 and 7 is at least as high as the growth poten- tial. 3.5 Restricting pig growth With respect to growth, the producer can distin- guish the cases of unrestricted growth and restrict- ed growth. Restricted growth (and restricted feed- ing) requires that the producer endogenously re- stricts the supply of nutrients in feed so that giving additional units of energy or protein to the animal can yield additional daily weight gain (see the growth model in Chapter 3.4). In the event of un- restricted growth (and unrestricted feeding) no To give the Gompertz weight function a form that is consistent with that of a transition equation, a logarithmic transformation of Equation 8 is done ﬁrst. Thus, the expression lnxτ i = lnαi – kiexp(–ϕiτ). Next, the transformation is differentiated with re- spect to time (i.e. ∂lnxτ i/∂τ). Then, the time deriva- tive is added to the current state, and the change is discretised as a relative change with respect to xτ i. 35 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 4 Precision feeding – a benchmark scenario also introduces the basic scenario by giving back- ground information and by deﬁning numerical val- ues for each of the model parameters. The basic scenario is presented in terms of the “average pig”. This implies that the pigs in a group are homoge- neous throughout the fattening period and that they are managed similarly. It is used as a benchmark and subsequent scenarios are compared to it. Char- acteristics of the average pig will serve as bench- marks that facilitate an analysis of the adjustments Vol. 15 (2006): Supplement 1. The yield of marketable meat of carcass live weight is computed with the formula: (12) φ(xt weight) = (1 – (θ121 – θ122xt weight)/100)xt weight , where θ121 and θ122 are parameters of the percent- age function. The percentage yield of meat at Vol. 15 (2006): Supplement 1. (11) Rt(xt,ut) = –(1 – ut cull)(ut soypt soy + ut barleypt barley + ut aminopt amino) + ut cull(φ(xt weight)pt meat(xt fat,xt lean) – pt weaner + pt subsidy), slaughter is generally 73–75% (e.g. Partanen et al. 1998, Perttilä et al. 2002, Partanen et al. 2003). The price of pig meat pt meat(xt fat,xt lean) follows current quality-adjusted meat pricing practices in Finland. The principle quality components subject to the adjustment are the share of red meat (Hen- nessy-scale) in the carcass and carcass weight upon slaughter. Although the share of red meat is not included in the instantaneous returns function, it implicitly affects the quality price premiums paid to producers. The following formula gives the share of red meat: where the current state vector xt includes animal weight measures as deﬁned above and exogenous prices (p’s). The quality-adjusted price of meat, pt meat(xt fat,xt lean), depends on the relative amounts of fatty and lean tissues as the slaughterhouses adjust the price for meat quality. pt weaner denotes the price of a weaned piglet, and pt subsidy is the slaughter pre- mium received for each slaughtered pig. Other in- put costs, such as veterinary and electricity costs, also affect the proﬁtability of pig farming. These costs are assumed to be constant over time. Time- constant costs affect the value of a capacity unit, but they do not affect optimal feeding and slaugh- ter patterns. Moreover, the impact of time-constant costs vanishes when value functions of alternative scenarios are compared. The function φ(xt weight) gives the amount of marketable meat as a function of the pig’s live weight. (13) ρ(xt lean,xt fat) = θ131 + θ132xt lean – θ133xt fat, where θ131, θ132 and θ133 are parameters of the func- tion. The complete functional characterisation of the model is a combination of several equations. Because these equations take various functional forms, it is not possible to fully certify that the concavity of production function will hold global- ly true. The amount of feed required for an addi- tional unit of growth, for instance, is approximate- ly linear. According to Jensen’s inequality theorem (for details, see Mas-Colell et al. 1995, p. 185– 186), lack of sufﬁcient concavity exposes the mod- el to the risk of having an unstable ‘bang-bang’ control pattern (Intriligator 2002, p. 358). A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. 3.6 One-period returns function Over the continuation region ut cull = 0, instantane- ous returns are equivalent to the variable cost of feeding the animal. The feed cost is the sum of the products of feed prices pt soy, pt barley and pt amino and feed quantities ut soy, ut barley and ut amino. When the pig is slaughtered, a producer obtains a return from marketing the pig for slaughter (salvage value) and incurs an expenditure from purchasing a new pig- let. Instantaneous cash ﬂows are evaluated dis- cretely at the same points of the state and control space where control and state variables are evalu- ated. Hence, Equation 11 gives the instantaneous cash ﬂow: Equation 9 also includes an expression for the daily growth potential of tissue component i={lean,fat}. This expression was utilised in Equa- tion 7. The expression is the time derivative that indicates the difference between the current state variable and value of the state variable in the next period. The result corresponds to the maximum daily growth of a tissue component that was used 36 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs g digestible lysine and 8.44 MJ energy. The corre- sponding values for one kilogram of barley are 81 g digestible protein, 2.6 g lysine and 9.07 MJ en- ergy (MTT 2004). that changes in price ratios or genetic characteris- tics cause in the optimal feeding and slaughter pat- terns and in the value of a capacity unit. The vari- ous components of the sensitivity analysis are pre- sented in Chapters 5–7. Synthetic amino acids supply only digestible amino acids. At ﬁrst, the supplement ut amino in- cludes lysine. When the amount of synthetic lysine increases above 0.8 g per kilogram of barley, the supplement also includes threonine. Finally, when the amount of synthetic amino acid supplement in- creases above 2 g lysine and 1 g threonine per kilo- gram of barley, the supplement also includes me- thionine. The price of the supplement is adjusted accordingly. The maximum supplement is limited to 2.7 g lysine, 1.3 g threonine and 0.8 g methio- nine per kilogram of barley. Balancing the mix of amino acids such that lysine always limits protein synthesis implies that when a feed contains less than 5% soy meal, pigs cannot fully utilise a lysine supplement above 2 g per kilogram of barley. The potential beneﬁts of increased ﬂexibility of feeding technology (Boland et al. 1999, Cam- pos 2003, p. 80) lie in the ability of a ﬂexible feed- ing technology to take into account the marginal value of the pig’s carcass quality and its biological nutrient requirements at various stages of growth in greater detail than an inﬂexible technology. As these requirements depend on animal’s stage of maturity and genotype, it is important to consider the beneﬁts from using precision feeding technolo- gies. Estimating the beneﬁts of precision feeding in- cludes comparing results achieved with alternative combinations of feeding and slaughter patterns, and then choosing the patterns that maximise the value of a capacity unit for a given genotype of a pig and for a given feeding technology. Thereafter, the solution to the problem includes comparing the results of the benchmark scenario and the two- phase feeding scenario in order to observe the ef- fect of the technology. This approach is designed to allow a producer to apply the results to any con- venient precision feeding technique. 4.1 Data and scenarios The theoretical optimisation model developed in Chapter 3 is now applied to empirical data in order to examine the research problems. This chapter fo- cuses on the ﬁrst research problem, which is to es- timate how much a pig meat producer can beneﬁt from using a ﬂexible feeding technology in com- parison with the two-phase feeding. This chapter 37 Vol. 15 (2006): Supplement 1. markets between January 2001 and June 2003. This period includes extreme ﬂuctuations of the price of pig meat. The prices of pig meat, piglets (estimated price for a 25 kg piglet) and barley are based on statistics provided by the Information Centre of Ministry of Agriculture and Forestry (Tike 2002, 2003). The piglet price is linked to the price of pig meat, and the price of barley includes a treatment cost of approximately €0.01. The av- erage price of pig meat was €0.04–0.05 below the prices of the benchmark scenario in January and March 2005 (Tike 2005, Tike 2005b). In February and March 2005, however, the price of piglets as well as to the price of barley was 5–10% lower than in the benchmark scenario (Käytännön Maa- mies 2005, p. 70–75). estimate the initial body composition of their live littermates. One female and one castrated male piglet from 22 litters were weaned at the age of 5 weeks and reared until the age of 168 days. Live weight was recorded weekly. Pigs were fed ac- cording to appetite in groups of two pigs, applying three-phase feeding (200 g, 160 and 150 g crude protein and 13.3, 10.2 and 8.9 g lysine per kg dry matter in phase I, II and III, respectively). Half of the carcass of each pig was dissected using same method as used in the Finnish station test proce- dure (see e.g. Faba 2004). Carcasses were dissect- ed to three fractions and their chemical composi- tion was analysed. Based on the experiments pre- sented by Sévon-Aimonen (2001), the parameters of the growth curves were estimated in 2004 for this study, with minor corrections2 for the present study, at the animal level and with the non-linear least squares method. The estimates of maturing rate reported in Table 1 are identical to those pre- sented by Sévon-Aimonen (2001), and the differ- ence between the estimates of mature weight is less than 2%. Despite considerable genetic varia- bility within a group of pigs, the estimates are ex- pected to be consistent. Sévon-Aimonen (2001), for instance, reported that the pigs in any given group are heterogenous particularly with respect to mature weight of fatty tissue. p The marginal quality adjustments for the price of pig meat are continuous linear approximations of actual slaughterhouse price lists. The actual price lists apply discrete marginal quality adjust- ments. 2 The estimation results are still unpublished. Detailed estimates are available from Marja-Liisa Sévon-Aimo- nen. 3 Eeva-Liisa Paju, Raisio Group Plc., personal commu- nication 3 October 2003 4 €1 equals to $0.85–1.15. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs The tech- niques range from animal speciﬁc feeding ma- chines to having two feed stocks and two feed pipes that mix feed immediately prior to feeding. pp g p g y In the subsequent economic analysis, the pa- rameters of the growth model (see Table 1) are taken as given and known to the decision maker. Hence, the problem is not to estimate these param- eters but to estimate how genotype and market pa- rameters affect pig production management and the value of a capacity unit. Other parameters ex- cept the constraints on growth potential and the parameters of energy to protein growth are inde- pendent of genotype. The parameter values of ma- turing rate and mature weight are means of the parameter values of female and castrated male pigs, respectively (Table 1). Marja-Liisa Sevón- Aimonen, a researcher in the MTT Animal Pro- duction Research unit, has estimated the parame- ters based on a dataset of animal experiments pre- sented by Sévon-Aimonen (2001). The experi- ments were conducted at the Agri-Food Research Finland (MTT) Pig Research station in Hyvinkää on pigs obtained from 22 litters. Of the model variables, the generic control variable of slaughtering the pig ut cull is a binary de- cision variable having the value ut cull = 1 at slaugh- ter and value ut cull = 0 otherwise. Producer can de- liver pigs to slaughter every day. After the delivery, the pen is kept empty 24 hours so that the new pig- let enters the capacity unit on the day after slaugh- ter. The importance of optimising the timing of slaughter is examined in Chapter 7. The amount ut barley of barley in the diet is measured as grams per day, whereas the amount ut soy of soy meal used in these computations is a ratio of kilograms of soy to kilograms of barley. These feeds are used because they are among the most important feed ingredi- ents used to fatten pigs in Finland. One kilogram of soy meal supplies 403 g digestible protein, 23.9 The pigs in the experiment were born between December 1998 and March 1999. Dams were crossbreeds of Finnish Yorkshire and Landrace and genetically at the same level as sows in pro- duction stock. Sires were purebred AI-boars of the same breeds. In total 14 piglets were dissected and chemically analysed at the age of 3 days in order to 38 Vol. 15 (2006): Supplement 1. Linearisation improves the continuity and concavity properties of the model. Linear adjust- ments also illustrate producer’s valuation of the meat quality as a continuous variable. The slopes of adjustments in the benchmark scenario were ob- tained from selected Finnish slaughterhouse price lists. The base price is paid for carcasses that weigh 75–85 kg and contain 59% red meat. The marginal discount for the meat price is €0.02 for each ad- ditional kilogram deviating from the target weight range. Each additional percentage below 59% re- sults in additional price discount of €0.02 per kilo- gram of meat. Each additional percentage above 59% results in price premium of €0.02 per kilo- gram of meat. Information on parameter values of Equations 6 and 7 was also obtained from Marja-Liisa Sevón- Aimonen. In addition, loss ratio of meat upon slaughter and share of red meat in carcass were es- timated from the growth experiment data. In the basic scenario, where characteristics of female and male pigs are pooled, the minimum fat to lean growth ratio is λ = 1. This represents the ratio of commercial castrated male pigs as described in Whittemore (1998, p. 70). The prices of synthetic amino acids in Table 2 were obtained from Raisio Group Plc.3, one of the major feed manufacturers in Finland. The price of soy meal was derived from the Chicago Board of Trade futures price (USD) (Futurestrading 2003) by converting it to euros at the ofﬁcial exchange rates4 (Bank of Finland, 2003), and adding a con- In addition to the production function, prices (Table 2) are an important element of the model. The base price of pig meat and input prices in the benchmark scenario are the averages of minimum and maximum prices observed in the Finnish meat 39 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Table 1. The values of parameters used in the basic scenario, their standard error and a brief description of the variables related to the parameters. Parameter Description of related variable Value Standard error θ51 Weight of water in the carcass1) 5.107 0.652(1000 θ53/1000) θ52 Weight of ash in the carcass1) 0.195 0.026(1000 θ54/1000) θ53 Weight of water in the carcass1) 0.871 0.006 θ54 Weight of ash in the carcass1) 0.951 0.016 θ61 Lysine for maintenance2) 0.000 NA θ62 Conversion of one kilogram of lysine to protein tissue3) 8.333 NA θ63 Lysine for maintenance2) 0.75 NA θ71 Conversion of one MJ of energy to fatty tissue4) 0.025 NA θ72 Energy for body maintenance5) 0.026 NA θ73 Energy for body maintenance5) 0.6 NA θ74 Energy for protein growth at the potential6) 0.24 NA θ75 Adjustment of energy to protein growth based on the level of lean tissue6) 0.36 NA θ76 Adjustment of energy to protein growth based on the level of lean tissue6) 23.6 NA θ77 Adjustment of energy to protein growth based on the level of fatty tissue6) 39.3 NA θ78 Subtraction of maintenance energy from energy used to protein growth6) 0.102 NA θ79 Subtraction of maintenance energy from energy used to protein growth6) 1.02 NA αfat Mature weight of fatty tissue1) 52.85 15 ϕfat Maturing rate of fatty tissue1) 0.014 0.002 αlean Mature weight of lean tissue1) 29.69 5.3 ϕlean Maturing rate of lean tissue1) 0.014 0.002 θ121 Loss of meat at slaughter (constant)1) 30.81 NA θ122 The effect of live weight on the loss of meat at slaughter1) 0.04 NA θ131 The share of red meat in the carcass (constant)1) 56 3.005 θ132 Increase in the share of red meat due to lean tissue1) 0.749 0.19 θ133 Decrease in the share of red meat due to fatty tissue1) 0.443 0.048 NA not available. 1) Estimated from the growth experiment data by Marja-Liisa Sévon-Aimonen, MTT Animal Production Research. For g ( ) ( ) 4) One kilogram of fat contains 39.3 MJ energy and one kilogram of protein contains 23.6 MJ energy (e.g. Whittemore 1998, p. 281). 5) C lli t l (2001) Collin et al. (2001). 6) Derived using the results of Quiniou et al. (1999) and estimation results from the Finnish growth experiment. stant transportation cost to the futures price. Anonymous 2002b, Anonymous 2002c, Anony- mous 2002d, MMM 2004, p. 100–103, MKL 2003, p. 32). In 2005, the slaughter premium was lower than in the benchmark scenario. 5) Collin et al. (2001). 4.2 Estimation method at time t = T, the procedure selects a state of nature (a node) from all possible combinations (nodes) of lean and fatty tissue of a pig. Next, the procedure computes returns for all feasible values of the con- trol variable and thereafter chooses the option that maximises returns for the particular node. The re- turns are the sum of the t+1-period value function and the instantaneous returns function (Equation 4; maximised returns) for particular values of con- trol variables. Connections between the states of nature over time are obtained from the transition equations. Similar computations of returns and choices of the optimal decision are repeated sepa- rately for all state nodes at time t = T. Thereafter, the next-period value function is updated to corre- spond the current value function, and time index t is updated one period backwards in time (i.e. t = T–1). Then, the steps of computing the returns and choosing the optimal control values are repeated for all state nodes at time t, after which the value function is again updated and the time index pro- ceeds one step backwards in time. These steps are repeated until the time index becomes t = 0. The ﬁnal step is to print out the optimal decision path and the value function over time when initial state of nature is given. Appendix A provides further de- The optimisation problem is modelled in an opti- mal stopping framework by ﬁnite horizon recur- sive dynamic programming. The optimal controls and the value function are solved numerically by iterating on the Bellman equation. The iteration procedure utilises the value function iteration (for details, see Ljunqvist and Sargent 2000, p. 32) and the grid search method. The model optimises feed- ing and slaughter patterns conditional on the as- sumption that producer continues production. The value function indicates whether it is more proﬁt- able to exit the industry than to continue produc- tion. Producer should continue production only if the sum of the value function minus the omitted production costs is positive. Before initialising the procedure, the length of the planning horizon T is set long enough so that the feeding and slaughter patterns converge during the iteration process. The procedure locally line- arises the state and control spaces. A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Table 2. Price parameters Price parameter Symbol Parameter value Pig meat, €/kg 1) pt meat 1.39 +/– Price for each additional red meat percentage 0.02 – Discount for each kilogram of excess weight 0.02 Slaughter premium, €/carcass 2) pt subsidy 21.92 Piglet, €/piece3) pt weaner 61.07 Barley, €/kg pt barley 0.11 Soy meal, €/kg pt soy 0.31 Lysine, €/kg pt lysine 2.36 Methionine €/kg pt methionine 2.63 Threonine, €/kg pt threonine 5.25 Daily discount rate4) β 99.98% 1) The base price that is paid for a carcass weighting 76-85 kg and containing 59% red meat. Any deviation from these values results in a price premium or discount. 2) Paid for carcasses weighing at least 61 kg upon slaughter. 3) Estimated price of a 25 kg piglet. 4) Daily discount rate used in the analysis corresponds to 6% annual interest rate. Table 2. Price parameters Price parameter Symbol Parameter value Pig meat, €/kg 1) pt meat 1.39 +/– Price for each additional red meat percentage 0.02 – Discount for each kilogram of excess weight 0.02 Slaughter premium, €/carcass 2) pt subsidy 21.92 Piglet, €/piece3) pt weaner 61.07 Barley, €/kg pt barley 0.11 Soy meal, €/kg pt soy 0.31 Lysine, €/kg pt lysine 2.36 Methionine €/kg pt methionine 2.63 Threonine, €/kg pt threonine 5.25 Daily discount rate4) β 99.98% 1) The base price that is paid for a carcass weighting 76-85 kg and containing 59% red meat. Any deviation from these values results in a price premium or discount. 2) Paid for carcasses weighing at least 61 kg upon slaughter. 2) Paid for carcasses weighing at least 61 kg upon slaughter. 3) Estimated price of a 25 kg piglet. 3) Estimated price of a 25 kg piglet. 4) Daily discount rate used in the analysis corresponds to 6% annual interest rate. A G R I C U L T U R A L A N D F O O D S C I E N C E In ad- dition, producers receive a slaughter premium for each pig of at least 61 kg carcass weight upon slaughter. The premium varies according to region and year (Anonymous 2001, Anonymous 2002a, 40 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Define: - Input parameters - Control and state variables and their discrete spaces - The terminal value function - set t=T Trace the optimal control path using the optimised feeding and slaughter patterns Solve the optimal decision rules: 2. Compute returns for all feasible control options at the given node 4. Set t=t-1 3. Find the maximum returns for the given node Repeat until all nodes at t have been evaluated 1. Select a state of nature (a node) to be evaluated at the moment t Repeat until t=0 Fig. 8. Flow chart of the optimisation model. The model solves the optimal decisions and the value function when the cur- rent state of nature is given and time runs from t = T,..,0. Repeat until all nodes at t have been evaluated Trace the optimal control path using the optimised feeding and slaughter patterns Fig. 8. Flow chart of the optimisation model. The model solves the optimal decisions and the value function when the cur- rent state of nature is given and time runs from t = T,..,0. programming language (Aptech Systems 2003). The optimal decisions are solved for a time hori- zon of 1800 days (5 years). Within this period, the optimal feeding and slaughter patterns converged to the reported patterns, whereas the value func- tion required a much longer time period to con- verge. The value function was left unconverged because increasing the duration of the planning horizon did not alter the optimal control patterns but it did require more computational capacity than the current model. Kennedy (1986, p. 56) ar- gues that convergence of the optimal decisions for successive iterations is a good indication that the number of iterations (i.e. t) is sufﬁcient. Conver- gence of control variables was tested in two ways: 1) by verifying in selected scenarios that a signiﬁ- cant increase in the time horizon (until the value function had converged) had no impact on the con- trol patterns, and 2) that the model repeated the optimal feeding and slaughter pattern for at least 2–3 pigs close to the present. The results are there- fore suitable for comparison and ranking of sce- tails and the computer code of the optimisation procedure. 4.2 Estimation method It sets up dis- crete state and control spaces such that the state and control variables are evaluated at given points of the space (i.e. at given nodes). When initialised 41 Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. narios, whereas the value functions reported in this study are underestimated. where t is the time index, the term ut f denotes the optimal amount of marketable feed f in period t and for feed type f indicated by the superscript, and θ f 14j, j = 1,2,3,4, are parameters to be estimated. Since approximation is carried out only for the ﬁrst fattened and slaughtered pig, the time index t in Equation 14 counts the number of days elapsed from the beginning of the fattening period. When t = 1, the pig has a live weight of 25 kg (see Tables 1 and 2). All approximations are estimated using ordinary least squares regression and the Matlab econometrics toolbox (LeSage 2000). The amount of barley is evaluated at 50 g dis- crete ranges and the amount of soy is evaluated at the ranges of 20 g soy per kilogram of barley. The optimal level of amino acid supplement is iterated on a daily basis so that the other control variables are at the optimum. Then, the level is ﬁxed at the “optimal” level with respect to the amount of bar- ley. This approach keeps the number of control variables low enough in order to minimise the curse of dimensionality (Bellman and Dreyfus 1962, p. 323). The states of fatty and lean tissue are both evaluated approximately at 500 g ranges (37 nodes for xt lean and 52 nodes for xt fat). The state- control space approximates continuous space be- cause linear interpolation is carried out between the nodes. For more details, see the end of Step 4 in Appendix A. The curse of dimensionality can be a problem even if the state space is currently quite small, because control nodes and state nodes must be evaluated on all possible combinations of con- trol and state nodes and at each day of the planning horizon. Only slaughter decision is a binary con- trol variable. If feeding decisions are not opti- mised, then a standard PC could solve an individ- ual optimisation scenario in less that 60 seconds. The computations are carried out using the ac- tual numerical results. Hence, the approximations in Equation 14 are only to help graphical presenta- tion of the optimal feeding patterns. The layout of the Figures that illustrate the optimal feeding pat- terns is the following. A G R I C U L T U R A L A N D F O O D S C I E N C E The state space of a model needs to be sufﬁ- ciently large in order to ﬁnd the optimal path of control variables, and it needs to be sufﬁciently ﬁne across states so that the state and the control spaces do not themselves impose boundaries to the optimal solutions. When testing the model in year 2004, the grid was deﬁned by ﬁrst running the model with a rather coarse grid and then reﬁning the grid using the results as a reﬁnement indicator (cf. Kennedy 1986, p. 105). Care was taken to en- sure that neither the optimal state nor the optimal control space ever reached the maximal or mini- mal values given in the state and control space. Some coarseness still remains because a very ﬁne grid required considerable amount of computation capacity, but the remaining error has no impact on qualitative results, and only a small impact on the numerical results. The iterations are performed with a model (see Appendix A) developed in Gauss 6.0 for Windows 42 Vol. 15 (2006): Supplement 1. The optimised pattern (thin broken line) is visible behind the approximation (solid black line), and behind that is the approxi- mation of the benchmark scenario (grey line). When the broken or grey lines are invisible, the invisible parts of the line are graphically identical to the continuous black line (polynomial approxi- mation). Therefore, the Figures also show any er- ror caused by the approximation. The optimal solutions are characterised numer- ically as well. Monetary results over different time periods are converted to equivalent annual income (Brealey and Myers 2003, Chapter 6) using the re- sults for the pig that is closest to the present. This approximates the value function by carrying out a project appraisal type comparison. Monetary re- sults given for a 5-year planning period can be converted to annual values by multiplying them by 0.228. The optimal feeding and slaughter patterns are plotted graphically for individual pigs. In order to ﬁlter ﬂuctuations of the optimal ﬂexible feeding pattern due to the concavity problems, a polyno- mial approximation of the optimised feeding pat- tern is estimated for a single pig. The approxima- tion is a model that explains the daily amounts of soy meal and barley for each day of the fattening period. It utilises the fact that any nonlinear func- tion can be expressed using Taylor series expan- sion. In other words, the nonlinear function is lin- earised locally (Pindyck and Rubinfeld 1998, p. 267–268, Intriligator 2002, p. 329). Approxima- tions have a functional form that is ﬂexible enough to graphically illustrate the optimal feeding pattern of a single pig over time. The approximation takes the following form: The test of whether a producer can beneﬁt from applying a continuously adjusting feeding policy instead of a two-phase feeding policy is carried out by comparing the results of ﬂexible feeding policy to the result of the two-phase feeding. In the event of the two-phase feeding, a producer can choose the share of soy meal only twice for each slaugh- tered pig. The producer can adjust the amount of premixed feed (i.e. ﬁxed shares of barley, soy meal and amino acid supplement) each day, choose the live weight at which the share of soy meal in feed (14) ut f = θ f 141 + θ f 142t + θ f 143t2 + θ f 144t3, where f = {soy, barley}, 43 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs here to results where the optimal feeding and slaughter decisions at time t = 0 became stable over time and are unaffected by an increase in T. Convergence does not refer to the full convergence of both value function and control patterns. In the benchmark scenario, convergence suggests that it is optimal to slaughter the pig at 115 kg live weight after a feeding period of 105 days. Figure 9 illus- trates the regions of converged control patterns (Figures 9a and 9b), corresponding values of the state variables (Figures 9c, 9d and 9e) and the val- ue of a capacity (Figures 9f) unit for a ﬁnite plan- ning horizon of 1800 days (approximately 5 years). The convergence region is indicated by the narrow changes, and choose the timing of slaughter. The shares of soy meal and the switching weight are constant throughout the planning horizon. The op- timal two-phase feeding is iterated using a proce- dure which estimates the value of a capacity unit under optional soy feeding patterns and optional switching weights. 4.3 Results Decision rules converge to the optimal feeding and slaughter policy gradually. Convergence refers a) Share of soy meal in feed 0 5 10 15 20 25 1 201 401 601 801 1001 1201 1401 1601 1801 time in days % of feed b) Amount of barley in feed 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 201 401 601 801 1001 1201 1401 1601 1801 time in days kg feed per day c) Amount of lean tissue 0 5 10 15 20 25 1 201 401 601 801 1001 1201 1401 1601 1801 time in days kg d) Amount of fatty tissue 0 5 10 15 20 25 1 201 401 601 801 1001 1201 1401 1601 1801 time in days kg e) Live weight of a pig 0 20 40 60 80 100 120 1 201 401 601 801 1001 1201 1401 1601 1801 time in days kg f) Value function 0 100 200 300 400 500 600 700 800 1 201 401 601 801 1001 1201 1401 1601 1801 time in days € per capacity unit Fig. 9. Convergence regions (indicated by a small box having a width equal to the optimal length of the fattening period (i.e. 105 days) in each graph a-f) of the optimal control policy, deﬁned for the optimal feeding patterns (Figures a and b), the amount of lean tissue (c), amount of fatty tissue (d), live weight (e), and the value value of a capacity unit (f) over the entire optimisation period. When the amount of barley drops to zero, the pig is slaughtered and replaced with a new pig- let. Vol. 15 (2006): Supplement 1. box in each ﬁgure. As subsequent Figures report more detailed results, the purpose of Figure 9 is only to indicate the convergence of control pat- terns. In the subsequent analysis, feeding and growth patterns are reported for the converged policy only. The value of a capacity unit refers to the value of the value function at date t = 0 (Figure 9f). Annual approximations of the value function are based on the converged feeding and slaughter policy. The optimal amino acid supplements are 2.7 g of lysine, 0.8 g of methionine and 1.3 g of threo- nine per kilogram of barley. The efﬁciency of the lysine supplement decreases linearly when the share of soy in feed drops below 5% at the end of the fattening period. This means that when the share of soy meal drops to zero only 2 g of the sup- plement is available for growth. The adjustment allows only lysine to limit the protein synthesis. The optimal level of daily supplement is generally quite constant. Figure 10 reports the optimal amount of barley in the feed and the polynomial approximation thereof. The approximation estimates the optimal amount of barley quite well. The performance of the third-order approximation is quite similar, for instance, to that of the fourth-order approximation. The results suggest that the optimal diet of young pigs is protein-rich, whereas pigs close to slaugh- ter maturity have an energy-rich diet. In total, the pig eats 31.1 kg soy meal and 225.4 kg barley dur- ing the fattening period. The model recommends feeding 1.25 kg barley per day to 25 kg piglets. Thereafter, the amount of barley increases at a de- celerating rate. During the last 30 days of the fat- tening period, the amount of barley stabilises to 2.6–2.7 kg per day. Before slaughter, the amount of barley even decreases a little. Peaks in the dot- ted line in Figure 10 as well as subsequent ﬁgures are due to the fact that the production function is non-convex. In other words, production function does not imply globally decreasing marginal returns to input use (see discussion in Chapter 2.3.3). In pigs of 25 kg live weight, the share of soy meal is approximately 20% of the total amount of feed. Thereafter, the share of soy meal gradually decreases. After 85 days of fattening, the share drops below 6% (Figure 11). 4.3 Results a) Share of soy meal in feed 0 5 10 15 20 25 1 201 401 601 801 1001 1201 1401 1601 1801 time in days % of feed b) Amount of barley in feed 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 201 401 601 801 1001 1201 1401 1601 1801 time in days kg feed per day a) Share of soy meal in feed 0 5 10 15 20 25 1 201 401 601 801 1001 1201 1401 1601 1801 time in days kg b) Amount of barley in feed 0 5 10 15 20 25 1 201 401 601 801 1001 1201 1401 1601 1801 time in days kg d) Amount of fatty tissue f) Value function 0 100 200 300 400 500 600 700 800 1 201 401 601 801 1001 1201 1401 1601 1801 time in days € per capacity unit c) Amount of lean tissue e) Live weight of a pig 0 20 40 60 80 100 120 1 201 401 601 801 1001 1201 1401 1601 1801 time in days kg Fig. 9. Convergence regions (indicated by a small box having a width equal to the optimal length of the fattening period (i.e. 105 days) in each graph a-f) of the optimal control policy, deﬁned for the optimal feeding patterns (Figures a and b), the amount of lean tissue (c), amount of fatty tissue (d), live weight (e), and the value value of a capacity unit (f) over the entire optimisation period. When the amount of barley drops to zero, the pig is slaughtered and replaced with a new pig- let. 44 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Soy Fit of soy Fig. 11. The optimal share of soy meal (% of the total amount of feed) for the fattening period of a single pig, and polynomial ap- proximation (Fit of soy) of the optimal share of soy meal. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Fig. 11. The optimal share of soy meal (% of the total amount of feed) for the fattening period of a single pig, and polynomial ap- proximation (Fit of soy) of the optimal share of soy meal. Fig. 11. The optimal share of soy meal (% of the total amount of feed) for the fattening period of a single pig, and polynomial ap- proximation (Fit of soy) of the optimal share of soy meal. and formulation of the model. For small piglets (25–29 kg live weight) the model recommends an approximately 15% higher level of energy than the standard energy-rich diet. After 20 days fattening period (at 40 kg live weight), the model recom- mends energy feeding that is close to the stand- ards, whereas after 40 days of fattening (at 59 kg live weight and thereafter), the model suggests ad- justing the amount of energy below the standards (Figure 12). Thus, the pig eats, on the average, 2.40 fodder units per day. This is 0.05 fodder units less than even in the standard low energy diet. that it reaches the level of 7 g lysine per fodder unit at 100 kg live weight (85 days after 25 kg live weight). Thus, the model suggests a higher level of lysine in the middle of the fattening period than that recommended by MTT (2004). The model recommends slaughtering the pig at 115 kg live weight. Slaughter yields 85 kg of meat, of which 61.3% is red meat. Carcass leanness is above the average of Finnish pigs, because 59.4% of fattening pigs slaughtered in Finnish slaughter- houses in 2003 contained red meat less than 60.0% (Tike 2004a). Therefore, the model recommends controlling the feeding and the timing of slaughter so that the producer beneﬁts from the price pre- mium paid for lean carcasses. Vol. 15 (2006): Supplement 1. Since both the amount of soy meal and the amount of barley affect the percentage share of soy meal in feed, converting the share of soy to kilograms of soy meal changes the pattern. During the ﬁrst three weeks after 25 kg liveweight, the amount of soy increases from the initial amount of 330 g per day to 390 g per day. For the next three weeks, the amount of soy meal remains at approximately 400 g per day. Thereaf- ter, the amount of soy meal decreases gradually until three weeks before the slaughter, when it sta- bilises at close to 150 g per day. As Figures 12 and 13 indicate, the estimated feeding patterns in the benchmark scenario differ from the Finnish feeding standards. Optimised feeding patterns, however, are conditional on the relative prices of pig meat, feeds and other inputs, 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Barley Fit of barley Fig. 10. The optimal amount of barley (kg per day) fed during the fattening period; the polynomial approximation (Fit) for the opti- mal amount of barley. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Barley Fit of barley Fit of barley Fig. 10. The optimal amount of barley (kg per day) fed during the fattening period; the polynomial approximation (Fit) for the opti- mal amount of barley. time in days 45 Fig. 12. The amount of energy (fodder units per day) optimised by the model and as recommend- ed by MTT (2004) for the stand- ard energy-rich (high level) and low energy (low level) diets. A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. 0 2 4 6 8 10 12 1 11 21 31 41 51 61 71 81 91 101 time in days g lysine per fodder unit Two-phase feeding Three-phase feeding The model Fig. 13. The amount of lysine (g per fodder unit) estimated by the model and as recommended in Finnish two-phase and three- phase feeding standards (MTT 2004). Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. 0 2 4 6 8 10 12 1 11 21 31 41 51 61 71 81 91 101 time in days g lysine per fodder unit Two-phase feeding Three-phase feeding The model g lysine per fodder unit Fig. 13. The amount of lysine (g per fodder unit) estimated by the model and as recommended in Finnish two-phase and three- phase feeding standards (MTT 2004). Fig. 13. The amount of lysine (g per fodder unit) estimated by the model and as recommended in Finnish two-phase and three- phase feeding standards (MTT 2004). The model tential whereas the growth of fatty tissue is re- stricted below the potential. The average daily weight gain of lean tissue is 147 g, the average daily weight gain of fatty tissue is 167 g, and the average daily weight gain of the carcass is 868 g. The daily weight gain is high during the early stag- es of growth, and reaches the maximum value (ap- proximately 950 g per day) just before the growth of lean tissue starts decelerating (Figure 14). Re- stricting energy feeding strongly reduces the growth of live weight and fatty tissue in the second half of the fattening period, and helps to increase carcass leanness. value of income ﬂows to the capacity unit ob- tained under optimal feeding and slaughter pat- terns and discounted over the ﬁve years period. Each pig that is slaughtered under the optimal feeding and slaughter policy contributes €44.21 in value to the capacity unit. The annual equiva- lent value added is €150.33 per capacity unit. The optimised results are conditional on the growth parameters given in Table 1 and on the prices giv- en in Table 2 on pages 40–41. A G R I C U L T U R A L A N D F O O D S C I E N C E On pigs having less than 50 kg live weight, the optimal amount of lysine with respect to the amount of energy is close to the feeding standards (9.5 g lysine per fodder unit; see Figure 13). There- after, the amount of lysine decreases gradually so The result of the optimal feeding policy is that the lean tissue grows according to the growth po- 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days fodder units per day High level Low level The model Fig. 12. The amount of energy (fodder units per day) optimised by the model and as recommend- ed by MTT (2004) for the stand- ard energy-rich (high level) and low energy (low level) diets. fodder units per day time in days 46 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs of fattening, at a carcass weight of 85.3 kg with the carcass containing 60.8% red meat. A two-phase feeding technology restricts the producer to adjusting the share of soy meal in feed discretely only once during the fattening period. The model suggests that it is optimal to switch the share of soy from 17% to 6% at 75 kg live weight. The amount of barley increases from the initial amount of 1.4 kg to 2.2 kg just before adjusting the soy content of the feed. Then, the optimal amount of barley jumps to 3.0 kg, after which it gradually decreases to 2.6 kg (Figure 15). The value of a ca- pacity unit, however, remains almost constant when the feed composition is adjusted at 65–75 kg live weight, or even at 55–85 kg live weight. When adjusting the composition at 45–55 kg live weight, the feed is richer in soy meal than Figure 15 indi- cates (see Appendix B). The value of a capacity unit over the 5-year pe- riod decreases from €687.41 per capacity unit un- der the ﬂexible feeding technology to €678.23 per capacity unit under two-phase feeding. The model estimates annual returns from using the ﬂexible feeding technology instead of the two-phase feed- ing technology at €1.70 per capacity unit. The ﬁg- ure takes into account that the producer optimises both feed ratios and the timing of slaughter under the constraint that the share of soy meal in feed can be adjusted only once during the fattening period. Therefore, the optimal feeding patterns differ in Figure 15 from the recommendations (MTT 2004). The beneﬁts of ﬂexible feeding technology, how- ever, depend on price ratios. When the price of pig meat increases to €1.62, the optimal ﬂexible diet changes more rapidly over time than is illustrated in Figure 15. Simultaneously, annual returns from the ﬂexible feeding technology increase (see Ap- pendix B). The optimal shares of lysine in feed in the two- phase feeding are 8.9 g per fodder unit during the ﬁrst phase and 6.8 g per fodder unit during the sec- ond phase. After adjusting the feed composition, the share of energy in feed increases above the level of ﬂexible feeding technology. A G R I C U L T U R A L A N D F O O D S C I E N C E Since piglets and feeds generally pay more than 90% of the total variable cost of pig fattening (excl. cost of labour), the ﬁgures are mainly compensation for capital and labour inputs. The model optimises the cost of feed for a single fattened pig at €36.60, and the value of a slaughtered pig and slaughter premium at €142.21. The model estimates the value of a capacity unit to be €687.41. The value of a capacity unit refers to the value of the value function on the ﬁrst day of the planning horizon. It is the net present 47 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1 11 21 31 41 51 61 71 81 91 101 time in days kg/day Lean tissue+fatty tissue+water+ash Lean tissue+fatty tissue Lean tissue Fig. 14. The average daily growth of lean tissue, lean and fatty tis- sue, and live weight (includes fatty tissue, lean tissue, water and ash) for each 10 day period. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1 11 21 31 41 51 61 71 81 91 101 time in days kg/day Lean tissue+fatty tissue+water+ash Lean tissue+fatty tissue Lean tissue Lean tissue+fatty tissue+water+ash Lean tissue+fatty tissue Fig. 14. The average daily growth of lean tissue, lean and fatty tis- sue, and live weight (includes fatty tissue, lean tissue, water and ash) for each 10 day period. time in days 47 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 4.4 Summary mal slaughter weight later than when it is fed ac- cording to the growth potential. The optimal tim- ing of slaughter is such that the slaughterhouse price scheme gives no price discount to the carcass due to excessively low or high carcass weight. If other quality factors are held constant, a slightly higher slaughter weight would result in a price dis- count. The impact of restricting slaughter timing is examined in Chapter 7. The model indicates that pig meat producer can achieve higher returns per capacity unit when us- ing a ﬂexible feeding technology (precision feed- ing), which allows the adjustment of slaughter tim- ing and feed ratios according to the pig’s growth, than those offered by less ﬂexible two-phase feed- ing. The model allows producer to improve the ef- ﬁciency of input use and provides more options to control carcass quality under precision feeding than under phase feeding. The model is ﬂexible also in the sense that it allows the producer to choose the date when pigs are delivered to slaugh- ter. Based on the prices given in Table 2, an aver- age pig contributed €150.33 value per year to the capacity unit. This was €1.70 more than under op- timised two-phase feeding. This ﬁgure takes into account that producer optimised the two feeding phases, the timing at switching the feed, and the timing of slaughter. Under two-phase feeding, the model optimises the switch of the feed ratios approximately at 65– 75 kg live weight, whereas according to Finnish feeding recommendations the second phase begins already at 55 kg live weight. The optimal two- phase feeding is rich in protein during the early stages of growth (17% soy meal in feed in the ﬁrst phase), and thereafter rich in energy (6% soy meal in feed in the second phase). In the ﬁrst phase, the amount of protein is lower than the amount of pro- tein in the protein-rich diet recommended by MTT (2004). The optimal diet also includes decreasing the amount of energy with the approach of optimal slaughter maturity. Under ﬂexible feeding technology, the optimal diet of a young pig is characterised by protein-rich feed. While pig grows, the amount of barley in feed increases, but at decreasing rate. Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Under two- phase feeding, the pig is slaughtered after 101 days 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Two-phase feeding 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Benchmark Fig. 15. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in feed under optimised two-phase feeding and as approximated in the benchmark sce- nario. time in days time in days Two-phase feeding Benchmark Two-phase feeding Benchmark Fig. 15. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in feed under optimised two-phase feeding and as approximated in the benchmark sce- nario. 48 4.4 Summary Although young pigs require protein-rich feed to grow and to maintain carcass leanness, they also require quite large amounts of energy in order to keep the daily weight gain at a high level. Heavier pigs, on the other hand, need a large amount of energy for body maintenance and growth of tissue. During the last weeks before the slaughter, the amount of barley stabilises at 2.6–2.7 kg per day. Although the amount of soy meal increases in the beginning of the fattening period, the share of soy meal in feed decreases throughout the fattening period. Thus, the diet gradually changes from being a protein- rich diet to an energy-rich diet. In two-phase feeding the amount of energy in feed increases and the growth of fatty tissue in- creases when feed ratios change. As lean growth potential at the time of change is still quite high, the increase is necessary in order to maintain the level of lean growth with the new energy-rich feed. The producer also beneﬁts from continuing the ﬁrst phase until 65–75 kg live weight. The share of red meat in the carcass, and hence the price of pig meat, is lower under the two-phase feeding tech- nology than under the ﬂexible feeding technology. The growth rate of fatty tissue, however, is higher under two-phase feeding than under ﬂexible feed- ing technology. This decreases the length of the fattening period and increases the volume of an- nual meat production per capacity unit. The logic of the results is robust and holds quite generally, despite the fact that the results pre- sented in this study are conditional on prices and on the formulation of the optimisation model. This implies that the steepness and height of the feeding curves can vary depending on current markets and genotype of an animal. Hence, the results give best information on how changes in the model variables The optimal feeding pattern restricts growth so that lean tissue grows in accordance with the growth potential and fatty tissue grows, on the av- erage, 80 g (30%) below the potential. The adjust- ment of feeding is more tightly controlled when the pig is close to the optimal slaughter maturity than when it is young. As restricting growth de- creases daily weight gain, the pig reaches the opti- 49 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs p. 931). The inequality problem implies that a model having a convex production function, such as the Gompertz function, can produce a result in- dicating that it is optimal to split the total amount, say 4 kg, of feed given to animal on subsequent days into two portions, such that a larger amount, say 2.5 kg, of the energy-rich feed is given to the animal on the ﬁrst day, and a smaller amount, say 1.5 kg, of the energy-rich feed is given to the ani- mal on the second day. In the absence of the ine- quality problem, the amount of feed could be the same on both days. On the average, the inequality problem is cancelled out. (such as feeding technology) affect production management and the value of a capacity unit. Because the model at hand can use energy and protein for growth almost linearly after subtracting maintenance requirements, the optimal feed con- trol occasionally ﬂuctuates between the limiting constraints (‘bang-bang-control’, (cf. Intriligator 2002, p. 358)). This shows up as kinks in the feed- ing curves. The problem is related particularly to the question of whether it is proﬁtable to produce low-fat meat. The problem is due to the numerical iteration techniques and to the problem known as Jensen’s inequality (Mas-Colell p. 185–186 and Vol. 15 (2006): Supplement 1. feeding technology as described in Chapter 4.2. Thus, the only difference between the benchmark scenario and a market scenario is the difference in the speciﬁc price variable. and to the premium paid for pigs slaughtered in northern parts of the region C in 2003 (MKL 2003). In the second scenario, the premium is removed from the optimisation problem (i.e. decoupled). After optimising feeding and slaughter patterns with the prices given in Table 3, each scenario is compared to the benchmark scenario. Thereafter, own price and cross-price elasticity estimates for supply of pig meat, slaughter intensity, input use and changes in the value of a capacity unit are computed. Elasticity estimates are computed uti- lising the results provided by the optimisation model, and information on a given price parame- ter. Elasticity estimates are used to illustrate which factors have most signiﬁcant impact on input use, output and producer income. They are useful for instance when information on how the impacts of changes in various feed prices need to be com- pared. An elasticity estimate indicates a percent- age change in the observed variable when one of the prices increases by a percentage point and oth- The market scenarios focus on changes in the prices of pig meat, piglets or feeds, as well as to changes in slaughter premium. The parameter val- ues of the market scenarios are based on the same data as the prices in the benchmark scenario (see Chapter 4.1 and Table 3). Parameter values are ob- tained by adjusting the selected price to the highest price or price ratio observed in the data. In some scenarios, for instance when the variable is the price of pig meat, the relative price change can be quite large. In addition, one of the scenarios in- creases all feed prices by 10%, but maintains feed price ratios, and another scenario decreases the price of soy meal by 10%. Changes in slaughter premium are analysed in two scenarios. In the ﬁrst scenario, the premium increases from the premium paid for pigs slaughtered in subsidy regions A/B Table 3. Price parameters used in the market scenarios. Table 3. Price parameters used in the market scenarios. 5 Adjusting pig production management to market conditions feed ingredients. Since non-genetically modiﬁed soy meal is more expensive than genetically modi- ﬁed soy meal (e.g. Dros and Kriesch 2003, p. 10– 11), such regulations can affect the competitive- ness of domestic meat production by increasing the production cost of pig meat. Decoupling the slaughter premium, on the other hand, is the equiv- alent of completely removing the premium from the optimisation problem. Therefore, decoupling can affect producer incentives to invest in new production capacity in the long run even if pro- ducer can obtain an equivalent income as an in- come transfer. The problem arises because decou- pled transfer is independent of maintaining pro- duction. 5.1.1 Price movements The objective of Chapter 5 is to explore the prob- lem of how the optimal feeding and slaughter pat- terns change when prices of pig meat, feeds, pig- lets, carcass quality premiums or slaughter premi- um change (the second research problem). This problem is important because changes in the input and output prices of pig fattening can affect the optimality of feeding patterns and the timing of slaughter (Chavas et al. 1985, Kure 1997, Sipiläi- nen and Ryhänen 1996, p. 157–200). Therefore, the analysis of production decisions with respect to changes in relative prices can provide informa- tion on how producers can adjust production deci- sions according to markets and how market move- ments affect producer income. The effects of price changes are analysed by estimating the marginal change in slaughter and feeding decisions and the value of a capacity unit when the parameter value of one of the variables changes (ceteris paribus). In other words, all prices are ﬁrst ﬁxed to exactly the same values as in the benchmark scenario. Then, one of the prices is re- laxed and adjusted to an optional value. Finally, the optimal feeding and slaughter patterns and the value function are estimated under the ﬂexible The problem of price and subsidy ratios is in- teresting also from society’s point of view, because ethical and economic policy choices inﬂuence pro- ducer’ decisions. Regulations can, for instance, force producers to use non-genetically modiﬁed 50 g 6) Eeva-Liisa Paju, Raisio Group Plc, personal communication, 3 October 2003. p y p 2) Estimated price of a 25 kg piglet. Estimated from Tike (2002, 2003). 3) Slaughter premium paid for carcasses over 61 kg upon slaughter in 2003. Based on MKL (2003, p. 32). For further details, see Anonymous (2002a), Anonymous (2002c), Anonymous (2002d), Anonymous (2002b), Anonymous (2001), MMM (2004, p. 100–103). 4) Based on Tike (2002, 2003). 5.2.1 The effects of price changes The results suggest that market movements affect the allocation of feeds and the timing of slaughter (Figures 16–21, Table 5). When the price of pig meat increases from €1.39 to €1.62 (ceteris pari- bus), the optimal share of soy meal in feed de- creases and the amount of barley in feed increases. Young pigs, however, are fed with almost the same diet in both cases. The total amount of soy meal fed during the fattening period decreases by 9.92 kg, and the total amount of barley increases by 14.28 kg. After a 93 days fattening period, the pig eats 3.4 kg barley and 30 g soy meal in the €1.62 scenario (Figure 16). Synthetic amino acids are supplied at the same ratio with respect to the amount of barley in both cases. Because the share of energy in the diet increases, the growth of fatty tissue almost reaches the growth potential when the price of pig meat is €1.62. A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs er prices remain constant. An elasticity estimate εij for the change in quantity Qi of decision variable i with respect to the change of price Pj of input or output j is obtained using the formula (Chambers 1988, p. 135, Gravelle and Rees 1992, p. 367): The marginal effects of meat quality price ad- justments are analysed experimentally as in the event of price ratios in Chapter 5.1.1. Analysing the impacts of quality adjustments on optimal feeding and slaughter patterns is similar to chang- ing the slope of the adjustment for excess carcass weight, the share of red meat, or both, and position of the slope with respect to the benchmark scenar- io (ceteris paribus). This is due to the fact that quality price adjustments are linear approxima- tions of discrete pricing schemes. The effects of a red meat premium are analysed by increasing or decreasing the premium paid for each additional percentage of red meat. The effects of price adjust- ments based on carcass weight are analysed by in- creasing or decreasing the marginal discount per each additional kilogram that deviates from the target weight range. In addition, an option to de- crease slaughter weights by shifting the location of the target weight range is examined (Table 4). (15) εij = ∆Qi\|Pj = ∂lnQi , ∆Pj Qi ∂lnPj where ∆Qi denotes the change in the amount of the decision variable i when the price Pj of input or output j changes by ∆Pj euro. The elasticity esti- mates are normalised for a capacity unit. Changes in the supply of pig meat are meas- ured on the basis if the optimal slaughter weights, the amount of pig meat produced per year per ca- pacity unit, and the time required to reach slaugh- ter maturity. In addition, elasticity estimates meas- ure changes in the share of red meat in the slaugh- tered carcass. When computing elasticity esti- mates, the amount of feed input is evaluated as the total amount of barley or soy meal fed to an indi- vidual pig. Elasticity estimates also measure how price changes affect the value of a capacity unit, where the value is evaluated in terms of annual re- turns under optimised feeding and slaughter pat- terns. These data and corresponding price changes are obtained from the model as differences be- tween the scenarios. Vol. 15 (2006): Supplement 1. Price of Pig meat1) Piglet2) Premium3) Barley4) Soy meal5) Lysine6) Methionine6) Threonine6) €/kg €/piece €/carcass €/kg €/kg €/kg €/kg €/kg Scenario pt meat pt weaner pt subsidy pt barley pt soy pt lysine pt methionine pt threonine Benchmark 1.39 61.07 21.92 0.11 0.31 2.36 2.63 5.25 Price of pig meat increases4) 1.62 61.07 21.92 0.11 0.31 2.36 2.63 5.25 Slaughter premium increases3) 1.39 61.07 28.54 0.11 0.31 2.36 2.63 5.25 Slaughter premium removed 1.39 61.07 0 0.11 0.31 2.36 2.63 5.25 Price of piglets increases4) 1.39 67.96 21.92 0.11 0.31 2.36 2.63 5.25 Price of barley increases4) 1.39 61.07 21.92 0.12 0.31 2.36 2.63 5.25 Price of soy increases5) 1.39 61.07 21.92 0.11 0.34 2.36 2.63 5.25 Price of soy decreases by 10% 1.39 61.07 21.92 0.11 0.28 2.36 2.63 5.25 All feed prices increase 10% 1.39 61.07 21.92 0.12 0.34 2.59 2.89 5.78 1) The price paid for a carcass weighting 76–85 kg and containing 59% red meat. Any deviation from this quality results i i dj t t A dditi l t i t f d t i d dditi l kil f i ht p y p 2) Estimated price of a 25 kg piglet. Estimated from Tike (2002, 2003). 3) Slaughter premium paid for carcasses over 61 kg upon slaughter in 2003. Based on MKL (2003, p. 32). For further details, see Anonymous (2002a), Anonymous (2002c), Anonymous (2002d), Anonymous (2002b), Anonymous (2001), MMM (2004, p. 100–103). 4) Based on Tike (2002, 2003). 5) Estimated from Futurestrading (2003). 6) Eeva-Liisa Paju, Raisio Group Plc, personal communication, 3 October 2003. 51 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. Table 4. Meat pricing schemes in quality-adjusted meat pricing scenarios Red meat premium1) Weight discount2) Target weight2) Scenario € per % € per kg carcass weight, kg No adjustment 0 0 none Lean price is €0.01 0.01 0.02 85 Benchmark; lean price is €0.02 and weight price discount is €0.02 0.02 0.02 85 Lean price is €0.03 0.03 0.02 85 Lean price is €0.04 0.04 0.02 85 No weight price discount 0.02 0 none Target weight decreases by 5 kg 0.02 0.02 80 1) Red meat premium and lean price both refer to the same price adjustment, viz. price premium based on the share of red meat in carcass. 2) Target weight is an upper bound above which the weight discount applies. The lower bound is 10 kg below the upper bound. The discount scheme is a linear approximation of a discrete scheme. Each additional kilogram deviating from the target weight range results in additional price discount. Table 4. Meat pricing schemes in quality-adjusted meat pricing scenarios Red meat premium1) Weight discount2) Target weight2) Scenario € per % € per kg carcass weight, kg No adjustment 0 0 none Lean price is €0.01 0.01 0.02 85 Benchmark; lean price is €0.02 and weight price discount is €0.02 0.02 0.02 85 Lean price is €0.03 0.03 0.02 85 Lean price is €0.04 0.04 0.02 85 No weight price discount 0.02 0 none Target weight decreases by 5 kg 0.02 0.02 80 1) Red meat premium and lean price both refer to the same price adjustment viz price premium based on Table 4. Meat pricing schemes in quality-adjusted meat pricing scenarios 2) Target weight is an upper bound above which the weight discount applies. The lower bound is 10 kg below the upper bound. The discount scheme is a linear approximation of a discrete scheme. Each additional kilogram deviating from the target weight range results in additional price discount. per year) (Table 5). One of the reasons for these large positive income effects is that increasing the growth rate of pigs increases the amount of annu- ally marketed meat by 31.3 kg per capacity unit. ter maturity decreases by 11 days. Simultaneously, the optimal slaughter weight decreases by 830 g. In addition, the share of red meat in the carcass decreases by 3.2%. 5.1.2 Quality price premiums Chapter 5 estimates the marginal effects that changes in the quality premiums of pig meat have on optimal feeding and slaughter decisions and the value of a capacity unit. The marginal quality ad- justments reﬂect the value of improved or reduced carcass quality. Quality adjustments are of interest to both producers and meat processors. Producers can optimise the allocation of feeds, slaughter weight and carcass fatness according to the premi- ums whereas processors can control the volume and quality of pig meat through quality premiums and discounts. Thus, information on how price quality adjustments affect carcass weights can be useful when markets distortions occur or when the market needs to be balanced. When the price of pig meat increases from €1.39 to €1.62, the time required to reach slaugh- 52 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs increases from €22 to €28, the amount of soy meal fed during the fattening period decreases by 5.23 kg and the amount of barley increases by 1.07 kg. When the premium is €28, the amount of bar- ley, however, is sharply reduced the last two weeks before the slaughter (Figure 17). When the slaugh- ter premium increases from €22 to €28, length of the fattening period decreases by 7 days and the optimal slaughter weight decreases by 1.6 kg. Due to the energy-richer diet, the share of red meat in the carcass decreases by 1.5%. In addition, the value of a capacity unit increases by €97.08 over the period of 5 years (€12.58 additional income per year) (Table 5). slaughter weight decreases, and the share of red meat upon slaughter increases. The most signiﬁ- cant change occurs in the value of a capacity unit, which decreases by €307.53 (–45%) (Table 5). y When the price of piglets increases from €61 to €68, the total amount of soy meal fed during the fattening period increases by 1.62 kg, and the amount of barley fed during the fattening period decreases by 3.16 kg. The largest increase in the protein content of the feed is observed in the mid- dle of the fattening period (Figure 18). Increasing the piglet price increases the length of the fattening period by one day, but has no signiﬁcant effect on the optimal slaughter weight. Therefore, the share of red meat increases by 0.4% compared to the benchmark scenario. The result that changes in the price of piglets have only a small impact on the optimal decisions is affected by the facts that the meat pricing scheme guides slaughter weights and that fat-to-lean growth rate constraint mitigates against increasing carcass leanness. Finally, the value of a capacity unit over the 5-year period de- creases by €98.04 (€23.22 less income per year) compared to the benchmark scenario (Table 5). When the slaughter premium is completely re- moved, the total amount of soy meal fed during the fattening period increases by 2.42 kg and the total amount of barley decreases by 7.21 kg. The opti- mal feeding patterns are graphically very close to the scenario where the piglet price increases (see Figure 18), because the minimum fat to lean growth ratio is then a binding constraint. A G R I C U L T U R A L A N D F O O D S C I E N C E Although the carcass suffers from a price discount due to low share of red meat, the value of a capacity unit increases by €303.34 over a period of 5 years (€68.31 additional income A higher slaughter premium is paid to produc- ers in the northern parts of Finland than in south- ern parts of Finland. When the slaughter premium 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days € 1.62 Fit of €1.62 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day € 1.39 % of feed Fig. 16. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the price of pig meat is either €1.39 (benchmark scenario) or €1.62. The ﬁt of €1.62 represents a polynomial approximation of the optimised €1.62 scenario. 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Fig. 16. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the price of pig meat is either €1.39 (benchmark scenario) or €1.62. The ﬁt of €1.62 represents a polynomial approximation of the optimised €1.62 scenario. 53 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Removing the slaughter premium increases the length of the fat- tening period by one day. In addition, the optimal 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed € 28 Fit of €28 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day € 22 Fig. 17. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when slaughter premium is either €22 (benchmark scenario) or €28. The ﬁt of €28 represents a polynomial approximation of the €28 scenario. time in days Fig. 17. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when slaughter premium is either €22 (benchmark scenario) or €28. The ﬁt of €28 represents a polynomial approximation of the €28 scenario. 54 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Fit of €68 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day € 61 € 68 Fig. 18. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the price of piglets is either €61 (benchmark scenario) or €68. The ﬁt of €68 represents a polynomial approximation of the €68 scenario. Vol. 15 (2006): Supplement 1. time in days Fig. 18. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the price of piglets is either €61 (benchmark scenario) or €68. The ﬁt of €68 represents a polynomial approximation of the €68 scenario. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs A one percent increase in the price of pig meat, for in- stance, increases the amount of annually pro- duced meat (kg per capacity unit) by 0.66 per- cent. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs p g f g g g g p g 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed € 0.31 Fit of €0.34 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day € 0.31 Fig. 19. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the price of soy meal is either €0.31 (benchmark scenario) or €0.34. The ﬁt of €0.34 represents a polynomial approximation of the €0.34 scenario. time in days Fig. 19. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the price of soy meal is either €0.31 (benchmark scenario) or €0.34. The ﬁt of €0.34 represents a polynomial approximation of the €0.34 scenario. Table 5. Time required to reach the optimal slaughter maturity (duration of fattening period in days), carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value of a capacity unit (value function of a capacity unit) over the 5-year period in the market scenarios Table 5. Time required to reach the optimal slaughter maturity (duration of fattening period in days), carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value of a capacity unit (value function of a capacity unit) over the 5-year period in the market scenarios. Scenario Duration Carcass weight Red meat Value function days kg % € for the 5-year period Benchmark 105 85 61.3 687 High price of pig meat 94 84.2 58.1 991 Slaughter premium removed 106 84.4 62 380 High slaughter premium 98 83.4 59.8 784 High piglet price 106 85 61.7 589 High barley price 106 85.1 61.7 665 High soy meal price 102 84.4 60.7 674 The price of soy meal decreases by 10% 106 85.1 61.6 700 High price of all feeds 106 85.2 61.6 632 scenarios. Each line reports the percentage change in the amount of the decision variable given in the second line when the price given in the ﬁrst column increases by one percentage point. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs The shift towards energy-rich 55 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs When the price of barley increases from €0.11 to €0.12, the amount of soy meal fed during the fattening period increases by 1.46 kg, whereas the amount of barley decreases by 2.40 kg. Both opti- mised and approximated feeding patterns are graphically almost identical to those presented in Figure 18 for the case of increased price of piglets. Changes in the timing of slaughter, slaughter weight and carcass leanness are also of the same magnitude than in the case where the price of pig- let increased. The value of a capacity unit decreas- es by €22.10 over the period of 5 years (€4.99 less income per year) (Table 5). feed decreases the share of red meat in the carcass by 0.7%. The value of a capacity unit decreases by €13.47 over the period of 5 years (€3.24 less in- come per year) (Table 5). In contrast to this, the scenario with a 10% decrease in the price of soy meal generates approximately €3.33 less income per capacity unit per year than the benchmark sce- nario. When the prices of all feeds increase by 10%, the diet becomes richer in protein. The amount of soy meal in feed increases by 0.94 kg, whereas the amount of barley decreases by 0.56 kg. The feed- ing curves also shift only marginally. Particularly at the early stages of the fattening period, the curves are graphically almost the same as in the benchmark scenario. When the prices of all feeds increase by 10%, the length of the fattening period decreases by one day and the share of red meat in the carcass increases by 0.2%. The value of a ca- pacity unit decreases by €55.15 over the period of ﬁve years (€4.47 less income per year) compared to the benchmark scenario. When the price of soy meal increases from €0.31 to €0.34, the total amount of soy meal fed during the feeding period decreases by 2.43 kg and the total amount of barley increases by 1.14 kg. The share of soy meal in feed decreases, particu- larly in the second half of the fattening period (Figure 19). The length of the fattening period de- creases by 3 days and the optimal slaughter weight decreases by 0.6 kg. Vol. 15 (2006): Supplement 1. The share of red meat, however, responds very in- elastically to changes in the input prices. Length of fattening period is a discrete varia- ble. It measures the intensity at which producer makes slaughter and replacement decisions. The length of the fattening period decreases by 0.72% when the price of pig meat increases by one per- centage point. A one per cent increase in the slaughter premium shortens the fattening period only by 0.26% and one percent increase in the price of soy meal shortens the period by 0.31%. Changes in other input prices than that of soy meal increase the length of the fattening period only marginally. The amount of soy meal fed during the fatten- ing period increases elastically when the price of pig meat increases. A one percent increase in the price of soy meal decreases the amount of soy meal by 0.6% whereas an increase in the price of barley has approximately an opposite effect. The same factors that decrease the length of the fatten- ing period also decrease the amount of soy meal fed to a pig. The amount of barley increases almost unit elastically when the price of pig meat increases. The amount of barley responds less elastically to changes in feed prices than the amount of soy meal. When the price of barley increases by one percent, the amount of soy meal fed during the fat- tening period decreases by 0.33% and the amount of barley increases by 0.37%. An increase in all feed prices results in similar but less elastic chang- es in feeding than an increase in the price of barley only. Even if the length of the fattening period can be quite elastic with respect to price changes, slaughter weights respond very inelastically to price changes. Therefore, a larger supply effect is observed indirectly in the annual amount of pig meat produced. The annual production volume per capacity unit increases most elastically when the price of pig meat increases (elasticity estimate 0.66). This is due to several simultaneous changes in feeding and slaughter decisions. Furthermore, increasing either the price of soy meal or the slaughter premium by one per cent increases the amount of pig meat produced per annum by ap- proximately 0.2%. Another supply effect is ob- served in the share of red meat, which responds most elastically to changes in the price of pig meat. 5.2.2 Elasticity estimates for price movements Elasticity estimates allow the comparison of fac- tors that are measured using different scales. Ta- ble 6 reports own and cross-price elasticity esti- mates for major decisions involved in the market 56 are normalised for a capacity unit and computed from the converged feeding and slaughter patterns. The exceed a given threshold value and from above so that the daily weight gain cannot exceed the pig’s growth potential. The estimates are valid upon price increase and near the parameters used in the market scenarios. p y p g g g p estimates are bounded from below so that the growth of fatty tissue with respect to the growth of lean tissue must exceed a given threshold value and from above so that the daily weight gain cannot exceed the pig’s growth potential. The estimates are valid upon price increase and near the parameters used in the market scenarios. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs the growth potential. Thus, producer maximises daily weight gain. um increases. Producer income decreases most elastically when the price of piglets increases. Al- though income responds almost unit elastically to an increase in all feed prices, an increase only in the price of barley or soy meal decreases incomes quite inelastically. Removing the quality price adjustments in- creases the optimal slaughter weight by 6.9 kg and decreases the length of the fattening period by one day compared to the benchmark scenario. Simulta- neously, the annual amount of meat produced in- creases by 26.8 kg. Removing the quality price adjustment increases the value of a capacity unit by €26.80 (4%) over the 5-year period (Table 7), or €6.67 per year. Vol. 15 (2006): Supplement 1. Table 6 also shows elasticity estimates for pro- ducer income changes with respect to market movements. Income effects are measured using the value of the capacity unit. Producer incomes increase very elastically when the price of pig meat increases, but inelastically when slaughter premi- Table 6. Elasticity estimates of use of feeds, time required to reach slaughter maturity (duration), slaughter weight (weight), annual amount of meat produced (meat yield), share of red meat in the slaughtered carcass (%), and pig meat producer income effect (i.e. annual contribution to the value function) with respect to changes in the price of meat, feeds or piglets or in the slaughter premium. Percentage change in1) 1% increase in the price of Duration Weight Meat yield Red meat Soy meal Barley Income effect Pig meat –0.72 –0.06 0.66 –0.36 –1.78 1.12 2.44 Piglet 0.09 –0.01 –0.1 0.06 0.39 –0.22 –1.57 Barley 0.16 0 –0.15 0.09 0.6 –0.33 –0.55 Soy meal –0.31 –0.07 0.24 –0.12 –0.56 0.37 –0.23 All feeds 0.1 0.03 –0.07 0.04 0.22 –0.13 –0.93 Slaughter premium –0.26 –0.07 0.19 –0.09 –0.44 0.28 0.54 1) Th ti t li d f it it d t d f th d f di d l ht tt Th 57 5.2.3 The effects of quality price premiums When the price premium based on the share of red meat in the carcass decreases from €0.02 to €0.01, the amount of barley fed during the fatten- ing period increases by 16.62 kg (7%), and the amount of soy meal decreases by 10.36 kg (–33%) (Figure 21). Simultaneously, the length of the fat- tening period decreases by 11 days, the optimal slaughter weight decreases by 0.5 kg (–1%), and the share of red meat decreases by 3.4%. Although the value of a capacity unit decreases somewhat, the converged slaughter and feeding patterns can contribute slightly higher income after the price premium based on the share of red meat in the car- Producers can beneﬁt from adjusting feeding pat- terns and the timing of slaughter when marginal quality price adjustments increase (Table 7 on page 61). When both the red meat adjustment and the excess live weight adjustments are removed (i.e. set equal to zero), the amount of barley fed during the fattening period increases by 55.87 kg (25%) and the amount of soy meal decreases by 10.01 kg (–32%) (Figure 20) compared to the benchmark scenario (Figure 13). Due to the use of energy-rich feed ration when no quality adjustments are ap- plied, both fatty and lean tissue grow according to set equal to zero), the amount of barley fed during the fattening period increases by 55.87 kg (25%) and the amount of soy meal decreases by 10.01 kg (–32%) (Figure 20) compared to the benchmark scenario (Figure 13). Due to the use of energy-rich feed ration when no quality adjustments are ap- plied, both fatty and lean tissue grow according to 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Fit of no adjustments 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Benchmark No adjustments Fig. 20. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where no marginal meat quality adjust- ments to the price of pig meat are applied. Fit of no adjustments represents a polynomial approximation of the no adjustments scenario. 5.2.3 The effects of quality price premiums 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 % of feed 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 kg feed per day 15 10 time in days Fit of no adjustments Benchmark No adjustments Fig. 20. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where no marginal meat quality adjust- ments to the price of pig meat are applied. Fit of no adjustments represents a polynomial approximation of the no adjustments scenario. 58 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Lean price €0.01 Fit of lean price €0.01 Lean price €0.02 Fig. 21. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the marginal price adjustment based on the share of red meat in the carcass is either €0.02 (lean price €0.02) or €0.01 (lean price €0.01). The ﬁt of lean price €0.01 represents a polyno- mial approximation of the lean price €0.01 scenario. Vol. 15 (2006): Supplement 1. 1 11 21 31 41 51 61 71 81 91 101 time in days time in days Fig. 21. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the marginal price adjustment based on the share of red meat in the carcass is either €0.02 (lean price €0.02) or €0.01 (lean price €0.01). The ﬁt of lean price €0.01 represents a polyno- mial approximation of the lean price €0.01 scenario. increases by €31.42 (–5%) over the 5 years period (€10.01 additional income per year) when the amount of the red meat premium increases from €0.02 to €0.03. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Fit of lean price €0.03 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Lean price €0.02 Lean price €0.03 Fig. 22. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the marginal price adjustment based on the share of red meat in the carcass is either €0.02 (lean price €0.02) or €0.03 (lean price €0.03). The ﬁt of the lean price of €0.03 represents a polynomial approximation of the lean price €0.03 scenario. time in days Fig. 22. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) when the marginal price adjustment based on the share of red meat in the carcass is either €0.02 (lean price €0.02) or €0.03 (lean price €0.03). The ﬁt of the lean price of €0.03 represents a polynomial approximation of the lean price €0.03 scenario. barley decreases by 21.84 kg (–10%). These changes are mainly due to the fact that the fatten- ing period becomes 7 days shorter and slaughter weight decreases by 4.5 kg. In other words, chang- es in the composition of feed given to pigs of a certain weight are marginal, but the shorter fatten- ing period implies that more emphasis is put on the protein-rich diet that is fed at the early stages of the fattening period. In addition, the value of a ca- pacity unit decreases by €11.47 over the 5-year period (€1.63 less income per year). scheme due to excess carcass weight is an impor- tant signal in determining the slaughter weight. Completely removing the weight discount increas- es the optimal slaughter weight by 6.0 kg com- pared to the benchmark scenario. Simultaneously, the length of the fattening period increases by 9 days and the share of red meat decreases by 0.3%. Thus, the annual amount of pig meat produced per capacity unit increases by 20.7 kg. The target weight above 91.0 kg results in exactly the same slaughter weight as no weight discount at all. 5.2.3 The effects of quality price premiums cass has decreased from €0.02 to €0.01. Changes in the daily weight gains are similar to the case where the price quality adjustments were com- pletely removed. The length of the fattening period becomes dominant in the slaughter decision when the price premium based on the share of red meat in the carcass decreases from €0.02 to €0.01, and thus the amount of annually produced meat in- creases by 32.5 kg and the carcass becomes fattier (Table 7). Increasing the price adjustment based on the share of red meat from €0.02 to €0.04 results in almost similar changes in feeding and slaughter patterns than observed in the event of an increase from €0.02 to €0.03. Adjustments of feeding are only little larger when the red meat premium in- creases from €0.02 to €0.04 than those reported in Figure 22, because the minimum ratio of fat to lean growth is a binding constraint in both cases. Furthermore, when the red meat adjustment in- creases from €0.02 to €0.04, the value of a capac- ity unit over the 5-year period increases €65.49 above the benchmark scenario (€18.55 additional income per year) (Table 7). When the price adjustment based on the share of red meat in the carcass increases from €0.02 to €0.03, the amount of barley fed during the fatten- ing period decreases by 6.87 kg (–3%) and the amount of soy meal increases by 2.49 kg (8%). The amount of barley decreases particularly after one third of the fattening period has passed where- as the share of soy meal in feed increases also for young pigs (Figure 22). When the price premium for red meat increases from €0.02 to €0.03, the optimal share of red meat in the carcass increases by 0.6%. Producers also gain returns from produc- ing lean meat, because the value of a capacity unit An increase or a decrease in the discount due to excess carcass weight generally results in negligi- ble changes in feeding patterns, timing of slaugh- ter, slaughter weight, carcass leanness and value function. Nevertheless, the existence of a discount 59 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. Table 7. Time required to reach slaughter maturity (duration), carcass weight upon slaughter, share of red meat in the slaughtered carcass, and the value of a capacity unit (value function) over a 5-year period under alternative meat quality pricing scenarios. Duration Carcass weight Red meat Value function days kg % €/5 years No price premiums on carcass quality 104 91.9 57.5 714 Lean price is €0.01 94 84.5 58 685 Benchmark (lean price is €0.02 and excess weight price discount is €0.02) 105 85 61.3 687 Lean price is €0.03 106 84.5 62 719 Lean price is €0.04 106 84.4 62 753 Excess weight price discount is €0.00 114 91 61.1 694 Target weight decreases 5 kg 98 80.6 61.3 676 Table 7. Time required to reach slaughter maturity (duration), carcass weight upon slaughter, share of red meat in the slaughtered carcass, and the value of a capacity unit (value function) over a 5-year period under alternative meat quality pricing scenarios. and the timing of slaughter. In other words, the op- timal feeding patterns and the timing of slaughter correlate and are strongly linked to carcass quality, particularly carcass leanness. It is generally opti- mal to grow lean tissue according to the potential and control carcass quality by adjusting the growth rate of fatty tissue and the timing of slaughter. Re- ducing the growth of fatty tissue decreases the amount of barley in the feed and increases the use of soy meal. Hence, the protein lost when reducing the amount of barley is replaced by protein ob- tained from soy meal. that the effects that quality price adjustments have on carcass quality management are similar to those reported in Table 7. The meat pricing scheme used in the bench- mark scenario is a linear approximation of an ac- tual meat pricing scheme that measures carcass weight and leanness on a discrete scale. As optimi- sation with the discrete price scheme resulted in estimates that were similar to those reported in this chapter, linearisation causes no signiﬁcant bias to the results. A discrete price scheme can, however, affect management patterns. This is the case when the pricing scheme has a large ﬂat region, where no adjustments apply. A scheme that paid no pre- mium for carcasses containing 58–60% red meat, for instance, increased the amount of barley in feed. Vol. 15 (2006): Supplement 1. The growth of fatty tissue then reached growth potential, the fattening period became shorter and the share of red meat in the carcass de- creased when compared to the benchmark scenar- io. When the daily amount of feed is a function of time, it is possible to characterise marginal chang- es in feeding patterns due to price movements by the steepness and concavity of the slopes of the curves for the amount of barley and for the share of soy meal in the feed. When the growth of fatty tissue is controlled and incentives for unrestricted feeding decrease, the producer at ﬁrst increases the daily growth rate at the early stages of growth. When incentives to maximise live weight gain fur- ther increase, the producer gradually starts increas- ing the growth rates as well when pigs are close to the optimal slaughter maturity. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Re- moving the weight discount from the benchmark scenario decreases the total amount of soy meal fed during the fattening period by 0.1 kg and in- creases the amount of barley by 33.2 kg. There- fore, the share of protein in feed decreases and the amount of barley increases during the second half of the fattening period. The changes increase the value of the capacity unit by €6.7 over the period of 5 years. If the target weight shifts from 85 kg to 80 kg and simultaneously either the discount, due to ex- cess carcass weight changes, or the premium, due to the share of red meat changes, then the optimal timing of slaughter falls close to 80 kg carcass weight. The changes in feeding patterns are mainly due to the shorter fattening period and minor ad- justments before the slaughter, because feeding patterns during the ﬁrst 85 days of the fattening period are very similar at both target weights. Therefore, removing the difference between the benchmark scenario and the scenario where only the target weight range shifts downwards conﬁrms When the target weight range moves 5 kg downwards from 85 kg to 80 kg, the total amount of soy meal fed during the fattening period de- creases by 0.24 kg (–1%), and the total amount of 60 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs price increases the length of the fattening period and decreases the annual meat yield per capacity unit. As the changes required adjustments in the growth rates, a one percent increase in the piglet price increases the amount of soy meal fed during the fattening period by 0.39%, and decreases the amount of barley by 0.22%. Qualitatively, these impacts are equal to decreasing slaughter premi- um. Management patterns are affected by the fat- to-lean growth ratio constraint. Relaxing this con- straint would increase choice elasticities somewhat with respect to changes in the piglet price. ported in Table 6 suggest that producer responds to the increasing price of pig meat (ceteris paribus) by increasing the daily weight gain and decreasing the length of the fattening period. A one percent increase in the price of pig meat shortens the fat- tening period by 0.72%. As the amount of annually produced pig meat then increases, an increase in the price of pig meat price emphasises the volume of production. Increasing the daily weight gain re- quires increasing the amount of energy in feed, which decreases the share of red meat in carcass. Although the producer suffers from a price dis- count due to fatty carcass, the increase in the pro- duction volume and in the base price of pig meat increases the value of a capacity unit. Although the pig management that maximises the value of a capacity unit implicitly controls car- cass quality, the optimal slaughter weight depends primarily on the target weight range. In most sce- narios, the optimal slaughter weight is close to the point at which additional carcass weight reduces the price of pig meat. Therefore, decreasing the target weight range also decreases the optimal slaughter weight. Slaughter returns increase when slaughter pre- mium increases. Consequently, when the slaughter premium increases by one percent, the amount of meat produced per capacity unit per year increases by 0.19%, and length of the fattening period de- creases by 0.26%. Since the optimal slaughter weight is quite unaffected by the length of the fat- tening period, the diet becomes richer in energy when the slaughter premium increases. When the slaughter premium is removed from the optimisa- tion problem, annual meat production per capacity unit decreases by 5 kg and the value of a capacity unit drops to one half of the benchmark value. 5.3 Summary Comparisons of the optimal feeding and slaughter patterns give information on how price changes affect pig management decisions, but the optimal levels of feeding patterns in Figures 16–22 should be interpreted with the normative approach of the study in mind. The elasticity estimates re- The results presented in Chapter 5.2 indicate that pig meat producers can adjust feeding and slaugh- ter patterns to market conditions by controlling the share of protein and the amount of energy in feed, 61 6.1 Data and scenarios The problem confronting animal breeders is whether it is optimal to improve mature weight, maturing rate, or both. The problem is compli- cated by the fact that maturing rate correlates negatively with mature weight. When the maxi- mum daily growth rate is a function of time, the effects of mature weight and maturing rate on the growth rates of fatty tissue, lean tissue, and pig’s live weight can be determined on the basis of the concavity and height of the growth curve. When mature weight is constant and maturing rate in- creases, the daily growth rate increases, particu- larly at early stages of growth. Hence, the pig can quickly reach the speciﬁed slaughter weight. The growth rate of a pig having high maturing rate can be very high for a short period around the inﬂec- tion point, but thereafter the daily weight gain of the pig can decrease rapidly compared to that of a pig having low maturing rate. The daily weight gain can still be higher on a pig having high ma- turing rate than on a pig having low maturing rate (Whittemore 1998, p. 67, Emmans and Kyriaza- kis 1999). Chapter 6 focuses on the third research problem, which was to examine the effects of genotype on the optimal slaughter and feeding patterns, and the potential beneﬁts related to animals of improved genotype. The problem is related the producer’s ability to control genotype quality through animal breeding in the long run. Improvements in genetic characteristics of an animal are known to be able improve economic performance of pig meat pro- duction (Chavas et al. 1985, Boland et al. 1993). Even if improvements are once implemented, the potential for further improvements still exists. In the following analysis, the genotype of an animal is measured with parameters of mature weight and maturing rate. The parameters are re- lated to the pig’s genetic carcass composition and to the biological maximum of the daily weight gain of lean tissue, fatty tissue and live weight (see Equation 9 in Chapter 3.5). Maturing rate refers to the maximum rate of daily weight gain of a pig, whereas mature weight refers to the biological maximum weight that it can reach when it reaches maturity. An increase in either of the parameters, however, increases the maximum daily weight gain of the pig when other factors are held constant (Emmans and Kyriazakis 1999). Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Quality premiums based on the share of red meat in the carcass have more complex effects on carcass quality than weight discounts do. When the premium paid for an additional percentage point of red meat increases, the share of red meat in the carcass quickly increases. Since producing leaner carcasses requires decreasing the growth of fatty tissue, the optimal amount of barley in feed decreases. Protein content lost when decreasing the amount of barley is then replaced in favour of increased use of soy meal. Since growth rates then decrease, the length of the fattening period also in- creases. Therefore, an increase in the relative price premium paid for lean carcasses increases the share of red meat in the carcass at slaughter but decreases the amount of meat produced per capac- ity unit per year. Nevertheless, the value of a ca- pacity unit responds sluggishly to options in the feeding patterns. In general, producers respond to rising price of a feed by decreasing the amount of that particular feed and replacing the lost nutrients by increasing use of other feeds. This substitution effect is small- er when the prices of all feeds increase than when only the prices of selected feeds increases. Chang- es in feed prices have only a marginal effect on the length of the fattening period, slaughter weight and the share of red meat in slaughtered carcass. Nevertheless, increasing feed prices decreases the value of the capacity unit. On the other hand, when the price of soy meal drops by 10%, the producer is able to substitute soy meal for barley and syn- thetic amino acids. This together with decreased price of feed results in a €3.33 increase in the an- nual income of a capacity unit. Changes in the price variables generally result in large income effects. Income effects are meas- ured based on the value of a capacity unit. Particu- larly, when the price of pig meat is high, producers are able to earn high returns, whereas low prices result in signiﬁcantly smaller returns. A one per- cent increase in the price of pig meat increases An increase in piglet price decreases incentives for maximising daily weight gain of a pig instead of its carcass value. Thus, an increase in the piglet 62 Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. producer income by 2.44%, whereas a one percent increase in the slaughter premium increases pro- ducer income by 0.54%. Producer income responds inelastically also to changes in the prices of other inputs besides piglet price. Despite adjusting feed- ing and slaughter patterns, a one percent increase in the price of piglet decreases producer income by 1.57%. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs characteristics of piglets and variation of the char- acteristics. Both maturing rate and mature weight contribute to the index value, since daily weight gain, feed conversion, and meat quality all affect the value of the K-index (Faba 2004). In general, fatty and slowly growing pigs tend to have a low K-index. Lean and rapidly growing pigs tend to have a high K-index. The difference between high and low K-index pigs in the analysis is 10 K-index points. The difference in the average daily weight gain potential of the two genotypes is 38 g per day between 25–120 kg liveweight. Parameter values of K-index scenarios are based on the estimates for high and low breeding class reported by Sévon- Aimonen (2001). An animal’s genotype affects its potential to utilise energy and protein. It follows that full utili- sation of the economic potential of animals of im- proved genotype can require adjustments in pig management patterns. Particularly, this may re- quire adjusting the feeding and slaughter patterns according to genotype. Thus, it is essential to take into account the effects of efﬁcient input allocation in order to obtain consistent estimates of the value of animal breeding strategies. The value of genotype and the effects of ge- netic differences on the optimal feeding and slaughter patterns are analysed in six scenarios. The scenarios are, in general, derived from the benchmark scenario by changing the parameter value of maturing rate, mature weight, or both, so that desired marginal changes in genotype are ob- tained. The changes have implications for the ini- tial weights of lean and fatty tissue, which depend on these two parameters (Table 8). Prices, subsidy rates and estimation methods used in the genotype scenarios are the same as in the benchmark sce- nario unless otherwise mentioned. Animal breeding has progressed at the rate of approximately 4–6 K-index points per year. Thus, an improvement of 10 K-index points can be reached in 1.7–3.5 years5, 6. According to Faba (2000), the daily weight gain of pigs that partici- pated in the progeny test increased in 1990–1999, on the average, 7.9 g per year in Yorkshire pigs and 8.7 g in landrace pigs (genetically trended esti- mate). Simultaneously, feed conversion, which measures the amount of feed required to produce an additional kilogram of meat, decreased by 0.024 fodder units per kilogram per year (genetically trended estimate). 5 Marja-Liisa Sevón-Aimonen, MTT Animal Production Research, personal communication, 12 November 2004. 6.1 Data and scenarios Although the av- erage increase in live weight gain within a speci- ﬁed weight range can be the same in both cases, the shapes of the growth curves can be very differ- ent, and two pigs of equal live weight can have different amounts of lean and fatty tissue depend- ing on their mature weight and maturing rate. An increase in the mature weight of a pig in- creases both its daily growth rate and the biologi- cal maximum weight that it can reach when it be- comes adult (e.g. Schinckel and de Lange 1996, Whittemore 1998, p. 59–68, Emmans and Kyria- zakis 1999). Since an increase in mature weight only increases the growth rate throughout the fat- tening period, it can have a larger impact on the growth rate of older pigs than an increase in matur- ing rate. In contrast to this, the growth rate of young pigs can increase less than in the event of an increase in maturing rate. 63 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. Table 8. The parameter values of mature weight (βlean and βfat), maturing rate (αi) and the initial amounts of lean (x0 lean) and fatty tissues (x0 fat), and average daily weight gain potential (ADG between 25-120 kg) of an individual pig for split- sex feeding and genotype scenarios1). Parameter Scenario αi 2) βlean βfat x0 lean x0 fat ADG Benchmark 0.014 29.875 55.499 3.775 3.041 0.969 Female pig 3) 0.014 30.625 50.984 3.818 2.829 0.943 Castrated male pig 0.015 29.124 60.015 3.743 3.198 0.987 Low K-index 0.014 29.95 58.76 3.759 3.119 0.953 High K-index 0.014 30.93 53.87 3.797 2.931 0.991 Improved mature weight 0.014 33.045 63.941 3.775 3.041 1.072 Improved maturing rate 0.016 29.875 55.499 3.775 3.041 1.067 1) Parameter values of castrated male pig and female pig scenarios were estimated from the growth experiment data. The benchmark scenario was constructed from these two scenarios (see Chapter 4.1). Parameter values of K-index scenarios were obtained from Sévon-Aimonen (2001). The benchmark scenario was constructed from these two scenarios (see Chapter 4.1). Other scenarios were constructed from the benchmark scenario by adjusting the parameter values of the benchmark scenario in order to obtain desired marginal change in comparison with the benchmark scenario. 2) The value of the maturing rate parameter αi, where i={lean, fat}, is the same for both tissue components. 3) Female pigs have a minimum fat to lean growth ratio 0.9, whereas for castrated male pigs the ratio is 1.0 (Whittemore 1998, p. 90). Split-sex feeding scenarios solve the optimal feeding and slaughter patterns of female and cas- trated male pigs separately for both sexes. There- after, the analysis focuses on the effects of manag- ing pigs as a group where both sexes are fed and slaughtered similarly (pooled-sex feeding). Pooled feeding and slaughter patterns are selected so that the optimal feeding and slaughter pattern is either female or castrated male pig’s optimal feeding and slaughter pattern. Pooled feeding takes into ac- count differences between genotypes of pigs. It is conditional on the fact that pigs are provided con- ditions where feeding has no signiﬁcant impact on their behaviour. The beneﬁts of split-sex feeding are examined under two price scenarios, where the base price of pig meat is either €1.39 or €1.62 (otherwise prices are identical). These scenarios are run separately for both sexes. 6 Matti Puonti, Finnish Animal Breeding Association, personal communication, 15 November 2004. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs The share of red meat increased by 0.37% per year in Yorkshire pigs and by 0.29% per year in landrace pigs (Faba 2000). The ﬁrst set of scenarios compares animals that have either improved maturing rate or improved mature weight. In other words, these scenarios ex- amine the effects of an improvement in a single ge- netic characteristic on the value of a capacity unit. In both scenarios, the biological maximum of pig’s daily liveweight gain between 20–120 kg live weight is approximately 1.07 kg. This is 100 g more than the daily weight gain potential in the bench- mark scenario. In the improved mature weight sce- nario, the growth potential increases steadily at all stages of the growth. In the improved maturing rate scenario, the maximum growth of a young piglet increases more than the maximum growth of a pig that is close to the optimal slaughter maturity. Hence, the growth curve has increased concavity with respect to time. At the maximum growth rate, the pig having an improved mature weight can reach approximately the same share of red meat than the pig having an improved maturing rate. At the rate reported in Faba (2000), improving the daily weight gain of pigs by 100 g requires 11–13 years of genetic progress. Nevertheless, it is possible to change the growth curve to have simi- lar shape as the scenarios in Table 8 in 3.5 years by breeding either mature weight or maturing rate only. Due to the strong negative correlation be- tween mature weight and maturing rate, this could imply signiﬁcant drawbacks in the other compo- nent of the growth curve, and hence, smaller in- crease in the daily weight gain5. The direct cost of animal breeding in Finland is approximately €1.75 million per year. At the rate of 12 g additional daily weight gain per year, the The second set of scenarios includes a com- parison between pigs having a low K-index and a high K-index. The K-index is based on a progeny test. It measures the fertility of a sow using the 64 6.2.1 The effects of animal genotype When mature weight increases so that the average daily growth potential of live weight between 20– 120 kg live weight increases 100 g above the benchmark scenario, the pig eats 5.28 kg (17%) more soy meal and 20.84 kg (–9%) less barley dur- ing the fattening period than in the benchmark sce- nario. As suggested by the increased concavity of the curve representing the share of soy meal in feed (Figure 23), the share of soy meal in feed increases particularly in the middle of the fattening period. The changes in feeding patterns are linked to the increased growth potential of lean tissue and to the result that the optimal slaughter maturity is reached 9 days earlier than in the benchmark scenario. When the maturing rate increases so that the daily live weight growth potential increases, on the average 100 g above the benchmark scenario (be- tween 20–120 kg live weight) the amount of soy meal fed during the fattening period increases by 3.04 kg (10%), whereas the amount of barley de- creases by 17.42 kg (–8%). The curve illustrating the daily amount of barley shifts upwards, whereas the share of soy meal in feed increases at the early stages of growth, but decreases when pigs ap- proach optimal slaughter maturity (Figure 24). Thus, the pattern of protein feeding is linked to the genotype of the animal. The increase in mature weight has only a small impact on the optimal slaughter weight, whereas the optimal share of red meat in the carcass in- creases by 0.4% compared to the benchmark sce- Despite the fact that the fattening period is 10 days shorter in the increased maturing rate scenar- io than in the benchmark scenario, differences in carcass quality at slaughter are negligible. The 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Fit of mature weight 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Benchmark Improved mature weight Fig. 23. Vol. 15 (2006): Supplement 1. In pooled-sex feeding, the beneﬁts are normalised so that both sexes contribute 50% of returns for a capacity unit. The parameter values of female and castrated male pigs were estimated from growth experiment data by Marja-Liisa Sévon-Aimonen, MTT Animal Production Research (see Chapter 4.1). cost of animal breeding equals at least €145,833 per additional gram of weight gain, or at least €291,666 per additional point of K-index6. The third set of scenarios (split-sex feeding) focuses on the animal’s phenotype and the beneﬁts of ﬂexible feeding technology on female and cas- trated male pigs. Since phenotype is an observable feature, producer can visually conclude genetic characteristics typical to each pig. Castrated male pigs, for instance, are fattier and grow faster than female pigs when they are fed according to the bi- ological growth potential. Therefore, female pig- lets can have improved carcass composition when compared to castrated male piglets at the same live weight. Due to genetic variations among pigs, pro- ducers may beneﬁt from a ﬂexible feeding tech- nology that allows pigs to be grouped into batches according to their gender and then, to feed and slaughter female and castrated male pigs separate- ly (cf. Kure 1997, Boland et al. 1999). Thus, the name split-sex feeding. 65 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 6.2 Results nario. When compared to the benchmark scenario, the increase in mature weight increases the value of a capacity unit by €91.70 (13%) over the 5-year period. This implies that the converged slaughter and feeding policy contributes annually €25.05 in additional value to the capacity unit (Table 9). 6.2.1 The effects of animal genotype The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where an increase in maturing rate in- creases the daily weight gain potential by 100 g. The ﬁt of the maturing rate represents a polynomial ap- proximation of the improved maturing rate scenario. Fig. 24. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where an increase in maturing rate in- creases the daily weight gain potential by 100 g. The ﬁt of the maturing rate represents a polynomial ap- proximation of the improved maturing rate scenario. Genetic improvements that increase the K-in- dex of a pig affect maturing rate, mature weight and the initial composition of the pig. Pigs having a high K-index are fed 2.37 kg more soy meal and 9.37 kg less barley during the fattening period than in the benchmark scenario. Even if differences in energy feeding of pigs of a given age are quite small, high K-index pigs can eat more protein than benchmark pigs (Figure 25). The fattening period is 4 days shorter for pigs having high K-index than in the benchmark scenario. Pigs having a high K- index contribute €38 (6%) more value to the ca- pacity unit than pigs in the benchmark scenario (Table 9). higher growth rate of the pig in the increased ma- turing rate scenario than in the benchmark scenario implies that the value of a capacity unit increases by €84.93 (12%) over the 5 years period (Table 9). Under converged feeding and slaughter patterns this also means that the improved maturing rate increases the value annually contributed to a ca- pacity unit by €23.54 when compared to the benchmark scenario. The increase is somewhat less than in the event of increased mature weight. Pricing meat on the basis of carcass merit in- creases the economic importance of carcass com- position. Therefore, if the price of pig meat in- creases or if the red meat premium decreases (ce- teris paribus), beneﬁts from genetic improvements of growth rate can increase compared to those ﬁg- ures reported in Table 9. 6.2.1 The effects of animal genotype The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where an increase in mature weight in- creases the maximum daily weight gain by 100 g above the benchmark scenario. The ﬁt of the mature weight is a polynomial approximation of the improved mature weight scenario. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 kg feed per day 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 % of feed kg feed per day 10 time in days Benchmark Improved mature weight Fit of mature weight Fig. 23. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where an increase in mature weight in- creases the maximum daily weight gain by 100 g above the benchmark scenario. The ﬁt of the mature weight is a polynomial approximation of the improved mature weight scenario. 66 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Fit of maturating ratio 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Benchmark Improved maturating ratio Fig. 24. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where an increase in maturing rate in- creases the daily weight gain potential by 100 g. The ﬁt of the maturing rate represents a polynomial ap- proximation of the improved maturing rate scenario. Vol. 15 (2006): Supplement 1. 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 % of feed time in days time in days Fit of maturating ratio Fig. 24. 6.2.1 The effects of animal genotype In addition, the minimum requirement for the fat to lean growth ratio can in- ﬂuence the optimal feeding patterns and the value of a capacity unit. The sensitivity analysis in Ap- pendix D, however, shows that the value of im- proved mature weight is higher than the value of improved maturing rate. Pigs having a low K-index are fed with 0.44 kg less soy meal and 5.25 kg more barley during the fattening period than in the benchmark scenario. The share of soy meal in feed decreases below the benchmark scenario, particularly when the pig is young (Figure 26). Since pigs having a high K-in- dex are leaner and grow faster than the pigs having a low K-index, differences in feeding patterns due to K-index value of pig show up particularly in the 67 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed High K-index Fit of high K-index 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Benchmark Fig. 25. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where the pig has a high K-index. The ﬁt of the K-index is a polynomial approximation of the high K-index scenario. time in days time in days Benchmark Benchmark Fit of high K-index Fig. 25. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where the pig has a high K-index. The ﬁt of the K-index is a polynomial approximation of the high K-index scenario. (-4%) less value to the capacity unit than the pigs in the benchmark scenario. The equivalent annual value of a genetic improvement of 10 K-index points is €14.51 per capacity unit. share of soy meal in feed (Figures 26 and 27). The fattening period of pigs of low K-index is a few days longer than the period in the benchmark sce- nario. Pigs having a low K-index contribute €26 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Fit of low K-index 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Benchmark Low K-index Fig. 26. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where the pig has a low K-index. The ﬁt of the low K-index is a polynomial approximation of the low K-index scenario. 6.2.2 Split-sex feeding7 In this study, the differences between female and castrated male pigs are considered in the form of differences in mature weight and maturing rate. Female piglets are leaner than castrated male pigs at the same live weight, but castrated male pigs have the potential to grow at a faster rate than fe- male pigs. When the producer optimises the feed- ing and slaughter patterns of female pigs (the grey line in Figure 27; see also Figure A4 in Appendix C), the total amount of soy meal fed during the fat- tening period is 2.49 kg (8%) higher, and the amount of barley is 8.58 kg (–4%) lower than in the benchmark scenario. The share of soy meal in feed increases throughout the fattening period, ex- cluding the last few weeks before slaughter. The length of the fattening period is the same in both cases, but female pig has a lower slaughter weight than in the benchmark scenario. In addition, the share of red meat in female pigs is 0.8% lower than In this study, the differences between female and castrated male pigs are considered in the form of differences in mature weight and maturing rate. Female piglets are leaner than castrated male pigs at the same live weight, but castrated male pigs have the potential to grow at a faster rate than fe- male pigs. When the producer optimises the feed- ing and slaughter patterns of female pigs (the grey line in Figure 27; see also Figure A4 in Appendix C), the total amount of soy meal fed during the fat- tening period is 2.49 kg (8%) higher, and the amount of barley is 8.58 kg (–4%) lower than in the benchmark scenario. The share of soy meal in feed increases throughout the fattening period, ex- cluding the last few weeks before slaughter. The length of the fattening period is the same in both cases, but female pig has a lower slaughter weight than in the benchmark scenario. In addition, the share of red meat in female pigs is 0.8% lower than The optimal feeding patterns of a castrated male pig (the grey line in Figure 28; see also Fig- ure A5 in Appendix C) and the benchmark pig are quite similar. 7 Part of the material reported in this chapter was pre- sented in preliminarily form at the European Workshop for Decision Problems in Agriculture and Natural Resources held 27–28 September 2004 in Silsoe, United Kingdom. Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. Table 9. Time required to reach slaughter maturity (duration), carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value of a capacity unit (value function) over the period of 5 years under alternative genotype scenarios1). Scenario Duration Carcass weight Red meat Value function days kg % €/5 years Benchmark 105 85 61.3 687 Improved mature weight2) 96 85.2 61.7 779 Improved maturing rate2) 95 84.8 61.3 772 High K- index3) 101 85 61.6 725 Low K-index3) 109 85.3 61.4 661 1) The changes in the genotype are observed through the parameter values of mature weight (βlean and βfat) and maturing rate (αi). 2) The scenarios increase the average biological maximum daily weight gain by 100 g between 20 and 120 kg live weight when compared to the bechmark scenario. 3) The difference between pigs of high K-index and pigs of low K-index in 10 K-index points. Table 9. Time required to reach slaughter maturity (duration), carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value of a capacity unit (value function) over the period of 5 years under alternative genotype scenarios1). red meat in the slaughtered carcass, and value of a capacity unit (value function) over the period of 5 years under alternative genotype scenarios1). Scenario Duration Carcass weight Red meat Value function days kg % €/5 years Benchmark 105 85 61.3 687 Improved mature weight2) 96 85.2 61.7 779 Improved maturing rate2) 95 84.8 61.3 772 High K- index3) 101 85 61.6 725 Low K-index3) 109 85.3 61.4 661 1) The changes in the genotype are observed through the parameter values of mature weight (βlean and βfat) and maturing rate (αi). β g The scenarios increase the average biological maximum daily weight gain by 100 g between 20 and 120 kg live weight when compared to the bechmark scenario. 2) The scenarios increase the average biological maximum daily weight gain by 100 g between 20 and 120 kg live weight when compared to the bechmark scenario. g g p 3) The difference between pigs of high K-index and pigs of low K-index in 10 K-index points. g g p The difference between pigs of high K-index and pigs of low K-index in 10 K-index points. in the benchmark scenario. Vol. 15 (2006): Supplement 1. The value of a capacity unit over the 5-year period is €8.12 higher than in the benchmark scenario (€2.75 per year) (Table 10). Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 % of feed 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 kg feed per day % of feed time in days Fig. 26. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the scenario where the pig has a low K-index. The ﬁt of the low K-index is a polynomial approximation of the low K-index scenario. 68 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Table 10. Time required to reach slaughter maturity (duration), carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years in the benchmark scenario, the female pig scenario and the castrated male pig scenario when the price of pig meat is set at Table 10. Time required to reach slaughter maturity (duration), carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years in the benchmark scenario, the female pig scenario and the castrated male pig scenario when the price of pig meat is set at €1.39. Scenario Duration Carcass weight Red meat Value function days kg % €/5 years Benchmark 105 85 61.3 687 Female pig 105 84.3 62.1 696 Castrated male pig 107 85.2 61.3 674 the price of pig meat is high, the producer can fully utilise the growth potential of castrated male pigs, and still beneﬁt from the leanness of female pigs. of initial leanness is also visible in the feeding curves (Figures 27 and 28), where female pigs can utilise high levels of protein at early stages of growth. In pooled-sex feeding, the producer feeds all pigs using the same feeding and slaughter pattern instead of ﬁrst separating them by sex and then feeding and slaughtering both sexes separately. Pooled-sex feeding patterns in Table 12 are the same optimal feeding and slaughter patterns that were reported in Tables 10 and 11 with the excep- tion that each of the patterns is applied simultane- ously on both female and castrated male pigs. Un- der pooled-sex feeding both female and castrated male pigs contribute 50% of their returns to a ca- pacity unit. g When the price of pig meat increases from €1.39 to €1.62, which is the only difference be- tween scenarios in Tables 10 and 11, producer has incentives to grow pigs almost according to their growth potential. The price increase leads the pro- ducer to reduce the amount of soy meal fed to the female pig during the fattening period by 9.11 kg, and to increase the amount of barley by 17.50 kg (Figure 27). For castrated male pigs, the price change leads the producer to reduce the total amount of soy meal fed during the fattening period by 12.42 kg and to increase the amount of barley by 12.52 kg (Figure 28). 6.2.2 Split-sex feeding7 The total amount of soy meal fed to a castrated male pig during the fattening period is 0.18 kg lower, and the amount of barley is 2.40 kg higher in the castrated male pig scenario than in the benchmark scenario. Thus, the difference is mainly due to the fact that the fattening period is longer on castrated male pigs than in the bench- mark scenario. The value of a capacity unit with a castrated male pig over the 5-year period is €13.08 lower than in the benchmark scenario (Table 10). The constraint on the minimum fat-to-lean growth ratio is binding, particularly during the medium stages of growth. The diet fed to female pigs is richer in protein than that fed to castrated male pigs. In addition, the fattening period is shorter for female pigs than for castrated male pigs. This is due to the fact that the initial state of nature is different for female pigs than for castrated male pigs. Female pigs gain an advantage through the length of the fattening pe- riod because of their leanness and because of the price premium paid for lean carcasses. The effect 69 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs For castrated male pigs the price increase leads the producer to decrease the length of the fattening period almost twice as much as for female pigs. Thus, after this price in- crease, the fattening period is 5 days shorter for castrated male pigs than for female pigs (Table 11). When feeding and slaughter patterns are pooled, it is optimal to feed and slaughter female and castrated male pigs according to the optimal policy for female pigs. When the price of pig meat is €1.39, the producer’s expected annual beneﬁts from split-sex feeding are €1.89 per capacity unit. Then, only castrated male pigs suffer from inefﬁ- cient input allocation. Similarly, when the price of pig meat is €1.62, the expected beneﬁts from split- sex feeding are €1.55 per capacity unit per year. If the producer uses the optimal feeding patterns of castrated male pig, then expected value of a capac- ity unit is signiﬁcantly lower than when using the optimal female pig’s feeding policy. The share of red meat in the carcass decreases more for castrated male pigs than for female pigs when the price of pig meat increases from €1.39 to €1.62. In addition, the value of a capacity unit increases more than 43% for both sexes (Tables 10 and 11). Differences in the daily amounts of feeds between the sexes are larger when the base price of pig meat is €1.62 than when the price is €1.39. Particularly at the higher price and a few weeks before the optimal slaughter time, the diet fed to female pigs is richer in protein than that fed to cas- trated male pigs (Figures 27 and 28). Thus, when When the price of pig meat is €1.39, losses from pooled-sex feeding are mainly due to the fact that castrated male pig is slaughtered prematurely; and when the price of pig meat is €1.62, due to the fact that the slaughter of castrated male pig delays. Thus, income from the castrated male pig then suf- 70 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 0 20 25 1 11 21 31 41 51 61 time in days % of feed Fit of €1.62 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day €1.39 5 10 15 71 81 91 101 €1.62 Fig. 27. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) for female pigs, when the base price of pig meat is either €1.39 or €1.62. The ﬁt of €1.62 represents a polynomial approximation of the scenario where pig meat price is €1.62 and the pig is female pig. Vol. 15 (2006): Supplement 1. time in days time in days Fig. 27. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) for female pigs, when the base price of pig meat is either €1.39 or €1.62. The ﬁt of €1.62 represents a polynomial approximation of the scenario where pig meat price is €1.62 and the pig is female pig. cause the optimal feeding pattern of female pigs is characterised by protein-rich feeding. fers from a quality price discount due to low car- cass weight or due to fatty and overweight carcass. Nevertheless, producer can reduce an increase in carcass fatness due to pooled feeding patterns, be- The optimal feeding and slaughter patterns can exist between the two options considered in Table 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Fit of €1.62 1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day €1.39 €1.62 Fig. 28. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) for castrated male pigs, when the base price of pig meat is either €1.39 or €1.62. The ﬁt of €1.62 represents a polynomial approximation of the scenario where pig meat price is €1.62 and the pig is castrated male pig. A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Table 11. Time required to reach slaughter maturity (duration), carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years for the benchmark scenario, female pig, and castrated male pig when the price of pig meat is set at €1.62. Scenario Duration Carcass weight Red meat Value function days kg % €/5 years Benchmark at €1.62 94 84.2 58.1 991 Female pig 97 84.4 59.4 994 Castrated male pig 92 83.7 57.1 979 Table 12. The expected value of a capacity unit when all the units have female pigs or castrated male pigs, or when half of the units have female pigs, and when the price of pig meat is either €1.39 or €1.62.1) Scenario Share of female pigs Value Optimal policy % of capacity units €/year Price of pig meat is €1.39 Female pig 100 152.56 female Castrated male pig 0 143.56 castrated male Split-sex feeding 50 148.06 both separately Pooled feeding 50 146.17 female Price of pig meat is €1.62 Female pig 100 220.48 female Castrated male pig 0 217.27 castrated male Split-sex feeding 50 218.88 both separately Pooled feeding 50 217.33 female 1) The values are not directly comparable to the estimates reported in other tables. Table 12. The expected value of a capacity unit when all the units have female pigs or castrated male pigs, or when half of the units have female pigs, and when the price of pig meat is either €1.39 or €1.62.1) Scenario Share of female pigs Value Optimal policy % of capacity units €/year Price of pig meat is €1.39 Female pig 100 152.56 female Castrated male pig 0 143.56 castrated male Split-sex feeding 50 148.06 both separately Pooled feeding 50 146.17 female Price of pig meat is €1.62 Female pig 100 220.48 female Castrated male pig 0 217.27 castrated male Split-sex feeding 50 218.88 both separately Pooled feeding 50 217.33 female 1) The values are not directly comparable to the estimates reported in other tables. Table 12. The expected value of a capacity unit when all the units have female pigs or castrated male pigs, or when half of the units have female pigs, and when the price of pig meat is either €1.39 or €1 62 1) 12. A G R I C U L T U R A L A N D F O O D S C I E N C E An analysis where producer can shift the feed ratios and the timing of slaughter marginally from the female pig’s optimal pattern towards castrated male pig’s optimal pattern resulted approximately in 50% lower beneﬁts in both scenarios reported in Table 12. In such a case, the optimal pattern is also close to female pig’s patterns. The analysis suggest that when the restricted feeding is applied, the quality of a carcass is valuable, and the timing of pooled slaughter is particularly important. The choice of feeding pattern at the margin is particu- larly important when the pigs are fed according to their growth potential. Then, differences in feed- ing patterns between sexes are larger and the daily weight gain is larger than when energy supply is restricted. Furthermore, energy-rich feeding can reduce carcass value through excess weight and fatness more than protein-rich feeding. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 % of feed 1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 kg feed per day % of feed time in days Fig. 28. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) for castrated male pigs, when the base price of pig meat is either €1.39 or €1.62. The ﬁt of €1.62 represents a polynomial approximation of the scenario where pig meat price is €1.62 and the pig is castrated male pig. Fig. 28. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) for castrated male pigs, when the base price of pig meat is either €1.39 or €1.62. The ﬁt of €1.62 represents a polynomial approximation of the scenario where pig meat price is €1.62 and the pig is castrated male pig. 71 Vol. 15 (2006): Supplement 1. Pig genotype also affects the optimal timing of slaughter. When the price of pig meat is €1.62, the pigs are fed according to their growth potential, and the female pigs are slaughtered 5 days later than the castrated male pigs. On the other hand, when prices of the benchmark scenario are used, the castrated male pigs are slaughtered 2 days later than the female pigs. This is affected by the ini- tially higher fat content and higher growth poten- tial of a castrated male pig than of a female pig. If the initial composition is the same for both sexes, the fattening period of female pigs would be long- er than the period of castrated male pigs. The dif- ference in pig’s genetic characteristics is also ob- servable in the share of red meat in the carcass. Despite restricting the growth rates of both the fe- male pigs and the castrated male pigs, the female pigs have higher share of red meat upon slaughter. The difference in carcass leanness is even larger when the pigs are fed according to growth poten- tial than when the feeding is restricted. weight increases, the concavity of the curve illus- trating the share of soy meal (or protein) in the feed as a function of the animal’s age increases. In addition, the share of soy meal in the feed then re- mains high until the inﬂection point of the growth curve is passed. Thereafter the share of soy meal in the feed decreases rapidly. When the maturing rate increases, the curve illustrating the share of soy meal in the feed approaches linearity, and steep- ness of the curve increases. In other words, the share of protein in the feed is high for young pigs, but decreases quickly when pigs become older. An increase in mature weight or maturing rate shortens the fattening period, whereas the slaugh- ter weights are almost unaffected. In addition, an increase in mature weight increases carcass lean- ness and carcass value. At prices used in the bench- mark scenario, the model estimates that a 100 g increase in the daily weight gain increases the val- ue of a capacity unit by 12–13% (€85–95 per ca- pacity unit over the period of 5 years). In general, an increase in mature weight beneﬁts the producer slightly more than an increase in mature weight. 6.3 Summary Producers are able to gain additional value from the capacity unit by improving the genetic proper- ties of fattening pigs. In order to gain full advan- tage from such improvements, the feeding and slaughter patterns are adjusted according to geno- type. When maturing rate or mature weight of an animal increases, the total amount of barley fed during the fattening period generally decreases and the total amount of soy meal increases. The changes in feeding patterns are mainly due to the facts that the growth curve, which is a function of time, either shifts upwards or becomes steeper, or that the concavity of the curve increases. Changes in mature weight and maturing rate affect feeding patterns differently. When mature 72 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. 7 The importance of slaughter timing8 mals. As a carcass becomes heavier when the tim- ing of slaughter is postponed and a heavy carcass generally contains more fat than a light carcass (Whittemore 1998, p. 53), delayed slaughter re- sults in quality price discounts and decreases in value. If the expected delay of the timing of slaugh- ter increases, producer losses can increase also for other reasons than reduced carcass quality. For in- stance, pigs in overpopulated pens can suffer from increased stress, behavioural problems, respiratory diseases and mortality, or reduced weight gain (cf. Kornegay and Knotter 1984, Heikkonen 1998). Fortunately, producers can reduce decreases in carcass value by restricting feeding so that growth rates decrease. Restricted feeding, however, pro- vides only limited options for minimising a de- crease in carcass value unless information on delay is available well before the optimal timing of slaughter. In the previous chapters, a producer was able to fully control the timing of slaughter. Due to exog- enous constraints, however, a producer may not always control the exact delivery date of pigs. This may result in slaughter delays beyond the opti- mum. The delay can be relatively short, such as few days, or quite long, such as several weeks. This chapter examines the last research problem, the importance of the timing of slaughter, using two examples: a short delay of the timing of slaughter (slaughterhouse logistics problem) and a long delay of the timing of slaughter (animal movement restrictions problem). In the previous chapters, a producer was able to fully control the timing of slaughter. Due to exog- enous constraints, however, a producer may not always control the exact delivery date of pigs. This may result in slaughter delays beyond the opti- mum. The delay can be relatively short, such as few days, or quite long, such as several weeks. This chapter examines the last research problem, the importance of the timing of slaughter, using two examples: a short delay of the timing of slaughter (slaughterhouse logistics problem) and a long delay of the timing of slaughter (animal movement restrictions problem). The problem of delayed slaughter is interest- ing, because delayed slaughter can reduce the quality of meat, and hence reduce income. Re- duced meat quality decreases the quality of the meat processor’s raw material and hence affects the competitiveness of the industry. The problem is largely a co-ordination problem. 8 This chapter is based on model similar to Niemi, J.K., Pietola, K. & Sevón-Aimonen, M.-L. 2004. Hog producer income losses under contagious animal disease restric- tions. Acta Agriculturae Scandinavica Section C – Food Economics 1: 185–194. This chapter extends the analysis and includes a revised meat pricing system in contrast with the earlier paper. Vol. 15 (2006): Supplement 1. The producer beneﬁts from separating the feeding and slaughter patterns of the female and the castrated male pigs. The beneﬁts are estimated at €1.89 when the price of pig meat is €1.39, and at €1.55 when the price of pig meat is €1.62. The beneﬁts increase when separating feeding and slaughter by sex is able to improve the accuracy of the timing of slaughter with respect to the slaugh- ter weight or carcass leanness, or when the separa- tion improves precision of nutrient supply. There- fore, when pooled-sex feeding results in either premature or delayed slaughter, the beneﬁts of split-sex feeding increase if a quality-adjusted price premium is related to the change. In addition, when the feeding and slaughter patterns signiﬁ- cantly increase carcass fatness, producer suffers price discounts due to fatty carcass, and the bene- ﬁts from precision feeding increase. Thus, when feeding patterns are pooled, using the optimal feeding patterns of the female pigs to feed both sexes rather than the optimal feeding patterns of the castrated male pigs contribute higher returns to the capacity unit. In addition, pigs having a higher K-index con- tribute higher value to the capacity unit than pigs having low K-index. The value of improving ge- netic characteristics of a pig by 10 K-index points is €64 per capacity unit over a period of 5 years. In feeding patterns, the most signiﬁcant difference is that particularly at the beginning of the fatten- ing period, pigs having a high K-index require feed richer in protein than that required by pigs having low K-index. In addition, pigs having a high K-index are able to reach the optimal slaugh- ter maturity 8 days earlier than pigs having a low K-index. The optimal diet of the female pigs is richer in protein than the diet of the castrated male pigs. Fe- male pigs eat more soy meal than castrated male pigs, whereas castrated male pigs eat more barley than female pigs. In addition, the share of soy meal decreases at a slower rate for the female pigs than for the castrated male pigs. The differences in feeding are more visible when the producer has in- centives to feed the pig according to its growth potential than when feed supply is restricted below the stomach capacity. Although carcass merits based meat pricing emphasises the value of carcass composition, sen- sitivity analysis provides results that are in line 73 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs with those of basic genotype scenarios. Sensitivity analysis (see Appendix D) illustrates that if the price of pig meat increases or if the price of red meat decreases (ceteris paribus), the beneﬁts from genetic improvements may increase. In addition to the growth potential, the minimum requirement for fat-to-lean growth affects the optimal feeding patterns as well as the value of growth. Vol. 15 (2006): Supplement 1. Vol. 15 (2006): Supplement 1. they can fully optimise the timing of slaughter and the quality of meat. hazard problems would be a scheme that provides individual producer with incentives to minimise losses due to maintenance feeding by increasing the probability of having his/her herd infected in order to gain quick access to slaughter. Even if such behaviour is prohibited by law, producer may have incentives to behave so that he/she uninten- tionally increases the probability of spreading the disease into his/her herd or from his/her herd to other herds. Another example could be a producer who minimises the risk of losing animals by mar- keting them for slaughter prematurely (cf. swine inﬂuenza application by Toft et al. (2005) who ﬁnd the optimal slaughter pattern and animal disease policy to correlate). When such decisions are made under uncertainty, information on increased ani- mal disease risk could increase the number of ani- mal transports throughout the country. The second example involves government ani- mal disease regulations, which can result in long delays of the timing of slaughter. Contagious dis- eases, such as classical swine fever and foot and mouth disease, can cause catastrophic losses to in- dividual livestock producers, producers as a group, other stakeholders, as well as to taxpayers (Saatkamp et al. 2000). The European Union is therefore prepared to eradicate most contagious diseases quickly once they are introduced into member countries. Standard procedures applied to eradicate highly contagious animal diseases and prevent further spread of the infections include culling infected herds, disinfecting the premises and imposing restrictions on animal movement (e.g. European Council 2001). Restrictions on ani- mal movement can force producers to feed ready- to-slaughter pigs even if the farm is uninfected. Farms can be quarantined, for instance, due to sus- picion of the presence of a contagious animal dis- ease on the farm itself or in the immediate vicinity. Animal movement restrictions are still necessary, because they can prevent new infections in cases where infected animals are transported without ob- serving signs of an infection. Even if the producer cannot fully determine the exact timing of the slaughter in the above exam- ples, he/she can minimise the losses due to delayed timing of slaughter. First, the producer can try to decrease the animal’s growth rate and to slow down the increase in carcass fatness by adjusting feeding. 9 In the event of measures taken to eradicate an animal disease, ‘cull’ should be used instead of the term ‘slaugh- ter’ whenever government authorities order animals to be culled and disposed of. 7 The importance of slaughter timing8 If co-ordination fails, the quality of pig meat can be low despite the fact that the slaughterhouse can control the meat supply via quality premiums and discounts. There- fore, it is important to have information on how pig meat producers can take the probability for de- layed slaughter into account, and how this affects the value of a capacity unit. When both the pro- ducer and the slaughterhouse have sufﬁcient infor- mation for determining the exact slaughter date, In the ﬁrst example, a slaughterhouse organises the transport of pigs for slaughter so that the trans- port date can be any date in a given interval. This can result in short delays in the timing of slaughter of individual pigs, because the slaughter date is ex- ogenously given to the producer and because it is revealed only after, or upon, marketing the ani- 74 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. 7.1.2 Modelling the value of slaughter timing The scenarios include two options, viz. exoge- nously delayed slaughter and premature slaughter. The options are studied by imposing restrictions on the timing of slaughter. The producer’s problem is to maximise the expected value of a capacity unit. Therefore, when the slaughter date is exoge- nously given, the objective includes minimising losses due to reduced carcass quality. In the ﬁrst option, the producer feeds the pig until slaughter becomes feasible and then slaughters the pig pro- vided that it is mature enough. In the second op- tion, the producer slaughters the pig immediately and the capacity unit then remains idle for a speci- ﬁed number of days. Both cases are modelled in the event of a short delay and long delay, and opti- misation is conditional on the expected duration of the restrictions imposed on slaughter. Finally, the discussion extends the analysis to how uncertainty regarding the imposition the restrictions affects the maximised value of a capacity unit when producer must choose either premature slaughter or delayed slaughter (which may never turn out to be delayed) before uncertainty is revealed. After carrying out the expectations operation, the next-period value function becomes: (17) Et(Vt+1(xt+1)) = Prt+1Vt+1 cull(xt+1) + (1 – Prt+1)Vt+1 feed(xt+1), t = 0,1,…,T, (1 – Prt+1)Vt+1 feed(xt+1), t = 0,1,…,T, where Prt+1 denotes the probability that the slaugh- ter becomes feasible before the next-period t+1, Vt+1 cull(xt+1) denotes the state-speciﬁc next-period value function when the slaughter is feasible, and Vt+1 feed(xt+1) denotes the state-speciﬁc next-period value function when the restrictions on the slaugh- ter continue. The difference in the two state-spe- ciﬁc value functions is that only Vt+1 cull(xt+1) allows the producer to slaughter the animals. Therefore, the expectations operator is the weighted sum of two state-speciﬁc value functions and the weights are the corresponding probabilities that a delay will occur. Under restricted slaughter, the binary slaughter variable is exogenously restricted at ut cull = 0. Thus, the optimal choice is nested and conditional such that uncertainty over outcomes arises from the unobservability of the state of na- ture. As opposed to the studies where instantane- ous returns are unobservable, in this study instan- taneous returns are observable to the producer. Hence, the state-speciﬁc expected value of future net returns is not observable in the current period t, but it is observable in the future (cf. Rust 1987, Keane and Wolpin 1994). Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs subject to: xt+1 = g(xt,ut,εt) (transition equations) x0 given (initial state given) VT+1(xT+1) given (the terminal value), subject to: xt+1 = g(xt,ut,εt) (transition equations) x0 given (initial state given) VT+1(xT+1) given (the terminal value), fore, the decision has to be made according to ex- pected outcomes. Producer then minimises losses from possible delay of the timing of slaughter ei- ther by allowing a certain loss due to premature slaughter or by running the risk of losses due to delayed slaughter. As Kure (1997) observes, the cost of early slaughter increases if the supply of new piglets to the capacity unit is ﬁxed in such a way that the capacity unit temporarily becomes idle. where the value function Vt is the function of the current state vector xt, t is the time index, ut is the control vector, Rt(.) is the instantaneous net return function, β is the discount factor, Et is the expecta- tions operator, Vt+1(.) denotes the next-period value function, and g(.) is a function determining the transition equation. The only differences between Equations 4 and 16 are the expectations operator, the possibility of removing restrictions on slaugh- ter indicated by εt in the transition equation, and the fact that the state vector now includes informa- tion on slaughter restrictions. This modiﬁcation implies that instantaneous returns in Equation 16 are certain, whereas future returns are uncertain. Vol. 15 (2006): Supplement 1. Therefore, unbiased estimates of producer income losses due to delayed slaughter require in- formation on the expected duration of the delay even when the timing of culling is exogenously de- termined. Second, the producer can take into ac- count the probability of delayed timing of slaugh- ter and consider marketing the pigs prematurely. In the event of animal disease restrictions, this entails either the moral hazard problem or the sufﬁciently large probability that the farm will be placed under restrictions. Although individual producers may face losses due to disease eradication measures, such meas- ures can beneﬁt society or producers collectively as a group (Mangen et al. 2002). Furthermore, eco- nomic incentives can affect the outcome of an eradication policy (cf. Kuchler and Hamm 2000). These observations imply that cost efﬁcient eradi- cation of a contagious animal disease requires choosing policies that minimise losses to society, conditional on the requirement that all agents (such as pig meat producers) who can avoid infecting their herd or can otherwise reduce the outbreak have incentives to do so. If an exogenous date of slaughter cannot be re- vealed to producer until after he/she has chosen when to market the pigs, then he/she has to decide under uncertainty whether to market pigs prema- turely. In other words, events resulting in delayed slaughter may never come to pass even if a deci- sion is made to slaughter9 pigs prematurely. There- The problem of authorities is to design com- pensation schemes that provide all producers in- centives to choose preferred management practices and thus avoid the problem of moral hazard (for further details on the moral hazard problem, see Salanié (2005, p. 119–160)). An example of a ﬂawed compensation scheme suffering from moral 75 (16) Vt(xt) = max ut {Rt(xt,ut) + βEt(Vt+1(xt+1))}, t = 0,1,…,T A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Vol. 15 (2006): Supplement 1. Producer income losses arise not only as a con- sequence of delayed slaughter timing, but also by overcrowding pens as a results of the delay. There- fore, the instantaneous return function (Equation 11) of a capacity unit becomes: the losses because it takes more than 5 years for the value function to converge. It does not affect the difference between delayed slaughter and pre- mature slaughter scenarios, because both scenarios discount income ﬂows obtained after lifting the re- strictions similarly. Restrictions on slaughter are imposed and the losses are estimated only for the delayed slaughter of the pig that currently com- prises the capacity unit. When slaughter becomes feasible in the next period, producer slaughters the pig (provided that it is mature enough to be slaugh- tered) and thereafter follows an unrestricted opti- mal slaughter policy. Since producer can have in- centives to reduce the amount of feed to such an extent that animal welfare is endangered, the mini- mum daily weight gain of a pig must remain above 0.5 kg. The limit is based on discussions with vet- erinarians and animal scientists. (18) Rt(xt,ut) = – (1 – ut cull)(ut soypt soy + ut barleypt barley + ut aminopt amino) + ut cull(φ(xt weight)pt meat(xt fat,xt lean) – Ct pcc(xt weight) – Ct mor(xt weight) – pt weaner + pt subsidy), where exogenous prices (p’s), the price of pig meat (pt meat(xt fat,xt lean)), and the measure of meat yield are used as in Equation 11. The function Ct pcc(xt weight) gives the value of excess meat lost through partial carcass condemnations, and Ct mor(xt weight) gives losses due to increased mortality. In addition, the animals consume more feed when slaughter is de- layed. Economic effects of farm overpopulation are estimated as a function of the pig’s live weight using the basic formula of allometry. In other words, overpopulation losses occur when the opti- mal (unrestricted) slaughter weight is exceeded and the losses increase with live weight because heavy animals require more pen area than light animals. The model is normalised for an average pig. The average pig represents an individual pig or a group of homogeneous pigs. Restrictions on slaughter are imposed, or op- tionally, the pigs are slaughtered prematurely at different live weights. 7.1.2 Modelling the value of slaughter timing The problem is modelled by introducing sto- chasticity into the optimisation problem and re- stricting the slaughter of an individual pig so that it is exogenously given. The economic model now follows a Bellman equation of the form: 76 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs tion, and 3.2% for the scenario with 31 days ex- pected duration. meal in feed and the amount of barley in feed be- low the levels of the benchmark scenario immedi- ately after obtaining the information on restrictions and thereafter, maintaining the reduced feed lev- els. When the restrictions are imposed for 31 days, the results suggest decreasing the share of soy meal in the feed more than in the 24 days scenario (Figure 29). If the expected duration of the restric- tion period were more than 31 days, restricting of soy feeding would be stronger than in the scenarios in Figure 29. If the expected duration were long enough, all protein could be supplied through the least expensive feed (barley). On the other hand, if the restrictions are imposed on animals heavier than 99 kg for 24 days, then the optimal slaughter date is closer to the present than in the 99 kg sce- nario and therefore, the amount of soy meal is re- duced more than as illustrated in the 24 days sce- nario in Figure 29. The kinks in the feeding curves are due to the fact that the underlying production function is non-convex. This causes irregularity of control variables at certain nodes of the state space. In addition, discretisation of the model has a very small impact on the curves. In the event of animal disease restrictions, the minimum duration of the restriction period is 45 days. This represents the estimated minimum du- ration for a farm located in the protection zone of a classical swine fever outbreak (European Council 2001). After a restriction period of 45 days, prob- abilities for the slaughter to become feasible be- fore the next day are 50% for the scenario with an expected duration of 47 days, 14% for the scenario with an expected duration of 52 days, 7.1% for the scenario with an expected duration of 59 days, and 4.8% for the scenario with an expected duration of 66 days. In premature slaughter scenarios, the animals are slaughtered prematurely and the capacity unit remains idle until the slaughter restrictions expire. Thus, supply for piglets is ﬁxed, and expected du- ration of the idle capacity follows a similar jump process as in the expected duration of the restric- tions. These scenarios also allow the case to be examined in which new piglets are obtained im- mediately after premature slaughter. Vol. 15 (2006): Supplement 1. The weights and corre- sponding dates are 93 kg (4 weeks before the opti- mal slaughter date), 99 kg (3 weeks before the op- timal slaughter date), 105 kg (2 weeks before the optimal slaughter date), 111 kg (1 week before the optimal slaughter date), and 116 kg (at the optimal slaughter date). Therefore, the expected delay of slaughter depends on both the age of an animal when the restrictions are imposed and on the ex- pected duration of the restriction period. Using the results obtained by Heikkonen (1998), Ct mor(xt weight) triggers when the pen area re- quirement per overweight pig is at least 10% more than the area allocated for pigs at the optimal slaughter weight. The cost of mortality is a quad- ratic function of live weight such that each addi- tional kilogram of live weight increases the cost by €0.0125 and that the cost per unit of live weight increases simultaneously by €0.0015. Additional meat lost in partial carcass condemnations is esti- mated based on Tuovinen (1994). Using the base price of pig meat, the value of condemned meat increases at the rate of €0.0498 per each addition- al unit of the allocation factor (xt weight)0.67. The loss factors are scaled according to the quality-adjusted price of pig meat. The expected duration of the restriction period follows a jump process and can be either certain or uncertain. Expected duration computed as in Dixit and Pindyck (1994, p. 85–87), is 0–66 days begin- ning from t = 0. In uncertain cases, the maximum duration is 120 days. In the event of slaughter- house logistics problems, restrictions are imposed for 0–31 days. When the expected duration of the restrictions is certain (deterministic), the slaughter date is known after imposing the restrictions. When the expected duration is uncertain (stochas- tic), the probabilities for the slaughter to become feasible before the next day are 33% for the sce- nario with an expected duration of 3 days, 10% for the scenario with 10 days expected duration, 5.9% for the scenario with 17 days expected duration, 4.2% for the scenario with 24 days expected dura- The model is applied to unrestricted slaughter and to restricted (i.e. delayed) slaughter scenarios. The losses are normalised by estimating them over a planning horizon of 5 years. This underestimates 77 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Producer in- come from premature slaughter equals the return that he/she receives when marketing the pig at the moment the restrictions on slaughter are imposed. Even if Figure 29 illustrates the patterns of re- stricted feeding, there are additional options to de- crease growth rates. This is the case when the pro- ducer has incentives to feed the pigs according to the growth potential, as in the scenario where the price of pig meat was €1.62 (see Chapter 5). This is due to the fact that under unrestricted feeding daily growth rates are higher than under restricted feeding. In the scenario where the price of pig meat was €1.62 the pig reached the optimal slaughter weight approximately two weeks after 99 kg live weight, and the optimal diet contained 3.0–3.5 kg barley and 2–4% soy meal. Therefore, introducing a delay beyond the optimal timing of slaughter would result in stronger adjustments than in the example given in Figure 29. A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. 0 5 10 15 20 25 1 11 21 31 time in days 24 days 31 days 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 time in days kg feed per day No delay % of feed Fig. 29. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in feed when slaughter is not delayed and when the pig can be slaughtered either 24 days or 31 days after 99 kg live weight (the live weight at day zero of the Figure). Fig. 29. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in feed when slaughter is not delayed and when the pig can be slaughtered either 24 days or 31 days after 99 kg live weight (the live weight at day zero of the Figure). per day) increases somewhat. When the duration of the restriction period increases, the share of soy meal in feed decreases, and the share of red meat and daily weight gain further decreases. The result for increased share of soy meal in feed is valid for scenarios where expected duration of the restric- tion period is short and the animals are young such that producer is able to adjust the slaughter weight close to the target weight. Thus, if information on the delayed timing of slaughter is obtained just few days before the optimal slaughter date, producer has hardly any options to adjust the slaughter weight below the target weight. tained at 116 kg, and the timing of slaughter is de- layed by 3 days, the producer suffers €3.44 income loss due to decreased carcass quality and mainte- nance feeding. When the delay increases to 10 days, the loss increases to €9.76, and when the de- lay increases to 17 days, the loss increases to €16.42 (Figure 30). The timing of the arrival of information on de- lay is important. A G R I C U L T U R A L A N D F O O D S C I E N C E When restrictions on slaughter are imposed on young pigs, the producer has more time to minimise losses though feeding than when the restrictions are imposed on pigs that are ap- proaching slaughter maturity. Therefore, income losses increase when the pigs affected by the re- strictions become larger. When restrictions on slaughter are imposed for 10 days at 111 kg live weight, income losses due to delayed slaughter are €4.21 per capacity unit. When the same restric- tions are imposed at 116 kg, the losses per capacity unit are €5.55 higher than in the 111 kg case. On the other hand, when the restrictions are imposed so that slaughter becomes feasible 3 days after the optimal slaughter date and the restrictions are im- posed at 111 kg live weight, the loss is €0.77 high- er than when the restrictions are imposed at 116 kg (Figure 30). 7.2.1 Adjusted feeding patterns when the slaughter is delayed Producers can minimise income losses due to de- layed slaughter by adjusting feeding patterns. This chapter illustrates adjusted feeding pattern using selected examples and parameter values of the benchmark scenario. In the example, the restric- tions are imposed at 99 kg live weight (21 days before the optimal slaughter date). Expected dura- tion of the restrictions is certain and it is set at ei- ther 24 or 31 days. When the pig is initially fed according to its growth potential, and slaughter is restricted, for instance, for 24 days, the producer can decrease the amount of barley and increase the share of soy meal in feed so that the feeding patterns approxi- mately follow the 24 days scenario illustrated in Figure 29. Therefore, the amount of soy meal (kg When restrictions are imposed for 24 days, the model recommends decreasing the share of soy 78 A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding 4.45 4.27 8.87 4.12 9.11 15.04 4.21 9.79 16.16 23.03 3.44 9.76 16.42 23.49 31.07 0 10 20 30 40 50 60 3 10 17 24 31 Expected duration of the delay €/animal Delay imposed at 93 kg Delay imposed at 99 kg Delay imposed at 105 kg Delay imposed at 111 kg Delay imposed at 116 kg Fig. 30 when t delayed the del Inform ceived is eithe kg or 1 allowe sibility Fig. 30. Expected income losses when the timing of slaughter is delayed and expected duration of the delay is known in advance. Information about the delay is re- ceived when the pig’s live weight is either 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the slaughter is allowed 3–31 days after the pos- sibility of a delay is observed. Delay imposed at 93 kg Delay imposed at 99 kg Delay imposed at 105 kg Delay imposed at 111 kg Delay imposed at 116 kg When the exact timing of delayed slaughter is uncertain, i.e. when the expected duration of the restriction period is uncertain, producer income losses generally increase. This holds particularly for the cases where pigs are young and the expect- ed duration of the restriction period increases. Un- certainty implies that the pig can be delivered for slaughter before or after the expected duration of the restriction period expires. Thus, when restric- tions are imposed for 10 days for instance, the pro- ducer is expected to suffer income losses in all scenarios (Figure 31). restrictions are imposed at 116 kg (slaughter de- lays with certainty). If the expected duration of the restriction period increases to 10 days, for instance in the 111 kg scenario, then the losses increase by €1.33 when uncertainty over expected duration is introduced. Thus, the cost of uncertainty increases when the probability of delayed slaughter increas- es (Figure 31). Instead of taking the risk of delayed slaughter due to logistic problems, producer has an option to market the pigs for slaughter prematurely. Then producer allows certain losses due to premature slaughter in order to avoid losses due to delayed slaughter. When a pig is slaughtered at the optimal slaughter weight, producer receives €143.67 in- come from marketing the pig for slaughter. 7.2.2 Income losses in the event of a short slaughter delay Producers suffer income losses when the timing of slaughter is exogenously delayed beyond the opti- mum. The losses increase when the expected delay increases. The results suggest that the loss due to an additional day of delay is smaller when the ex- pected delay is short than when expected delay is long. When expected duration of the restriction pe- riod is certain, information on restrictions is ob- 79 A G R I C U L T U R A L A N D F O O D S C I E N C E The ﬁgure includes income from the meat and slaugh- ter subsidy but excludes the cost of a replacement animal. In scenarios where the pig is slaughtered prematurely, returns from the slaughter are When expected duration of restrictions in- creases, or when pigs facing the restrictions be- come younger, the introduction of uncertainty over the expected duration of the restriction period in- creases the losses. For example, uncertainty in- creases losses by €0.69 per capacity unit when restrictions are imposed for 3 days at 111 kg live weight, and by €0.02 per capacity unit when the 80 0.00 0.70 3.43 7.56 12.31 0.01 1.38 5.14 10.13 15.57 0.07 2.72 7.73 13.61 19.72 0.69 5.54 11.84 18.67 25.53 3.46 10.10 17.37 24.90 32.27 0 10 20 30 40 50 60 3 10 17 24 31 Expected duration of the delay €/animal Delay imposed at 93 kg Delay imposed at 99 kg Delay imposed at 105 kg Delay imposed at 111 kg Delay imposed at 116 kg Fig. 31. Expected income losses when the timing of slaughter may be delayed and the expected du- ration of the delay is uncertain. Information about the delay is re- ceived when the pig’s live weight is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the slaughter is ex- pected to become feasible 3–31 days after being notiﬁed of the possible delay. 0.00 0.70 3.43 7.56 12.31 0.01 1.38 5.14 10.13 15.57 0.07 2.72 7.73 13.61 19.72 0.69 5.54 11.84 18.67 25.53 3.46 10.10 17.37 24.90 32.27 0 10 20 30 40 50 60 3 10 17 24 31 Expected duration of the delay €/animal Delay imposed at 93 kg Delay imposed at 99 kg Delay imposed at 105 kg Delay imposed at 111 kg Delay imposed at 116 kg 0.00 0.70 3.43 7.56 12.31 0.01 1.38 5.14 10.13 15.57 0.07 2.72 7.73 13.61 19.72 0.69 5.54 11.84 18.67 25.53 3.46 10.10 17.37 24.90 32.27 0 10 20 30 40 50 60 3 10 17 24 31 Expected duration of the delay €/animal Delay imposed at 93 kg Delay imposed at 99 kg Delay imposed at 105 kg Delay imposed at 111 kg Delay imposed at 116 kg Fig. 3 when be de ration Inform ceive is 93 116 k pecte days possib Fig. 31. Fig. 31. Expected income losses when the timing of slaughter may be delayed and the expected du- ration of the delay is uncertain. Information about the delay is re- ceived when the pig’s live weight is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the slaughter is ex- pected to become feasible 3–31 days after being notiﬁed of the possible delay. Fig. 31. Expected income losses when the timing of slaughter may be delayed and the expected du- ration of the delay is uncertain. Information about the delay is re- ceived when the pig’s live weight is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the slaughter is ex- pected to become feasible 3–31 days after being notiﬁed of the possible delay. Vol. 15 (2006): Supplement 1. €138.37 when the pig is slaughtered at 111 kg live weight (one week before the optimal slaughter date), €131.85 when the pig is slaughtered at 105 kg live weight (two weeks before the optimal slaughter date), €121.70 when the pig is slaugh- tered at 99 kg live weight (three weeks before the optimal slaughter date), and €108.94 when the pig is slaughtered at 93 kg live weight (four weeks be- fore the optimal slaughter date). discounted future returns, each additional day of the restriction period increases the losses almost linearly in the very short run. An additional week of idle production capacity increases the losses by €2.89–€2.92 in all scenarios where the expected duration is certain (Figure 32). When uncertainty about the expected duration of idle production capacity is introduced (Figure 33), the capacity unit can remain idle for a shorter, a longer or for exactly the same time than when expected duration is certain (Figure 32). Introduc- ing uncertainty over the expected duration of the idle production capacity decreases producer’s ex- pected income losses because he/she can then ben- eﬁt from a possibility to restock the capacity unit before the expected duration expires. When ex- pected duration is 17 days or fewer, the difference between deterministic and stochastic scenarios is less than €0.10 per capacity unit. Introducing un- certainty over the expected duration of idle capac- ity decreases expected losses in the 24 days sce- nario by €0.45 per capacity unit, and in the 31 days scenario by €1.45 per capacity unit. Increas- ing the expected duration of idle production capac- ity from 3 days to 10 days increases the losses by €2.91 per capacity unit. Thereafter, the value of each additional week decreases so that increasing expected duration for instance from 24 to 31 days increases the losses only by €1.89 per capacity unit. A prematurely slaughtered carcass is lighter than a carcass slaughtered at the optimal slaughter weight. In addition, the producer obtains more in- come from subsequent pigs when a new piglet is obtained immediately after the slaughter than when a new piglet is obtained after lifting the re- strictions. When the producer slaughters the pig prematurely at 111 kg live weight and obtains a new piglet immediately, his/her income loss is €0.36 per capacity unit. Similarly, slaughter at 105 kg live weight results in €1.11 income loss. A G R I C U L T U R A L A N D F O O D S C I E N C E Expected income losses when the timing of slaughter may be delayed and the expected du- ration of the delay is uncertain. Information about the delay is re- ceived when the pig’s live weight is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the slaughter is ex- pected to become feasible 3–31 days after being notiﬁed of the possible delay. 80 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. A G R I C U L T U R A L A N D F O O D S C I E N C E Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 13.42 16.33 19.19 21.70 23.60 6.38 9.29 12.15 14.66 16.56 1.93 4.85 7.70 10.22 12.11 1.18 4.09 6.95 9.47 11.36 0.81 3.72 6.58 9.10 10.99 0 10 20 30 40 50 60 3 10 17 24 31 Expected duration of idle capacity €/animal Slaughter at 93 kg Slaughter at 99 kg Slaughter at 105 kg Slaughter at 111 kg Slaughter at 116 kg Fig. 33. Expec when the pig maturely and piglet is acqui lay. The expec idle production tain. Live weig slaughter is 93 111 kg or 116 ed duration o capacity is 1–3 13.42 16.33 19.19 21.70 23.60 6.38 9.29 12.15 14.66 16.56 1.93 4.85 7.70 10.22 12.11 1.18 4.09 6.95 9.47 11.36 0.81 3.72 6.58 9.10 10.99 0 10 20 30 40 50 60 3 10 17 24 31 Expected duration of idle capacity €/animal Fig. 33. Expected income losses when the pig is slaughtered pre- maturely and the replacement piglet is acquired only after a de- lay. The expected duration of the idle production capacity is uncer- tain. Live weight upon premature slaughter is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the expect- ed duration of idle production capacity is 1–31 days. 10 17 24 Expected duration of idle capacity For example, when the restrictions are imposed at 93 kg live weight and the expected duration of the restriction period increases from 47 to 52 days, producer income losses increase by €3.37 (€0.67 per additional day) per capacity unit. A similar in- crease in expected duration of the restrictions at 116 kg live weight increases the losses by €4.27 (€0.85 per additional day). On the other hand, when the restrictions are imposed at 93 kg live weight and the expected duration of the restriction period increases from 52 to 59 days, the losses in- crease by €5.09 (€0.73 per additional day) (Fig- ure 34). 7.2.3 Income losses in the event of a long slaughter delay The effect of uncertainty on producer’s income losses is important in the event of a long restriction period. When animal disease regulations prohibit transport of animals for slaughter for several weeks, producer’s income losses increase signiﬁ- cantly compared to losses with shorter delays. When the expected duration of the restrictions im- posed on the timing of slaughter increases by one day, the producer’s expected income losses in- crease by €0.67–0.85 per additional day. The cost effect of each additional day increases when the pig’s live weight increases, and decreases when the expected duration of the restriction period in- creases. The losses due to delayed slaughter are signiﬁ- cantly related to the fact that a producer can mini- mise a decrease in carcass quality by adjusting feeding. The losses increase if the feeding technol- ogy used on the farm does not allow adjustment of 12.56 15.93 21.02 26.02 17.24 20.88 26.31 31.57 22.58 26.53 32.24 37.70 28.91 33.08 38.98 44.55 34.99 39.26 45.24 50.88 0 10 20 30 40 50 60 47 52 59 66 Expected duration of the delay €/animal Delay imposed at 93 kg Delay imposed at 99 kg Delay imposed at 105 kg Delay imposed at 111 kg Delay imposed at 116 kg 12.56 15.93 21.02 26.02 17.24 20.88 26.31 31.57 22.58 26.53 32.24 37.70 28.91 33.08 38.98 44.55 34.99 39.26 45.24 50.88 0 10 20 30 40 50 60 47 52 59 66 Expected duration of the delay €/animal Delay imposed at 93 kg Delay imposed at 99 kg Delay imposed at 105 kg Delay imposed at 111 kg Delay imposed at 116 kg Fig. 34. Expected income losses when the timing of culling can be delayed and the expected dura tion of the delay is uncertain, bu at least 45 days. Information about the delay is observed when the pig’s live weight is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and slaughter is expected to become feasible 45–66 days after the ob serving the potential for delay. 82 12.56 15.93 21.02 26.02 17.24 20.88 26.31 31.57 22.58 26.53 32.24 37.70 28.91 33.08 38.98 44.55 34.99 39.26 45.24 50.88 0 10 20 30 40 50 60 47 52 59 66 Expected duration of the delay €/animal Delay imposed at 93 kg Delay imposed at 99 kg Delay imposed at 105 kg Delay imposed at 111 kg Delay imposed at 116 kg Fig. 34. Vol. 15 (2006): Supplement 1. The quality-adjusted price of pig meat decreases rapidly when the weight of a slaughtered pig falls below the target weight range. Therefore, estimat- ed income loss from slaughter at 99 kg is €5.55 and at 93 kg it is €12.59. When a producer slaughters a pig prematurely and the capacity unit remains idle until the restric- tions imposed on slaughter are lifted (i.e. piglet supply is ﬁxed), his/her income loss is the higher the longer the expected duration of the restriction period is. Since additional losses depend only on 81 13.42 16.33 19.25 22.15 25.05 6.38 9.29 12.21 15.11 18.01 1.93 4.85 7.76 10.67 13.57 1.18 4.10 7.01 9.92 12.82 0.81 3.73 6.64 9.55 12.45 0 10 20 30 40 50 60 3 10 17 24 31 Expected duration of idle capacity €/animal Slaughter at 93 kg Slaughter at 99 kg Slaughter at 105 kg Slaughter at 111 kg Slaughter at 116 kg Fig. 32. Expected income losses when a pig is slaughtered prema- turely and a replacement piglet is acquired only after a delay. The expected duration of the idle pro- duction capacity is known in ad- vance. Live weight upon prema- ture slaughter is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the ex- pected duration of idle produc- tion capacity is 3–31 days. 13.42 16.33 19.25 22.15 25.05 6.38 9.29 12.21 15.11 18.01 1.93 4.85 7.76 10.67 13.57 1.18 4.10 7.01 9.92 12.82 0.81 3.73 6.64 9.55 12.45 0 10 20 30 40 50 60 3 10 17 24 31 Expected duration of idle capacity €/animal Slaughter at 93 kg Slaughter at 99 kg Slaughter at 105 kg Slaughter at 111 kg Slaughter at 116 kg Fig. 32. Expected income losses when a pig is slaughtered prema- turely and a replacement piglet is acquired only after a delay. The expected duration of the idle pro- duction capacity is known in ad- vance. Live weight upon prema- ture slaughter is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the ex- pected duration of idle produc- tion capacity is 3–31 days. Expected duration of idle capacity 81 81 Vol. 15 (2006): Supplement 1. feeding or if the adjustments are costly. In the 99 kg 24 days scenario, for instance, the adjustments have the potential to reduce the losses from €6.85 to €4.27 per capacity unit. In other words, adjust- ments have the potential to decrease the losses by 38%, compared to the situation where producer has no option to adjust feeding. This result is based on an additional simulation where producer fol- lows the same feeding pattern as he/she would have followed if no restriction on slaughter were imposed. For the time after optimal slaughter, the feeding pattern remains the same as immediately before the optimal slaughter date. The adjustments decrease price discounts due to excess carcass weight. Thus, relative beneﬁts of restricted feeding can be quite large when producer has an option to control the value of a carcass. to the delay by adjusting feeding patterns accord- ing to the expected duration of the delay. The ad- justment minimises price discounts due to excess carcass weight and may help to reduce decrease in carcass fatness. If producer can foresee the delay well before it realises and the slaughter moment and the delay is just few days, then the delay has little impact on the optimal feeding patterns. The impact that a delay has on feeding patterns and the value function can be large if information on the delay is obtained close to the optimal unrestricted slaughter timing. On the other hand, if the delivery schedule is ﬁxed such that slaughter is allowed e.g. every 7 days, then the model usually recommends that the growth rates of fatty tissue be adjusted so that the slaughter weight at the ﬁxed date is close to the optimum of the ﬂexible delivery schedule, to reduce slaughter weight a little, or both. When the animals are slaughtered prematurely in the response to animal disease restrictions (Fig- ure 35), a large share of the losses is due to the large expected duration of idle production capacity (cf. Figure 33 vs. Figure 35). Even if the low value of a carcass contributes losses, relative differences in losses of alternative expected duration and live weight scenarios are smaller in Figure 35 than in Figure 33 (slaughterhouse logistics problem). When the timing of slaughter is delayed for a short time, the producer reduces the amount of en- ergy in feed in order to decrease the growth rate of fatty tissue. Vol. 15 (2006): Supplement 1. Simultaneously, the share of soy meal in feed increases if producer initially has incen- tives to feed the pig according to its growth poten- tial and if the pig can be slaughtered close to the target weight range. This result is due to the fact that when the pig is fed according to growth poten- tial, the initial amount of energy feed is higher than when restricted feeding is initially applied. Feed- ing adjustments decrease the daily weight gain and thus minimise the meat price discounts due to the fact that the slaughter weight exceeds the target weight and due to the low share of red meat in car- cass. 7.2.3 Income losses in the event of a long slaughter delay Expected income losses when the timing of culling can be delayed and the expected dura- tion of the delay is uncertain, but at least 45 days. Information about the delay is observed when the pig’s live weight is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and slaughter is expected to become feasible 45–66 days after the ob- serving the potential for delay. Fig. 34. Expected income losses when the timing of culling can be delayed and the expected dura- tion of the delay is uncertain, but at least 45 days. Information about the delay is observed when the pig’s live weight is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and slaughter is expected to become feasible 45–66 days after the ob- serving the potential for delay. Fig. 34. Expected income losses when the timing of culling can be delayed and the expected dura- tion of the delay is uncertain, but at least 45 days. Information about the delay is observed when the pig’s live weight is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and slaughter is expected to become feasible 45–66 days after the ob- serving the potential for delay. Expected duration of the delay 82 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs When expected duration of the delay increases or when animals under slaughter restrictions ap- proach the optimal slaughter time, the share of soy meal in feed increases. Since both daily gain of live weight and growth of lean tissue then de- crease, the adjustment minimises price discounts due to heavy carcass and losses due to costly pro- tein feeding. The degree of restricting is con- strained by the minimum fat to lean growth ratio and a minimum daily weight gain of 0.5 kg. Hence, inﬂexibility of feeding technology constrains feed- ing decisions. ration of the idle capacity is certain, the losses range from €0.81 to €25.05 per capacity unit. When uncertainty over the expected duration is in- troduced, the losses for similar scenarios range from €0.81 to €23.60 per capacity unit. Finally, when animal disease restrictions are imposed at 93–116 kg live weight, such that the capacity unit remains idle for 47–66 days, the losses range from €19.05 to €37.71 per capacity unit. Losses due to premature slaughter increase as prematurely slaughtered animals are younger. Losses increase also as the expected duration of idle capacity unit increases. Therefore, a ﬁxed or restricted piglet supply increases producer’s in- come losses. If the optimal slaughter is not feasible (or is unlikely) upon a possibility for a premature slaughter, then an option for premature slaughter can have positive value. Even if ﬂexible feeding technology provides potential to reduce producer income losses due to exogenously delayed timing of slaughter, the re- maining losses are still considerable. When restric- tions are imposed at 93–116 kg live weight for 3– 31 days, and the expected duration of the restric- tion period is certain, the losses range from zero to €31.07. When uncertainty over the expected dura- tion is introduced, the losses for similar scenarios range from zero to € 32.27 per capacity unit. Fi- nally, when animal disease restrictions are imposed at 93–116 kg live weight for 47–66 days, the losses due to maintenance feeding range from €12.56 to €50.88 per capacity unit. Since differences in expected losses between delayed slaughter and premature slaughter scenar- ios are considerable, even a small probability that slaughter will be delayed is able to provide incen- tives for premature slaughter. 7.3 Summary If slaughter of a pig is delayed beyond the opti- mum, then producer can minimise the losses due 31.66 33.72 36.35 37.71 24.62 26.68 29.31 30.67 20.18 22.24 24.86 26.23 19.42 21.48 24.11 25.48 19.05 21.11 23.74 25.11 0 10 20 30 40 50 60 47 52 59 66 Expected duration of idle capacity €/animal Slaughter at 93 kg Slaughter at 99 kg Slaughter at 105 kg Slaughter at 111 kg Slaughter at 116 kg 31.66 33.72 36.35 37.71 24.62 26.68 29.31 30.67 20.18 22.24 24.86 26.23 19.42 21.48 24.11 25.48 19.05 21.11 23.74 25.11 0 10 20 30 40 50 60 47 52 59 66 Expected duration of idle capacity €/animal Slaughter at 93 kg Slaughter at 99 kg Slaughter at 105 kg Slaughter at 111 kg Slaughter at 116 kg 31.66 33.72 36.35 37.71 24.62 26.68 29.31 30.67 20.18 22.24 24.86 26.23 19.42 21.48 24.11 25.48 19.05 21.11 23.74 25.11 0 10 20 30 40 50 60 47 52 59 66 Expected duration of idle capacity €/animal Slaughter at 93 kg Slaughter at 99 kg Slaughter at 105 kg Slaughter at 111 kg Slaughter at 116 kg Fig. 35. Expected income losses when the pig is slaughtered pre- maturely and the replacement piglet is acquired only after a de- lay. Expected duration of idle production capacity is uncertain, but at least 45 days. Live weight upon premature slaughter is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the expected dura- tion of idle production capacity is 47–66 days. Fig. 35. Expected income losses when the pig is slaughtered pre- maturely and the replacement piglet is acquired only after a de- lay. Expected duration of idle production capacity is uncertain, but at least 45 days. Live weight upon premature slaughter is 93 kg, 99 kg, 105 kg, 111 kg or 116 kg, and the expected dura- tion of idle production capacity is 47–66 days. Expected duration of idle capacity 83 8.1 Management implications The results suggest that pig meat producers have substantial opportunities to increase returns from pig farming by improving and utilising genetic production potential of their stock. In order to fully utilise this production potential, producer must ad- just feeding and slaughter decisions particularly with respect to genotype of an animal, maturity stage, and market conditions. Hence, he/she can beneﬁt from increased ﬂexibility of feeding tech- nology. Previous studies support this view (Glen 1983, Chavas et al. 1985, Boland et al. 1999, Cam- pos 2003). The analysis is carried out with a structural- form optimisation model that characterises the growth mechanism of a pig. In other words, the focus is on its nutritional requirements. It should be noted that the result of this study give informa- tion on the mechanism of adjusting feeding and slaughter patterns in cases where producer knows the state of nature and genotype of a pig (i.e. pro- duction technology). The implications of uncer- tainty and biological variability in pig management have been explored in detail in previous studies (see e.g. Broekmans 1992, Burt 1993, Jorgensen 1993, Boland et al. 1996, Kure 1997, Roemen and de Klein 2000, White et al. 2004). The beneﬁts of precision feeding arise from the ability of technological ﬂexibility to avoid wasting nutrients while still fully utilising the economic potential of the genotype. The beneﬁts can be par- ticularly large if the initial technology is very in- ﬂexible, such as one-phase feeding. Another criti- cal factor that affects the beneﬁts is the extent to which ﬂexible feeding technology improves pro- ducer options to control carcass quality. If im- provements are large (small) then beneﬁts can also be large (small). The aggregate returns at the farm level are quite large despite the fact that individual returns per animal can be small. Therefore, addi- tional returns obtained from using a ﬂexible feed- ing technology can sufﬁce to cover the cost of in- troducing the precision feeding technology. If The conclusions focus on where the results give best information, viz. comparing the results between scenarios and understanding the effects that changes in the parameters of the mechanical growth model have on the outcomes. The results can be used to compare and rank reported scenari- os despite the fact that the value function estimated for a period of 1800 days underestimates the true value of a capacity unit over a longer time period. 8 Conclusions and discussion This chapter draws general conclusions focusing ﬁrst on herd management aspects and then on the policy implications of the results. The contribution of this study to the literature of pig management is that the analysis takes into account simultaneity of feeding and slaughter decisions, which are tradi- tionally separated in economic analyses, and links them explicitly to carcass quality. In addition to having an option to control composition and amount of feed, the model allows producer to con- trol carcass quality. Hence, it focuses on character- ising production technology. The issue is econom- ically important, because carcass quality deter- mines quality price premiums paid for a carcass and it can therefore have large impact on returns to capacity unit. p. 66). In individual scenarios, the value function and management patterns should be evaluated conditional on the underlying data and on the fact that in the model producer can control feeding and slaughter according to pig’s genotype. These data give information on the markets, interest rate, planning horizon, production technology, geno- type and other factors that are taken into account in the analysis and that affect the results. A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs In the event of a slaughterhouse logistics problem, such that ex- pected duration of the restriction period is certain, the results of 111 kg scenario provide producer in- centives to slaughter the pigs prematurely if the idleness of capacity unit is expected to last at least 10 days. The same applies in the 105 kg and 31 days scenario. Premature slaughter at 105 kg mini- mises losses even when expected duration of the restriction period is 17 days, provided that the ca- pacity unit is restocked immediately. When ex- pected duration of the restriction period is uncer- tain, incentives for premature slaughter increase, particularly in the 99 kg, 105 kg and 111 kg sce- narios. The losses increase rapidly when information on delay of the timing of slaughter is provided closer to the slaughter optimal moment. Similarly, the losses increase when expected duration of the delay increases, and when uncertainty over ex- pected duration of the restriction period is intro- duced. In such cases, the losses can be very large or quite small depending on whether slaughter is allowed before the expected duration expires. In all scenarios, the major determinant for the losses is that quality-adjusted value of pig meat decreases when pigs become overweight. In the event of an animal disease related re- striction on the timing of slaughter, producer has incentives to market the pigs prematurely when restrictions are imposed on pigs of 105 kg live weight or heavier. Also when the restrictions are imposed at 99 kg live weight and expected dura- tion of the restriction period is 66 days, there are incentives for premature slaughter. A producer is able to avoid delayed slaughter when he/she has an option to slaughter the pigs prematurely. The value of a prematurely slaugh- tered carcass is lower than the value of an opti- mally slaughtered carcass. When the pigs are slaughtered at 93–116 kg live weight, the capacity unit remains idle for 3–31 days, and expected du- 84 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs just feeding and slaughter patterns accordingly. Comparison with the studies of Hansen (1992) and Parsons et al. (2004) suggests that this approach can give an optimistic view on how accurately pro- ducer can observe genotypes of animals and how he/she can reach desired proﬁt-maximising carcass composition through continuous adjustment of feeding. The model used in this study eliminates extreme carcass quality control options by allow- ing producer not to feed very large amounts of feed or diets having very high protein content. At 110 kg live weight the difference in carcass lean- ness between pigs fed with protein-rich feed and energy-rich feed in this study is therefore 2.9%. In the study of Parsons et al. (2004), the difference in carcass leanness at 110 kg live weight between pigs fed with protein-rich feed and energy-rich feed is approximately 2.2%. The results are in line with the results of Affentranger et al. (1996), Ram- aekers et al. (1996) and Quiniou et al. (1999), who observed restricted energy feeding to be able to in- crease the share of lean meat tissue in carcass by 2-3% when compared to energy-rich diets, such as ad libitum feeding. technological options for precision feeding are available at a cost-efﬁcient price, producers should consider them as an option to conventional tech- nologies. When examined according to the maturity stage of a pig of given genotype, the optimal preci- sion feeding pattern is generally in line with the results of previous studies (Dent et al. 1970, Sonka et al. 1976, Glen 1983, Chavas et al. 1985). The degree of adjustment during the fattening period is, however, larger in this study than in similar studies. Chavas et al. (1985), for instance, estimat- ed that the share of protein in feed remains con- stant until the ﬁnishing period, after which it de- creases linearly, whereas in this study, the share of protein in feed decreases throughout the fattening period. In addition, Chavas et al. (1985) observed no decrease in the amount of energy-rich feed at the end of the fattening period. These differences are due to differences in feed characteristics and functional speciﬁcations, and due to the fact that this study splits animal’s live weight into fatty and lean tissue. Animal’s genotype may also have driv- en a wedge between the results of the two studies. 8.1 Management implications The validity of the comparison is due to the re- quirement that benchmark scenario and other sce- narios challenging it should be compared with the same units of measure (Robinson and Barry 1996, 85 Vol. 15 (2006): Supplement 1. breeding programmes, then the results encourage producers to focus on improving mature weight rather than maturing rate. Even if carcass quality matters, the beneﬁts of improved genotypes seem to relate to the growth rates. This is due to the fact that the difference between increasing the mature weight and the maturing rate is in the way that the daily growth rates increase. It could also be beneﬁ- cial to aim at improving simultaneously both ma- ture weight and maturing rate. Simultaneous in- creases in both properties could lead to piglets having high growth rates and the continuation of the high lean growth on pigs approaching slaugh- ter. This could allow producer to gain synergetic beneﬁts from both improvements. In fact, Emmans and Kyriazakis (1999) suggest that animal breed- ing programmes aiming at leaner pigs often result in improvements in both mature weight and matur- ing rate of lean tissue. to group pigs so that pigs in a pen have no large differences in their growth potential and that the pigs can maintain carcass leanness around slaugh- ter weight. It is also likely that a group of pigs should follow the same feeding regimen as the animal that has the highest lean growth potential, because otherwise the genetic potential of these animals might not be fully utilised. In order to reach this conclusion, producer should have sufﬁ- cient information on genotypes of pigs, genotypes should be similar enough, and he/she should pro- vide pigs an environment in which restricted feed- ing has insigniﬁcant impact on their behaviour. In two-phase feeding, the composition of the diet is adjusted discretely. This can result either in decreased daily weight gain or in supplying nutri- ents that cannot be utilised. The major technologi- cal advantage of precision feeding is that it can avoid such large changes in the composition of the diet. Therefore, genotype constraints that control the quality of carcass are more restricting under two-phase feeding than under a ﬂexible feeding technology, and precision feeding can provide a producer with improved tools to adjust manage- ment patterns to changing meat markets and ani- mal qualities. Vol. 15 (2006): Supplement 1. Although the optimal management patterns de- pend on the genotype of an animal and the options available to producers to monitor and control growth, the results suggest that revisions in feed- ing recommendations to increase the amount of lysine in the ﬁrst phase of three-phase feeding, and to expand switching the feed ration at no sooner than 55 kg live weight are economically justiﬁed (cf. Tuori et al. 2002 vs. MTT 2004). Nevertheless, the results reported here suggest that the producer can obtain higher returns per capacity unit per year when further adjusting feed composition and by continuing the protein-rich diet until 65–75 kg live weight. If the phase is switched within this weight range, the value of a capacity unit is quite stable. This could be due to the fact that the inﬂection point of the Gompertz function is within this range. Another explanation could be that energetic efﬁ- ciency of feed is almost linear in the feasible re- gion (cf. Black 1995, Emmans and Kyriazakis 1995). Boland et al. (1999) also noted that the rev- enue curves are ﬂat near the optimum. The results suggest that improvements in ani- mal’s genotype can contribute large economic beneﬁts to the capacity unit. This result is in line with the ﬁndings of Chavas et al. (1985), Boland et al. (1993) and Pietola and Sévon-Aimonen (2002). Even if the price of pig meat affects the value of genetic improvements, relative returns to improved genotypes are quite independent of the price of pig meat. The beneﬁts of genetic improvements are related to the fact that increased growth rates in- crease slaughter density and decrease the amount of feed required to produce a kilogram of pig meat. Above all, genetic improvements that increase lean growth can provide value added. This is in line with the results of earlier studies (Boland et al. 1993, Boland et al. 1996), although these ﬁndings emphasise the amount of feed required for a kilo- gram of lean meat more strongly than those of pre- vious studies. These results support the observation by Boland et al. (1999) that the maximisation of weight gain only is not a valid criterion for choos- ing the optimal management practices in pig fat- tening. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs The optimal management solutions can have important practical implications, because the mod- el can explicitly link feed components to carcass composition and meat quality. Application of feed- ing and slaughter patterns presented in this study requires that producer has sufﬁcient information on the state of nature and genotype of a pig (i.e. the ability to monitor pigs well) and that he/she can control its growth. The general principles of pro- tein and energy feeding are applicable regardless of whether premix or other feeds are used instead of soy meal, barley or amino acid supplements. In- dependent of genotypes, the optimal feed ration for young pigs is rich in protein. When pigs ap- proach their optimal slaughter maturity, the opti- mal daily allowance of both energy and feed bulk increases, and the share of protein in feed decreas- es. In contrast with some previous studies, this study emphasises that obtaining full beneﬁts from genetic improvements requires adjusting the feed- ing patterns according to genotype. The results suggest that pigs having a genetic tendency to high growth rates of lean tissue should be fed with a feed that is richer in protein than the feed of pigs having tendency to high growth rates of fatty tis- sue. The segregation between genotypes can con- tinue until late stages of growth because pigs hav- ing high mature weight of lean tissue have poten- tial to utilise large amounts of protein-rich feed until very late stages of the fattening period. Kure (1997) and Boland et al. (1999) also observed that segregated management is at least as economical as pooled pig management. Returns to segregated feeding increase when the genetic differences between the growth rates of lean tissue of animals increase. This is due to the fact that producers can avoid quality-adjusted price discounts due to the carcass weight and carcass leanness, particularly when genetic differences are large. Therefore, in pooled feeding it is important This study supports the view that feeding af- fects carcass leanness even if the pig’s genotype largely determines differences in leanness. The analysis focuses on cases where producer can ob- serve genotype and state of nature of a pig and ad- 86 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs study. Sipiläinen and Ryhänen (1996) also come to the same conclusion. The problem is related to the fact that producer’s incentives to enhance output quality increase when either the proﬁtability of marketing a ﬁxed quantity of output decreases or the proﬁtability of high marketing frequency de- creases. This is the case when the price of output quantity decreases, the price of output quality in- creases, or the production cost of high quality out- put decreases. In other words, in order to produce leaner meat, the producer must decrease the daily weight gain below the potential by reducing the growth of fatty tissue. This requires increasing the share of soy meal in feed in order to maintain the amount of protein in feed. Thus, less meat is pro- duced per day. that the producer can optimise production by re- placing an input, which price increases, with other inputs. The substitution effect is larger when the price of a single input increases than when the price of a group of inputs, such as all feeds, in- creases. In contrast to Kure (1997), who found little ﬁ- nancial leeway for taking on-farm measures to op- timise carcass leanness, this study suggests that the beneﬁts of optimising carcass leanness can be quite large, particularly under suitable market con- ditions. One explanation for this could be that this study emphasises the importance of managerial ef- forts to utilise improved growth rates and carcass quality premiums while allowing considerable ﬂexibility in feeding technology. Methodological differences may also have driven a wedge between the results of the two studies. This study used known biological characteristics along with opti- mised feeding and slaughter patterns of a pig, whereas Kure (1997) included biological variation of pigs into his model and optimised marketing de- cisions for a group of pigs. Our results suggest that the choice between restricted and unrestricted en- ergy feeding is particularly critical for the price of red meat with respect to the price of pig meat. When this price ratio falls below 0.013 in the benchmark scenario, the proﬁtability of producing lean meat quickly decreases, and hence maximis- ing production volumes can be more important than the quality of pig meat. Also large ﬂat regions in the quality-adjusted price scheme provide in- centives to maximise daily weight gain. Vol. 15 (2006): Supplement 1. This is due to the fact that restricted feed- ing is generally the optimal feeding pattern in this If measures to increase mature weight and ma- turing rate both carry the same cost in animal 87 Vol. 15 (2006): Supplement 1. producer incentives to invest in new production capacity (i.e. it decreases returns to cover the in- vestment cost), because the producer has no obli- gation to produce pig meat if he/she desires to have the decoupled payment. As a result, the net income that depends on the number of capacity units de- creases signiﬁcantly. Therefore, decoupling can increase the number of producers that do not invest in new production capacity but exit the industry, and thus decoupling can decrease the amount of pig meat produced in Finland in the long run. On the other hand, when the production capacity is given or when the payment is dependent on the ca- pacity unit (e.g. when investments are subsidised), decoupling can have only a small impact on pro- duction capacity. Then decoupling can even in- crease producer income, because he/she can ex- tract additional income from adjusting meat qual- ity and still obtain a decoupled income transfer as long as the income from markets exceeds the vari- able cost of production (cf. the exit criterion by Ross (1980)). expected delay will be extended, can result that premature slaughter will maximise the value of a capacity unit. Thus, the optimal delivery policy is affected by risk factors that can reduce slaughter income, such as the risk that the slaughterhouse might be late in transporting the animals. The gen- eral result regarding marketing income risk is in line with that reported for various risk manage- ment problems (e.g. Broekmans 1992, Toft et al. 2005). As the option of premature marketing may be unavailable, the results suggest an optional way to minimise losses due to maintenance feeding. This option, which previous studies do not examine, is to reduce the amount of energy in feed immedi- ately after obtaining the information on the delay. Thus, when producer can control carcass quality, he/she can minimise income losses from excess carcass weight and decreasing carcass leanness. In addition, if excess weight gain is sufﬁciently large (i.e. the delay is long), then he/she minimises the weight gain and the losses due to costly mainte- nance and protein feeding. The results suggest that the slaughter premium has a rather small impact on output per capacity unit as long as the producer has no incentive to change his/her behaviour by adjusting the length of the fattening period. Vol. 15 (2006): Supplement 1. Although a slaughter pre- mium fulﬁls the conditions required for a lump sum transfer (for details, see Mas-Colell et al. 1995, p. 524) better than a price support, an in- crease in slaughter premium increases producer incomes inelastically. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Output quality is at the core of the logic behind adjusting feeding and slaughter decisions to suit market conditions. The outcome is that elasticity estimates of the length of the fattening period and of the annual volume of meat production correlate negatively and they can have widely different val- ues. Our elasticity estimates have signs similar to those of Chavas et al. (1985) and Boland et al. (1993). Even if the producer responds inelastically to changes in most input and output prices, the re- sponses here are generally more elastic than those reported in previous studies. This is due to the fact that the connection between decomposition of feed and decomposition of carcass quality is made ex- plicit in this study but not in all previous studies. Also the fact that the state and genotype of a pig are known in this study, and the fact that there are differences between studies in costs that are taken into account may have increased responses. Even if carcass leanness matters, our results clearly show that the optimal timing of slaughter is when carcass weight is close to the upper limit of the target weight range. Hence, the optimal slaugh- ter weight is quite ﬁxed. Several studies share this insight (Jolly et al. 1980, Ross 1980, Kure 1997). The comparison above holds particularly for the length of the fattening period and for manage- ment responses with respect to the price of pig meat. Chavas et al. (1985), for instance, report that management patterns respond more elastically than in this study only in response to an increase in the price of piglet. Low management response with respect to the price of piglet in this study could be due to the impact of the minimum fat-to- lean growth rate constraint and the meat pricing scheme. Elasticity estimates also show commonly accepted input substitution effects. This implies An exception to the slaughter rule becomes rel- evant when producer cannot fully control for the timing of slaughter of an individual animal. When there is a risk that slaughter will be delayed be- yond the optimum, producer has incentives to slaughter prematurely the pigs that are within the target weight range. For such animals, even a small probability (>5%) of delayed slaughter, or that an 88 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs leanness of carcasses compared to other options. From the pig meat producer’s point of view, the decrease in the growth potential or leanness (cete- ris paribus) cannot be very large because, as Cha- vas et al. (1985) and Boland et al. (1999) already noted, the technological and economic possibili- ties of animal genetics are very large. and synthetic amino acids for soy meal. From the economic point of view, such regulations will re- sult in reduced demand for domestic products when some consumers shift to less expensive for- eign products. Thus, regulations that increase pro- duction costs in Finland decrease the competitive- ness of the Finnish pig meat industry and increase pig meat imports, unless producers can obtain val- ue added from the markets. The results suggest that quality adjustments of the price of pig meat provide slaughterhouses with an efﬁcient tool to control carcass quality. This ﬁnding is due to the fact that whenever a carcass weight-based price discount exists, it is dominant in optimising slaughter weights. Boland et al. (1993), Sipiläinen and Ryhänen (1996) and Kure (1997) also show that meat pricing affects the slaughter weights. In contrast to previous studies, this study suggests that the most effective way to control the supply of pig meat is to adjust simulta- neously both the target weight and price premium paid for red meat. This is so because as the produc- er’s incentives to increase carcass leanness in- crease, the length of the fattening period increases, and the annual production volume of pig meat per capacity unit also increases. Particularly, produc- ers who introduce a ﬂexible feeding technology have increased options to control carcass quality. Hence, the efﬁciency of lean meat pricing in con- trolling meat quantities depends how ﬂexible pro- ducers are in controlling carcass quality of a pig. Full utilisation of quality-adjusted pricing schemes also requires that both producers and slaughter- houses have sufﬁcient and symmetric information on how incomes and pig management respond to changes in quality premiums. The ﬁndings of this study suggest that efforts made to increase the competitiveness of pig meat production, particularly by increasing the techno- logical productivity of pig fattening, provide value added. For instance, 10% increase in the K-index of a pig increases the value of a capacity init by approximately the same percentage. 8.2 Policy implications The results support the view that changes in input prices and output prices have large income effects to producers. Several studies share this result (Chavas et al. 1985, Boland et al 1993, Sipiläinen and Ryhänen 1996, Kure 1997, Boland et al. 1999). These results join those of prior research in show- ing that providing a producer with the option of dynamically adjusting management decisions can result in higher management response elasticities and lower income elasticities with respect to price changes than when no such option exists. The elas- ticity estimates imply that depressions and peaks in the price of pig meat have a particularly strong impact on producer income. Therefore, producers and policy makers should extract value added from the markets whenever it is available. Another important policy issue arises in con- nection with regulations that prohibit producers from using certain inputs. If domestic regulations prohibit the use of genetically modiﬁed soy meal and foreign regulations do not, domestic producers have to pay a higher input price for soy feed than some of their competitors. The results suggest that pig meat producers as a group suffer approximate- ly €2 million per year (€0.01 per kilogram of pig meat) in higher production costs when using ge- netically non-modiﬁed feed instead of less expen- sive modiﬁed feed. Hence, farms having high in- put prices suffer from this competitive disadvan- tage when compared to farms beneﬁting from low input prices even though they can substitute barley The results suggest that completely decoupling the slaughter premium from production decreases 89 Vol. 15 (2006): Supplement 1. stead of continuing. Furthermore, as Meuwissen (1999) and Meuwissen et al. (2003) note, the groups which are responsible for actions and their consequences should be agreed before the emer- gency situation occurs in order to avoid misunder- standings and actions due to missing or imperfect information. meat sold to slaughterhouses. The rules could in- clude deﬁnition of the date at which pigs are col- lected for slaughter if the producer markets them today. In the event of contagious animal diseases, transporting animals can reduce the effectiveness of a disease eradication policy if the transports in- crease the risk of additional infections. Because the expected delay of the timing of slaughter can be very lengthy, even a small probability of a delay can provide the producer with incentives to mini- mise potential costs. In infection areas, premature slaughter implies either a moral hazard problem or that producer suspects that animal movement re- strictions will be imposed on his/her farm. In such circumstances, the producer can increase society’s expected losses by marketing the animals in ad- vance, before the restrictions are imposed. Kuchler and Hamm (2000) and Toft et al. (2005) also have suggested, although in a different context, that management patterns are correlated with animal disease control policies. Hence, any compensation independent of whether it is obtained from the markets or from the government should be condi- tional on non-infectious management practices. Thus the compensation scheme should provide the producer sufﬁcient incentives to minimise the probability of his/her herd being infected. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Hence, the average rate of genetic progress raises the quality- adjusted value of a capacity unit (ceteris paribus) by almost 6% per year. On the other hand, assum- ing that the total number of capacity units with im- proved pigs is 600,000, improving the daily weight gain potential by 100 g at ﬁxed prices, and equals approximately €150 million per year in additional returns to Finnish pig meat producers as a group. Even if this large beneﬁt requires adjustments in both genetics and management, the beneﬁts seem to exceed the costs. The advantage of these tech- nologies is that they provide more or less perma- nent improvements in the efﬁciency of input use whereas the breeding effort is required only once (although breeding takes several years). These technologies, however, provide only a temporary competitive advantage to producers because pro- ducers with high costs can obtain the same tech- nology over time. Thus, maintaining the competi- tive advantage requires continuous effort. Since the producer cannot always fully control the timing of slaughter, failures to optimise the timing of slaughter can reduce the performance of quality-adjusted pricing schemes. This is due to the fact that the producer can minimise income losses due to delayed slaughter by marketing the animals prematurely. The result holds particularly when pigs are within the target weight range and producer cannot reduce growth rates enough through feeding. Therefore, it is important to have well deﬁned rules for the timing of collection of animals for slaughter. Such rules can provide pro- ducer higher income and enhance the quality of One of the options to improve the genotype of pigs is to improve the productivity of sows. Al- though this study focuses on pig fattening, some implications relate to piglet production. For in- stance, if it is possible to decrease the price of a piglet by increasing the number of weaned piglets per sow, then pig meat producers can gain a com- petitive advantage from having access to less ex- pensive inputs. Such an increase can be proﬁtable even if decreasing the piglet price implies minor reductions in the daily growth potential of pigs or 90 8.3 Suggestions for further research The approach used in this study allows us to solve numerically a general dynamic programming problem that cannot be solved analytically. The postulated model takes into account the dynamic and simultaneous nature of pig meat production as suggested by Chavas et al. (1985). It maximises internal rate of return to the capacity unit and al- lows producer to adjust production according to the current pig quality. One major advantage of the structural-form model presented here is that it can explicitly link feed components with carcass com- position and meat quality. As opposed to reduced- form models, this analysis examines marginal changes in cases where genotype and state of a pig is known. The implications of such issues as het- erogeneity of a pig group and unknown production technology have been examined in previous stud- ies (such as Jorgensen 1993, Boland et al. 1996, Kure 1997, White et al. 2004). Further studies could extend this analysis to deal with cases in- volving unknown genotypes. Asymmetric information or the increased risk of infection in regions having an intensive contact structure can exacerbate the incentive problem. One solution to the incentives’ problem could be to take the effect of a reduction in the price of pig meat due to decreased carcass quality into account, and thus losses due to maintenance feeding, when designing compensation and restriction schemes for heavy animals. Another option could be to of- fer the producer an option to sell the animals in a buy-out scheme for economic welfare reasons dur- ing the restriction period. This would both allow epidemiologically safe culling and stabilise pro- ducer income. This study, however, does not focus on the question of whether society should organise such an income stabilisation scheme. In each case, special attention should be paid to producers keep- ing heavy animals (>100 kg) in stock, because they would be better off to interrupt farming in- The results are conditional on the functions, prices and growth parameters that are used in the model, and on the ability of producer to control carcass quality of individual pigs on a daily basis. They give information to producers and policy planners on how pig management patterns and the value of a capacity unit change in reaction to changes in relative prices, animal quality, animal growth rates, technology and constraints related to pig fattening. A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 15 (2006): Supplement 1. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs ignoring these variables by adhering to globally applicable optimal feed levels. marginal energetic efﬁciency decreases above maintenance requirements. One of the potential future applications of this model is in the analysis of production and market- ing contracts in pig production. The model can be extended to include the effects of price uncertainty, price volatility, and other production costs than piglet and feed costs even if the current analysis excludes these effects (for a pig management ap- plication under price uncertainty, see Burt 1993). Another extension might involve analyses in which prices of inputs and outputs are mutually correlat- ed and vary simultaneously but price sets are ran- dom. According to statistics presented in Tike (2003) and elsewhere, however, the price of pig meat in Finland is quite stable. In 2001–2003 the standard deviation of the change in the average price of pig meat over the period of 3 months was approximately €0.036. Furthermore, as some slaughterhouses announce prices in advance for a given period, producers may have quite consistent price expectations in the short run. This helps in planning and implementing feeding patterns. As the growth equations used in this model are combinations of several functions, it is impossible to ensure that the production function is globally concave. This concavity problem resulted in ir- regular shapes of optimised feeding curves in some scenarios. These irregularities were probably due to the fact that marginal returns from producing an additional unit of lean meat or fatty meat were, in some cases, non-decreasing. A simulation of pig growth in selected scenarios using optional man- agement patterns, however, veriﬁes that under given model formulation, any potential bias of es- timated management patterns is negligible. Boland et al. (1999, p. 92) also noted that their model was not globally concave. For this reason, they per- formed a heuristic matrix test. The results of this test allowed them to conclude that their optimal solution is likely to be also a global optimum. One optional modelling approach to ensure concavity of the production function could be to estimate the transition equations so that they have well deﬁned microeconomic form, such as in Chavas et al. (1985) or Campos (2003). The fact that the results are estimated only for a period of 5 years underestimates the value func- tions. 8.3 Suggestions for further research This information provides them with alternatives to adjust feeding regimens instead of 91 Vol. 15 (2006): Supplement 1. cy of the model could be an approach in which the terminal value of an inﬁnite process is received af- ter marketing the current batch, and where the so- lution to the optimisation problem is obtained by repeating a procedure that updates the terminal value (for details, see Kure 1997). count the case of unobservable parameters would allow a comparison between full-information man- agement pattern and a limited-information man- agement pattern. The uncertainty aspect includes at least two scenarios. In the ﬁrst scenario, pro- ducer is unable to observe the true current state of nature. In the second scenario, he/she is unable to observe the next-period state of nature (i.e. sto- chastic growth pattern). Further research is also needed on appetite control. Chavas et al. (1985, p. 642) discuss a sim- ilar problem. Information is required particularly on how low the ratios of protein to energy in feed can be under suitable markets without affecting the efﬁciency of feed conversion. Such information could give further insights into the beneﬁts of re- stricted feeding, and the results of this study could be validated with a growth experiment. This study estimated the optimal feeding and slaughter patterns for an individual pig. Although the results provide information on how non-opti- mal management affects the value of a capacity unit, further research is needed to extend the anal- ysis to large heterogeneous groups of pigs. The problem is interesting because, as Kure (1997) and Jorgensen (1993) point out, variation of the pig’s genetic characteristics can decrease the value of information about large groups of pigs. Taking into account heterogeneity of pigs is important and it requires including the distribution of the states of nature in the model. Such an extension is analyti- cally equivalent to optimising the current model, but it exposes the model to the problem of the curse of dimensionality. Unexploited opportunities may also exist to reduce the problem of the curse of dimensionality by using a more efﬁcient dy- namic programming algorithm or by enhancing the technical performance of the current program. One option to improve the computational efﬁcien- When optimising production, producers fre- quently use feed inputs efﬁciency as a measure. Feed efﬁciency is the ratio of the amount of feed required to produce one kilogram of meat. Feed efﬁciency is unable to take the interaction between carcass quality and feeding adequately into ac- count (cf. Vol. 15 (2006): Supplement 1. discussion by Boland et al. 1993, Cam- pos and Andersson 2003, p. 43). This study sug- gests that the problem is that when feeding is re- stricted, feed efﬁciency is lower than when feeding is unrestricted. Even if restricted feeding is opti- mal, the feed efﬁciency may indicate that the pro- duction is inefﬁcient. Consequently, the measure should be further improved so that it takes the rela- tive shares of lean and fatty tissue into account. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs This kind of normalisation has no impact on which scenario producer prefers if he/she is al- lowed to choose between them, because the sce- narios are compared using the same units of meas- ure. However, it would be more useful to extend the length of the estimation period such that the value function also converges. Another issue related to model validity is the consistency of growth parameters. In empirical studies where feeding is unrestricted (ad libitum), it is important to know whether the pig’s growth potential actually restricts pig’s growth. If not, then feeding (or some other factor, such as climate) actually limits the growth, and the data cannot be used to estimate the pig’s growth potential. The parameters used in this study, however, are expect- ed to be valid. One of the strengths of this study is that the parameters of the model are known. In other words, the problem was to estimate pig pro- duction management patterns conditional on pa- rameters, not to determine the parameters them- selves. Therefore, the results provide the best in- formation on how the producer can adjust manage- ment decisions when the values of the parameters change. Future applications could also examine the case where the genotype and the state of nature of a pig are unknown. As the results of Parsons et al. (2004) suggest, this could affect producer’s op- tions to control carcass quality. Taking into ac- As choice of functional forms is important for modelling, assumptions regarding concavity and decreasing marginal product are occasionally dis- cussed in the literature. Concavity can be particu- larly important when the choice is made between restricted and unrestricted feeding. In pig fatten- ing, the main problem is that biological studies generally postulate pigs to respond linearly or al- most linearly to increasing amounts of feed in the diet (cf. Black 1995). This can result in a corner solution or unstable decision variables in optimisa- tion models. Hence, it increases the importance of growth restrictions. Nevertheless, Emmans and Kyriazakis (1995) critisise the lack of empirical biological evidence to support the assumption that 92 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs 28. Cited 9 August 2004. Available on the Internet: ftp:// ftp.dina.kvl.dk/pub/Staff/Anders.R.Kristensen/dina-re- ports/notat7/notat7.pdf 28. Cited 9 August 2004. Available on the Internet: ftp:// ftp.dina.kvl.dk/pub/Staff/Anders.R.Kristensen/dina-re- ports/notat7/notat7.pdf Anonymous 2002d. Valtioneuvoston asetus vuodelta 2002 maksettavasta pohjoisesta tuesta annetun valtioneu- voston asetuksen muuttamisesta. Cited 11 November 2004. Available on the Internet: http://www.ﬁnlex.ﬁ/ﬁ/ laki/alkup/2002/20020213. Burt, O.R. 1965. Optimal replacement under risk. Journal of Farm Economics 47: 324–346. Arsenos, G., Emmans, G.C. & Kyriazakis, I. 2000. Variation between individuals and the consequences for diet se- lection by groups of animals. Animal Behaviour 60: 811–820. Burt, O.R. 1982. Dynamic programming: Has its day ar- rived? Western Journal of Agricultural Economics 7: 381–394. Burt, O.R. 1993. Decision rules for the dynamic animal feeding problem. American Journal of Agricultural Eco- nomics 75: 190–202. Agricultural Research Council 1981. The nutrient require- ments of pig. Farnham Royal: Commonwealth Agricul- tural Bureaux. 307 p. Campbell, R.G., Taverner, M.R. & Curic, D.M. 1983. The in- ﬂuence of feeding level from 20 to 45 kg live weight on the performance and body composition of female and entire male pigs. Animal Production 36:193. Aptech Systems 2003. Gauss for Windows, programming language, v. 6.0. Maple Valley, WA: Aptech Systems inc. Bank of Finland 2003. Ofﬁcial exchange rates. Cited 11 No- vember 2004. Available on the Internet:www.suomen- pankki.ﬁ. Campos, M. 2003. The economics of technologies in Swed- ish pig production. Doctoral thesis. Agra Universitatis Agriculturae Sueciae: Agraria 236. 101p. p Bellmann, R. 1957. Dynamic programming. New Jersey: Princeton University Press. 339 p. Campos, M. & Andersson, H. 2003. The value of animal welfare improving technologies in Swedish pig produc- tion. In: Campos, M. The economics of technologies in Swedish pig production. Doctoral thesis. Agra Universi- tatis Agriculturae Sueciae: Agraria 236: 29–49. Bellman, R.E. & Dreyfus S.E. 1962. Applied dynamic pro- gramming. New Jersey: Princeton University Press. 363 p. g g Chambers, R.G. 1988. Applied production analysis – A dual approach. Cambridge: Cambridge University Press. 331 p. Bertsekas, D.P. 1995a. Dynamic programming and optimal control. Volume 1. Belmont: Athena Scientiﬁc. 387 p. Bertsekas, D.P. 1995a. Dynamic programming and optimal control. Volume 1. Belmont: Athena Scientiﬁc. 387 p. Bertsekas, D.P. 1995b. Dynamic programming and optimal control. Volume 2. Belmont: Athena Scientiﬁc. 292 p. Bertsekas, D.P. 1995b. Dynamic programming and optimal control. Volume 2. Belmont: Athena Scientiﬁc. 292 p. Chavas, J.P., Klieberstein, J. & Crenshaw, T.D. 1985. Model- ling dynamic agricultural response: The case of swine production. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs American Journal of Agricultural Econom- ics 67: 636–646. Black, J. 1997. A dictionary of economics. New York: Oxford University Press. 512 p. Black, J.L. 1995. Modelling energy metabolism in the pig – critical evaluation of a simple reference model. In: Moughan, J.P., Verstegen, M.W.A. and Visser-Reyn- eveld, M.I. (eds.). Modelling growth in the pig. Wagenin- gen: Wageningen Pers. EAAP (European Association of Animal Production) publication 78: 87–12. Collin, A., van Milgren, J., Dubois, S. & Noblet, J. 2001. Ef- fect of high temperature and feeding level on energy utilization in piglets. Journal of Animal Science 79: 1849–1857. Danske slagterier 2001. Fokus på fodringstrategi til slagtes- vin. Copenhagen: Danske Slagterier. 4 p. Cited 11 No- vember 2004. Available on the Internet:www.dan- skeslagterier.dk. Black, J.L. 1988. Animal growth and its regulation. Journal of Animal Science 66 (supplement 3): 1–22. Black, J.L., Campbell, R.G., Williams, I.H., James, K.J. & Davies, G.T. 1986. Simulation of energy and amino acid utilisation in the pig. Research and Development in Agriculture 3: 121–145. Dent, J.B., Blair, R., English, P.R. & Raeburn, J.R. 1970. Protein, lysine and feed intake level effects on pig growth III. Regression analysis and economic aspects. Journal of Agricultural Science, Cambridge 75: 189– 205. Boehlje, M.D. & Eidman, V.R. 1984. Farm Management. New York: John Wiley & Sons. 806 p. Dillon, J.L. & Anderson, J. 1990. The analysis of response in crop and livestock production. 3rd edition. New York: Pergamon Publishing. 251 p. Boland, M.A. Foster, K.A. & Preckel, P.V. 1998. Economic analysis of phosphorous-reducing technologies in pork production. Journal of Agricultural and Resource Eco- nomics 23: 468–482. Dixit, A.K. & Pindyck, R.S. 1994. Investment under uncer- tainty. New Jersey: Princeton University Press. 468 pp. Boland, M.A., Foster, K.A. & Preckel, P.V. 1999. Nutrition and the economics of swine management. Journal of Applied and Agricultural Economics 31: 83–96 Dros, J.M. & Kriesch, M. 2003. GMO free soy for Europe – Quick scan on demand and arguments. Amsterdam: Aidenvironment. 76 p. Cited 13 November 2004. Avail- able on the Internet: http://www.genet-info.org/-docu- ments/GM%20_free_Soy_for_Europe.pdf. Boland, M.A., Foster, K.A., Preckel, P.V. & Schnickel, A.P. 1996. Analyzing pork carcass evaluation technologies in a swine bioeconomic model. Journal of Production Agriculture 9: 45–49. Emmans, G.C. 1995. Ways of describing pig growth and food intake using equations. Pig News and Information 16: 113–116. Boland, M.A., Preckel, P.V. & Schnickel, A.P. 1993. Optimal hog slaughter weights under alternative pricing sys- tems. References 11 November 2004. Available on the Internet: http:// www.ﬁnlex.ﬁ/ﬁ/laki/alkup/2002/20021083. 11 November 2004. Available on the Internet: http:// www.ﬁnlex.ﬁ/ﬁ/laki/alkup/2002/20021083. Affentranger, P., Gerwig, C., Seewer, G.J.F., Schwörer, D. & Künzi, N. 1996. Growth and carcass characteristics as well as meat and fat quality of three types of pigs under different feeding regimens. Livestock Production Scien- ce 45: 187–196. Anonymous 2002b. Valtioneuvoston asetus 212/2002 vuodelta 2002 maksettavasta Etelä-Suomen kansalli- sesta tuesta annetun valtioneuvoston asetuksen 5 §: n muuttamisesta. Cited 11 November 2004. Available on the Internet: http://www.ﬁnlex.ﬁ/ﬁ/laki/alkup/2002/ 20020212. Anonymous 2001. Laki maa- ja puutarhatalouden kansalli- sista tuista 28.12.2001/1559. Cited 11 November 2004. Available on the Internet: http://www.ﬁnlex.ﬁ/ﬁ/laki/ alkup/2001/20011559. Anonymous 2002c. Valtioneuvoston asetus vuodelta 2002 maksettavasta pohjoisesta tuesta. Cited 11 November 2004. Available on the Internet: http://www.ﬁnlex.ﬁ/ﬁ/ laki/alkup/2002/20020023. Anonymous 2002a. Valtioneuvoston asetus 1083/2002 vuodelta 2002 maksettavasta pohjoisesta tuesta anne- tun valtioneuvoston asetuksen muuttamisesta. Cited 93 Vol. 15 (2006): Supplement 1. Huirne, R.B.M. 1990. Computerized management support for swine breeding farms. PhD thesis. Wageningen: De- partment of Farm Management, Wageningen Agricul- tural University. 165 p. Emmans, G. C., & I. Kyriazakis. 1999. Growth and body composition. In: Kyriazakis, I. (ed.). A quantitative biol- ogy of the pig. Wallingford, Oxon, UK: CAB Internation- al. p.181–197. European Council 2001. Council directive 2001/89/EC of 23 October 2001 on Community measures for the con- trol of classical swine fever. Ofﬁcial Journal of the Euro- pean Communities L316: 5–35. Huirne, R.B.M., Dijkhuizen, A.A., van Beek, P. & Hendriks, Th.H.B. 1993. Stochastic dynamic programming to support sow replacement decisions. European Journal of Operational Research 67: 161–171. of Operational Research 67: 161–171. Eurostat 2004. NewCronos database. Cited 12 May 2004. Available on the Internet: http://europa.eu.int/comm/ eurostat/newcronos/reference/display.do?screen=welc omeref&open=/agric/agri&language=en&product=EU_ MAIN_TREE&root=EU_MAIN_TREE&scrollto=0 Huirne, R.B.M., Dijkhuizen, A.A., van Beek, P. & Renkema, J.A. 1997. Dynamic programming to optimize treatment and replacement decisions. In: Dijkhuizen, A.A. & Mor- ris, R.S. Animal health economics: principles and ap- plications. Sydney: Post Graduate Foundation in Vet- erinary Science. p. 85–97. Faba 2000. Kotieläinjalostuksen tilastokirja 2000 – Animal breeding statistics 2000. Vantaa, Finland: The Finnish Animal Breeding Association. 65 p. Cited 12 November 2004. Available on the Internet: http://www.faba.ﬁ/leh- det/julkaisut/tilastokirja.pdf. y p Intriligator, M.D. 2002. Mathematical optimisation and eco- nomic theory. Classics in applied mathematics 39. Philadelphia: Society for industrial and applied mathe- matics. 508 p. Faba 2004. Kantakoe. Cited 11 November 2004. Cited 25 August 2004. Available on the Internet: http://www.faba. ﬁ/palvelut/sikatilat/kantakoe/. Jolly, R.W., Sather, A.P., Patterson, R.D., Sonntag, B.H., Martin, A.H. & Freeden, H.T. 1980. Alternative market weights for swine: production economics. Journal of Animal Science 51: 804–810. Fawcett, R.H., Whittemore, C.T. & Rowland, C.M. 1978. To- wards the optimal nutrition of fattening pigs: Part II – Least cost growth and use of chemical value on diet formula- tion. Journal of Agricultural Economics 29: 175–182. Jorgensen, E. 1993. The inﬂuence of weighting precision on delivery in slaughter pig production. Acta Agriculturae Scandinavica, Section A: Animal Science 43: 181–189. Feinerman, E. & Siegel, P.B. 1988. A dynamic farm-level planning model for beef feedlot production marketing. Journal of Agricultural Economics 39: 413–429. Kamien, M.I. & Schwartz, N.L. 1992. Dynamic optimization: The calculus of variations and optimal control in eco- nomics and management. Advanced Textbooks in Eco- nomics 31. Amsterdam: Elsevier Science B.V. 377 p. Fuller, M.F., McWilliam, R., Wang, T.C. & Giles, L.R. 1989. Vol. 15 (2006): Supplement 1. The optimum dietary amino acid pattern for growing pigs. 2. Requirements for maintenance and for tissue protein accretion. British Journal of Nutrition 62, 255– 267. Kanis, E. 1988. Food intake capacity in relation to breeding and feeding of growing pigs. PhD thesis. Wageningen: University of Wageningen. 129 p. Käytännön Maamies 2005. Hintaseuranta. p. 71–75. Futurestrading 2003. Chicago board of trade futures price quotations for soy meal. Cited 7 October 2004. Availa- ble on the Internet: www.futurestrading.com. Keane, M.P. & Wolpin, K.I. 1994. The solution and estima- tion of discrete choice dynamic programming models by simulation and interpolation: Monte Carlo evidence. The Review of Economics and Statistics 76: 648–672. y p The Review of Economics and Statistics 76: 648–672. Giesen, G.W.J., Baltussen, W.H.M., & Oenema, J. 1988. Optimalisering van het aﬂeveren van mestvarkens. Den Haag: Lanbouw-Economisch Institut. 74 p. (In Dutch). Kennedy 1972 A model for determining optimal marketing and feeding policies of beef cattle. Journal of Agricul- tural Economics 23: 147–160. Glen, J.J. 1983. A dynamic programming model for pig pro- duction. Journal of Operational Research Society vol. 34, 6, 511–519. Kennedy, J.O.S. 1986. Dynamic programming: Applications to agriculture and natural resources. London: Elsevier Applied Science Publishers. 341 p. Gravelle, H. & Rees, R. 1992. Microeconomics. 2nd edition. London: Longman group. 752 p. Kennedy, J.O.S., Rofe, B.H., Grieg, I.D. & Hardaker, J.B. 1976. Optimal feeding policies for broiler production: an application of dynamis programming. Australian Jour- nal of Agricultural Economics 20: 19–32. Heady, E.O. & Dillon, J.L. 1972. Agricultural production functions. 5th edition. Ames: Iowa State University Press. 667 p. Kornegay, E.T. & Notter, D.R. 1984. Effects of ﬂoor space and number of pigs per pen on performance. Pig News and Information 5: 23–33. Hayashi, F. 2000. Econometrics. New Jersey: Princeton University Press. 683 p. Kristensen, A. R. 1987. Optimal replacement and ranking of dairy cows determined by a hierarchic Markov process. Livestock Production Science 16: 131–144. Heikkonen, H. 1998. Kasvatustiheyden vaikutus lihasikojen päiväkasvuun, sairastuvuuteen ja lääkityksen tarpee- seen. Syventävien opintojen tutkielma. Helsinki: Hel- singin yliopisto, Eläinlääketieteellinen tiedekunta, Koti- eläinhygienia. 41 p. Kristensen, A.R. 2003. Information from on-line weight as- sessment for optimal selection of slaughter pigs from market. In: EFITA 2003 Conference, 5–9 July 2003, De- brecen, Hungary. p. 754–759. Cited 11 June 2004. Available on the Internet: http://www.date.hu/eﬁta2003/ mainstream.php. Houben, E.H.M, Huirne, R.M.B., Dijkhuizen, A.A. & Kristen- sen, A.R. 1994. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Journal of Applied and Agricultural Economics 25: 148–163. Emmans, G.C. 1999. Energy ﬂows. In: Kyriazakis, I. (ed.). A quantitative biology of the pig. Wallingford, Oxon, UK: CAB International. p. 363–377. Brealey, R.A. & Myers, S.C. 2003. Principles of corporate ﬁnance. Boston: Irwin McGraw-Hill. 1071 p. Emmans, G.C. & Kyriazakis, I. 1995. The idea of optimisa- tion in animals: uses and dangers. Livestock Produc- tion Science 44: 189–197. Broekmans, J.E. 1992. Inﬂuence of price ﬂuctuations on delivery strategies for slaughter pigs. Dina Notat 7: 1– 94 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs MTT:n selvityksiä 86. 82 p. Cited 20 January 2005. Available on the Internet: http://www.mtt.ﬁ/mtts/pdf/ mtts86.pdf MTT:n selvityksiä 86. 82 p. Cited 20 January 2005. Available on the Internet: http://www.mtt.ﬁ/mtts/pdf/ mtts86.pdf Kure, H. 1997. Marketing management support in slaughter pig production. PhD thesis. Copenhagen: The Royal Veterinary and Agricultural University. 108 p. Cited 9 August 2004. Available on the Internet: http://www.dina. kvl.dk/publications/all_publications.html. Nemhauser, G.L. 1966. Introduction to dynamic program- ming. New York: John Wiley. 256 p. Kyriazakis, I. & Emmans, G.C. 1999. Voluntary food intake and diet selection. In: Kyriazakis, I. (ed.). A quantitative biology of the pig. Wallingford, Oxon, UK: CAB Interna- tional. p. 363–377. Nielsen, B.K., Kristensen, A.R. & Thamsborg, S.M. 2004. Optimal decisions in organic steer production – a mod- el including winter feed level, grazing strategy and slaughtering policy. Livestock Production Science 88: 239–250. LeSage 2004. Econometrics toolbox for Matlab. Cited 11 November 2004. Available on the Internet: http://www. spatial-econometrics.com. Partanen, K., Siljander-Rasi, H., Alaviuhkola, T. & van Gilse van der Pals, N. 1998. Utilisation of reactive lysine from meat and bone meals of different ash content by grow- ing-ﬁnishing pigs. Agricultural and Food Science in Fin- land 7: 1–12. Lizardo, R., van Milgren, J., Mourot, J., Noblet J. & Bon- neau, M. 2002. Nutritional model of fatty acid composi- tion in the growing ﬁnishing pig. Livestock Production Science 75: 167–182. Partanen, K., Alaviuhkola, T, Siljander-Rasi, H. & Suomi, K. 2003. Faba beans in diets for growing-ﬁnishing pigs. Agricultural and Food Science in Finland 12: 35–48. Ljunqvist, L. & Sargent, T.J. 2000. Recursive Macroeco- nomic theory. Cambridge: MIT Press. 701 p. Parsons, D.J., Schoﬁeld, C.P., Green, D.M. & Whittemore, C.T. 2004. Real-time control of pig growth through an integrated management system. In: Proceedings of EWDA-04 European workshop for decision problems in agriculture and natural resources. Silsoe, England, 27–28 September 2004. p. 109–120. Lloyd, F.W., Harsh, S.B., Kaneene, J.B., Schwad, G.D., Thacker, B.J. & Thulin, A.J. 1994. Development of a computerized systems model for health management decision support in growing hogs. Preventive Veteri- nary Medicine 19: 249–265 y Mangen, M-J, Nielen, M. & Burrell, A.M. 2002. Simulated effect of pig-population density on epidemic size and choice of control strategy for classical swine fever epi- demics in The Netherlands. Preventive Veterinary Medicine 56: 141–163. Patience, J.F. 1996. Precision in swine feeding programs: An integrated approach. Animal Feed Science Techno- logy 59: 137–145. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Perttilä, S., Siljander-Rasi, H., Partanen, K., Alaviuhkola, T., Suomi, K. & Valaja, J. 2002. Apparent ileal amino acid digestibility and the nutritive value of the triticale culti- vars Moreno and Ulrika for growing-ﬁnishing pigs. Agri- cultural and Food Science in Finland 11: 93–105. Mas-Colell, A., Whinston, M.D. & Green, J.R. 1995. Micro- economic theory. New York: Oxford University Press. 981 p. Meuwissen, M.P.M. 1999. Insurance as a risk management tool for European agriculture. PhD thesis. Wageningen: Wageningen Agricultural University. 117 pp. Pietola, K. & Sevón-Aimonen, M.-L. 2002. Eläinjalostus tuottaa lisäarvoa Suomen sikatalouteen. In: toim. An- neli Hopponen. Maataloustieteen Päivät 2002 [verkko- julkaisu]. Suomen maataloustieteellisen seuran tiedote 18: [4 p.]. Cited 11 November 2004. Available on the Internet: http://www.agronet.ﬁ/maataloustieteellinen- seura/julkaisut/esit/50pietola.pdf Meuwissen, M.P.M., Van Asseldonk, M.A.P.M. & Huirne, R.B.M. 2003. Alternative risk ﬁnancing instruments for livestock epidemics. Agricultural Systems 75: 305– 322. Moughan, P.J. 1995. Modelling metabolism in the pig – First principles. In: Moughan, J.P., Verstegen, M.W.A. & Visser-Reyneveld, M.I. (eds.). Modelling growth in the pig. Wageningen: Wageningen Pers. EAAP (European Association of Animal Production) publication 78: 59– 70. Pihamaa, P. & Pietola, K. 2002. Optimal beef cattle man- agement under agricultural policy reforms in Finland. Agricultural and Food Science in Finland 11: 3–12. Pindyck, R.S. & Rubinfeld, D.L. 1998. Econometric models and economic forecasts. 4th edition. Boston: Irwin Mc- Graw-Hill. 634 p. Mourits, M.C.M., Galligan, D.T., Dijkhuizen, A.A. & Huirne, R.B.M. 2000. Optimization of dairy heifer management decisions based on production conditions of Pennsyl- vania. Journal of Dairy Science 83: 1989–1997. Quiniou, N., Noblet, J., Dourmad, J.-Y. & van Milgren, J. 1999. Inﬂuence of energy supply on growth character- istics and consequences for growth modelling. Live- stock Production Science 60: 317–328. Mourits, M.C.M., Huirne, R.B.M., Dijkhuizen, A.A., Kris- tensen, A.R. & Galligan, D.T. 1999. Economic optimiza- tion of dairy heifer management decisions. Agricultural Systems 21: 17–31. Ramaekers, P.J.L., Swinkels, J.W.G.M., Huiskes, J.H., Ver- stegen, M.W.A. , Den Hartog, L.A. & Van der Peet- Schwering, C.M.C. 1996. Performance and carcass traits of individual pigs housed in groups as affected by ad libitum and restricted feeding. Livestock Production Science 47: 43–50. MKL 2003. Mallilaskelmia maataloudesta 2003. Vantaa, Finland: Maaseutukeskusten liitto. 43 p. MMM 2004. Hakuopas 2004. Helsinki: Ministry of Agricul- ture and Forestry. 167 p. Cited 11 November 2004. Available on the Internet: http://www.mmm.ﬁ/tuet/ ohjeet_oppaat_tiedonannot/oppaat/2004/Hakuopas_ 2004.pdf. Rantala, J. 2004. Missä painossa siat teuraaksi? Lihatalous 62 (6): 18–19. Roemen, J. Vol. 15 (2006): Supplement 1. Optimal replacement of mastitic cows determined by hierarchic Markov process. Journal of Dairy Science 77: 2975–2994. Howard, R.A. 1960. Dynamic programming and Markov processes. Cambridge, Massachusetts: MIT Press. 136 p. Kuchler, F. & Hamm, S. 2000. Animal disease incidence and indempity eradication programs. Agricultural Econom- ics 22: 299–308. 95 Vol. 15 (2006): Supplement 1. related. Lleida: Institut de Recerca i Technologia Agro- alimentaries & Universitat de Lleida. p. 107–116. Tike 2004a. Quality classiﬁcation of slaughter animals 2003. Helsinki: Information Centre of the Ministry of Ag- riculture and Forestry. Agricultural Statistical Bulletin 3/2004. related. Lleida: Institut de Recerca i Technologia Agro- alimentaries & Universitat de Lleida. p. 107–116. Ross, R.W. 1980. The use of a production function to max- imise proﬁts in feeder hog enterprise. Canadian Jour- nal of Agricultural Economics 28: 33–37. Tike 2004b. Kotieläinten lukumäärät 1.12.2003. The number of livestock at 1.12.2003. Helsinki: Information Centre of the Ministry of Agriculture and Forestry. Agricultural Statistical Bulletin 4/2004. 12 p. Rust, J. 1987. Optimal replacement of GMC bus engines: An empirical model of Harold Zurcher. Econometrica 55: 999–1033. Robinson, L.J. & Barry, P.J. 1996. Present value models and investment analysis. Northport, Alabama: The Aca- demic Page. 661 p. Tike 2005. Tietokappa. Helsinki: Information Centre of the Ministry of Agriculture and Forestry. Monthly Review of Agricultural Statistics 4/2005. 47 p. Tike 2005b. Teurastamotilasto. Maaliskuu 2005. Helsinki: Information Centre of the Ministry of Agriculture and Forestry. 1 p. Rydstedt, C. & Andersson, H. 1993. Optimala strategier i specialiserad slaktsvinsproduction. Uppsala: Swedish university of agricultural sciences. Report 62. 48 p. Toft, N, Kristensen, A.R. & Jorgensen, E. 2005. A frame- work for decision support related to infectious diseases in slaughter pig fattening units. Agricultural Systems 85: 120–137. Saatkamp, H, Berentsen, P.B.M & Horst, H.S. 2000. Eco- nomic aspects of the control of classical swine fever outbreaks in the European Union. Veterinary Microbiol- ogy 73: 221–237 Salanié, B. 2005. The economics of contracts: a primer. Cambridge, Massachusetts: The MIT Press. 244 p. Toft, N. 2000. Elements of support systems in pig produc- tion. PhD thesis. Copenhagen: The Royal Veterinary and Agricultural University. Dina research report no. 90. 92 p. Cited 9 August 2004. Available on the Internet: http://www.dina.kvl.dk/publications/all_publications. html Sargent, T.J. 1987. Dynamic macroeconomic theory. Cam- bridge: Harvard University Press. 369 p. Schinckel, A.P. & de Lange, C.F.M. 1996. Characterization of growth parameters needed as inputs for pig growth models. Journal of Animal Science 74: 2021–2036. Tuppi, K. 2004. Tuotanto- ja talousseurannan tulokset 2003. Sikatalouden tulosseminaari, Tampere 10.6.2004. Pro- Agria Maaseutukeskusten liitto ja MTT sikatalous. Schoﬁeld, C.P., Marchant, J.A., White, R.P., Brandl, N. & Wilson, M. 1999. Monitoring pig growth using a proto- type imaging system. Journal of Agricultural Engineer- ing Research 72: 205–210. Vol. 15 (2006): Supplement 1. Tuori, M., Kuoppala, K., Valaja, J., Aimonen, E., Saarisalo, E. & Huhtanen, P. 2002. Rehutaulukot ja ruokintasuosi- tukset: märehtijät–siat–siipikarja-turkiseläimet-hevoset. Helsinki: University of Helsinki. 88 p. Cited 17 August 2004. Available on the Internet: http://www.mtt.ﬁ. Seierstad, A. & Sudsaetter, K. 1993. Optimal control theory with economic applications. Amsterdam: Elsevier sci- ence publishers B.V. 445 p. Tuovinen, V.K, Gröhn, Y.K., Straw, B.E. & Boyd, R.D. 1992. Feeder unit environmental factors associated with par- tial carcass condemnations in market swine. Preven- tive Veterinary Medicine 12: 175–195. Sevón-Aimonen, M.-L. 2001. The parameters of growth curve and composition of growth for Finnish pigs. In: Book of Abstracts of the 52nd Annual Meeting of the European Association for Animal Production: Book of abstracts No. 7. Budapest, Hungary 26–29 August 2001. Wageningen: Wageningen Pers. p. 290. Valaja, J. 1992. The effect of supplementation of liquid lysine to barley distillers solids diet on the performance and carcass quality of pigs. Agricultural and Food Sci- ence in Finland 1: 599–567. Sipiläinen, T. & Ryhänen, M. 1996. EU-jäsenyyden vaikutus sianlihan tuotantoon. In: Ylätalo, M. (ed.). Maatalousyri- tysten sopeutuminen EU:ssa vallitseviin hintasuhteisiin – Tuotanto- ja kustannusteoreettinen tarkastelu kasvin- viljelyyn ja kotieläintuotantoon sovellettuna. Helsinki: University of Helsinki. Department of Economics and Management Publications 12. p.157–200. Valaja, J., Alaviuhkola, T. & Suomi, K. 1993. Reducing crude protein content with supplementation of synthetic lysine and threonine in barley-rapeseed meal-pea diet for growing pigs. Agricultural and Food Science in Finland 2: 117–123. Valaja, J., Alaviuhkola, T., Suomi, K. & Immonen, I. 1992. Compensatory growth after feed restriction during the rearing period in pigs. Agricultural and Food Science in Finland 1: 15–20. Sonka, S.T., Heady, E.O. & Dahm, P.F. 1976. Estimation of gain isoquants and a decision model application for swine production. American Journal of Agricultural Economics 58: 466–474. Stokey, N.L. & Lucas, R.E. with the collaboration of Pres- cott, E.C. 1989. Recursive methods in economic dy- namics. Cambridge: Harvard University Press. 588 p. Valaja, J., Siljander-Rasi, H., Alaviuhkola, T. & Rantanen, A. 1996. Lysine supplementation of barley wet distillers’ solids diets for growing pigs. Agricultural and Food Sci- ence in Finland 5: 157–166. Talpaz, H., Hurwitz, S., de la Torre, J.R. & Sharpe, P.J.H. 1988. Economic optimization of a growth trajectory for broilers. American Journal of Agricultural Economics 80: 382–390. Vargas, B., Herrero, M. & van Arendonk, J.A.M. 2001. Inter- actions between optimal replacement policies and feeding strategies in dairy herds. Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs & de Klein, J. 2000.An optimal marketing strat- egy for porkers with differences in growth rates and dependent prices. In: Plà, L.M. & Pomar, J. (eds.). Pro- ceedings of the international symposium on pig herd management modelling and information technoloqies MTT 2004. Rehutaulukot ja ruokintasuositukset 2004: mä- rehtijät – siat – siipikarja – turkiseläimet – hevoset. Feed tables and feeding recommendations 2004. 96 Niemi, J.K. Optimising feeding and slaughter decisions regarding pigs Whittemore, C.T. & Fawcett, R.H. 1976. Theoretical aspect of a ﬂexible model to simulate protein and lipid growth in pigs. Animal Production 22: 87–96. formance of growing/ﬁnishing pigs. Animal Science 78: 409–418. formance of growing/ﬁnishing pigs. Animal Science 78: 409–418. Whittemore, C. T. 1983. Development of recommended en- ergy and protein allowances for young pigs. Agricultural Systems 11: 159–186. Whittemore, C.T., Tullis, J.B. & Emmans, G.C. 1988. Protein growth in pigs. Animal Production 46: 437–455. Whittemore, C.T. 1998. The science and practice of pig pro- duction. Essex: Longman group. 661 p. Williams, N.H., Cline, T.R., Schinckel, A. P. & Jones, D. J. 1994. The impact of ractopamine, energy intake, and dietary fat on ﬁnisher pig growth performance and car- cass merit. Journal of Animal Science 72:3152–3162. Whittemore, C.T. 2004. Production control systems for pigs. London swine conference, Ontario, April 2004. 8 p. Yager, W.A., Green, R.C. & Burt, O. 1980. Optimal policies for marketing cull cows. American Journal of Agricultu- ral Economics 62: 456–467. Whittemore, E.C., Kyriazakis, I., Tolkamp, B.J. & Emmans, G.C. 2002. The short term feeding behaviour of grow- ing pigs fed foods in different bulk content. Physiology & Behaviour 76: 131–141. Vol. 15 (2006): Supplement 1. Livestock Production Science 69: 17–31. Tike 2002. Tietokappa. Helsinki: Information Centre of the Ministry of Agriculture and Forestry. Monthly Review of Agricultural Statistics 3/2002. 47 p. Varian, H.R. 1984. Microeconomic analysis. New York: W. W. Norton & Company 506 p. Tike 2003. Tietokappa. Helsinki: Information Centre of the Ministry of Agriculture and Forestry. Monthly Review of Agricultural Statistics 8/2003. 47 p. White, R.P., Schoﬁeld, C.P., Green, D.M., Parsons, D.J. & Whittemore, C.T. 2004. The effectiveness of a visual image analysis (VIA) system for monitoring the per- 97 Vol. 15 (2006): Supplement 1. ohran määrä vakiintuu 2,6–2,7 kg:an päivässä päivässä jo noin kuukausi ennen teurastusta. Lisäksi sialle anne- taan aminohappotäydennyksenä 2,7 g lysiiniä, 0,8 g me- tioniinia ja 1,3 g treoniinia/1 kg ohraa. valkuaispitoisuus pidetään melko korkea jopa yli kasva- tusjakson puolivälin, ja ohran määrä rehussa nousee melko suoraviivaisesti. Sen sijaan samaan päiväkasvuun yltävillä sioilla, joiden valkuaisen kasvukyky laskee no- peasti, rehun valkuaispitoisuus laskee melko nopeasti siitäkin huolimatta, että ne tarvitsevat alkukasvatuksessa runsaasti valkuaispitoista rehua. Perusskenaariossa sika saavuttaa 115 kg teuraspai- non (vastaa noin 85 kg ruhopainoa) 105 päivässä. Pe- russkenaariossa sikapaikka tuottaa vuodessa noin 150 € korvauksen tuotantoon sitoutuneelle työlle, pääomalle ja muille tuotannontekijöille kuin porsaille ja rehulle. Tar- kennettuun kaksivaiheruokintaan verrattuna joustava ruokintateknologia antaa 1,70 € lisätuoton sikapaikkaa kohti vuodessa. Tämän ohella lisätuottoja saadaan sika- kohtaiseen ruokintaan siirtymisestä ja kaksivaiheruokin- nan tarkentamisesta. Tarkennetussa kaksivaiheruokin- nassa sikoja ruokitaan alkukasvatusrehulla 65–75 kg elopainoon asti ja rehun määrää (muttei koostumusta) muutetaan sian kasvun mukaisesti. Tulokset viittaavat siihen, että nopeakasvuiset ja vä- härasvaiset siat voivat antaa hidaskasvuisiin ja helposti rasvoittuviin sikoihin verrattuna jopa yli 20 euron lisä- tuoton lihasikapaikkaa kohti vuodessa. Perusskenaarion hintasuhteilla esimerkiksi 10 K-indeksipisteen parannus sian perimässä tuottaa vuodessa 14,51 € lisätuoton sika- paikkaa kohti. Sian biologisen maksimipäiväkasvun noustessa elopainovälillä 20–120 kg keskimäärin 100 g saadaan sikapaikkaa kohti kohti vuodessa 23,54 € lisä- tuotto. Lisätuotto on hieman suurempi, mikäli päiväkas- vua nostettaessa panostetaan niin sanotun aikuistumis- suhteen sijasta aikuispainon nostamiseen. Hyödyt johtu- vat osittain siitä, että keskimääräistä korkeamman ai- kuispainon omaavat siat ovat rasvattomampia kuin muut siat. Koska geneettinen parannus on jokseenkin pysyvä, edellä mainittuja lisätuottoja voidaan saada pitkältä ai- kaväliltä. Eläinjalostuksen tulosten täysimääräinen hyö- dyntäminen kuitenkin edellyttää tuotantopanosten käy- tön tehostamista sian perintötekijöiden mukaan. Tämä johtuu erityisesti siitä, että nopeakasvuisen ja vähäras- vaisen sian tuotanto-ominaisuudet saadaan täysin hyö- dynnettyä vain, mikäli lihakkaan ruhon tuottamiseen tarvittavan valkuaisen määrää rehussa muutetaan sian kasvukyvyn mukaan. Lähtötietojen vaikutusta tuloksiin tarkasteltiin ver- taamalla perusskenaarion (luku 4) tuloksia vaihtoehtois- ten skenaarioiden (luvut 5–7) tuloksiin. Tulosten mu- kaan taloudellisesti optimaalinen lihasian ruokinta riip- puu markkinatilanteesta. Malli suosittaa ruokkimaan si- kaa vapaalla ruokinnalla ja energiavaltaisella rehulla silloin, kun sianlihan hinta on korkea suhteessa punai- sesta lihasta maksettavaan laatulisään. Tällöin ruokinta maksimoi sian päiväkasvun. Muissa tapauksissa malli suosittaa rajoittamaan energiaruokintaa. Tällöin ruho kasvaa lihakkaammaksi kuin vapaalla ruokinnalla. Ra- joitetun ruokinnan kannattavuus johtuukin siitä, että tuotantokierron pidentymisestä huolimatta tuottaja voi lisätä sikapaikan tuottoa nostamalla ruhon laatukorjattua arvoa ruokinnan avulla. SELOSTUS Jarkko K. Niemi MTT (Maa- ja elintarviketalouden tutkimuskeskus), Taloustutkimus Tässä tutkimuksessa tarkasteltiin tarkennetun ruokinnan ja eläinaineksen perinnöllisen laadun parantamisen ta- loudellisia hyötyjä suomalaisilla lihasioilla. Tarkenne- tulla ruokinnalla tarkoitetaan sialle annettavan rehun määrän ja koostumuksen sovittamista sian kasvunvai- heen, perinnöllisten tekijöiden ja hintasuhteiden mukaan niin, että sika voi hyödyntää mahdollisimman tehok- kaasti rehussa olevan energian ja valkuaisen. Lihasiko- jen taloudellisesti optimaaliseen ruokintaan vaikuttavat monet eri tekijät, kuten sian perimä, markkinatilanne ja sika-aineksen vaihtelu. Tämä tutkimus tarjoaa ensisijas- sa tietoa siitä, miten sianlihan tuottaja voi sopeuttaa sian ruokintaa ja teurastuksen ajoitusta tuotantopanosten ja lihan hintasuhteiden, eläimen perinnöllisen tason (mm. päiväkasvu ja ruhon rasvoittumisherkkyys) ja tuotanto- teknologian joustavuuden muuttuessa. Tutkimustuloksia tulisikin tarkastella ensisijaisesti vertaamalla tarkastel- tuja skenaarioita keskenään, sillä tulokset johtuvat erois- ta skenaarioiden lähtötiedoissa. ruokinta optimoitiin niin, että ne antoivat sikapaikalle mahdollisimman suuren tuoton. Tarkastelussa käytetty ruokinta perustui ohra-soijarouhe-aminohappotäyden- nys-seokseen, jonka määrä ja koostumus optimoitiin erikseen kasvatusjakson jokaiselle päivälle. Ruokinta- ja teurastuspäätöksiä määritettäessä otettiin huomioon se, miten sian perimän, ruokinnan ja teurastuksen ajoituk- sen välinen yhteys vaikuttaa ruhon laatuun ja sikapaikan tuottoarvoon. Lisäksi otettiin huomioon se, miten sian syöntikyky ja ravinteiden tarve rajoittavat tuottajan mahdollisuuksia valita rehun koostumus ja rehuannok- sen määrä. Tulokset viittaavat siihen, että lihasikaa kannattaa pääsääntöisesti ruokkia rajoitetulla energiaruokinnalla. Siksi on tärkeää, että rehu sisältää sian tarpeeseen näh- den riittävästi valkuaista. Optimoidun ruokinnan mukai- sesti toimittaessa nuorta lihasikaa ruokitaan korkean valkuaispitoisuuden omaavalla rehulla ja lähes teuras- kypsää sikaa korkean energiapitoisuuden omaavalla re- hulla. Perusskenaariossa (luku 4) soijarouheen osuus rehussa laskee joustavasti niin, että 25 kg painavan por- saan rehuannoksessa soijarouhetta on noin 20 % ja teu- raskypsällä sian annoksessa vajaat 5 %. Samalla ohran määrä rehussa lisääntyy. Porsaan rehuannos sisältää 1,25 kg ohraa päivässä ja teuraskypsän sian rehuannos noin 2,60 kg ohraa päivässä. Tutkimuksen perusskenaariossa Tutkimusmenetelmänä käytettiin numeerista dynaa- misen ohjelmoinnin mallia. Malli simuloi yksittäisen sian päiväkasvua ja optimoi ruokinnassa käytettävän re- hun koostumuksen ja määrän sekä teurastuksen ajoituk- sen annettujen lähtötietojen (rehujen, porsaan ja lihan hinnat, rehujen laatu, sian perimä) määräämissä rajoissa. Sian teuraspaino, ruhon koostumus teurastushetkellä ja 98 Vol. 15 (2006): Supplement 1. Rehun valkuaispitoisuutta kan- nattaa lisätä myös silloin, kun kaikkien rehujen hinnat tai teuraspalkkio laskevat. Sen sijaan vain rehun oman hinnan noustessa sitä kannattaa korvata muilla rehuilla. Energiaruokintaa vähennettäessä tulisi huolehtia siitä, että rehun sisältämän valkuaisen määrä ei laske energia- pitoisen rehun määrää vähennettäessä. Toisin sanoen energiapitoisen rehun määrää vähennettäessä myös re- huannoksen sisältämän valkuaisen määrä vähenee, mikä tulisi korvata lisäämällä valkuaispitoisen rehun määrää. Tulokset viittaavat siihen, että sika kannattaa teuras- taa teuraspainon ollessa mahdollisimman lähellä kor- keimman hintaportaan yläpäätä. Siten lihan hinnoittelun muuttuessa myös optimaalinen teuraspaino muuttuu. Jos teurastus jostain syystä uhkaa viivästyä niin että sika kasvaa ”ylipainoiseksi”, tuottajan kannattaa markkinoi- da sika ennenaikaisesti. Tulos johtuu siitä, että ylipainoi- sen sian lihasta maksetaan alennettua kilohintaa. Mikäli sika voidaan teurastaa ennenaikaisesti korkeimman hin- taportaan sisällä, tuottaja välttää lihan hintaan tehtävän laatuvähennyksen ja ennenaikaisen teurastuksen mene- tys jää melko pieneksi. Tämä pätee erityisesti silloin, kun uusi sikaerä saadaan ilman tavanomaista pitempää viivettä heti teurastuksen jälkeen. Jos teurastus kuiten- kin viivästyy, tuottaja voi minimoida laatutappioiden riskin vähentämällä energiaruokintaa. Tällöin hän estää ruhoa rasvoittumasta. Lisäksi tuottaja voi vähentää re- hun kokonaismäärää, jolloin hän minimoi ruhon painon vuoksi lihan hintaan tehtävän laatuhintavähennyksen suuruuden. Taloudellisesti optimaalinen lihasian ruokinta riip- puu myös sika-aineksen perimästä (genotyypistä). Tu- losten mukaan sikoja, joilla on perinnöllinen taipumus nopeaan päiväkasvuun ja jotka rasvoittuvat helposti, kannattaa ruokkia energiapitoisemmalla rehulla kuin si- koja, joilla on taipumus hitaaseen päiväkasvuun ja jotka säilyttävät lihakkuutensa melko hyvin. Tässä tutkimuk- sessa ei kuitenkaan tarkastella tilanteita, joissa peräk- käisten sikaerien perinnöllinen taso vaihtelee voimak- kaasti. Lihakkuutensa hyvin säilyttävillä sioilla rehun 99 Appendix A: Computer code of the basic model This appendix illustrates functioning of the com- puter code. The optimisation problem is solved recursively by iterating on the Bellman’s equation. sions, the live weight of an animal, is a function of the other state variables, and hence redundant. In addition, the state vector includes several scalars such as input and output prices. Furthemore, the typical model is deﬁned in a four dimensional con- trol space, where three of the control variables re- late to the feed levels. The fourth control variable is the binary slaughter variable, which collapses other controls to scalar when executed. This appendix illustrates functioning of the com- puter code. The optimisation problem is solved recursively by iterating on the Bellman’s equation. The iteration is carried out by discretising the state and control spaces, and thereafter solving the opti- mal controls separately at each point of the state space and at every period of the planning horizon. As Figure A1 illustrates, the model is deﬁned in a three dimensional state space. One of the dimen- The iteration is carried out by discretising the state and control spaces, and thereafter solving the opti- mal controls separately at each point of the state space and at every period of the planning horizon. As Figure A1 illustrates, the model is deﬁned in a three dimensional state space. One of the dimen- The amount of lean tissue (xt lean) The amount of fatty tissue (xt fat) Ɣ (xt fat, xt lean) The amount of soy meal (ut soy) The amount of barley (ut barley) The amount of amino acid supplementation (ut aminos) Fig. A1. The typical model is deﬁned in a three dimensional state space, where one dimension is redundant due to the rela- tionship xt weight(xt fat,xt lean), and in a four dimensional control space, where feed allocations determine three of the dimensions and the binary slaughter variable determines the fourth dimension. The amount of lean tissue (xt lean) The amount of fatty tissue (xt fat) The amount of barley (ut barley) The amount of barley (ut barley) Fig. A1. The typical model is deﬁned in a three dimensional state space, where one dimension is redundant due to the rela- tionship xt weight(xt fat,xt lean), and in a four dimensional control space, where feed allocations determine three of the dimensions and the binary slaughter variable determines the fourth dimension. Fig. A1. Appendix A: Computer code of the basic model The typical model is deﬁned in a three dimensional state space, where one dimension is redundant due to the rela- tionship xt weight(xt fat,xt lean), and in a four dimensional control space, where feed allocations determine three of the dimensions and the binary slaughter variable determines the fourth dimension. The solutions are obtained using backwards re- cursion. This implies that the model solves the op- timal controls of the ﬁnal period t = T ﬁrst. There- after, the model solves the optimal optimal control for each period t = t–1 until the time index reaches period t = 0. At each combination of the points of the state space and the time space, the solution en- tails that the model evaluates the value of a capac- ity unit for all feasible values of the control varia- bles. After the optimal solutions are obtained, the model prints out the optimal control patterns and the value function throughout the time (Figure A2). The computer code below follows a pattern similar to that shown in Figure A2. The text be- tween /* and / is commented text separated from the computer code. 100 Figure A2. Fig. A2. Flow diagram of the model. A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Appendix A: Computer code of the basic model Initialisation Step 1: Define input parameters: -Meat pricing scheme and input prices -Feed characteristics -Animal’s genetic characteristics -State and control variables and initial states of nature Step 2: Set up space grids: -State spaces (lean tissue, fatty tissue, live weight) -Control spaces (barley, soy meal, amino acid supplementation, binary slaughter variable) -Quality adjusted meat price and loss ratio of meat upon slaughter at each state of nature Step 3: Specify the terminal value function Step 5: Trace the optimal control paths: -Give the initial state of nature for t=1 -Use the optimal feeding and slaughter decisions from step 4 to simulate pig growth -For each t=1,…,T, find the corresponding values of the value function -Print the optimal paths of the state variables, the optimal controls and the value function over time Terminate computation and save the results reported at step 5 Step 4: Solve the optimal decision rules: -Set up computational variables and matrices and begin with t=T For each control node, repeat: -Compute instantaneous returns at each node of the control space -Solve daily growth of tissue components and live weight -Consider whether the constraints are binding at any control -Solve the next-period state of nature for each node of the control spaces -Find the next-period value function and the value of a capacity unit Repeat step 4 for each time period t=T,…,0 For each given state of nature: -Solve the controls that maximise the value of a capacity unit -Save the optimal controls and corresponding value function Repeat for all state nodes t=t-1 if t>0 if t=0 If all state nodes at time t evaluated Figure A2 Appendix A: Computer code of the basic model Initialisation Step 1: Define input parameters: -Meat pricing scheme and input prices -Feed characteristics -Animal’s genetic characteristics -State and control variables and initial states of nature Step 2: Set up space grids: -State spaces (lean tissue, fatty tissue, live weight) -Control spaces (barley, soy meal, amino acid supplementation, binary slaughter variable) -Quality adjusted meat price and loss ratio of meat upon slaughter at each state of nature Step 3: Specify the terminal value function Step 5: Trace the optimal control paths: -Give the initial state of nature for t=1 -Use the optimal feeding and slaughter decisions from step 4 to simulate pig growth -For each t=1,…,T, find the corresponding values of the value function -Print the optimal paths of the state variables, the optimal controls and the value function over time Step 4: Solve the optimal decision rules: -Set up computational variables and matrices and begin with t=T For each control node, repeat: -Compute instantaneous returns at each node of the control space -Solve daily growth of tissue components and live weight -Consider whether the constraints are binding at any control -Solve the next-period state of nature for each node of the control spaces -Find the next-period value function and the value of a capacity unit Repeat step 4 for each time period t=T,…,0 For each given state of nature: -Solve the controls that maximise the value of a capacity unit -Save the optimal controls and corresponding value function Repeat for all state nodes t=t-1 if t>0 if t=0 If all state nodes at time t evaluated Appendix A: Computer code of the basic model Appendix A: Computer code of the basic model Terminate computation and save the results reported at step 5 101 !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL ! ' 2 ) # 5 , 4 5 2 ! , ! . $ & / / $ 3 # ) % . # % !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL ! ' 2 ) # 5 , 4 5 2 ! , ! . $ & / / $ 3 # ) % . # % !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL ! ' 2 ) # 5 , 4 5 2 ! , ! . $ & / / $ 3 # ) % . # % !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL ! ' 2 ) # 5 , 4 5 2 ! , ! . $ & / / $ 3 # ) % . # % !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL Appendix B: Sensitivity analysis of two-phase feeding Appendix B: Sensitivity analysis of two-phase feeding A3 represents the optimal feeding patterns under two-phase feeding and ﬂexible feeding technology, when producer has incentives to feed the pig ac- cording to its natural growth potential. This scenario is parameterised as the market scenario where the price of pig meat was €1.62. In other words, when the price of pig meat is €1.62 the value of a capac- ity unit for the two-phase feeding in Figure A3 is €979 over the period of 5 years. The corresponding value for the ﬂexible feeding technology is €991. This appendix presents a sensitivity analysis of the two-phase feeding technology. Table A1 illustrates the value of a capacity unit and an approximation of the optimal share of soy meal in both phases of the two-phase feeding when the feed ration is switched at live weight of 45–120 kg. As the pig is always slaughtered below 120 kg live weight, switching at 120 kg live weight in practice is the equivalent of using the same feed ration throughout the fattening period (i.e. one-phase feeding). In addition, Figure Table A2. The value function and approximated optimal share of soy meal in feed under two-phase feeding before (ﬁrst phase) and after (second phase) switching the feed ratios, when the share of soy meal is adjusted once at 45– 120 kg live weight, and the price of pig meat is €1.62. Table A1. The value function and approximated optimal share of soy meal in feed under two-phase feeding before (ﬁrst phase) and after (second phase) switching the feed ratios, when the share of soy meal is adjusted once at 45– 120 kg live weight. Switching weight kg First phase % soy meal Second phase % soy meal Value function € 45 19% 9% 673 55 18% 7% 677 65 18% 7% 678 75 17% 6% 678 85 15% 6% 677 95 14% 4% 673 120) 14% 4% 658 ) Switching the feed ratios at 120 kg live weight is equal to one-phase feeding, because the pig is slaughtered at 116 kg live weight. ! ' 2 ) # 5 , 4 5 2 ! , ! . $ & / / $ 3 # ) % . # % !PPENDIX!#OMPUTERCODEOFTHEBASICMODEL A G R I C U L T U R A L A N D F O O D S C I E N C E Appendix B: Sensitivity analysis of two-phase feeding Appendix B: Sensitivity analysis of two-phase feeding Switching weight kg First phase % soy meal Second phase % soy meal Value function € 45 17% 7% 974 55 15% 7% 976 65 15% 6% 979 75 15% 6% 978 85 14% 4% 977 95 11% 4% 973 120) 9% 2% 965 *) Switching the feed ratios at 120 kg live weight is equal to one-phase feeding, because the pig is slaughtered at 116 kg live weight. 0 2 4 6 8 10 12 14 16 18 20 1 11 21 31 41 51 61 71 81 91 101 time in days Two phase 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Flexible % of feed Fig. A3. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in feed under two-phase feeding and as approximated in the benchmark scenario of the ﬂexible feeding technology. The price of pig meat in both scenarios is €1.62. 0 2 4 6 8 10 12 14 16 18 20 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 kg feed per day time in days time in days Fig. A3. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in feed under two-phase feeding and as approximated in the benchmark scenario of the ﬂexible feeding technology. The price of pig meat in both scenarios is €1.62. 116 A G R I C U L T U R A L A N D F O O D S C I E N C E Appendix C: Feeding patterns of female pigs and castrated male pigs Appendix C: Feeding patterns of female pigs and castrated male pigs This appendix presents the optimised feeding pat- terns for female and castrated male pigs, and their position with respect to the benchmark scenario. The Figures are based on optimising feeding and slaughter patterns separately for female pigs and castrated male pigs. The only difference between these scenarios and the benchmark scenario is that the parameters of the growth potential of the pig and the minimum fat-to-lean growth constraint are given gender-speciﬁc values. The price of pig meat in these scenarios is €1.39. The polynomial ap- proximations presented in Figures A4 and A5 cor- respond to the €1.39 curves reported in Figures 28 and 29 in Chapter 6. 1 11 21 31 41 51 61 71 81 91 101 time in days Female pig Fit of female pig 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Benchmark 0 5 10 15 20 25 % of feed 1 11 21 31 41 51 61 71 81 91 101 0 5 10 15 20 25 % of feed 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 kg feed per day time in days Fig. A4. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the female pig scenario. The ﬁt of female pig represents a polynomial approximation of the female pig scenario. 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Fit of castrated male pig 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Benchmark Castrated male pig Fig. A5. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the castrated male pig scenario. The ﬁt of castrated male pig represents a polynomial approximation of the castrated male pig scenario. Appendix D: Sensitivity analysis for genotype scenarios Appendix D: Sensitivity analysis for genotype scenarios trated male pigs. The reduced ratios are for pigs of improved genotype (Whittemore 1998, p. 68–70 and 557–558). In other words, the change allows the producer to increase the degree of restricting of feeding. The change increases the convexity of the optimal feeding pattern. As the constraint is locally binding in the benchmark scenario, the change in- creases the share of soy meal in feed and decreases the amount of barley. Notably, the pattern of change in feed ratios is larger for young pigs than for pigs close to the optimal slaughter maturity. This appendix presents sensitivity analyses with respect to constraints used in the model, and additional analyses of the effects of price changes. The results below utilise the growth parameters of the genotype and split-sex feeding scenarios. The price ratios are exactly the same as those used in corresponding market scenarios in Chapter 5. In addition, Figures A6 and A7 represent changes in feeding patterns when the constraint on the mini- mum growth ratio of fat to lean is reduced by 0.2 units below the initial values for female and cas- 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Mi ti 0 8 Fit f i ti 0 8 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Min ratio 0.8 Fit of min ratio 0.8 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Min ratio 1 Fig. A6. The effect of reducing the minimum growth ratio from 1 to 0.8 on the optimal feeding patterns for castrated male pigs. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 kg feed per day % of feed 15 time in days time in days Min ratio 1 Min ratio 1 Fig. A6. The effect of reducing the minimum growth ratio from 1 to 0.8 on the optimal feeding patterns for castrated male pigs. for castrated male pigs. spect to price changes for female and castrated male pigs when changes in price ratios are similar to the market scenarios. Appendix C: Feeding patterns of female pigs and castrated male pigs 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 % of feed 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 kg feed per day time in days time in days Fit of castrated male pig Benchmark Castrated male pig Benchmark Castrated male pig Fig. A5. The amount of barley (kg per day, left panel) and the share of soy meal (% of the total amount of feed, right panel) in the benchmark scenario and in the castrated male pig scenario. The ﬁt of castrated male pig represents a polynomial approximation of the castrated male pig scenario. 117 A G R I C U L T U R A L A N D F O O D S C I E N C E Appendix D: Sensitivity analysis for genotype scenarios Appendix D: Sensitivity analysis for genotype scenarios Tables A6, A7, A8 and A9 repeat similar sensitivity analyses for pigs having a low K-index, a high K-index, improved maturing rate and improved mature weight. The effects of changes in the minimum fat to lean growth ratio on the numerical results are re- ported in Table A3 for all genotype and split-sex feeding scenarios. The results utilise price ratios similar to those in the benchmark scenario. Tables A4 and A5 represent sensitivity analyses with re- 118 118 A G R I C U L T U R A L A N D F O O D S C I E N C E Appendix D: Sensitivity analysis for genotype scenarios Table A4. Time required to reach slaughter maturity, carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years for castrated male pigs under alternative price parameters. The price ratios used in the scenarios correspond to those used in the market scenarios in Chapter 5 Scenario Time to slaughter Carcass weight Red meat Value function days kg % € per 5 years Benchmark price ratios 107 85.6 61.3 674 High price of pig meat 91 83.7 57.2 979 High price of piglet 107 85.1 61.7 578 High price of barley 107 85.3 61.5 652 High price of soy 103 84.8 60.5 661 High price of all feeds 107 85.3 61.5 620 High slaughter premium 99 84.6 59.3 771 Table A5. Time required to reach slaughter maturity, carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years for female pigs under alternative price parameters. The price ratios used in the scenarios correspond to those used in the market scenarios in Chapter 5 price parameters. The price ratios used in the scenarios correspond to those used in the market scenarios in Chapter 5. Scenario Time to slaughter Carcass weight Red meat Value function days kg % € per 5 years Benchmark price ratios 104 84.3 62.1 696 High price of pig meat 96 84.4 59.4 994 High price of piglet 108 85.4 62.8 599 High price of barley 104 83.8 62.4 674 High price of soy 104 85.1 61.7 681 High price of all feeds 104 83.8 62.4 641 High slaughter premium 100 84.4 60.7 791 Table A6. Time required to reach slaughter maturity, carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years under alternative price parameters for pigs having improved mature weight. Appendix D: Sensitivity analysis for genotype scenarios Appendix D: Sensitivity analysis for genotype scenarios Appendix D: Sensitivity analysis for genotype scenarios Fig. A7. The effect of reducing the minimum growth ratio from 0.9 to 0.7 on the optimal feeding patterns for female pigs. 0 5 10 15 20 25 1 11 21 31 41 51 61 71 81 91 101 time in days % of feed Min ratio 0.7 Fit of min ratio 0.7 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 time in days kg feed per day Min ratio 0.9 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 11 21 31 41 51 61 71 81 91 101 kg feed per day % of feed time in days time in days Min ratio 0.9 Min ratio 0.7 Fig. A7. The effect of reducing the minimum growth ratio from 0.9 to 0.7 on the optimal feeding patterns for female pigs. Table A3. Time required to reach slaughter maturity, carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years under alternative growth ratio constraints in genotype and split-sex feeding scenarios. Scenario Time to slaughter Carcass weight Red meat Value function days kg % €/5 years Castrated male pig, ratio is 1 107 85.6 61.3 674 Castrated male pig, ratio is 0.8 107 84.5 61.9 676 Female pig, ratio is 0.9 104 84.3 62.1 696 Female pig, ratio is 0.7 108 85.1 63 698 Improved mature weight, ratio is 1 95 85.2 61.7 779 Improved mature weight, ratio is 0.8 98 85.1 63 784 Improved maturing rate ratio is 1 94 84.8 61.3 772 Improved maturing rate, ratio is 0.8 95 84 62.1 775 Low K-index, ratio is 1 108 85.3 61.4 661 Low K-index, ratio is 0.8 108 83.8 62.2 664 High K-index, ratio is 1 100 85 61.6 725 High K-index, ratio is 0.8 104 85.1 62.9 729 Table A3. Time required to reach slaughter maturity, carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years under alternative growth ratio constraints in genotype and split-sex feeding scenarios. 119 A G R I C U L T U R A L A N D F O O D S C I E N C E Appendix D: Sensitivity analysis for genotype scenarios Appendix D: Sensitivity analysis for genotype scenarios Table A7. Time required to reach slaughter maturity, carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years under alternative price parameters for pigs having improved maturing rate. The price ratios used in the scenarios correspond to those used in the market scenarios in Chapter 5 p Scenario Time to slaughter Carcass weight Red meat Value function days kg % € per 5 years Benchmark price ratios 94 84.8 61.3 772 High price of pig meat 85 84.6 58 1107 High price of piglet 96 85 61.8 664 High price of barley 96 85.2 61.7 750 High price of soy 92 84.2 60.9 756 High price of all feeds 95 84.9 61.6 713 High slaughter premium 88 83.4 59.8 879 Table A8. Time required to reach slaughter maturity, carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years under alternative price parameters for pigs having a low K-index. The price ratios used in the scenarios correspond to those used in the market scenarios in Chapter 5. p Scenario Time to slaughter Carcass weight Red meat Value function days kg % € per 5 years Benchmark price ratios 108 85.3 61.4 661 High price of pig meat 96 84.9 57.7 956 High price of piglet 108 84.6 61.7 566 High price of barley 108 85 61.6 639 High price of soy 104 83.6 61 648 High price of all feeds 108 85 61.6 607 High slaughter premium 100 83.2 59.9 755 Table A9. Time required to reach slaughter maturity, carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years under alternative price parameters for pigs having a low K-index. The price ratios used in the scenarios correspond to those used in the market scenarios in Chapter 5 Table A9. Time required to reach slaughter maturity, carcass weight upon slaughter, share of red meat in the slaughtered carcass, and value function over the period of 5 years under alternative price parameters for pigs having a low K-index. Appendix D: Sensitivity analysis for genotype scenarios The price ratios used in the scenarios correspond to those used in the market scenarios in Chapter 5 Scenario Time to slaughter Carcass weight Red meat Value function days kg % € per 5 years Benchmark price ratios 95 85.2 61.7 779 High price of pig meat 85 84.4 58.2 1108 High price of piglet 95 85 61.8 671 High price of barley 95 85.1 61.8 757 High price of soy 95 85.5 61.6 762 High price of all feeds 95 85.1 61.8 720 High slaughter premium 95 85.6 61.4 884 120 A G R I C U L T U R A L A N D F O O D S C I E N C E Appendix D: Sensitivity analysis for genotype scenarios Appendix D: Sensitivity analysis for genotype scenarios The price ratios used in the scenarios correspond to those used in the market scenarios in Chapter 5 p Scenario Time to slaughter Carcass weight Red meat Value function days kg % € per 5 years Benchmark price ratios 100 85 61.6 725 High price of pig meat 92 84.8 58.9 1037 High price of piglet 100 84.7 61.8 623 High price of barley 100 84.8 61.7 703 High price of soy 100 85.2 61.4 710 High price of all feeds 100 84.9 61.7 669 High slaughter premium 97 85.1 60.5 825 121
https://openalex.org/W2913733485	https://www.frontiersin.org/articles/10.3389/fpsyg.2019.00174/pdf	English	null	Improving Cognitive Performance of 9–12 Years Old Children: Just Dance? A Randomized Controlled Trial	Frontiers in psychology	2,019	cc-by	11,969	ORIGINAL RESEARCH published: 06 February 2019 doi: 10.3389/fpsyg.2019.00174 Vera van den Berg1, Emi Saliasi1, Renate H. M. de Groot2,3, Mai J. M. Chinapaw1 and Amika S. Singh1* 1 Department of Public and Occupational Health, Amsterdam Public Health Research Institute, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 2 Welten Institute – Research Centre for Learning, Teaching and Technology, Open University of the Netherlands, Heerlen, Netherlands, 3 Department of Complex Genetics, School for Nutrition, Toxicology and Metabolism, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, Netherlands Exercise is assumed to have positive effects on children’s cognitive performance. However, given the inconclusive evidence for the long-term effects of exercise, it is difﬁcult to advice schools on what speciﬁc exercise programs can improve children’s cognitive performance. In particular, little is known about the effects of small exercise programs that may be feasible in daily school practice. Therefore, we assessed the effects of a 9-weeks program consisting of daily exercise breaks on children’s cognitive performance, aerobic ﬁtness and physical activity levels. We conducted a cluster- randomized controlled trial in 21 classes of eight Dutch primary schools. A total of 512 children aged 9–12 years participated. The exercise intervention had a duration of 9 weeks and consisted of a daily 10-min classroom-based exercise break of moderate to vigorous intensity. Before and after the intervention, we used four cognitive tasks (i.e., the Attention Network Test, Stroop test, d2 test of attention and Fluency task) to measure children’s cognitive performance in domains of selective attention, inhibition and memory retrieval. In addition, we measured aerobic ﬁtness with a Shuttle Run test and physical activity during school hours by accelerometers. We analyzed data using mixed models, adjusting for baseline scores, class and school. After 9 weeks, there were no intervention effects on children’s cognitive performance or aerobic ﬁtness. Children in the intervention group spent 2.9 min more of their school hours in moderate to vigorous physical activity as compared to the children in the control group. In conclusion, daily 10-min exercise breaks in the classroom did not improve, nor deteriorate cognitive performance in children. The exercise breaks had no effect on children’s ﬁtness, and resulted in 2.9 min more time spent in moderate to vigorous physical activity during school hours. Daily exercise breaks can be implemented in the classroom to promote children’s physical activity during school time, without adverse effect on their cognitive performance. Edited by: Henning Budde, Medical School Hamburg, Germany Reviewed by: Sebastian Ludyga, Universität Basel, Switzerland Annalisa Setti, University College Cork, Ireland Correspondence: Amika S. Singh a.singh@vumc.nl Correspondence: Amika S. Singh a.singh@vumc.nl Specialty section: This article was submitted to Movement Science and Sport Psychology, a section of the journal Frontiers in Psychology Received: 28 August 2018 Accepted: 18 January 2019 Published: 06 February 2019 Abbreviations: MVPA, moderate to vigorous physical activity; PA, physical activity. ywords: physical activity, exercise, selective attention, inhibition, memory, aerobic ﬁtness, MVPA, children Improving Cognitive Performance of 9–12 Years Old Children: Just Dance? A Randomized Controlled Trial Vera van den Berg1, Emi Saliasi1, Renate H. M. de Groot2,3, Mai J. M. Chinapaw1 and Amika S. Singh1* INTRODUCTION Furthermore, several acute and long-term studies have shown that cognitively demanding exercise (e.g., coordinative exercise, team games) can improve cognitive performance to a higher extent than mere repetitive aerobic exercise (e.g., Budde et al., 2008; Koutsandreou et al., 2016; Schmidt et al., 2016), likely due to the inherent motor and cognitive demands (Best, 2010; Tomporowski et al., 2015). Acute cognitively demanding exercise requires high cognitive eﬀort due to exercise complexity and changing circumstances, which may provide long-term improvement of self-control capacities and cognitive functioning (Best, 2010; Audiﬀren and André, 2015). This type of exercise could also result in higher intervention compliance, since challenge and variety seem important for children’s exercise motivation (e.g., Martins et al., 2015). Recently, an international expert panel indicated that there is a need for more well-designed, randomized controlled (RCT) trials to gain better insight in the causal eﬀects of exercise on cognition (Singh et al., 2018). In addition, the experts highlighted the importance of elucidating the characteristics of exercise interventions that may improve cognitive performance. Due to substantial heterogeneity in interventions (e.g., duration, frequency, content), it is diﬃcult to advise schools on the optimal form of exercise interventions to improve children’s cognitive performance (Donnelly et al., 2016; Watson et al., 2017; Singh et al., 2018). The vast majority of studies that examined the long-term eﬀects of exercise on cognitive performance of children have implemented extensive exercise interventions with durations of 30–60 min per session, mostly delivered three to ﬁve times a week (Donnelly et al., 2016; Alvarez-Bueno et al., 2017; Singh et al., 2018). However, it seems unlikely that such time-consuming programs will be implemented on a large scale in real-life daily school practice. Several qualitative studies have reported that time constraints are perceived as a major barrier that limit the opportunities for physical activity and exercise in schools (Howie et al., 2014b; McMullen et al., 2014; Stylianou et al., 2015; Dinkel et al., 2017; van den Berg et al., 2017). In addition, teachers indicate that it would only be feasible to implement short exercise bouts with a maximum duration of 5–10 min (Howie et al., 2014b; van den Berg et al., 2017). To the best of our knowledge, no previous studies investigated the long-term eﬀects of short exercise breaks (i.e., 10 min) in the classroom on cognitive performance of preadolescents (aged 9–12 years). Costigan et al. INTRODUCTION 2014; Donnelly et al., 2016; Ludyga et al., 2016). However, it is still unclear whether these acute eﬀects accumulate over time, i.e., if implementing short exercise bouts on a regular basis can improve children’s cognitive performance after weeks or months. The assumed positive relationship between exercise and cognitive performance is widely used to advocate in favor of increasing exercise opportunities in schools (e.g., Erwin et al., 2012; Webster et al., 2015; Savina et al., 2016). In particular, since cognitive performance in domains such as selective attention, inhibition, working memory and cognitive ﬂexibility, has been shown to be important for children’s academic performance (Stevens and Bavelier, 2012; Jacob and Parkinson, 2015). However, recent systematic reviews and meta-analyses have shown that the evidence for the long-term eﬀects of structured exercise programs on children’s cognitive performance is inconclusive; some studies report positive eﬀects, while others report no eﬀects (see for reviews Donnelly et al., 2016; Li et al., 2017; Watson et al., 2017; Singh et al., 2018). Nevertheless, it can be concluded that increasing the time spent on exercise in school at the cost of academic lessons does not negatively impact children’s cognitive performance (Donnelly et al., 2016; Singh et al., 2018). Several potential mechanisms underlying the eﬀects of exercise on cognition have been discussed in the literature. For example, acute eﬀects of exercise have been related to increased blood ﬂow (Ogoh and Ainslie, 2009), increased release of neurotrophic factors, such as brain derived neurotropic factor (BDNF) and insulin-like growth factor-1 (Piepmeier and Etnier, 2015), increased arousal levels (McMorris and Hale, 2015), and increased activity in certain brain areas (Budde et al., 2008). Mechanisms of chronic exercise eﬀects include increased availability of growth factors (e.g. BDNF), development of new blood vessels and neurons, changes in brain volume, increased eﬃciency of neural networks, and increased physical ﬁtness (see for reviews Hillman et al., 2008; Huang et al., 2014; Fernandes et al., 2017). In addition, chronic exercise is suggested to improve self-control, which is important for self-regulation and functioning of higher cognitive functions (Audiﬀren and André, 2015). Some of the above-mentioned mechanisms may explain cumulative eﬀects of acute exercise. For example, acute exercise- induced elevations of BDNF have been shown to be augmented by repeated exercise, resulting in increased resting levels of BDNF important for cognitive improvements and brain changes (see for a review Huang et al., 2014). Citation: van den Berg V, Saliasi E, de Groot RHM, Chinapaw MJM and Singh AS (2019) Improving Cognitive Performance of 9–12 Years Old Children: Just Dance? A Randomized Controlled Trial. Front. Psychol. 10:174. doi: 10.3389/fpsyg.2019.00174 Keywords: physical activity, exercise, selective attention, inhibition, memory, aerobic ﬁtness, MVPA, children Abbreviations: MVPA, moderate to vigorous physical activity; PA, physical activity. Abbreviations: MVPA, moderate to vigorous physical activity; PA, physical activity. February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 1 Exercise Breaks and Cognition van den Berg et al. Frontiers in Psychology \| www.frontiersin.org INTRODUCTION (2016) examined the eﬀects of two 8-week interventions, in which 8- to 10-min exercise bouts consisting of (1) high intensity aerobic exercises or (2) high intensity combined aerobic and strength exercises were implemented three times per week in 14–16 years old adolescents. The exercise bouts were implemented once a week during recess and twice a week as part of the regular physical education (PE) classes. The authors found no signiﬁcant diﬀerences in executive functioning between the intervention groups and the control group that followed the regular PE classes (Costigan et al., 2016). Little contrast in the amount of additional exercise in the three groups and the absence of measures to compare adolescent’s physical activity levels limit conclusions about the exercise related eﬀects on cognitive performance. Furthermore, the authors indicated that the relatively small sample (N = 65) Previous studies have focused on the acute, or immediate, eﬀects of relatively short exercise bouts on cognitive performance, such as attention, inhibition and working memory (e.g., Niemann et al., 2013; Howie et al., 2015; van den Berg et al., 2016). Several systematic reviews and meta-analyses concluded that overall, single moderate to vigorous exercise bouts with a minimum duration of 10 min can have small to moderate acute positive eﬀects on children’s classroom behavior (i.e., time-on-task) (Watson et al., 2017; Daly-Smith et al., 2018), selective attention (Chang et al., 2012; Janssen et al., 2014; de Greeﬀet al., 2018), and executive functioning (Chang et al., 2012; Verburgh et al., February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 2 Exercise Breaks and Cognition van den Berg et al. from one secondary school limits the generalizability of their results (Costigan et al., 2016). all children participated in the intervention/control program as part of the regular school curriculum. Permission of at least one parent/caregiver and children of 12 years and older was required to participate in the measurements. We received informed consent of 512 children (93%), who were included in the study. The study was approved by the Medical Ethical Committee of the VU University Medical Center Amsterdam [2014.363]. To ﬁll this gap, we conducted a cluster RCT trial to investigate the eﬀects of a 9-week exercise break program on cognitive performance of 9–12 years old Dutch primary school children. Procedure We used G∗power 3.1.9.2 (Faul et al., 2007) to calculate the required sample size. In line with earlier studies and meta- analytic ﬁndings, we expected to ﬁnd a small to medium eﬀect of our exercise intervention on children’s cognitive performance (e.g., Schmidt et al., 2015; Costigan et al., 2016; Vazou et al., 2016). The sample size calculation revealed that we needed to include a total of 404 participants (N = 202 per group) to detect a small to medium eﬀect (f = 0.18) of the intervention on children’s cognitive performance, with a power of 95% (two-sided testing at α = 0.05). Before the experiment, we trained the research team to conduct the measurements following a standardized protocol. We visited each class six times (see Figure 1). The ﬁrst visit consisted of a familiarization session in which we introduced the study and explained all measurement procedures. Children received detailed instructions about four cognitive tasks and practiced all tasks to make sure they understood them well. Furthermore, children ﬁlled out a demographics questionnaire and we measured their baseline height, weight, and ﬁtness. During the second visit, we conducted the baseline measurements (pretest) in which the cognitive tasks and a questionnaire were administered. We randomly assigned the children in each class to group A and B, stratiﬁed by gender (10–15 children per group, depending on class size). Group A started in the classroom where two paper-and-pencil cognitive tasks and the questionnaire were administered, while group B started in a separate room where two computerized cognitive tasks were administered on laptops. Halfway the test session, the groups switched rooms and continued with the other half of the measurements. In one school (N = 2 classes) there was no private room available, so we administered the paper-and-pencil tasks with the entire class, and divided the classroom in two testing areas for the laptop tasks and questionnaire. The week after the baseline measures, the classes started with the 9-week intervention/control program. The third and fourth visit were scheduled during the intervention period to: (1) hand out accelerometers in a subgroup of children, and (2) measure the exercise intensity of one exercise break in the INTRODUCTION The intervention consisted of one daily, classroom-based 10-min exercise break in which children were asked to mimic dance movements (i.e., aerobic exercise with coordinative and cognitive demands). The intervention was implemented within the school curriculum, as it has been shown that curricular exercise programs can result in stronger eﬀects on cognition compared to programs that are implemented outside school hours (Alvarez- Bueno et al., 2017). Moreover, Dutch teachers have indicated that classroom-based physical activity is most feasible in daily school practice (van den Berg et al., 2017). Study Design, Randomization and Blinding We conducted a cluster RCT. An independent statistician randomly assigned the participating classes to the intervention (N = 11) or control group (N = 10). Randomization was performed in R using block randomization with blocks of size 2. The randomization was stratiﬁed by school and grade for the schools in which multiple classes of the same grade took part. Randomization for the remaining schools was done by randomly assigning the 5th grade to one of the two conditions (with the 6th grade automatically receiving the alternative). This procedure ensured that in each school there were both control and intervention classes and that number of control and intervention classes was balanced between the two grades. The randomization took place after the pretest measurements to ensure that all children, teachers and researchers were blinded. Two members of the research team remained blinded the entire experiment and acted as test administrators at the posttest measurements. We examined the eﬀects of the intervention on selective attention, inhibition, and semantic memory retrieval, since these cognitive domains are associated with children’s academic performance (Rueda et al., 2010; Stevens and Bavelier, 2012). As secondary outcomes, we measured children’s aerobic ﬁtness and their physical activity levels during school hours. Given the earlier reported acute eﬀects of short exercise bouts, we hypothesized that implementing a daily exercise break will have a positive eﬀect on children’s cognitive performance after 9 weeks. Frontiers in Psychology \| www.frontiersin.org February 2019 \| Volume 10 \| Article 174 Recruitment of Participants We approached a convenience sample of regular primary schools from the network of our research group by email and personal contact. Twenty-three schools across the Netherlands received an information letter and were asked to respond if they were interested to participate. We included schools that were willing to participate with a minimum of two classes. For feasibility reasons, we decided to stop the inclusion after eight schools agreed to participate. Two schools declined due to busy school schedules and one school was excluded since they had only one class available. Twelve schools did not respond, but were neither followed-up since we reached the required sample size with schools that responded to our ﬁrst invitation. All children in grades 5 and 6 (N = 549) were invited to participate. Children and their parents/caregivers received an information letter about the study, including an informed consent form. In consultation with the schools it was decided that February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 3 Exercise Breaks and Cognition van den Berg et al. FIGURE 1 \| Overview and timeline of the study. FIGURE 1 \| Overview and timeline of the study. intervention classes. After the intervention period, we conducted the post-intervention measurements (ﬁfth visit: posttest), which were identical to the pretest and scheduled at the same day of the week and time of the day. To avoid contamination of the eﬀects by possible acute exercise eﬀects, we instructed all teachers not to perform an exercise break on the measurements days. During the sixth visit we measured children’s ﬁtness again. After the experiment, all children received a small symbolic present for their participation. via a secured webpage. The exercise breaks were performed in the classroom and we asked teachers to make sure that all children kept moving. The exercise break program was performed in addition to the regular PE classes. g The exercise breaks were selected based on a pilot study, in which we tested 83 Just Dance videos once (100%) or twice (67%) in 31 grades 5 and 6 of 14 primary schools (unpublished data). Children (N = 766) wore heart rate monitors to determine the mean exercise intensity of each video and we asked them to rate the videos on being fun and diﬃcult, respectively. In addition, we observed the feasibility of performing the dances in the classroom. Recruitment of Participants The pilot resulted in the selection of 55 suitable videos that were used to compose 45 diﬀerent exercise breaks used in the current study. To ensure variety in the program, each exercise break consisted of a unique combination of three videos. Each video returned two or three times during the 9 weeks period, each time combined with two other videos. Intervention Program The intervention lasted 9 weeks and consisted of one moderate to vigorous intensity exercise break per school day. The intervention duration was chosen for feasibility reasons. A period of 9 weeks best ﬁtted the school’s year schedules and was relatively short, by which we aimed to keep compliance high during the entire intervention period. p Each exercise break lasted approximately 10 min and consisted of three ‘Just Dance’ videos (Ubisoft, free available via YouTube). In the videos, a ﬁgure performs a dance which the children are asked to mimic. Our choice for Just Dance videos and the exercise duration of 10 min was based on the acute exercise literature combined with the preferences of Dutch teachers and children in the upper grades of primary school. The exercise literature indicates that moderate-to-vigorous exercise bouts need to have a duration of at least 10 min to exert acute cognitive improvements (Howie et al., 2014a, 2015; Daly-Smith et al., 2018). Teachers have indicated that they prefer additional PA in school to be classroom-based, easy to implement (i.e., requiring little preparation time) and up to a maximum of 10 min (van den Berg et al., 2017). Accordingly, many teachers in the Netherlands already use Just Dance in the classroom setting. A recent study of our group revealed that one of the ideas of children to increase PA in school, that matched the preferences of teachers, is to implement short exercise breaks during classroom time, for example Just Dance (van den Berg et al., 2018). Teachers received an instruction sheet with a password to access the exercise breaks Frontiers in Psychology \| www.frontiersin.org Control Program The control program consisted of nine educational lessons, lasting 10–15 min, one for each week of the experiment. The lessons were unrelated to the core school curriculum. We composed the lessons using information and educational videos on topics related to the body, exercise and/or sports for 9–12 year olds (free available online; see for example NTR, 2012). During six lessons children watched a short 5-min video (e.g., about agility in gymnasts, the role of balance in sports, or endurance in runners) and were asked to answer four to ﬁve questions based on what they learned from watching the video. In three lessons, children were asked to read one page of written information, for example about bones, joints and muscles in the body or about the respiratory system, and to answer ﬁve questions based on this information. The teacher discussed the answers with the entire class at the end of each lesson. Teachers received an instruction card including a password to access a secured webpage with the instructions, videos, worksheets and answers for each lesson. During the February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 4 Exercise Breaks and Cognition van den Berg et al. experiment, children in the control group followed the regular PE classes. of the maximum heart rate: (mean heart rate/maximum heart rate)∗100. The maximum heart rate was measured during the Shuttle Run test (see Aerobic Fitness). d2 Test of Attention The d2 test was used to measure selective attention (Brickenkamp and Oosterveld, 2012). The construct validity of the d2 test has been rated as suﬃcient (Egberink and Vermeulen, 2018c). The reliability has been rated as good, with moderate to high test- retest reliability in 10–13 years old Dutch children (r = 0.79–0.83) (Brickenkamp and Oosterveld, 2012; Egberink and Vermeulen, 2018c). We measured children’s body height (cm) and weight (kg) in sport clothes without shoes, using a Leicester Height Measure Mk II (Harlow Healthcare, United Kingdom) and a Seca weighting scale (Seca Instruments, Frankfurt, Germany). The Body Mass Index (BMI) of each child was calculated with the formula: [weight (kg)/height (m)2]. The d2 test consists of one page with fourteen lines, each consisting of 47 characters ‘d’ and ‘p’ with one to four dashes displayed above and/or below. We instructed the children to mark as much letters ‘d’ with a total of two dashes (‘d2’) as possible, while ignoring the other characters. They had to work from the left to the right, with a time limit of 20 s per line. The test instructor gave a signal when to continue with the next line. The total test lasted 4 min and 40 s. Fluency Task We used a paper-and-pencil version of the Verbal Fluency task (Mulder et al., 2006) to measure semantic memory retrieval performance. The validity and reliability of the Verbal Fluency task has been shown suﬃcient in children and adolescents (Korkman et al., 1998; Egberink and Vermeulen, 2018b). Intervention Integrity Each class received a calendar-poster that was attached to the classroom wall and remained visible during the intervention. We asked teachers and children to put a sticker on the poster each time they performed an exercise break (intervention group) or an educational lesson (control group). The poster served as a reminder to implement the program, as well as a measure of intervention integrity. We calculated the percentage of exercise breaks that were conducted, with 45 exercise breaks equaling 100% implementation. Halfway the intervention, we asked teachers to report potential implementation problems. In case of problems, we gave advice and encouraged teachers to implement as many exercise breaks or educational lessons as possible. We used the concentration performance (i.e., number of correctly marked d2’s minus the number of incorrectly marked characters) as dependent variable, since this is an objective measure of selective attention (Brickenkamp and Oosterveld, 2012). Primary Outcomes: Cognitive Performance Children self-reported their birth date, gender and sports participation. The questions on sports participation were derived from the ENERGY-child questionnaire, showing good to excellent test-retest reliability (ICC’s: 0.68–1.00) and moderate to excellent construct validity (ICC’s: 0.51–1.00) (Singh et al., 2011). The Dutch version of the Harter’s Self Perception Proﬁle for Children was administered to measure children’s perceived competence in ﬁve domains (scholastic, social, athletic, physical appearance, behavioral conduct) and their perceived global self- worth (Veerman et al., 1997). This questionnaire has been shown to have suﬃcient construct validity and good test–retest reliability (ICC’s ≥0.84) in 8–14 years old Dutch children (Muris et al., 2003; Egberink and Vermeulen, 2018a). Parents self-reported their highest completed educational level, which was used as a proxy measure of socio-economic status. We measured cognitive performance with two paper-and-pencil tasks, i.e., the d2 Test of Attention and the Fluency Task, and two computerized tasks, i.e., the Stroop Color-Word task and Attention Network Task (ANT) using E-prime 1.2 Software (Psychology Software Tools, Pittsburgh, PA, United States). During the pre- and posttest, children received standardized verbal and written instructions and made a few practice trials (d2 test, Stroop, ANT). Two trained and blinded test instructors gave task instructions for all tests and kept track of time in case of the d2 test and Fluency task. During the tests, two to three members of the research team each supervised a small group of children and made notes. We instructed the children to work quietly, individually, and as fast and accurately as possible. The order in which the tests were administered was counterbalanced and randomized, stratiﬁed by gender, grade and intervention/control group. The order of tests was identical during the pre- and posttest and each child made all tests on the same laptop. We asked teachers to provide standardized test scores of the children on reading comprehension, orthography and arithmetic. Scores were obtained from the standardized and norm-referenced CITO test battery (Hollenberg and Van Der Lubbe, 2017), which most schools in the Netherlands administer twice a year to assess and track children’s academic performance. After the experiment, teachers provided information on children with special educational needs (e.g., ADHD, autism spectrum disorders, learning disorders). Exercise Break Intensity We assessed the intensity of a subsample of exercise breaks by monitoring heart rate (11 exercise breaks; one per intervention class). All children were ﬁtted with a Polar H7 Bluetooth heart rate monitor that was connected to the Polar Team App (Polar Electro Oy, Finland) in which the mean heart rate of each child was stored. Exercise intensity was calculated as percentage Children were instructed to write down as many words as possible in the category ‘animals’ within 60 s. The total number of correct words was used as dependent variable. February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 5 Exercise Breaks and Cognition van den Berg et al. Secondary Outcomes Aerobic Fitness We conducted a Shuttle Run test to assess children’s aerobic ﬁtness (Léger et al., 1988). Due to the limited dimensions of the sports halls, all children performed the test over a distance of 18 m instead of 20 m. The highest completed stage was recorded with an accuracy of a half stage and was used to estimate children’s VO2max (Léger et al., 1988). All children were familiar with the test and were encouraged by the research team to exert maximum performance. Children wore heart rate monitors (Polar H7, Polar Team App) to determine their maximum heart rate. Sets of ﬁve horizontal black arrows pointing to the right or left were presented on a white 15-inch laptop screen. Children were instructed to identify the direction of the middle arrow (the ‘target’), by pressing the right mouse button for the right direction and the left mouse button for the left direction. The central target was ‘ﬂanked’ by two lateral arrows on the left and on the right, pointing either in the same direction (congruent; >>>>> or <<<<<) or in the opposite direction (incongruent; >><>> or <<><<). A ﬁxation cross remained visible in the middle of the screen during the task. In two-third of the trials, a warning cue (∗) was presented for 200 ms either above or below (spatial cue) or at the place of the ﬁxation cross (center cue) before the stimuli appeared. The total task lasted approximately 12 min and contained three blocks of 48 trials, with 1-min breaks in between. Physical Activity Levels We measured children’s PA during the intervention period with GT3x ActiGraph accelerometers (De Vries et al., 2009). In each class, we randomly selected a subgroup of 11–19 children that were asked to wear the device during waking hours for seven consecutive days, including the weekend (mean of 15 children per class; total n = 330). We gave children verbal instructions on how to wear the device and provided them and their parents/caregivers with an information sheet including a web-link to an online instruction video. ActiLife 6.13.3 software (ActiGraph, LCC.) was used to initialize the accelerometers and for processing the data (epoch = 15 s). We calculated the mean reaction time (correct responses only) and accuracy (proportion of correct responses) by the formulas of Fan et al. (2007): Alerting eﬀect = (SCORE no cue – SCORE center cue); Orienting eﬀect = (SCORE center cue - SCORE spatial cue); Conﬂict eﬀect (executive control) = (SCORE incongruent - SCORE congruent). Larger reaction time scores indicate better alerting and orienting performance, while a smaller value indicates better conﬂict performance. For accuracy, a larger value indicates better alerting performance, a larger negative value better orienting performance, and a smaller negative value better conﬂict performance. Reaction times faster than 200 ms were considered as incorrect and excluded from the data analysis (Fan et al., 2007). g We calculated children’s PA levels during school hours only. We included children in the data analysis when they wore the accelerometer at least 4 week days (Yildirim et al., 2011). We created a time ﬁlter for each school to extract only the exact school hours for analysis (e.g., 08:30 a.m. to 15:00 p.m.). Recess time was included in the analyses, because this is part of a regular school day for both intervention and control group. Non-wear time was deﬁned as having 20 min consecutive zero’s (Yildirim et al., 2011). We used the cut points of Evenson (Evenson et al., 2008) to estimate the time spent in sedentary (0–100 cpm), light (101–2295 cpm), moderate (2296–4011 cpm) and vigorous intensity activity (>4012 cpm), which have been shown to most accurately classify PA intensity levels in children and adolescents (Trost et al., 2011). Stroop Color-Word Task We used a computerized Stroop Color-Word task to assess children’s inhibitory performance. Computerized versions of the Stroop have been shown to have moderate to good test-retest reliability in children (r = 0.50–0.80) (Penner et al., 2012). During the task, a color-word (the Dutch word for BLUE, GREEN, or RED) was presented on a 15-inch white laptop screen. In the congruent conditions, the color-word was displayed in a similar text color as the meaning of the word (e.g., the word BLUE displayed in a blue text color). In the incongruent conditions, the text color diﬀered from the meaning of the color-word (e.g., GREEN written in a red text color). Children were instructed to press the button ‘1,’ ‘2’ or ‘3’ at the left side of the key board that corresponded to the text color of the color-word. A ﬁxation cross was presented for 1000 ms, followed by the color-word that was presented for 2500 ms. After a child responded, the color-word disappeared. The inter stimuli interval was 4000 ms. The answer Frontiers in Psychology \| www.frontiersin.org Attention Network Task options, 1 = GREEN, 2 = BLUE, 3 = RED, remained visible at the bottom of the screen. The task consisted of 105 trials and lasted approximately 9 min. We used the short version of the ANT to assess the eﬃciency of three attentional networks: alerting (i.e., achieving and maintaining an alert state), orienting (i.e., selection of information from sensory input) and executive control (i.e., resolving conﬂict among responses) (Fan et al., 2002, 2007). Several studies have recommended the use of the ANT in children, as it has been shown a valid instrument to measure their attentional performance (Rueda et al., 2004; Forns et al., 2014). The task was downloaded from the website of the Sackler Institute for Developmental Psychobiology (Sackler Institute for Developmental Psychobiology [SIDP], 2016). We calculated the interference score as dependent variable by subtracting the scores of the incongruent from the congruent conditions for both reaction time (correct responses only) and accuracy rates. A smaller interference score indicates better inhibition. Intervention Integrity and Exercise Break Intensity The median of implemented exercise breaks was 89%, which corresponds to 4.4 exercise breaks per week during the 9-week intervention (range: 49–98% across classes). The mean exercise intensity of the subsample of tested exercise breaks was 60% (SD 8.5) of the maximum heart rate. g We also found no eﬀects on children’s aerobic ﬁtness, which may be explained by our minimal exercise intervention. This ﬁnding is in line with several systematic reviews reporting that school-based exercise interventions with long durations and high frequencies are needed to improve children’s aerobic ﬁtness (e.g., Kriemler et al., 2011; Dobbins et al., 2013; Braaksma et al., 2018). Another reason for the lack of cognitive eﬀects might be due to the coordinative requirements of our exercise breaks. Our exercise breaks may have been (too) diﬃcult for the children, thus limiting the time they were active at moderate-to-vigorous intensity, which has been suggested to be important to exert cognitive eﬀects (Chang et al., 2012; McMorris and Hale, 2012). Furthermore, high diﬃculty levels might have led to substantial cognitive demands/eﬀort during the exercise breaks, depleting children’s cognitive resources and hindering improvements in We also found no eﬀects on children’s aerobic ﬁtness, which may be explained by our minimal exercise intervention. This ﬁnding is in line with several systematic reviews reporting that school-based exercise interventions with long durations and high frequencies are needed to improve children’s aerobic ﬁtness (e.g., Kriemler et al., 2011; Dobbins et al., 2013; Braaksma et al., 2018). RESULTS Our results are in line with the study of Costigan et al. (2016) who assessed the eﬀect of two 8-week exercise interventions, consisting of short exercise bouts that were implemented three times a week, on executive functioning in adolescents. Although there were several diﬀerences between our study and the study of Costigan and colleagues, such as the sample size (512 versus 65), setting in which the exercise bouts were implemented (classroom versus during recess and PE), exercise intensity (moderate versus high) and age of the participants (9–12 years versus 14– 16 years), there were also similarities. In both studies, the exercise intervention lasted approximately 2 months and consisted of bouts of approximately 10 min. Our ﬁndings do not conﬁrm our hypothesis that acute eﬀects of short exercise bouts on cognition accumulate over time. It is possible that exercise sessions of longer duration are needed to have beneﬁcial eﬀects on cognitive outcomes. In this respect, Ludyga and colleagues, who evaluated an 8-week school-based exercise program in which children performed a daily 20-min exercise bout, reported improvements in working memory (Ludyga et al., 2018b) and inhibition (Ludyga et al., 2018a). Furthermore, a longer intervention period than 9 weeks might be needed to ﬁnd eﬀects of 10-min exercise bouts. Study Population and Descriptive Characteristics A total of 510 children between 9 and 12 years old completed the trial (n = 2 lost to follow-up). The number of children included in the data analyses ranged from 448 to 467, depending on the outcome variable (Figure 2). A ﬂow diagram including the numbers and reasons for exclusion can be found in Figure 2. In addition to common reasons for exclusion (e.g., absence during the pre- or posttest, missing arithmetic score), we excluded 13 children from the d2 test analysis due to a technical mistake in the test administration by one of the test instructors. Baseline characteristics of the control and intervention group were similar, except for age and arithmetic performance (see Table 1). There were no signiﬁcant diﬀerences between the groups in pretest scores on any of the outcome variables. Data Analysis We performed all statistical analyses in SPSS version 22.0 (IBM SPSS Statistics). Independent t-tests and Chi-square tests were used to compare baseline values of the control and intervention group. To test the eﬀect of the intervention, we conducted a separate mixed-model analysis for each cognitive outcome and for aerobic ﬁtness (VO2max). The mixed-model included the cognitive outcome or VO2max as dependent variable and group (i.e., control or intervention) as ﬁxed factor. Class and February 2019 \| Volume 10 \| Article 174 6 Exercise Breaks and Cognition van den Berg et al. An overview of the mean scores, regression coeﬃcients, 95% conﬁdence intervals, and p-values can be found in Table 2. An overview of the mean scores, regression coeﬃcients, 95% conﬁdence intervals, and p-values can be found in Table 2. school were included as random intercepts. Covariates were the pretest score on the dependent variable, age and/or arithmetic performance. The latter two were included because of group diﬀerences at baseline and their expected relationship with the dependent variables. Diﬀerences in PA levels between the intervention and control group were also analyzed by mixed- models, with group as ﬁxed factor, class and school as random intercepts, and total wear time as covariate. The level of signiﬁcance was set at α < 0.05. DISCUSSION Daily exercise breaks did not improve nor harm children’s selective attention, inhibition and semantic memory retrieval performance as compared to the control group. Likewise, there were no eﬀects on children’s aerobic ﬁtness. Children that followed the intervention spent about 3 min more of their school hours in moderate to vigorous PA per day than the children in the control group. PA Levels A total of 312 children (95%) had valid wear time and were included in the data analysis. Children in the intervention group spent on average signiﬁcantly more minutes of their school hours in moderate PA (1.7 min) and moderate to vigorous PA (MVPA; 2.9 min) per day as compared the control group, adjusted for total wear time, class and school (see Table 3). We used an intention-to-treat approach, including all children that participated in the study in the data analyses. However, children with a missing pre- or posttest score of the dependent variables or with a missing score on a covariate, were excluded from the respective analysis. In addition, children who did not fully understand or follow the test instructions [i.e., having accuracy rates below chance level (<50%) in the ANT or Stroop task, or indicated by a note of the researchers] were excluded from the respective analysis. Intervention Effects: Cognitive Performance and Fitness Control group (n = 249) Intervention group (n = 263) p-value Age (years) 10.9 (0.7) 10.8 (0.6) 0.01∗ Special educational needs (%) 13 19 0.06 Sex (%, boys/girls) 53/47 54/46 0.79 Parental educational level (%) (n = 228) (n = 244) 0.25 -low 0 0.8 -low to medium 3.6 1.9 -medium 24.9 28.9 -high 63.1 61.2 Height (cm) (n = 244) (n = 259) 148.7 (7.2) 147.5 (7.6) 0.06 Weight (kg) (n = 238) (n = 258) 38.4 (6.9) 37.7 (7.3) 0.26 BMI (n = 238) (n = 258) 17.3 (2.3) 17.2 (2.3) 0.78 Academic performance (n = 242/244/238) (n = 255/258/258) -reading comprehension 42.6 (15.1) 40.7 (16.2) 0.17 -orthography 140.2 (7.4) 140.1 (7.0) 0.88 -arithmetic 102.1 (14.7) 98.0 (13.9) 0.00∗ Sports participation (hours per week) (n = 244) (n = 258) 3.4 (2.0) 3.3 (1.9) 0.78 Self-competence (n = 236/237) (n = 255/257) -scholastic 17.3 (3.8) 16.9 (3.5) 0.22 -social 19.1 (3.5) 18.8 (3.4) 0.36 -athletic 19.1 (3.5) 18.9 (3.4) 0.53 -physical appearance 20.0 (3.7) 19.9 (3.8) 0.98 -behavioral conduct 18.3 (3.0) 18.3 (2.9) 0.79 -self-worth 20.7 (3.1) 20.5 (3.6) 0.46 VO2max, pretest (ml/kg/min) (n = 236) (n = 253) 48.1 (5.0) 48.0 (5.0) 0.84 d2 test, pretest (n = 237) (n = 258) 133.9 (22.9) 132.1 (22.2) 0.38 Fluency, pretest (n = 240) (n = 258) 10.7 (3.3) 10.8 (3.3) 0.80 Stroop interference, pretest (n = 236) (n = 254) -reaction time (ms) 46.2 (69.2) 41.1 (80.2) 0.45 -accuracy (%) −1.0 (5.4) −1.7 (5.2) 0.14 ANT pretest, reaction time (ms) (n = 236) (n = 253) -Alerting 25.6 (37.3) 24.6 (35.5) 0.78 -Orienting 58.0 (36.5) 61.1 (39.6) 0.37 -Conﬂict 108.4 (45.4) 105.3 (41.5) 0.43 ANT pretest, accuracy (%) (n = 236) (n = 253) -Alerting 0.7 (5.1) 0.8 (4.9) 0.80 -Orienting −1.5 (4.3) −1.3 (4.7) 0.70 -Conﬂict −5.3 (5.0) −5.9 (5.8) 0.27 ∗Signiﬁcant group-difference, p < 0.05. TABLE 1 \| Descriptive characteristics (means and standard deviations) and group differences. ANT pretest, accuracy (%) -Alerting -Orienting -Conﬂict ∗Signiﬁcant group-difference, p < 0.05. meta-analyses reported positive chronic eﬀects of cognitively demanding exercise programs (e.g., Alvarez-Bueno et al., 2017; de Greeﬀet al., 2018). Therefore, we recommend future research to examine how exactly the eﬀects of acute and chronic cognitively demanding exercise relate to each other. Furthermore, research is needed to gain more insight in the optimal dose of the cognitive demands, taking into account children’s motor- and cognitive cognition after exercise. Intervention Effects: Cognitive Performance and Fitness We found no signiﬁcant diﬀerences between the intervention and control group in any of the cognitive outcomes, after controlling for pretest score, age, arithmetic performance, class and school. Children in both groups showed similar patterns of change from pre- to posttest. Thus, the exercise intervention did not improve cognitive performance of the children as compared to the control group. We found no intervention eﬀect on aerobic ﬁtness either. February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 7 van den Berg et al. Exercise Breaks and Cognition FIGURE 2 \| CONSORT ﬂow diagram: progress of participants through the trial. ∗Children can be excluded for one or more reasons; e.g., a child with a missing arithmetic score can also have a missing pretest score. d2, d2 test; FT, Fluency task; ST, Stroop test; ANT, Attention Network Test; SR, Shuttle Run test. FIGURE 2 \| CONSORT ﬂow diagram: progress of participants through the trial. ∗Children can be excluded for one or more reasons; e.g., a child with a missing arithmetic score can also have a missing pretest score. d2, d2 test; FT, Fluency task; ST, Stroop test; ANT, Attention Network Test; SR, Shuttle Run test. February 2019 \| Volume 10 \| Article 174 8 Exercise Breaks and Cognition van den Berg et al. TABLE 1 \| Descriptive characteristics (means and standard deviations) and group differences. Frontiers in Psychology \| www.frontiersin.org Intervention Effects: Cognitive Performance and Fitness This claim ﬁnds support in some earlier acute studies that reported no eﬀects of classroom-based cognitive demanding exercise bouts on selective attention (van den Berg et al., 2016), updating and inhibition (Jäger et al., 2015; Egger et al., 2018), and even negative eﬀects on shifting (Egger et al., 2018) as compared to low cognitive demanding aerobic exercise in children and young adolescents. In contrast, recent February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 9 Exercise Breaks and Cognition van den Berg et al. TABLE 2 \| Test performance (means, standard errors, and [95% conﬁdence intervals]) and statistics of the mixed model analyses for cognitive performance and ﬁtness. Dependent variable (posttest) Control group Intervention group Regression coefﬁcient (SE) 95% conﬁdence interval p d2 test 151.2 (0.86) 152.5 (0.80) 1.3 (1.5) −1.9 – 4.4 0.42 (n = 448) [149.5; 152.9] [151.0; 154.1] Fluency 11.9 (0.17) 11.7 (0.16) −0.2 (0.2) −0.7 – 0.2 0.33 (n = 467) [11.6; 12.2] [11.4; 12.0] Stroop interference, reaction time (ms) 41.1 (4.4) 33.4 (4.2) −7.7 (6.0) −19.6 – 4.1 0.20 (n = 461) [32.6; 49.7] [25.2; 41.6] Stroop interference, accuracy (%) −1.8 (0.4) −1.3 (0.3) 0.5 (0.5) −0.5 – 1.5 0.29 (n = 461) [−2.5; −1.1] [−2.0; −0.6] ANT alerting, reaction time (ms) 23.5 (2.0) 23.5 (1.9) 0.0 (2.8) −5.4 – 5.4 0.99 (n = 459) [19.6; 27.4] [19.7; 27.2] ANT orienting, reaction time (ms) 61.6 (2.1) 61.5 (2.0) 1.9 (3.3) −4.5 – 8.3 0.57 (n = 459) [57.5; 65.6] [57.6; 65.4] ANT conﬂict, reaction time (ms) 95.6 (2.3) 92.3 (2.2) −3.9 (3.7) −11.1 – 3.4 0.29 (n = 459) [91.1; 100.1] [88.0; 96.7] ANT alerting, accuracy (%) 1.0 (0.3) 1.2 (0.3) 0.1 (0.5) −0.8 – 1.0 0.85 (n = 459) [0.4; 1.7] [0.5; 1.8] ANT orienting, accuracy (%) −1.5 (0.3) −1.4 (0.3) 0.1 (0.5) −0.8 – 1.1 0.77 (n = 459) [−2.2; −0.9] [−2.0; −0.7] ANT conﬂict, accuracy (%) −6.4 (0.4) −5.9 (0.3) 0.6 (0.5) −0.4 – 1.6 0.24 (n = 459) [−7.1; −5.7] [−6.6; −5.2] VO2max (ml/kg/min) 48.8 (0.2) 48.9 (0.2) 0.1 (0.3) −0.6 – 0.7 0.77 (n = 448) [48.4; 49.2] [48.5; 49.3] TABLE 2 \| Test performance (means, standard errors, and [95% conﬁdence intervals]) and statistics of the mixed model analyses for cognitive performance and ﬁtness. TABLE 3 \| Physical activity levels during school hours (means, standard deviations, [95% conﬁdence intervals]) and statistics of the mixed-model analyses. Intervention Effects: Cognitive Performance and Fitness Control (n = 144) Intervention (n = 168) Regression coefﬁcient 95% conﬁdence interval p Sedentary (minutes/day) 228.9 (2.0) 224.6 (1.8) −2.6 (4.3) −11.9 – 6.7 0.56 [225.1; 232.8] [221.0; 228.2] Light PA (minutes/day) 98.2 (1.6) 99.4 (1.5) −0.3 (3.5) −7.9 – 7.4 0.95 [95.0; 101.4] [96.5; 102.4] Moderate PA (minutes/day) 12.6 (0.4) 14.4 (0.3) 1.7 (0.7) −0.3 – 3.2 0.02∗ [11.8; 13.3] [13.7; 15.1] Vigorous PA (minutes/day) 8.1 (0.4) 9.3 (0.3) 1.2 (0.7) −0.2 – 2.6 0.09 [7.3; 8.8] [8.7; 10.0] MVPA (minutes/day) 20.6 (0.7) 23.8 (0.6) 2.9 (1.3) 0.2 – 5.6 0.04∗ [19.3; 22.0] [22.5; 25.0] ∗Signiﬁcant, p < 0.05; PA, physical activity; MVPA, moderate to vigorous physical activity. PA levels include the exercise break time in the intervention group. nﬁdence intervals]) and statistics of the mixed-model analyses. l activity; MVPA, moderate to vigorous physical activity. PA levels include the exercise break time in the intervention group. development, and exercise characteristics such as diﬃculty, duration and intensity (Pesce et al., 2013; Egger et al., 2018). aerobic exercise breaks per day for a period of 9 months as compared to children who performed two 5-min exercise bouts in which academic content was integrated (Fedewa et al., 2018). On the other hand, Ahamed et al. (2007) found that implementing a daily 15-min classroom-based exercise break for 16 months did not improve academic performance in children aged 9– 11 years. Given the inconsistent ﬁndings, more insights need to be gained on the relevance of implementing short exercise breaks for academic purposes. Therefore, we recommend researchers to (1) combine acute as well as long-term measures; (2) include cognitive- as well as academic outcomes; and (3) include an inactive control group. Although the exercise breaks in our study did not result in improvements in cognitive domains of attention, inhibition and memory retrieval, it might be that short exercise bouts contribute to improved academic performance (e.g., maths or language scores) in the long-term via increasing children’s learning eﬃciency and academic engagement (e.g., improved classroom behavior, motivation) in the lessons following the exercise bouts (Owen et al., 2016). Long-term eﬀects of exercise interventions with a relatively short bout duration on academic performance in children are inconsistent. A recent study of Fedewa et al. Frontiers in Psychology \| www.frontiersin.org Intervention Effects: Cognitive Performance and Fitness (2018) reported small improvements in reading performance of 8–11 year olds who participated in two 5-min Our results further revealed that children who participated in the exercise breaks spent 2.9 min more of their school February 2019 \| Volume 10 \| Article 174 10 Exercise Breaks and Cognition van den Berg et al. hours in MVPA per day compared to children in the control group. Our ﬁndings are in line with an earlier study that found that implementing three 5-min classroom-based exercise breaks per day, increased schoolchildren’s MVPA levels (Drummy et al., 2016). Hence, these results suggest that implementing short exercise breaks in the classroom are one promising way to promote PA in children. The additional time spent in MVPA during school hours in our study, however, does not equal the length of the exercise breaks, i.e., 10 min MVPA per day. This might be due to an underestimation of MVPA during dance movements using accelerometers (van Ekris, personal communication). On the other hand, it could be that children were not (moderate to vigorously) active the entire exercise break. In this respect, our heart rate data showed that the mean intensity of the exercise breaks was at the lower boundary of MVPA (i.e., 60% HR max), indicating that it may be diﬃcult to reach or sustain moderate to vigorous intensity levels in a classroom setting. The low exercise intensity could also be a reason for not ﬁnding improvements in cognitive performance (McMorris and Hale, 2012). in academic lessons. Furthermore, PA is suggested to have beneﬁcial eﬀects for mental health (e.g., depression), well- being, mood, self-esteem, motivation, and social connectedness (Lubans et al., 2016; Biddle et al., 2018). However, the eﬀects of short exercise bouts on before mentioned outcomes is still unknown (Poitras et al., 2016). Therefore, we recommend including these outcome measures in future research on the eﬀects of short exercise bouts. As such, we can gain deeper insight in the beneﬁts of short exercise bouts on several domains important to children’s (academic) development, and thereby strengthening the relevance of short exercise bouts in school. Our study has several strengths, such as the RCT design, substantial sample size, blinded test administrators, use of objective measurement instruments, high compliance and a high implementation rate. Though, our study had also some limitations. Our population consisted of children of parents with a relatively high educational level, which limits the generalizability of the results. Intervention Effects: Cognitive Performance and Fitness In addition, we have no baseline accelerometer-based measure of PA. However, the intervention and control classes were equally distributed within each school, i.e., representing a similar population, and did not diﬀer on important descriptive characteristics, such as sports participation, aerobic ﬁtness and parental educational level. Another limitation is that we did not assess children’s PA behavior outside school hours. It could be that exercise breaks inﬂuenced children’s PA behavior outside school, for example if children liked the Just Dance videos they could have decided to perform them during leisure time as well. Lastly, for practical reasons we used a paper-and-pencil version of the verbal ﬂuency task which adds a motor component to the task. Therefore, the test outcomes also depend on writing speed and the length of the chosen words. In addition, we used a computerized Stroop task which measures interference eﬀects to a somewhat lower extent than interference eﬀects measured by the original oral version of the Stroop task (Penner et al., 2012). ( , ) Our results have several implications for practice and future research. First, it is important to be aware of the apparent gap between research and practice. Although we found no eﬀects of daily exercise breaks on children’s cognitive performance, teachers have indicated that they experience improved classroom behavior and performance when using short exercise breaks in the classroom throughout the school year (e.g., Carlson et al., 2015). Therefore, it might be important to consider using more ecological valid measurement instruments, such as systematic observations, teacher logs and/or tasks that mimic curricular activities as a more appropriate representation of classroom- related performance (Khan and Hillman, 2014). In addition, measures of academic engagement and enjoyment of academic lessons can provide important additional information as these factors may have a role in the relationship of exercise and cognitive/academic performance (Owen et al., 2016). Second, the number of exercise breaks implemented in our study was relatively high (median of 4.4 per week), suggesting that 10-min exercise breaks in the classroom are feasible to implement in school practice. However, the controlled setting and reminders during the experiment (i.e., poster-calendar, email contact and visits by the researchers) have likely inﬂuenced these outcomes. Future studies should therefore evaluate the feasibility of the long-term implementation of short exercise breaks in real- life school practice. Third, it is important to notice that children’s cognitive performance did not deteriorate either. CONCLUSION In sum, we found that implementing a daily 10-min exercise break for a period of 9 weeks in the classroom had no eﬀects on cognitive performance and aerobic ﬁtness of 9–12-year old children. The exercise breaks brought about 3 min more MVPA during school hours. Therefore we conclude that schools can implement the seemingly feasible daily exercise breaks in the classroom to promote PA in children without adverse eﬀects on their cognitive performance. Intervention Effects: Cognitive Performance and Fitness We can therefore conclude that implementing exercise breaks on a daily basis, instead of devoting this time to academic tasks, has no adverse eﬀect on children’s cognitive performance. Lastly, an increase of 3 min MVPA induced by the exercise breaks is small. However, implementing short exercise breaks can be a relatively feasible and easy manner to start increasing PA opportunities in school. In order to increase minutes of MVPA during a school day, we recommend to complement exercise breaks in the classroom with other short and feasible exercise interventions in school, e.g., during recess or integrated Frontiers in Psychology \| www.frontiersin.org REFERENCES Characteristics of physical activity interventions and eﬀects on cardiorespiratory ﬁtness in children aged 6-12 years-A systematic review. J. Sci. Med. Sport 21, 296–306. doi: 10.1016/j.jsams.2017.07.015 Egberink, I. J. L., and Vermeulen, C. S. M. (2018b). COTAN Beoordeling 2007, Woord Fluency Test [COTAN Review 2007, Word Fluency Task]. Available at: www.cotandocumentatie.nl Egberink, I. J. L., and Vermeulen, C. S. M. (2018c). COTAN Beoordeling 2012, d2 Aandachts- en Concentratietest voor Kinderen en Adolescenten [COTAN Review 2012, d2 test of Attention for Children and Adolescents]. Available at: www.cotandocumentatie.nl Brickenkamp, R., and Oosterveld, P. (2012). d2 Aandachts- en Concentratietest: Handleiding [d2 Test of Attention: User Manual]. Amsterdam: Hogrefe. Budde, H., Voelcker-Rehage, C., Pietrabyk-Kendziorra, S., Ribeiro, P., and Tidow, G. (2008). Acute coordinative exercise improves attentional performance in adolescents. Neurosci. Lett. 441, 219–223. doi: 10.1016/j. neulet.2008.06.024 Egger, F., Conzelmann, A., and Schmidt, M. (2018). The eﬀect of acute cognitively engaging physical activity breaks on children’s executive functions: too much of a good thing? Psychol. Sport Exerc. 36, 178–186. doi: 10.1016/j.psychsport.2018. 02.014 Carlson, J. A., Engelberg, J. K., Cain, K. L., Conway, T. L., Mignano, A. M., Bonilla, E. A., et al. (2015). Implementing classroom physical activity breaks: associations with student physical activity and classroom behavior. Prev. Med. 81, 67–72. doi: 10.1016/j.ypmed.2015.08.006 Erwin, H., Fedewa, A., Beighle, A., and Ahn, S. (2012). A quantitative review of physical activity, health, and learning outcomes associated with classroom- based physical activity interventions. J. Appl. Sch. Psychol. 28, 14–36. doi: 10. 1080/15377903.2012.643755 Chang, Y. K., Labban, J. D., Gapin, J. I., and Etnier, J. L. (2012). The eﬀects of acute exercise on cognitive performance: a meta-analysis. Brain Res. 1453, 87–101. doi: 10.1016/j.brainres.2012.02.068 Evenson, K. R., Catellier, D. J., Gill, K., Ondrak, K. S., and Mcmurray, R. G. (2008). Calibration of two objective measures of physical activity for children. J. Sports Sci. 26, 1557–1565. doi: 10.1080/02640410802334196 Costigan, S. A., Eather, N., Plotnikoﬀ, R. C., Hillman, C. H., and Lubans, D. R. (2016). High-intensity interval training for cognitive and mental health in adolescents. Med. Sci. Sports Exerc. 48, 1985–1993. doi: 10.1249/MSS. 0000000000000993 Fan, J., Byrne, J., Worden, M. S., Guise, K. G., Mccandliss, B. D., Fossella, J., et al. (2007). The relation of brain oscillations to attentional networks. J. Neurosci. 27, 6197–6206. doi: 10.1523/JNEUROSCI.1833-07.2007 Daly-Smith, A. J., Zwolinsky, S., Mckenna, J., Tomporowski, P. D., Defeyter, M. A., and Manley, A. (2018). REFERENCES sensors in youth: a systematic update. Med. Sci. Sports Exerc. 41, 818–827. doi: 10.1249/MSS.0b013e31818e5819 sensors in youth: a systematic update. Med. Sci. Sports Exerc. 41, 818–827. doi: 10.1249/MSS.0b013e31818e5819 Ahamed, Y., Macdonald, H., Reed, K., Naylor, P. J., Liu-Ambrose, T., and Mckay, H. (2007). School-based physical activity does not compromise children’s academic performance. Med. Sci. Sports Exerc. 39, 371–376. doi: 10.1249/01.mss. 0000241654.45500.8e Dinkel, D., Schaﬀer, C., Snyder, K., and Lee, J. M. (2017). They just need to move: teachers’ perception of classroom physical activity breaks. Teach. Teach. Educ. 63, 186–195. doi: 10.1016/j.tate.2016.12.020 Dobbins, M., Husson, H., Decorby, K., and Larocca, R. L. (2013). School- based physical activity programs for promoting physical activity and ﬁtness in children and adolescents aged 6 to 18. Cochrane Database Syst. Rev. 2:CD007651. doi: 10.1002/14651858.CD007651. pub2 Alvarez-Bueno, C., Pesce, C., Cavero-Redondo, I., Sanchez-Lopez, M., Martinez- Hortelano, J. A., and Martinez-Vizcaino, V. (2017). The eﬀect of physical activity interventions on children’s cognition and metacognition: a systematic review and meta-analysis. J. Am. Acad. Child Adolesc. Psychiatry 56, 729–738. doi: 10.1016/j.jaac.2017.06.012 Donnelly, J. E., Hillman, C. H., Castelli, D., Etnier, J. L., Lee, S., Tomporowski, P., et al. (2016). Physical activity, ﬁtness, cognitive function, and academic achievement in children: a systematic review. Med. Sci. Sports Exerc. 48, 1197– 1222. doi: 10.1249/MSS.0000000000000901 Audiﬀren, M., and André, N. (2015). The strength model of self-control revisited: linking acute and chronic eﬀects of exercise on executive functions. J. Sport Health Sci. 4, 30–46. doi: 10.1016/j.jshs.2014.09.002 Drummy, C., Murtagh, E. M., Mckee, D. P., Breslin, G., Davison, G. W., and Murphy, M. H. (2016). The eﬀect of a classroom activity break on physical activity levels and adiposity in primary school children. J. Paediatr. Child Health 52, 745–749. doi: 10.1111/jpc.13182 Best, J. R. (2010). Eﬀects of physical activity on children’s executive function: contributions of experimental research on aerobic exercise. Dev. Rev. 30, 331–551. doi: 10.1016/j.dr.2010.08.001 Biddle, S. J. H., Ciaccioni, S., Thomas, G., and Vergeer, I. (2018). Physical activity and mental health in children and adolescents: an updated review of reviews and an analysis of causality. Psychol. Sport Exerc. (in press). doi: 10.1016/j. psychsport.2018.08.011 Egberink, I. J. L., and Vermeulen, C. S. M. (2018a). COTAN Beoordeling 1998, Competentie Belevingsschaal Voor Kinderen [COTAN Review 1998, Self Perception Proﬁle for Children]. Available at: www.cotandocumentatie.nl Braaksma, P., Stuive, I., Garst, R. M. E., Wesselink, C. F., Van Der Sluis, C. K., Dekker, R., et al. (2018). FUNDING This study is part of the SMART MOVES! project, funded by the Netherlands Organization for Scientiﬁc Research (NWO; Project No. 328-98-003). AUTHOR CONTRIBUTIONS VvdB, ES, RdG, MC, and AS conceived and designed the study. VvdB and ES acquired the data. VvdB analyzed the data. VvdB, MC, and AS interpreted the data analyses. VvdB drafted the manuscript. ES, RdG, MC, and AS contributed to critical revision of the draft. All authors read and approved the ﬁnal manuscript. ACKNOWLEDGMENTS We would like to thank all schools, teachers, and children who participated in this study. ETHICS STATEMENT This study was carried out in accordance with the recommendations of the Medical Ethical Committee of the VU University Medical Center, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 11 Exercise Breaks and Cognition van den Berg et al. approved by the Medical Ethical Committee of the VU University Medical Center. approved by the Medical Ethical Committee of the VU University Medical Center. REFERENCES Available at: https://schooltv.nl/video/lenigheid-hoe-word- je-leniger/ [accessed February 2016]. Ogoh, S., and Ainslie, P. N. (2009). Cerebral blood ﬂow during exercise: mechanisms of regulation. J. Appl. Physiol. 107, 1370–1380. doi: 10.1152/ japplphysiol.00573.2009 Janssen, M., Toussaint, H. M., Van Mechelen, W., and Verhagen, E. A. (2014). Eﬀects of acute bouts of physical activity on children’s attention: a systematic review of the literature. Springerplus 3:410. doi: 10.1186/2193-1801-3-410 Owen, K. B., Parker, P. D., Van Zanden, B., Macmillan, F., Astell-Burt, T., and Lonsdale, C. (2016). Physical activity and school engagement in youth: a systematic review and meta-analysis. Educ. Psychol. 51, 129–145. doi: 10.1080/ 00461520.2016.1151793 Khan, N. A., and Hillman, C. H. (2014). The relation of childhood physical activity and aerobic ﬁtness to brain function and cognition: a review. Pediatr. Exerc. Sci. 26, 138–146. doi: 10.1123/pes.2013-0125 Penner, I. K., Kobel, M., Stocklin, M., Weber, P., Opwis, K., and Calabrese, P. (2012). The Stroop task: comparison between the original paradigm and computerized versions in children and adults. Clin. Neuropsychol. 26, 1142– 1153. doi: 10.1080/13854046.2012.713513 Korkman, M., Kirk, U., and Kemp, S. L. (1998). NEPSY. A Developmental Neuropsychological Assessment. San Antonio, TX: The Psychological Corporation. Koutsandreou, F., Wegner, M., Niemann, C., and Budde, H. (2016). Eﬀects of motor versus cardiovascular exercise training on children’s working memory. Med. Sci. Sports Exerc. 48, 1144–1152. doi: 10.1249/MSS.0000000000000869 Pesce, C., Crova, C., Marchetti, R., Struzzolino, I., Masci, I., Vannozzi, G., et al. (2013). Searching for cognitively optimal challenge point in physical activity for children with typical and atypical motor development. Ment. Health Phys. Activ. 6, 172–180. doi: 10.1016/j.mhpa.2013.07.001 Kriemler, S., Meyer, U., Martin, E., Van Sluijs, E. M., Andersen, L. B., and Martin, B. W. (2011). Eﬀect of school-based interventions on physical activity and ﬁtness in children and adolescents: a review of reviews and systematic update. Br. J. Sports Med. 45, 923–930. doi: 10.1136/bjsports-2011-090186 Piepmeier, A. T., and Etnier, J. L. (2015). Brain-derived neurotrophic factor (BDNF) as a potential mechanism of the eﬀects of acute exercise on cognitive performance. J. Sport Health Sci. 4, 14–23. doi: 10.3389/fpsyt.2017. 9.00191 Léger, L. A., Mercier, D., Gadoury, C., and Lambert, J. (1988). The multistage 20 meter shuttle run test for aerobic ﬁtness. J. Sport Sci. 6, 93–101. doi: 10.1080/ 02640418808729800 Poitras, V. J., Gray, C. E., Borghese, M. M., Carson, V., Chaput, J. P., Janssen, I., et al. (2016). REFERENCES Systematic review of acute physically active learning and classroom movement breaks on children’s physical activity, cognition, academic performance and classroom behaviour: understanding critical design features. BMJ Open Sport Exerc. Med. 4:e000341. doi: 10.1136/bmjsem-2018-000341 Fan, J., Mccandliss, B. D., Sommer, T., Raz, A., and Posner, M. I. (2002). Testing the eﬃciency and independence of attentional networks. J. Cogn. Neurosci. 14, 340–347. doi: 10.1162/089892902317361886 Faul, F., Erdfelder, E., Lang, A.-G., and Buchner, A. (2007). G∗Power 3: a ﬂexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods 39, 175–191. doi: 10.3758/BF0319 3146 de Greeﬀ, J. W., Bosker, R. J., Oosterlaan, J., Visscher, C., and Hartman, E. (2018). Eﬀects of physical activity on executive functions, attention and academic performance in preadolescent children: a meta-analysis. J. Sci. Med. Sport 21, 501–507. doi: 10.1016/j.jsams.2017.09.595 Fedewa, A. L., Fettrow, E., Erwin, H., Ahn, S., and Farook, M. (2018). Academic- based and aerobic-only movement breaks: are there diﬀerential eﬀects on physical activity and achievement? Res. Q. Exerc. Sport 89, 153–163. doi: 10. 1080/02701367.2018.1431602 De Vries, S. I., Van Hirtum, H. W., Bakker, I., Hopman-Rock, M., Hirasing, R. A., and Van Mechelen, W. (2009). Validity and reproducibility of motion February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 12 Exercise Breaks and Cognition van den Berg et al. Fernandes, J., Arida, R. M., and Gomez-Pinilla, F. (2017). Physical exercise as an epigenetic modulator of brain plasticity and cognition. Neurosci. Biobehav. Rev. 80, 443–456. doi: 10.1016/j.neubiorev.2017.06.012 Ludyga, S., Gerber, M., Kamijo, K., Brand, S., and Puhse, U. (2018b). The eﬀects of a school-based exercise program on neurophysiological indices of working memory operations in adolescents. J. Sci. Med. Sport 21, 833–838. doi: 10.1016/ j.jsams.2018.01.001 Forns, J., Esnaola, M., López-Vicente, M., Suades-González, E., Alvarez- Pedrerol, M., Julvez, J., et al. (2014). Supplemental material for the n-back test and the attentional network task as measures of child neuropsychological development in epidemiological studies. Neuropsychology 28, 519–529. doi: 10.1037/neu0000085 Martins, J., Marques, A., Sarmento, H., and Carreiro Da Costa, F. (2015). Adolescents’ perspectives on the barriers and facilitators of physical activity: a systematic review of qualitative studies. Health Educ. Res. 30, 742–755. doi: 10.1093/her/cyv042 McMorris, T., and Hale, B. J. (2012). Diﬀerential eﬀects of diﬀering intensities of acute exercise on speed and accuracy of cognition: a meta-analytical investigation. Brain Cogn. 80, 338–351. doi: 10.1016/j.bandc.2012.09.001 Hillman, C. H., Erickson, K. I., and Kramer, A. F. (2008). REFERENCES Be smart, exercise your heart: exercise eﬀects on brain and cognition. Nat. Rev. Neurosci. 9, 58–65. doi: 10.1038/nrn2298 Hollenberg, J., and Van Der Lubbe, M. (2017). Toetsen op School Primair Onderwijs. Arnhem: Cito. McMorris, T., and Hale, B. J. (2015). Is there an acute exercise-induced physiological/biochemical threshold which triggers increased speed of cognitive functioning? A meta-analytic investigation. J. Sport Health Sci. 4, 4–13. doi: 10.1016/j.jshs.2014.08.003 Howie, E. K., Beets, M. W., and Pate, R. R. (2014a). Acute classroom exercise breaks improve on-task behavior in 4th and 5th grade students: a dose–response. Ment. Health Phys. Act. 7, 65–71. doi: 10.1016/j.mhpa.2014.05.002 McMullen, J., Kulinna, P., and Cothran, D. (2014). Chapter 5 physical activity opportunities during the school day: classroom teachers’ perceptions of using activity breaks in the classroom. J. Teach. Phys. Educ. 33, 511–527. doi: 10.1123/ jtpe.2014-0062 Howie, E. K., Newman-Norlund, R. D., and Pate, R. R. (2014b). Smiles count but minutes matter: responses to classroom exercise breaks. Am. J. Health Behav. 38, 681–689. doi: 10.5993/AJHB.38.5.5 Howie, E. K., Schatz, J., and Pate, R. R. (2015). Acute eﬀects of classroom exercise breaks on executive function and math performance: a dose-response study. Res. Q. Exerc. Sport 86, 217–224. doi: 10.1080/02701367.2015.1039892 Mulder, J. L., Dekker, P. H., and Dekker, R. (2006). Woord-Fluency Test / Figuur- Fluency Test: Handleiding [Word-Fluency Task / Figure-Fluency Task: User Manual]. Leiden: PITS. Muris, P., Meesters, C., and Fijen, P. (2003). The self-perception proﬁle for children: further evidence for its factor structure, reliability, and validity. Pers. Individ. Dif. 35, 1791–1802. doi: 10.1016/S0191-8869(03)00004-7 Huang, T., Larsen, K. T., Ried-Larsen, M., Moller, N. C., and Andersen, L. B. (2014). The eﬀects of physical activity and exercise on brain-derived neurotrophic factor in healthy humans: a review. Scand. J. Med. Sci. Sports 24, 1–10. doi: 10.1111/sms.12069 Niemann, C., Wegner, M., Voelcker-Rehage, C., Holzweg, M., Arafat, A. M., and Budde, H. (2013). Inﬂuence of acute and chronic physical activity on cognitive performance and saliva testosterone in preadolescent school children. Ment. Health Phys. Activ. 6, 197–204. doi: 10.1016/j.mhpa.2013.08.002 Jacob, R., and Parkinson, J. (2015). The potential for school-based interventions that target executive function to improve academic achievement. Rev. Educ. Res. 85, 512–552. doi: 10.3102/0034654314561338 Jäger, K., Schmidt, M., Conzelmann, A., and Roebers, C. M. (2015). The eﬀects of qualitatively diﬀerent acute physical activity interventions in real-world settings on executive functions in preadolescent children. Ment. Health Phys. Activ. 9, 1–9. doi: 10.1016/j.mhpa.2015.05.002 NTR (2012). Schooltv. REFERENCES Cognitively engaging chronic physical activity, but not aerobic exercise, aﬀects executive functions in primary school children: a group-randomized controlled trial. J. Sport Exerc. Psychol. 37, 575–591. doi: 10.1123/jsep.2015-0069 Vazou, S., Pesce, C., Lakes, K., and Smiley-Oyen, A. (2016). More than one road leads to Rome: a narrative review and meta-analysis of physical activity intervention eﬀects on cognition in youth. Int. J. Sport Exerc. Psychol. doi: 10.1080/1612197X.2016.1223423 [Epub ahead of print]. Singh, A. S., Saliasi, E., Van Den Berg, V., Uijtdewilligen, L., De Groot, R. H. M., Jolles, J., et al. (2018). Eﬀects of physical activity interventions on cognitive and academic performance in children and adolescents: a novel combination of a systematic review and recommendations from an expert panel. Br. J. Sports. Med. doi: 10.1136/bjsports-2017-098136 [Epub ahead of print]. Veerman, J. W., Straathof, M. A. E., Treﬀers, A., Van Den Bergh, B. R. H., and Ten Brink, L. T. (1997). Competentie-Belevingsschaal voor Kinderen: Handleiding [Self-Perception Proﬁle for Children: User Manual]. Amsterdam: Pearson Benelux B.V. Singh, A. S., Vik, F. N., Chinapaw, M. J. M., Uijtdewilligen, L., Verloigne, M., Fernández-Alvira, J. M., et al. (2011). Test-retest reliability and construct validity of the ENERGY-child questionnaire on energy balancerelated behaviours and their potential determinants: the ENERGY-project. Int. J. Behav. Nutr. Phys. Activ. 8:136. doi: 10.1186/1479-5868-8-136 Verburgh, L., Konigs, M., Scherder, E. J., and Oosterlaan, J. (2014). Physical exercise and executive functions in preadolescent children, adolescents and young adults: a meta-analysis. Br. J. Sports Med. 48, 973–979. doi: 10.1136/ bjsports-2012-091441 Watson, A., Timperio, A., Brown, H., Best, K., and Hesketh, K. D. (2017). Eﬀect of classroom-based physical activity interventions on academic and physical activity outcomes: a systematic review and meta-analysis. Int. J. Behav. Nutr. Phys. Act 14:114. doi: 10.1186/s12966-017-0569-9 Stevens, C., and Bavelier, D. (2012). The role of selective attention on academic foundations: a cognitive neuroscience perspective. Dev. Cogn. Neurosci. 2(Suppl. 1), S30–S48. doi: 10.1016/j.dcn.2011.11.001 Stylianou, M., Kulinna, P. H., and Naiman, T. (2015). ‘. . .because there’s nobody who can just sit that long’. Eur. Phys. Educ. Rev. 22, 390–408. doi: 10.1177/ 1356336X15613968 Webster, C. A., Russ, L., Vazou, S., Goh, T. L., and Erwin, H. (2015). Integrating movement in academic classrooms: understanding, applying and advancing the knowledge base. Obes. Rev. 16, 691–701. doi: 10.1111/obr.12285 Tomporowski, P. D., Mccullick, B., Pendleton, D. M., and Pesce, C. (2015). Exercise and children’s cognition: the role of exercise characteristics and a place for metacognition. J. REFERENCES Systematic review of the relationships between objectively measured physical activity and health indicators in school-aged children and youth. Appl. Physiol. Nutr. Metab. 41, S197–S239. doi: 10.1139/apnm-2015- 0663 Li, J. W., O’connor, H., O’dwyer, N., and Orr, R. (2017). The eﬀect of acute and chronic exercise on cognitive function and academic performance in adolescents: a systematic review. J. Sci. Med. Sport 20, 841–848. doi: 10.1016/ j.jsams.2016.11.025 Rueda, M. R., Checa, P., and Rothbart, M. K. (2010). Contributions of attentional control to socioemotional and academic development. Early Educ. Dev. 21, 744–764. doi: 10.1080/10409289.2010.510055 Lubans, D., Richards, J., Hillman, C. H., Faulkner, G., Beauchamp, M., Nilsson, M., et al. (2016). Physical activity for cognitive and mental health in youth: a systematic review of mechanisms. Pediatrics 138:e20161642. doi: 10.1542/peds. 2016-1642 Rueda, M. R., Fan, J., Mccandliss, B. D., Halparin, J. D., Gruber, D. B., Lercari, L. P., et al. (2004). Development of attentional networks in childhood. Neuropsychologia 42, 1029–1040. doi: 10.1016/j.neuropsychologia.2003. 12.012 Ludyga, S., Gerber, M., Brand, S., Holsboer-Trachsler, E., and Puhse, U. (2016). Acute eﬀects of moderate aerobic exercise on speciﬁc aspects of executive function in diﬀerent age and ﬁtness groups: a meta-analysis. Psychophysiology 53, 1611–1626. doi: 10.1111/psyp.12736 Sackler Institute for Developmental Psychobiology [SIDP] (2016). Attention Network Task. Available at: https://www.sacklerinstitute.org/cornell/assays_ and_tools/ant/jin.fan/ [accessed June 2016]. Ludyga, S., Gerber, M., Herrmann, C., Brand, S., and Pühse, U. (2018a). Chronic eﬀects of exercise implemented during school-break time on neurophysiological indices of inhibitory control in adolescents. Trends Neurosci. Educ. 10, 1–7. doi: 10.1016/j.tine.2017.11.001 Savina, E., Garrity, K., Kenny, P., and Doerr, C. (2016). The beneﬁts of movement for youth: a whole child approach. Contemp. Sch. Psychol. 20, 282–292. doi: 10.1007/s40688-016-0084-z February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 13 Exercise Breaks and Cognition van den Berg et al. Schmidt, M., Benzing, V., and Kamer, M. (2016). Classroom-based physical activity breaks and children’s attention: cognitive engagement works! Front. Psychol. 7:1474. doi: 10.3389/fpsyg.2016.01474 van den Berg, V., Vos, E., De Groot, R. H. M., Singh, A. S., and Chinapaw, M. J. (2018). Untapped resources: 10- to 13-year-old primary schoolchildren’s views on additional physical activity in the school setting: a focus group study. Int. J. Environ. Res. Public Health 15:E2713. doi: 10.3390/ijerph15122713 Schmidt, M., Jager, K., Egger, F., Roebers, C. M., and Conzelmann, A. (2015). Frontiers in Psychology \| www.frontiersin.org REFERENCES Sport Health Sci. 4, 47–55. doi: 10.1016/j.jshs.2014. 09.003 Yildirim, M., Verloigne, M., De Bourdeaudhuij, I., Androutsos, O., Manios, Y., Felso, R., et al. (2011). Study protocol of physical activity and sedentary behaviour measurement among schoolchildren by accelerometry–cross- sectional survey as part of the ENERGY-project. BMC Public Health 11:182. doi: 10.1186/1471-2458-11-182 Trost, S. G., Loprinzi, P. D., Moore, R., and Pfeiﬀer, K. A. (2011). Comparison of accelerometer cut points for predicting activity intensity in youth. Med. Sci. Sports Exerc. 43, 1360–1368. doi: 10.1249/MSS.0b013e31820 6476e 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. van den Berg, V., Saliasi, E., De Groot, R. H., Jolles, J., Chinapaw, M. J., and Singh, A. S. (2016). Physical activity in the school setting: cognitive performance is not aﬀected by three diﬀerent types of acute exercise. Front. Psychol. 7:723. doi: 10.3389/fpsyg.2016.00723 Copyright © 2019 van den Berg, Saliasi, de Groot, Chinapaw and Singh. 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) and the copyright owner(s) 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. van den Berg, V., Salimi, R., De Groot, R. H. M., Jolles, J., Chinapaw, M. J. M., and Singh, A. S. (2017). “It’s a battle.. You want to do it, but how will you get it done?”: teachers’ and principals’ perceptions of implementing additional physical activity in school for academic performance. Int. J. Environ. Res. Public Health 14:E1160. doi: 10.3390/ijerph14101160 February 2019 \| Volume 10 \| Article 174 Frontiers in Psychology \| www.frontiersin.org 14
https://openalex.org/W2569921743	https://europepmc.org/articles/pmc5245841?pdf=render	English	null	Impact of Brazilian Papers in Cardiology and Cardiovascular Sciences in the Last Decade	Arquivos Brasileiros de Cardiologia	2,017	cc-by	1,644	Luiz Felipe P. Moreira Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina - Universidade de São Paulo - USP, São Paulo, SP – Brazil Luiz Felipe P. Moreira Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina - Universidade de São Paulo - USP, São Paulo, SP – B Luiz Felipe P. Moreira Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina - Universidade de São Paulo - USP, São Paulo, SP – Brazil ‘myocardial revascularization’ and ‘atrial fibrillation’ published in Brazilian journals are not different from those of articles published in other countries.3 Aiming to expand this analysis, we assessed 968 articles in cardiology indexed in the Web of Science, written exclusively by Brazilian authors and published between 2010 and 2014. During the last decade, there has been a significant increase in the number of Brazilian publications in cardiology and cardiovascular sciences in important international citation indexing platforms. This occurred in Brazil and in most Latin American countries at approximately 13% per year between 1999 and 2008 according to the study by Colantionio et al.1 This represents nearly 3% of all articles published in international journals indexed in the Web of Science platform, maintained by Thompson-Reuters, and Scopus-Scimago, provided by Elsevier. We considered the mean citation index of the 30 journals with the highest number of articles published, and assessed correlations between the number of times these articles were cited and the impact factor of the journals in which these articles were published. There was a weak correlation between the mean number of times these papers were cited within two years of publication and the impact factor of the journals, especially in higher impact journals (Figure 2). Although most of our indexed articles have been published in international journals, the citation indexes achieved by Brazilian and Latin American authors are usually lower than those from countries with higher income or higher Human Development Index.1 This is even more evident for studies conducted in national institutions in comparison with those developed with some degree of international cooperation. These findings confirm that, despite recent advances, the citation indexes obtained from Brazilian authors are still lower than the mean international index. Besides, they highlight the importance to analyze the citation indexes of each study. These are currently available in the main international citation indexing systems that, in turn, provide an online, daily update of these parameters. For this reason, the ‘value’ of a publication may not be related to the journal impact. Luiz Felipe P. Moreira Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina - Universidade de São Paulo - USP, São Paulo, SP – Brazil In fact, the citation index of scientific publications has been used by most of Brazilian fostering agencies and the Lattes Platform, a database of Brazilian researchers’ resumes. As compared with international citations, using the data from Scimago country ranking,2 the mean citation index of Brazilian papers in cardiology has progressively increased from less than 0.5 to nearly 0.65 in the last ten years (Figure 1). Today, this index is similar to that of countries like Japan, South Korea and China. Advances in the quality of Brazilian papers in cardiology has occurred along with the improvement of Brazilian researchers’ qualification thanks to Master and Doctoral degree programs and incentive programs for scientific research supported by agencies and national medical societies. On the other hand, approximately 35% of Brazilian papers in cardiology or cardiovascular sciences have been published in Arquivos Brasileiros de Cardiology or other Brazilian journals, and less than 50% of papers in journals with an impact factor greater than 1.6. On the other hand, one of the main ranking criteria of graduate programs in Brazil, used by the Coordination for the Improvement of Higher Education Personnel (CAPES) of the Ministry of Health, is based on Qualis system. Qualis classifies scientific production of graduate programs according to the impact factors of journals in which the papers are published, without taking into consideration the indexes of each publication. Thus, a revision of this method is required to promote adequate fostering of research and incentive for the publication of Brazilian papers in internationally indexed journals. Previously, we have reported that the citation indexes of studies on major subjects in cardiovascular sciences, such as A positive attitude towards the progress of scientific research in Brazil also depends on the maintenance of government and private funding to scientific research, on the expansion of training programs on clinical and laboratory studies, and improvement of Master’s and Doctoral programs. Besides, the development of clinical trials and multicenter or multinational studies on major cardiovascular diseases by our centers and medical societies4,5 represent important initiatives of great impact and an adaptation of scientific knowledge to national conditions. Editorial Mailing address: Luiz Felipe P. Moreira • Av. Dr. Enéas Carvalho Aguiar, 44, 2º andar, bloco 2, sala 13, Cerqueira César. Postal Code 05403-000, São Paulo, SP - Brazil E-mail: luiz.moreira@incor.usp.br Keywords 1.5 1.0 0.5 0.0 2006 Mean citation index per country/ mean international citation index 2008 2010 2012 2014 2016 Spain South Korea Germany China Japan United States Brazil Figure 1 – Relationship between the mean citation indexes of papers published in the fields of cardiology and cardiovascular sciences in each country and the mean Figure 1 – Relationship between the mean citation indexes of papers published in the fields of cardiology and cardiovascular sciences in each country and the mean international citation index in the period from 2006 to 2016. Figure 2 – Correlation between mean number of citations obtained from 968 articles of Brazilian authors published in 30 journals in cardiology and cardiovascular sciences within 2 years of their publication and the impact factor in which these articles were published. 8 6 4 2 0 0 Impact factor (journal) r = 0.406 (0.053 a 0.668) Number of citation (mean) / article 4 2 6 8 Figure 2 Correlation between mean number of citations obtained from 968 articles of Brazilian authors published in 30 journals in cardiology and cardiovascular 8 6 4 2 0 0 Impact factor (journal) r = 0.406 (0.053 a 0.668) Number of citation (mean) / article 4 2 6 8 r = 0.406 (0.053 a 0.668) Figure 2 – Correlation between mean number of citations obtained from 968 articles of Brazilian authors published in 30 journals in cardiology and cardiovascular sciences within 2 years of their publication and the impact factor in which these articles were published. Keywords Cardiovascular Diseases; Cardiology; Periodicals as Topic; Bibliometrics. Mailing address: Luiz Felipe P. Moreira • Av. Dr. Enéas Carvalho Aguiar, 44, 2º andar, bloco 2, sala 13, Cerqueira César. Postal Code 05403-000, São Paulo, SP - Brazil E-mail: luiz.moreira@incor.usp.br DOI: 10.5935/abc.20170005 DOI: 10.5935/abc.20170005 Editorial Moreira Impact of Brazilian Papers 1.5 1.0 0.5 0.0 2006 Mean citation index per country/ mean international citation index 2008 2010 2012 2014 2016 Spain South Korea Germany China Japan United States Brazil Figure 1 – Relationship between the mean citation indexes of papers published in the fields of cardiology and cardiovascular sciences in each country and the mean international citation index in the period from 2006 to 2016. Moreira Impact of Brazilian Papers Editorial 1.5 1.0 0.5 0.0 2006 Mean citation index per country/ mean international citation index 2008 2010 2012 2014 2016 Spain South Korea Germany China Japan United States Brazil Figure 1 – Relationship between the mean citation indexes of papers published in the fields of cardiology and cardiovascular sciences in each country and the mean international citation index in the period from 2006 to 2016. Figure 2 – Correlation between mean number of citations obtained from 968 articles of Brazilian authors published in 30 journals in cardiology and cardiovascular sciences within 2 years of their publication and the impact factor in which these articles were published. 8 6 4 2 0 0 Impact factor (journal) r = 0.406 (0.053 a 0.668) Number of citation (mean) / article 4 2 6 8 1.5 1.0 0.5 0.0 2006 Mean citation index per country/ mean international citation index 2008 2010 2012 2014 2016 Spain South Korea Germany China Japan United States Brazil Figure 1 – Relationship between the mean citation indexes of papers published in the fields of cardiology and cardiovascular sciences in each country and the mean international citation index in the period from 2006 to 2016. 5. Moreira LF. Implementation of multicenter records in the therapeutic cardiovascular assessment in Brazil. Arq Bras Cardiol. 2013;101(6):478-9. 4. Gomes WJ, Kalil RA, Jatene FB. Brazilian Registry of Cardiovascular Surgery in Adults Fully Operational. Braz J Cardiovasc Surg. 2016;31(2):II. 3. Moreira LF. Is there any Influence of Journal Impact Factor on the Citation Index of Articles Published in Brazilian Journals with International Scope? Arq Bras Cardiol. 2015;105(1):1-2. 4. Gomes WJ, Kalil RA, Jatene FB. Brazilian Registry of Cardiovascular Surgery in Adults Fully Operational. Braz J Cardiovasc Surg. 2016;31(2):II. 5. Moreira LF. Implementation of multicenter records in the therapeutic cardiovascular assessment in Brazil. Arq Bras Cardiol. 2013;101(6):478-9. 2. SCImago (2007). SJR – SCImago Journal & Country Rank. [Accessed in 2016 Dec 10]. Available from: http://www.scimagojr.com/countryrank. php?area=2700&category=2705 3. Moreira LF. Is there any Influence of Journal Impact Factor on the Citation Index of Articles Published in Brazilian Journals with International Scope? Arq Bras Cardiol. 2015;105(1):1-2. 1. Colantonio LD, Baldridge AS, Huffman MD, Bloomfield GS, Prabhakaran D. Cardiovascular research publications from Latin America between 1999 and 2008. A bibliometric study. Arq Bras Cardiol. 2015;104(1):5-15. References 1. Colantonio LD, Baldridge AS, Huffman MD, Bloomfield GS, Prabhakaran D. Cardiovascular research publications from Latin America between 1999 and 2008. A bibliometric study. Arq Bras Cardiol. 2015;104(1):5-15. 3. Moreira LF. Is there any Influence of Journal Impact Factor on the Citation Index of Articles Published in Brazilian Journals with International Scope? Arq Bras Cardiol. 2015;105(1):1-2. 2. SCImago (2007). SJR – SCImago Journal & Country Rank. [Accessed in 2016 Dec 10]. Available from: http://www.scimagojr.com/countryrank. php?area=2700&category=2705 Arq Bras Cardiol. 2017; 108(1):1-2 2
W2520567119.txt	https://www.medienpaed.com/article/download/391/391	de		Entgrenzung aus zwei Perspektiven: Jugendforschung und Medienpädagogik	MedienPädagogik	2,016	cc-by	2,440	www.medienpaed.com ISSN 1424-3636 Rezensionen Ulrike Becker, Henrike Friedrichs, Friederike von Gross und Sabine Kaiser (Hrsg.) Ent-Grenztes Heranwachsen Wiesbaden: VS Verlag für Sozialwissenschaften, 2016. 315 Seiten ISBN: 978-3-658-09793-6 (E-Book), 978-3-658-09792-9 (Print) 9,99 € (Print), 29,69 € (E-Book) This work is licensed under a Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/ Entgrenzung aus zwei Perspektiven: Jugendforschung und Medien- pädagogik   Genau wie jede Gesellschaft sind die in ihr lebenden Individuen stetigen Wandlungsprozessen unterworfen. Das Heranwachsen einzelner Generati- onen findet jeweils mit Veränderung statt. Die neue Generation wächst in einer veränderten Lebenswelt auf und hat mehr oder andere Freiheiten oder Begrenzungen, als es die vorherige hatte. Besonders der mediale Wandel beinhaltet nicht nur technische Neuerungen, sondern eröffnet Kindern und Jugendlichen Zugang zu neuen Welten in beispielsweise Computerspielen und sozialen Netzwerken. Dies birgt vielleicht Unsicherheiten und Gefahren, aber auch Herausforderungen und Möglichkeiten. Die Heranwachsenden interagieren in digitalen Räumen und kommunizieren mit digitalen ‹Freun- den›. Viele (Lern-)Erfahrungen sammeln sie über den Kontakt zu Peers. Dabei nutzen sie unterschiedlichste Zugangsmöglichkeiten zu diesen Medi- enwelten (iPod, Smartphone, PC, Konsole, …). Mediale und non-mediale können miteinander Welten verschmelzen. So ist es stets von Interesse für Pädagogen/-innen, Soziologen/-innen und Eltern, diese neue Generation und ihre Lebensumstände zu analysieren und zu verstehen. Die Untersu- chung des Medienhandelns Heranwachsender bildet einen erheblichen Teil der Forschungsprojekte von Uwe Sander, Professor für Medienpädagogik und Jugendforschung an der Universität Bielefeld. Kontext Der vorliegende Sammelband entstand anlässlich des 60. Geburtstags von Uwe Sander. Wissenschaftler/innen, die als Kollegen/-innen oder Doktoran- den/-innen persönlich mit ihm zu tun haben/hatten und von ihm beeinflusst und begleitet wurden, beschäftigen sich aus verschiedenen Perspektiven mit https://doi.org/10.21240/mpaed/XX/2016.07.29.X Online publiziert: 29. Juli 2016 Rezensionen den Grenzen und Entgrenzungen im Aufwachsen von Kindern und Jugend- lichen. Deren Umwelt ist geprägt von gesellschaftlichen Veränderungspro- zessen, die eine Positionierung der eigenen Werte und Normen fordern, von Mobilisierung und Globalisierung sowie von einer zunehmenden Mediati- sierung. Entsprechend gross ist die inhaltliche Bandbreite der Artikel des Sammelbandes. Er enthält eine kritische Auseinandersetzung mit einzelnen Aspekten des Heranwachsens im 21. Jahrhundert, betrachtet die Medien- sozialisation Jugendlicher und gibt einen Ausblick auf die daraus entstehen- den Herausforderungen für Bildung und Erziehung. Aufbau und Inhalt Die eben genannten Aspekte bilden zugleich die drei inhaltlichen Schwer- punkte, in die der Sammelband gegliedert ist. Der erste Teil, «Heranwach- sen im 21. Jahrhundert zwischen Wandel und Beständigkeit», wird von den Autoren/-innen Sabine Andresen, Wilfried Ferchhoff und Bernd Dewe, Karin Wehmeyer, Marc Bienefeld und Oliver-Böhm-Kasper, Arne Schäfer und Matthias D. Witte sowie von Hans-Jürgen von Wensierski bestritten. Aktuelle erziehungswissenschaftliche Diskurse und empirische Forschung werden hier geschickt mit Uwe Sanders ganzheitlichem Blick auf Kinder und Ju- gendliche verknüpft. Der erste Beitrag von Sabine Andresen stellt kritische Anfragen an die Kind- heitsforschung. Unter dem Titel «Normierte Kindheit» fragt sie, wie Inklusion «im Sinne der Achtung der Würde aller Kinder und Jugendlichen» (S. 21) realisiert werden könne und welche fachlichen Herausforderungen daraus resultieren. Am Beispiel der krebskranken Hazel Grace werden Normen und Normalitätsvorstellungen infrage gestellt. Davon ausgehend widmet sie sich den Aspekten Vulnerabilität und Wohlbefinden in der Kindheit. Um diese besser zu verstehen, spricht sie sich für einen Perspektivwechsel aus und sieht neues Potenzial für die Kindheits- und Jugendforschung in einer Ver- schränkung der Sichtweisen der Kultur- mit der Sozialwissenschaft. Die Autoren Ferchhoff und Dewe rekonstruieren gesellschaftliche Struktur- entwicklungen in «Entstrukturierung und Entgrenzung der Jugendphase». Sie stellen die Jugend unter anderem unter den Gesichtspunkten Enttraditio- nalisierung und Individualisierung dar. Daraus resultiere eine Konzentration auf den Körper und das Körperbewusstsein der Heranwachsenden (vgl. S. 37), die zu neuen Ästhetisierungen, aber auch Selbstbewusstsein und Selbstkasteiung führe (vgl. S. 39). Des Weiteren stellen sie dar, dass Ju- gend abgekoppelt vom Alter auf dem Arbeitsmarkt und in der Gesellschaft www.medienpaed.com/rezensionen 2 / 8 Rezensionen hohes Ansehen geniesst und als erstrebens- und erhaltenswert gilt. Dadurch verschwimmt das Verhältnis der Generationen. Gekoppelt mit der Mediati- sierung übernehmen vermehrt Peers Vorbildfunktionen für gesellschaftliche Werte und pädagogische Zwecke. Das bedeutet, Jugendliche lernen in vie- len Fällen besser von Peers, da diese für sie verständlich erklären können. Ausserdem hilft die Vorbildfunktion der Peers den Jugendlichen, das Gelern- te vorbehaltlos anzunehmen. Karin Wehmeyer schliesst mit «Entgrenzte Jugend im begrenzten öffentli- chen Raum» an diese Überlegungen an. Auch sie sieht Tendenzen der Indi- vidualisierung, die einer Rollen- und Ordnungsorientierung im Wege stehen. Die Selbstfindung und -erfahrung ist ebenfalls durch eine Begrenzung der öffentlichen Räume gefährdet, in denen sich die Heranwachsenden aufhal- ten und bewegen. Diese Räume sind kontrolliert und verregelt. Die Jugend- phase findet zunehmend in institutionalisierter, kontrollierter und überwach- ter Umgebung statt. Darunter leidet nicht nur die Privatsphäre. Das unkon- trollierte Ausprobieren der eigenen Grenzen, das für die Aneignung und das Lernen wichtig ist, kann kaum noch stattfinden. Marc Bienefeld und Oliver-Böhm-Kasper stellen in «Jugend und Politik» empirische Befunde der Jugendforschung dar und betrachten sie vor dem Hintergrund der sich ständig weiter entwickelnden Medienwelt kritisch. Sie erklären, «dass das politische Interesse und die Neigung zur politischen Partizipation im Jugendalter periodischen Schwankungen unterworfen» sei (S. 84). Dies stehe im Gegensatz zur weit verbreiteten Annahme, dass die Jugend weder politisch interessiert noch engagiert sei. In ihrem Artikel for- dern sie eine zunehmende und verstärkte Kombination qualitativer und quantitativer Forschungsmethoden, um validere, differenziertere, objektivere und nach ihrer Ansicht richtigere Aussagen zu politischen Interessen und Partizipationsformen Jugendlicher treffen zu können. Um «Jugendrevolten in Europa: Gewalt und zerstörerische Wut als flüchtige Handlungsermächtigung» geht es im Beitrag Arne Schäfers und Matthias D. Wittes. Am Beispiel der Jugendriots in England 2011 und Schweden 2013 zeigen sie, wie sich Krisen der Erwachsenenwelt auf Jugendliche auswirken können und dass sie zu Revolten führen können. Gerade an Orten mit hoher Jugendarbeitslosigkeit treffen Heranwachsende konsumorientierter Gesell- schaften auf grosse Diskrepanzen. Krawalle und Revolten machen die Ju- gendlichen und ihre Nöte sichtbar und bieten ihnen zumindest kurzfristig eine Möglichkeit zur emotionalen Entladung sowie das Gefühl der Hand- lungsmacht. www.medienpaed.com/rezensionen 3 / 8 Rezensionen Die Schwierigkeiten einer Individualisierung, die sich einer sozialen Kontrolle und Anerkennung unterwerfen muss, stellt Hans-Jürgen von Wensierski in seinem, den ersten Teil des Sammelbandes abschliessenden, Artikel «Ho- mosexualität bei jungen Muslimen in Deutschland» dar. Dieser basiert auf einer von Wenierski selbst durchgeführten qualitativen Studie mit leitfaden- basierten Interviews. Ausserdem werden individuelle biografische Beispiele Betroffener vorgestellt. Er stellt einen «Trend zur Liberalisierung sexualmo- ralischer Überzeugungen» (S. 125) bei den jungen Muslimen fest. Insbe- sondere Frauen scheinen liberalere Vorstellungen zu haben, wogegen die in der Studie befragten religiösen jungen Männer Homosexualität als Lebens- entwurf strikt ablehnen. Damit bilden sie das «Schlusslicht» eines anschei- nend bereits begonnenen «kulturellen Wandels» (S. 126). Der zweite Teil des Sammelbandes ist überschrieben mit «Mediensozialisa- tion als Katalysator von Wandlungs- und Entgrenzungsprozessen». Alle Beiträge knüpfen an Uwe Sanders Forschungsprojekte zu Lebens- und Me- dienwelten Heranwachsender an, in denen er nicht nur die Frage nach dem Einfluss von Werbung auf Kinder gestellt hat, sondern auch nach der Be- deutsamkeit von Medien für den Bildungsprozess. Den Anfang macht Sonja Ganguin mit «Entgrenzung von Jugend und Arbeit im Kontext des medialen Wandels». Sie merkt an, dass die Jugendlichen sich stark individualisieren. Dadurch können sie das Gefühl eines Entgrenzt- Seins entwickeln. Ganguin geht der Frage nach, «was die Auflösung klarer Bezüge und Strukturen in der Wissensgesellschaft für heutige Jugendliche bedeutet» (S. 131). Sie betrachtet die Subjektivierung von Arbeit und den digitalen Medien in der Arbeitswelt und stellt heraus, dass als Ziel der Medi- enkompetenz Jugendlicher die «Teilhabe an der Gesellschaft, Übernahme von Verantwortung sowie die Gestaltung selbstorganisierter Lebensprozes- se sein» (S. 143) sollte. Gerade die Unbefangenheit und Experimentierfreu- de, mit der Jugendliche an die ‹neuen› Medien herangehen, bergen neue Möglichkeiten und Potenziale. Sabine Kaiser hält in ihrem Beitrag «Medienaneignung im Jugendalter» zu- nächst einleitend fest, dass «Jugendliche Lebenswelten […] Medienwelten» sind (S. 150). Zwischen sozialer Ungleichheit und Anerkennung von Hetero- genität diskutiert sie die Vielfalt der Erscheinungsformen jugendlichen Medi- enhandelns. Sie stellt ein Spannungsverhältnis zwischen Subjekten, Medien und Ungleichheitsstrukturen fest und den daraus resultierenden Bedarf neu- er Impulse für die Medienpädagogik. Sie fordert: «Eine Anerkennung von www.medienpaed.com/rezensionen 4 / 8 Rezensionen Heterogenität sollte nicht die Duldung von Benachteiligung aufgrund hetero- gener Lebenslagen transportieren, sondern stattdessen die Fluidizität […] betonen» (S. 163). Im darauffolgenden Beitrag Kira van Bebbers wird eine Medienerfahrung Jugendlicher quasi von innen betrachtet. Sie beschreibt eine empirische Studie zur Rezeption des «High School Musicals». Sie stellt dar, dass Ju- gendliche im Prozess der eigenen Identitätsbildung Personen brauchen, mit denen sie sich identifizieren und vergleichen können. Bei der rezeptiven Aneignung des fiktiven «High School Musicals» verschmelzen mediale mit non-medialen Welten. Die Autorin fordert, dass die Pädagogik sich intensiv mit den Sehgewohnheiten von Kindern und Jugendlichen auseinanderset- zen solle, um sich Anknüpfungspunkte an deren Vorbilder zur Identifikation zu verschaffen (S. 181). Renate Möller betrachtet das Smartphone als Leitmedium. Sie stellt eine «ambivalente Beziehung» (S. 198) zu diesem Gerät fest. Der Bildschirm des Smartphones habe das Potenzial, den realen Raum zu dominieren und lege fest, auf was der Nutzer seine Aufmerksamkeit zu richten habe (S. 189). Jugendliche wissen durchaus, dass es fraglich ist, immer und zu jeder Zeit Informationen abrufen zu können, und dass sie einem Aufmerksamkeits- und Reaktionszwang unterliegen. Dennoch können sie sich diesem «Sog des Monitors» (S. 189) nur schlecht entziehen. Der Forschungsschwerpunkt Uwe Sanders Kinder und Werbung wird in Sonja Krögers und Dorothee M. Meisters Beitrag «Kinder, Kommerzialisie- rung und (Online-)Werbung» fortgeführt. Sie halten fest, dass Werbung stets auf Kinder (in der Altersgruppe der 6- bis 13-Jährigen) wirkt. Selbst wenn diese eine kritische Einstellung gegenüber Werbung haben, kann nur von einer «vorkritischen Werbekompetenz» (S. 205) gesprochen werden. Die Autorinnen sprechen sich dafür aus, dass die Werbekompetenz Heranwach- sender gestärkt und Eltern sowie pädagogische Fachkräfte im Hinblick auf Medienkompetenz und durch Datenfülle gezielte Onlinewerbung sensibili- siert werden sollten. Der Sammelband schliesst mit dem dritten Teil, der Herausforderungen für Bildung und Erziehung darstellt. Zoë Clark und Holger Ziegler verstehen den Begriff Entgrenzung als Neu- ordnung und diskutieren in «Jugend, Capabilities und das Problem der Pä- dagogik» die Ansätze der Jugendforschung. Um die gegenwärtigen sozialen Bedingungen des Jungseins analysieren zu können, solle die Jugend als www.medienpaed.com/rezensionen 5 / 8 Rezensionen «Capability» verstanden werden. Das bedeutet, dass Jugendlichkeit (dazu gehören Zustände, Relationen und Praktiken) als Aspekt eines Lebens- standards verstanden wird. Dann wird geschaut, welche Jugendlichen die- sen Standard erfüllen und welche fern davon aufwachsen (S. 227). Der Capabilities-Ansatz fokussiere die Ungleichheit von Lebensaussichten und versuche zu begründen, «in welcher Hinsicht solche Ungleichheiten relevant und wie individuelle Lebensstandards zu evaluieren sind» (S. 226). Thomas Walden schliesst daran mit «Medienkompetenz 2.1» an. Er stellt die Frage, wie die Medienpädagogik mit der explosionsartig anfallenden Infor- mationsflut und den stetigen technischen Innovationen umgehen kann. Er stellt Dieter Baackes Medienkompetenzmodell vor und schliesst seine Über- legungen wie folgt ab: «Angesichts des Umfangs der Herausforderungen […] stellt sich die Frage, ob […] die […] Medienpädagogik […] als ein eige- nes Schulfach zu etablieren wäre» (S. 248). Damit würden Lehrer/innen und Schüler/innen die heutzutage erforderlichen Kompetenzen gezielt lehren und lernen können. Dass Jugendliche im Umgang mit Medien nicht nur Kompetenzen, sondern auch Schutz benötigen, stellt Thorsten Junge in seinem Artikel «Grenzenlo- se Mediennutzung? Jugendmedienschutz und Medienerziehung im digitalen Zeitalter» umfassend dar. Er betrachtet den institutionellen Jugendmedien- schutz kritisch und stellt fest, dass Jugendlichen, die problematische Medi- eninhalte konsumieren, ihre Grenzüberschreitung bewusst (gemacht) wer- den solle, damit sie ihr Verhalten reflektieren können. Der Jugendmedien- schutz muss natürlich Grenzen beinhalten. Werden diese jedoch überwun- den, sind die Heranwachsenden «mit einer Grenzenlosigkeit konfrontiert, deren Auswirkungen noch nicht präzise bestimmt werden können und die ihnen unter Umständen ungewollt zum Nachteil geraten» (S. 262). Auch das Phänomen Cybermobbing wird in diesem Artikel behandelt. Dass das Medienerziehungsverhalten bei Eltern, die selbst Computerspiele spielen oder mit diesen aufgewachsen sind, ein anderes ist als bei Eltern, die keine Erfahrungen damit haben, zeigen Henrike Friedrichs, Friederike von Gross und Katharina Herde in: Die Computerspielnutzung Heranwach- sender aus Elternsicht unter dem Blickwinkel der Habitustheorie. Sie stellen eine Erhebung vor, die sie in einem Forschungsprojekt unter der Leitung Uwe Sanders durchgeführt haben. Sie setzen sich mit Risikovorstellungen und Erziehungskonzepten der Eltern auseinander. Je medial erfahrener Eltern sind, desto eher erlauben sie Computerspiele. Dies bedeutet aber nicht, dass sie das Medium Computerspiel nicht kritisch betrachten. Es www.medienpaed.com/rezensionen 6 / 8 Rezensionen konnte gezeigt werden, dass in den meisten Familien die Altersvorgaben der USK berücksichtigt werden und dass der Einsatz von Computern als Baby- sitter eher abgelehnt wird. Die beiden letzen Beiträge des Sammelbandes beschäftigen sich mit der internationalen Jugendarbeit. Zuerst werden Europäische Perspektiven auf internationale Jugendarbeit kritisch hinterfragt von Judith Dubinski, Yasmine Chehata und Andreas Thimmel. In «‹Youth on the move›?! Mobilität und learning mobility» wird der Mobilitätsbegriff der Europäischen Kommission untersucht. Die Autoren stellen fest, dass es nicht empfehlenswert sei, die Begriffe internationale Jugendarbeit und Mobilität gleichzusetzen, wie es von der Arbeitsgemeinschaft für Kinder- und Jugendhilfe zur Internationalen Jugendarbeit in Deutschland sowie auf EU-Ebene probiert wird, sondern fordern eine kritisch-reflexive Verwendung dieser Begriffe (S. 304). Eine positiv besetze Mobilität aus ökonomischer Sicht, die Freiheit und wirtschaft- liches Vorankommen verspricht, kann nicht gleichgesetzt werden mit sozia- ler Mobilität, die auf Ungleichheit basieren kann oder auch physischer Mobi- lität, die aus Migration und Flucht bestehen kann. Im letzen Beitrag («Internationale Jugendarbeit») richtet Ulrike Becker einen Blick auf «Eine Analyse von Machtstrukturen unter intersektionaler Perspek- tive». Ent-Grenzung einer globalisierten Welt bietet Heranwachsenden Per- spektiven, Erfahrungen, Lernmöglichkeiten und kann ihr Selbstbewusstsein fördern. Dennoch sollten die Konzepte der internationalen Jugendarbeit sensibel sein und diversitätsbewusstere Perspektiven einnehmen. Jugendli- che Gruppen sind nicht homogen. Und gerade Kinder mit erhöhtem Förder- bedarf können von der internationalen Jugendarbeit profitieren. Somit kön- nen auch sie die Erfahrung der Ent-Grenzung machen. Fazit Die (digitalen) Medien entwickeln sich rasant, die Technik erfährt ständig neue Innovationsleistungen. Kinder und Jugendliche wachsen in einer – technisch gesehen – ganz anderen Welt auf, als ihre Eltern. Ausserdem müssen Heranwachsende sich in einer Welt zurechtfinden, die ihre Freihei- ten teilweise stark einschränkt – begrenzt;; die aber wiederum nahezu gren- zenlose Möglichkeiten in der Medienwelt bietet. Dabei darf nicht vergessen werden, dass Jugend keine homogene Gruppe ist, sondern dass sie ver- schiedenste soziale und individuelle Voraussetzungen in sich vereint. Gren- zen werden nicht nur innerhalb und ausserhalb von Medienwelten erfahren, sondern auch im Miteinander unter Jugendlichen (innerhalb der Peergroup) www.medienpaed.com/rezensionen 7 / 8 Rezensionen und in Hinblick auf die Erwachsenenwelt. Der Sammelband ehrt Uwe San- ders und führt seine Forschungen schlüssig fort. Er enthält interessante interdisziplinäre Beiträge, die sich umfassenden Themen widmen und neue Impulse für die Forschung bieten und auch fordern. Den unterschiedlichen Problemen, die einen Heranwachsenden heute be- oder entgrenzen, scheint dieser Sammelband gerecht zu werden. Nicht nur an die Wissenschaft, auch an die Pädagogik bzw. an alle, die mit Jugendlichen umgehen, wird die For- derung gestellt, sich selbst und die Jugend kompetent im Umgang mit Medi- en und den darin enthaltenen teilweise komplexen Welten zu schulen. Yvonne Stingel-Voigt www.medienpaed.com/rezensionen 8 / 8
https://openalex.org/W2982601966	https://digitalcommons.uri.edu/cgi/viewcontent.cgi?article=1966&context=oa_diss	English	null	The Methodological Characteristics of an Independent Living Skills Assessment Form for Adolescents in Placement	null	2,020	cc-by	35,091	The Methodological Characteristics of an Independent Living The Methodological Characteristics of an Independent Living Skills Assessment Form for Adolescents in Placement Skills Assessment Form for Adolescents in Placement Susan Maria Lotto University of Rhode Island Follow this and additional works at: https://digitalcommons.uri.edu/oa_diss Terms of Use All rights reserved under copyright. F39 • 1- 77 L-w • \C\93 Recommended Citation Recommended Citation Lotto, Susan Maria, "The Methodological Characteristics of an Independent Living Skills Assessment Form for Adolescents in Placement" (1993). Open Access Dissertations. Paper 954. https://digitalcommons.uri.edu/oa_diss/954 This Dissertation is brought to you by the University of Rhode Island. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of DigitalCommons@URI. For more information, please contact digitalcommons-group@uri.edu. For permission to reuse copyrighted content, contact the author directly. r,r,-,:_1 I~,,,.... ,._ 1 I ERSITY OF RHODE IS THE METHODOLOGICAL CHARACTERISTICS OF AN INDEPENDENT LIVING SKILLS ASSESSMENT FORM FOR ADOLESCENTS IN PLACEMENT BY SUSAN MARIA LOTTO BY SUSAN MARIA LOTTO ABSTRACT Due to a recent upsurge in interest over the success of preparing state supervised youth for independent living, New York State has revised the majority of its regulations concerning how adolescents in placement should be prepared for independent living. The new regulations require that all adolescents in placement, fourteen years of age and over, have their independent living skills periodically assessed. The present research study examined a 140 item inventory, designed to measure the independent living skills of adolescents in placement, in terms of its internal consistency and concurrent validity. In this study 210 subjects, who resided in either the group home or Residential Treatment Center level of care, were individually administered the 140-item Independent Living Skills Inventory for Adolescents (ILSFA). Subjects also had their global independent living skills independently appraised by their social workers. Based on New York State"s dictates concerning independent living skills, a 7-Factor Model was investigated in terms of identifying practical constructs. Principal Component Analyses, item analyses, and internal consistency analyses were used to develop a 44-item instrument with 2- Factors. This instrument measures information with face validity in the areas of Job Finding and Maintenance, Budgeting, Housecleaning, and Cooking / Care of Food / Nutrition. ACKNOWLEDGEMEN Special thanks to Dr. Paul Florin for his help as major professor. Many thanks to Dr. Peter Merenda for his methodological contributions. Many thanks to Dr. Marge Bumpus and Dr. Henry Biller for their special input into the subject of interest. And, finally, much appreciation to my parents and all of the staff and residents of St. Christopher/Jennie Clarkson' s Child Care Services and The Children's Village for their unmitigated cooperation, devotion, and support with the ILSFA. iii PREFACE The state regulations that dictate how adolescents in placement should be prepared for independent living have recently been changed in New York State. The new regulations, effective July 1, 1988, require that adolescents, 14 years of age and over, be provided with several different services which will assist them in preparing for independent living. More specifically, it is currently mandated in New York State that every adolescent 14 years of age and over must be provided with structured training and supervised performance in the following li ving skills: Job Search, Career Counseling, Locating Housing/Apartments, Budgeting of Money, Shopping, Cooking, and Housecleaning. It is also mandated that these adolescents" Independent Living Skills be periodically assessed by the Child Care Agencies that are responsible for them. Although there are several independent living skills assessment instruments, a review revealed that many are inappropriate for adolescents, particularly adolescents in placement. Furthermore, none have employed rigorous psychometric analyses during i nstrument development. This study attempts to contribute to the development o f an independent living skills instrumen t by employing several psychometric analyses to contribute to item selection and factor identification . iv TABLE OF CONTENTS Acknowledgement • . Pref ace .. ....... . . Table of Contents . List of Tables • . .. List of Figures ... .iii .iv • • V .vi .vii Literature Review. . .. . . . ... . .................................... .1-12 Method •• ..• • ... Subjects. Setting • . Materials. Procedure. Results .... Discussion. Appendix A. Appendix B. Appendix c. Appendix D. Appendix E. Appendix F . Appendix G. Bibliography. . 13-18 .13 -15 .15-16 .16-17 .17-18 .19 -48 .49 -54 .55-73 .74-75 . 76 -87 .88-89 .. 90 .. 91 . 92-109 . .. 110 LIST OF TABLES Demographic Data on Subjects' Sex .•..••.......•....•..•••..••...•••••.••• 1 Demographic Data on Subjects' Age •..•.•....•••.•.•••.••..••••.••..•••...• 2 Demographic Data on Subjects' Race ...••.••••.•••.•...•••••.••••....••.•.• 3 Post-Item Analysis Item within Sub-Category Internal Consistency Data •. •. 4 A Comparison of the Internal Consistency Data for Sub-Categories Pre-and-Post-Item Analysis •.•..••.••.•••...••..•••.••••...•••••..•.• 5 Post-Item Analysis Item Sub-Category within Category Internal Consistency Data .• • ....••••••.••..•...•••.•.••••••.•.••...•••. . .•••• 6 Post-Item Analysis Item within Category Internal Consistency Data • •...• •. 7 A Comparison of Internal Consistency Coefficients Pre-and-Post-Item Analysis •••.........•...••..•.....••..•.••..••.•.. 8 Post-Item Analysis PCA •..••.••...••....•.•.•.•.........•••••......•...• 9 Eigenvalue Listing with 7 Factors Extracted . ..•• • . •....••.. •.. • . . .••.... 10 Varimax Rotated Structure Matrix with 7 Factors Extracted ....• • .....•.•. 11 Post-Item Analysis Eigenvalue Table with 2 Factors Extracted ....•.• •.. .. 12 Varimax Rotated Factor Matrix with 2 Factors Extracted .••.••••. ..... .... 13 Structure Matrix of Factors with Oblique Rotation .....••.•..•...•••..... 14 Communalities on Factor Loading Data ..... . . .• . ..•.•...••..•.. . •••.••.• •• 15 PCA Performed Post-Item Analysis within Each Category ....• • .•.•• ....•••. 16 A Comparison of each of the ILSFA's PCA Factor Analyses ....... . . .. . ...• • 17 Post-Item Analysis Examination of S. W. Reliability Data •... •. .. .•. •.. .•• 18 Constructs of the ILSFA •....... . .... • ..••.•........•.........••......... 19 The Revised ILSFA According to a 2-Factor Model ................. : . .... .. 20 Pre-Item Analysis Item within Sub-Category Internal Consistency Data •... 21 Pre-Item Analysis Item within Category Internal Consistency Data •..•... . 22 Pre-Item Analysis Sub-Category within Category Internal Consistency Data ................................................... 23 PCA Performed on all 7 Categories Pre-Item Analysis ...•..............•. . 24 PCA Performed on all Sub-Categories Pre-Item Analysis ...........•...• . .. 25 Rotated PCA Analysis Pre-Item Analysis with 7 Factors Extracted .......•. 26 vi LIST OF FIGURES Scree Chart of the Post-Item Analysis Factor Analysis .••• • •• • ••..••.•. • •• l Scree Chart of the Pre-Item Analysis Factor Analysis • • •..•.•••••. • ••••••• 2 vii 1 1 Preparing foster adolescents to live on their own can be considered both a relativel y old and relatively new concept in child welfare practice. The fact that it is an old concept was evidenced in colonial times when orphaned children were prepared for adult life through the indenture system. The fact that it is a new concept is illustrated in the recent surge in agency programming of independent living for youths 14 to 21 who do not have the life skills or family support to make a successful transition to young adult life, and are becoming numbered among the homeless, mentally unstable, and criminal young adults. These agency programs are concerned that adolescents in placement be provided with structured training and assessment in the major areas of indep endent living. While most undomiciled youths were placed in foster home type settings during the 1920's and 1930's, a shortage of foster homes growing out of changes brought by World War II in the late 1940's, combined with a sharply delineated need for care of some nonconforming youngsters who could not live comforta bly in typical foster home settings, spurred a regrowth of group care. The trend toward group care for emotionally disturbed children emerged from the realization that children being referred for admission required not only nurt'urance, education, and kindly supervision, but special psychosocial help as a necessary step toward return to their own communities. This group or II in stitutional II care evolved into our present Residential Treatment and Group Home levels of restrictive care. A Residential Treatment Center is an institution for emotionally disturbed children who , because o f their own problems and disturbed relationships with other persons , cannot remain in their own homes for treatment - they need the more controlled and therapeutic environment the institution provides . Ideally, residential treatment provides a therapeutic milieu for the children under care. This practice is predicated on the belief that the daily living experiences of the children - their relationships with adults and with each other - may be as 2 significant for their emotional growth as the hours of clinical therapy they receive (Hylton, 1964). LIST OF FIGURES significant for their emotional growth as the hours of clinical therapy they receive (Hylton, 1964). According to the Child Welfare League of America, the function of a Residential Treatment Center is to provide treatment in a group care therapeutic environment that integrates daily group living, remedial education and treatment services on the basis of an individualized plan for each child, exclusively for children with severe emotional disturbances, whose parents cannot cope with them and who cannot be effectively treated in their own homes, in another family, or in other less intensive treatment-oriented child care facilities. Service elements include: 1) study and diagnosis to determine appropriate service and to develop a treatment plan for each child; 2) work in behalf of or directly with children and youths in a therapeutic milieu during placement ( including provision of group living facilities and the essentials of daily living, such as dental care, and child care supervision); 3) provision of treatment services as needed by each child (social work, psychiatry, psychology, remedial education); 4) work with parents while child is in placement; 5) postplacement activities during readjustment period (Child Welfare League of America, 1981). Many children entering residential placement and, in particular, those entering residential treatment, present a multiplicity of problems - interpersonal, familial, emotional, and educational. Some . commonly observed problems include poorly developed impulse controls, low self- image, poorly developed modulation of emotions, deficiencies in forming relationships, and special learning disabilities. Group homes - small informal homes located in neighborhoods away from the parent institutions - similarly offer a therapeutic transitional experience in preparing young adolescents and testing their readiness to move successfully back to their own homes and communities. A group home placement is also the planned care for adolescents until they attain the legal status of adulthood in order to meet their continuing need for. care and protection, when return to their own family is not possible and 3 adoption or foster family is not preferred by them or their families (Stone, 1987). LIST OF FIGURES According to the Child Welfare League of America, the function of a group home is to provide care and treatment in an agency-owned or operated facility that assures continuity of care and opportunity for community experiences, in combination with a planned gr oup living program and specialized services, for small groups of children and youths whose parents cannot care for them adequately and who, because of their age, problems, or stage of treatment, can benefit by such a program. Service elements include: 1) exploration to determine appropriate service, developme nt of a plan for services , and preparation for placement; 2) work in behalf o f or directly with children and youths during placement (including provision of facilities and the essentials of daily living, such as meals, clothing, arrangements for education, recreation, religion, medical - dental care; child care supervision; social work, psychiatry, psychology; special education, vocational and employment counseling; 3) work with parents while child is in placement; 4) postplace ment activities during readjustment period (Child Welfare League of America, 1981) . The number o f adolescents in foster care is steadil y increasing. This increase is attributable to a greater emphasis o n pro v iding placement as the least restrictive alternative for status offenders, an iQcrease in the number o f dependent adolescents due to unmarried pregnancy, an increase in the disintegra t i on o f the famil y unit due to a rise in alcohol / drug abuse, a greater emphasis on upholding child abuse / neglect laws, and the larger number of children who simply grew up in the syste m because no permanent plans were made for them at an earlier age (Stone, 198 7 ) . No rmal youngsters, t hat is t ho se who achie v e i ndependen ce with the help o f their own families, e ncounter certain kinds o f problems, and they meet these challenges wit h re sources generated by their own families . State-super v ised youth are not so lucky. Their problems are usually greater, but resources must be generated by the system and the caseworker, and these are minimal in comparison to those available to the normal youth. Normal youngsters also have more time and latitude to make mistakes than state-supervised youth, and usually have more emotional stability and better social skills because of their less disrupted lives (Simonitch, 1983). LIST OF FIGURES In addition, there is some research that suggests that the powerful nature of residential treatment in and of itself may actually be harmful to the disturbed client. Easson (1969) stresses this point, stating that the external structure can counter the healthy adolescent emancipation individualization process, perpetuating emotional dependence and confirming the client's view of himself as "crazy" or "bad". Writers such as Goffman (1961) have also drawn attention to the dangers of institutional life, such as depersonalization and iatrogenic disturbance, where disturbance is an adaptation to the culture of the hospital. The report "Children in Canada in Residential Care" (1971) states that often there is a lack of continuity of care - for example, a lack of specific discharge planning, and that as a result there is danger that the child will adapt to the needs of the institution while ties to the outside community are allowed to wither. This danger is increased further if the values and expectations of the residential unit deviate dramatically from those of society in general or the client's family in particular .( Johnson, 1982) . An additional issue that warrants consideration is the widespread belief concerning the strength and significance of the influence of the peer culture in the socialization of children. According to sociological theory, family and peers are the two primary groups that are responsible for a child's social development in America. Typically, the two primary groups complement each other in socializing children. Parents provide love, security, and indoctrination in social values, while the peer group provides friendship, normalizing and leveling influences and reality- testing experiences. The difficulty arises when parents fail to provide 5 5 basic structure and support so that children have to rely primarily on their peers for their upbringing. In most residential treatment centers and group homes, the prevailing practice is to place about 10-15 emotionally disturbed and/or delinquent youths together in a group living unit. Typically, two daytime child care counselors supervise each living unit. In this type of group living environment, youngsters inevitably have much more social interaction with each other than with adults. LIST OF FIGURES It is possible that extended exposure to a large number of socially deviant peers could have a detrimental effect on children, particularly on the values and skills that wil l later be needed when they are preparing for autonomous living (Schaefer, 1980) A key aspect of residential treatment for children is "Milieu Therapy" which can be defined as a scientific or systematic manipulation of the environment aimed at producing changes in the deviant behavior of patients. A basic difficulty that has been found with the milieu approach is that the peer group subcultures within the total milieu operate to undermine the prosocial values of the treatment staff. Regardless of institutional size, peer group communication patterns have been found to maintain the very attitudes and behaviors that led to institutionalization in the first place. Polsky (1959) and Cohen (1955) have graphically described the inner dynamics of how a delinquent sub-culture functions in a Residential Treatment Center. First, authoritarian power structure with the there appears to be a strong, brighter and stronger youths exploiting and tyrannizing the weaker ones. Status, masculine identification and social acceptance are the rewards for delinquent acts . Antisocial norms and values prevail, i.e., an eye-for-an-eye justice, materialistic values and a "take what you can get away with" orientation, as well as an avoidance of constructive activities or meaningful involvement in schools, girls, jobs, or community . Some believe that hostility is so pervasive in this culture that after a while it becomes an automatic response and is displaced readily upon any available target. Delinquent gang leaders are believed to serve as the chief source of 6 models for superego or conscience development. These leaders have learned to view social authorities as persecutory and punishing. As a result, the relationship between counselors and ch ildren is a deadly game, and the main rule is "beat them or they' 11 beat you." No one trusts here and everyone is hungry for love, acceptance, and affection. In addition to affect hunger, delinquent children reportedly exhibit a constant stimulus hunger. Turned off to school, reading, and other constructive activities, they attempt to relieve their intense feelings of boredom by the quest for excitement and adventure, usually antisocial in nature. LIST OF FIGURES In short, the predominant behavior of delinquent youth has been aptly described as malicious (enjoyment of the discomfort of others), negativistic (defined by its negativistic polarity to society's norms), hedonistic (pursuit of immediate pleasure rather than of rational, long-term goals), and non- utilitarian (no rhyme or reason to much of the behavior). (Schaefer, 1980) . In light of the above findings, it seems reasonable to assume that the peer culture within Residential Treatment Center's and group home environments operates to undermine the milieu therapy approach devised by staff. Indeed, it is quite possible, particularly in those institutions that are primarily custodial in nature, that the children may leave the institution even more delinquent than when they entered. Hence, it is easy to understand how the possible harmful effects of re.sidential treatment could be putting the youths from the foster care system at even a further disadvantage as compared to the adolescents who grow up in "normal" family settings. Although children from "normal" family situations may also encounter powerful negative influences from their peers, they also have the love and support from their families to counteract the possible deleterious effects. Since delinquent children "seek excitement and are turned off to constructive activities", it is reasonable to assume that they are not in active pursuit of developing their independent living skills . Instead, in most c ases they are developing attitudes and perceptions that run contrary 7 to the positive goal-minded and responsible attitudes that are requisite for developing independent living skills. The aforementioned difficulties are much more likely to be observed with state-supervised youth as opposed to with youths who are raised at home with their families within their respective communities. Following a New York State Court case, Palmer vs. Cuomo, in which six plaintiffs between the ages of 18 and 21 who were foster care recipients discharged from care to independent living, and also three recipients currently placed in group homes who expected to be discnarged, brought suit claiming lack of supervision of those discharged and lack of provision of discharge for the others, New York State's regulations concerning independent living skills training and preparation have become more stringent and complex. Such Independent Living Skills training programs need accurate assessment tools to measure youths' current fund of independent living skills as well as their progress (Stone, 1987). LIST OF FIGURES Such an Independent Living Skills assessment tool would be significant to Independent Living Skills training programs, and group home and residential treatment programs. Although the aforementioned Palmer vs. Cuomo Civil Rights case brought widespread attention to the fact that children who have been raised in the foster care system are becoming numbered among the homeless, mentally unstable, and criminal young adults, additional research projects have highlighted the defects in foster care systems with regard to preparing youths for independent living. Four studies have confirmed what has long been hinted in informal surveys of homeless populations: People formerly in foster care systems are vastly over-represented among the homeless. The studies conducted in different parts of the country found that the percentage ranged from 38 percent in Minneapolis to about 13 percent in rural Ohio. There are now more than 360,000 youths in foster care . Reportedly, about one-third of those are at risk of leading chaotic lives, ranging from homelessness to criminality. (Stone, 1987) A University of California at Berkeley research project studied 55 8 people who had left foster care in the San Francisco Bay area in the recent years. Twenty-nine percent reported that they had no home or were moving from week to week, while 39 percent said they sometimes or often had problems with housing. (Stone, 1987) Festinger (1983) studied case data and interviewed nearly 400 young adults who had been discharged from foster care - both family homes and group settings - in 1975 in New York City. She noted several problems specific to the foster care population. Although the proportion of white males from foster care who were employed did not differ from white males in the general population, proportionately more black males from foster care were unemployed than black males nationally. Those discharged from group care settings fared less well than those discharged from foster family homes. Of those from group settings, 19 percent more young men and nearly 31 percent more women were receiving public assistance than males and females their age in the general population. In general, those from group care faced greater hurdles than those discharged from foster homes. Festinger's stud y sho wed that 32.7 percent of the males and 4.9% of the females had been arrested o n charges of having committed a misdemeanor or felony. LIST OF FIGURES More of the men who were discharged from group settings (40.7 percent) tended t o have been arrested than men discharged from foster homes (28.5 percent), and they had been arrested more frequently. Festinger speculates that "i t .was more difficult for them to get settled f o llowing their discharge from care and they had less econ omic support to fall back on. Their connections to a famil y were more tenu ous, and more of them may h ave relied on peers as a central source of social support." (Festinger, 1983). However, there is no ev idence t hat the different settings c ontributed to antisocial behaviors and the populations in these two settings might have been different t o begin with . According to Rich Gordon, Executive Director o f Youth and Family Assistance, a pr iv ate gr oup in Redwoo d City, Calif ornia, that helps youths in need, the vast majority of 18 year-olds are not ready to live on their 9 own in the best of circumstances (Barden, 1991). He further points that since the youngsters in foster care have never had the kind of childhood that is considered normal, they have not had the opportunity to complete the task of being children, and yet in many states, the system demands they become independent at eighteen. Following the Palmer vs. Cuomo case, the New York State foster care system raised its age limit to 21 in the mid 1980's when advocates for the homeless won the civil lawsuit on behalf of a group of young people who had been discharged from the foster care system on their 18th birthdays and subsequently became homeless. Now, advocacy groups are pushing all states to raise their foster care age limits to 21. Congress last year raised the age limit to 21 on the Federal Foster Care Independent Living Program, a program that helps these youths learn to manage life on their own . The American Civil Liberties Union has begun pressing for expanded independent living plans in lawsuits aimed at forcing states to spend more money to improve their systems. (Barden, 1991). Given New York State• s recent mandates concerning training and assessing the independent living skills of children in placement, mental health professionals will need an expedient, reliable, and valid instrument to facilitate their jobs . LIST OF FIGURES Instruments that have been designed to assess individual competence are commonly used in the planning of appropriate educational and therapeutic programs for mentally _ retarded children. These instruments often measure an individua l• s adaptive behavior in everyday life situations. For example, the Vineland social Maturity Scale is a developmental schedule concerned with the individual's ability to look after his or her practica l needs and to take responsibility for common daily situations in life (Anastasi, 1982). A newer and more comprehensive instrument is the Adaptive Behavior Scale which measures the effectiveness of an individual in coping with the natural and social demands of his or her environment. Other instruments, such as the Cain-Levine Social Competency Scales (Anastasi, 1982) and the T.M.R. School Competency Scales (Anastasi, 1982), are especially designed 10 to assess the competency of trainable mentally retarded individuals. While the aforementioned instruments tap into such independent living skills as hygiene, socialization, and economic/shopping skills, these instruments would be inappropriate to use with foster care youth because the scales and instruments were primarily designed for mentally retardates and found to be most useful with these populations. In addition, the aforementioned instruments assess various types of "adaptive behaviors" which are not directly relevant to non-retarded adolescent foster care populations (i.e. language development, motor development, etc.). In much the same way, these instruments do not measure specific domains which are especially relevant to obt aining a comprehensive assessment of an adolescent's independent living skills (job search skills, locating housing skills, etc.) (Anastasi, 1982). Castellani (1988) examined assessment instruments from 11 different sources which purport to measure independent living skills. The instruments reviewed were typical of those developed by programs in New York and several other states. The major finding of the review was that the scope of available instruments varies considerably. While the broad skills areas assessed tend to be quite similar, some sources provide a single document, while others provide an entire package of forms. The instruments v ary primarily in the depth to which they explore life skill areas and in their basic approach or style of assessment . Howe.ver, they have not been as concerned with the statistical properties of the instrument, but more with the face validity . LIST OF FIGURES The "Life Skills Inventory Report for Youth in Foster Care" is an assessment instrument with a broad approach, focusing on 14 life skill areas (Dorothy Ansell and the Independent Living Skills Center, 1988). This instrument is sc ored by a checklist approach in which a co unselor, caseworker, foster parent, or other caregiver observes and rates an individual yo uth's skill level. This instrument does not come with an accompanying manual on its statistical properties . Other currently used assessment tools such as the PAYA (Preparing Adolescents for Young 11 Adulthood) engage the youth in the assessment process and in developing a plan for greater self-sufficiency in 10 life skill areas (Castellani, 1988). However, there is no accompanying manual for this tool as well. Only the "Tests for Everyday Living" has an accompanying manual listing the statistical properties of the assessment tool. All other instruments which were reviewed lacked such a manual. "Tests for Everyday Living (TEL) was developed by three staff members at the University of Oregon (Halpern, Irvin, and Landman, 1979). TEL is an instrument for measuring achievement in the life skills area, designed to be used at both the junior high and senior high school levels. The life skills measured by TEL are components of both regular and special education cu rri culums in the areas of career education, consumer economics, and health education. The statistical properties of the TEL listed in the manua l include the degree of internal consistency of each item with every other item and within the total test, the point-biserial correlation coefficient for each item with its test, difficulty level of each item percentage of students answering an item correctly, reliability coefficients, and content validity data. Content validity was used because "TEL is meant to be used as a domain-reference test." The authors suggest that TEL possesses adequate content validity because "content sampling and item selection procedures were clearly specified and defined through exhaustive reviews of the literature relevan~ to life skills education of adolescents" (Halpern, Irvin and Landman, 1979 ) .. While the coefficient alpha internal consistency reliability estimates are adequate, ranging from .7 2 to . 83, it is cited in the manual that "no content area was eliminated on statistical grounds alone". LIST OF FIGURES In addition, the fact that the TEL was standardized on youths from Oregon, California, and Alaska who were not living in foster care, and includes content and subcontent areas that are not directly relevant or useful to youths preparing to leave the foster care system, detracts from its usefulness with N. Y. c. adolescents in the N. Y. s. foster care sys te m. Moreover, the TEL is a multiple choice test and therefore does 12 not tap into a youth's fund of knowledge of independent living skills in the same manner that the present instrument's blank choice items are capable of doing during an assessment. In other words, the ILSFA is capable of tapping into a wider range of responses. This study attempted to contribute to the development of a sound instrument by examining the psychometric qualities of The Independent Living Skills Form for Adolescents ( ILSFA) . The ILSFA (Appendix A) consists of 140 face valid items which have been designed to assess the independent living skills of adolescents 14 years of age and over. The categories assessed by the ILSFA are six of those mandated by New York State (e.g. job search, locating housing/apartments, budgeting of money, shopping, cooking, housecleaning) and an additional miscellaneous category. Although items for the ILSFA were written to reflect six New York State categories, this study explored whether these categories were empirically defensible. This study also explored the concurrent validity of the ILSFA by comparing ILSFA data with ratings of social workers who provided an overall judgement of an adolescent"s skills in the six areas. 13 METHOD SUBJECTS The initial proposal of the present study indicated that 100 or more adolescents between the ages of 14 and 20, and who were residing in either a group home or the RTC level of care, would be the subjects under investigation . However, since the results of a power analysis indicated that a subject pool of 200 would be necessary, 210 students were used in the actual study. Due to the fact that some of the subjects did not complete their forms, 16 ILSFA forms were discarded . These 16 subjects did not complete their entire assessment forms for a variety of reasons including 1) an excessive amount of resistance towards being assessed which evolved into a complete or partial refusal to cooperate, 2) obvious cognitive deficits which significantly interfered with the subjects' comprehension of the presented material, or 3) emergency removals from their placement sites due to incarceration, hospitalization, or transfer to a different level o f restricti ve care. The population investigated includes 194 N.Y.C. youths, between the ages of 14 and 20, who resided in either group home or Residential Treatment Center settings. The two Child care Agencies that were used are considered to be typical and representative of New York State foster care agencies. Once a diagnosis and /or court decision has be en de termined regarding an appr opri ate level of care, there is a random ch ance that any given adolescent will be placed with any of the v arious Child . Care Agencies in New York State. The severity of the behavioral/emotional/ cognitive problems of residents who live in either group homes or the Residential Treatment Center level of care is not significantly different across agencies or institutions" locality. Although all of the subjects are N.Y.C. residents, the group home and institutional locations were distributed throughout the New York City/Westchester / Rockland County areas. Only t he forms of residents who were between the ages of 14 and 20 , and who had demographic data available, were used in the analysis. 14 The demographic data on the 194 subjects are as follows: The demographic data on the 194 subjects are as follows: TABLE 1 DEMOGRAPHIC DATA ON SUBJECTS' SEX Female Male Frequency 64 130 Percent 33 67 Thus, approximately 1/3 of the subjects analyzed in this study were female and 2/3 of the subjects were male. SUBJECTS DEMOGRAPHIC DATA ON SUBJECTS' SEX Female Male Frequency 64 130 Percent 33 67 DEMOGRAPHIC DATA ON SUBJECTS' SEX Female Male Frequency 64 130 Percent 33 67 Thus, approximately 1/3 of the subjects analyzed in this study were female and 2/3 of the subjects were male. The demographic data concerning age is as follows: TABLE 2 The demographic data concerning age is as follows: The demographic data concerning age is as follows: The demographic data concerning age is as follows: Age Frequency Percent 14 83 42.8 15 44 22.7 16 30 15.5 17 18 9.3 18 12 6.2 19 5 2.6 20 2 1.0 The following is a list of the racial/ethnic backgrounds of the subjects under investigation: DEMOGRAPHIC DATA ON SUBJECTS' RACE Race Frequency Percent Black 134 69.1 Hispanic 24 12.4 Puerto Rican 12 6.2 White 7 3.6 X 17 a.a 15 (The miscellaneous [X] category for ethnic origin includes those subjects of mixed ethnicity or who opted to omit that information from their questionnaire.) Participants were not evaluated until they had resided at their present placement site for at least six months. This was done to ensure that the subjects" social workers had enough familiarity with the subjects' strengths and weaknesses in order to complete an independent appraisal of their independent living skills in all of the major skill areas. As a result, the collection of data frequently needed to be temporarily suspended whenever there was a high turnover rate with the agency social workers. The ILSFA was administered in accordance with the agencies' overall independent living skills programs. In much the same way as the residents receive annual psychiatric and bi-annual psychological evaluations, they were not given any options concerning having their independent living skills assessed . However, it should be noted that all children and their legal guardians sign formal consent forms concerning treatment and evaluation procedures during the intake process into their respective agencies. In addition, both agencies involved have organized research committees that approved both the use of this instrument, in addition to the collection of anonymous data for future research purposes . Once the test results were presented in the form of a brief summary. of each resident's ILS strengths and weaknesses, identifying material was removed from all of the test data that would be used in future analysis. SUBJECTS Thus, this study itself used only the archived data, recorded in such a manner that the subjects cou ld not be identified directly or through identifiers linked to the subjects. SETTING The research was conducted at two non-profit child care agencies, at either their group home or residential locations. Both of these agencies have contracts with the City and State of New York to serve non-retarded emotionally disturbed chi ldren . Both agencies provide comprehensive 16 programs to help the child and his/her family or surrogate family define the difficulties they are encountering and restructure their relationships and behavior to enable them to cope more successfully with daily situations. In the cases where re-unification with the family is not a viable solution, both agencies provide comprehensive programs to prepare the adolescents for independent living. The basic treatment team for each adolescent is composed of the cottage staff, social worker, school staff, psychologist, psychiatrist, recreational staff, nursing/medical staff, ILS counselor, and at times, the parents or another significant family member . Treatment modalities are individualized and eclectic. Various therapies in use are milieu (a planned and semi-structured environment and a group living situation in which the individual's needs can be met), behavioral (based on learning theory and different forms of behavior modification programs), relationship (treatment which is oriented toward fostering a positive relationship between a child and his/her therapist or child care worker), and family (parent education and family counseling). The actual assessment took place in the clinical/staff offices of the institution or group home settings. The Child Care Worker Forms (Part B) were individually administered by one of the subjects' child care workers in their respective cottage or group home settings. MATERIALS The assessment tool was the individually administered Independent Living Skills Form for Adolescents (ILSFA). The ILSFA contains 140 items that are scored dichotomously. Topic areas such as Job Finding and Maintenance, Budgeting, Housing, Shopping, Cooking, Housecleaning, and Community Living were included in the test items, which were designed as face v alid items to assess the c ategories of independent living skills mandated by New York State. Another form (Appendix B) was used to obtain the social workers' global ratings of all o f the subjects' independent living skills in the major topic areas. 17 Finally, an answer key was developed by the examiner to assist in scoring the first 100 test items (Appendix C). SUBJECTS This answer key was used by the examiner after the entire test was administered. Prior to administering the ILSFA with the residents for testing purposes, four social work staff used the answer key to check the inter-rater reliability for scoring. The inter-rate reliability was .95. Noteworthy is the fact that during the construction of the ILSFA, a professor of education was consulted to ensure that the vocabulary used was on an approximate fourth grade level. In this way, the examiner was confident that the population under investigation could understand all of the inventory's inclusive vocabulary. PROCEDURE The investigator of this study administered the first 100 items of the ILSFA to all of the 210 subjects on an individual basis as part of her work duties in the agency . Each subject was informed about his/her scheduled testing session by the respective social worker. Each was told that the ILS assessment was an integral component of the agency's overall Independent Living Skills program. The administration of the ILSFA took approximately 45 minutes to 1 1/ 2 hours, depending upon the individual subjects" verbal productivity and language processing skills . With few exceptions, the entire ILSFA was administered in one day. However, on a few occasions the ILSFA's administration needed to be distribu~ed across two days. This occurred due to the subjects ' conflicting schedules with other agency appointments, subjects' resistance, and subjects ' short attention spans. Individual test items were administered verbally to ensure that reading limitations did not affect an individual's score. With the exception of four test items, the examiner wrote down the subjects' verbal responses. The four o ccasions in which sub j ects were expected to write their responses include 1) two items that require the subject to match various terms with their respectful definitions, and 2) the two test items in the Budgeting section that focus on checking accounts. With these two latter test items, the subjects' ability to 18 18 complete a personal check and checking register is inherent to the subject matter under investigation. The last forty items of the ILSFA (Part B) were administered individually to the subjects by one of their child care workers. These last forty items were always administered verbally and did not require any written response from the subject. SUBJECTS Part II of the ILSFA was consistently administered to the subjects within a one to three week time period following the investigator's administration of Part I. The ILSFA was consistently administered in the same manner to all subjects. The subjects' social workers were given the Social Worker's Global Rating Scale (Form B) to complete on each individual subject after the investigator administered Part I with a subject. The social workers were asked to return this form as soon as possible. With few exceptions, Form B was returned to the investigator within a one to two week period after Part I of the ILSFA was administered. The social workers were selected to rate the subjects on their global fund of independent living skills because they have historically been responsible for keeping abreast of the development of these skills as part of their job description. 19 RESULTS The first data analysis to be presented is the item analysis, which was conducted to shorten the original inventory in an attempt to increase the instrument's validity and reliability. Secondly, the reliability and validity data for the original 140-item assessment tool will be summarized, followed by a more comprehensive description of the psychometric properties of the ILSFA post-item analysis (after the original 140 item test was reduced to 96 test questions) . Finally, results from the post-item analysis Factor Analyses that suggest that the ILSFA is best described by a 2-Factor Model will be presented. ITEM ANALYSIS ITEM ANALYSIS In quest of an internally consistent and time efficient instrument, item analysis was performed by studying both the internal consistency and frequencies of correctly answered items. Item analysis makes it possible to shorten a test, and at the same time, to increase its validity and reliability (Anastasi, 1982). Given that the ILSFA' s purpose is to ascertain whether an individual youth has adequately mastered a basic fund of -knowledge about independent living, it is most beneficial to include the test items that contribute the most information about a youth's performance level. A perusal of the "percentage passing" data revealed that several of the items initially included in the ILSFA were nondiscriminating, and therefore contributed minimal or contradictory/confusing information about an individual's overall independent living skills. For the purpose of the present item analysis, all items that were passed by either 95% or 5% of the subjects examined were omitted. (Appendix D) . It is believed that the items in the 95% and 5% passing categories provide little information about individual differences concerning fund of independent living skills. Similarly, it is known that the closer an item approaches 1:00 or O, the less it affects the variability of test scores and contributes relatively nothing to the reliability or validity of the test (Anastasi, 1982). 20 Second, for the purposes of this item analysis, items that were passed by either 90% or 10% of the subjects examined were also omitted if their internal consistency data indicated that they contributed relatively nothing to the overall reliability of a particular ILS category (i.e. Job Finding, Budgeting, etc.). This was accomplished by looking at the value of the corrected item - total correlation data and by determining whether the deletion of the particular item would significantly impact upon the overall internal consistency (Kuder-Richardson #20). Following the aforementioned item analysis based on frequency, 114 of the original 140 items remained in the ILSFA. See Appendix E for a list of test items that either 90% or 10% of the subjects correctly passed . These items' internal consistency data are similarly listed to illustrate how the examiner decided whether or not to delete a particular item . ITEM ANALYSIS The rule of thumb that the examiner decided upon is that if a particularly test item had a percentage passed frequency of 90% or 10% in addition to a corrected item - total correlation coefficient of less than .30, the item was deleted. On the other hand, items with percentage passed frequencies of 90% or 10%, combined with corrected item - total correlation coefficient of greater than .3, were maintained . As a rule, these corrected item - total correlation coefficients that exceeded .3 also markedly decreased the overall internal consistency coefficient alphas (Kuder-Richardson #20) if deleted. The internal c onsist~ncy data that were analyzed include the analysis of individual test items into categories. Hence, according to the second item analysis, 10 more items were deleted. Another 9 items could have been deleted based on their poor discriminatory power alone, but were maintained due to the fact that they contributed to the overall internal consistency of a particular ILS category to a significant degree. A final item analysis was conducted on the basis of the internal c onsistency data alone. These data looked at particular test items within ILS categories. Items were deleted on the basis that their item / total 21 correlation coefficients were so small that their inclusion in the test actually lowered the overall internal consistency of the category under discussion. In other words, the alphas (Kuder-Richardson #20) for a particular ILS category increased when these items were removed. The data are located in Appendix F. An additional 4 items were deleted from the original instrument because if they had been retained, 4 of the remaining sub-categories would have been dichotomous and therefore inappropriate to be used in subsequent factor analyses. In all four cases, the test items contributed minimally to the overall internal consistency of the ILSFA. (These test items include P3Vl2, P4Vl7, P5Vl0, and P6Vl5.) As a result of all 4 methods of item analysis, 50 items were deleted from the original 140 item inventory. Thus, 90 items were retained. FURTHER ANALYSES CONDUCTED PRE-ITEM ANALYSIS The next section describes in detail the results of further analyses (e.g. internal consistency and factor analyses) which were conducted after 50 items were removed from the 140 item ILSFA pool. However, in order to allow comparison of how the removal of the items affected the results, analyses were also done with the entire set of 140 items. ITEM ANALYSIS These results are presented in detail in Appendix G and comparisons of pre and post item analysis results are mentioned in the next section. briefly summarized below. These results are As previously mentioned, the 140 items of the ILSFA are divided into seven categories, each with 20 items. These categories are Job Finding and Maintenance, Apartment Hunting, Budgeting, Shopping, Miscellaneous, Housecleaning, and Cooking/Care of Food/Nutrition . However, all ILSFA items are scored dichotomously and dichotomous items are inappropriate for the kinds of factor analytic analyses desired. Therefore, the 20 items within each category were further divided a posteriori into subcategories that appeared logically related. This would allow the items within each subcategory to be summed, producing non-dichotomous scores. Thirty-six subcategories were created . The range of items within a sub-category was 22 2 - 6 but most contained 4 or 5 items. The 36 internal consistency coefficients obtained for these sub-categories were consistency quite low, perhaps because they were of few items per category or perhaps because items were categorized into sub-categories incorrectly. In order to check for this possibility, internal consistency coefficients were done with the 20 items in each general category. The standardized item alphas here ranged from .57 to .91, with four of the seven categories above Nunnally's . 70 rule of thumb, suggesting that erroneous a priori distribution of items into sub-categories produced the low internal consistency coefficients of sub-categories. Internal consistency analysis was also performed on the sub- categories within categories. The number of sub-categories in a category ranged from 4-7 and three of the seven categories had internal consistency coefficients above .70. Principal Component Analysis was performed in two ways. First, with sub-categories within each category. Four categories had only factor and three categories had two factors, but in these cases the second factor added little variance . A Principal Component Analysis with all 36 sub- categories entered into the analysis revealed 2 factors similar to the two factors which are discussed below. POST-ITEM ANALYSIS INTERNAL CONSISTENCY ANALYSES As previously indicated, item analyses reduced the original data set from 140 to 90 items. The internal consistency and factor analyses data should be altered as a result of removing the SO items. The following analyses were performed post-item analysis . The following is a post-item analysis representation of the internal consistency of each of the sub-categories. In other words, items within sub-category data are being examined. 23 TABLE 4 POST-ITEM ANALYSIS ITEM WITHIN SUB-CATEGORY INTERNAL CONSISTENCY DATA Category 1 = Job Finding and Maintenance Sub-Category 1 = Job Application Forms and Procedures for Applying for a Job Sub-Category= Items PlVl PlV2 P1V8 PlVl0 Standardized Item Alpha= .49 Sub-Category 2 = Job Interviewing Skills Sub-Category= Items PlV4 PlVS PlV6 Standardized Item Alpha= .67 Sub-Category 3 = General Job-Related Knowledge and Terms Sub- category= Items PlVll P1V13 PlVlS P1Vl6 Standardized Item Alpha= .51 Sub-Category 4 = Job-Related Behavior Sub-Category= Items P1V18 P1V20 Standardized Item Alpha= .20 Category 2 = Apartment Hunting and Maintenance Sub-Category 1 = Procedures for Looking for an Apartment Sub- category= Category 1 = Job Finding and Maintenance Sub-Category 1 = Job Application Forms and Procedures for Applying for a Job Sub-Category= Items PlVl PlV2 P1V8 PlVl0 Standardized Item Alpha= .49 Category 2 = Apartment Hunting and Maintenance Sub-Category 1 = Procedures for Looking for an Apartment Sub- category= Items P2V3 P2Vl2 Standardized Item Alpha= .20 Sub-Category 3 = Apartment Related Items Sub-Category Items P2VS P2V10 P2V19 Standardized Item Alpha= .44 Sub-Category 4 = Utility and Protective Agencies Sub-Category = Items P2V8 P2V18 Standardized Item Alpha= .27 Sub-Category 5 = Apartment Living Sub-Category= Items P2V13 P2V14 Standardized Item Alpha .39 24 Sub-Category 6 = Apartment Maintenance Sub-Category= Items P2Vl5 P2Vl6 P2Vl7 Standardized Item Alpha= .02 Sub-Category 6 = Apartment Maintenance Sub-Category= Items P2Vl5 P2Vl6 Category 3 = Budgeting Sub-Category 1 = Checking Accounts Sub-Category= Items P3Vl P3V2 P3V4 P3VS Standardized Item Alpha= .54 Sub-Category 2 = Concept of Interest and Savings Accounts Sub- category= Items P3V7 P3V8 standardized Item Alpha= .31 Sub-Category 3 = How to Budget Sub - Category= Items P3V6 P3Vl5 P3Vl6 P3Vl7 Standardized Item Alpha= .41 Sub-Category 4 = Reading and Understanding Paychecks Sub-Category= Items P3V9 P3Vl4 P3Vl8 P3Vl9 Standardized Item Alpha= .36 Category 3 = Budgeting Category 3 = Budgeting Sub-Category 1 = Checking Accounts Sub-Category= Items P3Vl P3V2 P3V4 P3VS Standardized Item Alpha= .54 Sub-Category 2 = Concept of Interest and Savings Accounts Sub- category= Items P3V7 P3V8 standardized Item Alpha= .31 Sub-Category 4 = Reading and Understanding Paychecks Sub-Category= Items P3V9 P3Vl4 P3Vl8 P3Vl9 Standardized Item Alpha= .36 Category 4 = Shopping Sub-Category 1 = Methods and Implications of Comparative . Shopping Sub - Category= Items P4V4 P4V8 P4Vl3 P4Vl6 Standardized Item Alpha= .37 Sub-Category 2 = Warranties and consumer's Rights Sub - Category Items P4Vl P4Vl9 P4V20 Standardized Item Alpha= .26 Sub-Category 4 = Advertising Ploys Sub-Category= Items P4V6 P4V14 Standardized Item Alpha= .17 Category 4 = Shopping Sub-Category 1 = Methods and Implications of Comparative . Shopping Sub - Category= Items P4V4 P4V8 P4Vl3 P4Vl6 Standardized Item Alpha= .37 Sub-Category 2 = Warranties and consumer's Rights Sub - Category Items P4Vl P4Vl9 P4V20 Standardized Item Alpha= .26 Sub-Category 2 = Warranties and consumer's Rights Sub - Category Items P4Vl P4Vl9 P4V20 Standardized Item Alpha= .26 Sub-Category 4 = Advertising Ploys Sub-Category= Items P4V6 P4V14 25 Sub-Category 5 = Consumer Terminology. and Knowledge About Bargains Sub-Category= Items P4V5 P4Vl2 P4Vl5 P4Vl8 Standardized Item Alpha= .25 Category 5 = Miscellaneous Category 5 = Miscellaneous Sub-Category 1 = Transportation and Car Issues Sub-Category= Items P5V2 P5V3 Standardized Item Alpha= .31 Sub-Category 2 = Alcohol and Drug Issues Sub-Category Items P5V6 P5V9 Standardized Item Alpha= .22 Sub-Category 3 = Knowledge About Mature Adult Behavior Sub-Category= Items P5Vl6 P5V20 Standardized Item Alpha= .28 Sub-Category 4 = Sex Education Issues Sub-Category= Items P5Vl8 P5Vl9 Standardized Item Alpha= .53 Sub-Category 6 = Negotiating Leisure Activities Sub-Category= Items P5V8 P5Vl2 Standardized Item Alpha= .42 Category 6 = Housecleaning Sub-Category 1 = Fundamentals of Housecleaning Sub-Category= Items P6V2 P6Vl9 Standardized Item Alpha = .48 Sub-Categor y 3 = Household Safety Sub-Category = Items P6V7 Category 5 = Miscellaneous Sub-Category 1 = Transportation and Car Issues Sub-Category= Items P5V2 P5V3 Standardized Item Alpha= .31 Sub-Category 2 = Alcohol and Drug Issues Sub-Category Items P5V6 P5V9 Standardized Item Alpha= .22 Sub-Category 3 = Knowledge About Mature Adult Behavior Sub-Category= Items P5Vl6 P5V20 Standardized Item Alpha= .28 Sub-Category 4 = Sex Education Issues Sub-Category= Items P5Vl8 P5Vl9 Standardized Item Alpha= .53 Sub-Category 6 = Negotiating Leisure Activities Sub-Category= Items P5V8 P5Vl2 Standardized Item Alpha= .42 Sub-Category 1 = Transportation and Car Issues Sub-Category= Items P5V2 P5V3 Standardized Item Alpha= .31 Category 6 = Housecleaning Sub-Category 1 = Fundamentals of Housecleaning Sub-Category= Items P6V2 P6Vl9 Standardized Item Alpha = .48 Sub-Categor y 3 = Household Safety Sub-Category = Items P6V7 P6V8 P6Vl7 P6V20 Standardized Item Alpha = . 72 Category 6 = Housecleaning Sub-Category 1 = Fundamentals of Housecleaning Sub-Category= Items P6V2 P6Vl9 Standardized Item Alpha = .48 Sub-Categor y 3 = Household Safety Sub-Category = Items P6V7 P6V8 P6Vl7 P6V20 Standardized Item Alpha = . Category 5 = Miscellaneous 72 Category 6 = Housecleaning 26 Sub-Category 4 = Care of Clothing Sub-Category= Items P6V9 P6V10 P6Vll P6Vl3 P6V14 Standardized Item Alpha= .73 Category 7 = Cooking/Care of Food/Nutrition Sub-Category 1 = Nutrition Sub-Category= Items P7Vl P7V2 P7V3 P7V4 Standardized Item Alpha= .83 Sub-Category 2 = How to Cook and Prepare Food Sub-Category= Items P7V14 P7V15 P7V16 P7V18 Standardized Item Alpha .76 Sub-Category 3 = How to Shop for Food Sub-Category= Items P7VS P7V6 P7V7 P7V8 P7V9 Standardized Item Alpha= .79 Sub-Category 4 = Food Storage Sub-Category= Items P7V10 P7Vll Category 7 = Cooking/Care of Food/Nutrition Sub-Category 4 = Food Storage Sub-Category= Items P7V10 P7Vll P7V13 P7Vl 7 Standardized Item Alpha= .71 With the exception of the "Housecleaning" and "Cooking/Care of Food/Nutrition" sub-categories, the internal consistency coefficients obtained were consistently low. This is believed to be true because l) several of the subcategories were only comprised of two or three items, and 2) the subcategories were superimposed on the items a posteriori due to the examiner's desire not to do factor analyses with dichotomous items. A comparison of the internal consistency coefficients of each of the sub-categories will now be made pre and post-item analyses. 27 TABLE 5 A COMPARISON OF THE INTERNAL CONSISTENCY DATA FOR SUB-CATEGORIES PRE AND POST-ITEM ANALYSIS A COMPARISON OF THE INTERNAL CONSISTENCY DATA FOR SUB-CATEGORIES PRE AND POST-ITEM ANALYSIS Sub-Categories 1 2 3 4 Sub-Categories 1 2 3 4 5 6 Sub-Categories 1 2 3 4 5 Sub-Categories 1 2 3 4 5 Sub-Categories 1 2 3 4 5 6 7 Job Finding and Maintenance Pre-Item Analysis .48 .62 . 46 . 13 Apartment Hunting and Maintenance Pre-Item Analysis .26 .12 .44 .09 .39 .02 Budgeting Pre-Item Analysis .53 . 44 . 41 .36 .02 Shopping Pre-Item Analysis .35 .36 .02 .04 . 36 Miscellaneous Pre-Item Analysis .31 .22 .37 .53 .14 . 40 .10 Post-Item Analysis .49 .67 . 51 . 20 Post-Item Analysis .20 Omitted Completely .44 .27 .39 . 02 Post-Item Analysis .54 .31 .41 .36 Omitted Completely Post-Item Analysis .37 . 26 Omitted completely .17 . .25 Post-Item Analysis .31 .22 .28 .53 Omitted completely .42 Omitted Completely Sub-Categories 1 2 3 4 Job Finding and Maintenance Pre-Item Analysis .48 .62 . 46 . 13 Post-Item Analysis .49 .67 . 51 . Category 5 = Miscellaneous 20 Sub-Categories 1 2 3 4 5 6 Apartment Hunting and Maintenance Pre-Item Analysis .26 .12 .44 .09 .39 .02 Post-Item Analysis .20 Omitted Completely .44 .27 .39 . 02 28 Sub-Categories 1 2 3 4 5 Sub-Categories 1 2 3 4 Housecleaning Pre-Item Analysis .66 -.07 • 72 . 73 .26 Cooking/Care of Food/Nutrition Pre-Item Analysis .83 • 71 .79 .78 Post-Item Analysis .48 Omitted Completely • 72 .73 Omitted Completely Post-Item Analysis . 83 . 76 .79 • 71 Sub-Categories 1 2 3 4 5 Housecleaning Pre-Item Analysis .66 -.07 • 72 . 73 .26 Post-Item Analysis .48 Omitted Completely • 72 .73 Omitted Completely Sub-Categories 1 2 3 4 Cooking/Care of Food/Nutrition Pre-Item Analysis .83 • 71 .79 .78 Post-Item Analysis . 83 . 76 .79 • 71 Generally, the item analysis, which resulted in 50 items being omitted from the ILSFA, did not result in any significant changes with regard to the sub-categories' Internal Consistency data. The next internal consistency data to be re-examined following the item analyses, and the concomitant deletion of the 50 less discriminating test items, is the sub-category within category internal consistency analysis. In other words, the degree to which sub-categories in particular categories hang t ogether will be in vestigated. The data is as follows: TABLE 6 POST-ITEM ANALYSIS SUB-CATEGORY WITHIN CATEGORY INTERNAL CONSISTENCY DATA Sub-Categories 1 2 3 4 Job Finding and Maintenance Corrected Item - Total Correlation .60 .35 .so .33 Standardized Item Alpha .64 Alpha if Item Deleted . 42 . 62 .52 . 64 29 Sub-Categories 1 3 4 5 6 Apartment Hunting and Maintenance Corrected Item - Total Correlation .35 .38 .37 . 31 .34 Sub-Category #2 was omitted. Standardized Item Alpha= .60 Sub-Categories 1 2 3 4 Budgeting Corrected Item - Total Correlation . 45 .42 .46 .42 Sub-Category #5 was omitted. Standardized Item Alpha= .66 Sub-Categories 1 2 4 5 Shopping Corrected Item - Total Correlation .36 .32 .23 .23 Sub-Category #3 was omitted. Standardized Item Alpha= . 48 Sub-categories 1 2 3 4 6 Miscellaneous Corrected Item - Total Correlation . 20 .32 .44 .37 .42 Sub-Category #5 and #7 were omitted. Category 5 = Miscellaneous Standardized Item Alpha= .59 Alpha if Item Deleted .54 .52 .51 .55 .53 Alpha if Item Deleted .57 .60 .54 .57 Alpha if Item Deleted .33 .38 .45 .46 Alpha if Item Deleted .60 .54 .49 .52 .49 Sub-Categories 1 3 4 5 6 Apartment Hunting and Maintenance Corrected Item - Total Correlation .35 .38 .37 . 31 .34 Sub-Category #2 was omitted. Alpha if Item Deleted .54 .52 .51 .55 .53 30 Sub - Categories 1 3 4 Housecleaning Corrected Item - Total Correlation .63 .so .60 Sub - Category #2 and #5 were omitted. Standardized Item Alpha= .76 Alpha if Item Deleted • 63 . 70 . 53 Cooking/Care of Food/Nutrition Corrected Item - Alpha if Item Deleted .8 1 Sub-Categories 1 2 3 4 Total Correlation . 70 . 78 .77 . 65 Standardized Item Alpha= .87 . 78 . 79 .as Sub - Categories 1 3 4 Housecleaning Corrected Item - Total Correlation .63 .so .60 Sub - Category #2 and #5 were omitted. Standardized Item Alpha= .76 Alpha if Item Deleted • 63 . 70 . 53 Cooking/Care of Food/Nutrition Corrected Item - Alpha if Item Deleted .8 1 Sub-Categories 1 2 3 4 Total Correlation . 70 . 78 .77 . 65 Standardized Item Alpha= .87 . 78 . 79 .as The sub-category within category internal consiste ncy data revealed that two of the seven categories had internal consistency coefficients above .70 . They include the "Housecleaning" and "Cooking/Care of Food/Nutrition" categories. The final internal consistency data that will be examined will be the item within category reliability. In other words, the degree to which all of the items within each category hang together will be investigated. The data is as follows: TABLE 7 POST- ITEM ANALYSIS ITEM WITHIN CATEGORY INTERNAL CONSISTENCY DATA OST- ITEM ANALYSIS ITEM WITHIN CATEGORY INTERNAL CONSISTENCY DATA Sub-Item PlVl PlV2 PlV3 Omitted P1V4 PlVS PlV6 PlV7 Omitted PlV8 PlV9 Omitted PlVlO PlVll PlV12 Omitted Job Finding and Maintenance Corrected Item - Total Cor relati on .21 .42 .35 . 29 .37 .40 .52 .38 Alpha if Item Deleted .72 . 69 .70 • 71 .70 .70 .68 . 70 31 31 Sub-Item P1V13 P1V14 omitted PlV15 P1V16 P1V17 omitted PlV18 P1V19 Omitted PlV20 Corrected Item - Total Correlation .34 .41 .31 .21 .30 Standardized Item Alpha= .73 Alpha if Item Deleted .70 .69 • 71 .72 • 71 P1V16 P1V17 omitted PlV18 P1V19 Omitted PlV20 .31 .21 .30 Standardized Item Alpha= .73 • 71 .72 • 71 Apartment Hunting/Maintenance Sub-Item P2Vl omitted P2V2 omitted P2V3 P2V4 omitted P2V5 P2V6 omitted P2V7 omitted P2V8 P2V9 omitted P2V10 P2Vll Omitted P2V12 P2V13 P2Vl4 P2V15 P2V16 P2V17 P2Vl8 P2Vl9 P2V20 omitted Corrected Item - Total Correlation .36 .35 . 22 .26 . 18 .31 .22 .14 .23 . 19 .39 .34 Standardized Item Alpha= . 62 Sub-Item P3Vl P3V2 P3V3 Omitted P3V4 P3V5 P3V6 P3V7 P3V8 P3V9 P3V10 Omitted P3Vll Omitted P3V12 Omitted P3V13 Omitted P3Vl4 P3Vl5 P3Vl6 P3V17 P3V18 Budgeting Corrected Item - Total Correlation .42 .29 .4 1 .29 .34 .18 .45 .29 .38 .20 .26 .39 .22 Alpha if Item Deleted .57 .57 .60 .59 .60 .58 .60 .61 .60 .60 .56 .57 Alpha if Item Deleted .66 .68 . 66 .68 .67 . 69 .66 .68 .67 .69 .68 .67 .69 Standardized Item Alpha= .73 Apartment Hunting/Maintenance Apartment Hunting/Maintenance Sub-Item P2Vl omitted P2V2 omitted P2V3 P2V4 omitted P2V5 P2V6 omitted P2V7 omitted P2V8 P2V9 omitted P2V10 P2Vll Omitted P2V12 P2V13 P2Vl4 P2V15 P2V16 P2V17 P2Vl8 P2Vl9 P2V20 omitted Corrected Item - Total Correlation .36 .35 . 22 .26 . 18 .31 .22 .14 .23 . 19 .39 .34 Standardized Item Alpha= . 62 Alpha if Item Deleted .57 .57 .60 .59 .60 .58 .60 .61 .60 .60 .56 .57 Sub-Item P3Vl P3V2 P3V3 Omitted P3V4 P3V5 P3V6 P3V7 P3V8 P3V9 P3V10 Omitted P3Vll Omitted P3V12 Omitted P3V13 Omitted P3Vl4 P3Vl5 P3Vl6 P3V17 P3V18 Budgeting Corrected Item - Total Correlation .42 .29 .4 1 .29 .34 .18 .45 .29 .38 .20 .26 .39 .22 Alpha if Item Deleted .66 .68 . 66 .68 .67 . POST- ITEM ANALYSIS ITEM WITHIN CATEGORY INTERNAL CONSISTENCY DATA 69 .66 .68 .67 .69 .68 .67 .69 32 Sub-Item P3Vl9 P3V20 Omitted Corrected Item - Total Correlation .19 Standardized Item Alpha= .69 Sub-Item P4Vl P4V2 Omitted P4V3 Omitted P4V4 P4VS P4V6 P4V7 Omitted P4V8 P4V9 P4Vl0 P4Vll P4Vl2 P4Vl3 P4Vl4 P4Vl5 P4Vl6 Omitted Omitted Omitted P4Vl7 Omitted P4Vl8 P4Vl9 P4V20 Shopping corrected Item - Total Correlation .37 .23 .25 .17 . 30 .17 .25 .20 .21 .21 .02 .02 .27 Standardized Item Alpha= .53 Sub-Item PSVl Omitted PSV2 PSV3 PSV4 Omitted PSVS Omitted PSV6 PSV7 Omitted PSV8 PSV9 PSVl0 Omitted PSVll Omitted PSV12 PSV13 Omitted PSV14 Omitted PSVlS Omitted PSV16 PSV17 Omitted PSV18 PSV19 PSV20 Miscellaneous Corrected Item - Total Correlation .25 .11 .26 .39 .25 .33 .32 . 39 . 35 . 41 Standardized Item Alpha= .64 Alpha if Item Deleted .69 Alpha if Item Deleted .47 .so .so . 52 .49 .52 .so .51 .51 .51 .56 .ss .so Alpha if Item Deleted .61 .64 .62 .58 .61 .60 .60 . 58 . 69 . 60 Sub-Item P3Vl9 P3V20 Omitted Corrected Item - Total Correlation .19 Standardized Item Alpha= .69 Sub-Item P4Vl P4V2 Omitted P4V3 Omitted P4V4 P4VS P4V6 P4V7 Omitted P4V8 P4V9 P4Vl0 P4Vll P4Vl2 P4Vl3 P4Vl4 P4Vl5 P4Vl6 Omitted Omitted Omitted P4Vl7 Omitted P4Vl8 P4Vl9 P4V20 Shopping corrected Item - Total Correlation .37 .23 .25 .17 . 30 .17 .25 .20 .21 .21 .02 .02 .27 Standardized Item Alpha= .53 Alpha if Item Deleted .69 Alpha if Item Deleted .47 .so .so . 52 .49 .52 .so .51 .51 .51 .56 .ss .so Sub-Item PSVl Omitted PSV2 PSV3 PSV4 Omitted PSVS Omitted PSV6 PSV7 Omitted PSV8 PSV9 PSVl0 Omitted PSVll Omitted PSV12 PSV13 Omitted PSV14 Omitted PSVlS Omitted PSV16 PSV17 Omitted PSV18 PSV19 PSV20 Miscellaneous Corrected Item - Total Correlation .25 .11 .26 .39 .25 .33 .32 . 39 . 35 . 41 Standardized Item Alpha= .64 Alpha if Item Deleted .61 .64 .62 .58 .61 .60 .60 . 58 . 69 . 60 33 Sub - Item P6Vl omitted P6V2 P6V3 omitted P6V4 omitted P6VS omitted P6V6 omitted P6V7 P6V8 P6V9 P6V10 P6Vll P6V12 omitted P6V13 P6V14 P6Vl5 omitted P6V16 omitted P6V17 P6Vl8 Omitted P6V19 P6V20 Housecleaning Corrected Item - Total Correlation . 54 .40 .so .59 . 53 . 36 .52 . 41 . 62 .49 . 41 Standardized Item Alpha= . POST- ITEM ANALYSIS ITEM WITHIN CATEGORY INTERNAL CONSISTENCY DATA 82 Sub - Item P7Vl P7V2 P7V3 P7V4 P7VS P7V6 P7V7 P7V8 P7V9 P7V10 P7Vll P7V12 Omitted P7Vl3 P7V14 P7V15 P7V16 P7V17 P7V18 P7V19 Omitted P7V20 Omitted Cooking/Care of Food/Nutrition Corrected Item - Total Correlation . 60 .66 . 61 . 67 . 64 .64 . 62 .54 . 56 .49 .44 . 56 .41 .69 .75 .49 . 58 Standardized Item Alpha= .91 Alpha if Item Deleted . 79 .81 . 80 . 79 . 80 .81 . 80 .81 .79 .80 .81 Alpha if Item Deleted .91 .90 . 90 .90 .90 .90 .90 . 91 . 91 .91 .91 . 91 . 91 .90 . 90 .91 .9 1 The item with categ or y internal consistency data revealed that t hree of the seven categories had internal consistency coefficients above . 70. This suggests that erroneous a p riori distribution o f items into sub - Sub - Item P6Vl omitted P6V2 P6V3 omitted P6V4 omitted P6VS omitted P6V6 omitted P6V7 P6V8 P6V9 P6V10 P6Vll P6V12 omitted P6V13 P6V14 P6Vl5 omitted P6V16 omitted P6V17 P6Vl8 Omitted P6V19 P6V20 Housecleaning Corrected Item - Total Correlation . 54 .40 .so .59 . 53 . 36 .52 . 41 . 62 .49 . 41 Standardized Item Alpha= . 82 Alpha if Item Deleted . 79 .81 . 80 . 79 . 80 .81 . 80 .81 .79 .80 .81 . 54 . 79 Sub - Item P7Vl P7V2 P7V3 P7V4 P7VS P7V6 P7V7 P7V8 P7V9 P7V10 P7Vll P7V12 Omitted P7Vl3 P7V14 P7V15 P7V16 P7V17 P7V18 P7V19 Omitted P7V20 Omitted Cooking/Care of Food/Nutrition Corrected Item - Total Correlation . 60 .66 . 61 . 67 . 64 .64 . 62 .54 . 56 .49 .44 . 56 .41 .69 .75 .49 . 58 Standardized Item Alpha= .91 Alpha if Item Deleted .91 .90 . 90 .90 .90 .90 .90 . 91 . 91 .91 .91 . 91 . 91 .90 . 90 .91 .9 1 The item with categ or y internal consistency data revealed that t hree of the seven categories had internal consistency coefficients above . 70. This suggests that erroneous a p riori distribution o f items into sub - 34 34 categories produced the low internal consistency coefficients of sub- categories. categories produced the low internal consistency coefficients of sub- categories. POST- ITEM ANALYSIS ITEM WITHIN CATEGORY INTERNAL CONSISTENCY DATA It should be noted that the reason why the internal consistency coefficients data improve as we examine larger quantities of variables is that correlation coefficients always tend to increase as the number of items increase. Thus, as we move from examining items within sub- categories, to sub-categories within categories, to items within categories, the correlation coefficients, and corresponding alphas, progressively increase. For the purpose of examining how the standardized item alphas improved as the number o f variables increased and less discriminating items were removed, we will look at both the items within categories and sub-categories within categories data, both pre-and-post item analysis. Again, according to Nunnally's rule of thumb, standardized item alphas that are .70 or above will be considered significant for the purpose of this study. A COMPARISON OF INTERNAL CONSISTENCY COEFFICIENTS PRE AND POST-ITEM ANALYSIS Job Finding and Maintenance =Significant Alpha According to Nunnally Pre-Item Analysis Sub-Categories 1-4 Items PlVl - PlV20 .66 .72 Post Item Analysis 7 I tems Removed .64 . 73* Apartment Hunting and Mai ntena n ce Pre-Item Analysis Sub-Categories 1-6 Items P2Vl-P2 V20 . 56 . 57 Post Item Analysis 8 Items Removed . 60 . 62 . 73* . 62 35 Budgeting Pre-Item Analysis Post Item Analysis 6 Items Removed Sub-Categories 1-5 .73* .66 P3Vl-P3V20 . 73* .69 Shopping Pre-Item Analysis Post Item Analysis 7 Items Removed Sub-Categories 1-5 .52 .48 Items P4Vl-P4V20 .59 .53 Miscellaneous Pre-Item Analysis Post Item Analysis 10 Items Removed Sub-Categories 1-7 .61 .59 Items P5Vl-P5V20 .67 . 64 Housecleaning Pre-Item Analysis Post Item Analysis 9 Items Removed Sub-Categories 1-5 .85* . 76* Items P6Vl-P6V20 .84* . 82* Cooking/Care of Food/Nutrition Pre-Item Analysis Sub-Categories 1-4 Items P7Vl - P7V20 .86* .91* Post Item Analysis 3 Items Removed .87* .91* .91* When looking at the item within category internal consistency data, we find that 3 of the 7 categories are homogeneous at a significant level according to Nunnally's .70 rule of thumb. Although removing less discriminating items improved the alphas in some cases, the item analysis did not cause any category to be homogeneous that was not homogeneous before the item analysis was performed. 36 POST-ITEM ANALYSIS FACTOR ANALYSIS DATA The factor analysis data also changed once the item analysis was performed. Although it is more difficult to interpret, th e first factor analysis data to be examined is the Principal Component Analysis on the remaining 29 sub-categories that contain 90 test items. The first analysis that will be presented is the overall factor analysis that extracted factors until all of the variance was used up. TABLE 9 POST-ITEM ANALYSIS PCA Category l 2 3 4 5 6 7 8 9 10 11 12 13 Factor Analysis/Post-Item Analysis Eigenvalue 7.01 3.83 1.38 1.36 1.16 1.14 1.03 . 95 .91 .88 .83 . 81 .75 Pct. of Var. 24.2 13.2 4.8 4.7 4.0 3.9 3.6 3.3 3.1 3.0 2.8 2.8 2.6 Cum. Pct. 24.2 37.4 42.1 46.8 so.a 54.7 58.3 61.6 64.7 67.7 70.6 73.4 76 .0 As mentioned above, Table 9 depicts an overall factor analysis that extracted factors until the cumulative percent of variance used up equaled 100%. Since the percent of variance explained by factors - became ridiculously small as the number of factors extracted increased, only 13 factors are actually represented in Table 9. If the eigenvalue method is applied to the data, 7 components can be extracted. However, the eigenvalue method is not the preferred method due to the fact that this method often extracts individual factors or components that are not real. In other words, the eigenvalue rule tends to extract factors that do not account for much of the total variance. The a priori method and scree methods being used in this study concur. Thus, we will now examine the data with 7 components extracted once the matrix is rotated, using ob limin oblique rotation. As can be seen, once 37 the data are rotated, only the first 2 factors can be assumed to be real. TABLE 10 EIGENVALUE LISTING WITH 7 FACTORS EXTRACTED Communality • 68 . 53 .57 .54 .57 .48 .35 Factor 1 2 3 4 5 6 7 Eigenvalue 7.01 3 . 83 1.38 1.36 1.16 1.14 1.03 Pct. of Var. 24.2 13.2 4.8 4.7 4.0 3.9 3.6 Cum. Pct . 24.2 37.4 42.1 46.8 50.8 54.7 58.3 The following information includes (1) the scree chart of the Post- Item Analysis Factor Analysis and (2) the rotated structure matrix with 7 factors extracted. 38 Figure 1 Figure 1 Figure 1 8.00 ~---------- -- ---------, 7.00 6.00 en s.oo Q) :::, ~ 7.01 > 4.00 3. C Q) 0) W 3.00 2.00 1.00 1.14 0.75 0.40 Post-item Factor Analysis Scree Chart 0.18 Post-item Factor Analysis Scree Chart 39 VARIMAX ROTATED STRUCTURE MATRIX WITH 7 FACTORS EXTRACTED VARIMAX ROTATED STRUCTURE MATRIX WITH 7 FACTORS EXTRACTED SUB-CATEGORY FACTOR FACTOR 2 FACTOR 3 FACTOR 4 FACTOR 5 FACTOR 6 FACTOR 7 P1C3 .74 .22 · .32 P3C1 .72 .40 .36 - .31 P1C1 . 71 .36 .44 .35 -.44 P3C3 .67 .32 .27 .27 -.21 P2C3 .66 .22 .23 .24 P3C4 .60 .31 -.39 P4C2 .57 .29 .29 -.26 P4C5 .57 .2 1 .21 .33 P2C4 .42 .35 .38 · .21 P7C2 .22 .85 P7C3 .84 P7C1 .81 P7C4 .80 .27 P6C4 .23 .78 .28 P6C3 .76 .22 ·.28 P6C1 . 71 .43 P4C1 .35 . 71 P4C4 .68 .30 P2C5 .32 .55 - .23 .21 · .35 P1C4 .43 .45 .26 -.32 P5C3 .21 .20 .72 .20 · .34 P5C2 .37 . 71 P5C6 .33 .37 .50 .49 -.23 P5C1 .20 .80 P2C6 .30 .27 .27 .63 -.31 P5C4 .34 .23 .21 .48 .52 P2C1 .32 .23 • • 73 P1C2 .23 .27 .35 ·.68 P3C2 .40 .45 -.52 As can be seen from Table 11, the varimax rotated structure matrix with 7 factors extracted generated a complex factor matrix where several of the subcategories have moderately high facto r loadings on more than one factor. In addition, bipolar factors were generated, indicating that factors exist with substantial positive and negative loadings . Thus, it appea r s that the varimax rotation was unable to produce a factor mat r ix where the principles of simple struc t ure were followed . This is particularly evident by examining subcategories P2C4, PlC4, PSC6, and P3C2. All of these subcategories (variables) load moderately high on three or four factors. EIGENVALUE LISTING WITH 7 FACTORS EXTRACTED In fact, the latter two contain moderately high negative l o adings on at least one factor a s well. POST-ITEM ANALYSIS PCA WITH 2 FACTORS EXTRACTED Due to the fact that the previous PCA data strongly suggest t hat only tw o real factors can be extracted, the following PCA analysis will examine the 29 sub - categories post-item analysis with two factors 40 extracted. As can be seen from the following table, 37.4% of the variance can be accounted for by the two extracted factors . TABLE 12 can be accounted for by the two extracted factors . TABLE 12 POST-ITEM ANALYSIS EIGENVALUE TABLE WITH 2 FACTORS EXTRACTED Factor 1 2 Eigenvalue 7.01 3.83 Pct. of Var. 24 . 2 13.2 Cum. Pct. 24.2 37.4 POST-ITEM ANALYSIS EIGENVALUE TABLE WITH 2 FACTORS EXTRACTED Factor 1 2 Eigenvalue 7.01 3.83 Pct. of Var. 24 . 2 13.2 Cum. Pct. 24.2 37.4 The following table (Table 13) is the rotated factor matrix. As can be seen, the Varimax rotation produced a much clearer definition of variables in terms of the two factors . As shown, several of the ILSFA's items are quite "pure" in terms of loading high on one factor, but not the other. SUB-CATEGORIES PlCl P3Cl PlC3 P3C3 P4C2 P3C4 P2C3 PSC6 P2C4 PSC4 PlC2 P2Cl P3C2 PSC3 P4CS PSC2 P4Cl PlC4 P2CS P2C6 P4C4 PSCl P7C2 P7C4 P7C3 P6C4 P7Cl P6Cl P6C3 = Job Application Forms and Procedures for Applying to a Job = Checking Accounts = General Job-Related Knowledge and Terms = How to Budget = Warranties and Consumer ' s Rights = Reading and Understanding Pa ychecks = Apartment Related Items = Negotiating Leisure Activities = Utility and Protective Agencies = Sex Education Issues = Job Interviewing Skills = Procedures for Looking for an Apartment Concept of Interest and Savings Accounts = Knowledge About Mature Adult Behavior = Consumer Terminology and Knowledge About Bargains = Alcohol and Drug Issues = Methods and Implications o f Comparative Shopping = Job-Related Behavior Apartment Living = Apartment Maintenance Advertising Ploys = Transportati on and car Issues = How to Cook and Prepare Food Fo od Storage How to Shop for Food = Care of Cl othing Nutrition = Fundamentals o f Housecleaning Household Safety FACTOR 1 .81 . 70 .66 .66 .61 .56 .56 .53 .51 .50 .so .48 .47 .47 .46 .46 . 45 .44 .42 . EIGENVALUE LISTING WITH 7 FACTORS EXTRACTED 41 . 32 .27 .11 -.02 .06 .13 .13 .OS . 13 FACTOR 2 .12 .04 .06 .14 -.08 .06 .18 . 07 .01 .22 .08 .11 .. 12 .OS .18 .01 .14 -.02 .14 .28 -.09 .01 . 85 .82 .82 .79 . 78 . 74 . 73 SUB-CATEGORIES p Negotiating Leisure Activities 41 Whereas orthogonal rotations maintain the independence of factors; that is the angles between the a.xes are kept at 90 degrees, rotations in which the factor a.xes are allowed to form acute or obtuse angles are called oblique. Thus, orthogonal rotations assume that factors are uncorrelated, while obliqueness means that factors are correlated. According to Kerlinger, "there is no doubt that factor structures can be better fitted with oblique axes and the simple structured criteria better satisfied. (Kerlinger, 1973) Thus, an ob l ique rotation was also performed on the post-item analysis data with two factors extracted. The delta value for direct oblimin rotation was set at zero. Table 14 depicts the factor loadings. TABLE 14 STRUCTURE MATRIX OF FACTORS WITH OBLIQUE ROTATION SUB-CATEGORIES PlCl = P3Cl = P3C3 = PlC3 = P4C2 = P2C3 = P3C4 = PSC6 = PSC4 = P2C4 = PlC2 P2Cl = P3C2 = P4CS Job Application Forms and Procedures for Applying to a Job Checking Accounts How to Budget General Job-Related Knowledge and Terms Warranties and Consumer's Rights Apartment and Related Items Reading and Understanding Paychecks Negotiating Leisure Activities Sex Education Issues Utility and Protective Agencies Job Interviewing Skills Procedures for Looking for an Apartment Concept of Interest and Savings Accounts Consumer Terminology and Knowledge About Bargains PSC3 P4Cl = Knowledge About Mature Adult Behavior = Methods and Implications of Comparison Shopping PSC2 P2C6 P2CS PlC4 P4C4 PSCl P7C2 P7C3 P7C4 P6C4 P7Cl P6Cl P6C3 = Alcohol and Drug Issues Apartment Maintenance = Apartment Living = Job-Related Behavior = Advertising Ploys = Transportation and Car Issues = How to Cook and Prepare Food = How to Shop for Food Food Storage Care of Clothing Nutrition Fundamentals of Housecleaning = Household Safety FACTOR 1 .82 .70 . 67 .66 .59 .57 .56 .54 .52 .50 .50 .49 .48 .48 .47 .46 .46 .44 .44 .43 .30 .27 .21 .16 .08 .23 .23 .14 . 22 FACTOR 2 .20 .10 .20 .12 - .03 . 23 .11 .12 .27 .OS .13 .15 .1 7 . 22 .09 .18 .OS .32 .18 .02 -.06 . 03 . 8 5 . 82 .82 .80 .79 .74 .74 SUB-CATEGORIES 42 Since the data output from the orthogonal and oblique rotations do not significantly differ, and since oblique rotation is viewed as the preferred method, we will examine the oblique rotation data in terms of the factor loadings. According to Kerlinger, there is no generally accepted standard rule of factor loadings. A crude rule of thumb is to use the standard error of r - or, easier, to find the r that is significant for the N of the study. For example, with N = 200 an r of about .18 is statistically significant at the .01 level. Some factor analysis in some studies do not bother with factor loadings less than .30, or even .40 . Others do. Given the fact that the present analyses are exploratory in nature, the researcher has established the following criteria to differentiate between strong, moderate, and weak factors. (Kerlinger, 1973) Factor loadings .6 and higher= Strong Factor loadings from .4 - .59 = Moderate Factor loadings .39 and lower= Weak The following table (Table 15) will examine the sums of squares of the factor loadings, in addition to the common factor variance of the ILSFA. The entries in the last column are called commonalities, or hz's. They are the sums of squares of the factor loadings of a test or variable. From the sums of squares of the factor loadings, it will be determined whether the components have strong, moderate, or weak factor .l oadings. The aforementioned determination will be made by rounding off the square factor loadings to two decimals. COMMONALITIES ON FACTOR LOADINGS DATA ILSFA Sub-Category Factor 1 Factor 2 hz PlCl Strong .67 .04 .71 P3Cl Moderate .49 .01 . 50 P3C3 Moderate .45 .04 .48 PlC3 Moderate .44 .01 . 45 P4C2 Weak . 35 .DO .35 P2C3 Weak .33 .OS .38 P3C4 Weak . 32 .01 . 33 PSC6 Weak . There is one component extracted from this category . The final SUB-CATEGORIES 29 .01 .30 PSC4 Weak .27 .07 .34 43 ILSFA Sub-Category P2C4 P1C2 P2Cl P3C2 P4CS PSC3 P4Cl PSC2 P2C6 P2CS PlC4 P4C4 P5Cl P7C2 P7C3 P7C4 P6C4 P7Cl P6Cl P6C3 Weak Weak Weak Weak Weak Weak Weak Weak Weak Weak Weak Weak Weak Strong Strong Strong Strong strong Moderate Moderate Factor 1 .25 .25 .24 .23 .23 .22 .21 .21 .19 .19 .19 .09 .07 .04 .03 .01 .OS .05 .02 .OS Factor 2 .oo .02 .02 .03 .O S .01 .03 .00 .10 .03 .00 .oo .00 .73 .67 .67 .63 .62 .55 .55 .26 .27 .26 .26 .28 .23 .25 .22 .28 .22 .19 .10 .07 • 77 .70 .68 .68 .67 .57 .59 For the purpose of this study, only sub-categories that had strong or moderate factor loadings will be considered as psychometrically sound. The ILSFA has 11 sub-categories that fall into this category. For example, subcategories in Factor 1 include Job Application Forms and Procedures for Applying to a Job, General Job -Rel ated Knowledge and Terms, Checking Accounts, and How to Budget. Factor 2. includes Nutrition, How to Cook and Prepare Food, How to Shop fo r Food, Food Storage, Fundamentals of Housecleaning, Household Safety, and Care of Clothing. Inspection of the data also reveals that 18 of the 29 subcategories did not load high on either of the constructs. Another factor analysis to be investigated is the Principal Component Analysis performed with the sub-categories in each category following the item analysis. TABLE 16 PCA PERFORMED POST-ITEM ANALYSIS WITH EACH CATEGORY Job Finding Communality 1.00 1.00 1.00 1.00 and Maintenance Sub-Categories 1-4: Factor 1 2 3 4 Eigenvalue 1.96 .86 .74 .45 Pct. of Var. 49.0 21.4 18.4 11.2 Cum. Pct. 49.0 70.4 88 .8 100.0 There is one component extracted from this category . The final Another factor analysis to be investigated is the Principal Component Analysis performed with the sub-categories in each category following the item analysis. PCA PERFORMED POST-ITEM ANALYSIS WITH EACH CATEGORY Job Finding Communality 1.00 1.00 1.00 1.00 and Maintenance Sub-Categories 1-4: Factor 1 2 3 4 Eigenvalue 1.96 .86 .74 .45 Pct. of Var. 49.0 21.4 18.4 11.2 Cum. Pct. SUB-CATEGORIES 49.0 70.4 88 .8 100.0 h i d f hi h fi l PCA PERFORMED POST-ITEM ANALYSIS WITH EACH CATEGORY Job Finding Communality 1.00 1.00 1.00 1.00 and Maintenance Sub-Categories 1-4: Factor 1 2 3 4 Eigenvalue 1.96 .86 .74 .45 Pct. of Var. 49.0 21.4 18.4 11.2 Cum. Pct. 49.0 70.4 88 .8 100.0 There is one component extracted from this category . The final 44 statistics after the varimax rotation and principal components extracted a r e as follows: Communality .70 .37 . 56 33 Factor 1 Eigenvalue 1.96 Pct. of Var. 49 . 0 Cum. Pct . 49.0 Communality .70 .37 . 56 .33 Factor 1 Eigenvalue 1.96 Pct. of Var. 49 . 0 Cum. Pct . 49.0 Apartment Hunting and Maintenance Sub-Categories 1 , 3,4,5, and 6 : Apartment Hunting and Maintenance Sub-Categories 1 , 3,4,5, and 6 : Communality 1.00 1.00 1.00 1.00 1.00 Fa ctor 1 2 3 4 5 Eigenvalue 1.91 . 84 .80 .77 .68 Pct. of Var . 38 . 3 16.8 16.0 15.3 13 . 6 Cum. Pct. 38.3 55.1 71.0 86 . 4 100 . 0 There is one component extracted from this catego r y. The final following the rotation and principal components ext r acted a r e Communality . 38 . 42 .41 .32 .37 Factor 1 Eigen value 1. 91 Pct . of Var. 38 . 3 Cum. Pct • 38.3 Communality . 38 . 42 .41 .32 37 Factor 1 Eigen value 1. 91 Pct . of Var. 38 . 3 Cum. Pct • 38.3 Communality . 38 . 42 41 Factor 1 Eigen value 1. 91 Pct . of Var. 38 . 3 Cum. Pct • 38.3 Communality 1.00 1.00 1. 00 1.00 Factor 1 2 3 4 Eigenvalue 1.98 .77 .66 . 59 Pct . of Var . 49.6 19.1 16.4 14 . 9 Cum. Pct . 49.6 68.7 85 . 1 100 .0 Again, there is one compone nt extracted . The final statistics g p p p Communality .49 . 47 . 52 .49 Factor Eigenvalue 1.98 Pct. of Var. 49 . 6 Cum. Pct . 49.6 pp g g Communality Factor Eigenvalue 1.00 1 1.58 1.00 2 .97 1.00 3 . 77 1.00 4 .69 Pct. o f Var . 39.5 24 . 2 19.1 17 . 2 Cum. Pct . 39.5 63. 6 82 . SUB-CATEGORIES 8 100.0 Here, there is 1 c omponent extracted . The final statistics follo wing the varimax rotati on and principal components extracted are: 45 Communality: Factor Eigenvalue . 53 1 1.58 . 45 .31 . 29 Miscella ne ou s Sub - Categories 1 , 2,3,4, Pct . of 39.5 and 6 Var. Cum. Pct . 39 . 5 Miscella ne ou s Sub - Categories 1 , 2,3,4, and 6 Communality: Factor Eigenvalue 1. 00 1 1.94 1.0 0 2 1.00 1. 00 3 .74 1. 00 4 .67 1.00 5 . 64 Pct . of 38.9 20 . 1 14.9 13 . 4 12.8 Var. Cum. Pct . 38.9 58.9 73.8 87 . 2 100.0 th i s case, there ar e two components extracted. However, since In th i s case, there ar e two components extracted. However, since t he second component r ounded off to the tenth decimal is just one, it is suggested that the r e is only one component operating in this category . The fina l statistics following th e varimax rotation and principal components extracted are: Communality: Factor Eigenvalue . 84 1 1.94 . 59 2 1.00 . 55 .43 . 53 Pct . of 38.9 20.1 Var. Cum. Pct . 38 . 9 58.9 Housec l eaning Sub - Cat egories 1,3, and 4: Housec l eaning Sub - Cat egories 1,3, and 4: Housec l eaning Sub - Cat egories 1,3, Communality: Factor Eigenvalue 1.00 1 2 . 03 1.00 2 .60 1.00 3 .3 6 and 4: Pct . of 67.8 20.2 12.1 Var . Cum. Pct. 67.8 87.9 100.0 Here, there is 1 component ex t racted . The final statistics ng the varima x rotation and principa l components extracted are: Communality: .73 .56 .74 Factor 1 Eigenvalue 2.03 Pct. o f Var. 67 . 8 Cum. Pct. . 67 .8 Cooki ng/Care o f Food/Nutrition Sub- Categ o ries 1- 4 Communality: Fact or Eigen value Pct . o f Var . 1.00 1 2 . 88 71. 9 1.00 2 . 52 13.0 1.00 3 . 33 8.2 1.00 4 .27 6.8 Cum. Pct. 71.9 85 . 0 93 . 2 100.0 Again, t here is 1 component extracted. SUB-CATEGORIES In this case the final stics following the v arimax r otation and principal co mponents statistics following the v arimax r otation and principal co mponents extracted are: 46 Communality .68 .76 .64 Factor 1 Eigenvalue 2.87 Pct. of Var. 71.9 Cum. Pct. 71.9 Overall, 6 out of the 7 categories seem to be homogeneous. However, in the case where there is two factors, the second factor has an eigenvalue that is nearly at the value of one. Thus, the data suggests that all of the ILSFA's categories are basically measuring one distinct area. A comparison of each of the ILSFA's categories' factor analyses will be investigated to see if the item analysis improved the homogeneity of each of the categories under question. The final statistics for both the pre-and-post item analyses data will be presented. A COMPARISON OF EACH OF THE ILSFA's PCA FACTOR ANALYSES A COMPARISON OF EACH OF THE ILSFA s PCA FACTOR ANALYSES Pre - Post - Pre - Post - Pre - Post - Job Finding Category 1 Factor 2.02 Eigenvalue 1 Factor 1. 96 Eigenvalue Budgeting 1 Factor 2.39 Eigenvalue 1 Factor 1. 98 Eigenvalue Miscellaneous 2 Factors 2.19 1.09 = Eigenvalue 2 Factors 1.94 1.00 = Eigenvalue Apartment Hunting 2 Factors 1.96 1.02 = Eigenvalue 1 Factor 1.91 Eigenvalue Shopping 2 Factors 1.75 1.02 = Eigenvalue 1 Factor 1.58 = Eigenvalue Housecleaning 1 Factor 3.17 = Eigenvalue 1 Factor 2.03 = Eigenvalue Pre - 2.03 = Eigenvalue 47 Cooking/Care of Food/Nutrition 1 Factor Pre - 2.85 Eigenvalue 1 Factor Post - 2.87 Eigenvalue The final data to be examined following the item analysis are the social worker reliability data. As previously indicated, social workers were asked to rate each subject in terms of their abilities on a Likert- type Scale in 6 of the categories of independent living. (The Miscellaneous Category was not rated.) These ratings were then correlated with the subject's performance in each of the post-item analysis remaining 29 sub-categories. The correlations are as follows: TABLE 18 POST-ITEM ANALYSIS EXAMINATION OF SOCIAL WORKER RELIABILITY DATA POST-ITEM ANALYSIS EXAMINATION OF SOCIAL WORKER RELIABILITY DATA SWl SW2 SW3 SW4 SW5 SW6 Job Finding .25** Apartment Hunting .15 Budgeting . 19* Shopping .01 Housecleaning .14 Cooking .18* 1-Tailed Signif: p5 . significant degree with either the pre or post-analyses. SUB-CATEGORIES 0l pS , 001 Here we find that in 3 out of the 6 categories, Social Worker correlations reached a statistically significant level. While these correlations were significant in the Apartment Hunting and Maintenance Category prior to the item analyses, this category did not reach a level of significance post-item analyses. All of the other categories are quite similar pre- and post -i tem analysis . Thus, post-item analyses, we seem to find concurrent validity in the areas of Job Finding, Budgeting, and Cooking; whereas, we found concurrent reliability in the categories of Job Finding, Apartment Hunting, Budgeting, and Cooking during the analyses of the original 140 test items. Social workers ratings did not concur with subjects' performance in the Shopping and Housecleaning areas to a 48 48 49 DISCUSSION DISCUSSION As previously indicated, recent humanistic, political, and financial interest in the fate of children who grow up in the foster care system, has resulted in several significant changes in the laws and guidelines that dictate how agencies that are responsible for state supervised youth have to run their programs. More specifically, New York State currently mandates that every adolescent 14 years of age and over must be provided with structured training and periodic assessment in independent living. Although these regulations have been in existence over the last five years, up to the present time, agencies have been choosing assessment instruments in a haphazard, random manner, with little thought given to the psychometric properties of the instruments they have chosen to employ. The Independent Living Skills Form for Adolescents ( ILSFA) was specifically designed to assess the independent living skills of adolescents in placement and this study, to the investigator's knowledge, is the first to conduct extensive psychometric examination on such an instrument. The results of all of the statistics and other analyses performed on the data contribute a great fund of information about the instrument's methodological characteristics and practical usefulness as an instrument to be used in the future with adolescents in placement. At the broadest level, this study challenges the seven category approach established by New York State as representing independent living skills. While the ILSFA' s test items were constructed based on an intuitive face validity according to this 7 factor model, the factor analyses performed failed to confirm such a model. Instead, the exploratory PCA Factor Analysis suggests a 2 - Factor Model. Although an inspection of the data reveals that only 1/3 of the ILSFA's variance is accounted for by the two factors, it is simultaneously evident that these two factors are "real" and account for practically all of the explained variance, although this was not very much . An examination of the factor loadings data reveals some very useful pragmatic information about the underlying constructs of these two 50 factors. All of the subcategories that load heavily on component 1 are directly related to independent living skills that involve an individual's interaction with the community. On the other hand, Component 2 contains items that are directly related to domestic types of independent living skills. DISCUSSION This latter construct is very much akin to what the literature has been referring to as "Daily Living Skills" for years . It is possible that a 2-Factor Model also exists because what is really being tapped into are two different forms of skills. These include 1) pragmatic, performance type of skills, and 2) more conceptual thinking operations. In other words, whi le domestic-related skills tend to be more straightforward, pragmatic , and dependent upon gross motor coordination and learning through observation and practice, community-related skills tend to be more abstract, complex, people-oriented, and learned via a more multimodal approach to instruction. Thus, it appears that the didactic skills could be presented from a concrete didactic approach, while the community-related skills would need to be presented over a longer period of time, and in such a manner, that the new abstract material could be understood. It would be interesting to pursue this hypothesis with future research that compares I.Q. with scores earned on each of the components of the ILSFA. The two factor ILSFA model that emerged contains information from 4 out of the 7 of the original New York State categories. The fol .lowing is a list of the categories or topics (according to their face validity) in terms of the construct the data suggests that they fall under. The two factor ILSFA model that emerged contains information from 4 out of the 7 of the original New York State categories. The fol .lowing is a list of the categories or topics (according to their face validity) in terms of the construct the data suggests that they fall under. TABLE 19 CONSTRUCTS OF THE ILSFA Component 1 = Community-Related Independent Living Skills Job Finding and Maintenance Items Budgeting Items Component 2 = Domestic-Related Independent Living Skills Housecleaning Items Cooking / Care of Food/Nutrition Items The eleven subcategories which represent these four general categories contain the final forty-four items which will comprise the Component 1 = Community-Related Independent Living Skills Job Finding and Maintenance Items Budgeting Items Component 2 = Domestic-Related Independent Living Skills Housecleaning Items Cooking / Care of Food/Nutrition Items The eleven subcategories which represent these four general categories contain the final forty-four items which will comprise the Component 1 = Community-Related Independent Living Skills The eleven subcategories which represent these four general categories contain the final forty-four items which will comprise the 51 revised ILSFA . The items are presented in Table 20. TABLE 20 THE REVISED ILSFA ACCORDING TO THE 2 - FACTOR MODEL (Refer to Appendix A to see which test questions will be used in the revised version.) The Revised ILSFA Community-Related Independent Living Skills Job Finding Original Test Items #1, 2, 8, 10, 11, 13, 15, and 16 Budgeting Original Test Items #1 , 2, 4, 5, 6, 15, 16, and 17 Domestic-Related Independent Living Skills Housecleaning Original Test Items#2, 7, 8, 9, 10, 11, 13, 14, 17, 19, and 20 Domestic-Related Independent Living Skills Housecleaning Original Test Items#2, 7, 8, 9, 10, 11, 13, 14, 17, 19, and 20 Cooking/Care of Food/Nutrition Original Test Items #1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, and 18 Cooking/Care of Food/Nutrition Original Test Items #1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, and 18 Three of the four general categories in the Revised ILSFA, Job Finding and Maintenance, Housecleaning, and Cooking/Care of Food/Nutrition had internal consistency coefficie nts above .70, and the fourth, Budgeting, had an internal consistency coefficient of . 69. Further, three of these categories, Job Finding, Budgeting, and Cooking/Care of Food/Nutrition demonstrated concurrent validity through statistically significant, if weak relationships, with social workers' ratings. The three general categories whose subcategories loaded only weakly on Factor 1 and 2 were Apartment Hunting, Shopping, and Miscellaneous. Not surprisingly, the internal consistency data were poor for these categories and they demonstrated no significant relationship with the social workers' ratings. Future research should address the idea of constructing new items for these categories which would increase their internal consistency. There are several limitations of this study. It should be noted that the proposed study was limited to the exploration of the methodological characteristics of the ILSFA with the sample of adolescents who were in placement either in the group home or RTC level of care . Hence, generalizations about the validity of the ILSFA with adolescents 52 who are in foster homes, Division for Youth Facilities, or more intensive psychiatric placement may not be possible on the basis of this study. In addition, adolescents who live with their families and attend school in their respective community schools were not investigated in the present study. Hence, the data obtained from the present study cannot be applied to adolescents who are living in "normal" environments. However, the ILSFA can be administered to other kinds of adolescent populations in the future once statistical data for these populations is available. The Revised ILSFA Finally, the sample under present investigation included adolescents who were 14 years of age and over, New York City residents, and are currently residing in a Residential Treatment Center or Group Home located in the New York City/Westchester area. Therefore, it is assumed that the participating subjects were all exposed to inner city living and had similarly incorporated independent living skills which relate to inner city living into their repertoire of autonomous skills. For this reason, it is probably not advisable for the results of this study to be generalized to rural populations. Lastly, it is assumed that the Residential Treatment Centers in the aforementioned geographical area did not significantly differ in terms of structure, population served, or available clinical services. It is similarly assumed that the group home residences of different agencies did not significantly differ in terms of level of structure, populations served, and available clinical services . . Thus, it is believed that the subjects in this study were representative sample of the population of adolescents who reside in N. Y. C. / Westchester Residential Treatment Centers and Group Home residences. Another limitation of the present study resides in the fact that concurrent validity was measured by obtaining social workers' appraisals of the adolescents studied in the original six components of independent living, as prescribed by New York State's current description of independent living skills. Future research should be geared towards assessing whether social workers ' global ratings in the t wo construct areas {Domestic-Related and Community - Related Independent Living Skills) 53 are significantly and positively correlated with their scores in both of these areas . are significantly and positively correlated with their scores in both of these areas . An additional issue deser v ing of mention is the fact that administering the ILSFA on an individual basis is markedly more time consuming than administering the test to a group of youths at one time. (The Community-Related items could only be administered to a group since the Domestic-Related items require individualized administration by a child care worker who is familiar with the youth . ) Future research could examine whether youths" performance on the Community - Related test items vary from group to individualized administration . The Revised ILSFA On a positive note, the fact that the ILSFA was successfully reduced from a 140 to a 44-item instrument makes the administration of the ILSFA significantly more time efficient and less effected by adolescents ' deteriorating moods/attention s pans over time. Based on the fact that Form A of the ILSFA (100 items) took approximately 45 minutes to 1 1/2 hours to administer, it is estimated that the revised Form A (16 Community-Related items) will take 10 minutes to 20 minutes for its entire individualized administration . In sum, according to a 2 - Factor Model, which defines the ILSFA as an instrument that measures an adolescents' fund of Community and Domestic-related Independent Living Skills, 44 items of the original 140 item inventory can confidently be used in the form o f a revised -test t o assess these skills. The revised ILSFA contains individual items with face validity from 4 out of the 7 categories that New York State lists as important in terms o f a comprehensive definition of independent living skills. The three categories that do not have any items represented in the revised 2 - Factor Instrument include the "Apartment Hunting", "Shopping" and "Miscel l aneous" c ategories. Future research should be geared towards creating new test items wi th face validit y i n both of these areas and then performing statistical analyses o n them to obtain information about their psychometric properties. Hopefully, items can be constructed in these three areas that load heavily on Factor 1 or 2, and 54 54 possess adequate internal consistency and concurrent validity data. Overall, the present research has resulted in a methodologically sound 44 item instrument that measures Community and Domestic-Related Independent Living Skills that contains individual items with face validity in the areas of Job Finding and Maintenance, Budgeting, Housecleaning, and Cooking/Care of Food/Nutrition. 55 APPENDIX A Child's Name: ------------- ( l - 4) Date: ------('""1'""1---=-1-=-6_) _____ _ Sex: Race: Date of Birth: ---...,.(=5--,:-10="'"") -- Date of Admission: -----~-- (17-22) (23) --(,-,2:-,4,--"""2-=6.,....) ---- Age: ___ (~2~7~-~2=a...,.) __ _ To be completed by examiner with resident. APPENDIX A Child's Name: ------------- ( l - 4) Date: ------('""1'""1---=-1-=-6_) _____ _ Sex: Race: Date of Birth: ---...,.(=5--,:-10="'"") -- Date of Admission: -----~-- (17-22) (23) --(,-,2:-,4,--"""2-=6.,....) ---- Age: ___ (~2~7~-~2=a...,.) __ _ To be completed by examiner with resident. To be completed by examiner with resident. Form A - Part 1 - JOB SEARCH AND MAINTENANCE 1) Name at least three places where you can get information about jobs. score (29) 2) Name at least four t ypes of information / documents that you will need before you apply for a job. Score ---...,.(-=-3-=-o.,....) ___ _ 3) Before I apply for a job, I should find out about the goals and policies of the particular company/agency and understand what skills are being asked of me to fulfill the available position . True False Score ------ ----------- ( 3 l) 4) List three good things to do on a job inter v iew. Score -----,,..-,,.------ ( 3 2) 5) List three bad things to do on a job intervie w. Score ------,- ( =3=3.,...) ---- 6) Name three important things you would want to ask the person who interviews you for a job. Score ------,-(=3-=-4.,...) ---- 7) You should f o llow-up a j ob interview with a phone call or a letter thanking the interviewer for his or her time and sayin g that yo ur are still i nterested in the j ob . True False Score ------ ------ -----(~3~5=--) ---- 8) Define: U.S. Citizen S 1) Name at least three places where you can get information about jobs. (29) 3-=-o.,..._ 3) Before I apply for a job, I should find out about the goals and policies of the particular company/agency and understand what skills are being asked of me to fulfill the available position . True False Score ------ ----------- ( 3 l) 4) List three good things to do on a job inter v iew. Score -----,,..-,,.------ ( 3 2) 5) List three bad things to do on a job intervie w. Score ------,- ( =3=3.,...) ---- -=-6.,.. 56 9) You should complete a job application ve ry quickly and in a sloppy manner. 10) True False Score ------ ------ ------,-(=3=7.,..) ---- What kinds of information should be listed on a resume? least 3) (Name at score -----(~3~8~)----- 11) Do you know how to fill out a tax form? (W4-Form) How? Score ------,,....,,...----- ( 39) 12) If you have a disagreement with another employee, what should you do? Form A - Part 1 - JOB SEARCH AND MAINTENANCE Score ----(-4~0-) ___ _ 13) Match the following terms with their correct definitions: A) Time Clock B) Salary C) Hourly Wage D) A Fringe Benefit E) Work Week F) Overtime G) Full-time a) Health Insurance b) # of days you work in a week c) When you work 35+ hours per week d) Total Income e) Registers amount of hours you work in a day f) Amount of$ you earn per hour g) When you work above and beyond your regular work week Score ------,- (..,..4-=-l.,...) ---- 14) It is acceptable for a person to come ten minutes late to work on a daily basis. True ------ False Score __________ _ (42) 15) What are the advantages of being in a union? Score -------,-(--,-4-=3.,..) ___ _ ,,...--- ( 39) Match the following terms with their correct definitions: A) Time Clock B) Salary C) Hourly Wage D) A Fringe Benefit E) Work Week F) Overtime G) Full-time a) Health Insurance b) # of days you work in a week c) When you work 35+ hours per week d) Total Income e) Registers amount of hours you work in a day f) Amount of$ you earn per hour g) When you work above and beyond your regular work week 14) It is acceptable for a person to come ten minutes late to work on a daily basis. True ------ False Score __________ _ (42) 15) What are the advantages of being in a union? Score -------,-(--,-4-=3.,..) ___ _ 16) Define "Sick and Vacation Days." Score __________ _ (44) 17) You should be responsible and dependable on your job. True False Score ------ ------ ----------- (45) is acceptable for a person to come ten minutes late to work on aily basis. --- (45) 57 18) How much notice do you need to give to your employer when you are resigning from a job? 2 days 4 months __ _ 2-4 weeks --- 2 years Score ------,-( ..,..4.,,.6.,...) ---- 19) Good hygiene habits are important for keeping a job. True False Score -------,(--,4'""7,...,.) ___ _ 20) It is O.K. to make several personal phone calls from your job. True False Score ------ ----------- (48) 18) How much notice do you need to give to your employer when you are resigning from a job? Form A - Part 1 - JOB SEARCH AND MAINTENANCE 2 days 4 months __ _ 2-4 weeks --- 2 years Score ------,-( ..,..4.,,.6.,...) ---- 19) Good hygiene habits are important for keeping a job. True False Score -------,(--,4'""7,...,.) ___ _ 20) It is O.K. to make several personal phone calls from your job. True False Score ------ ----------- (48) ---- (48) 58 Date of Birth: (5-10) Social Worker: ---------- RTMENT HUNTING/MAINTENANCE with resident. an go about finding an apartment? Score ----------- (49) what do the following abbreviations South Side Vic. - 2 br apt., 1 bth. Near shop/trans. Call 381-2407 Score -------,-(=5=0.,....) ---- should ask about when looking at an about renting. Score ----(-=-5-=-1-> ---- enses when you first move into an ur of them. Score _____ (_5_2_) ____ _ Score ------,-(-=-5=1..,..> ---- t to live on his or her own , about how e least amount of necessary furniture beds, etc.? Score ---- (--,,5,....,4 :-) ___ _ same person would need to spend on plies: pots , pans, utensils, cups, and Score __________ _ ( 55) utilities and telephone services? score -----(__,5,_6:--,-) ---- 58 Child's Name: Date of Birth: ------------- ( l - 4) (5-10) Date: Social Worker: ----------------- ---------- FORMA PART 2 APARTMENTHUNTING/MAINTENANCE FORM A - PART 2 - APARTMENT HUNTING/MAINTENANCE To be completed by social worker with resident. l) Name at least two ways you can go about finding an apartment? Score ----------- (49) 2) In the classi fied ad below, what do the following abbreviations mean? l) Name at least two ways you can go about finding an apartment? Score ----------- (49) l) Name at least two ways you can go about finding an apartment? Score ----------- (49) 2) In the classi fied ad below, what do the following abbreviations mean? br = shop/trans = vie = South Side Vic. - 2 br apt., 1 bth. Near shop/trans. Call 381-2407 Score -------,-(=5=0.,....) ---- What are three things you should ask about when looking at an apartment that you're thinking about renting. 3) 4) 5) en a person first moves out to live on his or her own , about how h would it cost to buy the least amount of necessary furniture one person: sofas, chairs, beds, etc.? 8) --,-) 59 9) How much would you have to spend on gas, electricity, and telephone each month, if you were living modestly by yourself? Give a realistic example of each expense. 9) 10) Match the following: 10) Match the following: 10) 1. lease 2. security deposit 3. landlord 4. superintendent a. Money that is given as a pledge that will only be returned if property is not damaged. b. The owner of property that he/she leases or rents to another. b. The owner of property that he/she leases or rents to another. c. The person in an apartment building who is paid to handle day to day problems. d. A legal contract by which a person exchanges property for a period of time for rent Score ----- (58) 11) Provide a fair estimate of the amount of money you would need to make to live on your own in a modest studio apartment by yourself in New York.______________ Score __________ _ (59) make to live on your own in a modest studio apartment by yourself in New York.______________ Score __________ _ (59) 12) A landlord or landlady may legally refuse to rent to you if you do not have a steady source of income. True False score ------ _____ (_6_0_) ___ _ 13) A best fr i end always makes a good roommate . True ------ False Score __________ _ (61) 14) If you are paying the rent for an apartment on a regular monthl y basis, you have the right to blast your stereo and have wild parties. True ------ False Score __________ _ (62) 15) If you are renting an apartment and your plumbing system needs to be repaired, you are always responsible to pay for it. True False Score ------ ------ ----------- (63) 16) If you are renting an apartment that was recentl y painted an o ff- white col or and you want to paint your bedroom blue, yo u s hould expect the landlord t o pay for it . True False Score ------ ------ ---- (--=-6...,..4_) ___ _ 12) A landlord or landlady may legally refuse to rent to you if you do not have a steady source of income. (62) 15) If you are renting an apartment and your plumbing system needs to be repaired, you are always responsible to pay for it. --- (63) 16) If you are renting an apartment that was recentl y painted an o ff- white col or and you want to paint your bedroom blue, yo u s hould expect the landlord t o pay for it . 16) If you are renting an apartment that was recentl y painted an o ff- white col or and you want to paint your bedroom blue, yo u s hould expect the landlord t o pay for it . 16) If you are renting an apartment that was recentl y painted an o ff- white col or and you want to paint your bedroom blue, yo u s hould expect the landlord t o pay for it . (--=-6.._ 60 1 7) What would you do if your toilet were stopped up? Score __________ _ What would you do if all the lights in your apartment went off? __ _ Score -----.e=-,------ (65) 18) What is the thing to do if you feel you are being discriminated against in your efforts to rent an apartment? ____________ _ Score ----------- (66) 19) What does the term "absentee landlord" mean? ____________ _ Score ------,-(-=-6=7.,...) ---- 20) What is a rental application fee? ___________________ _ How much do you usually have to pay a real estate agent to help you get an apartment?___________ Score _____ (~ 6 ~ 8 .,..,...) ___ _ 1 7) What would you do if your toilet were stopped up? __________ _ What would you do if all the lights in your apartment went off? __ _ Score -----.e=-,------ (65) 18) What is the thing to do if you feel you are being discriminated against in your efforts to rent an apartment? ____________ _ Score ----------- (66) 19) What does the term "absentee landlord" mean? ____________ _ Score ------,-(-=-6=7.,...) ---- 20) What is a rental application fee? ___________________ _ How much do you usually have to pay a real estate agent to help you get an apartment?___________ Score _____ (~ 6 ~ 8 .,..,...) ___ _ 61 Child's Name: Dat e of Birt h : ---- (,-,,1--"""' 4_) ____ _ ----<--= 5,...._...., 1'""0=--) --- Da te : Social Worker: - - --------- ------ ---------- FORM A - PART 3 - BUDGETING Da te : Social Worker: - - --------- ------ ---------- FORM A - PART 3 - BUDGETING 1) You ju s t bought a new alarm clock on November 8, 1993 from Sears. The total cost of the clock is $3 1 .50 and yo u want to pay for it with a check. Us ing the blank check be low, show how you wou l d write a ch e c k to pay for the ala r m clock. 100 Pa y to the Or der of ________ __ _____________ $ _ _____ ---1 Green Meadow Bank item Score -- --(_,,1---,) _ ___ _ 2) How do you endorse a check? 3) 4) score ( 2) What is a checking register? What is a checking service charge/fee? Score (3) No . Date Check Issued To : Check Amount Deoosit 1 Balance 1 11/6/93 S200 . 00 a) Your check register currently has a balance of $200 . 00 . Score Using the Check Register, write in the check you wrote for the alarm clock . b) Now put thirty more dollars into your checking account on December 29th. Record this in the Check Register . c) What is your new balance? $ ________ __ _ _ Score - - -- -(-4,....) ____ _ 5) A "bounced" check means that you had enough money in your checking account to pay for what you bought. True False Score ---- - - ---- - - ---- (--::5,...,.) ____ _ FORM A - PART 3 - BUDGETING True False Score ------ ------ ----------- ( 7) 8) Define Interest. score ----(-=-8_) ___ _ 9) Name at least two types of information that are normally found on payroll checks. Score -------,-(-:-9-,-) ___ _ 10) What is a stop payment? ------------------------- Score ----(-=-1-=-o.,....> ___ _ 11) You receive more interest on savings accounts that require that you do not make withdrawals for a specified period of tim~. True False Score ------ ------ ----- (--,1,...,1 ,....,...) ---- 12) What is a credit card and how do credit card companies work with you? _________________________________ _ Score -------,- c-=-1=2.,...> ---- 13 > You will not maintain a good credit rating if you do not pay your bills on time. True False Score ------ ------ -------,-(-=-1-=-3.,....) ---- 14) When do we file income taxes? _____________________ _ Score ----------- ( l 4) 15) Money that you spend on recreation should be part of your monthly budget. True False Score ------ ------ -- --("""1"""s,...,) ___ _ 16) It is not a good idea to put aside your transportation money when you get paid. True ------ False ------ Score __________ _ (16) 6) You need to consider your monthly---,--------- and ______ _ when figuring out your monthly budget. Choose two words to complete the above sentences: interest; taxes; income; expenses; account. score desires; ----------- ( 6) 7) Most banks require that your have a minimum balance in your savings account to keep your account active. True False Score ------ ------ ----------- ( 7) 8) Define Interest. score ----(-=-8_) ___ _ 9) Name at least two types of information that are normally found on payroll checks. Score -------,-(-:-9-,-) ___ _ 10) What is a stop payment? ------------------------- Score ----(-=-1-=-o.,....> ___ _ 11) You receive more interest on savings accounts that require that you do not make withdrawals for a specified period of tim~. True False Score ------ ------ ----- (--,1,...,1 ,....,...) ---- 12) What is a credit card and how do credit card companies work with you? _________________________________ _ Score -------,- c-=-1=2.,...> ---- 13 > You will not maintain a good credit rating if you do not pay your bills on time. True False Score ------ ------ -------,-(-=-1-=-3.,....) ---- 14) When do we file income taxes? _____________________ _ Score ----------- ( l 4) 15) Money that you spend on recreation should be part of your monthly budget. FORM A - PART 3 - BUDGETING FORM A - PART 3 - BUDGETING FORM A - PART 3 - BUDGETING ) You ju s t bought a new alarm clock on November 8, 1993 from Sears. The total cost of the clock is $3 1 .50 and yo u want to pay for it with a check. Us ing the blank check be low, show how you wou l d write a ch e c k to pay for the ala r m clock. 100 1) 1) You ju s t bought a new alarm clock on November 8, 1993 from Sears. The total cost of the clock is $3 1 .50 and yo u want to pay for it with a check. Us ing the blank check be low, show how you wou l d write a ch e c k to pay for the ala r m clock. 100 Pa y to the Or der of ________ __ _____________ $ _ _____ ---1 Green Meadow Bank item Score -- --(_,,1---,) _ ___ _ 2) How do you endorse a check? 3) 4) score ( 2) What is a checking register? What is a checking service charge/fee? Score (3) No . Date Check Issued To : Check Amount Deoosit 1 Balance 1 11/6/93 S200 . 00 $ Green Meadow Bank item Score -- --(_,,1---,) _ ___ _ 2) How do you endorse a check? 3) score ( 2) What is a checking register? What is a checking service charge/fee? Score (3) (_,,1 ,) _ ___ _ 2) How do you endorse a check? 3) score ( 2) What is a checking register? What is a checking service charge/fee? Score 4) b) Now put thirty more dollars into your checking account on December 29th. Record this in the Check Register . c) What is your new balance? $ ________ __ _ _ Score - - -- -(-4,....) ____ _ A "bounced" check means that you had enough money in your checking account to pay for what you bought. 5) Score ---- (--::5,...,.) ____ _ ( ::5,_ 62 6) You need to consider your monthly---,--------- and ______ _ when figuring out your monthly budget. Choose two words to complete the above sentences: interest; taxes; income; expenses; account. score desires; ----------- ( 6) 7) Most banks require that your have a minimum balance in your savings account to keep your account active. FORM A - PART 3 - BUDGETING True False Score ------ ------ -- --("""1"""s,...,) ___ _ 16) It is not a good idea to put aside your transportation money when you get paid. True False Score 6) c-=-1=2- 15) Money that you spend on recreation should be part of your monthly budget. True False Score ------ ------ -- --("""1"""s,...,) ___ _ 16) It is not a good idea to put aside your transportation money when you get paid. True ------ False ------ Score __________ _ (16) 1 s,...,)_ (16) 63 1 7) What is a W2 Form? _____ _________________ _____ _ Score ------ ----- ( l 7) 18) For which item is the largest amount of money deducted from your pay? A. Federal Tax B. Social Security c. City Tax Score o. State Tax (18) 19) What is health insurance? Score (19) 20) Wher e would be a safe p l ace to keep your money? a. Hidden in your house b. In your wallet Score c. In the Bank (20) What is a W2 Form? _ 1 7) ( l 7) 18) For which item is the largest amount of money deducted from your pay? A. Federal Tax B. Social Security c. City Tax Score o. State Tax (18) 19) What is health insurance? Score (19) 20) Wher e would be a safe p l ace to keep your money? a. Hidden in your house b. In your wallet Score c. In the Bank (20) 18) 19) 20) 64 Child's Name: ----(_,,1,.... -"""' 4,....) ____ _ Date of Birth =----(--=s,...._--= 1--= 0...,.) __ _ Date: ________________ _ Social Worker: ---------- FORM A - PART 4 - SHOPPING l) What is "Consu mer 's Rights"? ---------------------- Score ____ (~ 2 ~ 1 ,....,....) ___ _ 2) A pair of Puma sneakers is the same price in all stores. True _____ _ False Score ----("""' 2=2c-:-) ___ _ 3) Describe the differences between a full warranty and a limited warranty. _______________________________ _ Score ---- (-:--:2:-::3,-:-)- --- 4) When items are put on sale they are usually of poorer quality than the items that are regularly priced. True ------ False ------ Score ____ ~( 2 ~ 4 .,..,..) ___ _ 5) What is a "rain check"? ________________________ _ Score ____ ~( 2=-=s~ )---- 6) Name at least two techniques that store clerks use to t r ick or "take in" consumers. 7) Which candy is the best buy? FORM A - PART 3 - BUDGETING #1 Item Price $1.00 Unit Price $1.60 per pound Candy 10 oz. sweety's Score ____ ~( 2 ~ 6 .,,..,....) ___ _ #2 Item Price .84 Tasty Treats Unit Price : $1.67 per pound 8 oz. Score ____ ~( 2 ~7:--:- )- --- 8) If you are going to make a big purchase, how should you go about determining how you can get the best buy for your money? (i . e . purchasing a VCR) Score _ ____ ~----- (28) Child's Name: ----(_,,1,.... -"""' 4,....) ____ _ Date of Birth =----(--=s,...._--= 1--= 0...,.) __ _ Date: ________________ _ Social Worker: ---------- FORM A - PART 4 - SHOPPING l) What is "Consu mer 's Rights"? ---------------------- Score ____ (~ 2 ~ 1 ,....,....) ___ _ 2) A pair of Puma sneakers is the same price in all stores. True _____ _ False Score ----("""' 2=2c-:-) ___ _ 3) Describe the differences between a full warranty and a limited warranty. _______________________________ _ Score ---- (-:--:2:-::3,-:-)- --- 4) When items are put on sale they are usually of poorer quality than the items that are regularly priced. True ------ False ------ Score ____ ~( 2 ~ 4 .,..,..) ___ _ 5) What is a "rain check"? ________________________ _ Score ____ ~( 2=-=s~ )---- 6) Name at least two techniques that store clerks use to t r ick or "take in" consumers. 7) Which candy is the best buy? #1 Item Price $1.00 Unit Price $1.60 per pound Candy 10 oz. sweety's Score ____ ~( 2 ~ 6 .,,..,....) ___ _ #2 Item Price .84 Tasty Treats Unit Price : $1.67 per pound 8 oz. Score ____ ~( 2 ~7:--:- )- --- 8) If you are going to make a big purchase, how should you go about determining how you can get the best buy for your money? (i . e . purchasing a VCR) S 10 Date: ________________ _ Social Worker: ---------- FORM A - PART 4 - SHOPPING l) What is "Consu mer 's Rights"? ---------------------- Score ____ (~ 2 ~ 1 ,....,....) ___ _ 2) A pair of Puma sneakers is the same price in all stores. True _____ _ False Score ----("""' 2=2c-:-) ___ _ 3) Describe the differences between a full warranty and a limited warranty. FORM A - PART 3 - BUDGETING _______________________________ _ Score ---- (-:--:2:-::3,-:-)- --- 4) When items are put on sale they are usually of poorer quality than the items that are regularly priced. True ------ False ------ Score ____ ~( 2 ~ 4 .,..,..) ___ _ 5) What is a "rain check"? ________________________ _ Score ____ ~( 2=-=s~ )---- 6) Name at least two techniques that store clerks use to t r ick or "take in" consumers. 7) Which candy is the best buy? #1 Item Price $1.00 Unit Price $1.60 per pound Candy 10 oz. sweety's Score ____ ~( 2 ~ 6 .,,..,....) ___ _ #2 Item Price .84 Tasty Treats Unit Price : $1.67 per pound 8 oz. Score ____ ~( 2 ~7:--:- )- --- 8) If you are going to make a big purchase, how should you go about determining how you can get the best buy for your money? (i . e . purchasing a VCR) FORM A - PART 4 - SHOPPING ~( 2 ~7:--:)- If you are going to make a big purchase, how should you go about determining how you can get the best buy for your money? (i . e . purchasing a VCR) 8) Score _ (28) 65 9) Store owners usually put attractive items in their show windows to get consumers to come inside. True False score ------ ------ -----.=-=------ (29) 10) Before making any significant purchases, you should find out what the store's policy is for returning merchandise. True False score ------ ------ ----<-=-3-=-o.,...) ---- 11) You should buy items because you like them and not because you really need them. True False Score ------ ------ ----(-=-3-=-l-) ---- 12) What does the term "lay-away" mean? S 9) -=---- (29) 10) Before making any significant purchases, you should find out what the store's policy is for returning merchandise. -o.,...) - l-) (32) 13) Brand name clothing/items are always of better quality than non- brand names. True False Score ------ ------ -----.=-=------ (33) 14) Most mail advertisements that offer to send you items for free are good examples of companies sincere generous natures. True False Score ------ ------ ----(-=-3..,..4-) ---- 15) When you are making a large purchase, it is usually cheaper to have it delivered to your home as opposed to bringing it home yourself. True False Score ------ ------ ----(--=3--=5,_) ___ _ 16) You can always find the best bargains at huge shopping malls. 20) With a full warranty, you must pay the manufacturer for repairing an item if you bought it broken. True False Score ------ ------ ----------- FORM A - PART 3 - BUDGETING True False Score ------ ------ ----(--::3,-,6,....,.) ___ _ 17) What is the current amount of sales tax in the City of New York? Score -----.=-=------ (37) 18) It is often cheaper to buy summer clothes after the summer season has just passed. True False Score ------ ------ --------,-(-=-3-=-a..,....) ___ _ 19) With a limited warranty, you must pay the postage and handling fees when sending the item to the manufacturer. True False Score ------ ------ ----------- -=---- (33) 14) Most mail advertisements that offer to send you items for free are good examples of companies sincere generous natures. 4-) - 15) When you are making a large purchase, it is usually cheaper to have it delivered to your home as opposed to bringing it home yourself. 16) You can always find the best bargains at huge shopping malls. 17) What is the current amount of sales tax in the City of New York? Score -----.=-=------ (37) 18) It is often cheaper to buy summer clothes after the summer season has just passed. True False Score ------ ------ --------,-(-=-3-=-a..,....) ___ _ 3-=-a..,._ 19) With a limited warranty, you must pay the postage and handling fees when sending the item to the manufacturer. Score - 66 20) With a full warranty, you must pay the manufacturer for repairing an item if you bought it broken. 20) Score - 67 Child ' s Name: Date of Birth: -----,---,.------- ( l - 4) ---------- ( S - l O) Date: Social Worker: ---------- FORM A - PART 5 - MISCELLANEOUS Child ' s Name: - FORM A - PART 5 - MISCELLANEOUS To be completed by social worker and child. 1) Name four types of transportation. a) ________________ _ b) ______________ _ c) ________________ _ d) ______________ _ score ----------- ( 4 l) 2) How do you get a bus or train schedule? Score ----------- (42) 3) You must have car insurance in order to drive your car . True False Score ------ ------ ----------- (43) 4) Give an estimate of how much car insurance costs i n your state for one year with minimum coverage when an individual is in his / her early twenties with no previous accidents. ______________ _ score ----------- (44) 5) How do you find a doctor? __________ -,,, ____________ _ Score ----(--,.4=5.,..) ___ _ 6) What are the signs of alcoh o lism ? FORM A - PART 3 - BUDGETING ___________________ _ Score ----------- (46) 7) What should you do if you t hought y ou were addicted to drugs or alcoh o l ? -------------------------------- score ----------- (47) 8) Describe how you would look up the name and telephone number o f a particular restaurant in the Yello w Pages ? ______________ _ Score ----------- (4 8) 9 ) When yo u drink alcohol yo u bec ome more alert and ener getic ? True ------ False ------ Score __________ _ (49) 10) Name t wo over the c ounter medications. What are they u sed f or? a) ___________________________________ _ b) ________________________________ _ Score __________ _ ( SO) FORM A - PART 5 - MISCELLANEOUS To be completed by social worker and child. 1) Name four types of transportation. a) ________________ _ b) ______________ _ c) ________________ _ d) ______________ _ score ----------- ( 4 l) 2) How do you get a bus or train schedule? Score ----------- (42) 3) You must have car insurance in order to drive your car . True False Score ------ ------ ----------- (43) 4) Give an estimate of how much car insurance costs i n your state for one year with minimum coverage when an individual is in his / her early twenties with no previous accidents. ______________ _ score ----------- (44) 5) How do you find a doctor? __________ -,,, ____________ _ Score ----(--,.4=5.,..) ___ _ 6) What are the signs of alcoh o lism ? ___________________ _ Score ----------- (46) 7) What should you do if you t hought y ou were addicted to drugs or alcoh o l ? -------------------------------- score ----------- (47) 8) Describe how you would look up the name and telephone number o f a particular restaurant in the Yello w Pages ? ______________ _ Score ----------- (4 8) 9 ) When yo u drink alcohol yo u bec ome more alert and ener getic ? True ------ False ------ Score __________ _ (49) 10) Name t wo over the c ounter medications. What are they u sed f or? a) ___________________________________ _ b) ________________________________ _ Score __________ _ ( SO) To be completed by social worker and child. Score _ ( SO) 68 11) When a medicine label says to t ak e a p i ll 2 x a day, a nd you fe e l ve r y sick , should you t ak e that pill four times a day? Yes No Score ---- -- ------ ----------- ( 5 l) 12) What ar e th r e e fr e e lei s ure ac ti vitie s ? 1. 2.-- - --- - - --------- Score 3 . - - - - -------- --- - - - - - ---,-( =52......,...) - -- - 13) Name at least fo ur o f t he things you need to do to plan a party . 1. 2.-- - - ---------- -- --------------- - -- - 3. FORM A - PART 5 - MISCELLANEOUS 4.------- - - --- ------------ - -- --- ----- 11) When a medicine label says to t ak e a p i ll 2 x a day, a nd you fe e l ve r y sick , should you t ak e that pill four times a day? Yes No Score ---- -- ------ ----------- ( 5 l) 12) What ar e th r e e fr e e lei s ure ac ti vitie s ? 1. 2.-- - --- - - --------- Score 3 . - - - - -------- --- - - - - - ---,-( =52......,...) - -- - 13) Name at least fo ur o f t he things you need to do to plan a party . 1. 2.-- - - ---------- -- --------------- - -- - 3. 4.------- - - --- ------------ - -- --- ----- 11) When a medicine label says to t ak e a p i ll 2 x a day, a nd you fe e l ve r y sick , should you t ak e that pill four times a day? 3 . - - - - -------- --- - - - - - ---,-( =52......,...) - -- - 13) Name at least fo ur o f t he things you need to do to plan a party . 1. 2.-- - - ---------- -- --------------- - -- - 3. 4.------- - - --- ------------ - -- --- ----- s core ---- <"""' 5""" 3,_> ___ _ 14) When you need to ask for help or emotional support from other peop l e t hat means you are a weak individual . True Fa l se Score ------ ------ ----- (54) 15) What should you do if you committed a c r ime and got arrested? _ __ _ Score ------,- < =5=5.,... > - --- 16) List at least three pro b lems you are lik el y to have when living on your own . 1. 2.-- - --- - --- ------- 3 . ----------------- Score ______ ____ _ (56) 17) It is important to be able t o control your temper i n social situations . True False Score (57) 18) Do you know how to prevent unwanted pregnancies? Name at least three methods of birth control. 1. 2.------ ----------- 3 . Name at least three responsibilities of a parent. 1. 2.------------------------------------ 3. -----------------------=s,....c_o_r_e ___________ _ -----e-(.,..60.,,....,....) ---- Name at least three responsibilities of a parent. FORM A - PART 5 - MISCELLANEOUS -- - - - ------ ------ Score ___ ____ ___ _ (58) 19) What are the signs of sexuall y transmitted d i seases? Name at leas t two . 1. 2.-------------- - ------- - ------ --- ---- Score -----,c-=5=9 ,...,. > __ _ _ <53,_> _ 16) List at least three pro b lems you are lik el y to have when living on your own . 1. 2.-- - --- - --- ------- 3 . ----------------- Score ______ ____ _ (56) 17) It is important to be able t o control your temper i n social situations . True False Score (57) 18) Do you know how to prevent unwanted pregnancies? Name at least three methods of birth control. 1. 2.------ ----------- 3 . -- - - - ------ ------ Score ___ ____ ___ _ (58) 19) What are the signs of sexuall y transmitted d i seases? Name at leas t two . 1. 2.-------------- - ------- - ------ --- ---- Score ----- c-=5=9 > 19) Score - 9 ,...>_ 69 20) .,,....,.. 70 Child's Name: Dat e of Birt h: --------,---,------- ( l - 4 ) -----=---,-,,----- - ( 5- 10) Dat e : Social Worker : - - -- - - -------- - - - ---------- FORM B - HOUSECLEANING Child's Name: - FORM B - HOUSECLEANING To be comple te d by c hi l d ca r e worker or exam i ner . It is r e commended that th e child c ar e work er invite the child to pa rticipat e in the ass e s sment pr oce s s. The ch i l d care wor ke r or exami ne r s hould ask t he r e side nt qu e stions that cl ari f y whether he or she knows the following facts . Wher e applicable , a s k resident to demonstra t e their knowledg e of a skil l thr ou gh body lan gua ge . l) Child knows how to mop and sweep a floor good unde r sta ndi ng of th e appro pri ate pr oducts to use on a floor. Ye s___ _ _ _ No __ __ _ thoroughly. Child ha s a equ i pment and c le a ning Score -------,-(-:-1-,-) _ __ _ l) Child knows how to mop and sweep a floor good unde r sta ndi ng of th e appro pri ate pr oducts to use on a floor. thoroughly. Child ha s a equ i pment and c le a ning 2) Child can clean and defrost a refrigerator. Chi l d understands what clean i ng pr oducts to use on a refrigerator. Ye s______ No_ _ ____ Score _ __ _ ~(~ 2 ~):---- - 3) Child knows how to clean a kitchen and a bathroom , with an adequate understand i ng of appropriate cleaning pro ducts. Yes ______ No______ Scor e _____ (~ 3 ~):- ---- 4) Child knows how to make a bed, demonstrating an adequate know l edge about how often bed linen should be chang e d. Yes _____ _ No______ Score _ __ _ _ (~ 4 ~):- - --- S) Child is able to use small kitchen appliances (i.e. toaster, can 6) opener). Yes - --- - - No -- - --- Child is able to use a vacuum cleaner. Yes______ No _____ _ Score ----- (~S-) ----- Score --- ----,-(-:-6 -,-) -- -- 7) Child understands the safety precautions involving elect r ical outlets and wires. FORM B - HOUSECLEANING Yes_ _ _ __ _ No_ __ ___ Score __ _____ ___ _ ( 7) 8) Child understands th e importance of storing flammable cleaning products in designated places . Yes______ No______ Score _____ (~S- ) ___ _ _ 9) Child is able to distinguish which types of clothing need to be dry cleaned . Yes No Score (9) 10) Child knows what colors get washed in hot or cold water . Yes No Score (10) 2) Child can clean and defrost a refrigerator. Chi l d understands what clean i ng pr oducts to use on a refrigerator. Ye s______ No_ _ ____ Score _ __ _ ~(~ 2 ~):---- - 3) Child knows how to clean a kitchen and a bathroom , with an adequate understand i ng of appropriate cleaning pro ducts. Yes ______ No______ Scor e _____ (~ 3 ~):- ---- 4) Child knows how to make a bed, demonstrating an adequate know l edge about how often bed linen should be chang e d. Yes _____ _ No______ Score _ __ _ _ (~ 4 ~):- - --- S) Child is able to use small kitchen appliances (i.e. toaster, can 6) opener). Yes - --- - - No -- - --- Child is able to use a vacuum cleaner. Yes______ No _____ _ Score ----- (~S-) ----- Score --- ----,-(-:-6 -,-) -- -- 7) Child understands the safety precautions involving elect r ical outlets and wires. Yes_ _ _ __ _ No_ __ ___ Score __ _____ ___ _ ( 7) 8) Child understands th e importance of storing flammable cleaning products in designated places . Yes______ No______ Score _____ (~S- ) ___ _ _ 9) Child is able to distinguish which types of clothing need to be dry cleaned . Yes No Score (9) 10) Child knows what colors get washed in hot or cold water . Yes No Score (10) (10) 71 11) Child has demonstrated that they can effectively use a wash i ng ma chine and dryer . Yes ______ No______ Scor e _____ (~l~l::-:- ) ___ _ 12) Chi l d has demonst r ated that they can i r on. Yes ______ No______ Score _____ (~l~ 2 ::-:- ) ---- 13) Child has some basic sewing skill s. FORM B - HOUSECLEANING Ye s No Score (13) 14) Child knows what the purposes of bleach are for the care of the i r clothes. Yes No Score (14) 15) Child knows what Te flon pots are and how to c l ean them. Yes __ __ __ No______ Score _____ (~l~S::-:-) ___ _ 16) Chi l d knows what the following c l eaning products are used for: a) Br illo/Scouring Pads b) Ajax/Cornet Cleans er c) Floor Wax d) Polish (Furni t ure) e) Disinfectant (i . e. Lyso l ) Yes No Score ----- - ------ ------, ~--- - - (16) (17) 18) Child knows how to change a light bulb; change a vacuum bag. Yes No Score (18) 19) Child knows how to clean an oven . Yes No Score (19) 20) Child knows what to do in case of an oven gas leak. Yes No Score (20) ~- (16) (20) 72 Child's Name: Date of Birth: -----,(-:-1---:-4-:--) ----- ------,----- ( 5 - 10) Date: ________________ _ Social Worker : _________ _ FORM B - COOKING/PURCHASING AND CARE OF FOOD/NUTRITION Child's Name: - Date: _ FORM B - COOKING/PURCHASING AND CARE OF FOOD/NUTRITION To be completed by child care worker or examiner. It is recommended that the child care worker invite the child to participate in the assessment process. The child care worker or examiner should ask the resident questions that clarify whether they know the following facts . l) Child is able to plan varied and balanced meals. Yes______ No______ Score ____ __,,...,.. ____ _ (21) 2) Child knows the four basic food groups and is able many servings of each group he/she should consume to in determine how one day. Yes______ No______ score ____ -,-,=-,----- (22) 3) Child understands the importance of consuming enriched with vitamins and minerals. foods that are Yes______ No______ Score _____ (~ 2 ~ 3 ...,...) ___ _ 4) Child understands which foods should be limited to maintain good health. Yes ------ No ------ Score ____ ~( 2 ~ 4 .,....,...) ___ _ 5) Child can shop for food, with an understanding of how the concept of comparative shopping for groceries. Yes______ No______ Score _____ (~ 2 ~ 5 ...,...) ___ _ 6) Child understands the practical usefulness of coupons and no frill brands. Yes No Score ------ ------ ----------- (26) 7) Child understands the concept of unit pricing with regard to determining the best buy. Yes______ No______ Score ____ ~(~ 2 ~ 7 ~),----- 8) Child understands why it is better to shop in a large supermarket as opposed to a small grocery store. Yes______ No______ Score ____ -=-=--:----- (28) 9) Child understands what a reasonable weekly food bill is for a single person. Yes No Score (29) 10) Child recognizes signs of food spoilage. Yes No Score (30) l) Child is able to plan varied and balanced meals. Yes______ No______ Score ____ __,,...,.. ____ _ (21) 2) Child knows the four basic food groups and is able many servings of each group he/she should consume to in determine how one day. Yes______ No______ score ____ -,-,=-,----- (22) 3) Child understands the importance of consuming enriched with vitamins and minerals. foods that are Yes______ No______ Score _____ (~ 2 ~ 3 ...,...) ___ _ 4) Child understands which foods should be limited to maintain good health. Yes ------ No ------ Score ____ ~( 2 ~ 4 .,....,...) ___ _ 5) Child can shop for food, with an understanding of how the concept of comparative shopping for groceries. FORM B - COOKING/PURCHASING AND CARE OF FOOD/NUTRITION Yes______ No______ Score _____ (~ 2 ~ 5 ...,...) ___ _ 6) Child understands the practical usefulness of coupons and no frill brands. Yes No Score ------ ------ ----------- (26) 7) Child understands the concept of unit pricing with regard to determining the best buy. Yes______ No______ Score ____ ~(~ 2 ~ 7 ~),----- 8) Child understands why it is better to shop in a large supermarket as opposed to a small grocery store. Yes______ No______ Score ____ -=-=--:----- (28) 9) Child understands what a reasonable weekly food bill is for a single person. Yes No Score (29) 10) Child recognizes signs of food spoilage. Yes No Score (30) ,, , (21) 73 11) Child understands which foods need to be frozen. Yes______ No______ Score _____ (_ 3 ~ 1 ,,..-) ___ _ 12) Child understands which foods need to be refrigerated. Yes______ No______ Score _____ (_ 3 ~ 2 ~),----- 13) Child knows how to store left-overs. Yes______ No _____ _ Score ----(:-::3,...,.3,...,...) ___ _ 14) Child understands the difference between frying, baking and broiling. Yes______ No______ Score __________ _ (34) 15) Child knows how to read and use recipes. Yes No Score ------ ------ ------=-=------ ( 35) 16) Child knows how to adequatel y measure ingredients. Yes______ No______ Score __________ _ (36) 17) Child knows how to defrost a refrigerator. Yes______ No______ Score __________ _ (37) 18) Child knows which foods must be rinsed prior to eating or cooking. Yes______ No______ Score _____ (_ 3 ~ 8 :-,-)---- 19) Child kno ws how to set a table. Yes No Score __________ _ (39) 20) Child knows how to clean up table and dishes after meals. Yes______ No______ Score _____ (_ 4 ~ 0 :-,-)---- 11) Child understands which foods need to be frozen. Yes______ No______ Score _____ (_ 3 ~ 1 ,,..-) ___ _ 12) Child understands which foods need to be refrigerated. Yes______ No______ Score _____ (_ 3 ~ 2 ~),----- 13) Child knows how to store left-overs. Yes______ No _____ _ Score ----(:-::3,...,.3,...,...) ___ _ 14) Child understands the difference between frying, baking and broiling. Yes______ No______ Score __________ _ (34) 15) Child knows how to read and use recipes. Yes No Score ------ ------ ------=-=------ ( 35) 16) Child knows how to adequatel y measure ingredients. Yes______ No______ Score __________ _ (36) 17) Child knows how to defrost a refrigerator. FORM B - COOKING/PURCHASING AND CARE OF FOOD/NUTRITION Yes______ No______ Score __________ _ (37) 18) Child knows which foods must be rinsed prior to eating or cooking. Yes______ No______ Score _____ (_ 3 ~ 8 :-,-)---- 19) Child kno ws how to set a table. Yes No Score __________ _ (39) 20) Child knows how to clean up table and dishes after meals. Yes______ No______ Score _____ (_ 4 ~ 0 :-,-)---- ( 40 74 74 Date -- I JOB SEARCH AND MAINTENANCE (An individual who is knowledgeable in this area would know about: where to look for a job; the job application process; job interviewing; job jargon; appropriate on-the-job behavior; work benefits; unions, payroll deductions.) I JOB SEARCH AND MAINTENANCE (An individual who is knowledgeable in this area would know about: where to look for a job; the job application process; job interviewing; job jargon; appropriate on-the-job behavior; work benefits; unions, payroll deductions.) II APARTMENT HUNTING/MAINTENANCE (An individual who is knowledgeable in this area would know about: how to find an apartment; renting terms and policies; roommates; initial and continued apartment care costs.) II APARTMENT HUNTING/MAINTENANCE (An individual who is knowledgeable in this area would know about: how to find an apartment; renting terms and policies; roommates; initial and continued apartment care costs.) II APARTMENT HUNTING/MAINTENANCE (An individual who is knowledgeable in this area would know about: how to find an apartment; renting terms and policies; roommates; initial and continued apartment care costs.) III BUDGETING (An individual who is knowledgeable in this area would know about: checking accounts, concept of interest and savings accounts; how to budget; reading and understanding paychecks; ·credit buying and ratings.) III BUDGETING (An individual who is knowledgeable in this area would know about: checking accounts, concept of interest and savings accounts; how to budget; reading and understanding paychecks; ·credit buying and ratings.) IV V VI ratings.) SHOPPING (An individual who is knowledgeable in this area would know about: methods and implications of comparative shopping; warranties and consumer's rights; unit pricing and sales tax; advertising ploys; and consumer terminology and knowledge about bargains.) HOUSECLEANING (An individual who is knowledgeable in this area would know about: the fundamentals of housecleaning; use of cleaning appliances; household safety; care of clothing; cleaning products and accessories.) COOKING/PURCHASING AND CARE OF FOOD/NUTRITION (An individual who is knowledgeable in this area would know about: nutrition; how to cook and prepare food; shopping for food; food storage.) RATINGS 1. Knows nothing/almost nothing 2. Has a slight knowledge 3. Has a moderate knowledge 4. Has a good knowledge S. APPENDIX B SOCIAL WORKERS GLOBAL RATINGS OF RESIDENTS' INDEPENDENT LIVING SKILLS All residents who are fourteen years of age and older need to have their independent living skills assessed. While a comprehensive assessment will be performed by an individual counselor, your global appraisal of each of the residents would be greatly appreciated . Please complete the demographic information on each resident, and then using the descriptors presented below, provide an appraisal of each of the skills listed below. Resident's Name ___________ _ Date of Birth _________ _ (5-10) Date (l-4) ---------::-,:---::-,,..,....-------- ( 11 - 16) Date of Admission. ___ --~_,,_._ (17-22) Resident's Name ___________ _ Date of Birth _________ _ (5-10) Date (l-4) ---------::-,:---::-,,..,....-------- ( 11 - 16) Date of Admission. ___ --~_,,_._ (17-22) Resident's Name __ RATING (23) (24) (25) (26) (27) (28) INDEPENDENT LIVING SKILLS ASSESSMENT FORM This form (Form A) should be completed by a trained examiner during an individualized session with a resident. The examiner should read each question aloud in a clear, articulate manner and copy down the resident's response. Actual scores should be determined in the resident's absence. There are six (6) areas of independent living skills which need to be assessed for each youth. They include: (1) Job Search and Maintenance, (2) Apartment Hunting and Maintenance, (3) Budgeting, (4) Shopping, (5) Housecleaning, and (6) Cooking/Purchasing and Care of Food/Nutrition. The other independent living skills, which are not easily classified, but serve to be integral components of a comprehensive I.L.S. assessment, will be classified into a seventh category entitled "Miscellaneous . " The Housecleaning, and Cook/Purchasing and Care of Food/Nutrition domains constitute Form B, and should preferably be administered by child care staff since they are most familiar with residents' housecleaning and cooking habits. EXAMINER'S RESPONSIBILITY Standard testing procedures must be followed with all residents to ensure that every resident has an equal opportunity to respond correctly to test items. Firstly, the examiner should organize all test materials before the testing session begins. Secondly, a quiet, comfortable room should be u sed for testing, and only the e xaminer and resident should be in the room where the testing takes place. Thirdly, make sure that auditory or perceptual problems, in addition to language difficulties or severe physical handicaps, do not interfere with the resident's ability t o comprehend orally presented inquiries. Fourthly, make sure that the residents know what they have to do before testing begins. Explain that there is no special need to prepare for the test and that they cannot fail the ILSFA and that its purpose is to help plan future training emphases. Although praise can be given for test performance in general, it should not be given for correctness of response. TESTING SCHEDULE Form A should be administered in one or two sessions. However, the testing can be extended to more sessions if necessary . Form B should be administered in one session. Normally, the entire battery should be completed within on week. Avoid scheduling tests on days just before or after vacations, important school functions, or immediately after strenuous physical exercise. Also, avoid causing residents to miss classes and activities they enjoy (i.e. art, recreation). The counselor should always be sensitive to resident fatigue. Although the tests are not timed, some reasonable time constraint should be followed. Date -- Knows everything/almost everything Please use the above rating scale and descriptions to evaluate each of the following skill domains: V SHOPPING (An individual who is knowledgeable in this area would know about: methods and implications of comparative shopping; warranties and consumer's rights; unit pricing and sales tax; advertising ploys; and consumer terminology and knowledge about bargains.) VI COOKING/PURCHASING AND CARE OF FOOD/NUTRITION (An individual who is knowledgeable in this area would know about: nutrition; how to cook and prepare food; shopping for food; food storage.) Please use the above rating scale and descriptions to evaluate each of the following skill domains: SKILL DOMAIN RATING I Job Search and Maintenance (23) II Apartment Hunting/Maintenance (24) III Budgeting (25) IV Shopping (26) V Housecleaning (27) VI Cooking/Purchasing and Care of Food/Nutrition (28) SKILL DOMAIN RATING I Job Search and Maintenance (23) II Apartment Hunting/Maintenance (24) III Budgeting (25) IV Shopping (26) V Housecleaning (27) VI Cooking/Purchasing and Care of Food/Nutrition (28) 76 APPENDIX C TESTING CAUTIONS Coaching - Examiners should not provide clues to help residents answer the test items . It is important that this is a test taking situation and not a learning activity. Correct responses should not be indicated. Examiners should also not explain words unless they are completely unrelated to the questi o n being asked by a particular i tem. Finally, voice changes and / or inflections on key words, such as "usually" or "never", should be avoided. 77 Use answer key to determine whether credit should be given to a particular response. Record child's responses while in session so score can be determined later. 78 FORM A - PART I - JOB SEARCH & MAINTENANCE KEY Questions - Each question receives 1-point credit. 1. Only give credit to a response which includes at least 3 of the following: l) 2) 3) 4) 5) 6) 7) 8) newspaper - classified section through a friend store windows - help wanted signs private or district employment agency bulletin boards (i.e. school) job placement site through your present job (promotion) personal contact - Yellow Pages 1 - Point 2. A correct response must include 4 of the following: 3. 1) working papers/birth certificate 2) social security care - and/or number 3) proof of educational status and/or educational background 4) addresses and phone numbers of personal references 5) addresses and phone numbers of supervisors at previous employment sites 6) If child responds "resume", only give credit if they are able to list the important information which is usually listed on 7) 8) 9) True resume. citizenship papers phone number of person to notify extracurricular activities in case of emergency 1 - Point 1 - Point 4. Correct response should include any form of non-verbal body language, projected attitude, or speech style. For example: l) 2) 3) 4) 5) 6) 7) 8) 9) shaking hands with interviewer having a polite and motivated attitude impressive attire being on time level of eye contact good body posture articulate speech / grammar responding in complete sentences asking appropriate questions 1 - Point 5. correct response can include any negative of Question #4 or: l) 2) 3) 4) 5) bringing a friend to the interview being unprepared chewing gum/smoking foul language or st reet jargon other logical statements 6. Correct answer should include any of the following: 1) salary 2) hours required 3) supervision 4) fringe benefits 5) i i 1 - Point Questions - Each question receives 1-point credit. Questions - Each question receives 1-point credit. 1. Only give credit to a response which includes at least 3 of the following: l) 2) 3) 4) 5) 6) 7) 8) newspaper - classified section through a friend store windows - help wanted signs private or district employment agency bulletin boards (i.e. school) job placement site through your present job (promotion) personal contact - Yellow Pages 1 - Point 1 - Point 1 - Point Correct response should include any form of non-verbal body language, projected attitude, or speech style. For example: 1 - Point 1 - Point correct response can include any negative of Question #4 or: 1 - Point 1 - Point 79 7. 8) 9) True chances for promotion or further training other logical facts 1 - Point 1 - Point 8. An adequate definition would connect the term U.S. citizen with native or nationalized person of the U.S. who owes allegiance to a government and is entitled to reciprocal protection from it. If resident responds "born here", question them further to give a more in depth response. 1 - Point 9. False 1 - Point 10. Correct response should include at least 3 of the following: 11. 12. 13. 14. 15. 16 . 1) 2) 3) 4) 5) 6) 7) 8) 9) Name Address - Current/Permanent Job Experience Educational Background Special Recognition or Honors Special interests/hobbies Career Objectives Extracurricular Activities References 1 - Point Correct response should indicate that child has knowledge about listing number of dependents, as well as an understanding that their taxes will be based on the number of dependents that they claim. 1 - Point Score only if child demonstrates such appropriate job relations tactics as 1) attempting to verbally settle conflicts and 2) taking matter up with supervisor. 1 - Point Score 1 point only A) Time Clock if child provides all of the correct matches. 8) C) D) E) F) G) Salary Hourly Wage A Fringe Benefit Work Week Overtime Full-time False = e - Registers amount of hrs. Questions - Each question receives 1-point credit. you work in a day d - Total Income = f - Amount of$ you earn per hour = a - Health Insurance = b - # of days you work in a week = g - When you work above & beyond your regular work week = c - When you work 35+ hours per week 1 - Point 1 - Point An adequate response should include at least one of the following advantages: l) you can take up grievances between you and your supervisor with a union representative, 2) you have input into your work policy, 3) a boss must substantiate his reasons for suspending somebody, 4) less room f or unequal treatment o f employees - fixed salary s cale, fringe benefits, vacation days, fixed overtime rules, etc., 5) negotiates raises with company, and 6) defends you in case you are being treated unfairly by employer . 1 - Point A scorable response should indicate that both "Sick" and "Vacation" days are paid days off which are usually earned after being on the job for a specified period of time. Answers such as "time off" are not acceptable. Child should indicate that Sick days are to be taken if the illness would hamper job performance or worsen a health condition and after advance notice has been given to an employer 7. 8) 9) True chances for promotion or further training other logical facts 8. An adequate definition would connect the term native or nationalized person of the U.S. who government and is entitled to reciprocal protec resident responds "born here", question them fur in depth response. 9. False 10. Correct response should include at least 3 of 11. 12. 13. 14. 15. 16 . 1) 2) 3) 4) 5) 6) 7) 8) 9) Name Address - Current/Permanent Job Experience Educational Background Special Recognition or Honors Special interests/hobbies Career Objectives Extracurricular Activities References Correct response should indicate that child listing number of dependents, as well as an under taxes will be based on the number of dependents Score only if child demonstrates such appropria tactics as 1) attempting to verbally settle conf matter up with supervisor. Questions - Each question receives 1-point credit. Score 1 point only A) Time Clock if child provides all of the 8) C) D) E) F) G) Salary Hourly Wage A Fringe Benefit Work Week Overtime Full-time False = e - Registers amount of day d - Total Income = f - Amount of$ you earn = a - Health Insurance = b - # of days you work = g - When you work above & work week = c - When you work 35+ h An adequate response should include at least o advantages: l) you can take up grievances b supervisor with a union representative, 2) you work policy, 3) a boss must substantiate his rea somebody, 4) less room f or unequal treatment salary s cale, fringe benefits, vacation days, f etc., 5) negotiates raises with company, and 6) you are being treated unfairly by employer . A scorable response should indicate that both " days are paid days off which are usually earned job for a specified period of time Answers su 1 - Point 1 - Point 1 - Point 1 - Point g 1 - Point 1 - Point 1 - Point 1 - Point 1 - Point Correct response should indicate that child has knowledge about listing number of dependents, as well as an understanding that their taxes will be based on the number of dependents that they claim. 1 - Point Score only if child demonstrates such appropriate job relations tactics as 1) attempting to verbally settle conflicts and 2) taking matter up with supervisor. 1 - Point 1 - Point 1 - Point A scorable response should indicate that both "Sick" and "Vacation" days are paid days off which are usually earned after being on the job for a specified period of time. Answers such as "time off" are not acceptable. Child should indicate that Sick days are to be taken if the illness would hamper job performance or worsen a health condition and after advance notice has been given to an employer. 16 . 80 80 17. 18. 19. 20. True 2-4 Weeks True False 1 - Point 1 - Point 1 - Point 1 - Point 17. 18. 19. 20. True 2-4 Weeks True False 81 FORM A - PART 2 - APARTMENT HUNTING/MAINTENANCE KEY 1. Correct response should include seeking information from at least two of the following: Newspaper; Community Bulletin Board, Real Estate Agency, Asking Landlords . 1 - Point 1. Correct response should include seeking information from at least two of the following: Newspaper; Community Bulletin Board, Real Estate Agency, Asking Landlords . 1 - Point 2. Score 1 point if completely correct. br = bedroom shop/trans= shopping/transportation vie= vicinity 2. Score 1 point if completely correct. br = bedroom shop/trans= shopping/transportation vie= vicinity 1 - Point 2. Score 1 point if completely correct. br = bedroom shop/trans= shopping/transportation vie= vicinity 1 - Point 3. Correct responses should include three or more of the issues mentioned below: pets; accessibility to transportation; questions about lease, utilities; accessibility to stores and other community resources; permission to have a roommate; questions abut rent; are stove and refrigerator included; safe neighborhood; furniture included . 1 - Point 4 . Correct response include: rent; security deposit; electricity; gas; telephone installation; necessary household items (i.e. plates, toilet items, basic foods) . 1 - Point 5. An adequate definition of security deposit should indicate that it is when the landlord is given an extra month's rent in advance . 1 - Point 6. Score if amount is within the range of $700 - $1500. 1 - Point 7. Score if amount is within range of $75 - $150. 1 - Point 8 . Child demonstrates adequate knowledge regarding obtaining utilities (i.e. calling or visiting utility offices, installation charges, previous credit, employment, or income information). 1 - Point 9 . Child provides fair estimate and is able to itemize each expense. Scorable responses should fall within the range of: gas - $18.00 - $30.00 bi-monthly or $9 . 00 - $50.00 monthly. 1 - Point 10. Correct response: l) D, 2) A, 3) B, 4) c 1 - Point 11. A correct response should fall within the range of $350 - $550 weekly . 1 - Point 12. True 1 - Point 13 . False 1 - Point 14. False 1 - Point 15. False 1 - Point 16. False 1 - Point 17. A correct response should reflect a knowledgeable understanding of appropriate problem-solving skills in these domains. More specifically, a scorable response for solving a stopped up toilet problem should include steps as using a plunger and call a plumber. FORM A - PART 2 - APARTMENT HUNTING/MAINTENANCE KEY A scorable response to the electrical failure issue should include turning off all electrical appliances, investigating fuse box and correcting blown fuse problem if applicable, and calling an electrician if problem is extensive. 1 - Point 1 - Point 3. Correct responses should include three or more of the issues mentioned below: 3. Correct responses should include three or more of the issues mentioned below: pets; accessibility to transportation; questions about lease, utilities; accessibility to stores and other community resources; permission to have a roommate; questions abut rent; are stove and refrigerator included; safe neighborhood; furniture included . 1 - Point 4 . Correct response include: rent; security deposit; electricity; gas; telephone installation; necessary household items (i.e. plates, toilet items, basic foods) . 1 - Point 5. An adequate definition of security deposit should indicate that it is when the landlord is given an extra month's rent in advance . 1 - Point 6. Score if amount is within the range of $700 - $1500. 1 - Point 7. Score if amount is within range of $75 - $150. 1 - Point 6. Score if amount is within the range of $700 - $1500. 1 - Point 7. Score if amount is within range of $75 - $150. 1 - Point 8 . Child demonstrates adequate knowledge regarding obtaining utilities (i.e. calling or visiting utility offices, installation charges, previous credit, employment, or income information). 1 - Point 8 . Child demonstrates adequate knowledge regarding obtaining utilities (i.e. calling or visiting utility offices, installation charges, previous credit, employment, or income information). 1 - Point 82 18. A correct response should include making complaints to City or County Housing Bureau, Bureau of Discrimination, NAACP, and seeking legal assistance. 1 - Point 19. Correct response should include a definition of term in addition to the significance of having an absentee landlord . An absentee landlord is a landlord who does not reside in the house or building in which he/she rents. Therefore, they are less available for complaints, repairs, and other problems. 1 - Point 19. Correct response should include a definition of term in addition to the significance of having an absentee landlord . An absentee landlord is a landlord who does not reside in the house or building in which he/she rents. Therefore, they are less available for complaints, repairs, and other problems. 1 - Point 20. 20. 18. A correct response should include making complaints to City or County Housing Bureau, Bureau of Discrimination, NAACP, and seeking legal assistance. 1 - Point FORM A - PART 2 - APARTMENT HUNTING/MAINTENANCE KEY A rental application fee is specific amount of money that is required in order for rental organization/office to process an application for housing. Amount of money to real estate broker should be the equivalent of one month's rent. 1 - Point 83 FORM A - PART 3 - BUDGETING - KEY FORM A - PART 3 - BUDGETING - KEY 1. Score if child completes check correctly. Pay to the Order of sears Thirtv one dollars Green Meadow Bank Item Re: Alarm Clock , / 1 - Points November 8 100 1993 S 31. so 50/100 2. By putting your signature as it is written on the check across the back of the check widthwise. 1 - Point 1. By putting your signature as it is written on the check across back of the check widthwise. 1 - Point 3. A correct response should closely resemble the following definitions: Checking Register= A register with columns in which all checking transactions are listed; Checking Service Charge/Fee= A fee (usually monthly) that the bank charges to keep your checking account active. l - Point y g l - Point y g l - Point Child completes check register accurately. 1 - Point 4. a) b) Child completes check register accurately. No. Date Check Issued 11/6/93 100 11/8/93 Sears 12/29 /93 Deoosit To: Check Amount S31.SO 1 - Point Deoosit Balance S 200.00 31.50 s 168.50 S30.00 30.00 s 198.50 4. Child completes check register accurately. 1 - Point c) 5 . 6. 7. New Balance= $198.50 False Income, Expense True 1 - Point 1 - Point 1 - Point 1 - Point 8. An adequate definition of interest includes a charge for borrowed money - generally a percentage of the amount borrowed and/or an 9. excess above what is due. 1 - Point A correct response can include hourly wage; taxes deducted, City); union dues deducted; account number; name of bank. your name; pay period; hours worked; (Social Security, Federal, State, insurance fees deducted; your job 1 - Po int 10. An adequate definition includes - a procedure you go through to keep the bank from paying a ch eck . 1 - Point 11. True 1 - Point 12. Child demonstrates adequate knowledge of credit system . A response should indicate that credit cards allow an individual to purchase things or borrow money and pay at a later date with interest. FORM A - PART 2 - APARTMENT HUNTING/MAINTENANCE KEY When you fill out a credit application, you sign an installment agreement. 1 - Point c) 5 . 6. 7. New Balance= $198.50 False Income, Expense True 1 - Point 1 - Point 1 - Point 1 - Point 8. An adequate definition of interest includes a charge for borrowed money - generally a percentage of the amount borrowed and/or an 9. excess above what is due. 1 - Point A correct response can include hourly wage; taxes deducted, City); union dues deducted; account number; name of bank. your name; pay period; hours worked; (Social Security, Federal, State, insurance fees deducted; your job 1 - Po int 10. An adequate definition includes - a procedure you go through to keep the bank from paying a ch eck . 1 - Point 11. True 1 - Point 12. Child demonstrates adequate knowledge of credit system . A response should indicate that credit cards allow an individual to purchase things or borrow money and pay at a later date with interest. When you fill out a credit application, you sign an installment agreement. 1 - Point 8. An adequate definition of interest includes a charge for borrowed money - generally a percentage of the amount borrowed and/or an excess above what is due. 1 - Point 1 - Point 84 13. 14. 15. 16. True January - April True False 1 - Point 1 - Point 1 - Point 1 - Point 17. An adequate definition would include "a form which lists your annual salary and tax deductions, place of employment, and social security number. 1 - Point 18. A 1 - Point 19. An adequate definition would include a company which pays all or a portion of your medical expenses. You receive this benefit by either you or your employer paying a check. 1 - Point 20. c - in the bank. 1 - Point 13. 14. 15. 16. True January - April True False 1 - Point 1 - Point 1 - Point 1 - Point 13. 14. 15. 16. True January - April True False 1 - Point 1 - Point 1 - Point 1 - Point 17. An adequate definition would include "a form which lists your annual salary and tax deductions, place of employment, and social security number. 1 - Point 18. A 1 - Point 19. FORM A - PART 4 - SHOPPING KEY FORM A - PART 4 - SHOPPING KEY FORM A - PART 4 - SHOPPING KEY 1. An adequate definition would indicate that they are the leg al rights you have as a purchaser of goods or services. 1 - Point 2. False 1 - Point 3. Correct response should include specifications of a limited war ranty and an understanding of benefits of full warr anty . A full warr a nty us ually means that a defective product will be fi xed or replaced within a re a sonable period of time at no cost to you . A limited warranty usually means that something is missing, i . e. labor, costs of parts, charge for handling, or the first purchaser will only be covered. 1 - Point 4. False 1 - Point 5. An adequate definition would define a raincheck as an ag r eement between a store and a consume r that the sto r e will sell the consumer an item at th e sale price as soon as a new sh i pment of the item arrives . 1 - Point 6 . correct response includes the bait and switch technique, complimenting consumer, pressuring consumer to buy a n item immediately, lowballing, loss leaders or conditional purchases. 1 - Point 7 . #1 1 - Point 8 . Correct response shou l d include description of comparison shopping techniques (i.e. comparing the prices in different stores of different brand names, checking the consumer magazines, etc.). 1 - Point 9. True 1 - Point 10 . True 1 - Point 11. False 1 - Point 12. An adequate definition would include "putting a deposit on merchandise to put on hold on its sale until you return with the full payment. 1 - Point 13. False 1 - Point 14 . False 1 - Point 15. False 1 - Point 16 . Fa l se 1 - Point 17. 8 1/4% or 8.25 l - Point 18 . True l - Point 19 . True 1 - Point 20 . False 1 - Point 1. An adequate definition would indicate that they are the leg al rights you have as a purchaser of goods or services. FORM A - PART 2 - APARTMENT HUNTING/MAINTENANCE KEY An adequate definition would include a company which pays all or a portion of your medical expenses. You receive this benefit by either you or your employer paying a check. 1 - Point 20. c - in the bank. 1 - Point equate definition would include "a form which lists your annual and tax deductions, place of employment, and social security 1 - Point 1 - Point 1 - Point 85 FORM A - PART 4 - SHOPPING KEY FORM A - PART 4 - SHOPPING KEY 1 - Point 1 - Point 1 - Point 1 - Point An adequate definition would define a raincheck as an ag r eement between a store and a consume r that the sto r e will sell the consumer an item at th e sale price as soon as a new sh i pment of the item arrives . 1 - Point 1 - Point 1 - Point 1 - Point 13. False 1 - Point 14 . False 1 - Point 15. False 1 - Point 16 . Fa l se 1 - Point 17. 8 1/4% or 8.25 l - Point 18 . True l - Point 19 . True 1 - Point 20 . False 1 - Point 86 FORM A - PART 5 - MISCELLANEOUS - KEY 1. Correct response should include following list: car, taxi, bus, motorcycle, bicycle. at least four train , plane, items from the boat, walking, 1 - Point 2. Correct response should illustrate that child has knowledge of transit authority, community transportation facilities, etc. Child should know that they can obtain schedules from bus/train stations, community information centers. 1 - Point 3. True 1 - Point 4 . A realistic estimate should be within the range of $800 - $2400 . 1 - Point 5. Correct response should include 1-AMA, 2- Recommendations from a friend/family, 3-Yellow Pages, 4-Clinic, 5-HIP Center, etc. 1 - Point 6. Correct response should include mentioning of the effects of alcohol on an individual's physical, emotional, social functioning , family and intellectual functioning. 1 - Point 7. Correct response should include seeking help from a substance abuse 8 . 9 . agency, doctor, therapist, friend, or clergy, etc . 1 - Point Correct response should include an adequate knowledge about yellow pages. For example, individual needs to look under a broader category as well as in alphabetical order. False demonstration of knowing that an for most services 1 - Point 1 - Point 10 . Correct response should include mentioning of two over-the - counter drugs (i . e . aspirin, cough medicine, allergy medicine, acne medicine, muscle ache relief, appetite suppressor, etc.) 1 - Point 11. No 1 - Point 12. Responses such as going to the beach, a park, a museum, bicycling on bike path, hiking, library, etc. are correct. 1 - Point 13 . FORM A - PART 4 - SHOPPING KEY Appropriate response should include alerting landlord / neighb ors, planning menu, planning guest list and inviting guests, planning music and / or activities, budgeting for party expenses, etc. 1 - Point 14. False 1 - Point 15. Responses should reflect the child's understanding of his rights , the Miranda Warnings and importance of legal counsel prior to making any statements. 1 - Point 16. Any logical difficulty should be recognized as correct. ( i.e. being a crime victim, sticking t o a budget, maintaining a job, maintaining a household, taking responsibilities for your actions, staying away from drugs / alcohol, getting along in a community, taking care of medical issues, self-discipline, etc.) 1 - Point 17. True 1 - Point 1. Correct response should include following list: car, taxi, bus, motorcycle, bicycle. at least four train , plane, items from the boat, walking, 1 - Point 2. Correct response should illustrate that child has knowledge of transit authority, community transportation facilities, etc. Child should know that they can obtain schedules from bus/train stations, i i f i 1 P i 1 - Point 4 . A realistic estimate should be within the range of $800 - $2400 . 1 - Point 5. Correct response should include 1-AMA, 2- Recommendations from a friend/family, 3-Yellow Pages, 4-Clinic, 5-HIP Center, etc. 1 - Point 6. Correct response should include mentioning of the effects of alcohol on an individual's physical, emotional, social functioning , family and intellectual functioning. 1 - Point 6. Correct response should include mentioning of the effects of alcohol on an individual's physical, emotional, social functioning , family and intellectual functioning. 1 - Point 7. Correct response should include seeking help from a substance abuse agency, doctor, therapist, friend, or clergy, etc . 1 - Point . Correct response should include seeking help from a substance abuse agency, doctor, therapist, friend, or clergy, etc . 1 - Point 8 . Correct response should include an adequate knowledge about yellow pages. For example, individual needs to look under a broader category as well as in alphabetical order. demonstration of knowing that an for most services 1 - Point 8 . Correct response should include an adequate knowledge about yellow pages. For example, individual needs to look under a broader category as well as in alphabetical order. 20. A response should be logical and realistic in order to be scored correct. A scorable response would indicate that parents provide shelter, food, medical services, affection, guidance, discipline, and love to their children. 1 - Point FORM A - PART 4 - SHOPPING KEY demonstration of knowing that an for most services 1 - Point 1 - Point 1 - Point ) 1 - Point ) 1 - Point 1 - Point 1 - Point 1 - Point 12. Responses such as going to the beach, a park, a museum, bicycling on bike path, hiking, library, etc. are correct. 1 - Point Responses such as going to the beach, a park, a museum, bicycling on ike path, hiking, library, etc. are correct. 1 - Point 1 - Point 1 - Point 1 - Point 1 - Point 1 - Point 1 - Point 87 18. Responses can include ·condoms, the pill, diaphram, IUD, Sponges, Spermicide, or rhythm method, and abstinence. 1 - Point 18. Responses can include ·condoms, the pill, diaphram, IUD, Sponges, Spermicide, or rhythm method, and abstinence. 1 - Point 18. Responses can include ·condoms, the pill, diaphram, IUD, Sponges, Spermicide, or rhythm method, and abstinence. 1 - Point 19. An adequate description would include 1) painful urination, 2) itching, 3) mucous discharge, 4) pus and/or blood in urine, 5) lymph glands in groin enlarge and become tender, chancre sores on genitalia or mouth region, 6) body rash, 7) hair loss, 8) wart/blisters or 9) finding lice or eggs. 1 - Point 19. An adequate description would include 1) painful urination, 2) itching, 3) mucous discharge, 4) pus and/or blood in urine, 5) lymph glands in groin enlarge and become tender, chancre sores on genitalia or mouth region, 6) body rash, 7) hair loss, 8) wart/blisters or 9) finding lice or eggs. 1 - Point 20. A response should be logical and realistic in order to be scored correct. A scorable response would indicate that parents provide shelter, food, medical services, affection, guidance, discipline, and love to their children. 1 - Point 20. A response should be logical and realistic in order to be scored correct. A scorable response would indicate that parents provide shelter, food, medical services, affection, guidance, discipline, and love to their children. 1 - Point 88 APPENDIX D Items that were omitted from the original inventory because either 95% or 5% of the subjects examined passed them as follows: g y 5% of the subjects examined passed them as follows: p = Category/ V = Test Item Pl = Job Finding Category P2 = Apartment Hunting category P3 = Budgeting Category P4 = Shopping Category PS = Miscellaneous Category P6 = Housecleaning Category P7 = Cooking/Nutrition/Storage of Food Items Deleted Value Fregyency PlV3 0 3 1 191 P1V9 0 1 1 193 PlV17 0 2 1 192 P1V19 0 7 1 187 P2V2 0 185 1 9 P2V9 0 187 1 7 P2V20 0 194 l 0 P3Vl3 0 8 1 186 P3V20 0 3 1 191 P4V2 0 3 1 191 P4V3 0 185 1 9 P4V9 0 2 1 192 P4V10 0 2 1 192 PSVl 0 2 1 192 PSVll 0 5 1 189 PSVl 7 0 5 1 189 0 = Incorrectly l = Correctly Category Percent 1.5 98.5 .s 99.5 1.0 99.0 3.6 96.4 95.4 4.6 96.4 3 . 6 100.0 4.1 95.9 1.5 98.5 1.5 98 . 5 95.4 4 .6 1.0 99.0 1.0 99.0 1.0 99.0 2 .6 97.4 2.6 97.4 p = Category/ V = Test Item Pl = Job Finding Category P2 = Apartment Hunting category P3 = Budgeting Category P4 = Shopping Category PS = Miscellaneous Category P6 = Housecleaning Category P7 = Cooking/Nutrition/Storage of Food 0 = Incorrectly Answered l = Correctly Answered Category 89 Items Deleted Value Frequency Percent P6Vl 0 5 2.6 1 189 97.4 P6V3 0 7 3.6 1 187 96.4 P6V4 0 8 4.1 1 186 95 . 9 P6V5 0 3 1.5 1 191 98.5 P6V6 0 2 1.0 1 192 99.0 P6V16 0 3 1.5 1 191 98.5 P6V18 0 8 4.1 1 186 95.9 P7Vl2 0 5 2.6 1 189 97.4 P7V19 0 4 2 . 1 1 190 97.9 P7V20 0 2 1.0 1 192 99.0 Items Deleted Value Frequency Percent P6Vl 0 5 2.6 1 189 97.4 P6V3 0 7 3.6 1 187 96.4 P6V4 0 8 4.1 1 186 95 . 9 P6V5 0 3 1.5 1 191 98.5 P6V6 0 2 1.0 1 192 99.0 P6V16 0 3 1.5 1 191 98.5 P6V18 0 8 4.1 1 186 95.9 P7Vl2 0 5 2.6 1 189 97.4 P7V19 0 4 2 . APPENDIX D 1 1 190 97.9 P7V20 0 2 1.0 1 192 99.0 90 . 90 . APPENDIX E APPENDIX E Corrected Item - Al:Qha if Value Fregyency Percent Total Correlation Item Deleted PlV4 0 16 8.2 .37 .70 Maintained 1 178 91.8 when Alpha PlV5 0 11 5.7 .32 .70 Maintained 1 183 94.3 when Alpha PlV7 0 17 8.8 .18 • 71 Deleted 1 177 91.2 when Alpha PlV14 0 12 6.2 .26 .70 Deleted 1 182 93.8 when Alpha P2Vl 0 19 9.8 .19 .54 Deleted 1 175 90.2 when Alpha P3V3 0 178 91.8 .23 .73 Deleted 1 16 8.2 when Alpha P3Vl0 0 176 90.7 .22 .73 Deleted 1 18 9 . 3 when Alpha P4Vll 0 16 8.2 .23 .57 Deleted 1 178 91.8 when Alpha PSV7 0 10 5.2 .24 .62 Deleted 1 184 94 . 8 when Alpha PSV13 0 10 5.2 .20 .62 Deleted 1 184 94.8 when Alpha PSV14 0 16 8.2 .18 .62 Deleted 1 178 91.8 when Alpha P5V20 0 10 5.2 . 42 . 60 Maintained 1 184 94 . 8 when Alpha P6V7 0 19 9.8 .39 .83 Maintained 1 175 90.2 when Alpha P6Vll 0 10 5.2 . 41 .83 Maintained 1 184 94.8 when Alpha P6Vl2 0 10 5.2 .28 .83 Deleted 1 184 94.8 when Alpha P6Vl4 0 16 8.2 .43 .83 Maintained 1 178 91.8 when Alpha P7Vl0 0 17 8 .8 .so .90 Maintained 1 177 91.2 when Alpha P7Vll 0 11 5.7 .46 .91 Maintained 1 183 94.3 when Alpha P7Vl3 0 17 8.8 .57 .90 Maintained 1 177 91.2 when Alpha Corrected Item - Al:Qha Value Fregyency Percent Total Correlation Item D .62 when Alpha is .63 .83 when Alpha is . 83 91 APPENDIX F Corrected Item - Items Deleted Total Correlation P1Vl2 . 02 P2V4 .06 P2V6 . 08 P2V7 .10 P2Vll . 01 P3Vll .14 P4V7 .11 P5V4 .01 P5V5 .00 P5Vl5 .11 Alpha if Item Deleted .73 when Alpha is . 71 .57 when Alpha is .56 .57 when Alpha is .56 .56 when Alpha is .56 .58 when Alpha is .56 .74 when Alpha is .74 .59 when Alpha is . 58 .65 when Alpha is .63 .65 when Alpha is . 63 .64 when Alpha is .64 .73 when Alpha is . 71 .73 when Alpha is . APPENDIX D 71 .57 when Alpha is .56 .57 when Alpha is .56 .56 when Alpha is .56 .58 when Alpha is .56 .74 when Alpha is .74 .59 when Alpha is . 58 .65 when Alpha is .63 .65 when Alpha is . 63 92 APPENDIX G INTERNAL CONSISTENCY DATA PRIOR TO ITEM ANALYSIS Three forms of internal consistency analyses were performed on the original data. For all of these analyses, Nunnally's rule of thumb was used which considers internal consistency coefficients of .70 and greater to be indicative of statistical reliability. (Nunnally, 1978). Since the data contains dichotomously - scored items, what is stated as Cronbach's alphas, are actually Kuder-Richardson's #20 coefficients. The first and least successful internal consistency analyses performed looked at each individual test item within sub-categories. In other words, these internal consistency analyses examined whether the individual test items actually tested what the sub-category stated it was testing. Each category was broken down into 4-6 sub-categories a priori based upon face validity, or in other words, the different logical components of the larger subject area. For example, it was predicted that the first Job Search and Maintenance Skills category was comprised of four smaller topic areas. They include: - Job Application Forms and Procedure for Applying for a Job - Job Interviewing Skills - Job Application Forms and Procedure for Applying for a Job - Job Interviewing Skills - Job Interviewing Skills - General Job-Related Knowledge and Terms - Job-Related Behavior - Job-Related Behavior The 20 o riginal i ndi v idual items from the Job Search and Maintenance Skills Category were then respectfully distributed into the sub-category with which they were most similar. The following is a listing of all of the predicted sub-categories of the ILSFA, i n addition t o t he i tems within sub- c ategory internal consistency data. APPENDIX G INTERNAL CONSISTENCY DATA PRIOR TO ITEM ANALYSIS 93 TABLE 21 PRE-ITEM ANALYSIS ITEMS WITHIN SUB-CATEGORY INTERNAL CONSISTENCY DATA Category I - Job Search and Maintenance Skills Job Application Forms and Procedures for Applying for a Job Sub-Category Items PlVl P1V2 PlV8 PlV9 PlVlO Standardized Item Alpha= .48 Job Interviewing Skills Sub-Category Items PlV4 PlVS PlV6 PlV7 Standardized Item Alpha= .62 General Job-Related Kno wledge and Terms Sub-Category Items PlV3 PlVll PlV13 PlVlS PlV16 Standardized Item Alpha= .46 Job Related Behavior Sub-Category Items P1Vl2 P1V14 P1V17 P1Vl8 P1V19 PlV20 Standardized Item Alpha= .13 Category II - Apartment Hunting/Maintenance Procedures for Looking for an Apartment Sub-Categor y Items P2Vl P2V2 P2V3 P2Vl2 Standardized Item Alpha 26 TABLE 21 PRE-ITEM ANALYSIS ITEMS WITHIN SUB-CATEGORY INTERNA Category I - Job Search and Mainten Job Application Forms and Procedures for Applying fo Items PlVl P1V2 PlV8 PlV9 PlVlO Standardized Item Alpha= .48 Job Interviewing Skills Sub-Category Items PlV4 PlVS PlV6 PlV7 Standardized Item Alpha= .62 General Job-Related Kno wledge and Terms Sub-Category Items PlV3 PlVll PlV13 PlVlS PlV16 Standardized Item Alpha= .46 Job Related Behavior Sub-Category Items P1Vl2 P1V14 P1V17 P1Vl8 P1V19 PlV20 Standardized Item Alpha= .13 Category II - Apartment Hunting/Main Procedures for Looking for an Apartment Sub-Categor Items P2Vl P2V2 P2V3 Category II - Apartment Hunting/Maintenance Procedures for Looking for an Apartment Sub-Categor y Items P2Vl P2V2 P2V3 P2Vl2 Standardized Item Alpha .26 94 Knowledge About Initial and Continued Apartment Care Costs Sub-Category Items P2V4 P2V6 P2V7 P2Vll Standardized Item Alpha= .12 Apartment Related Items Sub-Category Items P2VS P2Vl0 P2Vl9 P2V20 Standardized Item Alpha= .44 Utility and Protective Agencies Sub-Category Items P2V8 P2V9 P2V18 Standardized Item Alpha= .09 Apartment Living Sub-Category Items P2V13 P2V14 Standardized Item Alpha= . 39 Apartment Maintenance Sub-Category Items P2Vl5 P2V16 P2V1 7 Standardized Item Alpha= .02 categ or y III - Budgeting Checking Acc ounts Sub-Categ or y Items P3Vl P3V2 P3V3 P3V4 P3VS P3V10 Knowledge About Initial and Continued Apartment Care Costs Sub-Category Items P2V4 P2V6 P2V7 P2Vll Standardized Item Alpha= .12 Apartment Related Items Sub-Category Items P2VS P2Vl0 P2Vl9 P2V20 Standardized Item Alpha= .44 Utility and Protective Agencies Sub-Category Items P2V8 P2V9 P2V18 Standardized Item Alpha= .09 Apartment Living Sub-Category Items P2V13 P2V14 Standardized Item Alpha= . Consumer Terminology and Knowledge About Bargains Sub-Category APPENDIX G INTERNAL CONSISTENCY DATA PRIOR TO ITEM ANALYSIS 39 Apartment Maintenance Sub-Category Items P2Vl5 P2V16 P2V1 7 Standardized Item Alpha= .02 categ or y III - Budgeting Checking Acc ounts Sub-Categ or y Items P3Vl P2Vll Standardized Item Alpha= .12 Apartment Related Items Sub-Category Items P2VS P2Vl0 P2Vl9 P2V20 Standardized Item Alpha= .44 Utility and Protective Agencies Sub-Category Items P2V8 P2V9 P2V18 Standardized Item Alpha= .09 Apartment Living Sub-Category Items P2V13 P2V14 Standardized Item Alpha= . 39 Apartment Maintenance Sub-Category Items P2Vl5 P2V16 P2V1 7 Standardized Item Alpha= .02 categ or y III - Budgeting Checking Acc ounts Sub-Categ or y Items P3Vl P3V2 P3V3 P3V4 P3VS P3V10 Standardized Item Alpha= . 53 Concept of Interest and Sa v in g s Accounts Sub-Category Items P3V7 P3V8 P3Vll P3V20 Standardized Item Alpha .44 categ or y III - Budgeting Concept of Interest and Sa v in g s Accounts Sub-Category Items P3V7 P3V8 P3Vll P3V20 Standardized Item Alpha .44 How to Budget Sub-Category Items P3V6 P3V15 P3V16 P3V17 Standardized Item Alpha= .40 Reading and Understanding Paychecks Sub-Category Items P3V9 P3V14 P3V18 P3V19 Standardized Item Alpha= .36 Credit Buying and Rating Sub-Category Items P3V12 P3V13 Standardized Item Alpha= -.02 Category IV - Shopping Methods and Implications of Comparative Shopping Sub-Category Items P4V2 P4V4 P4V8 P4V13 P4V16 Standardized Item Alpha= .35 Warranties and Consumer"s Rights Sub-Category Items P4Vl P4V3 P4V19 P4V20 Standardized Item Alpha= .36 Unit Pricing and Sales Tax Sub-Category Items P4V7 P4Vl7 Standardized Item Alpha= .02 Advertising Ploys Sub-Category Items P4V6 P4V9 P4V14 Standardized Item Alpha= . APPENDIX G INTERNAL CONSISTENCY DATA PRIOR TO ITEM ANALYSIS 04 95 95 How to Budget Sub-Category Methods and Implications of Comparative Shopping Sub-Category Items P4V2 P4V4 P4V8 P4V13 P4V16 Standardized Item Alpha= .35 Methods and Implications of Comparative Shopping Sub-Category 96 Consumer Terminology and Knowledge About Bargains Sub-Category Items P4VS P4Vl0 P4Vll P4Vl2 P4Vl5 P4Vl8 standardized Item Alpha= .36 Items P4VS P4Vl0 P4Vll P4Vl2 P4Vl5 P4Vl8 standardized Item Alpha= .36 Category V - Miscellaneous Transportation and Car Issues Sub-Category Items PSVl PSV2 PSV3 PSV4 Standardized Item Alpha .31 Alcohol and Drug Issues Sub-Category Items PSV6 PSV7 PSV9 Standardized Item Alpha= .22 Knowledge About Mature Adult Behavior Sub-Category Items PSV16 PSVl 7 PSV20 Standardized Item Alpha= .37 Sex Education Issues Sub-Category Items PSV18 PSV19 Standardized Item Alpha= .53 Medical Issues Sub-Category Items PSVS PSVlO PSVll Standardized Item Alpha= .14 Negotiating Leisure Activities Sub-Category Items PSV8 PSV12 PSV13 Standardized Item Alpha = .40 Medical Issues Sub-Category 97 Knowledge of Emotional/Legal Supports in Community Sub-Category Items PSV14 PSVlS Standardized Item Alpha= .10 Category VI - Housecleaning Fundamentals of Housecleaning Sub-Category Items P6Vl P6V2 P6V3 P6V4 P6V19 Standardized Item Alpha= .66 Use of Cleaning Appliances Sub-Category Items P6VS P6V6 P6V12 Standardized Item Alpha= - . 07 Household Safety Sub-Category Items P6V7 P6V8 P6V17 P6V20 Standardized Item Alpha= .72 Care of Clothing Sub-Category Items P6V9 P6V10 P6Vll P6V13 P6V14 Standardized Item Alpha= .73 Knowledge of Cleaning Products and Accessories Sub-Category Items P6V15 P6V16 P6V18 Standardized Item Alpha . 26 Knowledge of Emotional/Legal Supports in Community Sub-Category Items PSV14 PSVlS Standardized Item Alpha= .10 Category VI - Housecleaning Fundamentals of Housecleaning Sub-Category Items P6Vl P6V2 P6V3 P6V4 P6V19 Standardized Item Alpha= .66 Use of Cleaning Appliances Sub-Category Items P6VS P6V6 P6V12 Standardized Item Alpha= - . 07 Household Safety Sub-Category Items P6V7 P6V8 P6V17 P6V20 Standardized Item Alpha= .72 Care of Clothing Sub-Category Items P6V9 P6V10 P6Vll P6V13 P6V14 Standardized Item Alpha= .73 Knowledge of Cleaning Products and Accessories Sub-Category Items P6V15 P6V16 P6V18 Standardized Item Alpha . APPENDIX G INTERNAL CONSISTENCY DATA PRIOR TO ITEM ANALYSIS 26 Knowledge of Emotional/Legal Supports in Community Sub-Category Household Safety Sub-Category Household Safety Sub-Category 98 category VII - Cooking/Care of Food/Nutrition Nutrition Sub-Category Items P7Vl P7V2 P7V3 P7V4 Standardized Item Alpha= .83 How to Cook and Prepare Food Sub-Category Items P7V14 P7V15 P7V16 P7V18 P7Vl9 P7V20 Standardized Item Alpha= .71 How to Shop for Food Sub-Category Items P7VS P7V6 P7V7 P7V8 P7V9 Standardized Item Alpha= .79 Food Storage Sub-Category Items P7V10 P7Vll P7V12 P7Vl3 P7V17 Standardized Item Alpha= . 78 category VII - Cooking/Care of Food/Nutrition Nutrition Sub-Category Items P7Vl P7V2 P7V3 P7V4 Standardized Item Alpha= .83 How to Cook and Prepare Food Sub-Category Items P7V14 P7V15 P7V16 P7V18 P7Vl9 P7V20 Standardized Item Alpha= .71 How to Shop for Food Sub-Category Items P7VS P7V6 P7V7 P7V8 P7V9 Standardized Item Alpha= .79 Food Storage Sub-Category Items P7V10 P7Vll P7V12 P7Vl3 P7V17 Standardized Item Alpha= . 78 category VII - Cooking/Care of Food/Nutrition As can be gleaned from the above Table, with the exception of the Cooking/Care of Food/Nutrition category, the internal consistency coefficients obtained from the remaining categories were consistently low. The original inventory's 7 categories were initially broken down into smaller sub-categories because it would not be statistically sound for dichotomous items to be used in statistical analyses. This indicates that the e x aminer's apriori choices about distributing individual items into smaller sub-categories were incorrect. It is possible that the individual items do not hang together within each category at all, and therefore should not be grouped together. However, it is also possible that the 99 examiner's decisions about which individual items should be grouped together to form specific sub-categories were erroneous, and therefore largely contributed to the low Standardized Item Alphas obtained . The following is an internal consistency analysis of items within categories. In other words, each of the seven major categories will be examined in terms of determining how well their 20 individual sub-items that comprise them hang together. PRE-ITEM ANALYSIS ITEM WITHIN CATEGORY INTERNAL CONSISTENCY DATA PRE-ITEM ANALYSIS ITEM WITHIN CATEGORY INTERNAL CONSISTENCY DATA Category 1 PlVl - P1V20 = Job Search and Maintenance Skills Category Standardized Item Alpha= .72 Category 1 PlVl - P1V20 = Job Search and Maintenance Skills Category Standardized Item Alpha= .72 Category 2 P2Vl - P2V20 = Apartment Hunting/Maintenance Category Standardized Item Alpha= .57 Category 3 P3Vl - P3V20 = Budgeting Category Standardized Item Alpha= .73 Category 4 P4Vl - P4V20 = Shopping Category Standardized Item Alpha= . 59 Category 5 P5Vl - P5V20 = Miscellaneous Category Standardized Item Alpha= .67 Category 6 P6Vl - P6V20 = Housecleaning Category Standardized Item Alpha= .84 Category 7 P7Vl - P7V20 = Cooking/Care of Food/Nutrition Category Standardized Item Alpha= .91 Category 7 P7Vl - P7V20 = Cooking/Care of Food/Nutrition Category Standardized Item Alpha= .91 The above findings suggest that Categories 1, 3, 6, and 7 have acceptable internal consistency coefficients according to Nunnally's .70 rule of thumb. This similarly indicates that the significantly lower findings associated with the items within sub-category data resulted from the erroneous a priori distribution of items into small sub-category groups. The final internal consistency analysis performed on the original data, which contains 7 main categories that are comprised of 20 individual 100 items each, examined each sub-category within each category. In other words, these standardized item alphas (Kuder-Richardson #20) are based upon the average correlation of all the sub-categories with one another within a particular category . TABLE 23 PRE-ITEM ANALYSIS SUB-CATEGORY WITHIN CATEGORY INTERNAL CONSISTENCY DATA PRE-ITEM ANALYSIS SUB-CATEGORY WITHIN CATEGORY INTERNAL CONSISTENCY DATA Within the Job Search and Maintenance Skills Category there are 4 sub-categories. They include: - Job Application Forms and Procedures for Applying for a Job - Job Application Forms and Procedures for Applying for a Job Job Interviewing Skills - Job Interviewing Skills - General Job-Related Knowledge and Terms - Job-Related Behavior - Job-Related Behavior Standardized Item Alpha= .66 Within the Apartment Hunting/Maintenance Category there are 6 sub- categories. They include: categories. They include: - Procedures for Looking for an Apartment - Knowledge about Initial and Continued Apartment Care Costs - Apartment - Related Issues - Utility and Protective Agencies - Apartment Living - Apartment Maintenance Standardized Item Alpha= .56 Within the Budgeting Category there are 5 sub-categories. PRE-ITEM ANALYSIS ITEM WITHIN CATEGORY INTERNAL CONSISTENCY DATA They include: - Checking Accounts - Concept of Interest and Savings Accounts - How t o Budget - How t o Budget - Reading and Understanding Pay Checks - Credit Buying and Rating Standardized Item Alpha= .73 101 Within the Shopping Category there are 5 sub-categories. They include: - Methods and Implications of Comparative Shopping - Warranties and Consumer's Rights - Unit Pricing and Sales Tax - Advertising Ploys - Consumer Terminology and Knowledge About Bargains Standardized Item Alpha= .52 Within the Miscellaneous Category there are 7 sub-categories. They include: include: - Transportation and Car Issues - Alcohol and Drug Issues - Knowledge About Mature, Adult Behavior - Sex Education Issues - Medical Issues - Negotiating Leisure Activities Knowledge of Emotional/Legal Supports in Community Standardized Item Alpha= .61 Knowledge of Emotional/Legal Supports in Community Standardized Item Alpha= .61 Within the Housecleaning Category there are 5 sub-categories. They include: include: - Fundamentals of Housecleaning - Use of Cleaning Appliances - Household Safety - care of Clothing - Knowledge of Cleaning Products and Accessories standardized Item Alpha= .85 102 Within the Cooking/Care of Food/Nutrition Category there are 4 sub- categories . They include: categories . They include: - Nutrition - How to Cook and Prepare Food - How to Shop for Food - Food Storage Standardized Item Alpha= .86 With regard to the sub-categories within category internal consistency analysis, there are three acceptable internal consistency coefficients according to Nunnally's .70 rule of thumb. This includes the internal consistency data obtained for categories 3,6, and 7. Inconsistent with the internal consistency data obtained by examining items within categories , the internal consistency data obtained by examining sub-categories within categories did not suggest acceptable internal consistency for the Job Search and Maintenance Skills Category ( . 66) . FACTOR ANALYSIS ON PRE-ITEM ANALYSIS DATA The next data analysis that was performed on the original data of 7 categories with 20 items each was a factor analysis. First, a Principal Component Analysis was performed with the sub-categories f o r each item. The results of these factor analysis were marginally promising in terms o f tapping into one major component in all of the 7 categories. TABLE 24 PCA PERFORMED ON ALL 7 CATEGORIES PRE-ITEM ANALYSIS A PERFORMED ON ALL 7 CATEGORIES PRE-ITEM ANALYSIS Category 1 Eigenvalue 2.02 .83 . 7 1 .44 Pct. o f Var. 50 . 4 20.8 17 . 7 11.1 Cum. Pct . 50.4 71.2 88.9 100.0 Thus, there appears to be only one significant component within the Job Finding / Maintenance Category Finding / Maintenance Category. The data concerning how much variance is predicted by this factor 103 Sub-Category 1 2 3 4 Communality • 69 .39 .58 .36 Category 2 Eigenvalue 1.96 1.02 . 87 .80 • 71 .65 Pct. of Var. 32.7 16.9 14.5 13.3 11.8 10.8 Cum. Pct. 32.7 49.6 64.1 77 . 1 89 . 2 100.0 Thus, there appear to be two major components operating within the ' Apartment Hunting/Maintenance Category. However, the first component is much stronger and meaningful than the second. Sub-Category 1 2 3 4 5 6 Communality .41 .93 .48 .so .28 .37 Category 3 Eigenvalue Pct. of Var. 47 . 9 16.1 14.5 12.8 2.39 . 80 .73 .64 .44 8.8 Cum. Pct. 47.9 64 . 0 14.5 12.8 8.8 Thus, there appears to be one primary component within the Budgeting Thus, there appears to be one primary component within the Category. Sub-Category 1 2 3 4 5 Communality .56 .38 . 49 .39 .58 Category 4 Eigenvalue 1.75 1.02 . 89 • 72 .63 Pct. of Var. 35.0 20.3 17.8 14.3 12.6 Cum. Pct. 35.0 55.3 73.1 87.4 100.0 Thus, the factor analyses suggest that there are t wo components 104 operating within the Shopping Category. However, as wi th the Apartment Hunting Category, the first component is markedly stronger and accounts for more of the variance than the second. operating within the Shopping Category. However, as wi th the Apartment Hunting Category, the first component is markedly stronger and accounts for more of the variance than the second. Communality .53 .so .48 •. 79 .47 Sub-Category 1 2 3 4 5 Communality .53 .so .48 •. 79 .47 category 5 Eigenvalue 2.19 1.09 .95 . 79 . 72 .70 .55 Pct. of Var. 31.3 15 . S 13.6 11.3 10.3 10.0 7.9 Cum. Pct. 31.3 46.9 60.5 71.7 82. 1 92.1 100.0 Thus, there appear to be two components operating within Thus, there appear to be two components operating within the Miscellaneous Category . However, the first component is stronger and accounts for more of the variance than the second. A PERFORMED ON ALL 7 CATEGORIES PRE-ITEM ANALYSIS pp p p g Miscellaneous Category . However, the first component is stronger and accounts for more of the variance than the second. Sub-Category 1 2 3 4 5 6 7 Communality .68 .40 .53 . 43 .17 .45 .60 Category 6 Eigenvalue 3.17 .79 .60 .33 .11 Pct. of Var. 63.3 15.8 12.1 6 . 6 2.3 Cum. Pct. 63.3 79.1 91.1 97 . 7 100.0 Thus, there appears to be one major component operating within the Budgeting Category. Sub -Categ ory 1 2 3 4 5 Communality .so .26 .42 .69 1.00 105 Category Eigenvalue Pct. of Var. Cum. Pct. 7 2.85 71.2 71.2 .53 13.3 84.5 .34 8.4 92.9 . 28 7.1 100.0 Thus, there appears to be one main component within the Cooking/care of Food/Nutrition Category. Sub-Category 1 2 3 4 Communality .68 .78 .76 .63 With the aforementioned data, the latent root criterion (Eigenvalue) was used to determine how many factors should be considered. (Hair, Anderson, and Tatham, 1987). However, it should be noted that Eigenvalues that are close to 1 are not very high, and are therefore n ot suggestive of clear-cut factors . In addition, the percentage of variance accounted for by these factors should be considered in determining the presence of actual factors. The next statistical data that was examined is the Principal Component Analysis (PCA) of all of the ILSFA • s sub-categories. An orthogonal rotation (Varimax) was employed to achieve the simplest and theoretically most meaningful factor pattern. (Hair , Anderson, and Tatham, 1987). TABLE 25 PCA PERFORMED ON ALL SUB-CATEGORIES PRE-ITEM ANALYSIS The data are as follows: Components that contribute up to 70% of the ILSFA's cumulative variance wi ll be listed. The remaining factors are quite small and therefore meaningless. Factor 1 2 3 4 5 6 Eigenvalue 8.18 4.46 1.56 1.54 1.31 1.26 Pct. of Var. 22.7 12. 4 4.3 4.3 3.6 3.5 Cum. p·ct . 22 . 7 35.1 39.5 43.7 47.4 50.9 106 Factor Eigenvalue Pct. of var. Cum. Pct. 7 1.21 3.4 54.2 8 1.06 3.0 57.2 9 1.05 2.9 60.1 10 1.00 2.8 62.9 11 .93 2.6 65.5 12 .91 2.5 68.0 13 .87 2.4 70.4 According to the latent root criterion, 10 components would be extracted since 10 components have eigenvalues over 1. However, the a priori criterion was utilized in determining the number of factors to be extracted since it is deemed to be an appropriate method when a hypothesis about the number of components to be extracted is being tested. (Hair, Anderson, Tatham, 1987) . Noteworthy is the fact that the Guttman-Kaiser rule has been shown to be faulty and leads to over-factoring. The Cattell Scree plot is said to reveal more "real" and meaningful components. In this case, the Scree Chart reveals 2 real components . In addition, in this case the scree test criterion for the number of components to be extracted does not coincide with the number of factors extracted by using the a priori criterion. (See Figure 2) 107 Figure 2 Figure 2 Cl) Q.) ::J ctS > C Q.) 0) ·-w Figure 2 9.00 ,---------- - -------- - - --- --, 8.18 8.00 7.00 6.00 5.00 4. 5 4.00 3.00 2.00 1.00 1.31 0.46 Pre-item Analysis Scree Chart 108 As can be seen from the eigenvalues of this Principal Component Analysis, there appears to be one very significant component, and one second strong, but lesser component, operating in the ILSFA. These findings shall be interpreted in the discussion section of this paper. However, it should be remembered that the PCA performed on the I LSFA following the item analysis is likely to generate a much clearer and meaningful factor pattern. Table 26 presents the rotated factor matrix that can be used to reflect the contents of the various 7 dimensions. PCA PERFORMED ON ALL SUB-CATEGORIES PRE-ITEM ANALYSIS Rotated Factor Matrix: FACTOR FACTOR 2 FACTOR 3 FACTOR 4 FACTOR 5 FACTOR 6 FACTOR 7 P6C5 .85 .27 P7C2 .84 P6C4 .81 P7C4 .80 P7C3 .80 P7C1 .78 P6C1 .77 P6C3 .74 .28 P1C3 .72 P3C1 .72 P3C5 .68 P3C3 .64 P2C3 .60 P1C1 .60 .39 .33 P4C2 .57 P3C4 .57 P4C5 .48 P1C4 .45 -.34 P3C2 .42 .40 .29 P1C2 .73 P5C3 .62 .28 P2C1 .35 .55 P5C6 .49 .32 .28 P5C2 .30 .40 P4C4 .65 P4C1 .31 .58 P5C5 .55 .32 .27 P2C5 .37 .43 P6C2 .36 .61 P2C4 .31 .27 .56 P5C4 .31 .44 P2C6 .27 .31 - .30 P4C3 .29 .69 P5C1 .27 .48 P5C7 .32 .28 - .47 P2C2 .75 109 Factor Transformation Matrix: FACTOR 1 FACTOR 2 FACTOR 3 FACTOR 4 FACTOR 5 FACTOR 1 . 54 .66 . 38 .25 .2 5 FACTOR 2 - .83 .44 .28 . 18 . 04 FACTOR 3 .04 -.24 . 59 - .31 -. 13 FACTOR 4 . 00 - . 47 .14 .84 .17 FACTOR 5 . 00 . 23 - .61 .15 .09 FACTOR 6 .OS .18 - .16 . 13 - .58 FACTOR 7 -.09 -.07 - .12 . 26 .74 110 BIBLIOGRAPHY Anastasi, A. (1982). Macmillan. Psychological Testing ( 5th Ed.) New York: Ansell, D. and the Independent Living Skills Center (1987). Lif Skill I t R t f Y th i F t C N Y k Ansell, D. and the Independent Living Skills Center (1987). Life Skills Inventory Report for Youth in Foster Care New York: Author Ansell, D. and the Independent Living Skills Center (1987). Life Skills Inventory Report for Youth in Foster Care New York: Au Barden, J.C. (1991, January 5). After Release from Foster Care, Many Turn to Lives on the Streets. The New York Times p.1. Castellani, J. (1988). Assessment Instruments Services. An Annotation and Analysis of Eleven Life Skill New York: New York State Department of Social Child Welfare League of America (1978). Standards for Group Home Services to Children New York: Child Welfare League of America. Cohen, A.K . (1955). Delinquent Boys : The Culture of the Gang. Illinois: The Free Press. Easson, W.M. (1969). The Severely Disturbed Adolescent: Inpatient, Residential and Hospital Treatment . New York: International University Press. Fe stinger, T. ( 1983) . University Press. No One Ever Asked Us . New York: Columbia Goffman, E. (1961). Asylums . PCA PERFORMED ON ALL SUB-CATEGORIES PRE-ITEM ANALYSIS New York: Doubleday Anchor Books. Hair, J., Anderson, R. and Tatham, R. (1987) . Multivariate Data Analysis New York: MacMillan. Halpern B., Irvin, L., and Landman, J. (1979) Tests for Everyday Living California: McGraw Hill. Hylton, L. F. (1964) The Residential Treatment Center - Children, Programs, and Costs New York: Child Welfare League of America. Johnson, S. ( 1982). Residential Treatment for Emotionally Disturbed Children and Adolescents: A review of the literature. Canada's Mental Health, 1, 5-8. Kerlinger, F. N. (1973). Foundations of Behavioral Research New York: Holt, Rinehart, and Winston. Nunnally, J.C. ( 1978). Psychometric Theory New York: McGraw Hill. Polsky, H. W. (1959) . Psychological Approach . Changing Delinquent Subcultures: Social Work, i, 3-15. A Social Schaefer, c. (1980). Treatment of Youth. The Impact of the Peer Culture in the Residential Adolescence, 15, 832-845. Simonitch, B. and Anderson, J . (1979). On Their Own. Children Today . Stone, Living H. D. ( 19 8 7) . Re adv, Set Go - An Agency Guide to Independent Washington, D.C.: Child Welfare League of America.
https://openalex.org/W2162328609	https://europepmc.org/articles/pmc3429492?pdf=render	English	null	LPS-Induced Lung Inflammation in Marmoset Monkeys – An Acute Model for Anti-Inflammatory Drug Testing	PloS one	2,012	cc-by	9,205	Abstract The Fraunhofer ITEM is a public non-profit research organisation doing contract research for e.g ech industry. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing Interests: Fraunhofer ITEM is a public non-profit research organisation doing contract research for e.g. pharmaceutical and biotech industry. The institution of JMH has received research grants from AstraZeneca, Novartis, Nycomed, and Pfizer to conduct clinical trials using LPS-induced inflammation. Encepharm is a research organisation doing contract research for e.g. pharmaceutical and biotech industry. The institution has received no grants to conduct preclinical trials using LPS-induced inflammation in marmoset monkeys. Encepharm confirms that this does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. CF declares affiliation to the company Klinikum Region Hannover GmbH. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: sascha.knauf@item.fraunhofer.de (SK); katherina.sewald@item.fraunhofer.de (KS) . These authors contributed equally to this work. . These authors contributed equally to this work. These inflammatory aspects of cytokine up-regulation can also be mimicked in in-vitro, ex vivo, as well as in vivo approaches by using infectious or environmental stimuli [4–8]. Especially the endotoxin lipopolysaccharide (LPS), which is part of the outer membrane of gram-negative bacteria, is one of the most potent immune- activating stimuli known. LPS induces a profound activation of the innate immunity via CD14 and Toll-like receptor (TLR) 4 that results in a strong inflammatory response due to activation of the transcription factor NF-kB [9;10]. LPS is, therefore, widely used to model features of inflammatory diseases in vitro as well as in vivo. LPS-Induced Lung Inflammation in Marmoset Monkeys – An Acute Model for Anti-Inflammatory Drug Testing Sophie Seehase1,2, Hans-Dieter Lauenstein1,2, Christina Schlumbohm3, Simone Switalla1, Vanessa Neuhaus1, Christine Fo¨ rster4, Hans-Gerd Fieguth5, Olaf Pfennig4, Eberhard Fuchs3, Franz- Josef Kaup2, Martina Bleyer2, Jens M. Hohlfeld1, Armin Braun1, Katherina Sewald1., Sascha Knauf1,2. 1 Airway Research, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany, 2 Pathology Unit, German Primate Center, Leibniz-Institute for Primate Research, Go¨ttingen, Germany, 3 Encepharm GmbH, Go¨ttingen, Germany, 4 Institute of Pathology, Klinikum Region Hannover Klinikum Nordstadt, Hannover, Germany, 5 Division of Thoracic Surgery, Klinikum Region Hannover Klinikum Oststadt-Heidehaus, Hannover, Germany LPS-Induced Lung Inflammation in Marmoset Monkeys – An Acute Model for Anti-Inflammatory Drug Testing Sophie Seehase1,2, Hans-Dieter Lauenstein1,2, Christina Schlumbohm3, Simone Switalla1, Vanessa Neuhaus1, Christine Fo¨ rster4, Hans-Gerd Fieguth5, Olaf Pfennig4, Eberhard Fuchs3, Franz- Josef Kaup2, Martina Bleyer2, Jens M. Hohlfeld1, Armin Braun1, Katherina Sewald1., Sascha Knauf1,2. 1 Airway Research, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany, 2 Pathology Unit, German Primate Center, Leibniz-Institute for Primate Research, Go¨ttingen, Germany, 3 Encepharm GmbH, Go¨ttingen, Germany, 4 Institute of Pathology, Klinikum Region Hannover Klinikum Nordstadt, Hannover, Germany, 5 Division of Thoracic Surgery, Klinikum Region Hannover Klinikum Oststadt-Heidehaus, Hannover, Germany August 2012 \| Volume 7 \| Issue 8 \| e43709 tation: Seehase S, Lauenstein H-D, Schlumbohm C, Switalla S, Neuhaus V, et al. (2012) LPS-Induced Lung Inflammation in Marmoset Mon Anti-Inflammatory Drug Testing. PLoS ONE 7(8): e43709. doi:10.1371/journal.pone.0043709 Abstract Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro- inflammatory cytokines including tumor necrosis factor-alpha (TNF-a) and macrophage inflammatory protein-1 beta (MIP- 1b) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-a, and MIP-1b. TNF-a release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-a secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC50). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-a and MIP-1b levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs. Citation: Seehase S, Lauenstein H-D, Schlumbohm C, Switalla S, Neuhaus V, et al. (2012) LPS-Induced Lung Inflammation in Marmoset Monkeys – An Acute Model for Anti-Inflammatory Drug Testing. PLoS ONE 7(8): e43709. doi:10.1371/journal.pone.0043709 Editor: Marco Idzko, University Hospital Freiburg, Germany Received January 5, 2012; Accepted July 25, 2012; Published August 28, 2012 Copyright: 2012 Seehase 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. nding: The work was funded by the Fraunhofer Society. The Fraunhofer ITEM is a public non-profit research organisation doing co armaceutical and biotech industry. The funders had no role in study design, data collection and analysis, decision to publish, or prepara funded by the Fraunhofer Society. LPS-Induced Lung Inflammation in Marmoset Monkeys Moreover, the cages were cleaned in weekly intervals and disinfected with water and Biguacid (Antiseptica, Polheim/ Brauweiler, Austria). Room temperature was maintained at 2661.5uC and the relative humidity was kept between 60 and 80%. Artificial light was set to give a cycle of 12 hours light and 12 hours darkness. Room air was changed approximately 8 times per hour and filtered adequately. All materials were changed regularly, cleaned and sterilized. In order to define hygiene status of the colony and to exclude infection with enteral pathogens, faecal samples were collected from the animal housing rooms for bacteriological analysis every three months. Animals received water and a pelletized marmoset diet (ssniffH Spezialdia¨ten GmbH, Soest, Germany) ad libitum. Water quality was controlled on a regular basis. they do not reflect all features of human, notably chronic, respiratory diseases. So far, corticosteroids have widely been used for the anti-inflammatory treatment of inflammatory lung diseases. However, they provide only little benefit in disease progression or mortality [13;14]. Thus, development of new, effective anti- inflammatory drugs is urgently needed. Particularly, antagonists or inhibitors targeting the mechanisms involved in recruitment and accumulation of inflammatory cells, including neutrophils, display promising options for therapeutic intervention in lung inflamma- tion [15]. In fact, a first success has been achieved with the highly potent phosphodiesterase-4 (PDE4) inhibitor roflumilast [11;16], which was extensively tested on acute respiratory LPS challenge models [4;8;11]. Preclinical testing of highly specific anti-inflammatory drugs requires valid translational animal models [17]. Yet, rodent models have commonly been used, even though they are often limited in reflecting the human pathology [18;19]. Rodents in contrast to non-human primates (NHP) or humans have less extensive airway branching and normally do not have respiratory bronchioles [19]. The close phylogenetic relationship between NHP and humans, and the resulting high homology to a variety of human target structures make NHP interesting for preclinical testing of newly developed drugs [18]. The New World monkey common marmoset (Callithrix jacchus), for instance, is widely used as a model of human inflammatory diseases of the central nervous system, such as experimental autoimmune encephalomyelitis (EAE) to model multiple sclerosis [20;21]. Because of its phylogenetic proximity to humans and its small body size of about 400 grams the marmoset monkey seems to be of high value for modeling human respiratory diseases. Furthermore, marmoset monkeys have already been used for inhalation studies [22;23]. Culture Media and Reagents Dulbecco’s Modified Eagle’s Medium Nutrient Mixture F-12 Ham (DMEM) with L-glutamine, 15 mM HEPES, pH 7.2–7.4 without phenol red and foetal calf serum was complemented with Table 1. Demographic data of the study population. Table 1. Demographic data of the study population. sham (n = 8) dxm (n = 7) rof (n = 7) Median age [years]4.060.6 3.960.3 4.460.1 Body weight [g] 440651 409625 429657 Sex, n Male 4 3 2 Female 4 4 5 Animal number #13901 a #13958 a #13859 a #14374 a #13763 a #13866 a #13952 a #13853 a #13605 a #14168 a #14168 b #13763 b #14299 a #13866 b #13863 b #13859 b #13853 b #13958 b #14374 b #12785 b #11595 b #13564 b Excluded #13605 b #13863 a #13526 a Animals were randomized in each of the two independent study cycles as indicated (a: first study cycle, b: second study cycle). Before each cycle a baseline BAL was performed 3 weeks before LPS challenge and served as control. Altogether, 3 animals had to be excluded. Data are given as mean 6 S.E.M., dxm = dexamethasone, rof = roflumilast. doi:10.1371/journal.pone.0043709.t001 LPS-Induced Lung Inflammation in Marmoset Monkeys The experiments were approved by the Lower Saxony Federal State Office for Consumer Protection and Food Safety, Germany (reference number AZ 33.11.42502-04-10/0032). All procedures, except oral administration of dexamethasone or roflumilast or sham, and blood withdrawal, were performed in anesthetized animals. Anesthesia was performed using a combination of AlfaxanH (Ve´toquinol, Ravensburg, Germany; 12–18 mg alfax- alon/kg i.m.), DiazepamH (Ratiopharm, Ulm, Germany; 0.5– 1.5 mg diazepam/kg i.m.), and RobinulH (Riemser, Greifswald, Germany; 0.02–0.05 mg glycopyrronium bromide/kg s.c.) [24]. Animals used for ex vivo experiments were euthanized during general anesthesia with sodium pentobarbital (NarcorenH, Merial GmbH, Hallbergmoos, Germany; 400 mg kg/bw i.v.) according to EU Guideline 2010/63/EU. Table 1 shows the animals used for the in vivo experiments. Lungs for ex vivo studies were used from animals with an average age of 662 years. All of them were part of control groups and were not pre-treated with any substances. The rationale of the present study was to establish a tired translational LPS-induced acute lung inflammation model in the marmoset monkey for preclinical testing of anti-inflammatory drugs. Both, an ex vivo and in vivo approach of LPS-induced acute inflammation were used to reflect inflammatory lung diseases. Firstly, we investigated whether marmoset precision-cut lung slices (PCLS) display a similar inflammatory response upon LPS exposure as seen in human PCLS studies [7]. Secondly, we analyzed the effect of an acute unilateral LPS challenge in marmoset monkeys. The study was designed close to a clinical trial conducted by our Clinical Airway Research department, where segmental LPS challenge was performed in healthy subjects after roflumilast treatment [11]. By using the PDE4 inhibitor roflumilast and for control the corticosteroid dexamethasone we investigated the therapeutic efficacy of immunosuppressive drugs ex vivo and in vivo against the acute LPS-induced inflammatory response. Introduction Inflammatory lung diseases including pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD) cause significant morbidity and mortality worldwide and display a major public health impact [1;2]. On cellular level, these respiratory diseases are based on inflammation which can be either acute or chronic. The inflammatory process is characterized by an increased expression of multiple cytokines and chemokines. In particular, activated macrophages and epithelial cells produce inflammatory mediators such as tumor necrosis factor alpha (TNF-a) and interleukin-1 beta (IL-1b) which in turn induce the attraction of neutrophils and the release of further cytokines including IL-6 [3]. Acute respiratory LPS challenge models in animals as well as in humans which are characterized by bronchoalveolar neutrophil influx and cytokine up-regulation, have extensively been used for the testing of new anti-inflammatory drugs [4;8;11;12], although August 2012 \| Volume 7 \| Issue 8 \| e43709 August 2012 \| Volume 7 \| Issue 8 \| e43709 1 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org LPS-Induced Lung Inflammation in Marmoset Monkeys LPS-Induced Lung Inflammation in Marmoset Monkeys 7.5% (w/v) sodium bicarbonate (all Sigma Aldrich, Mu¨nchen, Germany). DMEM and RPMI 1640 (Lonza, Verviers, Belgium) were supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma Aldrich). LPS (E. coli, serotype 0111:B4) was supplied in lyophilized form by Sigma Aldrich and dissolved in PBS (Lonza), pH 7.4. 7.5% (w/v) sodium bicarbonate (all Sigma Aldrich, Mu¨nchen, Germany). DMEM and RPMI 1640 (Lonza, Verviers, Belgium) were supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma Aldrich). LPS (E. coli, serotype 0111:B4) was supplied in lyophilized form by Sigma Aldrich and dissolved in PBS (Lonza), pH 7.4. collected after 24 hours of incubation, supplemented with 0.2% protease inhibitor cocktail, and stored at 280uC until further analysis. Moreover, PCLS were lysed with 1% Triton X-100 in PBS (1 h, 4uC) for measurement of intracellular cytokine levels. For the purpose of standardization the cytokine amount per approach was calculated in relation to the protein content. Human Donors Marmosets were health-screened in advance to study-start. Screening included an initial haemogram, blood chemistry, and bronchoalveolar lavage (BAL). The initial BAL served as an individual baseline control for each animal. After a recovery time of at least three weeks, animals were orally pre-treated with roflumilast (7 mg/kg bw) on five consecutive days according to Figure 1. Additionally, pre-treatment with dexamethasone (2 mg/ kg bw) served as treatment control, while animals receiving water served as positive control (sham). Roflumilast or dexamethasone were dissolved in 300 mL water and mixed with 700 mL of a nutritional substitution (Nutri-CalH, Albrecht, Aulendorf, Germany) to encourage unsolicited uptake by the animals. On day 5, animals were anaesthetized and received unilateral LPS administration, after a bronchoscope had been placed into the left bronchus under video control. A MicroSprayerH (PennCentury, Philadelphia, PA, USA) was inserted through the working channel of the bronchoscope and an aerosol of 100 mL saline (0.9% NaCl, Braun, Melsungen, Germany) containing a total dose of 500 ng LPS was administered. Eighteen hours later, animals were anaesthetized again and underwent another bronchoscopy for collection of BAL fluid from the challenged lung lobe. The experiments performed with human tissue were approved by the ethics committee of the Hannover Medical School. Patients gave written informed consent. Human lung lobes were obtained from patients who underwent lobectomy because of cancer. Only tumor-free lung tissue was used. Tissue was processed immediately on the day of resection. Table 2 characterizes the human lung tissue donors. The average age of patients was 60610 years (male and female), and 80% of them were smokers. Whole-blood Assay (WBA) Marmoset heparinised whole-blood was obtained from the German Primate Center. Whole-blood was incubated with RPMI (diluted to 1:10) containing the indicated concentrations of LPS under standard cell culture conditions (37uC, 5% CO2, and 95% humidified atmosphere). Dexamethasone (Ratiopharm, Ulm, Germany) was used for positive treatment control. Whole-blood cultures without addition of substances served as reference. After 24 hours of incubation, whole-blood cultures were centrifuged. Whole-blood culture supernatants were subsequently collected, supplemented with 0.2% protease inhibitor cocktail (Sigma Aldrich), and stored at 280uC for further analysis. BAL fluid was processed and analyzed for total and differential cell counts as well as cytokine levels. A third BAL was performed after a minimum of three weeks post LPS instillation to exclude long-term effects. The study was performed twice with an interval of 12 months to check reproducibility and aiming to reduce the number of animals. In each study cycle a baseline BAL representing baseline control was performed 3 weeks before LPS challenge. Furthermore, a power analysis was used to enhance the validity of the study. Animals in both cycles were randomized enabling the pooled analysis of both study cycles (Tab. 1). Animals Experiments were performed in adult common marmoset monkeys (Callithrix jacchus) at the German Primate Center, Goettingen, Germany. Animal care and housing conditions complied with the regulations of the European Parliament and the European Council Directive on the protection of animals used for scientific purposes (2010/63/EU) and also with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Briefly, animals were housed in pairs in standardized and commercially available wire mesh cages (cage size 806656150 cm, Bioscape, Castrop-Rauxel, Germany). In each cage a sleeping box and wooden sitting boards were installed. The floor under the cages was covered by paper sheets. Urine and faeces were removed daily by exchange of the paper sheets. August 2012 \| Volume 7 \| Issue 8 \| e43709 PLOS ONE \| www.plosone.org 2 LPS-Induced Lung Inflammation in Marmoset Monkeys Preparation of Precision-cut Lung Slices (PCLS) Preparation of Precision cut Lung Slices (PCLS) Human and marmoset PCLS were prepared as previously described [7;25]. Briefly, lungs or lung lobes were filled with warm (37uC), liquid, low gelling temperature agarose (1.5% (w/v), Sigma Aldrich) in DMEM. The agarose-filled tissue was allowed to solidify on ice. Tissue cores were prepared with a rotating sharpened metal tube (diameter 8 mm) and cut into sections of approximately 250 mm with a microtome (Krumdieck tissue slicer, Alabama Research and Development, Munford, AL, USA) in Earle’s Balanced Salt Solution (Sigma Aldrich). Thus, approxi- mately 300 PCLS per specimen were produced. PCLS were incubated in DMEM containing different concentrations of LPS (2.5 to 500 ng/mL LPS) under standard cell culture conditions. Additionally, PCLS were incubated with 0.6 to 310 nM roflumilast (DaxasH, Nycomed, Konstanz, Germany) and 100 ng/mL LPS to determine the half maximal inhibitory concentration (IC50) of roflumilast. Untreated PCLS were used as reference, dexamethasone-treated PCLS (50 mg/mL) served as treatment control. Approaches were performed in duplicates containing four PCLS of one specimen. Supernatants were Bronchoscopic Procedure for BAL Sample Retrieval p p Marmosets were food and water deprived for at least three hours prior to anesthesia. Animals were anesthetized as described above and placed into dorsal recumbency. The animal’s head was slightly tilted towards the neck. Body temperature was maintained at 37uC utilizing a heating pad. The mouth was opened with a 75- mm laryngoscope spatula (Heine Classic+H Miller 00 F.O., Herrsching, Germany), while larynx and trachea were brought into a horizontal line. Medical-silicon spray (Servoprax, Wesel, Germany) was carefully used to lubricate the distal part of the Table 2. Characterization of human lung tissue donors. Table 2. Characterization of human lung tissue donors. Donor Age Sex Location Smoke status Classification Cancer Type # 1 66 Female Left inferior lobe – – Adenocarcinoma #2 63 Male Right inferior lobe 20 py GOLD II Basaloid squamous cell carcinoma #3 65 Female Right inferior lobe 50 py GOLD I Squamous cell carcinoma #4 65 Male Medial lobe 40 py GOLD II Adenocarcinoma py = pack-years of smoking. doi:10.1371/journal.pone.0043709.t002 PLOS ONE \| www.plosone.org 3 August 2012 \| Volume 7 \| Issue 8 \| e43709 LPS-Induced Lung Inflammation in Marmoset Monkeys Figure 1. Study design of LPS-induced lung inflammation in marmoset monkeys. Animals were stimulated intrabronchially with 500 ng LPS instilled unilaterally after 5-day oral pre-treatment with either roflumilast (7 mg/kg bw) or dexamethasone (2 mg/kg bw). Sham-treated individuals receiving water were used as positive controls. Unilateral bronchoalveolar lavage (BAL) was performed 18 hours after LPS provocation. BAL fluid was analyzed for neutrophils, TNF-a and MIP-1b secretion. Baseline levels were determined three weeks before the first treatment and served as control. doi:10.1371/journal.pone.0043709.g001 Figure 1. Study design of LPS-induced lung inflammation in marmoset monkeys. Animals were stimulated intrabronchially with 500 ng LPS instilled unilaterally after 5-day oral pre-treatment with either roflumilast (7 mg/kg bw) or dexamethasone (2 mg/kg bw). Sham-treated individuals receiving water were used as positive controls. Unilateral bronchoalveolar lavage (BAL) was performed 18 hours after LPS provocation. BAL fluid was analyzed for neutrophils, TNF-a and MIP-1b secretion. Baseline levels were determined three weeks before the first treatment and served as control. doi:10 1371/journal pone 0043709 g001 correlation coefficient (rs). Dose-response curves were created using a nonlinear regression model of normalized values with variable slope. endoscope. The custom-made bronchoscope (OD 2.5 mm, length 250 mm, Karlheinz Hinze Optoengineering, Hamburg, Ger- many) was inserted into the trachea and placed into the bronchus principalis sinister. Statistics Data are expressed as mean 6 SEM or median, as indicated in the figures. By using GraphPad Prism 4.0 (GraphPad, San Diego, CA, USA) a one-tailed Mann-Whitney U-test was performed to answer the question, whether roflumilast treatment reduces LPS- induced inflammation. A Grubbs’ test with a significance level of 0.05 was performed to detect outliers. The number of subjects is indicated in the figure legends. Differences between treated samples and controls were considered statistically significant at a level of p,0.05. Correlations were evaluated using a linear regression analysis model combined with Spearman’s rank Bronchoscopic Procedure for BAL Sample Retrieval The left lung was flushed twice with 3 mL of warm saline solution. Finally, the bronchoscope was gently removed from the trachea. Oxygen saturation and heart rate were permanently monitored with a pulse oxymeter (Medair, Hudiksvall, Sweden). For the purpose of standardization, only the second BAL fluid was obtained. Additionally, a recovery volume of more than 2 mL was required. Analysis of Tissue Culture Supernatants and BAL y p Concentrations of TNF-a were quantified by Enzyme Linked Immunosorbent Assay (ELISA; New World monkey TNF- a ELISA, U-Cy-Tech, Utrecht, the Netherlands; human TNF- a DuoSet, R&D Systems, Wiesbaden-Nordenstadt, Germany) with a lower limit of detection of 5 pg/mL. Macrophage inflammatory protein-1beta (MIP-1b) was measured using Lumi- nex technology (Millipore, Schwalbach, Germany) with a lower limit of quantification of 0.64 pg/mL. Total protein concentra- tions of PCLS were determined with the Bicinchoininc acid (BCA) method using a commercial kit according to the manufacturer’s instructions (BCA, Protein Assay Kit, Pierce, Rockford, IL, USA). Total cell counts in BAL fluid were determined using a ScepterTM cell counter (Millipore, Schwalbach, Germany). Differential cell counts of 400 cells per slide were performed visually after Pappenheim staining. Results LPS Triggers an Increase in Pro-inflammatory Mediators in Marmoset Monkeys Ex Vivo – Correlation with Human Data The ability of marmoset monkeys to respond to the well- established immunomodulator LPS was first tested ex vivo in whole- blood cultures and vital lung tissue. Marmoset whole blood and PCLS were exposed to LPS, and the effect on cytokine release was determined. The LPS-induced acute inflammatory response in both blood cultures and vital lung tissue was characterized by rapid accumulation of pro-inflammatory cytokines such as TNF- a and MIP-1b. LPS significantly increased the release of TNF- a (control: 670 pg/mL vs. 500 ng/mL LPS: 16,700 pg/mL) and intracellular production of MIP-1b (control: 900 pg/mL vs. 500 ng/mL LPS: 12,600 pg/mL) in marmoset lung tissue (Fig. 2A, B). The half maximal effective concentration (EC50) was 22 ng/mL LPS for TNF-a and 5 ng/mL LPS for intracellular MIP-1b. Treatment with dexamethasone reduced LPS-elicited cytokine levels of TNF-a to 72% and of MIP-1b to 67% (Fig. 2C, D). LPS-induced TNF-a secretion in marmoset PCLS correlated significantly with LPS-induced TNF-a secretion in marmoset whole-blood cultures (Fig. 2E, rs = 1.0, p = 0.0004) and with LPS- induced TNF-a secretion in human PCLS (Fig. 2F, rs = 0.9, p = 0.01). Marmoset PCLS, however, showed a 50 times stronger TNF-a release to LPS than marmoset whole-blood cultures. No sex-specific differences in whole-blood cultures of female and male marmosets could be revealed (data not shown). Discussion The present study aimed to establish a tiered LPS-induced acute lung inflammation model in marmoset monkeys. Two major aspects were addressed: firstly, the suitability of marmoset PCLS exhibiting features of LPS-induced acute lung inflammation was evaluated. Secondly, based on the preliminary studies in PCLS the feasibility of using a single unilateral LPS provocation and subsequent lavage in marmoset monkeys in vivo combined with successful anti-inflammatory treatment was described. This tiered testing strategy might improve the prediction of possible efficacy of newly developed experimental therapeutics in human clinical trials. Compared with the positive control sham, pre-treatment with roflumilast resulted in a statistically significant decrease in absolute and relative neutrophil cell numbers in BAL (Fig. 4B, p = 0.04; Fig. 5A, p = 0.047, Fig. 6C). Moreover, relative but not absolute numbers of macrophages were significantly increased after roflumilast pre-treatment (Fig. 5B, p = 0.04; Fig. 4C, p = 0.09). LPS-induced lung inflammation models are widely used as short-term models with airway and parenchymal changes as well as inflammatory cytokine up-regulation to test drugs against respiratory diseases including ALI and COPD [26;27]. Especially rodent models are commonly used in this context, but their readouts are often not predictive enough due to the absence of human-relevant molecular specifications, such as crucial discre- pancies in receptor expression of immunity-related genes [18]. As a result, some pharmaceuticals are effective in rodents, but show no responses in humans, and vice versa. This was particularly fatal in the case of thalidomide, which was not teratogenic in rodents, but in NHP and in humans [28]. Therefore, alternatively to rodents further validation in phylogenetically most closely related animals is needed to translate findings to the human situation. So far, LPS-induced lung inflammation models have been established in cynomolgus macaques (Macaca fascicularis) [12]. This Old World monkey species is, however, more difficult to bread and to handle and thus very cost intensive. Contrary, the small New World monkey common marmoset is less demanding in this respect, and shows high homologies to humans, too [29]. Marmoset monkeys share 86% analogy in the amino acid residues of immunity related genes including IL-6 and IL-1b when compared to humans [18]. Additionally, marmoset monkeys exhibit strong analogies in Figure 3. Dose-response curves of roflumilast in marmoset and human PCLS. Marmoset and human PCLS were incubated with 100 ng/mL LPS and increasing concentrations of roflumilast (rof, 0.6– 310 nM) for 24 hours. Anti-inflammatory Treatment Reduces LPS-induced Changes in BAL Cell Composition Acute lung inflammation was induced by using an LPS dose of 500 ng, which was administered unilaterally by MicroSprayerH under bronchoscopic supervision. The study was performed in two cycles. The exact allocation of individual animals to treatment groups is shown in table 1. 9 out of 16 animals were used in both independent study cycles (Tab. 1). Altogether, two LPS challenges were found to be ineligible because of a BAL fluid recovery volume of less than 2 mL. In another LPS challenge bronchoscopy was not feasible because of an undersized throat (Tab. 1). Baseline lavages were performed before each study cycle revealing almost only macrophages (Fig. 4C, 5B, 6A). Therefore, results of both study cycles were pooled due to randomized character and identical performance of each cycle. Moreover, the observed effects were comparable between both cycles. LPS-Induced Lung Inflammation in Marmoset Monkeys between both species as well as to estimate a dose for in vivo usage. Roflumilast efficiently suppressed LPS-induced TNF-a secretion in marmoset and human PCLS, revealing IC50 values of 1.3 nM (pIC50 = 8.88) and 1.1 nM (pIC50 = 8.97), respectively (Fig. 3). Based on these data, the animals received roflumilast in a dose of 7 mg/kg bw, which is comparable to the dose used in humans [12]. Roflumilast pre-treatment had no influence on lymphocyte counts (Fig. 4D, 5C). between both species as well as to estimate a dose for in vivo usage. Roflumilast efficiently suppressed LPS-induced TNF-a secretion in marmoset and human PCLS, revealing IC50 values of 1.3 nM (pIC50 = 8.88) and 1.1 nM (pIC50 = 8.97), respectively (Fig. 3). Based on these data, the animals received roflumilast in a dose of 7 mg/kg bw, which is comparable to the dose used in humans [12]. Pre-treatment with the treatment control dexamethasone revealed also a reduction in neutrophil numbers (Fig. 4B, Fig. 5A, Fig. 6D) compared to the positive control sham. Additionally, there was no effect on macrophages and lymphocytes after dexamethasone pre-treatment (Fig. 4C, p = 0.23; Fig. 4D, p = 0.1). All changes in BAL fluid were fully reversible after a minimum of three weeks post LPS challenge and returned to baseline levels within this time period (data not shown). Moreover, analysis of haemograms showed a significant increase in blood neutrophils in all individuals 18 hours after LPS-challenge compared to baseline levels, but no increase in blood TNF-a was detectable (data not shown). Blood neutrophilia was fully reversible within three weeks after LPS challenge. In line with pre-studies in whole-blood cultures of male and female marmosets, no sex-specific differences within the groups could be revealed. Anti-inflammatory Treatment Reduces LPS-induced Changes in Cytokines in BAL Fluid The pro-inflammatory cytokines TNF-a and MIP-1b were measured to analyze the efficacy of the different treatments. On average, TNF-a and MIP-1b levels in BAL fluid were increased 10- and 200-fold, respectively, after LPS challenge (Fig. 7A, B). Roflumilast (p = 0.048) and dexamethasone (p = 0.036) pre-treat- ment reduced TNF-a levels significantly. Furthermore, there was a tendency towards attenuation of MIP-1b secretion. BAL was performed 18 hours after LPS challenge to monitor cellular changes and changes on cytokine levels. LPS induced a significant influx of inflammatory cells with increased total cell counts into the challenged lung lobe (Fig. 4A, p = 0.0007). Neutrophils showed the most pronounced increase after LPS challenge (Fig. 4B, 5A, 6B). In sham-treated animals (positive control) absolute macrophage (Fig. 4C, p = 0.26) and lymphocyte counts (Fig. 4D, p = 0.35) were not augmented. Relative macro- phage counts, but not lymphocyte counts, were significantly decreased (Fig. 5B, p,0.0001, 5C). Eosinophils were not observed. Therapeutic Intervention with the Phosphodiesterase-4 Inhibitor Roflumilast Prevents LPS-induced TNF-a Release in Marmoset and Human PCLS Dose-response curves of the PDE4 inhibitor roflumilast were created for marmoset and human PCLS to assess analogies August 2012 \| Volume 7 \| Issue 8 \| e43709 PLOS ONE \| www.plosone.org 4 Figure 2. LPS-dependent increase in cytokines and chemokines ex vivo. Ascending TNF-a (A) and intracellular MIP-1b (B) production after 24-hour incubation with increasing concentrations of LPS in marmoset PCLS. LPS-induced increase in TNF-a (C) and intracellular MIP-1b (D) in marmoset PCLS was significantly suppressed by dexamethasone (dxm). Marmoset PCLS and marmoset WBA (E) on the one hand and marmoset PCLS and human PCLS (F) on the other hand showed significant correlations for TNF-a secretion (Spearman’s rank correlation coefficient (rS) = 1.0 with p = 0.0004, and rs = 0.9 with p = 0.01, respectively). Symbols: #: 2.5 ng/mL LPS, %: 5 ng/mL LPS, .: 10 ng/mL LPS, n: 100 ng/mL LPS, e: 250 ng/mL LPS, x: 500 ng/mL LPS. Data are presented as mean 6 SEM, p,0.05, p,0.01, Mann-Whitney test (TNF-a: n = 6, MIP-1b: n = 4). Correlations were evaluated using a linear regression analysis model combined with Spearman’s rank correlation coefficient. NHP = non-human primate, int = intracellular. doi:10.1371/journal.pone.0043709.g002 LPS-Induced Lung Inflammation in Marmoset Monkeys LPS-Induced Lung Inflammation in Marmoset Monkeys Figure 2. LPS-dependent increase in cytokines and chemokines ex vivo. Ascending TNF-a (A) and intracellular MIP-1b (B) production after 24-hour incubation with increasing concentrations of LPS in marmoset PCLS. LPS-induced increase in TNF-a (C) and intracellular MIP-1b (D) in marmoset PCLS was significantly suppressed by dexamethasone (dxm). Marmoset PCLS and marmoset WBA (E) on the one hand and marmoset PCLS and human PCLS (F) on the other hand showed significant correlations for TNF-a secretion (Spearman’s rank correlation coefficient (rS) = 1.0 with p = 0.0004, and rs = 0.9 with p = 0.01, respectively). Symbols: #: 2.5 ng/mL LPS, %: 5 ng/mL LPS, .: 10 ng/mL LPS, n: 100 ng/mL LPS, e: 250 ng/mL LPS, x: 500 ng/mL LPS. Data are presented as mean 6 SEM, p,0.05, *p,0.01, Mann-Whitney test (TNF-a: n = 6, MIP-1b: n = 4). Correlations were evaluated using a linear regression analysis model combined with Spearman’s rank correlation coefficient. NHP = non-human primate, int = intracellular. doi:10.1371/journal.pone.0043709.g002 doi:10.1371/journal.pone.0043709.g002 August 2012 \| Volume 7 \| Issue 8 \| e43709 PLOS ONE \| www.plosone.org 5 LPS-Induced Lung Inflammation in Marmoset Monkeys Discussion The half maximal inhibitory concentrations were detected at 1.3 nM for marmoset and 1.1 nM for human (dashed lines). Data are presented as mean 6 SEM, marmoset: n = 5; human: n = 4. TNF-a concentration in supernatants was determined by ELISA. Marmoset = grey symbols, human = black symbols. doi:10.1371/journal.pone.0043709.g003 Figure 3. Dose-response curves of roflumilast in marmoset and human PCLS. Marmoset and human PCLS were incubated with 100 ng/mL LPS and increasing concentrations of roflumilast (rof, 0.6– 310 nM) for 24 hours. The half maximal inhibitory concentrations were detected at 1.3 nM for marmoset and 1.1 nM for human (dashed lines). Data are presented as mean 6 SEM, marmoset: n = 5; human: n = 4. TNF-a concentration in supernatants was determined by ELISA. Marmoset = grey symbols, human = black symbols. doi:10.1371/journal.pone.0043709.g003 August 2012 \| Volume 7 \| Issue 8 \| e43709 6 PLOS ONE \| www.plosone.org LPS-Induced Lung Inflammation in Marmoset Monkeys Figure 4. Changes in absolute cell numbers in bronchoalveolar lavage (BAL) fluid after LPS challenge. Sham-treated, dexamethasone (dxm)-treated, and roflumilast (rof)-treated marmosets were intrabronchially challenged with 500 ng LPS. Eighteen hours later, ipsilateral BAL was performed. Total cells (A), neutrophils (B), macrophages (C), and lymphocytes (D) were differentiated and quantified using light microscopy after Pappenheim staining. Data are presented as scatter dot plot with median, p,0.05, **p,0.001, one-tailed Mann-Whitney test against sham. doi:10.1371/journal.pone.0043709.g004 Figure 4. Changes in absolute cell numbers in bronchoalveolar lavage (BAL) fluid after LPS challenge. Sham-treated, dexamethasone (dxm)-treated, and roflumilast (rof)-treated marmosets were intrabronchially challenged with 500 ng LPS. Eighteen hours later, ipsilateral BAL was performed. Total cells (A), neutrophils (B), macrophages (C), and lymphocytes (D) were differentiated and quantified using light microscopy after Pappenheim staining. Data are presented as scatter dot plot with median, p,0.05, **p,0.001, one-tailed Mann-Whitney test against sham. doi:10.1371/journal.pone.0043709.g004 Figure 5. Changes in relative cell numbers in bronchoalveolar lavage (BAL) fluid after LPS challenge. Sham, dexamethason (dxm) and roflumilast (rof)-treated marmosets were intrabronchially challenged with 500 ng LPS. Eighteen hours later, an ipsilateral BAL was performed. Relative numbers of neutrophils (A), macrophages (B), and lymphocytes (C) were determined. Cells were quantified and differentiated using light microscopy after Pappenheim staining. Data are presented as scatter dot plot with median; p,0.05, **p,0.001, one-tailed Mann-Whitney test against sham. doi:10.1371/journal.pone.0043709.g005 Figure 5. Changes in relative cell numbers in bronchoalveolar lavage (BAL) fluid after LPS challenge. Discussion Sham, dexamethason (dxm) and roflumilast (rof)-treated marmosets were intrabronchially challenged with 500 ng LPS. Eighteen hours later, an ipsilateral BAL was performed. Relative numbers of neutrophils (A), macrophages (B), and lymphocytes (C) were determined. Cells were quantified and differentiated using light microscopy after Pappenheim staining. Data are presented as scatter dot plot with median; p,0.05, **p,0.001, one-tailed Mann-Whitney test against sham. doi:10.1371/journal.pone.0043709.g005 August 2012 \| Volume 7 \| Issue 8 \| e43709 PLOS ONE \| www.plosone.org 7 LPS-Induced Lung Inflammation in Marmoset Monkeys Figure 6. LPS-induced changes in bronchoalveolar lavage (BAL) fluid. Representative cytospots of BAL at x200 original magnification after Pappenheim staining. (A) Macrophages are the predominant cell type in unchallenged lung lobes. (B) LPS challenge induced strong neutrophilic influx in sham-treated animals. This effect could be significantly attenuated by (C) roflumilast and (D) dexamethasone pre-treatment. Scale bar = 50 mm. doi:10 1371/journal pone 0043709 g006 Figure 6. LPS-induced changes in bronchoalveolar lavage (BAL) fluid. Representative cytospots of BAL at x200 original magnification after Pappenheim staining. (A) Macrophages are the predominant cell type in unchallenged lung lobes. (B) LPS challenge induced strong neutrophilic influx in sham-treated animals. This effect could be significantly attenuated by (C) roflumilast and (D) dexamethasone pre-treatment. Scale bar = 50 mm. doi:10.1371/journal.pone.0043709.g006 m doi:10.1371/journal.pone.0043709.g006 500 ng LPS by smoking 3–4 cigarettes. Each cigarette contains 120664 ng bioactive LPS in mainstream smoke, which results in an estimated delivered amount of 2.4 mg LPS by smoking one pack of cigarettes per day [32]. This LPS dose is similar to the level of LPS exposure of cotton textile workers suffering from adverse health effects [33]. Moreover, human clinical trials most commonly used even higher concentrations varying between 40 and 100 mg LPS applied via inhalation for investigating anti- inflammatory effects of certain drugs [34–36]. In this context, the estimation of the deposited LPS dose is, however, difficult due to the fact that a part of the LPS gets lost due to exhalation. Additionally, in chronic disease potentially pathogenic bacteria colonize the airways of patients producing also a small amount of endotoxin, which contributes to the progression of the disease since LPS is active at femtomole levels [37]. In particular, Sethi and colleagues assessed an amount of 36 mEU/mL endotoxin by using the limulus ameobocyte assay corresponding to approxi- mately 3.6 pg/mL LPS which is thereby circa 6000-times lower than our instilled LPS dose [37]. Discussion Both, TNF-a levels and numbers of neutrophils were significantly diminished in the roflumilast-treated animals as well as in the treatment-control dexamethasone. Thus, in marmoset monkeys with LPS-induced lung inflammation, roflumilast acts as an anti-inflammatory agent like in humans [11]. The acute LPS challenge was used to model TNF-a and MIP- 1b increase as well as neutrophilia, which are hallmarks of complex respiratory diseases including ALI/ARDS and COPD. However, COPD represents a slowly progressive chronic re- spiratory disease implicating that only its pro-inflammatory features can be modeled in an acute approach. Additionally, COPD comprises airflow limitation and a variety of pathological changes in the peripheral airways and lung parenchyma such as chronic inflammation, tissue damage, emphysema resulting in airway fibrosis and alveolar destruction [45]. These features can only be mirrored in chronic studies which require LPS adminis- tration over several weeks. Appropriate studies have been performed in rodents and guinea pigs resulting in COPD- associated pathological changes such as enlarged air spaces, remodelled airways with thickened walls, and increased goblet cells [27;46;47]. 500 mg/kg bw injected intratracheally and 10 mg/mL LPS applied via inhalation to induce a profound lung inflammation [38–40]. that especially neutrophils influx represents a hallmark of in- flammation [11;45]. Both, TNF-a levels and numbers of neutrophils were significantly diminished in the roflumilast-treated animals as well as in the treatment-control dexamethasone. Thus, in marmoset monkeys with LPS-induced lung inflammation, roflumilast acts as an anti-inflammatory agent like in humans [11]. Furthermore, we could reveal a high and significant correlation between marmoset whole-blood cultures and marmoset lung tissue regarding TNF-a release. The TNF-a level in WBA was, however, 50-fold lower compared to PCLS, suggesting a higher number of responding cells in marmoset lung tissue. Our experiments have clearly demonstrated high correlation of marmoset and human lung tissue regarding the distinct production of TNF-a in response to LPS. Similarly to human PCLS [7], the corticosteroid dexamethasone showed anti-inflammatory effects in marmoset monkey PCLS regarding TNF-a and MIP-1b secretion. Thus, the discussed steroid resistance due to decreased binding affinity of the glucocorticoid receptor in other New World monkey species, such as the squirrel monkey (Samiri sciureus), is not an issue in the marmoset monkey [41]. The similarities in marmoset and human PCLS could further be confirmed by analogous pIC50 values (8.9 and 9.0, respectively) for the PDE4 inhibitor roflumilast. Discussion This is in line with previously shown pIC50 values of 7.7 in human WBA [42]. In fact, we previously used human PCLS as a physiologically relevant acute inflammation model showing high homology to the in vivo situation [7]. Because of the high analogies ex vivo [11;43], we decided to use the same dosage of roflumilast in our in vivo approach that was effective in human clinical trials. The acute LPS challenge was used to model TNF-a and MIP- 1b increase as well as neutrophilia, which are hallmarks of complex respiratory diseases including ALI/ARDS and COPD. However, COPD represents a slowly progressive chronic re- spiratory disease implicating that only its pro-inflammatory features can be modeled in an acute approach. Additionally, COPD comprises airflow limitation and a variety of pathological changes in the peripheral airways and lung parenchyma such as chronic inflammation, tissue damage, emphysema resulting in airway fibrosis and alveolar destruction [45]. These features can only be mirrored in chronic studies which require LPS adminis- tration over several weeks. Appropriate studies have been performed in rodents and guinea pigs resulting in COPD- associated pathological changes such as enlarged air spaces, remodelled airways with thickened walls, and increased goblet cells [27;46;47]. The use of a relatively poor characterized NHP species is an important limitation of the present model, which becomes particularly obvious in the restricted scope of marmoset-specific analytics. Reliable analysis of alterations on cytokine level only comprises TNF-a and MIP-1b levels, contrary to the variety on murine and human commercially available tools. Cell differenti- ation with common cytology methods is, however, possible. Moreover, the use of NHP species for biomedical research is often viewed critical due to ethical concerns [48]. But the evidence of the scientific value of marmoset monkeys as a predictor for efficacy and adverse effects in humans due to the closer similarities to human metabolism and enzyme structures compared to rodents, dogs, and pigs is increasing [49;50]. Furthermore, the marmoset monkey exhibits similar pharmacodynamic effects as humans [51]. Marmoset monkeys might, therefore, help to clarify In the present study, we performed a unilateral LPS challenge under bronchoscopic supervision in marmoset monkeys, followed by BAL 18 hours later. A similar approach has been used in human clinical trials. O’Grady et al. August 2012 \| Volume 7 \| Issue 8 \| e43709 Discussion In rodent models of ALI or ARDS even higher LPS doses have been used varying between anatomical and physiological aspects of the lung [25;30]. Marmoset monkeys are, therefore, a useful alternative to existing models in rodents and Old World primates for the development of novel drugs against human diseases. This is also reflected by the use of marmoset monkeys for toxicity testing. Canakinumab (IlarisH, Novartis Europharm Limited, Horsham, UK) a IL-1b blocker for example, was tested in marmoset monkeys due to a lack of cross-reactivity in rodents as well as in cynomolgus macaques [31]. Moreover, we recently demonstrated great analogies between marmoset and human lung tissue regarding mediator- induced bronchoconstriction [25]. Due to the urgent need of predictive animal models for human diseases, we investigated to what extent marmoset monkeys represent a suitable model of LPS- induced acute lung inflammation. We could show a dose-dependent increase of TNF-a release in PCLS after LPS stimulation showing an EC50 of 22 ng/mL. This concentration was utilized to deduce the instillation dose of 500 ng LPS for the in vivo approach by taking into account that the half volume of an adult marmoset lung is 25 mL. Since LPS is a contaminant in cigarette smoke, smokers inhale approximately August 2012 \| Volume 7 \| Issue 8 \| e43709 PLOS ONE \| www.plosone.org 8 LPS-Induced Lung Inflammation in Marmoset Monkeys Figure 7. Analysis of bronchoalveolar lavage (BAL) fluid. TNF-a content in BAL fluid of marmoset monkeys was significantly reduced in dexamethasone-treated (dxm, p = 0.04) and roflumilast-treated (rof, p = 0.049) animals in contrast to sham-treated individuals (A). MIP-1b concentrations in BAL fluid were reduced by trend in both treatment groups (sham vs. dxm: p = 0.27; sham vs. rof: p = 0.17) (B). Data are presented as scatter dot plot with median, p,0.05, **p,0.0.01, one-tailed Mann-Whitney test against sham. doi:10.1371/journal.pone.0043709.g007 Figure 7. Analysis of bronchoalveolar lavage (BAL) fluid. TNF-a content in BAL fluid of marmoset monkeys was significantly reduced in dexamethasone-treated (dxm, p = 0.04) and roflumilast-treated (rof, p = 0.049) animals in contrast to sham-treated individuals (A). MIP-1b concentrations in BAL fluid were reduced by trend in both treatment groups (sham vs. dxm: p = 0.27; sham vs. rof: p = 0.17) (B). Data are presented as scatter dot plot with median, p,0.05, ***p,0.0.01, one-tailed Mann-Whitney test against sham. doi:10.1371/journal.pone.0043709.g007 that especially neutrophils influx represents a hallmark of in- flammation [11;45]. References Neubert R, Neubert D (1997) Peculiarities and possible mode of actions of thalidomide. In: Kavlock RJ, Daston GP, editors. Drug toxicity in embryonic development. II ed. Berlin: Springer-Verlag. p. 41–119. 8. Tralau-Stewart CJ, Williamson RA, Nials AT, Gascoigne M, Dawson J, et al. (2011) GSK256066, an exceptionally high-affinity and selective inhibitor of phosphodiesterase 4 suitable for administration by inhalation: in vitro, kinetic, and in vivo characterization. J Pharmacol Exp Ther; 337(1): 145–54. 29. Mansfield K (2003) Marmoset models commonly used in biomedical research. Comparative Medicine; 53(4): 383–92. p p y and in vivo characterization. J Pharmacol Exp Ther; 337(1): 145–5 p 30. Barbier A, Bachofen H (2000) The lung of the marmoset (Callithrix jacchus): ultrastructure and morphometric data. Respiration Physiology; 120(2): 167–77. 9. Barnes PJ (2006) Transcription factors in airway diseases. Lab Invest; 86(9): 867– 72. 31. European Medicines Agency (2009) CHMP Assessment Report for Ilaris. Report No.: EMEA/H/C001109. 10. Fearon DT, Locksley RM (1996) The instructive role of innate immunity in the acquired immune response. Science;272(5258): 50–3. 32. Hasday JD, Bascom R, Costa JJ, Fitzgerald T, Dubin W (1999) Bacterial endotoxin is an active component of cigarette smoke. Chest; 115(3): 829–35. 11. Hohlfeld JM, Schoenfeld K, Lavae-Mokhtari M, Schaumann F, Mueller M, et al. (2008) Roflumilast attenuates pulmonary inflammation upon segmental endotoxin challenge in healthy subjects: A randomized placebo-controlled trial. Pulmonary Pharmacology & Therapeutics; 21(4): 616–23. 33. Kennedy SM, Christiani DC, Eisen EA, Wegman DH, Greaves IA, et al. (1987) Cotton dust and endotoxin exposure-response relationships in cotton textile workers. Am Rev Respir Dis; 135(1): 194–200. 12. Mitchell LA, Channell MM, Royer CM, Ryter SW, Choi AM, et al. (2010) Evaluation of inhaled carbon monoxide as an anti-inflammatory therapy in a nonhuman primate model of lung inflammation. Am J Physiol Lung Cell Mol Physiol; 299(6): L891–L897. p 34. Maris NA, de Vos AF, Dessing MC, Spek CA, Lutter R, et al. (2005) Antiinflammatory effects of salmeterol after inhalation of lipopolysaccharide by healthy volunteers. Am J Respir Crit Care Med; 172(7): 878–84. 13. Lamontagne F, Briel M, Guyatt GH, Cook DJ, Bhatnagar N, et al. (2010) Corticosteroid therapy for acute lung injury, acute respiratory distress syndrome, and severe pneumonia: a meta-analysis of randomized controlled trials. J Crit Care; 25(3): 420–35. 35. Michel O, Dentener M, Cataldo D, Cantinieaux B, Vertongen F, et al. References (2007) Evaluation of oral corticosteroids and phosphodiesterase-4 inhibitor on the acute inflammation induced by inhaled lipopolysaccharide in human. Pulm Pharmacol Ther; 20(6): 676–83. 14. Sharafkhaneh A, Southard JG, Goldman M, Uryniak T, Martin UJ (2011) Effect of budesonide/formoterol pMDI on COPD exacerbations: A double-blind, randomized study. Respir Med. 36. Thorn J, Rylander R (1998) Inflammatory response after inhalation of bacterial endotoxin assessed by the induced sputum technique. Thorax; 53(12): 1047–52. 37. Sethi S, Murphy TF (2008) Current Concepts: Infection in the Pathogenesis and Course of Chronic Obstructive Pulmonary Disease. New England Journal of Medicine; 359(22): 2355–65. y p 15. Cazzola M, Page CP, Calzetta L, Matera MG (2012) Emerging anti- inflammatory strategies for chronic obstructive pulmonary disease. Eur Respir J. 38. Faffe DS, Seidl VR, Chagas PS, Goncalves de Moraes VL, Capelozzi VL, et al. (2000) Respiratory effects of lipopolysaccharide-induced inflammatory lung injury in mice. Eur Respir J; 15(1): 85–91. 16. Fabbri LM, Beghe B, Yasothan U, Kirkpatrick P (2010) Roflumilast. Nat Rev Drug Discov; 9(10): 761–2. 17. Martinez FJ, Donohue JF, Rennard SI (2011) The future of chronic obstructive pulmonary disease treatment–difficulties of and barriers to drug development. Lancet; 378(9795): 1027–37. 39. Matthay MA, Zemans RL (2011) The acute respiratory distress syndrome: pathogenesis and treatment. Annu Rev Pathol; 6: 147–63. 18. Kohu K, Yamabe E, Matsuzawa A, Onda D, Suemizu H, et al. (2008) Comparison of 30 immunity-related genes from the common marmoset with orthologues from human and mouse. Tohoku Journal of Experimental Medicine; 215(2): 167–80. 40. Rocco PR, Momesso DP, Figueira RC, Ferreira HC, Cadete RA, et al. (2003) Therapeutic potential of a new phosphodiesterase inhibitor in acute lung injury. Eur Respir J; 22(1): 20–7. 41. Chrousos GP, Loriaux DL, Tomita M, Brandon DD, Renquist D, et al. (1986) The new world primates as animal models of glucocorticoid resistance. Adv Exp Med Biol; 196: 129–44. 19. Plopper CG, Hyde DM (2008) The non-human primate as a model for studying COPD and asthma. Pulmonary Pharmacology & Therapeutics; 21(5): 755–66. 42. Lunniss CJ, Cooper AW, Eldred CD, Kranz M, Lindvall M, et al. (2009) Quinolines as a novel structural class of potent and selective PDE4 inhibitors: optimisation for oral administration. Bioorg Med Chem Lett; 19(5): 1380–5. 20. Jagessar SA, Gran B, Heijmans N, Bauer J, Laman JD, et al. (2011) Discrepant Effects of Human Interferon-gamma on Clinical and Immunological Disease Parameters in a Novel Marmoset Model for Multiple Sclerosis. References sclerosis for immunotherapy development. J Neuroimmune Pharmacol; 5(2): 220–30. 1. de Marco R, Accordini S, Cerveri I, Corsico A, Anto JM, et al. (2007) Incidence of chronic obstructive pulmonary disease in a cohort of young adults according to the presence of chronic cough and phlegm. Am J Respir Crit Care Med; 175(1): 32–9. sclerosis for immunotherapy development. J Neuroimmune Pharmacol; 5(2): 220–30. 22. Bergers WW, van de Meent-van der Horst, Joosen MJ (2004) Respiration-based measurement of lung deposition of a fluorescent dextrane aerosol in a marmoset monkey. Inhal Toxicol; 16(3): 141–6. 2. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, et al. (2005) Incidence and outcomes of acute lung injury. N Engl J Med; 353(16): 1685–93. 23. Kurata Y, Takechi M, Toyota N, Tsuchitani M, Katoh M, et al. (1997) Four- week repeated inhalation study of HCFC 225ca and HCFC 225cb in the common marmoset. Toxicol Lett; 92(3): 209–19. 3. Barnes PJ (2004) Alveolar macrophages as orchestrators of COPD. COPD; 1(1): 59–70. 24. Whelan G, James MF, Samson NA, Wood NI (1999) Anaesthesia of the common marmoset (Callithrix jacchus) using continuous intravenous infusion of alphaxalone/alphadalone. Lab Anim; 33(1): 24–9. 4. Bundschuh DS, Eltze M, Barsig J, Wollin L, Hatzelmann A, et al. (2001) In vivo efficacy in airway disease models of roflumilast, a novel orally active PDE4 inhibitor. J Pharmacol Exp Ther; 297(1): 280–90. p 5. Hicks A, Kourteva G, Hilton H, Li H, Lin TA, et al. (2010) Cellular and molecular characterization of ozone-induced pulmonary inflammation in the Cynomolgus monkey. Inflammation; 33(3): 144–56. 25. Seehase S, Schleputz M, Switalla S, Matz-Rensing K, Kaup FJ, et al. (2011) Bronchoconstriction in nonhuman primates: a species comparison. J Appl Physiol; 111(3): 791–8. 26. Matute-Bello G, Frevert CW, Martin TR (2008) Animal models of acute lung injury. Am J Physiol Lung Cell Mol Physiol; 295(3): L379–L399. 6. Sandstrom T, Bjermer L, Rylander R (1992) Lipopolysaccharide (LPS) inhalation in healthy subjects increases neutrophils, lymphocytes and fibronectin levels in bronchoalveolar lavage fluid. Eur Respir J; 5(8): 992–6. 27. Wright JL, Cosio M, Churg A (2008) Animal models of chronic obstructive pulmonary disease. American Journal of Physiology-Lung Cellular and Molecular Physiology; 295(1): L1–L15. 7. Switalla S, Lauenstein L, Prenzler F, Knothe S, Forster C, et al. (2010) Natural innate cytokine response to immunomodulators and adjuvants in human precision-cut lung slices. Toxicol Appl Pharmacol. 28. Acknowledgments We are grateful to Dr. R. Kellner for statistical advice and to Dr. S. Voedisch for critical reading of the manuscript and helpful discussions. We wish to thank Dr. T. Becker, Dr. A. Schrod, and Dr. J. Ko¨nig for their veterinary assistance, as well as T. Meyer-Burhenne and S. Leineweber for their expert animal care. The technical assistance of N. Schminke, L. Hummel, and J. Krenzek is greatly appreciated. In conclusion, LPS-induced acute lung inflammation in marmoset monkeys displays a promising model with good cost- benefit ratio for testing new drugs of human respiratory diseases. Especially highly specific anti-inflammatory pharmaceuticals targeting pro-inflammatory cytokine secretion as well as neutro- philic influx might be tested in this model. Additionally, PCLS are a useful tool to estimate a dose range for in vivo usage. Thus, tiered testing in marmoset monkeys can be applied for the examination of potential new therapeutics. LPS-Induced Lung Inflammation in Marmoset Monkeys LPS-Induced Lung Inflammation in Marmoset Monkeys the scientific gap between commonly performed mouse models, human disease models, and human diseases [52]. Author Contributions Conceived and designed the experiments: S. Seehase HDL KS AB SK. Performed the experiments: S. Seehase S. Switalla VN HDL SK CS. Analyzed the data: S. Seehase. Contributed reagents/materials/analysis tools: CF CS EF FJK MB JMH HGF OP. Wrote the paper: S. Seehase KS SK AB. Conceived and designed the experiments: S. Seehase HDL KS AB SK. Performed the experiments: S. Seehase S. Switalla VN HDL SK CS. Analyzed the data: S. Seehase. Contributed reagents/materials/analysis tools: CF CS EF FJK MB JMH HGF OP. Wrote the paper: S. Seehase KS SK AB. Discussion were the first to show that local endotoxin-induced lung inflammation in healthy human subjects results in strong neutrophilic influx and increase in inflammatory mediators 6 hours after provocation [44]. Most pro- inflammatory cytokines, however, returned to baseline levels after 24 hours [44]. Additionally, Hohlfeld et al. used segmental endotoxin instillation for assessment of the PDE4 inhibitor roflumilast [11]. For the purpose of the present study, a time period of 18 hours was used, in order to cover cellular changes in BAL fluid and cytokine up-regulation in lung tissue due to the fact, August 2012 \| Volume 7 \| Issue 8 \| e43709 PLOS ONE \| www.plosone.org 9 48. Coffman RL, Hessel EM (2005) Nonhuman primate models of asthma. J Exp Med; 201(12): 1875–9. References J Neuroimmune Pharmacol. 43. Gross NJ, Giembycz MA, Rennard SI (2010) Treatment of chronic obstructive pulmonary disease with roflumilast, a new phosphodiesterase 4 inhibitor. COPD; 7(2): 141–53. 21. Kap YS, Laman JD, ’t Hart BA (2010) Experimental autoimmune encepha- lomyelitis in the common marmoset, a bridge between rodent EAE and multiple August 2012 \| Volume 7 \| Issue 8 \| e43709 August 2012 \| Volume 7 \| Issue 8 \| e43709 PLOS ONE \| www.plosone.org 10 46. Toward TJ, Broadley KJ (2001) Chronic lipopolysaccharide exposure on airway function, cell infiltration, and nitric oxide generation in conscious guinea pigs: effect of rolipram and dexamethasone. J Pharmacol Exp Ther; 298(1): 298–306. 45. Barnes PJ (2004) Mediators of chronic obstructive pulmonary disease. Pharmacological Reviews; 56(4): 515–48. 44. O’Grady NP, Preas HL, Pugin J, Fiuza C, Tropea M, et al. (2001) Local inflammatory responses following bronchial endotoxin instillation in humans. Am J Respir Crit Care Med; 163(7): 1591–8. LPS-Induced Lung Inflammation in Marmoset Monkeys LPS-Induced Lung Inflammation in Marmoset Monkeys LPS-Induced Lung Inflammation in Marmoset Monkeys 44. O’Grady NP, Preas HL, Pugin J, Fiuza C, Tropea M, et al. (2001) Local inflammatory responses following bronchial endotoxin instillation in humans. Am J Respir Crit Care Med; 163(7): 1591–8. 49. Igarashi T, Sakuma T, Isogai M, Nagata R, Kamataki T (1997) Marmoset liver cytochrome P450s: study for expression and molecular cloning of their cDNAs. Arch Biochem Biophys; 339(1): 85–91. 45. Barnes PJ (2004) Mediators of chronic obstructive pulmonary disease. Pharmacological Reviews; 56(4): 515–48. 50. Sakuma T, Igarashi T, Hieda M, Ohgiya S, Isogai M, et al. (1997) Marmoset CYP1A2: primary structure and constitutive expression in livers. Carcinogenesis; 18(10): 1985–91. 46. Toward TJ, Broadley KJ (2001) Chronic lipopolysaccharide exposure on airway function, cell infiltration, and nitric oxide generation in conscious guinea pigs: effect of rolipram and dexamethasone. J Pharmacol Exp Ther; 298(1): 298–306. 51. Smith D, Trennery P, Farningham D, Klapwijk J (2001) The selection of marmoset monkeys (Callithrix jacchus) in pharmaceutical toxicology. Labora- tory Animals; 35(2): 117–30. 47. Vernooy JH, Dentener MA, van Suylen RJ, Buurman WA, Wouters EF (2002) Long-term intratracheal lipopolysaccharide exposure in mice results in chronic lung inflammation and persistent pathology. Am J Respir Cell Mol Biol; 26(1): 152–9. 52. Sasaki E, Suemizu H, Shimada A, Hanazawa K, Oiwa R, et al. (2009) Generation of transgenic non-human primates with germline transmission. Nature; 459(7246): 523–U50. 48. Coffman RL, Hessel EM (2005) Nonhuman primate models of asthma. J Exp Med; 201(12): 1875–9. August 2012 \| Volume 7 \| Issue 8 \| e43709 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 11 11
https://openalex.org/W4283653929	https://www.researchsquare.com/article/rs-1729313/latest.pdf	English	null	Combustion of Al nanoparticles coated with NC/ethanol/ether molecules by equilibrium molecular dynamics simulations	Research Square (Research Square)	2,022	cc-by	11,129	Combustion of Al nanoparticles coated with NC/ethanol/ether molecules by equilibrium molecular dynamics simulations Xin Jin Harbin Engineering University Lei Wang Harbin Engineering University Pingan Liu (  liupingan631@126.com ) Harbin Engineering University Penghua Sui Harbin Engineering University Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/32 Page 1/32 Abstract Al nano-particle (ANP) have high reactivity, but they are easily inactivated by external oxidants. To improve its surface property, we coat ANPs with nitrocellulose (NC)/ethanol/ether solution. And MD simulation method is used to simulate the coating, ignition and combustion processes of ANPs under three different coating conditions. Our results show that the NC/ethanol/ether formed a dense coating layer on the surface of annealed ANPs and passivated ANPs through physical and chemical adsorption. The coating layer can block the contact between the active Al atoms and O2 molecules at low temperature. In ignition phase, NC/ethanol/ether coating layer can increase the density of O2 molecules around ANPs and the surface temperature of ANPs. At the same time, due to the desorption and diffusion of the coating layer exposed more reaction sites, ANPs have shorter ignition delay and lower ignition temperature. According to the change of atomic displacement, the combustion stage can be divided into three stages. The NC molecules can increase the combustion propagation speed, combustion time and efficiency of ANPs. Such improvement will enable ANPs to obtain better storage performance, combustion performance and play a stronger role in the field of energetic materials. 1. Introduction Energetic material is a kind of compound or mixture that containing explosive groups or combustibles and oxidants. Under certain external stimuli, it can conduct redox reactions alone and release a large amount of energy (usually accompanied by a large amount of gas and heat). As a typical energetic material, ANPs play an important role in propellants. ANPs have high specific surface area and reactivity due to the defects of bond state, electronic state and atomic coordination on the surface of nanoparticles. Using ANPs can greatly improve the combustion efficiency, the dynamic and static burning rate of high energy condensed system, improve the detonation heat and greatly reduce the activation energy of combustion system. However, those characteristics not only improve the application advantages of ANPs in propellants, but also bring difficulties in storage and processing. The newly prepared ANPs are easily oxidized or even spontaneous combustion in oxidation environment [1, 2]. To improve the surface properties of ANPs, researchers propose a concept of constructing stable envelope structure on particle surface. After cladding, ANPs have a passivation layer on the surface which isolate the contact between the external oxidation environment and the active Al atoms. By changing coating materials, we can get core-shell composite particles with different surface properties. As an important part of solid propellant, NC with high nitrogen content has the characteristics of water resistance, acid and alkali resistance, low cost and long storage life [3]. Moreover, the NC fiber bundle will undergo the process of swelling to dissolution in the solvent and the molecular chain is dispersed in the solvent to form a homogeneous sol. Therefore, preparing the stable structure core-shell composite aluminum particles with NC sol is of great significance for the storage of ANPs and its application in propellants [4]. Page 2/32 Most researchers used experimental methods to study NC/ANPs composite particles. Michael R. Zachariah et al. from the University of Maryland carry out a large number of studies on Al-NC composite particles prepared by electrostatic spray method [5]. They find the combustion efficiency of Al-NC composite particles prepared by electrostatic spray method is basically equivalent to that of non- agglomerated nano-aluminum powder in combustion performance. When the mass fraction of NC increased from 3–10%, the ignition delay time was shortened from 3.5 ms to 0.3 ms, and the combustion time was prolonged from 46 ms to 64 ms [6]. Wang H et al. 1. Introduction successfully prepare gel-like NC/ANPs composite particles by electrostatic spray method [7], and the mass fraction of NC is 1% -10%. The combustion performance test shows that the gel-like NC/ANPs composite particles have stronger combustion behavior than ANPs, and the 10% NC/ANPs composite particles show the longest combustion time (~ 64 ms) and the shortest ignition delay time (~ 0.26 ms). Yan et al. prepare uniform sized and porous NC/ANPs microspheres by electrostatic spray method. The NC/ANPs microspheres have high pressurization rate and combustion propagation rate [4]. Molecular dynamics (MD) simulation is an effective theoretical research mean from atomic scale. It has been successfully applied to explore the properties of ANPs. Alavi uses MD simulation to predict the melting points of ANPs and the simulation results are agreement with the experimental results [8]. Much work has been devoted to exploring the sintering of metal nanoparticles by molecular dynamics (MD) simulation [9–15]. Song et al. studied two different sizes of nickel nanoparticles through MD simulation [16], and found that the sintering of two different sizes of nanoparticles occurred in three stages closely related to surface melting. Liu et al. simulated the non-equilibrium molecular dynamics of the combustion of nano-Al particles coated with ethanol/ether molecules [17], and explored the combustion mechanism of coated ANPs. On the other hand, the sintering of nano powders and metal alloys has also been studied extensively in the experiments [18–24]. Sabzi et al. experimentally studied the effects of different sintering parameters on the microstructure evolution [18], density and hardness of nano-powder cemented carbide products. A.L. Ahmad et al. established the calculation model of horizontal transverse diffusion of protein in NC film [25], and determined the concentration gradient as the driving force of molecular diffusion. Dave T. F. Kuo et al. from Hong Kong City University use First-order model and Intra-Particle Diffusion model to calculate the adsorption and desorption kinetic parameters of NG and 2,4-dinitrotoluene in NC [26]. Waldemar A. Trzcinski et al. from Poland Warsaw Institute of Military Science and Technology derived the time-space resolution characteristics of NC-based composite reaction armor explosion through the modified Gurney model [27]. As far as we know, the research of NC and nano aluminum powder mixture system is mostly based on experimental methods, which is difficult to directly detect the reaction process and microscopic mechanism in the rapid response stage. 2.1 ReaxFF-lg long-range correction reaction force field In the current work, MD simulation is performed using a large-scale parallel atom/molecule simulator (LAMMPS [28]), and visualization post-processing is performed using VMD [29] and OVITO software. The core of MD simulation is the force field. Reasonable force field parameters greatly affect the accuracy and results of simulation. ReaxFF interatomic potential is a reaction force field based on bond-order (BO), which has been successfully applied to computational chemistry [30]. Different from the concept of atomic type in the classical force field, ReaxFF adopts bond-order (BO) formalism to evaluate the interactions between atoms including bond and long-range pair interactions. Eq. (1) presents the total energy in ReaxFF force field: Esystem = Ebond + Eover + Eunder + Elp + Eval + Etors + EvdWaals + ECoulomb 1 where denotes the total energy of the system. The bond ( ), overcoordination ( ), undercoordination ( ), lone pair ( ), valence angle ( ), dihedral twist angle ( ), van der Waals ( ), and Coulomb ( ) energy terms contribute to the total energy to vary degrees. In addition to and are non-bonding terms, other energies are expressed through BO. Esystem Ebond Eover Eunder Elp Eval Etors EvdWaals ECoulomb EvdWaals ECoulomb where denotes the total energy of the system. The bond ( ), overcoordination ( ), undercoordination ( ), lone pair ( ), valence angle ( ), dihedral twist angle ( ), van der Waals ( ), and Coulomb ( ) energy terms contribute to the total energy to vary degrees. In addition to and are non-bonding terms, other energies are expressed through BO. Esystem Ebond Eover Eunder Elp Eval Etors EvdWaals ECoulomb EvdWaals ECoulomb However, the first principle based on density functional theory (DFT) is often used to fit the results in the development of the ReaxFF reaction force field. This method will underestimate the van der Waals force of non-bonding molecules in the calculation process. Therefore, the ReaxFF reaction force field usually underestimates the calculation of equilibrium density when describing molecular systems [31]. In addition, the expression of ReaxFF force field has included the expression of van der Waals interaction. Therefore, it is necessary to introduce a new non-bonding term without affecting the fracture and formation of chemical bonds in the molecule, so that the ReaxFF force field can correct the long-range force and reduce the impact on covalent bonds. Liu et al. 1. Introduction Therefore, in order to explore the ignition and combustion mechanism of NC/ethanol/ether coated ANPs, MD simulations are carried out on three different models in this research. Radial distribution function (RDF), average displacement, mean square displacement Page 3/32 Page 3/32 (MSD) and other parameters will be quantitatively characterized to reveal the ignition and combustion mechanism of NC/ethanol/ether ternary coating ANPs. 2.2 Simulation system and setup This study includes two simulation scenarios. For the study on the pyrolysis mechanism of NC single chain discussed in section 3.1, the simulation box measured 25.792 × 25.792 × 25.792 Å3 and the density of NC system was set to be 1.6g/cm3 which is close to the actual density. Figure 1 (a) is a single NC molecular chain model and Fig. 1 (b) is an initial configuration snapshot of amorphous cell pyrolysis. The thermodynamic process of the system is as follows. Firstly, every atom is assigned a velocity to the specified temperature of 5 K in a Gaussian distribution, following which the system is relaxed under the canonical ensemble (NVT) at 5 K for 1 ps to obtain a reasonable configuration. Then, increasing the system temperature to 3000 K slowly at the heating rate of 0.03 K/fs. Finally, lasting for 100ps at 3000 K. The pyrolysis reaction started from the initial stage of heating. Using Berendsen control theory to control the temperature with 20 fs of the temperature resistance coefficient and 0.2 fs of the time step. For the cases of preparing and burning the ANPs, we use three ANPs models, which are passivated ANPs (sANPs), NC/ethanol/ether coating passivation ANPs (csANPs) and NC/ethanol/ether coating annealing ANPs (cANPs). The simulation box measured 110×110×110 Å3, ANPs are located in the center of the box and other molecules such as NC, ethanol, ether, oxygen is randomly distributed around ANPs. In our study, the ANP has a typical core-shell structure, which the Al core diameter is 4 nm and the oxide shell thickness is 0.5 nm. According to Liu et al., the melting point of ANPs changes linearly with diameter with diameter more than 2nm [33]. Considering the computational cost, 4nm ANP is sufficient to show the thermodynamic properties. The Al core and alumina shell models are cut from cubic crystals and assembled together. The fusion process which ensures close contact between the core and shell and eliminated surface stress. The basic idea is to relax the passivated particles at 50 K for 1.5 ps to obtain the stable configuration at this temperature; then, the particles are heated to 300 K at the heating rate of 1013 K/fs under the NVT ensemble, and the shell-core structure will gradually integrate during this process. Finally, the passivated particles were relaxed for 200 ps at 300 K to obtain the stable configuration at room temperature. 2.1 ReaxFF-lg long-range correction reaction force field use a low-gradient model (Low-Gradient) to correct the long-range effect of density functional calculation [32]. Based on this, the ReaxFF-lg long-range correction reaction force field is developed by using the similar model to correct the long-range effect of the ReaxFF force field. Here, we use the ReaxFF-lg long-range correction reaction force field for simulation calculation. The expression of total energy in ReaxFF-lg force field is described in Eq. (2): Elg system = Esystem + Elg Elg system = Esystem + Elg 2 2 Page 4/32 Page 4/32 Page 4/32 where denotes the lg long-range amendments. Its calculation uses the Low - Gradient model. Eq. (3) is the calculation formula of : Elg Elg Elg = − N ∑ ij,i<j Clg,ij r6 ij + d ⋅R6 eij 3 where is the distance between atoms and , and is the Van der Waals interaction equilibrium distance between atoms and , and is the correction coefficient of dispersion force, and is the scale factor rij Reij Clg,ij where is the distance between atoms and , and is the Van der Waals interaction equilibrium distance between atoms and , and is the correction coefficient of dispersion force, and is the scale factor. rij Reij Clg,ij 2.2 Simulation system and setup Figure 2 shows the initial configuration and optimized configuration of passivated ANPs. Page 5/32 In order to establish the cANPs model, we need to obtain the annealed ANPs model first. ANPs constructed directly by script commands in LAMMMPS are ideal spherical particles composed of FCC cells. To eliminate the edge effect of ANPs, annealing and recrystallization are necessary. The basic idea is that cutting out an aluminum particle with a diameter of 4 nm in the aluminum FCC crystal block, and the whole particles are relaxed at 1300 K to completely melt. Then, to get the annealed ANPs at 300 K as the initial model for subsequent simulation, the molten aluminum clusters were cooled and recrystallized. The whole annealing process is carried out under the NVT with EAM force field. Using the Verlet integral algorithm to solve the Newton motion equation. Using the Nose/Hoover theory to control the temperature. The temperature damping coefficient is selected as 100 fs. The accuracy and efficiency of recrystallization annealing are related to the selection of simulated force field and cooling rate. In our study, we choose 20 K/ns as the cooling rate, and the cooling rate is also verified to have good accuracy and economy [34]. Figure 3 shows the ANPs model before and after annealing and the crystal type distribution of the central section. 3. Results And Discussions The force field used in this study has been proved to be effective for the ethanol-ether-oxygen system [17]. However, NC molecules are quite different from ethanol and ether molecules. The chemical properties of ethanol are determined by hydroxyl groups, and ether are characterized by their self-propagating autooxidation (peroxidation) process [35]. The main oxidation product of gas phase ether is ether. However, NC molecules will undergo severe self-decomposition at higher temperature (573 K), and the decomposition products are complex. Therefore, it is necessary to simulate the thermal decomposition mechanism of NC molecules in ReaxFF MD simulations. The thermal decomposition reaction simulation settings are shown in section 2.2. The thermal decomposition temperature of NC single chain is about 1182 K during the heating process [36], which is obviously higher than the experimental temperature. It is because the thermal decomposition of NC is limited by the time scale of ReaxFF MD simulation, which has been confirmed in a large number of literatures. Figure 3 shows the variation curve of the number of key product molecules with time in the thermal decomposition process of NC single molecular chain model. The first meteorological product is NO2, which is consistent with the fact that the -NO2 base strength measured in the heating process of NC thermal decomposition experiment decreases first. In addition, we found that CH2O generated later than NO2. It is because CH2O is the decomposition product of O-NO2 group on the primary (C-6 position). This is consistent with the experimental results of Wang et al. To see this process more clearly, Fig. 4 shows a snapshot of the local structure of the decomposition process of the primary O-NO2 group in the simulation. The fracture of O-NO2 bond and the subsequent formation of CH2O can be clearly seen from Page 6/32 Page 6/32 Fig. 4. From Fig. 3, we can also find that the number of H2O, NO, CO2 and CO molecules began to increase and a small amount of HCOOH formation with the number of NO2 molecules reaching the peak. Fig. 4. From Fig. 3, we can also find that the number of H2O, NO, CO2 and CO molecules began to increase and a small amount of HCOOH formation with the number of NO2 molecules reaching the peak. In addition, there are many researchers interesting in the fracture of molecular chains during thermal decomposition of NC. Jutier et al. 3. Results And Discussions believed that the thermal decomposition of NC did not necessarily lead to the fracture of the chain [37]. However, Wang et al. believed that along with the denitrification process, the NC macromolecular chain would be decomposed into small molecules. In our study, we captured the fracture of C-O-C bond between NC molecular chains, as shown in Fig. 6. In the pyrolysis process of NC molecular single chain model, the interannular C-O-C bond will be broken, and NC is decomposed into small molecules from macromolecular chains. However, the C-O-C bond breakage in the NC molecular chain is relatively lagging behind, which supports Wang's view that NC macromolecular chains will be decomposed into small molecules along with the denitrification process. It also verifies the accuracy of using ReaxFF force field to simulate NC system. 3.2 Preparation of ANPs with NC/ethanol/ether As for the establishment of NC/ethanol/ether ternary coating ANPs model, the simulation box measured 120 × 120 × 120 Å3. The annealed and passivated ANPs established in section 2.2 are placed in the center of the box, and the ethanol ether solution molecules are placed in the surrounding area of ANPs. In addition, a vacuum region of 7 Å was set between NC/ethanol/ether solution molecules and ANPs as a buffer region. The volume ratio of ethanol molecule to ether molecule in NC/ethanol/ether solution was 1:1. Considering the ANPs used in our study is small, the number of molecules in NC/ethanol/ether solution needs to be limited within the range that ANPs can withstand, otherwise ANPs are prone to deformation. The specific values are shown in Table 1. The position distribution of ethanol ether solution molecules is completed by the Packmol program [38], so that it initially forms a nearly uniform mixed solution system. Table 1 Computational materials science Table 1 Computational materials science Table 1 Computational materials science In the coating process of passivated ANPs, the adsorption rates of ethanol and ether molecules are always slower than those of annealed ANPs. At the end of the coating, the number of ethanol molecules in the coating layer on the particle surface is about 287, the number of ether molecules is 135, and the ratio of ethanol/ether molecules is about 2.12. The number of ethanol ether molecules in the coating layer of passivated ANPs was significantly less than that of annealed ANPs, indicating that the compactness of coating layer on the surface of passivated ANPs was relatively poor. In the actual production process, after ANP particles and solution are fully mixed, more organic solutions are removed in the oven. Therefore, at the end of the cycle-coating process, an evaporation-elimination procedure is carried out to eliminate atoms that are not tightly adsorbed on the ANPs. Considering the real ignition environment is about 300 K, the evaporation-elimination simulation is carried out at 300 K. For the first time, manual filter is selected to remove the free complete solvent molecules in the box. Then, put the filtered ANPs in the center of cube box of 120 × 120 × 120 Å3, and to leave sufficient space for the release pressure of the coating layer, the environment was set as a vacuum. After each filtering simulation, the separated solvent molecules were filtered until no molecules were separated and the cycle ended. Figure 8 shows the molecular numbers of each component in coating layer after each filtering of cANP and csANP. From Fig. 8, we can find that whether csANP or cANP, a large number of ether molecules and a small amount of ethanol molecules are separated from the filtering simulation, and NC molecules remain stable. It is because different molecular structures and adsorption modes, ethanol is chemically adsorbed while ether is physically adsorbed, and ether molecules are more likely to escape in a dry environment. The final composite particles are shown in Fig. 9. In order to explore the relative structure between organic coating layer and ANPs, Fig. 10 shows the cross- sectional charge distribution and atomic potential energy distribution of the final configuration of cANPs. The Al atoms on the surface of ANPs are strongly positively charged, while the ethanol ether molecules infiltrated into the surface of ANPs are strongly negatively charged. Table 1 Computational materials science NC-alcohol ether solution molecular number for sANPs, csANPs and cANPs. NC-alcohol ether solution molecular number for sANPs, csANPs and cANPs. NC ethanol ether sANPs 0 0 0 csANPs 3 1000 562 cANPs 3 1000 562 The whole simulation process is carried out under the NVT ensemble. The ANPs temperature is set at 200 K, which can ignore the thermal vibration of the base atom. All aluminum atoms can retain the interaction with other atoms while fixing the site. The temperature of NC molecular chain was selected as 300 K to ensure the structural stability of NC molecular chain. The temperature of ethanol ether solution was NC ethanol ether sANPs 0 0 0 csANPs 3 1000 562 cANPs 3 1000 562 Th h l i l ti i i d t d th NVT bl Th ANP t t i t t 200 The whole simulation process is carried out under the NVT ensemble. The ANPs temperature is set at 200 h h h h l b f h b ll l h The whole simulation process is carried out under the NVT ensemble. The ANPs temperature is set at 200 K, which can ignore the thermal vibration of the base atom. All aluminum atoms can retain the interaction with other atoms while fixing the site. The temperature of NC molecular chain was selected as 300 K to ensure the structural stability of NC molecular chain. The temperature of ethanol ether solution was Page 7/32 Page 7/32 selected as 500 K, and the higher solution temperature could make up for the limitation of time scale, help us get similar simulation results to those in the experiment (time-scale: ms) within a reasonable time range (time-scale: ps). According to the study of Liu et al., 500 K does not break the results of organic molecules at short time [17], and can effectively inhibit the formation of hydrogen bonds, which is conducive to the development of coating. Berendsen control theory, which is similar to hot bath, is used in temperature control. The temperature damping coefficient is set to 25 fs. In consideration of the possible of dissociation and bonding in the package process, the time step is 0.25 fs, and the whole simulation lasts 100 ps. Figure 7 shows the molecular isothermal adsorption curve of NC/ethanol/ether solution coated annealing ANPs and passivation ANPs. Table 1 Computational materials science A new bond pair is formed between the Al atoms on the particle surface and the ethanol ether molecules, and the charge transfer also occurs between the bonding atoms. Therefore, this layer is a chemical adsorption layer. From Fig. 10, we can also find that the atomic potential energy of NC/ethanol/ether solution in the chemical adsorption layer is Page 8/32 Page 8/32 Page 8/32 low, and the adsorption configuration is more stable. The outside ethanol ether molecule has a weak charge, indicating that the molecules outside the coating layer interact through non-bonding forces such as Van der Waals force and Coulomb force, so this layer is a physical adsorption layer. The atomic potential energy in the physical cladding layer is high, and the structure is relatively unstable. NC/ th l/ th l ti i till th lt f th bi ti f h i l d ti d h i l low, and the adsorption configuration is more stable. The outside ethanol ether molecule has a weak charge, indicating that the molecules outside the coating layer interact through non-bonding forces such as Van der Waals force and Coulomb force, so this layer is a physical adsorption layer. The atomic potential energy in the physical cladding layer is high, and the structure is relatively unstable. NC/ethanol/ether solution is still the result of the combination of physical adsorption and chemical adsorption. In addition, the O and N atoms in the NC molecular chain also have strong charge values, which also reveals the hydrogen bond between the ethanol ether molecule and the NC molecular chain. y p y p y potential energy in the physical cladding layer is high, and the structure is relatively unstable. NC/ethanol/ether solution is still the result of the combination of physical adsorption and chemical adsorption. In addition, the O and N atoms in the NC molecular chain also have strong charge values, which also reveals the hydrogen bond between the ethanol ether molecule and the NC molecular chain. We calculate the radial distribution functions of different atom pairs, as shown in Fig. 11, to quantitatively study hydrogen bonding between the surface structure of ANPs and NC/ethanol/ether solution of the final configuration ANPs. From Fig. Table 1 Computational materials science 11 (a), we can find that the surface O atoms of ANPs are mostly located near the Al atom about 1.95 Å, indicating that the outermost Al atoms of ANPs tend to form an alumina layer provided by organic inclusion. The difference between the first peak values of g (Al-EO) and g (Al-DO) curves of NC/ethanol/ether coating was small, indicating that the number of ethanol molecules and ether molecules involved in the chemical adsorption of ANPs surface was similar. In addition, the peak value of g (Al-NO) was relatively low, and the first peak was located near 1.8 Å, indicating that the part of NC molecular chain involved in chemical adsorption was less, and it was more inclined to adsorb on the surface of ANPs by chemical adsorption at one end or one section of the molecular chain. The adsorption strength was stronger than ethanol ether molecules. The other segments were adsorbed on the surface of ANPs by physical adsorption through the traction of the chemical adsorption section and hydrogen bonding. Figure 11 (b) calculated the radial distribution function of H-O and H-N atomic pairs between NC molecular chains and ethanol ether molecules. From Fig. 11 (b) we can find that the ethanol molecule is more likely to form hydrogen bonds with the NC molecular chain. The reason is that the polar-OH in the ethanol molecule and the polar-OH and-ONO2 groups in the NC molecular chain are more likely to form hydrogen bonds through Coulomb interaction. Ether molecule is not easy to form hydrogen bonds in the NC molecular chain, which can prevent large agglomeration and make the NC molecular chain dissolve smoothly to form a homogeneous solution. molecular chain are more likely to form hydrogen bonds through Coulomb interaction. Ether molecule is not easy to form hydrogen bonds in the NC molecular chain, which can prevent large agglomeration and make the NC molecular chain dissolve smoothly to form a homogeneous solution. To analyze the adsorption behavior of NC molecular chains on the surface of csANPs intuitively, Fig. 12 is the structural snapshot of the final configuration of csANPs. The -NO2 and -OH groups in NC chains are adsorbed on the surface of ANPs, accompanied by the dissociation of -NO2 and -OH groups. The atoms in the separated-NO2 and-OH groups will not be further separated, and it is difficult to penetrate into the passivated ANPs. Table 1 Computational materials science Therefore, NC molecular chains are still adsorbed on the surface of passivated ANPs by physical and chemical adsorption. Active Al atom is the main reactant of fuel. After ANP is completely coated, the content of active Al is a very important detection index. Table 2 shows the content of active Al atoms in three models. According to the oxidation degree of Al atom, we divide it into active Al atom, oxidation potential Al atom and oxidation Al atom. For passivated ANPs, the Al atom with charge less than the average charge in the nuclear region is defined as the active Al atom; Al atom whose charge is more than the average charge in the shell is defined as oxidation Al atom; the charge between the average charge in the core region and Page 9/32 Page 9/32 Page 9/32 shell region is defined as oxidation potential Al atom. For the cANPs model, due to there isn't passivation treatment and the concept of average shell charge is not available, the Al atom whose charge is less than 0.5 is defined as active Al atom. Al atom with charge more than 1 is defined as oxidized Al atom; the charge between 0.5 and 1 is defined as oxidation potential Al atom. Table 2 shows that NC/ethanol/ether coating treatment could reduce the content of oxidation Al atom. However, no matter which model, the content of unoxidized Al atom is above 70%. Table 2 The Al atomic activity for sANPs, csANPs and cANPs. active Al atom oxidation potential Al atom oxidation Al atom sANPs 27.4% 42.6% 30.0% csANPs 22.1% 51.8% 26.1% cANPs 52.00% 38.20% 9.80% 3.3 MD simulations of heating and combustion processes 3.3.1 Heating period 3.3 MD simulations of heating and combustion processes 3.3.1 Heating period 3.3.1 Heating period After the evaporation-elimination procedure, the ignition and combustion stage can be carried out. In order to verify the effect of NC/ethanol/ether coating layer on the heating and combustion performance of ANPs, we set up three combustion models of sANPs, csANPs and cANPs. In the heating period, the three ANPs models were put in a 110 × 110 × 110 Å3 box, and 1200 O2 molecules were randomly put around the ANPs. The specific parameters of the heating model are as follows: Firstly, the heating system is relaxed at 300 K, and then the system temperature is increased to 1000 K in 70 ps under the NVT ensemble. According to the ignition characteristics of ANPs, 1000K is enough to ignite ANPs. At present, although there are many controversies about the ignition mechanism of ANPs, the diffusion movement of atoms is considered to be one of the important ideas about the ignition mechanism of ANPs [39, 40]. Figure 13 calculated the mean square displacement curves of ethanol, ether and NC atoms during heating period of csANPs and cANPs. At the initial heating period (– 20 ps), the atomic mobility of each component increased slowly, and the coating layer won't parsing clearly at low temperature. After 20 ps, the atomic mobility of ether and ethanol began to increase rapidly. At this time, the ether and ethanol molecules began to parsing and diffuse rapidly, while the parsing and diffusion of NC lagged significantly. In addition, ether molecules showed higher mobility during ignition, indicating that the parsing and diffusion of ether were earlier than ethanol. Due to the strong of physical adsorption in csANPs coating layer, csANPs coating layer atoms have higher mobility than cANPs. In order to observe the diffusion movement of each component atom intuitively in heating period, Fig. 14 shows the central section configuration diagram of sANPs, csANPs and cANPs systems at different Page 10/32 Page 10/32 Page 10/32 ignition times. At the initial heating period (20 ps), O2 molecules adsorbed on the surface of ANPs gradually, but it was difficult to penetrate the oxide/coating layer. Because of coating layer, csANPs and cANPs can adsorb more O2 molecules around them than sANPs. It means organic coating layer was beneficial to the adsorption of O2 molecules. With the increase of temperature (20–40 ps), the Al core atoms begin to melt, and the melting temperature of Al core decreases significantly. 3.3.1 Heating period With the melting of Al core, the O atom in the oxide shell (sO) begins to diffuse inward, while the Al atom in the aluminum core (cAl) begins to diffuse outward. As for cANPs, due to without oxide layer, organic coating layer atoms began inward diffuse at this stage, which makes the surface structure of ANPs looser. At 40–70 ps, the molecules ethanol and ether molecules in the coating layer were obviously exfoliated, while the NC macromolecular chain was still around the surface of ANPs although it showed a certain degree of detachment. With the detachment of coating layer, O2 molecules were further adsorbed on the surface of ANPs even diffused into ANPs, which promoted the ignition of ANPs. The pyrolysis behavior of NC molecular chains during heating period is also interesting. Figure 15 shows the change rates of C-O and N-O bond pairs number with time in the heating period of NC. At about 25 ps (549 K), the NC molecular chains in the coating layer of csANPs and cANPs began to pyrolysis. NC pyrolysis also releases heat, increases the surface temperature of ANPs particles which can shorten the ignition time of ANPs. Figure 16 shows the MSD curves of cAl atoms during heating period of sANPs, csANPs and cANPs. At the initial ignition stage, the MSD curve of cAl atom in csANPs and cANPs was lower than that of sANPs, which means NC/ethanol/ether coating can improve the stability of active Al core atoms at low temperature. After that, the MSD curves of cAl atoms in csANPs and cANPs increased rapidly, and exceeded sANPs at about 22 ps and 15 ps respectively. With the increase of temperature, the active Al core in csANPs and cANPs showed higher atomic mobility. High atomic mobility makes the active cAl atoms in ANPs diffuse to the surface and react with external O2 molecules faster, which also reveals one of the reasons why NC/ethanol/ether coating layer can shorten the ignition delay of ANPs. The direct contact reaction of external O2 molecules with active Al atoms in ANPs is usually considered to be an important indicator of ANPs ignition. Figure 17 shows the variation of the O atoms' number in the range 2 Å of ANPs surface during heating period. At the beginning of ignition, the oxygen adsorption amount of csANPs and cANPs was much lower than sANPs. 3.3.2 Combustion period In the combustion period, the simulation box is expanded to 150 × 150 × 150 Å3, and the ignition process parameters are same with 3.3.1. Due to the research background is the improvement of aluminum- containing propellant, the combustion temperature of aluminum-containing propellant in the combustion chamber can stabilized at about 3000 K continuously. Therefore, after the heating period, the ensemble is changed from NVE to NVT, and the simulation temperature is set at 3000 K [41]. The three models are stabilized at 3000 K for combustion simulation. Figure 19 shows the rectangular central cross-sectional views along the Y-axis and the atomic displacement scatter plots at different times of the three systems' constant-temperature combustion at different times (with the atomic positions 5 ps before this time as a reference). The width of rectangular section is 15 Å, the transverse coordinate of scatter plot corresponds to the radial distribution along the Y axis, and the ordinate represents the absolute displacement of each atom before 5 ps. According to the absolute value of atomic displacement in 5 ps, the displacement scatter diagram of combustion system can roughly divide into gas phase region and solid-liquid phase region roughly. For sANPs and csANPs, because 3000 K is much higher than the melting point of Al core and oxide shell, the cAl atom in the central region and the sO atom in the oxide shell diffuse rapidly and the shell-core structure completely disappear at 10 ps. Figure 19 (a) shows that the displacement of cAl and sO atoms tend to the same level at 10 ps, indicating that there is an almost uniform distribution of cAl-sO mixed configuration in sANPs. In this stage, the sAl atoms are still mainly distributed in the outer oxide shell region, resulting the movement ability of the external O2 molecules is strong, it is mainly adsorbed in the oxide shell region, which is difficult to penetrate into the inner layer of sANPs. At this stage, due to the inward diffusion of sO, releasing more partial coordination of sAl, which makes it exhibit excellent oxidation potential. After 20 ps, sO and eO atoms began to diffuse to the inner region of the particles, and the combustion began to propagate inward and formed a diffusion-dominated reaction zone. Finally, sO and eO atoms propagate to the core area of particles, and all components gradually tend to be uniform. 3.3.1 Heating period With the increase of temperature, the oxygen adsorption amount of csANPs and cANPs quickly rising and exceeded the sANPs. Which further proof that NC/ethanol/ether coating will inhibit external O2 molecules contact with ANPs at low temperature, and promote external O2 molecules adsorption and reaction with ANPs at high temperature. Figure 18 shows the radial distribution function of cAl-eO atom during ignition period. The RDF curves in sANPs, csANPs and cANPs systems have the same trend over time. From Fig. 18, the first peak is about 1.9 Å, corresponding to the Al-O bond length. At 0–20 ps, the first peak value of csANPs was significantly lower than sANPs, indicating that the coating inhibits the external O2 molecules contact with Al core Page 11/32 Page 11/32 atoms at low temperatures. With the increase of temperature, the first peak value of csANPs increased rapidly and exceeded sANPs. It is because at low temperature, the O2 molecules adsorbed by the coating layer react rapidly with the reaction sites released on the surface of ANPs after coating layer resolution, which also reveals another important mechanism of coating layer shortening the ignition delay of ANPs. Finally, with the increase of temperature, RDF curve appeared obvious second and third peaks. It shows that the reaction between O2 and cAl atoms gradually diffuses inward, forming a long-range ordered oxidation structure. 3.3.2 Combustion period In addition, after 20 ps, although the displacement of each component in sANPs also gradually tends to be consistent, the number of external O2 molecules continues to decline. So, at this time, sANPs enter the uniform combustion stage, and the combustion is still ongoing. Due to the influence of reaction temperature, the whole sANPs finally showed a uniform liquid structure. From the Fig. 19 (b), for csANPs, the NC/ethanol/ether coating layer was rapidly desorbed at 3000 K, most of which diffused to the gas Page 12/32 Page 12/32 phase region, and only a small part was directly involved in the surface reaction of csANPs and reacted with sAl atoms. Before 10 ps only a few O2 molecules can react with sAl atoms, however the combustion process of particles will not be slowed down due to the NC molecules get heat and rapid pyrolysis releases heat. After 20 ps, sAl atoms began to diffuse to the inner region, and also led to the inward diffusion of eO and organic atoms (mainly EO and DO atoms), entering the combustion propagation stage. Compared with sANPs, the solid-liquid phase structure of csANPs is more complex due to the participation of NC/ethanol/ether coating layer components. At the later stage of combustion, csANPs transformed into Al-O-C mixed structure. In the central section configuration diagram at 60 ps, C atoms are mainly distributed in the outer layer of particles, forming a large number of Al-C bonds. It is also consistent with the reports on the formation of carbon inclusion products in Al-containing propellants combustion experiments [42]. In addition, the atomic displacement scatter plot also shows that the displacement of each component in the csANPs system doesn't decrease in the sANPs system at the later stage of combustion, but shows an upward trend, which further indicates that the diffusion and pyrolysis of NC/ethanol/ether coating layer will accelerate the multiphase reaction and promote csANPs combusting more sufficient. From the Fig. 19 (c), the combustion process of cANPs is much faster than sANPs and csANPs. Due to the Al atoms in the oxidation shell provided by cANPs still have strong oxidation potential, the outside Al atoms outside react violently at the beginning of combustion, resulting in a rapid decrease in the density inside the particles, and even appear a cavity phenomenon at 10 ps. 3.3.2 Combustion period In addition, the H atoms infiltrated in the coating process will generate H2 molecules in ANPs firstly, and then release to the gas phase region to participate in gas phase combustion. Because cANPs don't have the oxide layer, the atom diffusivity is stronger, showing higher combustion propagation rate and combustion efficiency. At the late combustion stage, cANPs were also transformed into Al-O-C mixture, which was consistent with csANP. The diffusion and pyrolysis of NC/ethanol/ether coating layer accelerate the multiphase reaction and improve the combustion performance of cANPs. In order to further quantitatively analyze the ANPs component distribution in the combustion process, Fig. 20 shows the number of O atoms and C atoms adsorbed with time in the 2 Å range of Al atom. At the early stage of combustion, the O atom adsorption rate of sANPs and cANPs was almost the same, slightly faster than csANPs. The reason is that the sO atoms in the sANPs oxidation shell diffuse inward rapidly at the early stage of combustion, and the Al-rich environment is formed on the particle surface, thus adsorbing the external O2 molecules rapidly and generating oxidation reaction. However as for cANPs, due to the cladding layer involved in the surface reaction, the initial O2 adsorption rate slowed down. As for cANPs, due to the insufficient coordination number of Al atoms in the oxide layer, they also have the power to absorb external O2 molecules rapidly. On the other hand, at the end of combustion, the oxygen adsorption rate of sANPs tends to be constant, while the adsorption rate of cANPs begins to decrease, and csANPs even begin to release some adsorbed O atoms. It is because that the further combination of Al-C bonds in the later stage, occupying the outer space of ANPs. The C atom adsorption curve also shows that csANPs and cANPs adsorb large amounts of C atoms at the early stage of combustion and increase steadily at the later stage. Therefore, C atom not only is a high-quality Page 13/32 Page 13/32 additional fuel source for gas phase reaction, but also participates in the solid-liquid phase combustion reaction outside ANPs directly. Figure 21 shows the potential energy curves of sANPs, csANPs and cANPs during combustion. Compared with sANPs, the decline rate of potential energy curve in the early stage of the system was similar for csANPs and cANPs, and higher than sANPs significantly. 3.3.2 Combustion period It indicated that csANPs and cANPs had a more intense combustion reaction. The slope of the potential energy curve of the sANPs tended to be constant in the late stage, and entered a stable and uniform combustion stage. On the other hand, the interesting thing is that the potential curves of csANPs and cANPs tend to flatten at about 55 ps and 30 ps respectively, while the potential curves of cANPs begin to fall again at about 65 ps. The reason is that the existence of NC/ethanol/ether coating layer causes the gas phase combustion in csANPs and cANPs systems, and the gas phase combustion in cANPs system starts earlier. The breaking of the bonds of organic molecules such as NC leads to the system potential energy gradual even increase, while the large generation of gaseous molecules leads to the system potential energy continuous decrease. In addition, the potential energy curves of csANPs and cANPs also revealed that the existence of NC/ethanol/ether coating layer not only brought the occurrence of multiphase reactions, but also increased the reaction time of ANPs combustion system. It will make the combustion of ANPs more fully and the energy release time longer, also improving the application potential in propellants. 4. Conclusions In this study, we used MD simulations to simulate three models: passivated ANPs, NC/ethanol/ether coating passivated ANPs and NC/ethanol/ether coating annealed ANPs. The analysis was carried out from the organic coating process to the ignition and combustion stages. The simulation results show that the ReaxFF-lg long-range correction reaction force field can not only capture the accurate changes of products, but also accurately capture the formation and fracture of bonds in the reaction process. During the pyrolysis of NC single-chain molecules, the C-O-C bond between the rings will break. And NC will decompose from the macromolecular chain into small molecules, while the C-O-C bond fracture in the NC molecular chain is relatively lagging behind, which supports the research view of Wang et al., and also proves the applicability of the ReaxFF-lg long-range correction reaction force field in this study. In the preparation of NC/ethanol/ether coated ANPs model, we found that NC/ethanol/ether solution formed a dense coating layer on the surface of annealed ANPs and passivated ANPs, which was the result of the combined action of physical and chemical adsorption. The inner side of the coating layer is a chemical adsorption layer composed of surface Al atoms and coating solution atoms. The outer side of the coating layer is a physical coating layer composed of organic molecules and residues under the action of Coulomb force and van der Waals force. In the ignition and combustion simulation of the three models, we found that at low temperature, the coating layer blocked the contact between the active Al core atoms and the external O2 molecules, and increased the O2 molecular density around ANPs through adsorption. With the increase of ignition temperature and the accumulation of NC pyrolysis energy release, the NC/ethanol/ether coated ANPs have higher surface temperature, and the active Al atoms in the aluminum Page 14/32 Page 14/32 core can diffuse to the particle surface faster. At the same time, the desorption and diffusion of organic coating layer exposed more reaction sites, and the high-density O2 molecules around ANPs were easier to adsorb to the surface for reaction. Thus, NC/ethanol/ether coated ANPs have shorter ignition delay and lower ignition temperature. In the combustion stage, ANPs experienced surface oxidation/core melting diffusion, combustion inward propagation and uniform combustion three stages. CRediT authorship contribution statement Xin Jin: Writing - editing. Lei Wang: Writing - original draft & review & editing. Pingan Liu: Conceptualization, Methodology, Resources, Funding acquisition. Penghua Sui: Formal analysis, Software. Declarations The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 4. Conclusions After combustion beginning, the NC/ethanol/ether coating layer desorbs and diffuses to the gas phase region rapidly, and a small part is involved in the surface oxidation of ANPs directly. The energy released by NC pyrolysis and gas phase reaction promotes the atom diffusion in ANPs and accelerates the combustion propagation rate. In addition, the C atoms in the coating layer is not only a high-quality additional fuel source for the gas phase reaction, but also participates in the solid-liquid phase combustion reaction outside ANPs directly, increasing the combustion time and efficiency of ANPs. References 1. Liangui Guo, Wulin Song, Mulin Hu, Changsheng Xie, Xia Chen. Preparation and reactivity of aluminum nanopowders coated by hydroxyl-terminated polybutadiene (HTPB) [J]. Applied Surface Science. 2008, 254: 2413–2417. 2. Sundaram D S, Puri P, Yang V.Pyrophoricity of nascent and passivated aluminum particles at nano- scales[J].Combustion & Flame,2013, 160 (9): 1870–1875. 3. Junqing Yang, Xuedong Gong, Guixiang Wang. Density functional theory and molecular dynamic investigations on the energetic and mechanical properties of nitrocellulose/nitroglycerin/ Page 15/32 Page 15/32 Page 15/32 pentaerythritol diazido dinitrate composites [J]. Polymer Composites. 2017, 38: 192–198. 4. Yan T, Ren H, Li Y, et al.Tailoring Structural Energetics for Enhanced Reactivity of Nano-Aluminum Particles Based Microspheres[J].Advanced Engineering Materials,2019, 21 (8). 5. Rohit J. Jacob, Boran Wei, Michael R. Zachariah. Quantifying the enhanced combustion characteristics of electrospray assembled aluminum mesoparticles [J]. Combustion and Flame. 2016, 167: 472–480. 6. Haiyang Wang, Guoqiang Jian, Shi Yan, Jeffery B Delisio, Chuan Huang, Michael R Zachariah. Electrospray formation of gelled nano-aluminum microspheres with superior reactivity [J]. ACS Applied Materials & Interfaces. 2013, 5: 6797–6801. 7. Wang H, Jian G, Yan S, et al.Electrospray formation of gelled nano-aluminum microspheres with superior reactivity[J].Acs Appl Mater Interfaces,2013, 5 (15): 6797–6801. 8. S. Alavi, D.L. Thompson, Molecular dynamics simulations of the melting of alu-minum nanoparticles, J. Phys. Chem. A 110 (4) (2006) 1518–1523. 8. S. Alavi, D.L. Thompson, Molecular dynamics simulations of the melting of alu-minum nanoparticles, J. Phys. Chem. A 110 (4) (2006) 1518–1523. 9. E. El Koraychy, M. Meddad, M. Badawi, M. Mazroui, Sintering and deposition of homo- and heteronanoparticles of aluminum and nickel on aluminum (100) substrate, Chem. Phys. 541 (2021) 9, 9. E. El Koraychy, M. Meddad, M. Badawi, M. Mazroui, Sintering and deposition of homo- and heteronanoparticles of aluminum and nickel on aluminum (100) substrate, Chem. Phys. 541 (2021) 9, https://doi.org/10.1016/j chemphys.2020.111037. 10. J.S. Raut, R.B. Bhagat, K.A. Fichthorn, Sintering of aluminum nanoparticles: a molecular dynamics study, Nanostruct. Mater. 10 (5) (1998) 837–851, https://doi.org/10.1016/s0965-9773(98)00120-2. 10. J.S. Raut, R.B. Bhagat, K.A. Fichthorn, Sintering of aluminum nanoparticles: a molecular dynamics study, Nanostruct. Mater. 10 (5) (1998) 837–851, https://doi.org/10.1016/s0965-9773(98)00120-2. 11. H.L. Zhu, R.S. Averback, Sintering processes of two nanoparticles: a study by molecular-dynamics, Phil. Mag. Lett. 73 (1) (1996) 27–33, https://doi.org/10.1080/095008396181073. 11. H.L. Zhu, R.S. Averback, Sintering processes of two nanoparticles: a study by molecular-dynamics, Phil. Mag. Lett. 73 (1) (1996) 27–33, https://doi.org/10.1080/095008396181073. 12. S. Arcidiacono, N.R. Bieri, D. Poulikakos, C.P. References 15 (6) (2018) 1350–1357, https://doi.org/10.1111/ijac.12905. 20. M. Sabzi, S.H.M. Anijdan, M. Asadian, The effect of substrate temperature on microstructural evolution and hardenability of tungsten carbide coating in hot filament chemical vapor deposition, Int. J. Appl. Ceram. Technol. 15 (6) (2018) 1350–1357, https://doi.org/10.1111/ijac.12905. 21. S.H.M. Anijdan, M. Sabzi, M. Asadian, H.R. Jafarian, Effect of sub-layer temperature during HFCVD process on morphology and corrosion behavior of tungsten carbide coating, Int. J. Appl. Ceram. Technol. 16 (1) (2019) 243–253, https://doi.org/10.1111/ijac.13087. 21. S.H.M. Anijdan, M. Sabzi, M. Asadian, H.R. Jafarian, Effect of sub-layer temperature during HFCVD process on morphology and corrosion behavior of tungsten carbide coating, Int. J. Appl. Ceram. Technol. 16 (1) (2019) 243–253, https://doi.org/10.1111/ijac.13087. 22. I. Danilenko, M. Lakusta, L. Loladze, G. Volkova, I. Popov, V. Glazunova, T. Konstantinova, Effect of alumina added by mechanical mixing and co-doping on the densification mechanisms of zirconia nanoparticles at the initial stage of sintering, Results Phys. 19 (2020) 10, https://doi.org/10.1016/j.rinp.2020.103495. 23. M. Sabzi, S.M. Far, S.M. Dezfuli, Characterization of bioactivity behavior and corrosion responses of hydroxyapatite-ZnO nanostructured coating deposited on NiTi shape memory alloy, Ceram. Int. 44 (17) (2018) 21395–21405, https://doi.org/10.1016/j.ceramint.2018.08.197. 23. M. Sabzi, S.M. Far, S.M. Dezfuli, Characterization of bioactivity behavior and corrosion responses of hydroxyapatite-ZnO nanostructured coating deposited on NiTi shape memory alloy, Ceram. Int. 44 (17) (2018) 21395–21405, https://doi.org/10.1016/j.ceramint.2018.08.197. 24. M. Sabzi, S.M. Dezfuli, Deposition of Al2O3 ceramic film on copper-based heterostructured coatings by aluminizing process: study of the electrochemical responses and corrosion mechanism of the coating, Int. J. Appl. Ceram. Technol. 16 (1) (2019) 195–210, https://doi.org/10.1111/ijac.13072. 24. M. Sabzi, S.M. Dezfuli, Deposition of Al2O3 ceramic film on copper-based heterostructured coatings by aluminizing process: study of the electrochemical responses and corrosion mechanism of the coating, Int. J. Appl. Ceram. Technol. 16 (1) (2019) 195–210, https://doi.org/10.1111/ijac.13072. 25. A.L. Ahmad, S.C. Low, S.R. Abd. Shukor, W.J.N. Fernando, A. Ismail. Hindered diffusion in lateral flow nitrocellulose membrane: Experimental and modeling studies [J]. Journal of Membrane Science. 2010, 357: 178–184. 25. A.L. Ahmad, S.C. Low, S.R. Abd. Shukor, W.J.N. Fernando, A. Ismail. Hindered diffusion in lateral flow nitrocellulose membrane: Experimental and modeling studies [J]. Journal of Membrane Science. 2010, 357: 178–184. 26. Dave T.F. Kuo, Michael Simini, Herbert E. Allen. Sorption and desorption kinetics of nitroglycerin and 2,4-dinitrotoluene in nitrocellulose and implications for residue-bound energetic materials [J]. Water Research. 2018, 128: 138–147. 27. Waldemar A. Trzcinski, Stanisław Cudzilo, Sławomir Dyjak, Leszek Szymanczyk. References Grigoropoulos, On the coalescence of gold nanoparticles, Int. J. Multiphas. Flow 30 (7–8) (2004) 979–994, https://doi.org/10.1016/j.ijmultiphaseflow.2004.03.006. 13. P.X. Song, D.S. Wen, Molecular dynamics simulation of the sintering of metallic nanoparticles, J. Nanoparticle Res. 12 (3) (2010) 823–829, https://doi.org/10.1007/s11051-009-9718-7. 13. P.X. Song, D.S. Wen, Molecular dynamics simulation of the sintering of metallic nanoparticles, J. Nanoparticle Res. 12 (3) (2010) 823–829, https://doi.org/10.1007/s11051-009-9718-7. 14. J.P. Liu, M.J. Wang, P.G. Liu, R.C. Sun, Y.X. Yang, G.W. Zou, Molecular dynamics study of sintering of Al nanoparticles with/without organic coatings, Comput.Mater. Sci. 190 (2021) 10, https://doi.org/10.1016/j.commatsci.2020.110265. 14. J.P. Liu, M.J. Wang, P.G. Liu, R.C. Sun, Y.X. Yang, G.W. Zou, Molecular dynamics study of sintering of Al nanoparticles with/without organic coatings, Comput.Mater. Sci. 190 (2021) 10, https://doi.org/10.1016/j.commatsci.2020.110265. 15. J. Nandy, S. Sahoo, N. Yedla, H. Sarangi, Molecular dynamics simulation of coalescence kinetics and neck growth in laser additive manufacturing of aluminum alloy nanoparticles, J. Mol. Model. 26 (6) (2020) 14, https://doi.org/10.1007/s00894-020-04395-4. 15. J. Nandy, S. Sahoo, N. Yedla, H. Sarangi, Molecular dynamics simulation of coalescence kinetics and neck growth in laser additive manufacturing of aluminum alloy nanoparticles, J. Mol. Model. 26 (6) (2020) 14, https://doi.org/10.1007/s00894-020-04395-4. 16. Pengxiang Song,Dongsheng Wen, Molecular dynamics simulation of the sintering of metallic Nanoparticles, J Nanopart Res (2010) 12:823–829,DOI 10.1007/s11051-009-9718-7 16. Pengxiang Song,Dongsheng Wen, Molecular dynamics simulation of the sintering of metallic Nanoparticles, J Nanopart Res (2010) 12:823–829,DOI 10.1007/s11051-009-9718-7 17. Junpeng Liu, Pingan Liu, Mengjun Wang, Wenchao Wang, Fangwei Lv, Ruochen Sun,Yanxi Yang, Combustion of Al nanoparticles coated with ethanol/ether molecules by non-equilibrium molecular dynamics simulations, Materials Today Communications. 22 (2020) 100819, https://doi.org/10.1016/j.mtcomm.2019.100819. Page 16/32 18. M. Sabzi, S.H.M. Anijdan, M. Ghobeiti-Hasab, M. Fatemi-Mehr, Sintering variables optimization, microstructural evolution and physical properties enhancement of nano-WC ceramics, J. Alloys Compd. 766 (2018) 672–677, https://doi.org/10.1016/j.jallcom.2018.07.006. 19. M. Sabzi, S.M. Dezfuli, A study on the effect of compositing silver oxide nanoparticles by carbon on the electrochemical behavior and electronic properties of zinc-silver oxide batteries, Int. J. Appl. Ceram. Technol. 15 (6) (2018) 1446–1458, https://doi.org/10.1111/ijac.13047. 19. M. Sabzi, S.M. Dezfuli, A study on the effect of compositing silver oxide nanoparticles by carbon on the electrochemical behavior and electronic properties of zinc-silver oxide batteries, Int. J. Appl. Ceram. Technol. 15 (6) (2018) 1446–1458, https://doi.org/10.1111/ijac.13047. 20. M. Sabzi, S.H.M. Anijdan, M. Asadian, The effect of substrate temperature on microstructural evolution and hardenability of tungsten carbide coating in hot filament chemical vapor deposition, Int. J. Appl. Ceram. Technol. References Experimental and theoretical investigation of a model reactive armour with nitrocellulose and cellulose composites [J]. Central European Journal of Energetic Materials. 2013, 10: 191–207. 27. Waldemar A. Trzcinski, Stanisław Cudzilo, Sławomir Dyjak, Leszek Szymanczyk. Experimental and theoretical investigation of a model reactive armour with nitrocellulose and cellulose composites [J]. Central European Journal of Energetic Materials. 2013, 10: 191–207. 28. Plimpton Steve. Fast Parallel Algorithms for Short-Range Molecular Dynamics [J]. Journal of Computational Physics, 1995, 117(1): 1–19. 28. Plimpton Steve. Fast Parallel Algorithms for Short-Range Molecular Dynamics [J]. Journal of Computational Physics, 1995, 117(1): 1–19. 29. Hunphrey W., Dalke, A., Schulten, K. VMD:Visual Molecular Dynamics [J]. Mol Graphics, 1996, 14: 33–38. 29. Hunphrey W., Dalke, A., Schulten, K. VMD:Visual Molecular Dynamics [J]. Mol Graphics, 1996, 14: 33–38. 30. A.C.T. van Duin, et al., CHAPTER 6 a Reaxff reactive force-field for proton transfer reactions in bulk water and its applications to heterogeneous catalysis, Computational Catalysis, The Royal Society of Chemistry, 2014, pp. 223–243. 30. A.C.T. van Duin, et al., CHAPTER 6 a Reaxff reactive force-field for proton transfer reactions in bulk water and its applications to heterogeneous catalysis, Computational Catalysis, The Royal Society of Chemistry, 2014, pp. 223–243. Page 17/32 Page 17/32 31. Strachan A, Kober E M, Duin A V, et al.Thermal decomposition of RDX from reactive molecular dynamics[J].Journal of Chemical Physics,2005, 122 (5): 54502. 32. Liu Y, Goddard W A.First-Principles-Based Dispersion Augmented Density Functional Theory: From Molecules to Crystals[J].Journal of Physical Chemistry Letters,2010, 1 (17): 2550–2555. 33. Junpeng Liu, Mengjun Wang, P. Liu, Molecular dynamical simulations of melting Al nanoparticles using a reaxff reactive forcefield, Mater. Res. Express 5 (6) (2018). 34. Pingan Liu, Junpeng Liu, Mengjun Wang, Ignition and combustion of nano-sized aluminum particles: A reactive molecular dynamics study, Combustion and Flame 201 (2019) 276–289 34. Pingan Liu, Junpeng Liu, Mengjun Wang, Ignition and combustion of nano-sized aluminum particles A reactive molecular dynamics study, Combustion and Flame 201 (2019) 276–289 35. S.D. Tommaso, et al., Oxidation mechanism of diethyl ether: a complex process for a simple molecule, Phys. Chem. Chem. Phys. 13 (32) (2011) 14636–14645. 36. Zheng, Mei, Feng-Qi, et al.Reactive molecular dynamics simulation of thermal decomposition for nano-aluminized explosives[J].Physical chemistry chemical physics: PCCP,2018. 36. Zheng, Mei, Feng-Qi, et al.Reactive molecular dynamics simulation of thermal decomposition for nano-aluminized explosives[J].Physical chemistry chemical physics: PCCP,2018. 37. References Jutier, Jj, Harrison, et al.A NONISOTHERMAL FOURIER-TRANSFORM INFRARED DEGRADATION STUDY OF NITROCELLULOSES DERIVED FROM WOOD AND COTTON[J].J APPL POLYM SCI,1987, 1987,33(4) (-): 1359–1375. 38. Martínez L, Andrade R A, Birgin E G, et al.PACKMOL: A package for building initial configurations for molecular dynamics simulations[J].Journal of Computational Chemistry,2010, 30 (13): 2157–2164. 38. Martínez L, Andrade R A, Birgin E G, et al.PACKMOL: A package for building initial configurations for molecular dynamics simulations[J].Journal of Computational Chemistry,2010, 30 (13): 2157–2164. 39. Trunov M A, Schoenitz M, Dreizin E L.Effect of polymorphic phase transformations in alumina layer on ignition of aluminum particles[J].Combustion Theory and Modelling,2006, 10 (4): 603–623. 39. Trunov M A, Schoenitz M, Dreizin E L.Effect of polymorphic phase transformations in alumina layer on ignition of aluminum particles[J].Combustion Theory and Modelling,2006, 10 (4): 603–623. 40. Chakraborty P, Zachariah M R.Do nanoenergetic particles remain nano-sized during combustion? [J].Combustion & Flame,2014, 161 (5). 40. Chakraborty P, Zachariah M R.Do nanoenergetic particles remain nano-sized during combustion? [J].Combustion & Flame,2014, 161 (5). 41. Doi R, Nakagaki M, Kuwahara T, et al. Surface Temperature of Agglomerated Aluminum Particle in the Reaction Zone of AP/HTPB Composite Propellants[C].Aiaa/sae/asee Joint Propulsion Conference,2013. 41. Doi R, Nakagaki M, Kuwahara T, et al. Surface Temperature of Agglomerated Aluminum Particle in the Reaction Zone of AP/HTPB Composite Propellants[C].Aiaa/sae/asee Joint Propulsion Conference,2013. 42. Ao W, Liu P, Yang W.Agglomerates, smoke oxide particles, and carbon inclusions in condensed combustion products of an aluminized GAP-based propellant[J].Acta Astronautica,2016, 129 (dec.): 147–153. 42. Ao W, Liu P, Yang W.Agglomerates, smoke oxide particles, and carbon inclusions in condensed combustion products of an aluminized GAP-based propellant[J].Acta Astronautica,2016, 129 (dec.): 147–153. Figures Figures Page 18/32 Page 18/32 Figure 1 (a) NC single molecular chain model, (b) NC amorphous cell pyrolysis simulation snapshot of initial configuration structure. Figure 1 (a) NC single molecular chain model, (b) NC amorphous cell pyrolysis simulation snapshot of initial configuration structure. (a) NC single molecular chain model, (b) NC amorphous cell pyrolysis simulation snapshot of initial configuration structure. Page 19/32 Figure 2 (a) the initial configuration and (b) optimized configuration of core-shell ANP used in this paper. The Al core diameter 4 nm and the thickness of oxide shell is 0.5 Å. Atoms are colored by atom type: blue and dark gray for shell and core Al atoms, yellow for O atoms. Figure 2 Figure 2 (a) the initial configuration and (b) optimized configuration of core-shell ANP used in this paper. The Al core diameter 4 nm and the thickness of oxide shell is 0.5 Å. Atoms are colored by atom type: blue and dark gray for shell and core Al atoms, yellow for O atoms. (a) the initial configuration and (b) optimized configuration of core-shell ANP used in this paper. The Al core diameter 4 nm and the thickness of oxide shell is 0.5 Å. Atoms are colored by atom type: blue and dark gray for shell and core Al atoms, yellow for O atoms. (a) the initial configuration and (b) optimized configuration of core-shell ANP used in this paper. The Al core diameter 4 nm and the thickness of oxide shell is 0.5 Å. Atoms are colored by atom type: blue and dark gray for shell and core Al atoms, yellow for O atoms. (a) the initial configuration and (b) optimized configuration of core-shell ANP used in this paper. The Al core diameter 4 nm and the thickness of oxide shell is 0.5 Å. Atoms are colored by atom type: blue and dark gray for shell and core Al atoms, yellow for O atoms. Page 19/32 Figure 3 (a) Initial model (b) melting model (c) particle configuration of annealing model and (d) initial model (e) Figure 3 Figure 3 (a) Initial model (b) melting model (c) particle configuration of annealing model and (d) initial model (e) melting model (f) crystal type distribution of particle center section of annealing model. (a) Initial model (b) melting model (c) particle configuration of annealing model and (d) initial model (e) melting model (f) crystal type distribution of particle center section of annealing model. Page 20/32 Figure 4 Figure 4 Time-dependent curves of molecular number of key products during thermal decomposition of NC single molecular chain model. Figure 5 Figure 5 Snapshot of thermal decomposition process of O-NO2 bond. Snapshot of thermal decomposition process of O-NO2 bond. Snapshot of thermal decomposition process of O-NO2 bond Page 21/32 Page 21/32 Page 21/32 Figure 6 Snapshot of C-O-C bond decomposition over local structure in thermal decomposition process of NC single chain model Figure 6 Figure 6 Figure 6 Snapshot of C-O-C bond decomposition over local structure in thermal decomposition process of NC single chain model. Snapshot of C-O-C bond decomposition over local structure in thermal decomposition process of NC single chain model. Figure 7 Molecular isothermal adsorption curves during annealing ANPs coated with NC/ethanol/ether solution and passivation ANPs. Figure 7 Molecular isothermal adsorption curves during annealing ANPs coated with NC/ethanol/ether solution and passivation ANPs. Molecular isothermal adsorption curves during annealing ANPs coated with NC/ethanol/ether solution and passivation ANPs. Page 22/32 Figure 8 (a) cANP and (b) csANP filter out the molecular number of each component in the simulated envelope each time. Figure 8 Figure 8 (a) cANP and (b) csANP filter out the molecular number of each component in the simulated envelope each time. Figure 9 (a) cANP and (b) csANP final configurations. Figure 9 (a) cANP and (b) csANP final configurations. Figure 9 (a) cANP and (b) csANP final configurations. Figure 9 (a) cANP and (b) csANP final configurations. Page 23/32 Figure 10 The charge coloring diagram and atomic potential energy coloring diagram of the central section of cANPs’ the final configuration. Figure 10 The charge coloring diagram and atomic potential energy coloring diagram of the central section of cANPs’ the final configuration. The charge coloring diagram and atomic potential energy coloring diagram of the central section of cANPs’ the final configuration. Page 24/32 Page 24/32 Figure 11 Radial distribution function curves of related atom pairs in cANPs final configuration. (a) Al-NO, Al-EO and Al-DO atom pairs. (b) NH-EO, NH-DO, EH-NO, DH-NO, EH-NN and DH-NN atom pairs. NO: the O atom in NC molecular chain, EO: the O atom in ethanol molecules, DO: the O atom in ether molecules, NE: the H atom in NC molecular chain, EE: the H atom in ethanol molecules, DE: the H atom in ether molecules, NN: the N atom in NC molecular chain. Figure 11 Figure 11 Radial distribution function curves of related atom pairs in cANPs final configuration. (a) Al-NO, Al-EO and Al-DO atom pairs. (b) NH-EO, NH-DO, EH-NO, DH-NO, EH-NN and DH-NN atom pairs. NO: the O atom in NC molecular chain, EO: the O atom in ethanol molecules, DO: the O atom in ether molecules, NE: the H atom in NC molecular chain, EE: the H atom in ethanol molecules, DE: the H atom in ether molecules, NN: the N atom in NC molecular chain. Figure 12 Local Patterns of NC Adsorption in Simulated Final Configurations of csANPs. Local Patterns of NC Adsorption in Simulated Final Configurations of csANPs. Local Patterns of NC Adsorption in Simulated Final Configurations of csANPs. Page 25/32 Page 25/32 Figure 13 (a) csANPs, (b) cANPs mean square displacement curve of components in heating period. Figure 13 (a) csANPs, (b) cANPs mean square displacement curve of components in heating period. Page 26/32 Figure 14 sANPs、csANPs and cANPs configuration diagram of central section during heating period. Figure 14 sANPs、csANPs and cANPs configuration diagram of central section during heating period. Page 27/32 Page 27/32 Page 27/32 Page 27/32 Figure 15 The change rate curves of C-O and N-O bond pairs with time during NC ignition process in csANPs and cANPs models. Figure 15 The change rate curves of C-O and N-O bond pairs with time during NC ignition process in csANPs and cANPs models. Page 28/32 Figure 16 Figure 16 Mean square displacement diagram of cAl atom during ignition for sANPs、csANPs and cANPs. Mean square displacement diagram of cAl atom during ignition for sANPs、csANPs and cANPs. Figure 17 The adsorption quantity of eO atoms changing with time during heating period for sANPs、csANPs and cANPs. Figure 17 The adsorption quantity of eO atoms changing with time during heating period for sANPs、csANPs and ANP The adsorption quantity of eO atoms changing with time during heating period for sANPs、csANPs and cANPs. Page 29/32 Page 29/32 Page 29/32 Figure 18 Radial distribution function diagram of cAl-eO atom pair during heating period for (a) sANPs、(b) csANPs and (c) cANPs. Figure 18 Radial distribution function diagram of cAl-eO atom pair during heating period for (a) sANPs、(b) csANPs and (c) cANPs. Page 30/32 Figure 19 The snapshots of the rectangular central section along the Y axis and the atomic displace plot during the constant temperature combustion period. (a) sANPs、(b) csANPs and (c) cA Figure 19 The snapshots of the rectangular central section along the Y axis and the atomic displacement scatte plot during the constant temperature combustion period. (a) sANPs、(b) csANPs and (c) cANPs. Atomic coloring by atomic type. Grey: cAl atoms, yellow: O atoms, blue: sAl atoms, black: C atoms, purple: eO atoms, green: H atoms, red: O atoms. Figure 19 Figure 20 Curve of atomic adsorption quantity with time during combustion for (a) sANPs, csANPs, cANPs and (b) csANPs, cANPs. Curve of atomic adsorption quantity with time during combustion for (a) sANPs, csANPs, cANPs and (b) Figure 21 Curve of potential energy change during combustion for sANPs, csANPs and cANPs. Figure 19 The snapshots of the rectangular central section along the Y axis and the atomic displacement scatter plot during the constant temperature combustion period. (a) sANPs、(b) csANPs and (c) cANPs. Atomic coloring by atomic type. Grey: cAl atoms, yellow: O atoms, blue: sAl atoms, black: C atoms, purple: eO atoms, green: H atoms, red: O atoms. Page 31/32 Page 31/32 Figure 20 Curve of atomic adsorption quantity with time during combustion for (a) sANPs, csANPs, cANPs and (b) csANPs, cANPs. Figure 20 Figure 21 Curve of potential energy change during combustion for sANPs, csANPs and cANPs. Curve of potential energy change during combustion for sANPs, csANPs and cANPs. Page 32/32 Page 32/32
https://openalex.org/W2947675942	https://europepmc.org/articles/pmc6499953?pdf=render	English	null	An evaluation of efficacy of the auto-dissemination technique as a tool for Aedes aegypti control in Madeira, Portugal	Parasites & vectors	2,019	cc-by	9,176	© The Author(s) 2019. 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. An evaluation of efficacy of the auto‑dissemination technique as a tool for Aedes aegypti control in Madeira, Portugal Gonçalo Seixas1†, Richard E. L. Paul2,3†, Bianca Pires1, Gonçalo Alves1, Ana de Jesus1, Ana‑Clara Silva4, Gonçalo Seixas1†, Richard E. L. Paul2,3†, Bianca Pires1, Gonçalo Alves1, Ana de Jesus1, Ana‑Cla Gregor J. Devine5† and Carla A. Sousa1† Abstract Background: The frequency and intensity of arboviral epidemics is steadily increasing and posing an intractable public health burden. Current vector control methods are proving ineffectual and despite progress in the develop‑ ment of high technology approaches, there is an urgent need for the development of tools for immediate imple‑ mentation. Several studies suggest that the auto-dissemination of pyriproxyfen (PPF) is a promising new approach to larviciding although there is little detail on the conditions under which it is optimally effective. Here, we evaluate the efficacy of the approach in urban and rural sites in Madeira, Portugal. Results: Auto-dissemination of PPF through adapted Biogents Sentinel traps (BGSTs) resulted in a modest but consistent impact on both juvenile and adult mosquito populations, but with considerable spatial heterogeneity. This heterogeneity was related to the distance from the BGST dissemination station as well as the local density of adult mosquitoes. There was evidence that the impact of PPF was cumulative over time both locally and with gradual spatial expansion. Conclusions: The density of adult mosquitoes and the spatial distribution of dissemination devices are key factors in mediating efficacy. In addition, urban topography may affect the efficiency of auto-dissemination by impeding adult mosquito dispersal. Further studies in a range of urban landscapes are necessary to guide optimal strategies for the implementation of this potentially efficacious and cost-effective approach to larviciding. Keywords: Auto-dissemination, Pyriproxyfen, Europe, Aedes Parasites & Vectors Parasites & Vectors Seixas et al. Parasites Vectors (2019) 12:202 https://doi.org/10.1186/s13071-019-3454-3 Correspondence: greg.devine@qimrberghofer.edu.au; casousa@ihmt.unl.pt †Gonçalo Seixas, Richard E. L. Paul, Gregor J. Devine and Carla A. Sousa contributed equally to this work 1 UEI Parasitologia Médica, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da Junqueira 100, 1349‑008 Lisbon, Portugal5 Mosquito Control Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia Full list of author information is available at the end of the article Evaluation of Biogents Sentinel (BGS) traps as dissemination stations To ensure the suitability of adapted Biogents-Sentinel traps (BGSTs) for the dissemination of PPF, a small-scale proof of principle was carried-out under laboratory con- ditions. A prototype had previously been successfully field-tested in Peru (GJ Devine, unpublished data). A BGST with a fine mesh catch bag, but without the cone net that normally impedes escape, working in 1 hour on/ off cycles, was placed in an isolated room (9 m2, 25 ± 2 °C, 12 h Light:Dark photocycle). The BGST capture bag was first treated with fluorescent dust (DayGlo Color Corp., Cleveland, OH, USA) to simulate PPF particles (20– 30 µm in diameter). A total of 50 mosquitoes (25 unfed females and 25 males), Funchal strain, F1 generation, were released into the room. After 24 h, resting mosqui- toes were captured individually using mouth aspirators. Contamination with fluorescent dust was observed using a stereomicroscope under a UV light (Fig. 1). All mosqui- toes contaminated with dust must have entered, and then escaped from the adapted BGST. Pyriproxyfen (PPF) is a WHO-approved pupacide that can be used in drinking water and is recommended for use in conventional larviciding programmes against con- tainer breeding mosquitoes such as Ae. aegypti [13, 14]. It is a synthetic analogue of juvenile hormone and, at minis- cule doses, it prevents larval and pupal development and affects female fertility and male spermiogenesis [15, 16]. The potential of PPF as an auto-dissemination tool has been proven in a variety of small-scale trials in Peru and Italy [11, 17] and more recently at a larger scale in the Amazon [18]. The auto-dissemination of PPF co-opts the innate behaviors of container-breeding mosquitoes to distrib- ute this chemical to their aquatic habitat. Mosquitoes exposed to a surface contaminated with PPF subse- quently spread the pupacide to their own breeding sites during oviposition [11, 17]. The exposure of the adult mosquito population is achieved through the use of arti- ficial structures, called dissemination stations, which lure mosquitoes seeking oviposition or resting sites. Contami- nated mosquitoes then transport the particles of PPF on their body and legs to the containers that they visit subse- quently. This strategy may be particularly effective for Ae. aegypti because it may lay its eggs in several sites (skip oviposition), thus allowing greater breeding site coverage [17, 19]. Background of 17 other conditions, including upper respiratory infec- tions and hepatitis B [1]. Epidemics are complicated by overloaded public health infra-structures, ineffective intervention strategies and collateral economic damage through lost employment and negative impacts on tour- ism, an industry that many countries rely upon [2]. Mosquito-borne infections are a major public health burden. Amongst these, arboviruses are imposing con- siderable immediate and long-term pathological and socio-economic problems in many countries. The burden of arboviral disease is higher than the combined impact The management of these diseases in urban settings is particularly difficult as human populations and their associated mosquito habitats have created environments that are almost impossible to treat with traditional insec- ticidal interventions. Aedes aegypti, the major urban vector of arboviruses, is superbly adapted to man-made environments, transmitting dengue, chikungunya and Zika viruses. Uncontrolled, unplanned towns and cit- ies and the detritus of our “throw-away” society form an Correspondence: greg.devine@qimrberghofer.edu.au; casousa@ihmt.unl.pt †Gonçalo Seixas, Richard E. L. Paul, Gregor J. Devine and Carla A. Sousa contributed equally to this work 1 UEI Parasitologia Médica, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da Junqueira 100, 1349‑008 Lisbon, Portugal5 Mosquito Control Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia Full list of author information is available at the end of the article Correspondence: greg.devine@qimrberghofer.edu.au; casousa@ihmt.unl.pt †Gonçalo Seixas, Richard E. L. Paul, Gregor J. Devine and Carla A. Sousa contributed equally to this work 1 UEI Parasitologia Médica, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da Junqueira 100, 1349‑008 Lisbon, Portugal5 Mosquito Control Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia Full list of author information is available at the end of the article Seixas et al. Parasites Vectors (2019) 12:202 Page 2 of 13 number of devices can contaminate a much wider habitat [11, 20]. optimal transmission environment and a major challenge to arbovirus management. The increasing frequency and amplitude of arboviral epidemics even in Europe bears testament to the scale of the threat [3–5]. Here we describe a 2-year study on the efficacy of PPF in reducing populations of Ae. aegypti using an auto- dissemination strategy implemented at two contrasting sites, rural and highly urban, in Madeira, Portugal. Methodsh This study aimed to assess the efficacy of adapted Bio- gents-Sentinel traps (Biogents, Regensburg, Germany) for the auto-dissemination of pyriproxyfen in reducing both juvenile and adult Aedes aegypti populations in a rural and urban site in Madeira and to assess factors affecting efficacy. Background These studies were conducted in 2015–2016, precipitated by the 2012–2013 dengue outbreak on Madeira that resulted in > 2000 cases [3]. Despite progress in the development of vaccines for chikungunya, dengue and Zika [6–8], no current candi- dates are likely to have general application in endemic areas and it is generally agreed that an integrated approach with a significant role for vector control will be needed [9, 10]. Unfortunately, current mosquito control techniques are proving ineffectual and there remains an urgent need for the development of interventions that could be reasonably implemented across the environ- ments and epidemiological contexts that arboviruses inhabit. Achieving sufficient coverage of aquatic habi- tats, mosquito populations or indoor resting areas by any intervention is proving impossible in many urban transmission settings [11]. Insecticide-based control pro- grammes are also threatened by the evolution and spread of mosquitoes that are resistant to the very limited set of chemistries that we currently rely upon [12]. Evaluation of Biogents Sentinel (BGS) traps as dissemination stations This technique may complement source reduc- tion and larviciding campaigns by efficiently targeting the most productive containers (because the mosquito chooses and then contaminates its own breeding sites). It is potentially a more effective intervention than adult lethal traps, because its impact is amplified between the dissemination devices and the breeding sites - a small Auto‑dissemination studies The ABS were removed when none of the orig- inal sentinel cohort remained. We then proceeded to the next phase. The duration of the phases differed according to the rate of immature development and hence the time to the collection of the last pupae in the ABS. In Paul do Mar, the duration of the phases was 6 days (pre-treat- ment), 9 days (T1 treatment) and 29 days (T2 treatment); in Funchal the durations were 9, 11 and 13 days, respec- tively. When the treatment phases ended, the BGSTs were fitted with new catch bags (without PPF), the cone of Funchal strain, F1 generation, reared in the insectary. These larvae act as sentinels for the transfer of PPF to the ABS. In Funchal, 13 adapted BGSTs were placed in an area of 125,600 m2 (approximately one per 200 m radius) and 45 ABS distributed through a wider area (Fig. 2b). The auto-dissemination study comprised four phases: a pre-treatment phase to measure adult mosquitoes in BGSTs and larval mortality in ABS; two PPF treatment phases (T1 and T2) using the same BGSTs but adapted for auto-dissemination of PPF to measure larval mortal- ity in ABS and a final post-treatment assessment of mos- quito density using the BGSTs as in the pre-treatment phase. During the treatment phases, the catch bags of adapted BGSTs were dusted with a 10% PPF formulation. The traps were set to a one hour on/off cycle. The ABS were used to monitor larval mortality in the presence or absence of PPF-treated BGSTs. In each of the treatment phases (pre-treatment, T1, T2 and post-treatment) new ABS were placed in the same locations. Larval develop- ment in the ABS was observed at 48 h intervals. All live pupae were transferred to cups of uncontaminated water and taken to the laboratory to record emergence or death. All dead larvae and pupae were also removed and recorded. The ABS were removed when none of the orig- inal sentinel cohort remained. We then proceeded to the next phase. The duration of the phases differed according to the rate of immature development and hence the time to the collection of the last pupae in the ABS. In Paul do Mar, the duration of the phases was 6 days (pre-treat- ment), 9 days (T1 treatment) and 29 days (T2 treatment); in Funchal the durations were 9, 11 and 13 days, respec- tively. Auto‑dissemination studies When the treatment phases ended, the BGSTs were fitted with new catch bags (without PPF), the cone funnel was replaced and the traps were run constantly to assess adult mosquito numbers per day for a further week. Independently, ovitraps (14 in Paul do Mar and 78 in Funchal) were monitored throughout the two sites to provide an additional measure of mosquito abundance throughout the year. There was some loss of ABS in each treatment phase due to interference by domestic animals and on some occasions fewer than 20 larvae were recovered from each ABS suggesting larval death, decomposition and/or can- nibalism. Mortality analyses were calculated using only those mature larvae/pupae that were recovered and fol- lowed to emergence or death in the laboratory. Auto‑dissemination studies Two PPF auto-dissemination studies were carried out in Madeira, one in the parish of Paul do Mar, Calheta County (a coastal village with 868 residents), and the other the following year (2015) in the parish of Imacu- lado Coração de Maria, Funchal (an urban area with 6207 residents). In Paul do Mar, seven adapted BGSTs (powered by battery and described above) were deployed (approximately one per 200 m radius) and 37 artificial breeding sites (ABS), were distributed throughout the study area of 27,138 m2 (Fig. 2a). Each ABS consisted of a 12 cm diameter container filled with 250 ml of dechlorin- ated tap water, a cat food pellet and 20 third-instar larvae Seixas et al. Parasites Vectors (2019) 12:202 Page 3 of 13 Fig. 1 Mosquitoes captured from a room containing an adapted BGST. Their contamination with fluorescent dust demonstrates their exposure and subsequent release from these devices Fig. 1 Mosquitoes captured from a room containing an adapted BGST. Their contamination with fluorescent dust demonstrates their exposure and subsequent release from these devices of Funchal strain, F1 generation, reared in the insectary. These larvae act as sentinels for the transfer of PPF to the ABS. In Funchal, 13 adapted BGSTs were placed in an area of 125,600 m2 (approximately one per 200 m radius) and 45 ABS distributed through a wider area (Fig. 2b). The auto-dissemination study comprised four phases: a pre-treatment phase to measure adult mosquitoes in BGSTs and larval mortality in ABS; two PPF treatment phases (T1 and T2) using the same BGSTs but adapted for auto-dissemination of PPF to measure larval mortal- ity in ABS and a final post-treatment assessment of mos- quito density using the BGSTs as in the pre-treatment phase. During the treatment phases, the catch bags of adapted BGSTs were dusted with a 10% PPF formulation. The traps were set to a one hour on/off cycle. The ABS were used to monitor larval mortality in the presence or absence of PPF-treated BGSTs. In each of the treatment phases (pre-treatment, T1, T2 and post-treatment) new ABS were placed in the same locations. Larval develop- ment in the ABS was observed at 48 h intervals. All live pupae were transferred to cups of uncontaminated water and taken to the laboratory to record emergence or death. All dead larvae and pupae were also removed and recorded. Evaluation of the adapted Biogents Sentinel traps (BGST) as dissemination stations Evaluation of the adapted Biogents Sentinel traps (BGST) as dissemination stations Of the 50 mosquitoes released in the room, 42 mosqui- toes (25 females and 17 males) were recaptured. By expo- sure to UV light, it was confirmed that all 42 mosquitoes were marked with fluorescent dust (Fig. 1) and had thus visited the BGST, become contaminated with powder and been released. Statistical analyses I d In order to compare the effects of PPF auto-dissemi- nation on adult catches pre- and post-treatment using standard BGSTs, we used Generalized Linear Mixed Models (GLMM) using Genstat v. 15 [21] to examine individual BGSTs and overall catches. Period (pre- or post-treatment) was fitted as an explanatory variable and BGSTs as a random variable in a log-linear mixed model. Only totals per period were analysed. The impact of auto- dissemination on juvenile survival in the ABS was ana- lysed by mixed model logistic regression, fitting period (pre-treatment, T1 or T2) as an explanatory factor, indi- vidual ABS as a random variable and juvenile mortality as the number of dead juveniles over the total number of collected juveniles per ABS. Only totals per period were analysed for the Paul do Mar study site; temporal infor- mation on larval mortality was available for Funchal but only analysed temporally in the SaTScan analysis. Seixas et al. Parasites Vectors (2019) 12:202 Page 4 of 13 Fig. 2 BGST dissemination sites (blue) and ABS (yellow) in Paul do Mar (a) and Funchal (b) Seixas et al. Parasites Vectors (2019) 12:202 Page 5 of 13 Results This was performed using Kulldorffʼs scan statistic in SaTScan (v.9.1.1) (http://www.satscan.org/) [22]. A dis- crete Poisson model was used to analyse the spatial dis- tribution of adult mosquito catches (females and males were combined) in standard, non-adapted BGSTs and larval/pupal mortality in ABS. The program compares the occurrence of adult mosquito catches (or larvae/ pupae dead) over the number of sites (BGST or ABS) inside a randomly generated cluster circle compared to the rest of the population. The unit of analysis was the sampling site (BGST or ABS). An infinite number of cluster circles are generated with a maximum diameter set to values between 10–250 m, adapted to the area of study. Clusters represented hot or cold spots represent- ing greater or less than expected adult mosquito numbers or juvenile mortality. Only clusters with no geographical overlap were accepted. A relative risk of observed lower (cold spot) or higher (hot spot) numbers of events (here adult mosquitoes or larval mortality) than expected from the whole study area is calculated and a likelihood ratio test performed. For BGST catches only a spatial analysis was performed. For Funchal, information on larval/pupal mortality was available every 48 h and thus a spatio-tem- poral analysis was performed. Additional analyses on lar- val/pupal mortality were performed fitting BGST adult mosquito catches in the nearest BGST as a covariate. A Bonferroni correction was applied when multiple analy- ses were performed on the same data set. Table 2 Percent of ABS that had < 5%, 5–50% and > 50% juvenile mortality according to treatment period Table 2 Percent of ABS that had < 5%, 5–50% and > 50% juvenile mortality according to treatment period Percentage juvenile mortality 0–5% 5–50% 50–100% Pre-treatment 81.3 18.7 0 T1 54.6 21.2 24.2 T2 26.9 34.6 38.5 Auto‑dissemination Paul do Mar BGST adult catches RR P-value Pre-treatment 1 4 0 97 25.57 7.1 10−16 2 2, 5, 6 0.043 34 76.71 0.31 4.4×10−11 3 3 0 10 25.57 0.36 0.002 Post-treatment: no significant clusters Table 1 Spatial hot and cold spots of adult mosquito catches in Paul do Mar Notes: Shown are the BGST ID numbers associated with the hot and cold spots (with area of hot/cold spots given) and the observed (Obs.) and expected (Exp.) numbers caught from those traps with associated relative risk (RR) and P-values Fig. 4 Larval and pupal mortality during pre-treatment, T1 and T2 periods (Paul do Mar site). Proportion refers to the larval and pupal mortality that occurred among the sentinel larval cohorts that were used to seed the artificial breeding sites (ABS). Mean proportions and standard errors calculated from the fitted GLMM model are shown. Significant differences between treatment phases are noted Fig. 4 Larval and pupal mortality during pre-treatment, T1 and T2 periods (Paul do Mar site). Proportion refers to the larva that occurred among the sentinel larval cohorts that were used to seed the artificial breeding sites (ABS). Mean proportion calculated from the fitted GLMM model are shown. Significant differences between treatment phases are noted Fig. 4 Larval and pupal mortality during pre-treatment, T1 and T2 periods (Paul do Mar site). Proportion refers to the larval and pupal mortality that occurred among the sentinel larval cohorts that were used to seed the artificial breeding sites (ABS). Mean proportions and standard errors calculated from the fitted GLMM model are shown. Significant differences between treatment phases are noted and low mosquito densities observed in specific BGSTs (Fig. 2). These differences were unstable and disappeared during the post-treatment period. Table 2 Percent of ABS that had < 5%, 5–50% and > 50% juvenile mortality according to treatment period Auto‑dissemination Paul do Mar BGST adult catches Adult Ae. aegypti mosquito numbers captured in the BGSTs (Fig 2a) decreased between the pre-treatment and post-treatment periods, due to a sharp reduction in males (χ2 1 = 14.5, P = 0.001) (Fig. 3). There was variation among catches from the seven BGSTs, but again only for male mosquitoes (χ2 6 = 38.97, P = 0.019). The mean num- ber of eggs per surveillance ovitrap per week (over the 14 ovitraps spread in and around the study area) varied in the pre-treatment weeks between 1 and 18 and between 1 and 17 in the post-treatment period, suggesting that overall the female mosquito abundance was low but com- parable in the two periods.i Spatial analyses revealed significant heterogeneity in BGST catches, with greater than or less than expected adult numbers in some clusters when compared to the study site overall. This was most notable during the pre- treatment period (Table 1) and largely reflected the high Fig 3 Aedes aegypti caught by BGSTs per day in the pre- and post-treatment periods (Paul do Mar site) Mean numbers and standard errors are Fig. 3 Aedes aegypti caught by BGSTs per day in the pre- and post-treatment periods (Paul do Mar site). Mean numbers and standard errors are shown. Significant differences between pre- and post-treatments are noted (ns: not significant, P < 0.01) Fig. 3 Aedes aegypti caught by BGSTs per day in the pre- and post-treatment periods (Paul do Mar site). Me shown. Significant differences between pre- and post-treatments are noted (ns: not significant, P < 0.01) Fig. 3 Aedes aegypti caught by BGSTs per day in the pre- and post-treatment periods (Paul do Mar site). Mean numbers and standard errors are shown. Significant differences between pre- and post-treatments are noted (ns: not significant, P < 0.01) Seixas et al. Parasites Vectors (2019) 12:202 Page 6 of 13 Table 1 Spatial hot and cold spots of adult mosquito catches in Paul do Mar Notes: Shown are the BGST ID numbers associated with the hot and cold spots (with area of hot/cold spots given) and the observed (Obs.) and expected (Exp.) numbers caught from those traps with associated relative risk (RR) and P-values Cluster BGST Area radius (km) Obs. Exp. Artificial breeding sites (ABS) ‑ impacts on juvenilesh Artificial breeding sites (ABS) ‑ impacts on juvenilesh i Thirty-seven ABS each seeded with 20 larvae were dis- tributed throughout the study site (Fig 2a). Mortality rates of juvenile stages (larvae and pupae) were recorded during three periods: pre-treatment, T1 and T2. Juve- nile mortality increased from 2.7% (SE 1.3) in the pre- treatment period to 23.1% (SE 3.3) in T1 and 38.4% (SE 4.7) in T2 (χ2 2 = 13.07, P = 0.002) (Fig. 4). In T1, almost all mortality occurred in ABS located near to BGST dis- semination stations. In T2, juvenile mortality was more geographically widespread. There was significant varia- tion in the impact of PPF on ABS: from 0–100% juvenile mortality (Table 2). To explore whether adult mosquito density affected juvenile mortality in the ABS and contributed to the observed spatial heterogeneity, we analysed the impact of adult mosquito abundance from the nearest adapted BGST to each ABS (using total pre-treatment adult counts for T1 and total post-treatment adult counts for T2). Whilst overall there was no impact of adult Seixas et al. Parasites Vectors (2019) 12:202 Page 7 of 13 periods (Total: χ2 1 = 23.5, P < 0.001; female: χ2 1 = 23.1, P < 0.001; male: χ2 1 = 4.74, P = 0.045) (Fig. 5b). There was considerable variation in catch numbers among the thir- teen BGSTs (χ2 12 = 80.6, P < 0.001). The mean number of eggs in the 4 ovitraps sited in the treatment area varied between 60 (pre-treatment) and 50 (post-treatment). In greater Funchal the other 74 ovitraps yielded between 30 eggs per trap (pre-treatment) to 20 (post-treatment). This suggests that overall mosquito abundance remained similar between pre- and post-treatment periods and that observed decreases in adults in the treatment area were not due to a universal temporal effect. mosquito densities on adjacent juvenile mortality rates, there was a near-significant association of higher adult densities with higher T2 juvenile mortality (Log-linear regression χ2 1 = 3.65, P = 0.068). Spatial analyses revealed greater and less than expected juvenile mortality in the ABS during pre-treatment and treatment periods (Table 3). A single ABS with 50% mor- tality was responsible for a hot spot in the pre-treatment period. During the T1 period the spatial variation in mortality rates increased with four distinct hot and cold spots. Artificial breeding sites (ABS) ‑ impacts on juvenilesh At the end of the T2 period there remained sig- nificant spatial variation but at different sites. Indeed, one cluster that showed zero mortality in the T1 period became a mortality hot spot during the T2 period. There was a notable impact of adult mosquito density on larval and pupal mortality and the significance of the hot and cold spots was reduced or even lost when adult density was fitted as a covariate (Table 3). This suggests that adult mosquito density is contributing to the spatial patterns of mortality in the ABS. f As observed in Paul do Mar, spatial analyses revealed heterogeneity in adult mosquito captures with BGSTs (Table 4). Hot and cold spots were in the same areas for Aedes and Culex. During the post-treatment phase, the number, importance and size of these clusters was reduced. The inclusion of pre-treatment adult mosquito density as a covariate further reduced spatial heteroge- neity in post-treatment catch rates. This suggests that, as found for juvenile mortality rates in Paul do Mar, varia- tion in adult mosquito densities affects the efficiency of PPF dissemination with measurable effects even in the adult mosquito population. Auto‑dissemination Funchal BGST adult catches Adult Ae. aegypti numbers captured in the BGSTs (Fig 2b) decreased significantly between pre- and post- treatment periods (Total: χ2 1 = 9.13, P = 0.009; female: χ2 1 = 7.46, P = 0.015; male: χ2 1 = 3.74, P = 0.073) (Fig. 5a). In contrast to Paul do Mar where there were almost no Culex spp., in Funchal Culex spp. mosquitoes were pre- sent and also decreased between pre- to post-treatment Notes: Shown are observed (Obs.) and expected (Exp.) mortality rates (numbers of dead larvae/pupae) in significant hot or cold spots during pre-treatment, T1 and T2 periods, with and without BGST adult mosquito catches fitted as a covariate. ABS ID numbers, area covered by hot/cold spot, and relative risk (RR) with associated P-values are presented Artificial breeding sites (ABS) Juvenile mortality increased from 1.2% (SE 0.7) in the pre-treatment period to 17.3% (SE 2.2) during T1 and 21.6% (SE 2.4) in T2 (χ2 2 = 25.77, P < 0.001) (Fig. 6). Despite these significant impacts, many ABS were largely Table 3 Hot and cold spots of juvenile mortality in Paul do Mar Notes: Shown are observed (Obs.) and expected (Exp.) mortality rates (numbers of dead larvae/pupae) in significant hot or cold spots during pre-treatment, T1 and T2 periods, with and without BGST adult mosquito catches fitted as a covariate. ABS ID numbers, area covered by hot/cold spot, and relative risk (RR) with associated P-values are presented Cluster ABS code Area radius (km) Obs. Exp. RR P-value Pre-treatment 1 14 0 10 0.91 17.7 7.9 × 10−8 T1 1 20–30, 32 0.120 21 58.62 0.24 3.1 × 10−10 2 37 0 20 2.82 8.11 5.4 × 10−10 3 2, 15, 16, 31, 33–35 0.026 65 28.95 3.32 5.4 × 10−10 4 1, 3–5, 10, 11 0.084 0 20.30 0 1.9 × 10−9 With BGST pre-treatment adult catches as covariate 1 1, 3–5, 10, 11 0.084 0 17.00 0 2 × 10−7 2 37 0 20 4.00 5.67 5.3 × 10−7 T2 1 3–5, 11, 12, 37 0.058 51 28.50 2.36 0.00063 2 21, 29, 32 0.025 2 15.37 0.12 0.00066 3 15, 34, 36 0.048 7 22.73 0.27 0.0028 With BGST post-treatment catches as covariate: no clusters Seixas et al. Parasites Vectors (2019) 12:202 Page 8 of 13 unaffected. Twenty-three of the 43 ABS were < 50 m from a BGST dissemination site (ABS 1–18, 38–43) while the remainder (ABS 19–37, 44–45) were further from dis- semination stations (see Fig. 2b). Those ABS that were far from the BGST dissemination sites had significantly vs Far, from dissemination site, with individual ABS fitted as a random factor: χ2 1 = 11.6, P = 0.001; odds ratio 0.22, 95% CI: 0.09–0.52) (Fig. 7). Spatial analyses of juvenile mortality in the 43 ABS identified no clusters of high or low mortality rates dur Fig. 5 Mosquitoes caught by BGSTs per day in the pre- and post-treatment periods (Funchal site). Mean numbers and standard errors are shown. a Ae. aegypti. b Culex spp. Significant differences between pre- and post-treatments are noted (ns: not significant, P < 0.05, P < 0.01, P < 0.001) Fig. Artificial breeding sites (ABS) RR P-value Aedes Pre-treatment 1 1 0 42 11.60 4.62 1.9 × 10−11 2 3–5, 8, 9, 11 0.048 42 69.69 0.45 0.00021 Post-treatment 1 1, 7, 12 0.043 48 20.10 4.10 8 × 10−9 Post-treatment with pre-treatment Aedes as covariate 1 1, 7, 12 0.043 48 31.00 2.23 0.0085 Culex Pre-treatment 1 1–3, 13 0.065 72 32.90 4.63 5.2 × 10−13 2 4, 5, 8–11 0.062 14 49.40 0.18 1.3 × 10−11 Post-treatment 1 1, 2, 4, 7, 12 0.047 24 12.30 4.80 0.001 Post-treatment with pre-treatment Culex as covariate - no clusters detected Table 4 Spatial hot and cold spots of adult mosquito catches (Aedes and Culex) pre- and post-treatment in Funchal Cl t BGST A di (k ) Ob E RR ot and cold spots of adult mosquito catches (Aedes and Culex) pre- and post-treatment in Funchal Notes: Shown are observed (Obs.) and expected (Exp.) adult mosquito catches with and without BGST pre-treatment catches fitted a area covered by the hot/cold spot, relative risk (RR) and associated P-values are given Notes: Shown are observed (Obs.) and expected (Exp.) adult mosquito catches with and without BGST pre-treatment catches fitted as a covariate. BGST ID numbers, area covered by the hot/cold spot, relative risk (RR) and associated P-values are given Fig. 6 Larval and pupal mortality in ABS in the pre-treatment, T1 and T2 periods (Funchal site). Proportion refers to the larval and pupal mortality that occurred among the sentinel larval cohorts that were used to seed the artificial breeding sites (ABS). Mean proportions and standard errors calculated from the fitted GLMM model are shown. Significant differences between treatments phases are noted Fig. 6 Larval and pupal mortality in ABS in the pre-treatment, T1 and T2 periods (Funchal site). Proportion refers to the larval and pupal mortality that occurred among the sentinel larval cohorts that were used to seed the artificial breeding sites (ABS). Mean proportions and standard errors calculated from the fitted GLMM model are shown. Significant differences between treatments phases are noted T2 and involved the ABS furthest from the dissemination stations. ABS from days 5 to 7 of this treatment phase; these ABS were all close to BGST dissemination stations. There was also one cold spot that included the two ABS (nos 44 and 45) that were far from any dissemination station. Artificial breeding sites (ABS) 5 Mosquitoes caught by BGSTs per day in the pre- and post-treatment periods (Funchal site). Mean numbers and standard errors are shown. a Ae aegypti b Culex spp Significant differences between pre- and post-treatments are noted (ns: not significant P<0 05 P<0 01 P<0 001) Fig. 5 Mosquitoes caught by BGSTs per day in the pre- and post-treatment periods (Funchal site). Mean numbers and standard errors are shown. a Ae. aegypti. b Culex spp. Significant differences between pre- and post-treatments are noted (ns: not significant, P < 0.05, P < 0.01, P < 0.001) unaffected. Twenty-three of the 43 ABS were < 50 m from a BGST dissemination site (ABS 1–18, 38–43) while the remainder (ABS 19–37, 44–45) were further from dis- semination stations (see Fig. 2b). Those ABS that were far from the BGST dissemination sites had significantly lower larval mortality rates in the treatment phases (GLMM Logistic regression of impact of distance, Near vs Far, from dissemination site, with individual ABS fitted as a random factor: χ2 1 = 11.6, P = 0.001; odds ratio 0.22, 95% CI: 0.09–0.52) (Fig. 7). ) ( g ) Spatial analyses of juvenile mortality in the 43 ABS identified no clusters of high or low mortality rates dur- ing the pre-treatment phase (Table 5). In T1, by contrast, peak juvenile mortality occurred in an area covering 18 Seixas et al. Parasites Vectors (2019) 12:202 Page 9 of 13 Table 4 Spatial hot and cold spots of adult mosquito catches (Aedes and Culex) pre- and post-treatment in Funchal Notes: Shown are observed (Obs.) and expected (Exp.) adult mosquito catches with and without BGST pre-treatment catches fitted as a covariate. BGST ID numbers, area covered by the hot/cold spot, relative risk (RR) and associated P-values are given Cluster BGST Area radius (km) Obs. Exp. Artificial breeding sites (ABS) RR P-value Day of treatment Pre-treatment phase Space-time: no clusters Space only: no clusters T1 phase Space-time analysis 1 1, 3, 7–12, 14–18, 22–25, 43 0.088 93 40 6.16 1 × 10−17 5 to 7 Space only analysis 1 1, 3, 7–12, 14–18, 22–25, 43 0.088 100 61.3 4.06 3.8 × 10−10 2 38, 39 0.0045 0 8.4 0 0.0043 T2 phase Space-time analysis 1 1–4, 6–12, 14–18, 38, 39, 43 0.12 55 18.5 3.82 8.5 × 10−10 1 to 3 2 27, 33–37 0.086 4 27.2 0.13 1.8 × 10−5 6 to 10 3 19–21, 23, 28 0.025 1 13.7 0.07 0.0097 6 to 8 Space only analysis 1 1–4, 6–11, 14–18, 38–40, 43 0.11 99 57.8 2.54 6.8 × 10−8 2 34, 36 0.013 4 22.6 0.16 6.6 × 10−5 Fig. 7 Larval and pupal mortality dependent on distance from dissemination station (Funchal site). Proportion refers to the larval and pupal mortality that occurred among the sentinel larval cohorts that were used to seed the artificial breeding sites (ABS). Mean proportions and standard errors calculated from the fitted GLMM model are shown. Red bars include ABS far from (> 50 m) a dissemination site; blue bars include ABS near to (< 50 m) a dissemination site. Significant differences between near and far sites are noted (ns: not significant, P < 0.05, *P < 0.01) Fig. 7 Larval and pupal mortality dependent on distance from dissemination station (Funchal site). Proportion refers to the larval and pupal mortality that occurred among the sentinel larval cohorts that were used to seed the artificial breeding sites (ABS). Mean proportions and standard errors calculated from the fitted GLMM model are shown. Red bars include ABS far from (> 50 m) a dissemination site; blue bars include ABS near to (< 50 m) a dissemination site. Significant differences between near and far sites are noted (ns: not significant, P < 0.05, *P < 0.01) Table 5 Juvenile mortality hot and cold spots during the pre-treatment, T1 and T2 periods Notes: Shown are observed (Obs.) and expected (Exp.) mortality rates (numbers of dead larvae) in significant hot and cold spots. ABS ID numbers, area covered, relative risk (RR) with associated P-values and associated timepoints are given Cluster ABS Radius (km) Obs. Exp. Artificial breeding sites (ABS) The hotspot was again observed during days 1 to 3 of T2 and two new cold spots were identified from day 6 onwards of We assessed the impact of adult abundance (monitored through BGST catches) on the ABS mortality rates. Those ABS distant from BGST dissemination stations (ABS nos 19–37, 44 and 45) were excluded from the analysis, We assessed the impact of adult abundance (monitored through BGST catches) on the ABS mortality rates. Those ABS distant from BGST dissemination stations (ABS nos 19–37, 44 and 45) were excluded from the analysis, Seixas et al. Parasites Vectors (2019) 12:202 Page 10 of 13 b f th l ti l ti b t di t l l l Th i d l “ l i d” Fig. 7 Larval and pupal mortality dependent on distance from dissemination station (Funchal site). Proportion refers to the larval and pupal mortality that occurred among the sentinel larval cohorts that were used to seed the artificial breeding sites (ABS). Mean proportions and standard errors calculated from the fitted GLMM model are shown. Red bars include ABS far from (> 50 m) a dissemination site; blue bars include ABS near to (< 50 m) a dissemination site. Significant differences between near and far sites are noted (ns: not significant, P < 0.05, **P < 0.01) Table 5 Juvenile mortality hot and cold spots during the pre-treatment, T1 and T2 periods Notes: Shown are observed (Obs.) and expected (Exp.) mortality rates (numbers of dead larvae) in significant hot and cold spots. ABS ID numbers, area covered, relative risk (RR) with associated P-values and associated timepoints are given Cluster ABS Radius (km) Obs. Exp. Artificial breeding sites (ABS) RR P-value Day of treatment Pre-treatment phase Space-time: no clusters Space only: no clusters T1 phase Space-time analysis 1 1, 3, 7–12, 14–18, 22–25, 43 0.088 93 40 6.16 1 × 10−17 5 to 7 Space only analysis 1 1, 3, 7–12, 14–18, 22–25, 43 0.088 100 61.3 4.06 3.8 × 10−10 2 38, 39 0.0045 0 8.4 0 0.0043 T2 phase Space-time analysis 1 1–4, 6–12, 14–18, 38, 39, 43 0.12 55 18.5 3.82 8.5 × 10−10 1 to 3 2 27, 33–37 0.086 4 27.2 0.13 1.8 × 10−5 6 to 10 3 19–21, 23, 28 0.025 1 13.7 0.07 0.0097 6 to 8 Space only analysis 1 1–4, 6–11, 14–18, 38–40, 43 0.11 99 57.8 2.54 6.8 × 10−8 2 34, 36 0.013 4 22.6 0.16 6.6 × 10−5 Notes: Shown are observed (Obs.) and expected (Exp.) mortality rates (numbers of dead larvae) in significant hot and cold spots. ABS ID numbers, area covered, relative risk (RR) with associated P-values and associated timepoints are given at local scales. There remained only one “unexplained” coldspot during the early stages of treatment phase 1 (Table 6). This lends further support to the importance of adult mosquito density in efficiently disseminating PPF. because of the clear negative correlation between dis- tance and juvenile mortality (Fig. 7). Incorporation of adult catches as a covariate of ABS mortality explained the majority of the spatial variation in juvenile survival Seixas et al. Parasites Vectors (2019) 12:202 Page 11 of 13 Table 6 Juvenile mortality hot and cold spots in ABS proximal to BGSTs Notes: Shown are observed (Obs.) and expected (Exp.) mortality rates (numbers of dead larvae/pupae) in hot and cold spots with and without adult densities from closest BGST catch points fitted as a covariate. ABS ID numbers, area covered and relative risk (RR) with associated P-values are given. For space-time clusters, associated treatment day is also shown Cluster ABS Radius (km) Obs. Exp. Artificial breeding sites (ABS) RR P-value Day of treatment T1 phase Space only, no covariate 1 38, 39 0.0045 0 7.5 0 0.0043 Space with BGST pre count as covariate: no cluster Space-time no covariate 1 6, 16–18 0.021 24 5.9 5.23 2.8 × 10−6 5 2 1, 8–12, 14, 15, 43 0.051 0 9.8 0 0.0035 6 Space-time with BGST pre count as covariate 1 9, 10, 18 0.082 0 12.1 0 4.7 × 10−4 3 to 4 T2 phase Space only, no covariate: no clusters Space with BGST post count as covariate: no cluster Space-time no covariate 1 1, 4–7, 16, 38–42 0.065 84 28.2 3.98 1 × 10−17 7 2 8–10, 43 0.120 0 30.5 0 3.6 × 10−12 7 Space-time with BGST post count as covariate: no cluster Table 6 Juvenile mortality hot and cold spots in ABS proximal to BGSTs Notes: Shown are observed (Obs.) and expected (Exp.) mortality rates (numbers of dead larvae/pupae) in hot and cold spots with and without adult densities from closest BGST catch points fitted as a covariate. ABS ID numbers, area covered and relative risk (RR) with associated P-values are given. For space-time clusters, associated treatment day is also shown spp. adults. It is conceivable that the abundant Culex spp. population was compensating for the low density of the target species, Ae. aegypti, in Funchal, leading to dissemi- nation of PPF to natural oviposition sites other than our ABS, which were not designed to measure impacts on Culex spp. Across all trials there was distinct spatial het- erogeneity in the impact of PPF dissemination on juvenile mortality rates and adult abundance. Spatial variation in juvenile mortality decreased over time, suggesting that impact became more universal as the number of dis- semination events and ABS coverage increased. Notably, spatial variation in juvenile mortality became negligible once spatial variation in adult abundance was taken into account (i.e. dissemination impacts are related to adult density and, presumably, the number of contamination events). The higher juvenile mortality rates and higher adult densities in Paul do Mar also suggest that dissemi- nation works better when there are more adult mosqui- toes. Proximity to a dissemination site also had a very significant effect on juvenile mortality over the short and long terms, suggesting a significant influence of urban topography and mosquito dispersal on dissemination and coverage.h Discussion Despite continued interest in the auto-dissemination concept as a complementary, potentially highly efficient larviciding tool, we have a very limited understanding of how to optimize its operation and deployment. One of the largest scale trials yet conducted [18] set 1000 sim- ple dissemination stations at an approximate density of 1 every 100 meters (1/10,000 m2). Distance between these stations and sentinel larval habitats was not recorded although at least some sentinel habitats were clearly placed in the immediate vicinity of the dissemination sta- tions. No direct measure of impact on adult mosquito density was made. We explicitly evaluated the efficacy of adapted BGSTs as auto-dissemination stations for PPF when placed at a low density (approximately 1 every 200 meters). The relationship between their impact and their proximity to sentinel habitats was carefully recorded for a short period of time, in areas of low adult Ae. aegypti abundance on the island of Madeira, Portugal. As shown by others [11, 17, 18], brief deployment of PPF reduced juvenile mosquito survival and impacts appeared to accumulate during continuous deployment. We also measured the impact on the adult mosquito pop- ulation and found a significant reduction of males and females. Larvicide/pupacide efficacy was higher during the trial conducted at higher mosquito abundance (Paul do Mar), but the impact on the adult mosquito popula- tion was lower than in the low abundance setting of Fun- chal, where there was also a measurable impact on Culex The main challenge in the implementation of vector control measures is to achieve sufficient coverage of the mosquito population (i.e. aquatic habitats treated, houses sprayed, LLINs used). Although source reduction and the application of larvicides is a key tenet of urban mosquito control [23], it is challenging in highly urbanized areas Page 12 of 13 Page 12 of 13 Seixas et al. Parasites Vectors (2019) 12:202 Seixas et al. Parasites Vectors (2019) 12:202 because of the difficulty in identifying and treating myr- iad aquatic habitats. The auto-dissemination technique, in which mosquitoes contaminate their own aquatic hab- itats through their resting and oviposition behaviours is a potentially powerful way of overcoming those challenges [11]. available, but these need to be evaluated under compa- rable field conditions. Funding d d b Funded by a European Union FP7 grant through the DENFREE consortium (European Union 7th FP, grant 282378). Fundação para a Ciência e Tecnologia (FCT), through GHTM - UID/Multi/04413/2013 has partially funded the work and GS is supported by a FCT doctoral grant (SFRH/BD/98873/2013). Availability of data and materials Data supporting the conclusions of this article are included within the article. The datasets used and/or analysed during the present study are available from the corresponding author upon reasonable request. In addition to the problems posed by urban topogra- phy, consideration of the attractiveness and transfer effi- ciency of the dissemination stations is needed [26]. There are currently many experimental and commercial devices Conclusions h l h Whilst the complexity of systems seems overwhelm- ing, there is consistent evidence that auto-dissemination of PPF works. The next steps should focus on improv- ing implementation, with its use at the right place and the right time. There has been a call for more intelligent use of currently effective insecticides, and auto-dissem- ination is a clear example. Optimal intervention strat- egies will vary from site to site and the combination of auto-dissemination with other interventions is likely. To date, almost all auto-dissemination studies focus on PPF because of its mammalian safety and its unique toxic- ity at parts per trillion but, in the future, other agents such as insect specific viruses that can infect aquatic life stages [27] or other biological control agents [28] may be considered. The extent to which the auto-dissemination technique is limited by the efficacy of dissemination tools, their spatial deployment patterns in urban environments, and their impact on the productivity of aquatic habi- tats and adult populations, needs to be addressed before the utility and cost-efficacy of the paradigm can be fully assessed. In particular, we need some universal algorithm for deployment across endlessly variable urban habitats. Numerous studies have addressed the effect of urbaniza- tion on mosquito dispersal and population dynamics [25] but, in brief, many facets of the mosquito life-cycle (mat- ing, resting, oviposition, biting rate, survival) are directly influenced by the urban environment (topography, infra- structure, housing). In short, there can be consider- able very localized heterogeneity in the suitability of the urban environment for mosquito production, mosquito dispersal, and therefore auto-dissemination as a control measure. Discussion Our choice of adapted BGSTs as a dissemination device, and of a uniform 10% PPF for- mulation (specifically manufactured for the purpose of auto-dissemination trials) was an attempt to remove the idiosyncrasies of hand-milling 0.5 WG formulations and improve on the use of roughly treated buckets as dissemi- nation stations [11, 18]. The BGST is widely perceived as the most effective trapping tool on the market, which suggests that our adapted version should be effective as a “lure and release” device. Our trials continue to demonstrate this potential but highlight, for the first time, the barriers to successful optimization. Local structural topography will impact significantly on adult mosquito flight range and hence PPF dispersal capacity. The limited flight range of Ae. aegypti is well described and further constrained by the urban landscape [24]. This urban heterogeneity will have a significant impact upon the efficacy of PPF delivery to the aquatic habitat and needs to be considered when optimizing the design of auto-dissemination trials. Furthermore, because it is the adult mosquito that transmits the pathogens, measures of impact on adult mosquito populations are necessary. Our trial demon- strated modest but consistent decreases in adult abun- dance at both trial sites. This is a crucial first proof prior to implementation of more extensive epidemiological studies that will ascertain the entomological and epide- miological efficacy of auto-dissemination. Target species abundance, the existence of non-targets co-opted into the auto-dissemination process, distance between aquatic sites and dissemination stations, and urban topography will all contribute to the substantial variation in efficacy noted between our results and those reported in other urban trials [18]. A limitation of all auto-dissemination studies to date is the use of sentinel juvenile habitats to monitor efficacy. When measuring impacts in these habitats alone, we have no idea whether the observed impacts underestimate the true power of the technique (are most contaminated adults choosing sites other than those being monitored?). Our study is the first to have demonstrated an impact on adult abun- dance, the ultimate and most important entomological measure of impact. Abbreviations BGST Bi t S BGST: Biogents Sentinel traps; PPF: pyriproxyfen; ABS: artificial breeding sites; GLMM: Generalized Linear Mixed Models; CI: confidence interval. References 1. Shepard DS, Coudeville L, Halasa YA, Zambrano B, Dayan GH. Eco‑ nomic impact of dengue illness in the Americas. Am J Trop Med Hyg. 2011;84:200–7. 20. Devine GJ, Killeen GF. The potential of a new larviciding method for the control of malaria vectors. Malar J. 2010;9:142. 2. Shepard DS, Undurraga EA, Betancourt-Cravioto M, Guzman MG, Halstead SB, Harris E, et al. Approaches to refining estimates of global burden and economics of dengue. PLoS Negl Trop Dis. 2014;8:e3306. 2. Shepard DS, Undurraga EA, Betancourt-Cravioto M, Guzman MG, Halstead SB, Harris E, et al. Approaches to refining estimates of global burden and economics of dengue. PLoS Negl Trop Dis. 2014;8:e3306. 21. VSN International. Genstat for Windows. 19th ed. Hemel Hempstead, UK: VSN International; 2017. 22. Kulldorff M, Heffernan R, Hartman J, Assunção R, Mostashari F. A space- time permutation scan statistic for disease outbreak detection. PLoS Med. 2005;2:e59. 3. Sousa CA, Clairouin M, Seixas G, Viveiros B, Novo MT, Silva AC, et al. Ongo‑ ing outbreak of dengue type 1 in the Autonomous Region of Madeira, Portugal: preliminary report. Eurosurveillance. 2012;17:20333. 3. Sousa CA, Clairouin M, Seixas G, Viveiros B, Novo MT, Silva AC, et al. Ongo‑ ing outbreak of dengue type 1 in the Autonomous Region of Madeira, Portugal: preliminary report. Eurosurveillance. 2012;17:20333. 23. WHO. Dengue—guidelines for diagnosis, treatment, prevention and control. Geneva: World Health Organization; 2009. 4. Calba C, Guerbois-Galla M, Franke F, Jeannin C, Auzet-Caillaud M, Grard G, et al. Preliminary report of an autochthonous chikungunya outbreak in France, July to September 2017. Eurosurveillance. 2017;22:39. 4. Calba C, Guerbois-Galla M, Franke F, Jeannin C, Auzet-Caillaud M, Grard G, et al. Preliminary report of an autochthonous chikungunya outbreak in France, July to September 2017. Eurosurveillance. 2017;22:39. 24. Barbu CM, Hong A, Manne JM, Small DS, Quintanilla Calderón JE, Sethura‑ man K, et al. The effects of city streets on an urban disease vector. PLoS Comput Biol. 2013;9:e1002801. y 5. Bordi L, Carletti F, Lalle E, Colavita F, Meschi S, Di Caro A, et al. Molecular characterization of autochthonous chikungunya cluster in Latium region, Italy. Emerg Infect Dis. 2018;24:178–80. 5. Bordi L, Carletti F, Lalle E, Colavita F, Meschi S, Di Caro A, et al. Molecular characterization of autochthonous chikungunya cluster in Latium region, Italy. Emerg Infect Dis. 2018;24:178–80. 25. LaDeau SL, Allan BF, Leisnham PT, Levy MZ. The ecological foundations of transmission potential and vector-borne disease in urban landscapes. Competing interests 12. Maciel-de-Freitas R, Avendanho FC, Santos R, Sylvestre G, Araújo SC, Lima JB, et al. Undesirable consequences of insecticide resistance following Aedes aegypti control activities due to a dengue outbreak. PLoS ONE. 2014;9:e92424. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 13. WHO. Pesticides and their application for the control of vectors and pest of public health importance. 6th ed. Geneva: World Health Organization; 2006. Author details 1 UEI Parasitologia Médica, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da Junqueira 100, 1349‑008 Lisbon, Portugal. 2 Functional Genetics of Infectious Diseases Unit, Department of Genomes and Genetics, Institut Pasteur, 75015 Paris, France. 3 Centre National de la Recherche Scientifique (CNRS), Génomique évolutive, modélisation et santé UMR 2000, 75724 Paris Cedex 15, France. 4 Unidade de Engenharia Sanitária, Departamento de Pro‑ moção e Proteção da Saúde, Instituto de Administração da Saúde e Assuntos Sociais, IP-RAM, Funchal, Madeira, Portugal. 5 Mosquito Control Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia. 14. WHO. Guidelines for drinking-water quality. 4th ed. Geneva: World Health Organization; 2011. 15. Invest JF, Lucas JR. Pyriproxyfen as a mosquito larvicide. In: Robinson WH, Bajomi D, editors. Proceedings of the sixth international conference on urban pests, London. Veszprém: OOK-Press Kft; 2008. p. 239–46. urban pests, London. Veszprém: OOK-Press Kft; 2008. p. 239–46 16. Ohba SY, Ohashi K, Pujiyati E, Higa Y, Kawada H, Mito N, et al. The effect of pyriproxyfen as a “population growth regulator” against Aedes albopictus under semi-field conditions. PLoS ONE. 2013;8:e67045. i 17. Caputo B, Ienco A, Cianci D, Pombi M, Petrarca V, Baseggio A, et al. The “auto-dissemination” approach: a novel concept to fight Aedes albopictus in urban areas. PLoS Negl Trop Dis. 2012;6:e1793. Received: 7 December 2018 Accepted: 22 April 2019 Received: 7 December 2018 Accepted: 22 April 2019 18. Abad-Franch F, Zamora-Perea E, Luz SLB. Mosquito-disseminated insecticide for citywide vector control and its potential to block arbovirus epidemics: entomological observations and modeling results from Ama‑ zonian Brazil. PLoS Med. 2017;14:e1002213. 19. Reiter P. Oviposition, dispersal, and survival in Aedes aegypti: implica‑ tions for the efficacy of control strategies. Vector Borne Zoonotic Dis. 2007;7:261–73. 8. Larocca RA, Abbink P, Peron JP, Zanotto PM, Iampietro MJ, Badamchi- Zadeh A, et al. Vaccine protection against Zika virus from Brazil. Nature. 2016;536:474–8. Consent for publication Not applicable. Consent for publication Not applicable. y g 10. Paul R, Sousa CA, Sakuntabhai A, Devine GJ. Mosquito control might not bolster imperfect dengue vaccines. Lancet. 2014;384:1747–8. 10. Paul R, Sousa CA, Sakuntabhai A, Devine GJ. Mosquito contr bolster imperfect dengue vaccines. Lancet. 2014;384:1747–8. 11. Devine GJ, Perea EZ, Killeen GF, Stancil JD, Clark SJ, Morrison A. Using adult mosquitoes to transfer insecticides to Aedes aegypti larval habitats. Proc Natl Acad Sci USA. 2009;106:11530–4. 7. Ramsauer K, Schwameis M, Firbas C, Müllner M, Putnak RJ, Thomas SJ, et al. Immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial. Lancet Infect Dis. 2015;15:519–27. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. 9. Knerer G, Currie CS, Brailsford SC. Impact of combined vector-control and vaccination strategies on transmission dynamics of dengue fever: a model-based analysis. Health Care Manag Sci. 2013;18:205. Authors’ contributions GS, GA, BP and ADJ performed the field studies. GJD and CAS designed the study and wrote the paper. REP analysed the data and wrote the paper. ACS provided on site logistical assistance. All authors read and approved the final manuscript. Page 13 of 13 Page 13 of 13 Seixas et al. Parasites Vectors (2019) 12:202 Seixas et al. Parasites Vectors References Funct Ecol. 2015;29:889–901. 6. Capeding MR, Tran NH, Hadinegoro SR, Ismail HI, Chotpitayasunondh T, Chua MN, et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer- masked, placebo controlled trial. Lancet. 2014;384:1358–65. 6. Capeding MR, Tran NH, Hadinegoro SR, Ismail HI, Chotpitayasunondh T, Chua MN, et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer- masked, placebo controlled trial. Lancet. 2014;384:1358–65. 26. Devine G. Auto-dissemination of pyriproxyfen for the control of con‑ tainer-inhabiting mosquitoes—a progress review. Outlooks Pest Manag. 2016;27:164–7. 7. Ramsauer K, Schwameis M, Firbas C, Müllner M, Putnak RJ, Thomas SJ, et al. Immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial. Lancet Infect Dis. 2015;15:519–27. 27. Carlson J, Suchman E, Buchatsky L. Densoviruses for control and genetic manipulation of mosquitoes. Adv Virus Res. 2006;68:361–92. 28. Schlein Y, Pener H. Bait-fed adult Culex pipiens carry the larvicide Bacillus sphaericus to the larval habitat. Med Vet Entomol. 1990;4:283–8. 8. Larocca RA, Abbink P, Peron JP, Zanotto PM, Iampietro MJ, Badamchi- Zadeh A, et al. Vaccine protection against Zika virus from Brazil. Nature. 2016;536:474–8.
https://openalex.org/W2117770204	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0112793&type=printable	English	null	View-Based Organization and Interplay of Spatial Working and Long-Term Memories	PloS one	2,014	cc-by	10,525	Abstract Space perception provides egocentric, oriented views of the environment from which working and long-term memories are constructed. ‘‘Allocentric’’ (i.e. position-independent) long-term memories may be organized as graphs of recognized places or views but the interaction of such cognitive graphs with egocentric working memories is unclear. Here we present a simple coherent model of view-based working and long-term memories, together with supporting evidence from behavioral experiments. The model predicts (i) that within a given place, memories for some views may be more salient than others, (ii) that imagery of a target square should depend on the location where the recall takes place, and (iii) that recall favors views of the target square that would be obtained when approaching it from the current recall location. In two separate experiments in an outdoor urban environment, pedestrians were approached at various interview locations and asked to draw sketch maps of one of two well-known squares. Orientations of the sketch map productions depended significantly on distance and direction of the interview location from the target square, i.e. different views were recalled at different locations. Further analysis showed that location-dependent recall is related to the respective approach direction when imagining a walk from the interview location to the target square. The results are consistent with a view-based model of spatial long-term and working memories and their interplay. Citation: Ro¨hrich WG, Hardiess G, Mallot HA (2014) View-Based Organization and Interplay of Spatial Working and Long-Term Memories. PLoS ONE 9(11): e112793. doi:10.1371/journal.pone.0112793 Editor: Paul Graham, University of Sussex, United Kingdom Editor: Paul Graham, University of Sussex, United Kingdom Received July 9, 2014; Accepted October 17, 2014; Published November 19, 2014 Received July 9, 2014; Accepted October 17, 2014; Published November 19, 2014 Copyright: 2014 Ro¨hrich et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Ro¨hrich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which pe ribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was carried out at the Department of Biology of the University of Tu¨bingen. Introduction As we walk through an environment, we constantly keep track of objects, landmarks, and path opportunities around us. This environmental information forms a working memory of surround- ing space for which Loomis, Klatzky, and Giudice [1] suggested the term ‘‘spatial image’’. Local, ego-centric representations of space have been studied in many contexts, including among others sensori-motor integration, visual scene recognition, and spatial cognition. Tatler and Land [2] and Land [3] review a large body of evidence on ego-centric visual representation supporting the stability of perception across eye-movements as well as eye-hand coordination with and without locomotion of the body. The representation considered by Tatler and Land [2] extends around the agent up to about the size of a room in an indoor environment. A similar spatial working memory including also a mechanism for spatial updating has been suggested by Byrne, Becker, and Burgess [4]. The notion of the spatial image [1] is slightly more general in that it may include knowledge from other (non-visual) modalities and extends to more distant spaces, which may be out of sight even if the observer would turn his or her head accordingly. Information from distant locations beyond the current sensory horizon can originate from two sources, i.e. long-term memory of Abstract WGR received additional support from the Werner Reichardt Center for Integrative Neuroscience (CIN) at the University of Tu¨bingen. The CIN is an Excellence Cluster funded by the Deutsche Forschungsgemeinschaft (www. dfg.de) within the Excellence Initiative under grant number EXC 307. Additional support was provided by the German Federal Ministry of Education and Research (www.bmbf.de) within the Tu¨bingen Bernstein Center for Computational Neuroscience under grant number 01GQ1002A. 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. * Email: hanspeter.mallot@uni-tuebingen.de distant places, or spatial updating if the distant place had been visited before and was since maintained in working memory. Wolfgang G. Ro¨ hrich, Gregor Hardiess, Hanspeter A. Mallot* Cognitive Neuroscience Unit, Department of Biology, University of Tu¨bingen, Tu¨bingen, Germany November 2014 \| Volume 9 \| Issue 11 \| e112793 A view-based model of spatial working and long-term memories One obvious inter- pretation of this finding is that recall from long-term memory goes into some sort of spatial image, or working memory centered at the observer’s imagined position and that it is the left side of this representation which is affected by neglect. The concept of view-based representations of navigational space has been developed by Scho¨lkopf and Mallot [26] and used in robot simulations [39] and models of hippocampal processing [40]. Behavioral evidence for view-based navigation in humans has been presented by [41–43]. View specific neuronal activity has been reported e.g. from the monkey parahippocampal formation [44] or the human retrosplenial cortex [30]. Spatial memory systems may differ in the reference system employed to organize spatial information. Perception is ego- centric and so is the assumed spatial image [1,2,23]. In perspective taking, route planning, and mental travel, ego-centric memories centered at imagined positions may also exist. The reciprocal term, allocentric, is harder to define. Summarizing discussions e.g. by Klatzky [24], Burgess [23], and Mallot and Basten [25], we define an allocentric memory as one that does not change as the observer moves. Note that this definition does not refer to coordinate systems or global anchor points. Indeed knowledge such as distances between places as well as oriented views and their relation to other oriented views qualifies as allocentric memory in this sense, because it can be carried around and remains useful without a need for movement-dependent changes or transforma- tions. Almost as a corollary to this definition, long-term memories will always be allocentric, while working memories involving automated spatial updating will be not. In the Model section, we describe the view-graph [26] as an allocentric data structure for spatial long-term memory that lends itself easily to interactions with ego-centric working memories. The central spatial concept of the view-based framework is the view, i.e. an image or early visual representation of a sector or angle of the environment taken at a position x~(x1,x2) and with a viewing direction Q; we denote the view by v(x,Q). It need not be limited by the visual perimeter, but may also contain information from beyond the current visual horizon, encoded in an egocentric way, see, for example, Tatler and Land [2]. The simplest long- term memory of a place xo is then a collection of views taken at that place, fv(xo,Qi),i~1, . . . A view-based model of spatial working and long-term memories In the interplay between spatial working and long-term memories, the encoding, or data-format, used by each memory structure is of great importance. Recall from long-term memory into spatial working memory, i.e. between allocentric and ego- centric representations, is often thought to require a coordinate transform, which is certainly true if spatial information is explicitly represented in the form of coordinates. However, in a view-based account, an allocentric, long-term representation of place may even be a view or a collection of views which were egocentric when first perceived and stored, but are now carried around for reference. Simply enough, transformation of this view-based allocentric representation into an egocentric one amounts to picking a particular view which corresponds to the current viewing direction and loading this view into working memory, e.g. for comparison to the currently visible view of the present place. As a result, places would be recognized by view matching [34], similar to the snapshot algorithms discussed in insects [35]. In addition to simple matching, a process of view transformation might be involved, allowing the prediction of nearby or intermediate views from stored ones, as has been suggested for robot applications [36]. Such a mechanism seems to be required also in the pointing task studied by Giudice et al. [20], involving both long-term and working memories. In pose-invariant object recognition, view interpolation is a well-established mechanism [37,38]. In a study by Basten, Meilinger, and Mallot [21], visitors of the University restaurant of the University of Tu¨bingen were asked to draw sketches of the ‘‘Holzmarkt’’, a central and familiar downtown square about two kilometers away. Drawings were rated for orientation and a clear preference for the southward view was found, depicting a landmark church building on top of a hill. However, when subjects had been asked prior to the sketching task to imagine walking a route passing by the target square in one of two opposite directions, drawings in the respective viewing direction became significantly more frequent. The authors concluded that mental travel activated a view-dependent (‘‘ego’’- centric with respect to the imagined travel) representation of the target square which later primed the sketching process. A particularly interesting case for the present discussion is representational neglect studied by Bisiach and Luzzatti [22], which shows that (at least in patients suffering from hemilateral neglect), recall of spatial long-term memories depends on the subject’s imagined position and orientation. Multiple representations of space Multiple representations of space have been suggested for a number of reasons. One issue is the problem of scale which may vary from centimeters in manipulation tasks to thousands of kilometers in way-finding. Gru¨sser [5] distinguishes a (mostly metrical) grasp space, a near- and a far-distance action space, and a visual background. Montello [6] presented a classification of ‘‘psychological spaces’’ also based on scale, in which the spatial image discussed here is somewhere between ‘‘vista space’’ (what is currently visible) and ‘‘environmental space’’ (the area a subject is used to navigate in). The distinction between working and long-term memories of space is grounded both in behavioral and neurophysiological data [7,8]. Spatial working memory tasks which are largely indepen- dent of spatial long-term memories include spatial sequence learning such as walking versions of the Corsi block-tabbing task [9], perspective taking and spatial updating [10], walking without vision [11], path integration [12,13], path-planning in multi-local tasks [14], etc. Interactions of spatial working and long-term memories are crucial in way-finding, i.e. the planning of novel November 2014 \| Volume 9 \| Issue 11 \| e112793 1 PLOS ONE \| www.plosone.org View-Based Organization and Interplay of Spatial Memories paths from known segments [15–17], spatial imagery [18], direction giving, and other tasks. Wang and Brockmole [19] studied spatial updating, a typical working memory task, in nested environments and concluded that spatial updating acts differently on close (the surrounding room) and distant (the outdoor buildings) environments. Giudice, Klatzky, Bennett, and Loomis [20] addressed the interaction of long-term and working memories in a pointing task involving the angle between items stored in the different memory systems. November 2014 \| Volume 9 \| Issue 11 \| e112793 A view-based model of spatial working and long-term memories Map source: OpenStreetMap contributors. doi:10.1371/journal.pone.0112793.g001 When asked to imagine a nearby target place xt, subjects will recall from memory one of the stored views v(xt,Qj) of this place. In spatial working memory, only the views contained in the outward neighborhood of vo will be present. Therefore, if recall is based on working memory content, the view obtained when (mentally) traveling from the current ‘‘here’’ to the target place will be selected. In this case, we predict that in visual recall of a target place, the recalled viewing direction will depend on interview location. If, however, recall is based solely on long-term memory, one of the known views of the target place will be selected independent of interview location. neighboring place, as it appears when arriving from the starting location. As was demonstrated by Scho¨lkopf and Mallot [26], the resulting view graph contains sufficient information for route planning between connected views. As a model of spatial working memory, we suggest a sub-graph of the full view-graph, consisting of the current view corresponding to the observer’s current position and orientation, and the views reachable from this current view in a small number of steps s, i.e. the outward neighborhood Ns(vo). Note that the graph links are directed, allowing to distinguish an outward neighborhood (views reachable from vo) from an inward neighborhood (views from which vo can be reached). In Figure 1c, we show the one-step (s~1) outward neighborhood of view 1 of place B. As the observer moves, the current view will change and so will its outward neighborhood represented in working memory. This may be achieved by repeatedly refreshing the neighborhood from long- term memory, i.e. loading the appropriate sub-graph after each movement step. Alternatively, or on smaller scales, one could think of some sort of ego-motion driven image transformation (spatial updating) within working memory. We indicate this possibility by adding a polar coordinate grid to working memory in Figure 1c. In our experiment, we cannot distinguish between refreshing from long-term memory and spatial updating within working memory. See [20] for an experiment directly addressing this problem. For the analysis of the data presented below, we introduce the following notation: Let pi,t(Q) denote the probability that the recalled view of target place xt has the orientation Q, given that the interview location is xi. A view-based model of spatial working and long-term memories ,ng where the index i enumerates the individual viewing directions and n is the total number of views stored for the particular place (see Figure 1a). The views may be overlapping and the distribution of viewing directions Qi may be anisotropic. If, for example, one particular view of a place is especially salient, we may model this by assuming that multiple copies of this view, or largely overlapping adjacent views, will be included in the place representation. In analogy to object representation, such views might be called ‘‘canonical’’ for the respective place. In addition to the views themselves, we assume that the adjacencies of views are also represented in the place code. The views together with their adjacencies thus form a simple view- graph with a ring-topology. As in [26], the adjacency links will be labelled with action codes such as ‘‘turn left’’, or ‘‘turn right 40 degrees’’. Over the past decade, imaging studies have identified an extensive network of cortical and subcortical brain areas involved in a variety of spatial behaviors. Tasks involving an interplay of spatial long-term and working memories have been shown to recruit structures such as the retrosplenial cortex as well as medial temporal lobe [27–30]. More on the visual side, scene recognition as well as imagery of out-of-sight places or perspectives has been related to various parts of the parietal cortex and transverse occipital sulcus [31–33]. From this place representation, a long-term memory of a larger environment, i.e. a cognitive map, can be built as a full view-graph and used for way-finding (see Figure 1b). For multiple places, interplace view adjacencies have to be stored as ‘‘action labels’’ representing egocentric locomotor actions such as ‘‘walk straight from here’’ or ‘‘follow the street from here’’. In these action labels, ‘‘here’’ refers to a view from the current place assuming the observer’s current heading. The link will end at a view of a PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e112793 November 2014 \| Volume 9 \| Issue 11 \| e112793 2 View-Based Organization and Interplay of Spatial Memories Figure 1. View-based model of spatial long-term memory. Upper case letters A, B, C denote places, numbers (1–4) denote views visible at each place. E.g., view A3 depicts a church building when standing at the ‘‘Holzmarkt’’ (A), facing south. A view-based model of spatial working and long-term memories a) Place representation composed of a collection of directional views (1–4) obtained at a place A. Views may be represented multiply, or overlapping, allowing to represent viewing direction in a population code. The size of the circles indicates the frequency with which each view is stored, or the likelihood that it is read out in recall. (Tu¨bingen Holzmarkt icons are sections of a panoramic image retrieved with permission from www.kubische-panoramen.de.) b) View-graph of 12 views (A1-C4) belonging to three places. Within each place, views are linked by turning movements. Views of different places are linked by movements involving translations. Note that these links are unidirectional; for example a path from A to B starts from view A3, while the return from B to A will end on A1. c) A view-based model of spatial working memory is obtained by extracting a sub-graph from the total view-graph. It contains the current view (B1) which also marks the current observer position and forward direction, and its outward neighborhood of order 1, i.e., the directly adjacent views (A1, B2, B4, C1). Outward neighborhoods of higher orders may also be represented but are not shown in the figure. The polar grid is added to indicate that metric updating may take place in the working memory, which, however, does not play a role in the experiment reported in this paper. Map source: OpenStreetMap contributors. doi:10.1371/journal.pone.0112793.g001 View-Based Organization and Interplay of Spatial Memories Figure 1. View-based model of spatial long-term memory. Upper case letters A, B, C denote places, numbers (1–4) denote views visible at each place. E.g., view A3 depicts a church building when standing at the ‘‘Holzmarkt’’ (A), facing south. a) Place representation composed of a collection of directional views (1–4) obtained at a place A. Views may be represented multiply, or overlapping, allowing to represent viewing direction in a population code. The size of the circles indicates the frequency with which each view is stored, or the likelihood that it is read out in recall. (Tu¨bingen Holzmarkt icons are sections of a panoramic image retrieved with permission from www.kubische-panoramen.de.) b) View-graph of 12 views (A1-C4) belonging to three places. Within each place, views are linked by turning movements. Views of different places are linked by movements involving translations. November 2014 \| Volume 9 \| Issue 11 \| e112793 A view-based model of spatial working and long-term memories Note that these links are unidirectional; for example a path from A to B starts from view A3, while the return from B to A will end on A1. c) A view-based model of spatial working memory is obtained by extracting a sub-graph from the total view-graph. It contains the current view (B1) which also marks the current observer position and forward direction, and its outward neighborhood of order 1, i.e., the directly adjacent views (A1, B2, B4, C1). Outward neighborhoods of higher orders may also be represented but are not shown in the figure. The polar grid is added to indicate that metric updating may take place in the working memory, which, however, does not play a role in the experiment reported in this paper. Map source: OpenStreetMap contributors. doi:10.1371/journal.pone.0112793.g001 Figure 1. View-based model of spatial long-term memory. Upper case letters A, B, C denote places, numbers (1–4) denote views visible at each place. E.g., view A3 depicts a church building when standing at the ‘‘Holzmarkt’’ (A), facing south. a) Place representation composed of a collection of directional views (1–4) obtained at a place A. Views may be represented multiply, or overlapping, allowing to represent viewing direction in a population code. The size of the circles indicates the frequency with which each view is stored, or the likelihood that it is read out in recall. (Tu¨bingen Holzmarkt icons are sections of a panoramic image retrieved with permission from www.kubische-panoramen.de.) b) View-graph of 12 views (A1-C4) belonging to three places. Within each place, views are linked by turning movements. Views of different places are linked by movements involving translations. Note that these links are unidirectional; for example a path from A to B starts from view A3, while the return from B to A will end on A1. c) A view-based model of spatial working memory is obtained by extracting a sub-graph from the total view-graph. It contains the current view (B1) which also marks the current observer position and forward direction, and its outward neighborhood of order 1, i.e., the directly adjacent views (A1, B2, B4, C1). Outward neighborhoods of higher orders may also be represented but are not shown in the figure. The polar grid is added to indicate that metric updating may take place in the working memory, which, however, does not play a role in the experiment reported in this paper. A view-based model of spatial working and long-term memories Let further Lt(Q) and Wi,t(Q) denote the probability densities of recalling a view Q if recall is from long-term or working memory, respectively. Note that the working memory contribution depends on interview location, whereas the long-term memory contribution does not. We expect that Wi,t(Q) is a peaked distribution with a maximum at the approach direction from interview location xi to target place xt. In the data analysis, we will identify the approach direction with the air-line direction between the two places, November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org 3 View-Based Organization and Interplay of Spatial Memories View-Based Organization and Interplay of Spatial Memories 2. City maps of Tu¨ bingen with interview locations and target places (‘‘Holzmarkt’’ & ‘‘Marktplatz’’). a) Distant w locations (North, East, South, West) were located in small shopping areas about 2 km away from the target squares, which were wn area (red square). b) Close-up view of the downtown area of Tu¨bingen. Blue: Interview locations (A–J) and target place for ex arkt’’). Green: Interview locations (A–H) and target place for experiment 2 (‘‘Marktplatz’’). Map source: OpenStreetMap contrib 371/journal.pone.0112793.g002 NE \| www plosone org 4 November 2014 \| Volume 9 \| Issue 11 Figure 2. City maps of Tu¨ bingen with interview locations and target places (‘‘Holzmarkt’’ & ‘‘Marktplatz’’). a) Distant (suburban) interview locations (North, East, South, West) were located in small shopping areas about 2 km away from the target squares, which were inside the downtown area (red square). b) Close-up view of the downtown area of Tu¨bingen. Blue: Interview locations (A–J) and target place for experiment 1 (‘‘Holzmarkt’’). Green: Interview locations (A–H) and target place for experiment 2 (‘‘Marktplatz’’). Map source: OpenStreetMap contributors. doi:10.1371/journal.pone.0112793.g002 November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org 4 View-Based Organization and Interplay of Spatial Memories Figure 3. Examples of sketches of the ‘‘Holzmarkt’’ from four participants. The blue arrows indicate the orientation the sketch was rated in. Note the inscriptions ‘‘Stiftskirche’’ or ‘‘Kirche’’ referring to the landmark church building located at this square (see also view A3 in Figure 1a). The parallel lines mark a flight of stairs leading from the square to the church, the circles mark a fountain at the Western side of the square. doi:10.1371/journal.pone.0112793.g003 Figure 3. Examples of sketches of the ‘‘Holzmarkt’’ from four participants. A view-based model of spatial working and long-term memories The blue arrows indicate the orientation the sketch was rated in. Note the inscriptions ‘‘Stiftskirche’’ or ‘‘Kirche’’ referring to the landmark church building located at this square (see also view A3 in Figure 1a). The parallel lines mark a flight of stairs leading from the square to the church, the circles mark a fountain at the Western side of the square. doi:10.1371/journal.pone.0112793.g003 where p:,t(Q) denotes the average view distribution over all interview locations. From this, we will calculate an estimate for the working memory contribution as Qi,t~atan2(xt{xi), ð1Þ ð1Þ where atan2 is the inverse tangent function with two arguments. For the distribution of the recalled view orientations, we obtain Wi,t(Q)!pi,t(Q){p:,t(Q)zc, ð4Þ ð4Þ pi,t(Q)~aLt(Q)z(1{a)Wi,t(Q), ð2Þ where c is a constant reflecting the non-zero average of the working memory distributions. In the analysis of the experimental data, orientations are sampled to the four cardinal directions (N, E, S, W). The constant c cancels out in the calculation of the circular vectors following Equation 5 below. In analyses of the distribution Wi,t(Q) this constant is important to avoid negative values; it can be set to 0:25. The proportionality factor in Equation 4 will be ignored in the sequel. ð2Þ where Lt(Q) and Wi,t(Q) and are the long-term and working memory contributions, respectively and a is a mixing factor varying between 0 and 1. It reflects the relative strength of long- term and working memory components in the recall. We expect that a is less than 1 for interview sites close to the target place and 1 for distant interview locations. If, for a given target place, the interview locations are spaced regularly around this place, the average of the Wi,t(Q) will approach the uniform distribution, (1=n) Pn i~1 Wi,t(Q)&1=2p and we may estimate the long-term memory contributions as Material and Methods Passers-by at 14 locations in Tu¨bingen (see below and Figure 2) where approached during day time and asked ‘‘if they would participate in a quick interview for a navigational study’’. They were informed about the type of the collected data and the general aLt(Q)&p:,t{ 1{a 2p , ð3Þ ð3Þ PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 View-Based Organization and Interplay of Spatial Memories e 4. Sketch orientation frequencies for drawing the ‘‘Holzmarkt’’. a) Orientation frequencies of the near interview locations (A–J). The ed frequencies differed significantly from each other. b) Orientation frequencies of the distant interview locations (North to West). c, d) Average ation frequencies with standard deviation of the near and distant condition, respectively. The y-axis shows the frequency of sketch map ations, the x-axis the rated orientation (North, East, South, West). 1371/journal.pone.0112793.g004 Figure 4. Sketch orientation frequencies for drawing the ‘‘Holzmarkt’’. a) Orientation frequencies of the near interview locations (A–J). The obtained frequencies differed significantly from each other. b) Orientation frequencies of the distant interview locations (North to West). c, d) Average orientation frequencies with standard deviation of the near and distant condition, respectively. The y-axis shows the frequency of sketch map orientations, the x-axis the rated orientation (North, East, South, West). doi:10.1371/journal.pone.0112793.g004 Figure 4. Sketch orientation frequencies for drawing the ‘‘Holzmarkt’’. a) Orientation frequencies of the near interview locations (A–J). The obtained frequencies differed significantly from each other. b) Orientation frequencies of the distant interview locations (North to West). c, d) Average orientation frequencies with standard deviation of the near and distant condition, respectively. The y-axis shows the frequency of sketch map orientations, the x-axis the rated orientation (North, East, South, West). doi:10.1371/journal.pone.0112793.g004 locations in walking distance (about 150 m) to but out of sight of the target square ‘‘Holzmarkt’’ (‘‘near’’ condition; see Figure 3). Care was taken to approach participants walking in different directions. Approach was from sideways with respect to the participant’s heading. Upon being approached, participants stopped but did not change their general body orientation. Also during recall, no regular turning movements of the participants were observed. procedure. About one third agreed to participate (verbal informed consent) as was documented by their later participation in the interview. Participants were not asked for their names and accordingly were not required to give their consent in writing. Material and Methods Participants were free to terminate their participation at any time, simply by walking away. The informed consent procedures adheres to the guidelines of the Declaration of Helsinki, approval by the local ethics committee was not required. The sketches were categorized for orientation (North, East, South or West up) by three independent raters. From the 335 drawings 331 were judged identically (99%) with a chance- corrected inter-rater reliability of k~0:98. A small number of sketches was consistently rated diagonal; in these cases, the number 0.5 was added to the two adjacent directions. Only the 331 identically judged drawings were analyzed further (254 near condition, 77 distant condition). The mean age of the 331 participants whose maps were included was 33:36 years, their average time of residency in Tu¨bingen was 12:9 years, their own judgment of local knowledge and general navigation skills was 5:9 and 6:2, respectively, both on a scale between 1 and 9 with 1~ very poor and 9~ very good. Participants were requested to ‘‘sketch the layout of the Holzmarkt’’ (timber market), a well-known down-town square, on an A4 sheet of paper. After sketching, they were asked for their age, years of residency in Tu¨bingen, own judgment of general navigation skills, and own judgment of local knowledge (see below). Only sketches by subjects who had lived in Tu¨bingen for more than two years were analyzed further. In total, these were 335 adults (161 male, 174 female). An interview and sketch map production took less than two minutes in total. Examples of sketch maps appear in Figure 3. Interviews took place outdoors, either at one of four distant locations in small suburban shopping areas about 2 km away from the target square (‘‘distant’’ condition) or at one of ten downtown November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org 6 View-Based Organization and Interplay of Spatial Memories Figure 5. Downtown map of Tu¨ bingen with interview locations (A–J) for the near condition and target square ‘‘Holzmarkt’’. The vectors show the average sketch map orientation at the respective interview site. At seven (blue circles) out of ten near sites sketch orientations were found to point from the interview location in the direction of the target square (pv0:05 or better). At one location, a strong tendency was indicated (A, cyan, p~0:051). For two locations (F,G; red), no significant orientation effect could be found. Results Orientation frequencies of the sketches of the ten downtown and four suburban interview locations are shown in Figure 4; for the orientation counts, see Figure S1. The distributions obtained at the near locations differ significantly from each other (x2(27,N~254)~88:036; pv0:001) indicating that recalled view orientation depends on interview location. For the distant locations, no differences between the histograms could be found. The average distributions for near and distant interview sites are shown separately in Figure 4. These distributions are significantly different from each other (x2(3,N~331)~12:654; pv0:01) though comparable in shape. Orientation frequencies of the sketches of the ten downtown and four suburban interview locations are shown in Figure 4; for the orientation counts, see Figure S1. The distributions obtained at the near locations differ significantly from each other (x2(27,N~254)~88:036; pv0:001) indicating that recalled view orientation depends on interview location. For the distant locations, no differences between the histograms could be found. The average distributions for near and distant interview sites are shown separately in Figure 4. These distributions are significantly different from each other (x2(3,N~331)~12:654; pv0:01) though comparable in shape. The orientation vectors obtained from the location-dependent components of the downtown interview locations (Equation 5) are plotted in Figure 5 superimposed on a map of Tu¨bingen showing the target and interview locations. An overall tendency of the vectors to point to the target square is clearly apparent. wi~ (ei{e) { (wi{w) (ni{n) { (si{s) : ð5Þ ð5Þ In order to test this tendency, we calculated the angular deviation between the location dependent orientation vectors and the theoretical air-line vector obtained for each interview location by subtracting the coordinates of the target square (defined as the center of gravity of the blue area in Figure 5) from the coordinates of the interview sites (Equation 1). For each interview location, the deviation or bias of the data from a uniform distribution towards the theoretical direction was tested with the circular V-test [45,46], taking into account the vector length as a measure of concentration. The deviations towards the theoretical direction are The orientation of these vectors is an estimator of the circular mean of the working memory distribution Wi,t(Q) from Equation 4. The length is a measure of concentration of this distribution related to the circular variance [45,46]. A long vector means more concentration (more coherent sketch orientations) and stronger differences from the average (long-term memory) distribution. Material and Methods Vector length ranges from zero to one (radius of circle) and is a measure of concentration of the location-dependent vectors. Map source: Open-StreetMap contributors. doi:10.1371/journal.pone.0112793.g005 Figure 5. Downtown map of Tu¨ bingen with interview locations (A–J) for the near condition and target square ‘‘Holzmarkt’’. The vectors show the average sketch map orientation at the respective interview site. At seven (blue circles) out of ten near sites sketch orientations were found to point from the interview location in the direction of the target square (pv0:05 or better). At one location, a strong tendency was indicated (A, cyan, p~0:051). For two locations (F,G; red), no significant orientation effect could be found. Vector length ranges from zero to one (radius of circle) and is a measure of concentration of the location-dependent vectors. Map source: Open-StreetMap contributors. doi:10.1371/journal.pone.0112793.g005 For each interview location i, relative frequencies of ratings for the four cardinal directions were calculated and denoted as (ni,ei,si,wi) for North, East, South, and West. Average frequencies were also calculated separately of the ten ‘‘near’’ and the four ‘‘distant’’ interview locations and denoted as (ni,ei,si,wi). In the next step, the average frequencies from the ‘‘near’’ interview locations were subtracted from each of the local histograms of the ‘‘near’’ condition. Similarly, the average frequencies for the four distant interview locations were subtracted from the distant histograms. We refer to the results as the ‘‘location-dependent components’’ and consider them as an estimator of local working memory content, according to Equation 4. Finally, these location- depenedent components were transformed into location-depen- dent orientation vectors November 2014 \| Volume 9 \| Issue 11 \| e112793 Discussion The data presented in this study indicate that visuo-spatial recall of out-of-sight places does not occur with a random or fixed orientation but that recall orientation depends on both target and interview location. Figure 6 shows the location-dependent vectors rotated such that the theoretical direction for each interview location appears in upwards direction. For this sample of 10 vectors, we again applied the circular V-test, this time with the 0-degree-vector as a theoretical direction. For the overall sample, bias towards the theoretical direction was significant with (V(N~254)~0:234; u~5:276; pv0:001). The target square effect suggests a non-isotropic representation of each target square in long-term memory. For the distant (suburban) interview locations, orientation distributions were found that equal the average distributions taken over all near (downtown) locations. We therefore conclude that the target square dependence is underlying all our measurements and is modulated by interview location-dependent effects visible only for the downtown interview locations. The average view distribution for the ‘‘Holzmarkt’’ square (Exp. 1) is strongly peaked with a ‘‘canonical view’’ in southward direction, depicting a landmark church building on top of a hillock. In contrast, the view distribution for the ‘‘Marktplatz’’ (Exp. 2) is more isotropic, probably reflecting the more balanced salience of the surrounding houses. These differences are probably related to the specific topography of each place. The ‘‘Holzmarkt’’ is rising to the South, with a prominent church building on top. Approaches from behind the church (Northwards) are almost impossible and very rarely walked. Drawings with the church on top might thus be favored by familiarity, alignment with environmental axes and the fact that uphill buildings will appear on top of the sketching paper. In contrast, the salience of the buildings surrounding the ‘‘Marktplatz’’ (Exp. 2) is much more balanced. The ‘‘Marktplatz’’ is also rising to the South, but the most prominent building, the city hall, appears not on top but on the Western side. Also, approaches from all directions are possible and frequently walked. Still, a peak in the experimental data towards ‘‘South’’ and ‘‘West’’ is apparent here, too. We suggest that the long-term memory of For the four distant interview locations no such orientation effect could be found (V(N~77)~0:038; u~0:477; p~0:317). Results Orientation frequencies of the sketches of the eight near interview locations differed significantly from each other (x2(21,N~220)~95:457; pv0:001). For the distant locations, no difference between the histograms could be found (Figure 7). Also, there was no significant difference between the near and distant average frequencies (x2(3,N~306)~3:986; p~0:263). As shown in Figure 8, the majority of the location-dependent vectors of the near condition point towards the ‘‘Marktplatz’’ (center of gravity of green area in Figure 8). A significant bias of sketch orientations towards the air-line direction to the target square (center of gravity of the green area Figure 8) for six of the eight interview locations could be revealed by a circular V-test. The sample of eight location-dependent vectors, rotated to align their respective air-line directions, also showed a highly significant bias towards the theoretical direction at zero degrees (V(N~220)~0:343; u~7:203; pv0:001) (Figure 9). Figure 6. Location-dependent vectors from Fig. 5, rotated to align the air-line directions from all interview locations to 0 degrees (letters indicate interview locations). Vectors are significantly biased towards the theoretical direction (green line, pv0:001). Vector length reaches from zero to one (radius of circle) and is a measure of concentration of the location-dependent vectors. doi:10.1371/journal.pone.0112793.g006 No bias could be detected for the three distant interview locations (V(N~86)~0:099; u~1:295; p~0:098). significant (pv0:05 or better) for seven out of ten interview locations, and marginally significant for an additional one (p~0:051). For two interview locations (F and G in Figure 5), no significant deviation from uniformity could be demonstrated. significant (pv0:05 or better) for seven out of ten interview locations, and marginally significant for an additional one (p~0:051). For two interview locations (F and G in Figure 5), no significant deviation from uniformity could be demonstrated. Figure 6 shows the location-dependent vectors rotated such that the theoretical direction for each interview location appears in upwards direction. For this sample of 10 vectors, we again applied the circular V-test, this time with the 0-degree-vector as a theoretical direction. For the overall sample, bias towards the theoretical direction was significant with (V(N~254)~0:234; u~5:276; pv0:001). Experiment 2 – ‘‘Marktplatz’’ To test the robustness of the findings of the first experiment with respect to other target squares, we chose another well-known square and repeated the previous experiment. View-Based Organization and Interplay of Spatial Memories View-Based Organization and Interplay of Spatial Memories Figure 6. Location-dependent vectors from Fig. 5, rotated to align the air-line directions from all interview locations to 0 degrees (letters indicate interview locations). Vectors are significantly biased towards the theoretical direction (green line, pv0:001). Vector length reaches from zero to one (radius of circle) and is a measure of concentration of the location-dependent vectors. doi:10.1371/journal.pone.0112793.g006 participants (131 male, 175 female) whose maps were included was 37:4 years, their average years of residency in Tu¨bingen was 12:7, their own judgment of local knowledge was 3:4 (with 1~ very poor and 9~ very good) and own judgment of how often they frequent the ‘‘Marktplatz’’ was 3:0, with 1~ very rarely and 9~ very often. Average orientation frequencies for the near and distant conditions were calculated and subtracted from the histogram of the near and distant interview locations, respectively, yielding the location-dependent components of each distribution. Results Short vectors would result from sketch orientations that are similar to the long-term memory content. November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org 7 Material and Methods Eight new interview locations around the ‘‘Marktplatz’’ (market square) were selected for the near condition (Figure 2b, green). For the distant condition the same locations as in experiment 1 were used except for the southern one, which we did not again get access to. 330 passers-by agreed to participate. The procedure was the same as in experiment 1. Sketches were again categorized for orientation (North, East, South or West up) by three independent raters. From the 330 drawings 306 were judged identically (93%) with a chance- corrected inter-rater reliability of k~0:93. Only the 306 identically judged drawings were analyzed further (220 near condition, 86 distant condition). The mean age of the 306 November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org 8 View-Based Organization and Interplay of Spatial Memories Figure 7. Sketch orientation frequencies for drawing the ‘‘Marktplatz’’. a) Orientation frequencies of the near interview locations (A–H). The obtained frequencies differed significantly from each other. b) Orientation frequencies of the distant interview locations (North, East and West). No significant difference could be found. c, d) Average orientation frequencies with standard deviation of the near and distant condition, respectively. The y-axis shows the frequency of sketch map orientations, the x-axis the rated orientation (North, East, South, West). doi:10.1371/journal.pone.0112793.g007 Figure 7. Sketch orientation frequencies for drawing the ‘‘Marktplatz’’. a) Orientation frequencies of the near interview locations (A–H). The obtained frequencies differed significantly from each other. b) Orientation frequencies of the distant interview locations (North, East and West). No significant difference could be found. c, d) Average orientation frequencies with standard deviation of the near and distant condition, respectively. The y-axis shows the frequency of sketch map orientations, the x-axis the rated orientation (North, East, South, West). doi:10.1371/journal.pone.0112793.g007 Figure 7. Sketch orientation frequencies for drawing the ‘‘Marktplatz’’. a) Orientation frequencies of the near interview locations (A–H). The obtained frequencies differed significantly from each other. b) Orientation frequencies of the distant interview locations (North, East and West). No significant difference could be found. c, d) Average orientation frequencies with standard deviation of the near and distant condition, respectively. The y-axis shows the frequency of sketch map orientations, the x-axis the rated orientation (North, East, South, West). doi:10.1371/journal.pone.0112793.g007 to the target square. November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org Material and Methods A view of the target square, oriented in the current approach direction, thus seems to be activated in a spatial working memory either by automated spatial updating when walking in the city, or by a mental travel initiated when asked to draw the sketch, or by both effects (see Figure 1b,c). Spatial updating itself could again be achieved by two mechanisms, either image transformation as discussed in view-based object recogni- tion [38] or by refreshing working memory from long-term memory. either square is organized as a collection of discrete views (Figure 1a), sampling the various viewing directions with variable resolution much as has been suggested for view-dependence in face recognition [37]. Allocentric place memory might therefore be organized as a population code of orientation-specific memories. Indeed, neuronal specificities for views of places have been reported in the medial temporal lobe, see for example [47,48]. The formation of one or several canonical views of a place requires further study, concerning potential relationships to canonical views of landmark objects and the selection of one view or another as canonical. Reasons for selection might include: Distinctiveness to other places, availability and distribution of local landmarks, geometric layout, visual salience of objects, path options and functionality, or intrinsic axes of the environment [49]. In the introduction, we presented a view-based model of spatial recall predicting that the directional distributions of recalled sketch maps are a mixture of a fixed long-term memory distribution and a set of position dependent working memory distributions (Equation 3). As a direct test of this model, we performed a maximum likelihood analysis assuming for the orientation histograms a multinomial distribution with four possible outcomes (N, E, S, W) and theoretical probabilities alkz(1{a)wik, where k numbers the four possible outcomes and (l1, . . . ,l4) are the class averages over all interview locations, i.e. the assumed long-term memory contributions. The log likelihood function reads The distribution of recalled views depends also on interview location as was revealed by Chi-Squared tests on the orientation histograms. For the near (downtown) interview locations each local distribution is biased towards a preferred orientation roughly corresponding to the air-line direction from the interview location November 2014 \| Volume 9 \| Issue 11 \| e112793 November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org 9 View-Based Organization and Interplay of Spatial Memories Figure 8. Material and Methods Downtown map of Tu¨ bingen with target square ‘‘Marktplatz’’, near interview locations (A–H) and location-dependent vectors drawn at these locations. Vectors at six (blue circles) out of eight interview sites point towards the target square (pv0:05 or better). For two locations (C, D; red), no significant orientation effect could be found. Vector length reaches from zero to one (radius of circle) and is a measure of concentration of the location-dependent vectors. Map source: OpenStreetMap contributors. doi:10.1371/journal.pone.0112793.g008 Figure 8. Downtown map of Tu¨ bingen with target square ‘‘Marktplatz’’, near interview locations (A–H) and location-dependent vectors drawn at these locations. Vectors at six (blue circles) out of eight interview sites point towards the target square (pv0:05 or better). For two locations (C, D; red), no significant orientation effect could be found. Vector length reaches from zero to one (radius of circle) and is a measure of concentration of the location-dependent vectors. Map source: OpenStreetMap contributors. doi:10.1371/journal.pone.0112793.g008 Figure 9. Location-dependent vectors from Fig. 8, rotated to align the air-line directions from all interview locations to 0 degrees (letters indicate interview location). Vectors are signif- icantly biased towards the theoretical direction (green line, pv0:001). Vector length reaches from zero to one (radius of circle) and is a measure of concentration of the location-dependent vectors. doi:10.1371/journal.pone.0112793.g009 Figure 9. Location-dependent vectors from Fig. 8, rotated to align the air-line directions from all interview locations to 0 degrees (letters indicate interview location). Vectors are signif- icantly biased towards the theoretical direction (green line, pv0:001). Vector length reaches from zero to one (radius of circle) and is a measure of concentration of the location-dependent vectors. doi:10.1371/journal.pone.0112793.g009 Figure 10. Likelihood analysis of the mixing model. Yellow: Distant locations, relative likelihood peaks for a~1, indicating that orientation distributions do not depend on air-line direction. Blue and green: Near locations, relative likelihood peaks at av1, indicating the orientation distributions do depend on air-line direction in this condition. The black markers indicate with a with 99% confidence intervals. The y-axis shows the relative log likelihood LL(a){LL(a), the x-axis the mixing factor for working and long-term memory contributions. doi:10.1371/journal.pone.0112793.g010 Figure 10. Likelihood analysis of the mixing model. Yellow: Distant locations, relative likelihood peaks for a~1, indicating that orientation distributions do not depend on air-line direction. Blue and green: Near locations, relative likelihood peaks at av1, indicating the orientation distributions do depend on air-line direction in this condition. Material and Methods The black markers indicate with a with 99% confidence intervals. The y-axis shows the relative log likelihood LL(a){LL(a), the x-axis the mixing factor for working and long-term memory contributions. doi:10.1371/journal.pone.0112793.g010 Figure 10. Likelihood analysis of the mixing model. Yellow: Distant locations, relative likelihood peaks for a~1, indicating that orientation distributions do not depend on air-line direction. Blue and green: Near locations, relative likelihood peaks at av1, indicating the orientation distributions do depend on air-line direction in this condition. The black markers indicate with a with 99% confidence intervals. The y-axis shows the relative log likelihood LL(a){LL(a), the x-axis the mixing factor for working and long-term memory contributions. doi:10.1371/journal.pone.0112793.g010 Figure 10. Likelihood analysis of the mixing model. Yellow: Distant locations, relative likelihood peaks for a~1, indicating that orientation distributions do not depend on air-line direction. Blue and green: Near locations, relative likelihood peaks at av1, indicating the orientation distributions do depend on air-line direction in this condition. The black markers indicate with a with 99% confidence intervals. The y-axis shows the relative log likelihood LL(a){LL(a), the x-axis the mixing factor for working and long-term memory contributions. d i 10 1371/j l 0112793 010 Figure 9. Location-dependent vectors from Fig. 8, rotated to align the air-line directions from all interview locations to 0 degrees (letters indicate interview location). Vectors are signif- icantly biased towards the theoretical direction (green line, pv0:001). Vector length reaches from zero to one (radius of circle) and is a measure of concentration of the location-dependent vectors. doi:10.1371/journal.pone.0112793.g009 doi:10.1371/journal.pone.0112793.g010 November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 10 View-Based Organization and Interplay of Spatial Memories connections to the target places existed while the experiment took place. LL(a)~ X I i~1 (ci X 4 k~1 nik log (alkz(1{a)wik)), ð6Þ ð6Þ The theoretical account presented in the Introduction is clearly able to explain our data. In addition, the findings by Basten et al. [21] on view-based priming of recall by mental travel also fit into the overall scheme. In this study, all interviews were carried out at a distant location (the North location in Fig. 3b) and simple recall of the ‘‘Holzmarkt’’ square revealed the same view preference reported here. Mental travel across the ‘‘Holzmarkt’’, however, primed view-specific recall in the direction of travel, indicating that mental travel, just as actual walking in downtown Tu¨bingen, activates view-specific working memories. Material and Methods where nik is the number of orientations k found at interview location i and the constant ci is the log of the multinomial coefficient for the local orientation distribution. Theoretical estimates for the working memory contributions at each interview location are derived from the local air-line directions Qi (Equation 1). The theoretical outcome probabilities for the assumed working memory distributions were set to wi1~cz0:5 max (0, sin Qi), wi2~cz0:5 max (0, cos Qi), wi3~cz0:5 max (0,{ sin Qi), and wi4~cz0:5 max (0,{ cos Qi), where c~1{0:5(D sin QiDzD cos QiD) is a constant assuring that the four probabilities will add to 1. This distribution has the circular mean Qi and variance 0:5, which reasonably approximates the location-dependent components shown in Figures 5 and 8. Alternative models of spatial working memory not based on views but on object representations and maps have been presented by [1,2,4]. While our data do not strictly rule out these models, they make clear that representations of places are not unique entities that are always activated in their entirety, but that parts of place representations can play independent roles in spatial recall. Such parts are oriented and have therefore been referred to as ‘‘views’’ in this paper. Alternatively, such parts could be landmarks or houses located at one side of a square, or names or other properties of such landmarks or houses, as might have been the case in the experiments reported by Bisiach and Luzzatti [22]. The considered parts of place representations are view-like in two respects: First, the target square effect (canonical view) shows that oriented parts of a place representation can be anisotropically distributed. Second, priming by spatial nearness activates oriented parts of the representation of places, not place representations in their entirety. This finding is in line with previous results of [41] who showed that associative landmark usage depends on oriented parts of place representations rather than on representations of entire places. Overall, we suggest that oriented ‘‘views’’ form a separate level of granularity in spatial representation that can be activated whenever view-specific information is required. Figure 10 shows the relative log likelihood LL(a){LL(a) as a function of the mixing parameter a separately for the near and far interview locations in both experiments. For the ‘‘far’’ cases, the maximum likelihood estimator a is 1, i.e. adding position- dependent working memory contributions to the model does not improve likelihood in these cases. Supporting Information No location-dependent effect was found for the distant (suburban) interview locations. We conclude that in these cases, recall did not depend on working memory processes such as spatial updating or mental travel. Of course, other working memory effects might still be involved. Since we used only two distance conditions, downtown and suburban, we cannot decide how far the location-dependent effect extends around the target place or if there is a gradual decay as could be modelled by a distance- dependent factor a in Equation 2. It is clear, however, that the effect extends over tens to hundreds of meters which seem to be included in spatial working memory. Figure S1 Sketch orientation counts. The figure shows the counts for the sketch orientations for all interview locations and both experiments as determined by the rating process. In rare cases, where all raters agreed that the orientation was between two cardinal directions (e.g., SW), a count of 0:5 was added to each of the adjacent cardinal directns (e.g., S and W). The rightmost column of the table shows the airline directions from interview location to goal, computed according to Equation 1. (XLS) Material and Methods In contrast, for the ‘‘near’’ cases, the maximum likelihood estimates lie between 0:6 and 0:7; the horizontal lines in the plot are 99% confidence intervals. A likelihood ratio test for a~1 vs. av1 is significant with pv10{16 for the ‘‘near’’ cases in either experiment. The model with the location-dependent working memory component thus significantly improves the fit of the data. We cannot decide from our data whether recall bias is strictly toward the air-line direction or toward the actual entry view obtained when walking to the target place along the street network, although in a view-based account, the latter seems more plausible. Indeed, this might have been the problem with the interview location D in experiment 2 from which two roughly equidistant routes to the target place exist, each with opposite entry directions into the target square. November 2014 \| Volume 9 \| Issue 11 \| e112793 References Proceedings of the National Academy of Sciences of the United States of America 110: 10371–10378. 8. Chen GF, King JA, Burgess N, O’Keefe J (2013) How vision and movement combine in the hippocampal place code. Proceedings of the National Academy of Sciences of the United States of America 110: 378–383. 32. Nasr S, Devaney KJ, Tootell RBH (2013) Spatial encoding and underlying circuitry in scene-selective cortex. Neuroimage 83: 892–900. y g 33. Schindler A, Bartels A (2013) Parietal cortex codes for egocentric space beyond the field of view. Current Biology 23: 177–182. 9. Piccardi L, Bianchini F, Argento O, De Nigris A, Maialetti A, et al. (2013) The walking Corsi test (WalCT): standardization of the topographical memory test in an Italian population. Neurological Sciences 34: 971–978. gy 34. Gillner S, Weiß AM, Mallot H (2008) Visual place recognition and homing in the absence of feature-based landmark information. Cognition 109: 105–122. 10. Farrell MJ, Robertson IH (1998) Mental rotation and the automatic updating of body-centered spatial relationships. Journal of Experimental Psychology: Learning, Memory and Cognition 24: 227–233. 35. Cartwright BA, Collett TS (1982) How honey bees use landmarks to guide their return to a food source. Nature 295: 560–564. 11. Philbeck JW, Loomis JM (1997) Comparison of two indicators of perceived egocentric distance under full-cue and reduced-cue conditions. Journal of Experimental Psychology: Human Perception and Performance 23: 72–75. 36. Moller R, Krzykawski M, Gerstmayr L (2010) Three 2D-warping schemes for visual robot navigation. Autonomous Robots 29: 253–291. 37. Bu¨lthoff HH, Edelman SY, Tarr MJ (1995) How are 3-dimensional objects represented in the brain? Cerebral Cortex 5: 247–260. 12. Loomis JM, Klatzky RL, Golledge RG, Cicinelli JG, Pellegrino JW, et al. (1993) Nonvisual navigation by blind and sighted: Assessment of path integration ability. Journal of Experimental Psychology: General 122: 73–91. 38. Ullman S, Basri R (1991) Recognition by linear combinations of models. IEEE Transactions on Pattern Analysis and Machine Intelligence 13: 992–1006. y g 39. Franz MO, Scho¨lkopf B, Mallot HA, Bu¨lthoff HH (1998) Learning view graphs for robot navigation. Autonomous Robots 5: 111–125. 13. Wolbers T, Wiener JM, Mallot HA, Bu¨chel C (2007) Differential recruitment of the hippocampus, medial prefrontal cortex and the human motion complex during path integration in humans. Journal of Neuroscience 27: 9408–9416. 40. Acknowledgments Another parameter in addition to the mere distance could be regionalization and spatial hierarchies. In virtual environments, navigators were shown to prefer routes that cross fewer region boundaries over equidistant routes through multiple regions [17]. In this experiment, regions were defined by the semantic class of landmark objects. In a pointing experiment, Wang and Brockmole [50] demonstrate that information from nested environments may be kept separate in spatial representations. In the city environ- ments used in the present study, there are various configurations of buildings, roads, shops, etc. which segregate the environment into quarters, districts, neighborhoods, etc. Therefore it seems possible that the extension of spatial working memory is defined by region boundaries rather than by metric distance. This might also explain the results for the interview locations F and G in experiment 1 and C in experiment 2: They were probably attributed to the region ‘‘riverfront’’ and not ‘‘downtown’’, and therefore no or only weak This work was carried out at the Department of Biology of the University of Tu¨bingen. WGR received additional support from the Werner Reichardt Center for Integrative Neuroscience (CIN) at the University of Tu¨bingen. The CIN is an Excellence Cluster funded by the Deutsche Forschungsgemeinschaft (www.dfg.de) within the Excellence Initiative under grant number EXC 307. Additional support was provided by the German Federal Ministry of Education and Research (www.bmbf.de) within the Tu¨bingen Bernstein Center for Computational Neuroscience under grant number 01GQ1002A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are grateful to Niklas Binder and Julia Mayer for help with the collection and analysis of the data. November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org 11 View-Based Organization and Interplay of Spatial Memories References Gaussier P, Revel A, Banquet JP, Babeau V (2002) From view cells and place cells to cognitive map learning: processing stages of the hippocampal system. Biological Cybernetics 86: 15–28. g p g J 14. Hardiess G, Basten K, Mallot HA (2011) Acquisition vs. memorization trade-offs are modulated by walking distance and pattern complexity in a large scale copying paradigm. PlosONE 6(4). g y 41. Mallot HA, Gillner S (2000) Route navigation without place recognition: what is recognized in recognition–triggered responses? Perception 29: 43–55. py g p g ( ) 15. Hartley T, Maguire EA, Spiers HJ, Burgess N (2003) The well-worn route and the path less traveled: distinct neural bases of route following and wayfinding in humans. Neuron 37: 877–888. 42. Pickup LC, Fitzgibbon AW, Glennerster A (2013) Modelling human visual navigation using multiview scene reconstruction. Biological Cybernetics 107: 449–464. 16. Meilinger T, Knauff M, Bu¨lthoff HH (2008) Working memory in wayfinding – a dual task experiment in a virtual city. Cognitive Science 32: 755–770. 43. Wang RF, Spelke ES (2002) Human spatial representation: insights from animals. Trends in Cognitive Sciences 6: 376–382. 17. Wiener JM, Mallot HA (2003) ‘Fine-to-coarse’ route planning and navigation in regionalized environments. Spatial Cognition and Computation 3: 331–358. 44. Furuya Y, Matsumoto J, Hori E, Boas CV, Tran AH, et al. (2014) Place-related neuronal activity in the monkey parahippocampal gyrus and hippocampal formation during virtual navigation. Hippocampus 24: 113–130. 18. Mellet E, Bricogne S, Crivello F, Mazoyer B, Denis M, et al. (2002) Neural basis of mental scanning of a topographic representation built from a text. Cerebral Cortex 12: 1322–1330. 45. Batschelet E (1981) Circular Statistics in Biology. London: Academic Press. 19. Wang RF, Brockmole JR (2003) Simultaneous spatial updating in nested environments. Psychonomic Bulletin & Review 10: 981–986. 46. Berens P (2009) Circstat: A MATLAB toolbox for circular statistics. Journal of Statistical Software 31. 20. Giudice NA, Klatzky RL, Bennett CR, Loomis JM (2013) Combining locations from working memory and long-term memory into a common spatial image. Spatial Cognition and Computation 13: 103–128. 47. Epstein RA (2008) Parahippocampal and retrosplenial contributions to human spatial navigation. Trends in Cognitive Sciences 12: 388–396. 48. Ison MJ, Mormann F, Cerf M, Koch C, Fried I, et al. (2011) Selectivity of pyramidal cells and interneurons in the human medial temporal lobe. Journal of Neurophysiology 106: 1713–1721. p g p 21. Author Contributions Conceived and designed the experiments: WGR GH HAM. Performed the experiments: WGR. Analyzed the data: WGR HAM. Wrote the paper: WGR GH HAM. References editors, Spatial Cognition. An Interdisciplinary Approach to Representing and Processing Spatial Knowledge, Berlin: Springer Verlag, number 1404 in Lecture Notes in Artficial Intelligence. pp. 1–17. 1. Loomis JM, Klatzky RL, Giudice NA (2013) Representing 3D space in working memory: Spatial images from vision, hearing, touch, and language. In: Lacey S, Lawson R, editors, Multisensory Imagery: Theory and Applications, New York: Springer. pp. 131–156. g pp 25. Mallot HA, Basten K (2009) Embodied spatial cognition: biological and artificial systems. Image and Vision Computing 27: 1658–1670. g pp 25. Mallot HA, Basten K (2009) Embodied spatial cognition: p g pp 2. Tatler BW, Land MF (2011) Vision and the representation of the surroundings in spatial memory. Philosophical Transactions of the Royal Society (London) B 366: 596–610. systems. Image and Vision Computing 27: 1658–16 y g p g 26. Scho¨lkopf B, Mallot HA (1995) View–based cognitive mapping and path planning. Adaptive Behavior 3: 311–348. p , ( ) planning. Adaptive Behavior 3: 311–348. p g p 27. Bird CM, Burgess N (2008) The hippocampus and memory: insights from spatial processing. Nature Reviews Neuroscience 9: 182–194. p g p 27. Bird CM, Burgess N (2008) The hippocampus and mem 3. Land MF (2014) Do we have an internal model of the outside world? Philosophical Transactions of the Royal Society (London) B 369. g ( ) pp p processing. Nature Reviews Neuroscience 9: 182–194. p g 28. Ranganath C, Ritchey M (2012) Two cortical syste 28. Ranganath C, Ritchey M (2012) Two cortical systems for memory-guided behaviour. Nature Reviews Neuroscience 13: 713–726. 4. Byrne P, Becker S, Burgess N (2007) Remembering the past and imagining the future: A neural model of spatial memory and imagery. Psychological Review 114: 340–375. behaviour. Nature Reviews Neuroscience 13: 713–726 29. Vann SD, Aggleton JP, Maguire EA (2009) What does the retrosplenial cortex do? Nature Reviews Neuroscience 10: 792–802. 5. Gru¨sser OJ (1982) Space perception and the gazemotor system. Human Neurobiology 1: 73–76. 30. Wolbers T, Bu¨chel C (2005) Dissociable retrosplenial and hippocampal contributions to successful formation of survey representations. The Journal of Neuroscience 25: 3333–3340. gy 6. Montello DR (1993) Scale and multiple psychologies of space. Lecture Notes in Computer Science 716: 312–321. 31. Lambrey S, Doeller C, Berthoz A, Burgess N (2012) Imagining being somewhere else: Neural basis of changing perspective in space. Cerebral Cortex 22: 166– 174. 7. Carruthers P (2013) Evolution of working memory. View-Based Organization and Interplay of Spatial Memories References Basten K, Meilinger T, Mallot HA (2012) Mental travel primes place orientation in spatial recall. Lecture Notes in Artificial Intelligence 7463: 378–385. p g p 21. Basten K, Meilinger T, Mallot HA (2012) Mental travel primes pl 49. Mou WM, McNamara TP (2002) Intrinsic frames of reference in spatial memory. Journal of Experimental Psychology: Learning, Memory and Cognition 28: 162–170. 22. Bisiach E, Luzzatti C (1978) Unilateral neglect of representational space. Cortex 14: 129–133. 23. Burgess N (2006) Spatial memory: how egocentric and allocentric combine. Trends in Cognitive Sciences 10: 551–557. 50. Wang RXF, Brockmole JR (2003) Human navigation in nested environments. Journal of Experimental Psychology: Learning, Memory and Cognition 29: 398– 404. 24. Klatzky RL (1998) Allocentric and egocentric spatial representations: Defini- tions, distinctions, and interconnections. In: Freksa C, Habel C, Wender KF, November 2014 \| Volume 9 \| Issue 11 \| e112793 PLOS ONE \| www.plosone.org 12
https://openalex.org/W4306756219	https://www.frontiersin.org/articles/10.3389/fphy.2022.1036557/pdf	English	null	Multiple radionuclide identification using deep learning with channel attention module and visual explanation	Frontiers in physics	2,022	cc-by	7,030	TYPE Original Research PUBLISHED 19 October 2022 DOI 10.3389/fphy.2022.1036557 TYPE Original Research PUBLISHED 19 October 2022 DOI 10.3389/fphy.2022.1036557 TYPE Original Research PUBLISHED 19 October 2022 DOI 10.3389/fphy.2022.1036557 multiple radionuclide identiﬁcation, deep learning, channel attention module, visual explanation, gamma-ray spectrum, nuclear safety OPEN ACCESS EDITED BY Jorge Segovia, Universidad Pablo de Olavide, Spain REVIEWED BY Kaibo Shi, Chengdu University, China Herun Yang, Institute of Modern Physics, Chinese Academy of Sciences (CAS), China Xiaodong Zhang, North China Electric Power University, China Chengdu University, China Herun Yang, Institute of Modern Physics, Chinese Academy of Sciences (CAS), China Xiaodong Zhang, North China Electric Power University, China Yu Wang, Quanhu Zhang, Qingxu Yao, Yonggang Huo, Man Zhou and Yunfeng Lu As a rapid and automatic method, multiple radionuclide identiﬁcation using deep learning has drawn wide interest from researchers in the ﬁeld of nuclear safety and nuclear security. However, the network model in deep learning often appears in the form of a black box, which makes it difﬁcult for people to understand its decision-making basis. It is necessary to develop an interpretable deep learning model for multiple nuclide identiﬁcation. In the work on nuclide identiﬁcation using deep learning, very few interpretable studies have been conducted. In this paper, channel attention weights are used for interpretable radionuclide identiﬁcation for the ﬁrst time. We propose a multiple radionuclide identiﬁcation method using deep learning with channel attention module and visual explanation. A dataset of gamma spectra simulated by Geant4 was created, containing 256 combinations of 8 radionuclides. These gamma spectra were used to train using a convolutional neural network (CNN) with a channel attention module. The obtained accuracies on training, validation, and test sets are 97.8%, 97.6%, and 97.1%, respectively. The result of interpretation of spectral features show that based on the channel attention module, the CNN can make full use of the feature information of the photoelectric peak and Compton edge and suppress the background and noise interference. In addition, the t-distributed stochastic neighbor embedding (t-SNE) method was used to visualize the inner working process of the CNN and visually illustrate the correctness of feature extraction. This research will promote the application of artiﬁcial intelligence algorithms in nuclide identiﬁcation instruments. COPYRIGHT © 2022 Wang, Zhang, Yao, Huo, Zhou and Lu. 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) and the copyright owner(s) 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. Multiple radionuclide identiﬁcation using deep learning with channel attention module and visual explanation OPEN ACCESS EDITED BY Jorge Segovia, Universidad Pablo de Olavide, Spain REVIEWED BY Kaibo Shi, Chengdu University, China Herun Yang, Institute of Modern Physics, Chinese Academy of Sciences (CAS), China Xiaodong Zhang, North China Electric Power University, China CORRESPONDENCE Quanhu Zhang, zhangqh_102@sina.com SPECIALTY SECTION This article was submitted to Nuclear Physics, a section of the journal Frontiers in Physics RECEIVED 04 September 2022 ACCEPTED 28 September 2022 PUBLISHED 19 October 2022 CITATION Wang Y, Zhang Q, Yao Q, Huo Y, Zhou M and Lu Y (2022), Multiple radionuclide identiﬁcation using deep learning with channel attention module and visual explanation. Front. Phys. 10:1036557. doi: 10.3389/fphy.2022.1036557 OPEN ACCESS KEYWORDS Frontiers in Physics 01 frontiersin.org Wang et al. 10.3389/fphy.2022.1036557 Introduction algorithm, the inner working process of the neural network is visualized. algorithm, the inner working process of the neural network is visualized. The main contributions of the paper are listed here: Airborne radionuclide identiﬁcation is an important technology in the Comprehensive Nuclear-Test-Ban Treaty [1, 2]. In order to improve the coverage and efﬁciency of the monitoring, it is necessary to analyze the gamma-ray spectrum measured in a short period of time. With this limit, the counting ﬂuctuation error of the spectrum is large, and the photoelectric peak is easily overwhelmed by the background. In addition, the calibration of detector is prone to deviation due to temperature changes. In this case, it is difﬁcult to identify radionuclides by traditional methods. • The created dataset includes 256 combinations of 8 nuclides. Data augmentation was performed on the gamma spectra of the dataset. • We analyzed the interpretation results of the neural network by the channel attention weights. It was found that the neural network can use the photoelectric peak and Compton edge in the spectrum to identify the nuclide while excluding the inﬂuence of background and noise. while excluding the inﬂuence of background and noise. • The data distribution was visualized by t-SNE. The results show that the channel attention module is able to improve the performance of the neural network. Some of the latest works with machine learning methods for nuclide identiﬁcation have achieved rapid, real-time, and automatic results. Intelligent nuclide identiﬁcation methods have received more and more attention. Bayesian inference [3], support vector machines [4], and artiﬁcial neural networks [5–11] are applied to nuclide identiﬁcation and quantiﬁcation. Based on artiﬁcial neural networks, some variants of neural networks, such as convolutional neural network (CNN) [12–15], hybrid neural network [16], and generative adversarial network [17], have been applied to nuclide identiﬁcation. The performance of these methods on nuclide identiﬁcation tasks is better than that of traditional methods. Convolutional neural network model The proposed model of the neural network is mainly composed of an input layer, an output layer, three convolution modules, and a channel attention module, as shown in Figure 1. However, intelligent identiﬁcation methods, especially deep learning models, have a problem that plagues researchers because of their “black box” features. It is difﬁcult for deep neural networks to give a basis for decisions, which leads to a lack of trust for researchers [18, 19]. Although CNN has the ability to extract features, it must be ensured that the network can correctly and effectively utilize the extracted key features from the gamma spectrum. Among the deep learning studies related to radionuclide identiﬁcation, few works have investigated the interpretability of neural networks for gamma spectral analysis. The interpretability can help people understand the process about how a neural network processes the gamma spectrum and increase people’s interest and conﬁdence in using neural networks for nuclide identiﬁcation. The input to the neural network is the entire gamma spectrum (1024 × 1). It is followed by three convolutional modules, each of which contains three convolutional layers and a max-pooling layer. The convolution layer is responsible for extracting the features of the input gamma-ray spectrum, and the max pooling layer reduces the dimension of the data and the number of parameters, which can improve the training speed. The number of convolution kernels increases with the deepening of the network. In fact, processing gamma spectra with a one-dimensional convolution kernel can be regarded as a ﬁltering operation, and different convolution kernels correspond to the features of different channel outputs. Different features are of different importance to the identiﬁcation of radionuclides. The features that are more representative of the characteristics of nuclides should be paid attention to. Considering the above reasons, a channel attention module is added after the highest convolutional module. Attention mechanism has become a research hotspot in the ﬁeld of deep learning in recent years. The idea of the attention mechanism comes from the attention mechanism of the human brain. The human brain will consciously ignore unimportant information and focus on information closely related to the target. Applying attention to the CNN structure can improve the ability of feature maps to encode key information and improve the learning effect of the middle layer [20, 21]. The use of attention mechanisms to explain the internals of neural networks is a hot research topic. Geant4 simulation The deep learning model requires a large amount of data for training. The Geant4 simulation toolkit [24] was used to create the data, i.e., the gamma-ray spectra. First, a Φ3″ × 3″ NaI (Tl) detector was modeled using Geant4, as shown in Figure 3. The detector is mainly composed of NaI (green), a magnesium oxide ﬁlm (purple), and Al protective shell (orange). The red balls represent point radionuclide sources. The green rays are gamma rays emitted by the sources. Creation of the dataset Finally, the channel attention weights are multiplied by each feature to get the features with the channel attention weights. The output layer follows the global average pooling layer. It consists of 9 neurons, whose output value is between 0 and 1 due to the limitation of the sigmoid activation function. Its physical meaning is the probability of the existence of 8 nuclides and the related background. Channel attention module The inspiration for using the channel attention module comes from Squeeze-and-Excitation Networks [20]. The channel attention module can calculate the importance of features extracted by different convolution kernels. In general, it is divided into three steps: squeeze, excitation, and reweight. Speciﬁcally, data compression is ﬁrst performed on the data of each channel, and the one-dimensional data output by each channel is compressed into In this study, we propose a CNN model with a channel attention module [20]. A gamma spectroscopy dataset was established for multiple radionuclide identiﬁcation. We try to explain and understand how CNN processes features using channel attention. By using the t-distributed stochastic neighbor embedding (t-SNE) [22] dimensionality reduction Frontiers in Physics 02 frontiersin.org Wang et al. 10.3389/fphy.2022.1036557 FIGURE 1 Schematic diagram of the proposed model. FIGURE 2 Schematic diagram of channel attention operation. FIGURE 1 Schematic diagram of the proposed model. FIGURE 2 Schematic diagram of channel attention operation. FIGURE 2 Schematic diagram of channel attention operation. a single value, corresponding to Fsq(·) in Figure 1. Then use the shared Multi-layered perceptron (MLP) to perform the excitation operation to generate a corresponding channel attention weight for each channel. Channel attention weights are used to represent the importance of features output by different channels. Figure 2 is a speciﬁc schematic diagram of the channel attention operation. The feature maps are input to the average pooling layer and the max pooling layer, respectively, both of which are connected to the shared MLP. The shared MLP is a fully connected network with three layers, and the number of neurons in the middle layer network is less than that of the two side layers. The two outputs of the shared neural network are connected together, and after passing through the sigmoid activation function, the channel attention weights are obtained. size, and the number of neurons of the shared MLP. A reasonable setting of hyperparameters is conducive to better performance of the neural network. After some experience attempts, more suitable hyperparameters are explored. The ﬁnal neural network hyperparameters are shown in Table 1. Table 2 is a model summary of the network. The loss function of the neural network is the categorical cross- entropy function. The neural network weights are updated iteratively based on the training data using the Adam optimizer. The running environment of the program is python3.8.12 and tensorﬂow2.5.0-GPU [23]. Determination of hyperparameters The neural network model proposed in this paper has the following hyperparameters: The number of ﬁlters, the size of the kernel, the activation function, the initial learning rate, the batch The total number of channels is 1,024, and the step size is 2 keV. For 137Cs, the energy resolution at 0.662 keV is 7.5%. The G4EmStandardPhysics was added to the physis list to simulate the Frontiers in Physics Frontiers in Physics 03 frontiersin.org Wang et al. 10.3389/fphy.2022.1036557 TABLE 1 Hyperparameters of the proposed neural network. TABLE 1 Hyperparameters of the proposed neural network. Hyperparameters of the model Value Number of ﬁlters 64, 64, 64, 128, 128, 128, 256, 256, 256 Size of the kernel 3 Activation function ReLU Initial learning rate 2 × 10–4 Batch size 128 The number of neurons of the shared MLP 85,256 TABLE 2 Model summary of the CNN with channel attention module. Layer (type) Output shape Param Connected to input_1 (InputLayer) [(None, 1,024, 1)] 0 conv1d (Conv1D) (None, 1,022, 64) 256 input_1 [0][0] conv1d_1 (Conv1D) (None, 1,020, 64) 12,352 conv1d [0][0] conv1d_2 (Conv1D) (None, 1,018, 64) 12,352 conv1d_1 [0][0] max_pooling1d (MaxPooling1D) (None, 339, 64) 0 conv1d_2 [0][0] conv1d_3 (Conv1D) (None, 337, 128) 24,707 max_pooling1d [0][0] conv1d_4 (Conv1D) (None, 335, 128) 49,280 conv1d_3 [0][0] conv1d_5 (Conv1D) (None, 333, 128) 49,280 conv1d_4 [0][0] max_pooling1d_1 (MaxPooling1D) (None, 111, 128) 0 conv1d_5 [0][0] conv1d_6 (Conv1D) (None, 109, 256) 98,560 max_pooling1d_1 [0][0] conv1d_7 (Conv1D) (None, 107, 256) 196,864 conv1d_6 [0][0] conv1d_8 (Conv1D) (None, 105, 256) 196,864 conv1d_7 [0][0] max_pooling1d_2 (MaxPooling1D) (None, 35, 128) 0 conv1d_8 [0][0] channel_avgpool (GlobalAveragePooling1D) (None, 256) 0 max_pooling1d_2 [0][0] channel_maxpool (GlobalMaxPooling1D) (None, 256) 0 max_pooling1d_2 [0][0] channel_fc1 (Dense) (None, 85) 21,845 channel_avgpool [0][0], channel_maxpool [0][0] channel_fc2 (Dense) (None, 256) 22,016 channel_fc1 [0][0], channel_fc1 [1][0] add (Add) (None, 256) 0 channel_fc2 [0][0], channel_fc2 [1][0] channel_sigmoid (Activation) (None, 256) 0 add [0][0] channel_reshape (Reshape) (None, 1, 256) 0 channel_sigmoid [0][0] tf.math.multiply (TFOpLambda) (None, 35, 256) 0 max_pooling1d_2 [0][0], channel_reshape [0][0] global_average_pooling1d (GlobalMaxPooling1D) (None, 256) 0 tf.math.multiply [0][0] dense (Dense) (None, 9) 2,313 global_average_pooling1d [0][0] TABLE 2 Model summary of the CNN with channel attention module. very different from the energy actually measured and recorded by the detector. The energy deposition simulated by Geant4 is processed with Gaussian broadening, and Eqs 1, 2 are the broadening formulas. physical process of photoelectric reaction. Determination of hyperparameters The G4RadioactiveDecayPhysics and G4DecayPhysics were added to the physis list to simulate the physical process of the decay of radioactive sources. FWHM a + b E0 + cE2 0 (1) E FWHM 2 2 ln 2 √ g + E0 (2) (1) Frontiers in Physics Gaussian broadening (2) Geant4 can only obtain the deposition energy of particles in the detector and cannot simulate the response process of the detector. The energy deposition simulated by Geant4 is Where FWHM is the full width at half maximum. a, b, c, are the corresponding coefﬁcients, which are determined by the frontiersin.org Frontiers in Physics Frontiers in Physics 04 Wang et al. 10.3389/fphy.2022.1036557 ﬁtting of the gamma-ray spectrum measured in practice. E0 is the deposition energy simulated by Geant4. g is a random variable that obeys a standard normal distribution, that is g ~ N(0, 1). E is the expanded energy. Figure 4 is a comparison of the gamma-ray spectra of 137Cs and 60Co mixed nuclides before and after energy broadening. The broadened gamma-ray spectrum has a lower resolution at the photoelectric peak, which is more in line with the actual situation. Referring to the four types of common radioisotopes listed in the IAEA technical guidance reference manual for nuclear security [25], a dataset of gamma spectra simulated by Geant4 was created. It contains 256 combinations of 8 radionuclides, they are, 241Am, 133Ba, 57Co, 60Co, 137Cs, 131I, 40K, and 235U. A background gamma-ray spectrum obtained from a long-term measurement is used as a FIGURE 3 Detector model established with Geant4. FIGURE 4 Gamma spectra before and after Gaussian broadening. FIGURE 5 Template for sampling simulation and simulated background spectrum. FIGURE 3 Detector model established with Geant4. Detector model established with Geant4. ﬁtting of the gamma-ray spectrum measured in practice. E0 is the deposition energy simulated by Geant4. g is a random variable that obeys a standard normal distribution, that is g ~ N(0, 1). E is the expanded energy. Figure 4 is a comparison of the gamma-ray spectra of 137Cs and 60Co mixed nuclides before and after energy broadening. The broadened gamma-ray spectrum has a lower resolution at the photoelectric peak, which is more in line with the actual situation. Referring to the four types of common radioisotopes listed in the IAEA technical guidance reference manual for nuclear security [25], a dataset of gamma spectra simulated by Geant4 was created. It contains 256 combinations of 8 radionuclides, they are, 241Am, 133Ba, 57Co, 60Co, 137Cs, 131I, 40K, and 235U. A background gamma-ray spectrum obtained from a long-term measurement is used as a FIGURE 4 Gamma spectra before and after Gaussian broadening. FIGURE 5 Template for sampling simulation and simulated background spectrum. Gaussian broadening FIGURE 4 Gamma spectra before and after Gaussian broadening. FIGURE 5 Template for sampling simulation and simulated background spectrum. ﬁtting of the gamma-ray spectrum measured in practice. E0 is the deposition energy simulated by Geant4. g is a random variable that obeys a standard normal distribution, that is g ~ N(0, 1). E is the expanded energy. Figure 4 is a comparison of the gamma-ray spectra of 137Cs and 60Co mixed nuclides before and after energy broadening. The broadened gamma-ray spectrum has a lower resolution at the photoelectric peak, which is more in line with the actual situation. Referring to the four types of common radioisotopes listed in the IAEA technical guidance reference manual for nuclear security [25], a dataset of gamma spectra simulated by Geant4 was created. It contains 256 combinations of 8 radionuclides, they are, 241Am, 133Ba, 57Co, 60Co, 137Cs, 131I, 40K, and 235U. A background gamma-ray spectrum obtained from a long-term measurement is used as a Frontiers in Physics 05 Frontiers in Physics frontiersin.org Wang et al. 10.3389/fphy.2022.1036557 FIGURE 6 Example of the data augmentation; (A) spectra with different gross counts, (B) spectra with different radionuclide activity ratios, (C) spectra with different magnitude of shift. FIGURE 6 Example of the data augmentation; (A) spectra with different gross counts, (B) spectra with different radionuclide activity ratios, (C) spectra with different magnitude of shift. template. The background gamma-ray spectrum is simulated based on this template, as shown in Figure 5. ratios of different nuclides were simulated by changing the number of particles. Figure 6B shows the comparison of the activity ratios of 137Cs and 60Co at 4 and 0.25, respectively. In our dataset, the activity ratios of individual nuclides ranged from 0.2 to 5. Finally, the shift of the spectra was simulated by means of interpolation, as shown in Figure 6C. The magnitude of shift was randomly selected between 0.9 and 1.1. Data augmentation The dataset needs to contain gamma spectra simulated in different situations to prevent overﬁtting when the neural network training data. In deep learning, this practice is called data augmentation. It was realized with three methods: Randomize the gross counts, randomize the radionuclide activity ratio, and perform the spectrum shift. For each combination of radionuclides, 1,000 spectra were simulated. Referring to the proportion of the small sample dataset [26], the dataset were divide into training set, validation set, and test set according to the ratio of 3:1:1. Figure 6 shows the comparison of 60Co and 137Cs mixed nuclide gamma-ray spectra after data augmentation. Speciﬁcally, ﬁrst, the gross counts of the gamma-ray spectrum were randomly selected between 103 −105 following a logarithmic distribution. Figure 6A shows the gamma-ray spectra of different total counts after count normalization. Second, randomize the radionuclide activity ratio. The spectrum of mixed nuclides is a composite spectrum formed by linearly superimposing the spectra of single nuclides. The activity Frontiers in Physics Training results of neural networks 241Am 133Ba 57Co 60Co 137Cs 131I 40K 235U Background Spectrum labels 0 1 0 1 1 0 1 0 1 Identiﬁcation output of spectrum a 0.03 0.98 0.08 0.78 0.79 0.12 0.56 0.01 1 Identiﬁcation output of spectrum b 0.01 0.92 0.01 0.86 0.90 0.01 0.32 0.01 0.99 Identiﬁcation output of spectrum c 0.01 0.98 0.01 0.90 0.95 0.01 0.78 0.01 1 FIGURE 7 Training accuracy and validation accuracy during model training. The training accuracy and veriﬁcation accuracy of each epoch of training in the training process are shown in Figure 7. The accuracy of training increases rapidly in the early stage and reaches saturation in the late stage. During training, the learning rate was halved every 20 epochs. This allows us to quickly adjust the neural network parameters at the beginning of training and ﬁne-tune the parameters at the end of training. In the later stage of training, it is necessary to slow down the speed of neural network updating weights to make the accuracy reach a stable value. This is the reason why the ﬂuctuation of accuracy in the early training period is larger than that in the late training period, as shown in Figure 7. The basis for stopping training is that the accuracy of the validation set no longer increases for 10 consecutive epochs. In the local magniﬁcation of Figure 7, the accuracy of the validation set does not grow after the 56th epoch. The accuracy of the validation set was no more than 98.3% in epochs 56 to 66, so the training was stopped at epoch 66 to prevent overﬁtting. To highlight the role of the channel attention module. We train the network without adding channel attention. During training, we still use the metrics we deﬁned. Table 4 shows Training accuracy and validation accuracy of CNN with channel attention and without channel attention at the end of the training. Compared with the CNN without channel attention module, the CNN with channel attention module improves the training accuracy and validation accuracy. identiﬁcation. TensorFlow’s own metrics, such as accuracy and binary_accuracy, do not meet our requirements. So, we deﬁne our own metrics. To describe our own deﬁned metrics, we use Table 3 as an example. Table 3 lists the labels of the three gamma spectra for a certain combination and the identiﬁcation output of spectra. Training results of neural networks Only when all existing nuclides are accurately identiﬁed, and no excess nuclides are incorrectly identiﬁed can the neural network be considered to have successfully completed the 06 frontiersin.org 10.3389/fphy.2022.1036557 Wang et al. The training accuracy and veriﬁcation accuracy of ea epoch of training in the training process are shown Figure 7. The accuracy of training increases rapidly in early stage and reaches saturation in the late stage. Dur training, the learning rate was halved every 20 epochs. T allows us to quickly adjust the neural network parameters at beginning of training and ﬁne-tune the parameters at the end training. In the later stage of training, it is necessary to slow do the speed of neural network updating weights to make accuracy reach a stable value. This is the reason why ﬂuctuation of accuracy in the early training period is lar than that in the late training period, as shown in Figure 7. The basis for stopping training is that the accuracy of validation set no longer increases for 10 consecutive epochs. the local magniﬁcation of Figure 7, the accuracy of the validat set does not grow after the 56th epoch. The accuracy of validation set was no more than 98.3% in epochs 56 to 66, so training was stopped at epoch 66 to prevent overﬁtting. To highlight the role of the channel attention module W TABLE 3 Labels and identiﬁcation output for the three gamma spectra. 241Am 133Ba 57Co 60Co 137Cs 131I 40K 235U Backgrou Spectrum labels 0 1 0 1 1 0 1 0 1 Identiﬁcation output of spectrum a 0.03 0.98 0.08 0.78 0.79 0.12 0.56 0.01 1 Identiﬁcation output of spectrum b 0.01 0.92 0.01 0.86 0.90 0.01 0.32 0.01 0.99 Identiﬁcation output of spectrum c 0.01 0.98 0.01 0.90 0.95 0.01 0.78 0.01 1 FIGURE 7 Training accuracy and validation accuracy during model training. TABLE 3 Labels and identiﬁcation output for the three gamma spectra. TABLE 3 Labels and identiﬁcation output for the three gamma spectra. TABLE 3 Labels and identiﬁcation output for the three gamma spectra. Training results of neural networks In addition, comparing Figures 9A,C does not have the noise of the statistical ﬂuctuation in Figure 9A, which is due to the automatic n operation in the neural network. Neural network features such as photoelectric peaks, Compton backgrounds in gamma spectra, but what is mo may be how to use these features in a targeted ma Table 5 shows the channel attention weights o network for these three features. Due to the limi sigmoid activation function, the attention constrained to be between 0 and 1. The chann weight of feature map 1 and feature map 2 is weight of feature map 3 is 0. This indicates tha network considers photoelectric peaks and Compto important information for nuclide identiﬁcation. information is interfering information which does n TABLE 4 Training accuracy and validation accuracy of CNN with channel attention and without. Training accuracy (%) Validation a CNN with channel attention 97.8 97.6 CNN without channel attention 97.0 95.1 FIGURE 8 Identiﬁcation accuracy of the spectra with different numbers of nuclides in the test set. TABLE 4 Training accuracy and validation accuracy of CNN with channel attention and without. Training accuracy (%) see the characteristics of the photoelectric peaks of 137Cs and 60Co. In Figure 9C, one may perceive not only the characteristics of the photoelectric peaks of 137Cs and 60Co, but also the related Compton edge. In Figure 9D, the main features come from the background. In addition, comparing Figures 9A,C, Figure 9C does not have the noise of the statistical ﬂuctuation distribution in Figure 9A, which is due to the automatic noise ﬁltering operation in the neural network. Neural networks can extract features such as photoelectric peaks, Compton edges, and backgrounds in gamma spectra, but what is more important may be how to use these features in a targeted manner. FIGURE 8 Identiﬁcation accuracy of the spectra with different numbers of nuclides in the test set. FIGURE 8 Identiﬁcation accuracy of the spectra with different numbers of nuclides in the test set. Table 5 shows the channel attention weights of the neural network for these three features. Due to the limitation of the sigmoid activation function, the attention weights are constrained to be between 0 and 1. The channel attention weight of feature map 1 and feature map 2 is 1, and the weight of feature map 3 is 0. Interpretation of spectral features by channel attention weights CNN relies on convolutional layers to extract features from spectral data. The deeper the convolutional layers, the more abstract the features are. These features may represent different spectral peaks, including but not limited to photoelectric peaks, Compton edges, and background. We hope that through the channel attention module, the neural network can assign different attention weights to these features so as to better utilize the features that are beneﬁcial to nuclide identiﬁcation. Training results of neural networks This indicates that the neural network considers photoelectric peaks and Compton edges to be important information for nuclide identiﬁcation. Background information is interfering information which does not require the attention of the neural network. This is in line with the logic of people identifying nuclides. People mainly rely on photoelectric peaks to identify nuclides rather than relying on the characteristics of the background. Here, the neural network automatically completes the gamma spectrum analysis work. Only spectra and labels are input, then the neural network completes the nuclide identiﬁcation work that is consistent with people’s cognitive logic. time to identify each spectrum is 0.285 ms. The proposed method is a rapid and automatic method for nuclide identiﬁcation. Training results of neural networks The three gamma spectra have the same labels and are named spectrum a, spectrum b, and spectrum c respectively. Then use different metrics to calculate the accuracy to illustrate the difference between different metrics. In total, gamma-ray spectra for 256 combinations were simulated. Let the number of existing nuclides is i(0 ≤i ≤8), then the number of cases follows the combination Ci 8. i 0 means only background exists. When the test set is input into the trained neural network, the total accuracy is 97.1%. Figure 8 shows the accuracy of a different number of nuclides in the test set. When the number of nuclides is between 1-7, the accuracy gradually decreases with the increase of the number of nuclides. Even so, all of them are at least 90%. According to the JJF 1687- 2018 measurement standard [27], our proposed algorithm can be applied to radionuclide identiﬁcation instruments. Compared with the CNN without the channel attention module, the performance of the CNN with channel attention module is better on the test set. We set 0.5 as the threshold for the network to judge the existence of nuclides. When the output value of a certain output channel is greater than 0.5, the network considers that the nuclide exists. Among the identiﬁcation output of spectra, for the identiﬁcation output of spectrum b, the neural network did not recognize the existence of 40K, but the rest of the prediction results were correct. According to the metrics we deﬁned, the accuracy should be 66.6% at this time. However, the binary_accuracy that comes with tensorﬂow will calculate the accuracy for each output channel separately. At this time, the accuracy is 26/27 × 100% 96.3%, which is obviously a wrong calculation result. The accuracy directly affects the adjustment of parameters during training, which is why it is necessary to strictly deﬁne the metrics. The network is trained and tested on a notebook PC with AMD Ryzen 7 5800H, a Nvidia GeForce RTX 3060 Laptop GPU and 16 GB RAM in 64-bit Windows 11. In the test set, the average Frontiers in Physics 07 frontiersin.org Wang et al. 10.3389/fphy.2022.1036557 see the characteristics of the photoelectric peaks 60Co. In Figure 9C, one may perceive not only the c of the photoelectric peaks of 137Cs and 60Co, but als Compton edge. In Figure 9D, the main features co background. Feature visualization using t-distributed stochastic neighbor embedding In order to further analyze the process of feature extraction by the neural network, the features of the gamma spectra of four nuclide combinations in the test set were selected for visualization. Each combination contains two nuclides, they are, 241Am + 133Ba, 57Co+60Co, 137Cs + 131I, 40K + 235U. t-SNE is a nonlinear dimensionality reduction method. Despite the high computational cost, t-SNE is still effective for visualizing high- dimensional data containing multiple manifolds simultaneously. The t-SNE algorithm was used to convert gamma spectral features in high-dimensional space into vector data in two- dimensional space. The transformed two-dimensional vector preserves the structure of the high-dimensional space. Figure 9 shows several feature maps of gamma spectroscopy for several different convolution kernels in the highest convolutional layers. Figure 9A is the original gamma spectrum input to the neural network. Figures 9B–D are several feature maps, which are named feature maps 1, 2, and 3, respectively. Although they do not include all feature maps, they are still representative. These feature maps may be abstract to people, but they are still understandable. In Figure 9B, one may Frontiers in Physics 08 frontiersin.org Wang et al. 10.3389/fphy.2022.1036557 FIGURE 9 Examples of mixed nuclides of 60Co and 137Cs; (A) original gamma-ray spectrum, (B) feature map 1, (C) feature map 2, (D) feature map 3. URE 9 mples of mixed nuclides of 60Co and 137Cs; (A) original gamma-ray spectrum, (B) feature map 1, (C) feature map 2, (D) feature map 3 FIGURE 10 Results of feature visualization using t-SNT; (A) Visualization of the outputs of channel_maxpool in Table 2, (B) global_average_pooling1d in Table 2, (C) dense in Table 2. FIGURE 10 Results of feature visualization using t-SNT; (A) Visualization of the outputs of channel_maxpool in Table 2, (B) global_average_pooling1d in Table 2, (C) dense in Table 2. 09 Frontiers in Physics frontiersin.org 10.3389/fphy.2022.1036557 10.3389/fphy.2022.1036557 Wang et al. layers, which contain information such as photoelectric peaks, Compton edges, and background. The channel attention module allows the neural network to make full use of the characteristic information, such as the photoelectric peak and Compton edge of the spectrum while suppressing the background and noise interference. This is in line with people’s basic logical knowledge when identifying nuclides because people mainly rely on the photoelectric peaks in the spectrum to identify different nuclides. Furthermore, t-SNE was used to visualize feature extraction. Author contributions This paper proposes a convolutional neural network with a channel attention module for multiple radionuclide identiﬁcation. The NaI detector model was modeled by Geant4. The gamma-ray spectra of multiple nuclides were simulated. Data augmentation is achieved by randomizing the gross counts, randomizing the radionuclide activity ratio, and performing the spectrum shift. With the data augmentation, a gamma-ray spectral dataset that can be used for multiple radionuclide identiﬁcation has been obtained. During training, a gradually decreasing learning rate method was used to stabilize the training results and an early stopping method to prevent overﬁtting. We strictly deﬁne metrics to calculate accuracy. Only when all the nuclides present are accurately identiﬁed and no redundant nuclides are misidentiﬁed can the neural network be considered to have successfully completed the identiﬁcation. The obtained accuracies on training, validation, and test sets are 97.8%, 97.6%, and 97.1%, respectively. YW Simulated the data and drafted the manuscript. YW and YL performed the analysis. QZ Guided the study and analyzed the results. QY and YW designed the research. YH and MZ reviewed the manuscript. All authors have read and approved the content of the manuscript. Data availability statement The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author. Feature visualization using t-distributed stochastic neighbor embedding By visualizing the feature data distribution, one may ﬁnd that using the channel attention module is able to enhance the training effect, and also the process of processing the data inside the neural network is explainable. These results vividly illustrate the correctness of feature extraction. TABLE 5 Channel attention weights corresponding to different feature maps. Feature map number Channel attention weight Feature map 1 1 Feature map 2 1 Feature map 3 0 The data distribution of the average pooling layer before the channel attention, the global average pooling layer after the channel attention, and the output layer of the neural network were analyzed. Figures 10A–C correspond to the outputs of channel_maxpool, global_average_pooling1d and dense in Table 2, respectively. For each combination of nuclides, there are 200 gamma spectra as test samples, corresponding to 200 discrete points for each color in Figure 10. The 2D vector data after t-SNE dimensionality reduction were normalize, so the coordinates on the graph were constrained to be between 0 and 1. In general, the closer the points of the same color are on the graph, the better the classiﬁcation performance of the neural network. Compared to the data distributions in Figure 10A, all points of the same color are clustered together in Figure 10B, which proves the effectiveness of the channel attention module. The data distribution in Figure 10C visually shows the recognition results of the neural network. Figure 10A–C visually shows the process of processing data inside the neural network. Future work will build a database on measured gamma-ray spectra, which is a long-term effort. With the development of deep learning, more and more new neural network models have appeared. It is interesting work to explore whether new network models are suitable for nuclide identiﬁcation. Future work may include the use of transformer models and reinforcement Learning represented by Q-learning [28–30] for nuclide identiﬁcation. Conﬂict of interest 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. References J Anal Spectrom (2012) 27:432–9. doi:10.1039/C2JA10230G 20. Hu J, Shen L, Albanie S, Sun G, Wu E. Squeeze-and-excitation networks. arXiv (2019). doi:10.48550/arXiv.1709.01507 6. Bobin C, Bichler O, Lourenço V, Thiam C, Thévenin M. Real-time radionuclide identiﬁcation in γ-emitter mixtures based on spiking neural network. Appl Radiat Isot (2016) 109:405–9. doi:10.1016/j.apradiso.2015.12.029 21. Woo S, Park J, Lee J-Y, Kweon IS. Cbam: Convolutional block Attention module (2022). In: V Ferrari, M Hebert, C Sminchisescu, Y Weiss, editors. Computer vision – ECCV 2018 lecture notes in computer science. Cham: Springer International Publishing. p. 3–19. doi:10.1007/978-3-030-01234-2_1 7. Kamuda M, Stinnett J, Sullivan CJ. Automated isotope identiﬁcation algorithm using artiﬁcial neural networks. IEEE Trans Nucl Sci (2017) 64:1858–64. doi:10. 1109/TNS.2017.2693152 22. van der Maaten LJP, Hinton GE. Visualizing high-dimensional data using t-SNE. J Machine Learn Res (2008) 9:2579–605. 8. Van Hiep C, Hung DT, Anh NN, Giang NN, Khang PD, Hai NX, et al. Nuclide identiﬁcation algorithm for the large-size plastic detectors based on artiﬁcial neural network. IEEE Trans Nucl Sci (2022) 69:1203–11. doi:10.1109/TNS.2022.3173371 23. Abadi M, Agarwal A, Barham P, Brevdo E, Chen Z, Citro C, et al. TensorFlow: Large-scale machine learning on heterogeneous distributed systems. arXiv (2016). doi:10.48550/arXiv.1603.04467 9. Qi S, Wang S, Chen Y, Zhang K, Ai X, Li J, et al. Radionuclide identiﬁcation method for NaI low-count gamma-ray spectra using artiﬁcial neural network. Nucl Eng Techn (2022) 54:269–74. doi:10.1016/j.net.2021.07.025 24. Agostinelli S, Allison J, Amako K, Apostolakis J, Araujo H, Arce P, et al. Geant4—A simulation toolkit. Nucl Instr Methods Phys Res Section A: Acc Spectrometers, Detectors Associated Equipment (2003) 506:250–303. doi:10.1016/ S0168-9002(03)01368-8 10. Kim J, Park K, Cho G. Multi-radioisotope identiﬁcation algorithm using an artiﬁcial neural network for plastic gamma spectra. Appl Radiat Isot (2019) 147: 83–90. doi:10.1016/j.apradiso.2019.01.005 25. IAEA. Technical and functional speciﬁcations for border monitoring equipment. Vienna: INTERNATIONAL ATOMIC ENERGY AGENCY (2006). 11. Zhang C, Hu G, Luo F, Xiang Y, Ding G, Chu C, et al. Identiﬁcation of SNM based on low-resolution gamma-ray characteristics and neural network. Nucl Instr Methods Phys Res Section A: Acc Spectrometers, Detectors Associated Equipment (2019) 927:155–60. doi:10.1016/j.nima.2019.02.023 26. N Jankowski, W Duch, K Gra̧bczewski, editors. Meta-learning in computational intelligence. Berlin, Heidelberg: Springer Berlin Heidelberg (2011). doi:10.1007/978-3-642-20980-2 27. JJF. Calibration speciﬁcation for hand-held radiation monitors for detection and identiﬁcation of radionuclides. (2018). 12. Liang D, Gong P, Tang X, Wang P, Gao L, Wang Z, et al. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. To the best of our knowledge, this paper is the ﬁrst to use the channel attention module for multiple radionuclide identiﬁcation. It is proved that the channel attention weights are able to explain how the neural network analyses the gamma-ray spectrum. The CNN extracts the features of the gamma spectrum through convolutional Frontiers in Physics 10 frontiersin.org Wang et al. Wang et al. 10.3389/fphy.2022.1036557 References 16. Galib SM, Bhowmik PK, Avachat AV, Lee HK. A comparative study of machine learning methods for automated identiﬁcation of radioisotopes using NaI gamma-ray spectra. Nucl Eng Techn (2021) 53:4072–9. doi:10.1016/j.net.2021. 06.020 1. Aarnio PA, Ala-Heikkilä JJ, Hakulinen TT, Nikkinen MT. The nuclide identiﬁcation system SHAMAN in the veriﬁcation of the comprehensive nuclear-test-ban treaty. J Radioanal Nucl Chem (2001) 248:587–93. doi:10.1023/ A:1010607820953 1. Aarnio PA, Ala-Heikkilä JJ, Hakulinen TT, Nikkinen MT. The nuclide identiﬁcation system SHAMAN in the veriﬁcation of the comprehensive nuclear-test-ban treaty. J Radioanal Nucl Chem (2001) 248:587–93. doi:10.1023/ A:1010607820953 2. Aarnio PA, Ala-Heikkilä JJ, Ansaranta TK, Hakulinen TT. Analysis pipeline for air ﬁlter gamma-ray spectra from the CTBT veriﬁcation network. J Radioanal Nucl Chem (2005) 263:253–7. doi:10.1007/s10967-005-0045-x 17. Wu S, Tang X, Gong P, Wang P, Liang D, Li Y, et al. Peak-searching method for low count rate γ spectrum under short-time measurement based on a generative adversarial network. Nucl Instr Methods Phys Res Section A: Acc Spectrometers, Detectors Associated Equipment (2021) 1002:165262. doi:10.1016/j.nima.2021. 165262 2. Aarnio PA, Ala-Heikkilä JJ, Ansaranta TK, Hakulinen TT. Analysis pipeline for air ﬁlter gamma-ray spectra from the CTBT veriﬁcation network. J Radioanal Nucl Chem (2005) 263:253–7. doi:10.1007/s10967-005-0045-x 3. Kim J, Lim KT, Kim J, Kim Y, Kim H, Cho G. Quantiﬁcation and uncertainty analysis of low-resolution gamma-ray spectrometry using Bayesian inference. Nucl Instr Methods Phys Res Section A: Acc Spectrometers, Detectors Associated Equipment (2020) 953:163144. doi:10.1016/j.nima.2019.163144 18. Gomez-Fernandez M, Wong W-K, Tokuhiro A, Welter K, Alhawsawi AM, Yang H, et al. Isotope identiﬁcation using deep learning: An explanation. Nucl Instr Methods Phys Res Section A: Acc Spectrometers, Detectors Associated Equipment (2021) 988:164925. doi:10.1016/j.nima.2020.164925 4. Hata H, Yokoyama K, Ishimori Y, Ohara Y, Tanaka Y, Sugitsue N. Application of support vector machine to rapid classiﬁcation of uranium waste drums using low-resolution γ-ray spectra. Appl Radiat Isot (2015) 104:143–6. doi:10.1016/j. apradiso.2015.06.030 4. Hata H, Yokoyama K, Ishimori Y, Ohara Y, Tanaka Y, Sugitsue N. Application of support vector machine to rapid classiﬁcation of uranium waste drums using low-resolution γ-ray spectra. Appl Radiat Isot (2015) 104:143–6. doi:10.1016/j. apradiso.2015.06.030 19. Wang Y, Yao Q, Zhang Q, Zhang H, Lu Y, Fan Q, et al. Explainable radionuclide identiﬁcation algorithm based on the convolutional neural network and class activation mapping. Nucl Eng Techn (2022). doi:10.1016/ j.net.2022.08.011 5. Aitkenhead MJ, Owen M, Chambers DM. Use of artiﬁcial neural networks in measuring characteristics of shielded plutonium for arms control. 30. Shi K, Cai X, She K, Zhong S, Soh Y, Kwon O. Quantized memory proportional–integral control of active power sharing and frequency regulation in island microgrid under abnormal cyber attacks. Appl Energ (2022) 322:119540. doi:10.1016/j.apenergy.2022.119540 References Rapid nuclide identiﬁcation algorithm based on convolutional neural network. Ann Nucl Energ (2019) 133:483–90. doi:10.1016/j.anucene.2019.05.051 28. Peng Z, Luo R, Hu J, Shi K, Ghosh BK. Distributed optimal tracking control of discrete-time multiagent systems via event-triggered reinforcement learning. IEEE Trans Circuits Syst (2022) 69:3689–700. doi:10.1109/TCSI.2022. 3177407 13. Daniel G, Ceraudo F, Limousin O, Maier D, Meuris A. Automatic and real- time identiﬁcation of radionuclides in gamma-ray spectra: A new method based on convolutional neural network trained with synthetic data set. IEEE Trans Nucl Sci (2020) 67:644–53. doi:10.1109/TNS.2020.2969703 29. Peng Z, Luo R, Hu J, Shi K, Nguang SK, Ghosh BK. Optimal tracking control of nonlinear multiagent systems using internal reinforce Q-learning. IEEE Trans Neural Netw Learn Syst (2022) 33:4043–55. doi:10.1109/TNNLS. 2021.3055761 14. Koo BT, Lee HC, Bae K, Kim Y, Jung J, Park CS, et al. Development of a radionuclide identiﬁcation algorithm based on a convolutional neural network for radiation portal monitoring system. Radiat Phys Chem (2021) 180:109300. doi:10. 1016/j.radphyschem.2020.109300 30. Shi K, Cai X, She K, Zhong S, Soh Y, Kwon O. Quantized memory proportional–integral control of active power sharing and frequency regulation in island microgrid under abnormal cyber attacks. Appl Energ (2022) 322:119540. doi:10.1016/j.apenergy.2022.119540 15. Ryu J, Park C, Park J, Cho N, Park J, Cho G. Development of neural network model with explainable AI for measuring uranium enrichment. IEEE Trans Nucl Sci (2021) 68:2670–81. doi:10.1109/TNS.2021.3116090 Frontiers in Physics 11 frontiersin.org
https://openalex.org/W4378347620	https://bibliotecadigital.ipb.pt/bitstream/10198/28474/3/PM.pdf	English	null	30th European Congress on Obesity (ECO 2023)	Obesity facts	2,023	cc-by	2,082	PL2 – Obesity and Sustainability PL2 – Obesity and Sustainability PL2 Innovative and sustainable approaches to tackling obesity, other noncommunicable diseases and their risk factors in the WHO European Region Wickramasinghe, K.; Whiting, S.; Dagless, S.; Rippin, H.; Andreeva, O.; Malykh, R.; Williams, J. WHO European Office for the Prevention and Control of Noncommunicable Diseases, Division of Country Health Programmes, WHO European Region, Copenhagen, Denmark 1 Please note: By default, authors did not disclose any conflicts of interest unless explicitly stated in the abstract. PL1 Social justice and health equity Marmot, M. Department of Epidemiology and Public Health, UCL, London, England Department of Epidemiology and Public Health, UCL, London, England Taking action to reduce health inequalities is a matter of social justice. In developing strategies for tackling health inequalities we need to confront the social gradient in health not just the difference between the worst off and everybody else. There is clear evidence when we look across countries that national policies make a difference and that much can be done in cities, towns and local areas. But policies and interventions must not be confined to the health care system; they need to address the conditions in which people are born, grow, live, work and age. The evidence shows that economic circumstances are important but are not the only drivers of health inequalities. Tackling the health gap will take action, based on sound evidence, across the whole of society. Conclusion: With repeated statements made by Member States to WHO governing bodies to link the climate change agenda with NCD and/or obesity, this remains a priority area to develop further. This requires fur- ther analysis to elucidate the role of wider systems such as urban planning and One Health. The mapping will highlight synergies between obesity and sustainable development goals and promote the identification of co-benefits, win-win, and sustainable solutions. This will facilitate policy development and identify entry points to advance this agenda. Abstracts Abstracts Obes Facts 2023;16(suppl 1):1–351 DOI:10.1159/000530456 Obes Facts 2023;16(suppl 1):1–351 DOI:10.1159/000530456 Published online: May 12, 2023 PL3 – Clustering of Obesity Phenotypes - Learnings from Diabetes Wickramasinghe, K.; Whiting, S.; Dagless, S.; Rippin, H.; Andreeva, O.; Malykh, R.; Williams, J. PLENARY LECTURES implementation. For HSD, these laid the foundation for the development of seven workstreams to address its multidimensional nature. For climate change, a systems approach was employed to map the links between cli- mate change and NCD in collaboration with Queen’s University, Belfast in the first expert meeting, with future collaboration between the group of experts agreed during this initial meeting. Wednesday, 17 May 2023 © 2023 S. Karger AG, Basel PL1 – Obesity and Inequality: where are we and how should we proceed? PL1 – Obesity and Inequality: where are we and how should we proceed? p g g g Results: For HSD, initiatives and outputs have comprised a suite of tools to strengthen surveillance and build capacity of Member States, enabling them to create their own national HSD packages. To date, these have included a new modelling tool for the analysis of the health, environ- mental and affordability implications of diets and dietary change, a food procurement manual for officers at public institutions and a manual for systems thinking. For climate change and NCD prevention, an initial causal loop diagram has been produced. Causal links identified as being associated with obesity include transportation, air pollution, heatwaves and coldwaves via impacts on physical activity levels. This emerging pro- gramme will respond with a solutions-oriented map which can be used as an advocacy tool. Commonalities identified between the HSD and climate change workstreams is the need to develop nutrition and sustainability indicators, which will also be explored further. Eating behavior features in persons with cardiac pathology Eliashevich, S. O.1; Orekhova, A. V.1; Filichkina, E. M.2; Yarovaya, E. B.2; Drapkina, O. M.3 Introduction: The prevalence of Metabolic Syndrome (MetS) has been increasing exponentially in the Portuguese population. Primary preven- tion using physical activity (PA), exercise and healthy lifestyles seems to be limited. Thus, current study aims to present the design and preliminary findings of a pilot community intervention for patients with MetS. 1Laboratory of eating behavior study and correction, Department of fundamental and practical aspects of obesity, National Medical Research Center for Therapy and Preventive Medicine, Moscow, Russia 1Laboratory of eating behavior study and correction, Department of fundamental and practical aspects of obesity, National Medical Research Center for Therapy and Preventive Medicine, Moscow, Russia 2Laboratory of Biostatistics, Department of Epidemiology of Chronic Noncommunicable Diseases, National Medical Research Center for Therapy and Preventive Medicine, Moscow, Russia 3Department of fundamental and practical aspects of obesity, National Medical Research Center for Therapy and Preventive Medicine, Moscow, Russia Laboratory of Biostatistics, Department of Epidemiology of Chronic Noncommunicable Diseases, National Medical Research Center for Therapy and Preventive Medicine, Moscow, Russia i Methods: A randomized controlled trial was conducted between April and July 2022 with patients with MetS, from a Northeaster Portuguese primary health care. Eight participants completed the three-month inter- vention program, 6 women (51.0±6.4 years) and 2 men (46.5±4.9 years). The program included: (1) evaluation and prescription of PA (steps/ day and floors/day); (2) nutritional and dietary support; (3) strength training sessions; (3) blood testing; (4) anthropometric and body com- position assessment. A pre- and post-intervention follow up was con- ducted. Garmin®F 745 were used for PA prescription and assessment. PA targets per day were: number of steps ≥10 000; uphill walking ≥10 floors; and minutes of intensity ≥150 per week. Nutritional and dietary evaluation was recorded by completing the food frequency question- naire with subsequent recommendation of changes to healthy diet. The designed strength training program was: (i) frequency of 2x/week; (ii) 8 to 10 exercises, in order to work the main muscle groups; (iii) 2 sets of 10 to 12 repetitions; (iv) 60 to 70% of one maximum repetition. Blood samples evaluated glycated hemoglobin (HbA1c), fating glucose (FG), tri- glycerides (TG), high-density lipoprotein (HDL) cholesterol, total choles- terol (TC), systolic (SBP) and diastolic blood pressure (DBP). Body weight (kg), lean body mass (kg) and fat mass percentage (%) was evaluated by bio-impedance scale (Tanita MC 780-P MA®). PO2.002 Conclusions: The effect of the implemented pilot community intervention program was greater in the participants who followed the program more rigorously. Additionally, the strength training may have been important in increasing lean body mass. The inclusion of PA, strength training and nutritional support in primary health care, through a supervised program, seems to be a key strategy to reduce the risk associated with MetS and delay the complications associated with cardiometabolic diseases. Conclusions: The effect of the implemented pilot community intervention program was greater in the participants who followed the program more rigorously. Additionally, the strength training may have been important in increasing lean body mass. The inclusion of PA, strength training and nutritional support in primary health care, through a supervised program, seems to be a key strategy to reduce the risk associated with MetS and delay the complications associated with cardiometabolic diseases. Downloaded from http://karger.com/ofa/article-pdf/16/Suppl. 1/1/3867019/000530456.pdf by guest on 30 May 2023 Background and Aims: Nowadays there are a lot of radical dietary shifts in many developing nations are supplanting traditional patterns of eat- ing on diet high in animal products and refined carbohydrates and low in whole grains, fruits, and vegetables. Additionally, the proportion of elderly is increasing trend is paralleled by an increase in chronic diseases, such as cardiovascular disease. A limited number of studies have inves- tigated the evaluation of eating behavior in the group of patients with cardiac disease. The aim of this study was validation of the Dutch Eating Behavior Questionnaire (DEBQ) in group of patients with cardiac disease. Methods: 137 patients who received preventive counseling in the hospital from February 17, 2022 to August 16, 2022 were consecutively included in the study. The median age was: 61.00 [48.00; 67.00] years, 60 men (44%) and 77 women (56%). The median of body mass index was 34.34 [32.01;36.98]. The Dutch Eating Behavior Questionnaire scoring was used to assess the characteristics of eating behaviors among the group of patients with cardiac disease. Results: Eating disorders were identified in 98 (71.5%) patients, with 34 (24.8 %) patients having two combinations of types of deviation. There were also patients who had 3 types of disorders at once (12 (8.8%)). t Results: Eating disorders were identified in 98 (71.5%) patients, with 34 (24.8 %) patients having two combinations of types of deviation. There were also patients who had 3 types of disorders at once (12 (8.8%)). Figure 1 shows that the emotional type of eating disorder is often pre- dominant in women (Figure 1), according to the DEBQ questionnaires ( p<0.001).i i Results: Regarding PA parameters at the end of the three-month pro- gram, the average number of daily steps was 11 818, the number of daily floors was 15.35 and moderate to vigorous PA was 253 minutes/week. An improvement in pre- and post-intervention blood concentrations and pressure was observed, namely in HbA1c (-7.4%±15.2), FG (-4.9%±14.4), HDL (-4.8%±9.7), TC (-1.9%±19.9), SBP (-15.6%±10.7) and DBP (-12.7%±9.3). All participants registered a weight loss (-3.4%±2.0), with an increase in lean mass (-4.5%±2.0) and a decrease in fat mass (-8.7%±6.3). Except for one participant, all improved in heart rate recovery after exer- cise (19.3%±28.2), suggesting an improvement in cardiovascular capacity. Obes Facts 2023;16(suppl 1):1–351 DOI: 10.1159/000530456 Physical activity, strength training and nutritional support in patients with metabolic syndrome from a Northeaster Portuguese primary health care: A pilot community intervention program Bragada, J. A.1; Teixeira, J. A.2; Bragada, J. P.3; Duarte, C. M.1; Pontes, J. H.1; Saldanha, L. S.1; Magalhães, P. M.1 1Department of Sport Science, Polytechnic Institute of Bragança, Portugal (IPB) 2Department of Sport Science, Polytechnic Institute of Guarda, Portugal (IPG) 3North East Local Health Unit – Health Care Unit of Santa Maria, Portugal (ULSNE) Precision medicine in diabetes and obesity WHO European Office for the Prevention and Control of Noncommunicable Diseases, Division of Country Health Programmes, WHO European Region, Copenhagen, Denmark Introduction: Obesity remains a significant public health issue in the European region, affecting 60% of adults and nearly 30% of chil- dren. The WHO European Office for the Prevention and Control of Noncommunicable Diseases (NCD Office) is identifying and promoting innovative and sustainable approaches to tackle obesity and other NCD. In October 2019, the NCD Office hosted an expert meeting to explore how to promote healthy and sustainable diets (HSD), which led to the establishment of workstreams. This was followed by an expert meeting in December 2022 to map the linkages and co-benefits between NCD prevention and the climate change agenda. More widely, the NCD Office leads several innovative projects related to strengthening implementation research capacity and providing practical tools on infant and young child nutrition. People are all different, and this is no different when we consider people living with diabetes and obesity, yet the current approaches to their man- agement tend to treat everyone the same. The field of precision medicine aims to recognise these differences – whether at the level of their pheno- type or at the molecular level. Faced with multiple, and increasing, treat- ment options as well as increasing healthcare costs there is a clear need to target therapy to maximise benefit and reduce harm for every patient with diabetes and obesity.h y This talk will discuss advances in precision medicine in diabetes and obe- sity. I will highlight recent work on how phenotypic variation matters, how we can cluster phenotypes and genotypes to inform on this variation, and the limitations of clustering. I will provide a framework for how we are implementing precision medicine in diabetes clinical care in Tayside, Scotland and how this might translate to care of people with obesity. Methods: Expert meetings have been held with key stakeholders to discuss and explore data availability, key linkages and gaps in research and policy © 2023 S. Karger AG, Basel Eating behavior features in persons with cardiac pathology MetS parameters was defined by joint interim statement (JIS) criteria (Alberti et al, 2009). 3Department of fundamental and practical aspects of obesity, National Medical Research Center for Therapy and Preventive Medicine, Moscow, Russia Downloaded from http://karger.com/ofa/article-pdf/16/Suppl. 1/1/3867019/000530456.pdf by guest on 30 May 2023 Figure 1 shows that the emotional type of eating disorder is often pre- dominant in women (Figure 1), according to the DEBQ questionnaires (* p<0.001).i Conclusion: It was identified the prevalence of the emotional type of eat- ing disorder in women which help to clinical psychologist to create the support group by DEBQ test. 116 Abstracts
https://openalex.org/W3124017631	https://gh.bmj.com/content/bmjgh/6/3/e004347.full.pdf	English	null	Small and sick newborn care during the COVID-19 pandemic: global survey and thematic analysis of healthcare providers’ voices and experiences	BMJ global health	2,021	cc-by	10,340	What is already known? ► ►The COVID-19 pandemic has disrupted health sys- tems worldwide; a recent global survey of 714 frontline maternal care providers reported effects on pregnancy, intrapartum and postpartum services; inadequate preparedness; and increased levels of stress among health personnel. Methods Using a widely disseminated online survey in three languages, we reached out to neonatal healthcare providers. We collected data on COVID-19 preparedness, effects on health personnel and on newborn care services, including kangaroo mother care (KMC), as well as disruptors and solutions. on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ from Original research Original research on October 23, 2024 by guest. http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 Small and sick newborn care during the COVID-19 pandemic: global survey and thematic analysis of healthcare providers’ voices and experiences BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloa on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ shed as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from Suman P N Rao ‍ ‍ ,1 Nicole Minckas,2 Melissa M Medvedev ‍ ‍ ,3,4 David Gathara,5 Prashantha Y N,1 Abiy Seifu Estifanos ‍ ‍ ,6 Alfrida Camelia Silitonga,7 Arun Singh Jadaun,8 Ebunoluwa A Adejuyigbe,9 Helen Brotherton,4,10 Sugandha Arya,11 Rani Gera,11 Chinyere V Ezeaka,12 Abdou Gai,10 Abebe Gebremariam Gobezayehu,13 Queen Dube,14 Aarti Kumar,15 Helga Naburi,16 Msandeni Chiume,17 Victor Tumukunde,18 Araya Abrha Medhanyie,19 Gyikua Plange-Rhule,20 Josephine Shabini,21 Eric O Ohuma ‍ ‍ ,4 Henok Tadele,22 Fitsum W/Gebriel,23 Amanuel Hadgu,19 Lamesgin Alamineh,13 Rajesh Mehta,24 Elizabeth Molyneux,14 Joy E Lawn ‍ ‍ ,4 on behalf of the COVID-19 Small and Sick Newborn Care Collaborative Group To cite: Rao SPN, Minckas N, Medvedev MM, et al. Small and sick newborn care during the COVID-19 pandemic: global survey and thematic analysis of healthcare providers’ voices and experiences. BMJ Global Health 2021;6:e004347. doi:10.1136/ bmjgh-2020-004347 Key questions Introduction The COVID-19 pandemic is disrupting health systems globally. Maternity care disruptions have been surveyed, but not those related to vulnerable small newborns. We aimed to survey reported disruptions to small and sick newborn care worldwide and undertake thematic analysis of healthcare providers’ experiences and proposed mitigation strategies. Handling editor Seye Abimbola ► ►Pandemic-associated disruptions are increasing neonatal mortality, yet small and sick newborn care is relatively new in global health and has not yet been included in global assessments of health ser- vice disruptions. ► ►Additional material is published online only. To view, please visit the journal online (http://dx.doi.org/10.1136/ bmjgh-2020-004347). Results We analysed 1120 responses from 62 countries, mainly low and middle-income countries (LMICs). Preparedness for COVID-19 was suboptimal in terms of guidelines and availability of personal protective equipment. One-third reported routine testing of all pregnant women, but 13% had no testing capacity at all. More than 85% of health personnel feared for their own health and 89% had increased stress. Newborn care practices were disrupted both due to reduced care-seeking and a compromised workforce. More than half reported that evidence-based interventions such as KMC were discontinued or discouraged. Separation of the mother– baby dyad was reported for both COVID-positive mothers (50%) and those with unknown status (16%). Follow-up care was disrupted primarily due to families’ fear of visiting hospitals (~73%). ► ►Keeping mothers and newborns together is a core aspect of respectful care and is particularly under threat during the pandemic, including for vulnerable newborns requiring kangaroo mother care (KMC). ► ►Keeping mothers and newborns together is a core aspect of respectful care and is particularly under threat during the pandemic, including for vulnerable newborns requiring kangaroo mother care (KMC). SPNR and NM are joint first authors. Received 2 November 2020 Revised 5 December 2020 Accepted 9 December 2020 What are the new findings? ► ►Our survey of 1120 respondents show that COVID-19 preparedness, particularly testing of pregnant women, availability of personal pro- tective equipment (PPE), and guidelines for small and sick newborn care, are suboptimal in all regions, with most health professionals reporting higher stress levels and 85% fearing for their own health. Maternity services were affected during initial stages of the COVID-19 pandemic, and an online survey with over 700 responses from maternity workers reported reduc- tions in antenatal and postnatal care, and a shift in birth location from hospital to home.14 Quality of care was also reported to be affected, most notably evidence-based, respectful care practices such as birth companions, family visitation, keeping newborns and mothers together and breastfeeding.14 Importantly, health personnel reported higher workload due to staff shortages and longer shifts, and increased levels of stress. This survey provided an extremely valuable picture of the challenges faced by maternity health providers during the COVID-19 pandemic. However, as noted by the authors, only 10 respondents worked in neonatal care.14 ► ►Reductions in hospital births and neonatal admissions were report- ed in all regions in addition to compromised newborn care due to women fearing to come to hospital, reallocation of personnel and/ or equipment from newborn units, and early discharge which was reported as the norm. on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ s 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from ► ►Two-thirds of workers stated they would not allow mothers whose SARS-CoV-2 status is positive or unknown to practice KMC, and >20% of workers would not allow KMC even among mothers test- ing negative. Study design, population and sampling neonatal deaths per 1000 live births by 2030.6 However, major inequities still lie between and within countries. Some countries are predicted to reach the SDG target over a century too late.2 This cross-sectional study used a methodology similar to that of the previous study on maternal health impacts of COVID-19.14 We targeted all relevant cadres working in newborn healthcare provision, including health providers (nurses, midwives, doctors and community health workers), public health professionals and policy- makers. We particularly focused on LMICs. The survey was distributed using professional member organisations and personal networks, and was widely disseminated through social media (eg, Twitter, WhatsApp, Facebook and Telegram) and at events (eg, webinars). Respond- ents were encouraged to share the survey with colleagues for snowball sampling. on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ m Newborn care is a well-recognised marker of high- quality care since it is exquisitely time-sensitive, and delays of minutes can lead to death. Keeping mothers and newborns together is a core aspect of evidence- based, respectful care, including for neonates who are born preterm (<37 completed weeks of gestation) or with LBW, or both.2 7 8 Facility-based care of small and sick newborns has been highlighted as having potential for high-impact (>757 000 lives per year) yet low coverage and suboptimal quality of care exist due to limited invest- ment in most low and middle-income countries (LMIC).9 er 23, 2024 by guest. Protected by copyright. ( ) The COVID-19 pandemic and associated nationwide restrictions risk reversing fragile gains for maternal and newborn health in the highest burdened settings.10 Newborns are among the most vulnerable to the indirect effects of the COVID-19 pandemic on healthcare provi- sion. Roberton and colleagues estimated that coverage disruptions of 9.8% to 51.9% over 6 months could result in 253 500 to 1 157 000 additional under-5 child deaths in LMICs, and these estimates did not include small and sick newborn care.11 An observational study in Nepal reported that institutional births were halved, while labour ward What do the new findings imply? ► ►Respondents reported mitigating strategies at all levels of care such as provision of adequate PPE for all health personnel; clearer guidance, particularly on non-separation of mothers and their new- borns; and higher-profile messaging on benefits and safety of early KMC during the pandemic. Small and sick newborns are among our most vulner- able citizens and have not yet been included in global assessments of disruptions during the pandemic. We conducted a global online survey to provide insights on disruptions to coverage and quality of small and sick newborn care, and to identify possible solutions to protect vulnerable newborns during the COVID-19 and similar future pandemics. ► ►Policymakers can and must do better to protect neonatal health services, support personnel and particularly ensure evidence-based practices for all mothers and newborns, including those who are SARS-CoV-2-positive. ► ►Our findings provide sobering insights into disrupted care for small and sick newborns across the globe; more implementation re- search is urgently needed, with evaluation of mitigation approaches and sharing what works to protect vulnerable newborns. ► ►Our findings provide sobering insights into disrupted care for small and sick newborns across the globe; more implementation re- search is urgently needed, with evaluation of mitigation approaches and sharing what works to protect vulnerable newborns. BMJ Global Health BMJ Global Health on October 23, 2024 by guest. Protected by http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from neonatal mortality increased threefold during the first 2 months of the COVID-19 lockdown.12 A comparative analysis of lives saved by kangaroo mother care (KMC) versus mortality risk due to COVID-19 among neonates weighing ≤2000 g showed that the benefit outweighs the risk by 65-fold to 630-fold.13 INTRODUCTION 23, 2024 by guest. Protected by copyright. Each year, 2.5 million newborns die within 28 days of birth,1 more than 80% of whom are low birth weight (LBW, <2500 g) and particularly vulnerable.2 Since the adop- tion of the Every Newborn Action Plan by all United Nations member states in 20143 with the first ever global neonatal mortality target in the Sustainable Development Goals (SDG),4 momentum has increased for ending preventable newborn mortality, although less emphasis has yet been placed on stillbirths.5 Over 78 high-burden countries have set national mortality targets to reach 12 or fewer For numbered affiliations see end of article. Correspondence to Dr Suman P N Rao; raosumanv@gmail.com © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. Conclusion Newborn care providers are stressed and there is lack clarity and guidelines regarding care of small newborns during the pandemic. There is an urgent need to protect life-saving interventions, such as KMC, threatened by the pandemic, and to be ready to recover and build back better. Correspondence to Dr Suman P N Rao; raosumanv@gmail.com 1 Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 Questionnaire The questionnaire was developed by a multidisciplinary team that included neonatologists, paediatricians, nurses, epidemiologists and public health professionals from diverse settings. Where relevant, we adapted from the maternal health survey.14 We collected data on respond- ents’ characteristics, including area of work, healthcare preparedness and responses to COVID-19, and the effect of the pandemic on health professionals and newborn care. We also requested respondents to list major disrup- tions that occurred in their work settings and solutions 2 Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 BMJ Global Health on October 23, 2024 b http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from that should be developed to overcome these disruptions. Embedded logic skips in the survey guaranteed that only relevant questions were asked. The questionnaire was developed in English and piloted by professionals from various settings to assess face validity and clarity of wording and answer options. The final version was trans- lated into Spanish and Bahasa (Indonesia). The English questionnaire can be found in online supplemental appendix 1. Information was presented on the landing website and respondents were requested to indicate consent by checking a box before participating. enablers/solutions to small and sick newborn care during the COVID-19 pandemic. We triangulated qualitative and quantitative data and present combined results to enrich interpretation. on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ th: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from Respondents’ characteristics A total of 1120 participants (after exclusions noted above) responded to the survey, spanning 62 countries. Africa and Asia had the largest numbers: 483 (43.1%) and 376 (33.6%), respectively. Overall, the majority of the respondents were healthcare professionals: nurses (43.4%, out of which 17.0% were neonatal nurses and 26.4% not specified) and paediatricians (17.7%); policy- makers and administrators constituted only 2.5% of the sample. Most respondents (>40%) across all the regions worked in tertiary level hospitals/organisations with the exception of Oceania and Southeast Asia (SEA), where 82.1% worked in primary-level or district-level facilities. Of the 997 respondents who provided direct newborn care, the majority from Africa worked in neonatal special care units (WHO level 2), while those from Asia, Latin America and the Caribbean, Europe and North America mainly provided care in neonatal intensive care units (WHO level 3).9 The characteristics and geographical distribution of respondents are provided in figure 1. Data processing, missing data and analysis We received 1389 responses between 13 July and 13 October. During data cleaning, we removed refusals to participate (n=47), responses with country missing (n=216) and those with missing answers on >90% of questions (n=6). Analysis involved descriptive statistics (frequencies and percentages) using STATA/SE V.14. Countries were aggregated by region (online supple- mental appendix 2). on October 23, 2024 by guest. Pro http://gh.bmj.com/ 347 on 14 March 2021. Downloaded from We conducted a qualitative analysis of free-text responses on disruptors and solutions to neonatal care. Disruptions and solutions were coded separately using NVivo V.12. Thematic analysis of free text was used to identify common themes among disruptions reported by respondents. For analyses of solutions, we conducted a framework analysis applying an adapted version of the socioecological model previously used for KMC quali- tative research (Brotherton, in press) (online supple- mental appendix 3). Results are presented according to the five levels of this conceptual framework: family/ caregivers, facility/ ward, health systems, community and policy. For each level, we identified disruptions and COVID-19 preparedness for neonatal care COVID-19 testing was not routinely available for preg- nant women and great variations across regions were observed (table 1). While the WHO suggests that testing protocols for a pregnant woman depends on where she lives, it recommends that symptomatic pregnant women on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ from Figure 1 Distribution of survey respondents by country, region, occupation and hospital context. NICU, neonatal intensive care unit; NSCU, neonatal special care unit; SEA, Southeast Asia. Figure 1 Distribution of survey respondents by country, region, occupation and hospital context. NICU, neonatal intensive care unit; NSCU, neonatal special care unit; SEA, Southeast Asia. Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 3 Table 1 COVID-19 preparedness among neonatal providers and its impact on healthcare personnel and health systems, by region Africa N=334 n (%) LatAm and Caribbean N=86 n (%) Asia (excl. COVID-19 preparedness for neonatal care SEA) N=300 n (%) Oceania and SEA N=66 n (%) North America and Europe N=21 n (%) Total* N=807 n(%) Test for SARS-CoV-2 available at admission for delivery Routinely for all women 30 (9.0) 25 (30.1) 180 (62.3) 43 (67.2) 8 (38.1) 286 (36.2) Only for women with risk factors 81 (24.3) 14 (16.9) 36 (12.5) 8 (12.5) 6 (28.6) 145 (18.4) Only for women with symptoms or contact history 147 (44.1) 37 (44.6) 53 (18.3) 0 (0.0) 5 (23.8) 242 (30.6) Only for elective caesarean section 2 (0.6) 0 (0.0) 5 (1.7) 3 (4.6) 0 (0.0) 10 (1.3) Never available 73 (21.9) 7 (8.4) 15 (5.2) 10 (15.4) 2 (9.5) 107 (13.5) Sign-posted area for SARS-CoV-2 screening available in facility 254 (76.3) 80 (94.1) 217 (82.8) 48 (73.9) 18 (85.7) 617 (80.6) Isolation areas for suspected and confirmed SARS-CoV-2 cases available in facility 296 (88.6) 83 (97.7) 199 (75.7) 36 (56.1) 17 (89.6) 631 (82.) Sufficient PPE items† Gloves 162 (49.7) 66 (79.5) 212 (75.3) 14 (21.5) 17 (85.0) 471 (59.9) N95 masks 37 (11.7) 44 (52.4) 114 (39.7) 8 (13.3) 10 (50.0) 213 (27.6) Eye protection 42 (13.1) 46 (55.4) 99 (35.0) 11 (17.7) 13 (65.0) 210 (27.3) Aprons or gowns 84 (26.3) 57 (69.7) 142 (48.5) 10 (16.4) 14 (70.0) 308 (39.9) Sanitisers 156 (48.2) 65 (77.4) 230 (79.0) 14 (21.9) 16 (84.2) 481 (61.4) Respondents’ source of information on small and sick newborn care during the COVID-19 pandemic Institution provided updated guidelines 81 (24.6) 25 (29.4) 65 (22.0) 6 (9.2) 7 (33.3) 184 (23.1) Personally searched for information 224 (67.0) 52 (61.1) 158 (55.0) 15 (23.8) 14 (70.0) 463 (58.6) Received information from colleagues or professional bodies 226 (67.6) 45 (52.9) 179 (62.3) 36 (57.1) 14 (70.0) 500 (63.3) Received information from hospital or public health authorities 241 (72.1) 56 (65.8) 183 (63.7) 14 (22.2) 16 (84.2) 510 (64.6) Respondents’ level of knowledge on care of newborns born to SARS-CoV-2 confirmed or suspected mothers Very clear 62 (18.6) 19 (22.1) 36 (12.0) 10 (15.4) 4 (19.0) 131 (16.3) Mostly clear, but some areas of concern remain 111 (33.2) 38 (44.2) 126 (42.0) 34 (52.3) 7 (33.3) 316 (39.2) Somewhat clear, but major issues remain 77 (23.1) 16 (18.6) 43 (14.3) 6 (9.2) 3 (14.3) 145 (18.0) Some points clear but not confident 50 (15.0) 9 (10.5) 73 (24.3) 11 (16.9) 5 (23.8) 148 (18.4) Not at all clear 34 (10.2) 4 (4.7) 22 (7.3) 4 (6.2) 2 (9.5) 66 (8.2) Respondents’ work affected by COVID-19 292 (86.9) 74 (86.1) 237 (79.3) 48 (72.7) 19 (95.0) 670 (83.0) Respondents' changes in practice due to COVID-19 Reduced working hours 93 (27.6) 18 (20.6) 44 (14.7) 6 (9.0) 2 (9.5) 163 (20.1) Always use PPE 263 (78.2) 63 (72.4) 205 (68.5) 50 (75.7) 18 (85.7) 599 (74.0) Avoid practices that can increase transmission risk 271 (80.6) 54 (62.0) 161 (53.8) 14 (21.2) 11 (52.3) 511 (63.1) No change in practice 16 (4.7) 3 (3.4) 37 (12.3) 1 (1.5) 0 (0) 57 (7.0) Respondents’ fear for own health 314 (93.2) 78 (89.7) 231 (77.0) 58 (87.9) 15 (75.0) 696 (85.9) Respondents’ higher stress level 305 (90.8) 80 (93.0) 256 (86.2) 58 (89.2) 19 (95.0) 718 (89.3) Differential number of missing values by variable.i on October 23, 2024 by guest. i atin America; PPE, personal protective equipment; SEA, Southeast Asia. Disruptions for neonatal care provision and processes g The inadequate or erratic supply of personal protec- tive equipment (PPE) was a frequently noted barrier; sanitiser and gloves were the most readily available, with 61.4% and 59.9% of respondents, respectively, reporting consistent access whenever these supplies were needed. There was a clear shortage of N95 masks and eye shields/ protectors, with only 27.6% and 27.3% of respondents, respectively, reporting their availability at all times. Avail- ability of PPE varied widely across regions, with Africa and SEA/Oceania being the most affected. Lack of PPE prevented providers from having close contact with mothers and their babies due to the risk of SARS-CoV-2 transmission. A paediatrician from Tanzania highlighted, ‘the increased demand for PPE for nurses going to get expressed milk from quarantined mothers’. p p p Substantial disruptions in the use and delivery of care were reported (table 2), with families reluctant to access and stay in facilities. Reductions in hospital births and neonatal admissions of more than 25% were reported by 25% and 20% of respondents, respectively, with larger reductions occurring in Asian countries (35% and 27%). Changes to newborn care were noted to include reallocation of unit space (14.6%) and reassignment of staff from newborn care to COVID-19-related or other duties (18.9%). Oxygen supplies for newborn care were also reported to be compromised. In addition, requests for early discharge by families were widely reported by respondents, resulting in 43.8% of babies being discharged earlier than usual. A Kenyan paediatrician described an increase in ‘anxiety of mothers due to worry of contracting COVID while in hospital’, leading many fami- lies to request early discharge. A respondent from the Dominican Republic wrote, ‘The KMC ward has been closed because it shared the space with the COVID area’. A paedia- trician from South Africa wrote, ‘A COVID outbreak in mother lodger and KMC wards resulted in closure of wards, and delay and hesitancy in reopening’. Limited space was noted as a barrier to maintaining social distancing measures in many settings and additionally affected the quality of neonatal care in facilities. A paediatrician from Kenya reported, ‘[Babies are] sharing incubators, radiant warmers and phototherapy spaces’. Lack of clarity regarding evidence-based maternal/ newborn care guidelines was frequently noted by respon- dents, with only 16.3% reporting ‘very clear’ knowl- edge regarding care of neonates born to mothers with confirmed or suspected COVID-19. Experiences and voices from neonatal care providers Experiences and voices from neonatal care providers Most respondents’ (83.0%) work has been affected by COVID-19, with 89.3% reporting higher than usual stress levels and 85.9% fearing for their own health. Across regions, 93.2% of African respondents reported fearing for their own health, while 93.0% of respondents in Latin America and the Caribbean reported higher than usual stress levels (table 1). Stress was primarily related to staff shortages due to infection, self-isolation or reallocation to COVID-19 wards, which resulted in an increased workload among the remaining neonatal care providers. A neonatologist from Ecuador reported that 40% of personnel working in his/her facility contracted SARS-CoV-2, ‘affecting the number of personnel and the working hours’. Most health providers reported apprehension related to COVID-19, including fear of contracting and spreading the virus and ‘fear and panic for the unknown’ (nurse, Nigeria). Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 COVID-19 preparedness for neonatal care Protected http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 4 BMJ Global Health on October 23, 2024 by guest. Protected by http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from should be prioritised. Lack of testing was flagged as a major challenge by many of the respondents. Around one-third of the respondents providing facility-based care reported that SARS-CoV-2 testing was routinely avail- able for pregnant women admitted for delivery (36.2%) and those with symptoms or contact history (30.7%). Testing of pregnant women admitted for delivery was reported as unavailable by 21.9% of respondents from Africa. Even when testing was available, the lag between testing and results impacted clinical management deci- sions. A neonatal nurse from India wrote, ‘When babies are admitted in SNCU [special newborn care unit], we don't treat them until [we receive] their COVID-19 test’. Similar experi- ences were reflected by other respondents who explained that practices such as KMC are postponed until maternal COVID-19 status is known. Although testing availability fluctuated across regions, 80.6% of respondents reported that facilities had sign-posted areas for COVID-19 screening and isolation. Disruptions for neonatal care provision and processes http://gh.bmj.com/ d from Changes to KMC practice were reported by 528 respondents due to embedded skip logic within the survey. KMC, kangaroo mother care; NICU, neonatal intensive care unit; NSCU, neonatal special care unit. 3, 2024 by guest. Protected by copyright. 85% of respondents before the pandemic, compared with 55% during the pandemic. Changes to KMC prac- tice included reduced duration of skin-to-skin contact (26.5%), earlier discharge (30.8%) and full disruption of KMC services (7%). Respondents mentioned concerns for the continuation of KMC in the community following discharge, especially as counselling and assistance to mothers had been reduced due to competing activities among healthcare providers or social distancing meas- ures. A paediatrician from Nicaragua explained, ‘We limit [KMC] training to mothers only, fathers are being excluded resulting in suboptimal monitoring of infant growth stem- ming from lack of comprehensive examinations. Commu- nity health workers conducting neonatal home visits described being rejected by families. An Indian outreach worker wrote, ‘[The] child’s family members refused me to come (in)to their home because they said ‘you [are] working out of your home and may be exposed to the pandemic’’. Disruptions for neonatal care provision and processes doi:10.1136/bmjgh-2020-004347 BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 M Table 2 Effect of the COVID-19 pandemic on small and sick newborn care, KMC practice, facility visitation and follow-up care Total, n (%) Neonatal inpatient care during COVID-19 pandemic (n=623) Newborn unit admission capacity reduced 229 (36.7) Newborn unit/KMC areas are reallocated (for COVID-19 care or other care) 91 (14.6) Newborn unit/KMC staff are reallocated (for COVID-19 care or other areas) 118 (18.9) Babies are discharged earlier than usual 273 (43.8) Women/families refuse to stay in facilities that are marked as COVID-19 treatment centres 197 (31.6) KMC practice during COVID-19 pandemic (n=623) KMC ward admission capacity reduced 172 (27.6) Health workers more hesitant to promote KMC 150 (24.0) Women/families more hesitant to practise KMC 105 (16.8) Counselling/support focus shifted from KMC to hand hygiene, masks and social distancing 237 (38) KMC practised with improved hand and respiratory hygiene (ie, masks and tissues) 321 (51.5) Changes to KMC practice* (n=528) KMC practice has stopped 37 (7.0) KMC is practised as normal 292 (55.3) KMC is practised, but the daily duration of skin-to-skin contact is reduced 140 (26.5) KMC is initiated and babies are discharged home early 163 (30.8) KMC is practised in another area 23 (4.4) NSCU/NICU visitation during COVID-19 pandemic (n=593) Mother and family allowed as usual 68 (11.4) Mothers are permitted except for those who are COVID-19-positive 304 (51.2) Mother and family are not permitted 24 (4.0) Family members (other than mother) are not permitted 266 (44.8) Visiting hours are restricted 237 (39.9) Follow-up care during COVID-19 pandemic (n=435) Reduced space for follow-up clinic 100 (22.9) Less staff to conduct follow-up clinic 142 (32.6) Fewer appointments for each newborn 211 (48.5) Follow-up schedule has been changed 178 (40.9) Women/families reluctant to attend follow-up due to fear of infection 319 (73.3) Reduced attendance due to logistical reasons (eg, public transport disruptions) 247 (56.7) Home visits disrupted 95 (21.8) Telephone follow-up visits have been started. 160 (36.7) Changes to KMC practice were reported by 528 respondents due to embedded skip logic within the survey. KMC, kangaroo mother care; NICU, neonatal intensive care unit; NSCU, neonatal special care unit. on October 2 http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Disruptions for neonatal care provision and processes Sources of profes- sional information on hospital care of small and sick newborns varied greatly and across regions (table 1). Institutional guidelines for small and sick newborn care and KMC were reported as available by 23.1% of respon- dents. Information on COVID-19 was mainly derived from internet searches (58.6%), colleagues and profes- sional bodies (63.3%), and hospital or public health authorities (64.6%). on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ m 23, 2024 by guest. Protected by copyright. p py p At the hospital level, newborn follow-up care has been seriously affected by the pandemic (table 2), with 48.5% of respondents reporting fewer appointments per newborn and 32.6% reporting less staff to conduct follow-up clinics. Attendance has been interrupted by logistical challenges related to lockdown restrictions, such as decreased avail- ability of public transport (56.7%) and financial constraints. In addition, 73.3% of respondents indicated that families were reluctant to attend follow-up appointments due to fear of COVID-19. Many respondents are conducting follow-up by telephone to mitigate the impact of these disruptions, Knowledge gaps for the wider community and families regarding COVID-19 were also reported to affect access to care and duration of hospitalisation. Some respon- dents suggested that changes in behaviours emerged from fears caused by lack of knowledge or awareness regarding transmission risk and hygiene practices, with others noting that ‘Families don't want to stay in hospital even after receiving counselling’ (neonatal nurse, India). A community health worker from India reported, ‘In the pandemic, baby’s family [may] refuse to go to the hospital; they say, ‘our child will [get] sick [due] to Corona’’. 5 Rao SPN, et al. BMJ Global Health 2021;6:e004347. Disruptions for neonatal care provision and processes Downloaded from Table 2 Effect of the COVID-19 pandemic on small and sick newborn care, KMC practice, facility visitation and follow-up care Total, n (%) Neonatal inpatient care during COVID-19 pandemic (n=623) Newborn unit admission capacity reduced 229 (36.7) Newborn unit/KMC areas are reallocated (for COVID-19 care or other care) 91 (14.6) Newborn unit/KMC staff are reallocated (for COVID-19 care or other areas) 118 (18.9) Babies are discharged earlier than usual 273 (43.8) Women/families refuse to stay in facilities that are marked as COVID-19 treatment centres 197 (31.6) KMC practice during COVID-19 pandemic (n=623) KMC ward admission capacity reduced 172 (27.6) Health workers more hesitant to promote KMC 150 (24.0) Women/families more hesitant to practise KMC 105 (16.8) Counselling/support focus shifted from KMC to hand hygiene, masks and social distancing 237 (38) KMC practised with improved hand and respiratory hygiene (ie, masks and tissues) 321 (51.5) Changes to KMC practice (n=528) KMC practice has stopped 37 (7.0) KMC is practised as normal 292 (55.3) KMC is practised, but the daily duration of skin-to-skin contact is reduced 140 (26.5) KMC is initiated and babies are discharged home early 163 (30.8) KMC is practised in another area 23 (4.4) NSCU/NICU visitation during COVID-19 pandemic (n=593) Mother and family allowed as usual 68 (11.4) Mothers are permitted except for those who are COVID-19-positive 304 (51.2) Mother and family are not permitted 24 (4.0) Family members (other than mother) are not permitted 266 (44.8) Visiting hours are restricted 237 (39.9) Follow-up care during COVID-19 pandemic (n=435) Reduced space for follow-up clinic 100 (22.9) Less staff to conduct follow-up clinic 142 (32.6) Fewer appointments for each newborn 211 (48.5) Follow-up schedule has been changed 178 (40.9) Women/families reluctant to attend follow-up due to fear of infection 319 (73.3) Reduced attendance due to logistical reasons (eg, public transport disruptions) 247 (56.7) Home visits disrupted 95 (21.8) Telephone follow-up visits have been started. 160 (36.7) *Changes to KMC practice were reported by 528 respondents due to embedded skip logic within the survey. KMC, kangaroo mother care; NICU, neonatal intensive care unit; NSCU, neonatal special care unit. on October 2 http://gh.bmj.com/ MJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ h: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from on October 23, 2024 by guest. Protected by copyright. Disruptions for KMC practice and respectful In settings where visitations were still ongoing, many respondents expressed concern that family members were not following proper infection prevention and control (IPC) measures, such as hand hygiene, mask wearing and social distancing.15 At the family and caregivers’ level, most solutions aimed at promoting the safe continuation of lifesaving newborn interventions. A major theme was the need to increase awareness of caregivers with an emphasis on IPC, such as social distancing, correct mask wearing and hygiene practices. Many respondents stressed the impor- tance of counselling sessions with mothers and families to educate them on adequate hygiene and safety proto- cols to increase their confidence to initiate and continue KMC and breastfeeding, even after discharge. Better education regarding follow-up care and reduction of transmission was also proposed to enable continued care and attendance at follow-up visits. At the facility level, most solutions aimed at guaran- teeing IPC measures, including intensifying the use of PPE, hand hygiene practice and increasing the number of cleaning staff. The allocation of isolation wards to separate Covid-19 suspected and positive babies and mothers was also proposed; a neonatologist from India suggested to create ‘isolation wards for suspect or definite [COVID-19] cases separately. If space is a problem, then at least a barrier to separate between two cots’. Avoiding over- crowding and respecting social distancing measures were mentioned frequently because of lack of space, and many recommended shifting to remote patient monitoring. A neonatal nurse from India suggested to ‘install CCTV cameras in wards so that we [providers] can observe the patients even maintaining social distancing’. Another solution that emerged was the need for increased education to health- care workers with updated guidelines and clear protocols related to KMC and care of small and sick newborns, also suggesting that these trainings could serve as opportuni- ties to provide emotional support to staff. A neonatolo- gist wrote, ‘daily meetings with frontline workers could provide an opportunity to address problems [faced during practice]’. on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ m yg g g KMC practice varied greatly by maternal SARS-CoV-2 status: positive, negative, suspected COVID-19 or unknown (table 3). Routine KMC practice (with or without a face mask) was reported by 79.2% of respon- dents for SARS-CoV-2-negative mothers and 32.4% for SARS-CoV-2-positive mothers. Disruptions for KMC practice and respectful Early discharge and fear of providers to come into close contact with mothers were major barriers to the practice of KMC. KMC was reported to be practised routinely by Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 6 BMJ Global Health Table 3 Care of small newborns during the COVID-19 pandemic, by maternal SARS-CoV-2 status Mother SARS- CoV-2-positive N=519 n (%) Mother suspected with SARS-CoV-2 N=543 n (%) Mother SARS- CoV-2 unknown N=672 n (%) Mother SARS- CoV-2-negative N=664 n (%) Routine KMC practice (with or without mask) 168 (32.4) 196 (36.1) 413 (61.5) 526 (79.2) Mother and baby stay together, direct breast feeding but no prolonged skin-to-skin contact 93 (17.9) 112 (20.6) 151 (22.5) 89 (13.4) Mother and baby separated, except during breast feeding 70 (13.5) 67 (12.3) 48 (7.1) 25 (3.8) Mother and baby separated, expressed breastmilk feeding by uninfected caregiver 127 (24.5) 116 (21.4) 47 (7.0) 19 (2.9) Mother and baby separated, formula feeding by uninfected caregiver (no breastmilk) 61 (11.8) 52 (9.6) 13 (1.9) 5 (0.8) KMC, kangaroo mother care. on October 23, 2024 by guest. Protected by c http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from are of small newborns during the COVID-19 pandemic, by maternal SARS-CoV-2 status on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from disruptions/underlying challenges and reported solu- tions by these levels is given in figure 2. by effects of the pandemic’. Restrictions in visitation poli- cies and family involvement in the provision of care for small and sick newborns impacted the normal practice of KMC, with family members not present to act as surro- gates or provide support to mothers. Access and visiting hour limitations were widely reported. A paediatrician from Indonesia wrote, ‘Mothers rarely come to the perinatal room for routine KMC, as regulation limits people to enter the perinatal room from outside hospital’. Access to neonatal units was restricted, with 51.2% of respondents reporting that only mothers (except those positive for SARS-CoV-2) were permitted access and 11.4% reporting that fami- lies had usual access. Disruptions for KMC practice and respectful For SARS-CoV-2-positive mothers, almost 12% of respondents reported that they totally separated the baby from the mother and fed the baby formula milk. About 10% reported that they did the same for COVID-19 suspect mother–baby dyads. Breast- feeding disruptions were also highlighted, with practices including counselling, milk expression and human milk banking either stopped or continued at limited capacity during the pandemic. 23, 2024 by guest. Protected by copyright. Reported solutions and enablers Respondents suggested possible solutions to maintain service delivery for the small and sick newborn during the COVID-19 pandemic, with 55.4% of respondents reporting that they were currently implementing at least one of these solutions. We present their solutions by the levels of our conceptual framework: family and caregivers, facility and ward, health system, community and policy level (online supplemental appendix 3). A synthesis of At the level of health systems planning, many responses focused on increasing the number of personnel, reaching universal testing, ensuring supply provision and 7 Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 BMJ Global Health Figure 2 Synthesis of disruptions and underlying challenges, and analyses of solutions and enablers according to levels of care conceptual framework. LMIC, lowand middle-income country; KMC, kangaroo mother care; SSNC, small and sick newborn care. on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ ealth: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from Figure 2 Synthesis of disruptions and underlying challenges, and analyses of solutions and enablers according to levels of care conceptual framework. LMIC, lowand middle-income country; KMC, kangaroo mother care; SSNC, small and sick newborn care. on October 23, 2024 by guest. Protect http://gh.bmj.com/ on 14 March 2021. Downloaded from globe. We analysed responses from 1120 individuals in 62 countries from all world regions, encompassing all relevant cadres working in neonatal inpatient care as well as policymakers and public health professionals. The COVID-19 pandemic has caused serious disruptions at all levels of healthcare and particularly in LMICs, where the vast majority of newborn deaths occur. Our data suggest that coverage reductions for care at birth, and even more so for small and sick newborn care, continue at 20%–35%, although most of these countries were no longer in ‘hard lockdowns’. expanding infrastructure. In order to overcome health systems shortages such as limited personnel or reduc- tion of space, respondents suggested innovative solu- tions like showing, ‘videos demonstrating [practices] such as handwashing, breastfeeding and spoon feeding’, or opening a ‘neonatal COVID-19 query helpline’. Many respondents encouraged the use of telemedicine and mobile health to maintain care coverage without overstretching available resources. A doctor recommended, ‘Telephonic assistance or support should be provided after discharge from hospital as duration of stay is reduced so they may have not learnt proper KMC or breastfeeding technique’. Reported solutions and enablers g These results underline the additional stressors of a pandemic overlaid on already overstretched health systems, especially in Africa and South Asia.16 Even 9 months into the pandemic, with the world facing a second wave, testing of pregnant women was reported to be unavailable by more than one in five African respon- dents. Even when testing was available, the lag between testing and results delayed or impacted safe clinical management. Gaps in PPE provision have been high- lighted previously, including from high-income coun- tries.17 Lack of availability of PPE was a major stressor for health personnel in our study. Respondents underlined the imperative for a coordi- nated pandemic response including community inter- ventions and greater government involvement. Many proposed the use of community sensitisation campaigns on COVID-19, using television or radio programmes to provide audiovisual education and tackle stigmatisation. Others recommended governmental policies to guar- antee healthcare accessibility to vulnerable populations or to ensure provision of essential supplies, such as PPE. An administrator from the Philippines suggested that ‘local government should provide transportation for mothers and newborns in order to increase access to health services’. More- over, a paediatrician from Tanzania highlighted the need to ‘encourage local industry to manufacture affordable PPE’. r 23, 2024 by guest. Protected by copyright. The three-delay model18 is useful to explain how the pandemic results in disruptions,19 with delays in the decision to seek healthcare, delays in access and delays in receiving timely, high-quality care once at the facility. Delays in seeking or reaching care may be caused by fear of acquiring infection in hospitals and/or logistical effects of lockdown policies, such as curfews and trans- port restrictions.16 Pre-existing societal barriers also compound care-seeking for small and sick newborns and women with complications. Fatalism regarding newborns, Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 DISCUSSION Newborn care is a relative newcomer to global health,23 with programmatic focus only just beginning for hospital- based care of small and sick newborns.9 The WHO recently released standards for small and sick newborn care in the context of universal health coverage.24 Since this care is recent and considered low priority, with care- givers lacking power, in many facilities, the pandemic response has shifted essential equipment and workforce to other wards. Protecting and maintaining the staff, equipment and supplies in newborn units was strongly voiced among respondents in this survey. on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ m This study has several strengths. It is the first COVID-19 survey to focus on small and sick newborns—the most vulnerable users of any health system. It provides valu- able insights into the specific disruptors of already tenuous care at different levels of the health system and potential solutions, which would help policymakers and administrators protect and sustain services. Learnings from this study would provide some guidance to make health systems more resilient to future pandemics. One limitation is that the online survey was only available in three languages, which could have affected the repre- sentativeness of respondents in some regions, as well as those working in extremely rural and remote areas where internet access is limited. However, we do have responses from 62 countries across all regions. Respondents from LMICs constitute the majority of responses, although we note that this may not be a major limitation consid- ering LMICs have the greatest burden (98%) of neonatal deaths. During the 3 months the survey was open, coun- tries were in different phases of the pandemic, and this may have affected responses. However, this could provide a diverse and comprehensive picture, which may be useful as the pandemic is still far from over and many countries Keeping mothers and their newborns together is fundamental for respectful and effective care. Our data show this principle is being disrupted for the COVID- positive mother and her newborn, and even for COVID- negative mother–baby dyads. KMC coverage is generally low, although increasing evidence shows that more rapid progress in scaling up is possible.25 The pandemic has further decreased KMC coverage, which could have a serious impact on survival of small newborns and could threaten global targets. DISCUSSION While the direct mortality risk from SARS-CoV-2 infec- tion in newborns is low, indirect effects of the COVID-19 pandemic are already increasing neonatal mortality in hospitals.12 Our survey provides sobering insights into disruption to care for small and sick newborns across the Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 8 BMJ Global Health on October 23, 2024 by g http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from of the COVID-19 pandemic. Neonatal care, like care at birth, necessitates close interaction of health personnel with mothers and their babies, and trust, based on effec- tive testing of both families and staff and provision of adequate PPE. A review examining COVID-19 guidelines on neonatal care for 17 countries highlighted variable quality and unsustainability of evidence,26 leading to uncertainties in policy and programmes. especially those who are born preterm, is common in many cultures.20–22 In the context of the COVID -19 pandemic, even when women seek care, early discharge before full clinical stabilisation and preparedness is more common due to lack of family support and fear of SARS-CoV-2 infection. Improving community awareness about safety measures in health facilities, education and teleconsultation for follow-up care are some of the solu- tions that were suggested to reduce the occurrence and impact of these disruptions. of the COVID-19 pandemic. Neonatal care, like care at birth, necessitates close interaction of health personnel with mothers and their babies, and trust, based on effec- tive testing of both families and staff and provision of adequate PPE. A review examining COVID-19 guidelines on neonatal care for 17 countries highlighted variable quality and unsustainability of evidence,26 leading to uncertainties in policy and programmes. especially those who are born preterm, is common in many cultures.20–22 In the context of the COVID -19 pandemic, even when women seek care, early discharge before full clinical stabilisation and preparedness is more common due to lack of family support and fear of SARS-CoV-2 infection. Improving community awareness about safety measures in health facilities, education and teleconsultation for follow-up care are some of the solu- tions that were suggested to reduce the occurrence and impact of these disruptions. on October 23, 2024 by guest. Protected by copyright. http://gh.bmj.com/ h: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. DISCUSSION Downloaded from p y p g Our results suggest that evidence-based care is being affected adversely, with two-thirds of providers reporting they would not allow mothers with confirmed or suspected SARS-CoV-2 infection to practise routine KMC, and nearly one-quarter reporting they would not allow breastfeeding. Health workers seemed unsure of guide- lines for small and sick newborn care, even for breast- feeding, despite global educational campaigns by the WHO, UNICEF and others.27 28 Breastmilk is unlikely to be a route of SARS-CoV-2 transmission.29–31 Chambers and colleagues detected SARS-CoV-2 RNA in one of 64 milk samples from 18 infected women, with a negative viral culture for the positive sample, suggesting that RNA does not represent replication-competent virus.29 A case series reported that 4 of 12 neonates born to SARS-CoV-2- infected mothers tested positive within 48 hours of birth, after maternal symptom onset, of whom one was fed posi- tive milk and subsequently tested negative despite exclu- sively breastfeeding while the mother was infected.30 A study among 185 neonates of infected mothers in Mumbai, India, reported that 12 (7%) tested positive for SARS-CoV-2 while bedding -in with their mothers, all of whom were healthy and thriving on exclusive breastmilk through 2 months of age.32 Available evidence suggests the benefits of breastfeeding on infant health, growth and development substantially outweigh the potential risk of SARS-CoV-2 transmission. p p At the facility level, barriers to effective care include shortages of nurses and doctors whose function is further affected by an almost universal increase in stress, anxiety and fear, a recurring theme during previous outbreaks and this pandemic too. In the global maternity care survey, 90% reported increased stress.14 Increased stress stems from shortage of staff, overwork and lack of PPE. A recent survey of >2700 healthcare professionals from 60 countries found that half reported work-related burnout, as defined by a single measure of emotional exhaustion, and two-thirds indicated work impacting their quality of life and household activities during the pandemic.17 Avail- ability of adequate PPE was associated with a 32% reduc- tion in reported burnout among 314 respondents from LMICs.17 Provision of PPE and mental health support were recommended as potential solutions. Governments should perhaps consider prioritising provision of masks, eye shields and other PPE rather than items such as sani- tisers, which can be replaced with soap and water hand washing. CONCLUSION The COVID-19 pandemic has disrupted small and sick newborn care, including KMC, as well as caused high levels of stress among neonatal care providers. This paper sheds light on these effects and provides insights for poli- cymakers. Management and allocation of newborn unit staff and essential equipment and supplies, including PPE, can and should be improved urgently. More atten- tion must be placed on ensuring evidence-based prac- tices, such as breastfeeding and KMC for all women and babies, including among SARS-CoV-2-positive mothers who are well enough. As a global health community, we need to act to protect the most vulnerable and prevent reversals of hard-earned gains in newborn survival, as well mitigate the wider impact on women, families and national development. Collaborators COVID-19 Small and Sick Newborn Care Collaborative Group:Helen Brotherton and Abdou Gai (Early KMC; The Gambia); Irene Agyeman, Gyikua Plange-Rhule, Sam Newton, and Naana Wireko-Brobby (Immediate KMC; Ghana); Sugandha Arya, Rani Gera and Harish Chellani (Immediate KMC; India); Queen Dube and Kondwani Kawaza (Immediate KMC; and NEST360° Malawi); Ebunoluwa Adejuyigbe, Henry Anyabolu, and Osagie Ugowe (Immediate KMC; Nigeria); Augustine Massawe and Helga Naburi (Immediate KMC; Tanzania); David Gathara and Grace Irimu (NEST360° Kenya); Msandeni Chiume and Elizabeth Molyneux (NEST360° Malawi); Chinyere Ezeaka (NEST360° Nigeria); Nahya Salim and Josephine Shabini (NEST360° Tanzania); James Cross and Eric Ohuma (NEST360° UK); Joy E Lawn (NEST360° UK, OMWaNA, Operationalizing kangaroo Mother care among low birth Weight Neonates in Africa; Uganda); Ivan Mambule, Melissa Medvedev, Cally Tann, Victor Tumukunde, and Peter Waiswa (OMWaNA, Operationalizing kangaroo Mother care among low birth Weight Neonates in Africa; Uganda); Lamesgin Alamineh, Abiy Seifu Estifanos, Abebe Gebremariam Gobezayehu, Amanuel Hadgu, Araya Abrha Medhanyie, Henok Tadele, and Fitsum Woldegebriel (Scale-up KMC; Ethiopia); Arun Singh Jadaun, Sarmila Mazumder, Nita Bhandari (Scale-up KMC; Haryana, India); Suman Rao PN (Immediate KMC; WHO and Scale-up KMC; Karnataka, India); Troy Cunningham, Prathibha Rai, and Prashantha YN (Scale-up KMC; Karnataka, India); Aarti Kumar, Rashmi Kumar, Ved Prakash, Vinay Pratap Singh and Vishwajeet Kumar (Scale-up KMC; Uttar Pradesh, India); Rajesh Mehta, Nicole Minckas, Alfrida Camelia Silitonga (WHO). on October 23, 2024 by guest. Protect http://gh.bmj.com/ on 14 March 2021. Downloaded from DISCUSSION Further, KMC continuity is adversely affected by restricted visitation polices, discharge before babies meet discharge criteria and limited post-discharge care. Strengthening counselling for KMC and breastfeeding and improving awareness of personal precautions could mitigate these effects to some extent. Unambiguous guidelines are urgently needed regarding small and sick newborn care in the context 23, 2024 by guest. Protected by copyright. 9 Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 BMJ Global Health BMJ Global Health on October 23, 2024 by guest. Protected by c http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from 21Bagamoyo Research and Training Centre, Ifakara Health Institute, Bagamayo, Tanzania 21Bagamoyo Research and Training Centre, Ifakara Health Institute, Bagamayo, Tanzania are encountering varying waves of COVID-19. The survey was designed to highlight experiences of neonatal care providers, particularly those in hospitals; however, the voices of mothers, families and wider communities are also crucial. Our collaborative group is undertaking a multi-country qualitative study to better understand the perspectives of health workers, especially at the commu- nity level, as well as families of small and sick newborns. are encountering varying waves of COVID-19. The survey was designed to highlight experiences of neonatal care providers, particularly those in hospitals; however, the voices of mothers, families and wider communities are also crucial. Our collaborative group is undertaking a multi-country qualitative study to better understand the perspectives of health workers, especially at the commu- nity level, as well as families of small and sick newborns. 22Department of Pediatrics and Child Health, Addis Ababa University, Addis Ababa, Ethiopia 23Hawassa University, Hawassa, Ethiopia 23Hawassa University, Hawassa, Ethiopia 24Regional Office, South East Asia, World Health Organisation, New Delhi, India Twitter Melissa M Medvedev @melissacmorgan, Eric O Ohuma @ohumaeric and Joy E Lawn @joylawn Acknowledgements We thank Rajiv Bahl and Sachiyo Yoshida from WHO HQ for their support. We also thank Adesh Sirjusingh, Adnan Mirza, Adwoa Pokua Boakye-Yiadom, Catherine Chaguza, Cecilia W. Ndungu, Constance Malanga, Daniel Larry Gunya, Emma Sacks, Evelyne Assenga, Ezra Ogundare, Gift Masiye, Grace Zakeyu, Ireen Phiri, Khumbo Mbale, Lina Yasin Amin, Lloyd Tooke, Marlon Timothy, Melese Gabure Shukulo, Michael Aondoaseer, Olabisi Dosunmu, Pratima Anand, Sam Newton, Sharon Owusu Danquah, Wilson Okot, Zainab Mwaka Kioni, all of the survey respondents, and the many colleagues around the world who supported the dissemination of the survey. Author affiliations 1 om/ 9Department of Pediatrics and Child Health, Obafemi Awolowo University, Ile-Ife, Nigeria 9Department of Pediatrics and Child Health, Obafemi Awolowo University, Ile-Ife, Nigeria Tropical Medicine, Fajara, The Gambia 11Department of Pediatrics, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India 12 12Department of Pediatrics, University of Lagos and Lagos State University Teaching Hospital, Lagos, Nigeria 12Department of Pediatrics, University of Lagos and Lagos State Univer 2024 by guest. Protected by copyright. Map disclaimer The depiction of boundaries on this map does not imply the expression of any opinion whatsoever on the part of BMJ (or any member of its group) concerning the legal status of any country, territory, jurisdiction or area or of its authorities. This map is provided without any warranty of any kind, either express or implied. 13Emory University Ethiopia, Addis Ababa, Ethiopia 13Emory University Ethiopia, Addis Ababa, Ethiopia 14Department of Pediatrics and Child Health, University of Malawi College of 14Department of Pediatrics and Child Health, University of Malawi College of Medicine and Queen Elizabeth Central Hospital, Blantyre, Malawi 15Community Empowerment Lab, Lucknow, India 16Department of Pediatrics and Child Health, Muhimbili University of Health and Competing interests None declared. Competing interests None declared. Patient consent for publication Not required. Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 Author affiliations 1 1Department of Neonatology, St. John's Medical College Hospital, Bangalore, India 2Institute for Global Health, University College London, London, UK 1Department of Neonatology, St. John's Medical College Hospital, Bangalore, India 2Institute for Global Health, University College London, London, UK 3Department of Pediatrics, University of California San Francisco, San Francisco, California, USA 4Maternal, Adolescent, Reproductive and Child Health (MARCH) Centre, London School of Hygiene & Tropical Medicine, London, UK 4Maternal, Adolescent, Reproductive and Child Health (MARCH) Centre, London School of Hygiene & Tropical Medicine, London, UK Maternal, Adolescent, Reproductive and Child Health (MARCH) Centre, London School of Hygiene & Tropical Medicine, London, UK 5Kenya Medical Research Institute-, Wellcome Trust Research Program, Nairobi, Kenya Contributors SR and JEL conceived the study and developed the survey, with substantial input from NM, MMM and DG. All authors reviewed and helped disseminate the survey. NM analysed the data, with input from SR, MMM and JEL. SR, NM, MMM, DG and JEL wrote the manuscript. All authors interpreted the data and critically revised the manuscript. 6Department of Reproductive, Family and Population Health Health, Addis Ababa University, Addis Ababa, Ethiopia 6Department of Reproductive, Family and Population Health, Health, Addis Ababa University, Addis Ababa, Ethiopia 7Department of Reproductive, Maternal, Newborn, Child and Adolescent Health, i World Health Organization, Country Office Indonesia, Jakarta, Indonesia 8 Funding During the conduct of this study, MMM was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (#K23HD092611) and JEL and NEST360 colleagues were supported by the NEST360° COVID grant funding from the Bill & Melinda Gates Foundation and Elma Philanthropies via Rice University. The funders had no role in the study design, data collection, data analysis, data interpretation or writing of the report. SR, NM, MMM and JEL had full access to all the data and had the final responsibility for the decision to submit for publication. 8Centre for Health Research and Development, Society for Applied Studies, New Delhi, India on October 23, 2024 by guest. Protected by copyright. REFERENCES 1 United Nations Inter-agency Group for Child Mortality Estimation. Levels & Trends in Child Mortality: Report 2020. New York: UNICEF, 2020. https://data.unicef.org/resources/levels-and-trends-in-child- mortality/ ; 22 Morgan MC, Nambuya H, Waiswa P, et al. Kangaroo mother care for clinically unstable neonates weighing ≤2000 g is it feasible at a hospital in Uganda? J Glob Health 2018;8:010701. p g 23 Darmstadt GL, Kinney MV, Chopra M, et al. Who has been caring for the baby? Lancet 2014;384:174–88. 2 Lawn JE, Blencowe H, Oza S, et al. Every newborn: progress, priorities, and potential beyond survival. Lancet 2014;384:189–205. 3 WHO UNICEF E b i l d bl 24 WHO. Standards for improving the quality of care for small and sick newborns in health facilities. Geneva: WHO, 2020. https://www.who. int/publications/i/item/9789240010765 3 WHO, UNICEF. Every newborn: an action plan to end preventable deaths. Geneva: WHO, 2014. https://www.who.int/maternal_child_ adolescent/newborns/every-newborn/en/ 3 WHO, UNICEF. Every newborn: an action plan to end preventable deaths. Geneva: WHO, 2014. https://www.who.int/maternal_child_ adolescent/newborns/every-newborn/en/ 25 Medhanyie AA, Alemu H, Asefa A, et al. Kangaroo mother care implementation research to develop models for accelerating scale- up in India and Ethiopia: study protocol for an adequacy evaluation. BMJ Open 2019;9:e025879. 4 United Nations. Sustainable development goals. Available: https:// www.un.org/sustainabledevelopment/sustainable-development- goals/ [Accessed 1 Nov 2020]. 5 United Nations Inter-agency Group for Child Mortality Estimation. A neglected tragedy: the global burden of stillbirths. New York: UNICEF, 2020. https://data.unicef.org/resources/a-neglected- tragedy-stillbirth-estimates-report/ 5 United Nations Inter-agency Group for Child Mortality Estimation. A neglected tragedy: the global burden of stillbirths. New York: UNICEF, 2020. https://data.unicef.org/resources/a-neglected- tragedy-stillbirth-estimates-report/ 26 Yeo KT, Oei JL, De Luca D, et al. Review of guidelines and recommendations from 17 countries highlights the challenges that clinicians face caring for neonates born to mothers with COVID-19. Acta Paediatr 2020;109:2192–207. 6 WHO, UNICEF. Every newborn progress report 2019. Geneva: WHO, 2020. https://www.who.int/publications/i/item/9789240005082 6 WHO, UNICEF. Every newborn progress report 2019. Geneva: WHO, 2020. https://www.who.int/publications/i/item/9789240005082 27 WHO. Frequently asked questions: breastfeeding and COVID-19 for health care workers. Available: https://www.who.int/docs/default- source/maternal-health/faqs-breastfeeding-and-covid-19.pdf? sfvrsn=d839e6c0_5 [Accessed 24 Jun 2020]. 7 Blencowe H, Krasevec J, de Onis M, et al. National, regional, and worldwide estimates of low birthweight in 2015, with trends from 2000: a systematic analysis. Lancet Glob Health 2019;7:e849–60. [ ] 28 UNICEF, International Pediatric Association. Joint IPA-UNICEF COVID-19 information brief: epidemiology, spectrum, and impact of COVID-19 on children, adolescents, and pregnant women. Available: https://www.unicef.org/bulgaria/sites/unicef.org.bulgaria/files/2020- 07/UNICEF-IPA_COVID_brief_WEB_29June_ENG.pdf [Accessed 20 Jul 2020]. BMJ Global Health on October 23, 2024 by guest. Protected by http://gh.bmj.com/ BMJ Glob Health: first published as 10.1136/bmjgh-2020-004347 on 14 March 2021. Downloaded from Data availability statement Data are available upon request. Anonymised data analysed in the current study will be made available from the corresponding author on reasonable request. 13 Minckas N, Medvedev MM, Adejuyigbe E, et al. Preterm care during the COVID-19 pandemic: a comparative risk analysis of neonatal deaths averted by kangaroo mother care versus mortality due to SARS-CoV-2 infection. EClinicalMedicine 2021. (In press) Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. 14 Semaan A, Audet C, Huysmans E, et al. Voices from the frontline: findings from a thematic analysis of a rapid online global survey of maternal and newborn health professionals facing the COVID-19 pandemic. BMJ Glob Health 2020;5:e002967. 15 WHO Reproductive Health Library. Who recommendation on respectful maternity care during labour and childbirth. Geneva: WHO, 2018. https://extranet.who.int/rhl/topics/preconception- pregnancy-childbirth-and-postpartum-care/care-during-childbirth/ who-recommendation-respectful-maternity-care-during-labour-and- childbirth 16 GRID COVID-19 Study Group. Combating the COVID-19 pandemic in a resource-constrained setting: insights from initial response in India. BMJ Glob Health 2020;5:e003416. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. 17 Morgantini LA, Naha U, Wang H, et al. Factors contributing to healthcare professional burnout during the COVID-19 pandemic: a rapid turnaround global survey. PLoS One 2020;15:e0238217. p g y 18 Thaddeus S, Maine D. Too far to walk: maternal mortality in context. Newsl Womens Glob Netw Reprod Rights 1991;36:22–4. ORCID iDs 19 Calvello EJ, Skog AP, Tenner AG, et al. Applying the lessons of maternal mortality reduction to global emergency health. Bull World Health Organ 2015;93:417–23. Suman P N Rao http://orcid.org//0000-0002-9721-0308 Melissa M Medvedev https://orcid.org/0000-0003-3457-8452 Abiy Seifu Estifanos http://orcid.org/0000-0002-6833-9823 Eric O Ohuma http://orcid.org/0000-0002-3116-2593 Joy E Lawn https://orcid org/0000 0002 4573 1443 20 Waiswa P, Nyanzi S, Namusoko-Kalungi S, et al. 'I never thought that this baby would survive; I thought that it would die any time': perceptions and care for preterm babies in eastern Uganda. Trop Med Int Health 2010;15:1140–7. Joy E Lawn https://orcid.org/0000-0002-4573-1443 21 Morgan MC, Dyer J, Abril A, et al. Barriers and facilitators to the provision of optimal obstetric and neonatal emergency care and to the implementation of simulation-enhanced mentorship in primary care facilities in Bihar, India: a qualitative study. BMC Pregnancy Childbirth 2018;18:420. on October 23, 2024 by guest. Protect http://gh.bmj.com/ on 14 March 2021. Downloaded from Patient consent for publication Not required. 17Department of Pediatrics, Kamuzu Central Hospital, Lilongwe, Malawi 18 17Department of Pediatrics, Kamuzu Central Hospital, Lilongwe, Malawi Ethics approval This study was approved by the Institutional Ethics Committee of St. John’s Medical College (Bangalore, India) on 7 August 2020 (number 198/2020), by Addis Ababa University Institutional Review Board (protocol number: 068/20/SPH) and the Ethics Review Committee of the Centre of Health Research and Development, Society for Applied Studies (number SAS/ERC/KMC COVID Study/2020). 19School of Public Health, Mekelle University College of Health Sciences, Mekelle, Ethiopia 20School of Medicine and Dentistry, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Provenance and peer review Not commissioned; externally peer reviewed. 10 Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347 BMJ Global Health REFERENCES y y 8 Chawanpaiboon S, Vogel JP, Moller A-B, et al. Global, regional, and national estimates of levels of preterm birth in 2014: a systematic review and modelling analysis. Lancet Glob Health 2019;7:e37–46. g y 9 WHO, UNICEF. Survive and thrive: transforming care for every small and sick newborn. Geneva: WHO, 2019. https://www.unicef.org/ media/58076/file 29 Chambers C, Krogstad P, Bertrand K, et al. Evaluation for SARS-CoV-2 in breast milk from 18 infected women. JAMA 2020;324:1347–8. 10 Graham WJ, Afolabi B, Benova L, et al. Protecting hard-won gains for mothers and newborns in low-income and middle-income countries in the face of COVID-19: call for a service safety net. BMJ Glob Health 2020;5:e002754. 30 Bertino E, Moro GE, De Renzi G, et al. Detection of SARS-CoV-2 in milk from COVID-19 positive mothers and follow-up of their infants. Front Pediatr 2020;8:597699. 11 Roberton T, Carter ED, Chou VB, et al. Early estimates of the indirect effects of the COVID-19 pandemic on maternal and child mortality in low-income and middle-income countries: a modelling study. Lancet Glob Health 2020;8:e901–8. 31 Lugli L, Bedetti L, Lucaccioni L, et al. An uninfected preterm newborn inadvertently fed SARS-CoV-2-Positive breast milk. Pediatrics 2020;146:e2020004960.i 12 Kc A, Gurung R, Kinney MV, et al. Effect of the COVID-19 pandemic response on intrapartum care, stillbirth, and neonatal mortality outcomes in Nepal: a prospective observational study. Lancet Glob Health 2020;8:e1273–81. 32 Kalamdani P, Kalathingal T, Manerkar S, et al. Clinical profile of SARS-CoV-2 infected neonates from a tertiary government hospital in Mumbai, India. Indian Pediatr 2020;57:S097475591600250. 32 Kalamdani P, Kalathingal T, Manerkar S, et al. Clinical profile of SARS-CoV-2 infected neonates from a tertiary government hospital in Mumbai, India. Indian Pediatr 2020;57:S097475591600250. 11 Rao SPN, et al. BMJ Global Health 2021;6:e004347. doi:10.1136/bmjgh-2020-004347
https://openalex.org/W4223444957	https://figshare.com/articles/media/Psychological_Pain_among_Divorced_women_in_Ramallah_and_Al-Bireh_Governorate/19642875/1/files/34888284.pdf	English	null	Psychological Pain among Divorced women in Ramallah and Al-Bireh Governorate	Humanities & Social Sciences reviews	2,022	cc-by	3,259	Research question As a result, this study needs to answer the following question: As a result, this study needs to answer the following question:  Are there differences in the arithmetic averages in the psychological pain of divorced women in Ramallah and Al- Bireh governorates?  Are there differences in the arithmetic averages in the psychological pain of divorced women in Ramallah and Al- Bireh governorates? Copyright @Author Applications of this study: The researchers expected to apply the research in centers, institutions, and societies focusing on issues for divorced women for psychological and social rehabilitation programs through activities aimed at enhancing their self-confidence. Novelty/Originality of this study: As a result, divorce is a threatening, frightening, and troubling factor for women that causes stress and frustration, particularly in the early post-divorce years. Novelty/Originality of this study: As a result, divorce is a threatening, frightening, and troubling factor for women that causes stress and frustration, particularly in the early post-divorce years. Research aims This study aimed to identify the differences in psychological pain means among divorced women in Ramallah and Al- Bireh governorates based on the study variables INTRODUCTION Divorce is one of the most critical social and psychological issues threatening the family and society. Most divorcees feel that they will solve their difficulties after divorce; this idea is incorrect. Divorce leads to many social, psychological, and emotional problems, including feelings of loneliness, isolation, anxiety, sadness, profound psychological pain, depression, and a poor capacity to adjust to new circumstances. The choice to divorce also hurts the family, causing the family to split apart (Baweh, 2017), and the difficulties of sadness and psychological pain do not diminish with time; instead, they rise and expand, hurting (15% to 25%) of those who may lose. Deep sadness appears alongside depression with a ratio of (20%-45%), and post-traumatic stress disorder with (35%-50%) (Shear et al., 2005). There are many symptoms associated with intense sadness that may last longer. Many international studies have emphasized the psychological repercussion of divorce. Dykstra and Fokkema (2007) and Ben-Zur (2012) demonstrated that divorce causes psychological incontinence, as most divorced women suffer anxiety and depression. Van Tilburg and his colleagues (2015) discovered an increase in self-centeredness among divorced women. Divorced women suffer from extreme introversion, which leads to an increasing feeling of psychological loneliness over time. Psychological loneliness is the starting point for many of the problems and psychological disorders that divorced women suffer from (Zahar&Kausar,2014; Knöpfli et al.,2016). Psychological Pain among Divorced women in Ramallah and Al-Bireh Governorate Email: 1obraighith@birzeit.edu, 2*orimawi@staff.alquds.edu Humanities & Social Sciences Reviews eISSN: 2395-6518, Vol 10, No 2, 2022, pp 38-42 Humanities & Social Sciences Reviews eISSN: 2395-6518, Vol 10, No 2, 2022, pp 38-42 Abstract Purpose of the study: This study aimed to assess the psychological pain level among divorced women in Ramallah and Al-Bireh Governorate based on various variables. Methodology: The researchers used a descriptive approach to achieve the study goals. The study's sample consisted of (67) divorced women as the researchers used the affordable method to choose using the participants. They also used the Person correlation coefficient with the total score of the instrument to test the instrument's validity. The results showed that all of the questionnaire's items had statistical significance. Cite this article Brighieth, M., & Rimawi, O. (2022). Psychological Pain among Divorced women in Ramallah and Al-Bireh Governorate: Psychological Pain among Divorced women . Humanities & Social Sciences Reviews, 10(2), 38-42. https://doi.org/10.18510/hssr.2022.1026 Findings: The current study's findings revealed a moderate psychological pain level among divorced women in Ramallah and Al-Bireh Governorate, with a ratio of (70.2%). The results showed no statistically significant differences and differences in the means of the participants' responses due to the study's variables (age, academic level, duration of marriage, occupational status, and post-divorce Period). The population of the study and its sample: The study's population included all divorced women in Ramallah and Al-Bireh Governorate. The participants were chosen from the study's sample in an affordable method since it was difficult to access all participants. Some of them refused to participate and collaborate. Table 1 shows the distribution of the participants based on their demographic variables. Table 1: Participants’ distribution due to their demographic variables Table 1: Participants’ distribution due to their demographic variables Variable Level No. Percentage Age under 25 years 18 26.9 26-35 years 13 19.4 36-45 years 13 19.4 Above 45 years 23 34.3 Academic qualification Below Tawjihi 29 43.3 Tawjihi- B.A. 25 37.3 Higher than B.A. 13 19.4 Marriage duration Less than four years 22 32.8 5-10 years 32 47.8 More than 10 years 13 19.4 Occupational status Employed 28 41.8 Unemployed 39 58.2 Post-divorce period 1-5 years 27 40.3 6-10 years 29 43.3 More than ten years 11 16.4 The approach of the study To fulfil the study's objectives, the researchers employed the descriptive approach, which is an approach that investigates a phenomenon, event, or issue from which we can gather the information that answers the research questions without the researcher's intervention. Instrumentation After reviewing many previous research and instruments, and the educational studies related to the issue of the current study, the researchers used the scale of Al-Kubaisi et al. (2015), which consisted of 30 items. The researcher applied the instrument to the participants. After collecting the questionnaires from the participants and ensuring that the participants completed them accurately, the results discovered that only (67) questionnaires were valid to be statistically analyzed. The questionnaire items score on a 1-5 scale (from 1=Not at all to 5= Absolutely) LITERATURE REVIEW The previous studies by Al-Badayna and Al-qotitat (2018) in Palestine and neighboring Arab countries found that divorced women face stigma, have low self-esteem, and are less adaptable than married women. Anxiety about children's 38 \| Visit HSSR at https://mgesjournals.com/hssr/ 38 \| Visit HSSR at https://mgesjournals.com/hssr/ Humanities & Social Sciences Reviews eISSN: 2395-6518, Vol 10, No 2, 2022, pp 38-42 future was ranked first, followed by suffering from social, economic, and psychological problems came in second place (Abu Darwish et al., 2016). According to the findings of Abdullah (2018), divorce was the first source of mental health imbalance among divorced women. The results also revealed statistically significant differences in the mental health level of divorced women based on age, the residence of children after divorce, the standard of living, and academic qualifications of divorced women. According to Alawneh (2019), the majority of divorced women in Palestine were young women under 30 years old, with a ratio of 68%, but 59% of divorced women had no children or only one, with low academic qualifications represented by less than an intermediate diploma, and the majority of them were unemployed. Al-Ali and his colleagues (2021) discovered statistically significant differences in the psychological resilience level of divorced women owing to the age group differences in favor of those under 30 years and the difference in academic level in favor of a university degree and above. According to Nasser and Rimawi (2022), there are no differences in the existential vacuum among battered married women based on academic qualifications. Divorce reflects one of the social problems that threaten divorced people's lives, families, and societies. It is also one of the problems that have psychological ramifications for the divorced woman, primarily because of the increase in divorce percentage in Palestine as it reaches 19.4% according to the (BCPS) in the year 2020. This study is significant because it addresses the psychological pain of divorced women due to unstable economic, social, and political conditions that cast a shadow on the lives of individuals, families, and societies, affecting them negatively in general and on families and divorced women in particular. This fact necessitates the conduct of such investigations as soon as possible. FINDINGS / RESULTS The results also showed differences in the participant's responses on the psychological pain level among divorced women in Ramallah and Al-Bireh Governorate due to the variables (age, academic qualification, duration of marriage, and post- divorce Period). Data Analysis The researchers extracted the means and the standard deviations for each item of the scale after verifying the validity and reliability of the study's instrument and ensuring its validity for statistical analysis using Cronbach Alpha, t-test, One Way ANOVA, and the statistical packages (SPSS). FINDINGS / RESULTS The results showed that the mean of the total score for psychological pain level among divorced women in Ramallah and Al-Bireh Governorate was (3.51) and the standard deviation was (0.78), indicating a moderate level of psychological pain among divorced women in Ramallah and Al-Bireh Governorate with a ratio of (70.2%). The item "I see that the law is exclusively against the simple person of the people" got the highest mean (4.01), followed by "I blame myself for the prior mistakes I committed" (4.00). The items "I feel sad and miserable" and "My appetite is not what it used to be" had the lowest mean of (2.90). : Results of T-test analysis of variance for the differences due to the variable (occupational status) Variable Level No. Mean S.D T Sig. Occupational status Employed 28 3.62 0.86 1.35 0.18 Unemployed 39 3.36 0.64 ble 2: Results of T-test analysis of variance for the differences due to the variable (occupational status) Table 2 shows that the values of "T" revealed no statistically significant differences between the total scores. The results also show differences in the participant's responses to the psychological pain level among divorced women in Ramallah and Al-Bireh Governorate due to the variable (occupational status). Table 3: Results of One Way Anova analysis of variance for the differences due to the variables (age, academic level, duration of marriage, and post-divorce period) ults of One Way Anova analysis of variance for the differences due to the variables (age, academic level, duration of marriage, and post-divorce period) Variable Level No. Mean S.D F Sig. Age under 25 years 18 3.91 0.25 2.17 0.10 26-35 years 13 3.57 0.62 36-45 years 13 3.35 0.77 Above 45 years 23 3.25 1.08 Academic qualification Below Tawjihi 29 3.36 0.49 0.28 0.75 Tawjihi- B.A. 25 3.54 0.98 Above B.A 13 3.55 0.64 Marriage duration Less than four years 22 3.46 0.90 0.96 0.38 5-10 years 32 3.43 0.74 More than ten years 13 3.78 0.61 Post-divorce period 1-5 years 27 3.58 0.69 0.32 0.72 6-10 years 29 3.55 0.60 More than ten years 11 3.41 0.93 Table 3 shows that the values of "F" revealed no statistically significant differences between the total scores. Instrument validity A group of specialized and experienced arbitrators confirmed the instrument's validity. The researchers calculated the Pearson correlation coefficient for the questionnaire items and the total instrument's score to verify the instrument's 39 \| Visit HSSR at https://mgesjournals.com/hssr/ Humanities & Social Sciences Reviews Humanities & Social Sciences Reviews eISSN: 2395-6518, Vol 10, No 2, 2022, pp 38-42 eISSN: 2395-6518, Vol 10, No 2, 2022, pp 38-42 validity. The results revealed a statistical significance in all the questionnaire items, indicating an internal consistency between them. Instrument reliability The researchers verified the instrument reliability by calculating the reliability of the total score of the reliability coefficient of the study items using Cronbach's Alpha reliability equation. The total score for the psychological pain level was (0.95), indicating that this tool has the reliability that meets the study's purposes. DISCUSSION / ANALYSIS The findings revealed high divorced women's responses to the factor of psychological pain, especially in the age group under 25 years. This result is consistent with the requirements of the developmental-developmental stage. At this age, women tend to cling to life, considering the concept of happiness culturally is more closely related to women's correlation with the element of safety. Thus, marriage as a social institution that protects women from all forms of oppression and violence reinforces stability that embodies security. The psychological pain indicator reflects a core issue that responds to the loss of those needs stem from the divorce. This result is consistent with Al-Ali et al. (2021). The same applies to women who responded to the psychological pain indicator for its relation to the academic qualification variable, where education is the main component of a sense of "self-efficacy" and social efficiency alike. The psychological immunity, where females have a level of education, is an 40 \| Visit HSSR at https://mgesjournals.com/hssr/ Humanities & Social Sciences Reviews Humanities & Social Sciences Reviews eISSN: 2395-6518, Vol 10, No 2, 2022, pp 38-42 economic and financial immunity, which gives women a greater sense of economic security, especially in the absence or absence of the head of the family. This result is consistent with Alawneh's (2019) and Al-Ali et al. (2021). The study's findings also show that divorced women who get married for more than ten years are more likely to experience psychological pain. This fact does not contradict human nature in general, particularly women because they belong to the family environment as a place of warmth, affection, and serenity. Therefore, the Period (10 years More than) is sufficient for the divorced woman so that she was able to cope and familiarize within a social institutional structure, that is the marriage. Once she is removed from this institution, whether for social or psychological reasons, she loses all her needs, which strongly justifies that feeling of psychological pain as a reaction that may continue for months or years after leaving the family as a social system. Remarkably, 28 of the divorced women who took part in the study showed a high level of psychological pain as a result of their unemployment. CONCLUSION Regardless of the chronological age difference between the participants, psychological pain and its emotional symptoms are a core component for divorced women. Women's intensive emotional aspects and attachment to the marital relationship affected them more than men. The quality of the marriage before divorce also influences how the divorce news is received. Women who have been in a bad marriage are more open to the idea of divorce than those who had a good marriage life but separated for some reason, leaving them discontented with life after the divorce. As a result, divorce is a threatening, worrying, stressful, and frustrating factor for women, particularly in the first post-divorce years. Thus, we strongly recommend that divorced women participate in psychosocial rehabilitation programs through activities for reinforcing their self-esteem. AUTHOR’S CONTRIBUTION Brighieth Mohammed's contribution was as follows: concept, data acquisition, analysis, as well as inte Rimawi Omar's contribution was as follows: concept, analysis and interpretation. Rimawi Omar's contribution was as follows: concept, analysis and interpretation. Rimawi Omar's contribution was as follows: concept, analysis and interpretation. DISCUSSION / ANALYSIS Interpretatively, this result is a main focus in the context of the divorce and its impact on the psychological state of women, in that the divorced woman, in the prevailing culture, is exposed after being divorced to a form of "societal blame" even if she is correct, because she is a woman living in a male-style society, and thus a work place is a space in which a woman may be exposed to several questions that may be difficult to answer, such as why she got divorced. The societal blame placed on her by male and female coworkers in the workplace is an additional psychological burden that may make her to leave the job and become, in her opinion, a burden on a society that rejects her because she is divorced, This is consistent with the study of Al-Badayna and Al-qotitat (2018). The variable "post- divorce period" from one to five years is also one of the striking variables in this study, as 27 of the participating divorced women showed high statistical rates on the psychological pain items, and this is because the first post-divorce Period is shocking for women in terms of reorganizing their cognitive structures on a new lifestyle completely different from the one they lived or adapted within. Some memories and feelings are still invading her cognitive scope that concerning marriage as a "protective and secure" social institution in the societal-cultural concept, especially since in the early post-divorce periods she struggles with a society that views her as incompetent in maintaining her family and home, This is consistent with the study of Abu Darwish et al. (2016). LIMITATION AND STUDY FORWARD As the researchers confined this study to a specific geographic location, they suggested other segments of society and a much bigger sample size to generalize the study results. ACKNOWLEDGEMENT ACKNOWLEDGEMENT The authors thank the research participants for taking part in this study. 41 \| Visit HSSR at https://mgesjournals.com/hssr/ CONFLICT OF INTEREST AND ETHICAL STANDARDS The authors declare that they have no conflicting interests. REFERENCES 1. Abdullah, N. (2018). Psychological Health among a Sample of Divorced Women in the Iraqi Governorate of Halabja: an Islamic view. Journal of Arts, Literature, Humanities, and Sociology: Emirates College of Educational Sciences, 24(1), 169 - 186. 2. Abu Darwish, M., Abu Tayeh, A., Al-Hrob, A., Al-Tarawni, I. (2016). Divorced and Widow Women's Traits in the Governorates of Southern Jordan and the Problems They Face. Journal of Studies and Research: the University of Djelfa, 23(1), 274 - 289. y j , ( ), 3. Al-Ali, N., Al-Omran, J. (2021). Psychological Resilience and its Relationship to Methods of Coping with Pressures among Divorced Bahraini Women. Egyptian Journal of Psychological Studies: Egyptian Association for Psychological Studies, 31(111), 471-506. y g 4. Alawneh, A. (2019). Divorce in Palestinian Society and its Relationship to Some Variables from Divorced Women's Point of View in the City of Ramallah during the Period (2010-2019): a critical field study. Journal of Human and Social Sciences 3(10), 29 - 60. Humanities & Social Sciences Reviews eISSN: 2395-6518, Vol 10, No 2, 2022, pp 38-42 Humanities & Social Sciences Reviews eISSN: 2395-6518, Vol 10, No 2, 2022, pp 38-42 Humanities & Social Sciences Reviews eISSN: 2395-6518, Vol 10, No 2, 2022, pp 38-42 5. Al-Badayna, D., Al-Qutaitat, M. (2018). Problems and Pressures Faced by Divorced Women before Entering Jordan. Journal of Studies and Research: the University of Djelfa, 33(2), 463-483 6. Al-Kubaisi, A., Al-Obaidi, A. (2015). Psychological Pain among Faculty Members at the University of Anbar. Anbar University. Journal of Human Sciences: Anbar University 2(1), 407 - 440. 7. Bawih, N. (2017). Quality of Divorced Women's Life: An analytical study. Journal of Human and Social Sciences: Kasdi Merbah University 30(1), 205-272. y ( ) Ben-Zur, H. (2012). Loneliness, optimism, and well-being among married, divorced, and wido individuals. The Journal of Psychology, 146(1-2), 23-36. https://doi.org/10.4324/9781315873367 9. Dykstra, P. A., & Fokkema, T. (2007). Social and emotional loneliness among divorced and married men and women: Comparing the deficit and cognitive perspectives. Basic and applied social psychology, 29(1), 1-12. https://doi.org/10.1080/01973530701330843 10. Knöpfli, B., Cullati, S., Courvoisier, D. S., Burton-Jeangros, C., & Perrig-Chiello, P. (2016). Marital breakup in later adulthood and self-rated health: a cross-sectional survey in Switzerland. International journal of public health, 61(3), 357-366. doi:10.1111/j.1741-3737.2000.01269.x p ( ) j 11. Nasser A., Rimawi O. (2022). Existential Vacuum and its Relationship with Family-based Support among Battered Women. Social Science and Humanities Journal, 6 (1), 2524- 2530. 12. Palestinian Central Bureau of Statistics (2020). Indicators of Marriage and Divorce in Palestine, date: 12-09-2021. https://2u.pw/z3KVG p p 13. Shear, K., Frank, E., Houck, P. R., & Reynolds, C. F. (2005). Treatment of complicated grief: a randomized controlled trial. Jama, 293(21), 2601-2608. https://doi.org/10.1007/978-3-319-07109-1_15 14. Van Tilburg, T., Aartsen, M., & van der Pas, S. (2015). Loneliness after divorce: A cohort comparison among Dutch young-old adults. European Sociological Review, 31(3), 243-252. https://doi.org/10.1093/esr/jcu086 y g p g j 15. Zafar, N., & Kausar, R. (2014). Emotional and Social Problems in Divorced and Married Women. FWU Journal of Social Sciences, 8(1).27-50. 42 \| Visit HSSR at https://mgesjournals.com/hssr/
https://openalex.org/W2556936510	https://www.nature.com/articles/srep37161.pdf	English	null	Hydrogen-bond potential for ice VIII-X phase transition	Scientific reports	2,016	cc-by	8,144	Hydrogen-bond potential for ice VIII-X phase transition OPEN Xi Zhang1, Shun Chen2 & Jichen Li2 Repulsive force between the O-H bonding electrons and the O:H nonbonding pair within hydrogen bond (O-H:O) is an often overlooked interaction which dictates the extraordinary recoverability and sensitivity of water and ice. Here, we present a potential model for this hidden force opposing ice compression of ice VIII-X phase transition based on the density functional theory (DFT) and neutron scattering observations. We consider the H-O bond covalent force, the O:H nonbond dispersion force, and the hidden force to approach equilibrium under compression. Due to the charge polarization within the O:H-O bond, the curvatures of the H-O bond and the O:H nonbond potentials show opposite sign before transition, resulting in the asymmetric relaxation of H-O and O:H (O:H contraction and H-O elongation) and the H+ proton centralization towards phase X. When cross the VIII-X phase boundary, both H-O and O:H contract slightly. The potential model reproduces the VIII-X phase transition as observed in experiment. Development of the potential model may provide a choice for further calculations of water anomalies. received: 31 May 2016 accepted: 25 October 2016 Published: 14 November 2016 The physical origin and theoretical reproduction of the measured anomalies of water and ice remains a great chal- lenge1–9. For example, under compression of the O:H-O bond, the H-O covalent bond becomes longer while the total O–O distance is shortened, leading to the proton centring in the O–O of ice VIII-X phase transition2,10–14. The behaviour of the proton is so strange that thermal fluctuation15 and quantum effect of nuclei16 were both con- sidered contributing to the ambiguous behaviour. Unlike other materials whose phonons were harden by external pressure, ice-VIII demonstrated the anomalous softening of the H-O vibration mode at frequency greater than 3000 cm−1 while stiffening of the O:H vibration mode at frequency lower than 400 cm−1 17–20. Ice melts under compression and freezes again when the pressure is relieved, evidencing extraordinary recoverability of O:H-O bond21. Hydrogen bond interaction potential, typical double-well potentials of the symmetrical22 and the asym- metrical23 forms, is still under debate. An accurate description of the H-bonding between water-water molecules is widely recognised as the crucial factor in the understanding of water anomalies9,24–26. Progresses has been made by considering the flexibility of the covalent bonding in water/ice in models such as the flexible-SPC27 and TIP4PF28. Kumagai et al. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 31 May 2016 accepted: 25 October 2016 Published: 14 November 2016 Scientific Reports \| 6:37161 \| DOI: 10.1038/srep37161 Hydrogen-bond potential for ice VIII-X phase transition OPEN have pro- posed a flexible KKY potential which has three separate terms: VHO, VHH and VOO in order to account the intra- and inter-molecular interactions separately11 and adopted by our previous MD calculation29. However, since those models have not distinguished O–O linked by O:H-O or not, the reproduction of the proton centring of ice VIII-X has seldom been reported. Ab initio MD (AIMD) enabled people to use the ab initio-derived force field, though time-expensive. Bernasconi et al.15,30 reproduced the ice VIII/VII/X phase transition by AIMD-derived lattice relaxation and infrared spectra. Sugimura et al.9,11 determined the intermediate structure during the ice VII/X phase transition using AIMD. Katoh et al.31,32 confirmed the fast protonic diffusion coefficient at high tem- perature molecular phase of ice VII. However, previous researches mostly focused on the structure determination or proton transferring, while the mechanism of the proton centring and the cooperative relaxation of the O:H and H-O are not fully understood. A potential model for time-saving calculations of the proton centring in the ice VIII/X phase transition is still lacking.i p g Recent progresses find that the hidden force33, i.e. the repulsive force between the H-O bonding electrons and the O:H nonbonding pair within a O:H-O bond, should play a significant role in various anomalies of water and ice, such as extraordinary recoverability, skin lubricity, etc.33–36. For example, in the temperature range from 250 K to 277 K, H-O covalent bond exhibit the normal thermal expansion, while O:H nonbond shrinks due to the reducing repulsive force, leading to the contraction of the total O–O distance34,36. At the skin of ice, due to 1Institute of Nanosurface Science and Engineering & Guangdong Provincial Key Laboratory of Micro/Nano Optomechatronics Engineering, Shenzhen University, Guangdong, 518060, China. 2School of Physics and Astronomy, the University of Manchester, Manchester, M13 9PL, UK. Correspondence and requests for materials should be addressed to X.Z. (email: zh0005xi@szu.edu.cn) or J.L. (email: j.c.li@manchester.ac.uk) Scientific Reports \| 6:37161 \| DOI: 10.1038/srep37161 1 1 www.nature.com/scientificreports/ Figure 1. (a) Hidden force model for O: H-O bond. xH and xC are the O atomic position counting from x = 0 (H atom). fOO is the hidden repulsive force. fP is the external compression force. fH and fC are the recovery force of O:H and O-H, in value of slope of the potential curves (blue dash lines) of VH and VC. Star represents the curvature turning point. Hydrogen-bond potential for ice VIII-X phase transition OPEN (b) Two stages of O:H-O relaxation under compression. Stage I (ice VIII): A small external force compresses the O:H at first since O:H interaction is weaker than O-H. foo is then increased to extend O-H. Stage II (ice X): under a large external force, fH goes across the curvature turning point, leading to the shrinks of both O:H and O-H. Figure 1. (a) Hidden force model for O: H-O bond. xH and xC are the O atomic position counting from x = 0 (H atom). fOO is the hidden repulsive force. fP is the external compression force. fH and fC are the recovery force of O:H and O-H, in value of slope of the potential curves (blue dash lines) of VH and VC. Star represents the curvature turning point. (b) Two stages of O:H-O relaxation under compression. Stage I (ice VIII): A small external force compresses the O:H at first since O:H interaction is weaker than O-H. foo is then increased to extend O-H. Stage II (ice X): under a large external force, fH goes across the curvature turning point, leading to the shrinks of both O:H and O-H. the decrease of the molecular coordination, H-O covalence bond contracts spontaneously to lower the cohesive energy, and hence O:H nonbond is polarized, leading to the surface lubricity35,37. This force is beyond the con- ventional intra- and inter-molecular interactions but depending on the existence of the O:H-O link, i.e. if there is no O:H-O bond like between two O atoms in difference sublattice in ice VIII, hidden force (3 times stronger than Van de Vaals force) interaction would not show38. However, it has still seldom reported to address the hidden force of O:H-O bond in a H-bond model, although the force plays a significant role in altering the properties of water and ice. In this paper, in order to investigate the strange behaviour of proton centring in phase VIII-X transition, we clarified a hidden force model considering the quantum interactions between electron clouds of covalent bond and nonbond and reproduced the anomalous behaviour of proton in ice VIII-X transition. Potential Model Proposed p Based on our extensive investigation of water anomalies21,34–36, we proposed hidden force model for dynamics of the “O: H-O” bond under external pressures, as shown in Fig. 1a. In the model, “O: H-O” bond is similar to be connected by three “non-harmonic converted to anharmonic springs”. Black spring represents covalent bonding of H-O (denoted as subscript C); Grey spring represents van der Waals interaction between lone pair (blue cir- cles) and proton of O:H (denoted as subscript H); Blue spring represents repulsive interaction between bonding electron pair and lone pair (denoted as subscript OO, but the interaction is not the conventional O–O coulomb repulsion). A water molecule is surrounded by four nearest neighbours (Pauling’s ice rule). Four identical O: H-O forms a tetrahedron as the basic unit of water structure (Supplementary Fig. S1). The bond length (x), forces (f) and potential(V) of O:H, H-O and O–O need to reach to a balance under external force (fp). Figure 1b shows two stages of O:H-O relaxation under compression.ih g p In stage I, a small external force compresses the O:H at first since O:H interaction is weaker than O-H. The repulsive foo then is increased by the contraction of O…O, leading to H-O extension. In stage II, under a large fp, fH goes across the curvature turning point, leading to the shrinks of both O:H and O-H. Intermediate phases exist during the transition where proton disordered symmetrization occurs with delocalized proton in a rela- tively broad potential well. The delocalization of proton (or proton-transfer) may attributed to quantum effect of nuclei16,39, or to thermal fluctuations obtained by AIMD15. In this work, we focus on the proton centring due to electron interactions within the O:H-O bond. Without external force, taking centre proton as reference, three springs can reach to equilibrium with their forces along the directions as indicated in Fig. 1a. Due to the foo, both O atoms are pushed away a little from their lowest-energy positions in the potentials of isolated bonds. Thus, the initial potential recovery forces fC and fH will both point inward to center proton; while repulsive force fOO will point outward. Potential Model Proposed Supposing ≅ + x x x OO H C, at equilibrium, total forces (force values) added on O atoms should both be zero:     + − = − + = f x x f x f x f x x ( ) ( ) 0 ( ) ( ) 0 (1) OO C H C C H H OO C H 0 0 0 0 0 0 (1) Scientific Reports \| 6:37161 \| DOI: 10.1038/srep37161 2 www.nature.com/scientificreports/ where, xC0 and xH0 (length values in positive numbers) are the equilibrium position of O atoms counting form x = 0. Thus, in equilibrium without external force, where, xC0 and xH0 (length values in positive numbers) are the equilibrium position of O atoms counting form x = 0. Thus, in equilibrium without external force, = f x f x ( ) ( ) (2) C C H H 0 0 = f x f x ( ) ( ) C C H H 0 0 (2) Considering an identical force fp at both sides, the forces satisfy: Considering an identical force fp at both sides, the forces satisfy:     + − − = + − + = f x x f x f f f x f x x ( ) ( ) 0 ( ) ( ) 0 OO C H C C p p H H OO H C if δ δ δ δ = + = + + = + x x x x f x f x x x x x , ( ) ( ) (3) C C H H C C H H C H C H 0 0 0 0 (3) Supposing the displacement δx is small enough to consider k as a constant, since = = − ∂ ∂ ∂ ∂ k f x V x 2 2 , combining eq. (2) and eq. (3), we can get: δ δ = k k x x (4) C H C H δ δ = k k x x C H C H (4) Eq. (4) tells us: i) The values of δxC and δxH should be determined by the potential forms of VH and VC. Since O:H bond is much longer and weaker than H-O bond, VH is more flat (smaller curvature) than VC, i.e. \|kC\| >\| kH\|. Thus, the value of δxH is always larger than δxC. Potential Model Proposed Both the work of fP and the change of VC provide energy for increase of VH and work of fOO: i) Stage I (ice VIII): in quasi-static process, external force contracts the O:H bond but extends the H-O bond with the total O–O length shortened, when kC ∙ kH < 0. Both the work of fP and the change of VC provide energy for increase of VH and work of fOO: ∫ ∫ ∫ δ δ δ δ δ δ ⋅ + + + = + + + + δ δ δ f x x V x x dr V x x x dr V x x dr ( ) ( ) ( ) ( ) (5) P C H x C C C x OO OO C H x H H H 0 0 0 C OO H (5) ii) Stage II (ice X): external force is strong enough to make xH shrink over the curvature turning point (star in Fig. 1) and kC ∙ kH > 0. External force contract both segments, pushing O atoms back towards the ener- gy-lowest positions, compressing all the three springs. In this case, fP and energy increases for both segments provides the sharp increase of the VOO: ii) Stage II (ice X): external force is strong enough to make xH shrink over the curvature turning point (star in Fig. 1) and kC ∙ kH > 0. External force contract both segments, pushing O atoms back towards the ener- gy-lowest positions, compressing all the three springs. In this case, fP and energy increases for both segments provides the sharp increase of the VOO: ii) Stage II (ice X): external force is strong enough to make xH shrink over the curvature turning point (star in Fig. 1) and kC ∙ kH > 0. External force contract both segments, pushing O atoms back towards the ener- gy-lowest positions, compressing all the three springs. Potential Model Proposed i) The values of δxC and δxH should be determined by the potential forms of VH and VC. Since O:H bond is much longer and weaker than H-O bond, VH is more flat (smaller curvature) than VC, i.e. \|kC\| >\| kH\|. Thus, the value of δxH is always larger than δxC. H y g C ) If δxC and δxH have opposite signs, i.e. xC and xH do not both increase or decrease together, kH and kC must have opposite sign with each other, which means the curvatures of potentials are opposite, as shown in Fig. 1a ii) If δxC and δxH have opposite signs, i.e. xC and xH do not both increase or decrease together, kH and kC must have opposite sign with each other, which means the curvatures of potentials are opposite, as shown in Fig. 1a. iii) If δxC and δxH have same signs, i.e. xC and xH both increase or decrease together, kH and kC must have the same sign. ii) If δxC and δxH have opposite signs, i.e. xC and xH do not both increase or decrease together, kH and kC must have opposite sign with each other, which means the curvatures of potentials are opposite, as shown in Fig. 1a. iii) If δxC and δxH have same signs, i.e. xC and xH both increase or decrease together, kH and kC must have the pp g , p pp , g i) If δxC and δxH have same signs, i.e. xC and xH both increase or decrease together, kH and kC must have the same sign. iii) If δxC and δxH have same signs, i.e. xC and xH both increase or decrease together, kH and kC must have same sign. Due to the charge polarization within the O:H-O bond, the curvature of the H-O potential has the opposite sign with O:H potential in the low-pressure region, leading to the contraction of O:H bond but extension of H-O bond and the proton centring. Hence, for O: H-O bond relaxation under external fp, there are two stages as shown in Fig. 1b: i) Stage I (ice VIII): in quasi-static process, external force contracts the O:H bond but extends the H-O bond with the total O–O length shortened, when kC ∙ kH < 0. Potential Model Proposed (b) Force constants of O-H, O:H, O–O in O:H-O bonding and O–O’ without bonding. www.nature.com/scientificreports/ Figure 2. (a) Structure of ice VIII. Two nested sublattices connected by green and purple lines respectively are shown. The oxygen atoms in the same sublattice were denoted as O and in the other sublattice as O’. Deformation electron density was plotted as blue isosurface. (b) DFT-derived bond length of O:H(xH) and O-H(xC) versus pressure. The changing trend of xH and xC falls into two stages. Figure 2. (a) Structure of ice VIII. Two nested sublattices connected by green and purple lines respectively are shown. The oxygen atoms in the same sublattice were denoted as O and in the other sublattice as O’. Deformation electron density was plotted as blue isosurface. (b) DFT-derived bond length of O:H(xH) and O-H(xC) versus pressure. The changing trend of xH and xC falls into two stages. Figure 3. (a) Phonon spectra of ice VIII measured using inelastic neutron scattering and calculated uing DFT. (b) Force constants of O-H, O:H, O–O in O:H-O bonding and O–O’ without bonding. Figure 3. (a) Phonon spectra of ice VIII measured using inelastic neutron scattering and calculated uing DFT. (b) Force constants of O-H, O:H, O–O in O:H-O bonding and O–O’ without bonding. Ice VIII has a cubic structure containing two sets of interpenetrated ice sub-lattices as shown in Fig. 2a. Hence, each water molecule has 8 nearest neighbours, 4 of them are H-bonded and the others are not, at almost the same O–O distance. The oxygen atoms in the same sublattice were denoted as O and in the other sublattice as O’. Blue isosurface of deformation electron density (density difference before and after bonding,) indicates the position of the bonding electron pair and the O:H nonbond pair. The charge distribution in a water molecule was polarized in the tetrahedral directions by the O:H-O bond. Figure 2b shows the pressure-dependent bond length of O:H(xH) and O-H(xC). Proton centring is obtained as pressure increases, in accord with the experimental observation2,12 and other DFT calculation33. The changing trends of xH and xC verify the two stages of O:H-O under compression. xH shrinks and xC extends in stage I. As pressure increases, xH and xC both shrink in stage II like normal material.hh Ice VIII has a cubic structure containing two sets of interpenetrated ice sub-lattices as shown in Fig. 2a. Potential Model Proposed Hence, each water molecule has 8 nearest neighbours, 4 of them are H-bonded and the others are not, at almost the same O–O distance. The oxygen atoms in the same sublattice were denoted as O and in the other sublattice as O’. Blue isosurface of deformation electron density (density difference before and after bonding,) indicates the position of the bonding electron pair and the O:H nonbond pair. The charge distribution in a water molecule was polarized in the tetrahedral directions by the O:H-O bond. Figure 2b shows the pressure-dependent bond length of O:H(xH) and O-H(xC). Proton centring is obtained as pressure increases, in accord with the experimental observation2,12 and other DFT calculation33. The changing trends of xH and xC verify the two stages of O:H-O under compression. xH shrinks and xC extends in stage I. As pressure increases, xH and xC both shrink in stage II like normal material. The DFT calculation can reproduce phonon spectrum for Ice VIII as shown in Fig. 3a. The simulation repro- duces the main features of inelastic neutron scattering (INS) spectrum24, such as the three peaks in the trans- lational region (<50 meV) and the two sharp peaks of the librational region at about 65 meV. The small peak in the right hand side of librational region at about 100 meV was not shown in the INS spectra due to large Debye-Waller effect in the measured spectra which smeared the spectra dramatically at higher energy transfer. Force constants kC, kH and kOO were also obtained from the calculation of phonon spectrum of the ice VIII and are plotted as a function of pressure in Fig. 3b. Apart from the trend for the kC, kB to merge at xOO = 2.3 Å the force constants between the H-bonded O–O atoms kOO also shows a rapid increase under the external pressure. For the non H-bonded O…O’ atoms between the two sub-lattices of ice VIII, a new force constant, kOO’, was obtained which remains at small values around kH. This implies that the O–O interaction only becomes relevant The DFT calculation can reproduce phonon spectrum for Ice VIII as shown in Fig. 3a. The simulation repro- duces the main features of inelastic neutron scattering (INS) spectrum24, such as the three peaks in the trans- lational region (<50 meV) and the two sharp peaks of the librational region at about 65 meV. Potential Model Proposed In this case, fP and energy increases for both segments provides the sharp increase of the VOO: ∫ ∫ ∫ δ δ δ δ δ δ ⋅ + + + + + = + + δ δ δ f x x V x x dr V x x dr V x x x dr ( ) ( ) ( ) ( ) (6) P C H x H H H x C C C x OO OO C H 0 0 0 H C OO (6) Calculations and Verifications. To verify and quantify our potential model, we performed density func- tional theory (DFT) calculation using CASTEP. The generalized gradient approximation (GGA) function of PW91, HCTH and RPBE were used to describe the exchange–correlation effects (results show little differences among the different functions and hence the PW91 results were presented here). The van der Waals force was also examed by adopting the DFT-D, its effect to the stability of the ice structures has reported, a small effect of red-shifts (~2 meV) for the main peaks of the vibrational spectra was observed. The phonon spectra were calcu- lated using the CASTEP module with finite displacement method. The force constants produced by the CASTEP for the phonon spectra calculations were obtained from the output files. The force for the atom i, fi (= dE/dri), is simply the derivative of the total energy E. By applying a second small displacement for the atom j, the force constant kij (= dU/dridrj) is obtained for the pair atoms i and j. A force constant matrix k for the unit cell is a 3 N× 3 N matrix (where N is total number of atoms in the unit cell) and were constructed based on above procedure. Scientific Reports \| 6:37161 \| DOI: 10.1038/srep37161 3 entificreports/ I VIII h bi i i f i d i b l i h i Fi 2 H Figure 2. (a) Structure of ice VIII. Two nested sublattices connected by green and purple lines respectively are shown. The oxygen atoms in the same sublattice were denoted as O and in the other sublattice as O’. Deformation electron density was plotted as blue isosurface. (b) DFT-derived bond length of O:H(xH) and O-H(xC) versus pressure. The changing trend of xH and xC falls into two stages. Figure 3. (a) Phonon spectra of ice VIII measured using inelastic neutron scattering and calculated uing DFT. Potential Model Proposed the electron clouds were accumulated in the line of O:H-O, leading to the increase of the repulsive force between bonding pair and nonbonding pair of O–O and the decrease of the interaction between O and O’. Potential Model Parametrization. Fitting from DFT-derived k-x results of stage I of P-dependent IceVIII Fig. 3b), we get the relations among ki and xi (I = H, C, OO) according to our potential model,       =       =    . −. − . . − . .    ( ) ( ) k k k a b a b a b x x x x x x 1 1 1 3 96125 8 91589 289 37672 325 26263 21 26174 67 04064 1 1 1 (7) H C OO H H C C OO oo H C OO H C OO (7) where we add negative sign on kH since DFT only take the positive square root value. Based on k-x curve, we fit the expression of f-x and V-x curves (Supplementary Eq. S1 and S2). The shapes of f-x and V-x are plotted in Fig. 4.h where we add negative sign on kH since DFT only take the positive square root value. Based on k-x curve, we fit the expression of f-x and V-x curves (Supplementary Eq. S1 and S2). The shapes of f-x and V-x are plotted in Fig. 4. In stage I, as fp increases, fH = fC should also increases according to Eq.(3). Thus, xH decreases while xC increases as indicated by black arrows in Fig. 4a. In stage II, when xH decreases to about 1.1 and reach to the curvature turning point, fH changes to decrease and xC will decreases back (as shown by blue arrows) to make fC = fH. The absolute value of repulsive force fOO always increases as distance xOO decreses. We focus on the parametrization of stage I in this work since it is more interesting than the normal compression contraction in stage II where we add negative sign on kH since DFT only take the positive square root value. Based on k-x curve, we fit the expression of f-x and V-x curves (Supplementary Eq. S1 and S2). The shapes of f-x and V-x are plotted in Fig. 4. Potential Model Proposed The small peak in the right hand side of librational region at about 100 meV was not shown in the INS spectra due to large Debye-Waller effect in the measured spectra which smeared the spectra dramatically at higher energy transfer. Force constants kC, kH and kOO were also obtained from the calculation of phonon spectrum of the ice VIII and are plotted as a function of pressure in Fig. 3b. Apart from the trend for the kC, kB to merge at xOO = 2.3 Å the force constants between the H-bonded O–O atoms kOO also shows a rapid increase under the external pressure. For the non H-bonded O…O’ atoms between the two sub-lattices of ice VIII, a new force constant, kOO’, was obtained which remains at small values around kH. This implies that the O–O interaction only becomes relevant Scientific Reports \| 6:37161 \| DOI: 10.1038/srep37161 4 www.nature.com/scientificreports/ Figure 4. The DFT-fitting curves of bond length x-dependent (a), fC and fH, (b), fOO, (c), VC and VH, and (d), VOO according to water model. Black arrows indicate the opposite relaxation in length of xC and xH in stage I; Blue arrows indicate the both decreasing in length of xC and xH in stage II. Figure 4. The DFT-fitting curves of bond length x-dependent (a), fC and fH, (b), fOO, (c), VC and VH, and (d), VOO according to water model. Black arrows indicate the opposite relaxation in length of xC and xH in stage I; Blue arrows indicate the both decreasing in length of xC and xH in stage II. when the two O atoms are linked by an H atom and hence this large value of kOO is not due to the simple coulomb interaction between O atoms. As shown by the isosurface in Fig. 2a, charge distribution was polarized by O:H-O bonding, i.e. the electron clouds were accumulated in the line of O:H-O, leading to the increase of the repulsive force between bonding pair and nonbonding pair of O–O and the decrease of the interaction between O and O’. when the two O atoms are linked by an H atom and hence this large value of kOO is not due to the simple coulomb interaction between O atoms. As shown by the isosurface in Fig. 2a, charge distribution was polarized by O:H-O bonding, i.e. Potential Model Proposed In stage I, as fp increases, fH = fC should also increases according to Eq.(3). Thus, xH decreases while xC increases as indicated by black arrows in Fig. 4a. In stage II, when xH decreases to about 1.1 and reach to the curvature t i i t f h t d d ill d b k ( h b bl ) t k f f Th h In stage I, as fp increases, fH = fC should also increases according to Eq.(3). Thus, xH decreases while xC increases as indicated by black arrows in Fig. 4a. In stage II, when xH decreases to about 1.1 and reach to the curvature turning point, fH changes to decrease and xC will decreases back (as shown by blue arrows) to make fC = fH. The absolute value of repulsive force fOO always increases as distance xOO decreses. We focus on the parametrization of stage I in this work since it is more interesting than the normal compression-contraction in stage II. g g p g Hence, for a given fP, there is only one set of solutions of xH and xC obtained in Fig. 5. Figure 5a shows that, for a given xH, fH = fC gets the xC. Then, Fig. 5b shows that, fOO can be calculated by xH + xC and fP is correlated with xH by fp = fOO−fH. Hence, we have obtained the fP-xH curve in Fig. 5b, from which a given external fP can determine the relaxation of xH and xC. Thus, the correlations among external compression force, the length relaxation of H-O and O:H bond, the forces of O-H, O:H and O:O are all determined. Figure 5b also indicates that, as fP increases, fOO rises much more significantly than fH. Thus, fOO is indeed a hidden force that should not be ignored. fOO gi y fHh fOO g Based on our potential model, foo, fH and fp are functions of xH and xC determined by Supplementary Eq. S1 and Fig. 4. We run the time-dependent dynamic process of external pressure on O:H-O bond in Fig. 6. The dynamic equation can be expressed as: Scientific Reports \| 6:37161 \| DOI: 10.1038/srep37161 5 www.nature.com/scientificreports/ ificreports/ Figure 5. (a) Correlation between xH and xC. (b) Correlation of xH, fP. fH, and fOO. Under external fp, the length and force relaxations inside the O:H-O bonding are all determined. Figure 6. Potential Model Proposed Time-dependent relaxation dynamics of O:H-O bond with the same initial lengths and different external forces. (a) xH relaxes with time and (b), xC relaxes with time under fp = 0.1, 0.5 and 1.5 eV/Å. The initial lengths are 1.9 and 0.98269 at 0 external force, which give initial energies for bonds to oscillate around the equbrillium point under pressures. xH and xC under fp = 0.5 and 1.5 eV/Å were taken average. Figure 5. (a) Correlation between xH and xC. (b) Correlation of xH, fP. fH, and fOO. Under external fp, the length and force relaxations inside the O:H-O bonding are all determined. Figure 5. (a) Correlation between xH and xC. (b) Correlation of xH, fP. fH, and fOO. Under external fp, the length and force relaxations inside the O:H-O bonding are all determined. Figure 6. Time-dependent relaxation dynamics of O:H-O bond with the same initial lengths and different external forces. (a) xH relaxes with time and (b), xC relaxes with time under fp = 0.1, 0.5 and 1.5 eV/Å. The initial lengths are 1.9 and 0.98269 at 0 external force, which give initial energies for bonds to oscillate around the equbrillium point under pressures. xH and xC under fp = 0.5 and 1.5 eV/Å were taken average. Figure 6. Time-dependent relaxation dynamics of O:H-O bond with the same initial lengths and different external forces. (a) xH relaxes with time and (b), xC relaxes with time under fp = 0.1, 0.5 and 1.5 eV/Å. The initial lengths are 1.9 and 0.98269 at 0 external force, which give initial energies for bonds to oscillate around the equbrillium point under pressures. xH and xC under fp = 0.5 and 1.5 eV/Å were taken average. µ µ      = \| \| −\| \| −\| \| = \| \| −\| \| −\| \| = . = . = = = = d x dt f f f d x dt f f f x x t v v t 1 9, 0 98269 ( 0) 0 ( 0) (8) H OO H P C OO C P H C H C 2 2 2 2 µ µ      = \| \| −\| \| −\| \| = \| \| −\| \| −\| \| = . = . Potential Model Proposed = = = = d x dt f f f d x dt f f f x x t v v t 1 9, 0 98269 ( 0) 0 ( 0) (8) H OO H P C OO C P H C H C 2 2 2 2 (8) The initial condition of xH and xC are the lengths (Å) without pressure. The partial differential equation is solved by 4th order Runge-Kutta method (Supplementary Method).f The initial condition of xH and xC are the lengths (Å) without pressure. The partial differential equation is solved by 4th order Runge-Kutta method (Supplementary Method).f Figure 6 shows the xH and xC relaxation with the same initial lenghs and different external forces under fp = 0.1, 0.5 and 1.5 eV/Å. The initial lengths are bond lengths at fp = 0, which give energies to atoms to oscillate. If the process is slow, i.e. initial coordinates are around the stable point, the oscillation will decrease. xH and xC under fp = 0.5 and 1.5 eV/Å were taken average as ∑x t t ( ( ))/ t C H 0 / . The results show that, as fp increases, xH decreases from 1.890 to 1.811 and to 1.645 Å in average, while xC increases from 0.983 to 0.992 and to 1.015 Å in average, agreeing well with proton centring obtained both in DFT calculation and experimental observation2,12. Figure 7 a and b show the time-dependent dynamics of O:H-O bond in the quasi-static process under fp from 0.1 to 0.5 eV/Å. The initial lengths at each fp were taken as the average bond lengths of the previous fp. Hence, bond length relaxes more stably with time. Figure 7c shows the average bond lengths obtained from the quasi-static states at different fp. The dynamic curves oscillated stably raise the reliability of the average lengths. Results show that as fp increases, O:H bond length decreases and H-O bond length increases, approaching to equal at about fp = 6.91 eV/Å. the Therefore, upon compression, in the equilibrium status of O:H-O, proton and oxygen shift both towards the other oxygen while proton shift a little to the weaker O:H part. Besides, O:H vibrates much slower than H-O bond due to its weaker force constant, as indicated by the frequency of the relax- ation curves. Scientific Reports \| 6:37161 \| DOI: 10.1038/srep37161 6 www.nature.com/scientificreports/ Figure 7. References 1. Goncharov, A. F., Struzhkin, V. V., Mao, H.-k. & Hemley, R. J. Raman Spectroscopy of Dense H2O and the Transition to Symmetri Hydrogen Bonds. Phys. Rev. Lett. 83, 1998 (1999). y g y , ( ) 2. Benoit, M., Marx, D. & Parrinello, M. Tunnelling and zero-point motion in high-pressure ice. Nature 392, 258–261 (1998). 3 F k J W M & O k T H MICROSCOPY Wh i d N 464 38 39 d i 10 1038/46 y g y 2. Benoit, M., Marx, D. & Parrinello, M. Tunnelling and zero-point motion in high-pressure ice. Nature 392, 258–261 (1998). 3 Frenken J W M & Oosterkamp T H MICROSCOPY When mica and water meet Nature 464 38–39 doi: 10 1038/464038 2. Benoit, M., Marx, D. & Parrinello, M. Tunnelling and zero-point motion in high-pressure ice. Nature 392, 258–261 (1998). 3. Frenken, J. W. M. & Oosterkamp, T. H. MICROSCOPY When mica and water meet. Nature 464, 38–39, doi: 10.1038/464038 ( ) , , , , g p g p , ( ) 3. Frenken, J. W. M. & Oosterkamp, T. H. MICROSCOPY When mica and water meet. Nature 464, 38–39, doi: 10.1038/464038 (2010). ( ) 4. Headrick, J. M. et al. Spectral signatures of hydrated proton vibrations in water clusters. Science 308, 1765–1769, doi: 10.1126/ science.1113094 (2005). 4. Headrick, J. M. et al. Spectral signatures of hydrated proton vibrations in water clusters. Science 308, 1765–1769, doi: 10.1126/ science.1113094 (2005).h 5. Gregory, J. K., Clary, D. C., Liu, K., Brown, M. G. & Saykally, R. J. The Water Dipole Moment in Water Clusters. Science 275, 814–817 doi: 10.1126/science.275.5301.814 (1997). 6. Bjerrum, N. Structure and properties of ice. Science 115, 385–390 (1952). j p p 7. Soper, A. K., Teixeira, J. & Head-Gordon, T. Is ambient water inhomogeneous on the nanometer-length scale? Proc. Natl. Acad. Sci USA. 107, E44–E44, doi: 10.1073/pnas.0912158107 (2010). 7. Soper, A. K., Teixeira, J. & Head-Gordon, T. Is ambient wate 7. Soper, A. K., Teixeira, J. & Head-Gordon, T. Is ambient water inhomogeneous on the n USA. 107, E44–E44, doi: 10.1073/pnas.0912158107 (2010). USA. 107, E44–E44, doi: 10.1073/pnas.0912158107 (2010). p 8. Zha, C.-S., Hemley, R. J., Gramsch, S. A., Mao, H.-k. & Bassett, W. A. Optical study of H2O ice to 120GPa: Dielectric function, molecular polarizability, and equation of state J. Chem. Phys. 126, 074506, doi: 10.1063/1.2463773 (2007). p y q y 9. Potential Model Proposed Time-dependent dynamics of O:H-O bond in the quasi-static process, with different initial bond lengths. (a) xH relaxes with time and (b), xC relaxes with time under fp from 0.1 to 0.5 eV/Å. The initial lengths at each fp were taken as the average bond lengths of the previous fp. Hence, the oscillations of bonds were largely reduced. (c), the average bond lengths derived at fp from 0.1 to 6.91 eV/Å. Proton centring occurs near fp = 6.91 eV/Å. Figure 7. Time-dependent dynamics of O:H-O bond in the quasi-static process, with different initial bond lengths. (a) xH relaxes with time and (b), xC relaxes with time under fp from 0.1 to 0.5 eV/Å. The initial lengths at each fp were taken as the average bond lengths of the previous fp. Hence, the oscillations of bonds were largely reduced. (c), the average bond lengths derived at fp from 0.1 to 6.91 eV/Å. Proton centring occurs near fp = 6.91 eV/Å. The potential model of O:H-O can be further applied to water molecules and crystal structures considering the interactions of H—H and O–O’. VOO’ follows the weak dispersion interaction as indicated by the force con- stant developed by DFT in Fig. 3b, since electron cloud distributes mostly on the line of O:H-O bond as shown in Fig. 2a. Current potential model provides an efficient tool to investigate bond length relaxation, time-dependent proton dynamics, mass density and intermediate structure determination of dense ice phases. Conclusion DFT and neutron scattering observations have enabled a O:H-O bond potential for the ice VIII-X phase tran- sition under compression. Before transition, H-O undergoes elongation and the O:H compression because of the O:H-O bond segmental disparity and O-O repulsion; after transition, both H-O and O:H undergoes slight contraction because of the excessive compression. The effectiveness of current potential model was illustrated when dealing with H-bonded systems under pressure. More importantly, this additional force could provide the necessary mechanism to explain a range of water anomalies, for instance the complex phase diagram, i.e. its morphism, this is probably because the hydrogen bonds become very easy to buckle under the extra strong O–O interaction if the O:H-O is not in straight line. This hidden force, fOO has often been overlooked experimentally in the past, perhaps because it is easily concealed by the fH and fC. The hidden force potential model reproduced the contraction of O:H bond and extension of H-O bond and the proton centralization under compression. The potential model to develop further can serve as a key for theoretical reproduction of the O:H-O bond asymmetric relaxation under external stimuli. References Benoit, M., Romero, A. H. & Marx, D. Reas g g y g g y 31. Cavazzoni, C. et al. Superionic and Metallic States of Water and Ammonia at Giant Planet Conditions. Science 283, 44–46, doi: 10.1126/science.283.5398.44 (1999).f 2. Eriko Katoh, H. Y., H. Fujihisa, M. Sakashita & K. Aoki. Protonic Diffusion in High-Pressure Ice VII. Science 295, 1264–1266, doi 10.1126/science.1067746 (2002).h 33. Sun, C. Q., Zhang, X. & Zheng, W. The hidden force opposing ice compression. Chemical Science 3, 1455–1460 (2012). h 4. Huang, Y. et al. Hydrogen-bond relaxation dynamics: Resolving mysteries of water ice. Coord. Chem. Rev. 285, 109–165, doi 10.1016/j.ccr.2014.10.003 (2015).hh 5. Zhang, X. et al. Water Nanodroplet Thermodynamics: Quasi-Solid Phase-Boundary Dispersivity. The Journal of Physical Chemistry B 119, 5265–5269, 10.1021/acs.jpcb.5b00773 (2015). jp 36. Zhang, X. et al. Water’s phase diagram: From the notion of thermodynamics to hydrogen-bond cooperativity. Prog. Solid State Chem. 43, 71–81, doi: 10.1016/j.progsolidstchem.2015.03.001 (2015). 36. Zhang, X. et al. Water’s phase diagram: From the notion of thermodynamics to hydrogen-bond cooperativity. Prog. Solid State Chem. 43, 71–81, doi: 10.1016/j.progsolidstchem.2015.03.001 (2015). j p g ( ) 7. Zhang, X., Huang, Y., Ma, Z., Niu, L. & Sun, C. Q. From ice superlubricity to quantum friction: Electronic repulsivity and phononic elasticity. Friction 3, 294–319, 10.1007/s40544-015-0097-z (2015).h y 38. Shun, C., Zhiping, X. & Jichen, L. The observation of oxygen—oxygen interactions in ice. New Journal of Physics 18, 023052, (2016). 39 H C P & R í R P th i t l i l ti f i VII P d t t ff t Ch Ph 461 125 136 d i 38. Shun, C., Zhiping, X. & Jichen, L. The observation of oxygen—oxygen interactions in ice. New Journal of Physics 18, 023052, (2016). 39. Herrero, C. P. & Ramírez, R. Path-integral simulation of ice VII: Pressure and temperature effects. Chem. Phys. 461, 125–136, doi: 38. Shun, C., Zhiping, X. & Jichen, L. The observation of oxygen—oxygen interactions in ice. New Journal of Physics 18, 023052, (2016). 39. Herrero, C. P. & Ramírez, R. Path-integral simulation of ice VII: Pressure and temperature effects. Chem. Phys. 461, 125–136, doi: 10.1016/j.chemphys.2015.09.011 (2015). 38. Shun, C., Zhiping, X. & Jichen, L. The observation of oxygen—oxygen interactions in ice. New Journal of Physics 18, 023052, (2016). 39. Herrero, C. P. & Ramírez, R. Path-integral simulation of ice VII: Pressure and temperature effects. Chem. Phys. 461, 125–136, doi: 10.1016/j.chemphys.2015.09.011 (2015). Acknowledgements g We would like to thank ISIS of Rutherford-Appleton Laboratory for the access of neutron scattering facilities. The authors thank the support of the National Nature Science Foundation of China (No. 51605306), NSF of Guangdong (No. 2016A030310060), Guangdong Innovation Youth Fund (No. 2015KQNCX144), NSF of SZU (No. 201528, No. 827000131), and Shenzhen foundation fund (No. JCYJ20160427105015701). We would like to thank ISIS of Rutherford-Appleton Laboratory for the access of neutron scattering facilities. The authors thank the support of the National Nature Science Foundation of China (No. 51605306), NSF of Guangdong (No. 2016A030310060), Guangdong Innovation Youth Fund (No. 2015KQNCX144), NSF of SZU (No. 201528, No. 827000131), and Shenzhen foundation fund (No. JCYJ20160427105015701). Author Contributions X.Z. proposed the theoretical model. X.Z. and S.C. performed the calculation. S.C. and J.C.L. did the inelastic neutron scattering measurements. X.Z. and J.C.L wrote the main manuscript text. All authors reviewed the manuscript. References Iitaka, T., Fukui, H., Li, Z., Hiraoka, N. & Irifune, T. Pressure-induced dissociation of water molecules in ice VII. Sci Rep 5, 12551 doi: 10.1038/srep12551 (2015).l p 0. Loubeyre, P., LeToullec, R., Wolanin, E., Hanfland, M. & Husermann, D. Modulated phases and proton centring in ice observed by X-ray diffraction up to 170 GPa. Nature 397, 503–506 (1999). yf p ( ) 11. Sugimura, E. et al. Compression of H2O ice to 126 GPa and implications for hydrogen-bond symmetrization: Synchrotron x-ray diffraction measurements and density-functional calculations. Physical Review B 77, 214103 (2008).i f 2. Kang, D., Dai, J., Hou, Y. & Yuan, J. Structure and vibrational spectra of small water clusters from first principles simulations. J Chem. Phys. 133, 014302 (2010).h y 13. Holzapfel, W. B. On the Symmetry of the Hydrogen Bonds in Ice VII. The Journal of Chemical Physics 56, 712–715 (1972).h y y y gh f y 14. Bhatt, H. et al. Hydrogen Bond Symmetrization in Glycinium Oxalate under Pressure. The Journal of Physical Chemistry B 120, 851–859, 10.1021/acs.jpcb.5b11507 (2016). jp 5. Bernasconi, M., Silvestrelli, P. L. & Parrinello, M. Ab Initio Infrared Absorption Study of the Hydrogen-Bond Symmetrization in Ice Phys. Rev. Lett. 81, 1235–1238 (1998). y 16. Meng, X. et al. Direct visualization of concerted proton tunnelling in a water nanocluster. Nat Phys 11, 235–239, doi: 10.1038/ nphys3225 (2015).f 17. Yoshimura, Y., Stewart, S. T., Somayazulu, M., Mao, H. & Hemley, R. J. High-pressure x-ray diffraction and Raman spectroscopy of ice VIII. J. Chem. Phys. 124, 024502, doi: 10.1063/1.2140277 (2006). J y , ( ) 8. Pruzan, P. et al. Phase diagram of ice in the VII–VIII–X domain. Vibrational and structural data for stronglycompressed ice VIII. J Raman Spectrosc. 34, 591–610 (2003). Scientific Reports \| 6:37161 \| DOI: 10.1038/srep37161 7 www.nature.com/scientificreports/ 19. Song, M., Yamawaki, H., Fujihisa, H., Sakashita, M. & Aoki, K. Infrared absorption study of Fermi resonance and hydrogen-b symmetrization of ice up to 141 GPa. Physical Review B 60, 12644 (1999). 19. Song, M., Yamawaki, H., Fujihisa, H., Sakashita, M. & Aoki, K. Infrared absorption study of Fermi resonance and hydrogen bond symmetrization of ice up to 141 GPa. Physical Review B 60, 12644 (1999). symmetrization of ice up to 141 GPa. Physical Review B 60, 12644 (1999). l d d d d l b d h h ( ) y p y ( ) 20. Santra, B. et al. References Hydrogen Bonds and van der Waals Forces in Ice at Ambient and High Pressures. Phys. Rev. Lett. 107, 185701 (2 21. Zhang, X. et al. Ice Regelation: Hydrogen-bond extraordinary recoverability and water quas Scientific Reports 5, 13655, doi: 10.1038/srep13655 (2015).h 1. Zhang, X. et al. Ice Regelation: Hydrogen-bond extraordinary recoverability and water quasisolid-phase-boundary dispersivity Scientific Reports 5, 13655, doi: 10.1038/srep13655 (2015).h Scientific Reports 5, 13655, doi: 10.1038/srep13655 (2015).h fi p p ( ) 22. Teixeira, J. High-pressure physics - The double identity of ice X. Nature 392, 232–233 (1998). fi p p 22. Teixeira, J. High-pressure physics - The double identity of ice X. Nature 392, 232–233 (1998). g p p yh y 3. Kumagai, T. Direct observation and control of hydrogen-bond dynamics using low-temperature scanning tunneling microscopy Prog. Surf. Sci. 90, 239–291, doi: 10.1016/j.progsurf.2015.04.001 (2015). h 23. Kumagai, T. Direct observation and control of hydrogen-bond dynamics using low-tempera Prog. Surf. Sci. 90, 239–291, doi: 10.1016/j.progsurf.2015.04.001 (2015). g f j p g 24. Li, J. & Ross, D. K. Evidence for two kinds of hydrogen bond in ice. Nature 365, 327–329 (1993). y g 25. Zhang, L.-J., Wang, J., Luo, Y., Fang, H.-P. & Hu, J. A novel water layer structure inside nanobubbl Science and Techniques 25, 060503, doi: 10.13538/j.1001-8042/nst.25.060503 (2014).f 25. Zhang, L.-J., Wang, J., Luo, Y., Fang, H.-P. & Hu, J. A novel water layer structure inside nanobubbles at room temperature N Science and Techniques 25, 060503, doi: 10.13538/j.1001-8042/nst.25.060503 (2014).f 26. Gaffney, K. J., Ji, M., Odelius, M., Park, S. & Sun, Z. H-bond switching and ligand exchange dynamics in aqueous ionic solution. Chem. Phys. Lett. 504, 1–6, doi: 10.1016/j.cplett.2011.01.063 (2011). f Chem. Phys. Lett. 504, 1–6, doi: 10.1016/j.cplett.2011.01.063 (2 y j p ( ) 27. Toukan, K. & Rahman, A. Molecular-dynamics study of atomic motions in water. Physical Review B 31, 2643–2648 (1985). k l bl d l f l l d l f l d h h 28. Lawrence, C. P. & Skinner, J. L. Flexible TIP4P model for molecular dynamics simulation of liquid water. Chem. Phys. Lett 842–847, doi: 10.1016/S0009-2614(03)00526-8 (2003). 842–847, doi: 10.1016/S0009-2614(03)00526-8 (2003) 29. Burnham, C. J., Li, J. C. & Leslie, M. Molecular Dynamics Calculations for Ice Ih. The Journal of Physical Chemistry B 6192–6195, doi: 10.1021/jp9632596 (1997). jp 30. Benoit, M., Romero, A. H. & Marx, D. Reassigning Hydrogen-Bond Centering in Dense Ice. Phys. Rev. Lett. 89, 145501 (2002) 30. Additional Information upplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepihi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Zhang, X. et al. Hydrogen-bond potential for ice VIII-X phase transition. Sci. Rep. 6, 37161; doi: 10.1038/srep37161 (2016). How to cite this article: Zhang, X. et al. Hydrogen-bond potential for ice VIII-X phase transition. Sci. Rep. 6 37161; doi: 10.1038/srep37161 (2016). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. 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https://openalex.org/W3123163513	https://aura.abdn.ac.uk/bitstream/2164/15717/1/Schoumacher_et_al_SR_TheRostroCaudal_VoR.pdf	English	null	The rostro-caudal gradient in the prefrontal cortex and its modulation by subthalamic deep brain stimulation in Parkinson’s disease	Scientific reports	2,021	cc-by	12,177	www.nature.com/scientificreports www.nature.com/scientificreports The rostro‑caudal gradient in the prefrontal cortex and its modulation by subthalamic deep brain stimulation in Parkinson’s disease OPEN F. Konrad Schumacher1,2,3,4,10,11, Lena V. Schumacher1,3,5,10, Florian Amtage1,3,10,11, Andreas Horn6, Karl Egger2,3,10, Tobias Piroth1,7,10, Cornelius Weiller1,3,10,11, Björn O. Schelter8,11, Volker A. Coenen9,10,11 & Christoph P. Kaller1,2,3,10,11* Deep brain stimulation of the subthalamic nucleus (STN-DBS) alleviates motor symptoms in Parkinson’s disease (PD) but also affects the prefrontal cortex (PFC), potentially leading to cognitive side effects. The present study tested alterations within the rostro-caudal hierarchy of neural processing in the PFC induced by STN-DBS in PD. Granger-causality analyses of fast functional near- infrared spectroscopy (fNIRS) measurements were used to infer directed functional connectivity from intrinsic PFC activity in 24 PD patients treated with STN-DBS. Functional connectivity was assessed ON stimulation, in steady-state OFF stimulation and immediately after the stimulator was switched ON again. Results revealed that STN-DBS significantly enhanced the rostro-caudal hierarchical organization of the PFC in patients who had undergone implantation early in the course of the disease, whereas it attenuated the rostro-caudal hierarchy in late-implanted patients. Most crucially, this systematic network effect of STN-DBS was reproducible in the second ON stimulation measurement. Supplemental analyses demonstrated the significance of prefrontal networks for cognitive functions in patients and matched healthy controls. These findings show that the modulation of prefrontal functional networks by STN-DBS is dependent on the disease duration before DBS implantation and suggest a neurophysiological mechanism underlying the side effects on prefrontally-guided cognitive functions observed under STN-DBS. Many aspects of human behavior rely on the prefrontal cortex (PFC) and its interactions with a variety of other cortical and subcortical brain structures1. The PFC is directly connected to the subthalamic nucleus (STN)2–4, a prime target of deep brain stimulation (DBS) in Parkinson’s disease. Although STN-DBS is primarily used to target the motor circuit of cortex-basal ganglia interactions, changes of activity and behavior in associative loops have been widely observed, studied and described5–8. Specifically, STN-DBS intervenes in a complex network of cortico-subcortical pathways, providing a variety of potential routes to interfere with remote brain areas9. Given that STN-DBS is assumed to shift the balance between inhibitory and excitatory network activity to restore functionality of the diseased motor system10,11, currents spreading beyond the stimulation target can likely com- promise the balance in associative and limbic cortico-basal ganglia loops connected with the STN12. Although the 1Department of Neurology, Medical Center, University of Freiburg, Breisacher Str. 64, 79106 Freiburg, Germany. 2Department of Neuroradiology, Medical Center, University of Freiburg, Freiburg, Germany. Resultsh The analysis workflow, including the measurement protocol and illustration of fNIRS channel positions, is sum- marized in Fig. 1. The main analyses were conducted by means of two linear mixed-effects models; the first model investigated whether STN-DBS (compared ON vs. OFF) exerted an influence on the rostro-caudal connectivity in the lateral PFC, while the second model (additionally including data from the second ON measurement, cf. Figure 1A) served as an experimental validation of the main finding of a stimulation-induced alteration of the rostro-caudal connectivity. An overview of the fixed and random effects structures of Models 1 and 2 is sum- marized in Table 1. The concise report of the results below is complemented by a comprehensive report of the respective statistical indices underlying significant effects, estimates, and contrasts in the Supplementary Table S2. Additionally, control analyses including healthy adults (Supplementary Model S1, Supplementary Fig. S2), assess- ing the role of the rostro-caudal gradient for cognitive functions (Supplementary Model S2, Supplementary Fig. S3), and investigating the role of gray matter volume as a proxy of disease-induced cortical atrophy for directed functional connectivity (Supplementary Model S3) are available in the Supplementary Information. STN‑DBS modulates hierarchical processing in the PFC (Model 1). In Model 1 we tested the hypothesis that STN-DBS has an impact on rostro-caudally directed interactions in the PFC. Understand- ing remote effects of STN-DBS is however challenged by the multitude of variables that add to the outcome of the treatment, including e.g. the patient’s preoperative clinical status14, stimulation parameters37, electrode positions38, and concomitant drug treatment39,40. Moreover, higher age and longer disease duration have been discussed to be disadvantageous for the clinical outcome of STN-DBS34. The initial fixed effects structure of model 1 (Table 1) therefore included the following factors of interest: stimulation state (ON vs. OFF), direction of influences (rostro-caudal vs. caudo-rostral), and hemisphere (ipsilateral vs. contralateral with respect to the hemisphere of disease onset) and all possible interactions between this set of within-subject-factors as well as each of the following covariates: (I) age at disease onset (initial diagnosis), (II) disease duration before DBS implantation, (III) time since DBS implantation (I–III summing up to the chronological age), (IV) dopamin- ergic medication in terms of the levodopa equivalent daily dose (LEDD, calculated according to Tomlinson et al.41), and (V) stimulation intensity in terms of the volume of activated tissue (VAT). www.nature.com/scientificreports/ benefits of STN-DBS for the alleviation of motor symptoms are unquestioned13–15, cognitive and psychiatric side effects like impaired verbal fluency16–18, impulsive behavior19 and even a possibly increased suicidal tendency20 have been reported. These impairments indicate far-reaching implications of modulating basal-ganglia networks, suggesting that STN-DBS may impact on the functional integrity of the PFC12,21. Directly assessing DBS-induced neurophysiological changes in the PFC may hence substantially advance our understanding of remote effects beyond the targeted stimulation sites. y g Several models of human PFC functioning have suggested a rostro-caudal hierarchical organization22–24. In these, it is assumed that information processing in rostral PFC precedes and determines processing in caudal PFC23. Indeed, as shown by functional magnetic resonance imaging (fMRI) and lesion studies, rostral parts of the lateral PFC get gradually more involved when cognitive demands become increasingly abstract, interdependent, and temporally extended25–27. Moreover, recent evidence suggests that the upper end of this processing cascade resides in the mid-dorsolateral PFC rather than in the frontal pole28–31.h The PFC directly projects into the STN by the associative hyperdirect pathway: Fibers of the corticospinal tract send axon collaterals to the associative part of the nucleus3,32,33. Moreover, with disease progression, frontal areas involved in both motor and associative loops show increased atrophy which has recently been linked to the degree of clinical improvement of Parkinson’s patients under STN-DBS34. Thus, the outcome of STN-DBS signifi- cantly relies on the integrity of the PFC, which is possibly influenced by the stimulation of the limbic hyperdirect pathway that was recently found to be confluent with the superolateral medial forebrain bundle in humans2. In consequence, the remote impact of STN-DBS on cortical regions is likely to differ between Parkinson’s patients that undergo surgery early and late after disease onset. g g y yt Based on the assumption of rostro-caudally directed interactions within the PFC22–24, we therefore directly assessed STN-DBS effects on the integrity of this prefrontal hierarchical network by comparing the rostro-caudal gradient of neural processing in the lateral PFC, estimated from intra-individual functional measurements of Parkinson’s patients in different DBS states—initially in the ON state, followed by steady-state OFF, and thereafter again ON DBS. To this end, we used a recently established approach on estimating directed functional connectiv- ity based on multi-channel near-infrared spectroscopy (fNIRS) measurements that provide a high temporal and a sufficient spatial resolution to reveal the spatiotemporal evolution of directed neural activity within the lateral PFC (e.g. www.nature.com/scientificreports/ Refs31,35,36). Thus, the present approach evaluates the functional integrity of those cortical regions that are fundamental for higher-order cognitive functions. In addition, neuropsychological assessments were used to establish a link between the estimated prefrontal hierarchical network properties and cognitive functioning. The rostro‑caudal gradient in the prefrontal cortex and its modulation by subthalamic deep brain stimulation in Parkinson’s disease OPEN 3Freiburg Brain Imaging Center, University of Freiburg, Freiburg, Germany. 4Faculty of Biology, University of Freiburg, Freiburg, Germany. 5Medical Psychology and Medical Sociology, University of Freiburg, Freiburg, Germany. 6Department of Neurology, Movement Disorders and Neuromodulation Unit, Charité, University Medicine Berlin, Berlin, Germany. 7Kantonsspital Aarau, Aarau, Switzerland. 8Institute for Complex Systems and Mathematical Biology, University of Aberdeen, Aberdeen, UK. 9Department of Stereotactic and Functional Neurosurgery, Medical Center, University of Freiburg, Freiburg, Germany. 10Faculty of Medicine, University of Freiburg, Freiburg, Germany. 11BrainLinks‑BrainTools Cluster of Excellence, University of Freiburg, Freiburg, Germany. *email: christoph.kaller@uniklinik‑freiburg.de \| https://doi.org/10.1038/s41598-021-81535-7 Scientific Reports \| (2021) 11:2138 www.nature.com/scientificreports/ Resultsh This extensive model was successively reduced by non-significant fixed effects terms to yield a parsimonious model42. Multicollinearity was controlled using the variance inflation factor (VIF, all VIF < 3.6)43,44. Correlation coefficients for the covari- ates are provided in Supplementary Table S3.iif p pp y Confirming the assumed rostro-caudal hierarchical organization of the PFC, the significant main effect for direction revealed that influences in rostro-caudal direction were generally higher than caudo-rostral influences (F(1,1995.4) = 110.9, p < 0.001, Fig. 2). As STN-DBS is known to have side effects on cognitive functions subserved by the PFC16,19, we hypothesized that the prefrontal rostro-caudal gradient could be compromised ON compared to OFF stimulation. While the respective two-way interaction between stimulation state (ON vs. OFF) and the direction of influences (rostro-caudal/caudo-rostral) was not statistically significant on its own (F(1,1995.4) = 0.6, p = 0.454), it was dependent on the disease duration before DBS implantation (F(1,1995.4) = 5.8, p = 0.016). This https://doi.org/10.1038/s41598-021-81535-7 Scientific Reports \| (2021) 11:2138 \| www.nature.com/scientificreports/ rostral caudal caudal rostral DC DBS ON DBS OFF DBS ON 2h Healthy controls: 12 min. resting-state PD patients: 3 x 12 min. resting-state: detector source oxy deoxy Channels Channels Channels Channels DC Connectivity matrix rostral caudal caudal rostral Influences Influences 3cm Multichannel fNIRS measurements Spectral estimation of directed interactions correlation- based artifact correction standardization Peak extraction between .06 and .12 Hz Selection of directed connections along rostro-caudal axis Averaging across participants Visualization Linear mixed effects modeling Pre- processing a b c d f e g Figure 1. Scheme of the analysis workflow followed in the present study. Patients watched muted parts of a nature documentary during fNIRS measurements (a). Artifact correction67 was applied, resulting in perfectly anticorrelated signals of oxygenated and deoxygenated hemoglobin (b). The time-series were standardized before estimation of directed coherence (DC) as a measure of directed interactions between fNIRS channels69 (c). The maximum DC value in the frequency band between 0.06 and 0.12 Hz was extracted to yield a 2-dimensional, directed connectivity matrix for each measurement (d). According to models postulating a predominant flow of information from rostral to caudal PFC the influences between PFC regions should be significantly stronger in rostral-to-caudal direction than in caudal-to-rostral direction. Visualization g Selection of directed connections along rostro-caudal axis e Selection of directed connections along rostro-caudal axis e g Figure 1. Scheme of the analysis workflow followed in the present study. Patients watched muted parts of a nature documentary during fNIRS measurements (a). Artifact correction67 was applied, resulting in perfectly anticorrelated signals of oxygenated and deoxygenated hemoglobin (b). The time-series were standardized before estimation of directed coherence (DC) as a measure of directed interactions between fNIRS channels69 (c). The maximum DC value in the frequency band between 0.06 and 0.12 Hz was extracted to yield a 2-dimensional, directed connectivity matrix for each measurement (d). According to models postulating a predominant flow of information from rostral to caudal PFC the influences between PFC regions should be significantly stronger in rostral-to-caudal direction than in caudal-to-rostral direction. This prediction was tested by analyzing connections between directly neighboring channels along four rostro-caudal streams comprising four channels each (black and gray arrows in panel e) and covering most of the lateral surface of the PFC in each hemisphere (e). The distinction between rostro-caudal and caudo-rostral connections was represented in the model by the factor direction of influences (f). Influences in both directions were averaged across patients and projected separately on a standard brain surface (g). Model Fixed effects terms Random intercepts 1: ON and OFF measurements dir. × hemisphere × stim. × age at disease onset + dir. × hemisphere × stim. × disease duration before DBS implantation + dir. × hemisphere × stim. × time since DBS implantation + dir. × hemisphere × stim. × LEDD + dir. × hemisphere × stim. × VAT Stream × participant Level × participant 2: ON, OFF & 2nd ON measurements dir. × stim. × disease duration before DBS implantation Stream × participant Level × participant Model Fixed effects terms Random intercepts 1: ON and OFF measurements dir. × hemisphere × stim. × age at disease onset + dir. × hemisphere × stim. × disease duration before DBS implantation + dir. × hemisphere × stim. × time since DBS implantation + dir. × hemisphere × stim. × LEDD + dir. × hemisphere × stim. × VAT Stream × participant Level × participant 2: ON, OFF & 2nd ON measurements dir. × stim. × disease duration before DBS implantation Stream × participant Level × participant Table 1. Initial fixed and random effects structure of each model. N.B. Resultsh This prediction was tested by analyzing connections between directly neighboring channels along four rostro-caudal streams comprising four channels each (black and gray arrows in panel e) and covering most of the lateral surface of the PFC in each hemisphere (e). The distinction between rostro-caudal and caudo-rostral connections was represented in the model by the factor direction of influences (f). Influences in both directions were averaged across patients and projected separately on a standard brain surface (g). DBS ON DBS OFF DBS ON 2h Healthy controls: 12 min. resting-state PD patients: 3 x 12 min. resting-state: detector source oxy deoxy 3cm Multichannel fNIRS measurements correlation- based artifact correction standardization Pre- processing a b Channels Channels Spectral estimation of directed interactions c Healthy controls: 12 min. resting-state PD patients: 3 x 12 min resting state: detector source oxy deoxy 3cm Multichannel fNIRS measurements a Channels Channels DC Connectivity matrix Peak extraction between .06 and .12 Hz d Spectral estimation of directed interactions c a rostral caudal caudal rostral DC DBS ON DBS OFF DBS ON 2h Healthy controls: 12 min. resting-state PD patients: 3 x 12 min. resting-state: detector source oxy deoxy Channels Channels Channels Channels DC Connectivity matrix rostral caudal caudal rostral Influences Influences 3cm directed interactions correlation- based artifact correction standardization and .12 Hz Selection of directed connections along rostro-caudal axis Averaging across participants Visualization Linear mixed effects modeling f e g Figure 1. Scheme of the analysis workflow followed in the present study. Patients watched muted parts of a nature documentary during fNIRS measurements (a). Artifact correction67 was applied, resulting in perfectly h DBS ON DBS OFF DBS ON 2h p g rostral caudal caudal rostral DC rostral caudal caudal rostral Influences Influences Selection of dir along rost Averaging across participants Visualization Linear mixed effects modeling f e g Selection of directed connections along rostro-caudal axis e Visualization g The significant main effect for direction in the mixed effects model showed that influences from rostral to caudal PFC were stronger than from caudal to rostral (top panel). For cortical projections (bottom panel), directed coherence (DC) values were averaged across hemispheres and projected onto the cortical surface to represent the influences from channels (black dots) toward caudally (left brain) and rostrally (right brain) neighboring channels as indicated by arrows. Darker red colors signify stronger influences in terms of higher DC values. Bars in the top panel represent least square means; error bars indicate 95% confidence intervals; n = 24. Besides VAT, LEDD was also positively associated with the strength of the rostro-caudal gradient (F(1,1995.4) = 9.6, p = 0.002). However, this two-way interaction between LEDD and the direction of influences was mostly driven by the simple effect of influences in one direction, i.e. LEDD was negatively correlated with caudo-rostrally directed influences, while the relationship between influences in rostro-caudal direction and LEDD failed to reach significance (Fig. 5, Supplementary Table S2).l gi g pp y Albeit weak, a third two-way interaction was present between hemisphere and the direction of influences (F(1,1995.4) = 4.6, p = 0.033). The rostro-caudal gradient appeared to be steeper in the ipsilateral than in the contralateral hemisphere with respect to the hemisphere of disease onset. This within-patient effect hence cor- roborates the enhancing between-patient effect of the VAT on the rostro-caudal gradient, as VATs were larger in the ipsilateral than in the contralateral hemisphere (mean VAT in ipsilateral hemisphere: 230 mm3; mean VAT in contralateral hemisphere: 174 mm3, t(22) = 2.0, p = 0.053). Thus, the hemispheric differences in the gradient may be introduced by the asymmetric stimulation intensities.iif y y y Taken together, the key finding of Model 1 constituted a significant modulation effect of STN-DBS on the rostro-caudally directed interactions in the PFC that was moderated by the patients’ disease duration before DBS implantation. Given that disease duration before DBS implantation was highly correlated with the overall disease duration (Pearson’s r = 0.79, p < 0.001), we conducted a control analysis and refitted the model with (I) the variables disease duration before DBS implantation and time since DBS implantation substituted by the overall disease duration and (II) with all three time-related covariates (age at disease onset, disease duration before DBS implantation and time since DBS implantation) substituted by chronological age. Visualization g Exhaustive models were successively reduced by non-significant higher-order terms to yield a parsimonious model (see Supplementary Table S2 for significant effects). Continuous predictors are underlined. All lower-order terms were included. Random effects were not reduced. dir. direction of influences, LEDD levodopa equivalent daily dose, stim. stimulation state (ON, OFF, and, in model 2, 2nd ON), VAT volume of activated tissue. three-way interaction was primarily driven by influences in rostro-caudal direction (Fig. 3 top row, Supplemen- tary Table S2): Patients who underwent DBS implantation early after disease onset showed stronger rostro-caudal influences and a steeper gradient ON compared to OFF stimulation. In contrast, in patients who received DBS implantation at longer disease durations the rostro-caudal gradient was diminished ON stimulation. Moreover, there was no significant relation between the disease duration before DBS implantation and the gradient in the OFF state (Fig. 3 middle row). g Model 1 further revealed that the prefrontal gradient heavily depended on the stimulation intensity (F(1,1995.4) = 15.6, p < 0.001), with a larger VAT being associated with both stronger rostro-caudal and weaker caudo-rostral influences (Fig. 4, Supplementary Table S2). This two-way interaction between VAT and the direc- tion of influences was independent of whether STN-DBS was turned ON or OFF and persisted after stimulation was switched off for 2 h. https://doi.org/10.1038/s41598-021-81535-7 Scientific Reports \| (2021) 11:2138 \| www.nature.com/scientificreports/ .2 .3 .4 .5 .0 .2 .4 .6 Effect of Direction p < .001 rostral caudal caudal rostral Directed Coherence (DC) DC Figure 2. Granger-causality analysis of directed interactions reveals the rostro-caudal hierarchical organization in the PFC. The significant main effect for direction in the mixed effects model showed that influences from rostral to caudal PFC were stronger than from caudal to rostral (top panel). For cortical projections (bottom panel), directed coherence (DC) values were averaged across hemispheres and projected onto the cortical surface to represent the influences from channels (black dots) toward caudally (left brain) and rostrally (right brain) neighboring channels as indicated by arrows. Darker red colors signify stronger influences in terms of higher DC values. Bars in the top panel represent least square means; error bars indicate 95% confidence intervals; n = 24. Directed Coherence (DC) Figure 2. Granger-causality analysis of directed interactions reveals the rostro-caudal hierarchical organization in the PFC. Visualization g Neither overall disease duration nor chronological age exerted any significant moderator effect (all p > 0.05) on the directed interactions in the PFC, demonstrating that the impact of STN-DBS on the rostro-caudal gradient was indeed specifically depend- ent on the disease duration before DBS implantation. Remote effects of STN‑DBS on PFC are reproducible (Model 2). As an experimental validation, in Model 2 we addressed the reproducibility of the modulation effect of STN-DBS on the rostro-caudally directed interactions in the PFC and its dependence on the disease duration before DBS implantation. To this end, the examination followed a fixed protocol with the initial ON measurements always being followed by the subse- quent OFF measurements. Precluding that the stimulation-dependent results in Model 1 were simply driven by carry-over effects would hence require to demonstrate that the dependence of the STN-DBS effects on the disease duration before DBS implantation was reversible and would reappear after stimulation was switched on again. In consequence, we fitted Model 2 to the data of the ON, OFF, and ON2 measurements and, as a fixed https://doi.org/10.1038/s41598-021-81535-7 Scientific Reports \| (2021) 11:2138 \| www.nature.com/scientificreports/ .1 .2 .3 .4 .5 5 10 15 20 .1 .2 .3 .4 .5 Direction rostral caudal caudal rostral Directed Coherence (DC) .1 .2 .3 .4 .5 rostral caudal caudal rostral rostral caudal caudal rostral rostral caudal caudal rostral Directed Coherence (DC) Directed Coherence (DC) F F O S B D h 2 N O S B D N O S B D Disease duration before DBS implantation (years) 1st third [3.9, 8.2] years .2 .3 .4 .5 DC 2nd third [8.9, 13.2] years 3rd third [13.9, 20] years Model estimates Averaged estimates of directed interactions Disease duration before DBS implantation (years) .2 .3 .4 .5 DC .2 .3 .4 .5 DC Figure 3. The effect of DBS on rostro-caudally directed interactions in the PFC depends on the disease duration before DBS implantation. The relationship between the rostro-caudal gradient and the disease duration before DBS implantation in the ON stimulation state (top row, n = 24) shows that patients who received DBS early in the course of their disease had a strong gradient that was even stronger than in the OFF stimulation state (middle row, n = 22). Visualization g The later in the individual course of the disease patients underwent implantation, the more influences in rostro-caudal direction declined, such that patients who received DBS late after disease onset had a clearly diminished gradient ON stimulation compared to OFF stimulation. The replication of this time-dependent impact of DBS stimulation on the rostro-caudal hierarchical organization of the PFC in the second ON stimulation measurement (bottom row, n = 18) further confirmed this dependence (Model 2, see below). The projection of directed coherence (DC) values on the cortical surface was done analogous to Fig. 2 Averaged estimates of directed interactions Averaged estimates of directed interactions rostral caudal caudal rostral DC caudal rostral rostral caudal rostral caudal rostral caudal F F O S B D DC caudal rostral caudal rostral caudal rostral .1 .2 .3 .4 .5 5 10 15 20 rostral caudal caudal rostral Directed Coherence (DC) N O S B D Disease duration before DBS implantation (years) 1st third [3.9, 8.2] years 2nd third [8.9, 13.2] years 3rd third [13.9, 20] years Disease duration before DBS implantation (years) .2 .3 .4 .5 DC rostral caudal caudal rostral Disease duration before DBS implantation (years) 1st third [3.9, 8.2] years 2nd third [8.9, 13.2] years 3rd third [13.9, 20] years .2 .3 .4 .5 DC rostral caudal DC 3rd third [13.9, 20] years Disease duration before DBS implantation (years) 1st third [3.9, 8.2] years 2nd third [8.9, 13.2] years 3rd third [13.9, 20] years Disease duration before DBS implantation (years) 1st third [3.9, 8.2] years 2nd third [8.9, 13.2] years 3rd third [13.9, 20] years Disease duration before DBS implantation (years) Figure 3. The effect of DBS on rostro-caudally directed interactions in the PFC depends on the disease duration before DBS implantation. The relationship between the rostro-caudal gradient and the disease duration before DBS implantation in the ON stimulation state (top row, n = 24) shows that patients who received DBS early in the course of their disease had a strong gradient that was even stronger than in the OFF stimulation state (middle row, n = 22). The later in the individual course of the disease patients underwent implantation, the more influences in rostro-caudal direction declined, such that patients who received DBS late after disease onset had a clearly diminished gradient ON stimulation compared to OFF stimulation. www.nature.com/scientificreports/ www.nature.com/scientificreports/ .2 .3 .4 .5 DC .1 .2 .3 .4 .5 100 200 300 400 Directed Coherence (DC) Volume of activated tissue averaged across hemispheres (mm³) Direction rostral caudal caudal rostral Model estimates Averaged estimates of directed interactions Volume of activated tissue averaged across hemispheres (mm³) 1st third [78, 113] mm³ 2nd third [117, 213] mm³ 3rd third [216, 425] mm³ rostral caudal caudal rostral Figure 4. Rostro-caudally directed interactions in the PFC increase with stimulation intensity. The prediction by the linear mixed effects model showed a steep increase of influences from rostral to caudal prefrontal cortex (PFC) (black line) with larger volumes of activated tissue (VAT) and stimulation strength. At the same time, caudo-rostrally directed influences weakened with stimulation strength (gray line). These diverging trends can also be observed from the projected directed coherence (DC) values, where colors indicate the strength of influences between neighboring channels in rostro-caudal (top row) and caudo-rostral direction (bottom row). Sources of influences are marked by black dots. The projection of DC values was done separately for three sub-groups, split at the terciles of VAT. The most striking difference between patients with small and large VATs appears in influences exerted by the mid-lateral on the caudal PFC. Model predictions in the left panel are shown with 95% non-simultaneous confidence bands; n = 23. DC .1 .2 .3 .4 .5 100 200 300 400 Directed Coherence (DC) Direction rostral caudal caudal rostral Model estimates Volume of activated tissue averaged across hemispheres (mm³) Averaged estimates of directed interactions Averaged estimates of directed interactions .2 .3 .4 .5 DC Volume of activated Averaged 1st third [78, 113] mm³ .2 .3 .4 .5 Volume of activated tissue averaged across hemispheres (mm³) Averaged estimates of directed interactions 1st third [78, 113] mm³ 2nd third [117, 213] mm³ 3rd third [216, 425] mm³ rostral caudal caudal rostral 2nd third DC Volume of activated tissue averaged across hemispheres (mm³) 1st third [78, 113] mm³ 2nd third [117, 213] mm³ 3rd third [216, 425] mm³ averaged across hemispheres (mm³) averaged across hemispheres (mm³) Figure 4. Rostro-caudally directed interactions in the PFC increase with stimulation intensity. The prediction by the linear mixed effects model showed a steep increase of influences from rostral to caudal prefrontal cortex (PFC) (black line) with larger volumes of activated tissue (VAT) and stimulation strength. At the same time, caudo-rostrally directed influences weakened with stimulation strength (gray line). www.nature.com/scientificreports/ These diverging trends can also be observed from the projected directed coherence (DC) values, where colors indicate the strength of influences between neighboring channels in rostro-caudal (top row) and caudo-rostral direction (bottom row). Sources of influences are marked by black dots. The projection of DC values was done separately for three sub-groups, split at the terciles of VAT. The most striking difference between patients with small and large VATs appears in influences exerted by the mid-lateral on the caudal PFC. Model predictions in the left panel are shown with 95% non-simultaneous confidence bands; n = 23. .1 .2 .3 .4 .5 200 600 1000 1400 Direction rostral caudal caudal rostral Levodopa equivalent daily dose (mg/d) .2 .3 .4 .5 DC Directed Coherence (DC) Levodopa equivalent daily dose (mg/d) 1st third [150, 510] mg/d 2nd third [520, 661] mg/d 3rd third [680, 1606] mg/d Model estimates Averaged estimates of directed interactions rostral caudal caudal rostral Figure 5. Dopaminergic medication increases the rostro-caudal gradient in the PFC by diminishing rostrally directed influences. The enhancing effect of the levodopa equivalent daily dose (LEDD) on the rostro-caudal gradient was mainly driven by a strong decrease of influences in caudo-rostral direction (bottom row of projections). Analogous to Figs. 3 and 4, the projection of directed coherence (DC) values was done separately for three sub-groups, split at the terciles of LEDD. Model predictions are plotted with 95% non-simultaneous confidence bands; n = 24. .1 .2 .3 .4 .5 200 600 1000 1400 Direction rostral caudal caudal rostral Levodopa equivalent daily dose (mg/d) Directed Coherence (DC) Model estimates Averaged estimates of directed interactions Averaged estimates of directed interactions .2 .3 .4 .5 DC Levodopa equivalent daily dose (mg/d) 1st third [150, 510] mg/d 2nd third [520, 661] mg/d 3rd third [680, 1606] mg/d Averaged estimates of directed interactions rostral caudal caudal rostral Model estimates Directed Coherence (DC) DC Levodopa equivalent p q daily dose (mg/d) Figure 5. Dopaminergic medication increases the rostro-caudal gradient in the PFC by diminishing rostrally directed influences. The enhancing effect of the levodopa equivalent daily dose (LEDD) on the rostro-caudal gradient was mainly driven by a strong decrease of influences in caudo-rostral direction (bottom row of projections). Analogous to Figs. 3 and 4, the projection of directed coherence (DC) values was done separately for three sub-groups, split at the terciles of LEDD. Visualization g The replication of this time-dependent impact of DBS stimulation on the rostro-caudal hierarchical organization of the PFC in the second ON stimulation measurement (bottom row, n = 18) further confirmed this dependence (Model 2, see below). The projection of directed coherence (DC) values on the cortical surface was done analogous to Fig. 2 but separately for three sub-groups, split at the terciles of the disease duration before DBS implantation. Model predictions are plotted with 95% non-simultaneous confidence bands. https://doi.org/10.1038/s41598-021-81535-7 Scientific Reports \| (2021) 11:2138 \| Discussionh The present study revealed that STN-DBS in Parkinson’s patients significantly modulates the hierarchical organi- zation of the PFC. Most crucially, this remote network effects of STN-DBS on the PFC depended on disease progression and hence possibly on the level of sustained integrity of nigro-striatal and fronto-striatal circuits: Patients who underwent DBS implantation earlier after disease onset had a stronger rostro-caudal gradient of directed interactions ON versus OFF stimulation. In contrast, patients who received a DBS implantation after longer disease durations showed diminished rostro-caudally directed interactions ON compared to OFF stimula- tion. Importantly, this effect was clearly reproducible: In the transition when stimulation was switched ON again after steady-state OFF, the dependence of the rostro-caudal gradient in the PFC on the disease duration before DBS implantation re-emerged in the same systematic fashion as before (Fig. 3). The role of the basal ganglia for the rostro‑caudal hierarchy in the PFC. One hypothesis is that the STN—as a major input nucleus of the basal ganglia—plays an important role in integrating sensorimotor information and regulating the activity of two opposing signaling pathways: the direct, movement-facilitating pathway and the indirect, movement-inhibiting pathway9,45. Loss of dopaminergic neurons in the substantia nigra pars compacta in Parkinson’s disease leads to an excessive activity of the STN and an imbalance in favor of the indirect pathway causing hypokinetic symptoms9. DBS is known to elicit action potentials in afferent and efferent axons37 thus not only exciting downstream neurons in basal ganglia output nuclei but also anti- dromically modulating cortical neurons projecting (as part of the hyperdirect pathway) to the STN46. Although the STN was previously assumed to receive cortical projections only from motor cortices47 a recent tracer study in macaques revealed topographic projections from various PFC regions (including the dorsolateral PFC) to the dorsomedially located associative functional zone of the STN48 which could also be reproduced in human imag- ing studies2,3,49. Antidromic activation of the limbic hyperdirect pathway may therefore contribute to the pre- sent stimulation-induced changes in the hierarchical organization of the PFC, affecting cognitive functions4,50. Alternatively, remote STN-DBS effects on the PFC may be mediated through the downstream nigro-thalamo- cortical pathway37—in particular as the impact of STN-DBS on rostro-caudally directed prefrontal interactions depended on the disease duration before DBS implantation and therefore on the disease progression at the time STN-DBS was initially administered. www.nature.com/scientificreports/ tion closely resembled the pattern observed in Model 1, it hence demonstrated its immediate reproducibility (F(2,2949.6) = 3.2, p = 0.041). Post-hoc comparisons further confirmed that for influences in rostro-caudal direc- tion the effect of disease duration before DBS implantation in the ON and ON2 state was significantly different from the OFF state, while there was no significant difference between the ON and the ON2 state (Fig. 3, Supple- mentary Table S2). Regarding caudo-rostral influences, there was no significant simple effect of disease duration before DBS implantation in any stimulation state.l f p y In summary, the modulation of directed influences between PFC sub-regions by STN-DBS and its modera- tion by disease duration before DBS implantation was not only replicable, but also specific to the predominant rostro-caudal direction within the PFC and specific for ON states, arguing for a significant stimulation-induced network effect of STN-DBS on PFC integrity. Supplemental control analyses. Healthy participants that were matched to PD patients in terms of age and sex (Supplementary Fig. S1) were assessed in two supplemental analyses. Supplementary Model S1 revealed that the rostro-caudal connectivity gradient was also apparent in healthy controls (Supplementary Fig. S2). Sup- plementary Model S2 included both PD patients and matched healthy controls and demonstrated that the rest- ing-state connectivity gradient within the rostral PFC was associated with performance in a prefrontally-guided cognitive planning task as well as a more general measure of global cognitive ability (Supplementary Fig. S3). Thus, the present approach consistently detects a rostro-caudal functional network in the PFC that underlies higher cognitive abilities. Another supplemental control analysis testing the predictability of the directed func- tional connectivity by gray matter volume in the PFC revealed no significant interaction effects, precluding that the variance in the rostro-caudal gradients within the functional networks was significantly driven by differences in cortical atrophy (Supplementary Model S3). www.nature.com/scientificreports/ Model predictions are plotted with 95% non-simultaneous confidence bands; n = 24. effect, included the respective three-way interaction between stimulation state, direction of influences and dis- ease duration before DBS implantation as well as all corresponding lower-order terms. As depicted in Fig. 3, the negative correlation between the disease duration before DBS implantation and the rostro-caudal gradient was present in the ON state and absent in the OFF state. Most importantly, it reemerged in the ON2 state (Fig. 3, bottom row), within only a few minutes after the stimulation was switched on again. As the three-way interac- effect, included the respective three-way interaction between stimulation state, direction of influences and dis- ease duration before DBS implantation as well as all corresponding lower-order terms. As depicted in Fig. 3, the negative correlation between the disease duration before DBS implantation and the rostro-caudal gradient was present in the ON state and absent in the OFF state. Most importantly, it reemerged in the ON2 state (Fig. 3, bottom row), within only a few minutes after the stimulation was switched on again. As the three-way interac- https://doi.org/10.1038/s41598-021-81535-7 Scientific Reports \| (2021) 11:2138 \| t hence demonstrated its imm r confirmed that for influences i in the ON and ON2 state was ce between the ON and the ON re was no significant simple ef ween PFC sub-regions by STN only replicable, but also specif N states, arguing for a significan nts that were matched to PD pa lemental analyses. Supplementa ent in healthy controls (Supplem hed healthy controls and demo associated with performance in e of global cognitive ability (Su audal functional network in th nalysis testing the predictability aled no significant interaction e ional networks was significantly tients significantly modulates th ffects of STN-DBS on the PFC ntegrity of nigro-striatal and fr sease onset had a stronger rost t ti t h i d D www.nature.com/scientificreports/ www.nature.com/scientificreports/ Interpreting the effects of VAT and LEDD is limited by the lack of data from patients OFF medication or before DBS implantation. A stronger gradient may in fact be the cause for rather than the result of stronger stimulation amplitudes (i.e. patients having a stronger gradient might need a stronger stimulation to alleviate motor symptoms). The persistent VAT effect OFF stimulation may support this argument but it may likewise reflect plastic changes in network organization or sustained shifts in neurochemical homeostasis56. lt Finally, the magnitude of the VAT estimates (Supplementary Table S1) indicates that the stimulation directly affected subcortical structures other than the STN in some patients. Besides stimulation intensities, taking loca- tions of active electrode contacts into account in future studies is therefore vital to understand the role of this excessive stimulation. Limitations. A possible limitation of the present approach constitutes its reliance on hemodynamic low-fre- quency oscillations as systemic physiological oscillations (e.g. Mayer-waves57) may partly contribute to the signal variance in the frequency band used here. However, there is increasing evidence that this signal component reflects neuronal activity58 and conveys information about functional connectivity59,60. In addition, we recently demonstrated the robustness of the present approach against physiological noise35. The network connections reconstructed here are hence unlikely to reflect mere physiological artifacts but instead represent (direct or indi- rect) signaling pathways between neuronal populations. ) g g p y p p A pivotal question that remains is whether the disruption of the prefrontal hierarchy by STN-DBS is indeed causal for cognitive decline. The present findings suggest that receiving STN-DBS in a more advanced stage of Parkinson’s disease may increase the risk of cognitive side-effects because the stimulation disturbs the hierarchical organization in the PFC. Furthermore, the relevance of this hierarchical organization in the PFC for cognitive functioning is demonstrated in the Supplementary Model S2. Yet, the present data do not allow to conclude that effects of STN-DBS on cognitive performance are mediated by its remote effects on the PFC. Resolving this issue would require to simultaneously conduct cognitive assessments and fNIRS measurements repeatedly ON and OFF STN-DBS, which would however likely be confounded by the patients’ motor symptoms and challenged by psychometric issues61. www.nature.com/scientificreports/ not confirmed in humans so far52, it has been discussed that DBS implantation may have a slowing effect on the progression of neurodegeneration51. Finally, a brain in a healthier state may simply cope with and adapt to an extensive intervention like DBS better than a more depleted one as, for instance, the motor, cognitive, and psychiatric symptoms of Parkinson’s disease are alleviated by STN-DBS when applied early in the course of the disease13,15,52. In line with this, younger patients are known to recover better and have less cognitive decline after surgery than the elderly54. As STN-DBS electrodes were implanted through prefrontal entry points the penetration of the PFC during STN-DBS surgery may also impact on PFC network integrity. However, the present moderation of the rostro- caudal gradient by disease duration before DBS implantation was reversible and only present under active stimulation. It is hence unlikely to be introduced by the surgery itself. Nevertheless, the lesion of the STN caused by electrode implantation is known to have a temporary effect that clinically mimics the effect of stimulation55. This so-called stun effect is a transient phenomenon which is apparent within the first postoperative weeks but usually regresses before initial activation of the stimulator. Preliminary results from a follow-up study indicate that the stun effect caused by STN-DBS surgery indeed attenuates the rostro-caudal gradient post- compared to pre-DBS surgery only in patients with long disease durations but not in patients with short disease durations (F. K. Schumacher, V. A. Coenen, C. P. Kaller, unpublished data) reproducing the moderating effect of disease duration before DBS implantation on the rostro-caudal gradient ON stimulation in the current study. Effects of stimulation intensities and dopamine level. VAT and LEDD both had a general amplify- ing effect on the strength of the prefrontal gradient. However, the effect of VAT was driven by both increas- ing rostro-caudal influences and decreasing caudo-rostral influences, while the effect of LEDD solely relied on decreasing influences in caudo-rostral direction (Figs. 4, 5). The mechanisms behind both effects are hence likely to be distinct40. Moreover, shifts in network states induced by the systemic application of dopamine are mainly dosage dependent, whereas the VAT effect on cortical networks is probably transduced through parts of the basal ganglia-cortical loops and may also rely on resonance mediated by the direct, indirect, and limbic hyperdirect pathway2,45. www.nature.com/scientificreports/ y y Although PD patients were assessed on medication adhering to their usual medication to ensure dopamine levels being as stable and as physiological as possible, it cannot be fully excluded that changes in dopamine over time may have at least partly contributed to the differences ON vs. OFF STN-DBS observed in Model 1. However, these STN-DBS effects on the functional network structure in the PFC were replicated in the second (later) ON stimulation measurement (Model 2; cf. Fig. 3 top and bottom row), thus strongly suggesting that the modulation of the rostro-caudal gradient in the PFC was driven by the experimental variation of the STN stimulation and not by changes in dopamine levels over time. Discussionh In Parkinson’s disease dopamine depletion often progresses from sensori- motor towards associative/cognitive basal ganglia-thalamocortical circuits and affects cortical afferents in caudal PFC earlier than in rostral PFC5,39. The enhancement of directed interactions within lateral PFC by STN-DBS may thus be mediated through the intact associative circuits in early-stage Parkinson’s disease, while STN-DBS has debilitating effects on PFC integrity as soon as disease progression has reached associative circuits. Disease progression impacts on prefrontal effects of STN‑DBS. The present results provide new, complementary evidence suggesting that implantation at an earlier stage of Parkinson’s disease may reduce the risk of DBS-induced detrimental cognitive and psychiatric side effects on prefrontal functioning. As the STN- DBS effect on directed prefrontal interactions depended neither on the duration of stimulation, nor on the over- all disease duration, nor on the chronological age but only on the disease duration before DBS implantation, this raises the question whether STN-DBS itself or the reduction of dopaminergic medication following STN-DBS may exert preserving effects on the associative basal ganglia-thalamocortical loops51. The stimulation-induced decrease of the rostro-caudal gradient with increasing disease duration before DBS implantation suggests that STN-DBS intervention at early-stage Parkinson’s disease may contribute to sustained PFC integrity and that the timing of implantation is of essence for the overall outcome of DBS52. When patients notice first symptoms of Parkinson’s disease, at least 50% of neurons in the substantia nigra are already lost53. Animal models of Parkin- son’s disease further show that STN-DBS can have neuroprotective effects in the substantia nigra51. Although https://doi.org/10.1038/s41598-021-81535-7 Scientific Reports \| (2021) 11:2138 \| www.nature.com/scientificreports/ Methods and materialsh Cognitive functioning of patients and healthy controls was assessed with the Tower of London planning task63 (see Supplementary Methods for details) which substantially relies on the PFC64; planning accuracy was used to predict resting-state directed functional connectivity in Supplementary Model S2 to address the role of prefrontal networks for cognitive abilities. Acquisition of fNIRS data. Multi-channel fNIRS was used to record brain activity in the PFC (Fig. 1a) and to explicitly test the hypothesis of rostro-caudally directed interactions as it sampled activation in the PFC at a sufficient temporal resolution of 10 Hz65 and at a spatial resolution that ensured separation of rostral, mid- dle, and caudal PFC (Fig. 1e). FNIRS data was acquired using an ETG-4000 optical topography system (Hitachi Medical Systems, Japan). Spatial optode arrangement was derived from the system’s 3 × 11 grid configuration consisting of 17 emitters and 16 detectors. We modified this probe set by placing 12 emitters and 13 detectors on the forehead (interoptode distance of 3 cm resulting in a diagonal channel distance of 2.1 cm), resulting in 38 channels evenly distributed over the PFC (Fig. 1e). Unused emitter optodes were covered by black caps to avoid crosstalk; recordings were performed in a room without windows and the room lights were switched off during measurements. Grid placement over PFC was standardized across patients (I) by aligning the grid center to the sagittal midline and (II) by positioning the lower center optode at a distance of about 1.5 cm above the nasion. Presentation of the nature video and on-/offset of simultaneous fNIRS recordings were controlled by NBS Presentation software (version 12.2; Neurobehavioral Systems Inc., CA). To prevent artifacts during fNIRS measurement due to head movements, participants’ heads were stabilized using a chin rest. Raw data of light intensity changes were converted into hemoglobin concentration changes by in-house Matlab software (version 2012b, The MathWorks, Natick, MA, USA, unpublished tool box) using the modified Beer-Lambert law66. Due to the absorption of interfering hairs or saturation of detectors some channels did not contain any signal. The respective time series were interpolated from the surrounding channels using the Matlab 4 griddata method. This affected a total of 15 channels in 9 datasets (out of 2432 channels in 64 datasets; 0.6% of channels). In order to remove motion-induced artifacts, we applied the correlation-based correction method developed by Cui et al.67. Methods and materialsh The study protocol was approved by the ethics committee of the University of Freiburg (vote 410/11) and reg- istered at German Clinical Trials Register (DRKS, www.drks.de, identifier DRKS00003530, date of registration: 10/02/2012). The study was carried out in accordance with the Declaration of Helsinki and the guidelines of the local ethics committee on research involving human participants. All participants gave written informed consent prior to participation. Participants and procedures. Twenty-six patients with idiopathic Parkinson’s disease and implanted STN-DBS participated in the present study. To control for hemispheric differences in disease severity, two patients that had no record of the side of disease onset were excluded from the present analyses. Following a neuropsychological assessment (including the Tower of London task, see below), patients were rated by the UPDRS-III and underwent the fNIRS measurements. After the first fNIRS measurement (12 min, ON stimu- lation), the stimulator was switched off for approximately 2 h. Of the 24 included patients (6 females, mean age ± SD 61.5 ± 9.9 years), 2 dropped out during this period. The second fNIRS measurement was conducted OFF stimulation for 12 min and was preceded by another UPDRS-III assessment. Four further patients dropped out after the measurement OFF stimulation. In order to capture the transition from OFF to ON stimulation, we conducted a third measurement immediately after the stimulator was switched on again. Taken together, three 12-min fNIRS measurements were acquired (Fig. 1a): first ON stimulation (n = 24), thereafter in steady-state OFF stimulation (after 2 h of rest without stimulation; n = 22) and finally in the transition state immediately after the stimulator was switched back on (ON2, n = 18). During all measurements, patients were watching muted movie parts from the nature documentary ‘Earth’62; the order of the movie parts was balanced across patients. To ensure dopamine levels to be as stable and as physiological as possible, patients were told to adhere to their usual medication on the day of study participation and were examined during medical ON periods (see Supplemental Table S1 for patient characteristics; cf. Fig. 1a for experimental workflow). As a control group, 24 healthy adults were matched to the patients in terms of age and sex (Supplementary Fig. S1) and underwent a 12-min fNIRS measurement during passive watching of a nature documentary (see Supplementary Model S1 for results and details of healthy controls). Conclusionh The present study not only provides novel insights into the remote network effects of STN-DBS but also offers a completely new perspective on the potential neurophysiological mechanisms underlying cognitive and psychiat- ric side effects of STN-DBS in Parkinson’s disease. Specifically, we demonstrated that the rostro-caudal hierarchy in the PFC is compromised by STN stimulation in patients who underwent electrode implantation after longer disease duration. In contrast, stimulation enhanced the prefrontal hierarchy in patients who received DBS early in the course of their disease. In addition, stimulation intensities and dopaminergic drug dosages predicted the strength of the prefrontal hierarchy. Taken together, by allowing to directly monitor the remote network effects https://doi.org/10.1038/s41598-021-81535-7 Scientific Reports \| (2021) 11:2138 \| www.nature.com/scientificreports/ of STN-DBS the novel approach applied here might provide a promising opportunity for future refinements of DBS in the STN and beyond in terms of a methodological foundation for individually tailored optimization and adjustment of stimulation parameters. of STN-DBS the novel approach applied here might provide a promising opportunity for future refinements of DBS in the STN and beyond in terms of a methodological foundation for individually tailored optimization and adjustment of stimulation parameters. Methods and materialsh Renderings of electrode positions in relation to the STN are provided in Supplementary Fig. S4, showing that implanted electrodes mainly resided within the posterior, i.e. the motor subdivision of the STN. Estimation of the volume of activated tissue (VAT). To construct a conductor model of the DBS elec- trode and surrounding tissue, the Medtronic DBS electrode 3391 model was discretized into voxels of 0.22 mm isotropic size. The electrode defined the mid-axis of a volume 101 × 101 × 90 voxels in size. The rest of the space was filled with gray and white matter. Gray matter was defined by the structures STN, red nucleus and internal/ external pallidum as specified by the DISTAL atlas3 which exactly corresponds to the ICBM 2009b nonlin- ear template used for normalization. The remaining space was declared as white matter. The volume was con- verted into a hexaedral mesh using FieldTrip software74; www.fieldtriptoolbox.org). Conductivities of 0.33 and 0.14 S/m were assigned to gray and white matter respectively. These values are commonly used in neuroscientific modeling studies75. For the platinum/iridium contacts and insulated parts of the electrodes, values of 108 S/m and 10–16 S/m were used, respectively. A forward model to obtain the voltage distribution was solved using the SimBio toolbox (Ref.76; https://www.mrt.uni-jena.de/simbio/index.php/Main_Page). In case of monopolar stimulation, the surface of the cubic model was used as the cathode. This generated a gradient denoting the volt- age distribution for each hexaedral element of the model. The gradient was thresholded for magnitudes > 0.2 V/ mm2 to define the VAT. The VATs in both hemispheres were substantially correlated (Pearson’s r = 0.55, p = 0.007) and were therefore averaged and used as a single predictor in the statistical analysis. Statistical analyses. DC and between-subject covariates were analyzed in linear mixed effects models using the lme4 package77 (version 1.1–14) for R statistics (version 3.4.2; http://cran.r-project.org). According to extant models of prefrontal organization22,23, we expected influences predominantly in rostro-caudal direc- tion and therefore selected the connections between 4 rostro-caudally adjacent fNIRS channels in 4 parallel rows within each hemisphere for statistical analyses (i.e., resulting in 12 connections per hemisphere; Fig. 1e). Hemispheres were classified into ‘ipsilateral’ and ‘contralateral’ with respect to the hemisphere of disease onset. Two linear mixed effects models were computed as summarized in Table 1. Models were analyzed following a top-down procedure42: Higher-order fixed-effects interaction terms that did not reach significance (i.e. Methods and materialsh This method not only effectively removes motion artifacts, but also increases the contrast-to-noise ratio. The resulting data for oxygenated and deoxygenated hemoglobin are perfectly anticorrelated and therefore have identical spectral properties. The time series were standardized for Granger-causality analysis. We refrained from applying further artifact correction methods like filtering or resampling, because such data preprocessing has been shown to produce false positives in Granger-causality analysis68. Furthermore, we used a frequency- domain measure of Granger-causality (see below) that allowed avoiding frequency bands prone to physiological noise introduced by respiration and heart-beat (see also Refs.31,35). Further information on the validity of the here applied fNIRS approach on directed functional connectivity can be found in Refs.31,35. Analyses of directed interactions. Directed coherence (DC)69 as an implementation of Granger-cau- sality70 was used to capture the intrinsic functional organization of the PFC and directed interactions between its rostral and caudal parts. DC was estimated using the frequency domain multivariate analyses toolbox (www. fdm.uni-freiburg.de/Toolboxes/fdma-toolbox). For DC calculation, a vector autoregressive model was fitted https://doi.org/10.1038/s41598-021-81535-7 Scientific Reports \| (2021) 11:2138 \| www.nature.com/scientificreports/ with a model order of 20, corresponding to the past 2 s of the time-series and providing a frequency resolution of the DC estimate of 0.5 Hz. Note, however, that for the Fourier transformation the autoregression coefficients were zero-padded to the length of the time series in order to smooth the spectral estimate. As functional con- nectivity between different brain areas is apparent in low frequency oscillations59, we chose the frequency band between 0.06 and 0.12 Hz and used the maximum DC value in this band for further analysis (see also Refs.31,35). DBS electrode localizations. Preoperative T2-weighted MRI and CT scans as well as postoperative axial slabs of CT scans focusing on the subcortical volume around the electrode tips were acquired. Details on imag- ing parameters varied across patients and can be requested from the authors. Using BRAINSfit software71 as implemented in 3D slicer (version 4.6.0; www.slicer.org), postoperative CTs were linearly co-registered and fused to preoperative CTs. The fused images were then again linearly co-registered to preoperative MRIs. Pre- operative MRIs were nonlinearly warped into International Consortium for Brain Mapping (ICBM) 152 2009b nonlinear asymmetric space using the SyN approach as implemented in Advanced Normalization Tools72. Using the Lead-DBS software73 (version 1.5.1; www.leadsuite.io), electrode positions were reconstructed hybridly in native and standard stereotactic space. Methods and materialsh p > 0.05) were identified using the anova method (Type III F-statistics with Satterthwaite’s approximation of degrees of freedom) implemented in the lmerTest package78 (version 2.0-33) and successively removed to yield a parsimo- nious model42 (see Table 1 for the highest-order terms of the initial models and Supplementary Table S2 for the significant highest-order terms of the parsimonious models). Models were fitted with random intercepts for (I) the interaction between participant and stream (identifying channel rows in rostro-caudal direction) and (II) the interaction between participant and level (identifying adjacent channel pairs—i.e. connections—along the rostro-caudal axis) using maximum likelihood estimation. Post-hoc comparisons and calculation of confidence bands were performed using the lsmeans package79 (version 2.27-2). Multiplicity adjustment was applied using Tukey’s method. Received: 28 May 2020; Accepted: 28 December 2020 Received: 28 May 2020; Accepted: 28 December 2020 References 1. Graybiel, A. M. & Mink, J. W. The basal ganglia and cognition. In The Cognitive Neurosciences (ed. Gazzaniga, M. S.) 565–585 (MIT Press, Cambridge, 2009).h www.nature.com/scientificreports/ Margulies, D. S. et al. Situating the default-mode network along a principal gradient of macroscale cortical organization. Proc Natl. Acad. Sci. U. S. A. 113, 12574–12579 (2016). 1. Schumacher, F. K., Schumacher, L. V., Schelter, B. O. & Kaller, C. P. Functionally dissociating ventro-dorsal components within the rostro-caudal hierarchical organization of the human prefrontal cortex. Neuroimage 185, 398–407 (2019). 2. McIntyre, C. C. & Hahn, P. J. Network perspectives on the mechanisms of deep brain stimulation. Neurobiol. Dis. 38, 329–337 (2010).f 3. Accolla, E. A. et al. Brain tissue properties differentiate between motor and limbic basal ganglia circuits. Hum. Brain Mapp. 35 5083–5092 (2014).f ( ) 34. Muthuraman, M. et al. Effects of DBS in parkinsonian patients depend on the structural integrity of frontal cortex. Sci. Rep. 7, 43571 (2017).h 5. Schumacher, F. K. et al. The impact of physiological noise on hemodynamic-derived estimates of directed functional connectivity Brain Struct. Funct. 224, 3145–3157 (2019). 35. Schumacher, F. K. et al. The impact of physiological noise on hemodynamic-derived estimates of directed functional connectivity. Brain Struct. Funct. 224, 3145–3157 (2019). 36 Medvedev, A V Does the resting state connectivity have hemispheric asymmetry? A near-infrared spectroscopy study Neuroimage 6. Medvedev, A. V. Does the resting state connectivity have hemispheric asymmetry? A near-infrared spectroscopy study. Neuroimage 85, 400–407 (2014). , ( ) 37. Kringelbach, M. L., Jenkinson, N., Owen, S. L. F. & Aziz, T. Z. Translational principles of deep brain stimulation. Nat. Rev. Neurosci. 8, 623–635 (2007). 8. Min, H.-K. et al. Dopamine release in the nonhuman primate caudate and putamen depends upon site of stimulation in the sub thalamic nucleus. J. Neurosci. 36, 6022–6029 (2016). 9. Cools, R. Dopaminergic modulation of cognitive function-implications for L-DOPA treatment in Parkinson’s disease. Neurosci Biobehav. Rev. 30, 1–23 (2006). 0. Zaidel, A., Bergman, H., Ritov, Y. & Israel, Z. Levodopa and subthalamic deep brain stimulation responses are not congruent. Mov Disord. 25, 2379–2386 (2010). 1. Tomlinson, C. L. et al. Systematic review of levodopa dose equivalency reporting in Parkinson’s disease. Mov. Disord. 25, 2649–2653 (2010).t 42. West, B. T., Welch, K. B. & Galecki, A. T. Linear Mixed Models: A Practical Guide Using Statistical Software (Chapman & Hall/CRC, Boca Raton, 2014). 43. Lüdecke, D., Makowski, D., Waggoner, P. & Patil, I. Performance: Assessment of regression models performance. CRAN https:// doi.org/10.5281/zenodo.3952174 (2020). g 4. James, G., Witten, D., Hastie, T. & Tibshirani, R. www.nature.com/scientificreports/ 25, pars internus in Parkinson’s disease: A meta-analysis. Neuropsyc 8. Witt, K. et al. Neuropsychological and psychiatric changes after deep brain stimulation for Parkinson’s disease: A randomised multicentre study. Lancet Neurol. 7, 605–614 (2008). y 9. Frank, M. J., Samanta, J., Moustafa, A. A. & Sherman, S. J. Hold your horses: Impulsivity, deep brain stimulation, and medication in parkinsonism. Science 318, 1309–1312 (2007). p 20. Voon, V. et al. A multicentre study on suicide outcomes following subthalamic stimulation for Parkinson’s disease. Brain 131, 2720–2728 (2008).f 21. Castrioto, A., Lhommée, E., Moro, E. & Krack, P. Mood and behavioural effects of subthalamic stimulation in Parkinson’s disease. Lancet Neurol. 13, 287–305 (2014).h 2. Koechlin, E., Ody, C. & Kouneiher, F. The architecture of cognitive control in the human prefrontal cortex. Science 302, 1181–1185 (2003). ( ) 23. Badre, D. & D’Esposito, M. Is the rostro-caudal axis of the frontal lobe hierarchical?. Nat. Rev. Neurosci. 10, 659–669 (2009). 24 F t J M Th P f t l C t (A d i P /El i A t d 2008) & D’Esposito, M. Is the rostro-caudal axis of the frontal lobe hierar 23. Badre, D. & D’Esposito, M. Is the rostro-caudal axis of the frontal lobe hierarchical?. Nat. Rev. Neurosci. 10, 659–669 (2009). 24. Fuster, J. M. The Prefrontal Cortex (Academic Press/Elsevier, Amsterdam, 2008). 23. Badre, D. & D Esposito, M. Is the rostro caudal axis of the frontal lobe hierarchical?. Nat. Rev. Neurosci. 10, 65 24. Fuster, J. M. The Prefrontal Cortex (Academic Press/Elsevier, Amsterdam, 2008).f p 24. Fuster, J. M. The Prefrontal Cortex (Academic Press/Elsevier, Amsterdam, 2008).f h 25. Christoff, K., Keramatian, K., Gordon, A. M., Smith, R. & Mädler, B. Prefrontal organization of cognitive control according to levels of abstraction. Brain Res. 1286, 94–105 (2009). 6. Azuar, C. et al. Testing the model of caudo-rostral organization of cognitive control in the human with frontal lesions. Neuroimage 84, 1053–1060 (2014). 7. Badre, D. & D’Esposito, M. Functional magnetic resonance imaging evidence for a hierarchical organization of the prefronta cortex. J. Cogn. Neurosci. 19, 2082–2099 (2007). g 28. Badre, D. & Nee, D. E. Frontal cortex and the hierarchical control of behavior. Trends Cogn. Sci. 22, 170–188 (2018).h 29. Nee, D. E. & D’Esposito, M. The hierarchical organization of the lateral prefrontal cortex. Elife 5, e12112 (2016). 29. Nee, D. E. & D’Esposito, M. The hierarchical organization of t h 0. References g 2. Coenen, V. A. et al. The anatomy of the human medial forebrain bundle: Ventral tegmental area connections to reward-associated subcortical and frontal lobe regions. NeuroImage Clin. 18, 770–783 (2018).i g g , ( ) 3. Ewert, S. et al. Toward defining deep brain stimulation targets in MNI space: A subcortical atlas based on multimodal MRI, histol- ogy and structural connectivity. Neuroimage 170, 271–282 (2018). 5. Redgrave, P. et al. Goal-directed and habitual control in the basal ganglia: Implications for Parkinson’s disease. Nat. Rev. Neurosci 11, 760–772 (2010). 6. Rodriguez-Oroz, M. C. et al. Initial clinical manifestations of Parkinson’s disease: Features and pathophysiological mechanisms Lancet. Neurol. 8, 1128–1139 (2009). Scientific Reports \| (2021) 11:2138 \| https://doi.org/10.1038/s41598-021-81535-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ y p 8. Vanegas-Arroyave, N. et al. Tractography patterns of subthalamic nucleus deep brain stimulation. Brain 139, 1200–1210 (2016).h 8. Vanegas-Arroyave, N. et al. Tractography patterns of subthalamic nucleus deep brain stimulation. Brain 139, 1200–1210 (2016). 9 Albin R L Young A B & Penney J B The functional anatomy of basal ganglia disorders Trends Neurosci 12 366–375 (1989) 8. Vanegas-Arroyave, N. et al. Tractography patterns of subthalamic nucleus deep brain stimulation. Brain 139, 1200–1210 (20 9. Albin, R. L., Young, A. B. & Penney, J. B. The functional anatomy of basal ganglia disorders. Trends Neurosci. 12, 366–375 (1 9. Albin, R. L., Young, A. B. & Penney, J. B. The functional anatomy of basal ganglia disorders. Trends Ne g yh y g g 0. Jahanshahi, M., Obeso, I., Rothwell, J. C. & Obeso, J. A. A fronto-striato-subthalamic-pallidal network for goal-directed and habitual inhibition. Nat. Rev. Neurosci. 16, 719–732 (2015).hf 1. Kahan, J. et al. Resting state functional MRI in Parkinson’s disease: The impact of deep brain stimulation on ‘effective’ connectivity Brain 137, 1130–1144 (2014).f ( ) 2. Volkmann, J., Daniels, C. & Witt, K. Neuropsychiatric effects of subthalamic neurostimulation in Parkinson disease. Nat. Rev Neurol. 6, 487–498 (2010). 13. Schüpbach, W. M. M. et al. Neurostimulation for Parkinson’s disease with early motor complications. N. Engl. J. Med. 368, 610 (2013). ( ) 14. Bronstein, J. M. et al. Deep brain stimulation for Parkinson disease. Arch. Neurol. 68, 165–171 (2011). 15. Lhommée, E. et al. Behavioural outcomes of subthalamic stimulation and medical therapy versus medical therapy alone for Par- kinson’s disease with early motor complications (EARLYSTIM trial): Secondary analysis of an open-label randomised trial. Lancet. Neurol. 17, 223–231 (2018). 16. Parsons, T. D., Rogers, S. A., Braaten, A. J., Woods, S. P. & Tröster, A. I. Cognitive sequelae of subthalamic nucleus deep b stimulation in Parkinson’s disease: A meta-analysis. Lancet Neurol. 5, 578–588 (2006). y 17. Combs, H. L. et al. Cognition and depression following deep brain stimulation of the subthalamic nucleus and globus pallidus pars internus in Parkinson’s disease: A meta-analysis. Neuropsychol. Rev. 25, 439–454 (2015). y 17. Combs, H. L. et al. Cognition and depression following deep brain stimulation of the subthalamic nucleus and globus pal p i t i P ki ’ di A t l i N p h l R 25 439 454 (2015) us in Parkinson’s disease: A meta-analysis. Neuropsychol. Rev. www.nature.com/scientificreports/ Lead-DBS: A toolbox for deep brain stimulation electrode localizations and visualizations. Neuroimage 107, 127–135 (2015).ft 74. Oostenveld, R., Fries, P., Maris, E. & Schoffelen, J.-M. FieldTrip: Open source software for advanced analysis of MEG, EEG, and invasive electrophysiological data. Comput. Intell. Neurosci. 2011, 1–9 (2011). y g 5. Buzsáki, G. Rhythms of the Brain. (Oxford University Press, Oxford, 2006). https://doi.org/10.1093/acprof:oso/9780195301 069.001.0001. 6. SimBio Development Group. SimBio: A generic environment for bio-numerical simulations. (2017). https://www.mrt.uni-jena de/simbio. Accessed 8 Aug 2017.ft g 7. Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67, 1–48 (2015). 8 K A B B kh ff P & H b B j Ch i R l T T i Li Mi d Eff M d l (2016) g 77. Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67, 1–48 (2015). 78. Kuznetsova, A., Bruun Brockhoff, P. & Haubo Bojesen Christensen, R. lmerTest: Tests in Linear Mixed Effects Models. (2016). 79. Lenth, R. V. Least-squares means: The R Package lsmeans. J. Stat. Softw. 69, 1–33 (2016). 77. Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67, 1–48 (2015). 78. Kuznetsova, A., Bruun Brockhoff, P. & Haubo Bojesen Christensen, R. lmerTest: Tests in Linear Mixed Effects Models. (2016).t Acknowledgementsh g The authors thank Benjamin Rahm (University of Freiburg) and Michael Fox (Harvard Medical School) for valu- able comments on a previous version of this manuscript. This work was supported by a grant of the BrainLinks- BrainTools Cluster of Excellence funded by the German Research Foundation (DFG, grant number EXC 1086) to C.P.K., F.A., T.P., B.O.S., C.W, and V.A.C.; A.H. was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, Emmy Noether Stipend 410169619 and 424778381 – TRR 295) as well as Deutsches Zentrum für Luft- und Raumfahrt (DynaSti grant within the EU Joint Programme Neurodegenerative Disease Research, JPND). g The authors thank Benjamin Rahm (University of Freiburg) and Michael Fox (Harvard Medical School) for valu- able comments on a previous version of this manuscript. This work was supported by a grant of the BrainLinks- BrainTools Cluster of Excellence funded by the German Research Foundation (DFG, grant number EXC 1086) to C.P.K., F.A., T.P., B.O.S., C.W, and V.A.C.; A.H. was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, Emmy Noether Stipend 410169619 and 424778381 – TRR 295) as well as Deutsches Zentrum für Luft- und Raumfahrt (DynaSti grant within the EU Joint Programme Neurodegenerative Disease Research, JPND). www.nature.com/scientificreports/ y g ( ) 1. Rabbitt, P. Methodologies and models in the study of executive function. In Methodology of Frontal and Executive Function (ed Rabbitt, P.) 1–38 (Psychology Press, 1997).i y gy 62. Fothergill, A. & Linfield, M. Earth. (Universum Film GmbH, Munich, 2007). y gy A. & Linfield, M. Earth. (Universum Film GmbH, Munich, 2007) 62. Fothergill, A. & Linfield, M. Earth. (Universum Film GmbH i 3. Kaller, C. P., Unterrainer, J. M., Kaiser, S., Weisbrod, M. & Aschenbrenner, S. Tower of London—Freiburg version. (Schuhfried Mödling, 2012). 64. Nitschke, K., Köstering, L., Finkel, L., Weiller, C. & Kaller, C. P. A Meta-analysis on the neural basis of planning: Activation likeli- hood estimation of functional brain imaging results in the Tower of London task. Hum. Brain Mapp. 38, 396–413 (2017).l 65. Roebroeck, A., Formisano, E. & Goebel, R. Mapping directed influence over the brain using Granger causality and fMRI. Neuroim- age 25, 230–242 (2005).l g 6. Delpy, D. T. et al. Estimation of optical pathlength through tissue from direct time of flight measurement. Phys. Med. Biol. 33 1433–1442 (1988). 67. Cui, X., Bray, S. & Reiss, A. L. Functional near infrared spectroscopy (NIRS) signal improvement based on negative correlation between oxygenated and deoxygenated hemoglobin dynamics. Neuroimage 49, 3039–3046 (2010).hfi 68. Florin, E., Gross, J., Pfeifer, J., Fink, G. R. & Timmermann, L. The effect of filtering on Granger causality based multivariate causality measures. Neuroimage 50, 577–588 (2010).l g 9. Schelter, B. et al. Testing for directed influences among neural signals using partial directed coherence. J. Neurosci. Methods 152 210–219 (2006). ( ) 0. Granger, C. W. J. Investigating causal relations by econometric models and cross-spectral methods. Econometrica 37, 424 (1969) 70. Granger, C. W. J. Investigating causal relations by econometric models and cross-spectral methods. Econometrica 37, 424 (1969). 71 J h H J l BRAINSFi M l i f i i id i i f h l b i 3D i i h i i h lki (2007) 0. Granger, C. W. J. Investigating causal relations by econometric models and cross-spectral methods. Econometrica 37, 424 (1969) 1. Johnson, H. J. et al. BRAINSFit: Mutual information rigid registrations of whole-brain 3D images, using the insight toolkit. (2007) g g g g g 72. Avants, B. B. et al. A reproducible evaluation of ANTs similarity metric performance in brain image registration. Neuroimage 54, 2033–2044 (2011). 3. Horn, A. & Kühn, A. A. Author contributions F.K.S., F.A., B.O.S., C.W., V.A.C., and C.P.K. designed the experiments. F.K.S. and L.V.S. performed the experi- ments. F.A., T.P., V.A.C, and K.E. provided patient care. F.K.S., L.V.S., B.O.S., A.H., and C.P.K. performed the analyses. F.K.S., L.V.S., and C.P.K. wrote the paper; the other authors commented on the drafts of the manuscript at all stages. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 50. Neumann, W.-J. et al. Functional segregation of basal ganglia pathways in Parkinson’s disease. Brain https://doi.org/10.1093/brain /awy206 (2018).t y 1. Wallace, B. A. et al. Survival of midbrain dopaminergic cells after lesion or deep brain stimulation of the subthalamic nucleus in MPTP-treated monkeys. Brain 130, 2129–2145 (2007). y 2. Desouza, R. M., Moro, E., Lang, A. E. & Schapira, A. H. V. Timing of deep brain stimulation in Parkinson disease: A need for reappraisal?. Ann. Neurol. 73, 565–575 (2013). g p reappraisal?. Ann. Neurol. 73, 565–575 (2013). pp 3. Cheng, H.-C., Ulane, C. M. & Burke, R. E. Clinical progression in Parkinson disease and the neurobiology of axons. Ann. Neurol 67, 715–725 (2010). 67, 715–725 (2010). 54. Carlson, J. D. et al. Postoperative delirium in Parkinson’s disease patients following deep brain stimulation surgery. J. Clin. Neurosci. 21 1192–1195 (2014) 4. Carlson, J. D. et al. Postoperative delirium in Parkinson’s disease patients following deep brain stimulation surgery. J. Clin. Neurosci 21, 1192–1195 (2014).h 5. Jech, R. et al. The subthalamic microlesion story in Parkinson’s disease: Electrode insertion-related motor improvement with relative cortico-subcortical hypoactivation in fMRI. PLoS One 7, (2012).t yp 6. Trager, M. H. et al. Subthalamic beta oscillations are attenuated after withdrawal of chronic high frequency neurostimulation in Parkinson’s disease. Neurobiol. Dis. 96, 22–30 (2016).h yp 56. Trager, M. H. et al. Subthalamic beta oscillations are attenuated after withdrawal of chronic Parkinson’s disease. Neurobiol. Dis. 96, 22–30 (2016).h 57. Julien, C. The enigma of Mayer waves: Facts and models. Cardiovasc. Res. 70, 12–21 (2006). 57. Julien, C. The enigma of Mayer waves: Facts and models. Cardiovasc. Res. 70, 12–21 (2006). h g y 8. Du, C., Volkow, N. D., Koretsky, A. P. & Pan, Y. Low-frequency calcium oscillations accompany deoxyhemoglobin oscillations in rat somatosensory cortex. Proc. Natl. Acad. Sci. U. S. A. 111, E4677–E4686 (2014). h 58. Du, C., Volkow, N. D., Koretsky, A. P. & Pan, Y. Low-frequency calcium oscillations accompany rat somatosensory cortex. Proc. Natl. Acad. Sci. U. S. A. 111, E4677–E4686 (2014). y 9. Biswal, B., Yetkin, F. Z., Haughton, V. M. & Hyde, J. S. Functional connectivity in the motor cortex of resting human brain using echo-planar MRI. Magn. Reson. Med. 34, 537–541 (1995).i p g 0. Sasai, S., Homae, F., Watanabe, H. & Taga, G. Frequency-specific functional connectivity in the brain during resting state revealed by NIRS. Neuroimage 56, 252–257 (2011). www.nature.com/scientificreports/ An Introduction to Statistical Learning. (Springer, New York, 2013). https://doi org/10.1007/978-1-4614-7138-7. g 45. Lee, H. J. et al. Activation of direct and indirect pathway medium spiny neurons drives distinct brain-wide responses. Neuron 91, 412–424 (2016).h 46. Gradinaru, V., Mogri, M., Thompson, K. R., Henderson, J. M. & Deisseroth, K. Optical deconstruction of parkinsonian neural circuitry. Science 324, 354–359 (2009). 47. Nambu, A., Takada, M., Inase, M. & Tokuno, H. Dual somatotopical representations in the primate subthalamic nucleus: Evidence for ordered but reversed body-map transformations from the primary motor cortex and the supplementary motor area. J. Neurosci. 16, 2671–2683 (1996).h ( ) 8. Haynes, W. I. A. & Haber, S. N. The organization of prefrontal-subthalamic inputs in primates provides an anatomical substrate for both functional specificity and integration: Implications for Basal Ganglia models and deep brain stimulation. J. Neurosci. 33 4804–4814 (2013). 48. Haynes, W. I. A. & Haber, S. N. The organization of prefrontal-subthalamic inputs in primates provides an anatomical substrate for both functional specificity and integration: Implications for Basal Ganglia models and deep brain stimulation. J. Neurosci. 33, 4804–4814 (2013). ( ) 9. Coenen, V. A. et al. Medial forebrain bundle stimulation as a pathophysiological mechanism for hypomania in subthalamic nucleus deep brain stimulation for Parkinson’s disease. 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https://openalex.org/W2340636203	https://are-journal.com/are/article/download/15/16	English	null	Sustainable use of land in danger of wind erosion in Ukraine: stakeholder engagement	Agricultural and resource economics	2,015	cc-by	11,597	Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com JEL: Q15, Q57 Anatoliy Kucher1, Iryna Kazakova1, Lesya Kucher2, Antonia Schraml3, Hekuran Koka4, Warren Priest5 1National Scientific Center «Institute for Soil Science and Agrochemistry Research named after O. N. Sokolovsky» Ukraine 2Kharkiv National Agrarian University named after V. V. Dokuchaev Ukraine 3Humboldt University of Berlin Germany 4Lezha Regional Development Agency Albania 5Independent scientist United Kingdom SUSTAINABLE USE OF LAND IN DANGER OF WIND EROSION IN UKRAINE: STAKEHOLDER ENGAGEMENT Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com JEL: Q15, Q57 Anatoliy Kucher1, Iryna Kazakova1, Lesya Kucher2, Antonia Schraml3, Hekuran Koka4, Warren Priest5 1National Scientific Center «Institute for Soil Science and Agrochemistry Research named after O. N. Sokolovsky» Ukraine 2Kharkiv National Agrarian University named after V. V. Dokuchaev Ukraine 3Humboldt University of Berlin Germany 4Lezha Regional Development Agency Albania 5Independent scientist United Kingdom SUSTAINABLE USE OF LAND IN DANGER OF WIND EROSION IN UKRAINE: STAKEHOLDER ENGAGEMENT Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com JEL: Q15, Q57 Анатолій Кучер1, Ірина Казакова1, Леся Кучер2, Антонія Шрамл3, Хікуран Кока4, Уорен Пріст5 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com 1Національний науковий центр «Інститут ґрунтознавства та агрохімії імені О. Н. Соколовського» Україна 2Харківський національний аграрний університет ім. В. В. Докучаєва Україна 3Університет Гумбольдта в Берліні Німеччина 4Агентство регіонального розвитку м. Лежа Албанія 5Незалежний учений Великобританія СТАЛЕ ВИКОРИСТАННЯ ДЕФЛЯЦІЙНО НЕБЕЗПЕЧНИХ ЗЕМЕЛЬ В УКРАЇНІ: ВЗАЄМОДІЯ СТЕЙКХОЛДЕРІВ Досліджено теоретичні й методичні аспекти ідентифікації, систематизації тейкхолдерів, визначення їх цілей і можливостей їх залучення для розробки перспективних аріантів сталого використання дефляційно небезпечних земель. Розглянуто різні методи налізу стейхолдерів (мапи, матриці). Розроблено план дій, механізми взаємодії та ндикатори вимірювання якості залучення стейкхолдерів. Ключові слова: вітрова ерозія, стале використання земель, аналіз скейкхолдерів, заємодія стейкхолдерів. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com SUSTAINABLE USE OF LAND IN DANGER OF WIND EROSION IN UKRAINE: STAKEHOLDER ENGAGEMENT In the modern world overcoming the problems of soil erosion and the transition to the sustainable use of land, especially land involved in agricultural production, is impossible without the broad involvement of all interested and stakeholding parties in solving these problems. Dialogue with stakeholders is a key element of effective management at individual farm and state levels. Currently in Ukraine, there is considerable scope and need for significant improvements in this area as only a small number of companies (industrial, commercial) involved in the agricultural sector have developed stakeholder engagement tools, and only one is actively involved in dialogue with stakeholders. This paper presents research focusing on the village of Pishcha in Volyn region, Ukraine. It examines the theoretical and methodological aspects of: identifying key stakeholders, determining their interests (explicit and implicit) and the possible impact of these on the project; project preparation and implementation; the identification of apparent or potential conflicts between the interests of different stakeholders, and the possibility of reconciling these interests and establishing constructive relationships between them; the forms of participation appropriate to each of the stakeholders at each stage of the project cycle; and, the possibilities for the monitoring and evaluation processes, especially participatory methods that look to involve interested parties. The paper also sets out a provisional budget (facilitation needed, personnel and staffing required) for and discusses the feasibility of the implementation of the engagement process of stakeholders for the promotion and roll out of promising options for the sustainable use of land in danger of wind erosion in Ukraine. Key words: wind erosion, sustainable use of land, stakeholders analysis, stakeholder engagement. Анатолій Кучер1, Ірина Казакова1, Леся Кучер2, Антонія Шрамл3, Хікуран Кока4, Уорен Пріст5 Vol. 1, No. 2, 2015 5 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com has a number of advantages, including: the cultivation of perennial grasses generates income from the sale of seed clover in the first year and hay in the following four years; through nitrogen fixing bacteria associated with the annual grasses improving the nitrogen levels in the soil; and, the cessation of the deflationary processes in the different soil types extant in the investigated areas and allowing for the steady build up of a fertile humus layer. The options and recommendations put forward in that paper for effective management for the sustainable use of soil resources that are exposed to wind erosion potentially involve a wide range of actors and stakeholders. Research on the identification and engagement of these stakeholders was performed and is presented in this paper. The purpose of the article is to identify and classify stakeholders, and determine the possibilities for engagement with and between stakeholders within the context of implementing measures that can help tackle or allow adaptation to the challenges of land degradation in Ukraine using the example of the use of land in danger of wind erosion in the Pishcha village. Results and discussion. This paper examines techniques to involve different stakeholders in the identification of appropriate methods for the sustainable use of land in danger of wind erosion that have the capacity to reverse land degradation trends in Pishcha village. It covers the setting of project goals, the planning of the stakeholder engagement process, the identification of relevant stakeholders, the implementation of the stakeholder engagement plan, using tried and tested tools and facilitation techniques, and the monitoring the engagement process in order to evaluate whether or not the desired goals of engagement have been achieved, including identifying the most appropriate indicators for the particular project. The first step in any stakeholder engagement process is to clearly define the goals that the process seeks to address. Only when these are clear is it possible to identify who has a stake in the achievement of these goals and who, as a result, needs to be involved [5]. СТАЛЕ ВИКОРИСТАННЯ ДЕФЛЯЦІЙНО НЕБЕЗПЕЧНИХ ЗЕМЕЛЬ В УКРАЇНІ: ВЗАЄМОДІЯ СТЕЙКХОЛДЕРІВ Досліджено теоретичні й методичні аспекти ідентифікації, систематизації стейкхолдерів, визначення їх цілей і можливостей їх залучення для розробки перспективних варіантів сталого використання дефляційно небезпечних земель. Розглянуто різні методи аналізу стейхолдерів (мапи, матриці). Розроблено план дій, механізми взаємодії та індикатори вимірювання якості залучення стейкхолдерів. Ключові слова: вітрова ерозія, стале використання земель, аналіз скейкхолдерів, взаємодія стейкхолдерів. Introduction and review of literature. Currently, the overlapping and interacting issues of understanding of the economic value of productive land, preventing the loss of natural capital, preserving ecosystem services, combatting and adapting to climate change, and addressing food, energy, and water security are being investigated by The Economics of Land Degradation (ELD) Initiative. Based on state of the art research provided by a world-wide network of researchers and practitioners, and with the aim of establishing a global approach for the analysis of the economics of land degradation, the ELD has produced a number of reports and practitioners manuals [1–3]. The analysis presented in this paper was produced as part of an ELD initiative. Soil degradation has been identified as one of the major threats to European soils. In the previous paper [4] the conditions under which wind erosion of soils and the potential soil loss caused by this problem in the Ukraine were described. The best options for the project area, from both an economic and an environmental point of view, to conserve and restore soil productivity on the land suffering from and at risk of wind erosion were identified. Three options to mitigate and compensate for the losses of organic matter and nutrients on the affected lands are assessed for the comparative effectiveness of the measures in the Pishcha village, Shatsky district, Volyn region during 2008–2012. The research identified that the best measure against soil deflation was changing the economic use of lands at risk of soil erosion by wind by the halting cattle grazing and the creating overseeding grass meadows. This option Vol. 1, No. 2, 2015 6 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com tackling land degradation lead to trade-offs between different ecosystem services and those with a stake or interest in those services [5]. This trade-off could be between short-term provisioning services (e.g. crop and animal production or extractive uses of forests), upon which the resource-dependent poor often depend for their livelihoods, and the protection and enhancement of regulating and supporting services (such as nutrient cycling and soil formation), which have the potential to reverse land degradation, contribute to Land Degradation Neutrality [1], and enhance resilience to climate change [6]. Given the challenges associated with stakeholder engagement, it is essential to be clear about the reasons for engaging in the first place in order to clearly understand the context in which these trade-offs need to be made. y Traditional top-down approaches to tackling land degradation have often failed to deliver the intended results [6, 7]. Frequently, these problems can be attributed to the lack of ownership over, or buy in to, the process by those who have the power to implement decisions, such as state actors or land owners. This lack of engagement with and ownership of the process may then lead to these groups delaying or preventing the implementation of decisions in order to preserve their perceived and limited interests, or just to be obstructive of something they have been excluded from. However, care should be taken in applying the widespread implicit expectation that more participation is generally better [8, 9], especially where resources for engagement are limited and/or the total number of individual stakeholders is too large to successfully manage the constructive engagement of. Some research has highlighted that the adoption of participatory methods should be optimized rather than maximized [10]. In this case, stakeholders can be seen as having an interest, either directly or indirectly, in the provision of ecosystem services and products generated by the project area. The, mainly pasture, land in Pishcha village provides several ecosystem goods and services, which can be classified as follows: supporting (e.g. nutrient cycle; soil formation; primary production); provisioning (e.g. food through grazing of cattle); and regulating (e.g., conserving and protecting soil, water, and air resources through natural processes, such as filtration, detoxification, etc.). The goods and services provided by the pasture ecosystem are shown in table 1. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com The key goal of this project was to investigate the ecological, social and economic feasibility of measures to compensate for the losses of organic matter and nutrients resulting from soil deflation on, and to maintain and enhance the ecosystem services provided by, the affected lands in the Pishcha village, Shatsky district, Volyn region. Several options were identified and investigated: anic fertilizers in the form of mixed manure and straw; 1) Organic fertilizers in the form of mixed manure and straw; 2) Fertilization using ammophos to raise phosphorus levels and kalimag-30 fo restoring potassium; 3) Changing the economic use of land – replacing the grazing of grasslands with overseeding creation of perennial grass meadows [4]. Land degradation, as a highly complex process that interacts with other biophysical and social processes, affects different stakeholders at different scales. Successfully tackling land degradation therefore requires engagement with diverse stakeholders, who often have conflicting priorities. For example, many approaches to Land degradation, as a highly complex process that interacts with other biophysical and social processes, affects different stakeholders at different scales. Successfully tackling land degradation therefore requires engagement with diverse stakeholders, who often have conflicting priorities. For example, many approaches to Vol. 1, No. 2, 2015 7 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Another key goal of the project is the improved co-production of knowledge of land degradation and measures for the protection of soils among scientists, local community members, technical advisors, administrators and policy makers to facilitate better joined up thinking and action. These different groups are «stakeholders» [6], as they do, literally, have something at stake currently, or in the future as current circumstances evolve or if changes are introduced. As stakeholders they can influence processes or be influenced by them [11, 12, 13] and should be included in the engagement planning. In instances where a number of competing goals are identified for a stakeholder engagement process, it is usually necessary to prioritize them. There are a number of transparent and participatory ways of doing this with stakeholders, ranging from simple voting and ranking exercises to more complex prioritization exercises [6]. Vol. 1, No. 2, 2015 8 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Table 1 Ecosystem goods and services of pasture ecosystems Ecosystem Good or Service Benefits Economic Environmental Social/Cultural Forage production for livestock Sale of feed Hay, forage production Biodiversity (species and habitat) Clean air and water Carbon sequestration Soil enrichment from certain plants Landscape value Open space Rural communities dependent on forage- livestock systems Livestock production for humans Sale of meat and fiber products Farming operations Economic base for rural communities Recycling of nutrients Biodiversity (species and habitat) Clean air and water Carbon sequestration Soil enrichment from certain plants Landscape value Open space Satisfaction derived from farming as a way of life Serenity of pastoral scenery Fishing, hunting, bird watching Sales of licenses, gear, guide services Access rights on private or public lands Promotion of healthy wildlife populations Maintenance of biodiversity Control of hunted populations Landscape value Pleasure derived from outdoor activities Opportunity to observe wildlife Clean water Meet needs of domestic, agricultural, and industrial uses Sale of bottled water Income from recreation Human health Aquatic habitat Drinking water for wildlife Rejuvenation of riparian areas Watershed function Landscape value Aesthetics of unpolluted water Pleasure derived from recreation Biofuel feedstocks Sale of feedstocks and resultant biofuel products (depending on feedstock): Biodiversity maintenance Soil enrichment Carbon sequestration Greenhouse gas mitigation Reduced dependence on fossil fuels Source: adapted from Sustainable Rangelands Roundtable, 2008. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Table 1 Source: adapted from Sustainable Rangelands Roundtable, 2008. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com international development and environmental non-governmental organizations, professional bodies; and, local communities. Academic researchers and research institutes are sometimes considered as stakeholders through their role in collecting and analyzing relevant data, and providing knowledge and understanding to feed into evidence-based discussions for decision-making by stakeholders [11]. Stakeholder analysis, looking at the differences and similarities between the various stakeholders in relation to the project goals, is an indispensable tool in the planning phase of a project. It can help involve relevant stakeholders by providing an understanding of who has a stake in the social and/or natural system affected by the decision or action, and the nature of their claims and inter-relationships between each other [6]. As there are many different stakeholders, acting at a range of levels involved in this case of soil erosion, it is crucial to identify their varying degrees of power they exert on the situation and the level of interest in the particular situation. In this case it was felt that the best tool for this was an Interest-Influence Matrix (fig.1) for assessing and categorizing each stakeholder, and starting to develop a strategy for how to involve the actors. In the Interest-Influence Matrix, Influence (= power) captures the capacity of a stakeholder to have an effect on the situation, either directly on the ground or through and with other stakeholders. Interest shows the level of priority that a stakeholder gives specifically to the situation in hand. Figure 1 shows how these criteria help to identify four types of stakeholders: Stakeholders with high levels of interest and influence (in the cloud) are termed key players, and priority should be given to engaging actively with this group as they have both the will and the capacity to affect the situation. Context setters are highly influential, but have little interest in the specific situation. Because of this, they may have significant influence over the success of the project goals, but may be difficult to constructively engage with. However, particular effort to engage this group in the process can be rewarding and may be necessary to produce the facilitating environment required to achieve the project goals or to roll positive results out more widely. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com The analysis begins with the identification of stakeholders and their relationship to the project goals (Annex A). p j g Methods for identifying stakeholders include: self-selection (e.g. in response to advertisements or announcements); written records or census data, which can also be used to categorize stakeholders by age, gender, religion or place of residence; oral or written accounts of major events that can help identify those stakeholders who were involved or affected; and, using a checklist of likely stakeholder categories [6]. As an example, the types of stakeholders identified by the ELD Initiative in relation to land management are: Governments, i.e. political decision makers and administrators, including national and sub-national government authorities and agencies; private businesses, including multinationals and other big corporations, small and medium enterprises, farmers and smallholders; civil society represented by local, national and Vol. 1, No. 2, 2015 9 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Subjects have high levels of interest in the project, but predominantly have low levels of influence, and, although by definition they are supportive, they are unlikely to be able to play a significant role in the broad implementation of the project goals. From the perspective of stakeholder engagement, these are often marginal and can be considered «hard to reach». The low level of influence held by this group is often used as a justification for excluding them from the research process. However, as they are often the most affected by localized changes they warrant special attention to secure their engagement and to empower them to engage as equals with more influential participants in the project and the achievement of its goals. This will improve overall buy-in to the project goals and potentially increase the chances of long term success, as well as offsetting their potential to become more influential by forming alliances with other more influential stakeholders, who may be looking to obstruct the project goals, through frustration with not having their voices heard in Vol. 1, No. 2, 2015 10 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com processes that affect their lives and livelihoods. processes that affect their lives and livelihoods. ocesses that affect their lives and livelihoods. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com appreciation of the relationships between stakeholders relations and their particular roles in regard to the issue at hand. This, in turn, allows for the development of stakeholder engagement strategies more likely to foster the successful and sustainable solutions. Applying this tool to the Pishcha village (Annex B), the 4Rs are:  Access to and use of land and/or pasture – these may be customary or purchased rights;  Access to the ecosystem services and/or goods of the land and/or pasture; and,  Social, economic and environmental functions related to the land held by groups. Fig.1. Interest-Influence Matrix of Pishcha land’s Stakeholders Source: authorsʼ research. Fig.1. Interest-Influence Matrix of Pishcha land’s Stakeholders Source: authorsʼ research. The crowd are stakeholders who have little interest in or influence over the project goals and there is little need to consider them in much detail or to engage with them [6]. In Pishcha village, it was identified that the most important groups are the Village Council (who allocate tenancies), the potential tenants (who may, in the future, become tenants of the land) and farmers. However, while all these stakeholder groups have a strong interest in delivering sustainable land use change to combat the adverse impacts of wind erosion in many ways their power is only theoretic, as the notional power that they possess to make the necessary decisions is not matched by having access to the resources to do so. Those stakeholder groups who do have the resources have little or no awareness of the situation and lack the power to specifically influence the situation at the study site. Stakeholder engagement needs to address these gaps. One way of extending the analysis provided by the matrix is via the 4Rs categorization. Using the 4Rs tool enables the mapping of the identified stakeholders according to their roles, which consist of rights, responsibilities, relationships and revenues. In combination with knowledge about power dynamics, provided by the interest-influence matrix, categorization using the 4Rs tool allows for a deeper Vol. 1, No. 2, 2015 11 Responsibilities:  Land management tasks, including monitoring and control, coordination, decision making, and measurement;  Implementing decisions on rules and procedures; and,  Abiding by sustainable land management legislation and guidance. Revenues (benefits):  Direct benefits arising from proceeds derived from the land resources accessed (from grazing, haymaking and/or other activities such as agrotourism); and,  Indirect benefits arising from land restoration and preservation, and the development of rural areas, including health preservation, preservation of the soil ecosystems and the realisation their functions, potential increases in customers, and the formation of raw materials base. Relationships, which are informed by:  Analysis of Interest-influence matrix; and, Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com conflicts between stakeholders. The results of social network analysis is often represented as a social network map [6]. conflicts between stakeholders. The results of social network analysis is often represented as a social network map [6]. For the study at hand, the method of an Actor-Linkage Matrix was found to be most appropriate. Using this approach (Table 2) allowed for the interactions between the different actors and organisations, which are central to effective innovation systems, to be explored and analyzed. In order to appreciate the patterns of interaction at play in the case study and incorporate these into the development of the stakeholder engagement plan, it is important to map stakeholder linkages and to understand and describe the nature and purpose of those linkages. In this matrix, all relevant actors in the sector are marked on both the first row and first column of the matrix. Each box in the matrix then represents whether the linkage between two actors or organisations is cooperative, complimentary, or conflictual.  Roles in project implementation.  Roles in project implementation. There are a range of methods available when trying to understand relationships between stakeholders. These include: There are a range of methods available when trying to understand relationships between stakeholders. These include:  Venn diagrams that can be used to visualize the relative influence and interest of different stakeholders. The size of circles refers to the relative influence of the stakeholder, and overlaps between circles express the overlapping interests of the different groups.  Actor-linkage matrices, which consists of a table (i.e. matrix) listing all of the stakeholders and providing descriptions of the type of interrelation between each of them.  Actor-linkage matrices, which consists of a table (i.e. matrix) listing all of the stakeholders and providing descriptions of the type of interrelation between each of them.  Social Network Analysis, which is a method that seeks to provide insights into the patterns of communication, trust and influence that exists between actors in social networks at work in the situation being looked at. It utilizes techniques that analyze the structure of social networks and map stakeholder perceptions and values, as well as approaches that assess and analyze Vol. 1, No. 2, 2015 12 Table 2 Table 2 Stakeholder Relationships within the Actor-Linkage Matrix Stakeholder Group Farmers Village council Potential tenants Local Population Green Agrotourism farmer organizations Agricultural enterprises Department of AgriDevelopment SPInstitute Ecological Inspectorate Ministries GSP IUCN ESBN Farmers - coop conf compl coop coop coop -/coop conf conf -/coop - - - Village council - coop coop coop compl coop compl - - comp - - - Potential tenants - conf conf coop conf coop conf conf - - - - Local Population - coop - - - - - - - - - Population that provide Green Agrotourism - coop coop coop - - coop - - - Farmer organizations - coop coop coop - coop - - - Agricultural enterprises - coop - - coop - - - Department of AgriDevelopment* - coop coop compl coop coop coop SPInstitute - compl compl/ coop coop coop coop Ecological Inspectorate - compl/ coop coop coop coop Ministries - coop coop coop GSP - coop coop IUCN - coop ESBN - Remarks. Department of Agricultural Development of Shatsky District State Administration & Department of Agricultural Development of Volyn Regional State Administration h h Remarks. Department of Agricultural Development of Shatsky District State Administration & Department of Agricultural Development of Volyn Regional State Administration Source: authorsʼ research Analysis of the Actor-Linkage Matrix provided a number of conclusions:  As farmers have a high interest but a low state of power it is crucial that there is cooperation and complementation with the main powerful stakeholders such as the Department of Agricultural Development and the Farmer Organizations.  Since potential tenants and land owners have higher power there is the need to address the potentially conflicting linkage between these stakeholders and the farmers, as only together are they able to effectively change the current agricultural practices (transfer of rights and best agricultural practice). Vol. 1, No. 2, 2015 13 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com  The Matrix indicates that potential conflicts are mainly associated with the potential tenants/land owners. When used conjunction with Actor-Linkage Matrix, using Knowledge Mapping allows for a clear picture of what stakeholder groups are present, and how they influence each other and the potential attainment of the desired sustainable land management goals through their communication, provision of information and decisions. The outcome of this analysis is presented in Figure 2. Fig. 2. Stakeholder Map and grouping of stakeholders according to their relationships Source: authorsʼ research based on [13]. In order to prioritize stakeholders for involvement in the stakehold Fig. 2. Stakeholder Map and grouping of stakeholders according to their relationships Source: authorsʼ research based on [13] relati Source: authorsʼ research based on [13]. [ ] In order to prioritize stakeholders for involvement in the stakeholder engagement process, Table 3 maps out the interest of the various stakeholders in the ecosystem services that the land is providing and that the proposed land use change and adoption of sustainable land management practices intends to conserve and enhance. They are Beneficiaries (B) of the ecosystem services, who may reap the benefits of the ecosystem services but may also suffer the costs of unsustainable land management and the resultant impacts of soil erosion, and those with an Interest (I) in ensuring the sustainable management of the land and the reduction of soil erosion. The stakeholdersʼ types of benefits/costs and interest are denoted by: income (Inc); Vol. 1, No. 2, 2015 14 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com and wellbeing (H&W); and information (Inf). Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Table 3 Stakeholder engagement prioritization Stakeholder / Beneficiary Groups Ecosystem Service Supporting Provisioning Regulating Cultural Nutrient Cycling Soil Formation Primary Production Food Fuel Fresh Water (sediment) Climate Regulation (Carbon) Water Regulation & Purification Disease Regulation (Dust control) Recreational, Spiritual and Educational Farmers B Inc B Inc B Inc B Inc B Inc B Inc B Inc H&W B Inc Village council I Gov I Gov I Gov I, Dev Gov I Gov I Gov I Gov I, Dev Gov Potential tenants B Inc B Inc B Inc B Inc B Inc B Inc B Inc B Inc Local Population B H&W B H&W B H&W B, Inc H&W B H&W Population that provides Green Agrotourism B Inc B H&W B Inc H&W Farmer organizations I Rep I Rep I Rep Agricultural enterprises B Inc B Inc Department of AgriDevelopment I Dev Reg I Dev Reg I Dev Reg I Dev Reg I Dev Reg I Dev Reg SPInstitute I Reg Inf I Reg Inf I Reg Inf I Reg Inf I Reg Inf I Reg Inf I Reg Inf Ecological Inspectorate I Reg Inf I Reg Inf I Reg Inf I Reg Inf I Reg Inf I Reg Inf I Reg Inf Ministries I Gov Reg I Gov Reg I Gov Reg I Gov Reg I Dev Reg I Gov Reg I Gov Reg I Gov Reg I Gov Reg I Gov Reg GSP I, Inf I, Inf IUCN I, Inf I, Inf ESBN I, Inf I, Inf Remarks. Department of Agricultural Development of Shatsky District State Administration & Department of Agricultural Development of Volyn Regional State Administration. Source: authorsʼ research. The major stakeholders of the land of Pishcha village Volynska Region North- Remarks. Department of Agricultural Development of Shatsky District State Administration & Department of Agricultural Development of Volyn Regional State Administration. Source: authorsʼ research. The major stakeholders of the land of Pishcha village, Volynska Region, North- West Ukraine were identified and divided into three levels with the assistance of expert assessment. The first level (micro/local), containing stakeholders representing private interests, local government and civil society, is key to the success of the The major stakeholders of the land of Pishcha village, Volynska Region, North- West Ukraine were identified and divided into three levels with the assistance of expert assessment. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com project and the adoption of proposed new approaches. These groups are characterized by their high interest in soil conservation and the restoration of the land. Almost all members of these groups can influence the land use change process. Farmers, the Local Population and Local Organisations can exercise moral pressure, but they haven’t the money to invest in making and adapting to change. The Village Council can receive state monies, but they are often slow to act and do not possess the requisite technical expertise. Potential tenants are mainly agricultural investors. They can be very difficult to engage as their identities are often not known until they make the investment. The next step is to design a stakeholder engagement process. The next step is to design a stakeholder engagement process. h b f k h ll i l d d i ll There are a number of key components that are usually included in a well- designed stakeholder engagement plan [14]: a description of the context; systematic identification and analysis of the interests and influence of stakeholders; the setting of engagement objectives and/or expected outcomes; the identification of appropriate engagement techniques or activities to meet the objectives; and, consideration and identification of risks and indicators to monitor the progress, ensuring that engagement activities are appropriately resourced and integrated with project management. The engagement process can be defined as the process through which the identified stakeholders have an opportunity to discuss and provide their perspective on possible options and pathways for action before decisions are made in order to inform which are best in the circumstances. In this case (table C.1 in Annexes), the process enables identified stakeholders to address current land use issues affecting them through (i) identifying more sustainable alternative land management practices from a pre-established list of options, and (ii) identifying suitable pathways to establish such sustainable alternative land management practices. The next step is defining roles and responsibilities for implementation, milestones, and identifying a realistic timeline for completion (table C.2) and identifying what types and levels of engagement require planning for with the different stakeholder groups (table C.3). An important part of the engagement plan is the development budget (table C.4), especially where finances are restricted or external funding is being sought to implement the work. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com The first level (micro/local), containing stakeholders representing private interests, local government and civil society, is key to the success of the Vol. 1, No. 2, 2015 15 driver. The emphasis on particular outcomes, rather than on the completion of activities, requires that program implementers monitor key outcome variables and make midstream corrections as necessary. Through facilitating a focus on specific expected outcomes, the results framework provides a strong tool for engaging stakeholders in thinking through the theory of change underpinning the intervention. Discussion centered on a results framework provides program staff and other stakeholders with the opportunity to clarify and adjust the development hypothesis, allowing partners to harmonize their efforts or to identify areas where additional program activities will be needed. Through adopting a participatory approach to discussions, using the results framework serves a critical role in building understanding, consensus and ownership around shared objectives and clarifying the different stakeholders' interpretations of the elements of the development hypothesis [15]. The first step to knowing whether engagement is working is to decide on the approach to take to monitoring and evaluation, including whether the intervention is primarily looking at the outcomes or process of engagement (or both). The goals of the intervention should be incorporated into the engagement plan for monitoring and evaluation. Developing appropriate indicators of progress and success is a key step when producing the framework, as they provide powerful tools for monitoring progress and evaluating the intervention processes and outcomes. A good indicator should, wherever possible, provide cost-effective, timely and accurate information with minimum effort [6]. In some cases, it may be possible to work with stakeholders to collect and analyze indicator data, which can reduce costs and improve the effectiveness of monitoring and evaluation. The potential benefits from this can be enhanced if the indicators are developed using participatory approaches and/or to have social appeal to and resonance with the stakeholders, which can build interest and buy in to the work [6]. The preferred format and level of detail for results frameworks vary according to the organizations involved in, and by the scope and scale of the intervention, but all include the same basic components to guide implementers in achieving, and evaluators in assessing, results. The example developed for this project-level intervention results frameworks is shown in Table C.5. Conclusions. There is a clear and demonstrable need for concerted action to be taken to address the ongoing issue of soil erosion resulting from wind in Ukraine. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com The budget for engagement should be proportional to the overall budget for the work and, if necessary, engagement activities should be reviewed and tailored to available resources. Finally, it is essential to answer the question «How will we know that the intervention has succeeded»? A well-conceived and simple results framework, clearly outlining the ultimate objectives of the intervention rather than simply listing implementation activities, processes, and inputs, allows for this question to be addressed. For stakeholder engagement, a results framework can be a useful management tool, with program implementation assessed in direct relationship to progress in achieving results, at the outputs, outcomes, and impact levels, with the strategic objective being the ultimate Vol. 1, No. 2, 2015 16 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com This paper has examined some of the underlying theory, the potential methodologies and how they could be methodically applied, and the feasibility of the stakeholder engagement necessary to support land use changes to improve resilience against wind erosion in Pishcha village, Shatsky district, Volyn region. While, in theory, the proposed work is possible there are a number of practical issues that require addressing before it is genuinely feasible. Practical scoping, through pilot projects, is required to fully assess the feasibility and efficacy of stakeholder engagement on the ground, and to adapt general methods to local conditions. g g g p g In respect to the scale of the intervention examined in this and the previous paper [4], where the project area of 5 hectares is very small, the costs for materials and human resources identified for stakeholder engagement are impractical and running this project is not cost effective. There could, however, be considerable benefits of scaling up the intervention as there would be little or no additional costs at the regional and national levels, and many opportunties for savings (per local area/community engaged) on the stakeholder engagement at the local level. It is also likely that scaling up the work would increase the robustness of any lessons learnt or best practise identified by the work. There are currently considerable barriers to accessing the funding necessary to run this project, in the form presented here or scaled up, from the State. and it is also unlikely that private funding can be obtained. Other potential sources of funding may be accessible and their criteria would inform the final project design. There are a number of ways to develop the work in order to improve the feasibility of the work and chances of securing funding: 1. There are other areas in the Volyn region that face similar wind erosion issues to the project area. Involving these other local communities in the research will increase cost effectiveness, and the strength and applicability of the analysis. It would allow for a more comprehensive assessment of the issues that require addressing, through a greater understanding of the commonalities and differences between a range of locations and contexts, and for the development of approaches with the potential for wider application. 2. The project could provide a greater emphasis on the development of green agritourism enterprises with the local populations. driver. The effective engagement of a range of stakeholders, with varying types and levels of interest, is vital in order to facilitate the joined up action and ensure the buy in necessary to identify and make the land use changes required on the scale that dealing with wind erosion necessitates. Work is needed to explore and stimulate effective mechanisms of interaction around the issues affecting soil erosion between the range of stakeholders in Ukraine, which, for various reasons, is currently extremely weak. Vol. 1, No. 2, 2015 17 Vol. 1, No. 2, 2015 References ELD Initiative (2015), Pathways and options for action and stakeholder engagement, based on the 2015 ELD Massive Open Online Course «Stakeholder Engagement». Practitioner’s Guide, available at: www.eld-initiative.org. 7. Cramb, R. A., Garcia, J. N. M., Gerrits, R. V. and Saguiguit, G. C. (1999), Smallholder adoption of soil conservation technologies: evidence from upland projects in the Philippines. Land Degradation & Development, vol. 10, pp. 405–423. 7. Cramb, R. A., Garcia, J. N. M., Gerrits, R. V. and Saguiguit, G. C. (1999), Smallholder adoption of soil conservation technologies: evidence from upland projects in the Philippines. Land Degradation & Development, vol. 10, pp. 405–423. p j pp g p pp 8. Arnstein, S. (1969), A Ladder of Citizen Participation. Journal of the American Institute of Planners, vol. 35(4), pp. 216–224. p j pp g p pp 8. Arnstein, S. (1969), A Ladder of Citizen Participation. Journal of the American Institute of Planners, vol. 35(4), pp. 216–224. f pp 9. Blackstock, K. L., Kelly, G. J. and Horsey, B. L. (2007), Developing and applying a framework to evaluate participatory research sustainability. Ecological Economics, vol. 60(4), pp. 726–742. 9. Blackstock, K. L., Kelly, G. J. and Horsey, B. L. (2007), Developing and applying a framework to evaluate participatory research sustainability. Ecological Economics, vol. 60(4), pp. 726–742. pp 10. Neef, A. and Dieter, N. (2011), Stakeholder participation in agricultural research projects: a conceptual framework for reflection and decision-making. Agriculture and Human Values, vol. 28.2, pp. 179–194. pp 10. Neef, A. and Dieter, N. (2011), Stakeholder participation in agricultural research projects: a conceptual framework for reflection and decision-making. Agriculture and Human Values, vol. 28.2, pp. 179–194. 11. Quillérou E. and Falk T. Stakeholder engagement and the Economics of Land Degradation (ELD) Initiative, аvailable at: www.eld-initiative.org 11. Quillérou E. and Falk T. Stakeholder engagement and the Economics of Land Degradation (ELD) Initiative, аvailable at: www.eld-initiative.org g g 12. Reed, M. S. (2008), Stakeholder participation for environmental management: A literature review. Biological Conservation, vol. 141, is. 10, pp. 2417–2431. 12. Reed, M. S. (2008), Stakeholder participation for environmental management: A literature review. Biological Conservation, vol. 141, is. 10, pp. 2417–2431. 13. Aaltonen, S. and Kreutz, E. (2009), Engage your stakeholders: stakeholder involvement toolkit for local authorities, available at: http://www.ubcenvironment.net/library/publication/engage-your-stakeholders-toolkit. 13. Aaltonen, S. and Kreutz, E. (2009), Engage your stakeholders: stakeholder involvement toolkit for local authorities, available at: http://www.ubcenvironment.net/library/publication/engage-your-stakeholders-toolkit. 14. Reed, M. and Attlee, A. References 1. ELD Initiative (2015), The value of land: Prosperous lands and positive rewards through sustainable land management, available at: www.eld-initiative.org. 1. ELD Initiative (2015), The value of land: Prosperous lands and positive rewards through sustainable land management, available at: www.eld-initiative.org. 2. Edward B. Barbier and Jacob P. Hochard (2014), Land Degradation, Less Favored Lands and the Rural Poor: A Spatial and Economic Analysis. A Report for the Economics of Land Degradation Initiative. Department of Economics and Finance, University of Wyoming, available at: www.eld-initiative.org. 2. Edward B. Barbier and Jacob P. Hochard (2014), Land Degradation, Less Favored Lands and the Rural Poor: A Spatial and Economic Analysis. A Report for the Economics of Land Degradation Initiative. Department of Economics and Finance, University of Wyoming, available at: www.eld-initiative.org. 3. ELD Initiative (2014), Principles of economic valuation for sustainable land management based on the Massive Open Online Course «The Economics of Land Degradation». Practitioner’s Guide, available at: www.eld-initiative.org. 3. ELD Initiative (2014), Principles of economic valuation for sustainable land management based on the Massive Open Online Course «The Economics of Land Degradation». Practitioner’s Guide, available at: www.eld-initiative.org. 4. Kucher, A., Kazakova, I., Kucher, L., Kozak, H., Schraml, A., Koka, H. and Priest, W. (2015), Economics of soil degradation and sustainable use of land in danger of wind erosion. Agricultural and Resource Economics: International Scientific E-Journal, [Online], vol. 1, no. 1, available at: www.are-journal.com. 4. Kucher, A., Kazakova, I., Kucher, L., Kozak, H., Schraml, A., Koka, H. and Priest, W. (2015), Economics of soil degradation and sustainable use of land in danger of wind erosion. Agricultural and Resource Economics: International Scientific E-Journal, [Online], vol. 1, no. 1, available at: www.are-journal.com. j 5. Reed, M., Stringer, L., Dougill, A., Perkins, J., Atlhopheng, J., Mulale, K. and Favretto, N. (2015), Reorienting land degradation towards sustainable land management: linking sustainable livelihoods with ecosystem services in rangeland systems. Journal of Environmental Management, vol. 151, pp. 472–485. 5. Reed, M., Stringer, L., Dougill, A., Perkins, J., Atlhopheng, J., Mulale, K. and Favretto, N. (2015), Reorienting land degradation towards sustainable land management: linking sustainable livelihoods with ecosystem services in rangeland systems. Journal of Environmental Management, vol. 151, pp. 472–485. y f g pp 6. ELD Initiative (2015), Pathways and options for action and stakeholder engagement, based on the 2015 ELD Massive Open Online Course «Stakeholder Engagement». Practitioner’s Guide, available at: www.eld-initiative.org. 6. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com There is considerable untapped tourism potential in Western Ukraine, helped by the absence of large industrial enterprises in the region and the retention of traditional extensive agricultural systems. Incorporating the potential scope to develop this business sector in the region could greatly alter the dynamics of the intervention and provide alternatives for change that stakeholders are interested in and that can deliver wider economic and environmental benefits. This could result in a widening of the potential funding streams available to deliver the work. 3. Collaboration with international partners facing similar and related issues (e.g. Spain and Greece in the E.U.) may allow for the development of a larger trans- national project that can attract funding from different sources to those currently available at the national level. Vol. 1, No. 2, 2015 18 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com References (2015), Knowledge exchange training for research impacts. Unpublished training manual, Sustainable Learning project and Living with Environmental Change programme. 2nd Ed 14. Reed, M. and Attlee, A. (2015), Knowledge exchange training for research impacts. Unpublished training manual, Sustainable Learning project and Living with Environmental Change programme. 2nd Ed Vol. 1, No. 2, 2015 19 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Vol 1 No 2 2015 20 Achieving Results: A How-to Guide. Independent Evaluation Group. The World Bank, Washington, DC. Annexes Table A Stakeholder Identification Stakeholder Name Type of Stakeholder Description Relationship to the land (Level/Role/Policy context) The 1st level (local) 1. Farmers / (households) Private Land user groups, concerned with: land Land user. May include key influencers. Uses land for household livelihoods and income generation. Villagers use land to graze cattle (without paying/commons) 2. Village council Local Government Land owner, concerned with: land, economically profitable exploitation, conservation, preservation. Land owner. Include key influencers. This land is reserve land, so the village council may transfer the land lease if a new tenant appears. 3. Potential tenants and landowner Private Concerned with: land economically profitable exploitation. May include key influencers. Uses land for income generation. They may begin to take rent pay for grazing cattle from population (farmers) or change the direction of its economic use. 4. Local Population Private Concerned with: conservation May include key influencers. Potential health problems from dust (air and water pollution) 5. Population that provides «green agrotourism» Private Concerned with: land, economically profitable exploitation, conservation. Land user/ Land owner. May include key influencers. Lack of grazing threatens the provision of services for tourists and the use of fresh organic milk 6. Local and/or national landowner/farmer organizations Civil Society Concerned with: land, economically profitable exploitation, conservation, preservation. Representation of landowners/farmers The 2nd level (regional, national) 1. Agricultural produce processors, enterprises from milk and meat cattle processing Private Concerned with conservation Lack of grazing causes a reduction in the supply of milk and meat for processing, which, in turn, could cause damage to the economy and food security 2. Department of Agricultural Development of Shatsky District State Administration & Department of Government Concerned with: land, economically profitable exploitation, conservation, preservation. Participate in the formulation and implementation of social and rural development policy in the countryside. Table A 20 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural Development of Volyn Regional State Administration 3. Volyn branch of state institution «Soils Protection Institute of Ukraine» Government/ research institute Concerned with: conservation, preservation The Institute carries out the development of proposals and implementation of united scientific-technical policy in the field of soil fertility, and the rational use and environmental safety of agricultural land. Its recommendations are desirable but not mandatory for implementation. 4. State Ecological Inspectorate of Ukraine (and its regional offices) and The State Agency of Land Resources of Ukraine Government Concerned with conservation SEI – supports the rational use, restoration and protection of natural resources, including land; SALR – ensures the implementation of measures aimed at the rational use and protection from harmful human impacts of land. 5. Ministry of ecology and natural resources of Ukraine & Ministry of Agrarian Policy and Food of Ukraine Government Concerned with conservation Formulating state policy in the areas of protection of the environment, including restoration and protection of land The 3rd level (global) 1. Global Soil Partnership (GSP) International environmental organizations Concerned with conservation Concerned with the environmental well- being of soils, including prevention of erosion and land degradation. 2. International Union for Conservation of Nature (IUCN) International environmental organizations Concerned with conservation Is committed to the conservation of nature and natural resources, preservation of ecosystem integrity, ensuring the use of natural resources in a sustainable and reasonable manner. 3. European Soil Bureau Network (ESBN) International research organizations Concerned with conservation The main tasks are to collect, harmonize, organize and distribute soil information for Europe. Source: authorsʼ research. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Table B Table B The «4 R's» stakeholder analysis methods Stakeholder Rights Responsibilities Revenues (benefits) Relationship The 1st level local) 1. Farmers / (households) User rights. Part access to pasture Abiding by rational land management guidance Direct benefits arising from proceeds from land resources accessed Defender Employees/ performers 2. Village council Owner rights. Access to land Decision making Abiding by sustainable land management legislation and guidance Implementing decisions on rules and procedures Monitoring and control Indirect benefits from preservation and development of rural areas Promoter Employees/ performers 21 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Coordination Decision making 3. Potential tenants and landowner User rights Potential access to pasture, land Abiding by sustainable land management guidance Potential direct benefits from land resources accessed Promoter Partners/ employees/ performers 4. Local Population None Part access to pasture Negotiate on behalf of the community Abiding by sustainable land management guidance Indirect benefits arising from preservation of the soil ecosystem and realization of ecosystem functions/services and health preservation) Defender Community 5. Population that provides «green agrotourism» None Part access to pasture, land Abiding by sustainable land management guidance Potential indirect benefits from an increase in customers and income Defender Partners 6. Local and/or national landowner/farmer organizations Negotiate on behalf of the community Implementing decisions on rules and procedures Coordination - Latent Community The 2nd level (regional, national) 1. Agricultural produce processors, enterprises from milk and meat cattle processing (business) Negotiate on behalf of the business - Indirect benefits from formation of raw materials base Latent Partners 2. Department of Agricultural Development of Shatsky District State Administration & Department of Agricultural Development of Volyn Regional State Administration None Negotiate on behalf of the community and/or government Implementing decisions on rules and procedures Monitoring and control Coordination Decision making Indirect benefits arising from preservation of the soil ecosystem and realization of ecosystem functions/services Apathetic Government 3. Volyn branch of state institution «Soils Protection Institute of Ukraine» None Access to land Negotiate on behalf of the government Supervision management Monitoring and control Implementing decisions on rules and procedures Decision making Measurement - Apathetic Partners 4. State Ecological Inspectorate of Ukraine (and its regional offices) and The State Agency of Land Resources of Ukraine None Access to land Negotiate on behalf of the government Supervision management Sanction Monitoring and control Measurement Decision making Implementing decisions on rules and procedures - Latent Partners 5. Table B Ministry of ecology and natural resources of Ukraine & Ministry of Agrarian Policy and Food of Ukraine None Negotiate on behalf of the government Supervision management Implementing decisions on rules and procedures monitoring and control Coordination Decision making Indirect benefits arising from preservation of the soil ecosystem and realization of ecosystem functions/services Latent Government The 3rd level (global) 1. Global Soil Partnership (GSP) None Negotiate on - Indirect benefits arising from preservation of the Apathetic Community Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com 22 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com 2. International Union for Conservation of Nature (IUCN) behalf of the global community - soil ecosystem and realization of ecosystem functions/services 3. Table B European Soil Bureau Network (ESBN) - Source: authorsʼ research Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Table C.1 on of the project, with objectives and expected outputs and outcomes Vol 1 No 2 2015 23 Table C.1 Description of the project, with objectives and expected outputs and outcomes Stake- holders Description of perceived area of interest Engagement outcomes Format (Mode of Channel/Communication Material to be prepared ahead Farmers Economic Exploitation Health & Wellbeing  We want them to change their use and management of pasture  We want them to understand what it means to them financially in lay manʼs terms  We want them to be aware of the wider issues  We want them to explain how they can influence and cooperation with the government  We want them to inform us of opportunities for and barriers against land use change  We want them to let us know what actions they take and how these work Letters to farmers with information and invitation to open meeting (outside farming hours and locally) to explain work and informing them that researchers will be visiting pasture in future Meeting to explain work, identify more interested farmers and, where possible make arrangements for meeting Interview with farmers at farms Letters and information Materials for meeting Semi-structured interview/questio nnaire for interview with farmers Village council Economic Exploitation  We want them to change land use  We want them to understand what it means to them financially in lay man's terms  We want them to be aware of the wider issues  We want them to inform us of opportunities for and barriers against land use change  We want them to let us know what actions they take and how these work Meeting to explain work, present results of previous research, request permission for researchers to visit pasture in future Phone contact, e-mail Regular Meeting Materials for meeting Potential tenants and landowner Economic Exploitation  We want to find a new responsible owners  We want them to understand what it means to them financially  We want them to be aware of the wider issues  We want them to let us know what plans they have and actions they want to take  We want make contact, agree Stakeholder conference that will include key stakeholders (representatives of farmers, council, scientist, farmer organizations) that will inform them of the work and allow for them to inform its development Materials for conference Semi-structured Vol. Table B 1, No. 2, 2015 23 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com on possible cooperate, and conduct observations and research Interviews with potential tenants and landowners interview/questio nnaire for interview with representatives Local Population Health & Wellbeing  We want them to be aware of the wider issues  We want explain how they can influence and cooperate with the government Letters to Population with information and invitation to open meeting to explain the project, informing them that researchers will be visiting, their role in the maintenance and preservation of the local environment, and the earning possibilities of land use change. Stakeholder conference for key stakeholders (representatives of farmers, council, scientist, farmer organizations) to inform them of the work and allow for them to inform its development Consultative meetings Letters and information Materials for conference Materials for meeting Population that provides «green agro- tourism» Economic Exploitation Health & Wellbeing  We want them to begin a new business on the base on sustainable land use  We want them to understand what it means to them financially in lay man's terms  We want explain how they can influence by and cooperation with government Local and/or national landowner/ farmer organiza- tions Information Representation We want them to let us know what actions they take and how these work Stakeholder conference that will include key stakeholders (representatives of farmers, council, scientist, farmer organizations) Materials for conference Agricultural produce processors, enterprises from milk and meat cattle processing (business) Economic  We want them to be aware of the wider issues  We want them to become sponsors of and investors in change land use  We want them to understand what it means to them financially, maybe in lay businessmanʼs terms Letters to Enterprises with information and invitation to open meeting to explain work Individual meetings to agree on cooperation and conducting observations and research with enterprises and farmers, council or farmer organizations Phone contact, e-mail Letters and information Volyn branch «Soils Protection Institute of Ukraine» Information Representation  We want them to monitors land use in village control  We want them provide information to local residents and local authorities about the state Consultative meetings Invitation letters and information Materials for meetings Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Vol. 1, No. Table B 2, 2015 24 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com State Ecological Inspectorate of Ukraine (regional offices) and Agency of Land Resources lands, their possible improvement alternatives use  We want them conducted an environmental audit Ministry of ecology and natural resources & Ministry of Agrarian Policy and Food of Ukraine Information Representation  We want them to perform their legislative functions and improve land legislation  We want them monitored the execution of orders, resolutions, etc.  We want them developed mechanism ecological insurance  We want them to initiate an environmental audit at local level Stakeholder conference for key stakeholders (representatives of farmers, council, scientist, farmer organizations) to inform them of the work and allow for them to inform its development Invitations and information Materials for conference Legislative briefings and proposals Departments of Agricultural Develop- ment 1. GSP  We want them to participate in securing funding.  We want them provide international assistance in specific projects (including «green agrotourism»).  We want them to carry out independent monitoring and provide general reports. e-mail Letters and information 2. IUCN 3. ESBN Source: authorsʼ research Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Table C.2 Engagement with Government groups will focus on ensuring they have the capacity to organize environmental monitoring, to cont the implementation of its orders, and, most importantly, to learn to pay attention to the needs of lower level stakeholders. The development and maintenance of internatio contacts is of key importance to creating pl for the future. The final Joint Report should be example for other communities faced wi similar issues and the development and of implementation for projects to protect soils S th ʼ h Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Engagement with the legislative process will be initiated and carried forward. Engagement with the legislative process will be initiated and carried forward. Engagement with Government groups will focus on ensuring they have the capacity to organize environmental monitoring, to control the implementation of its orders, and, most importantly, to learn to pay attention to the needs of lower level stakeholders. The development and maintenance of international contacts is of key importance to creating plans for the future. The final Joint Report should be example for other communities faced with similar issues and the development and of implementation for projects to protect soils. Table C.2 Table C.2 Timeline and Engagement Process Matrix Timeline Research Process (Objectives, Expected Output, Deadlines, etc.) The Why and The Facilitation Needed Engagement Process (Discussion and Activities) Managing the Stakeholders Participatory Process Beginning of the engagement process The initial phase should take no more than 6 months. During 1st and 2nd months initial engagement and explanatory work will be conducted with farmers and the village council, because they have to change plans of land use and prepare for spring sowing. We will engage all of the population and wider interests as we will need to find investors and cooperation opportunities in parallel to identifying farmers willing to change. At this stage it is necessary to establish contacts with all groups of stakeholders. to hold meetings, perform outreach, and complete questionnaires. It is necessary to identify potential investors and negotiate cooperation. During the engagement process Phase 1 The village council will be engaged in order to secure support for the change of land use. Environmental organizations will perform research on the soils and provision of ecosystem services. The team of scientists should develop and submit initial proposals for legislative change. In the second period the main interaction will occur between us, the village council and the government. Regular contact will be maintained with all participants. State environmental organizations will implement regular monitoring of changes in the quality of land and the agreements for its operation. Timeline and Engagement Process Matrix Vol. 1, No. 2, 2015 25 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Phase 2 At this time the main work in Volyn will be monitored by local government. Results will be shared twice a year. We will maintain contact (mail, phone) with the local population as technical consultants (and arrange site visits if necessary) Engagement with the legislative process wi be initiated and carried forward. End and follow up of engagement process Final analysis of the land use change, soils, and ecosystem services. Final interviews with selected stakeholders to understand social and economic framework for land use change decisions. Identify the benefits and costs generated by the project. Final presentation of project analysis and results to stakeholders through meetings, discussions and public conference. Source: authorsʼ research. Table C.3 Field observations and data obtained from farmers/reports from council Formation of real interest and motivation for soil protection activities and implementation of sustainable land use All the participants actively engage with soil protection activities, or they interact and respond to other proposals Interviews with stakeholders on motives and perceptions Improving knowledge on wind erosion, land degradation and ways to resisting these processes (for farmers, population and others) The brochure, covering the issues in clear language targeting farmers and the local population, reaches every household and 5 classes are held at local schools to raise awareness of the issues. Questionnaire on knowledge Assessment of innovations to reduce erosion Observation of behavior and knowledge transfer Raising the level of financial and political awareness (for farmers, population and others) All the relevant government stakeholders get engaged in the workshops. 5 media talk shows engaging politicians and donors are carried out. Amount of subsidies (support by local government population and farmers in new business) Improvement of living standards of the villagers (health, financial situation) Engagement of 3 big industry representatives who communicate with local farms on new product lines and markets. Engagement with local healthcare providers regarding health and well-being impacts. Quantitative data: income per household, other socio-economic factors, number of respiratory or other related diseases The growth of business activity of the villagers, opening of new businesses Stakeholders engage in creating local tourism initiative. Delivery of two educational field visits, one with tourism agencies and journalists, and one with industry representatives focused on local ecological/organic products Number of businesses engaged Wages Employment Table C.4 Example of personnel and other requirements to achieve the engagement process Major Activity Personnel needs (scientist appointments) Facilitation and Coordination Needs Other Needs Total Calculated costs, USD Beginning of the engagement process Design and sending letters to farmers with information and invitation to open meeting to explain work 1 Coordination print material, post, transport, meeting Analysis 3 Total 3903.6 During the engagement process Production costs for the change of economic use of land 1 year 3 Coordination Consultative meetings with Government and Private groups 1 Facilitation print material, transport, conferences Total 56717.8 End and follow up of engagement Final conference and meeting 3 Coordination Facilitation material, transport, conferences Total 1928.6 Total Calculated costs, USD 62550.0 Source: authorsʼ research. Table C.3 able C.3 Matrix for planning activities for different level of engagement (Evaluation scenarios) Timing Stakeholders Beginning of the engagement process During the engagement process End and follow up of engagement Phase 1 Phase 2 Farmers Collaborate Inform / Involve Inform / Involve Inform Village council Collaborate Collaborate Collaborate Collaborate Potential tenants and landowner Involve / Collaborate Involve / Collaborate Involve / Collaborate Involve / Collaborate Local Population Involve Involve Inform Inform Population that provides «green agrotourism» Involve Involve Involve Collaborate Local and/or national landowner/farmer organizations Inform/Involve Consult Consult Agricultural produce processors, enterprises from milk and meat cattle processing (business Inform/Involve Consult Consult Involve Soils Protection Institute of Ukraine, State Ecological Inspectorate of Ukraine and The State Agency of Land Resources Involve Collaborate Collaborate Collaborate Ministry of ecology and natural resources of Ukraine & Ministry of Agrarian Policy and Food of Ukraine Inform Consult Collaborate Collaborate Departments of Agricultural Development Consult Consult Consult International organization Inform Inform Notes: Inform – most basic level of engagement; Consult – specific questions are asked but not full discussion or interaction; Involve – more opportunity for discussion; Collaborate – involved to some extent in full decision making (Durham, 2014). Source: authorsʼ research. Matrix for planning activities for different level of engagement (Evaluation scenarios) Vol. 1, No. 2, 2015 26 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com j Table C.4 Example of personnel and other requirements to achieve the engagement process Major Activity Personnel needs (scientist appointments) Facilitation and Coordination Needs Other Needs Total Calculated costs, USD Beginning of the engagement process Design and sending letters to farmers with information and invitation to open meeting to explain work 1 Coordination print material, post, transport, meeting Analysis 3 Total 3903.6 During the engagement process Production costs for the change of economic use of land 1 year 3 Coordination Consultative meetings with Government and Private groups 1 Facilitation print material, transport, conferences Total 56717.8 End and follow up of engagement Final conference and meeting 3 Coordination Facilitation material, transport, conferences Total 1928.6 Total Calculated costs, USD 62550.0 Source: authorsʼ research. Table C.5 Setting Success Criteria and according Measures of Success Stakeholder Involvement Plan Objective Success Criteria and Measure of Success Changing use of pasture (for farmers and council) 60 % of the inhabitants participate at our meetings; 90 % are reached by our teams at home or other places. Table C.3 T bl C 5 Example of personnel and other requirements to achieve the engagement process Table C.5 Setting Success Criteria and according Measures of Success Stakeholder Involvement Plan Objective Success Criteria and Measure of Success Changing use of pasture (for farmers and council) 60 % of the inhabitants participate at our meetings; 90 % are reached by our teams at home or other places. Field observations and data obtained from farmers/reports from council Formation of real interest and motivation for soil protection activities and implementation of sustainable land use All the participants actively engage with soil protection activities, or they interact and respond to other proposals Interviews with stakeholders on motives and perceptions Improving knowledge on wind erosion, land degradation and ways to resisting these processes (for farmers, population and others) The brochure, covering the issues in clear language targeting farmers and the local population, reaches every household and 5 classes are held at local schools to raise awareness of the issues. Questionnaire on knowledge Assessment of innovations to reduce erosion Observation of behavior and knowledge transfer Raising the level of financial and political awareness (for farmers, population and others) All the relevant government stakeholders get engaged in the workshops. 5 media talk shows engaging politicians and donors are carried out. Amount of subsidies (support by local government population and farmers in new business) Improvement of living standards of the villagers (health, financial situation) Engagement of 3 big industry representatives who communicate with local farms on new product lines and markets. Engagement with local healthcare providers regarding health and well-being impacts. Quantitative data: income per household, other socio-economic factors, number of respiratory or other related diseases The growth of business activity of the villagers, opening of new businesses Stakeholders engage in creating local tourism initiative. Delivery of two educational field visits, one with tourism agencies and journalists, and one with industry representatives focused on local ecological/organic products Number of businesses engaged Wages Employment 27 Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Increasing the level of interaction and communication between stakeholders at the local level Establishment of local village forums gathering once a month where all residents and others are invited (and at least 50 % participate); setting up a weekly radio program in the local radio Number of interaction opportunities, e.g. at meetings, workshops etc. Table C.3 Increase of effectiveness of village control land use Establishment of local village forums gathering once a month where land use practices are discussed by all participants Hours spent by those responsible on controlling land use Participation at local village forums Rising effectiveness of perform legislative functions A conference where the local and central government representatives, local farmers' and other relevant associations, scientists, and other relevant actors participate Production of a joint proposal document to be presented to the legislative bodies Production and adoption of a collection of policy, awareness raising and guidance documents aimed at strengthening the soil protection Rising effectiveness of monitoring and the execution of orders, resolutions, etc. Establishment of local village forums gathering once a month, with participation of local government, and production of one document once a year on the effectiveness of the actions undertaken. Number of reported violations of legislation Number of fines Participation in preparation of documents Establishing the mechanism of ecological insurance Delivery of a workshop with the participation of ecological insurance specialists, government, scientists, and local farmers. Establishing the environmental audit at local and national level One workshop every six months for external experts to engage local government, central government representatives and local association representatives in order to discuss and facilitate the coordination of the audit Participation in workshops Establishing links with international organizations During the workshops, a database of interested international organizations is compiled. A conference with at least 60% of the organizations operational in Ukraine in the field of land management participating. All these organizations will receive a monthly, trimestral, or other periodic newsletters/updates. Number of international cooperations Source: authorsʼ research. Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com Agricultural and Resource Economics: International Scientific E-Journal www.are-journal.com How to cite this article? Як цитувати цю статтю? Стиль – ДСТУ: Kucher A. Sustainable use of land in danger of wind erosion in Ukraine: stakeholder engagement [Electronic resource] / A. Kucher, I. Kazakova, L. Kucher and other // Agricultural and Resource Economics : International Scientific E-Journal. – 2015. – Vol. 1. – No. 2. – pp. 5–28. – Mode of access : www.are-journal.com. Style – Harvard: Kucher, A., Kazakova, I., Kucher, L., Schraml, A., Koka, H. and Priest, W. (2015), Sustainable use of land in danger of wind erosion in Ukraine: stakeholder engagement. Agricultural and Resource Economics: International Scientific E- Journal, [Online], vol. 1, no. 2, available at: www.are-journal.com. Vol. 1, No. 2, 2015 28
https://openalex.org/W2549125766	https://discovery.ucl.ac.uk/1531006/1/ncomms13580.pdf	English	null	A DNA dual lock-and-key strategy for cell-subtype-specific siRNA delivery	Nature communications	2,016	cc-by	11,390	1 State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China. 2 Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, University College London, London WC1N 1AX, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.X.J. (email: hxju@nju.edu.cn). URE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications ARTICLE Received 29 Jan 2016 \| Accepted 17 Oct 2016 \| Published 24 Nov 2016 Received 29 Jan 2016 \| Accepted 17 Oct 2016 \| Published 24 Nov 2016 Received 29 Jan 2016 \| Accepted 17 Oct 2016 \| Published 24 Nov 2016 DOI: 10.1038/ncomms13580 A DNA dual lock-and-key strategy for cell-subtype-speciﬁc siRNA delivery Since cells express multiple surface receptors, simultaneously assessing multiple surface receptors to recognize speciﬁc disease cells and enhance diagnostic and therapic accuracy in similar cells should be a more practical and less risk approach12. Taking advantages of autonomous DNA strand displacement cascades reaction13, programmable dual parameters controlled DNA logic platform has been used for cancer cell recognition14 and photodynamic therapy15,16. However, the DNA logic platform has not been used for siRNA delivery due to the limitation of using toehold- mediated strand displacement cascade reaction as an efﬁcient delivery carrier. The precise delivery of siRNA to speciﬁc target cells is still an urgent need. self-assembled oligonucleotide nano vehicle (ONV) and a ‘dual lock-and-key’’ were designed to load siRNA for controllable siRNA delivery. The ONV structure conferred higher payload capacity, which signiﬁcantly increased cell uptake. Besides, different cell-recognition aptamers could be conveniently incorporated into ONV via hybridization, and the rigid tube-like structure improved resistance to nuclease degradation upon endocytosis25,26. p y The incorporation of two factors in a delivery system to function in a serial manner can improve the site-speciﬁc transport and lower the non-target cytotoxicity27. Here an auto-cleavable hairpin structure is used to modify the siRNA- loaded ONV (siRNA-ONV) and act as the ‘smart key’, and two kinds of aptamers, sgc8c and sgc4f16, are bound on cell surface to act as the ‘double locks’. The ‘locks’ can be opened sequentially by reacting with the ‘key’ in a serial manner. The speciﬁc double recognition mode controls the cell ‘locked-open’ status, and thus achieves cell-subtype-speciﬁc recognition and precise siRNA delivery. Upon reaction with Zn2 þ-dependent MNAzyme on sgc4f, the hairpin structure oligonucleotide in siRNA-ONV is auto-cleaved to form single strand, which activates the ‘smart key’ on site at cell membrane. The ‘locked’ hairpin structure of sgc8c is thus opened by hybridizing with the cleaved single-strand oligonucleotide in siRNA-ONV subsequently to mediate the precise delivery of siRNA into speciﬁc target cells. To the best of our knowledge, this is the ﬁrst try of precise siRNA delivery and gene silencing with the participation of multiple cell membrane receptors. Previously reported siRNA delivery methods all rely on only one receptor on cell surface7–10, therefore usually suffer from the high nonspeciﬁc interaction and off-target toxicity to other cells. A DNA dual lock-and-key strategy for cell-subtype-speciﬁc siRNA delivery The efﬁcient and precise delivery of siRNA to target cells is critical to successful gene therapy. While novel nanomaterials enhance delivery efﬁciency, it still remains challenging for precise gene delivery to overcome nonspeciﬁc adsorption and off-target effect. Here we design a dual lock-and-key system to perform cell-subtype-speciﬁc recognition and siRNA delivery. The siRNA is self-assembled in an oligonucleotide nano vehicle that is modiﬁed with a hairpin structure to act as both the ‘smart key’ and the delivery carrier. The auto-cleavable hairpin structure can be activated on site at target cell membrane by reacting with two aptamers as ‘dual locks’ sequentially, which leads to cell-subtype discrimination and precise siRNA delivery for high efﬁcient gene silencing. The success of this strategy demonstrates the precise delivery of siRNA to speciﬁc target cells by controlling multiple parameters, thus paving the way for application of RNAi in accurate diagnosis and intervention. 1 State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China. 2 Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, University College London, London WC1N 1AX, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.X.J. (email: hxju@nju.edu.cn). 1 NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 G e e s G ene interference technology that selectively silences gene expression and inhibits protein transcription by delivering small interference RNA (siRNA) in mammalian cells is becoming a promising approach for the precise treatment of human diseases, including cancer and metabolic, neurodegenera- tive and infectious diseases1,2. One of the key challenges to realize the broad clinical application of RNA interference (RNAi) therapy is its delivery speciﬁcity3–5. A cell-speciﬁc and efﬁcient delivery system is highly desired to improve selective cellular uptake, decrease the overall dosage of siRNAs and avoid nonspeciﬁc adsorption as well as minimize off-target silencing in non-target cells6. A number of ligands that selectively bind tissue-associated antigens have been explored for targeted siRNA delivery, including antibody–protamine fusion proteins7,8 and aptamer-siRNA chimeras9,10. However, most receptors are often shared by multiple types of cell, or a receptor overexpressed in diseased cells is also expressed at a low level in normal cells, therefore the single-receptor-targeted delivery system potentially results in off-target toxicities and serious complications11. A DNA dual lock-and-key strategy for cell-subtype-speciﬁc siRNA delivery In comparison with ‘single-parameter’-controlled siRNA delivery, the ‘dual lock-and-key’-controlled siRNA delivery system activates two recognition elements on site at target cell membrane just before the delivery process, therefore affords substantial improvement for delivery speciﬁcity and avoids off- target toxicities, which is of great importance to the application of RNAi in precise diagnosis and treatment. A variety of materials have been explored as siRNA delivery carriers, such as liposomes, cationic polyelectrolytes and inor- ganic nanoparticle17–19. However, these conventional delivery vehicles suffer from low loading efﬁciency, less cell-speciﬁc manner, complex surface modiﬁcation process and/or the damage of immunogenic response or toxicity20,21. Self-assembled DNA nanostructures can offer the advantages of ﬂexible design, controllable size and orientation, ease of bioconjugation and excellent biocompatibility, and have demonstrated potential application in biosensing and drug delivery22–24. Here a Results Preparation siRNA-ONV. The triangular rung units (TRUs) with two overhangs at each end were synthesized ﬁrst as building R2 C2 V1 siRNA with 3′ overhang siRNA-TRU a R1 R2 C2 V1 C1 siRNA with 3′ overhang siRNA-TRU Phi29 DNA primer Hairpin siRNA-ONV Hairpin siRNA Autocleave position “rA” b c Circular DNA template Aptamer sgc8c Lock Aptamer sgc4f Smart key Open Nucleus Nucleus Nucleus On-site autocleavage of siRNA-ONV siRNA-ONV delivery Figure 1 \| Schematic illustration of ‘dual lock-and-key’-controlled cell-subtype-speciﬁc siRNA delivery. (a) Self-assembly synthesis of siRNA-TRU repeating unit, (b) self-assembly synthesis of siRNA-ONV nanotube and (c) siRNA delivery principle. a R1 R2 C2 V1 C1 siRNA with 3′ overhang siRNA-TRU b a 9 Circular DNA template siRNA ONV c Aptamer sgc8c Lock Aptamer sgc4f Smart key Open Nucleus Nucleus Nucleus On-site autocleavage of siRNA-ONV siRNA-ONV delive c On-site autocleavage of siRNA-ONV siRNA-ONV delivery siRNA-ONV delivery Figure 1 \| Schematic illustration of ‘dual lock-and-key’-controlled cell-subtype-speciﬁc siRNA delivery. (a) Self-assembly synthesis of siRNA-TRU repeating unit, (b) self-assembly synthesis of siRNA-ONV nanotube and (c) siRNA delivery principle. Figure 1 \| Schematic illustration of ‘dual lock-and-key’-controlled cell-subtype-speciﬁc siRNA delivery. (a) Self-assembly synthesis of siRNA-TRU repeating unit, (b) self-assembly synthesis of siRNA-ONV nanotube and (c) siRNA delivery principle. NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications 2 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 ‘closed’ hairpin structure prevented the hybridization, demon- strated by two separate bands for the mixture solution (Supplementary Fig. 2a, lanes 4). The hairpin structure DNA primer had an auto-cleavable position, which could be autocatalytically cleaved by the Zn2 þ-dependent MNAzyme containing sgc4f aptamer to form DNA single strand. The cleaved single-strand DNA primer hybridized with sgc8c aptamer to open its hairpin structure, acting as a smart key to be activated on site by reacting sequentially with double locks sgc4f and sgc8c. Upon the addition of sgc4f aptamer in the mixture of sgc8c and DNA primer, the bands representing sgc8c and DNA primer dis- appeared, and a new band with much lower mobility appeared (Supplementary Fig. 2a, lanes 5), indicating the hybridization of single-strand DNA primer with sgc8c. The feasibility of the ‘dual lock-and-key’-controlled system was further conﬁrmed by observing ﬂuorescence recovery from self-quenched aptamer BHQ2-sgc8c. The hairpin structure aptamer sgc8c was labelled with Cy3 and BHQ2 on each stem terminus. Results 2b, it showed a lower-mobility band appeared in lane 3 after the RCA reaction, suggesting the formation of DNA backbone. When DNA backbone was mixed with the siRNA-TRU (lane 4, Fig. 2b), the band for siRNA-TRU disappeared and a strengthened band was obtained in the sample loading zone, showing the successful preparation of siRNA-ONV (Fig. 2b, lane 5). The siRNA-ONV structure was also characterized by atomic force microscopy (AFM). The siRNA-ONV tubes appeared to be quite rigid and well dispersed with an average length of 0.60±0.15 mm (Fig. 2c) (± indicates the length range of siRNA-ONV tubes according to 10 times replicate measurements), which consists with the previously reported self-assembly DNA nanotube structure28,29. Given that the siRNA-TRU is 14.3 nm in length30, 42±10 siRNA-TRU repeating units were counted for each siRNA-ONV, indicating the loading capacity of 84±20 siRNA for siRNA-ONV. Sgc8c and sgc4f aptamers are bound to their individual receptors expressed by different cells. Sgc8c binds to cell membrane receptor tyrosine protein kinase-7, while the cell membrane receptor for sgc4f is not yet identiﬁed16. After different cells were incubated with ﬂuorescein dye (FAM) labelled aptamers, strong FAM ﬂuorescence were observed from their corresponding target cells in ﬂow cytometric assay (Supplementary Fig. 3), which indicated human cervix carcinoma (HeLa) cells only bound sgc8c aptamer, and human Burkitt’s lymphoma (Ramos) cells only bound sgc4f aptamer, while both sgc8c and sgc4f aptamers were bound to human acute lymphoblastic leukaemia (CEM) cells, indicating the binding selectivity of sgc8c and sgc4f to their respective target cells. g p y To verify the serum stability of siRNA-ONV, Cy3-tagged siRNA and black hole quencher 2 (BHQ2)-tagged R1 and R2 oligonucleotides were used as components to assemble self- quenched siRNA-ONV (SQ-siRNA-ONV). The ﬂuorescence recovery of Cy3-siRNA from SQ-siRNA-ONV disassembly was measured in 10% fetal bovine serum (FBS) reaction buffer over 12 h (Supplementary Fig. 1a). Compared with the control couple of self-quenched double-strand siRNA (SQ-ds-siRNA), the ﬂuorescence recovery from SQ-siRNA-ONV was much less, demonstrating that ONV nanotube structure could protect siRNA from nuclease degradation. The melting temperature (Tm) of siRNA-ONV was 72.1 C (Supplementary Fig. 1b,c), demonstrating good thermal stability. y g g p g The ‘double locks’ controlled CEM cell-subtype-speciﬁc siRNA-ONV delivery was relied on the sequential opening of locks sgc8c and sgc4f (Fig. 1c). Results The ‘locked’ sgc8c aptamer kept hairpin structure in the absence of sgc4f aptamer or/and ‘smart key’ ONV, thus only very weak ﬂuorescence was observed. In the presence of both sgc4f and ONV, the mixture solution demonstrated strong ﬂuorescent intensity due to the opening of hairpin structure sgc8c aptamer and corresponding Cy3 ﬂuorescence recovery (Supplementary Fig. 2b). blocks for ONV (Fig. 1a)28. siRNAs were hybridized with the overhangs of TRU, and the synthesis of siRNA-TRU was conﬁrmed by 8% polyacrylamide gel electrophoresis (PAGE) experiment (Fig. 2a). Upon mixing seven oligonucleotides of C1, C2, V1, R1, R2, antisense siRNA and siRNA strands, a distinct bright band that migrated much slower than all components was observed (lane 8, Fig. 2a), indicating the successful synthesis of siRNA-TRU. The siRNA-TRUs were subsequently assembled with a long continuous DNA backbone strand produced by rolling circle ampliﬁcation (RCA) to form siRNA-ONV (Fig. 1b). The synthesis process of siRNA-ONV was characterized in Fig. 2b, it showed a lower-mobility band appeared in lane 3 after the RCA reaction, suggesting the formation of DNA backbone. When DNA backbone was mixed with the siRNA-TRU (lane 4, Fig. 2b), the band for siRNA-TRU disappeared and a strengthened band was obtained in the sample loading zone, showing the successful preparation of siRNA-ONV (Fig. 2b, lane 5). The siRNA-ONV structure was also characterized by atomic force microscopy (AFM). The siRNA-ONV tubes appeared to be quite rigid and well dispersed with an average length of 0.60±0.15 mm (Fig. 2c) (± indicates the length range of siRNA-ONV tubes according to 10 times replicate measurements), which consists with the previously reported self-assembly DNA nanotube structure28,29. Given that the siRNA-TRU is 14.3 nm in length30, 42±10 siRNA-TRU repeating units were counted for each siRNA-ONV, indicating the loading capacity of 84±20 siRNA for siRNA-ONV. blocks for ONV (Fig. 1a)28. siRNAs were hybridized with the overhangs of TRU, and the synthesis of siRNA-TRU was conﬁrmed by 8% polyacrylamide gel electrophoresis (PAGE) experiment (Fig. 2a). Upon mixing seven oligonucleotides of C1, C2, V1, R1, R2, antisense siRNA and siRNA strands, a distinct bright band that migrated much slower than all components was observed (lane 8, Fig. 2a), indicating the successful synthesis of siRNA-TRU. The siRNA-TRUs were subsequently assembled with a long continuous DNA backbone strand produced by rolling circle ampliﬁcation (RCA) to form siRNA-ONV (Fig. 1b). The synthesis process of siRNA-ONV was characterized in Fig. Results However, the single-membrane receptor-controlled delivery system could not provide sufﬁcient cell-subtype selectivity, ﬂuorescence from S-Cy3-siRNA-ONV was observed from both CEM and HeLa cells (Supplementary Fig. 6). Speciﬁcity of dual lock-and-key-controlled siRNA delivery. The cell-subtype recognition and siRNA-ONV delivery were further veriﬁed with three different cells, HeLa, Ramos and CEM cells (Fig. 3). The bright ﬂuorescence from Cy3-siRNA-ONV was only observed within CEM cells. Only little ﬂuorescence was observed from Ramos and HeLa cells, demonstrating the speciﬁc recognition and precise delivery of siRNA to target cells. To examine the possible off-target delivery through the diffusion of activated siRNA-ONV or direct physical contact of neighbouring cells, the mixture of HeLa and Ramos cells or HeLa and CEM cells was treated with Cy3-siRNA-ONV, respectively. Both HeLa and Ramos cells showed little ﬂuorescence, while CEM cells showed bright ﬂuorescence, indicating that proximity-based siRNA delivery was dominant. The ﬂuorescence intensity of CEM cells was 4.6-fold that of HeLa cells (Supplementary Fig. 5), suggesting that B82.1% of activated siRNA-ONV in the mixture was internalized in CEM cells. To further demonstrate the advantages of dual parameters controlled cell-subtype-speciﬁc recognition and delivery, we also designed a single-parameter- controlled delivery vehicle as a negative control Cy3-siRNA-ONV (S-Cy3-siRNA-ONV). The ONV nanotube was modiﬁed with a single-strand DNA oligonucleotide instead of auto-cleavable Internalization process of siRNA-ONV. To conﬁrm the integrity of siRNA-ONV nanostructures when they crossed through cell membrane, Cy3 and Cy5 were labelled on different siRNA-TRUs and the assembled siRNA-ONV was incubated with CEM cells for 2 h. The co-localization experiment showed that the ﬂuorescence from Cy3 and Cy5 appeared nearly in the same place (Fig. 4a). To study the internalization process of siRNA-ONV, co-staining experiment was further performed by staining lysosomes with LysoTracker Green and cell nucleus with 40,6-diamidino-2-phenylindole (DAPI). Cy3-siRNA-ONV was localized in lysosomes on the same sites after incubation with CEM cells for 2 h by the overlap of red (Cy3-siRNA-ONV) and green (lysosome) ﬂuorescence (Fig. 4b), indicating successful cellular internalization of the Cy3-siRNA-ONV through endo- cytosis. After incubation with Cy3-siRNA-ONV for 6 h, weaker ﬂuorescence of LysoTracker Green was observed (Fig. 4b), suggesting the spreading of LysoTracker Green from acidic lysosome to neutral cytoplasm32,33, since the staining of lysosome by the tracker was dependent on the acidicity34. These phenomena clearly demonstrated the efﬁcient translocation or escape of Cy3-siRNA-ONV from lysosomes into the cytoplasm due to the rupture of the lysosomal membrane33,35. Results During the endocytosis process, the rigid structure of self-assembled ONV nanotube could carry siRNA crossing through cell membrane and protect them from degradation29. The successful endosomal escape was also attributed to the rigid linear structure with high aspect ratio of ONV nanotube36–38. To demonstrate the rigid structure of ONV prompts endosomal escape process, a control Cy3-siRNA-ONV without rigid structure (Cy3-siRNA-ONV-NR) was synthesized by mixing siRNA-TRU with DNA backbone at a lower ratio of 0.5:1. No ﬂuorescence colour separation was observed after incubating Cy3-siRNA-ONV-NR with CEM cells for 6 h, indicating the failure of endosomal escape process in the absence of rigid structure (Fig. 4c). To further validate the endosomal escape, calcein was used to monitor the stability of lysosomes after Cy3-siRNA-ONV uptake32. After CEM cells were treated with the mixture of calcein and Cy3-siRNA-ONV for 6 h, they showed the spreading of calcein to the cytoplasm with strong calcein ﬂuorescence and yellow ﬂuorescence resulted from the overlap of red Cy3 and green calcein ﬂuorescence (Supplementary Fig. 7b), while the cells treated with calcein alone or the mixture of calcein and Cy3-siRNA-ONV-NR only showed the weaker ﬂuorescence inside the endosomes (Supplementary Fig. 7a,c). These results demonstrated the success of endosomal escape of Cy3-siRNA-ONV39,40. To evaluate the particular internalization pathway of the Cy3-siRNA-ONV into the cells, a series of inhibitors were employed to selectively block different internalization processes (Supplementary Fig. 8). Treatment of the cells with NaN3 and sucrose led to a 50–60% reduction in Cy3-siRNA-ONV uptake, suggesting Cy3-siRNA-ONV experienced the clathrin-dependent endocytosis pathway upon entering the cells. Fluorescence HeLa Ramos CEM HeLa+Ramos HeLa+CEM Bright field Merge Figure 3 \| Speciﬁcity of ‘dual lock-and-key’-controlled siRNA delivery. Confocal microscopy images of HeLa, Ramos, CEM cells, the mixture of HeLa and Ramos cells, and the mixture of HeLa and CEM cells after incubation with 100 nM Cy3-siRNA-ONV for 2 h. All the cells were pretreated with sgc8c and sgc4f aptamers. Scale bars, 50 mm. Ramos Figure 3 \| Speciﬁcity of ‘dual lock-and-key’-controlled siRNA delivery. Confocal microscopy images of HeLa, Ramos, CEM cells, the mixture of HeLa and Ramos cells, and the mixture of HeLa and CEM cells after incubation with 100 nM Cy3-siRNA-ONV for 2 h. All the cells were pretreated with sgc8c and sgc4f aptamers. Scale bars, 50 mm. Characterization of siRNA release. Results The ﬂuorescence from Cy3-labelled siRNA-ONV (Cy3-siRNA-ONV) was only observed within CEM cells in the presence of both sgc8c and sgc4f aptamers, while no ﬂuorescence was observed in the absence of either aptamer (Supplementary Fig. 4a). This result was also conﬁrmed by ﬂow cytometric assay (Supplementary Fig. 4b). Z-stack images of CEM cells were used to verify the internaliza- tion of Cy3-siRNA-ONV in CEM cells31. The ﬂuorescence signals from Cy3 and the nucleus staining were located at the same focal plane and exhibited a position-sensitive dependence, Mechanism of double locks controlled siRNA delivery. The feasibility of dual parameter sequentially controlled ‘lock- and-key’ system was veriﬁed with PAGE analysis by mixing the hairpin structure oligonucleotide (DNA primer) in ONV with the ‘locks’ (sgc8c and sgc4f aptamers) together and respectively (Supplementary Fig. 2a). Although sgc8c aptamer and DNA primer had the complementary sequences, their individual a 1 2 4 1 300 bp 100 bp 2 3 4 5 6 5 6 7 8 9 3 b c 300 bp 100 bp Figure 2 \| Characterization of siRNA-ONV self-assembly. (a) PAGE analysis of siRNA-TRU self-assembly. Lanes 1–9 represent C1, C2, V1, R1, R2, antisense siRNA, siRNA, the mixture of lines 1–7 and DNA ladder marker. (b) PAGE analysis of siRNA-ONV self-assembly. Lanes 1–6 represent DNA primer, circular DNA template, DNA backbone, siRNA-TRU, siRNA-ONV and DNA ladder marker. (c) AFM image of siRNA-ONV. Phase image: scale bar, 300 nm. c b Figure 2 \| Characterization of siRNA-ONV self-assembly. (a) PAGE analysis of siRNA-TRU self-assembly. Lanes 1–9 represent C1, C2, V1, R1, R2, antisense siRNA, siRNA, the mixture of lines 1–7 and DNA ladder marker. (b) PAGE analysis of siRNA-ONV self-assembly. Lanes 1–6 represent DNA primer, circular DNA template, DNA backbone, siRNA-TRU, siRNA-ONV and DNA ladder marker. (c) AFM image of siRNA-ONV. Phase image: scale bar, 300 nm. NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 demonstrating intracellular localization of Cy3-siRNA-ONV (Supplementary Fig. 4c). demonstrating intracellular localization of Cy3-siRNA-ONV (Supplementary Fig. 4c). demonstrating intracellular localization of Cy3-siRNA-ONV (Supplementary Fig. 4c). demonstrating intracellular localization of Cy3-siRNA-ONV (Supplementary Fig. 4c). hairpin structure oligonucleotide. The single-strand DNA oligonucleotide had the same sequence as the rest part of the auto-cleavable hairpin structure oligonucleotide after cleavage and could recognize lock aptamer sgc8c. NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 Cy3 Cy5 2 h a b c DAPI 2 h 6h Cy3 LysoTracker green DAPI Merge 2 h 6 h Merge Cy3 LysoTracker green DAPI Merge acterization of endosomal escape. (a) Confocal ﬂuorescence microscopic images of CEM cells incubated with Cy3/ ere labelled on different siRNA-TRU and self-assembled together to form Cy3/Cy5-siRNA-ONV. The cell nucleus wa ge shows co-localization of both ﬂuorophores in cytoplasm of CEM cells. (b–c) Co-staining of CEM cells with LysoT bated with (b) Cy3-siRNA-ONV and (c) Cy3-siRNA-ONV-NR for 2 and 6 h. Scale bars, 10 mm. Cy3 Cy5 2 h a b c DAPI 2 h 6h Cy3 LysoTracker green DAPI Merge 2 h 6 h Merge Cy3 LysoTracker green DAPI Merge terization of endosomal escape. (a) Confocal ﬂuorescence microscopic images of CEM cells incubated with Cy3/ e labelled on different siRNA-TRU and self-assembled together to form Cy3/Cy5-siRNA-ONV. The cell nucleus was shows co-localization of both ﬂuorophores in cytoplasm of CEM cells. (b–c) Co-staining of CEM cells with LysoTr ted with (b) Cy3-siRNA-ONV and (c) Cy3-siRNA-ONV-NR for 2 and 6 h. Scale bars, 10 mm. Cy3 Cy5 2 h a b DAPI 2 h Merge Cy3 LysoTracker green DAPI Merge a b Cy3 DAPI c 6h Cy3 LysoTracker green DAPI Merge 2 h 6 h Figure 4 \| Characterization of endosomal escape. (a) Confocal ﬂuorescence microscopic images of CEM cells incubated with Cy3/C Cy3 and Cy5 were labelled on different siRNA-TRU and self-assembled together to form Cy3/Cy5-siRNA-ONV. The cell nucleus was The merge image shows co-localization of both ﬂuorophores in cytoplasm of CEM cells. (b–c) Co-staining of CEM cells with LysoTra c Cy3 Figure 4 \| Characterization of endosomal escape. (a) Confocal ﬂuorescence microscopic images of CEM cells incubated with Cy3/Cy5-siRNA-ONV. Cy3 and Cy5 were labelled on different siRNA-TRU and self-assembled together to form Cy3/Cy5-siRNA-ONV. The cell nucleus was stained with DAPI. The merge image shows co-localization of both ﬂuorophores in cytoplasm of CEM cells. (b–c) Co-staining of CEM cells with LysoTracker Green and DAPI after incubated with (b) Cy3-siRNA-ONV and (c) Cy3-siRNA-ONV-NR for 2 and 6 h. Scale bars, 10 mm. These cells showed negligible ﬂuorescence after 6-h incubation, suggesting the integrity of SQ-siRNA-ONV during the endosomal escape process. However, after 24-h incubation, the ﬂuorescence of Cy3 was clearly observed in CEM cells, which demonstrated the successful disassembly of SQ-siRNA-ONV and release of siRNA in cells. Results In RNAi, the siRNA strands are generally loaded into the RNA-induced silencing complex, and the antisense strand is separated from sense strand to silence NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 Even at very high concentration of 0.5 mM ONV, CEM cells still kept 96.5% viability (Supplementary Fig. 10). In comparison, the cells treated with Lipo2000 and TKO only exhibited 87.1% and 89.6% viability, respectively. The non-cytotoxicity and good biocompatibility of ONV guaranteed its potential application in clinical intervention. were compared with those of untreated cells, the cells only treated with ds-siRNA, ONV complex and NCsiRNA-ONV. The ds-siRNA-, ONV complex- or NCsiRNA-ONV-treated cells did not show inhibition effect, and their cell proliferation percentages still remained at B100% (Supplementary Fig. 11a). In contrast, siRNA-ONV demonstrated signiﬁcant inhibition effect on cell proliferation, and the cell proliferation percentage decreased down to 55.7%, at the same level as 2nd-siRNA-ONV, siRNA- Lipo2000 or siRNA-TKO. Moreover, the inhibition percentage of cell proliferation for siRNA-ONV also showed dose-dependent, and the cell proliferation percentage decreased with the increasing siRNA loading (Supplementary Fig. 11b). To further evaluate the therapeutic effect of siRNA-ONV, the cell apoptosis was studied by ﬂow cytometric assay using the Annexin V-ﬂuorescein isothiocyanate (FITC)/propidium iodide (PI) apoptotic kit (Supplementary Fig. 11c). The apoptosis rate for cells treated with siRNA-ONV was 49.3%, comparable with 2nd-siRNA-ONV, siRNA-lipo2000 and siRNA-TKO, and B16 times improvement than ds-siRNA. These results demonstrated the feasibility of ‘dual lock-and-key’-controlled siRNA delivery as a promising approach for efﬁcient anticancer therapeutics. Gene silencing assay. Vascular endothelial growth factor (VEGF) has been identiﬁed as a mitogen and important regulator of angiogenesis, and appeared to be involved in the vascular phase of many different neoplastic diseases. It is a promising approach for human acute lymphoblastic leukaemia treatment through blocking VEGF expression to inhibit tumour vascularization and growth46,47. To evaluate the therapeutic effect of ‘dual lock-and- key’-controlled siRNA delivery, VEGF was selected as a model therapeutic target and the siRNA-ONV were delivered into target CEM cells against VEGF. After the CEM cells bound with sgc4f and sgc8c aptamers and incubated with 100 nM siRNA-ONV for 48 h, the VEGF messenger RNA (mRNA) and VEGF protein expression levels were determined by real-time PCR assay and enzyme-linked immunosorbent assay (ELISA), and the treatment efﬁciency was also compared with siRNA-loaded lipo2000 and TKO (siRNA-lipo2000 and siRNA-TKO). A negative control siRNA-ONV (NCsiRNA-ONV) and a second active VEGF siRNA-loaded ONV (2nd-siRNA-ONV) were also synthesized using negative control siRNA and another active siRNA with a different effective sequence to perform the comparison. As observed in Fig. ARTICLE 5, CEM cells treated with just ONV complexes, NCsiRNA-ONV or only ds-siRNA showed little inhibition of VEGF mRNA expression or VEGF protein synthesis. Both the mRNA and protein expression percentage were little decreased compared with CEM cells without any treatment (control). The ‘dual lock-and-key’-controlled delivery approach (siRNA-ONV) demonstrated effective VEGF gene silencing in target cells by inhibiting the VEGF mRNA expression down to 47.1% and VEGF protein production down to 49.1%48–50, which was comparable with Lipo2000 mediation transfection, TKO mediation transfection and 2nd-siRNA-ONV. Therapeutic efﬁcacy under serum condition. To evaluate the therapeutic efﬁcacy of siRNA-ONV and 2nd-siRNA-ONV under serum condition, the gene silencing and cell apoptosis assays were performed under both serum and serum-free conditions. The mRNA expression, protein secretion level and cell proliferation under serum condition showed consistent results with those in reaction buffer (Supplementary Fig. 12), suggesting that the serum did not affect the ‘dual lock-and-key’-controlled siRNA delivery process. In vivo therapeutic applicability. To demonstrate the in vivo inhibition of VEGF expression and the antitumour efﬁcacy of this strategy, the mixture of sgc4f and sgc8c aptamers was ﬁrst intratumorally injected into mice bearing CEM xenograft tumour for 30 min, and siRNA-ONV, NCsiRNA-ONV, reaction buffer, siRNA-Lipo2000 and siRNA-TKO were then intratumorally injected into these mice, respectively. SiRNA-Lipo2000-, siRNA- TKO- and siRNA-ONV-injected mice presented pronounced inhibition efﬁcacy towards tumour growth compared with the control and NCsiRNA-ONV groups (Fig. 6a). Although the cell experiments for performing siRNA-ONV-, siRNA-Lipo2000- and Cell apoptosis assay. MTT assay was also used to verify the inhibition effect of siRNA-ONV to CEM cell proliferation. After the CEM cells were incubated with siRNA-ONV, 2nd-siRNA- ONV, siRNA-Lipo2000 or siRNA-TKO for 2 h, their viabilities 100 80 60 40 20 0 a N.S. N.S. Control NCsiRNA-ONV ds-siRNA siRNA-Lipo2000 siRNA-TKO siRNA-ONV Control ONV NCsiRNA-ONV ds-siRNA siRNA-Lipo2000 siRNA-TKO siRNA-ONV 2nd-siRNA-ONV 2nd-siRNA-ONV ONV N.S. 100 80 60 40 20 0 b N.S. VEGF expression (%) mRNA expression (%) * * NS NS NS NS Figure 5 \| Silencing of target VEGF gene in CEM cells. (a) Real-time PCR characterization of mRNA expression for CEM cells incubated with ONV, NCsiRNA-ONV, ds-siRNA, siRNA-Lipo2000, siRNA-TKO, siRNA-ONV and 2nd-siRNA-ONV. (b) ELISA characterization of protein secretion levels for CEM cells incubated with ONV, NCsiRNA-ONV, ds-siRNA, siRNA-Lipo2000, siRNA-TKO, siRNA-ONV and 2nd-siRNA-ONV. The data error bars indicate means±s.d. (n ¼ 3). Po0.05 (two-tailed Student’s t-test). N.S. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 corresponding gene and suppress protein expression41. Here siRNA duplexes were released from ONV nanotube in the cytoplasm, which was demonstrated from the PAGE analysis of the mixture of siRNA-ONV and CEM cell lysate (Supplementary Fig. 9a)42,43, and then incorporated into the RNA-induced silencing complex before it participates in RNAi. This result was also conﬁrmed by ﬂuorescence spectra assay (Supplementary Fig. 9b,c). A strong ﬂuorescent of Cy3 was obtained after over 24-h incubation of SQ-siRNA-ONV with CEM cell lysates, indicating the successful SQ-siRNA-ONV disassembly and siRNA release in cell microenvironment. To further conﬁrm the successful siRNA release in the cytoplasm, SQ-siRNA-ONV was also incubated with CEM cells over 24 h (Supplementary Fig. 9d). Cytotoxicity assay. The cytotoxicity was evaluated with a standard 3-(4,5-Dimethylthiazol-2-yl)-2-diphenyltetrazolium bromide (MTT) assay at a series of ONV concentrations, and the results were compared with standard transfection agents, Lipofectamine 2000 (Lipo2000) and Trans IT-TKO (TKO)44,45. 5 NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications 5 ARTICLE Control ONV NCsiRNA-ONV ds-siRNA siRNA-Lipo2000 siRNA-TKO siRNA-ONV 2nd-siRNA-ONV 100 80 60 40 20 0 b N.S. VEGF expression (%) NS NS b 100 80 60 40 20 0 a N.S. Control NCsiRNA-ONV ds-siRNA siRNA-Lipo2000 siRNA-TKO siRNA-ONV 2nd-siRNA-ONV ONV N.S. mRNA expression (%) * NS NS a Figure 5 \| Silencing of target VEGF gene in CEM cells. (a) Real-time PCR characterization of mRNA expression for CEM cells incubated with ONV, NCsiRNA-ONV, ds-siRNA, siRNA-Lipo2000, siRNA-TKO, siRNA-ONV and 2nd-siRNA-ONV. (b) ELISA characterization of protein secretion levels for CEM cells incubated with ONV, NCsiRNA-ONV, ds-siRNA, siRNA-Lipo2000, siRNA-TKO, siRNA-ONV and 2nd-siRNA-ONV. The data error bars indicate means±s.d. (n ¼ 3). Po0.05 (two-tailed Student’s t-test). NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications 6 6 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 ARTICLE Tumour volume (cm3) Control 0 2 4 6 8 10 Treatment time (day) Control NCsiRNA-ONV siRNA-Lipo2000 siRNA-ONV b a siRNA-TKO 12 ** NCsiRNA-ONV siRNA-Lipo2000 siRNA-ONV siRNA-TKO 0.3 0.2 0.1 0.4 Figure 6 \| In vivo demonstration of therapeutic applicability via target VEGF gene silencing. (a) Change of CEM tumour volume after treatment with reaction buffer, NCsiRNA-ONV, siRNA-Lipo2000, siRNA-TKO or siRNA-ONV through intratumoral delivery. The data error bars indicate means±s.d. (n ¼ 5). Po0.05, Po0.01 (two-tailed Student’s t-test). (b) Expression of VEGF in CEM tumour tissue sections evaluated by immunoﬂuorescence staining. Tumour volume (cm3) Control 0 2 4 6 8 10 Treatment time (day) a 12 ** NCsiRNA-ONV siRNA-Lipo2000 siRNA-ONV siRNA-TKO 0.3 0.2 0.1 0.4 Control NCsiRNA-ONV b b b a NCsiRNA-ONV siRNA-ONV siRNA-Lipo2000 Treatment time (day) Figure 6 \| In vivo demonstration of therapeutic applicability via target VEGF gene silencing. (a) Change of CEM tumour volume after treatment with reaction buffer, NCsiRNA-ONV, siRNA-Lipo2000, siRNA-TKO or siRNA-ONV through intratumoral delivery. The data error bars indicate means±s.d. (n ¼ 5). Po0.05, *Po0.01 (two-tailed Student’s t-test). (b) Expression of VEGF in CEM tumour tissue sections evaluated by immunoﬂuorescence staining. siRNA-TKO-induced VEGF gene silencing did not show signiﬁcant difference (Fig. 5), in vivo experiments demonstrated slightly higher efﬁcacy (Po0.05) of siRNA-ONV than siRNA- Lipo2000 and siRNA-TKO for inhibiting tumour growth (Fig. 6a), suggesting that the high selectivity of ‘dual lock-and- key’ strategy enhanced in vivo antitumour activity. Consistently, the immunoﬂuorescence staining of tumour tissue section showed less VEGF protein expression in siRNA-ONV-treated mice than siRNA-Lipo2000-, siRNA-TKO-, NCsiRNA-ONV- and reaction buffer-treated mice (Fig. 6b), demonstrating the downregulated expression level of cytoplasmic VEGF. Discussion h d In this study, we design a ‘dual lock-and-key’ system for gene interference, which achieves cell-subtype-speciﬁc siRNA delivery with high gene silencing efﬁciency. The siRNA-ONV is prepared by assembling siRNA-TRU repeating units with a DNA backbone produced by RCA, which is used as both the ‘smart key’ and the delivery carrier. The rigid structure of ONV with the high aspect ratio protects siRNA from nuclease degradation (Fig. 2c; Supplementary Fig. 1) when it crosses through the cell membrane, and prompts endosomal escape in the cytoplasm (Fig. 4b). The average length of siRNA-ONV is 0.60±0.15 mm with high loading amount of siRNA up to 84±20 mm (Fig. 2c), offering high delivery efﬁciency. Two aptamers sgc8c and sgc4f that bind to different cell surface receptors act as ‘dual locks’ sequentially to react with ‘smart key’ siRNA-ONV, thus achieving high speciﬁc recognition and siRNA delivery. Both the aptamer ‘locks’ and the self-assembled oligonucleotide nanotube ‘smart key’ can be designed and conveniently synthesized by mature DNA self-assembly technique with high yield and low cost29,30. The speciﬁcity of ‘dual lock-and-key’-controlled siRNA delivery is veriﬁed by using HeLa and Ramos cells as controls. Both CEM and Ramos cells belong to lymphoma cell lines and display the same surface marker sgc4f, and CEM and HeLa cells display the same surface marker sgc8c (Supplementary Fig. 3). These three kinds of cells are indistinguishable for single- receptor-mediated recognition and delivery therapies, therefore multiple cell surface receptors are needed to participate in the delivery process to increase speciﬁcity and eliminate off-target toxicities. Through sequential reaction with aptamers sgc8c and sgc4f that, respectively, bind to two different cell membrane receptors, the siRNA is delivered into target CEM cell via the ‘dual lock-and-key’-controlled system (Supplementary Fig. 4). This strategy shows negligible off-target toxicity (Fig. 3), which overcomes potential risk in the conventional single-receptor- targeted delivery system (Supplementary Fig. 6). In this study, we design a ‘dual lock-and-key’ system for gene interference, which achieves cell-subtype-speciﬁc siRNA delivery with high gene silencing efﬁciency. The siRNA-ONV is prepared by assembling siRNA-TRU repeating units with a DNA backbone produced by RCA, which is used as both the ‘smart key’ and the delivery carrier. The rigid structure of ONV with the high aspect ratio protects siRNA from nuclease degradation (Fig. 2c; Supplementary Fig. 1) when it crosses through the cell membrane, and prompts endosomal escape in the cytoplasm (Fig. 4b). Discussion h d The average length of siRNA-ONV is 0.60±0.15 mm with high loading amount of siRNA up to 84±20 mm (Fig. 2c), offering high delivery efﬁciency. Two aptamers sgc8c and sgc4f that bind to different cell surface receptors act as ‘dual locks’ sequentially to react with ‘smart key’ siRNA-ONV, thus achieving high speciﬁc recognition and siRNA delivery. Both the aptamer ‘locks’ and the self-assembled oligonucleotide nanotube ‘smart key’ can be designed and conveniently synthesized by mature DNA self-assembly technique with high yield and low cost29,30. g p py In conclusion, we present a strategy of the ‘dual lock-and-key’ system with structure switchable smart key for cell-subtype- speciﬁc siRNA delivery and gene silencing. SiRNA-ONV is activated on site at CEM cell membrane by reacting with aptamer sgc4f, and subsequently reacts with sgc8c to achieve precise VEGF siRNA delivery. ONV is synthesized by DNA self-assembly technique with a convenient procedure, providing good biocom- patibility, high loading efﬁciency with no adverse side effects. The structure of ONV can protect siRNA from degradation during endocytic process and guarantee efﬁcient release from lysosomes. The self-assembled nanostructure vehicle could be conveniently functionalized and extended as a robust strategy for speciﬁc precise delivery of other functional nucleic acids or DNA-binding proteins. This ‘dual lock-and-key’ strategy provides impressive improvement over the single-receptor delivery system by increasing delivery speciﬁcity and inhibiting off-target cytotoxi- city, therefore is of great importance for siRNA-targeted delivery and tumour therapy. y g y The speciﬁcity of ‘dual lock-and-key’-controlled siRNA delivery is veriﬁed by using HeLa and Ramos cells as controls. Both CEM and Ramos cells belong to lymphoma cell lines and display the same surface marker sgc4f, and CEM and HeLa cells display the same surface marker sgc8c (Supplementary Fig. 3). These three kinds of cells are indistinguishable for single- receptor-mediated recognition and delivery therapies, therefore multiple cell surface receptors are needed to participate in the delivery process to increase speciﬁcity and eliminate off-target toxicities. Through sequential reaction with aptamers sgc8c and sgc4f that, respectively, bind to two different cell membrane receptors, the siRNA is delivered into target CEM cell via the ‘dual lock-and-key’-controlled system (Supplementary Fig. 4). This strategy shows negligible off-target toxicity (Fig. 3), which overcomes potential risk in the conventional single-receptor- targeted delivery system (Supplementary Fig. 6). ARTICLE endosomal escape, as well as longer circulation times than spherical structure36,38. Self-assembled DNA nanoribbon is rigid with high aspect ratio and has been conﬁrmed to assist endosomal escape in gene delivery37. The siRNA-ONV nanotube has the rigid and tube-like features with high aspect ratio (Fig. 2c) and demonstrates efﬁcient endosomal escape by the co-localization experiment with dyes LysoTracker Green and DAPI (Fig. 4b). In comparison, a control siRNA-ONV without rigid structure (siRNA-ONV-NR) shows the failure of endosomal escape (Fig. 4c), supporting the proposed mechanism of endosomal escape. Compared with other endosomal escape technique based on positively charged polymer51, self-assembled DNA nanostructure simpliﬁes synthesis steps and has better biocompatibility, therefore can be used as a universal delivery vehicle for siRNA, drug and protein for cancer therapy. NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications ARTICLE CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukaemia (ALL)) from KeyGEN Biotech and Ramos (CRL-1596, B-cell line, human Burkitt’s lymphoma) from Cobioer Biosciences (Nanjing, China) were cultured in RPMI 1640 medium containing 10% FBS and 100 U ml 1 penicillin–streptomycin at 37 C in a humidiﬁed incubator containing 5% CO2 and 95% air. Short tandem repeats (STR) proﬁling and mycoplasma testing were conducted for each cell line before use. Cell numbers were determined with a Petroff-Hausser cell counter (USA). Preparation of circular DNA template. A volume of 4.2 ml of 100 mM phosphorylated linear DNA and 4.2 ml of 100 mM ligation DNA was mixed and annealed at 95 C for 4 min. After the mixture was slowly cooled to room temperature over 2 h, 1 ml of T4 DNA ligase (400 U ml 1), 2 ml of 10 T4 DNA buffer and 8.6 ml of ultrapure water were added and the solution was incubated at 25 C for 16 h. The T4 DNA ligase was inactivated by heating at 65 C for 10 min. A volume of 4 ml of exonuclease I (20 U ml 1) and 4 ml of exonuclease III (100 U ml 1) was added in the following, the mixture was then incubated at 37 C for 8 h to degrade ligation DNA. After heating at 80 C for 15 min to denature the exonuclease I and exonuclease III, the circular DNA template was obtained and stored at 4 C before use. Characterization of aptamers speciﬁcity. Sgc8c and sgc4f aptamers were labelled with FAM to characterize the aptamer-binding speciﬁcity to different cells. FAM-labelled aptamers were incubated with CEM, Ramos and HeLa cells at 37 C for 30 min, and then subjected to ﬂow cytometric assay over FL1 channel. Confocal ﬂuorescence imaging and ﬂow cytometric assay. A concentration of 50 nM of sgc4f and sgc8c aptamer probes was incubated with 1 104 corresponding cells in reaction buffer for 30 min at room temperature. After the cells were centrifuged at 156g for 5 min and washed twice with the reaction buffer, 100 nM of Cy3-siRNA-ONV was added and incubated with the cells for 2 h at 37 C. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 TdT-30, Cy5-sense 50-Cy5-GGAGUACCCUGAUGAGAUCdTdT-30, antisense 50-GAUCUCAUCAGGGUACUCCdTdTCAAAUGGACCAAGGCCAG-30, 21 base antisense 50-GAUCUCAUCAGGGUACUCCdTdT-30; second active VEGF siRNA, sense 50-ACCUCACCAAGGCCAGCACdTdT-30, antisense 50-GUGCUG GCCUUGGUGAGGUdTdTCAAAUGGACCAAGGCCAG-30; negative control siRNA, sense 50-UUCUCCGAACGUGUCACGUdTdT-30 and antisense 50-ACG UGACACGUUCGGAGAAdTdTCAAAUGGACCAAGGCCA G-30. All of the DNA were synthesized and puriﬁed by Sangon Biotech Co., Ltd (Shanghai, China). Their sequences were listed in Supplementary Table 1. between Cy3 and BHQ2. Then, 50 nM of SQ-siRNA-ONV (100 nM ﬂuorophore concentration) and 100 nM of SQ-ds-siRNA were separately diluted with FBS to yield a ﬁnal concentration of 50 nM ﬂuorophore in 10% FBS 1 TAMg. The mixture was incubated at 37 C for 12 h, ﬂuorescence signal recovery was observed with the disassembly of SQ-siRNA-ONV nanotube and release of siRNA. The ﬂuorescence signal recovery was measured every hour with 510 nm excitation and 560 nm emission. Thermal stability of siRNA-ONV. The sample for thermal stability assay was prepared by mixing the 19 ml of 1 mM siRNA-ONV sample solution with 1 ml of 20 SYBR Green I in 0.2 ml PCR tubes (white). Fluorescence data for melting curves were obtained with the real-time PCR detection system (Bio-Rad) during the temperature increase from 30 to 90 C at 0.2 C s 1 and held for 15 s in every temperature point53. Apparatus. Absorption spectra were recorded on an UV-3600 UV–vis-NIR spectrophotometer (Shimadzu Company, Japan). The gel electrophoresis was performed on the DYCP-31BN Electrophoresis Analyser (Liuyi Instrument Company, China) and imaged on a Bio-Rad ChemDoc XRS (Bio-Rad, USA). Fluorescence spectra were measured on an F-7000 spectroﬂuorophotometer (HITACHI, Japan). Confocal ﬂuorescence imaging of cells was performed on a TCS SP5 confocal laser scanning microscope (Leica, Germany). Flow cytometric analysis was performed on a Coulter FC-500 ﬂow cytometer (Beckman-Coulter). MTT and ELISA assays were carried out on Hitachi/Roche System Cobas 6000 (680, Bio-Rad, USA). Real-time PCR and melting curve measurement were performed on the CFX96 touch real-time PCR detection system (Bio-Rad, USA). AFM imaging was performed under ambient conditions with an Agilent 5500 AFM/SPM system (USA). Cell culture. HeLa cells (KeyGEN Biotech, Nanjing, China) were cultured in DMEM supplemented with 10% FBS, 100 mg ml 1 streptomycin and 100 U ml 1 penicillin–streptomycin at 37 C in a humidiﬁed incubator containing 5% CO2 and 95% air. ARTICLE Cy5 was excited at 633 nm with a HeNe 633 laser and the emissions were collected from 650 to 700 nm. Cell co-localization assay. Cy3-labelled siRNA (Cy3-siRNA) and Cy5-labelled siRNA (Cy5-siRNA) were self-assembled with other DNA strand component to form siRNA-TRU, respectively, and the resulting Cy3-siRNA-TRU and Cy5-siRNA-TRU were mixed with equimolar DNA backbone to generate the Cy3/Cy5-siRNA-ONV. After loading with Cy3/Cy5-siRNA-ONV, 5.0 mg ml 1 DAPI was added in CEM cells and incubated 15 min for imaging. DAPI was excited with a violet 405-nm laser diode and the emission was collected from 450 to 500 nm. Cy3 was excited at 543 nm with a HeNe 543 laser and the emissions were collected from 560 to 620 nm. Cy5 was excited at 633 nm with a HeNe 633 laser and the emissions were collected from 650 to 700 nm. Preparation of siRNA-ONV. The ONV was prepared based on a modiﬁed assembly strategy28. In brief, TRU was synthesized by the equimolar mixing of strands C1, C2, V1, R1 and R2 with a ﬁnal concentration of 5 mM in 1 TAMg buffer. This mixture was annealed at 95 C for 4 min and cooled to room temperature over 2 h. The ONV was generated by equimolar mixing the TRU with DNA backbone for 2 h at room temperature. To generate siRNA-modiﬁed ONV (siRNA-ONV), the siRNA-TRU was prepared by mixing of strands V1, C1, C2, R1, R2, siRNA and antisense siRNA at a ratio of 1:1:1:1:1:2:2 and assembled with equimolar DNA backbone. The control Cy3-siRNA-ONV without rigid structure (Cy3-siRNA-ONV-NR) was prepared by mixing of siRNA-TRU with DNA backbone at a ratio of 0.5:1. Endosomal escape of siRNA-ONV. To visualize the co-localization of inter- nalized siRNA-ONV with endosomal compartments, the aptamer-bound CEM cells were incubated with Cy3-siRNA-ONV and Cy3-siRNA-ONV without rigid structure (Cy3-siRNA-ONV-NR) at siRNA concentration of 200 nM for 2 or 6 h at 37 C. Then, the cells were washed with PBS and stained with 100 nM of LysoTracker Green and 5 mg ml 1 of DAPI for 15 min for imaging. LysoTracker Green was excited at 488 nm with an argon ion laser and the emission was collected from 505 to 535 nm. Endosomal escape of siRNA-ONV. ARTICLE After washing twice to remove nonbinding Cy3-siRNA-ONV, the ﬂuorescence of cells was visualized from 550 to 610 nm on the confocal laser scanning microscope (CLSM; TCS SP5, Leica, Germany) with the excitation wavelength of 514 nm for Cy3. All images were digitized and analysed with Leica Application Suite Advanced Fluorescence software package. To conﬁrm inter- nalization of Cy3-siRNA-ONV in the CEM cells, the vertical section scanning along the z axis was performed. While moving the focal plane in incremental steps from the bottom to the top of the cell within a 12 mm focal plane distance at the z position, 20 confocal ﬂuorescence microscopy images were taken and recorded. The ﬂow cytometric assay was performed in the PBS and used FL2 channel. Preparation of DNA backbone. The DNA backbone was prepared by RCA52. A volume of 10 ml of 3 mM circular DNA template and 0.5 ml of 100 mM DNA primer was mixed and annealed at 95 C for 4 min. Then, the mixture was cooled to room temperature over 2 h and incubated with phi29 DNA polymerase (0.2 U ml 1), bovine serum albumin (0.4 mg ml 1) and dNTPs (0.1 mM) at 37 C for 5 h in 150 ml of 1 phi29 reaction buffer. After reaction, the mixture was incubated at 65 C for 10 min to denature the phi29 DNA polymerase, and then puriﬁed by ultraﬁltration (100,000 molecular weight cutoff membrane, Millipore) for three times to obtain the DNA backbone. DNA backbone has repeating strand segments with sequences complementary to TRU. The concentration of the repeat unit in DNA backbone was obtained by measuring ultraviolet absorbance at 260 nm and used as the concentration of DNA backbone29. Cell co-localization assay. Cy3-labelled siRNA (Cy3-siRNA) and Cy5-labelled siRNA (Cy5-siRNA) were self-assembled with other DNA strand component to form siRNA-TRU, respectively, and the resulting Cy3-siRNA-TRU and Cy5-siRNA-TRU were mixed with equimolar DNA backbone to generate the Cy3/Cy5-siRNA-ONV. After loading with Cy3/Cy5-siRNA-ONV, 5.0 mg ml 1 DAPI was added in CEM cells and incubated 15 min for imaging. DAPI was excited with a violet 405-nm laser diode and the emission was collected from 450 to 500 nm. Cy3 was excited at 543 nm with a HeNe 543 laser and the emissions were collected from 560 to 620 nm. Methods R Reagents. MTT and calcein were purchased from Sigma-Aldrich (USA). Phi29 DNA polymerase, T4 DNA ligase, exonuclease I, exonuclease III and dNTPs were purchased from New England Biolabs Ltd. Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences (USA). Anti-VEGF primary antibody (19003-1-AP) was purchased from Proteintech Group (USA) and 50 times diluted during experiments. FITC-conjugated secondary antibody (111-095-003) was purchased from Jackson ImmunoResearch (USA) and 100 times diluted during experiments. LysoTracker Green and DAPI were purchased from Invitrogen (Carlsbad, CA, USA). SYBR Green I was obtained from Generay Biotech Co., Ltd (China). PBS (pH 7.4) contained 136.7 mM NaCl, 2.7 mM KCl, 8.72 mM Na2HPO4 and 1.41 mM KH2PO4. The reaction buffer was prepared using the PBS containing 0.5 mM MgCl2 and 50 mM ZnCl2, which helped the efﬁcient binding between aptamers and receptors, as well as providing MNAzyme with Zn2 þ for auto- catalytical cleavage of hairpin structure from the ONV. TAMg buffer (1 ) was composed of 45 mM Tris and 7.6 mM MgCl2, with pH adjusted to 8.0 using glacial acetic acid. All other reagents were of analytical grade. All aqueous solutions were prepared using ultrapure water (Z18 MO, Milli-Q, Millipore). siRNAs were obtained from GenePharma Co. Ltd (Shanghai, China). The siRNA sequences were as follows: VEGF siRNA, Cy3-sense 50-Cy3-GGAGUACCCUGAUGAGAUCd The nanomaterials with rigid structure and high aspect ratio such as cerium oxide nanowires and polymer nanoneedles generally exhibit better capability of cellular internalization and 7 ARTICLE To visualize the co-localization of inter- nalized siRNA-ONV with endosomal compartments, the aptamer-bound CEM cells were incubated with Cy3-siRNA-ONV and Cy3-siRNA-ONV without rigid structure (Cy3-siRNA-ONV-NR) at siRNA concentration of 200 nM for 2 or 6 h at 37 C. Then, the cells were washed with PBS and stained with 100 nM of LysoTracker Green and 5 mg ml 1 of DAPI for 15 min for imaging. LysoTracker Green was excited at 488 nm with an argon ion laser and the emission was collected from 505 to 535 nm. PAGE analysis. Native polyacrylamide gel (8%) was prepared using 1 TBE buffer. The loading sample was prepared by mixing 7 ml DNA sample, 1.5 ml 6 loading buffer and 1.5 ml UltraPowerTM dye, and placed still for 3 min before injected into polyacrylamide hydrogel. The gel electrophoresis was run at 90 V for 60 min in 1 TBE buffer and scanned using a Molecular Imager Gel Doc XR. AFM imaging. A volume of 6 ml sample was deposited on freshly cleaved mica surface and incubated for 2 min. After wicked away with ﬁlter paper, 20 ml ultrapure water was added to wash the mica for two times. The mica was then dried with a nitrogen ﬂow and scanned in tapping mode. The endosomal escape was further demonstrated with calcein release assay by incubating the aptamer-bound CEM cells with the mixture of 25 mM calcein and Cy3-siRNA-ONV or Cy3-siRNA-ONV-NR for 6 h at 37 C and then washing these cells with PBS. Calcein was excited at 488 nm with an argon ion laser and the emission was collected from 500 to 530 nm. Serum stability of siRNA-ONV Serum stability of siRNA-ONV. To verify the serum stability of siRNA-ONV, Cy3-tagged siRNA and BHQ2-tagged R1 and R2 oligonucleotides were used as components to assemble SQ-siRNA-ONV. SQ-ds-siRNA was also synthesized by assembling Cy3-tagged siRNA and BHQ2-tagged R1 and R2, and used as control. Fluorescence from Cy3 was quenched in the beginning due to the close distance Endocytosis pathways of siRNA-ONV. The uptake inhibitors of 450 mM sucrose, 200 mg ml 1 genistein, 50 mM methyl-b-cyclodextrin (Me-b-CD), 50 nM wortmannin and 10 mM NaN3 were preincubated with CEM cells for 30 min to inhibit clathrin, caveolae, lipid raft, macropinocytosis and energy-dependent References u, D. B. et al. Single-stranded RNAs use RNAi to potently and a 1. Yu, D. B. et al. Single-stranded RNAs use RNAi to potently and allele- selectively inhibit mutant huntingtin expression Cell 150 895 908 (2012) 1. Yu, D. B. et al. Single-stranded RNAs use RNAi to potently and allele- selectively inhibit mutant huntingtin expression. Cell 150, 895–908 (2012). 2. Lima, W. F. et al. Single-stranded siRNAs activate RNAi in animals. Cell 150, 883–894 (2012). 3. Kanasty, R., Dorkin, J. R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967–977 (2013). 4. Petros, R. A. & DeSimone, J. M. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discov. 9, 615–627 (2010). . Petros, R. A. & DeSimone, J. M. Strategies in the design of na siRNA-Lipo2000, siRNA-TKO and siRNA-ONV at siRNA concentration of 200 nM or equivalent ONV concentration of 100 nM were incubated with CEM cells for 2 h. After siRNA was transfected into CEM cells, fresh culture medium containing 10% FBS was added and incubated with cells for 48 h. Then, the cell proliferation was evaluated with the MTT assay. 5. Kanasty, R. L., Whitehead, K. A., Vegas, A. J. & Anderson, D. G. Action and reaction: the biological response to siRNA and its delivery vehicles. Mol. Ther. 20, 513–524 (2012). 6. Lu, H. et al. Site-speciﬁc antibody polymer conjugates for siRNA delivery. J. Am. Chem. Soc. 135, 13885–13891 (2013). Gene silencing assay. CEM cells were seeded into a 24-well plate at 5 105 cells per well and incubated with 50 nM of sgc8c and sgc4f in 200 ml reaction buffer for 30 min. After the cells were washed with PBS, 100 nM of siRNA-ONV was added and incubated for 2 h at 37 C. Meanwhile, ONV, NCsiRNA-ONV, 2nd-siRNA- ONV, ds-siRNA, siRNA-Lipo2000 and siRNA-TKO at siRNA concentration of 200 nM or equivalent ONV concentration of 100 nM were incubated with CEM cells for 2 h as controls. After centrifugation to precipitate cells, the supernatant medium was replaced by a fresh culture medium containing 10% FBS and further cultured for 48 h. The supernatant of culture medium was collected and analysed by ELISA to quantify VEGF secreted from the cells. Total RNAs from the transfected CEM cells were extracted using the Trizol reagent (Invitrogen), and complementary DNA was generated using the PrimeScriptRT reagent kit (Takara). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 In vivo antitumour efﬁcacy. Speciﬁc pathogen-free female BALB/c nude mice, 5–6 weeks of age, were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences, and bred in an axenic environment. All animal procedures were in accordance with Institutional Animal Use And Care Regulations approved by the Model Animal Research Center of Nanjing University (MARC), which allowed the maximal diameter (length) of every tumour should not exceed 20 mm or the volume of every tumour should be o2.0 cm3. To establish a CEM tumour model, 1.0 106 CEM cells were subcutaneous injected into the selected position of the nude mice. When the tumour volumes reached to 75 mm3, the tumour-bearing mice were randomly divided into ﬁve groups and intratumorally injected with 50 ml reaction buffer containing 200 pmol sgc8c and 200 pmol sgc4f, and subsequently injected with 50 ml reaction buffer (control), NCsiRNA-ONV, siRNA-ONV, siRNA-Lipo2000 or siRNA-TKO at a dose of 500 pmol siRNA per mouse after 30 min. The injections were performed every other day for ﬁve times. The tumour sizes were measured every 2 days by a digital caliper for 12 days, and the tumour volumes were calculated as V ¼ (L W2)/2, where L and W were the length and width of the tumour, respectively. Four days after the ﬁnal injection, tumours were collected, washed by saline thrice, ﬁxed in the 10% neutral buffered formalin, processed routinely into parafﬁn, sectioned at 4 mm and incubated with anti-VEGF primary antibody at 37 C for 2 h. The tissue sections were washed with saline thrice, stained with FITC-conjugated secondary antibody and the nuclei were counterstained with DAPI. The stained tissue sections were observed with CLSM. endocytosis, respectively37,54. Then, the cells were loaded with Cy3-siRNA-ONV and the inhibitors were maintained throughout the experiments. After that, ﬂow cytometric analyses were performed to determine the uptake amount of Cy3-siRNA-ONV. endocytosis, respectively37,54. Then, the cells were loaded with Cy3-siRNA-ONV and the inhibitors were maintained throughout the experiments. After that, ﬂow cytometric analyses were performed to determine the uptake amount of Cy3-siRNA-ONV. Release of siRNA. siRNA release process was ﬁrst conﬁrmed in cell lysates. ARTICLE A total of 5 107 cells were washed twice with cold PBS (pH 7.4) and resuspended in 200 ml of hypotonic lysis buffer containing 20 mM HEPES-KOH, pH 7.4, 10 mM KOAc, 1.5 mM Mg(OAc)2, 5 mM dithiothreitol, 0.1% Tween-20 and 1 EDTA-free protease inhibitor cocktail. The mixture was incubated for 10 min on ice and centrifuged at 35,200g and 4 C for 20 min. The supernatant was collected and diluted to 200 ml as cell lysates for detection. A concentration of 50 nM SQ-siRNA-ONV with 100 nM ﬂuorophore concentration was added in cell lysates or 1 TAMg buffer. The mixture was incubated at 37 C for 24 h to measure the ﬂuorescence signal with 510-nm excitation and 560-nm emission. For PAGE assay, 4 ml cell lysate was incubated with 4 ml siRNA-ONV at 37 C for 1 h and subsequently resolved on a 8% native polyacrylamide gel as described above. To characterize the intracellular release of siRNA from siRNA-ONV, the To characterize the intracellular release of siRNA from siRNA-ONV, the SQ-siRNA-ONV was incubated with CEM cells for 24 h, and the ﬂuorescence recovery from released Cy3-siRNA after SQ-siRNA-ONV disassembly was imaged with CLSM. MTT assay. MTT assay was carried out to investigate the cytotoxicity of ONV. CEM cells (5 105 per well) were seeded into two 96-well plates in 200 ml reaction buffer containing 50 nM of sgc4f and sgc8c aptamers for 30 min. Then, the cells were washed with PBS and incubated with serial concentrations of the ONV for 2 h. The results were compared with commercial transfection agent, 0.4 ml of Lipo2000 and TKO. Cells incubated with PBS served as control. The cells were washed twice with PBS buffer in the following and 50 ml of 5 mg ml 1 MTT solution was added and incubated for 4 h. After removing medium, 150 ml of dimethylsulphoxide was added to dissolve the formazan crystals precipitates. After shaking the cell plate for 15 min, the optical density at a wavelength of 490 nm was measured with a Bio-Rad microplate reader. The relative cell viability (%) was calculated by (Atest/Acontrol) 100. Negative control siRNA-ONV (NCsiRNA-ONV) was synthesized by using negative control siRNA instead of VEGF siRNA for cell proliferation assay. NCsiRNA-ONV, ds-siRNA, Data availability. ARTICLE The authors declare that the data supporting the ﬁndings of this study are available within the article and its Supplementary Information ﬁles or from the author on request. References The synthesized complementary DNA was then run on the CFX96 real-time PCR detection system (Bio-Rad, USA) to calculate VEGF mRNA level of cellular. 7. Song, E. et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23, 709–717 (2005). 8. Yao, Y. D. et al. Targeted delivery of PLK1-siRNA by scFv suppresses her2 þ breast cancer growth and metastasis. Sci. Transl. Med. 4, 130ra48 (2012). g 9. McNamara, II J. O. et al. Cell type–speciﬁc delivery of siRNAs with t iRNA hi N t Bi t h l 24 1005 1015 (2006) 9. McNamara, II J. O. et al. Cell type–speciﬁc delivery of siRNAs with aptamer-siRNA chimeras. Nat. Biotechnol. 24, 1005–1015 (2006). 10. Dassie, J. P. et al. Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors. Nat. Biotechnol. 27, 839–846 (2009). chimeras promotes regression of PSMA-expressing tumors. Nat. Biotechnol. 27, 839–846 (2009). 11. Zhou, H., Jiao, P., Yang, L., Li, X. & Yan, B. Enhancing cell recognition by scrutinizing cell surfaces with a nanoparticle array. J. Am. Chem. Soc. 133, 680–682 (2011). 12. Kluza, E. et al. Synergistic targeting of av3 integrin and galectin-1 with heteromultivalent paramagnetic liposomes for combined MR imaging and treatment of angiogenesis. Nano Lett. 10, 52–58 (2010). treatment of angiogenesis. Nano Lett. 10, 52–58 (2010). 13. Rudchenko, M. et al. Autonomous molecular cascades for evaluation of cell surfaces. Nat. Nanotechnol. 8, 580–586 (2013). Cell apoptosis experiments. A total of 5.0 105 CEM cells were seeded in a 24-well plate in 200 ml reaction buffer containing 50 nM sgc8c and 50 nM sgc4f for 30 min at room temperature. The cells were washed with PBS in the following and incubated with 100 nM of siRNA-ONV for 2 h. The cells were also incubated with ONV, NCsiRNA-ONV, 2nd-siRNA-ONV, ds-siRNA, siRNA-Lipo2000 and siRNA-TKO at siRNA concentration of 200 nM or equivalent ONV concentration of 100 nM under the same condition as the controls. After centrifugation of cells, the supernatant medium were replaced with fresh culture medium containing 10% FBS and further incubated for 48 h. The resulting cells were collected and stained with the mixture of 5.0 ml Annexin V-FITC and 5.0 ml propidium iodide for 15 min, and then imaged with ﬂow cytometry over FL1 (Annexin V-FITC) and FL3 (PI) channels. 14. Douglas, S. M., Bachelet, I. & Church, G. M. Endocytosis pathways of siRNA-ONV. The uptake inhibitors of 450 mM sucrose, 1 NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13580 21. Dong, H. F. et al. Target-cell-speciﬁc delivery, imaging, and detection of intracellular microRNA with a multifunctional SnO2 nanoprobe. Angew. Chem. Int. Ed. 51, 4607–4612 (2012). 46. Fusetti, L. et al. Human myeloid and lymphoid malignancies in the non-obese diabetic/severe combined immunodeﬁciency mouse model: frequency of apoptotic cells in solid tumors and efﬁciency and speed of engraftment correlate with vascular endothelial growth factor production. Cancer Res. 60, 2527–2534 (2000). 22. Chen, Y. J., Groves, B., Muscat, R. A. & Seelig, G. DNA nanotechnology from the test tube to the cell. Nat. Nanotechnol. 10, 748–760 (2015). 23. Pei, H., Zuo, X. L., Zhu, D., Huang, Q. & Fan, C. H. Functional DNA nanostructures for theranostic applications. Accounts Chem. Res. 47, 550–559 (2014). 23. Pei, H., Zuo, X. L., Zhu, D., Huang, Q. & Fan, C. H. Functional DNA 47. Avramis, I. A., Laug, W. E., Sausville, E. A. & Avramis, V. I. Determination of drug synergism between the tyrosine kinase inhibitors NSC 680410 (adaphostin) and/or STI571 (imatinib mesylate, Gleevec) with cytotoxic drugs against human leukemia cell lines. Cancer Chemother. Pharmacol. 52, 307–318 (2003). nanostructures for theranostic applications. Accounts Chem. Res. 47, 550–559 (2014). 24. Lee, J. B., Hong, J., Bonner, D. K., Poon, Z. Y. & Hammond, P. T. Self-assembled RNA interference microsponges for efﬁcient siRNA delivery. Nat. Mater. 11, 316–322 (2012). 48. Yang, Z. Z., Li, J. Q., Wang, Z. Z., Dong, D. W. & Qi, X. R. Tumor-targeting dual peptides-modiﬁed cationic liposomes for delivery of siRNA and docetaxel to gliomas. Biomaterials 35, 5226–5239 (2014). 25. Lee, H. et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nat. Nanotechnol. 7, 389–393 (2012). 49. Han, L., Tang, C. & Yin, C. H. Enhanced antitumor efﬁcacies of multifunctional nanocomplexes through knocking down the barriers for siRNA delivery. Biomaterials 44, 111–121 (2015). 26. Zhu, G. Z. et al. Self-assembled, aptamer-tethered DNA nanotrains for targeted transport of molecular drugs in cancer theranostics. Proc. Natl Acad. Sci. USA 110, 7998–8003 (2013). 50. Li, J. J. et al. Aptamer-functionalized silver nanoclusters-mediated cell type-speciﬁc siRNA delivery and tracking. Chem. Sci. 4, 3514–3521 (2013). 27. Pacardo, D. B., Ligler, F. S. & Gu, Z. Programmable nanomedicine: synergistic and sequential drug delivery systems. Nanoscale 7, 3381–3391 (2015). 51. Sun, W. J. et al. Self-assembled DNA nanoclews for the efﬁcient delivery of CRISPR–Cas9 for genome editing. Angew. Chem. Int. Ed. Acknowledgements y 33. Park, S. J., Park, W. & Na, K. Tumor intracellular-environment responsive materials shielded nano-complexes for highly efﬁcient light-triggered gene delivery without cargo gene damage. Adv. Funct. Mater. 25, 3472–3482 (2015). 33. Park, S. J., Park, W. & Na, K. Tumor intracellular-environment responsive materials shielded nano-complexes for highly efﬁcient light-triggered gene delivery without cargo gene damage. Adv. Funct. Mater. 25, 3472–3482 (2015). 34. Mo, R., Jiang, T. Y., DiSanto, R., Tai, W. Y. & Gu, Z. ATP-triggered anticancer drug delivery. Nat. Commun. 5, 3364 (2014). This work was ﬁnancially supported by National Natural Science Foundation of China (91213301, 91413118, 21135002 and 21322506) and Priority development areas of The National Research Foundation for the Doctoral Program of Higher Education of China (20130091130005). This work was ﬁnancially supported by National Natural Science Foundation of China (91213301, 91413118, 21135002 and 21322506) and Priority development areas of The National Research Foundation for the Doctoral Program of Higher Education of China (20130091130005). 34. Mo, R., Jiang, T. Y., DiSanto, R., Tai, W. Y. & Gu, Z. ATP-triggered anticancer drug delivery. Nat. Commun. 5, 3364 (2014). 35. Conde, J., Oliva, N., Atilano, M., Song, H. S. & Artzi, N. Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment. Nat. Mater. 15, 353–363 (2016). ARTICLE 54, 12029–12033 (2015). y CRISPR–Cas9 for genome editing. Angew. Chem. Int. Ed. 54, 12029–12033 (2015). 28. Hamblin, G. D. et al. Simple design for DNA nanotubes from a minimal set of unmodiﬁed strands: rapid, room-temperature assembly and readily tunable structure. ACS Nano 7, 3022–3028 (2013). 52. Ge, J., Zhang, L. L., Liu, S. J., Yu, R. Q. & Chu, X. A highly sensitive target-primed rolling circle ampliﬁcation (TPRCA) method for ﬂuorescent in situ hybridization detection of microRNA in tumor cells. Anal. Chem. 86, 1808–1815 (2014). 29. Hamblin, G. D., Carneiro, K. M. M., Fakhoury, J. F., Bujold, K. E. & Sleiman, H. F. Rolling circle ampliﬁcation-templated DNA nanotubes show increased stability and cell penetration ability. J. Am. Chem. Soc. 134, 2888–2891 (2012). 30. Lau, K. L., Hamblin, G. D. & Sleiman, H. F. Gold nanoparticle 3D-DNA building blocks: high purity preparation and use for modular access to nanoparticle assemblies. Small 10, 660–666 (2014). 53. Ririe, K. M., Rasmussen, R. P. & Wittwer, C. T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154–160 (1997). p 31. Wu, P. W., Hwang, K., Lan, T. & Lu, Y. A DNAzyme-gold nanoparticle probe for uranyl ion in living cells. J. Am. Chem. Soc. 135, 5254–5257 (2013). 54. Liang, L. et al. Single-particle tracking and modulation of cell entry pathways of a tetrahedral DNA nanostructure in live cells. Angew. Chem. Int. Ed. 53, 7879–7884 (2014). y g 32. Martens, T. F., Remaut, K., Demeester, J., De Smedt, S. C. & Braeckmans, K. 32. Martens, T. F., Remaut, K., Demeester, J., De Smedt, S. C. & Braeckmans, K. Intracellular delivery of nanomaterials: how to catch endosomal escape in the act. Nano Today 9, 344–364 (2014). Intracellular delivery of nanomaterials: how to catch endosomal escape in the act. Nano Today 9, 344–364 (2014). Author contributions H.J. initiated the projects. K.R., Y.L., J.W. and H.J. conceived the experiments. K.R., Y.Z. and J.Z. performed the experiments. K.R., Y.L., M.Y. and H.J. analysed the data and wrote the manuscript. 36. Wang, X. Z. et al. Designed synthesis of CeO2 nanorods and nanowires for studying toxicological effects of high aspect ratio nanomaterials. ACS Nano 6, 5366–5380 (2012). 37. Chen, G. et al. Enzymatic synthesis of periodic DNA nanoribbons for intracellular pH sensing and gene silencing. J. Am. Chem. Soc. 137, 3844–3851 (2015). Additional information 38. Kolhar, P., Doshi, N. & Mitragotri, S. Polymer nanoneedle-mediated intracellular drug delivery. Small 7, 2094–2100 (2011). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications 39. Hu, Y. et al. Cytosolic delivery of membrane-impermeable molecules in dendritic cells using pH-responsive core-shell nanoparticles. Nano Lett. 7, 3056–3064 (2007). Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. References A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834 (2012). 15. You, M. X. et al. DNA ‘‘nano-claw’’: logic-based autonomous cancer targeting and therapy. J. Am. Chem. Soc. 136, 1256–1259 (2014). 16. You, M. X., Zhu, G. Z., Chen, T., Donovan, M. J. & Tan, W. H. Programmable and multiparameter DNA-based logic platform for cancer recognition and targeted therapy. J. Am. Chem. Soc. 137, 667–674 (2015). g py 17. Yan, M. et al. Single siRNA nanocapsules for enhanced RNAi delivery. J. Am. Chem. Soc. 134, 13542–13545 (2012). 18. Calabrese, C. M. et al. Biocompatible inﬁnite-coordination-polymer nanoparticle–nucleic-acid conjugates for antisense gene regulation. Angew. Chem. Int. Ed. 54, 476–480 (2015). 19. Xu, X. Y. et al. Enhancing tumor cell response to chemotherapy through nanoparticle-mediated codelivery of siRNA and cisplatin prodrug. Proc. Natl Acad. Sci. USA 110, 18638–18643 (2013). Therapeutic efﬁcacy assay under serum condition. To characterize the therapeutic efﬁcacy of siRNA-ONV and 2nd-siRNA-ONV under serum condition, the internalization of siRNA-ONV and 2nd-siRNA-ONV in CEM cells was performed in fresh culture medium containing 10% FBS, 0.5 mM MgCl2 and 50 mM ZnCl2, and then the resulting cells were evaluated with gene silencing and cell apoptosis assays. 20. Kim, H. S. et al. In vitro and in vivo gene-transferring characteristics of novel cationic lipids, DMKD (O,O0-dimyristyl-N-lysyl aspartate) and DMKE (O,O0-dimyristyl-N-lysyl glutamate). J. Controlled Release 115, 234–241 (2006). NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications URE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications 9 Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 40. Febvay, S., Marini, D. M., Belcher, A. M. & Clapham, D. E. Targeted cytosolic delivery of cell-impermeable compounds by nanoparticle-mediated, light- triggered endosome disruption. Nano Lett. 10, 2011–2019 (2010). How to cite this article: Ren, K. et al. A DNA dual lock-and-key strategy for cell-subtype-speciﬁc siRNA delivery. Nat. Commun. 7, 13580 doi: 10.1038/ncomms13580 (2016). 41. Cuellar, T. L. et al. Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB–siRNA conjugates. Nucleic Acids Res. 43, 1189–1203 (2015). 42. Kwok, T. et al. Reduction of gene expression by a hairpin-loop structured oligodeoxynucleotide: Alternative to siRNA and antisense. Biochim. Biophys. Acta 1790, 1170–1178 (2009). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 43. Asbroek, A. L. M. A. T., Groenigen, M. V., Nooij, M. & Baas, F. The involvement of human ribonucleases H1 and H2 in the variation of response of cells to antisense phosphorothioate oligonucleotides. Eur. J. Biochem. 269, 583–592 (2002). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 44. Liu, X. X. et al. Adaptive amphiphilic dendrimer-based nanoassemblies as robust and versatile siRNA delivery systems. Angew. Chem. Int. Ed. 53, 11822–11827 (2014). 45. Zhou, J. H. et al. Systemic administration of combinatorial dsiRNAs via nanoparticles efﬁciently suppresses HIV-1 infection in humanized mice. Mol. Ther. 12, 2228–2238 (2011). r The Author(s) 2016 10 NATURE COMMUNICATIONS \| 7:13580 \| DOI: 10.1038/ncomms13580 \| www.nature.com/naturecommunications
https://openalex.org/W2109098292	https://digital.csic.es/bitstream/10261/117106/1/life-05-01282-v2%20%281%29.pdf	English	null	Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120	Life	2,015	cc-by	9,081	Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120 fael Pernil, Silvia Picossi, Antonia Herrero, Enrique Flores * and Vicente Mariscal Life 2015, 5, 1282-1300; doi:10.3390/life5021282 Life 2015, 5, 1282-1300; doi:10.3390/life5021282 life ISSN 2075-1729 www.mdpi.com/journal/life OPEN ACCESS f ISSN 2075-1729 www.mdpi.com/journal/life Article 1. Introduction Cyanobacteria are a group of prokaryotic microorganisms characterized by their ability to fix CO2 at the expense of oxygenic photosynthesis. They represent a coherent phylogenetic group in spite of showing a very diverse morphology and having colonized many different natural habitats. Some cyanobacteria are true multicellular organisms, growing as chains of cells that communicate between them and divide labor between specialized cells. Under combined nitrogen deprivation, heterocyst-forming cyanobacteria present two cell types: vegetative cells that perform oxygenic photosynthesis and heterocysts that carry out N2 fixation [1]. These specialized cells rely on each other: heterocysts require photosynthate that is provided by vegetative cells, and heterocysts provide vegetative cells with fixed nitrogen [2,3]. Three substrates that have been proposed as molecules transferred from vegetative cells to heterocysts are glutamate, sucrose, and alanine [2,4–7]. Sucrose is a universal vehicle of reduced carbon in plants [8], and it seems to be important also in cyanobacteria [9]; alanine can be an immediate source of reducing power in the heterocyst, where it is metabolized by alanine dehydrogenase [6]; and glutamate is mainly synthesized in vegetative cells by glutamate synthase and used in the heterocysts by glutamine synthetase to produce glutamine [2,10]. Heterocysts accumulate cyanophycin, a non-ribosomically synthesized peptide made of aspartate and arginine (multi-L-arginyl-poly [L-aspartic acid]) [11]. Cyanophycinase produces β-aspartyl-arginine, which is hydrolyzed by isoaspartyl dipeptidase in the vegetative cells [3]. Thus, glutamine and β-aspartyl-arginine (and perhaps also aspartate and arginine) appear to be nitrogen vehicles from heterocysts to vegetative cells [3,12]. Two possible pathways for intercellular molecular exchange in heterocyst-forming cyanobacteria have been discussed [1,13–15]. One involves structures at the septum between cells, septal junctions, which may consist of protein complexes that traverse septal peptidoglycan [16]. Septal junctions appear to allow the exchange of small, water-soluble compounds between cells [17,18]. Possible components of these cell–cell connecting structures are the SepJ, FraC, and FraD proteins [19,20]. The other route for cell–cell communication is the continuous periplasm present in the cyanobacterial filament [13,21]. The exchange of a substance through this route would require export to the periplasm in the producing cell, diffusion through this compartment, and import into the recipient cell. Interestingly, the outer membrane, which is continuous along the filament, appears to be relatively impermeable for compounds such as sucrose and glutamate that are exchanged between cells [22]. Rafael Pernil, Silvia Picossi, Antonia Herrero, Enrique Flores * and Vicente Mariscal Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Américo Vespucio 49, E-41092 Seville, Spain; E-Mails: rafapernil@gmail.com (R.P.); silvia.picossi@ibvf.csic.es (S.P.); herrero@ibvf.csic.es (A.H.); vicente.mariscal@ibvf.csic.es (V.M.) * Author to whom correspondence should be addressed; E-Mail: eflores@ibvf.csic.es; Tel.: +34-95-448-9523; Fax: +34-95-446-0165. * Author to whom correspondence should be addressed; E-Mail: eflores@ibvf.csic.es; Tel.: +34-95-448-9523; Fax: +34-95-446-0165. Academic Editors: John C. Meeks and Robert Haselkorn Academic Editors: John C. Meeks and Robert Haselkorn Received: 18 March 2015 / Accepted: 20 April 2015 / Published: 23 April 2015 Received: 18 March 2015 / Accepted: 20 April 2015 / Published: 23 April 2015 Abstract: Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion. Life 2015, 5 1283 Keywords: ABC-type transporters; amino acid diffusion; cyanobacteria; membrane transport 1. Introduction Independently of the pathway that might be used for nutrient exchange, cytoplasmic membrane transporters appear to be important in the diazotrophic physiology of heterocyst-forming cyanobacteria, since some amino acid transport mutants of the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 (hereafter Anabaena) are impaired in diazotrophic growth [23]. Anabaena is able to take up from the external medium a broad range of amino acids independently of whether it has been grown with combined nitrogen or under diazotrophic conditions [23–28]. This ability has been ascribed to the activity of several transport systems, termed N-I, N-II, and Bgt, which 1284 Life 2015, 5 1285 In this work we have investigated additional Open Reading Frames (ORF) of the Anabaena genome encoding proteins that show homology with amino acid transporters. We generated inactivation mutants of these ORFs and studied their amino acid uptake activity. Moreover, with the aim of clarifying aspects of the loss of amino acids to the external medium, we set up an assay to quantify the release from the cells of an alanine analogue that cannot be metabolized. 2.1. Bacterial Strains and Growth Conditions Anabaena sp. (also known as Nostoc sp.) strain PCC 7120 was grown in BG11 (which contains NaNO3) [33] or BG110 (free of combined nitrogen) medium at 30 °C in the light (25–75 μmol m−2 s−1), in shaken (80–90 rpm) liquid cultures or in medium solidified with 1% Difco agar. When indicated, the medium was supplemented with 10 mM NaHCO3 and the cultures were bubbled with 1% CO2 (BG11C or BG110C). For the mutants described below, antibiotics were used at the following concentrations: streptomycin sulfate (Sm), 2–5 μg mL−1; spectinomycin dihydrochloride pentahydrate (Sp), 2–5 μg mL−1 and neomycin sulfate (Nm), 5–30 μg mL−1 for liquid cultures; and Sm, 5 μg mL−1; Sp, 5 μg mL−1 and Nm, 40 μg mL−1 for solid cultures. DNA from Anabaena sp. was isolated by the method of Cai and Wolk [34]. Escherichia coli DH5 was used for plasmid constructions. This strain and strains HB101 and ED8654, used for conjugation to Anabaena, were grown in LB medium, supplemented when appropriate with antibiotics at standard concentrations [35]. Life 2015, 5 Life 2015, 5 Life 2015, 5 show different specificities for amino acids [27,28]. N-I recognizes 20 proteinogenic amino acids except for aspartate and is the main transporter responsible for the uptake of hydrophobic amino acids, especially proline, although it also transports some other amino acids including glutamine and glutamate. N-II recognizes and transports mainly acidic (aspartate and glutamate) and neutral polar amino acids, again including glutamine. Finally, the Bgt system is a basic amino acid transporter that also contributes to the uptake of glutamine, an amino acid that can therefore be transported by the three identified transporters of Anabaena. These three systems are ABC-type uptake transporters, and their constituents are summarized in Table 1. Of note, the bgtA gene encodes an ATP-binding subunit that energizes transport by the N-II and Bgt systems [28]. Table 1. ABC-type amino acid uptake transporters of Anabaena sp. strain PCC 7120. The Transporter Classification Database (TCDB) family number [29,30] is indicated. The order in which the transported amino acids are presented for each transporter reflects the contribution of the corresponding transporter to the total uptake of the indicated amino acids by nitrate-grown Anabaena filaments; bold face, preferred amino acids. ATPase: ATP-binding and hydrolyzing protein; Orn: ornithine; PSB: periplasmic substrate-binding protein; TM: transmembrane (permease) protein. Cys, Trp, and Tyr have not been investigated in transport assays in Anabaena. Transporter TCDB # Transported amino acids ORF Gene Predicted gene product Ref. N-I 3.A.1.4 Pro, Phe, Leu, all1046 natA ATPase [27] Gly, Thr, Ala, alr1834 natB PSB Ser, Met, Asn, all1047 natC TM His, Orn, Gln, all1248 natD TM Glu all2912 natE ATPase N-II 3.A.1.3 Asp, Glu, Asn, alr4164 natF PSB [28] Gln, Met, Thr, alr4165 natG TM Ala, Ser, Gly, alr4166 natH TM His alr4167 bgtA ATPase Bgt 3.A.1.3 Lys, Arg, Orn, alr4167 bgtA ATPase [28] His, Gln alr3187 bgtB PSB and TM N-III 3.A.1.4 Gly, Pro, Glu, alr2535 natI PSB This work Phe, Leu, Ala, alr2536 natJ TM Gln alr2538 natK TM alr2539 natL ATPase alr2541 natM ATPase Some cyanobacterial mutants have been reported to accumulate amino acids in the culture medium [6,23,27,28,31,32]. In Anabaena, release of hydrophobic amino acids is a general feature of strains mutated in neutral amino acid transporters [23,27,28]. This is especially relevant under diazotrophic conditions, where alanine released from strains mutated in the N-I and N-II systems reaches a concentration of 0.1 mM in the medium [28]. Life 2015, 5 for all3551 (all primers contain BamHI restriction sites in, or close to, their 5' ends). Amplified fragments were cloned into vector pMBL-T (MBL) producing pCSVM1, pCSVM2, and pCSVM3, respectively, and then transferred as BamHI-ended fragments to BamHI-digested pCSV3 producing, respectively, pCSVM4, pCSVM5, and pCSVM6 (Smr Spr). 2. Oligodeoxynucleotide primers used in this work. Introduced restriction enzyme g sites are indicated in boldface. Table 2. Oligodeoxynucleotide primers used in this work. Introduced restriction enzyme cutting sites are indicated in boldface. Table 2. Oligodeoxynucleotide primers used in this work. Introduced restriction enzyme cutting sites are indicated in boldface. Primer Sequence (5'→3') alr2536-7120-1 GGA TCC GCT AAC GCT ACT TTG CCG alr2536-7120-2 GGA TCC GCA ACC CAA AGC CAA TC all0342-7120-1 GGA TCC GTT GAC CAA TAC CCT CAT GGC all0342-7120-2 GGA TCC GCT TGG AAG GTT ACA GGC alr3429-7120-1 GGA TCC GGG GTT TAA AGA TGC TGA CGG alr3429-7120-2 GGA TCC GAG GAT GTT CTC TCA CCC all1189-1 GGA TCC GGA AAC TCA CAG all1189-2 GCG GAT CCA GGA TAA TAG alr1538-1 GGA TCC TGG CTG TGT ATT TAG alr1538-2 GGA TCC TTT GGG CAG AAG all3551-1 GGA TCC AGC CCA ATA GTT G all3551-2 GGA TCC CTG CCA AAG AC AA-1 GAG CCA TAC AAG CTC TGA TTC ATG G AA-2 ACG CGA TCG CTG ACT CCT GCC Conjugation of Anabaena sp. strain PCC 7120 with E. coli HB101 carrying plasmids pCSR17, pCSR13, pCSR23, pCSS4, pCSVM4, pCSVM5, or pCSVM6 with helper and methylation plasmid pRL623 was effected by the conjugative plasmid pRL443, carried in E. coli ED8654, and performed as described previously [39]. Exconjugants were selected for their resistance to Sm and Sp, or to Nm; for the generation of the mutant CSS4, double recombinants were selected for their resistance to sucrose. The genetic structure and segregation of selected clones were studied by Southern analysis using as probes the cloned fragments from the corresponding mutated gene (see above). The probes were labeled with Ready-To-Go DNA Labeling Beads (−dCTP) kit (GE Healthcare). The mutants, which were homozygous for the mutated chromosomes in all cases, were named as follows: CSR6 from plasmid pCSR17, CSR1 from plasmid pCSR13, CSR3 from plasmid pCSR23, CSS4 from plasmid pCSS4, CSVM1 from plasmid pCSVM4, CSVM2 from plasmid pCSVM5, and CSVM3 from plasmid pCSVM6. 2.2. Plasmid Construction and Genetic Procedures For inactivation of alr2536, all0342, and alr3429, internal fragments of 552 bp, 596 bp, and 469 bp, respectively, were amplified by PCR using DNA from strain PCC 7120 as a template and primers alr2536-7120-1 and alr2536-7120-2 for alr2536, all0342-7120-1 and all0342-7120-2 for all0342, and alr3429-7120-1 and alr3429-7120-2 for alr3429 (all primers contain BamHI restriction sites in their 5' ends and are listed in Table 2). Amplified fragments were cloned into vector pGEM-T (Promega) in the case of alr2536 and all0342 or pGEM-T Easy (Promega) for alr3429, producing pCSR4, pCSR1, and pCSR19, respectively, and then transferred as BamHI-ended fragments to BamHI-digested pRL424 [36] producing pCSR17, pCSR13, and pCSR23, respectively (Nmr). For inactivation of alr1519, a 2.3 kb DNA fragment carrying the full ORF and flanking regions was amplified from genomic DNA from strain PCC 7120 using primers AA-1 and AA-2. The amplified fragment was inserted into vector pGEM-T (Promega) producing pCSS1. The C.S3 cassette (encoding Smr Spr) was excised from plasmid pRL463 [37] with BamHI and inserted into the BclI-digested pCSS1 to interrupt the ORF alr1519, obtaining plasmid pCSS2. The construct was transferred as a PvuII-ended fragment to the suicide plasmid pRL278 [38] digested with NruI producing pCSS4 (Smr Spr Nmr). For inactivation of all1189, alr1538, and all3551, internal fragments of 439 bp, 440 bp, and 417 bp, respectively, were amplified by PCR using DNA from strain PCC 7120 as a template and primers all1189-1 and all1189-2 for all1189, alr1538-1 and alr1538-2 for alr1538, and all3551-1 and all3551-2 1286 Life 2015, 5 2.4. Substrate Transport Assays Amino acid uptake assays were carried out at 30 °C in the light (175 μmol m−2 s−1). Amino acid uptake was determined as described previously [23] in 10-min transport assays with the indicated concentration of L-[U-14C]amino acid, in filaments grown in BG11 medium (supplemented with the appropriate antibiotic in the case of the mutants), washed and suspended in 25 mM N-tris(hydroxymethyl)-methylglycine (Tricine)-NaOH buffer (pH 8.1). Significance of the difference of uptake between a mutant and the wild type was assessed by unpaired Student’s t tests assuming a normal distribution of the data. Differences with P ≤ 0.05 are considered statistically significant. For solute release experiments, filaments grown in BG11 or BG110 medium were washed, suspended in 25 mM Tricine-NaOH buffer (pH 8.1), and incubated with 3 μM α-[1-14C]aminoisobutyric acid (AIB) for 40 or 90 min at 30 °C in the light (175 μmol m−2 s−1). At the time indicated, an excess of unlabeled substrate at a concentration of 1 mM was added. Samples were removed at different times after the addition of the unlabeled substrate and cell-associated radioactivity was determined after filtration and washing with about 3–5 mL of 5 mM Tricine-NaOH buffer (pH 8.1). To determine metabolites produced from the labeled substrate and released from the cells at the end of the experiments, samples of 0.2 mL of the cell suspension were centrifuged at 14,000× g for 1 min and supernatants were centrifuged again. Samples of the resulting solutions were applied to 0.1-mm-thick cellulose thin-layer chromatography (TLC) plates (20 cm × 20 cm; Merck, Darmstadt, Germany). Two-dimensional separation of amino acids was effected by using the following solvents: the first dimension solvent consisted of n-butanol-acetone-ammonium hydroxide-water (20:20:10:4, v/v/v/v), and the second dimension solvent consisted of isopropanol-formic acid-water (20:1:5, v/v/v). TLC plates were analyzed by electronic autoradiography using a two-dimensional scanner for β particles (Cyclone Plus Phosphor Imager, PerkinElmer, Waltham, MA, USA). Life 2015, 5 1287 Life 2015, 5 Life 2015, 5 liquid cultures. Protein concentration was determined by a modified Lowry procedure [40]. Chlorophyll a (Chl) content of cultures was determined by the method of Mackinney [41]. 2.3. Growth Tests and Sample Preparation Growth tests were carried out by spotting 2–7 µL of cell suspension containing 2.5–10 µg Chl mL−1 on agar plates of BG11 or BG110 medium. The plates were incubated at 30 °C in the light (25–40 µmol m−2 s−1) and observed over a period of two weeks. Growth rates in liquid medium were determined as previously described [23]. The growth rate constant (µ = ln2/td, where td is the doubling time) was calculated from the increase of protein content determined in 0.2 mL of samples of shaken Life 2015, 5 Life 2015, 5 1288 To inactivate this putative ABC-type transporter, plasmid pCSR17, based on pRL424, which carries the npt gene with no transcriptional terminators [36], was inserted through a single cross-over event into alr2536, producing the Anabaena mutant strain CSR6 (Figure 1A). Strain CSR6 grew well on solid medium with or without combined nitrogen (not shown). The uptake of some amino acids was studied in nitrate-grown filaments of strain CSR6 in comparison to the wild type. The mutant was mainly impaired in the transport of hydrophobic amino acids (54%–72% of wild-type activity), especially glycine, and impairment in the transport of glutamate and glutamine (63% and 77% of wild-type activity, respectively) was also observed (Table 3). These results suggest that some of the genes in the alr2535 to alr2541 gene cluster encode components of a transport system that can mediate the uptake of some amino acids in Anabaena, mainly hydrophobic amino acids. We have named this system N-III, and the corresponding genes natI to natM (Table 1). Figure 1. Insertional inactivation of ORFs alr2536, alr3429, all0342, alr1519, all1189, alr1538, and all3551. Schematic representation of the loci of alr2536 (A); alr3429 (B); all0342 (C); alr1519 (D); all1189 (E); alr1538 (F); and all3551 (G), with indication of the plasmids inserted to produce strains CSR6, CSR3, CSR1, CSS4, CSVM1, CSVM2, and CSVM3, respectively. Figure 1. Insertional inactivation of ORFs alr2536, alr3429, all0342, alr1519, all11 Figure 1. Insertional inactivation of ORFs alr2536, alr3429, all0342, alr1519, all1189, alr1538, and all3551. Schematic representation of the loci of alr2536 (A); alr3429 (B); all0342 (C); alr1519 (D); all1189 (E); alr1538 (F); and all3551 (G), with indication of the plasmids inserted to produce strains CSR6, CSR3, CSR1, CSS4, CSVM1, CSVM2, and CSVM3, respectively. Figure 1. Insertional inactivation of ORFs alr2536, alr3429, all0342, alr1519, all1189, alr1538, and all3551. Schematic representation of the loci of alr2536 (A); alr3429 (B); all0342 (C); alr1519 (D); all1189 (E); alr1538 (F); and all3551 (G), with indication of the plasmids inserted to produce strains CSR6, CSR3, CSR1, CSS4, CSVM1, CSVM2, and CSVM3, respectively. The search for genes encoding other putative components of ABC-type amino acid transport systems identified ORF alr3429, which is annotated as GlnH, a periplasmic glutamine-binding protein [43], but is most similar to Synechocystis GtrC, a periplasmic glutamate-binding protein [44]. Although genes encoding an ABC transporter are frequently clustered together, alr3429 upstream and downstream genes have no obvious relation to transport functions. 3.1. Predicted ABC-Type Amino Acid Transporters Blast searches of the Anabaena genome, using as queries the amino acid sequences of the permease subunits of the Anabaena ABC-type transporters N-I and N-II (Table 1), identified a new cluster of genes (alr2535 to alr2541) putatively encoding the elements of an additional ABC-type transporter for amino acids (Figure 1A). ORF alr2535 would encode a protein of 268 amino acid residues with homology to the periplasmic substrate-binding protein BraC of the branched-chain amino acid transport system of Pseudomonas aeruginosa [42]. ORFs alr2536 and alr2538 would encode 316 and 308 amino acid residue peptides, respectively, with homology to permeases NatC and NatD of the transport system N-I of Anabaena. ORF alr2539 and alr2541 would encode 259 and 264 amino acid residue peptides, respectively, showing homology with ATPases NatA and NatE of the N-I system. Two additional ORFs, alr2537 and asl2540, would encode hypothetical proteins with no homologues in the databases. Life 2015, 5 Life 2015, 5 combined nitrogen (not shown). The uptake rate of representative amino acids in this mutant was analyzed, in comparison with the wild type, in filaments that had been grown with nitrate as the nitrogen source. For the tested amino acids, the activity was within a range of about ±20% the wild-type activity (Table 3), giving no support for a specific role of Alr3429 in amino acid transport. Table 3. Amino acid transport activities in Anabaena sp. strain PCC 7120 and mutant strains. BG11-grown cells were used in 10-min transport assays with 10 μM [14C]amino acid substrate as described in the Experimental Section. Activities are given in nmol (mg Chl)−1 per 10 min; the mean ± standard deviation of the mean (SE) from the number of measurements indicated in parenthesis is presented. The percentage of the wild-type activity is also given for the mutants. Differences between mutant and wild-type activities were assessed by the Student’s t test (P indicated in each case). Life 2015, 5 Substrate Transport activity (nmol [mg Chl]−1) PCC 7120 Mutant strain (inactivated ORF) CSR6 (alr2536) CSR3 (alr3429) CSR1 (all0342) Mean ± SE (n) Mean ± SE (n) % (P) Mean ± SE (n) % (P) Mean ± SE (n) % (P) Basic L-Arg 125 ± 1.59 (25) 115 ± 16.4 (3) 92% (0.077) 148 ± 6.60 (4) 119% (0.187) 195 ± 12.5 (3) 156% (0.068) L-Lys 138 ± 1.55 (16) 151 ± 3.92 (2) 110% (0.167) 163 ± 6.15 (3) 118% (0.119) 140 ± 8.40 (2) 102% (0.882) L-His 88.0 ± 1.10 (16) 71.7 ± 4.80 (3) 81% (0.174) 90.0 ± 4.94 (4) 102% (0.863) 132 ± 5.28 (3) 150% (0.023) Acidic L-Asp 37.3 ± 0.39 (27) 33.5 ± 2.18 (3) 90% (0.440) 31.1 ± 1.67 (4) 83% (0.170) 38.8 ± 2.19 (4) 104% (0.768) L-Glu 10.5 ± 0.12 (25) 6.62 ± 0.70 (3) 63% (0.061) 10.2 ± 0.26 (4) 97% (0.721) 16.9 ± 1.56 (4) 161% (0.130) Neutral polar L-Gln 99.4 ± 0.90 (30) 77.0 ± 6.89 (3) 77% (0.189) 99.3 ± 6.31 (4) 100% (0.994) 158 ± 6.48 (4) 158% (0.014) L-Ser 218 ± 2.50 (10) 195 ± 2.05 (2) 90% (0.022) 191 ± 9.37 (3) 88% (0.237) 239 ± 34.3 (3) 110% (0.753) Hydrophobic L-Ala 192 ± 1.09 (20) 139 ± 13.4 (3) 72% (0.145) 203 ± 16.3 (4) 105% (0.772) 286 ± 6.03 (3) 149% (0.004) Gly 200 ± 3.30 (15) 107 ± 14.0 (3) 54% (0.039) 222 ± 8.84 (4) 111% (0.337) 252 ± 5.50 (3) 126% (0.007) L-Leu 103 ± 0.99 (17) 70.4 ± 5.34 (3) 68% (0.051) 117 ± 5.33 (4) 113% (0.315) 177 ± 12.0 (3) 171% (0.067) L-Pro 135 ± 1.99 (16) 82.8 ± 14.9 (3) 61% (0.174) 151 ± 10.0 (3) 112% (0.449) 198 ± 13.1 (4) 147% (0.089) L-Phe 118 ± 1.87 (16) 79.4 ± 1.78 (2) 67% (<0.001) 91.6 ± 7.79 (4) 78% (0.197) 109 ± 8.32 (3) 92% (0.607) Life 2015, 5 ORF alr3429 was inactivated by insertion of plasmid pCSR23 (based on pRL424) through a single cross-over event producing the Anabaena mutant strain CSR3 (Figure 1B). Strain CSR3 grew well on a solid medium with or without 1289 Life 2015, 5 Life 2015, 5 Life 2015, 5 resulting in retention of some amino acids in the cells, or that lack of All0342 facilitates uptake mediated by other transporters. ORF alr1519 encodes a predicted 456 amino acid peptide showing homology to transporters of the amino acid-polyamine-organocation (APC) family (TCDB 2.A.3 [30]) that includes solute:cation symporters and solute:solute antiporters [46,47]. ORF alr1519 was inactivated by insertion of the C.S3 cassette (Smr Spr [36]), producing the mutant strain CSS4 (Figure 1D). The growth rate of CSS4 mutant in liquid BG11 medium was similar to that of the wild type (µ, 0.65 day−1 for both strains). However, under diazotrophic conditions the growth rate of the mutant was about 75% that of the wild type (0.36 ± 0.04 day−1 for the CSS4 mutant versus 0.43 ± 0.04 day−1 for the wild type; n = 3). Amino acid uptake was then tested in filaments of strain CSS4 and wild-type Anabaena grown in the presence and absence of combined nitrogen (Table 4). We found only a slight increase in the transport of aspartate, but this alteration was hardly statistically significant. These results give no support for a specific role of Alr1519 in amino acid uptake. Table 4. Amino acid transport activities in Anabaena sp. strains PCC 7120 and CSS4. BG11C and BG110C-grown filaments were used in 10-min transport assays with 10 μM [14C]amino acid substrate as described in the Experimental Section. Activities are presented in nmol (mg Chl)-1 per 10 min; the mean ± standard deviation of the mean (SE) from the number of measurements indicated in parenthesis is presented. The percentage of the wild-type activities is also given for the mutant. Differences between the mutant and wild-type activities were assessed by the Student’s t test (P indicated in each case). 3.2. Predicted Amino Acid Transporters from Other Transporter Families In the genome of Anabaena, ORF all0342 encodes a predicted 437 amino acid peptide, annotated as H+/Na+-dependent glutamate and aspartate symporter [45], which belongs to the dicarboxylate/amino acid:cation symporter (DAACS) transporter family (TCDB family 2.A.23 [30]). ORF all0342 was inactivated by insertion through a single cross-over event of pCSR13 (based on pRL424), producing Anabaena mutant strain CSR1 (Figure 1C). Strain CSR1 grew well on a solid medium with or without combined nitrogen (not shown). Regarding amino acid uptake, strain CSR1 showed about a 50% increase compared to the wild type in the uptake of some amino acids from all the chemical groups tested (Table 3). This increase was statistically significant for histidine, glutamine, alanine, and glycine (P ≤ 0.05). These results suggest either that All0342 participates in amino acid export, its mutation 1290 Life 2015, 5 Substrate Transport activity (nmol [mg Chl]−1) BG11C BG110C PCC 7120 CSS4 (alr1519) PCC 7120 CSS4 (alr1519) Mean ± SE (n) Mean ± SE (n) % (P) Mean ± SE (n) Mean ± SE (n) % (P) Basic L-Arg 116 ± 7.64 (3) 113 ± 3.16 (2) 98% (0.864) 187 ± 8.96 (3) 167 ± 3.58 (2) 89% (0.327) Acidic L-Asp 30.3 ± 1.03 (4) 42.0 ± 3.85 (2) 139% (0.214) 62.1 ± 0.98 (4) 77.6 ± 3.57 (3) 125% (0.119) L-Glu 10.4 ± 1.48 (4) 10.3 ± 0.64 (3) 99% (0.970) 34.7 ± 1.63 (4) 32.2 ± 1.90 (3) 93% (0.619) Neutral polar L-Gln 118 ± 4.46 (3) 125 ± 2.15 (2) 106% (0.487) 216 ± 5.30 (3) 214 ± 2.06 (2) 99% (0.854) Hydrophobic L-Ala 224 ± 12.6 (3) 215 ± 17.8 (2) 96% (0.826) 309 ± 11.7 (3) 299 ± 14.0 (2) 108% (0.748) 3.3. Release of Amino Acids from Vegetative Cells 3.3. Release of Amino Acids from Vegetative Cells Anabaena mutants impaired in transport systems N-I and N-II release hydrophobic amino acids, especially alanine, to the culture medium [27,28]. To gain a better understanding of the kinetics of alanine release, the alanine analogue α-aminoisobutyric acid (AIB), which cannot generally be metabolized, was used. In the N-I mutant strain CSR11 (natD) [27], the rate of [14C]AIB uptake was 2.43 ± 0.48% (n = 3) that of the wild type, indicating that the N-I system is responsible for about 98% of the uptake of this compound in Anabaena. 1291 Life 2015, 5 Life 2015, 5 To assay the release of AIB in Anabaena, the following experiment was set up. Filaments grown in the presence of nitrate were incubated with 3 μM [14C]AIB for 40 to 90 min under transport assay conditions. Then, an unlabeled substrate was added at 1 mM concentration and the amount of [14C]AIB remaining in the cells was determined at different times. The release of [14C]AIB in response to the addition of unlabeled substrates could thus be quantified as the decrease in the amount of radioactivity remaining in the cells. In wild-type Anabaena grown with combined nitrogen and incubated with or without combined nitrogen for 24 h, significant release of [14C]AIB was observed in samples supplemented with unlabeled AIB or alanine, but not with a buffer (Figure 2A, B). Note that, at the concentrations used, [14C]AIB uptake likely became negligible when a competitor such as unlabeled AIB or alanine was added, but evidently not when only buffer (T25) was added. The 14C-labeled compound(s) released to the medium were analyzed by TLC and only one spot corresponding to AIB was detected (Figure 2C), consistent with the idea that AIB is not metabolized. Figure 2. AIB release in Anabaena. Filaments from BG11 medium (A) or incubated in BG110 medium for 24 h (B) were incubated in Tricine buffer with 3 μM [14C]AIB. After 40 min the filament suspensions were supplemented with 1 mM AIB or alanine in a small volume of buffer or, as a control, with the same volume of buffer (T25). Cell-associated 14C was measured in samples taken from the filament suspensions at the times indicated. (C) A sample of the medium from the end of the assay with BG11-grown cells incubated with [14C]AIB and supplemented with unlabeled AIB was subjected to TLC analysis; o: origin of the chromatography. Only one radioactive spot co-migrating with AIB was detected. Figure 2. AIB release in Anabaena. Filaments from BG11 medium (A) or incubated in BG110 medium for 24 h (B) were incubated in Tricine buffer with 3 μM [14C]AIB. After 40 min the filament suspensions were supplemented with 1 mM AIB or alanine in a small volume of buffer or, as a control, with the same volume of buffer (T25). Cell-associated 14C was measured in samples taken from the filament suspensions at the times indicated. Life 2015, 5 (C) A sample of the medium from the end of the assay with BG11-grown cells incubated with [14C]AIB and supplemented with unlabeled AIB was subjected to TLC analysis; o: origin of the chromatography. Only one radioactive spot co-migrating with AIB was detected. Figure 2. AIB release in Anabaena. Filaments from BG11 medium (A) or incubated in BG110 medium for 24 h (B) were incubated in Tricine buffer with 3 μM [14C]AIB. After 40 min the filament suspensions were supplemented with 1 mM AIB or alanine in a small volume of buffer or, as a control, with the same volume of buffer (T25). Cell-associated 14C was measured in samples taken from the filament suspensions at the times indicated. (C) A sample of the medium from the end of the assay with BG11-grown cells incubated with [14C]AIB and supplemented with unlabeled AIB was subjected to TLC analysis; o: origin of the chromatography. Only one radioactive spot co-migrating with AIB was detected. Figure 2. AIB release in Anabaena. Filaments from BG11 medium (A) or incubated in BG110 medium for 24 h (B) were incubated in Tricine buffer with 3 μM [14C]AIB. After 40 min the filament suspensions were supplemented with 1 mM AIB or alanine in a small volume of buffer or, as a control, with the same volume of buffer (T25). Cell-associated 14C was measured in samples taken from the filament suspensions at the times indicated. (C) A sample of the medium from the end of the assay with BG11-grown cells incubated with [14C]AIB and supplemented with unlabeled AIB was subjected to TLC analysis; o: origin of the chromatography. Only one radioactive spot co-migrating with AIB was detected. Life 2015, 5 Life 2015, 5 In the experiments of AIB release, we observed that the rate of release depended on the intracellular concentration of [14C]AIB reached in each particular cell suspension, which depended on the time at which unlabeled AIB was added (cell suspensions were assayed at the same growth stage in all experiments). Because the level of accumulated [14C]AIB was known for each assay, we could represent the rate of release as a function of the intracellular [14C]AIB concentration (see data for the wild type, PCC 7120, in Figure 4). A linear relation, y = 0.004x + b, where y is the release rate and x the intracellular concentration of [14C]AIB, could be established, with r2 coefficients of 0.779 for BG11-grown filaments and 0.913 for filaments that had been incubated in the absence of combined nitrogen (BG110 medium). Figure 4. Kinetics of AIB release. The rate of [14C]AIB release in response to the addition of 1 mM unlabeled AIB to filaments incubated in Tricine buffer with 3 μM [14C]AIB was measured. Unlabeled AIB was added after 40 to 90 min of incubation with [14C]AIB. (A) Nitrate-grown filaments. (B) Filaments incubated in BG110 medium for 24 h. Assuming an intracellular volume of 125 µL (mg Chl)−1, the range of intracellular [14C]AIB concentrations reached in the assays was from about 0.5 to 4 mM. Figure 4. Kinetics of AIB release. The rate of [14C]AIB release in response to the addition of 1 mM unlabeled AIB to filaments incubated in Tricine buffer with 3 μM [14C]AIB was measured. Unlabeled AIB was added after 40 to 90 min of incubation with [14C]AIB. (A) Nitrate-grown filaments. (B) Filaments incubated in BG110 medium for 24 h. A i i t ll l l f 125 L ( Chl)−1 th f i t ll l Figure 4. Kinetics of AIB release. The rate of [14C]AIB release in response to the addition of 1 mM unlabeled AIB to filaments incubated in Tricine buffer with 3 μM [14C]AIB was ure 4. Kinetics of AIB release. The rate of [14C]AIB release in response to the addition of 1 mM unlabeled AIB to filaments incubated in Tricine buffer with 3 μM [14C]AIB was measured. Unlabeled AIB was added after 40 to 90 min of incubation with [14C]AIB. (A) Nitrate-grown filaments. (B) Filaments incubated in BG110 medium for 24 h. Life 2015, 5 Life 2015, 5 1292 The release of [14C]AIB in response to the addition of different concentrations of AIB ranging from 0.1 to 3 mM was assayed with no significant differences in the rate of release (not shown). To test whether the transport system N-I or N-II is involved in the release of [14C]AIB, assays were performed with specific substrates of these transporters. Filaments were incubated in the presence of 3 μM [14C]AIB for 90 min, and the release of [14C]AIB in response to the addition of an excess of unlabeled AIB, proline (a specific substrate of system N-I), or aspartate (a specific substrate of system N-II) was determined (Figure 3). Whereas the addition of proline produced the release of [14C]AIB in a similar way to AIB, the addition of aspartate did not affect [14C]AIB uptake, which continued in the presence of aspartate. These results indicate that net [14C]AIB release is dependent on the addition of a substrate of transport system N-I. We suggest that [14C]AIB, accumulated within the cells, is released to some extent and then re-incorporated through the N-I transporter, and that saturation of this transporter by unlabeled AIB, alanine (Figure 2), or proline (Figure 3) impedes re-incorporation, permitting us to observe net release. Figure 3. Specificity of AIB release in Anabaena. Filaments grown in BG11 medium were incubated in Tricine buffer with 3 μM [14C]AIB. After 90 min the filament suspensions were supplemented with 1 mM AIB, proline, or aspartate in a small volume of buffer or, as a control, with the same volume of buffer (T25). Cell-associated 14C was measured in samples taken from the filament suspensions at the times indicated. Figure 3. Specificity of AIB release in Anabaena. Filaments grown in BG11 medium were incubated in Tricine buffer with 3 μM [14C]AIB. After 90 min the filament suspensions were supplemented with 1 mM AIB, proline, or aspartate in a small volume of buffer or, as a control, with the same volume of buffer (T25). Cell-associated 14C was measured in samples taken from the filament suspensions at the times indicated. 1293 Life 2015, 5 Life 2015, 5 in every case, providing no indication of the involvement in AIB release of the protein encoded by any of the mutated genes. Instead, we observed that the rate of [14C]AIB release in the mutants fitted the relation to intracellular [14C]AIB concentration previously established for the wild type. Adding the data from the mutants to those of the wild type, the same linear relation was observed with r2 coefficients of 0.818 for BG11-grown filaments and 0.764 for filaments that had been incubated in BG110 medium. The linear dependence of the rate of AIB release on the intracellular concentration of AIB suggests that the release takes place by diffusion. Additionally, the fact that b takes a negative value (−0.131 for BG11-grown cells and −0.080 for BG110-incubated cells) implies that there was an intracellular concentration threshold for the observation of [14C]AIB release. Assuming an intracellular volume of 125 µL (mg Chl)−1 [48,49], this threshold was in the range 0.17–0.28 mM. This concentration might correspond to AIB immobilized through its interaction with cellular components. Life 2015, 5 Assuming an intracellular volume of 125 µL (mg Chl)−1, the range of intracellular [14C]AIB concentrations reached in the assays was from about 0.5 to 4 mM. Blast searches of the Anabaena genome for genes homologous to exporters from the EamA (amino acid, metabolite efflux pumps) family (TCDB 2.A.7.3.2, which belongs to the Drug and Metabolite Exporter [DME] family, TCDB 2.A.7.3 [30]) identified three open reading frames: all1189, alr1538, and all3551. Each of these ORFs was inactivated by insertion through single cross-over of a plasmid based on pCSV3 (Smr Spr). Insertion of pCSVM4, pCSVM5, and pCSVM6 produced mutant strains CSVM1, CSVM2, and CSVM3, respectively (Figure 1). The three mutants grew similarly to the wild type both in the presence and in the absence of combined nitrogen (not shown). Activity of [14C]AIB release in response to the addition of unlabeled AIB was tested in mutant strains CSVM1, CSVM2, and CSVM3, as well as in CSR1, CSR6, and CSR3 described above. Significant export was observed 1294 Life 2015, 5 Life 2015, 5 of a mutant of this gene showed increased uptake of a number of amino acids. These results could be interpreted assuming that All0342 participates in amino acid export with a low specificity, since basic, neutral, and hydrophobic amino acids were affected. An effect on amino acid export implies that blocking export would result in increased retention within the cells of amino acids taken up by different transporters. Alternatively, All0342 might work in amino acid uptake competing with other transporters, so that the lack of All0342 would facilitate uptake mediated by those transporters. p p y p Release of amino acids to the culture medium is a characteristic feature of some amino acid transport mutants. In the Anabaena nat mutants the set of released amino acids includes alanine, glycine, isoleucine, leucine, phenylalanine, proline, tyrosine, and valine, which are transported mainly by system N-I [23,27]. Because the released amino acids are all hydrophobic, they may leak out of the cells by diffusion, and a function of the N-I system may be the recapture of these leaked amino acids [23,31,32]. In order to test this further, a method to study kinetically the release of AIB from vegetative cells has been set up. Release of AIB is observed only when the N-I system is engaged in the uptake of specific substrates of this transporter, but not when a specific substrate of the N-II system is added. Because the N-I system is responsible for 98% of the uptake of AIB, these data support a function of the N-I system in recapturing the leaked amino acids. With the aim of identifying possible molecular actors of the release of AIB from Anabaena cells, the kinetics of AIB release was studied in wild-type Anabaena and some of the mutants generated in this work. The rate of release of AIB directly depends on the intracellular concentration of the substrate, following a first-order equation, which is consistent with diffusion. This could be simple diffusion through the lipid bilayer [50,51] or facilitated diffusion [52]. None of the investigated mutants was, however, impaired in release of AIB and, indeed, all of them showed the same trend as the wild type. Thus, if release were facilitated, proteins encoded by genes other than those inactivated in this work would be involved. 4. Discussion In this work we have investigated new possible amino acid transporters encoded in the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. The amino acid transport systems previously known in this cyanobacterium are the ABC-type transporters N-I, N-II, and Bgt [23,27,28]. The genes encoding the elements of each of these transporters are summarized in Table 1. The N-I system is encoded by five genes, which are spread in the genome: natA (all1046) and natE (all2912) encode ATPases, natC (all1047) and natD (all1284) encode transmembrane (permease) proteins, and natB (alr1834) encodes a periplasmic substrate-binding protein [27]. The system N-II is encoded by a cluster of four genes: natF (alr4164) encodes a periplasmic substrate-binding protein, natG (alr4165) and natH (alr4166) encode transmembrane (permease) proteins, and bgtA (alr4167) encodes an ATPase [28]. The third one, the system Bgt, is the product of bgtB (alr3187), which encodes a permease protein with an extended periplasmic domain with homology to substrate binding proteins, and bgtA (alr4167), which encodes an ATPase that is shared with the system N-II [28]. Seven loci have been investigated here, but evidence for the involvement in amino acid transport was only found for two of them (see Results). A cluster of genes encoding the elements of a new ABC-type amino acid uptake transporter has been identified in this work: alr2535 encodes a predicted periplasmic binding protein, alr2536 and alr2538 encode predicted transmembrane (permease) proteins, and alr2539 and alr2541 encode predicted ATPases. As judged from the transport activities displayed by an alr2536 insertional mutant, this putative transporter can mediate uptake of glycine and other hydrophobic amino acids (Table 1). We have termed this amino acid transporter system N-III. Similarly to the previously characterized amino acid transport systems N-I and N-II of Anabaena, system N-III mainly transports one group of amino acids (hydrophobic amino acids), but has a relatively low specificity, being able to mediate as well the uptake of some other amino acids such as glutamate and glutamine. Nonetheless, system N-III is phylogenetically most similar to transporters in the Hydrophobic Amino Acid Uptake Transporter (HAAT) family (TCDB 3.A.1.4 [30]), as the N-I system is. Members of the DAACS family of transporters (TCDB 2.A.23 [30]) are involved in the uptake of different substrates, including amino acids, mediated by a solute:Na+ symport mechanism [45]. Anabaena ORF all0342 encodes a protein that is homologous to members of this family. 4. Discussion The analysis 1295 Life 2015, 5 Life 2015, 5 We suggest that hydrophobic amino acids can leak out from the Anabaena filaments to the culture medium and that the ABC-type transporter N-I is important for recapturing the amino acids that have been lost from the cells. This function could be especially relevant under diazotrophic conditions, in which nitrogen is at a premium. The genome of Anabaena is predicted to contain 6223 genes [53], of which 481 are predicted to encode transport proteins [54]. ABC-type transporters are particularly abundant in cyanobacteria as compared to other bacteria [55], and proteins constituting ABC-type transporters represent in Anabaena about 61% of the total number of membrane transporter proteins [54]. The ample use of ABC-type transporters in cyanobacteria may be related to the fact that in these organisms the bulk of energy conservation takes place in intracellular membranes, the photosynthetic thylakoids, which are separated from the cytoplasmic membrane. ATP generated in the thylakoids can then be used for cytoplasmic membrane transport directly by ABC-type transporters. Whereas many transporters in Anabaena, including ABC-type and other transporters, are involved in the uptake of inorganic nutrients [56], there are also many transporters that mediate the uptake of organic substances including, for instance, sugars [22,57] and oxo-acids [58]. The mainly photoautotrophic lifestyle of organisms such as Anabaena and most cyanobacteria may be therefore complemented by the ability to utilize organic compounds that could be found in their natural habitats. Life 2015, 5 Life 2015, 5 1296 5. Conclusions The genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains a large number of genes encoding membrane transport proteins, many of which are elements of ABC-type transporters. Here we identified a new ABC-type uptake transporter, system N-III, mainly involved in the uptake of hydrophobic amino acids. We also identified a membrane protein, All0342, homologous to transporters of the dicarboxylate/amino acid:cation symporter (DAACS) family, whose mutation results in an increased accumulation of a wide range of amino acids in uptake assays. Finally, studying α-aminoisobutyric acid (AIB) export, we found that release of hydrophobic amino acids from cyanobacterial cells likely takes place by means of diffusion. Acknowledgments This work was supported by the Plan Nacional de Investigación, Spain, co-financed by the European Regional Development Fund (grants BFU2005-07672, BFU2008-03811, and BFU2011-22762). Author Contributions All authors designed and interpreted experimental work; Rafael Pernil, Silvia Picossi and Vicente Mariscal performed experiments; Rafael Pernil, Antonia Herrero, Enrique Flores and Vicente Mariscal drafted the manuscript. All authors have read and approved the final manuscript. Conflicts of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. 5. López-Igual, R.; Flores, E.; Herrero, A. Inactivation of a heterocyst-specific invertase indicates a principal role of sucrose catabolism in heterocysts of Anabaena sp. J. Bacteriol. 2010, 192, 5526–5533. References 1. Flores, E.; Herrero, A. Compartmentalized function through cell differentiation in filamentous cyanobacteria. Nat. Rev. Microbiol. 2010, 8, 39–50. 2. Thomas, J.; Meeks, J.C.; Wolk, C.P.; Shaffer, P.W.; Austin, S.M. Formation of glutamine from [13N]ammonia, [13N]dinitrogen, and [14C]glutamate by heterocysts isolated from Anabaena cylindrica. J. Bacteriol. 1977, 129, 1545–1555. 3. Burnat, M.; Herrero, A.; Flores, E. Compartmentalized cyanophycin metabolism in the diazotrophic filaments of a heterocyst-forming cyanobacterium. Proc. Natl. Acad. Sci. USA 2014, 111, 3823–3828. 4. Jüttner, F. 14C-labeled metabolites in heterocysts and vegetative cells of Anabaena cylindrica filaments and their presumptive function as transport vehicles of organic carbon and nitrogen. J. Bacteriol. 1983, 155, 628–633. 5. López-Igual, R.; Flores, E.; Herrero, A. Inactivation of a heterocyst-specific invertase indicates a principal role of sucrose catabolism in heterocysts of Anabaena sp. J. Bacteriol. 2010, 192, 5526–5533. Life 2015, 5 1297 6. Pernil, R.; Herrero, A.; Flores, E. Catabolic function of compartmentalized alanine dehydrogenase in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 2010, 192, 5165–5172. 7. Vargas, W.A.; Nishi, C.N.; Giarrocco, L.E.; Salerno, G.L. Differential roles of alkaline/neutral invertases in Nostoc sp. PCC 7120: Inv-B isoform is essential for diazotrophic growth. Planta 2011, 233, 153–162. 8. Ayre, B.G. Membrane-transport systems for sucrose in relation to whole-plant carbon partitioning. Mol. Plant 2011, 4, 377–394. 9. Kolman, M.A.; Nishi, C.N.; Perez-Cenci, M.; Salerno, G.L. Sucrose in cyanobacteria: From a salt-response molecule to play a key role in nitrogen fixation. Life 2015, 5, 102–126. 10. Martín-Figueroa, E.; Navarro, F.; Florencio, F.J. The GS-GOGAT pathway is not operative in the heterocysts. Cloning and expression of glsF gene from the cyanobacterium Anabaena sp. PCC 7120. FEBS Lett. 2000, 476, 282–286. 11. Sherman, D.M.; Tucker, D.; Sherman, L.A. Heterocyst development and localization of cyanophycin in N2-fixing cultures of Anabaena sp. PCC 7120 (Cyanobacteria). J. Phycol. 2000, 36, 932–941. 12. Wolk, C.P.; Thomas, J.; Shaffer, P.W.; Austin, S.M.; Galonsky, A. Pathway of nitrogen metabolism after fixation of 13N-labeled nitrogen gas by the cyanobacterium, Anabaena cylindrica. J. Biol. Chem. 1976, 251, 5027–5034. 13. Mariscal, V.; Herrero, A.; Flores, E. Continuous periplasm in a filamentous, heterocyst-forming cyanobacterium. Mol. Microbiol. 2007, 65, 1139–1145. 14. Haselkorn, R. Cell-cell communication in filamentous cyanobacteria. Mol. Microbiol. 2008, 70, 783–785. 15. Mariscal, V.; Flores, E. Multicellularity in a heterocyst-forming cyanobacterium: Pathways for intercellular communication. Adv. Exp. Med. Biol. 2010, 675, 123–135. 16. Mariscal, V. Cell-cell joining proteins in heterocyst-forming cyanobacteria. Life 2015, 5 Life 2015, 5 1298 21. Flores, E.; Herrero, A.; Wolk, C.P.; Maldener, I. Is the periplasm continuous in filamentous multicellular cyanobacteria? Trends Microbiol. 2006, 14, 439–443. 21. Flores, E.; Herrero, A.; Wolk, C.P.; Maldener, I. Is the periplasm continuous in filamentous multicellular cyanobacteria? Trends Microbiol. 2006, 14, 439–443. 22. Nicolaisen, K.; Mariscal, V.; Bredemeier, R.; Pernil, R.; Moslavac, S.; López-Igual, R.; Maldener, I.; Herrero, A.; Schleiff, E.; Flores, E. The outer membrane of a heterocyst-forming cyanobacterium is a permeability barrier for uptake of metabolites that are exchanged between cells. Mol. Microbiol. 2009, 74, 58–70. 23. Montesinos, M.L.; Herrero, A.; Flores, E. Amino acid transport systems required for diazotrophic growth in the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 1995, 177, 3150–3157. 24. Flores, E.; Muro-Pastor, M.I. Uptake of glutamine and glutamate by the dinitrogen-fixing cyanobacterium Anabaena sp. PCC 7120. FEMS Microbiol. Lett. 1988, 56, 127–130. 25. Herrero, A.; Flores, E. Transport of basic amino acids by the dinitrogen-fixing cyanobacterium Anabaena PCC 7120. J. Biol. Chem. 1990, 265, 3931–3935. 26. Xu, P.; McAuley, P.J. Uptake of amino acids by the cyanobacterium Anabaena ATCC 27893. New Phytol. 1990, 115, 581–585. 27. Picossi, S.; Montesinos, M.L.; Pernil, R.; Lichtlé, C.; Herrero, A.; Flores, E. ABC-type neutral amino acid permease N-I is required for optimal diazotrophic growth and is repressed in the heterocysts of Anabaena sp. strain PCC 7120. Mol. Microbiol. 2005, 57, 1582–1592. 28. Pernil, R.; Picossi, S.; Mariscal, V.; Herrero, A.; Flores, E. ABC-type amino acid uptake transporters Bgt and N-II of Anabaena sp. strain PCC 7120 share an ATPase subunit and are expressed in vegetative cells and heterocysts. Mol. Microbiol. 2008, 67, 1067–1080. 29. Busch, W.; Saier, M.H., Jr. The transporter classification (TC) system. Crit. Rev. Biochem. Mol. Biol. 2002, 37, 287–337. 30. Transporter Classification Database. Available online: http://www.tcdb.org (accessed on 15 April 2015). 31. Labarre, J.; Thuriaux, P.; Chauvat, F. Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. strain 6803. J. Bacteriol. 1987, 169, 4668–4673. 32. Montesinos, M.L.; Herrero, A.; Flores, E. Amino acid transport in taxonomically diverse cyanobacteria and identification of two genes encoding elements of a neutral amino acid permease putatively involved in recapture of leaked hydrophobic amino acids. J. Bacteriol. 1997, 179, 853–862. 33. Rippka, R.; Deruelles, J.; Waterbury, J.B.; Herdman, M.; Stanier, R.Y. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 1979, 111, 1–61. References In The Cell Biology of Cyanobacteria; Flores, E., Herrero, A., Eds.; Caister Academic Press: Norfolk, UK, 2014; pp. 293–304. 17. Mullineaux, C.W.; Mariscal, V.; Nenninger, A.; Khanum, H.; Herrero, A.; Flores, E.; Adams, D.G. Mechanism of intercellular molecular exchange in heterocyst-forming cyanobacteria. EMBO J. 2008, 27, 1299–1308. 18. Merino-Puerto, V.; Schwarz, H.; Maldener, I.; Mariscal, V.; Mullineaux, C.W.; Herrero, A.; Flores, E. FraC/FraD-dependent intercellular molecular exchange in the filaments of a heterocyst-forming cyanobacterium, Anabaena sp. Mol. Microbiol. 2011, 82, 87–98. 19. Flores, E.; Pernil, R.; Muro-Pastor, A.M.; Mariscal, V.; Maldener, I.; Lechno-Yossef, S.; Fan, Q.; Wolk, C.P.; Herrero, A. Septum-localized protein required for filament integrity and diazotrophy in the heterocyst forming cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 2007, 189, 3884–3890. 20. Merino-Puerto, V.; Mariscal, V.; Mullineaux, C.W.; Herrero, A.; Flores, E. Fra proteins influencing filament integrity, diazotrophy and localization of septal protein SepJ in the heterocyst-forming cyanobacterium Anabaena sp. Mol. Microbiol. 2010, 75, 1159–1170. Life 2015, 5 1299 37. Frías, J.E.; Flores, E.; Herrero, A. Activation of the Anabaena nir operon promoter requires both NtcA (CAP family) and NtcB (LysR family) transcription factors. Mol. Microbiol. 2000, 38, 613–625. 38. Black, T.A.; Cai, Y.; Wolk, C.P. Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena. Mol. Microbiol. 1993, 9, 77–84. 39. Elhai, J.; Vepritskiy, A.; Muro-Pastor, A.M.; Flores, E.; Wolk, C.P. Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J. Bacteriol. 1997, 179, 1998–2005. 40. Markwell, M.A.K.; Hass, S.M.; Bieber, L.L.; Tolbert, N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 1978, 87, 206–210. 41. Mackinney, G. Absorption of light by chlorophyll solutions. J. Biol. Chem. 1941, 140, 109–112. 42. Hoshino, T.; Kose, K. Cloning and nucleotide sequence of braC, the structural gene for the leucine-, isoleucine-, and valine-binding protein of Pseudomonas aeruginosa PAO. J. Bacteriol. 1989, 171, 6300–6306. 43. Okamoto, S.; Yamanishi, Y.; Ehira, S.; Kawashima, S.; Tonomura, K.; Kanehisa, M. Prediction of nitrogen metabolism-related genes in Anabaena by kernel-based network analysis. Proteomics 2007, 7, 900–909. 44. Quintero, M.J.; Montesinos, M.L.; Herrero, A.; Flores, E. Identification of genes encoding amino acid permeases by inactivation of selected ORFs from the Synechocystis genomic sequence. Genome Res. 2001, 11, 2034–2040. 45. Slotboom, D.J.; Konings, W.N.; Lolkema, J.S. Structural features of the glutamate transporter family. Microbiol. Mol. Biol. Rev. 1999, 63, 293–307. 46. Wong, F.H.; Chen, J.S.; Reddy, V.; Day, J.L.; Shlykov, M.A.; Wakabayashi, S.T.; Saier, M.H., Jr. The amino acid-polyamine-organocation superfamily. J. Mol. Microbiol. Biotechnol. 2012, 22, 105–113. 47. Rudnick, G.; Krämer, R.; Blakely, R.D.; Murphy, D.L.; Verrey, F. The SLC6 transporters: Perspectives on structure, functions, regulation, and models for transporter dysfunction. Pflugers Arch. 2014, 466, 25–42. 48. Ihlenfeldt, M.J.A.; Gibson, J. CO2 fixation and its regulation in Anacystis nidulans (Synechococcus). Arch. Microbiol. 1975, 102, 13–21. 49. Raboy, B.; Padan, E. Active transport of glucose and α-methylglucoside in the cyanobacterium Plectonema boryanum. J. Biol. Chem. 1978, 253, 3287–3291. 50. Klein, R.A.; Moore, M.J.; Smith, M.W. Selective diffusion of neutral amino acids across lipid bilayers. Biochim. Biophys. Acta 1971, 233, 420–433. 51. Chakrabarti, A.C. Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation. Amino Acids 1994, 6, 213–229. 52. Krämer, R. Secretion of amino acids by bacteria: Physiology and mechanism. FEMS Microbiol. Life 2015, 5 34. Cai, Y.P.; Wolk, C.P. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J. Bacteriol. 1990, 172, 3138–3145. 35. Ausubel, F.M.; Brent, R.; Kingston, R.E.; Moore, D.D.; Seidman, J.G.; Smith, J.A.; Struhl, K. Current Protocols in Molecular Biology; Greene Publishing and Wiley-Interscience: New York, NY, USA, 2015. 36. Elhai, J.; Wolk, C.P. A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 1988, 68, 119–138. Life 2015, 5 © 2015 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 license (http://creativecommons.org/licenses/by/4.0/). Life 2015, 5 Rev. 1994, 13, 75–93. 53. CyanoBase. Available online: http://genome.microbedb.jp/cyanobase/Anabaena (accessed on 15 April 2015). 53. CyanoBase. Available online: http://genome.microbedb.jp/cyanobase/Anabaena (accessed on 15 April 2015). Life 2015, 5 1300 54. TransportDB. Available online: http://www.membranetransport.org/index_v2_rc1.html (accessed on 15 April 2015). 54. TransportDB. Available online: http://www.membranetransport.org/index_v2_rc1.html (accessed on 15 April 2015). 54. TransportDB. Available online: http://www.membranetransport.org/index_v2_rc1.html (accessed on 15 April 2015). 55. Paulsen, I.T.; Nguyen, L.; Sliwinski, M.K.; Rabus, R.; Saier, M.H., Jr. Microbial genome analyses: Comparative transport capabilities in eighteen prokaryotes. J. Mol. Biol. 2000, 301, 75–100. 56. Hahn, A.; Schleiff, E. The cell envelope. In The Cell Biology of Cyanobacteria; Flores, E., Herrero, A., Eds.; Caister Academic Press: Norfolk, UK, 2014; pp. 29–87. 57. Stebegg, R.; Wurzinger, B.; Mikulic, M.; Schmetterer, G. Chemoheterotrophic growth of the cyanobacterium Anabaena sp. strain PCC 7120 dependent on a functional cytochrome c oxidase. J. Bacteriol. 2012, 194, 4601–607. 58. Pernil, R.; Herrero, A.; Flores, E. A TRAP transporter for pyruvate and other monocarboxylate 2-oxoacids in the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 2010, 192, 6089–6092. © 2015 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 license (http://creativecommons.org/licenses/by/4.0/). © 2015 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 license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3127627215	https://tckh.daihoctantrao.edu.vn/index.php/sjttu/article/download/350/358	Latin	null	Fish inventory and fishing practices in Candaba swamp, Philippines: their implication to fishermen’s income	Khoa học	2,020	cc-by-sa	4,109	Fish inventory and fishing practices in Candaba swamp, Philippines: their implication to fishermen’s income Alfred R. Mallaria, Jacqueline V. Bagunua, Gerald M. Salasa a Pampanga State Agricultural University, Philippines Article info Abstract Recieved: 28/11/2019 Accepted: 10/3/2020 This study generally aimed to make an inventory of fishes in Candaba Swamp and documented the fishing practices of the local fishermen as they are related to their income. The study documented 14 different species of fishes, each representing 14 families in the Barangays of San Agustin and Dukma Paralaya. The fourteen species were the Elopshawaiensis, Glossobiusgiurus, Megalops cyprinoides,Cyprinuscarpio, Trichopodus pectoralis, Clariasbatrachus, Hypostomus plecostomus, Arius dispar, Hypophthalmichthys molitrix, Anabas testudineus, Carassiuscarasius, Labeorohita, Hypophthalmichthysnobilis, Oreochromis niloticus. The Inventory recorded the Tilapia,Common Carp and Hito species as the most dominant species in Candaba Swamp. Fishermen’s ages were between 20 to 89, most of them were between 50-59, used the “panti” (kitig) as fishing gear and with daily income ranging from 100 pesos to 1,500 pesos. The total income of the fisherman during the wet season is from 1,000 pesos to 10,000 pesos, and the daily expenses in each fishing trip is from 20 pesos to 100 pesos which is mostly used for gasoline. The fishermen’s income depended on the number of fishes they catch which was affected by changes in season. The Cost-Benefit-Analysis showed a net income Of P281.27 per day with a Return of Investment of 174.4%. Keywords: Fish inventory; fishing practices; fishing gear; “Panti” b fi i h LGU h i i d h Alfred R. Mallaria, Jacqueline V. Bagunua, Gerald M. Salasa a Pampanga State Agricultural University, Philippines This study generally aimed to make an inventory of fishes in Candaba Swamp and documented the fishing practices of the local fishermen as they are related to their income. The study documented 14 different species of fishes, each representing 14 families in the Barangays of San Agustin and Dukma Paralaya. The fourteen species were the Elopshawaiensis, Glossobiusgiurus, Megalops cyprinoides,Cyprinuscarpio, Trichopodus pectoralis, Clariasbatrachus, Hypostomus plecostomus, Arius dispar, Hypophthalmichthys molitrix, Anabas testudineus, Carassiuscarasius, Labeorohita, Hypophthalmichthysnobilis, Oreochromis niloticus. The Inventory recorded the Tilapia,Common Carp and Hito species as the most dominant species in Candaba Swamp. Fishermen’s ages were between 20 to 89, most of them were between 50-59, used the “panti” (kitig) as fishing gear and with daily income ranging from 100 pesos to 1,500 pesos. Fish inventory and fishing practices in Candaba swamp, Philippines: their implication to fishermen’s income The total income of the fisherman during the wet season is from 1,000 pesos to 10,000 pesos, and the daily expenses in each fishing trip is from 20 pesos to 100 pesos which is mostly used for gasoline. The fishermen’s income depended on the number of fishes they catch which was affected by changes in season. The Cost-Benefit-Analysis showed a net income Of P281.27 per day with a Return of Investment of 174.4%. season, benefiting the LGUs, the communities, and the migratory birds themselves (Torres et al. 2008). No.15_Mar 2020\|Số 15 – Tháng 3 năm 2020\|p.10-16 No.15_Mar 2020\|Số 15 – Tháng 3 năm 2020\|p.10-16 TẠP CHÍ KHOA HỌC ĐẠI HỌC TÂN TRÀO ISSN: 2354 - 1431 http://tckh.daihoctantrao.edu.vn/ TẠP CHÍ KHOA HỌC ĐẠI HỌC TÂN TRÀO ISSN: 2354 - 1431 http://tckh.daihoctantrao.edu.vn/ 2. Methodology Where: Where: 1. Introduction Candaba Swamp, popularly known as “Pinac,” is one of the most panoramic wetland here in our country situated in Candaba, Pampanga, Central Luzon, Philippines. Aside from being the location of one of the oldest settlements in Central Luzon, it is a famous site because of its diverse and multiform fauna and flora and its exotic ecosystem. Candaba swamp is known in the province of Bulacan and Pampanga because of its importance in agriculture, fisheries production, irrigation and its helps in retention of natural flood in the said province here in the Philippines. Tourism in the area has increased, especially during the migratory Presently, the swamp is utilized all throughout the year. During summer which is every January to May, the swamp is primarily used for rice and watermelon production. From June to December which is usually the rainy season, the swamp serves as a fishing ground and stopover of migratory birds from Siberia and China. (Melendres, 2014). Studies in Candaba Swamp were undertaken on the different species of fishes (Bautista & Barila, 2013); Heavy metal analysis (Kabigting, 2013); Cytogenetic Studies of The waters (Dela Cruz, Ruel, 2012); and 10 A.R. Mallari et al/ No.15_Mar 2020\|p.10-16 A.R. Mallari et al/ No.15_Mar 2020\|p.10-16 Mollusk Taxonomy (Mallari and Bagunu, 2013). Literatures in fish biodiversity in the area are very limited, and even, non-reported nor published. fins were stretched to determine the number and then the scales of each specimen were also counted. 2.1. Research Design Pi= Proportion of Total Pi= Proportion of Total The study made use of the descriptive-survey method of research using an interview guide. The identification of the species made use of existing literatures based on their morphological characteristics. The identity and classification were validated by fishexperts from the University of the Philippines, Los Banos. Sample represented by species Sample represented by species S= Number of species (Species richness) E= S= Number of species (Species richness) S= Number of species (Species richness) Hmax = In(S) = maximum diversity possible Simple statistics such as frequency count and percentage was used to analyze the data from the questionnaire. 3.1. Inventory of fishes in the Candaba Swamp Collection of fish species in Candaba Swamp were based on the total number of fishes captured by the local fishermen. One sample each species group was collected to represent each group and was preserved using Taxidermy procedures for future documentation, identification and deposited at The Biology Museum of the College of Arts and Sciences, Pampanga State Agricultural University, Magalang, Pampanga. There were Fourteen (14) species identified and classified in Candaba Swamp. Among the 14 fish species, 5 of them are Cyprinidae (Common carp, Katkat,Pararak,Roho, and Tie fish) and the other 10 species are from different fish families, the Chanidae, Elopidae, Gobiidae, Osphronemidae, Clariidae, Loricariidae, Ariidae, Terapontidae, Anabantidae, and Cichlidae. In terms of their Genus,two of them have the same genus which is Hypophthalmichthys (Katkat and Tie fish) and the others are from Elops, Glossobius, Megalops, Cyprinus, Trichopodus, Clarias, Hypostomus, Arius, Anabas, Carrassius, Labeo, and lastly Oreochromis (Table 1). 2.2.5. Diversity Index and Statistical Analysis of Data 2.2.5. Diversity Index and Statistical Analysis of Data The Shannon-Weiner Index to determine species diversity was used with the following formula. Shannon- Weiner Index H= ∑ [(Pi) x In (Pi)] Where: Shannon- Weiner Index H= ∑ [(Pi) x In (Pi)] Where: Shannon- Weiner Index H= ∑ [(Pi) x In (Pi)] Where: Shannon- Weiner Index H= ∑ [(Pi) x In (Pi)] Where: Pi= Proportion of Total Sample represented by species S= Number of species (Species richness) E= Hmax = In(S) = maximum diversity possible Shannon- Weiner Index H= ∑ [(Pi) x In (Pi)] Where: Pi= Proportion of Total Sample represented by species S= Number of species (Species richness) E= Hmax = In(S) = maximum diversity possible 2.2.4. Ecological Factors This study is important in providing data on fish inventory in the San Agustin and Dukma Paralaya barangays in Candaba Swamp, Pampanga.The implications of this inventory to the income of the fishermen will also be useful as basis for policy recommendation in the area. This may also serve as a reference material to those who want to know how Candaba Swamp is very important to the livelihood of fishermen in the barangay of San Agustin and Dukma Paralaya. It will also contribute further to the existing knowledge in academic and research endeavors with regard to Biodiversity conservation. The researcher used an YSI Multi Parameter Test Probe and Hach Ammonia Test Kit from the Bureau of Fisheries and Aquatic Resources (BFAR) to determine the environmental factors such a pH, Temperature, Conductivity, Salinity, Turbidity, Dissolved Oxygen and Depth. 2.2.1. Key informant interview The Cost Benefit Analysis (CBA) and Return of Investment (ROI) were computed. The formula as follows: Interviews were conducted among the fishermen to elicit their fishing practices. Stratified method was used in determining the sample respondents and in the collection of data thru the survey given to the fishermen in the Candaba swamp. Thirty Percent (30%) of the total population of 519 fishermen served as respondents of the study. CBA = Sales – Expenses = Net Income 3.2.2. Fishing Practices of the Fishermen 3.2.2. Fishing Practices of the Fishermen 3.2.2.1. Fishing time of Respondents 3.2.1. Socio-Demographic Profile Of The Fishermen-Respondents As shown in Figure 3, most respondents (41.3%) have fishing time starting at around 2:01 to 4:00 in the morning. Thirty two percent starts at 4:01 to 6:00 in the morning, thirdly, 14% fish between 12:00 to 2:00 in the morning the rest starts between 6:00 in the morning to 12:00 noon. 3.2.1.1. Age of Fishermen-Respondents Figure 1 displays the ages of fishermen in Candaba, Swamp. The highest percentage (28%) of age is found within 50-59 age brockets and the lowest percentage (2.6%) is found in the 80-89 age brocket. Majority of the fishermen is from 50 to 50 of age because this are the person who did not finish High school to find a decent job , and the lowest percentage are from 80 years and above because they are too old to have a job for their family. The table shows that more fishermen start fishing very early in the morning because they believe that if they fish early in the morning they have the possibility to catch more fishes to be sold in the market. Figure 3. Fishing time of the Respondents 3.2.2.2. Fishing duration of fishermen Figure 1. Distributions of Respondents according to age Figure 3. Fishing time of the Respondents 3.2.2.2. Fishing duration of fishermen 3.2.2.2. Fishing duration of fishermen As shown in Figure 4, most fishermen have fishing duration within 4-6 hours. This was followed by 26.6% of the fishermen second for 7-10 hours; 1-3 with a 19.3 percent; and lastly 11 hours and more with a 12.6 percent of the respondents. Figure 1. Distributions of Respondents according to age 3.2.1.2. Livelihood of Fishermen-Respondent 2.2.3. Identification Procedure Identification of fish species in Candaba Swamp was done with the help of a fish expert from UP Los Banos. The species was laid on a flat surface with a ruler below for documentation and measurement. The Table 1. Fish species collected in Candaba Swamp 11 A.R. Mallari et al/ No.15_Mar 2020\|p.10-16 A.R. Mallari et al/ No.15_Mar 2020\|p.10-16 3.2. Socio-demographic and fishing practices of the fishermen - respondents is a favored environment for fishing because of the retention of natural flood in the said area. This justify why fishing of freshwater fishes is one of the source of income of the people in Candaba swamp. 3.2. Socio-demographic and fishing practices of Figure 2.Presents alternative sources of income aside from fishing Figure 2.Presents alternative sources of income aside from fishing Figure 2.Presents alternative sources of income aside from fishing 3.2. Socio-demographic and fishing practices of the fishermen - respondents 3.2.2.4. Fish Catch and Price Per Kilogram Figure 6. How the harvest used by the respondents 3.2.2.6. Harvest Seller Figure 6. How the harvest used by the respondents As shown in Table 2, the most abundant fish species caught by the fishermen is Tilapia with the average frequency of 58 and 702 kilogram, number 2 is Common carp with the average frequency of 44 and with a 744 kilogram, and the number 3 with an average frequency of 27 with a 187 kilogram. 3.2.1.2. Livelihood of Fishermen-Respondent As shown in Figure 2, the livelihood of the fishermen is farming comprising 22%, second is construction with 8%, third is vendor with 4.6%, fourth is a truck driver with 3.3%, and fifth is otherswith 2.6% and lastly, the barangay tanod with 0.6% then the 58.6% are just fishermen and no other livelihood. Most of the fishermen spend 4 hours, at least, in swamps so that they could capture more. Majority are fishermen because it is a common knowledge that Candaba swamp during the wet season 12 A.R. Mallari et al/ No.15_Mar 2020\|p.10-16 Table 2. Fish Catch and price per kilogram Table 2. Fish Catch and price per kilogram Figure 4. Number of hours spent for fishing 3 2 2 3 Fi hi G d b Fi h Figure 4. Number of hours spent for fishing 3.2.2.3. Fishing Gear used by Fishermen 3.2.2.3. Fishing Gear used by Fishermen Most of the fishermen used the fishinggear Pantiwith the highest percentage of 43.3 percent. For one thing, it catches more fish as compared to lambat and other fishing gears. Panti is easy to repair by hand once it gets broken. Lastly, it is more convenient to use because fishermen set the panti or kitig in swamps and wait until the gear catch a fish. 3.2.2.6. Harvest Seller Figure 7, shows who sells the harvest, The family members such as their spouses and also their children got the highest percentage of 79.3 percent, while the fishermen has a 20 percent and lastly friend with a o.6 percent. The fishermen caught Tilapia more than the Common carp and Hito. The Common carp is a lot heavier than the Tilapia, because common carp fishes are bigger in size.But when it comes to the price, fishermen have higher income from Tilapia because it is more expensive than the common carp and hito though hito commands higher price than the common carp. Figure 7. Harvest Sellers 3.2.2.7. Income of the Fishermen During Wet Season Figure 7. Harvest Sellers 3.2.2.7. Income of the Fishermen During Wet Season 3.2.2.7. Income of the Fishermen During Wet 3.2.2.5. Use of Harvest Figure 5, showed how the fishermen used their harvest. Ninety six (96) percent sold their harvest, and three (3) percent use them for food consumption and sold in market, and 2 percent used their harvest for food consumption only. Figure 5. Fishing gear that being used by the Respondents 3 2 2 4 Fi h C h d P i P Kil Figure 5. Fishing gear that being used by the Respondents Figure 6. How the harvest used by the respondents 3 2 2 6 H S ll Figure 5. Fishing gear that being used by the Respondents 4. Summary, conclusion, recommendation 4. Summary, conclusion, recommendation 3.2.2.8. Perception of Fishermen on their income 3.2.2.8. Perception of Fishermen on their income Shown inFigure8 is the sufficiency of the income of the fishermen for their family. Most of them answered the 2 or less sufficient with the 40 percent, while in the 3 or sufficient has a 35.3 percent, in 1 or not sufficient has a 18.6 percent, 4 or very sufficient with a 6 percent and lastly 5 or more sufficient has 0 percent because there’s no fishermen answered that their income is more sufficient for their family. 3.2.2.9. Cost-Benefit Analysis (CBA) & Return of Investment (ROI) 3.2.2.9. Cost-Benefit Analysis (CBA) & Return of Investment (ROI) 3.2.2.9. Cost-Benefit Analysis (CBA) & Return of Investment (ROI) Table 3 shows the Average Net Income per Day of the local fishermen of Candaba Swamp, Pampanga. It consists their Sales which is the Average sales of the fishermen is 442.90 pesos if the fishes is sold in the market. While in their Daily Average Expenses which is 161.63 pesos it consists the Gasoline for fishing, Food, and other Daily needs of their family each day, and lastly the Average Net Income of the individual fishermen is 281.27 pesos which is qualified to the Daily Minimum Wage of an individual person. According to National Wages and Productivity Commission from the Department of Labor and Employment the minimum wage in Region 3 starts from 274 pesos to 400 pesos (NWPC-DOLE, 2018). Daily income total income [during weseas Daily income total income [during weseas Daily income total income [during weseas Total expenses per day during wet season] Figure 8. The Daily Income, Total Income and the Total Expenses each fishing trip of the fishermen during Wet season. Table 3. Cost-Benefit Analysis Table 3. Cost-Benefit Analysis Total expenses per day during wet season] Figure 8. The Daily Income, Total Income and the Total Expenses each fishing trip of the fishermen during Wet season. Total expenses per day during wet season] Figure 8. The Daily Income, Total Income and the Total Expenses each fishing trip of the fishermen during Wet season. ROI (Return on Investment) = (Net Profit/ Cost of Investment) x 100 = 174.4 = (Net Profit/ Cost of Investment) x 100 = 174.4 Season Figure 7 shows the income per day of the fishermen most of them has an income of 100 to 1,500 pesos in daily fishing. The Total income during wet season is 73.3 percent the range of their income starts from 1,000 to 10,000 pesos during wet, 20.6 percent starts for 13 A.R. Mallari et al/ No.15_Mar 2020\|p.10-16 10,001 to 20,000 pesos and some of them are 20,001 and above. Then the Total expenses per day of the fishermen 68.6 percent has the daily expenses of 20 to 100 pesos, 17.3 percent has the daily expenses from 101 to 200 pesos, 8 percent has no daily expenses, 2.6 percent from 201 to 300 pesos, 2 pecent from 301 to 400 pesos, 0.6 percent from the 401 to 500 pesos same with 601 to 700 pesos. Figure 8. Fishermen’s perceptions on the sufficiency of income Daily income total income [during weseas Figure 8. Fishermen’s perceptions on the sufficiency of income 4.2. Conclusions The conclusions of the study are as follows: 4.2.1. Among the fourteen collected fish species, the most abundant were Common Carp (Cyprinus carpio), Tilapia (Oreochromis niloticus), Hito (Hypostomus plecostomus) 4.2.1. Among the fourteen collected fish species, the most abundant were Common Carp (Cyprinus carpio), Tilapia (Oreochromis niloticus), Hito (Hypostomus plecostomus) 4.2.1. Among the fourteen collected fish species, the most abundant were Common Carp (Cyprinus carpio), Tilapia (Oreochromis niloticus), Hito (Hypostomus plecostomus) 4.1. Summary of Findings Based on the data gathered, the researcher came up with the following findings: 4.1.1. There were fourteen species of fishes collected in the barangay of San Agustin and Dukma Paralaya. 14 A.R. Mallari et al/ No.15_Mar 2020\|p.10-16 4.1.2. The most abundant fish species in the area is the Common Carp. 4.3.4. Income of the local fishermen should be conducted During the Wet season and Dry Season for comparison. 4.1.3. Most of the fishermen were between20 up to 89 years.They generally used the fishing gear Panti or Kitig. Acknowledgement: To the Southeast Asian Regional Center for Graduate Study and Research in Agriculture (SEARCA) for the funding support and to the University of the Philippines School of Environmental Science and Management (UPLB- SESAM). 4.1.4. The daily income of the fisherman mostly ranged from 100 pesos to 1,500 pesos, The total income of the fisherman during the wet season is from 1,ooo pesos to 10,000 pesos, and the daily expenses in each fishing trip is from 20 pesos to 100 pesos mostly used for the gasoline.As to the Cost Benefit Analysis, the average daily net net income of each fisherman is 281.27 pesos and the Return of Investment is 174.41%. Erratum: “Fish inventory and fishing practices of farmers in Candaba Swamp, Philippines: their implication to fishermen’s income” Alfred D. Mallari, Jacqueline V. Bagunu, Gerald M. Salas. Scientific Journal of Tan Trao University, No. 15, March 2020; DOI: https://doi.org/10.51453/2354-1431/2020/350. Following publication of this paper, the journal was alerted that the authors and the acknowledgement of the published International Journal was incomplete and that it should include the author Gerald M. Salas as added accordingly and the acknowledgment as above. 4.2. Conclusions The conclusions of the study are as follows: REFERENCES 4.2.2. There are a lot of fishing gears of the local fishermen like Bukatut, Kimput, Kuryenti, Mandali, Panti/Kitig, Screen, Skylab. 1. Bautista, M. & Barila, J.V (2003), An inventory of the different species of Fishes in Candaba Swamo, Pampanga. BS Thesis, PAC, Magalang, Pampanga. 1. Bautista, M. & Barila, J.V (2003), An inventory of the different species of Fishes in Candaba Swamo, Pampanga. BS Thesis, PAC, Magalang, Pampanga. 4.2.3. The income of the fishermen during wet season it is not enough according to the respondents. 4.2.3. The income of the fishermen during wet season it is not enough according to the respondents. 4.2.3. The income of the fishermen during wet season it is not enough according to the respondents. But based on the Cost Benefit Analysis and Return of Investment it is just enough on the Minimum wage of an individual. 2. Kabigting, C. (2013), Heavy metal analysis on the gills female tilapia in selected areas of Candaba Swamp, Pampanga. BS Thesis, PAC, Magalang, Pampanga 2. Kabigting, C. (2013), Heavy metal analysis on the gills female tilapia in selected areas of Candaba Swamp, Pampanga. BS Thesis, PAC, Magalang, Pampanga But based on the Cost Benefit Analysis and Return of Investment it is just enough on the Minimum wage of an individual. But based on the Cost Benefit Analysis and Return of Investment it is just enough on the Minimum wage of an individual. 4.2.4. The abundance affects the income of the fishermen depending to their daily catch if there are a lot of fish catch it will be enough for their daily needs. 4.2.4. The abundance affects the income of the fishermen depending to their daily catch if there are a lot of fish catch it will be enough for their daily needs. 3. Mallari, M. & Bagunu, J.V. (2003), Taxonomy and Ecology of Molusks in Candaba Swamp, Candaba, Pampanga. BS Thesis, PAC, Magalang, Pampanga. 4.3. Recommendations Based on the results gathered, the following recommendations are here forwarded: 4.3. Recommendations Based on the results gathered, the following recommendations are here forwarded: 4.3. Recommendations Based on the results gathered, the following recommendations are here forwarded: 4. Melendres, R. (2014), The utilization of Candaba Swamp from prehistoric to present time: Evidences from Archaeology, History and Ethnography. 4. Melendres, R. (2014), The utilization of Candaba Swamp from prehistoric to present time: Evidences from Archaeology, History and Ethnography. 4.3.1. Bảng thống kê và tập quán đánh bắt cá ở đầm lầy Candaba, Philippines: ý nghĩa của chúng đối với thu nhập của ngư dân Alfred R. Mallari, Jacqueline V. Bagunu, Gerald M. Salas Alfred R. Mallari, Jacqueline V. Bagunu, Gerald M. Salas REFERENCES Further studies to identify and classify fish species thriving in the unexplored areas of Candaba, Swamp shall be conducted. 5. Torres, D. (2008), Interventions for wetland conservation in the Pampanga river basin and Candaba wetlands Philippines. 5. Torres, D. (2008), Interventions for wetland conservation in the Pampanga river basin and Candaba wetlands Philippines. 4.3.2. Yearly survey on the population of fish species must be conducted to find out if new fish species are present in the other part of Candaba Swamp. 4.3.2. Yearly survey on the population of fish species must be conducted to find out if new fish species are present in the other part of Candaba Swamp. 4.3.3. Fish inventory on the different sites should be conducted to know if there are different species on the other sites of Candaba Swamp. 4.3.3. Fish inventory on the different sites should be conducted to know if there are different species on the other sites of Candaba Swamp. 15 A.R. Mallari et al/ No.15_Mar 2020\|p.10-16 Tóm tắt Nghiên cứu này nhằm mục đích tạo ra một kho cá ở đầm lầy Candaba và ghi lại các hoạt động đánh bắt của ngư dân địa phương vì chúng có liên quan đến thu nhập của họ. Nghiên cứu đã ghi nhận 14 loài cá khác nhau, mỗi loài đại diện cho 14 họ ở Barangays của San Agustin và Dukma Paralaya. Mười bốn loài là Elopshawaiensis, Glossobiusgiurus, Megalops cyprinoides, Cyprinuscarpio, Trichopodus pectoralis, Clariasbatrachus, Hypostomus plecostomus, Arius dispar, Hypophthalmichthys molitrix, Anabas testudineus, Carassiuscarasius, Labeorohita, Hypophthalmichthysnobilis, Oreochromis niloticus. Bảng thống kê đã ghi nhận các loài cá rô phi, cá chép và cá Hito là những loài chiếm ưu thế nhất ở đầm lầy Candaba. Độ tuổi của ngư dân là từ 20 đến 89, hầu hết trong số họ là từ 50-59, đã sử dụng các “panti” (kitig) làm ngư cụ và thu nhập hàng ngày từ 100 peso đến 1.500 peso. Tổng thu nhập của ngư dân trong mùa mưa là từ 1.000 peso đến 10.000 peso và chi phí hàng ngày trong mỗi chuyến đi câu là từ 20 peso đến 100 pê sô, chủ yếu được sử dụng cho xăng dầu. Thu nhập của ngư dân phụ thuộc vào số lượng cá mà họ đánh bắt bị ảnh hưởng bởi những thay đổi trong mùa. Phân tích lợi ích chi phí cho thấy thu nhập ròng P281,27 mỗi ngày với tỷ lệ hoàn vốn đầu tư là 174,4%. 16
https://openalex.org/W4206603709	https://link.springer.com/content/pdf/10.1007%2F978-3-030-31846-8_10.pdf	English	null	Future Directions	Language, cognition and mind	2,020	cc-by	1,227	© The Author(s) 2020 A. Brasoveanu and J. Dotlaˇcil, Computational Cognitive Modeling and Linguistic Theory, Language, Cognition, and Mind 6, https://doi.org/10.1007/978-3-030-31846-8_10 Chapter 10 Future Directions Where do we go from here? We will keep this short because, if the reader has made it this far, the answer really is: in whatever direction the reader’s research inter- ests lie. The main purpose of this book was to introduce a general framework and workﬂow that enables us to enhance competence theories with fully speciﬁed per- formance/processing components. The resulting competence-performance theories can furthermore be embedded in Bayesian models, which enables us to ﬁt them to data and quantitatively compare them in a systematic fashion. This Bayes+ACT- R+formal linguistics workﬂow of model development is in principle applicable to linguistic accounts of many syntactic and/or semantic phenomena—if suitable data can be obtained from properly designed experiments, which is far from trivial. This being said, we think there are ﬁve speciﬁc directions worth pursuing in the near future: i. add more structure to the Bayesian models, for example, random effects for participants, grouping participants according to their strategies in self-paced reading tasks, etc.; ii. data-driven modeling: hand-coding models for speciﬁc experiments does not scale up well, and we should ﬁnd ways to leverage syntactically and seman- tically annotated corpora to make the process of building ACT-R models for speciﬁc tasks and experiments more automatic and data-driven, and more easily comparable across tasks/experiments; iii. enrich the range of studied semantic phenomena—quantiﬁers, scope, binding, questions, attitude verbs, modals—and the range of semantic representations that are considered—(trees of) variable assignments in addition to or instead of DRSs, compositionally assembled higher-order terms in a suitable logical system etc. © The Author(s) 2020 A. Brasoveanu and J. Dotlaˇcil, Computational Cognitive Modeling and Linguistic Theory, Language, Cognition, and Mind 6, https://doi.org/10.1007/978-3-030-31846-8_10 283 283 © The Author(s) 2020 A. Brasoveanu and J. Dotlaˇcil, Computational Cognitive Modeling and Linguistic Theory, Language, Cognition, and Mind 6, https://doi.org/10.1007/978-3-030-31846-8_10 284 10 Future Directions – relatively modest extensions of this framework could be used to build on the wealth of experimental results gathered in the last ten years or so and explicitly model and ﬁt to data different theories of presupposition projection, scalar implicature computation etc., not to mention the large amount of experimental data about syntactic phenomena that is available in the literature; iv. provide a framework for integrating and comparing models and theories of lan- guage interpretation that have been developed in largely disparate traditions up to this point: iv. Chapter 10 Future Directions In addition, there are several ways in which ACT-R is showing its age for modeling natural language interpretation: • it has a rule-ordering architecture that effectively employs transformational models of the kind generative linguistics used in the ‘60s and ‘70s, and that we moved away from; • it has a fairly strict ban on hierarchical structures, rather than a softer one that would allow but penalize them, e.g., the way a probabilistic context free grammar penalizes deeper trees; • the underlying logic for facts/chunks is the logic of feature structures, basically a modal logic with features as modal operators and values as (atomic) non-modal sentence variables; in semantics, we have moved away from this type of the- ory construction with ‘local’-perspective logics, and more towards the ‘global’- perspective of classical (many-sorted) ﬁrst-order or higher-order logic, which makes integrating ACT-R and formal semantics somewhat awkward. However, ACT-R is a widely used hybrid (symbolic and subsymbolic) cognitive architecture and as such, it was the obvious choice for a framework in which to build mechanistic processing models and integrated competence-performance theories for natural language interpretation. As computational cognitive modeling for natural language phenomena develops further, we expect to see a critical reevaluation of a variety of architectural assumptions that we took for granted in the present work. However, ACT-R is a widely used hybrid (symbolic and subsymbolic) cognitive architecture and as such, it was the obvious choice for a framework in which to build mechanistic processing models and integrated competence-performance theories for natural language interpretation. As computational cognitive modeling for natural language phenomena develops further, we expect to see a critical reevaluation of a variety of architectural assumptions that we took for granted in the present work. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), 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 chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. Chapter 10 Future Directions provide a framework for integrating and comparing models and theories of lan- guage interpretation that have been developed in largely disparate traditions up to this point: – for example, the rise of distributional semantics and neural-network mod- eling work in formal semantics (Bowman 2016; McNally and Boleda 2017 amongothers),andlinguisticsmoregenerally,raisesarangeofquestionsabout what the appropriate division of labor is in natural language interpretation between symbolic and subsymbolic components; our Bayes+ACT-R+formal linguistics framework enables us to explore a range of hybrid models that would integrate both perspectives and that can be quantitatively compared, for example, models in which more of the cognitive heavy-lifting is performed either by symbolic components (chunks, rules) or subsymbolic components (base/spreading activation, rule utilities); see, for example, Marcus (2018) for a recent discussion of and arguments for hybrid (symbolic and subsymbolic) architectures; – a speciﬁc example would involve exploring hybrid representations for lexi- cal items that would encode both structural information (like we have done throughout this book) and quantitative information, e.g., dense word embed- dings of the kind proposed in Mikolov et al. (2013) or Pennington et al. (2014); these dense word embeddings could be used to modulate spreading activation for lexical items or for larger phrasal units; – incorporating drift-diffusion models (Ratcliff 1978; Ratcliff et al. 2017) into ACT-R (cf. (Van Maanen et al. (2012))) and compare the resulting model(s) of language comprehension with other commonly used modeling choices; – yet another possibility is to systematically investigate rule learning for natural language interpretation: rules throughout this book were hand-coded, and no theory for how new rules are generated was put forth; this is a common feature of ACT-R modeling, but not a deﬁning and necessary one: ACT-R does have a system for rule learning (production compilation) and we could go further by hypothesizing ‘rule-generating’ mechanisms; y yp g g g – similarly, ACT-R has a system for rule utility learning, but recent advances in reinforcement learning might contribute new insights to this component of the cognitive architecture. v. onthecomputationalside,makeimprovementstoenablefasterestimationofpos- terior distributions for pyactr model parameters, e.g., by emulating pyactr models with neural networks, Gaussian Processes or other kinds of models; solu- tions along these lines could also enable us to do Approximate Bayesian Com- putation (ABC), that is, likelihood-free Bayesian inference for simulation-based 10 Future Directions 285 models with intractable likelihoods, e.g., ACT-R models with various stochastic components turned on. Chapter 10 Future Directions If material is not included in the chapter’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.
https://openalex.org/W3110227194	https://www.matec-conferences.org/10.1051/matecconf/202032905001/pdf	English	null	Designing a Tracked Running Gear of a Radio-Controlled Harvester	MATEC web of conferences	2,020	cc-by	4,824	Vladislav Klubnichkin1, Evgeny Klubnichkin1, Maxim Yakovlev1, Vladimir Makarov2, and Vladimir Belyakov2 1Mytishchi branch of the Moscow State Technical University n.a. N.E. Bauman, 14100 Mytishchi, 1 1-st Institutskaya str., 1, Russian Federation 2Nizhny Novgorod State Technical University n.a. R.E. Alekseev, 603950 Nizhny Novgorod, Minin str., 24, Russian Federation 1Mytishchi branch of the Moscow State Technical University n.a. N.E. Bauman, 14100 Mytishchi, 1-st Institutskaya str., 1, Russian Federation 2 Abstract. This work describes a model of a running gear of the designed tracked radio-controlled harvester intended for clean cutting which has been developed in the applied software package “Universal Mechanism” with use of the “Tracked Vehicles” module. It offers basic characteristics, justifies selection of variable parameters of the model. It describes modelling options of a model travel over variable irregularities, presents the obtained tractive effort torques on sprockets and loads acting on the support rollers. MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 https://doi.org/10.1051/matecconf/202032905001 Designing a Tracked Running Gear of a Radio- Controlled Harvester Vladislav Klubnichkin1, Evgeny Klubnichkin1, Maxim Yakovlev1, Vladimir Makarov2, and Vladimir Belyakov2 2 Designing a running gear When considering different designs of the tracked running gears of the timber harvesting equipment, we became interested in the principle of operation of a running gear made by Onezhsky tractor plant as it is one of the most reliable and simple in operation (fig.4). Fig. 4. Tractor Onezhets. Fig. 4. Tractor Onezhets. A tracked running gear of the designed radio-controlled harvester rests on the spring- lever-centerpoint suspension. It includes four bogies, two on each side of the vehicle. A bogie comprises a lever, an equalizer, a spring element and support rollers. A lever is attached by one side to the vehicle’s frame and by another side to the equalizer, two support rollers are attached to the equalizer axles. The running gear totally comprises 8 support rollers, 2 idler wheels with a spring-type tension devices, 2 driving sprockets and 2 tracks 450 mm wide. Design of a tracked running gear were presented on figure 5. The total weight of the tracked harvester is about 10 tons. Fig. 4. Tractor Onezhets. A tracked running gear of the designed radio-controlled harvester rests on the spring- lever-centerpoint suspension. It includes four bogies, two on each side of the vehicle. A bogie comprises a lever, an equalizer, a spring element and support rollers. A lever is attached by one side to the vehicle’s frame and by another side to the equalizer, two support rollers are attached to the equalizer axles. The running gear totally comprises 8 support rollers, 2 idler wheels with a spring-type tension devices, 2 driving sprockets and 2 tracks 450 mm wide. Design of a tracked running gear were presented on figure 5. The total weight of the tracked harvester is about 10 tons. Fig. 5. Design of a tracked running gear. The main results of the research are related to determining kinematics and dynamics of the tracked running gear of the radio-controlled harvester [13-17]. We have generated a 3D model of the harvester with a tracked running gear in the applied software package “Universal Mechanism” [18]. Ready for modelling simulator of a tracked radio-controlled harvester presented on figure 6. Fig. 5. Design of a tracked running gear. The main results of the research are related to determining kinematics and dynamics of the tracked running gear of the radio-controlled harvester [13-17]. 1 Introduction Today one of the topical issues of the forest industry is how to make harvesting equipment more capable of passing any terrain, more stable and more forest eco-friendly [1-3]. With that they shall demonstrate reasonable performance. Soil disturbance and vibration of an operator’s workplace are the main problems. Soil becomes strongly compacted under heavy weight of the harvesting equipment (fig. 1 and fig. 2). This results in rutting that may lead to linear erosion [4-7]. Besides, strong vibration of a timber-harvesting vehicle has a negative effect on the operator’s health [8, 9]. Fig. 1. Skid trail during passage of a timber-harvesting vehicle. Fig. 1. Skid trail during passage of a timber-harvesting vehicle. © 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/). https://doi.org/10.1051/matecconf/202032905001 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 Fig. 2. Skid trail after passage of a timber-harvesting vehicle. In forest conditions the wheeled running gear is second to the tracked gear in off-road performance (fig. 3). Besides, the tracked gear inflicts less disturbance to soil [10-12]. In this regard designing radio-controlled tracked timber-harvesting vehicles is topical. Fig. 2. Skid trail after passage of a timber-harvesting vehicle. In forest conditions the wheeled running gear is second to the tracked gear in off-road performance (fig. 3). Besides, the tracked gear inflicts less disturbance to soil [10-12]. In this regard designing radio-controlled tracked timber-harvesting vehicles is topical. In forest conditions the wheeled running gear is second to the tracked gear in off-road performance (fig. 3). Besides, the tracked gear inflicts less disturbance to soil [10-12]. In this regard designing radio-controlled tracked timber-harvesting vehicles is topical. In forest conditions the wheeled running gear is second to the tracked gear in off-road performance (fig. 3). Besides, the tracked gear inflicts less disturbance to soil [10-12]. In this regard designing radio-controlled tracked timber-harvesting vehicles is topical. Fig. 3. Side-by-side comparison of off-road performance of the tracked and wheeled running gears. Fig. 3. Side-by-side comparison of off-road performance of the tracked and wheeled running gears 2 2 2 https://doi.org/10.1051/matecconf/202032905001 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 2 Designing a running gear We have generated a 3D model of the harvester with a tracked running gear in the applied software package “Universal Mechanism” [18]. Ready for modelling simulator of a tracked radio-controlled harvester presented on figure 6. 3 3 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 https://doi.org/10.1051/matecconf/202032905001 Fig. 6. Ready for modelling simulator of a tracked radio-controlled harvester. Fig. 6. Ready for modelling simulator of a tracked radio-controlled harvester. 3 Simulation modelling of a tracked harvester A number of tests have been performed for correct experiments of travel of a tracked radio- controlled harvester model, including a balance test, a track tension test and an initial speed calculation test. They are resulted in loads acting on the running gear of the designed model as well as a model and its components position in space at the beginning of travel. This allows for maximum accurate results of the model travel research. 3.1 A balance test This is a test intended for eliminating transitional processes noted at the beginning of modelling due to inaccurate initial position of the model components. On figure 7 presented position of the tracked assembly components before and after a test. a b Fig. 7. Position of the tracked assembly components before (a) and after (b) a test. The test results are presented graphically in figures 8 and 9. This is a test intended for eliminating transitional processes noted at the beginning of modelling due to inaccurate initial position of the model components. On figure 7 presented position of the tracked assembly components before and after a test. a a b Fig. 7. Position of the tracked assembly components before (a) and after (b) a test. The test results are presented graphically in figures 8 and 9. Fig. 7. Position of the tracked assembly components before (a) and after (b) a test. The test results are presented graphically in figures 8 and 9. 4 https://doi.org/10.1051/matecconf/202032905001 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 Fig. 8. Drop in kinetic energy observed during a test. X-axis displays time (sec), y-axis displays total kinetic energy (J). Fig. 8. Drop in kinetic energy observed during a test. X-axis displays time (sec), y-axis displays total kinetic energy (J). Fig. 8. Drop in kinetic energy observed during a test. X-axis displays time (sec), y-axis displays total kinetic energy (J). Fig. 9. Loads acting on support rollers observed during a test. X-axis displays time (sec), y-axis displays load (N) acting on the support rollers. Fig. 9. Loads acting on support rollers observed during a test. X-axis displays time (sec), y-axis displays load (N) acting on the support rollers. Fig. 9. Loads acting on support rollers observed during a test. X-axis displays time (sec), y-axis displays load (N) acting on the support rollers. The graphs demonstrate that during the first second the transitional processes attenuate and a model takes a balanced state which was to be achieved. 3.3 An initial speed calculation test This is a test which simulates beginning of straight travel of a model. It helps to avoid strong load jumps and torques and obtain accurate results in further model travel experiments. On figure 12 were presented relationship between speed of a left and right driving sprockets and time. Fig. 12. Relationship between speed of a left and right driving sprockets (wheel) and time. X-axis displays time (sec), y-axis displays track travel speed (m/s). Fig. 12. Relationship between speed of a left and right driving sprockets (wheel) and time. X-axis displays time (sec), y-axis displays track travel speed (m/s). The graph shows that a model accelerates to 3.6 m/s for 5 sec and a speed curve gets straightened. 3.2 A track tension test This is a test intended to determine required tension of a track. Tension can be increased by elongation of a model of a tension device. The test results are presented as graphs of relationship between tension rod elongation and time (fig. 10) and between track tension force and time (fig. 11). Fig. 10. Relationship between elongation of the left and right tension rods and time. X-axis displays time (sec), y-axis displays tension device elongation (mm). Fig. 10. Relationship between elongation of the left and right tension rods and time. X-axis displays time (sec), y-axis displays tension device elongation (mm). 5 5 https://doi.org/10.1051/matecconf/202032905001 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 Fig. 11. Relationship between tension force of the LH and RH tracks and time. X-axis displays time (sec), y-axis displays track tension force (N). Fig. 11. Relationship between tension force of the LH and RH tracks and time. X-axis displays tim (sec), y-axis displays track tension force (N). Fig. 11. Relationship between tension force of the LH and RH tracks and time. X-axis displays time (sec), y-axis displays track tension force (N). The graph shows that in the course of the test the tension rod has elongated by 25 mm for 5 sec and track tension force has slightly increased. 2) Vehicle speed: 3.6 km/h 4 Simulation modelling of a tracked harvester linear travel In order to find capabilities of the designed model, simulation modelling of a tracked harvester travel has been done for the following cases: over sinusoidal irregularities and single irregularities which simulate travel over fallen trees [19]. Experiment conditions: Experiment conditions: 1) Vehicle speed: 3.6 km/h 1) Vehicle speed: 3.6 km/h Surface characteristics: sinusoidal irregularities with an amplitude of 0.2 m, wave length of 15 m and difference in wave periods of 5 m 2) V hi l d 3 6 k /h Surface characteristics: sinusoidal irregularities with an amplitude of 0.2 m, wave length of 15 m and difference in wave periods of 5 m Surface characteristics: sinusoidal irregula of 15 m and difference in wave periods of 5 m 2) Vehicle speed: 3.6 km/h 6 6 https://doi.org/10.1051/matecconf/202032905001 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 Surface characteristics: single obstacles 0.1 m, 0.2 m, 0.3 h and 0.4 m high 3) Vehicle speed: 7.2 km/h Surface characteristics: single obstacles 0.1 m, 0.2 m, 0.3 h and 0.4 m high 3) Vehicle speed: 7.2 km/h 3) Vehicle speed: 7.2 km/h Surface characteristics: single obstacles 0.1 m, 0.2 m, 0.3 m and 0.4 m high 4.1 Modelling of travel over sinusoidal irregularities Green color shows loads acting on the first support roller, red – on the second one, blue – on the third one and grey – on the fourth one. During modelling it has been found out that loads acting on the support rollers did not exceed 21 5 KN and their average value was within 10 KN A sprocket torque has reached TMTE 2020 Fig. 15. Loads acting on support rollers of the LH track. X-axis displays time (sec), y-axis displays load (N) acting on support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. Fig. 15. Loads acting on support rollers of the LH track. X-axis displays time (sec), y-axis displays load (N) acting on support rollers. Green color shows loads acting on the first support roller, purple on the second one, blue – on the third one and red – on the fourth one. Fig. 15. Loads acting on support rollers of the LH track. X-axis displays time (sec), y-axis displays load (N) acting on support rollers. Green color shows loads acting on the first support roller, purple on the second one, blue – on the third one and red – on the fourth one. Fig. 15. Loads acting on support rollers of the LH track. X-axis displays time (sec), y-axis displays load (N) acting on support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. Fig. 16. Loads acting on a support roller of the RH track. X-axis displays time (sec), y-axis displays loads (N) acting on a support roller. Green color shows loads acting on the first support roller, red – on the second one, blue – on the third one and grey – on the fourth one. Fig. 16. Loads acting on a support roller of the RH track. X-axis displays time (sec), y-axis displa loads (N) acting on a support roller. Green color shows loads acting on the first support roller, red h d bl h hi d d h f h Fig. 16. Loads acting on a support roller of the RH track. X-axis displays time (sec), y-axis displays loads (N) acting on a support roller. 4.1 Modelling of travel over sinusoidal irregularities Green color shows loads acting on the first support roller, red – on the second one, blue – on the third one and grey – on the fourth one. During modelling it has been found out that loads acting on the support rollers did not exceed 21.5 KN, and their average value was within 10 KN. A sprocket torque has reached 1.4 KNm. 4.1 Modelling of travel over sinusoidal irregularities Vehicle speed: 3.6 km/h Vehicle speed: 3.6 km/h Surface characteristics: sinusoidal irregularities with an amplitude of 0.2 m, wave length of 15 m and difference in wave periods of 5 m. of 15 m and difference in wave periods of 5 m. On figure 13 were presented a model at travel over sinusoidal irregularities. Fig. 13. A model at travel over preset surface. The experiment results are presented as graphs of torques occurring on the driving sprockets (fig. 14) and loads acting on the support rollers (fig. 15 and fig. 16) during a model travel over a surface with sinusoidal irregularities. Fig. 14. Torques on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket and purple color – on the right sprocket. On figure 13 were presented a model at travel over sinusoidal irregularities. Fig. 13. A model at travel over preset surface. Fig. 13. A model at travel over preset surface. The experiment results are presented as graphs of torques occurring on the driving sprockets (fig. 14) and loads acting on the support rollers (fig. 15 and fig. 16) during a model travel over a surface with sinusoidal irregularities. Fig. 14. Torques on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket and purple color – on the right sprocket. Fig. 14. Torques on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket and purple color – on the right sprocket. 7 7 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 https://doi.org/10.1051/matecconf/202032905001 Fig. 15. Loads acting on support rollers of the LH track. X-axis displays time (sec), y-axis displays load (N) acting on support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. Fig. 16. Loads acting on a support roller of the RH track. X-axis displays time (sec), y-axis displays loads (N) acting on a support roller. 4.2 Modelling of travel over single obstacles During operation a timber-harvesting vehicle shall be capable of negotiating variable obstacle, including fallen trees, stubs, pits, etc. In this regard it is necessary to conduct an experiment of modelling negotiating of such obstacles. A surface with single obstacles 100 mm, 200 mm, 300 mm, and 400 mm high simulating fallen trees has been selected for this experiment. On figure 17 were presented a model at travel over single obstacles. Case 1: vehicle speed of 3.6 km/h 8 8 https://doi.org/10.1051/matecconf/202032905001 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 Fig. 17. A model during travel over preset surface. The experiment results are presented as graphs of torques occurring on the driving sprockets (fig. 18) and loads acting on the support rollers (fig. 19 and fig. 20) during a model travel over surface with single obstacles. Fig. 18. Torques acting on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket, purple – on the right one. Fig. 17. A model during travel over preset surface. Fig. 17. A model during travel over preset surface. Fig. 17. A model during travel over preset surface. The experiment results are presented as graphs of torques occurring on the driving sprockets (fig. 18) and loads acting on the support rollers (fig. 19 and fig. 20) during a model travel over surface with single obstacles. Fig. 18. Torques acting on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket, purple – on the right one. Fig. 19. Loads acting on the support rollers of the LH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. Fig. 17. A model during travel over preset surface. The experiment results are presented as graphs of torques occurring on the driving ockets (fig. 18) and loads acting on the support rollers (fig. 19 and fig. 20) during a del travel over surface with single obstacles. The experiment results are presented as graphs of torques occurring on the driving sprockets (fig. 18) and loads acting on the support rollers (fig. 19 and fig. 4.2 Modelling of travel over single obstacles 20) during a model travel over surface with single obstacles. Fig. 18. Torques acting on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket, purple – o the right one. Fig. 18. Torques acting on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket, purple – the right one. g q g g g p p y ( ) y displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket, purple – on the right one. Fig. 19. Loads acting on the support rollers of the LH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. Fig. 19. Loads acting on the support rollers of the LH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support ll l h d bl h hi d d d h f h Fig. 19. Loads acting on the support rollers of the LH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. 9 9 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 https://doi.org/10.1051/matecconf/202032905001 Fig. 20. Loads acting on the support rollers of the RH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, red – on the second one, blue – on the third one and grey – on the fourth one. Fig. 20. Loads acting on the support rollers of the RH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, red – on the second one, blue – on the third one and grey – on the fourth one. 4.2 Modelling of travel over single obstacles During modelling it has been found out that loads acting on the support rollers reached 35 KN in average, and their average value was within 15 KN. Case 2: vehicle speed of 7.2 km/h Case 2: vehicle speed of 7.2 km/h The experiment results are presented as graphs of torques occurring on the driving sprockets (fig. 21) and loads acting one the support rollers (fig. 22 and fig. 23) during a model travel over surface with single obstacles. Fig. 21. Torques on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket, purple – on the right one. Fig. 22. Loads acting the support rollers of the LH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. Fig. 21. Torques on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket, purple – on the right one. Fig. 21. Torques on the left and right driving sprockets. X-axis displays time (sec), y-axis displays a tractive effort torque (Nm). Green color shows a torque on the left sprocket, purple – on the right one. Fig. 22. Loads acting the support rollers of the LH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. Fig. 22. Loads acting the support rollers of the LH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. Fig. 22. Loads acting the support rollers of the LH track. X-axis displays time (sec), y-axis displays loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, purple – on the second one, blue – on the third one and red – on the fourth one. 10 https://doi.org/10.1051/matecconf/202032905001 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 Fig. 23. Loads acting on the support rollers of the RH track. X-axis shows time (sec), y-axis shows loads (N) acting on the support rollers. 5 Outcomes The modelling has provided the results of loads acting on the support rollers at respective modes of travel, as well as speed and torques occurring on the driving sprockets, which in future will help to choose optimal transmission for a tracked harvester. The running gear model of the designed tracked radio-controlled harvester for clean cutting which has been developed in the applied software package “Universal Mechanism” with use of the “Tracked Vehicles” module has proved its efficiency through tests. The results of the given study have been obtained with financial support of the grants of the President of the Russian Federation № MD-226.2020.8. Case 2: vehicle speed of 7.2 km/h Green color shows loads acting on the first support roller, red – on the second one, blue – on the third one and grey – on the fourth one. During modelling it has been found out that loads acting on the support rollers reached in average 40 KN and their average value was within 18 KN. Fig. 23. Loads acting on the support rollers of the RH track. X-axis shows time (sec), y-axis shows loads (N) acting on the support rollers. Green color shows loads acting on the first support roller, red – on the second one, blue – on the third one and grey – on the fourth one. During modelling it has been found out that loads acting on the support rollers reached in average 40 KN and their average value was within 18 KN. During modelling it has been found out that loads acting on the support rollers reached in average 40 KN and their average value was within 18 KN. References 1. M. Cambi, G. Certini, F. Neri, E. Marchi, The impact of heavy traffic on forest soils. Forest Ecology and Management. Vol. 338, P. 124-138 (2015) 2. L.A. Goberman, V.A. Goberman, I.P. Ksenevich, Ground trailer transport systems. Technical and economic bases of designing machines and processes. Methodological aspects of project activities and decision making. Vol. 3. Encyclopedia. Engineering, Moscow. Pages 788 (2003) 3. R. Horn, J. Vossbrink, S. Peth, S. Becker, Impact of modern forest vehicles on soil physical properties. Forest Ecology and Management. Vol. 248, Issues 1–2, P. 56-63 (2007) 4. A. Bodin, Development of a tracked vehicle to study the influence of vehicle parameters on tractive performance in soft terrain. Journal of Terramechanics, Vol. 36, Issue 3, P. 167–181 (1999) 5. V. Makarov, A. Markovnina, A. Papunin, et al, Study of uneven surfaces distribution on forestry roads, Journal of Physics: Conference Series (2019) 6. J.Y. Wong, Theory of Ground Vehicles, third ed. John Wiley & Sons, NY, ISBN 0-471- 35461-9 (2001) 7. J.Y. Wong, Terramechanics and Off-Road Vehicle Engineering, 2nd Edition. Pages 488 (2009) 11 MATEC Web of Conferences 329, 05001 (2020) ICMTMTE 2020 https://doi.org/10.1051/matecconf/202032905001 8. A.M. Kochnev, G.M. Anisimov, Tests of harvesting machines, SPb.: SPbGLTA (200 9. S. Rakheja, M.F.R. Afonso, S. Sankar, Dynamic analysis of tracked vehicles with trailing arm suspension and assessment of ride vibrations, International Journal of Vehicle Design, Vol.13, No.1 (1992) 10. V.M. Kotikov, Impact harvesting machines on forest soils, Dissertation of the doctor of technical sciences, Moscow state forest university, Pages 214 (1995) 11. A.A. Silaev, Spectral theory of suspension of transport vehicles, Mechanical Engineering, Moscow. Pages 192 (1972) 12. N.A. Zabavnikov, Fundamentals of the theory of transport tracked vehicles, Moscow, Mashinostroenie, Pages 448 (1975) 13. V. Klubnichkin, E. Klubnichkin, Research of kinematics and dynamics of tracked timber harvesting vehicle running gear. Proceedings of the 48th International Symposium on Forestry Mechanization, P. 315–320, ISBN 978-3-900932-29-9 (2015) 14. Said Al-Milli, Lakmal D. Seneviratne, Kaspar Althoefer, Track–terrain modelling and traversability prediction for tracked vehicles on soft terrain, Journal of Terramechanics, Volume 47, Issue 3, P. 151–160 (2010) 15. E.E. Klubnichkin, V.E. Klubnichkin, Increasing durability of the undercarriage of the tracked timber harvesting machine, Proc. of the 13th ISTVS Europ. Conf. P. 200–210 (2015) 16. V. Klubnichkin, E. Klubnichkin G. References Kotiev, Developing a method of theoretical evaluation of the tracked timber harvesting machine undercarriage element loading, 19th International and 14th European-African Regional Conference of the ISTVS (2017) 17. A. Dhir, S. Sankar, Analytical track models for ride dynamic simulation of tracked vehicles, Journal of Terramechanics, Vol. 31, Issue 2, P. 107–138 (1994) 18. D.J. Pogorelov, Computer simulation of the dynamics of technical systems using the software package "Universal mechanism", J. of Comp. and Inform. Techn. 4, P. 27–34 (2005) 19. V.E. Klubnichkin, E.E. Klubnichkin, V.S. Makarov, D.V. Zezyulin, A.V. Redkozubov, V.V. Belyakov, Modelling of tracked vehicles’ travel over forest roads, Works of NNSTU n.a. R.E. Alekseev, No. 1, P. 171-176 (2016) 12
https://openalex.org/W1965530610	https://europepmc.org/articles/pmc3257616?pdf=render	English	null	Osteoporosis Prevention—A Worthy and Achievable Strategy	Nutrients	2,010	cc-by	972	Howard A. Morris 1,2 1 School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5001, Australia; E-Mail: howard.morris@health.sa.gov.au 2 Chemical Pathology Directorate, SA Pathology, Adelaide, South Australia 5000, Australia Received: 22 September 2010 / Accepted: 14 October 2010 / Published: 20 October 2010 1 School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5001, Australia; E-Mail: howard.morris@health.sa.gov.au 2 Chemical Pathology Directorate, SA Pathology, Adelaide, South Australia 5000, Australia Received: 22 September 2010 / Accepted: 14 October 2010 / Published: 20 October 2010 This special issue of Nutrients records seven of the presentations made to the very successful meeting titled “Osteoporosis Prevention: A Workshop on Calcium, Vitamin D and other Nutritional Aspects” held in Adelaide, Australia on 5 and 6 March 2010 [1-7]. Seventy six delegates attended from across Australia and New Zealand to review the current evidence that dietary calcium intake, vitamin D status, other nutrients and exercise play a significant role in bone mineral homeostasis and act to prevent the development of osteoporosis. The Workshop promoted the concept that osteoporosis is a predictable and preventable disease and that significant benefit would be achieved to reduce the incidence of osteoporosis and the risk of fractures from nutrition and life style activities. Such an achievement will not only save considerable pain, suffering and morbidity but will also have a major financial benefit for the healthcare system for which the cost of treatment for osteoporotic fractures already amounts to billions of dollars. A concrete proposal arising from the Workshop was the adoption by way of signing a petition addressed to the Federal Australian Government stating the following: We recommend to the Screening Subcommittee of the Australian Population Health Development Principal Committee (APHDPC) of the Australian Health Minister's Advisory Council that all women should be screened for bone density at the menopause with the aim of reducing post-menopausal bone loss and age-related fractures by adopting lifestyle measures, particularly diet and exercise. The session of the Workshop devoted to discussion of this statement agreed that the strongest evidence that lifestyle measures could improve bone status and prevent fractures was in postmenopausal women, since this is the time at which bone loss starts. Nutrients 2010, 2, 1073-1074; doi:10.3390/nu2101073 Nutrients 2010, 2, 1073-1074; doi:10.3390/nu2101073 nutrients ISSN 2072-6643 www.mdpi.com/journal/nutrients OPEN ACCESS Nutrients 2010, 2 Nutrients 2010, 2 again at 5 and/or 10 years later. The actual interventions to be recommended and the optimal practices to ensure the highest adoption rate of such lifestyle measures would need further research and review. Professor B E Christopher Nordin was a driving force for instigating and organizing the Workshop. Speakers acknowledged his major contribution to osteoporosis research over some 60 years. In particular he was recognized for his inspirational leadership for research and advocacy of the importance of calcium deficiency in the etiology of postmenopausal osteoporosis. His prodigious productivity as a medical researcher includes his outstanding publishing output of over 500 scientific papers and his achievement of an h-index of 67 (Web of Science) with 8 of these publications accumulating over 200 citations. This volume is dedicated to Professor Chris Nordin in recognition of his immense contribution to osteoporosis research and its prevention. Howard A. Morris 1,2 It was agreed that women with below average Bone Mineral Density at the hip or spine, as indicated by a T-score below zero, were the most likely to benefit from lifestyle advice to preserve bone and reduce the risk of fractures. Issues identified for further investigation included whether it could be beneficial to measure bone density 1074 © 2010 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 license (http://creativecommons.org/licenses/by/3.0/). References 1. Nowson, C.A. Prevention of fractures in older people with calcium and vitamin D. Nutrients 2010, 2, 975-984. 2. Winzenberg, T.; Oldenberg, B.; Jones, G. Bone Density Testing: An Under-Utilised and Under-Researched Health Education Tool for Osteoporosis Prevention? Nutrients 2010, 2, 985-996. 2. Winzenberg, T.; Oldenberg, B.; Jones, G. Bone Density Testing: An Under-Utilised and Under-Researched Health Education Tool for Osteoporosis Prevention? Nutrients 2010, 2, 985-996. 3. Nordin, B.E.C. Evolution of the Calcium Paradigm: The Relation between Vitamin D, Serum Calcium and Calcium Absorption. Nutrients 2010, 2, 997-1004. 3. Nordin, B.E.C. Evolution of the Calcium Paradigm: The Relation between Vitamin D, Serum Calcium and Calcium Absorption. Nutrients 2010, 2, 997-1004. 4. Daly, R.M. Independent and Combined Effects of Exercise and Vitamin D on Muscle Morphology, Function and Falls in the Elderly. Nutrients 2010, 2, 1005-1017. 4. Daly, R.M. Independent and Combined Effects of Exercise and Vitamin D on Muscle Morphology, Function and Falls in the Elderly. Nutrients 2010, 2, 1005-1017. 5. Sanders, K.M.; Stuart, A.L.; Kotowicz, M.A.; Nicholson, G.C. Annual Feedback Is an Effective Tool for a Sustained Increase in Calcium Intake among Older Women. Nutrients 2010, 2, 1018-1025. 6. Morris, H.A.; O’Loughlin, P.D.; Anderson, P.H. Experimental Evidence for the Effects of Calcium and Vitamin D on Bone: A Review. Nutrients 2010, 2, 1026-1035. 6. Morris, H.A.; O’Loughlin, P.D.; Anderson, P.H. Experimental Evidence for the Effects of Calcium and Vitamin D on Bone: A Review. Nutrients 2010, 2, 1026-1035. 7. Meng, X.; Kerr, D.A.; Zhu, K.; Devine, A.; Solah, V.; Binns, C.W.; Prince, R.L. Calcium Intake in Elderly Australian Women Is Inadequate. Nutrients 2010, 2, 1036-1043. 7. Meng, X.; Kerr, D.A.; Zhu, K.; Devine, A.; Solah, V.; Binns, C.W.; Prince, R.L. Calcium Intake in Elderly Australian Women Is Inadequate. Nutrients 2010, 2, 1036-1043. © 2010 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 license (http://creativecommons.org/licenses/by/3.0/). © 2010 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 license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W4311145592	https://link.springer.com/content/pdf/10.1007/s12517-022-10995-6.pdf	English	null	Missing rainfall data estimation—an approach to investigate different methods: case study of Baghdad	Arabian journal of geosciences	2,022	cc-by	10,781	ERROR: type should be string, got "https://doi.org/10.1007/s12517-022-10995-6\nArabian Journal of Geosciences (2022) 15:1740 https://doi.org/10.1007/s12517-022-10995-6\nArabian Journal of Geosciences (2022) 15:1740 ORIGINAL PAPER \t Nadhir Al‑Ansari \n\t\nnadhir.alansari@ltu.se; mukhaladabdullah@gmail.com Abstract The missing of the meteorological data in Iraq is common due to malfunction of measuring devices, security status, and \nhuman effects. The study tested 17 missing precipitation data estimation methods in Baghdad city as a case study, where, all \nthe surrounding stations around Baghdad experienced the missing of data for various reasons, and some of the missing data \nare for a full year record. The methods examined in this study are based on different approaches, some of the methods are \nbased upon the distances to the targeted station, others are upon regression factors, and there are also methods that combine \nseveral factors. There are also other types of missing data filling methods which depend on imputation and artificial intel-\nligence. The investigation of the most accurate method to find the missing data will assist researchers and decision makers to \nfill the gap in their analysis in one of the most vulnerable countries in terms of drought and climate changes impacts. Results \nshowed that Expectation Maximization (EM) method utilization has the best results with the least errors, and Multiple Linear \nRegression (MLR) method was ranked the second best method. In general, all of the applied methods had resulted acceptable \ninterpolations, and it was clear that the combined methods have low significance on the results in comparison with others. All of these findings are limited to the study area meteorological and spatial conditions. Keywords Missing precipitation · Expectation maximization · Multiple linear regression · Baghdad precipitation · Arid \nzones precipitation Keywords Missing precipitation · Expectation maximization · Multiple linear regression · Baghdad precipitation · Arid \nzones precipitation Missing rainfall data estimation—an approach to investigate different \nmethods: case study of Baghdad Mukhalad Abdullah1,2 · Nadhir Al‑Ansari2 Received: 21 July 2021 / Accepted: 12 November 2022 \n© The Author(s) 2022\n/ Published online: 30 November 2022 Responsible Editor: Broder J. Merkel 2\t\nLulea University of Technology, Lulea, Sweden 1\t\nBaghdad, Iraq Introduction years 2003 and 2004, most of the data are missed even \nin Baghdad station. Another example was in the period \nfrom 2014 to 2017; the meteorological stations in the \nwestern and northern parts of the country had stopped \nrecording the data((IMOaS) 2021). Baghdad station was selected as a case study in this research. Baghdad is the capital city and also the largest city in Iraq; it \nis also the main location of human activities in the Mesopo-\ntamian plain. The major irrigation projects are within Bagh-\ndad area (Abdullah and Al-Ansari 2021), where the accuracy \nof climate records is far important to plan and enhance irri-\ngation practices; also, this area is the hub of transportation \ninfrastructures in the country. The finding of the most suiting algorithm to perform the \nfilling of missing rainfall data is essential. It is worth men-\ntioning also that the climate and geospatial characteristics \nof Baghdad are greatly approaching other parts of Mesopo-\ntamia; thus, it can reliably extrapolate the results in other \nareas. Iraqi Meteorological Organization and Seismol-\nogy (IMOaS) is the official authority that manages the \nmeteorological and seismology data. It is common to \nfind missing data for many reasons, for instance, in the Many methods were successfully tested and adopted in \nother parts of the world, where the missing data filling mod-\nels are based on several concepts, which are mainly the cor-\nrelation with the surrounding stations, the spatial analysis \nwith the surrounding stations, and the artificial intelligence. i\nSeveral efforts were made by researchers to predict \nthe missing rainfall data, as it is one of the common \nproblems. Tropical Rainfall Measuring Mission (TRMM) \nestimates were analyzed; the method showed some limi-\ntation near the water bodies and when the precipitation (0121 3456789)\n3 1740 Page 2 of 17 Arab J Geosci (2022) 15:1740 is lower than 200 mm annually, but rather that, this pro-\nvides results with a correlation coefficient that might \nreach 0.91 (Abdulrazzaq 2020). This method also pro-\nvides some acceptable prediction in a specific circum-\nstance, but with overestimation during dry months at dif-\nferent locations (Abdulrida and Al-Jumaily 2016). surface-sea ice system designed to provide the best esti-\nmate of the state of these coupled domains over the \ntargeted period. The system has several strengths and \nlimitations; one of these limitations is the few relative \nevaluations that had been conducted. Methodology and data In order to examine the hypothesized missing data in the \nanalysis, Baghdad station was considered the target station \nwhere the missing occurred. Also, figures and conclusions \nwere shown for the time period from 1988 to 1995, and \nthe years 1982 and 2000, where these years are the wettest \nand driest, respectively, in terms of the time duration of \nthe study. Introduction The analysis was made for the period of 35 years, from \n1980 to 2014. Figure 1 and Table 1 show the statisti-\ncal and spatial characteristics of the study area stations. Twelve stations were gathered; these stations are Razazza, \nJurf Al-Sakhar, Latifiyah, Suwairah, Falluja, Abu Ghraib, \nBaghdad, Nahrawan, Tharthar, Taji, Rashidiyah, and \nKhan Saad. Baghdad station is considered in this study \nthe target station, it lies in the center of the area, and \nit is the most important one; there is no difference in \nelevation for most of the stations, except Tharthar stations \nwhich have an elevation of 82 m above sea level (a.s.l.). Also, the maximum diagonal distance between stations \nis 108 km, while the closest distance is around 29 km \nbetween Baghdad and Abu Ghraib stations. Figure 1 \nshows the geographical location of the study area stations. Artificial neural network (ANN) method to test the \nestimation of four different stations, which are Basra, \nBaghdad, Mosul, and Rutba, had shown good results (Al-\nSalihi et al. 2013); testing of this method also applied in \nSulaymaniyah city, northeast Iraq, and for the period of \n2013–2018, the results of ANN provide a good estima-\ntion up to 91.5% of accuracy (Murad and Jaff 2020). Isohyet method is adopted to estimate the missing rainfall \ndata in Nineveh governorate, which includes Mosul City. The study was made for 8 weather stations, during 20 years \nfrom 2000 to 2019. The results are promising and generally \ngood (Alozeer 2020). Study area Baghdad station was chosen as a case study where it is located \nin Iraq. Iraq is located in the Middle East covering an area \nof 438,320 km2. The climate of Iraq is mainly continental, \nsubtropical semi-arid type, with the north and north-eastern \nmountainous regions having a Mediterranean climate. In most \nof the country territories, the rainfall is very seasonal, which \nstarts in the winter from December to February. Regarding \nthe north and northeast of the country, the rainfall starts from \nNovember to April. Average annual rainfall is about 216 mm; \nit varies from 1200 mm in the northeast to less than 100 mm \nover 60% of the country in the south. Regarding the tempera-\ntures, winters are cool to cold; the temperature in the day is \n16 °C and dropping at night to 2 °C with a possibility of frost. Summers are dry and hot to extremely hot, with a shade tem-\nperature of over 43 °C during July and August, yet dropping \nat night to 26 °C (Frenken 2009). There are 17 methods performed to analyze the proposed \nmissing data findings, among which are simple and compli-\ncated methods, and some were made with help of computer \nsoftware. Arithmetic average (AA) This method is one of the easiest and most widely used in \nhydrologic application. It is simply the mean of the sur-\nrounding stations to the targeted area in the study zone. According to Linsley et al. (1975), AA method will yield a \ngood estimate in flat country; the surrounding stations are \nuniformly distributed, and the individual stations do not vary \nso far from the mean. Use of this method is limited when the \ntopography is more complicated; Eq. (1) as following is the \nformula of the AA method: This method is one of the easiest and most widely used in \nhydrologic application. It is simply the mean of the sur-\nrounding stations to the targeted area in the study zone. According to Linsley et al. (1975), AA method will yield a \ngood estimate in flat country; the surrounding stations are \nuniformly distributed, and the individual stations do not vary \nso far from the mean. Use of this method is limited when the \ntopography is more complicated; Eq. (1) as following is the \nformula of the AA method: As shown in Fig. 1, several stations exist and are man-\naged by IMOaS; unfortunately, many of the date were \nmissed in these stations, and some data are scattered and \nunreliable. Data for the years 2003 and 2004 are mostly \nnot available, and the data for the years 2014 to 2017 \nwere missed in Ramadi station. (1)\nY =\n∑n\ni=1Xi\nn Y =\n∑n\ni=1Xi\nn The resort to another alternative was to compile the \nraw from the online database of Texas A&M University \n(globalweather.tamu.edu/). The data are generated from \nclimate forecast system reanalysis (CFSR); this tool is a \nglobal, high-resolution, coupled atmosphere–ocean-land (1) where: Y is the missing value at the target station, Xi is the \nmeasured value of the ith surrounding station, and n is the \nnumber of these stations. 1 3 Page 3 of 17 1740 Arab J Geosci (2022) 15:1740 Fig. 1 Geographical location of the stations within the study area Fig. 1 Geographical location of the stations within the study area Fig. Linear regression (LR) Simply, this method is used to establish a linear relation \nbetween the targeted station and the most correlated nearby \nstation in terms of statistics (Armanuos et al. 2020). Once \nthe linear equation is derived, the estimated values can \nbe calculated using this formula. In this study, the linear \nequation was established between Abu Ghraib station and \nBaghdad Station, as the correlation between both is the \nhighest. The formula of this method is as follows: Correlation coefficient weighted (CCW) According to Teegavarapu and Chandramouli (2005), this \nmethod yields a better result as long as the correlation \nbetween the target and surrounding stations is higher. This \nmethod gives the weight of the ratio of the correlation \ncoefficient. Since the correlation factors between Baghdad \nstation and the other stations are almost above 0.9, then, a \npromising result is expected. The formula of CCW method \nis as follows: Arithmetic average (AA) 1 Geographical location of the stations within the study area Table 1 Statistical and spatial characteristics of the stations within the study area\nBased on monthly data\nStation\nLatitude (N)\nLongitude (E)\nElevation (m \na.s.l.)\nAverage rain-\nfall (mm)\nMaximum \nrainfall (mm)\nMinimum \nrainfall (mm)\nStandard \ndeviation\nRazazza\n32°56′24.37″\n43°45′0.00″\n54\n7.7\n109.0\n0.0\n13.7\nJurf Al-Sakhar\n32°56′24.37″\n44° 3′45.00″\n30\n7.5\n80.5\n0.0\n12.8\nLatifiyah\n32°56′24.37″\n44°22′30.00″\n26\n7.6\n86.8\n0.0\n12.4\nSuwairah\n32°56′24.37″\n44°41′15.00″\n29\n9.1\n92.9\n0.0\n14.2\nFalluja\n33°15′8.64″\n43°45′0.00″\n38\n8.6\n121.4\n0.0\n14.7\nAbu Ghraib\n33°15′8.64″\n44° 3′45.00″\n33\n8.5\n111.2\n0.0\n14.4\nBaghdad\n33°15′8.64″\n44°22′30.00″\n33\n9.2\n82.3\n0.0\n14.5\nNahrawan\n33°15′8.64″\n44°41′15.00″\n36\n11.7\n101.3\n0.0\n17.2\nTharthar\n33°33′52.57″\n43°45′0.00″\n82\n9.5\n88.9\n0.0\n14.7\nTaji\n33°33′52.57″\n44° 3′45.00″\n42\n9.9\n95.4\n0.0\n15.3\nRashidiyah\n33°33′52.57″\n44°22′30.00″\n35\n11.6\n98.2\n0.0\n17.2\nKhan Saad\n33°33′52.57″\n44°41′15.00″\n38\n14.7\n118.2\n0.0\n21.1 Table 1 Statistical and spatial characteristics of the stations within the study area 1740 Page 4 of 17 Arab J Geosci (2022) 15:1740 Normal ratio (NR) (USDA) to estimate the missing rainfall data by consider-\ning the reciprocal of the inverse of distances between the \ntarget station and the surrounding stations (Barbalho et al. 2014). The formula of IDW method is as follows: (USDA) to estimate the missing rainfall data by consider-\ning the reciprocal of the inverse of distances between the \ntarget station and the surrounding stations (Barbalho et al. 2014). The formula of IDW method is as follows: The NR method is applied when the annual mean of any sur-\nrounding station is no more than 10% of that for the target sta-\ntion; this method was adopted by the US National Weather \nServices (Anderson 1972); this method was firstly proposed by \nPaulhus and Kohler (1952), where the ratios between the tar-\ngeted station and surrounding stations are the weighting factor \nas in equation below: (5)\nY =\n\u001fn\ni=1\n⎡\n⎢\n⎢\n⎢⎣\n\u001b\n1\ndk\ni\n\u001a\n∑n\ni=1\n\u001b\n1\ndk\ni\n\u001a\n⎤\n⎥\n⎥\n⎥⎦\nXi Services (Anderson 1972); this method was firstly proposed by \nPaulhus and Kohler (1952), where the ratios between the tar-\ngeted station and surrounding stations are the weighting factor \nas in equation below: (5) where: di is the distance from the target station to the ith \nsurrounding station, and k is the distance of friction varying \nfrom 1 to 6; in this study, k was assumed to equal 1. (2)\nY = 1\nn\n∑n\ni=1\nNs\nNi\nX\ni (2) where Ns is the mean of available rainfall data at the target \nstation, Ni is the mean of the available rainfall data at the \nith surrounding stations, and n is the number of surrounding \nstations. Although, some stations in the study have a mean \ndifference by more than 10% of Baghdad station, but these \nstations were considered in the calculation to examine the \nlimitation of this criterion within the Baghdad area. Normal ratio with geographical coordinates (NRGC) This method is adopted to combine the weighting factors of \nmean ratios and geographical coordinates; some researchers \nfind a slightly better accuracy when employing this method \n(Armanuos et al. 2020). The formula of NRGC method is \nas follows: (4)\nY =\n\u001fn\ni=1\n⎡\n⎢\n⎢\n⎢⎣\n\u001b\n1\nx2\ni +y2\ni\n\u001a\u001b\nNs\nNi\n\u001a\n∑n\ni=1\n\u001b\n1\nx2\ni +y2\ni\n\u001a\u001b\nNs\nNi\n\u001a\n⎤\n⎥\n⎥\n⎥⎦\nXi (7)\nY = a + bXi Y = a + bXi (7) (4) where: Y is the estimated rainfall data of the targeted station, \nand Xi is the observed rainfall value of the neighboring sta-\ntion; a is the intercept, and b is the regression coefficient. Geographical coordinates (GC) Regarding the geographical coordinates method, it is weighting \nof the vertical and horizontal coordinates with reference to the \ntotal of all surrounding stations around target station (Yozgatligil \net al. 2012). The inputs as in the equation below are the latitude \nand longitude of the stations; the GC method formula is: (6)\nY =\n\u001fn\ni=1\n\u001e\n\u001dri\n\u001c\n∑n\ni=1\n\u001dri\n\u001c\n\u001a\nXi (6) (3)\nY =\n\u001fn\ni=1\n⎡\n⎢\n⎢⎣\n1\nx2\ni +y2\ni\n∑n\ni=1\n1\nx2\ni +y2\ni\n⎤\n⎥\n⎥⎦\nXi where: ri is the Pearson correlation coefficient (rPearson) \nbetween the target station and each surrounding station. (3) where: xi and yi are the longitude and latitude of the ith sur-\nrounding station. Modified correlation coefficient with inverse distance \nweighting (MCCIDW) The IDW and CCW methods are combined in a single formula \n[18]. The MCCIDW method gives a power for the correla-\ntion coefficient and the distance which is symbolled p, ranging \nfrom 1 to 6 (Armanuos et al. 2020), and for the purpose of \ncalculation, p is considered to be 1. The formula of MCCIDW \nmethod is as follows: Multiple imputation (MI) This method was first introduced by Rubin (1988) in 1988. It is based on the distribution of imputation that reflects \nuncertainty of the missing data, in order to overcome the \nunderestimation of single imputation (Sattari and Rezaza-\ndeh Joudi 2016). There are different software applications \nto perform this method. In this study, SPSS Statistics soft-\nware was adopted to conduct the missing data calculations. Modified old normal ratio with inverse distance (ONRID) This method is adopted by the UK Meteorological Office \nto calculate missing data of meteorological components \nwhere the comparison was held with one of a single nearby \nstation (Armanuos et al. 2020)(Kashani and Dinpashoh \n2011). Since Abu Ghraib station has the highest correla-\ntion with Baghdad station, so this station was adopted for \nthe application of the UK method. The estimated values \nwere calculated by multiplying the values in Abu Ghraib \nstation by the ratio of mean rainfall of Abu Ghraib station \nto that of Baghdad station. As in the previous method, this method adopted another \napproach by combining the effect of distance and mean ratios \nbetween stations (Azman, Zakaria, & Ahmad Radi, 2015; \nSyed Jamaludin et al. 2008); the formula of this method is as \nfollows: (10)\nY =\n\u001fn\ni=1\n⎡\n⎢\n⎢\n⎢⎣\n\u001b\nNs\nNi .d−2\ni\n\u001a\n∑n\ni=1\n\u001b\nNs\nNi .d−2\ni\n\u001a\n⎤\n⎥\n⎥\n⎥⎦\nXi (10) Nonlinear iterative partial least squares (NIPALS) algorithm \nfor missing data (NIPALS) The NIPALS method was first introduced by Wold (1968). The algorithm of this method is to calculate the slope of \nthe least squares line that crosses the origin of the points \nof the observed data. where eigenvalues are determined \nby the variance of the NIPALS components. In this study, \nSPSS Statistics software was adopted to conduct the miss-\ning data calculations using NIPALS method. (9)\nY =\n\u001fn\ni=1\n⎡\n⎢\n⎢\n⎢⎣\n\u001b rp\ni\ndp\ni\n\u001a\n∑n\ni=1\n\u001b rp\ni\ndp\ni\n\u001a\n⎤\n⎥\n⎥\n⎥⎦\nXi (9) Multiple linear regression (MLR) This method has been widely used, since it was first intro-\nduced by the United States Department of Agriculture MLR is based on the same concept as (LR) method, but \nthe modification with this method is that the regression 3 1 Page 5 of 17 1740 Arab J Geosci (2022) 15:1740 is linked with all other stations in the study area (Teega-\nvarapu, 2009). The factors were calculated using Excel-\nMicrosoft Office software. The formula for MLR is as \nfollows: (Yozgatligil et al. 2012). It is an iterative method both \nfor the estimation of mean values and covariance matri-\nces from incomplete data (Schneider 2001). In this study, \nSPSS Statistics software was adopted to perform EM \nmethod. is linked with all other stations in the study area (Teega-\nvarapu, 2009). The factors were calculated using Excel-\nMicrosoft Office software. The formula for MLR is as \nfollows: (8)\nY = b0 +\n∑n\ni=1biXi (8) Closest station method (CSM) where: Y is the estimated rainfall data at the target station, \nXi is the observed rainfall value of the ith surrounding sta-\ntion, bi are the regression coefficients of the ith surrounding \nstations, and n is the number of the surrounding stations. This is the simplest and easiest method to predict the miss-\ning data of meteorological factors. After analyzing the long \nrecords of data, the missing values are replaced with the data \nfrom a nearby station that has the highest correlation coef-\nficient (Bárdossy and Pegram 2014; Kanda et al. 2017). In \nthe case under this study, the station with the best correla-\ntion is Abu Ghraib station. Also, it is worth to mention that \nthis method is named with different jargons, but has the same \nalgorithm. Modified normal ratio based on square root distance \n(MNR‑T) This method was first proposed by Tang et al. (1996); it also \ncombines the weighting of mean rations and the distance \nto target station as in the ONRID method, but with another \nformulation. The MNR-T formula is as follows: (12)\nY =\n\u001fn\ni=1\nNs\nNi\n. ⎛\n⎜\n⎜\n⎜\n⎜⎝\n\u001b\nd\n1\np\ni\n\u001a\n∑n\ni=1\n\u001b\nd\n1\np\ni\n\u001a\n⎞\n⎟\n⎟\n⎟\n⎟⎠\nXi (12) where: the power of the distance p ranges from 1.5 to 2, \nwhere for the purpose of calculations, p is considered to be \nequal to 1.75. Normal ratio inverse distance weighting with correlation \n(NRIDC) This method was first proposed by Dempster et al. (1977); \nEM method is a multilayer perceptron type neural net-\nwork and multiple imputation strategy using Monte Carlo \nMarkov Chain based on expectation–maximization In this method, a new combination is proposed by Azman \net al. (2015), by considering the superimposition of NR, 1 3 1740 Page 6 of 17 Arab J Geosci (2022) 15:1740 Table 3 Results of Pettit’s test to examine data homogeneity\nStation\nPettit's test\nDegree of \nconfidence\np value\nRisk of rejecting \nH0 (%)\nRazazza\n0.759\n75.92\n95%\nJurf Al-Sakhar\n0.879\n87.86\n95%\nLatifiyah\n0.849\n84.88\n95%\nSuwairah\n0.748\n74.84\n95%\nFalluja\n0.671\n67.1\n95%\nAbu Ghraib\n0.878\n87.76\n95%\nBaghdad\n0.662\n66.22\n95%\nNahrawan\n0.536\n53.6\n95%\nTharthar\n0.441\n44.08\n95%\nTaji\n0.402\n40.16\n95%\nRashidiyah\n0.268\n26.76\n95%\nKhan Saad\n0.191\n19.14\n95% Table 3 Results of Pettit’s test to examine data homogeneity fi\nRazazza\nJurf Al-Sakhar\nLatifiyah\nSuwairah\nFalluja\nAbu Ghraib\nBaghdad\nNahrawan\nTharthar\nTaji\nRashidiyah\nKhan Saad\nRazazza\n1.00\n0.97\n0.87\n0.78\n0.97\n0.97\n0.91\n0.81\n0.89\n0.91\n0.86\n0.78\nJurf Al-Sakhar\n1.00\n0.96\n0.87\n0.94\n0.97\n0.95\n0.87\n0.88\n0.91\n0.88\n0.82\nLatifiyah\n1.00\n0.96\n0.85\n0.91\n0.96\n0.93\n0.83\n0.87\n0.87\n0.85\nSuwairah\n1.00\n0.78\n0.83\n0.92\n0.96\n0.78\n0.81\n0.85\n0.86\nFalluja\n1.00\n0.98\n0.92\n0.82\n0.95\n0.96\n0.90\n0.81\nAbu Ghraib\n1.00\n0.97\n0.88\n0.93\n0.97\n0.93\n0.86\nBaghdad\n1.00\n0.96\n0.89\n0.95\n0.96\n0.93\nNahrawan\n1.00\n0.83\n0.88\n0.94\n0.96\nTharthar\n1.00\n0.98\n0.91\n0.85\nTaji\n1.00\n0.97\n0.90\nRashidiyah\n1.00\n0.97\nKhan Saad\n1.00 IDW, and CCW in the same formula. The formula of NRIDC \nmethod is as follows: IDW, and CCW in the same formula. The formula of NRIDC \nmethod is as follows: (11)\nY =\n\u001fn\ni=1\n⎡\n⎢\n⎢\n⎢⎣\n\u001b\nrp\ni . Ns\nNi .d−2\ni\n\u001a\n∑n\ni=1\n\u001b\nrp\ni . Ns\nNi .d−2\ni\n\u001a\n⎤\n⎥\n⎥\n⎥⎦\nXi (11) where the power of the correlation coefficient P should be \nmore than 4. Metrics of performance UK method (UK); l. expecta-\nion maximization (EM); m. closet station method (CSM); n. modified correlation coefficient \nwith inverse distance weight-\nng (MCCIDW); o. modified \nold normal ration with inverse \ndistance (ONRID); p. normal \nation inverse distance weight-\nng with correlation (NRIDC); \nq. modified normal ration \nbased on square root distance \nMNR-T)\n(a)\n(b)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nAA Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNR Method\n(c)\n(d)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nGC Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNRGC Method (a)\n(b)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nAA Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNR Method\n(c)\n(d)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nGC Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNRGC Method (a)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nAA Method Fig. 3 Time series comparison \nof monthly precipitation predic-\ntion with measured values for \nthe years 1988 to 1995), meth-\nods abbreviation as following: \na. Arithmetic average (AA); \nb. normal ration (NR); c. geo-\ngraphical coordinates (GC); d. normal ration with geographical \ncoordinates (NRGC); e. inverse \ndistance weighted (IDW); f. correlation coefficient weighted \n(CCW); g. linear regression \n(LR); h. multiple linear regres-\nsion (MLR); i. multiple imputa-\ntion (MI); j. nonlinear iterative \npartial least square (NIPALS); \nk. UK method (UK); l. expecta-\ntion maximization (EM); m. closet station method (CSM); n. modified correlation coefficient \nwith inverse distance weight-\ning (MCCIDW); o. modified \nold normal ration with inverse \ndistance (ONRID); p. normal \nration inverse distance weight-\ning with correlation (NRIDC); \nq. Metrics of performance In order to evaluate the performance of each of 17 pro-\nposed methods in this study, several error measurements \nwere conducted to find the error between the predicted 1 3 Page 7 of 17 1740 Arab J Geosci (2022) 15:1740 Fig. 2 p values of the tested \nstations\n0\n0.1\n0.2\n0.3\n0.4\n0.5\n0.6\n0.7\n0.8\n0.9\n1\nRazazza\nJurf Al-Sakhar\nLatifiyah\nSuwairah\nFalluja\nAbu Ghraib\nBaghdad\nNahrawan\nTharthar\nTaji\nRashidiyah\nKhan Saad\np-values\nVariable\np-values(Pettitt) Fig. 2 p values of the tested \nstations p-values(Pettitt) is very common in meteorological application, and it is \nvery similar to (MAE); coefficient of efficiency (CE), \noutput values of CE range from − 1 to + 1, the value of \n1.0 shows a perfect estimation, while on the contrary, as \napproaching − 1, means not a good estimation (Kashani and observed values. This study uses six methods: mean \nabsolute error (MAE) which is one of the measures of \nerror, the output varies from 0 to ∞, less values mean \nbetter results (Azman et al. 2015)(C. Willmott et al. 2009); root mean square error (RMSE): this measure Method\nMAE\nRMSE\nCE\nS-index\nSS\nR pearson\nAA\n1.189\n2.338\n0.974\n0.993\n0.974\n0.987\nNR\n1.170\n2.408\n0.972\n0.993\n0.972\n0.986\nGC\n1.188\n2.343\n0.974\n0.993\n0.974\n0.987\nNRGC\n1.176\n2.419\n0.972\n0.993\n0.972\n0.986\nIDW\n1.095\n2.079\n0.979\n0.995\n0.979\n0.991\nCSM\n1.760\n3.736\n0.933\n0.983\n0.933\n0.968\nCCW\n1.178\n2.313\n0.974\n0.994\n0.974\n0.988\nLR\n2.061\n3.646\n0.936\n0.983\n0.936\n0.968\nMLR\n0.245\n0.455\n0.999\n1.000\n0.999\n1.000\nUK\n1.742\n3.735\n0.933\n0.983\n0.933\n0.968\nMCCIDW\n1.088\n2.062\n0.980\n0.995\n0.980\n0.991\nONRID\n1.353\n2.429\n0.972\n0.993\n0.972\n0.990\nNRIDC\n1.347\n2.417\n0.972\n0.993\n0.972\n0.991\nMNR-T\n1.287\n2.665\n0.966\n0.991\n0.966\n0.983\nMI\n0.328\n0.959\n0.980\n0.995\n0.980\n0.995\nNIPALS\n0.344\n1.234\n0.967\n0.992\n0.967\n0.984\nEM\n0.061\n0.209\n0.999\n1.000\n0.999\n1.000 1740 Page 8 of 17 Arab J Geosci (2022) 15:1740 Fig. 3 Time series comparison \nof monthly precipitation predic-\nion with measured values for \nhe years 1988 to 1995), meth-\nods abbreviation as following: \na. Arithmetic average (AA); \nb. normal ration (NR); c. geo-\ngraphical coordinates (GC); d. normal ration with geographical \ncoordinates (NRGC); e. inverse \ndistance weighted (IDW); f. correlation coefficient weighted \nCCW); g. linear regression \nLR); h. multiple linear regres-\nion (MLR); i. multiple imputa-\nion (MI); j. nonlinear iterative \npartial least square (NIPALS); \nk. Metrics of performance modified normal ration \nbased on square root distance \n(MNR-T) (b)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNR Method (c)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nGC Method (d)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNRGC Method 1 3 Page 9 of 17 1740 Arab J Geosci (2022) 15:1740 (e)\n(f)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nIDW Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nCCW Method\n(h)\n(g)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nLR Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMLR Method\nFig. 3 (continued) (e)\n(f)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nIDW Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nCCW Method\n(h)\n(g)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nLR Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMLR Method Fig. 3 (continued) Fig. Metrics of performance 3 (continued) (e)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nIDW Method 1 3 \n(e)\n(f)\n0\n10\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nYEAR\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nCCW Method\n(h)\n(g)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nLR Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMLR Method (f)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nCCW Method (g)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nLR Method (h)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMLR Method 3 Arab J Geosci (2022) 15:1740 1740 Page 10 of 17 (i)\n(j)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMI Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNIPALS Method\n(k)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nUK Method\n(l)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nEM Method\nFig. 3 (continued) (i)\n(j)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMI Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNIPALS Method Fig. Metrics of performance 3 (continued) (i)\n(j)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMI Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNIPALS Method\n(k)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nUK Method\n(l)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nEM Method (i)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMI Method (j)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNIPALS Method (k)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nUK Method (l)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nEM Method (l) 1 3 Page 11 of 17 1740 Arab J Geosci (2022) 15:1740 (m)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nCSM Method\n(n)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMCCIDW Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nONRID Method\n(o)\n(p)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNRIDC Method\nFig. 3 (continued) (m)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nCSM Method\n(n)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMCCIDW Method\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nONRID Method\n(o)\n(p)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNRIDC Method Fig. Examining of raw data To determine how far the studied stations are statistically \ncorrelated, Table 2 below shows the rPearson coefficient \nbetween stations. The targeted station in this study is Bagh-\ndad station, mostly the correlation is above 0.9, and 6 sta-\ntions correlation are above 0.95. Abu Ghraib station has \nthe largest value; further it is the closest station to Baghdad \nstation in terms of distance, while Tharthar station has the \nlowest value of correlation, which is 0.89. Generally, rPear-\nson coefficients between stations are above 0.8, except 3 \ncases, which are Razazza and Suwairah, Razazza and Khan \nSaad, and Tharthar and Suwairah. All of the last mentioned \ncases have the same value of 0.78. The largest correlation \nvalue in the table is 0.98 between Abu Ghraib and Falluja \nstations. It can be concluded that the distance has the larg-\nest effect on the value of the correlation factor, where the \nlater varies inversely with distance, keeping in mind that \nstation elevations within Baghdad have variance. The above mentioned metrics formulas are shown below \nin Eqs. (13) to (17): (13)\nMAE = 1\nn\n∑n\ni=1\|\|yi −xi\|\| (13) (14)\nRMSE =\n√\n∑n\ni=1\n(yi −xi\n)2\nn (14) CE = 1 −\n∑n\ni=1\n\u001eyi −xi\n\u001d2\n∑n\ni=1\n\u001eyi −y\u001d2 (15)\nCE = 1 −\n∑n\ni=1\n\u001eyi −xi\n\u001d2\n∑n\ni=1\n\u001eyi −y\u001d2 (15) (16)\nS index = 1 −\n∑n\ni=1\n\u001eyi −xi\n\u001d2\n∑n\ni=1\n\u001e\u001c\u001cyi −y\u001c\u001c + \u001c\u001cxi −y\u001c\u001c\n\u001d2\n(17)\nSS = 1 −\n∑n\ni=1\n\u001exi −yi\n\u001d2\n∑n\ni=1\n\u001e\ny −yi\n\u001d2 (16)\nS index = 1 −\n∑n\ni=1\n\u001eyi −xi\n\u001d2\n∑n\ni=1\n\u001e\u001c\u001cyi −y\u001c\u001c + \u001c\u001cxi −y\u001c\u001c\n\u001d2 In order to examine the homogeneity of the data \n(monthly precipitation data), Pettit’s test was performed \nutilizing (XLSTAT) software: in this test, the null hypoth-\nesis H0: Data are homogeneous, and alternative hypothesis \nHa: There is a date at which there is a change in the data. When the computed p value is greater than the significance \nlevel alpha = 0.05, one cannot reject the null hypothesis \nH0. Table 3 shows the results of Pettit’s test; the highest \np value was observed at Abu Ghraib station with a value \nof 0.878, while the lowest p value was observed at Khan \nSaad station with value of 0.191. Examining of raw data For all stations, and since \np values are greater than 0.05, one cannot reject the null \nhypothesis; therefore, and according to Pettit’s test, data \nfor the examined stations are homogenous. Figure 2 also \nshows the diagram of p values for the tested stations. (16) (17) Metrics of performance 3 (continued) (m)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nCSM Method (n)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMCCIDW Method 0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nONRID Method\n(o) (p)\n0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nNRIDC Method (p) 1 3 1740 Page 12 of 17 Arab J Geosci (2022) 15:1740 0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMNR-T Method\n(q)\nFig. 3 (continued) Fig. 3 (continued) 0\n10\n20\n30\n40\n50\n60\n70\n1988\n1989\n1990\n1991\n1992\n1993\n1994\n1995\nRAINFALL (MM)\nYEAR\nMeasured\nMNR-T Method\n(q)\nFig. 3 (continued) therefore, the comparisons were performed between the \npredicted values and the measured values. therefore, the comparisons were performed between the \npredicted values and the measured values. and Dinpashoh, 2011); similarity index (S-index), the \nvalues of similarity index range from 0 to 1, where the \nvalue of 1 means perfect results (Willmott 1981); skill \nscore (SS), which is another index of efficiency, where \nthe output ranges from 0 to 1, the value of 1 is perfect \nresults, while as approaching 0, there is a drop in the effi-\nciency of matching (Carvalho et al. 2016); rPearson coef-\nficient which is very common in statistical application. Results and discussion First, raw data were tested to examine the homogeneity \nand correlation between the stations within the command \narea. The main goal of this paper is to examine differ-\nent methods of missing precipitation data estimation; 3 Page 13 of 17 1740 Arab J Geosci (2022) 15:1740 Arab J Geosci (2022) 15:1740\t\nComparisons between 17 methods\nAs stated in this paper, 17 missing data methods were \napplied; the target station was Baghdad station, which \nis about in the center of the study area. Some methods \nare simple like CSM and AA; others are depending on \nspatial characteristics, averages, and regression with \nother stations. In addition, some methods are combin-\ning the weighting factors of 2 or 3 characteristics in one \nmethod, such as ONRID method. Also, some methods, \nlike EM and MI, employ artificial intelligence, where it \nwas computed using advanced software. Results and discussion In this study, it \nwas assumed that all the data of Baghdad station were \nmissed, i.e., the monthly data from 1980 to 2014; the \n(a)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nAA Method\n(b) \n(c) \n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nNR Method\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nGC Method\n(d)\n(e) \n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nNRGC Method\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nIDW Method\n(f)\n(g) \n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nCCW Method\n0\n20\n40\n60\n80\n100\n120\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nLR Method\n(h)\n(i)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nMLR Method\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nMI Method\n(j)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nNIPALS Method\nFig. 4 Comparison of monthly precipitation prediction with meas-\nured values for the year 1982, methods abbreviation as following: \na. Arithmetic average (AA); b. normal ration (NR); c. geographical \ncoordinates (GC); d. normal ration with geographical coordinates \n(NRGC); e. inverse distance weighted (IDW); f. Results and discussion correlation coeffi-\ncient weighted (CCW); g. linear regression (LR); h. multiple linear \nregression (MLR); i. multiple imputation (MI); j. nonlinear iterative \npartial least square (NIPALS); k. UK method (UK); l. expectation \nmaximization (EM); m. closet station method (CSM); n. modified \ncorrelation coefficient with inverse distance weighting (MCCIDW); \no. modified old normal ration with inverse distance (ONRID); p. nor-\nmal ration inverse distance weighting with correlation (NRIDC); q. Results and discussion modified normal ration based on square root distance (MNR-T)\nPage 13 of 17 1740 (a)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nAA Method (b) \n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nNR Method\n(d)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nNRGC Method (a)\n(c) \n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nGC Method (c) (f)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nCCW Method (e) \n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nIDW Method (e) (g) \n0\n20\n40\n60\n80\n100\n120\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nLR Method (h)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nMLR Method (h) (g) (i)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nMI Method (j)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nNIPALS Method (i) Fig. 4 Comparison of monthly precipitation prediction with meas-\nured values for the year 1982, methods abbreviation as following: \na. Arithmetic average (AA); b. normal ration (NR); c. geographical \ncoordinates (GC); d. normal ration with geographical coordinates \n(NRGC); e. inverse distance weighted (IDW); f. correlation coeffi-\ncient weighted (CCW); g. Results and discussion linear regression (LR); h. multiple linear \nregression (MLR); i. multiple imputation (MI); j. nonlinear iterative partial least square (NIPALS); k. UK method (UK); l. expectation \nmaximization (EM); m. closet station method (CSM); n. modified \ncorrelation coefficient with inverse distance weighting (MCCIDW); \no. modified old normal ration with inverse distance (ONRID); p. nor-\nmal ration inverse distance weighting with correlation (NRIDC); q. modified normal ration based on square root distance (MNR-T) Comparisons between 17 methods 4 (continued) (k) \n0\n20\n40\n60\n80\n100\n120\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nUK Method (l)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nEM Method (l) (k) \n(m) \n0\n20\n40\n60\n80\n100\n120\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nCSM Method ( )\n(n)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nMCCIDW Method (n) (n)\n(p)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nNRIDC Method (m) \n(o)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nONRID Method (q)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nMNR-T Method Fig. 4 (continued) Fig. 4 (continued) calculations of error indexes are based on these results. The exception was with MI, NIPALS, and EM methods, \nwhere the calculation was made for the years 1988 to \n1995, as there is a need to have some existing values to \nperform these methods. no tangible difference in comparison with other methods \nthat depend on a single factor, which are NR, GC, IDW. On \nanother hand, the multiple regression method (MLR) has a \nbetter result than the single linear regression (LR). In Figs. 3, 4 and 5, the results were visualized for a \nselected year to show how each method is performing. Figure 3 shows the time series of monthly precipitation \nfor the years 1988 to 1995; it is easily noticed that all \nmethods have a good matching with the measured data \nin Baghdad station, even with peak values. Table 4 shows the results of performance metrics of the \napplied methods. Comparisons between 17 methods other stations. In addition, some methods are combin-\ning the weighting factors of 2 or 3 characteristics in one \nmethod, such as ONRID method. Also, some methods, \nlike EM and MI, employ artificial intelligence, where it \nwas computed using advanced software. In this study, it \nwas assumed that all the data of Baghdad station were \nmissed, i.e., the monthly data from 1980 to 2014; the As stated in this paper, 17 missing data methods were \napplied; the target station was Baghdad station, which \nis about in the center of the study area. Some methods \nare simple like CSM and AA; others are depending on \nspatial characteristics, averages, and regression with 1 3 Arab J Geosci (2022) 15:1740 1740 Page 14 of 17 (k) \n0\n20\n40\n60\n80\n100\n120\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nUK Method\n(l)\n(m) \n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nEM Method\n0\n20\n40\n60\n80\n100\n120\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nCSM Method\n(n)\n(o) \n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nMCCIDW Method\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nONRID Method\n(p)\n(q)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nNRIDC Method\n0\n10\n20\n30\n40\n50\n60\n70\n80\n90\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 1982 \nMeasured\nMNR-T Method\nFig. Comparisons between 17 methods In this table, EM is the best method in \nterms of CE, S-index, SS, and rPearson metrics, and it has \nthe lowest values of MAE and RMSE. MLR has the same \nperformance, but it has a bit higher MAE and RMSE. In general, all the methods have good estimation of \nmissing data in Baghdad station; all can be adopted with \nthe acceptable level of trust. The lowest values were \nfounded with CSM, LR, and UK methods, where all has \na regression factor of 0.968, and values of MAE and \nRMSE are more than 1.7 and 3.6, respectively. However, \nthese indices seem good. Figures 4 and 5 show comparison between the meas-\nured and the predicted values in the 17 methods for \nthe years 1982 and 200, respectively. The year 1982 \nwas selected as it is the wettest year during the study \nperiod, while the year 2000 is the driest year during \nthe study period. Again, good results were observed, \nexcept at some peaks with some methods in the dry year \n2000, but for the year 1982, graphs were showing good \nestimations. The methods of combined weighting factors, which \nare NRGC, MCCIDW, ONRID, NRIDC, MNR-T made 3 Arab J Geosci (2022) 15:1740 Page 15 of 17 1740 (\n)\nConclusions\nSeveral conclusions can be derived from this study. The \nmethods that yield the best result with the least error \nare EM, then MLR methods. Generally, all the 17 meth-\nbetween the methods that employ a single factor, such as \nlocation mean value, with that employing several com-\nbined factors, where this is limited with study area in the \nBaghdad zone. Comparisons between 17 methods Errors of the predictions increase as the \nvalues of precipitation in the area decrease, where this \n(a)\n(b)\n0\n2\n4\n6\n8\n10\n12\n14\n16\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nAA Method\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nNR Method\n(c) \n(d)\n0\n2\n4\n6\n8\n10\n12\n14\n16\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nGC Method\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nNRGC Method\n(e) \n(f)\n0\n2\n4\n6\n8\n10\n12\n14\n16\n18\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nIDW Method\n0\n2\n4\n6\n8\n10\n12\n14\n16\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nCCW Method\n(g) \n(h)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nLR Method\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nMLR Method\n(i)\n(j)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nMI Method\n0\n2\n4\n6\n8\n10\n12\n14\n16\n18\n20\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nNIPALS Method\nFig. 5 Comparison of monthly precipitation prediction with meas-\nured values for the year 2000, methods abbreviation as following: \na. Arithmetic average (AA); b. normal ration (NR); c. geographical \ncoordinates (GC); d. normal ration with geographical coordinates \n(NRGC); e. inverse distance weighted (IDW); f. correlation coeffi-\ncient weighted (CCW); g. linear regression (LR); h. multiple linear \nregression (MLR); i. multiple imputation (MI); j. Comparisons between 17 methods nonlinear iterative \npartial least square (NIPALS); k. UK method (UK); l. expectation \nmaximization (EM); m. closet station method (CSM); n. modified \ncorrelation coefficient with inverse distance weighting (MCCIDW); \no. modified old normal ration with inverse distance (ONRID); p. nor-\nmal ration inverse distance weighting with correlation (NRIDC); q. modified normal ration based on square root distance (MNR-T)\ng (a)\n0\n2\n4\n6\n8\n10\n12\n14\n16\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nAA Method (b)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nNR Method (c) \n0\n2\n4\n6\n8\n10\n12\n14\n16\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nGC Method (d)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nNRGC Method (d) (c) (e) \n0\n2\n4\n6\n8\n10\n12\n14\n16\n18\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nIDW Method (f)\n0\n2\n4\n6\n8\n10\n12\n14\n16\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nCCW Method (e) (f) (g) \n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nLR Method (h)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nMLR Method (g) (i)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nMI Method (j)\n0\n2\n4\n6\n8\n10\n12\n14\n16\n18\n20\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nNIPALS Method (i) (j) Fig. Comparisons between 17 methods 5 Comparison of monthly precipitation prediction with meas-\nured values for the year 2000, methods abbreviation as following: \na. Arithmetic average (AA); b. normal ration (NR); c. geographical \ncoordinates (GC); d. normal ration with geographical coordinates \n(NRGC); e. inverse distance weighted (IDW); f. correlation coeffi-\ncient weighted (CCW); g. linear regression (LR); h. multiple linear \nregression (MLR); i. multiple imputation (MI); j. nonlinear iterative partial least square (NIPALS); k. UK method (UK); l. expectation \nmaximization (EM); m. closet station method (CSM); n. modified \ncorrelation coefficient with inverse distance weighting (MCCIDW); \no. modified old normal ration with inverse distance (ONRID); p. nor-\nmal ration inverse distance weighting with correlation (NRIDC); q. modified normal ration based on square root distance (MNR-T) Conclusions 5 (continued) (k) \n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nUK Method (l)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nEM Method (n)\n0\n2\n4\n6\n8\n10\n12\n14\n16\n18\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nMCCIDW Method (m) \n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nCSM Method (n) (p)\n0\n5\n10\n15\n20\n25\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nNRIDC Method (o) \n0\n5\n10\n15\n20\n25\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nONRID Method (p) (q)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nMNR-T Method (q) Fig. 5 (continued) Fig. 5 (continued) MAH and NAA; writing—review and editing, MAH and NAA; visu-\nalization, MAH and NAA; all authors have read and agreed to the \npublished version of the manuscript. MAH and NAA; writing—review and editing, MAH and NAA; visu-\nalization, MAH and NAA; all authors have read and agreed to the \npublished version of the manuscript. to the nature of the Baghdad area, where the topogra-\nphy is flat. Also, differences were observed between the \nresults of the tested methods in other researchers, but \nwith more complicated terrain. Funding Open access funding provided by Lulea University of \nTechnology. These methods will be useful as Baghdad location \nconsidered within the drylands, where most of the previ-\nous tested methods showed comparatively less accuracy \nin the arid region in the middle and south of Iraq during \nthe dry years, as well as the observed overestimates dur-\ning the dry conditions. Also, it might be essential to con-\nsider to future data gathering, where it was expected that \nthe climate change and rainfall trend variations might \nbring other facts. Conclusions between the methods that employ a single factor, such as \nlocation mean value, with that employing several com-\nbined factors, where this is limited with study area in the \nBaghdad zone. Errors of the predictions increase as the \nvalues of precipitation in the area decrease, where this \nwas noticed in the results’ comparison of the dry year \n2000. In general, these good results might be attributed Several conclusions can be derived from this study. The \nmethods that yield the best result with the least error \nare EM, then MLR methods. Generally, all the 17 meth-\nods produce good predictions of the proposed missing \ndata. Also, there are no tangible significant differences 1 3 Arab J Geosci (2022) 15:1740 1740 Page 16 of 17 (k) \n(l)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nUK Method\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nEM Method\n(m) \n(n)\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nCSM Method\n0\n2\n4\n6\n8\n10\n12\n14\n16\n18\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nMCCIDW Method\n(o) \n(p)\n(q)\n0\n5\n10\n15\n20\n25\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nONRID Method\n0\n5\n10\n15\n20\n25\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nNRIDC Method\n0\n2\n4\n6\n8\n10\n12\n14\nJAN\nFEB\nMAR\nAPR\nMAY\nJUN\nJUL\nAUG\nSEP\nOCT\nNOV\nDEC\nRAINFALL (MM)\nMONTHS OF THE YEAR 2000 \nMeasured\nMNR-T Method\nFig. Author contributions Conceptualization, MAH and NAA; methodol-\nogy, MAH and NAA; software, MAH and NAA; validation, MAH and \nNAA; formal analysis, MAH and NAA; investigation, MAH and NAA; \ndata curation, MAH and NAA; writing—original draft preparation, References (IMOaS), Iraqi Meteorological Organization and Seismol-\nogy (2021) Unpublished Data of Meteorological Stations in \nIraq Linsley Jr RK, Kohler MA, Paulhus JL (1975) Hydrology for engi-\nneers. McGraw Hillf q\nAbdullah M, Al-Ansari N (2021) Irrigation projects in Iraq. J Earth \nSci Geotech Eng 11(2):35–160. https://doi.org/10.47260/\njesge/1123 Murad S, Jaff Y (2020) Comparable investigation for rainfall fore-\ncasting using different data mining approaches in Sulaymaniyah \ncity in Iraq. Int J Environ Sci Technol. https://doi.org/10.18488/\njournal.72.2020.41.11.18 Abdulrazzaq Z (2020) The feasibility of using TRMM satellite data for \nmissing terrestrial stations in Iraq for mapping the rainfall contour \nlines. Civ Eng Beyond Limits 1:15–19. https://doi.org/10.36937/\ncebel.2020.003.003 Paulhus JL, Kohler MA (1952) Interpolation of missing precipitation \nrecords. J Monthly Weather Review 80(8):129–133 Rubin DB (1988) An overview of multiple imputation. Proceedings \nof the survey research methods section of the American statisti-\ncal association. Citeseer, pp 79–84f Abdulrida MA, Al-Jumaily K (2016) Comparisons of monthly rainfall \ndata with satellite estimates of TRMM 3B42 over Iraq. Int J Sci \nRes Publications 6(1):494–501 Sattari M, Rezazadeh Joudi A (2016) Assessment of different meth-\nods for estimation of missing data in precipitation studies. Hydrol Res 48. https://doi.org/10.2166/nh.2016.364 Al-Salihi AM, Al-Lami AM, Mohammed AJ (2013) Prediction of \nmonthly rainfall for selected meteorological stations in Iraq using \nback propagation algorithms. J Environ Sci Technol 6(1):16–28. https://doi.org/10.3923/jest.2013.16.28 Schneider T (2001) Analysis of incomplete climate data: estima-\ntion of mean values and covariance matrices and imputation \nof missing values. J Clim 14:853–871. https://doi.org/10.1175/\n1520-0442(2001)014%3c0853:AOICDE%3e2.0.CO;2 Alozeer A (2020) Estimation of mean areal rainfall and missing data \nby using GIS in Nineveh, Northern Iraq. Iraqi Geological Jour-\nnal 53:93–103. https://doi.org/10.46717/igj.53.1E.7Ry-2020-07.\n07 Syed Jamaludin SS, Deni S, Jemain A (2008) Revised spatial weight-\ning methods for estimation of missing rainfall data. Asia-Pac J \nAtmos Sci 44:93–104 Anderson EA (1972) National weather service river forecast system \nforecast procedures. NOAA Tech Memo NWS HYDRO-14 Tang W, Kassim A, Abubakar S (1996) Comparative studies of \nvarious missing data treatment methods—Malaysian experi-\nence. Atmos Res 42:247–262. https://doi.org/10.1016/0169-\n8095(95)00067-4 Armanuos A, Al-Ansari N, Yaseen Z (2020) Cross assessment of \ntwenty-one different methods for missing precipitation data \nestimation. Atmosphere 11:1–35. https://doi.org/10.3390/\natmos11040389 Teegavarapu R (2009) Estimation of missing precipitation records \nintegrating surface interpolation techniques and spatio-tem-\nporal association rules. J Hydro 11.https://doi.org/10.2166/\nhydro.2009.009 Azman MA-Z, Zakaria R, Ahmad Radi NF (2015) Estimation of miss-\ning rainfall data in Pahang using modified spatial interpolation \nweighting methods. AIP Conference Proceedings 1643(2015):65–\n72. Declarations Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attri-\nbution 4.0 International License, which permits use, sharing, adapta-\ntion, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, \nprovide a link to the Creative Commons licence, and indicate if changes \nwere made. The images or other third party material in this article are \nincluded in the article's Creative Commons licence, unless indicated \notherwise in a credit line to the material. If material is not included in \nthe article's Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will Author contributions Conceptualization, MAH and NAA; methodol-\nogy, MAH and NAA; software, MAH and NAA; validation, MAH and \nNAA; formal analysis, MAH and NAA; investigation, MAH and NAA; \ndata curation, MAH and NAA; writing—original draft preparation, 1 3 Page 17 of 17 1740 Arab J Geosci (2022) 15:1740 1740 need to obtain permission directly from the copyright holder. To view a \ncopy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Kanda N, Negi H, Shekhar M, Rishi M (2017) Performance of various \ntechniques in estimating missing climatological data over snow-\nbound mountainous areas of Karakoram Himalaya. Meteorol Appl \n25. https://doi.org/10.1002/met.1699fi Kashani M, Dinpashoh Y (2011) Evaluation of efficiency of dif-\nferent estimation methods for missing climatological data. Stoch Environ Res Risk Assess 26. https://doi.org/10.1007/\ns00477-011-0536-y References https://doi.org/10.1063/1.4907426 Teegavarapu R, Chandramouli V (2005) Improved weighting meth-\nods, deterministic and stochastic data-driven models for estima-\ntion of missing precipitation records. J Hydrol 312:191–206. https://doi.org/10.1016/j.jhydrol.2005.02.015 Barbalho F, Silva G, Formiga K (2014) Average rainfall estimation: \nmethods performance comparison in the Brazilian semi-arid. J \nWater Resour Prot 06:97–103. https://doi.org/10.4236/jwarp.\n2014.62014i Willmott C, Matsuura K, Robeson S (2009) Ambiguities inherent in \nsums-of-squares-based error statistics. Atmos Environ 43:749–\n752. https://doi.org/10.1016/j.atmosenv.2008.10.005 Bárdossy A, Pegram G (2014) Infilling missing precipitation records—\na comparison of a new copula-based method with other tech-\nniques. J Hydrol 519:1162–1170. https://doi.org/10.1016/j.jhydr\nol.2014.08.025 Willmott CJ (1981) On the validation of models. J Physical Geography \n2(2):184–194 Wold HOA (1968) Nonlinear estimation by iterative least square \nprocedures Carvalho J, Nakai A, Monteiro JE (2016) Spatio-temporal modeling \nof data imputation for daily rainfall series in homogeneous zones. Revista Brasileira De Meteorologia 31:196–201. https://doi.org/\n10.1590/0102-778631220150025 Yozgatligil C, Aslan S, Iyigun C, Batmaz I (2012) Comparison of miss-\ning value imputation methods in time series: the case of Turkish \nmeteorological data. Theoret Appl Climatol 112. https://doi.org/\n10.1007/s00704-012-0723-x Dempster A, Laird N, Rubin D (1977) Maximum likelihood \nfrom incomplete data via the EMalgorithm. J Roy Stat Soc \n39(1):1–38 Frenken K (2009) Irrigation in the Middle East region in figures \nAQUASTAT Survey-2008. Water Reports. Food and Agricul-\nture Organization of the United Nations, Rome 1 1 3 3"
https://openalex.org/W4220947096	https://bmcgeriatr.biomedcentral.com/track/pdf/10.1186/s12877-022-02917-8	English	null	Variations in physical activity and sedentary behavior during and after hospitalization in acutely admitted older medical patients: a longitudinal study	BMC geriatrics	2,022	cc-by	9,259	Jawad et al. BMC Geriatrics (2022) 22:209 https://doi.org/10.1186/s12877-022-02917-8 Open Access Abstract Background: Inactivity is frequent among older patients during hospitalization. It is unknown how patients’ daily activity pattern (diurnal profile) vary between hospitalization and after discharge. This study aims to describe and compare the distribution of physical activity and sedentary behavior in acutely hospitalized older patients during hospitalization and after discharge. Methods: We included data on 80 patients (+65 years) admitted with acute medical illness from the STAND-Cph trial. Physical activity and sedentary behavior were measured as daily number of steps, uptime (walking/standing) and sedentary behavior (lying/sitting) with an activity monitor (activPAL3, PAL Technologies Ltd). The patients wore the monitor for three periods of one week: during hospitalization, after discharge, and four weeks after discharge. Results: The patients’ median age was 80 years [IQR: 75;88], 68% were female and the median De Morton Mobility Index (DEMMI) was 57 [IQR: 48;67]. The daily median uptime was 1.7 h [IQR: 1;2.8] during hospitalization, 4.0 h [IQR: 2.7;5.4] after discharge and 4.0 h [IQR: 2.8;5.8] four weeks after discharge. The daily median number of steps was 728 [IQR: 176;2089], 2207 [IQR: 1433;3148], and 2622 [IQR: 1714;3865], respectively, and median daily sedentary behavior was 21.4 h (IQR: 20.7;22.4), 19.5 h (IQR: 18.1;21.0) and 19.6 h (IQR: 18.0;20.8), respectively. During hospitalization, a small activity peak was observed between 9-11 AM without any notable variation after. At discharge and four weeks after discharge, a peak in physical activity was seen between 9-12 AM and at 5 PM. Conclusion: Older hospitalized patients spend most of their time being sedentary with their highest activity between 9-11 AM. Daily activity doubles after discharge with one extra peak in the afternoon. Daily routines might be disrupted, and older patients have the potential to be more physically active during hospitalization. Interventions that encourage physical activity during hospitalization are warranted. Keywords: Accelerometer, physical activity, sedentary, hospitalization, post-acute care, older adults © 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. Variations in physical activity and sedentary behavior during and after hospitalization in acutely admitted older medical patients: a longitudinal study ker Nawfal Jawad1,2,3, Janne Petersen1,4,5, Ove Andersen1,2,3 and Mette Merete Pedersen1,3 Introduction Lack of physical activity among patients aged 65 years or older is linked to several adverse health outcomes [1], such as chronic conditions, cardiovascular disease, dia- betes [2], dementia [3], poor quality of life [4], cancer [2], rehospitalization [5] and mortality [6–9]. Worldwide, the proportion of older adults over 65 years of age is growing Correspondence: Baker.jawad@regionh.dk 3 Department of Clinical Medicine, University of Copenhagen, Nørre Allé 20, DK‑2200 Copenhagen‑N, Denmark 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. Jawad et al. BMC Geriatrics (2022) 22:209 Page 2 of 10 rapidly [10]. Similarly, hospitalization rates are expected to continue to increase [11, 12]. For instance, in 2018, persons aged 65 years and older accounted for more than 40% of Denmark’s acute hospitalizations [13]. The increasing demand for healthcare in this segment of the population is a global phenomenon [12]. Consequently, attention is increasingly being given to factors contrib- uting to more effective interventions and care for older (65+) patients during illness [14–17]. However, hospitali- zation is reported as an independent risk factor for loss of the ability to walk [18], loss of independence [19, 20], and functional decline after discharge, potentially leading to a higher level of sedentary behavior [18, 20, 21]. Mul- tiple studies have assessed physical activity and sedentary behavior among older hospitalized patients [22] and have shown that older adults, including those who are able to walk independently [23–26], are inactive during the entire hospitalization period. The mean uptime (standing or walking) is reported to be 70 min. per day [22]. Also, a larger prospective study by Zisberg et al. 2007 [27] , assessing the effect of hospitalization-care processes in 330 hospitalized older adults age 70+, found clear dis- ruptions to the self-reported frequency, duration, and timing of patients’ basic daily routines during hospitali- zation compared to pre-hospitalization. Assessment of physical activity Physical activity in the form of hourly number of steps taken, time spent standing, walking, and sitting/lying (sedentary behavior) was assessed with an activity moni- tor (activPAL3™, PAL Technologies Ltd., Glasgow, UK). The patients were asked to wear the monitor at three time points: from the time of inclusion until discharge, for one week immediately after discharge, and for one week 4 weeks after discharge. An investigator attached the activPAL3™ to the patient´s right thigh halfway between the spina iliaca anterior superior and the patella. For the attachments and detachments after discharge, an investigator visited the patients in their own homes. The monitor was covered in Tegaderm™ transparent water- proof film (3 M, Maplewood, MN, USA) and attached to the patient by a PALstickie™ (dual-layer hydrogel adhe- sive pad). Hereafter, the activPAL3™ was covered by Leukomed® T transparent film (BNS medical, Hamburg, Germany) to enable the patients to wear the monitor while showering. The activPAL3™ was programmed to record continuously for 7 days at 20 Hz. Recording was started shortly before attachment to the patient and the start time and date were noted in a data log along with the time and date of attachment, non-wearing (reported by patient or clinical staff), and detachment of the moni- tor. After detachment, data were downloaded to a com- puter using the activPAL3™ Professional software version 7.2.32. We regarded a day to extend from 12:00 A.M. The increasing demand for healthcare in this segment of the population is a global phenomenon [12]. Consequently, attention is increasingly being given to factors contrib- uting to more effective interventions and care for older (65+) patients during illness [14–17]. However, hospitali- zation is reported as an independent risk factor for loss of the ability to walk [18], loss of independence [19, 20], and functional decline after discharge, potentially leading to a higher level of sedentary behavior [18, 20, 21]. Mul- tiple studies have assessed physical activity and sedentary behavior among older hospitalized patients [22] and have shown that older adults, including those who are able to walk independently [23–26], are inactive during the entire hospitalization period. The mean uptime (standing or walking) is reported to be 70 min. per day [22]. Also, a larger prospective study by Zisberg et al. 2007 [27] , assessing the effect of hospitalization-care processes in 330 hospitalized older adults age 70+, found clear dis- ruptions to the self-reported frequency, duration, and timing of patients’ basic daily routines during hospitali- zation compared to pre-hospitalization. Preservation of basic daily routines is highly important, and promotes functional status, quality of sleep, and wellbeing [28–35]. Interestingly, Monk et al, assessed the lifestyle regular- ity in 100 healthy subjects with a mean age 31, and 104 seniors with mean age 79 years, found that irregulari- ties in routines were linked to adverse outcomes such as depression, poor sleep quality and unhealthy aging [36, 37]. However, during hospitalization a hospital depart- ment’s routines may conflict with older patients’ routines. Therefore, an objective evaluation of the impact of hospi- talization on patients’ activity patterns is needed. To the best of our knowledge, there are no previous longitudi- nal studies investigating physical activity and sedentary behavior with repeated objective measurement, focus- ing on pattern variation during hospitalization and after discharge. Hence, the aim of this study was to assess and compare daily and hourly patterns in step count, time spent in uptime and sedentary behavior during hospi- talization and after discharge in a group of older patients (+65) using accelerometers. Preservation of basic daily routines is highly important, and promotes functional status, quality of sleep, and wellbeing [28–35]. Interestingly, Monk et al, assessed the lifestyle regular- ity in 100 healthy subjects with a mean age 31, and 104 seniors with mean age 79 years, found that irregulari- ties in routines were linked to adverse outcomes such as depression, poor sleep quality and unhealthy aging [36, 37]. However, during hospitalization a hospital depart- ment’s routines may conflict with older patients’ routines. Therefore, an objective evaluation of the impact of hospi- talization on patients’ activity patterns is needed. To the best of our knowledge, there are no previous longitudi- nal studies investigating physical activity and sedentary behavior with repeated objective measurement, focus- ing on pattern variation during hospitalization and after discharge. Hence, the aim of this study was to assess and compare daily and hourly patterns in step count, time spent in uptime and sedentary behavior during hospi- talization and after discharge in a group of older patients (+65) using accelerometers. Methods access [38]. Briefly, the primary aim of the STAND-Cph trial was to investigate the effect of supervised, progres- sive strength training and post-training protein supple- mentation during and after hospitalization on mobility in older patients (≥ 65 years) admitted with acute medi- cal illness. All included patients were home dwelling and were excluded on the following criteria: terminal illness; in treatment for diagnosed cancer; diagnosis of Chronic Obstructive Pulmonary Disease (COPD) and partici- pation in a COPD rehabilitation program; inability to speak or understand Danish; inability to cooperate in tests/exercises; transfer to the intensive care unit; isola- tion-room stay; expected hospitalization lasting < 24 h; or inability to stand [38]. The patients were randomized to either the control group or the intervention group. This study involved patients allocated to the control group, who received routine care during hospitalization and following discharge [38]. The STAND-cph trail has been approved by the Ethics Committee of the Capital Region of Denmark (H-2-2012-115) and by the Danish Data Protection Agency (2007-58-0015). All participants gave written informed consent before participation, and the study was conducted in line with the Declaration of Helsinki. rapidly [10]. Similarly, hospitalization rates are expected to continue to increase [11, 12]. For instance, in 2018, persons aged 65 years and older accounted for more than 40% of Denmark’s acute hospitalizations [13]. Design and patientsh This study is based on data from the randomized, con- trolled STAND-Cph trial, which recruited patients from September 2013 to September 2018 at Copenhagen Uni- versity Hospital Hvidovre in Denmark and in the patients’ own homes. A full trial protocol is available with open Jawad et al. BMC Geriatrics (2022) 22:209 Jawad et al. BMC Geriatrics (2022) 22:209 Page 3 of 10 Orientation-Memory-Concentration test (OMC) [54], and habitual physical activity by a four-level self-reported questionnaire [55, 56]. until 12:00 A.M. to optimize the number of full days with 24 h of measurement and to avoid half-day meas- urements, as patients were usually enrolled in the study in the morning. This was in accordance with a previous observational study prior to this randomized clinical trial [24]. To avoid the inclusion of distorted days in the anal- ysis, only patient-days with more than 20 h of measure- ments were included [38]. Also, in the analysis we only included the first 6 days of hospitalization because very few patients were hospitalized for more than 6 days. Assessment of patient characteristics After inclusion, baseline assessments were performed by an investigator. At discharge and four weeks after, the patients were reassessed in their own homes. The follow- ing descriptive variables were collected on admission: age, sex, weight (BMI), living status (marital status, type of residence, and living alone), co-morbidities, admission diagnose, history of smoking, use of ambulatory aids, use of municipal assistance and length of stay. The following were assessed at all assessments: Mobility by the New Mobility Score (NMS) (recall of mobility 2 weeks before admission and on the day of admission) [46] and the De Morton Mobility Index (DEMMI). The NMS is a self- report assessment of a person’s ability to perform indoor walking, outdoor walking and shopping and the level of assistance needed with a composite score of 0-9. The DEMMI is a valid and reliable mobility tool assessing bed mobility, chair mobility, static and dynamic balance, and walking [47–50]. The DEMMI is scored from 0 to 100 points with 100 points reflecting a high level of mobil- ity and a score below 62 is considered limited mobility [51]; Activities of Daily Living (ADL) by the Bartel Index 20, which is scored between 0 and 20 with 20 reflecting no disability in ADL [52]; habitual gait speed (m/s) on a 4-m course [53]; cognitive impairment by the Short Data management and analysish The collected data were double entered into Epidata Entry 3.1 by the first and last author and two assistants. Data from the activPAL3™ monitors were downloaded using activPAL™ Professional software version 7.2.32. The 15s Epoch files were used for analyses and were transferred to SAS Enterprise Guide 7.1 (SAS Institute Inc., Cary, NC, USA) and merged with data log information on wear time to assure that only wear time was included in the analyses. Data on time spent standing and walking were combined as time spent upright. From the 15s Epoch files both data on hourly and daily average were derived. Normally distributed data are presented as means with standard deviations and non-normally distributed data as medians with interquartile ranges. Categorical data are presented as frequencies with percentages. To determine changes in uptime (standing/walking), sedentary behav- ior (time spent lying/sitting) and steps, we used a mixed model using the SAS procedure PROC MIXED to calcu- late differences between hospitalization, discharge, and 4 weeks after discharge. When performing the analysis, we log transformed data for steps since these were only log-normally distributed. The chi-squared (χ2) test and the Student´s t test were used to determine differences between patients included in the analysis and those who dropped out with regards to sex, age and DEMMI-score. ActivPal3™ has been shown to be valid and reliable for measuring posture and purposeful walking in both young people and older adults [39, 40]. However, the moni- tor has limited reliability for measuring not purposeful walking and small steps movement. Validation of the first generation monitor ActivPAL reported less reliable data on walking speed of 0.45 m/s or lower [41, 42], which is likely the case for the ActivPal3™ as well since there is a good agreement between the first and second generation of monitors [43]. In a previous study from our hospital in 317 older medical patients, 46% walked at a speed below 0.67 m/s, and 34% at a speed below 0.56 m/s [44]. There- fore, as stated in our protocol paper [38], to account for possible inability of the monitor to distinguish between standing and walking at slow walking speed, time spent in walking and standing were combined into one cat- egory, uptime [45]. Baseline characteristics In the STAND-Cph trial, 80 patients were randomized to the control group and thereby included in this study. The characteristics of the patients are shown in (Table 1). The patients´ median age was 80.9 years (IQR: 75;88), 68% were women, 98% were living at home, and 68% were liv- ing alone. At baseline, the median habitual walking speed was 0.67 (IQR: 0.48;0.87), the median DEMMI score was 57 (IQR: 48;67), and the median Bartel-20 score was 19 (IQR: 18;20). Thirty-one percent used assistive devices for walking and 37.5 % received social support services from the municipality. Prior to hospitalization, 46% of the patients were active two hours or more of per day based on self-report. The median length of stay was 4 days (IQR: 2;6.5), the median prevalence of co-morbidities was 4 (IQR: 3;5), and 41% were admitted to the hospital with respiratory symptoms. A total of 28 patients were lost to follow-up between hospitalization and 4 weeks after discharge. The rea- sons for missing data were: withdrawal from the study because of tiredness (n=10), withdrawal of consent (n=7), Jawad et al. BMC Geriatrics (2022) 22:209 Page 4 of 10 Table 1 Baseline characteristics of patients. Results are expressed as median (interquartile range) for continuous variables and as number of participants (percentage) for categorical variables. 1: Body mass index (kg/m2). 2: De Morton Mobility Index. 3: Orientation-Memory-Concentration test. Baseline variables N Overall Demographic characteristics Age (median, IQR) 80 80.9 (75;88) Sex (female, %) 80 55 (68%) Length of stay (median, IQR) 80 4 (2;6.5) BMI1 (median, IQR) 80 26 (22.6;30.1) Comorbidities (median, IQR)) 80 4 (3;5) Living alone: (number, %) 80 54 (68%) Self-reported activity level prior to admission: 80 <2 hours (N, %) 43 (54%) 2-4 hours (N, %) 26 (33%) >4 hour (N, %) 11 (13%) New mobility score (median, IQR) Fourteen days prior to admission 80 7 (6;9) Admission 80 6 (4;9) DEMMI2 (median, IQR) 79 57 (48;67) BARTEL-20 (median, IQR) 79 19 (18;20) OMC 3 (median, IQR) 68 23 (18;26) Walking speed m/s (median, IQR) 80 0.67(0.48;0.87) Smoking: 80 64 (80%) Smoking (no. yes, %) 13 (16.25) Previous (no. yes, %) 51 (70.8%) Assistive device (no., %) 80 26 (31%) Walking stick 21 (26%) Crutches 5 (6%) Use of municipal help 80 30 (37.5%) Personal assistance (no. yes, %) 7 (8.8%) Food service (no. yes, %) 13 (16.3%) Cleaning (no. Hourly variation Th h l Results are expressed as median (interquartile range) for continuous variables and as number of participants (percentage) for categorical variables. 1: Body mass index (kg/m2). 2: De Morton Mobility Index. 3: Orientation-Memory-Concentration test. The hourly variations in steps and uptime and seden- tary behavior are presented in Figures 1,2 and 3, respec- tively. The figures show the activity distribution within eighteen hours of monitoring (nighttime not included in the figures). During hospitalization, no notable variation in steps, uptime and sedentary behavior per hour was seen after 9 AM apart from a small two-hour peak in steps and uptime (Figures 1 and 2) and decline in sedentary time (Figure 3) at 10 - 11 AM. After dis- charge and 4 weeks after discharge, the patients’ diur- nal profiles changed and the patients took more steps at all hours and spent more time in uptime than during hospitalization with most activity occurring between 9 and 12 AM and with an additional activity peak at 5-6 PM. During hospitalization, between 10–11 AM, when readmission with apoplexy (n=5), severe pain (n=3), can- cer (n=1), abdominal surgery (n=1), and loss of contact with the subject (n=1). There were no significant baseline differences in age, sex, Barthel-score or DEMMI-score between patients lost to follow-up and patients remaining in the study. Baseline characteristics yes, %) 10 (12.5%) Admission diagnosis 80 Pulmonary 33 (41%) Cardiovascular 19 (24%) Neurological 12 (15%) Other 16 (20%) Table 1 Baseline characteristics of patients. accelerometer during hospitalization, 49 after discharge, and 43 patients four weeks after discharge. The distribu- tion of collected activPAL3 data from the three assess- ment timepoints is summarized in Supplementary 1. y In total, 21.216 h of patient activity were recoded (5710 h during hospitalization, 8232 h after discharge and 7224 h four weeks after discharge). The median number of steps taken per day was 728 (IQR: 176;2089) during hospitalization, 2207 (IQR: 1433;3148) after discharge and 2622 (IQR:1714;3864) four weeks after discharge. The median time per day spent in uptime was 1.7h (IQR:1.0;2.8) during hospitalization, 4.0 h (IQR: 2.7;5.4) after discharge, and 4.0 h (IQR: 2.8;5.8) four weeks after discharge. For sedentary behavior the median time per day was 21.4 h (IQR: 20.7;22.4) during hospitalization, 19.5 h (IQR: 18.1;21.0) after discharge and 19.6 h (IQR: 18.0;20.8) four weeks after discharge. After discharge, the patients’ number of steps and uptime significantly increased and sedentary behavior significantly decreased compared to hospitalization (p<0.0001). the steps num- ber increased by 204 % [149;271%], the mean uptime increased with 1.87 h [1.5;2.24] and the mean seden- tary time decreased with 1.89 h [- 2.28; -1.50]. Overall, also a significant increase was seen between discharge and four weeks after discharge (p<0.01). The number of steps increased by 28 % [10 %; 50 %], uptime increased with 0.57 h [0.28;0.87], and sedentary time decreased with 0.48 h [-0.79; -0,67]. When looking at the patients’ DEMMI score, those with limited mobility (DEMMI ≤ 62 had a relatively higher increase in activity between hospitalization and discharge compared to those with non-limited mobility (DEMMI > 62) (242 % versus 172 %, Table 2). Also, an increase between discharge and 4 weeks after discharge was only seen in those with a DEMMI >62 (Table 2). Variation in steps, uptime and sedentary behavior measured during hospitalization, after discharge and after 4 weeks.i Changes between test times are expressed as means with 95% Confidence interval (CI). relative changes are expressed as percentage for steps and absolute changes for uptime and sedentary behavior; * p<0.0001 for the difference between baseline (hospitalization) and discharge;** p<0.01 for the difference between discharge (baseline) and four weeks after discharge. Table 2: Results for daily number of steps, time spent in uptime and sedentary behavior during hospitalization, after discharge and four weeks after discharge. The results for taken number of steps and time (h) spent in uptime/sedentary are expressed as medians with first and third quartile. Changes between test times are expressed as means with 95% Confidence interval (CI). relative changes are expressed as percentage for steps and absolute changes for uptime and sedentary behavior; * p<0.0001 for the difference between baseline (hospitalization) and discharge;** p<0.01 for the difference between discharge (baseline) and four weeks after discharge. Fig. 1 Number of steps taken per hour from 6 am to 11 pm. Boxplots illustrating lower quartile, median, upper quartile and extremes per hour during hospitalization (blue), after discharge (green) and four weeks after discharge (red). The lines (blue, red, green) connect the medians during the day Fig. 1 Number of steps taken per hour from 6 am to 11 pm. Boxplots illustrating lower quartile, median, upper quartile and extremes per hour during hospitalization (blue), after discharge (green) and four weeks after discharge (red). The lines (blue, red, green) connect the medians during the day the patients’ exhibited the least sedentary behavior, the median time in minutes per hour for sedentary behav- ior was 53.5 minutes (IQR: 44.3;60). After discharge and four weeks after discharge, between 10 – 11 AM, when the patients’ exhibited the least sedentary behav- ior the patients’ median time spent in sedentary behav- ior was 43 minutes (IQR: 28.2;55.1). Variation in steps, uptime and sedentary behavior measured during hospitalization, after discharge and after 4 weeks.i Sixty-five patients wore the accelerometer at one or more assessment timepoints. In total, 48 patients wore the Jawad et al. BMC Geriatrics (2022) 22:209 Page 5 of 10 Table 2 Number of steps, time spent in uptime and sedentary behavior during the three periods. Table 2: Results for daily number of steps, time spent in uptime and sedentary behavior during hospitalization, after discharge and four weeks after discharge. The results for taken number of steps and time (h) spent in uptime/sedentary are expressed as medians with first and third quartile. Changes between test times are expressed as means with 95% Confidence interval (CI). relative changes are expressed as percentage for steps and absolute changes for uptime and sedentary behavior; * p<0.0001 for the difference between baseline (hospitalization) and discharge;** p<0.01 for the difference between discharge (baseline) and four weeks after discharge. Test time: Patients (N) Steps (number per day) Uptime (hours per day) Sedentary behavior (hours per day) Hospitalization 48 728 (IQR: 176;2089) 1.7 h (IQR: 1.0;2.8) 21.4 h (IQR: 20.7;22.4) Discharge 49 2207 (IQR: 1433;3148) 4.0 h (IQR: 2.7;5.4) 19.5 h (IQR: 18.1;21.0) Four weeks after discharge 43 2622(IQR: 1714;3865) 4.0 h (IQR: 2.8;5.8) 19.6 h (IQR: 18.0;20.8) Change from hospitalization to discharge 204 % [CI: 149 ;271%] * 1.87 h [CI: 1.5;2.24] * -1.89 h [CI: - 2.28; -1.50] * DEMMI > 62 28 172 % [CI: 108; 257 %] * 1.02 h [CI: 0.83;1.22] * -1,23 h [CI: -1.80; -0.67] * DEMMI ≤ 62 37 242 % [CI: 157; 353 %] * 2.86 h [CI: 2-43;3.29] * -3.10 h [CI: -3.60; -2,58] * Change from discharge to four weeks after 28 % [CI: 10 %; 50 %] 0.57 h [CI: 0.28;0.87] -0.48 h [CI: -0.79; -0,67] DEMMI > 62 28 62 % [CI: 34; 96 %] 0.23 h [CI: 0.09;0.37] -0.69 h [CI: -1.11; -0.27] DEMMI ≤ 62 37 -2 % [CI: -24; 26 %] 0.28 h [CI: -0.08;0.65] -0.09 h [CI: -0.50; -0.33] ** Table 2 Number of steps, time spent in uptime and sedentary behavior during the three periods. Table 2: Results for daily number of steps, time spent in uptime and sedentary behavior during hospitalization, after discharge and four weeks after discharge. The results for taken number of steps and time (h) spent in uptime/sedentary are expressed as medians with first and third quartile. Discussion the patients’ exhibited the least sedentary behavior, the median time in minutes per hour for sedentary behav- ior was 53.5 minutes (IQR: 44.3;60). After discharge and four weeks after discharge, between 10 – 11 AM, when the patients’ exhibited the least sedentary behav- ior the patients’ median time spent in sedentary behav- ior was 43 minutes (IQR: 28.2;55.1). In this study, physical activity and the diurnal profile of activity during and up to one month after hospitaliza- tion was investigated with an accelerometer in a group of older patients hospitalized for acute illness. During hospitalization, the patients spent more time engaged in sedentary behavior and took fewer steps and were Jawad et al. BMC Geriatrics (2022) 22:209 Jawad et al. BMC Geriatrics Page 6 of 10 Fig. 2 Time spent standing or walking (uptime) in minutes per hour from 6 am to 11 pm. Boxplots illustrating lower quartile, median, upper quartile and extremes in minutes per hour during hospitalization (blue), after discharge (green) and four weeks after discharge (red). The lines connect the medians during the day Fig. 3 Time spent sitting or lying (sedentary behavior) in minutes per hour from 6 am to 11 pm. Boxplots with lower quartile, median, upper quartile and extremes in minutes per hour during hospitalization (blue), after discharge (green) and four weeks after discharge (red). The lines connect the medians during the day Fig. 2 Time spent standing or walking (uptime) in minutes per hour from 6 am to 11 pm. Boxplots illustrating lower quartile, median, upper quartile and extremes in minutes per hour during hospitalization (blue), after discharge (green) and four weeks after discharge (red). The lines connect the medians during the day Fig. 2 Time spent standing or walking (uptime) in minutes per hour from 6 am to 11 pm. Boxplots illustrating lower quartile, median, upper quartile and extremes in minutes per hour during hospitalization (blue), after discharge (green) and four weeks after discharge (red). The lines connect the medians during the day Fig. 3 Time spent sitting or lying (sedentary behavior) in minutes per hour from 6 am to 11 pm. Boxplots with lower quartile, median, upper quartile and extremes in minutes per hour during hospitalization (blue), after discharge (green) and four weeks after discharge (red). The lines connect the medians during the day Fig. 3 Fig. Diurnal profiles A second important finding was that during hospitaliza- tion only one minor peak in uptime occurred at 9 AM. This suggests that the patients only get out of the bed in the morning, maybe for the morning toileting or break- fast, and spent most time engaged in sedentary behav- ior for the rest of the day. However, right after discharge and four weeks later, two peaks in activity occurred at similar timepoints throughout the day. A study from Germany, by Mai et al. [61] analyzed the diurnal physi- cal activity profile in 149 non-hospitalized, chronically ill community-dwelling individuals older than 70 years. The participants were instructed to wear a pedometer on six consecutive days. Consistent with our study, they identi- fied two peaks. However, their timing for the peaks was advanced one-two hours compared with our results, one at 10-11 AM and a second at 3-4 PM. They found sex, age, morbidity, and season to have no moderating effects and that limited mobility was the only factor that significantly moderated the profile, reducing the number of peaks to one [61]. In our sample, the median DEMMI-score was 57 (reflecting limited mobility) on admission. Thus, lim- ited mobility can also be the reason for the lack of a sec- ond peak of activity in our group during hospitalization since the median DEMMI score in the included patients was 57 on admission, which reflect the patients are lim- ited in their mobility and have increased reliance on care and caregivers [51]. In contrast to our study, a recent study from Switzerland by Tasheva et al. [62], found three peaks of physical activity during the day: between 8-10 AM, at 12 PM, and at 6 PM. Tasheva et al. assessed the distribution of physical activity levels continuously dur- ing the hospital stay by a wrist accelerometer in 177 old (+65) patients hospitalized for acute medical illness. The authors proposed that older inpatients are primar- ily active during meals, as reflected in the three peak times. Although the patients in our study did receive three meals per day, this was not reflected in their peak times and may indicate that meals were consumed close to or in bed, and that the patients in the study by Tasheva et al. had the possibility to consume their meals in e.g. a dining room at the hospital. Diurnal profiles Moreover, we consider the above-mentioned barriers to be explanations for the lack of a second peak during hospitalization in our study. The diurnal profiles of the patients after discharge and Discussion 3 Time spent sitting or lying (sedentary behavior) in minutes per hour from 6 am to 11 pm. Boxplots with lower quartile, median, upper quartile and extremes in minutes per hour during hospitalization (blue), after discharge (green) and four weeks after discharge (red). The lines connect the medians during the day Jawad et al. BMC Geriatrics (2022) 22:209 Jawad et al. BMC Geriatrics (2022) 22:209 Jawad et al. BMC Geriatrics (2022) 22:209 Page 7 of 10 less physically active than after discharge. The included patients took less than 900 steps per day and were there- fore at risk of hospitalization-associated functional decline [16]. Also, during hospitalization the diurnal profiles for steps and uptime had no notable variation in activity after 9 AM. In contrast, we found a diurnal pro- file for steps and uptime after discharge and four weeks after discharge with most physical activity occurring between 9 AM-12 PM, and 5-6 PM and with the patients being more physically active throughout the day than during hospitalization. for mobility in older medical patients were the provision of excessive service and care by the department, a culture of bed rest, and lack of encouragement by health care professionals to motivate the patients to increase activity. Also, Stefansdottir et al. reported that the staff brought food, beverages, and clothes to patients, including those who were able to get out of bed and walk. Levels of activity During hospitalization, the patients spent a median of 21.4 h of the day engaged in sedentary behavior and spent a median of 1.7 hours upright. This is consistent with a review of studies in acute-care settings in which the daily time spent lying or sitting accounted for 89–99%, and the duration of uptime was 1–2 hours per day [22]. After dis- charge a doubling in uptime was seen. Our findings are consistent with a recent study by Kolk et al. [57], which aimed to measure the number of steps taken per day in hospital and up to one week after discharge in 188 old (+70) acutely hospitalized patients, with Fitbit Flex worn on the wrist. In contrast, their study did not measure the distribution of physical activity and sedentary through the day. However, comparable findings were seen. Kolk et al. showed a doubling in steps only one day after dis- charge compared with one day prior to discharge - from 945 steps (IQR: 367;1943) to 1750 (IQR:675;4114). Pre- sumably, the relatively high level of activity after dis- charge in both our study and the study by Kolk et al. indicate a recovery of activity the first week after dis- charge. This doubling in the duration of uptime, and the consequent decrease in sedentary time after discharge, suggests that sedentary behavior is a result of a culture of bed rest at the departments and that the patients could potentially be more physical active. This is well in line with recent studies from our department. In an ethno- graphic study with observations of daily practice in the departments [58], mobility of older medical patients was found to be dependent on the health professionals’ different cultural models, which ended up blurring the responsibility for ensuring patient mobility and ended up restricting patient mobility. Also, Pedersen et al. 2020 conducted a qualitative study that investigated facilita- tors and barriers for mobility during hospitalization, by semi-structed interviews with twelve physicians at two medical departments and Stefánsdóttir et al. explored 20 old (+65) medical patients’ experiences with mobility during hospitalization more generally, and with an inter- vention to increase in-hospital mobility. Pedersen et al. 2020 [59] and Stefánsdóttir et al. [60] found that barriers Jawad et al. BMC Geriatrics (2022) 22:209 Jawad et al. Levels of activity BMC Geriatrics (2022) 22:209 Page 8 of 10 and delegated to all health professionals as suggested in recent recommendations for physical activity [15]. four weeks after discharge show that the patients started physical activity one hour earlier and had higher levels of physical activity in the morning and afternoon hours. These findings are consistent with those of Zisberg et al. [28], who showed that the timing of getting dressed in the morning moved an hour and a half during hospitaliza- tion, and most basic activities were reduced in frequency and duration. Conclusionh This study showed that in older acutely admitted adults the diurnal activity profile during hospitalization was dis- tinct from the diurnal profile when the patients returned home. During hospitalization, the patients took fewer steps, spent less time standing and walking, and spent more of their time engaged in sedentary behavior. The first week after discharge, the patients doubled their time spent standing and walking and lowered their time spent in sedentary behavior, suggesting that sedentary behavior is a result of a culture of bed rest in the hospital. There- fore, general mobility regimes and motivation should be provided to all patients during acute hospitalization. Strengths and limitationsh This study’s major strength was the longitudinal meas- urement of physical activity during hospitalization, at discharge, and at four weeks after discharge in a hetero- geneous cohort of older adults hospitalized for acute illness. This study has some limitations. First, physi- cal activity was not recorded prior to hospitalization. An objective assessment of physical activity prior to hospitalization would give a more sufficient picture of the impact of hospitalization on physical activity. How- ever, this would require assessments of a broad range of older adults to ensure that some of those who are hos- pitalized were assessed prior to hospitalization. There- fore, self-report on pre-hospitalization activity is easier to collect. Secondly, we assessed the number of steps using the activPAL3™ activity monitor. The first genera- tion of activPAL had an uncertainty in detecting walking at speeds less than 0.45 m/s [40, 41] and not purposeful walking. It is uncertain if the activPAL3™ has the same issues, however a study has reported a similarity between the two generations of activPAL accelerometers [43]. At baseline, the patients in the current study had a median walking speed of 0.67m/s (IQR: 0.48;0.87), and it is there- fore likely that walking and steps were underestimated in the patients who had a gait speed below the first quartile. However, we considered that an underestimation would affect the absolute level of steps, but not affect the distri- bution of activity throughout the day, which is one of the reasons why we looked at uptime (walking and standing), we therefore believe that the diurnal physical activity profiles are a true reflection of the included older adults’ patterns of daily physical activity. Daily routines fi d Our findings indicate that older patients could poten- tially be more physically active during hospitalization and emphasize the need for interventions that encourage more physical activity during hospitalization. It could be in the form of simple routine activities, such as patients eating their breakfast out of bed in a common room and changing clothes by themselves. Another starting point for the effort to increase patients’ physical activity dur- ing hospitalization could be to encourage physical activ- ity around 10-12 AM, and 5-6 PM, which would be more consistent with their habitual behavior. It is known that older patients are vulnerable to disturbances in their routines [63]. Thus, suggested efforts should be made to re-establish routines among those at risk of loss of func- tional decline. Since our results showed a wide range in steps, uptime and sedentary behavior, a personal- ized intervention strategy would be a reasonable means of optimizing physical activity during hospitalization. Recently, guidelines on physical activity for admitted older patients have also highlighted the importance of integrating physical activity throughout daily care, with a focus on functionality and activities of daily living; and bearing in mind that it is important for patients and staff to share the responsibility of promoting physical activ- ity and minimizing sedentary behavior [15]. An alterna- tive method for providing clinical care to a segment of this group of older patients has recently been suggested in a systematic review [64], which found that hospital- at-home (HaH) treatment may be a clinically effective approach and suggested that this treatment method may result in less functional decline in patients than the tra- ditional ward-based treatment method. However, further research is needed, and the implementation of this alter- native method of treatment (HaH) would necessitate sig- nificant changes to the current practice as well as time, it can takes several years making structural changes in the healthcare systems. In the short term, a relevant indica- tor is needed to identify patients at a high risk of inac- tivity during their time in the hospital. In the long term, systematic changes in the hospital environment and care setting are needed where the responsibility to encour- age physical activity should be a shared responsibility Abbreviation IQR: interquartile range; BMI: body mass index; DEMMI: De Morton Mobility Index; MSE: Mini-Mental State Examination; NMS: New mobility score. Availability of data and materials The data supporting the conclusions of this article are included within the article. The datasets analysed in this study are not publicly available due to regulations set out by the European and Danish Data Protection Agency regarding data anonymization but are available from the corresponding author on reasonable request. 16. Agmon M, et al. Association Between 900 Steps a Day and Func- tional Decline in Older Hospitalized Patients. JAMA Intern Med. 2017;177(2):272–4. 17. Cortes OL, Delgado S, Esparza M. Systematic review and meta-analysis of experimental studies: In-hospital mobilization for patients admitted for medical treatment. J Adv Nurs. 2019;75(9):1823–37. Declarations 18. Mahoney JE, Sager MA, Jalaluddin M. New walking dependence associ- ated with hospitalization for acute medical illness: incidence and signifi- cance. J Gerontol A Biol Sci Med Sci. 1998;53(4):M307–12. Funding h d This study was funded by the Danish Ministry of Health (grant number 9170); Danish Regions/The Danish Health Confederation; the Lundbeck Foundation (UCSF) (grant numbers FP 07/2012, FP 48/2012 and FP 61/2013); the Research Foundation of Hvidovre Hospital; the Capital Region of Copenhagen; and The Danish Foundation for Research in Physiotherapy. The funding bodies have had no authority over study design, collection and interpretation of data, or the writing of the manuscript. 12. de Meijer C, et al. The effect of population aging on health expenditure growth: a critical review. Eur J Ageing. 2013;10(4):353–61. 12. de Meijer C, et al. The effect of population aging on health expenditure growth: a critical review. Eur J Ageing. 2013;10(4):353–61. 13. 2018, S.D., Statistics Denmark 2018, INDO1: admission by region, diagnosis,age and sex, viewed 13. December 2020, < https://www.stati stikbanken.dk/IND01. 2018. 13. 2018, S.D., Statistics Denmark 2018, INDO1: admission by region, diagnosis,age and sex, viewed 13. December 2020, < https://www.stati stikbanken.dk/IND01. 2018. 14. Krumholz HM. Post-hospital syndrome--an acquired, transient condition of generalized risk. N Engl J Med. 2013;368(2):100–2. 14. Krumholz HM. Post-hospital syndrome--an acquired, transient condition of generalized risk. N Engl J Med. 2013;368(2):100–2. 15. Baldwin CE, et al. Recommendations for older adults’ physical activity and sedentary behaviour during hospitalisation for an acute medical illness: an international Delphi study. Int J Behav Nutr Phys Act. 2020;17(1):69. Consent for publication 21. Zisberg A, et al. Hospital-associated functional decline: the role of hospitalization processes beyond individual risk factors. J Am Geriatr Soc. 2015;63(1):55–62. References 1. Musich S, et al. The Frequency and Health Benefits of Physical Activity for Older Adults. Popul Health Manag. 2017;20(3):199–207. 1. Musich S, et al. The Frequency and Health Benefits of Physical Activity for Older Adults. Popul Health Manag. 2017;20(3):199–207. The online version contains supplementary material available at https://doi. org/10.1186/s12877-022-02917-8. 2. World Health, O. Global action plan on physical activity 2018–2030: more active people for a healthier world. Geneva: World Health Organization; 2018. Acknowledgment 4. Camboim FENDF, et al. Benefits of physical activity in the third age for the quality of life. Journal of Nursing Ufpe. Online. 2017;11:2415–22. g We thank Thomas Bandholm for his contribution to the study design, the Emergency Department at Copenhagen University Hospital Amager and Hvidovre, Denmark, and the staff at Department of Clinical Research, Copen- hagen University Hospital Amager and Hvidovre, Copenhagen, Denmark for the work during data collection. 5. Fisher SR, et al. Inpatient Walking Activity to Predict Readmission in Older Adults. Archives of physical medicine and rehabilitation. 2016;97(9 Suppl):S226–31. 5. Fisher SR, et al. Inpatient Walking Activity to Predict Readmission in Older Adults. Archives of physical medicine and rehabilitation. 2016;97(9 Suppl):S226–31. Older Adults. Archives of physical medicine and rehabilitation. 2016;97(9 Suppl):S226–31. 6. Ostir GV, et al. Mobility activity and its value as a prognostic indicator of survival in hospitalized older adults. J Am Geriatr Soc. 2013;61(4):551–7. 6. Ostir GV, et al. Mobility activity and its value as a prognostic indicator of survival in hospitalized older adults. J Am Geriatr Soc. 2013;61(4):551–7. 7. Buchman AS, et al. Total daily physical activity and longevity in old age. Arch Intern Med. 2012;172(5):444–6. 7. Buchman AS, et al. Total daily physical activity and longevity in old age. Arch Intern Med. 2012;172(5):444–6. ESM 1. 3. Livingston G, et al. Dementia prevention, intervention, and care. Lancet. 2017;390(10113):2673–734. Authors’ contributions MMP and JP, designed the original study. MMP was the primary investiga- tor and project leader and responsible for patient recruitment and data management for the STAND-Cph trial. BNJ, MMP, OA and JP designed these secondary analyses. BNJ and MMP performed the data management. BNJ was responsible for analyzing the data in this study under supervision of MMP and JP. BNJ wrote the first manuscript draft. Hereafter, all authors BNJ, MMP, OA and JP revised the manuscript critically. All authors approved the final version to be submitted for publication. 8. Lee, I.M., et al., Association of Step Volume and Intensity With All-Cause Mortality in Older Women. JAMA Intern Med, 2019. 8. Lee, I.M., et al., Association of Step Volume and Intensity With All-Cause Mortality in Older Women. JAMA Intern Med, 2019. 9. Hupin D, et al. Even a low-dose of moderate-to-vigorous physical activity reduces mortality by 22% in adults aged ≥60 years: a systematic review and meta-analysis. Br J Sports Med. 2015;49(19):1262–7. 9. Hupin D, et al. Even a low-dose of moderate-to-vigorous physical activity reduces mortality by 22% in adults aged ≥60 years: a systematic review and meta-analysis. Br J Sports Med. 2015;49(19):1262–7. y p 10. United Nations, D.o.E.a.S.A., World Population Prospects: The 2015 Revi- sion, Key Findings and Advance Tables. 2015. 10. United Nations, D.o.E.a.S.A., World Population Prospects: The 2015 Revi- sion, Key Findings and Advance Tables. 2015. 11. Wu, X., C.-k. Law, and P.S. Yip, A Projection of Future Hospitalisation Needs in a Rapidly Ageing Society: A Hong Kong Experience. International Journal of Environmental Research and Public Health, 2019. 16(3). The authors declare that they have no competing interests. 22. Fazio S, et al. How much do hospitalized adults move? A systematic review and meta-analysis. Appl Nurs Res. 2020;51:151189. Ethics approval and consent to participate All participants gave written informed consent before participation and the study procedures were approved by the Ethics Committee of the Capital Region of Denmark (H-2-2012-115) and by the Danish Data Protection Agency (2007-58-0015). 19. Covinsky KE, et al. Loss of independence in activities of daily living in older adults hospitalized with medical illnesses: increased vulnerability with age. J Am Geriatr Soc. 2003;51(4):451–8. 20. Dharmarajan K, et al. Disability and Recovery After Hospitalization for Medical Illness Among Community-Living Older Persons: A Prospective Cohort Study. J Am Geriatr Soc. 2020;68(3):486–95. Abbreviation IQR: interquartile range; BMI: body mass index; DEMMI: De Morton Mobility Index; MSE: Mini-Mental State Examination; NMS: New mobility score. Page 9 of 10 Jawad et al. BMC Geriatrics (2022) 22:209 Jawad et al. BMC Geriatrics Author details 1 Department of Clinical Research, Copenhagen University Hospital Amager and Hvidovre, Copenhagen, Denmark. 2 The Emergency Department, Copen- hagen University Hospital, Amager and Hvidovre, Copenhagen, Denmark. 3 Department of Clinical Medicine, University of Copenhagen, Nørre Allé 20, DK‑2200 Copenhagen‑N, Denmark. 4 Center for Clinical Research and Pre- vention, Copenhagen University Hospital, Bispebjerg and Frederiksberg, Copenhagen, Denmark. 5 Section of Biostatistics, Department of Public Health, University of Copenhagen, Copenhagen, Denmark. 23. Brown CJ, et al. The underrecognized epidemic of low mobility during hospitalization of older adults. J Am Geriatr Soc. 2009;57(9):1660–5. 24. Pedersen MM, et al. Twenty-four-hour mobility during acute hos- pitalization in older medical patients. J Gerontol A Biol Sci Med Sci. 2013;68(3):331–7. 25. Villumsen M, et al. Very Low Levels of Physical Activity in Older Patients During Hospitalization at an Acute Geriatric Ward: A Prospective Cohort Study. J Aging Phys Act. 2015;23(4):542–9. y y 26. Fisher SR, et al. Ambulatory activity of older adults hospitalized with acute medical illness. Journal of the American Geriatrics Society. 2011;59(1):91–5. Received: 1 September 2021 Accepted: 4 March 2022 Received: 1 September 2021 Accepted: 4 March 2022 Page 10 of 10 Page 10 of 10 Jawad et al. BMC Geriatrics (2022) 22:209 52. Collin C, et al. The Barthel ADL Index: a reliability study. Int Disabil Stud. 1988;10(2):61–3. 27. Zisberg A, Gur-Yaish N. Older adults’ personal routine at time of hospitali- zation. Geriatr Nurs. 2017;38(1):27–32. 53. Guralnik JM, et al. Lower extremity function and subsequent disability: consistency across studies, predictive models, and value of gait speed alone compared with the short physical performance battery. J Gerontol A Biol Sci Med Sci. 2000;55(4):M221–31. 28. O’Conor R, et al. Daily Routine: Associations With Health Status and Urgent Health Care Utilization Among Older Adults. Gerontologist. 2019;59(5):947–55. 29. Clark, F., et al., Occupational therapy for independent-living older adults. A randomized controlled trial. JAMA, 1997. 278(16): p. 1321-6. 54. Katzman R, et al. Validation of a short Orientation-Memory-Concentration Test of cognitive impairment. Am J Psychiatry. 1983;140(6):734–9. 30. Clark F, et al. Embedding health-promoting changes into the daily lives of independent-living older adults: long-term follow-up of occupational therapy intervention. J Gerontol B Psychol Sci Soc Sci. 2001;56(1):P60–3. 30. Clark F, et al. Embedding health promoting changes into the daily lives of independent-living older adults: long-term follow-up of occupational therapy intervention J Gerontol B Psychol Sci Soc Sci 2001;56(1):P60–3 55. Schnohr P, Scharling H, Jensen JS. Author details Changes in leisure-time physical activ- ity and risk of death: an observational study of 7,000 men and women. Am J Epidemiol. 2003;158(7):639–44. 31. Foldvari M, et al. Association of muscle power with functional status in community-dwelling elderly women. J Gerontol A Biol Sci Med Sci. 2000;55(4):M192–9. 56. Saltin B, Grimby G. Physiological analysis of middle-aged and old former athletes. Comparison with still active athletes of the same ages. Circula- tion. 1968;38(6):1104–15. 32. Resnick B, Galik E, Boltz M. Function focused care approaches: litera- ture review of progress and future possibilities. J Am Med Dir Assoc. 2013;14(5):313–8. 57. Kolk D, et al. Factors Associated with Step Numbers in Acutely Hospital- ized Older Adults: The Hospital-Activities of Daily Living Study. J Am Med Dir Assoc. 2021;22(2):425–32. 33. Resnick B, et al. Dissemination and Implementation of Function Focused Care for Assisted Living. Health Educ Behav. 2016;43(3):296–304. 58. Kirk JW, et al. Disentangling the complexity of mobility of older medical patients in routine practice: An ethnographic study in Denmark. PLoS One. 2019;14(4):e0214271. g 34. Zisberg A, Gur-Yaish N, Shochat T. Contribution of routine to sleep quality in community elderly. Sleep. 2010;33(4):509–14. 59. Pedersen MM, et al. Is Promotion of Mobility in Older Patients Hospital- ized for Medical Illness a Physician’s Job?-An Interview Study with Physi- cians in Denmark. Geriatrics (Basel, Switzerland). 2020;5(4):74. 35. Ludwig FM. How routine facilitates wellbeing in older women. Occupa- tional Therapy International. 1997;4(3):215–30. 36. Monk TH, et al. Age-related differences in the lifestyle regularity of seniors experiencing bereavement, care-giving, insomnia, and advancement into old-old age. Chronobiol Int. 2006;23(4):831–41. 60. Stefánsdóttir N, et al. Older medical patients’ experiences with mobility during hospitalization and the WALK-Copenhagen (WALK-Cph) interven- tion: A qualitative study in Denmark. Geriatr Nurs. 2020;42(1):46–56. g 37. Monk TH, et al. The relationship between lifestyle regularity and subjec- tive sleep quality. Chronobiol Int. 2003;20(1):97–107. 61. Mai A, et al. Diurnal profiles of pedometer-determined physical activity in chronically ill and mobility-limited older adults: a cross-sectional study. BMC Public Health. 2014;14:1268. 38. Pedersen MM, et al. Supervised progressive cross-continuum strength training compared with usual care in older medical patients: study protocol for a randomized controlled trial (the STAND-Cph trial). Trials. 2016;17:176. 62. Tasheva P, et al. Accelerometry assessed physical activity of older adults hospitalized with acute medical illness - an observational study. BMC Geriatr. 2020;20(1):382. 39. Bourke A, Ihlen E, Helbostad J. Publisher’s Note 42. Kanoun N. Validation of the ActivPAL Activity Monitor as a Measure of Walking at Pre-determined Slow Walking Speeds in a Healthy Population in a Controlled Setting’. Reinvention. Journal of Undergraduate Research. 2009;2(2). Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 43. Sellers C, et al. Agreement of the activPAL3 and activPAL for characteris- ing posture and stepping in adults and children. Gait & Posture. 2016;48. 44. Bodilsen AC, et al. Prediction of Mobility Limitations after Hospitalization in Older Medical Patients by Simple Measures of Physical Performance Obtained at Admission to the Emergency Department. PLoS One. 2016;11(5):e0154350. 45. Pedersen, M.M., et al., A randomized controlled trial of the effect of supervised progressive cross-continuum strength training and protein supplementation in older medical patients: the STAND-Cph trial. Trials, 2019. 20(1): p. 655-655. 46. Kristensen MT, Foss NB, Kehlet H. Timed Up and Go and New Mobility Score as predictors of function six months after hip fracture. Ugeskr Laeger. 2005;167(35):3297–300. g 47. de Morton NA, Davidson M, Keating JL. The de Morton Mobility Index (DEMMI): an essential health index for an ageing world. Health Qual Life Outcomes. 2008;6:63. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 48. Davenport SJ, de Morton NA. Clinimetric properties of the de Morton Mobility Index in healthy, community-dwelling older adults. Arch Phys Med Rehabil. 2011;92(1):51–8. 49. de Morton NA, Lane K. Author details Validation of the activPAL in Free-Living and Laboratory Scenarios for the Measurement of Physical Activity. Step- ping, and Transitions in Older Adults. 2019:1–8. 63. Stewart NH, Arora VM. Sleep in Hospitalized Older Adults. Sleep medicine clinics. 2018;13(1):127–35. 40. Sellers C, et al. Validity and reliability of the activPAL3 for measuring pos- ture and stepping in adults and young people. Gait & Posture. 2015;43. 64. Scott J, et al. A systematic review of the physical activity levels of acutely ill older adults in Hospital At Home settings: an under-researched field. Eur Geriatr Med. 2020:1–12. 41. Taraldsen K, et al. Evaluation of a body-worn sensor system to measure physical activity in older people with impaired function. Phys Ther. 2011;91(2):277–85. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Publisher’s Note Validity and reliability of the de Morton Mobility Index in the subacute hospital setting in a geriatric evaluation and man- agement population. J Rehabil Med. 2010;42(10):956–61. 50. de Morton NA, Davidson M, Keating JL. Validity, responsiveness and the minimal clinically important difference for the de Morton Mobility Index (DEMMI) in an older acute medical population. BMC Geriatr. 2010;10:72. 51. Macri EM, et al. The de morton mobility index: normative data for a clini- cally useful mobility instrument. J Aging Res. 2012;2012:353252.
https://openalex.org/W2053815405	https://escholarship.org/content/qt33r0r0sx/qt33r0r0sx.pdf?t=pd1nsi	English	null	Dynamics of Single-Species Population Growth: Stability or Chaos?	Ecology	1,981	cc-by	222	Peer reviewed Peer reviewed UC Irvine Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Dynamics of Single‐Species Population Growth: Stability or Chaos? Permalink https://escholarship.org/uc/item/33r0r0sx Journal Ecology, 62(5) ISSN 0012-9658 Authors Mueller, Laurence D Ayala, Francisco J Publication Date 1981-10-01 DOI 10.2307/1937278 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Dynamics of Single‐Species Population Growth: Stability or Chaos? Permalink https://escholarship.org/uc/item/33r0r0sx Journal Ecology, 62(5) ISSN 0012-9658 Authors Mueller, Laurence D Ayala, Francisco J Publication Date 1981-10-01 DOI 10.2307/1937278 Copyright Information This work is made available under the terms of a Creative Commons Attribu availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Dynamics of Single‐Species Population Growth: Stability or Chaos? Permalink https://escholarship.org/uc/item/33r0r0sx Journal Ecology, 62(5) ISSN 0012-9658 Authors Mueller, Laurence D Ayala, Francisco J Publication Date 1981-10-01 DOI 10.2307/1937278 Copyright Information This work is made available under the terms of a Creative Commons Att availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Powered by the California Digital Library University of California eScholarship.org
https://openalex.org/W2951266246	https://revistas.ulima.edu.pe/index.php/Ingenieria_industrial/article/download/3111/3021	es		Congratulaciones a la revista "Ingeniería Industrial"	Ingeniería industrial	1,993	cc-by	0
https://openalex.org/W4312057759	https://bg.copernicus.org/preprints/bg-2022-179/bg-2022-179.pdf	English	null	Comment on bg-2022-179	null	2,022	cc-by	19,150	Key drivers of the annual carbon budget of biocrusts from various 1 climatic zones determined with a mechanistic data-driven model 2 1 Key drivers of the annual carbon budget of biocrusts from various 1 climatic zones determined with a mechanistic data-driven model 2 Yunyao Ma1, Bettina Weber2,3, Alexandra Kratz3, José Raggio4, Claudia Colesie5, Maik Veste6,7, 3 Maaike Y.Bader8, and Philipp Porada1 4 1Institute of Plant Science and Microbiology, Universität Hamburg, 22609 Hamburg, Germany 5 2Institute of Plant Sciences, Department of Biology, University of Graz, 8010 Graz, Austria 6 3Department of Multiphase Chemistry, Max Planck Institute for Chemistry, 55128 Mainz, Germany 7 4Farmacología, Farmacognosia y Botánica Department, Complutense University of Madrid, 28040 Madrid, Spain 8 5School of Geosciences, University of Edinburgh, EH9 3FF Edinburgh, United Kingdom 9 6Institute of Environmental Sciences, Brandenburg University of Technology Cottbus-Senftenberg, 03046 Cottbus, Germany 10 7CEBra - Centre for Energy Technology Brandenburg e.V., 03042 Cottbus, Germany 11 8Faculty of Geography, University of Marburg, 35032 Marburg, Germany 12 13 Correspondence to: Yunyao Ma (yunyao.ma@uni-hamburg.de) 14 Abstract. Biocrusts are a worldwide phenomenon, contributing substantially to ecosystem functioning. Their growth and 15 survival depend on multiple environmental factors, including climatic conditions. While the physiological responses of 16 biocrusts to individual environmental factors have been examined in laboratory experiments, the relative importance of these 17 factors along climatic gradients is largely unknown. Moreover, it is not fully understood how acclimation of biocrusts may 18 alter the relative impacts of certain factors. We aim here at determining the relative effects of environmental factors on 19 biocrusts along climatic gradients, using the carbon balance of biocrust organisms as a measure of their performance. 20 Additionally, we explore the role that seasonal acclimation plays in the carbon balance of biocrusts. We applied a data- 21 driven mechanistic model at six study sites along a climatic gradient to simulate the annual carbon balance of biocrusts 22 dominated by different lichen and moss species. Furthermore, we performed several sensitivity analyses to investigate the 23 relative importance of driving factors, thereby including the impacts of acclimation. Our modelling approach suggests 24 substantial effects of light intensity and relative humidity in temperate regions, while air temperature has the strongest 25 impact at alpine sites. In drylands, ambient CO2 concentration and also the amount of rainfall are important drivers of the 26 carbon balance of biocrusts. Seasonal acclimation is a key feature, mostly in temperate regions, affecting biocrust 27 functioning. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 1 Introduction 33 Non-vascular photoautotrophs, such as lichens, mosses, eukaryotic algae and cyanobacteria, together with heterotrophic 34 microorganisms form biological soil crusts (biocrusts) (Belnap et al., 2016). Biocrusts occur in various environments across 35 the globe and are especially ubiquitous in environments where low water availability inhibits the development of vascular 36 plants (Belnap et al., 2004; Lange et al., 1998a; Samolov et al., 2020). They provide a wide range of important ecosystem 37 functions, such as build-up of soil organic carbon and nutrients (Chamizo et al., 2012; Dümig et al., 2014; Ferrenberg et al., 38 2018). Biocrusts contribute substantially to ecosystem carbon fluxes at the global scale (Elbert et al., 2012). Especially in 39 desert ecosystems, biocrusts can be a major contributor to the annual CO2 uptake (Tucker et al., 2019; Wohlfahrt et al., 2008). 40 Cyanobacteria, which are common components of biocrusts, either free-living or associated with mosses and lichens, 41 facilitate biotic nitrogen fixation and may subsequently increase carbon sequestration by enhancing the concentration of 42 photosynthesis-related enzymes in top soils (Ackermann et al., 2012; Evans and Lange, 2003). 43 Owing to their importance in ecosystem functioning, studies on growth and survival of biocrusts are crucial. Growth depends 44 on the long-term carbon balance (hereafter, C balance), which corresponds to the (accumulated) net carbon flux across the 45 system boundaries including all relevant carbon gains and losses. In order to ensure survival, any species needs to achieve a 46 positive C balance in the long-term, while negative values may occur for short periods. Acknowledging the importance of C 47 balance, an increasing number of studies have investigated the long-term C balance of individual non-vascular organisms as 48 well as biocrust communities, and their environmental drivers. An annual carbon budget based on measured field data was 49 reported in the study of Lange (2003b) on the crustose lichen Lecanora muralis growing on a rock surface in a temperate 50 climate of southern Germany. Furthermore, Büdel et al. (2018) measured the annual C balance for a cyanobacteria- 51 dominated biocrust in an Australian dry savannah ecosystem. Several other studies obtained long-term, large-scale values of 52 the C balance by scaling up short-term, local measurements of CO2 exchange rate under natural field conditions (Lange et al., 53 1994; Zotz et al., 2003). Key drivers of the annual carbon budget of biocrusts from various 1 climatic zones determined with a mechanistic data-driven model 2 We conclude that climate change, which may lead to warmer and, in some regions, drier air, will potentially 28 have large effects on long-term carbon balances of biocrusts at global scale. Moreover, we highlight the key role of seasonal 29 1 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. acclimation, which suggests that the season and timing of collecting and monitoring biocrusts should be given additional 30 consideration in experimental investigations, especially when measurements are used as the basis for quantitative estimates 31 and forecasts. 32 1 Introduction 33 For an estimation of the global C balance of cryptogamic covers, which include biocrusts, 54 conversion factors based on the maximum photosynthesis rate have been suggested as a best-guess solution (Elbert et al., 55 2012). However, there are some drawbacks to these approaches for acquiring a C balance at both organism and community 56 scale. First, the measurement of the long-term continuous CO2 exchange rate of an individual organism or biocrust 57 community has technical limitations and is highly time- and resource-consuming. Second, upscaling via extrapolation may 58 result in bias in annual C balance estimation if the length and the frequency of sampling cannot capture the full variability of 59 CO2 exchange throughout the year (Bader et al., 2010). Moreover, using empirical approaches alone, it is difficult to 60 2 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. understand the underlying mechanisms of how climatic conditions affect individual physiological processes, and 61 consequently which role these processes play in the observed changes in C balance and growth at the individual as well as 62 community level. Such approaches are thus subject to large uncertainty when used for projections of C balance under climate 63 change. 64 Most studies on the relationships between C balance and environmental factors for biocrusts are based on laboratory 65 experiments (e.g. Coe et al., 2012; Cowan et al., 1992; Lange et al., 1998a) or direct field measurements in situ over short 66 periods of time (e.g. Brostoff et al., 2005; Lange et al., 1994). From this work cited above, it has been recognized that the 67 type and temporal pattern of water supply, temperature, radiation, and also CO2 concentration are among the factors that are 68 crucial for the C balance of biocrusts. While highest values of productivity in biocrusts are achieved when the environmental 69 factors are in their optimal range, it has been found that biocrusts under field conditions are also able to achieve maximum 70 activity and thus, potential productivity, under sub-optimal conditions of temperature and light (Colesie et al., 2016; Raggio 71 et al., 2017, 2014). It is largely unknown, however, which relative importance each of these factors has for the long-term C 72 balance of biocrusts under natural field conditions, and if the importance of factors shows a spatial and temporal pattern. 1 Introduction 33 To complement 86 our analyses using the data-driven model, a process-based dynamic non-vascular vegetation model, called LiBry, was 87 employed as an supporting tool (Porada et al., 2013). 88 1 Introduction 33 In 73 addition, seasonal acclimation of photosynthetic and respiratory properties of species to intra-annually varying climate 74 factors found by several studies (e.g. Gauslaa et al., 2006; Lange and Green, 2005; Wagner et al., 2014) may substantially 75 affect biocrust C balance, thus leading to further complexity in the spatio-temporal relations between C balance and 76 environmental factors. One of the few experimental studies investigating biocrust acclimation potential to changing 77 temperatures has found threshold temperatures for the survival of lichen species (Colesie et al., 2018) but the overall extent 78 of the impact is poorly understood. 79 Here, we applied a mechanistic data-driven model to (a) complement empirical estimates of the annual C balance of 80 biocrusts and (b) to address the knowledge gaps concerning the relative importance of different environmental factors for the 81 C balance along climatic gradients, thereby accounting for the role of seasonal acclimation. The advantage of this modelling 82 approach is that it can predict at high temporal resolution the dynamic C balance of biocrust organisms for given locations by 83 simulating the physiological processes driven by environmental factors. The model allows for a deeper mechanistic 84 understanding of the C balance of biocrusts through factorial experiments and sensitivity analyses regarding parameters and 85 individual environmental factors, which would be impractical to realize in field or laboratory experiments. To complement 86 our analyses using the data-driven model, a process-based dynamic non-vascular vegetation model, called LiBry, was 87 employed as an supporting tool (Porada et al., 2013). 88 Here, we applied a mechanistic data-driven model to (a) complement empirical estimates of the annual C balance of 80 biocrusts and (b) to address the knowledge gaps concerning the relative importance of different environmental factors for the 81 C balance along climatic gradients, thereby accounting for the role of seasonal acclimation. The advantage of this modelling 82 approach is that it can predict at high temporal resolution the dynamic C balance of biocrust organisms for given locations by 83 simulating the physiological processes driven by environmental factors. The model allows for a deeper mechanistic 84 understanding of the C balance of biocrusts through factorial experiments and sensitivity analyses regarding parameters and 85 individual environmental factors, which would be impractical to realize in field or laboratory experiments. 2.1 Study sites 104 The mean 112 precipitation in these three sites is around 550 mm (Büdel et al., 2014; Diez et al., 2019). Site A1 is located in a humid alpine 113 region with a mean annual precipitation between 1750 and 2000 mm, of which more than 70% are snowfall; the A1 site is 114 covered by snow for at least 200 days per year (Büdel et al., 2014). More detailed site descriptions are provided in the 115 corresponding studies cited above. 116 2 Material and Methods 89 We simulated the C balance of biocrusts from six climatically different study sites in a semi-empirical way using a data- 90 driven model. The model simulates photosynthetic rate based on the Farquhar photosynthesis model developed by Farquhar 91 and von Caemmerer (1982) and respiration rate based on Q10 relationship. The C balance is computed as the difference of 92 3 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. photosynthesis and respiration accumulated over a given time period. In the model, both photosynthesis and respiration 93 depend on surface temperature, relative water saturation, and the activity of the biocrust, which are all simulated in a coupled 94 way via the surface energy balance as a function of climate input data. Photosynthesis additionally depends on ambient CO2 95 concentration. 96 To calibrate the model, we first determined soil and land surface properties that are required for the coupled energy and 97 water balance in the model through fitting simulated to measured surface temperature patterns. Then, we parameterized the 98 physiological properties of the organisms using measured relationships between net photosynthesis and light intensity, water 99 content, and temperature. Finally, we validated the model with regard to the water content or activity patterns of biocrusts 100 and compared the modelled CO2 assimilation rate to measured values. The data sets used for calibration and validation of the 101 model as well as basic climate conditions of each site are described in Table 1. Sites were listed in ascending order of total 102 annual precipitation based on measured data. 103 2.1 Study sites 104 In our study we considered six sites, namely two dryland sites at Almeria (Spain) and Soebatsfontein (South Africa; 105 hereafter D1 and D2); three temperate sites at Gössenheim (Germany), Öland (Sweden), and Linde (Germany; Hereafter T1, 106 T2, and T3); and one alpine site at Hochtor (Austria; Hereafter A1) (Table 1). These sites were chosen based on data 107 availability and because they cover a broad range of different climatic conditions. To our knowledge, the necessary empirical 108 data regarding climatic conditions, species physiological characteristics, and status such as activity, which is used to estimate 109 C balance, have been monitored in only a small number of experiments, including the six study sites chosen here. 110 In our study we considered six sites, namely two dryland sites at Almeria (Spain) and Soebatsfontein (South Africa; 105 hereafter D1 and D2); three temperate sites at Gössenheim (Germany), Öland (Sweden), and Linde (Germany; Hereafter T1, 106 T2, and T3); and one alpine site at Hochtor (Austria; Hereafter A1) (Table 1). These sites were chosen based on data 107 availability and because they cover a broad range of different climatic conditions. To our knowledge, the necessary empirical 108 data regarding climatic conditions, species physiological characteristics, and status such as activity, which is used to estimate 109 C balance, have been monitored in only a small number of experiments, including the six study sites chosen here. 110 Sites D1 and D2 are characterized by a semi-arid climate with mean annual precipitation of less than 250 mm, but a wet 111 winter season (Büdel et al., 2014; Haarmeyer et al., 2010). Sites T1, T2, and T3 have a temperate climate. The mean 112 precipitation in these three sites is around 550 mm (Büdel et al., 2014; Diez et al., 2019). Site A1 is located in a humid alpine 113 region with a mean annual precipitation between 1750 and 2000 mm, of which more than 70% are snowfall; the A1 site is 114 covered by snow for at least 200 days per year (Büdel et al., 2014). More detailed site descriptions are provided in the 115 corresponding studies cited above. 116 Sites D1 and D2 are characterized by a semi-arid climate with mean annual precipitation of less than 250 mm, but a wet 111 winter season (Büdel et al., 2014; Haarmeyer et al., 2010). Sites T1, T2, and T3 have a temperate climate. 2.2.2 Dynamic biocrust variables 126 Surface temperature data are available for all sites. Biocrust activity was either monitored directly (binary: active or not 127 active) using a continuous chlorophyll fluorescence monitoring system (Raggio et al., 2014, 2017), or indirectly via the 128 electrical conductivity of the substrate (BWP, Umweltanalytische Produkte GmbH, Cottbus, Germany; Weber et al., 2016; 129 M. Veste, unpublished data). For site D2, the biocrust water content was calculated instead of activity based on electrical 130 conductivity. Due to snow covering the measuring instruments, data of site A1 only covers the time from August to October. 131 Samples from both lichen- and also moss-dominated biocrusts were measured at all sites, except for site T3 where four 132 BWPs were mostly located in moss-dominated biocrusts. At site D2, additionally cyanolichen- and cyanobacteria-dominated 133 biocrusts were monitored. The measured surface temperature, water content, and activity data at all sites were then 134 aggregated to data with a temporal resolution of one hour. 135 We did not directly use the observed surface temperature and activity as forcing data for the model since these properties are 136 strongly linked to water saturation (and CO2 diffusivity). Input data of water saturation, however, were not available at most 137 sites. Although the overall patterns of simulated and observed surface temperature match well (see below, Sect. 2.3), 138 inconsistencies would likely occur at hourly resolution if simulated dynamic water content was used in the model together 139 with observed temperature and activity. Hence, the time-series of surface temperature and water saturation data at all sites 140 were estimated based on a simulation of the energy and water balance. The activity of the organisms was then approximated 141 via the empirical equations describing the link between water saturation and metabolic activity (see Porada et al., 2013). 142 Furthermore, ambient CO2 concentration was assumed to be constant at 400 ppm. 143 For validation of C balance, we used data of the on-site CO2-exchange rate of different biocrust types (lichen- and moss- and 144 also cyano-dominated biocrusts; the latter composed of cyanolichen and cyanobacteria) that were measured by a portable 145 gas exchange system at several time intervals from November 4th to 8th at site D2 (Tamm et al., 2018). For the other sites, 146 additional field measurements of CO2-exchange were not available. 147 2.2.1 Climatic variables 118 The proposed data-driven model for estimating the annual C balance of dominant biocrust types at each site was forced by 119 hourly microclimatic variables. The forcing data sets of the data-driven model include photosynthetically active radiation 120 (PAR), long-wave radiation (near-infrared), relative air humidity, air temperature, wind speed, rainfall, and snowfall. All the 121 microclimatic variables were measured on-site by climate stations with a temporal resolution of 10 min (5 min in A1 and D1; 122 data available at http://www.biota-africa.org; Raggio et al., 2017; M. Veste, unpublished data), except for long-wave 123 4 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. radiation and snowfall, which were taken from ERA5 dataset (https://www.ecmwf.int/en/forecasts/datasets/reanalysis- 124 datasets/era5). Then all these microclimate data were aggregated to data with hourly temporal resolution. 125 radiation and snowfall, which were taken from ERA5 dataset (https://www.ecmwf.int/en/forecasts/datasets/reanalysis- 124 datasets/era5). Then all these microclimate data were aggregated to data with hourly temporal resolution. 125 2.2.3 Photosynthesis response and water storage 148 5 For all sites, CO2 exchange measurements under controlled conditions in the laboratory or in the field (site T3) were 149 conducted using a mobile gas exchange system GFS 3000 (Walz GmbH, Effeltrich, Germany) with an infrared-gas analyzer 150 to explore the physiological characteristics of samples of different biocrust types (same as those measured for validation; 151 main species see Table 1; Diez et al., 2019; Raggio et al., 2018; Tamm et al., 2018). Net photosynthesis was measured at 152 different ranges of light intensity, water content, and temperature. Light response curves, for instance, were determined at 153 optimum water saturation and 15 °C, water response curves were measured at 400 µmol m-2 s-1 and 15 °C at sites D1, T1, T2, 154 and A1 (Raggio et al., 2018). Moreover, the maximum water storage capacity (MWC) of the samples was quantified in the 155 5 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. laboratory for samples from sites D1, T1, T2, and A1 (Raggio et al., 2018), whereas the MWC at site D2 was approximated 156 as the maximum value when measuring water response curves (Tamm et al., 2018; Weber et al., 2012). MWC at site T3 was 157 estimated as the value of the same genus measured in Hamburg, Germany (Cladonia portentosa and Polytrichum formosum, 158 Petersen et al., in prep.). MWC was acquired since it is one of the essential parameters of the model to convert the specific 159 water content in mm to relative water saturation required by the model used here. 160 6 , p p ) q p p water content in mm to relative water saturation required by the model used here. 2.2.3 Photosynthesis response and water storage 148 169 The set of parameters that corresponded to minimum differences between simulated and measured values (visual assessment) 170 was used in the data-driven model. The calibration results of surface temperature and the photosynthesis response curves at 171 site T2 are shown in Fig. 1 and Fig. 2, respectively. The results of dominant species at other sites are shown in Fig. S1 and 172 S2. 173 The daily surface temperature was simulated accurately (visual comparison) except for site T3 where the temperature during 174 cold seasons was underestimated, and at site D1 the peak temperature within a day in hot seasons was underestimated (Fig. 175 S1) The peak in surface temperature occurred too early by around 3 hours at site T1 and T2 but the magnitude of the peak 176 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. References Raggio et al., 2018 Tamm et al., 2018; Weber et al., 2012 Raggio et al., 2018 Raggio et al., 2018 Veste, unpublished data; Diez et al. 2019 Raggio et al., 2018 2.3 Parameterization of the data-driven model 163 2.3.1 Abiotic surface properties 164 Several abiotic parameters of the data-driven model describing soil and land surface properties, such as roughness length or 165 soil thermal conductivity, were required to simulate the energy and water balance. These parameters were obtained by fitting 166 the daily and diurnal surface temperature patterns of lichen-dominated biocrust at all sites except for site T3. At site T3, we 167 compared the surface temperature patterns of simulated moss-dominated biocrusts to data collected by sensors in four 168 locations. 169 The set of parameters that corresponded to minimum differences between simulated and measured values (visual assessment) 170 was used in the data-driven model. The calibration results of surface temperature and the photosynthesis response curves at 171 site T2 are shown in Fig. 1 and Fig. 2, respectively. The results of dominant species at other sites are shown in Fig. S1 and 172 S2. 173 The daily surface temperature was simulated accurately (visual comparison) except for site T3 where the temperature during 174 cold seasons was underestimated, and at site D1 the peak temperature within a day in hot seasons was underestimated (Fig. 175 S1). The peak in surface temperature occurred too early by around 3 hours at site T1 and T2, but the magnitude of the peak 176 corresponded well to the measured data (Fig. 2.2.3 Photosynthesis response and water storage 148 160 Table 1: Properties of the study sites and data which are available (+ sign) for calibration and validation of the data-driven 161 model 162 Site Almeria, Spain Soebatsfontein, South Africa Gössenheim, Bavaria, Germany Öland, Sweden Linde, Brandenburg, Germany Hochtor, Austria Code D1 D2 T1 T2 T3 A1 Climate semi-arid semi-arid Temperate Temperate Temperate Alpine Measured annual rainfall [mm] 110 141 424 441 449 744 Dominant species at the site Psora decipiens, Didymodon rigidulus Psora decipiens, Psora crenata, Ceratodon purpureus, Collema coccophorum Psora decipiens, Trichostomun crispulum Psora decipiens, Tortella tortuosa Cladonia furcata, Polytrichum piliferum Psora decipiens, Tortella rigens Data for Calibration Laboratory CO2 exchange response curves Light, water, temperature Light, water, temperature Light, water, temperature Light, water, temperature Light, temperature Light, water, temperature Surface temperature + + + + + + Data for Validation Water content - + - - - - Activity + - + + + + CO2 exchange on site - + - - - - Table 1: Properties of the study sites and data which are available (+ sign) for calibration and validation of the data-driven 161 model 162 udy sites and data which are available (+ sign) for calibration and validation of the data-driven 6 Data for Calibration Laboratory CO2 exchange response curves Light, water, temperature Light, water, temperature Light, water, temperature Light, water, temperature Light, temperature Light, water, temperature Surface temperature + + + + + + Data for Validation Water content - + - - - - Activity + - + + + + CO2 exchange on site - + - - - - References Raggio et al., 2018 Tamm et al., 2018; Weber et al., 2012 Raggio et al., 2018 Raggio et al., 2018 Veste, unpublished data; Diez et al. 2019 Raggio et al., 2018 2.3 Parameterization of the data-driven model 163 2.3.1 Abiotic surface properties 164 Several abiotic parameters of the data-driven model describing soil and land surface properties, such as roughness length or 165 soil thermal conductivity, were required to simulate the energy and water balance. These parameters were obtained by fitting 166 the daily and diurnal surface temperature patterns of lichen-dominated biocrust at all sites except for site T3. At site T3, we 167 compared the surface temperature patterns of simulated moss-dominated biocrusts to data collected by sensors in four 168 locations. 2.2.3 Photosynthesis response and water storage 148 1 and S1). Therefore, in general, the fitting of the surface temperature patterns 177 was satisfactory. 178 Several abiotic parameters of the data-driven model describing soil and land surface properties, such as roughness length or 65 soil thermal conductivity, were required to simulate the energy and water balance. These parameters were obtained by fitting 66 the daily and diurnal surface temperature patterns of lichen-dominated biocrust at all sites except for site T3. At site T3, we 67 compared the surface temperature patterns of simulated moss-dominated biocrusts to data collected by sensors in four 68 locations. 69 The set of parameters that corresponded to minimum differences between simulated and measured values (visual assessment) 170 was used in the data-driven model. The calibration results of surface temperature and the photosynthesis response curves at 171 site T2 are shown in Fig. 1 and Fig. 2, respectively. The results of dominant species at other sites are shown in Fig. S1 and 172 S2. 173 The daily surface temperature was simulated accurately (visual comparison) except for site T3 where the temperature during 174 cold seasons was underestimated, and at site D1 the peak temperature within a day in hot seasons was underestimated (Fig. 175 S1). The peak in surface temperature occurred too early by around 3 hours at site T1 and T2, but the magnitude of the peak 176 corresponded well to the measured data (Fig. 1 and S1). Therefore, in general, the fitting of the surface temperature patterns 177 was satisfactory. 178 The daily surface temperature was simulated accurately (visual comparison) except for site T3 where the temperature during 174 cold seasons was underestimated, and at site D1 the peak temperature within a day in hot seasons was underestimated (Fig. 175 S1). The peak in surface temperature occurred too early by around 3 hours at site T1 and T2, but the magnitude of the peak 176 corresponded well to the measured data (Fig. 1 and S1). Therefore, in general, the fitting of the surface temperature patterns 177 was satisfactory. 178 7 7 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 179 Figure 1: Calibration results of abiotic parameters of the data-driven model by fitting the daily (left panel) and diurnal (right 180 panel) patterns of surface temperature at site T2. 2.2.3 Photosynthesis response and water storage 148 Right: (a) to (d) represent the patterns of average hourly surface 181 temperature from January to March (JFM), April to June (AMJ), July to September (JAS), and October to December (OND), 182 respectively. 183 179 Figure 1: Calibration results of abiotic parameters of the data-driven model by fitting the daily (left panel) and diurnal (right 180 panel) patterns of surface temperature at site T2. Right: (a) to (d) represent the patterns of average hourly surface 181 temperature from January to March (JFM), April to June (AMJ), July to September (JAS), and October to December (OND), 182 respectively. 183 Figure 1: Calibration results of abiotic parameters of the data-driven model by fitting the daily (left panel) and diurnal (right 180 panel) patterns of surface temperature at site T2. Right: (a) to (d) represent the patterns of average hourly surface 181 temperature from January to March (JFM), April to June (AMJ), July to September (JAS), and October to December (OND), 182 respectively. 183 2.3.2 Biocrust physiological properties 184 198 lichen-dominated biocrust samples to measurements at site T2. (a): net photosynthesis rate in response to light at optimum 196 water content and 15 °C. (b): net photosynthesis rate in response to temperature at 1200 µmol m-2 s-1 light and optimal water 197 content. (c): net photosynthesis rate in response to relative water saturation at 400 µmol m-2 s-1 light and 15 °C. 198 lichen-dominated biocrust samples to measurements at site T2. (a): net photosynthesis rate in response to light at optimum 196 water content and 15 °C. (b): net photosynthesis rate in response to temperature at 1200 µmol m-2 s-1 light and optimal water 197 content. (c): net photosynthesis rate in response to relative water saturation at 400 µmol m-2 s-1 light and 15 °C. 198 Since the measured values between replicates showed large variation, in particular the water and temperature-dependent data, 199 as illustrated by the coloured points in Fig. 2, we fitted the curves to the average values of all replicates. The calibration 200 results showed that visually and overall, the photosynthetic curves could be parameterized to fit the measurements well, 201 given that different samples were used for measuring responses to different driving factors, and considering the 202 methodological differences between light and water response curve measurements. However, the water responses were least 203 well-fitted, especially at high water contents, because the measured photosynthesis response rate can become negative at 204 high water saturation, but it is impossible to simulate negative photosynthesis rates with the Farquhar photosynthetic model 205 for the light and CO2 conditions of the laboratory setup. Under these conditions, photosynthesis is larger than respiration. 206 Thus, even under low diffusivity, caused by high water saturation, there will be no net diffusion of CO2 from the thallus into 207 the ambient air. Furthermore, matching the simulated temperature-response of net photosynthesis to the measured data under 208 cool conditions turned out to be difficult for samples from site T1 and A1 (Fig. S2). There were too few data points in the 209 measured temperature response data set to constrain the optimum temperature and temperature relation (see Fig. 2), but the 210 fitting turned out well because the simulated optimum temperature for net photosynthesis at site T1 was coincidentally close 211 to the value of 17 °C reported by Colesie et al. for this site. (2014; see Fig. S2). 212 2.3.2 Biocrust physiological properties 184 Furthermore, several parameters required by the Farquhar photosynthesis scheme and the respiration scheme were directly 185 measured or calculated from gas exchange data sets, such as MWC of the thallus, optimum water content, the optimum 186 temperature for gross photosynthesis, reference maintenance respiration rate, and the Q10 value of respiration. Since the 187 temperature range at all sites except D2 was too small to capture the optimum temperature reliably, it was approximated 188 from the measured data set as the average surface temperature during active periods. In addition, the optimum temperature 189 was also constrained by fitting the Farquhar equations to photosynthesis curves, as related to environmental factors light, 190 water content, and temperature. Such fitting method was also used to obtain some other photosynthesis-related parameters of 191 organisms, such as molar carboxylation and oxygenation rate of RuBisCO (Vcmax, Vomax), respiration cost of RuBisCO 192 enzyme (p_rr), and water saturation at which organisms become active (satmin). 193 8 8 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 194 Figure 2: Calibration of photosynthesis parameters of the model by fitting photosynthesis response curves of moss- and 195 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 4 194 Figure 2: Calibration of photosynthesis parameters of the model by fitting photosynthesis response curves of moss- and 195 lichen-dominated biocrust samples to measurements at site T2. (a): net photosynthesis rate in response to light at optimum 196 water content and 15 °C. (b): net photosynthesis rate in response to temperature at 1200 µmol m-2 s-1 light and optimal water 197 t t ( ) t h t th i t i t l ti t t ti t 400 l -2 -1 li ht d 15 °C 198 Figure 2: Calibration of photosynthesis parameters of the model by fitting photosynthesis response curves of moss- and 195 Figure 2: Calibration of photosynthesis parameters of the model by fitting photosynthesis response curves of moss- and 195 lichen-dominated biocrust samples to measurements at site T2. (a): net photosynthesis rate in response to light at optimum 196 water content and 15 °C. (b): net photosynthesis rate in response to temperature at 1200 µmol m-2 s-1 light and optimal water 197 content. (c): net photosynthesis rate in response to relative water saturation at 400 µmol m-2 s-1 light and 15 °C. 2.4 Validation of the data-driven model 213 9 9 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. The water saturation and activity estimated by the data-driven model were validated by comparing with the daily and diurnal 214 patterns of measured activity (5 sites, not at site D2) and water content data (only at D2). Furthermore, the C balance 215 estimated by the data-driven model was validated by comparison to the in situ measured net CO2 exchange rate of moss, 216 lichen, and cyanocrust-dominated biocrusts. These data were sampled at site D2 by removing the soil respiration rate, 217 predicted by means of a fitted regression (Weber et al., 2012). Since data on water saturation were available, measured PAR, 218 surface temperature, and water content were used to simulate the C balance using the data-driven model, in contrast to the 219 setup described above. The activity, however, was calculated in the same way as described in the setup. Moreover, the 220 parameters of the model were the same as the calibrated ones of the corresponding biocrust types. 221 2.5.2 Effect of seasonal acclimation 247 Another sensitivity analysis was performed for site T1 to investigate the impact of seasonally acclimatized properties on 248 carbon assimilation. The properties were varied based on the literature. Respiration of lichens was found to acclimate to 249 seasonal changes in temperature (Lange and Green, 2005). Moreover, under low light, organisms showed shade-adapted 250 physiological characteristics with low PAR compensation and saturation points (LCP and LSP; Green and Lange, 1991). 251 Thus, under low light conditions, the organisms have a stronger ability to utilize low light intensities for photosynthesis. 252 These properties can be expressed by certain parameters of the data-driven model. For instance, the respiration rate is 253 determined by the parameter metabolic respiration cost per surface area (Resp_main); LCP and LSP can be affected by 254 changing the slope of the photosynthesis-light relations through light absorption fraction in cells (extL); LCP and LSP can 255 also be modified via the ratio of Jmax to Vcmax (jvratio) as it influences the value of light use efficiency at unsaturated light. 256 With higher efficiency, the light required to reach the saturated light level declines. Jmax is a crucial parameter quantifying 257 the maximum rate of electron transport in the light-dependent reactions of photosynthesis, Vcmax describes the maximum 258 rate of carboxylation of RuBisCO in the Calvin Cycle of photosynthesis (Walker et al., 2014). Accordingly, rather than 259 keeping all parameters fixed throughout the simulation period of the data-driven model, in the sensitivity analysis, the 260 physiological parameters were set to another set of values in the winter months. We analyzed the lichen- and moss- 261 dominated biocrusts at site T1 as an example, because the measured time-series activity showed that in temperate sites such 262 as T1, the organisms were active most of the time, and thus the C balance would be more sensitive to the modifying 263 properties. 264 In the sensitivity analysis, these calibrated physiological parameters of the data-driven model were varied for the non- 265 growing months to adapt to the climatic conditions because the organisms at site T1 were collected in their growing seasons. 266 Specifically, in an hourly simulation during September and December, January, and February, the parameter Resp_main was 267 reduced to half to lighten the respiratory cost for the samples collected at site T1. The size of extL was doubled to increase 268 the slope of photosynthesis-light relations. 2.5.1 Effects of environmental factors 223 (3): 241 ��= �� + ��, (2) 242 (2) 10 ��= �� =1 � �� σ , (3) 243 where Nab (increase) and Nab (decrease) are the normalized C balance of increasing or decreasing the environmental factor a 244 in climatic region b, respectively. Nab is thus the change amplitude of normalized C balance of environmental factor a in 245 climatic region b. 246 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. ��= �� =1 � �� σ , (3) 243 (3) where Nab (increase) and Nab (decrease) are the normalized C balance of increasing or decreasing the environmental factor a 244 in climatic region b, respectively. Nab is thus the change amplitude of normalized C balance of environmental factor a in 245 climatic region b. 246 2.5.1 Effects of environmental factors 223 To investigate the role that environmental factors, namely air temperature (Tair), light intensity (Light), ambient CO2 224 concentration (CO2), and different types of water sources play in regulating the C balance of biocrusts, sensitivity analyses 225 were conducted using our data-driven model for lichen-dominated biocrusts from all study sites. The different types of water 226 sources include rainfall (Rain) and non-rainfall water inputs such as dew and water vapor, which are also determined by 227 relative air humidity (Rhum). 228 All the environmental factors were reduced and increased by half (+/- 50%), except for air temperature and relative humidity. 229 The air temperature differences varied by 5 K and relative humidity by 20%. Moreover, relative humidity was constrained 230 between 0 and 100% when the varied relative humidity exceeded this range. The annual C balance with changing 231 environmental factors was then normalized for comparing the relative importance of factors among climate zones following 232 Eq. (1): 233 ��= ��−�� , (1) 234 ��= ��−�� , (1) 234 ��= ��−�� , (1) (1) where Cij is the C balance of factor j under operation i, and Cj is the original C balance of factor j where Cij is the C balance of factor j under operation i, and Cj is the original C balance of facto 5 where Cij is the C balance of factor j under operation i, and Cj is the original C balance of factor j. 235 Then the positive normalized C balance would show an increased annual C balance with varying environmental factors, and 236 thus more carbon accumulation. Moreover, the size of the normalized C balance is proportional to the magnitude of change 237 of the C balance when certain environmental factors change. Therefore, a larger normalized C balance also demonstrates a 238 larger change in annual C balance, and thus a larger effect of this environmental factor. 239 To interpret the spatial distribution of the importance of different environmental factors on C balance, the relative 240 importance of each factor in the given climatic region was calculated following Eq. (2) and Eq. (3): 241 To interpret the spatial distribution of the importance of different environmental factors on C balance, the relative 240 importance of each factor in the given climatic region was calculated following Eq. (2) and Eq. 2.5.2 Effect of seasonal acclimation 247 In addition, the parameter jvratio was doubled as well to enhance the light use 269 efficiency. 270 11 11 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. LiBry is a process-based dynamic global vegetation model (DGVM) specific to non-vascular vegetation. The model mim 272 environmental filtering in the real world by simulating many different functional strategies and selecting those wh 273 maintain a positive C balance under the respective climatic conditions. The strategies are characterized by a combination 274 11 physiological and morphological parameters. More information about the model is briefly described in the Appendix, a 275 a full detailed description can be found in Porada et al. (2013, 2019). For this study, the LiBry model was run for 300 ye 276 with repeated microclimate forcing data of one year from the six study sites, calibrated abiotic parameters same as the da 277 driven model, and initially generated 1000 strategies. C balance and dynamics of the surface cover of the strategies w 278 simulated until a steady state was reached, so that the final successful strategies were those where long-term biomass valu 279 were positive. Moreover, at the end of the simulation, the average values of functional traits were estimated by weighting 280 surviving strategies based on their relative cover. The (hypothetical) strategy characterized by these average values is cal 281 average strategy. The strategy with the largest cover area is called dominant strategy. 282 Furthermore, we compared the physiological parameters of the average strategy and the selected dominant strategies to 283 ones of organisms in the field by means of their respective photosynthesis response curves. This comparison can verify the 284 balance estimated by the data-driven model from a reversed perspective as the strategies were freely selected by the LiB 285 model based on their C balance, without prescribing values based on site level observations. 286 3 Results 287 3.1 Data-driven model 288 3.1.1 Validation of the data-driven model 289 LiBry is a process-based dynamic global vegetation model (DGVM) specific to non-vascular vegetation. The model mimics 272 environmental filtering in the real world by simulating many different functional strategies and selecting those which 273 maintain a positive C balance under the respective climatic conditions. The strategies are characterized by a combination of 274 11 physiological and morphological parameters. 2.5.2 Effect of seasonal acclimation 247 More information about the model is briefly described in the Appendix, and 275 a full detailed description can be found in Porada et al. (2013, 2019). For this study, the LiBry model was run for 300 years 276 with repeated microclimate forcing data of one year from the six study sites, calibrated abiotic parameters same as the data- 277 driven model, and initially generated 1000 strategies. C balance and dynamics of the surface cover of the strategies were 278 simulated until a steady state was reached, so that the final successful strategies were those where long-term biomass values 279 were positive. Moreover, at the end of the simulation, the average values of functional traits were estimated by weighting all 280 surviving strategies based on their relative cover. The (hypothetical) strategy characterized by these average values is called 281 average strategy. The strategy with the largest cover area is called dominant strategy. 282 Furthermore, we compared the physiological parameters of the average strategy and the selected dominant strategies to the 283 ones of organisms in the field by means of their respective photosynthesis response curves. This comparison can verify the C 284 balance estimated by the data-driven model from a reversed perspective as the strategies were freely selected by the LiBry 285 model based on their C balance, without prescribing values based on site level observations. 286 12 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 0 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 3: Validation of the estimated daily (left panel) and diurnal (right panel) patterns of activity of lichen- and moss- 291 Figure 3: Validation of the estimated daily (left panel) and diurnal (right panel) patterns of activity of lichen- and moss- 291 dominated biocrusts at site T3. The simulated patterns of activity were compared to measured data by four sensors at 292 Figure 3: Validation of the estimated daily (left panel) and diurnal (right panel) patterns of activity of lichen- and moss- 291 dominated biocrusts at site T3. The simulated patterns of activity were compared to measured data by four sensors at 292 different locations. Right: (a) to (d) represent the patterns of average hourly activity from January to March (JFM), April to 293 June (AMJ), July to September (JAS), and October to December (OND), respectively. 294 dominated biocrusts at site T3. 2.5.2 Effect of seasonal acclimation 247 The simulated patterns of activity were compared to measured data by four sensors at 292 different locations. Right: (a) to (d) represent the patterns of average hourly activity from January to March (JFM), April to 293 June (AMJ), July to September (JAS), and October to December (OND), respectively. 294 In general, the simulated daily and diurnal patterns of activity (water content at site D2) fit the measurements reasonably 295 well in magnitude (Fig. 3 and Fig. S3). However, our fitting resulted in a more dampened diurnal activity pattern simulated 296 by the model, and the activity at night and in the morning was underestimated during several seasons at sites D1, T1, T2 and 297 A1. In addition, both the daily and diurnal activity during April and June at site T1 were underestimated. Furthermore, water 298 content was overestimated for moss-dominated biocrust, especially when there was a large amount of water input at D2, 299 although the patterns corresponded well to the measured data for all three biocrust types (Fig. S3). This overestimation may 300 have resulted from the bias in measured MWC of samples used for constraining the water content in the model. 301 The comparison of simulated and on-site measured CO2 exchange rates of three biocrust types (moss, lichen, and cyanocrust 302 composed of cyanolichens and cyanobacteria) at site D2 showed mismatches, especially when water saturation was at both 303 ends of the gradient (Fig. 4). The CO2 exchange rate at high water content was overestimated compared to the measurements. 304 Moreover, there were large variations in measurements of respiration and CO2 exchange rate as water content was low and 305 thus simulated CO2 exchange rate was zero. Excluding the values at both ends of water content (0.58 and 1.74 mm for moss; 306 0.22 and 0.68 mm for lichen; 0.26 and 0.65 mm for cyanocrust), the accuracy of the model predictions was improved (root 307 mean square error (rmse) decreased from 1.45 to 1.36 for moss, 1.27 to 0.71 for lichen, and 0.92 to 0.87 for cyanocrust). 308 The comparison of simulated and on-site measured CO2 exchange rates of three biocrust types (moss, lichen, and cyanocrust 302 composed of cyanolichens and cyanobacteria) at site D2 showed mismatches, especially when water saturation was at both 303 ends of the gradient (Fig. 4). The CO2 exchange rate at high water content was overestimated compared to the measurements. Furthermore, the simulations were similar to measurements in magnitude. Therefore, despite the large variation, we are 309 confident about the general validity of the model. 310 Furthermore, the simulations were similar to measurements in magnitude. Therefore, despite the large variation, we are 309 confident about the general validity of the model. 310 Furthermore, the simulations were similar to measurements in magnitude. Therefore, despite the large variation, we are 09 confident about the general validity of the model. 10 g y 311 Figure 4: Validation of the photosynthesis and respiration scheme of the data-driven model through comparison of modelled 312 and measured CO2 exchange rate of moss, lichen, and cyanocrust given the measured water content, surface temperature, 313 PAR, and calculated activity. Observational data were collected in November in 2013 at site D2. The 1:1 line is shown in 314 black. 315 Figure 4: Validation of the photosynthesis and respiration scheme of the data-driven model through comparison of modelled 312 and measured CO2 exchange rate of moss, lichen, and cyanocrust given the measured water content, surface temperature, 313 PAR, and calculated activity. Observational data were collected in November in 2013 at site D2. The 1:1 line is shown in 314 black. 315 e 4: Validation of the photosynthesis and respiration scheme of the data-driven model through com Figure 4: Validation of the photosynthesis and respiration scheme of the data-driven model through comparison of modelled 312 and measured CO2 exchange rate of moss, lichen, and cyanocrust given the measured water content, surface temperature, 313 PAR, and calculated activity. Observational data were collected in November in 2013 at site D2. The 1:1 line is shown in 314 black. 315 2.5.2 Effect of seasonal acclimation 247 304 Moreover, there were large variations in measurements of respiration and CO2 exchange rate as water content was low and 305 thus simulated CO2 exchange rate was zero. Excluding the values at both ends of water content (0.58 and 1.74 mm for moss; 306 0.22 and 0.68 mm for lichen; 0.26 and 0.65 mm for cyanocrust), the accuracy of the model predictions was improved (root 307 mean square error (rmse) decreased from 1.45 to 1.36 for moss, 1.27 to 0.71 for lichen, and 0.92 to 0.87 for cyanocrust). 308 The comparison of simulated and on-site measured CO2 exchange rates of three biocrust types (moss, lichen, and cyanocrust 302 composed of cyanolichens and cyanobacteria) at site D2 showed mismatches, especially when water saturation was at both 303 ends of the gradient (Fig. 4). The CO2 exchange rate at high water content was overestimated compared to the measurements. 304 Moreover, there were large variations in measurements of respiration and CO2 exchange rate as water content was low and 305 thus simulated CO2 exchange rate was zero. Excluding the values at both ends of water content (0.58 and 1.74 mm for moss; 306 0.22 and 0.68 mm for lichen; 0.26 and 0.65 mm for cyanocrust), the accuracy of the model predictions was improved (root 307 mean square error (rmse) decreased from 1.45 to 1.36 for moss, 1.27 to 0.71 for lichen, and 0.92 to 0.87 for cyanocrust). 308 13 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Furthermore, the simulations were similar to measurements in magnitude. Therefore, despite the large variation, we are 309 confident about the general validity of the model. 310 3.1.2 Estimated C balance by data-driven model 316 The simulated annual C balance of each collected biocrust type at each site is listed in Table. 2. The annual C balance of 317 lichen- and moss-dominated biocrusts at two dryland sites showed a small positive value. Moreover, a cyanocrust 318 additionally measured at site D2 showed a small net release of carbon in the model. 319 Table 2: Simulated annual carbon budgets of each biocrust type at all sites 320 Table 2: Simulated annual carbon budgets of each biocrust type at all sites 320 Lichen g C m-2 yr-1 Moss g C m-2 yr-1 Cyanocrust g C m-2 yr-1 D1 (Almeria) 3.8 3.2 D2 (Soebatsfontein) 0.7 6.3 -2.0 T1 (Gössenheim) -27.3 -28.6 14 14 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. T2 (Öland) -96.0 -63.9 T3 (Linde) 7.1 13.3 A1 (Hochtor) -11.4 6.2 Furthermore according to these data-driven model simulations despite the C balance of two biocrust types being positive at 1 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. T2 (Öland) -96.0 -63.9 T3 (Linde) 7.1 13.3 A1 (Hochtor) -11.4 6.2 Furthermore according to these data driven model simulations despite the C balance of two biocrust types being positive at 21 , g , p yp g p site T3, a large amount of carbon was lost at the sites T1 and T2 in temperate humid regions. These results imply that 322 according to the data-driven model, the biocrusts would not survive in the long-term at most of the temperate humid research 323 sites. At the alpine site A1, the moss-dominated biocrust showed a small positive C balance, whereas the lichen crust lost 324 carbon in a year with long periods of ice cover. 325 3.2 Dominant strategies selected by the LiBry Model 326 In general, the photosynthesis response curves of dominant and average strategies selected by the LiBry model did not fit 327 well to the measurements, especially at temperate site T2 (Fig. 5; the results for the other sites with negative C balance are 328 shown in Fig. S4). Specifically, the selected physiological traits which determine water and light acquisition of the dominant 329 and average strategies in LiBry differed from those of the collected samples at all sites. Compared to the measured samples, 330 the LiBry strategies showed markedly higher efficiency at low light intensity and faster activation. By design, the Libry 331 model selected strategies with a positive C balance in the long-term run, and thus the mismatches are consistent with the fact 332 that the data-driven model simulated negative C balances. 333 334 Figure 5: Comparison of net photosynthesis response of measured samples with simulated selected dominant and average 335 strategies of LiBry at site T2. (a): light response curve; (b): temperature response curve; (c): water response relation. The 336 Figure 5: Comparison of net photosynthesis response of measured samples with simulated selected dominant and average 335 strategies of LiBry at site T2. (a): light response curve; (b): temperature response curve; (c): water response relation. The 336 Figure 5: Comparison of net photosynthesis response of measured samples with simulated selected dominant and average 335 strategies of LiBry at site T2. (a): light response curve; (b): temperature response curve; (c): water response relation. The 336 15 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. colored points represent the measured CO2 exchange rates of moss and lichen, and the coloured lines correspond to the data- 337 driven model. The black lines show the photosynthesis response of the dominant strategy selected by the LiBry model 338 (dashed) and the average strategy (solid). The parameter values of the average strategy correspond to the average of all 339 surviving strategies. 340 3.3.1 Environmental factors 342 The environmental factors light intensity, CO2 concentration, air temperature, and various water sources, had different 343 effects on the C balance of lichens in different climate zones (Fig. 6). For all sites within a given climate zone, the effects of 344 different environmental factors on C balance were overall similar but showed an apparent larger variation at the temperate 345 site T3 in contrast to the other two temperate sites, and at site D2 compared to D1 (Fig. 6a). This may be due to 346 physiological differences of the investigated biocrust species between these sites and consequently variations in the 347 responses of net photosynthesis rate to temperature, water, and light between them (Fig. 2 and S2). 348 Furthermore, the spatial patterns of the relative importance of different environmental factors show that the factors which 349 have the strongest effects differ between climatic regions (Fig. 6b). 350 Furthermore, the spatial patterns of the relative importance of different environmental factors show that the factors which 349 have the strongest effects differ between climatic regions (Fig. 6b). 350 16 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 351 17 17 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 6: (a) The effects of environmental factors - CO2 concentration (CO2), relative air humidity (Rhum), rainfall amount 352 (Rain), air temperature (Tair) and light intensity (Light) on the annual C balance of lichens in different climate regions. The 353 altered annual C balance resulting from increasing or decreasing environmental factors is normalized by the C balance under 354 original environmental conditions. The colored columns indicate the average value of the normalized C balance at sites with 355 similar climate conditions. Various styles of black points indicate different sites. Positive normalized C balance implies that 356 the annual C balance increases with varying environmental factors and more carbon was accumulated in a year at the site, 357 and vice versa. A larger normalized C balance reflects that the C balance is more sensitive to the altering environmental 358 factor, and thus the environmental factor has a larger effect on C balance. (b) Relative importance of each environmental 359 factor compared to other factors across the climatic regions. 3.3.1 Environmental factors 342 Larger relative importance implies a more important effect the 360 factor has on the C balance compared to other factors in the given climatic region, and vice versa. 361 In general, air temperature and CO2 concentration were the most important drivers for C balance of biocrust organisms 362 between climate zones. Light intensity and relative humidity played a relevant role in impacting the C balance as well. 363 Rainfall amount had lower relative importance at all sites except dryland D2, where the effect of rainfall on C balance was 364 similar to other factors (Fig. 6a). Therefore, rainfall amount showed a maximum in relative importance in drylands, 365 compared to other regions. In general, the effect of the other water source, relative air humidity, was moderate but notable at 366 all climate zones, and is slightly larger in temperate region in comparison to other climate zones. Furthermore, the humidity 367 had a slightly larger impact on C balance in comparison to rainfall amount at all temperate and alpine sites (e.g., change 368 amplitude was 0.04 for rainfall and 0.44 for humidity at T1). Moreover, reduced humidity can have a positive effect on C 369 accumulation in these regions (e.g., the normalized C balance was 0.3 at T1 when relative humidity was reduced). In 370 drylands, however, the impacts of water sources on C balance varied between sites. The results showed that relative 371 humidity had a larger impact than rainfall amount at D1 while similar at D2. Reducing relative humidity had a positive effect 372 on C accumulation at D2 while C accumulation was reduced at D1 (normalized C balance is 2.90 at D2 and -0.13 at D1 373 when relative humidity decreases). 374 The ambient CO2 concentration was an essential factor for the C balance at all sites especially in drylands, resulting in 375 positive effects on C balance with increasing CO2. Furthermore, light intensity had a marked impact on the C budget at all 376 sites except for dryland D1, and it was relatively more important in temperate regions. At site T2, for example, the 377 normalized C balance was changed to -0.39 and to 0.19 for half and doubled light intensity, respectively. At these sites, the 378 normalized C balance increased with enhanced light intensity. 3.3.1 Environmental factors 342 At site D1, however, the values did not vary largely, and even 379 slightly more carbon was lost as the light levels increased (0.16 and -0.08 for half and doubled light intensity). Air 380 temperature had a large impact on C balance at all sites. Especially at alpine site A1, C balance decreased strongly as air 381 temperature raised by 5 K (normalized C balance of -13.59), and at all sites, the direction of the effect remained constant, 382 namely, warming decreased the C balance and vice versa. 383 18 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 3.3.2 Acclimation of physiological properties 384 The sensitivity analysis for acclimation showed a marked increase in annual productivity of lichen- and moss-dominated 385 biocrusts at site T1 (Fig. 7) when the seasonal acclimation of several physiological parameters was included in the model 386 (from -27.3 to 3.1 g C m-2 yr-1 and from -28.6 to 15.7 g C m-2 yr-1). 387 388 Figure 7: Comparison of accumulated annual C balance between simulations with dynamic parameters and fixed ones 389 of lichens and mosses at site T1. For the simulation with the fixed parameters, all parameters that have been calibrated 390 or measured remained constant throughout the simulation year. For the simulation with dynamic parameters at site T1, 391 parameter metabolic respiration cost per surface area (Resp_main) was reduced by half, light absorption fraction in 392 cells (extL) was doubled but restricted to one, the ratio of Jmax to Vcmax (jvratio) was increased by two times from 393 September to February. The values remained the same in other months as the ones prescribed in the simulation with 394 fixed parameters. 395 4 Discussion 396 4.1 Simulated C balance of data-driven model 397 Figure 7: Comparison of accumulated annual C balance between simulations with dynamic parameters and fixed ones 389 of lichens and mosses at site T1. For the simulation with the fixed parameters, all parameters that have been calibrated 390 or measured remained constant throughout the simulation year. For the simulation with dynamic parameters at site T1, 391 parameter metabolic respiration cost per surface area (Resp_main) was reduced by half, light absorption fraction in 392 cells (extL) was doubled but restricted to one, the ratio of Jmax to Vcmax (jvratio) was increased by two times from 393 September to February. The values remained the same in other months as the ones prescribed in the simulation with 394 fixed parameters. 395 19 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. The data-driven model aims to provide observation-based estimates of the carbon fluxes of non-vascular photoautotrophs 398 which may serve as approximation for the C balance of vegetation in biocrust-dominated ecosystems. At the two dryland 399 sites, the lichen- and moss-dominated biocrusts were estimated to be carbon sinks on an annual basis. 3.3.2 Acclimation of physiological properties 384 (2014) recorded that the biocrusts composed of lichens, 403 mosses, and cyanobacteria of the Mu Us Desert in China took up 3.46 to 6.05 g C m-2 yr-1. Brostoff et al. (2005) estimated a 404 larger carbon gain by lichen biocrust of 11.7 g C m-2 yr-1 in the Mojave Desert, USA. For cyanobacteria, an annual carbon 405 uptake of 0.02 to 2.3 g C m-2 was reported for deserts (Jeffries et al., 1993). The estimated C balance values in drylands 406 fluctuate relatively largely, but the magnitude is consistent with the simulated results by the data-driven model at D1 and D2. 407 For biocrust lichens growing on rock surface in a temperate grassland, Lange (2003b) measured an annual carbon gain of 408 21.49 g C m-2. Additionally, several studies estimated the carbon budget in humid tundra habitats. An amount of ~12–70 g C 409 m-2 yr-1 carbon was fixed by moss-dominated biocrust, for instance (Schuur et al., 2007). The magnitude of these values 410 corresponds to the estimation of the C balance at T3. However, the estimated annual carbon losses of lichens and mosses by 411 the data-driven model in temperate regions T1 and T2 should actually lead to the death of these organisms, which is not 412 consistent with their dominant abundance in the field and is much lower than published by previous studies. 413 The mismatches of trait values between strategies selected by the LiBry model and collected species indicate that strategies 414 corresponding to the collected species could not maintain a positive long-term C balance, and thus were not able to survive 415 in the LiBry model. This is consistent with the results of the data-driven model, which also simulates a negative C balance 416 for those trait values. Nevertheless, there are some uncertainties in the model simulation. A bias in model estimates could 417 result, for instance, from missing physiological processes and trade-offs. Potential reasons for the inconsistency between 418 models and observations are discussed further below. 419 3.3.2 Acclimation of physiological properties 384 As shown in the results, 400 lichens took up 3.8 and 0.7 g C m-2 yr-1, and mosses accumulated 3.2 and 6.3 g C m-2 yr-1 at site D1 and D2, respectively. 401 which may serve as approximation for the C balance of vegetation in biocrust-dominated ecosystems. At the two dryland 399 sites, the lichen- and moss-dominated biocrusts were estimated to be carbon sinks on an annual basis. As shown in the results, 400 lichens took up 3.8 and 0.7 g C m-2 yr-1, and mosses accumulated 3.2 and 6.3 g C m-2 yr-1 at site D1 and D2, respectively. 401 The estimated C balance at the two dryland sites is consistent with the magnitude of the annual C balance of different 402 biocrust types reported by various studies in arid habitats. Feng et al. (2014) recorded that the biocrusts composed of lichens, 403 mosses, and cyanobacteria of the Mu Us Desert in China took up 3.46 to 6.05 g C m-2 yr-1. Brostoff et al. (2005) estimated a 404 larger carbon gain by lichen biocrust of 11.7 g C m-2 yr-1 in the Mojave Desert, USA. For cyanobacteria, an annual carbon 405 uptake of 0.02 to 2.3 g C m-2 was reported for deserts (Jeffries et al., 1993). The estimated C balance values in drylands 406 fluctuate relatively largely, but the magnitude is consistent with the simulated results by the data-driven model at D1 and D2. 407 For biocrust lichens growing on rock surface in a temperate grassland, Lange (2003b) measured an annual carbon gain of 408 21.49 g C m-2. Additionally, several studies estimated the carbon budget in humid tundra habitats. An amount of ~12–70 g C 409 m-2 yr-1 carbon was fixed by moss-dominated biocrust, for instance (Schuur et al., 2007). The magnitude of these values 410 corresponds to the estimation of the C balance at T3. However, the estimated annual carbon losses of lichens and mosses by 411 the data-driven model in temperate regions T1 and T2 should actually lead to the death of these organisms, which is not 412 The estimated C balance at the two dryland sites is consistent with the magnitude of the annual C balance of different 402 biocrust types reported by various studies in arid habitats. Feng et al. 4.2 Potential factors influencing the C balance 420 Although the intra-annual 439 change in air CO2 concentration may be small in the field compared to other environmental factors, the increase of CO2 in 440 the atmosphere in recent decades (IPCC 2021) may alter the long-term C balance substantially. 441 Ambient CO2 concentration affects the gross photosynthesis rate to a large extent in the model. Although the intra-annual 439 change in air CO2 concentration may be small in the field compared to other environmental factors, the increase of CO2 in 440 the atmosphere in recent decades (IPCC 2021) may alter the long-term C balance substantially. 441 Ambient CO2 concentration affects the gross photosynthesis rate to a large extent in the model. Although the intra-annual 439 change in air CO2 concentration may be small in the field compared to other environmental factors, the increase of CO2 in 440 the atmosphere in recent decades (IPCC 2021) may alter the long-term C balance substantially. 441 Light intensity is one of the essential factors for photosynthesis as simulated by our model. According to our climate forcing 442 data, the mean value of radiation maxima in January is 244 µmol m-2 s-1 at T1 and 245 µmol m-2 s-1 at the alpine site. During 443 wintertime in temperate and alpine regions, light intensity may be lower than the light saturation point (Fig.2 and S2). 444 Therefore, light intensity is a limiting factor of photosynthetic carbon assimilation in these regions, and increasing light 445 intensity can thus promote carbon accumulation. However, the higher light intensity can raise the surface temperature and 446 thus lead to more evaporation. More water loss would result in lower water saturation and activity especially in drylands, 447 which could limit the net photosynthesis rate. Therefore, at dryland D1, the increasing light intensity has the opposite impact 448 compared with other sites. 449 Biocrust water content is regulated by both rainfall and non-rainfall water inputs. The relative importance of different water 450 sources in mediating C balance varies in the model. Precipitation amount was not a key factor affecting the simulated 451 biocrust performance at one of the arid sites, which is consistent with another study (Baldauf et al., 2020). Our results 452 suggest that at the other dryland site D2, however, the precipitation amount is very closely associated with the C balance of 453 lichens. 4.2 Potential factors influencing the C balance 420 Despite diverse climatic conditions, we found similarities regarding the dominant environmental factors controlling the C 421 balance. As shown in the results, CO2 and air temperature were two most important factors at all sites that impact C balances 422 in the model. Relative air humidity, partly precipitation, and light intensity were also essential for C balance. The relative 423 importance of these factors varied slightly among climatic regions and the effects of these factors on the C balance were 424 different in direction among sites. We cannot rule out that the magnitudes of changes in environmental factors that we 425 applied in the sensitivity analysis were not balanced, which may have led to an overestimation of the relative importance of 426 certain factors, such as air temperature, for instance, compared to the others. The spatial patterns across climate regions of a 427 given environmental factor, however, are not affected by this, which means that differences between climatic regions for a 428 given factor are most likely robust. Hence, rainfall and CO2 are likely to have the largest effect on C balance in drylands 429 20 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. compared to other regions, while air temperature is more relevant in alpine regions and light and relative air humidity have a 430 higher impact in temperate than in other regions. Nevertheless, we only studied the sensitivity of the C balance of biocrusts 431 dominated by the lichen Psora decipiens and Cladonia furcata (at T3), and there are variations between lichens of different 432 growth forms and between biocrust types. For example, cyanolichens increase in abundance with increasing rainfall, but 433 trebouxioid lichens have their physiological optimum in drier conditions (Phinney et al., 2021). Moreover, the impact of 434 precipitation on isidiate lichens is weaker than that of temperature (Phinney et al., 2021). 435 Our results suggest that warming can result in a large amount of carbon loss at all sites, with a particular large effect in the 436 alpine region. This can be explained by the overall less optimal water and temperature conditions associated with warming. 437 The simulated increasing respiratory costs with warming overcompensate gains in gross photosynthesis. 438 Ambient CO2 concentration affects the gross photosynthesis rate to a large extent in the model. 4.2 Potential factors influencing the C balance 420 Decreasing the amount of precipitation alters the activity and water saturation patterns of the investigated 471 organisms to a larger extent in D2 as compared to D1 (Fig. S5e and f). Therefore, the amount of precipitation has a relatively 472 larger impact on the simulated C balance at site D2. Several studies found strong effects of variations in the precipitation 473 pattern on biocrust C balance (such as Reed et al., 2012). Our simulation results thus highlight the need for combined 474 application of field experiments and data-driven modelling to improve our understanding of differential responses to 475 variation in precipitation. 476 Furthermore, our findings indicate that relative humidity plays an important role in mediating the C balance of lichens in arid 477 regions, which is consistent with the results of Baldauf et al. (2020). Our results also imply that relative humidity is a crucial 478 factor at humid sites. However, in contrast to rainfall, the non-rainfall water inputs, such as water vapor and dew, that 479 depend on relative humidity have contrasting impacts on the simulated C balances of temperate, alpine, and dryland 480 biocrusts (Fig. 6). Whereas the increase in relative humidity can reduce the annual carbon gain in temperate regions, it 481 causes an increase at D1. The possible reason for this opposite direction of effect could be that the effect of air humidity 482 depends on the moisture conditions of the site. Higher relative air humidity could enhance water vapor and dew uptake and 483 reduce evaporation. At humid temperate and alpine sites, this yields larger water saturation and more periods with extremely 484 high saturation at sub-optimal, light-limited conditions. However, in drylands, the non-rainfall water uptake in the form of 485 dew or water vapor is greatest before sunrise (Chamizo et al., 2021; Ouyang et al., 2017). Especially in coastal deserts (like 486 the Succulent Karoo) increased fog and nocturnal dewfall in combination with higher humidity and shading mainly lead to 487 prolonged activated periods in the early morning when the organisms start assimilating carbon (Veste and Littmann, 2006). 488 Moreover, the reduced evaporation mitigates effects of drying and inactivity of organisms that occur especially at midday. 489 These two processes consequently result in a markedly increased annual C balance in the model. 490 However, the beneficial impact of the increased humidity is not common in all drylands. 4.2 Potential factors influencing the C balance 420 Moreover, we found that the effect of the amount of precipitation is small in relative humid temperate and alpine 454 regions. The effect of precipitation on C balance depends on the change in relative water saturation that follows from 455 precipitation event sizes and patterns. In some cases, decreased precipitation leading to a lower water saturation of biocrusts 456 may facilitate photosynthetic carbon gain via increasing the CO2 diffusivity from the atmosphere into the chloroplast (Lange 457 et al., 1997). Nevertheless, reducing water saturation below a certain value can cause a decline in the duration of activity 458 (Proctor, 2001; Veste et al., 2008) which thus reduces carbon accumulation. Thus, there may be a rain threshold below which 459 decreasing rain may start having a negative effect on biocrust C balances. The threshold is likely species-specific as it is 460 associated with the water holding capacity of the organism. At arid region D1, despite the number of rainfall events being 461 21 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. lower, the size of many single events is still large. When rainfall input is varied in the model (decreased by half), the activity 462 and water saturation patterns of the studied organisms are similar to the ones with original rainfall (Fig. S5a and S5b). At the 463 temperate and alpine sites, although the reduction in rainfall lowered the saturation as well as the activity at many time 464 points (Fig. S5c and S5d), the organisms still remained active during most of the time (active 56% to 52% of the time during 465 one year as rainfall decreased by half) and even still fully active, since there was frequent and considerable rainfall in the 466 year of data collection. Thereby, the negative impact induced by reduced activity can be compensated by the positive impact 467 caused by reduced saturation. Thus, in the model, the impact of precipitation amount is small on C balance in these regions. 468 Moreover, the difference in effects of precipitation between two drylands may result from the different precipitation patterns. 469 The precipitation events are more evenly distributed throughout the year in D2 compared to D1, and many single events are 470 small in size. 4.2 Potential factors influencing the C balance 420 At site D2, our results showed an 491 apparent decrease in annual C balance with increased humidity. This could result from the different calculated reference 492 respiratory costs of the investigated organisms at these two sites from their photosynthesis in response to temperature data. 493 During nights with higher humidity, the surface temperature of organisms increases due to less evaporative cooling, which 494 22 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. increases the respiratory carbon loss at night. Moreover, higher humidity increases the activity and activates organisms that 495 are otherwise inactive at night (annual mean humidity at night is 66% at D1 and 70% at D2). Thus, more carbon will be lost 496 due to longer periods of respiration in the dark or at low light. The reference respiratory cost of the measured organisms at 497 D2 is much larger than at D1 (1.2 and 2.5 µmol m-2 s-1 at D1 and D2, respectively), so the respiration rate at D2 will be 498 larger than D1 under similar temperature conditions. This is supported by our results that also showed a larger yearly mean 499 respiration rate during the night at D2 (0.35 and 1.04 µmol m-2 s-1 at D1 and D2, respectively; the yearly air temperature is 500 12.5 and 14 °C at D1 and D2, respectively). Therefore, although more carbon is assimilated during the day due to higher 501 humidity in both drylands, more carbon is also lost during the night. The higher carbon loss at night at site D2 is larger than 502 at D1, since the organisms at D2 have a higher respiration rate than at D1. This may explain the decrease of the annual C 503 balance with increased air humidity at site D2 in the model. 504 4.3 Estimated negative C balance using the data-driven model 505 Under climate change conditions, the individual environmental factors will likely interact with each other to affect organisms 506 (e.g. Rillig et al., 2019). The critical role of the combination of optimal air temperature, water content, and light intensity for 507 the growth of biocrusts is also observed in various other studies (Büdel et al., 2018; Lange, 2003a; Lange et al., 1998b). 508 Overall, optimal conditions are always rare within a year, which was also described by Lange (2003b). In some cases, carbon 509 gains during the relatively optimal conditions may not be sufficient to compensate for losses under long-term harsh 510 conditions, such as autumn and winter at site T1, for instance. For this reason, the simulated C balance of mosses and lichens 511 in temperate humid regions was mostly negative. Given their survival under field conditions, there may be some 512 unconsidered mechanisms in the model that allow real biocrusts to persist under these unfavourable environmental 513 conditions. 514 Seasonal acclimation of physiological traits to the current climatic conditions may play an important role in regulating the C 515 balance at humid sites where the organisms are active throughout the year, such as site T1 (Fig. 7). It was observed, for 516 instance, that the respiration of lichens shows acclimation to seasonal changes in temperature, and the maximum CO2 517 exchange rate of the organisms remains steady throughout the year (Lange and Green, 2005). Gauslaa (2006) found a higher 518 chlorophyll a/b ratio in forest lichen with increasing light. Moreover, depression in quantum efficiency in summer under 519 extremely dry conditions has been observed (Vivas et al., 2017). These varied physiological properties of organisms within a 520 year could result in different photosynthesis and respiration rates, and thus different C balances in comparison to the ones 521 that cannot acclimate to the seasonal climate. The missing seasonal acclimation of physiological traits may explain why the 522 data-driven model estimated a negative C balance for biocrusts in humid regions. Also, the LiBry model does not account for 523 seasonal acclimation since the strategies are assumed to have constant functional properties throughout the simulation. 524 Therefore, this can partly account for the mismatch of traits of selected strategies by LiBry and observations. 525 4.4 Validation of the data-driven model 526 S2 f), pointing at a certain degree of metabolic activity in natural 545 conditions. Furthermore, the samples used for validation were different from the ones for calibration, which can also lead to 546 inaccuracies. 547 Additionally, the ability of the model to capture seasonality variations of C balance, which have been shown by other studies 548 (Büdel et al., 2018; Lange, 2003a; Zhao et al., 2016), could not be evaluated here since the monitoring of C balance in the 549 field and collection of samples used for photosynthesis performance measurements were conducted only during October and 550 early November. 551 4.4 Validation of the data-driven model 526 23 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. The validation results of the model showed an overall good fit of daily and diurnal patterns of water content and activity (Fig. 527 3 and S3), and C balance at D2 (shown in Fig. 4) given the uncertainties in the data used to parameterize and evaluate the 528 model. This indicates that the data-driven model may be a reliable tool for C balance estimation, provided that a sufficient 529 amount of suitable forcing data is available. 530 A potential explanation for the general underestimation of activity at night and morning during several periods in a year is 531 the larger prescribed MWC and satmin of organisms in the model compared to those of the samples from the activity 532 measurements. Consequently, simulated saturation was lower, but minimal saturation for being active was higher than the 533 samples. Thus, the activity may have been underestimated at small water inputs such as dew and water vapor, which occur 534 mainly during the night and in the morning hours (Fig. S6). Moreover, underestimated activity in April and June at site T1 535 (Fig. S3 F(b)) may have resulted from a gap in rainfall measurements during this period. Not only rainfall amount but also 536 timing and frequency of rainfall events are essential for the physiological responses of biocrust communities (Belnap et al., 537 2004; Coe et al., 2012; Reed et al., 2012). Therefore, although the measured annual total amount of rainfall is reasonable 538 (424 mm at site T1), the missing rainfall during a series of days in summer at site T1 would lead us to incorrectly predict that 539 the biocrusts remain inactive on these days. 540 Moreover, the mismatch between modelled and observed CO2 assimilation rates at low or high water contents at site D2 (Fig. 541 4) may have partly resulted from the calibration procedure. In the calibration the simulated CO2 exchange rates were higher 542 than measurements when the saturation exceeded the optimum saturation and hardly showed any negative values at high 543 saturation (Fig. S2 f). In turn, the simulated CO2 exchange rates of biocrusts with an extremely low water content were zero 544 while the measurements showed negative values (see Fig. 4.5 Uncertainties of long-term C balance simulated by the data-driven model 552 566 Despite potential bias, this approach provides possibilities to predict the long-term C balance of biocrusts in the field across 563 various climate zones, and it enables us to analyse the mechanisms driving C balance. However, in the future, the model 564 needs to be calibrated with a larger number of samples collected and measured in various seasons to take the acclimation of 565 physiological properties into account. 566 4.5 Uncertainties of long-term C balance simulated by the data-driven model 552 Apart from the missing seasonal acclimation of physiological traits in the data-driven model, the estimated C balance may be 553 inaccurate due to potential bias in estimated relative water saturation, which partly depends on prescribed MWC, a 554 morphological model parameter that is obtained by measurements. We varied the MWC of lichen-dominated biocrust from 555 site T1 by half (+/- 50%) to examine how important uncertainty in this parameter is for the estimation of the C balance. The 556 outcome revealed that MWC has little effect on C balance (-25.0, -27.3, -28.3 g C m-2 yr-1 for reduced, original and increased 557 MWC). Therefore, the annual carbon estimation is robust to the uncertainties with regard to the prescribed MWC. 558 24 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Furthermore, the C balance estimated by the data-driven model could be affected by a bias in calibrated physiological 559 parameters for organisms from photosynthesis response curves. Not all organisms forming cryptogamic covers show the 560 same degree of depression in net photosynthesis at high water content. For instance, among lichens, there is a wide variation 561 in responses of net photosynthesis to water saturation (Lange et al., 1995), also between individuals (Fig. 2 and S2). 562 Furthermore, the C balance estimated by the data-driven model could be affected by a bias in calibrated physiological 559 parameters for organisms from photosynthesis response curves. Not all organisms forming cryptogamic covers show the 560 same degree of depression in net photosynthesis at high water content. For instance, among lichens, there is a wide variation 561 in responses of net photosynthesis to water saturation (Lange et al., 1995), also between individuals (Fig. 2 and S2). 562 Despite potential bias, this approach provides possibilities to predict the long-term C balance of biocrusts in the field across 563 various climate zones, and it enables us to analyse the mechanisms driving C balance. However, in the future, the model 564 needs to be calibrated with a larger number of samples collected and measured in various seasons to take the acclimation of 565 physiological properties into account. 5 Conclusions 567 While all environmental factors that were examined in our study may act as key drivers for the C balance of biocrusts, they 568 show distinct spatial patterns of their relative impacts. At alpine sites, air temperature is likely the most relevant factor. 569 Relative humidity and light may be relevant for the C balance mainly in humid temperate sites, which is not obvious. In 570 drylands, rainfall and also ambient CO2 are found to be additional relevant factors. Furthermore, the direction of effect of 571 relative humidity may vary between dryland and humid sites: the higher humidity can be beneficial for the C balance in arid 572 regions, whereas it induces carbon loss in humid temperate and alpine sites. However, these patterns in drylands depend on 573 the species characteristics and microclimatic conditions of the habitat. Therefore, for the generalization of the roles of water 574 types in various climatic zones, a larger number of different organisms at multiple sites needs to be studied. 575 Our study suggests that a better, more detailed understanding of the seasonal variation of physiological traits is necessary, as 576 acclimation may affect the C balance substantially. The season and timing of collecting and monitoring the species should be 577 considered in experimental studies, especially when the characteristics of species are the basis for further analyses and 578 forecasts to estimate the annual carbon budget. Additionally, integration of acclimation of physiological traits in models can 579 improve the accuracy in C balance estimation. 580 While all environmental factors that were examined in our study may act as key drivers for the C balance of biocrusts, they 568 show distinct spatial patterns of their relative impacts. At alpine sites, air temperature is likely the most relevant factor. 569 Relative humidity and light may be relevant for the C balance mainly in humid temperate sites, which is not obvious. In 570 drylands, rainfall and also ambient CO2 are found to be additional relevant factors. Furthermore, the direction of effect of 571 relative humidity may vary between dryland and humid sites: the higher humidity can be beneficial for the C balance in arid 572 regions, whereas it induces carbon loss in humid temperate and alpine sites. However, these patterns in drylands depend on 573 the species characteristics and microclimatic conditions of the habitat. 5 Conclusions 567 The research in Linde is funded by 594 Zwillenberg-Tietz Stiftung by a grant to MV. Research in South Africa was funded by the Federal Ministry of Education and 595 Research (BMBF), Germany, through its BIOTA project (promotion number: 01 LC 0024A), the German Research 596 Foundation (Project numbers WE 2393/2-1, WE 2393/2-2) and the Max Planck Society. The research was conducted with 597 Northern Cape research permits (No. 22/2008 and 38/2009) and the appendant export permits and lab facilities were 598 provided by Burkhard Büdel at the University of Kaiserslautern and Ulrich Pöschl at the Max Planck Institute for Chemistry 599 in Mainz. JR acknowledges the Research Projects SCIN (PRI-PIMBDV-2011-0874) and POLAR ROCKS (PID2019- 600 105469RB-C21), both funded by the Spanish Ministry of Science, the possibility of obtaining part of the data and analyzing 601 them respectively in the frame of this research. CC acknowledges funding support provided by a NERC Standard Grant 602 (NE/V000764/1) and the Feodor Lynen Research fellowship from the Alexander von Humboldt foundation. 603 Acknowledgments. This research is supported by the University of Hamburg. The research in Linde is funded by 594 Zwillenberg-Tietz Stiftung by a grant to MV. Research in South Africa was funded by the Federal Ministry of Education and 595 Research (BMBF), Germany, through its BIOTA project (promotion number: 01 LC 0024A), the German Research 596 Foundation (Project numbers WE 2393/2-1, WE 2393/2-2) and the Max Planck Society. The research was conducted with 597 Northern Cape research permits (No. 22/2008 and 38/2009) and the appendant export permits and lab facilities were 598 provided by Burkhard Büdel at the University of Kaiserslautern and Ulrich Pöschl at the Max Planck Institute for Chemistry 599 in Mainz. JR acknowledges the Research Projects SCIN (PRI-PIMBDV-2011-0874) and POLAR ROCKS (PID2019- 600 105469RB-C21), both funded by the Spanish Ministry of Science, the possibility of obtaining part of the data and analyzing 601 them respectively in the frame of this research. CC acknowledges funding support provided by a NERC Standard Grant 602 (NE/V000764/1) and the Feodor Lynen Research fellowship from the Alexander von Humboldt foundation. 603 Acknowledgments. This research is supported by the University of Hamburg. The research in Linde is funded by 594 Zwillenberg-Tietz Stiftung by a grant to MV. 5 Conclusions 567 Research in South Africa was funded by the Federal Ministry of Education and 595 Research (BMBF), Germany, through its BIOTA project (promotion number: 01 LC 0024A), the German Research 596 Foundation (Project numbers WE 2393/2-1, WE 2393/2-2) and the Max Planck Society. The research was conducted with 597 Northern Cape research permits (No. 22/2008 and 38/2009) and the appendant export permits and lab facilities were 598 provided by Burkhard Büdel at the University of Kaiserslautern and Ulrich Pöschl at the Max Planck Institute for Chemistry 599 in Mainz. JR acknowledges the Research Projects SCIN (PRI-PIMBDV-2011-0874) and POLAR ROCKS (PID2019- 600 105469RB-C21), both funded by the Spanish Ministry of Science, the possibility of obtaining part of the data and analyzing 601 them respectively in the frame of this research. CC acknowledges funding support provided by a NERC Standard Grant 602 (NE/V000764/1) and the Feodor Lynen Research fellowship from the Alexander von Humboldt foundation. 603 5 Conclusions 567 Therefore, for the generalization of the roles of water 574 types in various climatic zones, a larger number of different organisms at multiple sites needs to be studied. 575 Our study suggests that a better, more detailed understanding of the seasonal variation of physiological traits is necessary, as 576 acclimation may affect the C balance substantially. The season and timing of collecting and monitoring the species should be 577 considered in experimental studies, especially when the characteristics of species are the basis for further analyses and 578 forecasts to estimate the annual carbon budget. Additionally, integration of acclimation of physiological traits in models can 579 improve the accuracy in C balance estimation. 580 Mechanistic models, as an add-on to experimental approaches, are well suited to explore the responses of the C balance of 581 biocrusts to separate environmental factors, and the underlying mechanisms. In turn, models need to be constrained by 582 measurements. As a result, we recommend combining experiments, field investigations, and modelling approaches to acquire 583 a comprehensive understanding from all perspectives of how biocrusts respond to climate and, potentially, future climate 584 change. 585 Code and Data Availability Statement. Source code of the data-driven model, LiBry modelling results, R-scripts to run the 586 analysis in this manuscript are available in Zenodo repository at https://doi.org/10.5281/zenodo.6971250. Field and 587 laboratory data are available in the corresponding publications cited in the manuscript and also from the corresponding 588 author upon request. 589 25 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Author Contributions. YM and PP designed the study, BW, AK, MV and JR provided the observational data. YM did the 590 data processing, ran the model, YM, PP, CC, BW and MB did the data analysis and interpretation, YM wrote the manuscript 591 and all authors revised it. 592 Author Contributions. YM and PP designed the study, BW, AK, MV and JR provided the observational data. YM did the 590 data processing, ran the model, YM, PP, CC, BW and MB did the data analysis and interpretation, YM wrote the manuscript 591 and all authors revised it. 592 Conflict of Interest. The authors have no conflict of interest to declare. 593 Acknowledgments. This research is supported by the University of Hamburg. References 604 Ackermann, K., Zackrisson, O., Rousk, J., Jones, D. L. and DeLuca, T. H.: N2 Fixation in Feather Mosses is a Sensitive 605 Indicator of N Deposition in Boreal Forests, Ecosystems, 15(6), 986–998, doi:10.1007/s10021-012-9562-y, 2012. 606 Bader, M. Y., Zotz, G. and Lange, O. L.: How to minimize the sampling effort for obtaining reliable estimates of diel and 607 annual CO2 budgets in lichens, Lichenologist, 42(1), 97–111, doi:10.1017/S0024282909990338, 2010. 608 Baldauf, S., Porada, P., Maestre, F. T. and Tietjen, B.: Relative humidity predominantly determines long-term biocrust- 609 forming lichen cover in drylands under climate change, J. Ecol., 109(3), 1370–1385, doi:10.1111/1365-2745.13563, 2021. 610 Belnap, J., Phillips, S. L. and Miller, M. E.: Response of desert biological soil crusts to alterations in precipitation frequency, 611 Oecologia, 141, 306–316, doi:10.1007/s00442-003-1438-6, 2004. 612 Belnap, J., Weber, B. and Büdel, B.: Biological Soil Crusts as an organizing principle in drylands, in Biological soil crusts: 613 an organizing principle in drylands, pp. 3–13, Springer International Publishing., 2016. 614 Brostoff, W. N., Sharifi, M. R. and Rundel, P. W.: Photosynthesis of cryptobiotic soil crusts in a seasonally inundated 615 system of pans and dunes in the western Mojave Desert, CA: Field studies, Flora, 200(6), 592–600, 616 doi:10.1016/j.flora.2005.06.008, 2005. 617 Büdel, B., Colesie, C., Green, T. G. A., Grube, M., Lázaro Suau, R., Loewen-Schneider, K., Maier, S., Peer, T., Pintado, A., 618 Büdel, B., Colesie, C., Green, T. G. A., Grube, M., Lázaro Suau, R., Loewen-Schneider, K 618 26 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Raggio, J., Ruprecht, U., Sancho, L. G., Schroeter, B., Türk, R., Weber, B., Wedin, M., Westberg, M., Williams, L. and 619 Zheng, L.: Improved appreciation of the functioning and importance of biological soil crusts in Europe: The Soil Crust 620 International Project (SCIN), Biodivers. Conserv., 23(7), 1639–1658, doi:10.1007/s10531-014-0645-2, 2014. 621 Büdel, B., Williams, W. J. and Reichenberger, H.: Annual net primary productivity of a cyanobacteria-dominated biological 622 soil crust in the Gulf Savannah, Queensland, Australia, Biogeosciences, 15, 491–505, doi:https://doi.org/10.5194/bg-15-491- 623 2018, 2018. 624 Chamizo, S., Cantón, Y., Miralles, I. and Domingo, F.: Biological soil crust development affects physicochemical 625 characteristics of soil surface in semiarid ecosystems, Soil Biol. Biochem., 49, 96–105, doi:10.1016/j.soilbio.2012.02.017, 626 2012. 627 Chamizo, S., Rodríguez-Caballero, E., Moro, M. J. Elbert, W., Weber, B., Burrows, S., Steinkamp, J., Büdel, B., Andreae, M. O. and Pöschl, U.: Contribution of cryptogamic 647 Elbert, W., Weber, B., Burrows, S., Steinkamp, J., Büdel, B., Andreae, M. O. and Pöschl, U 47 References 604 and Cantón, Y.: Non-rainfall water inputs: A key water source for 628 biocrust carbon fixation, Sci. Total Environ., 792, 148–299, doi:10.1016/j.scitotenv.2021.148299, 2021. 629 Coe, K. K., Belnap, J. and Sparks, J. P.: Precipitation-driven carbon balance controls survivorship of desert biocrust mosses, 630 Ecology, 93(7), 1626–1636, doi:10.1890/11-2247.1, 2012. 631 Colesie, C., Green, T. G. A., Haferkamp, I. and Bu, B.: Habitat stress initiates changes in composition , CO 2 gas exchange 632 and C-allocation as life traits in biological soil crusts, ISME J., 8, 2104–2115, doi:10.1038/ismej.2014.47, 2014. 633 Colesie, C., Green, T. G. A., Raggio, J. and Büdel, B.: Summer Activity Patterns of Antarctic and High Alpine 634 Lichendominated Biological Soil Crusts — Similar But Different?, Arctic, Antarct. Alp. Res., 48(3), 449–460, 635 doi:10.1657/AAAR0015-047, 2016. 636 Colesie, C., Büdel, B., Hurry, V. and Green, T. G. A.: Can Antarctic lichens acclimatize to changes in temperature?, Glob. 637 Chang. Biol., 24(3), 1123–1135, doi:10.1111/gcb.13984, 2018. 638 Cowan, I. R., Lange, O. L. and Green, T. G. A.: Carbon-dioxide exchange in lichens: determination of transport and 639 carboxylation characteristics, Planta, 187(2), 282–294, doi:10.1007/BF00201952, 1992. 640 Diez, M., Wachendorf, M., & Stefan, T.: Modelling photosynthesis and carbon fluxes of moss-and lichen-dominated 641 biological soil crusts in temperate dry acid grasslands in Brandenburg, 49th Annual Meeting of the Ecological Society of 642 Germany, Austria and Switzerland, Münster, Germany, doi: 10.13140/RG.2.2.27244.51849, 2019. 643 Dümig, A., Veste, M., Hagedorn, F., Fischer, T., Lange, P., Spröte, R. and Kögel-Knabner, I.: Organic matter from 644 biological soil crusts induces the initial formation of sandy temperate soils, Catena, 122, 196–208, 645 doi:10.1016/j.catena.2014.06.011, 2014. 646 27 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. covers to the global cycles of carbon and nitrogen, Nat. Geosci., 5(7), 459–462, doi:10.1038/ngeo1486, 2012. 648 Evans, R. D. and Lange, O. L.: Biological Soil Crusts and Ecosystem Nitrogen and Carbon Dynamics, in Biological Soil 649 Crusts: Structure, Function, and Management, edited by J. Belnap and O. L. Lange, pp. 263–279, Springer Berlin Heidelberg, 650 Berlin, Heidelberg., 2003. 651 Farquhar, G. D. and von Caemmerer, S.: Modelling of Photosynthetic Response to Environmental Conditions, in 652 Physiological Plant Ecology II, edited by O. L. Lange, pp. 549–587, Springer, Berlin, Heidelberg., 1982. 653 Feng, W., Zhang, Y., Wu, B., Qin, S. IPCC, 2021: Climate Change 2021: The Physical Science Basis. Contribution of Working Group I to the Sixth Assessment 673 Report of the Intergovernmental Panel on Climate Change, edited by: Masson-Delmotte, V., P. Zhai, A. Pirani, S.L. Connors, 674 C. Péan, S. Berger, N. Caud, Y. Chen, L. Goldfarb, M.I. Gomis, M. Huang, K. Leitzell, E. Lonnoy, J.B.R. Matthews, T.K. 675 Maycock, T. Waterfield, O. Yelekçi, R. Yu, and B. Zhou. Cambridge University Press, Cambridge, United Kingdom and 676 New York, NY, USA, In press, doi:10.1017/9781009157896, 2021. 677 References 604 672 y , , , , g , , , , , , g , , , , p , , Dreber, N., Gibreel, T., Grohmann, C., Gröngröft, A., Haensler, A., Hanke, W., A., H., Husted, L. B., N., K. F., M., K., Krug, 663 Dreber, N., Gibreel, T., Grohmann, C., Gröngröft, A., Haensler, A., Hanke, W., A., H., Hu 663 Dreber, N., Gibreel, T., Grohmann, C., Gröngröft, A., Haensler, A., Hanke, W., A., H., Husted, L. B., N., K. F., M., K., Krug, 663 C. B., Labitzky, T., Linke, T., Mager, D., Mey, W., Muche, G., Naumann, C., Pellowski, M 64 Rutherford, M. C., Schneiderat, U., Strohbach, B. J., Vohland, K., Weber, B., Wesuls, D., Wisch, U., Zedda, L., Büdel, B., 665 Rutherford, M. C., Schneiderat, U., Strohbach, B. J., Vohland, K., Weber, B., Wesuls, D., Wisch, U., Zedda, L., Büdel, B., 665 Darienko, T., Deutschewitz, K., Dojani, S., Erb, E., Falk, T., Friedl, T., Kanzler, S.-E., Limpricht, C., Linsenmair, K. E., 666 Mohr, K., Oliver, T., Petersen, A., Rambold, G., Zeller, U., Austermühle, R., Bausch, J., Bösing, B. M., Classen, N., 667 Dorendorf, J., Dorigo, W., Esler, K. J., Etzold, S., G., A., G., L., Hecht, J., Hoyer, P., Kongor, R. Y., Lang, H., Lieckfeld, L. 668 A. B., Oldeland, J., Peters, J., Röwer, I. U., September, Z. M., Sop, T. K., van Rooyen, M. W., Weber, J., Willer, J. and 669 Jürgens, N.: The BIOTA Observatories., in Biodiversity in southern Africa. Volume I: Patterns at local scale - the BIOTA 670 Observatories, edited by N. Jürgens, D. H. Haarmeyer, Luther-Mosebach, D. J., M. J., Finckh, and U. Schmiedel, pp. 6–801, 671 Klaus Hess Publisher, Göttingen & Windhoek., 2010. 672 Darienko, T., Deutschewitz, K., Dojani, S., Erb, E., Falk, T., Friedl, T., Kanzler, S.-E., Limpricht, C., Linsenmair, K. E., 666 Darienko, T., Deutschewitz, K., Dojani, S., Erb, E., Falk, T., Friedl, T., Kanzler, S.-E., Lim 66 Mohr, K., Oliver, T., Petersen, A., Rambold, G., Zeller, U., Austermühle, R., Bausch 667 IPCC, 2021: Climate Change 2021: The Physical Science Basis. Contribution of Working Group I to the Sixth Assessment 673 Report of the Intergovernmental Panel on Climate Change, edited by: Masson-Delmotte, V., P. Zhai, A. Pirani, S.L. Connors, 674 C. Péan, S. Berger, N. Caud, Y. Chen, L. Goldfarb, M.I. Gomis, M. Huang, K. Leitzell, E. Lonnoy, J.B.R. Matthews, T.K. 675 Maycock, T. Waterfield, O. Yelekçi, R. References 604 and Lai, Z.: Influence of Environmental Factors on Carbon Dioxide Exchange in 654 Biological Soil Crusts in Desert Areas, Arid L. Res. Manag., 28(2), 186–196, doi:10.1080/15324982.2013.835006, 2014. 655 Ferrenberg, S., Faist, A. M., Howell, A. and Reed, S. C.: Biocrusts enhance soil fertility and Bromus tectorum growth, and 656 interact with warming to influence germination, Plant Soil, 429(1–2), 77–90, doi:10.1007/s11104-017-3525-1, 2018. 657 Gauslaa, Y., Lie, M., Solhaug, K. A. and Ohlson, M.: Growth and ecophysiological acclimation of the foliose lichen Lobaria 658 pulmonaria in forests with contrasting light climates, Oecologia, 147(3), 406–416, doi:10.1007/s00442-005-0283-1, 2006. 659 Green, T. G. A. and Lange, O. L.: Ecophysiological adaptations of the lichen genera pseudocyphellaria and sticta to south 660 temperate rainforests, Lichenol., 23(3), 267–282, doi:10.1017/S0024282991000427, 1991. 661 , g , p y g p g p yp temperate rainforests, Lichenol., 23(3), 267–282, doi:10.1017/S0024282991000427, 1991. 661 Haarmeyer, D. H., Luther-Mosebach, J., Dengler, J., Schmiedel, U., Finckh, M., Berger, K., Deckert, J., Domptail, S. E., 662 Haarmeyer, D. H., Luther-Mosebach, J., Dengler, J., Schmiedel, U., Finckh, M., Berger, K 62 Haarmeyer, D. H., Luther-Mosebach, J., Dengler, J., Schmiedel, U., Finckh, M., Berger, K., Deckert, J., Domptail, S. E., 662 Dreber, N., Gibreel, T., Grohmann, C., Gröngröft, A., Haensler, A., Hanke, W., A., H., Husted, L. B., N., K. F., M., K., Krug, 663 C. B., Labitzky, T., Linke, T., Mager, D., Mey, W., Muche, G., Naumann, C., Pellowski, M., Powrie, L. W., Pröpper, M., 664 Rutherford, M. C., Schneiderat, U., Strohbach, B. J., Vohland, K., Weber, B., Wesuls, D., Wisch, U., Zedda, L., Büdel, B., 665 Darienko, T., Deutschewitz, K., Dojani, S., Erb, E., Falk, T., Friedl, T., Kanzler, S.-E., Limpricht, C., Linsenmair, K. E., 666 Mohr, K., Oliver, T., Petersen, A., Rambold, G., Zeller, U., Austermühle, R., Bausch, J., Bösing, B. M., Classen, N., 667 Dorendorf, J., Dorigo, W., Esler, K. J., Etzold, S., G., A., G., L., Hecht, J., Hoyer, P., Kongor, R. Y., Lang, H., Lieckfeld, L. 668 A. B., Oldeland, J., Peters, J., Röwer, I. U., September, Z. M., Sop, T. K., van Rooyen, M. W., Weber, J., Willer, J. and 669 Jürgens, N.: The BIOTA Observatories., in Biodiversity in southern Africa. Volume I: Patterns at local scale - the BIOTA 670 Observatories, edited by N. Jürgens, D. H. Haarmeyer, Luther-Mosebach, D. J., M. J., Finckh, and U. Schmiedel, pp. 6–801, 671 Klaus Hess Publisher, Göttingen & Windhoek., 2010. References 604 Yu, and B. Zhou. Cambridge University Press, Cambridge, United Kingdom and 676 New York, NY, USA, In press, doi:10.1017/9781009157896, 2021. 677 28 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Jeffries, D. L., Link, S. O. and Klopatek, J. M.: CO2 fluxes of cryptogamic crusts: I. Response to resaturation, New Phytol., 678 125(2), 163–173, doi:10.1111/j.1469-8137.1993.tb03891.x, 1993. 679 Jeffries, D. L., Link, S. O. and Klopatek, J. M.: CO2 fluxes of cryptogamic crusts: I. Response to resaturation, New Phytol., 678 125(2), 163–173, doi:10.1111/j.1469-8137.1993.tb03891.x, 1993. 679 Lange, O. L.: Photosynthetic productivity of the epilithic lichen Lecanora muralis: Long-term field monitoring of CO2 680 exchange and its physiological interpretation. II. Diel and seasonal patterns of net photosynthesis and respiration, Flora, 198, 681 55–70, doi:10.1078/0367-2530-00100, 2003a. 682 Lange, O. L.: Photosynthetic productivity of the epilithic lichen Lecanora muralis: Long-term field monitoring of CO2 680 exchange and its physiological interpretation. II. Diel and seasonal patterns of net photosynthesis and respiration, Flora, 198, 681 55–70, doi:10.1078/0367-2530-00100, 2003a. 682 Lange, O. L.: Photosynthetic productivity of the epilithic lichen Lecanora muralis: Long-term field monitoring of CO2 683 exchange and its physiological interpretation. III. Diel, seasonal, and annual carbon budgets, Flora, 198(4), 277–292, 684 doi:10.1078/0367-2530-00100, 2003b. 685 Lange, O. L. and Green, T. G. A.: Lichens show that fungi can acclimate their respiration to seasonal changes in temperature, 686 Oecologia, 142(1), 11–19, doi:10.1007/s00442-004-1697-x, 2005. 687 Lange, O. L., Meyer, A., Zellner, H. and Heber, U.: Photosynthesis and Water Relations of Lichen Soil Crusts: Field 688 Measurements in the Coastal Fog Zone of the Namib Desert, Funct. Ecol., 8(2), 253, doi:10.2307/2389909, 1994. 689 Lange, O. L., Reichenberger, H. and Meyer, A.: High thallus water content and photosynthetic CO2 exchange of lichens. 690 Laboratory experiments with soil crust species from local xerothermic steppe formations in Franconia Germany Flecht 691 Lange, O. L., Reichenberger, H. and Meyer, A.: High thallus water content and photosynthetic CO2 exchange of lichens. 690 Laboratory experiments with soil crust species from local xerothermic steppe formations in Franconia, Germany, Flecht. 691 Follmann Contrib. to Lichenol. honour Gerhard Follmann. Geobot. Phytotaxon. Study Group, Univ. Köln, Köln, 139–153, 692 1995. 693 Lange, O. L., Reichenberger, H. and Meyer, A.: High thallus water content and photosynthetic CO2 exchange of lichens. References 604 690 Laboratory experiments with soil crust species from local xerothermic steppe formations in Franconia, Germany, Flecht. 691 Follmann Contrib. to Lichenol. honour Gerhard Follmann. Geobot. Phytotaxon. Study Group, Univ. Köln, Köln, 139–153, 692 Laboratory experiments with soil crust species from local xerothermic steppe formations in Franconia, Germany, Flecht. 691 Laboratory experiments with soil crust species from local xerothermic steppe formation 691 Follmann Contrib. to Lichenol. honour Gerhard Follmann. Geobot. Phytotaxon. Study Group, Univ. Köln, Köln, 139–153, 692 1995. 693 Lange, O. L., Belnap, J., Reichenberger, H. and Meyer, A.: Photosynthesis of green algal soil crust lichens from arid lands in 694 southern Utah, USA: Role of water content on light and temperature responses of CO2 exchange, Flora, 192(1), 1–15, 695 doi:10.1016/S0367-2530(17)30749-1, 1997. 696 Lange, O. L., Belnap, J. and Reichenberger, H.: Photosynthesis of the cyanobacterial soil-crust lichen Collema tenax from 697 arid lands in southern Utah, USA: Role of water content on light and temperature responses of CO2 exchange, Funct. Ecol., 698 12(2), 195–202, doi:10.1046/j.1365-2435.1998.00192.x, 1998a. 699 Lange, O. L., Hahn, S. C., Meyer, A. and Tenhunen, J. D.: Upland tundra in the foothills of the Brooks Range, Alaska, 700 U.S.A.: Lichen long-term photosynthetic CO2 uptake and net carbon gain, Arct. Alp. Res., 30(3), 252–261, 701 doi:10.2307/1551972, 1998b. 702 Ouyang, H., Lan, S., Yang, H. and Hu, C.: Mechanism of biocrusts boosting and utilizing non-rainfall water in Hobq Desert 703 of China, Appl. Soil Ecol., 120, 70–80, doi:10.1016/j.apsoil.2017.07.024, 2017. 704 Phinney, N. H., Gauslaa, Y., Palmqvist, K. and Esseen, P. A.: Macroclimate drives growth of hair lichens in boreal forest 705 canopies, J. Ecol., 109(1), 478–490, doi:10.1111/1365-2745.13522, 2021. 706 29 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Porada, P., Weber, B., Elbert, W., Pöschl, U. and Kleidon, A.: Estimating global carbon uptake by lichens and bryophytes 707 with a process-based model, Biogeosciences, 10, 6989–7033, doi:10.5194/bg-10-6989-2013, 2013. 708 Porada, P., Tamm, A., Kleidon, A., Pöschl, U. and Weber, B.: Global NO and HONO emissions of biological soil crusts 709 estimated by a process-based non-vascular vegetation model, Biogeosciences, 16(9), 2003–2031, 2019. 710 Porada, P., Tamm, A., Kleidon, A., Pöschl, U. and Weber, B.: Global NO and HONO emissions of biological soil crusts 709 estimated by a process-based non-vascular vegetation model, Biogeosciences, 16(9), 2003–2031, 2019. References 604 710 Proctor, M.: Patterns of desiccation tolerance and recovery in bryophytes, Plant Growth Regul., 35(2), 147–156, 711 doi:10.1023/A:1014429720821, 2001. 712 Raggio, J., Pintado, A., Vivas, M., Sancho, L. G., Büdel, B., Colesie, C., Weber, B., Schroeter, B., Lázaro, R. and Green, T. 713 G. A.: Continuous chlorophyll fluorescence, gas exchange and microclimate monitoring in a natural soil crust habitat in 714 Tabernas badlands, Almería, Spain: Progressing towards a model to understand productivity, Biodivers. Conserv., 23(7), 715 1809–1826, doi:10.1007/s10531-014-0692-8, 2014. 716 Raggio, J., Pintado, A., Vivas, M., Sancho, L. G., Büdel, B., Colesie, C., Weber, B., Schro 713 G. A.: Continuous chlorophyll fluorescence, gas exchange and microclimate monitoring 714 Raggio, J., Green, T. G. A., Sancho, L. G., Pintado, A., Colesie, C., Weber, B. and Büdel, B.: Metabolic activity duration can 717 be effectively predicted from macroclimatic data for biological soil crust habitats across Europe, Geoderma, 306, 10–17, 718 doi:10.1016/j.geoderma.2017.07.001, 2017. 719 Raggio, J., Green, T. G. A., Pintado, A., Sancho, L. G. and Büdel, B.: Environmental determinants of biocrust carbon fluxes 720 across Europe : possibilities for a functional type approach, Plant Soil, 429, 147–157 [online] Available from: 721 https://doi.org/10.1007/s11104-018-3646-1, 2018. 722 Reed, S. C., Coe, K. K., Sparks, J. P., Housman, D. C., Zelikova, T. J. and Belnap, J.: Changes to dryland rainfall result in 723 rapid moss mortality and altered soil fertility, Nat. Clim. Chang., 2(10), 752–755, doi:10.1038/nclimate1596, 2012. 724 Rillig, M. C., Ryo, M., Lehmann, A., Aguilar-Trigueros, C. A., Buchert, S., Wulf, A., Iwasaki, A., Roy, J. and Yang, G.: The 725 role of multiple global change factors in driving soil functions and microbial biodiversity, Science, 366(6467), 886–890, 726 doi:10.1126/science.aay2832, 2019. 727 Samolov, E., Baumann, K., Büdel, B., Jung, P., Leinweber, P., Mikhailyuk, T., Karsten, U. and Glaser, K.: Biodiversity of 728 algae and cyanobacteria in biological soil crusts collected along a climatic gradient in chile using an integrative approach, 729 Microorganisms, 8(7), 1–28, doi:10.3390/microorganisms8071047, 2020. 730 Schuur, E. A. G., Crummer, K. G., Vogel, J. G. and MacK, M. C.: Plant species composition and productivity following 731 permafrost thaw and thermokarst in Alaskan tundra, Ecosystems, 10(2), 280–292, doi:10.1007/s10021-007-9024-0, 2007. 732 Tamm, A., Caesar, J., Kunz, N., Colesie, C., Reichenberger, H. and Weber, B.: Ecophysiological properties of three 733 biological soil crust types and their photoautotrophs from the Succulent Karoo , South Africa, Plant Soil, 429, 127–146, 734 doi:10.1007%2Fs11104-018-3635-4, 2018. References 604 735 Tamm, A., Caesar, J., Kunz, N., Colesie, C., Reichenberger, H. and Weber, B.: Ecophysiological properties of three 733 biological soil crust types and their photoautotrophs from the Succulent Karoo , South Africa, Plant Soil, 429, 127–146, 734 doi:10.1007%2Fs11104-018-3635-4, 2018. 735 doi:10.1007%2Fs11104-018-3635-4, 2018. 735 30 https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/bg-2022-179 Preprint. Discussion started: 26 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Tucker, C. L., Ferrenberg, S. and Reed, S. C.: Climatic Sensitivity of Dryland Soil CO2 Fluxes Differs Dramatically with 736 Biological Soil Crust Successional State, Ecosystems, 22(1), 15–32, doi:10.1007/s10021-018-0250-4, 2019. 737 Veste, M. and Littmann, T.: Dewfall and its Geo-ecological Implication for Biological Surface Dewfall and its Geo- 738 ecological Implication for Biological Surface Crusts in Desert Sand Dunes ( North-western Negev , Israel ), J. Arid L. Stud., 739 16(3), 139–147, 2006. 740 Veste, M. and Littmann, T.: Dewfall and its Geo-ecological Implication for Biological Surface Dewfall and its Geo- 738 ecological Implication for Biological Surface Crusts in Desert Sand Dunes ( North-western Negev , Israel ), J. Arid L. Stud., 739 16(3), 139–147, 2006. 740 Veste, M., Heusinkveld, B. G., Berkowicz, S. M., Breckle, S.-W., Littmann, T. and Jacobs, A. F. G.: Dew formation and 741 activity of biological soil crusts, in Arid Dune Ecosystems – The Nizzana Sands in the Negev Desert, Ecological Studies 200, 742 edited by S.-W. Breckle, A. Yair, and M. Veste, pp. 305–318, Springer., 2008. 743 Vivas, M., Pintado, A. and Sancho, L. G.: F v / F m acclimation to the Mediterranean summer drought in two sympatric 744 Lasallia species from the Iberian mountains, Lichenol., 49(2), 157–165, doi:10.1017/S0024282917000032, 2017. 745 Wagner, S., Zotz, G. and Bader, M. Y.: The temperature acclimation potential of tropical bryophytes, Plant Biol., 16(1), 746 117–124, doi:10.1111/plb.12037, 2014. 747 Walker, A. P., Beckerman, A. P., Gu, L., Kattge, J., Cernusak, L. A., Domingues, T. F., Scales, J. C., Wohlfahrt, G., 748 Wullschleger, S. D., Woodward, F. I. and Walker, A. P.: The relationship of leaf photosynthetic traits – V cmax and J max – 749 to leaf nitrogen , leaf phosphorus , and specific leaf area : a meta-analysis and modeling study, Ecol. Evol., 4(16), 3218–3235, 750 doi:10.1002/ece3.1173, 2014. 751 Walker, A. P., Beckerman, A. P., Gu, L., Kattge, J., Cernusak, L. A., Domingues, T. F., Scales, J. C., Wohlfahrt, G., 748 Wullschleger, S. D., Woodward, F. References 604 I. and Walker, A. P.: The relationship of leaf photosynthetic traits – V cmax and J max – 749 to leaf nitrogen , leaf phosphorus , and specific leaf area : a meta-analysis and modeling study, Ecol. Evol., 4(16), 3218–3235, 750 doi:10.1002/ece3.1173, 2014. 751 Weber, B., Graf, T. and Bass, M.: Ecophysiological analysis of moss-dominated biological soil crusts and their separate 752 components from the Succulent Karoo , South Africa, Planta, 236, 129–139, doi:10.1007/s00425-012-1595-0, 2012. 753 Weber, B., Berkemeier, T., Ruckteschler, N., Caesar, J., Heintz, H., Ritter, H. and Braß, H.: Development and calibration of 754 a novel sensor to quantify the water content of surface soils and biological soil crusts, Methods Ecol. Evol., 7(1), 14–22, 755 doi:10.1111/2041-210X.12459, 2016. 756 Wohlfahrt, G., Fenstermaker, L. F. and Arnone III, J. A.: Large annual net ecosystem CO2 uptake of a Mojave Desert 757 ecosystem, Glob. Chang. Biol., 14(7), 1475–1487, doi:10.1111/j.1365-2486.2008.01593.x, 2008. 758 Zhao, Y., Zhang, Z., Hu, Y. and Chen, Y.: The seasonal and successional variations of carbon release from biological soil 759 crust-covered soil, J. Arid Environ., 127, 148–153, doi:10.1016/j.jaridenv.2015.11.012, 2016. 760 Zotz, G., Schultz, S. and Rottenberger, S.: Are tropical lowlands a marginal habitat for macrolichens? Evidence from a field 761 Zotz, G., Schultz, S. and Rottenberger, S.: Are tropical lowlands a marginal habitat for macrolichens? Evidence from a field 761 study with Parmotrema endosulphureum in Panama, Flora, 198(1), 71–77, doi:10.1078/0367-2530-00077, 2003. 762 study with Parmotrema endosulphureum in Panama, Flora, 198(1), 71–77, doi:10.1078/0367-2530-00077, 2003. 762 763 31 31
https://openalex.org/W2149138985	https://academicjournals.org/journal/AJPP/article-full-text-pdf/E1AEFD226932.pdf	English	null	The efficacy of premedication with ibuprofen, gelofen and acetaminophen in the depth of anesthesia in mandibular molars with irreversible pulpitis	African journal of pharmacy and pharmacology	2,013	cc-by	4,235	Accepted 27 May, 2013 Pain control is one of the major aspects in dental practice. Dental pain can usually be controlled using different techniques such as local anesthesia and medications. Acetaminophen is a pain reliever and fever reducer. However, it shows no effect on inflammation. Therefore, non-steroidal anti-inflammatory drugs are currently used to control inflammation and pain. This study sought to compare the efficacy of premedication with ibuprofen, gelofen and acetaminophen in the depth of anesthesia in mandibular molars with irreversible pulpitis. In this double-blind randomized controlled trial, 60 patients with at least one mandibular molar with symptoms of irreversible pulpitis requiring root canal therapy were recruited. Another tooth in the same quadrant was selected as the control. Patients were randomly allocated to one of the following groups: ibuprofen 400 mg, gelophen 400 mg, acetaminophen 325 mg, and placebo 500 mg. The medications were taken 30 min prior to local anesthesia and the cold test and electric pulp test (EPT) were repeated for the test teeth and the control teeth after 10 min with the development symptoms of anesthesia. Access preparation was then initiated and the patients were asked to quantify the level of pain during exposure of the dentin and pulp using a visual analogue scale (VAS). Data was analyzed using analysis of variance (ANOVA), and repeated measure ANOVA, P < 0.05 were deemed significant. There were significant differences between the mean baseline VAS score, and the mean VAS score recorded at the time of dentin and pulp exposure however revealed a significant difference in the VAS score only at the time of dentin exposure among the study groups (P < 0.005). No significant difference was observed in the mean VAS score at the time of pulp exposure among groups (P = 0.076). The EPT was significantly higher after the test compared with the baseline (P = 0.421). Premedication with ibuprofen and gelofen have significant effect in the depth of anesthesia in mandibular molars with irreversible pulpitis, and significantly decreased VAS but placebo and acetaminophen are functionally alike and had no significant effect. Key words: Irreversible pulpitis, pain, root canal therapy. Zahra Sadat Madani1, Azam Haddadi1, Aliakbar Moghadamnia2, Hamideh Alipour3 and Ali Bijani4 1Dental School, Babol University of Medica Sciences, Babol, Iran. 2Pharmacology Department, Babol University of Medical Sciences, Babol, Iran. 3General Dentist, Babol University of Medical Sciences, Babol, Iran. 4Non-communicable pediatric diseases research center, Babol University of Medical Sciences, Babol, Iran. 1Dental School, Babol University of Medica Sciences, Babol, Iran. 2 1Dental School, Babol University of Medica Sciences, Babol, Iran. 2Pharmacology Department, Babol University of Medical Sciences, Babol, Iran. 3General Dentist, Babol University of Medical Sciences, Babol, Iran. 4Non-communicable pediatric diseases research center, Babol University of Medical Sciences, Babol, Iran. 1Dental School, Babol University of Medica Sciences, Babol, Iran. 2Pharmacology Department, Babol University of Medical Sciences, Babol, Iran. 3General Dentist Babol University of Medical Sciences Babol Iran Dental School, Babol University of Medica Sciences, Babol, Iran. 2Pharmacology Department, Babol University of Medical Sciences, Babol, Iran. 3G l D i B b l U i i f M di l S i B b l I , y , , 2Pharmacology Department, Babol University of Medical Sciences, Babol, Iran. 3 2Pharmacology Department, Babol University of Medical Sciences, Babol, Iran. 3General Dentist, Babol University of Medical Sciences, Babol, Iran. gy p , y , , 3General Dentist, Babol University of Medical Sciences, Babol, Iran. General Dentist, Babol University of Medical Sciences, Babol, Iran. 4Non-communicable pediatric diseases research center, Babol University of Medical Scien , y , , 4Non-communicable pediatric diseases research center, Babol University of Medical Sciences, Babol, Iran. , y , , mmunicable pediatric diseases research center, Babol University of Medical Sciences, Bab African Journal of Pharmacy and Pharmacology African Journal of Pharmacy and Pharmacology MATERIALS AND METHODS Moreover, pain and anxiety is associated with increased stress, which may compromise the patient’s health lead- ing to problems such as syncope (Eli, 2003; Maggirias and Locker, 2002). Sudden, severe pain may induce substantial changes in the cardiovascular, respiratory, endocrine and neurogenic systems, resulting in severe medical conditions (Kaviani et al., 2011). Moreover, pain and anxiety is associated with increased stress, which may compromise the patient’s health lead- ing to problems such as syncope (Eli, 2003; Maggirias and Locker, 2002). Sudden, severe pain may induce substantial changes in the cardiovascular, respiratory, endocrine and neurogenic systems, resulting in severe medical conditions (Kaviani et al., 2011). This was adouble-blind randomized controlled clinical trial. The study population consisted of 60 patients presenting to the Endodontics Department in Babol School of Dentistry, Babol, North Iran. The patients were to have at least one mandibular tooth with symptoms of irreversible pulpitis (spontaneous or nocturnal pain). Patients with history of systemic diseases, consumption of any type of analgesic agent at least 12 h prior to the study, any contrain- dication towards the use of analgesic agents, hypersensitivity to lidocaine 2% with 1:80000 epinephrine or any of the infiltration techniques, advanced periodontal diseases and periapical radiolucency associated with the study teeth and teeth with extensive restorations or previous endodontic treatments, were excluded from the study. Patients provided informed consent followed by a complete medical and dental history and clinical examination. ( ) In endodontic therapy, inferior alveolar nerve block (IANB) is the standard method of local anesthesia in the mandibular teeth. Nevertheless, clinical studies have demonstrated a notable range of failure (7 to 77%) for this technique. Moreover, the success rate of anesthesia in teeth with irreversible pulpitis is less than normal pulp (Aggarwal, et al., 2009; Tortamano et al., 2009). Accord- ing to Hargreaves and Keiser (2002), some of the factors which may account for this failure include: anatomic factors, acute anaphylaxis, the effect of inflammation on the pH of the tissue, the effect of inflammation in blood circulation, the effect of inflammation on central hypersensitivity and its effect on the nociceptors. The pulp vitality tests including electric pulp test (EPT) and cold test were then performed for each study tooth and a control tooth from the same quadrant. For the EPT, tooth paste was placed between the tip of the pulp tester (COXO Medical Instrument Co, LTD) and the tooth. MATERIALS AND METHODS The tip of the pulp tester was placed on an area of sound enamel at the incisal third of the tooth where there was no contact with the gingiva, restoration or any cracks in the enamel. As soon as the patient reacted to the stimulus (feeling of heat or tinkling), the test was over. The EPT and cold test results were recorded for each study tooth and the respective control tooth. The patients were asked to determine their level of pain before and after taking the medications based on a visual analogue scale (VAS: 0 cm = no pain, 0 to 3: mild pain (patient can feel the pain, however does not become uncomfortable), 3 to 6: moderate pain (the pain is irritating but tolerable), 6 to 10: severe pain (the pain is intolerable) (Ianiro et al., 2007), > 10: very severe pain). Activation of nociceptors in the presence of inflamma- tion is one of the strongest theories explaining the reduced efficacy of anesthesia. Inflammatory mediators reduce the stimulation threshold in nociceptor neurons to a level at which the slightest stimulators induce a severe neurogenic response (Goodis et al., 2006). This inflammatory process occurs as a result of the production of prostaglandins (PGs) as the end point product of the metabolism of arachidonic acid through the cyclooxygenase pathway (COX). PGs then result in increased sensation of pain by increasing the sensitivity of the nerve endings to bradykinin and histamine (Dray, 1995). The medications included acetaminophen 325 mg (Kharazmi, Iran), ibuporfen 400 mg (Roozdaru, Iran), gelofen 400 mg (Zakaria, Iran), and placebo (capsule of 500 mg glucose). All medications were prepared identically with similar sizes and shapes in the Department of Pharmacology, Babol University of Medical Sciences and were presented to the patients as blue gelatinous capsules. The capsules were placed in identical envelopes and coded, yielding a total of 60 envelopes (four groups of 15 each). Researchers have studied different ways to achieve better anesthesia in teeth with pulpitis. Premedication with analgesic agents has been proposed as one of the alternatives with controversial results. Modarresi et al. (2006) and Parirokh et al. (2010) have reported a considerable improvement in the level of anesthesia when patients receive analgesics prior to IANB for the treatment of teeth with irreversible pulpitis. On the other hand, Ianiro et al. MATERIALS AND METHODS (2007) failed to show any significant difference between the case and control groups, although their observations were suggestive of a tendency toward better clinical results in the medication group. The patients were randomly divided into four groups of 15 and were asked to take one capsule of the same drug group. The patients and the operator were blind to this procedure. After 30 min, one cartridge (1.8 mm) of 2% lidocaine, with 1:80000 epinephrine (Pharmaceutical Mfg.Co. Daroupakhsh, Iran), was injected using the IANB technique. Patients who failed to meet the clinical signs of anesthesia after 10 min, that is tingling in the lower lip and the tip of the tongue, or those who reacted to the stimulus made by an explorer between the lateral incisor and the canine, were excluded. With the advent of signs of anesthesia, EPT and cold test recordings were repeated for the test and study teeth. Access preparation was then initiated and the quality and quantity of pain during dentin and pulp exposure were recorded using the VAS. g p Several other researchers including Aggarwal et al. (2009), Oleson et al. (2010) and Simpson et al. (2011) reported an insignificant improvement in the success of IANB accompanied by premedication with analgesics in treating irreversible pulpitis. Numerous analgesics are used for pain control in endodontics. Non-narcotic analgesics including acetaminophen and non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and gelofen are among these drugs. We aimed to compare the efficacy of premedication with three different analgesics (Ibuprofen 400 mg, gelofen 400 mg, and acetaminophen 325 mg) in the anesthetic depth of IANB in teeth with irreversible pulpitis. g p p p g Data were submitted to the statistical package for social sciences (SPSS) software version 18, and analysis was performed using the following tests: Analysis of variance (ANOVA) and repeated measure ANOVA. P < 0.05 was considered as significant. INTRODUCTION an important aspect of a successful dental procedure. Optimum pain management results in building up trust and facilitates the entire procedure (Walton et al., 2008). Pain is on of the most unpleasant feelings that a human being may experience throughout life. Pain control, especially in the early phases of endodontic treatment, is Corresponding author. E-mail: Haddadi_azam@yahoo.com. 1842 Afr. J. Pharm. Pharmacol. Afr. J. Pharm. Pharmacol. MATERIALS AND METHODS RESULTS This double blind clinical study included 60 patients (30 men and 30 women), with ages ranging between 14 to 55 years (25.69 ± 9.50 years). Table 1 summarizes patients’ age and sex as well as their pain level prior to treatment. Statistical tests failed to show any significant difference in Madani et al. 1843 Table 1. Comparison between the groups in terms of age, sex and pain at baseline. Table 1. Comparison between the groups in terms of age, sex and pain at baseline. Group Placebo Acetaminophen Ibuprofen Gelofen Total P-value Age (mean±SD) 22.80±8.53 24.53±7.44 26.47±10.58 28.80±10.91 25.65±9.50 0.353 Gender eFameF 7 6 10 7 30 0.494 Male 8 9 5 8 30 Pain at baseline (mean±SD) 7.52±2.01 8.18±1.89 6.16±2.40 6.52±1.46 7.09±2.08 0.027* SD = Standard deviation, Significant. Drugs Figure 1. Comparison between baseline VAS scores and VAS scores at the time of dentin and pulp exposure. Group Placebo Acetaminophen Ibuprofen Gelofen Total P-value Age (mean±SD) 22.80±8.53 24.53±7.44 26.47±10.58 28.80±10.91 25.65±9.50 0.353 Gender eFameF 7 6 10 7 30 0.494 Male 8 9 5 8 30 Pain at baseline (mean±SD) 7.52±2.01 8.18±1.89 6.16±2.40 6.52±1.46 7.09±2.08 0.027 SD = Standard deviation, *Significant. Drugs Figure 1. Comparison between baseline VAS scores and VAS scores at the time of dentin and pulp exposure. Figure 1. Comparison between baseline VAS scores and VAS scores at the time of dentin and pulp exposure. any of these factors between the groups. ANOVA reveal- ed a significant difference in the mean VAS scores at baseline and during access preparation (dentin and pulp exposure) between the groups. However, accordingly, this marked difference was only seen at the time of dentin exposure (P = 0.005) and not pulp exposure (P = 0.076). endodontic treatment. The success rate among the placebo, acetaminophen, ibuprofen and gelofen groups was 20, 13.33, 66.66, and 46.66%, respectively. Table 2 presents the success and failure rates of IANB at the time of dentin and pulp exposure among the study groups. The mean EPT values increased in all groups after taking the test medications and the injection, however, accord- ing to the ANOVA and repeated measure ANOVA, this increase was not deemed significant (P = 0.421) (Figure 2). In the acetaminophen group, the mean VAS score showed a reduction rate of 70.4 and 54.52% at the time of dentin and pulp exposure, respectively compared with the baseline. RESULTS In the Ibuprofen group, the reduction rate at the time of dentin and pulp exposure was 100 and 82.14%, respectively; and in the gelofen group, this reduction rate was 100% at the time of dentin exposure and 70.39% at the time of pulp exposure. The reduction rate in the placebo group at the time of dentin exposure was 92.95% and at the time of pulp exposure was 55.85% compared to the baseline (Figure 1). DISCUSSION The present study investigated the effect of premedical- tion with 325 mg dose of acetaminophen, 400 mg dose of gelofen and 400 mg dose of ibuprofen in the depth of anesthesia in mandibular molars with irreversible pulpitis. EPT and cold test were used to evaluate the sensitivity of The success rate of anesthesia was defined by complete elimination of pain or slight pain during the 1844 Afr. J. Pharm. Pharmacol. Table 2. The success and failure rates of IANB at the time of dentin and pulp exposure among the study groups. Group Success (%) Failure (%) Dentin exposure Pulp exposure Placebo 3 (20) 1 (6.66) 11 (73.33) Acetaminophen 2 (13.33) 5 (33.33) 8 (53.33) Ibuprofen 10 (66.66) 0 5 (33.33) Gelofen 7 (46.66) 0 8 (53.33) Drugs Figure 2. Mean EPT values in all groups after taking the test medications and the infiltration. Table 2. The success and failure rates of IANB at the time of dentin and pulp exposure among the study groups. Table 2. The success and failure rates of IANB at the time of dentin and pulp exposure among the study groups. Drugs Figure 2. Mean EPT values in all groups after taking the test medications and the infiltration Figure 2. Mean EPT values in all groups after taking the test medications and the infiltration. after third molar surgical extraction. Mehlisch (2002) stated that in cases of mild to moderate pain, acetami- nophen remains the most appropriate medication of choice. Seymour et al., (1996) studied the efficacy of three doses of ibuprofen (200, 400 and 600 mg) in reducing post surgical pain. Their findings suggested that the 600 mg dose renders more successful pain reduction. Based on the doses used in the literature and the doses available in the pharmaceutical market in Iran, we provided each patient with onecapsule of 325 mg acetaminophen, 400 mg ibuprofen and 400 mg gelofen. Lidocaine 2% with 1:80000 epinephrine is one of the most common local anesthetic agents used in dentistry as well as many other studies (Modaresi et al., 2006; Ianiro et al., 2007; Mikesell et al., 2005; Claffey et al., 2004). Therefore, one cartridge (1.8 ml) of this agent was used. Mikesell et al. (2005) and Claffey et al. (2004) showed no significant difference in the success of IANB using lidocaine and articaine. teeth at base line and after the intervention. REFERENCES Aggarwal V, Jain A, Debipada K (2009). Anesthetic efficacy of supplemental buccal and lingual infiltrations of articaine and lidocaine following an inferior alveolar nerve block in patients with irreversible pulpitis. J. Endod. 35:925–929. Bjornsson G, Haanaes H and Skoglund L(2003a). Naproxen 500 mg bid versus acetaminophen 1000 mg qid:effect on swelling and other acute postoperative events after bilateral third molar surgery. J. clin. pharm. 43:849-858. p Bjornsson G, Haanaes H, Skoglund L (2003b). A randomized, double- blinded crossover trial. paracetamol 1000 mg four times daily vs ibuprofen 600 mg: effect on swelling and other postoperative events after third molar surgery. J Clin Pharm 2003;55:405–412 The mean EPT readings at baseline were 32.87 and 40.46 in the case and control teeth, respectively. These readings increased to 53.22 and 57.37 in teeth with irreversible pulpitis and healthy teeth. These changes may explain why inflammation affects the depth of anesthesia. After taking the placebo agent and the IANB, the mean EPT readings in the control teeth and inflammed teeth was 62.07 and 51.47, respectively. This increase was solely attributed to the anesthetic agent, and the readings in the inflamed teeth marked the failure of local anesthetic agent in inducing a deep anesthesia in the presence of inflammation. Certosimo AJ, Archer RD (1996). A clinical evaluation of the electric pulp tester as an indicator of local anesthesia.Oper Dent. 21(1):25- 30. Claffey E, Reader A, Nusstein J, Beck M, Weaver J (2004). Anesthetic efficacy of articaine for inferior alveolar nerve blocks in patients with irreversible pulpitis. J. Endod. 30(8):568-571 p p ( ) Dray A (1995). Inflammatory mediators of pain. Br. J. Anaesth. 75:125– 131. Eli I, Schwartz-Arad D, Baht R, Ben-Tuvim H (2003). Effect of anxiety on the experience of pain in implant insertion. Clin. Oral Implants Res. 14:115-118. Goodis HE, Poon A, Hargreaves KM (2006). Tissue pH and temperature regulate pulpal nociceptors. J. Dent. Res. 85:1046– 1049. The increase in EPT readings in all groups after taking the premedication is indicative of the effect of premedication in increasing the depth of anesthesia. This increase was observed in all groups including the placebo group, with no significant difference. Modarresi et al. (2006) evaluated the effectiveness of premedication with 400 mg ibuprofen in comparison with acetaminophen codeine (600 mg acetaminophen + 40 mg codeine) 1 h prior to local anesthesia in treating irreversible pulpitis using EPT. DISCUSSION Cold test is a common method to evaluate pulp vitality. Petersson et al. (1999) reported 90% sensitivity for an accurate cold test compared to heat test (83%) and electric pulp test (84%). after third molar surgical extraction. Mehlisch (2002) stated that in cases of mild to moderate pain, acetami- nophen remains the most appropriate medication of choice. Seymour et al., (1996) studied the efficacy of three doses of ibuprofen (200, 400 and 600 mg) in reducing post surgical pain. Their findings suggested that the 600 mg dose renders more successful pain reduction. Certosimo and Archer (1996) demonstrated that EPT is a valuable tool in diagnosing the potential problems associated with local anesthesia in endodontic and restorative procedures. The present findings confirmed this conclusion. Based on the doses used in the literature and the doses available in the pharmaceutical market in Iran, we provided each patient with onecapsule of 325 mg acetaminophen, 400 mg ibuprofen and 400 mg gelofen. Lidocaine 2% with 1:80000 epinephrine is one of the most common local anesthetic agents used in dentistry as well as many other studies (Modaresi et al., 2006; Ianiro et al., 2007; Mikesell et al., 2005; Claffey et al., 2004). Therefore, one cartridge (1.8 ml) of this agent was used. Mikesell et al. (2005) and Claffey et al. (2004) showed no significant difference in the success of IANB using lidocaine and articaine. Belonging to the category of fast-acting analgesics with minimal adverse effects, acetaminophen, gelofen and ibuprofen were chosen to be evaluated in the present study. Researchers have studied the analgesic effects of a wide range of dosages for different medications. Bjornsson et al. (2003a,b) revealed that compared to 500 mg naproxen, premedication with 1000 mg acetamino- phen shows a significant reduction in pain up to 1 h post third molar surgical extraction. They further demonstrated that premedication with 600 mg ibuprofen and 1000 mg acetaminophen have similar effects in pain reduction g In another study by Ianiro et al. (2007), there was no Madani et al. Madani et al. 1845 significant difference in the success of IANB between the group taking 1000 mg acetaminophen and their counterparts taking 1000 mg acetaminophen + 600 mg ibuprofen. deemed insignificant, our observations were suggestive of a greater reduction in pain in the ibuprofen and gelofen group. ABBREVIATIONS On the other hand, Parirokh et al. (2010) and colleagues compared the efficacy of premedication with 600 mg ibuprofen, 75 mg indomethacin and placebo in 150 patients with irreversible pulpitis. Their findings showed that premedication with the test drugs signifi- cantly increased the success of IANB compared to the placebo group (P < 0.01). According to Parirokh et al. (2010), the severity of pulpal inflammation at the time of treatment may also account for the differences in the results. In their study, only patients with asymptomatic irreversible pulpitis (patients with delayed response to cold test and absence of spontaneous pain) were included in the study. In the present study, however, the study population consisted of patients with spontaneous pain who were referred to the endondontics clinic. ANOVA, Analysis of variance; EPT, electric pulp test; IANB, inferior alveolar nerve block; PGs, prostaglandins; COX, cyclooxygenase; NSAIDs, non-steroidal anti- inflammatory drugs; VAS, visual analogue scale. ANOVA, Analysis of variance; EPT, electric pulp test; IANB, inferior alveolar nerve block; PGs, prostaglandins; COX, cyclooxygenase; NSAIDs, non-steroidal anti- inflammatory drugs; VAS, visual analogue scale. DISCUSSION Seven patients in the acetaminophen group, four in the ibuprofen group, seven in the gelofen group, and nine patients in the placebo group received intrapulpal infiltration because of the pain during pulp exposure. Oleson et al. (2010) and Aggarwal et al. (2009) failed to show a significant difference in the success rate of IANB when the anesthesia was accompanied by premedication with two different doses of ibuprofen (600 and 800 mg). Likewise, Simpson et al. (2011) concluded that preme- dication with 800 mg ibuprofen +1000 mg acetaminophen has no significant effect in the success of IANB in patients with symptomatic irreversible pulpitis. Conclusion Premedication with ibuprofen 400 mg and gelofen 400 mg have significant effect in the depth of anesthesia in mandibular molars with irreversible pulpitis, and signifi- cantly decreased VAS, but placebo and acetaminophen 325 are functionally alike and had no significantly effect. Oleson et al. (2010) further stated that while Ibuprofen can inhibit the production of new prostaglandins, early production of other inflammatory mediators and the effects of pre-activated nociceptors are likely responsible for the high failure rate of anesthesia even after taking the analgesic agent. REFERENCES They revealed that both medications increased the depth of anesthesia, with no significant difference. Hargreaves KM, Keiser K (2002). Local anesthetic failure in endodontics: mechanisms and management. Endodontic Topics. 1:26-39. Ianiro SR, Jeansonne BG, McNeal SF, Eleazer PD (2007).The effect of preoperative acetaminophen or a combination of acetaminophen and ibuprofen on the success of inferior alveolar nerve block for teeth with irreversible pulpitis. JOE. 33:11–14. Kaviani N, Khademi A, Ebtehaj I, andMohammadi Z (2011) . The effect of orally administered ketamine on requirement for anesthetics and postoperative pain in mandibular molar teeth with irreversible pulpitis. J. Oral Sci. 53(4):461-465 Maggirias J, Locker D (2002). Psychological factors and perceptions of pain associated with dental treatment. Community Dent Oral Epidemiol. 30:151-159. p g Our findings resembled those of Modarresi et al. (2006). Although the difference between the groups was Afr. J. Pharm. Pharmacol. 1846 Mehlisch D (2002). The efficacy of combination analgesic therapy in relieving dental pain. J. Am. Dent Asso. 133(7):861-877. Simpson M, Drum M, Nusstein J, Reader A, Beck M (2011). Effect of Combination of Preoperative Ibuprofen/Acetaminophen on the Success of the Inferior Alveolar Nerve Block in Patients with Symptomatic Irreversible Pulpitis. JOE. 37, Number 5, May 2011. Mikesell P, Nusstein J, Reader A, Beck M, Weaver J (2005). A comparison of articaine and lidocaine for inferior alveolar nerve blocks. J. Endod. 31(4):265-270. Tortamano IP, Siviero M, Costa CG, Buscariolo IA, Armonia PL (2009). A comparison of the anesthetic efficacy of articaine and lidocaine in patients with irreversible pulpitis. J. Endod.35:165–168. ( ) Modaresi J, Dianat O, Mozayeni MA (2006). The efficacy comparison of ibuprofen, acetaminophen-codeine, and placebo premedication therapy on the depth of anesthesia during treatment of inflamed teeth. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 102:399–403. Walton RE, Reader A, Nusstein JM (2008). Local anesthesia. In: Torabinejad M, Walton RE, eds. Endodontics, Principles and Practice. 4th ed. St Louis, MO: Saunders Elsevier pp. 129–147. Oleson M, Drum M, Reader A, Nusstien J, Beck M (2010). Effect of preoperative ibuprofen on the success of the inferior alveolar nerve block in patients with irreversible pulpitis. J. Endod. 36:379–382. Parirokh M, Ashouri R, RekabiA, Nakhaee N, Pardakhti A, Askarifard S, Abbott PV (2010). The Effect of Premedication with Ibuprofen and Indomethacin on the Success of Inferior Alveolar Nerve Block. JOE. 9:36-39. Petersson K, Soderstrom C, Kiani-Anaraki M, Levy G (1999). REFERENCES Evaluation of the ability of thermal and electrical test to register pulp vitality. Endod. Dent. Traumatol. 15(3):127-131. ( ) Seymour RA, Ward-Booth P, Kelly PJ (1996). Evaluation of different doses of soluble ibuprofen and ibuprofen tablets in postoperative dental pain. Br. J. Oral MaxillofacSurg. 34(1):110-114.
https://openalex.org/W2613304515	https://dergipark.org.tr/tr/download/article-file/381826	Turkish	null	Chronic venous insufficiency and current treatment options	Ortadoğu tıp dergisi	2,017	cc-by	5,683	Öz Kronik venöz yetmezlik (KVY) hem kozmetik sebepler hem de komplikasyonları nedeniyle yaşam kalitesini düşü ren epidemiyolojik ve sosyoekonomik sonuçlarıyla önemli bir sağlık sorunudur. Konservatif tedavi ile semptomları azaltmak/kontrol altında tutmak ve komplikasyonları önlemek hedeflenirken, girişimsel tedavinin hedefi minimum komplikasyonla, venöz hipertansiyon kaynağının yok edilmesi, tüm varikozitlerin eksize edilmesi, tatminkar bir koz metik sonuç sağlamaktır. KVY tedavisi için klasik tedavi yöntemlerinin yanı sıra perkütan uygulamalar da yaygınlaş maya devam etmektedir. Endovenöz lazer ablasyon tedavisi, radyofrekans ablasyon tedavisi, buhar balsyon tedavisi, siyanoakrilat ile ablasyon tedavisi ve sklerozan madde ile ablasyon bu güncel yöntemlerin en bilinenlerindendir. Daha az invaziv olan bu yöntemler daha erken taburculuk ve daha konforlu bir tedavi süreci sunmaktadır. Ancak tek nik açıdan yeni zorluklar ve yüksek maliyetler bu güncel seçeneklerin rutin bir uygulama olmasını engellemektedir. Öte yandan nüks ve başarılı sonuç anlamında kısa ve orta dönem sonuçları literatürde açık cerrahiye benzer olarak bildirilmeye başlamış olsa da henüz uzun dönem sonuçları için net veriler yoktur. Sonuçta, KVY için altın standart hala klasik açık cerrahi teknik olsa da yakın gelecekte değişik perkütan yöntemlerin ağırlık kazanacağı muhtemeldir. AnahtarKelimeler: Kronik venöz yetmezlik skleroterapi endovenöz lazer tedavisi radyofrekans ablasyon siyanoakrilat Anahtar Kelimeler: Kronik venöz yetmezlik, skleroterapi, endovenöz lazer tedavisi, radyofrekans ablasyon, siyanoakrilat ov erleme d ov erleme d Chronic venous insufficiency and current treatment options Mehmet Kabalcı1, Turgut Kültür2 1Kırıkkale Üniversitesi Tıp Fakültesi Kalp ve Damar Hastalıkları AD, Kırıkkale, Türkiye 2Kırıkkale Üniversitesi Tıp Fakültesi Fiziksel Tıp ve Rehabilitasyon AD, Kırıkkale, Türkiye Geliş Tarihi:12.05.2016 Kabul Tarihi:15.05.2017 Doi: 10.21601/ortadogutip Mehmet Kabalcı1, Turgut Kültür2 1Kırıkkale Üniversitesi Tıp Fakültesi Kalp ve Damar Hastalıkları AD, Kırıkkale, Türkiye 2Kırıkkale Üniversitesi Tıp Fakültesi Fiziksel Tıp ve Rehabilitasyon AD, Kırıkkale, Türkiye Doi: 10.21601/ortadogutipdergisi.312129 Doi: 10.21601/ortadogutipdergisi.312129 Geliş Tarihi:12.05.2016 Kabul Tarihi:15.05.2017 Geliş Tarihi:12.05.2016 Kabul Tarihi:15.05.2017 Giriş Kronik venöz yetmezlik (KVY) sıklığının kadınlarda %1- 40 erkeklerde ise %1-27’dir [1] Yıllık artış oranı ise ka dınlarda %2,6 ve erkeklerde %1,9 olarak bildirilmiştir [2] Gelişmiş ve endüstriyel toplumlarda prevalansın daha faz la olduğu bildirilmiştir [3]. Hem kozmetik sebepler hem de komplikasyonları nede niyle yaşam kalitesini düşüren varislerin fizyopatolojisin de büyük oranda venöz yetmezlikler bulunur. Vena safena magna, vena safena parva ve dallarındaki yüzeyel venöz yetmezlikler ve derin venöz sistemle bağlantıyı sağlayan perforan venlerin yetmezlikleri sonucu gelişen venöz hi pertansiyon komplikasyonların oluşumunda önem taşır [4]. Sigara en önemli predispozanlardan biridir [5]. KVY için en önemli predispozan faktörler; genetik yatkınlık, uzun süre ayakta kalma, tromboflebit hikayesi, travma, obezite ve gebeliktir [6]. CEAP sınıflamasına göre en ileri evre olan venöz ülserlerin sıklığı ise %0,3’tür [7]. Venöz ülser tespit edilen hastalarda yapılan birçok çalışmada %60 ci varında perforan venöz yetmezlik gözlenmiştir [8]. Hem kozmetik sebepler hem de komplikasyonları nede niyle yaşam kalitesini düşüren varislerin fizyopatolojisin de büyük oranda venöz yetmezlikler bulunur. Vena safena magna, vena safena parva ve dallarındaki yüzeyel venöz yetmezlikler ve derin venöz sistemle bağlantıyı sağlayan perforan venlerin yetmezlikleri sonucu gelişen venöz hi pertansiyon komplikasyonların oluşumunda önem taşır [4]. En sık karşılaşılan şikayetlerden olan bacak ağrısı, bacakta ödem ve/veya ciltte görünen damarlar nedeniyle kliniğe başvuran hastaların fizik muayenesinde KVY’den şüp he edilmesi durumunda non-invaziv bir test olan doppler USG ile kesin tanı konulur [15]. KVY için geliştirilen ve CEAP olarak bilinen sınıflama bu hastalığın klinik (C), eti yolojik (E), anatomik (A) ve patofizyolojik (P) özellikleri ni tanımlamak için kullanılır [16]. Sigara en önemli predispozanlardan biridir [5]. KVY için en önemli predispozan faktörler; genetik yatkınlık, uzun süre ayakta kalma, tromboflebit hikayesi, travma, obezite ve gebeliktir [6]. CEAP sınıflamasına göre en ileri evre olan venöz ülserlerin sıklığı ise %0,3’tür [7]. Venöz ülser tespit edilen hastalarda yapılan birçok çalışmada %60 ci varında perforan venöz yetmezlik gözlenmiştir [8]. Abstract Chronic venous insufficiency (CVI) is an important health problem due to both epidemiological and socioeconomic out comes that decrease the quality of life due to both cosmetic reasons and complications. When aiming to reduce / control symptoms and prevent complications with conservative treatment, the goal of interventional treatment is to achieve a satisfactory cosmetic result with minimal complications, elimination of venous hypertension, excision of all varicosi ties. In addition to classical treatment methods percutaneous applications comes to be widespread. Endovenous laser ablation therapy, radiofrequency ablation therapy, steam ablation therapy, cyanoacrylate ablation therapy and sclerosing agent ablation are the best known of these current methods. These less invasive methods offer earlier discharge and more comfortable treatment. However, new technical challenges and high costs are the major problem of these new genera tion methods. On the other hand, short and mid-term results in terms of recurrence and successful outcome have been reported in the literature as similar to open surgery, but there is no clear data for long-term results yet. In conclusion, although the gold standard for CVI is still a classic open surgical technique it is likely that different per cutaneous methods will be more popular in the near future. Keywords: Chronic venous insufficiency, sclerotherapy, endovenous laser therapy, radiofrequency ablation, cyanoacrylate Keywords: Chronic venous insufficiency, sclerotherapy, endovenous laser therapy, radiofrequency ablation, cyanoacrylate 191 ORTADOGU MEDICAL JOURNAL 9 (4): 191-197, 2017 Kronik venöz yetmezlik ve güncel tedavi seçenekleri nedeniyle vasküler alandaki sıvının damar dışına kaçma sıyla oluşan ödem daha uzun süreler boyunca devam eder se şekilli elemanlar da ekstravaze olarak hemosiderin de polanması oluşur ve ciltte hiperpigmentasyon gelişir. Daha da uzun süre devam ederse kaşıntılı ve akıntılı dermatit ta kip eder ve tedavi edilmezse venöz ülser oluşumuna kadar ilerleyebilir. Ayrıca venöz obstrüksiyon genellikle bacak kramplarına sebep olabilir [14]. 1. Konservatif Tedavi Venöz ülserin düzenli kompresyon tedavisiyle 6 ayda %90 oranında tam iyileştiğini gösteren çalışmalar kompresyon tedavisinin ne denli önemli ve etkin olduğunu göstermektedir [20]. Ayrıca bu dönemde özellikle hastane de yatarak tedavi gören hastalar için kullanılan eksternal intermitant pnömotik kompresyon cihazı iyileşme sürecini hızlandırmak üzere kullanılan ve bacağı yukarıya doğru sı vazlayarak venöz kanın distalden proksimale doğru ilerle mesini kolaylaştıran eksternal basınç uygulamasıdır [21]. Farmakolojik tedavi venoaktif ve semptomatik ilaçlar olarak ele alınabilir. Venoaktif ilaçlar venotonik etki, an tiinflamatuvar etki, lenfatik drenaj etkisi, hemoreolojik etki ve kapiller kaçağı önleyici etkilerle oluşturmak için kullanılır. Venoaktif ilaçlar ödem, ağrı, yorgunluk hissi ve kramp gibi semptomlar için kullanılsa da venöz yetmezlik/ varisleri yok etme ve/veya kozmetik bir iyileşme gibi bir etkisi yoktur. Bitkisel kaynaklı olan Flavonoidler, hydrok sirutosidler, saponosidler (atkestanesi ekstraktları)’in yanı sıra ve sentetik olarak üretilen triterpen glikozide, okseru tin ve kalsiyum dobesilat en yaygın seçeneklerdir. Ancak yazık ki hiç birinin mucizevi sonuçları yoktur. Dolayısıy la ana tedavi seçeneği gibi düşünmekten ziyade önleyici tedavilere ve kompresyon tedavisine ek olarak kullanımı daha uygundur [11]. Diğer semptomatik ilaçlar arasında en yaygın olarak kullanılanlar temelde stazı azaltmak ve fle biti önlemek üzere tercih edilebilen antiagreganlar ve ağrı şikayetini azaltmak üzere tercih edilebilen analjeziklerdir. 2 Gi i i l T d i P küt C hi Yö t l uygulanan basınç desteğiyle dengelemek temel prensiptir. Ağrı ve cilt değişikliği gelişen hastaların 30-40 mmHg’lik çorapları düzenli olarak kullanımıyla tama yakın iyileşme mümkündür [18]. Fakat ne yazık ki kompresyon çorapları için hasta uyumu yüksek değildir. Bunun nedenleri arasın da ilk sırada sayılabileceklerden biri tedavinin çok uzun sürmesidir. Diğer önemli bir sebep de çorap tedavisinden vazgeçildiğinde hastalığın ve şikayetlerin kısa sürede ye niden eski haline dönmesidir. Üstelik dar kıyafetler bile pek çok insanı rahatsız ederken gün boyu bacağı önemli bir basınçla sarmalayan bir çorabın kabul görmesi kolay değildir. Dolayısıyla en az çorabın etkinliği kadar düşünül mesi gereken diğer bir husus hastanın uyum göstereceği bir çorap seçmektir. Tam gereken basınç olmasa bile biraz daha düşük bir basınç ve/veya tam bacak boyu olmasa bile en azından diz altı bir varis çorabı eğer hasta uyumu sağla yacaksa tedaviye olumlu katkı sağlayacağından tercih edil melidir. Aksi taktirde hiç giyilmeyen en uygun özellikteki çorabın tedaviye hiç katısı olmayacaktır. Netice itibariyle düzenli olarak varis çorabı kullanımı varis tedavisi için en temel yöntemlerin başında gelir [19]. ve aktif ülser (C6) arasındaki geçiş oldukça sık ve tekrarla yan niteliktedir. 1. Konservatif Tedavi KVY tanısı alan hastalar için konservatif ve/veya girişimsel tedavi yöntemleri mevcuttur. Konservatif tedavi ile semp tomları azaltmak/kontrol altında tutmak ve komplikasyon ları önlemek hedeflenir. Basınç artışını ve ödemi kontrol altında tutmak için yürüyüşün ve adımlamanın artırılması, uzun süre ayakta kalmanın azaltılması, bacak elevasyonu, kilo verilmesi, sıcaklıktan kaçınılması gibi temel önlemler konservatif tedavinin ilk aşamasıdır. Proksimal bölgede ba sıncı artıracak dar kıyafetlerden kaçınılması ve sigara kul lanımından uzak durulması da oldukça önemlidir [17]. Baldır kas pompası ve tek yönlü akıma izin veren kapak lar sayesinde her ayak bileği hareketi veya adım atma ile kan akımı distaldan kraniale doğru yer çekimine zıt yönde ve yüzeyden derine doğru seyreder [9]. KVY’yi oluşturan temel patoloji venöz basınçta artış, yani venöz hipertan siyondur [10]. Yüzeyel venler çaplarına göre spider ven (<1 mm), telenjektazi (1-4mm), pake (>4 mm) ve safenöz varisler olarak adlandırılabilir [11]. Damar duvar direnci azalmış venleri dışarıdan destekleme yi ve interstisyel alana sıvı kaçışını azaltmak için kullanı lan kompresyon çorabı tedavisi belki de bu hastalık için önerilebilecek en önemli yöntemdir [11]. Çünkü konjenital olan ve temelde venöz duvar direncinin zayıf olmasından kaynaklanan venöz çap artışı ve/veya venöz valv bozukluk ları hangi tedavi yöntemi kullanılırsa kullanılsın müdahale edilmeyen venlerde var olmaya ve nüksler halinde karşı mıza çıkmaya devam etmektedir. Dolayısıyla tanı konul duğu andan itibaren hastalığın ve patolojisinin ilerlemeden o aşamada kalmasını sağlamak önemli ölçüde kompresyon çorabıyla mümkün olabilmektedir. Venöz hipertansiyonun neden olduğu hidrostatik basınca karşı venleri dışarıdan Venöz hipertansiyonun etiyolojisinde venöz kapakların yetmezliği ve/veya venöz obstrüksiyon vardır. Uzun süre ayakta sabit durmak da bu hipertansiyonu daha da artırır [12]. Perforan venlerde de yetmezlik olması çok daha cid di ambulatuar venöz hipertansiyon sebebidir [10]. Fizik muayene ile hastalarda sadece basit telenjiektazi veya retiküler venler tespit edilebileceği gibi daha ciddi cilt değişiklikleri ve venöz ülserasyonlar da tespit edile bilir [13]. Dilate veya variköz venler nedeniyle özellikle ayakta durmakla artan ve istirahatla azalan ağrı en sık ve tanımlayıcı şikayetlerdendir. Perimalleolar bölgeden baş layan ve ayakta durmakla artan ödem de sık karşılaşılan fizik muayene bulgularındandır. İntravenöz basınç artışı 192 ORTADOĞU TIP DERGİSİ 9 (4): 191-197, 2017 Chronic venous insufficiency and current treatment options ve aktif ülser (C6) arasındaki geçiş oldukça sık ve tekrarla yan niteliktedir. Bu aşamada akut bir etkiden ziyade sıklık la karşılaşılan venöz ülserlerin nükslerini önlemek üzere sigaranın bırakılması önemlidir. Elbette uzun süre ayakta kalmak veya sıcak ortamda kalmak gibi diğer predispo zan faktörlerden uzaklaşmak da nüksleri azaltmak için önemlidir. 1. Konservatif Tedavi Bu aşamada akut bir etkiden ziyade sıklık la karşılaşılan venöz ülserlerin nükslerini önlemek üzere sigaranın bırakılması önemlidir. Elbette uzun süre ayakta kalmak veya sıcak ortamda kalmak gibi diğer predispo zan faktörlerden uzaklaşmak da nüksleri azaltmak için önemlidir. Venöz ülserin düzenli kompresyon tedavisiyle 6 ayda %90 oranında tam iyileştiğini gösteren çalışmalar kompresyon tedavisinin ne denli önemli ve etkin olduğunu göstermektedir [20]. Ayrıca bu dönemde özellikle hastane de yatarak tedavi gören hastalar için kullanılan eksternal intermitant pnömotik kompresyon cihazı iyileşme sürecini hızlandırmak üzere kullanılan ve bacağı yukarıya doğru sı vazlayarak venöz kanın distalden proksimale doğru ilerle mesini kolaylaştıran eksternal basınç uygulamasıdır [21]. Farmakolojik tedavi venoaktif ve semptomatik ilaçlar olarak ele alınabilir. Venoaktif ilaçlar venotonik etki, an tiinflamatuvar etki, lenfatik drenaj etkisi, hemoreolojik etki ve kapiller kaçağı önleyici etkilerle oluşturmak için kullanılır. Venoaktif ilaçlar ödem, ağrı, yorgunluk hissi ve kramp gibi semptomlar için kullanılsa da venöz yetmezlik/ varisleri yok etme ve/veya kozmetik bir iyileşme gibi bir etkisi yoktur. Bitkisel kaynaklı olan Flavonoidler, hydrok sirutosidler, saponosidler (atkestanesi ekstraktları)’in yanı sıra ve sentetik olarak üretilen triterpen glikozide, okseru tin ve kalsiyum dobesilat en yaygın seçeneklerdir. Ancak yazık ki hiç birinin mucizevi sonuçları yoktur. Dolayısıy la ana tedavi seçeneği gibi düşünmekten ziyade önleyici tedavilere ve kompresyon tedavisine ek olarak kullanımı daha uygundur [11]. Diğer semptomatik ilaçlar arasında en yaygın olarak kullanılanlar temelde stazı azaltmak ve fle biti önlemek üzere tercih edilebilen antiagreganlar ve ağrı şikayetini azaltmak üzere tercih edilebilen analjeziklerdir. Egzersiz tedavisi ile baldır kas pompasının aktifleştirilme si ile alt bölgelerde venöz kan birikmesini önlemek hedef lenir. Her kasılma ile venöz kan prokismale doğru pompa lanır ve kas gevşerken yerini distaldeki taze kana bırakmış olur. Ara ara adımlamak veya oturur pozisyondayken ayak bileği hareketi yapmak en kolay ve uygulanabilir baldır kas egzersizleridir. Her yarım saatte birkaç kez yapılacak bu egzersizler venöz dolaşıma ciddi katkı sağlar. Padberg ve ark. [9] baldır kas pompası rehabilitasyonunun ilerle miş KVY vakalarında bile cerrahi ve tıbbi tedaviye eklen mesiyle daha başarılı sonuçlar alınabileceğini bildirmiştir. Hastalarda CEAP sınıflamasına göre son evre sayılan genellikle iç malleol etrafında görülen aktif venöz ülser olması en sadece çok rahatsız edici bir durum değil ayın zamanda tedavi seçeneklerinin de en kısıtlı olduğu durum lardan biridir. Bu aşamada temel prensip genel yara temiz liği yaparak olaya enfeksiyon gibi ek sorunların da eklen mesini engellemek şeklindedir. Antimikrobiyal profilaksi ve/veya maserasyonu önlenmek için tıbbi yara örtüsü kul lanımı gerekebilir [19]. 193 ORTADOGU MEDICAL JOURNAL 9 (4): 191-197, 2017 1. Konservatif Tedavi Ayrıca ülser çevresindeki ödemi azaltmak üzere kompresyon tedavisi ve bacak elevasyonu yara iyileşmesine katkı sağlamaktadır. İyileşmiş ülser (C5) Egzersiz tedavisi ile baldır kas pompasının aktifleştirilme si ile alt bölgelerde venöz kan birikmesini önlemek hedef lenir. Her kasılma ile venöz kan prokismale doğru pompa lanır ve kas gevşerken yerini distaldeki taze kana bırakmış olur. Ara ara adımlamak veya oturur pozisyondayken ayak bileği hareketi yapmak en kolay ve uygulanabilir baldır kas egzersizleridir. Her yarım saatte birkaç kez yapılacak bu egzersizler venöz dolaşıma ciddi katkı sağlar. Padberg ve ark. [9] baldır kas pompası rehabilitasyonunun ilerle miş KVY vakalarında bile cerrahi ve tıbbi tedaviye eklen mesiyle daha başarılı sonuçlar alınabileceğini bildirmiştir. Hastalarda CEAP sınıflamasına göre son evre sayılan genellikle iç malleol etrafında görülen aktif venöz ülser olması en sadece çok rahatsız edici bir durum değil ayın zamanda tedavi seçeneklerinin de en kısıtlı olduğu durum lardan biridir. Bu aşamada temel prensip genel yara temiz liği yaparak olaya enfeksiyon gibi ek sorunların da eklen mesini engellemek şeklindedir. Antimikrobiyal profilaksi ve/veya maserasyonu önlenmek için tıbbi yara örtüsü kul lanımı gerekebilir [19]. Ayrıca ülser çevresindeki ödemi azaltmak üzere kompresyon tedavisi ve bacak elevasyonu yara iyileşmesine katkı sağlamaktadır. İyileşmiş ülser (C5) . Girişimsel Tedavi: Perkütan ve Cerrahi Yöntemler 2. Girişimsel Tedavi: Perkütan ve Cerrahi Yöntemler Girişimsel tedavinin hedefi minimum komplikasyonla, venöz hipertansiyon kaynağının yok edilmesi, tüm vari kozitelerin eksize edilmesi, tatminkar bir kozmetik sonuç sağlamaktır [22]. Hastaların ağrı ve rahatsızlık hissi gibi subjektif şikayetlerinin yanı sıra, USG’de izlenen valsal va manevrası ile uzun süreli reflü ve kapak yetmezliği ve venöz hipertansiyon bulguları girişimsel bir müdahale için endikasyon olsa da yöntem seçiminin hastaya göre karar verilmesi gerekmektedir [23]. 193 ORTADOGU MEDICAL JOURNAL 9 (4): 191-197, 2017 Kronik venöz yetmezlik ve güncel tedavi seçenekleri Çap artışı ve/veya valvüler yapısı bozulmuş venlerin ona rımı ne yazık ki mümkün değildir. Ancak oldukça yoğun bir ağ özelliği taşıyan venöz sistemden birkaç damarın eksilmesi venöz sirkülasyon açısından herhangi bir olum suzluk oluşturmaz. Dolayısıyla KVY için girişimsel nite likteki müdahalelerin hemen tümünde temel prensip hasar görmüş veya fonksiyonu bozulmuş yüzeyel venlerin sis temden çıkarılması şeklindedir. oluşturulan hasarın intima ve az miktarda medya tabakası ile internal elastik membranı da etkileyip adventisya taba kasının korunması hedeflenir [33]. EVLT VSM için yaygın olarak kullanılmasına rağmen, VSP ve perforan ven yet mezliklerinde kullanımı sık değildir. Girişim yeri olarak en sık yetmezlik bulunan safen venin hemen distali veya diz seviyesi tercih edilir. Diz bölgesinde hem ven çapı daha geniş ve düzgün seyirlidir hem de sinir hasarı riski daha azdır. USG eşliğinde iğne ponksiyonu ile kılavuz tel ven lümeninden SFJ’ye kadar ilerletilir. Daha sonra yine USG kontrolünde perivenöz kılıf içine izotonik mayi içinde ad renalin, bikarbonat ve lidokainden oluşan karışım, tümesan anestezik olarak uygulanır. Tümesan anestezi hem ağrıyı azaltmak hem de perivenöz dokuyu soğutmak ve daha iyi enerji transferi sağlamak için kullanılır [34]. Anestezi son rası kılavuz tel üzerinden termal enerjiyi dokuya ileten prob ilerletilir. EVLT için fiber optik uç, RF için radyo dalgaları üreten başlık, buhar enerjisi yöntemi için buhar başlığı gibi uçlar hedef bölgeye ulaştırılır. Bu aktif ucun SFJ’nin 1-2 cm distaline yerleştirilmesi çok önemlidir. Hem USG, hem de ciltten görülebilen kılavuz ışık probun konumunu belir lemeye yardımcı olur. Son olarak enerji kaynağı jeneratörü çalıştırılır ve kontrollü olarak intimal termal hasarın oluş ması sağlanır. Oluşturulan termal hasar, enerjinin uygula ma modu (sürekli veya aralıklı mod), jeneratör gücü (watt), akım süresi ve yüzey alanı başına uygulanan enerji (J/cm² ve J/cm) gibi parametrelerle ayarlanabilmektedir [35]. EVLT yönteminde lazer jeneratörü tarafından üretilen ışık enerjisi fiberoptik kablolar üzerinden dokuya iletilir[32]. RF yönteminde ultrasonik ses dalgaları bir prob ucunda ısıya dönüşerek dokuya iletilir. 85-90 oC ısı ile öncelikle subendotelyal kollajen doku hasarı hedeflenir [36]. . Girişimsel Tedavi: Perkütan ve Cerrahi Yöntemler Özellikle C5-C6 seviye lerindeki hastalar için önerilen bu seçenek maalesef uzun dönem sonuçları açısından yeterli başarıyı sağlayamamış ve güncel literatürler ışığında rutin bir seçenek değildir. Ancak seçilmiş hastalarda olumlu sonuçlar elde edildiği ni bildiren çalışmalar da mevcuttur [51]. Yine derin venöz sistem olaya dahil olduğunda özellikle proksimal venler deki darlıklar da olaya eşlik ettiğinde venöz geri dönüşü kolaylaştırmak adına darlık olan bölgede balonla dilate etme ve hatta stent yerleştirme gibi perkütan müdahaleler söz konusu olabildiği gibi bu yöntemlerden biriyle kalıcı başarı sağlanamadığında venöz baypas prosedürleri söz konusu olabilmektedir. Tek taraflı iliak ven tıkanıklıkla rında hasta olmayan taraf safen ven kullanılarak yapılan femorofemoral venöz bypass (Palma prosedürü) bu yön teme bir örnektir. Ancak hem venöz çapların geniş ve ve nöz akımın oldukça yavaş olmasından dolayı restenoz ve tromboz riskinin yüksek olması hem de venöz yapıdaki bir Yukarıda sözü edilen tedaviler daha çok yüzeyel venöz yet mezlik durumunda endikedir. Eğer derin venöz yetmezlik varsa yüzeyel venöz yetmezlik olsun olmasın yüzeyel ven dolaşımını derin sisteme daha çok yönlendirecek tedavi ler tabloyu daha da ağırlaştırır. Böyle bir durumda bilinen en başarılı tedavi kompresyon tedavisidir. Ayrı ayrı her iki sistemin de daha efektif çalışmasını ve kollabe olmuş venlerde staza maruz kalmış venöz kanın sirkülasyonunu sağlar. Öte yandan bu konservatif yönteme ilaveten yüze yel ve derin venöz sistemin distaldeki ilişkisini koparmak üzere, bazı perforan venlerin ligasyonunu anlamına gelen Linton ameliyatları geniş doku hasarına neden olduğu için yaygın kullanım alanı bulamamıştır. Aynı operasyonel tek niğin endoskopik olarak uygulandığı görece daha yaygın olan subfasiyal endoskopik perforan cerrahisi (SEPS) de rutin bir cerrahi seçenek değildir. Özellikle C5-C6 seviye lerindeki hastalar için önerilen bu seçenek maalesef uzun dönem sonuçları açısından yeterli başarıyı sağlayamamış ve güncel literatürler ışığında rutin bir seçenek değildir. Ancak seçilmiş hastalarda olumlu sonuçlar elde edildiği ni bildiren çalışmalar da mevcuttur [51]. Yine derin venöz sistem olaya dahil olduğunda özellikle proksimal venler deki darlıklar da olaya eşlik ettiğinde venöz geri dönüşü kolaylaştırmak adına darlık olan bölgede balonla dilate etme ve hatta stent yerleştirme gibi perkütan müdahaleler söz konusu olabildiği gibi bu yöntemlerden biriyle kalıcı başarı sağlanamadığında venöz baypas prosedürleri söz konusu olabilmektedir. Tek taraflı iliak ven tıkanıklıkla rında hasta olmayan taraf safen ven kullanılarak yapılan femorofemoral venöz bypass (Palma prosedürü) bu yön teme bir örnektir. . Girişimsel Tedavi: Perkütan ve Cerrahi Yöntemler Hipertonik sodyum klor solüsyonu, deterjanlar (sodyum tetradisil sülfat) gibi sklerozanlar var olsa da Türkiye de dahil en sık kullanı lan skleroterapotik ajan ise polidokanoldür [17]. Sklerozan maddelerin uygun olmayan yüksek konsantrasyonlarda kullanımı doku nekrozuna yol açabilir [39]. Nadir de olsa ortaya çıkabilen geçici ödem, ekimoz, ağrı, tromboflebit, hiperpigmentasyon, cilt nekrozu, DVT ve anafilaksi gibi komplikasyonlar bu tedavinin olumsuz yanlarındandır [22]. Başta siyanoakrilat olmak üzere çeşitli adheziv maddeler ile doku yapışıklıkları oluşturarak lümeni kapatmak veya kan kışını önleyecek şekilde lümeni doldurmak da diğer perkütan yöntemlerdendir [40]. Bütün bu yöntemlerin birbirine değişik üstünlükleri olduğu nu [37, 41- 43] veya aralarında belirgin fark olmadığını [44] gösteren çok sayıda yayın mevcuttur. Hatta bu yöntemle i ti hi l ü tü l [31 45] b Yüksek ligasyon ve skleroterapi kombinasyonu kozmetik avantajları ve daha düşük postoperatif morbiditeye sebep olması gibi nedenlerle tercih edilebilir. Hipertonik sodyum klor solüsyonu, deterjanlar (sodyum tetradisil sülfat) gibi sklerozanlar var olsa da Türkiye de dahil en sık kullanı lan skleroterapotik ajan ise polidokanoldür [17]. Sklerozan maddelerin uygun olmayan yüksek konsantrasyonlarda kullanımı doku nekrozuna yol açabilir [39]. Nadir de olsa ortaya çıkabilen geçici ödem, ekimoz, ağrı, tromboflebit, hiperpigmentasyon, cilt nekrozu, DVT ve anafilaksi gibi komplikasyonlar bu tedavinin olumsuz yanlarındandır [22]. uygulanabilmesi mini flebektominin önemli avantajlarıdır. Ancak diğer perkütan yöntemlerle de ilgili alana müdahale etmenin mümkün olabildiğini bildiren bazı çalışmalar var dır [47,48]. Yine de lokal tedavi olarak kullanılan en yaygın perkütan yöntem olan skleroterapinin hemen hiçbir anestezi ve ileri teknolojik ekipman gerektirmemesi en azından şim dilik skleroterapiyi rakipsiz kılmaktadır. Üstelik “Tessari yöntemi”olarak bilinen teknikle köpük oluşturulduğunda 4 kat daha etkili bir oklüzyon sağlanabilir [49,50]. Yukarıda sözü edilen tedaviler daha çok yüzeyel venöz yet mezlik durumunda endikedir. Eğer derin venöz yetmezlik varsa yüzeyel venöz yetmezlik olsun olmasın yüzeyel ven dolaşımını derin sisteme daha çok yönlendirecek tedavi ler tabloyu daha da ağırlaştırır. Böyle bir durumda bilinen en başarılı tedavi kompresyon tedavisidir. Ayrı ayrı her iki sistemin de daha efektif çalışmasını ve kollabe olmuş venlerde staza maruz kalmış venöz kanın sirkülasyonunu sağlar. Öte yandan bu konservatif yönteme ilaveten yüze yel ve derin venöz sistemin distaldeki ilişkisini koparmak üzere, bazı perforan venlerin ligasyonunu anlamına gelen Linton ameliyatları geniş doku hasarına neden olduğu için yaygın kullanım alanı bulamamıştır. Aynı operasyonel tek niğin endoskopik olarak uygulandığı görece daha yaygın olan subfasiyal endoskopik perforan cerrahisi (SEPS) de rutin bir cerrahi seçenek değildir. . Girişimsel Tedavi: Perkütan ve Cerrahi Yöntemler Buhar enerjisi yönteminde buhar jeneratörü tarafından oluşturu lan su buharı özel bir kateterden geçerek hedef dokuya iler letilir [37]. Operasyon sonrası rutin olarak elastik bandaj veya orta basınçlı kompresyon çorabı önerilmektedir. Bu prosedürler nedeniyle, safen sinir hasarına bağlı parestezi, ağrı, yüzeyel tromboflebit, DVT, yüzeyel dokuda yanık ha sarı, ekimoz, hematom gibi sorunlar ortaya çıkabilir [32]. Köpük tedavisi olarak da bilinen sklerozan maddenin ha vayla karıştırılarak ilgili vene uygulanması geçmişi daha eskiye dayanan ve perkütan uygulanan bir yöntemdir [38]. Cerrahi girişim önerilen hastaların %70’inde SFJ’de reflü mevcuttur [24]. Rekürren varikozitlerin birinci sebebi safeno femoral bileşkeye yeterli ve uygun olmayan yaklaşımdır [25]. Sadece derin ve yüzeyel venöz sistemi biribirine bağlayan ana yapı olan SFJ yok edilerek VSM üzerindeki volüm yükü azaltılmaya çalışıldığı yönteme “ligasyon” yada “yüksek ligasyon” adı verilir. Bu yöntemde yüzeydeki venöz akım perforan venler üzerinden derin venöz sisteme akmaya de vam etmektedir. Böylece daha sonraki muhtemel arteriyel bypass operasyonları için greft kaynağı olma özelliğini de korur. Oldukça basit ve pratik bir yöntem olsa da nüks ora nının diğer tekniklere göre daha yüksek olması (5 yıl içinde %42) [26] hastaları ve cerrahları başka arayışlara itmiştir. Daha sonra yüzeyel venöz sistemin ana yapıları olan VSM ve VSP aşırı rahatlıkla gözden çıkarılmıştır. Böylece ligas yona ilaveten bütün safen venlerin bir tel vasıtasıyla çı karıldığı cerrahi yöntem olan “stripping” tarif edilmiştir. Bu yöntemin CEAP sınıf 2-4 evrelerinde endike olan bu yöntem venöz hemodinamiye, derin venöz reflünün azal tılmasına, ülserin iyileşmesine katkı sağlar [27-29]. Nüks oranları ligasyon yöntemine göre yaklaşık %20 daha az [30] olmasına rağmen hastalığın genel karakteri yüzünden sıfırlanmış değildir. Teknolojinin ilerleyip yeni enstrumanla rın devreye girmesiyle safen venlerden başka yollarla kurtul manın da kapısı açılmıştır. İntimada ısı veya sklerozan mad de ile hasar oluşturmak veya yapıştırıcı maddeler ile lümeni kapatmak veya doldurmak bu yeni nesil yöntemlerdendir. Radyofrekans (RF ablasyon), lazer (EVLT: endo venöz la zer tedavisi) veya buhar enerjileri geniş kullanım alanı bul muş termal ablasyon yöntemleridir. Termal hasar ile direkt ve indirekt skar oluşumu, oklüzyon ve fibrozis sağlanır [31]. EVLT safenofemoral junction ve safen vende endotel ha sarı oluşturarak venöz oklüzyon sağlar [32]. Lazer, rad yofrekans veya diğer termal hasar oluşturan teknikler ile 194 ORTADOĞU TIP DERGİSİ 9 (4): 191-197, 2017 Chronic venous insufficiency and current treatment options Yüksek ligasyon ve skleroterapi kombinasyonu kozmetik avantajları ve daha düşük postoperatif morbiditeye sebep olması gibi nedenlerle tercih edilebilir. . Girişimsel Tedavi: Perkütan ve Cerrahi Yöntemler Ancak hem venöz çapların geniş ve ve nöz akımın oldukça yavaş olmasından dolayı restenoz ve tromboz riskinin yüksek olması hem de venöz yapıdaki bir Bütün bu yöntemlerin birbirine değişik üstünlükleri olduğu nu [37, 41- 43] veya aralarında belirgin fark olmadığını [44] gösteren çok sayıda yayın mevcuttur. Hatta bu yöntemle rin rutin cerrahiye olan üstün yanlarını [31,45] veya benzer olduğunu [46] gösteren yayınlar da mevcuttur. Yeni nesil yöntemlerin en önemli avantajı elbette perkütan uygula nabilmeleridir. Ancak geleneksel yöntemlere nazaran daha yüksek maliyetleri ve daha çok teknolojik altyapıya ihtiyaç duymaları ise en önemli dezavantajlarıdır. Üstelik neredey se bin yıllık geçmişi olan geleneksel yöntemlerin iyi bilinen uzun dönem sonuçlarına nazaran hemen hiçbirinin uzun dö nem sonuçları ve muhtemel komplikasyonları net değildir. Hem geleneksel hem de yeni nesil yöntemlerin ortak sorun lu kısmı ise kümülatif yaklaşımlarıdır. Mümkün olduğunca spesifik ve hedefe yönelik olarak tanımlanabilecek modern tıp anlayışına bu anlamda uygun değillerdir. Yani eğer ta miri mümkün değilse en azından sadece hasarlı kısımdan kurtulmak mümkünse neden tüm sistemden vazgeçmek ge rektiği sorusuna tatminkar bir cevap verememektedirler. Tanımlanışı oldukça eskiye dayanan ve daha çok kozmetik maksatlarla yapılan “mini flebektomi” denilen cerrahi yön temle de lokal olarak sadece hasar görmüş bölgedeki venler tığ benzeri aletlerle birkaç mm’lik kesilerle çıkarılabilmek tedir. Çok zaman kombine tedavi olarak VSM strippingi ne veya ablasyon yöntemlerine ilave olarak da uygulanır. Perkütan yöntemle yarışacak kadar küçük izler bırakması ve sağlam venleri yerinde bırakması ve lokal anesteziyle 195 ORTADOGU MEDICAL JOURNAL 9 (4): 191-197, 2017 Kronik venöz yetmezlik ve güncel tedavi seçenekleri 10. Kaira M, Gloviczki P. Endoscopic subfascial ligation of perfora ting veins. In: Ascher E, Hollier LH, Strandness DE Jr, Towne JB, Calligaro K, Kent KC, et al, eds. Haimovici’s vascular surgery. 5th ed. New York: Wiley-Blackwell; 2003. p. 1115-29. daralmanın distaldeki venöz hipertansiyonun gerçek sebe bi olduğunun objektif olarak ortaya konamaması her iki tedaviyi de çok tartışmalı bir hale getirmektedir. Kısa bir segmentte kapak yetmezliği olması durumun da açık yöntemle kapak tamiri veya eksternal destek uy gulanması yada başka bir venden alınan sağlam kapağın transpozisyonu yöntemleri tanımlanmıştır. Fakat rekürren tromboz, ve açıklık oranlarının düşük olması ve ve özel beceri gerektirmesi rutin kullanıma girmesini önlemiştir. 11. Turkish Association of Cardiovascular Surgery Peripheral Artery and Venous Disease Treatment Guide. (Türk Kalp Damar Cerra hisi Derneği Periferik Arter ve Ven Hastalıklari Tedavi Kılavuzu). Ankara: Öncü Basımevi 2008:97-123. 12. Pappas PJ, Duran WN, Hobson RW. Kaynaklar 16. Porter JM, Moneta GL. Reporting standards in venous disease: an update. International Consensus Committee on Chronic Venous Disease. J Vasc Surg 1995;21:635-45. 1. Yung-Feng Lo, Chih-Hsun Yang. Stripping And Ligation Of The Saphenous Vein. Semin Cutan Med Surg 2005;24:200-8. 1. Yung-Feng Lo, Chih-Hsun Yang. Stripping And Ligation Of The Saphenous Vein. Semin Cutan Med Surg 2005;24:200-8. 17. Köksal C, Alsalehi S, Kocamaz Ö, Sunar H. Treatment of Chronic Venous Insufficiency (Kronik venöz yetmezlik tedavisi). Kosuyo lu Heart J 2010;13:28-33. 2. Nicolaidesan, Allegra C, Bergan J, Bradbury A, Cairols M, Car pantier P And Et Al. Management Of Chronic Venous Disorders Of The Lower Limbs: Guidlines According To Scientific Evi-Den ce. Int Angiol 2008;27:1-59. 18. Motykie GD, Caprini JA, Arcelus JI, Reyna JJ, Overom E, Mokhtee D. Evaluation of therapeutic compression stockings in the treatment of chronic venous insufficiency. Dermatol Surg 1999;25:116-20. 3. Beaglehole R, Prior Ia, Salmond Ce, Davidson F. Varicose Veins İn The Southpacific Int J Epidemiol 1975; 4:295–299. 4. Scott Te, Lamorte Ww, Gorin Dr, Menzoian Jo. Risk Factors For Chronic Venous İnsufficiency: A Dual Case-Control Study. J Vasc Surg 1995;22:622-8. 19. Karlsmark T, Agerslev RH, Bendz SH, Larsen JR, Roed-Pe-tersen J, Andersen KE. Clinical performance of a new silver dressing, Contreet Foam, for chronic exuding venous leg ulcers. J Wound Care. 2003;12:351-4. 5. Petitti DB, Wingerd J, Pellegrin F, Ramcharan S. Oral Contracep tives, Smoking and Other Factors In Relation To Risk Of Venous Thromboembolic Disease. Am J Epidemiol 1978;480-5. 20. Mayberry JC, Moneta GL, Taylor LM, Porter JM. Fifteen year results of ambulatory compression therapy for chronic venous ul cers. Surgery 1991;109:575-81. 6. Allegra C, Antignani Pl, Carlizza A. Recurrent Varicose Veins Following Surgical Treatment: Our Experience With Five Years Follow-Up. Eur J Vasc Endovasc Surg 2007;33:751-6. 21. Margolis DJ, Bilker W, Santanna J, Baumgarten M. Venous leg ul cer: incidence and prevalence in the elderly. J Am Acad Dermatol 2002;46:381-6. 7. Fowkes Fg, Evans Cj, Lee Aj. Prevalence And Risk Factors For Chronic Venous İnsufficiency. Angiology 2001; 52:5-15. 22. Bergan JJ. Varicose veins, treatment by surgery and sclerotherapy. Rutherford. RB. Vascular surgery 2000;2007-20. 8. Gloviczki P, Rhodes Jm. Management Of Perforating Vein İn competence. In: Rutherford Rb, Editor. Rutherford’s Textbook On Vascular Surgery. 5th Ed. Philadelphia: W. B. Saunders; 2000. P.2021-36. 23. Bergan JJ. Varicose veins: treatment by intervention including scle rotherapy. In: Rutherford RB. Vascular Surgery. Sixth edition, volume two. . Girişimsel Tedavi: Perkütan ve Cerrahi Yöntemler Pathology and cellular physio logy of chronic venous insufficiency.In Gloviczki P, Yao JS, eds. Hand book of Venous Disorders 2nd ed. New York, NY: Arnold; 2001:58-67. Sonuç olarak; EVLT, RF, Steam, Mikrodalga gibi termal ablasyon tedavilerinin, sklerozan madde ile sağlanan kim yasal ablasyon yöntemlerinin, adheziv madde ile oklüzyon sağlanan yöntemlerin henüz uzun takip sonuçları açık ve net olmasa da bir kısmının kısa ve orta vadede klasik cer rahi yöntemlere benzer nüks, ve başarılı sonuç oranları bu yeni nesil ve daha az invaziv metodları artık kabul edilebi lir seçenekler arasına sokmuştur. 13. Chiesa R, Marone EM, Limoni C, Volontè M, Petrini O. Chronic venous disorders: correlation between visible signs, symptoms and presence of functional disease. J Vasc Surg 2007;46:322. 14. Shami SK, Sarin S, Cheatle TR, et al. Venous ulcers and the süper fisyal venous system. J Vasc Surg 1993;17:87-490. 15. Marston WA. PPG, APG, duplex: which noninvasive tests are most appropriate for the management of patients with chronic ve nous insufficiency? Semin Vasc Surg 2002;15: 13-20. Kaynaklar Philadelphia: WB Saunders Co; 2005; Section 21;157:2251-67. 9. Padberg F. The Physiology And Hemodynamics Of The Normal Venous Circulation.In Gloviczki P, Yao Js, Eds. Handbook Of Ve- Nous Disorders. 2nd Ed. New York, Ny: Arnold; 2001:25-35. 24. Garen G,Yellin AE, Primary varicose veins Topographic and he modynamic correlations. J Cardiovasc Surg 1990:31;672-9. 196 ORTADOĞU TIP DERGİSİ 9 (4): 191-197, 2017 196 ORTADOĞU TIP DERGİSİ 9 (4): 191-197, 2017 196 ORTADOĞU TIP DERGİSİ 9 (4): 191-197, 2017 Chronic venous insufficiency and current treatment options 25. Lapropoulos N, Touloupulos E, GiannoulesAD et al. Recur-rent varicose veins; investigations of the pattern and extent of ref-lux with color-şow duplex scanning. Surgery 1996:119;406-10.9. 40. Yasim A, Eroglu E, Bozoglan O, Mese B, Acipayam M, Kara H. A new non-tumescent endovenous ablation method for varicose vein treatment: Early results of N-butyl cyanoacrylate (VariClose®). Phlebology, 2016; 0268355516638577. 26. Recek C . Saphenofemoral junction ligation supplemented by pos toperative sclerotherapy: a review of long term clinical and he modynamic results. Vasc Endovasc Surg 2004;38:533-40. 41. Koramaz İ, Kılıç H, Gökalp F, Bitargil M, Bektaş N, Engin E, Bozkurt AK. Ablation of the great saphenous vein with nontumes cent n-butyl cyanoacrylate versus endovenous laser therapy. J Vasc Surg Venous and Lymphatic Disorders 2017. 27. Sarin S, Scurr JH, Coleridge Smith PD. Stripping of the long sap henous vein in the treatment of primary varicose veins. Br J Surg 1994;81:1455-8. 42. Gale SS, Lee JN, Walsh ME, Wojnarowski DL, Comerota AJ. A randomized, controlled trial of endovenous thermal ablation using the 810-nm wavelength laser and the ClosurePLUS radiofrequ ency ablation methods for superficial venous insufficiency of the great saphenous vein. J Vasc Surg 2010;52:645-50. 28. Padberg FT Jr, Pappas PJ, Araki CT, Back TL, Hobson RW. He modynamic and clinical improvement after superficial vein ablati on in primary combined insufficiency with ulceration. J Vasc Surg 1996;24:711-8. 43. Van den Bos R, Arends L, Kockaert M, Neumann M, Nijsten T. Endovenous therapies of lower extremity varicosities: a meta analysis. J Vasc Surg 2009;49:230-9. 29. MacKenzie RK, Allan PL, Ruckley CV, Bradbury AW. The effect of long saphenous vein stripping on deep venous reşux. Eur J Vasc Endovasc Surg 2004;28:104-7. 44. Brown KR, Rossi PJ. Superficial venous disease. Surg Clin North Am 2013; 93: 963-82. Gloviczki P, Gloviczki ML. Guidelines for the management of varicose veins. Phlebology 2012;27:2-9. 30. MC Mullin GM, Coloridge Smith P, Scurr JH: Objective as- sesment of high ligation without stripping the long saphenous vein, Br J Surg 1991;78:1139-42. 45. Min RJ, Khilnani N, Zimmet SE. Endovenous laser treatment of saphenous vein reflux: long term results. J Vasc Interv Radiol 2003;14:991-6. 31. Oğuzkurt L. Endovenous laser ablation for the treatment of varico se veins. Diagn Interv Radiol 2012;18:417-22. 32. Morrison N. Laser treatment of the incompetent sapheno-us vein. 196 ORTADOĞU TIP DERGİSİ 9 (4): 191-197, 2017 In: Gloviczki P, Dalsing MC, Eklöf B, Moneta GL, Wake-field TW (eds). Handbook of Venous Disorder. Third edition. American Ve nous Forum. London: Edward Arnold; 2009;37:418-28. 46. Mundy L, Merlin TL, Fitridge RA, Hiller JE. Systematic revi ew of endovenous laser treatment for varicose veins. Brit J Surg 2005;92,1189-94. 47. Timperman PE. Prospective evaluation of higher energy great saphenous vein endovenous laser treatment. J Vasc Interv Radiol 2005;16:791-4. 33. Pannier F, Rabe E. Endovenous laser therapy and radiofrequ ency ablation of saphenous varicose veins. J Cardiovasc Surg 2006;47:3-8. 48. Proebstle TM, Herdemann S. Early results and feasibility of in competent perforator vein ablation by endovenous laser treatment. Dermatol Surg 2007;33:162-8. 34. Van den Bos RR, Kockaert MA, Neumann HA, Nijsten T. Tech nical review of endovenous laser therapy for varicose veins. Eur J Vasc Endovasc Surg 2008;35:88-95. 49. Yamaki T, Nozaki M, Iwasaka S. Comparative study of duplex- guided foam sclerotherapy and duplex-guided liquid sclerotherapy for the treatment of superficial venous insufficiency. Dermatol Surg 2004;30:718-22. 35. Kabnick LS. Outcome of different endovenous laser wavelengths for great saphenous vein ablation. J Vasc Surg 2006;43:88-93. 36. Merchant RF and Kistner RL. Radiofrequency treatment of the incompetent saphenous vein. In: Gloviczki P, Dalsing MC, Eklöf B, Moneta GL, Wakefield TW. Handbook of Venous Disorder. Third edition. American Venous Forum. London: Edward Arnold; 2009;36:409-17. 50. Tessari L, Cavezzi A, Frullini A. Preliminary Experience with a New Sclerosing Foam in the Treatment of Varicose Veins. Derma tol Surg 2001;27:58-60. 51. Yin H, He H, Wang M, Li Z, Hu Z, Yao C, Chang G. Prospective Ran domized Study of Ultrasound-Guided Foam Sclerotherapy Combined with Great Saphenous Vein High Ligation in the Treatment of Severe Lower Extremity Varicosis. Ann Vasc Surg 2017;39:256-63. 37. Durukan AB. What is popular may no longer be popular: Cyano acrylate and other alternatives in surgical treatment of chronic ve nous insufficiency. Turk J Thoracic Cardiovasc Surg 2017;159-63. 38. Parsi K, Exner T, Connor D E, Ma D D, Joseph JE. In vitro effects of detergent sclerosants on coagulation, platelets and micropartic les. Eur J Vasc Endovasc Surg 2007;34:731-40. Sorumlu Yazar: Mehmet Kabalcı, Kırıkkale Üniversitesi Tıp Fakültesi Kalp ve Damar Cerrahisi AD, Kırıkkale, Türkiye E-mail: kabalci@hotmail.com Sorumlu Yazar: Mehmet Kabalcı, Kırıkkale Üniversitesi Tıp Fakültesi Kalp ve Damar Cerrahisi AD, Kırıkkale, Türkiye E-mail: kabalci@hotmail.com 39. Carlin MC, Ratz JL. Treatment of telengiectasias,comparison of sclerosing agents. J.Dermatol. Surg Oncol 1987:13;1181. 196 ORTADOĞU TIP DERGİSİ 9 (4): 191-197, 2017 197 ORTADOGU MEDICAL JOURNAL 9 (4): 191-197, 2017
https://openalex.org/W4361883104	https://figshare.com/articles/journal_contribution/Supplementary_Figure_3_from_Dual_CDK4_CDK6_Inhibition_Induces_Cell-Cycle_Arrest_and_Senescence_in_Neuroblastoma/22451169/1/files/39902253.pdf	English	null	Supplementary Figure 2 from Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma	null	2,023	cc-by	399	A S B MYCN BE2C IMR5 1643 SY5Y NGP KELLY LAN5 NLF NB69 NBSD NBLS SKNF1 EBC1 SKNAS NB16 RPE1 β-Actin Figure S3. MYCN expression is inversely correlated with LEE011 sensitivity. (A) High mRNA expression of MYCN correlates with low LEE011 IC50s (r = -0.55, p = 0.02). This correlation is further strengthened (r = -0 7 p = 0 003) by the removal of two outliers SKNSH (IC50 = 148 further strengthened (r 0.7, p 0.003) by the removal of two outliers, SKNSH (IC50 148 A S B MYCN BE2C IMR5 1643 SY5Y NGP KELLY LAN5 NLF NB69 NBSD NBLS SKNF1 EBC1 SKNAS NB16 RPE1 β-Actin Figure S3. MYCN expression is inversely correlated with LEE011 sensitivity. (A) High mRNA expression of MYCN correlates with low LEE011 IC50s (r = -0.55, p = 0.02). This correlation is further strengthened (r = -0 7 p = 0 003) by the removal of two outliers SKNSH (IC50 = 148 further strengthened (r 0.7, p 0.003) by the removal of two outliers, SKNSH (IC50 148 A S B MYCN BE2C IMR5 1643 SY5Y NGP KELLY LAN5 NLF NB69 NBSD NBLS SKNF1 EBC1 SKNAS NB16 RPE1 β-Actin Figure S3. MYCN expression is inversely correlated with LEE011 sensitivity. (A) High mRNA expression of MYCN correlates with low LEE011 IC50s (r = -0.55, p = 0.02). This correlation is further strengthened (r = -0 7 p = 0 003) by the removal of two outliers SKNSH (IC50 = 148 further strengthened (r 0.7, p 0.003) by the removal of two outliers, SKNSH (IC50 148 nM) and its subclone, SY5Y (IC50 = 154 nM). (B) Sensitive cell lines also tend to express higher levels of MYCN protein than resistant cell lines. A A A S B MYCN BE2C IMR5 1643 SY5Y NGP KELLY LAN5 NLF NB69 NBSD NBLS SKNF1 EBC1 SKNAS NB16 RPE1 β-Actin B Figure S3. MYCN expression is inversely correlated with LEE011 sensitivity. (A) High mRNA expression of MYCN correlates with low LEE011 IC50s (r = -0.55, p = 0.02). This correlation is further strengthened (r = -0 7 p = 0 003) by the removal of two outliers SKNSH (IC50 = 148 further strengthened (r 0.7, p 0.003) by the removal of two outliers, SKNSH (IC50 148 nM) and its subclone, SY5Y (IC50 = 154 nM). (B) Sensitive cell lines also tend to express higher levels of MYCN protein than resistant cell lines.
https://openalex.org/W3029408856	https://europepmc.org/articles/pmc7273487?pdf=render	English	null	Circulating Matrix Metalloproteinase-28 Levels Are Related to GRACE Scores and Short-Term Outcomes in Patients with Acute Myocardial Infarction	BioMed research international	2,020	cc-by	5,579	Hindawi BioMed Research International Volume 2020, Article ID 9206703, 8 pages https://doi.org/10.1155/2020/9206703 Hindawi BioMed Research International Volume 2020, Article ID 9206703, 8 pages https://doi.org/10.1155/2020/9206703 Hindawi BioMed Research International Volume 2020, Article ID 9206703, 8 pages https://doi.org/10.1155/2020/9206703 Research Article Circulating Matrix Metalloproteinase-28 Levels Are Related to GRACE Scores and Short-Term Outcomes in Patients with Acute Myocardial Infarction Ke Zhou,1,2 Yuanmin Li,3 Yawei Xu ,1 and Rong Guo 2 1Nanjing Medical University, Nanjing, Jiangsu 211166, China 2Department of Cardiology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China 3Department of Cardio-Thoracic Surgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China Correspondence should be addressed to Yawei Xu; xuyawei1960@126.com and Rong Guo; 09_doctorguo@tongji.edu.cn Received 31 January 2020; Revised 10 April 2020; Accepted 20 April 2020; Published 27 May 2020 Received 31 January 2020; Revised 10 April 2020; Accepted 20 April 2020; Published 27 May 2020 Academic Editor: Kimimasa Tobita Academic Editor: Kimimasa Tobita Copyright © 2020 Ke Zhou 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. Objective. To investigate the relationship between the level of matrix metalloproteinase-28 (MMP-28) in patients with acute myocardial infarction (AMI) and the global registry of acute coronary events (GRACE) scores as well as their short-term prognosis. Methods. Two hundred eleven patients with AMI were enrolled, and their basic clinical characteristics were collected for determining the GRACE score. We measured the plasma levels of MMP-28 and other biomarkers in the study population. The association of MMP-28 levels with cardiac events and cardiac deaths occurring within 30 days of discharge was evaluated with multivariable Cox proportional hazard models. Results. The MMP-28 levels were signiﬁcantly higher in patients with acute ST-elevation myocardial infarction (STEMI) than in patients with non-ST-elevation myocardial infarction (NSTEMI) (P < 0:01). Correlation analysis showed that the level of MMP-28 was positively correlated with the GRACE score in patients with AMI (R2 = 0:366, P < 0:05). Cox multivariate regression results showed that MMP-28 was associated with cardiovascular events during the hospitalization and 30 days after discharge (P < 0:01). In addition, Kaplan–Meier analysis showed that cardiac events and deaths were signiﬁcantly higher in patients with MMP-28 ≥1:21 ng/mL (all P < 0:01). Conclusion. There is a correlation between the plasma MMP-28 level and GRACE score in patients with AMI. MMP-28 is also associated with cardiovascular events and cardiovascular deaths during the hospitalization of patients and within 30 days of discharge. 2.2. Patient Selection 2.2.1. Inclusion Criteria. The patients with the following characteristics and clinical presentations were included in the study: (1) all the subjects met the latest diagnostic criteria for AMI [8, 9]. (2) The patient received emergency coronary intervention for the ﬁrst time. All patients with AMI were treated with standard percutaneous coronary intervention, and coronary angiography was performed through the radial artery or the femoral artery. After a clear diagnosis, balloon angioplasty or stent placement was performed at the prob- lematic coronary artery. (3) The selected subjects signed the informed consent form. 2.5. GRACE Score Analysis. Eight indices were collected: age of the patient, the HR, SBP, serum Cr, Killip class at the time of admission, cardiac arrest before hospitalization, ST seg- ment shift on the ECG, and elevated myocardial marker levels. The indices were scored using the GRACE software (http://www.outcome.org/grace). 2.6. Evaluation of Heart Function. Images of the standard long-axis section, short-axis section, and apical two- chamber view and four-chamber view were acquired using a Vivid-7 ultrasound system from GE (GE Healthcare, Pis- cataway, NJ, USA), and the left ventricular ejection fraction (LVEF) was measured using the Simpson’s method. 2.2.2. Exclusion Criteria. The patients with the following characteristics and clinical presentations were excluded from the study: (1) age < 30 years; (2) large-scale pulmonary embolism; (3) infectious diseases; (4) malignant tumors; (5) history of genetic cardiomyopathy; (6) combined pericardial disease, infective endocarditis, etc.; (7) severe liver and kid- ney dysfunction or coagulopathy; (8) pregnancy; and (9) other conditions that made the patient unsuitable for enroll- ment, as determined by the investigators. 2.2.2. Exclusion Criteria. The patients with the following characteristics and clinical presentations were excluded from the study: (1) age < 30 years; (2) large-scale pulmonary embolism; (3) infectious diseases; (4) malignant tumors; (5) history of genetic cardiomyopathy; (6) combined pericardial disease, infective endocarditis, etc.; (7) severe liver and kid- ney dysfunction or coagulopathy; (8) pregnancy; and (9) other conditions that made the patient unsuitable for enroll- ment, as determined by the investigators. 2.7. Follow-Up Questions. This study involved telephonic follow-up and/or face-to-face follow-up of all the selected patients for 30 days after discharge. The follow-up questions included cardiovascular deaths and cardiovascular events, including nonfatal recurrent myocardial infarction; nonfatal stroke; rehospitalization due to heart failure, angina pectoris, and arrhythmia; and revascularizations, including PCI and coronary artery bypass graft surgery (CABG). 2.3. Methods. 1. Introduction or may cooperate with other MMPs [5]. It has been reported that MMP-28 participates in the process of myocardial remodeling after myocardial infarction [6], and it has some correlation with the severity of coronary artery disease [7]. However, the role of MMP-28 in acute myocardial infarction and its correlation with coronary artery disease remain to be elucidated. Acute myocardial infarction (AMI) is a common cardiovas- cular disease [1]. Extracellular matrix is considered a dynamic constantly remodeling structure that plays a pivotal role in myocardial repair [2]. Matrix metalloproteinases (MMPs) play a pivotal role in postmyocardial infarction car- diac remodeling as well as in the development of adverse out- comes [3]. MMPs are a group of zinc ion-dependent proteases that degrade collagen and proteoglycans and play an important role in the development of atherosclerosis [4]. As a new member in the family of MMPs, MMP-28 may directly damage the matrix around cells through proteolysis Therefore, in this study, we investigated the relationship between the level of MMP-28 in AMI patients and the global registry of acute coronary events (GRACE) scores as well as their short-term prognosis. We aim to explore the clinical value of MMP-28 in the risk assessment and prognosis pre- diction for AMI. 2 BioMed Research International BioMed Research International 2.2. Patient Selection For patients with AMI, 5 mL of fasting venous blood was collected within 24h of the onset of the disease, before cardiac catheterization. In the normal control group, 5 mL of fasting venous blood was collected early in the morn- ing. The blood samples were placed at room temperature for 2 h. After centrifugation at 1,000 × g, the supernatant was collected and stored in a −80°C refrigerator. The samples were thawed at room temperature before the experiment and mixed thoroughly before measurement. The plasma levels of low-density lipoprotein cholesterol (LDL-C) were analyzed by using an enzymatic method (Roche Diagnostics); triglycerides were analyzed using an enzymatic-colorimetric assay. Serum N-terminal pro-B-type natriuretic peptide (NT-proBNP), hypersensitive C-reactive protein (hs-CRP), creatinine (Cr), hemoglobin A1c, and fasting blood glucose (FBG) were measured using a routine-automated technique in a laboratory at Shanghai Tenth People’s Hospital, China. The hs-cTnT was assayed using the Elecsys troponin T- high sensitive assay (Roche Diagnostics, Penzberg, DE). All the assays were carried out according to the manufacturer’s instructions. We also measured the heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) of the patients. 2.8. Statistical Analysis. All statistical analyses were carried out using SPSS version 17 (SPSS, Inc., Chicago, IL, USA). The data are represented as the means ± standard deviation. Student’s t-test or the Wilcoxon matched-pairs, signed- rank test was used to determine the diﬀerences between the groups for Gaussian-distributed data and non-Gaussian- distributed data, respectively. The χ2 test or the Fisher exact test was used to compare dichotomous data. Linear regres- sion was used for correlation analysis. The risks of cardiac events and cardiac deaths were assessed in a multivariate Cox proportional-hazard model to determine whether MMP-28 levels independently predicted the outcome. Other variables that were signiﬁcantly associated with the outcome were entered into the model in a step-wise procedure. Event- free survival curves were constructed using the Kaplan– Meier method and compared using the log-rank test. 2. Materials and Methods The level of MMP-28 was measured using the enzyme- linked immunosorbent assay (ELISA) kit by ELISA Genie (Dublin, Ireland) as per the manufacturer’s instructions. 2.1. Subjects. In this observational, retrospective, single- center study, we evaluated the prognostic value of plasma MMP-28 levels in patients with AMI. A total of 211 patients with AMI who were hospitalized in our center from January 2018 to December 2018 were selected. The study was obser- vational, and no intervention was given to the patients. The study was reviewed and approved by the ethics committee of the hospital. All the patients enrolled in the study provided informed consent. 2.4. Grading of Coronary Artery Stenosis. Coronary angiogra- phy was performed in all the AMI patients using the radial or femoral approach, and the degree of coronary artery disease was determined by two experienced interventional physi- cians. Among the coronary arteries, if only one major branch had a degree of stenosis > 50%, it was considered a single- branch disease; if two major branches had a degree of stenosis > 50%, it was considered a two-branch disease; if three or more major branches had a degree of stenosis greater than 50%, it was considered a three-branch disease. 3. Results 3.1. Baseline Data of the Patients. The baseline data of all the selected patients are shown in Table 1. The levels of NT- proBNP, hs-CRP, and MMP-28 were signiﬁcantly higher in BioMed Research International BioMed Research International 3 Table 1: Baseline characteristics of the included acute myocardial infarction (AMI) patients. Variables Control group (N = 50) Disease group STEMI (N = 142) NSTEMI (N = 69) Total (N = 211) Age (yrs) 34:2 ± 5:5 60:0 ± 8:1 59:4 ± 10:4 59:6 ± 9:7 Gender (M/F) 25/25 90/52 51/18 141/70 hs-cTnT (ng/mL) 0:04 ± 0:02 2:14 ± 1:87 1:89 ± 2:06 2:18 ± 2:20 NT-proBNP (pg/mL) 74:2 ± 52:3 1973:8 ± 1639:1∗ 744:8 ± 516:4∗ 1146:7 ± 1176:0 hs-CRP (mg/dL) 0:3 ± 0:2 7:5 ± 5:3∗ 4:4 ± 2:9∗ 5:4 ± 4:1 MMP-28 (ng/mL) 0:5 ± 0:3 4:3 ± 1:8∗ 1:4 ± 1:9∗ 2:4 ± 2:3 BMI (kg/m2) 23:3 ± 2:4 24:1 ± 2:1 24:2 ± 2:7 24:2 ± 2:5 Blood glucose proﬁles FBG (mmol/L) 5:4 ± 0:6 6:1 ± 1:2 6:3 ± 1:3 6:2 ± 1:3 HbA1C (%) 5:4 ± 0:6 6:3 ± 0:7 6:4 ± 0:7 6:4 ± 0:7 Lipid proﬁles TC (mmol/L) 4:8 ± 0:6 5:1 ± 0:7 5:0 ± 0:8 5:1 ± 0:7 LDL-C (mmol/L) 3:0 ± 0:5 3:0 ± 0:9 3:2 ± 1:0 3:1 ± 1:0 Heart rate (bpm) 77:6 ± 5:8 81:2 ± 16:2 76:3 ± 12:9 77:9 ± 14:2 SBP (mmHg) 128:4 ± 11:7 126:1 ± 22:9 135:5 ± 20:4 132:4 ± 21:6 DBP (mmHg) 64:1 ± 4:7 60:8 ± 10:9 73:6 ± 10:5 72:1 ± 10:9 LVEF (%) 66:5 ± 3:4 33:4 ± 7:4 51:6 ± 9:9 45:7 ± 12:5 eGFR (mL/min/1.73 m2) 92:3 ± 4:5 84:5 ± 9:8 87:2 ± 11:8 86:4 ± 11:2 hs-cTnT: hypersensitive cardiac troponin T; NT-proBNP: N-terminal pro-B-type natriuretic peptide; hs-CRP: hypersensitive C-reactive protein; BMI: body mass index; FBG: fasting blood glucose; HbA1C: hemoglobin A1c; TC: total cholesterol; LDL-C: low-density lipoprotein-cholesterol; SBP: systolic blood pressure; DBP: diastolic blood pressure; LVEF: left ventricular eject fraction; eGFR: estimated glomerular ﬁltration rate. ∗Signiﬁcant diﬀerences between two groups; P < 0:05. Table 1: Baseline characteristics of the included acute myocardial infarction (AMI) patients. 3. Results hs-cTnT: hypersensitive cardiac troponin T; NT-proBNP: N-terminal pro-B-type natriuretic peptide; hs-CRP: hypersensitive C-reactive protein; BMI: body mass index; FBG: fasting blood glucose; HbA1C: hemoglobin A1c; TC: total cholesterol; LDL-C: low-density lipoprotein-cholesterol; SBP: systolic blood pressure; DBP: diastolic blood pressure; LVEF: left ventricular eject fraction; eGFR: estimated glomerular ﬁltration rate. ∗Signiﬁcant diﬀerences between two groups; P < 0:05. Table 2: Coronary intervention data of the patients with acute myocardial infarction (AMI; N = 211). Data on coronary intervention N (%) Coronary angiography performed 205 (97.2) Time between admission and angiography (hours) ≤24 155 (73.5) >24–48 43 (20.4) >48 7 (3.3) Number of vessel disease 0 0 (0) 1 88 (41.7) 2 52 (24.6) 3 38 (18.0) LM 12 (5.7) LM+3 15 (7.1) Treatment Conservative 10 (4.7) PTCA/PCI 192 (90.9) CABG 3 (1.4) PTCA: percutaneous transluminal coronary angioplasty; LM: left main; PCI: percutaneous coronary intervention; CABG: coronary artery bypass grafting. patients with acute ST-elevation myocardial infarction (STEMI) than in patients with non-ST-elevation myocardial infarction (NSTEMI) (P < 0:01). There were signiﬁcant dif- ferences in HR, SBP, DBP, and LVEF between the two groups (P < 0:05, Table 1), whereas no signiﬁcant diﬀerence was noted in other parameters between the two groups (P > 0:05, Table 1). Table 2: Coronary intervention data of the patients with acute myocardial infarction (AMI; N = 211). 3.2. Coronary Angiography Results. All the patients received coronary angiography, which was performed using the Judkins technique. The angiography results were measured visually in combination with quantitative analysis on using a computer to determine the degree of coronary stenosis. The coronary angiography results showed that a 70% reduction in the internal diameter of the coronary arteries was signiﬁcant. According to the arteries and branches involved in stenosis, the condition was classiﬁed into left main branch and single-, double-, and triple-branch diseases, and the residual stenosis, thrombosis, dissection, perforation, calciﬁcation, and spasm were evaluated. The interventional surgery was considered successful if the residual stenosis was ≤10%, and there was no surgery-related myocardial infarction or death during the operation. The spe- ciﬁc results are presented in Table 2. PTCA: percutaneous transluminal coronary angioplasty; LM: left main; PCI: percutaneous coronary intervention; CABG: coronary artery bypass grafting. 3.3. GRACE Risk Stratiﬁcation, Plasma MMP-28 Levels of Patients, and Their Correlation. 3. Results The patients were divided into low-risk (N = 81), intermediate-risk (N = 83), and 4 BioMed Research International MMP-28 (ng/mL) GRACE score 0 2 4 6 8 Low risk (≤108) Moderate risk (109-140) High risk (>140) N = 47 N = 83 N = 81 ⁎ ⁎ (a) MMP-28 (ng/mL) GRACE score 0 2 4 6 8 10 0 50 100 150 200 250 R2 = 0.366, P < 0.001 (b) Figure 1: MMP-28 levels in the groups with diﬀerent risks and the correlation between MMP-28 and GRACE scores. (a) Plasma MMP-28 levels in the groups with diﬀerent risks. (b) MMP-28 levels were positively correlated with GRACE scores in patients with acute myocardial infarction. ∗P < 0:05. MMP-28 (ng/mL) GRACE score 0 2 4 6 8 10 0 50 100 150 200 250 R2 = 0.366, P < 0.001 (b) MMP-28 (ng/mL) GRACE score 0 2 4 6 8 Low risk (≤108) Moderate risk (109-140) High risk (>140) N = 47 N = 83 N = 81 ⁎ ⁎ (a) GRACE score (a) (b) Figure 1: MMP-28 levels in the groups with diﬀerent risks and the correlation between MMP-28 and GRACE scores. (a) Plasma MMP-28 levels in the groups with diﬀerent risks. (b) MMP-28 levels were positively correlated with GRACE scores in patients with acute myocardial infarction. ∗P < 0:05. MMP-28 (ng/mL) hs cTNT (ng/mL) 10 0 5 10 15 R2 = 0.331, P < 0.001 0 2 4 6 8 (b) MMP-28 (ng/mL) hs-CRP (mg/dL) 0 10 0 50 100 150 R2 = 0.346, P < 0.001 2 4 6 8 (a) MMP-28 (ng/mL) hs-cTNT (ng/mL) 10 0 5 10 15 R2 = 0.331, P < 0.001 0 2 4 6 8 (b) MMP-28 (ng/mL) NT-proBNP (pg/mL) 10 0 2000 4000 6000 8000 10000 R2 = 0.109, P < 0.001 0 2 4 6 8 (c) MMP-28 (ng/mL) LVEF 0 2 4 6 8 10 0.0 0.2 0.4 0.6 0.8 R2 = 0.403, P < 0.001 (d) Figure 2: Correlation between the levels of hs-CRP, hs-cTnT, NT-proBNP, LVEF, and MMP-28. Plasma MMP-28 was negatively correlated with hs-CRP (a), hs-cTNT (b), NT-proBNP (c), and LVEF (d) (P < 0:001). 4. Discussion Biomarkers related to AMI have been a topic of interest to cardiologists [10], and in recent years, many reports have focused on the diagnosis, risk stratiﬁcation, and prognosis of the disease [11]. This study is aimed at exploring the clin- ical value of MMP-28 in the risk assessment and prognosis prediction of patients with AMI (STEMI and NSTEMI). Our main ﬁndings are as follows: (1) MMP-28 is elevated to varying degrees in patients with AMI; (2) plasma MMP- 28 level correlates with the GRACE score in patients with AMI; and (3) MMP-28 level correlates with cardiovascular events and cardiovascular deaths during the hospitalization of AMI patients and within 30 days after discharge and can be considered a short-term prognosis predictor for patients with myocardial infarction. 95% CI: 95% conﬁdence interval; ∗P < 0:05; ∗∗P < 0:01. hs-CRP, hs-cTnT, and NT-proBNP of patients with AMI. The plasma MMP-28 level was found to be positively corre- lated with hs-CRP, hs-cTnT, and NT-proBNP (R2 = 0:346, R2 = 0:331, R2 = 0:109, P < 0:01; Figures 2(a)–2(c)). The MMP-28 levels were found to be negatively correlated with LVEF (R2 = 0:403, P < 0:01; Figure 2(d)). Atherosclerosis is an inﬂammatory pathological process. MMPs promote the rupture of atherosclerotic plaques by degrading the extracellular matrix. Usually, the occurrence of acute coronary syndrome is caused by the rupture of cor- onary atherosclerotic plaques, which leads to the thrombosis and occlusion of coronary arteries. The stability of coronary atherosclerotic plaques is aﬀected by the level of collagen [12]. Atherosclerotic plaque instability is the fundamental cause of acute coronary syndromes [13]. Whether plaques are prone to rupture depends mainly on their structure. Unstable plaques are characterized by large lipid cores, thin ﬁber caps, and are inﬁltrated by many inﬂammatory cells, such as monocytes, macrophages, and macrophage-derived foam cells [14]. Therefore, MMPs have been widely studied in the ﬁeld of atherosclerosis and AMI [15]. 3.5. Determination of the Cut-OﬀValue. To establish the optimal cut-oﬀvalue, coronary angiography, combined with hs-cTnT assessment, was used as the conﬁrmatory test for patients with AMI in reference to the plasma MMP-28 levels of 50 healthy individuals. Myocardial infarction was set to be 1, and nonacute myocardial infarction was set to be 0. The ROC curve was drawn where the y-axis represented sensitiv- ity and the x-axis represented speciﬁcity. The AUCROC was 0.835, and the 95% conﬁdence interval was 0.72–0.91. 3. Results Table 3: Results of the multivariate Cox proportional hazard analysis. results of Cox multivariate regression analysis showed that the MMP-28 level was associated with cardiovascular events during the hospitalization and 30 days after discharge (P < 0:01, Table 3); NT-proBNP and MMP-28 were closely associated with cardiovascular deaths during the hospitaliza- tion and 30 days after discharge (P < 0:05, Table 3). The remaining variables appeared to have little correlation with cardiovascular events and cardiovascular deaths. The Kaplan–Meier survival analysis showed that in the MMP-28 ≥1:21 ng/mL group, 17 patients had major cardio- vascular events and 14 cardiovascular deaths were reported. In the MMP-28 < 1:21ng/mL group, only one patient had a cardiovascular event, and no cardiovascular death was reported. There was a signiﬁcant diﬀerence between the two groups (P < 0:01, Figure 3). 3. Results MMP-28 (ng/mL) hs-CRP (mg/dL) 0 10 0 50 100 150 R2 = 0.346, P < 0.001 2 4 6 8 (a) R2 = 0.331, P < 0.001 hs-cTNT (ng/mL) (b) MMP-28 (ng/mL) LVEF 0 2 4 6 8 10 0.0 0.2 0.4 0.6 0.8 R2 = 0.403, P < 0.001 (d) MMP-28 (ng/mL) NT-proBNP (pg/mL) 10 0 2000 4000 6000 8000 10000 R2 = 0.109, P < 0.001 0 2 4 6 8 (c) LVEF Figure 2: Correlation between the levels of hs-CRP, hs-cTnT, NT-proBNP, LVEF, and MMP-28. Plasma MMP-28 was negatively correlated with hs-CRP (a), hs-cTNT (b), NT-proBNP (c), and LVEF (d) (P < 0:001). General linear regression analysis was performed based on the GRACE scores and MMP-28 levels. The results showed that the GRACE score of AMI patients had a signif- icant linear correlation with the plasma MMP-28 level, with an R2 of 0.366 (P < 0:05, Figure 1(b)). high-risk (N = 47) groups according to the GRACE score (Figure 1(a)), and the MMP-28 levels of the groups were 0:7 ± 1:5, 1:3 ± 1:1, and 4:5 ± 2:0 ng/mL, respectively (Figure 2(a)). Compared with the low-risk and intermediate-risk groups, the high-risk group had a signiﬁ- cantly higher MMP-28 level (P < 0:05 for both, Figure 1(a)). The diﬀerence in the MMP-28 levels between the intermediate-risk and the low-risk groups was also statis- tically signiﬁcant (P < 0:05, Figure 1(a)). 3.4. Relationship between Cardiac Function, Plasma MMP-28 Level, and Other Biomarkers. General linear correlation anal- yses were performed between the plasma MMP-28 level and BioMed Research International BioMed Research International 5 Table 3: Results of the multivariate Cox proportional hazard analysis. Outcome/variables Hazard ratio 95% CI P value Cardiac events Age (yrs) 1.02 0.97–1.08 0.39 Gender (M/F) 1.59 0.53–4.71 0.41 BMI (kg/m2) 1.16 0.97–1.39 0.10 NT-proBNP (pg/mL) 1.00 1.00–1.00 0.92 hs-CRP (mg/dL) 0.95 0.88–1.01 0.14 hs-cTNT (ng/mL) 0.81 0.63–1.06 0.12 eGFR (mL/min/1.73 m2) 1.02 0.97–1.07 0.48 Grace scores 1.01 0.99–1.04 0.34 LVEF (%) 0.06 0.00–32.59 0.37 MMP-28 (ng/mL) 1.64 1.14–2.36 0.008∗∗ In-hospital and 30-day cardiac mortality Age (yrs) 1.02 0.94–1.11 0.67 Gender (M/F) 1.05 0.22–1.23 0.95 BMI (kg/m2) 0.85 0.59–1.23 0.39 NT-proBNP (pg/mL) 1.00 1.00–1.00 0.009∗∗ hs-CRP (mg/dL) 1.00 0.98–1.03 0.98 hs-cTNT (ng/mL) 0.79 0.59–1.04 0.09 eGFR (mL/min/1.73 m2) 1.07 0.99–1.15 0.08 Grace scores 1.05 0.98–1.11 0.14 LVEF (%) 3.50 0.00–4997.72 0.74 MMP-28 (ng/mL) 1.85 1.08–3.16 0.02∗ 95% CI: 95% conﬁdence interval; ∗P < 0:05; ∗∗P < 0:01. 4. Discussion (b) Patients with high MMP-28 levels had signiﬁcantly higher rates of cardiac death than patients with low Cardiac deaths Time (days) Event free rates (%) 0 6 12 18 24 30 70 80 90 100 MMP-28 ≥ 1.21 ng/mL (N=122) MMP-28 < 1.21 ng/mL (N=89) Log-rank test, P < 0.001 Number at risk MMP-28 < 1.21 ng/mL MMP-28 ≥ 1.21 ng/mL 12 2 89 58 55 59 57 78 79 98 83 11 1 89 (b) 3: Kaplan–Meier analysis. (a) Kaplan–Meier curve showing the incidence of cardiac events, within 30 days after discharge in patients Event free rates (%) (b) Figure 3: Kaplan–Meier analysis. (a) Kaplan–Meier curve showing the incidence of cardiac events, within 30 days after discharge in patients with acute myocardial infarction. (b) Patients with high MMP-28 levels had signiﬁcantly higher rates of cardiac death than patients with low MMP-28 levels. tion. In this study, it was also observed that MMP-28 is closely related to the indices of cardiac function, such as NT-proBNP and LVEF in the population with myocardial infarction [6]. 28 protein is increased in malignant tumors and cancer cell lines [17–19]. However, studies on MMP-28 in the cardio- vascular ﬁeld are limited. Our study provides some clinical evidence and data on the relevance of MMP-28 in cardiovas- cular diseases. Liu et al. found that the level of MMP-28 increased in patients with stable coronary heart disease and was related to the severity of coronary artery disease, which led to the speculation that MMP-28 might be involved in atheroscle- rotic lesions [7]. Researchers have also suggested that it can be used as one of the markers in future research [23]. Zhan et al. examined the role of MMP-28 in patients with atrial ﬁbrillation, and the results suggest that MMP-28 is related to the left atrial diameter and the prognosis of heart failure [24]. These studies implicate the value of MMP-28 as a new biomarker for cardiovascular disease from diﬀerent perspec- tives. In our study, the level of MMP-28 was signiﬁcantly Ma et al. found that with the increase in age, the expres- sion of MMP-28 in the left ventricle increased by 42% in mouse models. When the MMP-28 gene was knocked out in mice, the levels of inﬂammatory factors, such as macro- phage inﬂammatory protein- (MIP-) 1α, MIP-1β, and MMP-9, in the left ventricle increased [22]. 4. Discussion When 1.21 ng/mL was used as the threshold, the sensitivity and speciﬁcity of MMP-28 were relatively higher, with values of 94% and 88%, respectively. Therefore, in the Kaplan–Meier survival analysis, the plasma MMP-28 levels in all the patients were classiﬁed with respect to the 1.21 ng/mL cut- oﬀvalue: ≥1.21 ng/mL group (N = 122, male/female = 83/39 ) and <1.21 ng/mL group (N = 89, male/female = 58/31). MMP-28 (an epithelial protease) is a protein with a molecular mass of 59kD and is the recently cloned human MMP [16]. It has been reported to play an important role in tumor progression [17–19]. MMP-28 contains a signal peptide sequence, a propeptide domain, a zinc-binding cata- lytic active site, and a C-terminal heme-binding protein-like domain [5]. MMP-28 is expressed in many normal tissues, such as the testis, small intestine, skin, and lungs, suggesting that it plays a key role in balancing the internal environment of the tissue [20]. Researchers have found that the expression of MMP-28 protein is higher in some tumors than in normal tissues [21]. Studies have shown that the expression of MMP- 3.6. Follow-Up Results and Kaplan–Meier Survival Analysis. All the patients in this study completed the follow-up analy- sis, with an average follow-up period of 24:1 ± 8:3 days. The 6 BioMed Research International Cardiac events Time (days) Event free rates (%) 12 18 24 30 60 70 80 90 100 MMP-28 ≥ 1.21 ng/mL (N=122) MMP-28 < 1.21 ng/mL (N=89) Log-rank test, P < 0.001 Number at risk MMP-28 < 1.21 ng/mL MMP-28 ≥ 1.21 ng/mL 108 89 58 55 59 57 78 78 97 83 103 89 0 6 (a) Cardiac deaths Time (days) Event free rates (%) 0 6 12 18 24 30 70 80 90 100 MMP-28 ≥ 1.21 ng/mL (N=122) MMP-28 < 1.21 ng/mL (N=89) Log-rank test, P < 0.001 Number at risk MMP-28 < 1.21 ng/mL MMP-28 ≥ 1.21 ng/mL 12 2 89 58 55 59 57 78 79 98 83 11 1 89 (b) –Meier analysis. (a) Kaplan–Meier curve showing the incidence of cardiac events, within 30 days after discharge in patients rdial infarction. 4. Discussion These results sug- gest that MMP-28 is involved in the regulation of myocardial inﬂammation and extracellular matrix responses. Subse- quently, Ma et al. found that knockout of the MMP-28 gene in mice resulted in a more signiﬁcant ventricular remodeling and deterioration of cardiac function after myocardial infarc- BioMed Research International 7 ican Heart Association,” Circulation, vol. 137, pp. e67–e492, 2018. elevated in the AMI group, and MMP-28 had a high correla- tion with hs-cTnT, hs-CRP, NT-proBNP, and LVEF. It is possible that the increased level of MMP-28 in peripheral blood may be one of the markers of plaque rupture or may be involved in the occurrence of AMI. In addition, the Cox risk regression analysis suggests that the level of MMP-28 is an independent risk factor for cardiovascular events and deaths in the short-term after AMI. [2] [2] U. S. Owolabi, A. R. Amraotkar, A. R. Coulter et al., “Change in matrix metalloproteinase 2, 3, and 9 levels at the time of and after acute atherothrombotic myocardial infarction,” Jour- nal of Thrombosis and Thrombolysis, vol. 49, no. 2, pp. 235– 244, 2020. [3] A. Yabluchanskiy, Y. Li, R. J. Chilton, and M. L. Lindsey, “Matrix metalloproteinases: drug targets for myocardial infarction,” Current Drug Targets, vol. 14, no. 3, pp. 276–286, 2013. Compared with coronary interventional therapy, moni- toring of the MMP-28 level in peripheral blood has the fol- lowing advantages: higher safety, less injury, lower risk, lower cost, and easier to perform. It can be used for early risk assessment and prognosis prediction for AMI, which are helpful in improving the clinical outcome. In addition, with further research, MMP-28 has the potential to be a target for drugs that interfere with plaque stability and can play a role in the early prevention of AMI, the reduction of the inci- dence of AMI, and targeted treatment. [4] V. A. Myasoedova, D. A. Chistiakov, A. V. Grechko, and A. N. Orekhov, “Matrix metalloproteinases in pro-atherosclerotic arterial remodeling,” Journal of Molecular and Cellular Cardi- ology, vol. 123, pp. 159–167, 2018. [5] S. A. Illman, J. Lohi, and J. Keski-Oja, “Epilysin (MMP- 28)—structure, expression and potential functions,” Experi- mental Dermatology, vol. 17, no. 11, pp. 897–907, 2008. [6] Y. Ma, G. V. Halade, J. Zhang et al., “Matrix metalloproteinase-28 deletion exacerbates cardiac dysfunction and rupture after myocardial infarction in mice by inhibiting M2 macrophage activation,” Circulation Research, vol. 112, no. 4, pp. 675–688, 2013. Data Availability [11] Y. Chen, Y. Tao, L. Zhang, W. Xu, and X. Zhou, “Diagnostic and prognostic value of biomarkers in acute myocardial infarction,” Postgraduate Medical Journal, vol. 95, no. 1122, pp. 210–216, 2019. All data generated or analyzed during this study are included in this published article. [12] P. Clancy, S. W. Seto, S. A. Koblar, and J. Golledge, “Role of the angiotensin converting enzyme 1/angiotensin II/angiotensin receptor 1 axis in interstitial collagenase expression in human carotid atheroma,” Atherosclerosis, vol. 229, no. 2, pp. 331– 337, 2013. 4. Discussion Finally, there are some limitations to this research. First, this was a single-center observational study, with a relatively small sample size. The results need to be veriﬁed with more samples and a larger cohort. In addition, there may be dynamic changes in the MMP-28 levels during the course of the disease, which may be related to the prognosis. More- over, the follow-up period of subjects in this study was rela- tively short, and a longer follow-up period may be required. [7] C. L. Liu, D. L. Shen, K. Zhu et al., “Characterization of interleukin-33 and matrix metalloproteinase-28 in serum and their association with disease severity in patients with coro- nary heart disease,” Coronary Artery Disease, vol. 25, no. 6, pp. 498–504, 2014. [8] K. Thygesen, J. S. Alpert, A. S. Jaﬀe et al., “ESC Scientiﬁc Doc- ument Group. Fourth universal deﬁnition of myocardial infarction,” European Heart Journal, vol. 40, no. 2019, pp. 237–269, 2018. Conflicts of Interest The authors declare that they have no conﬂicts of interest. Authors’ Contributions [13] M. J. Bom, D. J. van der Heijden, E. Kedhi et al., “Early detec- tion and treatment of the vulnerable coronary plaque: can we prevent acute coronary syndromes?,” Circulation: Cardiovas- cular Imaging, vol. 10, no. 5, 2017. Ke Zhou and Yuanmin Li contributed equally to this study. Ke Zhou and Yuanmin Li contributed equally to this study. 5. Conclusion There is a correlation between the plasma MMP-28 level and GRACE score in patients with AMI. MMP-28 is associated with cardiovascular events and cardiovascular deaths during the hospitalization of patients and within 30 days after dis- charge and can be considered a predictor for short-term prognosis in patients with myocardial infarction. [9] M. Roﬃ, C. Patrono, J.-P. Collet et al., “2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation,” Euro- pean Heart Journal, vol. 37, no. 3, pp. 267–315, 2016. [10] R. Body and C. Hendry, “Cardiac biomarkers in emergency care,” Cardiology Clinics, vol. 36, no. 1, pp. 27–36, 2018. Acknowledgments [14] J. F. Bentzon, F. Otsuka, R. Virmani, and E. Falk, “Mechanisms of plaque formation and rupture,” Circulation Research, vol. 114, no. 12, pp. 1852–1866, 2014. This work was supported by grants from the National Natu- ral Science Foundation of China (no. 81700378) and the Excellent Youth Scholars of Shanghai 10th People’s Hospital. [15] J. L. Johnson, “Metalloproteinases in atherosclerosis,” Euro- pean Journal of Pharmacology, vol. 816, pp. 93–106, 2017. [16] J. Lohi, C. L. Wilson, J. D. Roby, and W. C. Parks, “Epilysin, a novel human matrix metalloproteinase (MMP-28) expressed in testis and keratinocytes and in response to injury,” The Jour- nal of Biological Chemistry, vol. 276, no. 13, pp. 10134–10144, 2001. References [1] E. J. Benjamin, S. S. Virani, C. W. Callaway et al., “Heart dis- ease and stroke statistics-2018 update: a report from the Amer- BioMed Research International 8 [17] S. Suomela, V. Koljonen, T. Skoog, H. Kukko, T. Böhling, and U. Saarialho-Kere, “Expression of MMP-10, MMP-21, MMP- 26, and MMP-28 in Merkel cell carcinoma,” Virchows Archiv, vol. 455, no. 6, pp. 495–503, 2009. [18] S. A. Illman, K. Lehti, J. Keski-Oja, and J. Lohi, “Epilysin (MMP-28) induces TGF- mediated epithelial to mesenchymal transition in lung carcinoma cells,” Journal of Cell Science, vol. 119, no. 18, pp. 3856–3865, 2006. [19] V. O. Bister, M. T. Salmela, M. L. Karjalainen-Lindsberg et al., “Diﬀerential expression of three matrix metalloproteinases, MMP-19, MMP-26, and MMP-28, in normal and inﬂamed intestine and colon cancer,” Digestive Diseases and Sciences, vol. 49, no. 4, pp. 653–661, 2004. [20] U. R. Rodgers, L. Kevorkian, A. K. Surridge et al., “Expression and function of matrix metalloproteinase (MMP)-28,” Matrix Biology, vol. 28, no. 5, pp. 263–272, 2009. [21] X. Wang, K. Zhang, X. Chen, C. Zhao, and Z. Sun, “Epilysin is overexpressed in glioblastoma and related to clinical outcome of patients,” Medical Oncology, vol. 32, no. 1, p. 363, 2015. [22] Y. Ma, Y. A. Chiao, J. Zhang, A. M. Manicone, Y. F. Jin, and M. L. Lindsey, “Matrix metalloproteinase-28 deletion ampliﬁes inﬂammatory and extracellular matrix responses to cardiac aging,” Microscopy and Microanalysis, vol. 18, no. 1, pp. 81– 90, 2012. [23] A. C. Newby, “Proteinases and plaque rupture,” Current Opin- ion in Lipidology, vol. 25, no. 5, pp. 358–366, 2014. [24] G. Zhan, G. Wenhua, H. Jie et al., “Potential roles of circulating matrix metalloproteinase-28 (MMP-28) in patients with atrial ﬁbrillation,” Life Sciences, vol. 204, pp. 15–19, 2018.
https://openalex.org/W313448826	https://europepmc.org/articles/pmc3008128?pdf=render	Latin	null	4-Bromo-<i>N</i>-[4-(diethylamino)benzylidene]aniline	Acta crystallographica. Section E	2,010	cc-by	4,225	organic compounds organic compounds Triclinic, P1 a = 10.1863 (11) A˚ b = 12.3527 (13) A˚ c = 14.3400 (15) A˚ = 112.936 (2) = 92.986 (1) = 104.305 (1) V = 1587.8 (3) A˚ 3 Z = 4 Mo K radiation = 2.58 mm1 T = 298 K 0.45 0.39 0.38 mm Data collection Bruker SMART CCD diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.390, Tmax = 0.440 8347 measured reﬂections 5530 independent reﬂections 2795 reﬂections with I > 2(I) Rint = 0.031 Reﬁnement R[F 2 > 2(F 2)] = 0.048 wR(F 2) = 0.115 S = 1.02 5530 reﬂections 365 parameters H-atom parameters constrained max = 0.45 e A˚ 3 min = 0.55 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Xiao-Fang Li College of Automation & Electronic Engineering, Qingdao University of Science & Technology, Qingdao 261500, People’s Republic of China Correspondence e-mail: lixiaofangqd@163.com 365 parameters H-atom parameters constrained max = 0.45 e A˚ 3 min = 0.55 e A˚ 3 Received 8 August 2010; accepted 20 August 2010 Key indicators: single-crystal X-ray study; T = 298 K; mean (C–C) = 0.006 A˚; R factor = 0.048; wR factor = 0.115; data-to-parameter ratio = 15.2. Data collection: SMART (Bruker, 1997); cell reﬁnement: SAINT (Bruker, 1997); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXTL. The asymmetric unit of the title compound, C17H19BrN2, contains two independent molecules. The dihedral angles between the two benzene rings in are 60.4 (2) and 61.0 (2). Related literature For applications of Schiff base compounds, see: Yu et al. (2007). For related structures, see: You et al. (2004); Yu et al. (2007); Zhang (2010). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: LH5115). Experimental Crystal data C17H19BrN2 Mr = 331.25 4-Bromo-N-[4-(diethylamino)benzyl- idene]aniline Xiao-Fang Li Acta Cryst. (2010). E66, o2417 Bruker (1997). SMART and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. You, X.-L., Lu, C.-R., Zhang, Y. & Zhang, D.-C. (2004). Acta Cryst. C60, o693– o695. Yu, Y. Y., Zhao, G. L. & Wen, Y. H. (2007). Chin. J. Struct. Chem. 26, 1359– 1362. Zhang F G (2010) Acta Cryst E66 o382 Yu, Y. Y., Zhao, G. L. & Wen, Y. H. (2007). Chin. J. Struct. Chem. 26, 1359– 1362. References Bruker (1997). SMART and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Bruker (1997). SMART and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. You, X.-L., Lu, C.-R., Zhang, Y. & Zhang, D.-C. (2004). Acta Cryst. C60, o693– o695. You, X.-L., Lu, C.-R., Zhang, Y. & Zhang, D.-C. (2004). Acta Cryst. C60, o693– o695. Yu, Y. Y., Zhao, G. L. & Wen, Y. H. (2007). Chin. J. Struct. Chem. 26, 1359– 1362. Yu, Y. Y., Zhao, G. L. & Wen, Y. H. (2007). Chin. J. Struct. Chem. 26, 1359– 1362. Experimental Zhang, F.-G. (2010). Acta Cryst. E66, o382. Crystal data C17H19BrN2 Mr = 331.25 Xiao-Fang Li o2417 Acta Cryst. (2010). E66, o2417 doi:10.1107/S1600536810033726 Comment Schiff base compounds have been used as fine chemicals and medical substrates. They are important ligands in coordination chemistry due to their ease of preparation (Yu et al., 2007). In this paper, the crystal structure of the title compound is reported. The asymmetric unit of the title compound contains two independent molecules (Fig. 1). The dihedral angles between the two benzene rings in each molecule are 60.4 (2) ° and 61.0 (2) °. Bond lengths and angles are comparable to those observed for 4- chloro-N-[4-(dimethylamino)benzylidene]aniline (You, et al., 2004) and 4-Chloro-N-[4-(diethylamino)benzylidene]aniline (Zhang, 2010). Experimental A mixture of 4-(diethylamino)benzaldehyde (0.01 mol) and 4-bromobenzenamine (0.01 mol) in ethanol (10 ml) was refluxed for 2 h. After cooling, filtration and drying, the title compound was obtained. 10 mg of the title compound was dissolved in 15 ml ethanol, and the solution was kept at room temperature. The single-crystal suitable for X-ray determination was obtained by evaporation from ethanol solution of the title compound after a week. supplementary materials supplementary materials Acta Cryst. (2010). E66, o2417 [ doi:10.1107/S1600536810033726 ] Acta Cryst. (2010). E66, o2417 [ doi:10.1107/S1600536810033726 ] supplementary materials Acta Cryst. (2010). E66, o2417 [ doi:10.1107/S1600536810033726 ] Figures Fig. 1. The asymmetric unit of the title compound, drawn with 30% probability ellipsoids. 4-Bromo-N-[4-(diethylamino)benzylidene]aniline Refinement H atoms were initially located from difference maps and then refined in a riding model with C—H = 0.93–0.97 Å and Uiso(H) = 1.2Ueq(C) or 1.5Ueq(methyl C). 4-Bromo-N-[4-(diethylamino)benzylidene]aniline Crystal data C17H19BrN2 Mr = 331.25 Triclinic, P1 Hall symbol: -P 1 a = 10.1863 (11) Å b = 12.3527 (13) Å sup-1 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.048 wR(F2) = 0.115 S = 1.02 5530 reflections 365 parameters 0 restraints Primary atom site location: structure-invariant direct methods Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.048 wR(F2) = 0.115 S = 1.02 5530 reflections 365 parameters 0 restraints Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.048 wR(F2) = 0.115 S = 1.02 5530 reflections 365 parameters 0 restraints Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained H-atom parameters constrained w = 1/[σ2(Fo 2) + (0.046P)2] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.45 e Å−3 Δρmin = −0.55 e Å−3 w = 1/[σ2(Fo 2) + (0.046P)2] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.45 e Å−3 Δρmin = −0.55 e Å−3 w = 1/[σ2(Fo 2) + (0.046P)2] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.45 e Å−3 Δρmin = −0.55 e Å−3 supplementary materials c = 14.3400 (15) Å µ = 2.58 mm−1 α = 112.936 (2)° T = 298 K β = 92.986 (1)° Block, light yellow γ = 104.305 (1)° 0.45 × 0.39 × 0.38 mm V = 1587.8 (3) Å3 c = 14.3400 (15) Å µ = 2.58 mm−1 α = 112.936 (2)° T = 298 K β = 92.986 (1)° Block, light yellow γ = 104.305 (1)° 0.45 × 0.39 × 0.38 mm V = 1587.8 (3) Å3 c = 14.3400 (15) Å α = 112.936 (2)° β = 92.986 (1)° γ = 104.305 (1)° V = 1587.8 (3) Å3 µ = 2.58 mm−1 T = 298 K Block, light yellow 0.45 × 0.39 × 0.38 mm µ = 2.58 mm−1 T = 298 K Block, light yellow 0.45 × 0.39 × 0.38 mm Data collection Bruker SMART CCD diffractometer 5530 independent reflections Radiation source: fine-focus sealed tube 2795 reflections with I > 2σ(I) graphite Rint = 0.031 Detector resolution: 9 pixels mm-1 θmax = 25.0°, θmin = 1.6° φ and ω scans h = −11→12 Absorption correction: multi-scan (SADABS; Sheldrick, 1996) k = −14→14 Tmin = 0.390, Tmax = 0.440 l = −17→13 8347 measured reflections Data collection Bruker SMART CCD diffractometer Radiation source: fine-focus sealed tube graphite Detector resolution: 9 pixels mm-1 φ and ω scans Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.390, Tmax = 0.440 8347 measured reflections 5530 independent reflections 2795 reflections with I > 2σ(I) Rint = 0.031 θmax = 25.0°, θmin = 1.6° h = −11→12 k = −14→14 l = −17→13 Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.390, Tmax = 0.440 8347 measured reflections Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. supplementary materials supplementary materials C22 0.0708 (4) 0.2051 (4) 0.3388 (3) 0.0484 (11) C23 0.2142 (4) 0.2194 (4) 0.3527 (3) 0.0501 (11) H23 0.2670 0.2413 0.3083 0.060* C24 0.2755 (4) 0.2016 (4) 0.4297 (3) 0.0508 (11) H24 0.3694 0.2111 0.4363 0.061* C25 −0.1419 (4) 0.1959 (4) 0.2406 (3) 0.0645 (13) H25A −0.1652 0.2360 0.1986 0.077* H25B −0.1738 0.2303 0.3054 0.077* C26 −0.2177 (5) 0.0589 (4) 0.1868 (4) 0.0831 (16) H26A −0.1875 0.0241 0.1222 0.125* H26B −0.3147 0.0477 0.1752 0.125* H26C −0.1984 0.0189 0.2291 0.125* C27 0.0858 (5) 0.2632 (4) 0.1898 (3) 0.0614 (13) H27A 0.1768 0.3158 0.2263 0.074* H27B 0.0396 0.3115 0.1683 0.074* C28 0.1001 (6) 0.1581 (5) 0.0958 (4) 0.0974 (18) H28A 0.1378 0.1053 0.1163 0.146* H28B 0.1602 0.1899 0.0571 0.146* H28C 0.0115 0.1123 0.0540 0.146* C29 0.4486 (4) 0.1290 (4) 0.6724 (3) 0.0477 (11) C30 0.5184 (4) 0.0424 (4) 0.6583 (3) 0.0576 (12) H30 0.5236 −0.0093 0.5919 0.069* C31 0.5808 (4) 0.0306 (4) 0.7406 (3) 0.0550 (12) H31 0.6247 −0.0298 0.7299 0.066* C32 0.5762 (4) 0.1106 (4) 0.8388 (3) 0.0515 (11) C33 0.5105 (5) 0.1977 (4) 0.8546 (3) 0.0561 (12) H33 0.5086 0.2511 0.9212 0.067* C34 0.4466 (5) 0.2079 (4) 0.7729 (3) 0.0565 (12) H34 0.4016 0.2678 0.7846 0.068* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Br1 0.1166 (6) 0.0896 (5) 0.0824 (4) 0.0230 (4) −0.0006 (3) 0.0539 (3) Br2 0.0902 (4) 0.0839 (4) 0.0634 (3) 0.0281 (3) 0.0118 (3) 0.0378 (3) N1 0.053 (3) 0.051 (2) 0.058 (2) 0.008 (2) 0.009 (2) 0.024 (2) N2 0.058 (3) 0.057 (3) 0.070 (3) 0.004 (2) −0.001 (2) 0.035 (2) N3 0.059 (3) 0.056 (2) 0.050 (2) 0.017 (2) 0.014 (2) 0.0225 (19) N4 0.048 (3) 0.053 (2) 0.065 (2) 0.016 (2) 0.014 (2) 0.026 (2) C1 0.056 (3) 0.042 (3) 0.057 (3) 0.013 (3) 0.018 (2) 0.016 (2) C2 0.050 (3) 0.045 (3) 0.048 (3) 0.015 (3) 0.012 (2) 0.020 (2) C3 0.059 (3) 0.039 (3) 0.054 (3) 0.007 (3) 0.014 (2) 0.020 (2) C4 0.050 (3) 0.043 (3) 0.055 (3) 0.001 (2) 0.003 (2) 0.020 (2) C5 0.055 (3) 0.044 (3) 0.051 (3) 0.015 (3) 0.011 (2) 0.020 (2) C6 0.052 (3) 0.045 (3) 0.073 (3) 0.002 (3) 0.013 (3) 0.032 (3) C7 0.046 (3) 0.058 (3) 0.065 (3) 0.009 (3) 0.008 (2) 0.028 (3) C8 0.062 (4) 0.066 (4) 0.076 (3) 0.020 (3) 0.006 (3) 0.037 (3) C9 0.111 (5) 0.076 (4) 0.090 (4) 0.023 (4) −0.017 (3) 0.029 (3) Special details sup-2 Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-2 supplementary materials Br1 0.63511 (6) 0.55016 (5) 0.91153 (4) 0.0924 (2) Br2 0.66046 (6) 0.09334 (5) 0.95086 (4) 0.0763 (2) N1 0.3683 (4) 0.6583 (3) 0.5818 (3) 0.0545 (10) N2 −0.0254 (4) 0.6568 (3) 0.2131 (3) 0.0618 (10) N3 0.3916 (4) 0.1413 (3) 0.5863 (3) 0.0548 (10) N4 0.0089 (4) 0.2236 (3) 0.2607 (3) 0.0546 (10) C1 0.2611 (5) 0.5804 (4) 0.5184 (3) 0.0527 (12) H1 0.2260 0.5062 0.5235 0.063 C2 0.1917 (5) 0.6014 (4) 0.4395 (3) 0.0473 (11) C3 0.0608 (5) 0.5270 (4) 0.3888 (3) 0.0519 (11) H3 0.0204 0.4622 0.4055 0.062* C4 −0.0118 (4) 0.5440 (4) 0.3156 (3) 0.0521 (11) H4 −0.1001 0.4924 0.2849 0.062* C5 0.0457 (5) 0.6385 (4) 0.2864 (3) 0.0497 (11) C6 0.1787 (5) 0.7138 (4) 0.3369 (3) 0.0568 (12) H6 0.2206 0.7771 0.3189 0.068* C7 0.2474 (5) 0.6970 (4) 0.4109 (3) 0.0570 (12) H7 0.3342 0.7505 0.4437 0.068* C8 −0.1677 (5) 0.5849 (4) 0.1678 (3) 0.0656 (14) H8A −0.2121 0.6319 0.1430 0.079* H8B −0.2156 0.5705 0.2205 0.079* C9 −0.1800 (6) 0.4626 (5) 0.0798 (4) 0.0963 (18) H9A −0.1391 0.4763 0.0251 0.144* H9B −0.2753 0.4171 0.0554 0.144* H9C −0.1335 0.4167 0.1033 0.144* C10 0.0409 (5) 0.7398 (4) 0.1669 (3) 0.0661 (13) H10A 0.0001 0.7054 0.0951 0.079* H10B 0.1376 0.7448 0.1702 0.079* C11 0.0271 (6) 0.8664 (4) 0.2190 (4) 0.0936 (18) H11A −0.0683 0.8619 0.2198 0.140* H11B 0.0650 0.9143 0.1825 0.140* H11C 0.0757 0.9045 0.2883 0.140* C12 0.4256 (4) 0.6269 (4) 0.6563 (3) 0.0465 (11) C13 0.4527 (4) 0.5157 (4) 0.6331 (3) 0.0558 (12) H13 0.4283 0.4558 0.5659 0.067* C14 0.5154 (5) 0.4918 (4) 0.7080 (3) 0.0608 (13) H14 0.5339 0.4170 0.6915 0.073* C15 0.5501 (4) 0.5810 (4) 0.8077 (3) 0.0557 (12) C16 0.5253 (4) 0.6932 (4) 0.8328 (3) 0.0565 (12) H16 0.5484 0.7521 0.9003 0.068* C17 0.4664 (4) 0.7169 (4) 0.7578 (3) 0.0533 (12) H17 0.4533 0.7936 0.7741 0.064* C18 0.2677 (5) 0.1487 (4) 0.5796 (3) 0.0580 (12) H18 0.2170 0.1400 0.6296 0.070* C19 0.2032 (5) 0.1699 (4) 0.4987 (3) 0.0500 (11) C20 0.0633 (5) 0.1592 (4) 0.4867 (3) 0.0579 (12) H20 0.0122 0.1399 0.5329 0.070* C21 −0.0015 (5) 0.1758 (4) 0.4104 (3) 0.0572 (12) H21 −0.0952 0.1675 0.4055 0.069* sup-3 supplementary materials Atomic displacement parameters (Å2) sup-4 supplementary materials su C10 0.070 (4) 0.069 (4) 0.067 (3) 0.023 (3) 0.011 (3) 0.033 (3) C11 0.108 (5) 0.078 (4) 0.115 (4) 0.040 (4) 0.038 (4) 0.050 (4) C12 0.042 (3) 0.046 (3) 0.053 (3) 0.010 (2) 0.014 (2) 0.023 (2) C13 0.069 (3) 0.048 (3) 0.049 (3) 0.015 (3) 0.015 (2) 0.019 (2) C14 0.078 (4) 0.048 (3) 0.065 (3) 0.025 (3) 0.027 (3) 0.027 (3) C15 0.058 (3) 0.061 (3) 0.063 (3) 0.019 (3) 0.021 (2) 0.038 (3) C16 0.056 (3) 0.058 (3) 0.051 (3) 0.015 (3) 0.013 (2) 0.019 (2) C17 0.052 (3) 0.048 (3) 0.061 (3) 0.018 (2) 0.018 (2) 0.021 (3) C18 0.062 (3) 0.053 (3) 0.049 (3) 0.009 (3) 0.016 (3) 0.016 (2) C19 0.055 (3) 0.048 (3) 0.043 (3) 0.014 (2) 0.014 (2) 0.015 (2) C20 0.052 (3) 0.065 (3) 0.061 (3) 0.020 (3) 0.029 (2) 0.027 (3) C21 0.047 (3) 0.065 (3) 0.065 (3) 0.020 (3) 0.024 (3) 0.029 (3) C22 0.045 (3) 0.037 (3) 0.055 (3) 0.012 (2) 0.009 (2) 0.011 (2) C23 0.043 (3) 0.051 (3) 0.052 (3) 0.010 (2) 0.015 (2) 0.019 (2) C24 0.043 (3) 0.048 (3) 0.055 (3) 0.015 (2) 0.010 (2) 0.013 (2) C25 0.046 (3) 0.065 (4) 0.087 (3) 0.019 (3) 0.010 (3) 0.034 (3) C26 0.054 (3) 0.066 (4) 0.120 (4) 0.010 (3) 0.004 (3) 0.036 (3) C27 0.067 (3) 0.061 (3) 0.060 (3) 0.021 (3) 0.009 (3) 0.028 (3) C28 0.098 (5) 0.089 (4) 0.079 (4) 0.015 (4) 0.029 (3) 0.013 (3) C29 0.045 (3) 0.042 (3) 0.054 (3) 0.007 (2) 0.017 (2) 0.020 (2) C30 0.068 (3) 0.057 (3) 0.048 (3) 0.022 (3) 0.023 (2) 0.018 (2) C31 0.060 (3) 0.050 (3) 0.062 (3) 0.021 (3) 0.024 (2) 0.026 (3) C32 0.058 (3) 0.044 (3) 0.050 (3) 0.009 (2) 0.010 (2) 0.020 (2) C33 0.069 (3) 0.044 (3) 0.049 (3) 0.014 (3) 0.023 (2) 0.013 (2) C34 0.068 (3) 0.047 (3) 0.057 (3) 0.025 (3) 0.023 (2) 0.018 (3) Geometric parameters (Å, °) Br1—C15 1.893 (4) C14—C15 1.382 (6) Br2—C32 1.891 (4) C14—H14 0.9300 N1—C1 1.281 (5) C15—C16 1.381 (6) N1—C12 1.414 (5) C16—C17 1.367 (5) N2—C5 1.367 (5) C16—H16 0.9300 N2—C8 1.459 (5) C17—H17 0.9300 N2—C10 1.476 (5) C18—C19 1.444 (5) N3—C18 1.290 (5) C18—H18 0.9300 N3—C29 1.414 (5) C19—C24 1.382 (5) N4—C22 1.378 (5) C19—C20 1.394 (5) N4—C27 1.472 (5) C20—C21 1.356 (5) N4—C25 1.476 (5) C20—H20 0.9300 C1—C2 1.443 (5) C21—C22 1.402 (5) C1—H1 0.9300 C21—H21 0.9300 C2—C3 1.387 (5) C22—C23 1.422 (5) C2—C7 1.399 (6) C23—C24 1.358 (5) C3—C4 1.363 (5) C23—H23 0.9300 C3—H3 0.9300 C24—H24 0.9300 C4—C5 1.397 (5) C25—C26 1.526 (6) C4—H4 0.9300 C25—H25A 0.9700 C5—C6 1.405 (6) C25—H25B 0.9700 sup-5 supplementary materials supplementary materials 1.350 (5) C26—H26A 0.9300 C26—H26B 0.9300 C26—H26C 1.515 (6) C27—C28 0.9700 C27—H27A 0.9700 C27—H27B 0.9600 C28—H28A 0.9600 C28—H28B 0.9600 C28—H28C 1.494 (6) C29—C30 0.9700 C29—C34 0.9700 C30—C31 0.9600 C30—H30 0.9600 C31—C32 0.9600 C31—H31 1.384 (5) C32—C33 1.405 (5) C33—C34 1.385 (5) C33—H33 0.9300 C34—H34 118.0 (4) C15—C16—H16 121.6 (4) C16—C17—C12 122.2 (4) C16—C17—H17 115.9 (3) C12—C17—H17 119.3 (4) N3—C18—C19 123.0 (4) N3—C18—H18 120.2 (4) C19—C18—H18 116.7 (3) C24—C19—C20 123.6 (4) C24—C19—C18 118.2 C20—C19—C18 118.2 C21—C20—C19 115.8 (4) C21—C20—H20 120.5 (4) C19—C20—H20 123.7 (4) C20—C21—C22 123.4 (4) C20—C21—H21 118.3 C22—C21—H21 118.3 N4—C22—C21 120.3 (4) N4—C22—C23 119.8 C21—C22—C23 119.8 C24—C23—C22 121.1 (4) C24—C23—H23 122.2 (4) C22—C23—H23 116.6 (4) C23—C24—C19 122.0 (4) C23—C24—H24 119.0 C19—C24—H24 119.0 N4—C25—C26 121.8 (4) N4—C25—H25A 119.1 C26—C25—H25A 119.1 N4—C25—H25B 112.8 (4) C26—C25—H25B 1.350 (5) C26—H26A 0.9 0.9300 C26—H26B 0.9 0.9300 C26—H26C 0.9 1.515 (6) C27—C28 1.5 0.9700 C27—H27A 0.9 0.9700 C27—H27B 0.9 0.9600 C28—H28A 0.9 0.9600 C28—H28B 0.9 0.9600 C28—H28C 0.9 1.494 (6) C29—C30 1.3 0.9700 C29—C34 1.3 0.9700 C30—C31 1.3 0.9600 C30—H30 0.9 0.9600 C31—C32 1.3 0.9600 C31—H31 0.9 1.384 (5) C32—C33 1.3 1.405 (5) C33—C34 1.3 1.385 (5) C33—H33 0.9 0.9300 C34—H34 0.9 118.0 (4) C15—C16—H16 12 121.6 (4) C16—C17—C12 12 122.2 (4) C16—C17—H17 11 115.9 (3) C12—C17—H17 11 119.3 (4) N3—C18—C19 12 123.0 (4) N3—C18—H18 11 120.2 (4) C19—C18—H18 11 116.7 (3) C24—C19—C20 11 123.6 (4) C24—C19—C18 12 118.2 C20—C19—C18 12 118.2 C21—C20—C19 12 115.8 (4) C21—C20—H20 11 120.5 (4) C19—C20—H20 11 123.7 (4) C20—C21—C22 12 123.4 (4) C20—C21—H21 11 118.3 C22—C21—H21 11 118.3 N4—C22—C21 12 120.3 (4) N4—C22—C23 12 119.8 C21—C22—C23 11 119.8 C24—C23—C22 12 121.1 (4) C24—C23—H23 11 122.2 (4) C22—C23—H23 11 116.6 (4) C23—C24—C19 12 122.0 (4) C23—C24—H24 11 119.0 C19—C24—H24 11 119.0 N4—C25—C26 11 121.8 (4) N4—C25—H25A 10 119.1 C26—C25—H25A 10 119.1 N4—C25—H25B 10 112.8 (4) C26—C25—H25B 10 sup-6 C6—C7 1.350 (5) C26—H26A C6—H6 0.9300 C26—H26B C7—H7 0.9300 C26—H26C C8—C9 1.515 (6) C27—C28 C8—H8A 0.9700 C27—H27A C8—H8B 0.9700 C27—H27B C9—H9A 0.9600 C28—H28A C9—H9B 0.9600 C28—H28B C9—H9C 0.9600 C28—H28C C10—C11 1.494 (6) C29—C30 C10—H10A 0.9700 C29—C34 C10—H10B 0.9700 C30—C31 C11—H11A 0.9600 C30—H30 C11—H11B 0.9600 C31—C32 C11—H11C 0.9600 C31—H31 C12—C13 1.384 (5) C32—C33 C12—C17 1.405 (5) C33—C34 C13—C14 1.385 (5) C33—H33 C13—H13 0.9300 C34—H34 C1—N1—C12 118.0 (4) C15—C16—H16 C5—N2—C8 121.6 (4) C16—C17—C12 C5—N2—C10 122.2 (4) C16—C17—H17 C8—N2—C10 115.9 (3) C12—C17—H17 C18—N3—C29 119.3 (4) N3—C18—C19 C22—N4—C27 123.0 (4) N3—C18—H18 C22—N4—C25 120.2 (4) C19—C18—H18 C27—N4—C25 116.7 (3) C24—C19—C20 N1—C1—C2 123.6 (4) C24—C19—C18 N1—C1—H1 118.2 C20—C19—C18 C2—C1—H1 118.2 C21—C20—C19 C3—C2—C7 115.8 (4) C21—C20—H20 C3—C2—C1 120.5 (4) C19—C20—H20 C7—C2—C1 123.7 (4) C20—C21—C22 C4—C3—C2 123.4 (4) C20—C21—H21 C4—C3—H3 118.3 C22—C21—H21 C2—C3—H3 118.3 N4—C22—C21 C3—C4—C5 120.3 (4) N4—C22—C23 C3—C4—H4 119.8 C21—C22—C23 C5—C4—H4 119.8 C24—C23—C22 N2—C5—C4 121.1 (4) C24—C23—H23 N2—C5—C6 122.2 (4) C22—C23—H23 C4—C5—C6 116.6 (4) C23—C24—C19 C7—C6—C5 122.0 (4) C23—C24—H24 C7—C6—H6 119.0 C19—C24—H24 C5—C6—H6 119.0 N4—C25—C26 C6—C7—C2 121.8 (4) N4—C25—H25A C6—C7—H7 119.1 C26—C25—H25A C2—C7—H7 119.1 N4—C25—H25B N2—C8—C9 112.8 (4) C26—C25—H25B sup-6 supplementary materials 109.0 H25A—C25—H25B 109.0 C25—C26—H26A 109.0 C25—C26—H26B 109.0 H26A—C26—H26B 107.8 C25—C26—H26C 109.5 H26A—C26—H26C 109.5 H26B—C26—H26C 109.5 N4—C27—C28 109.5 N4—C27—H27A 109.5 C28—C27—H27A 109.5 N4—C27—H27B 113.0 (4) C28—C27—H27B 109.0 H27A—C27—H27B 109.0 C27—C28—H28A 109.0 C27—C28—H28B 109.0 H28A—C28—H28B 107.8 C27—C28—H28C 109.5 H28A—C28—H28C 109.5 H28B—C28—H28C 109.5 C30—C29—C34 109.5 C30—C29—N3 109.5 C34—C29—N3 109.5 C29—C30—C31 118.3 (4) C29—C30—H30 123.5 (4) C31—C30—H30 118.1 (4) C32—C31—C30 121.2 (4) C32—C31—H31 119.4 C30—C31—H31 119.4 C33—C32—C31 118.9 (4) C33—C32—Br2 120.5 C31—C32—Br2 120.5 C32—C33—C34 121.1 (4) C32—C33—H33 119.3 (3) C34—C33—H33 119.7 (3) C33—C34—C29 119.5 (4) C33—C34—H34 120.2 C29—C34—H34 179.3 (4) C29—N3—C18—C19 −165.6 (4) N3—C18—C19—C24 12.7 (7) N3—C18—C19—C20 −0.3 (6) C24—C19—C20—C21 178.2 (4) C18—C19—C20—C21 1.3 (6) C19—C20—C21—C22 4.7 (6) C27—N4—C22—C21 −168.3 (4) C25—N4—C22—C21 −174.5 (4) C27—N4—C22—C23 12.5 (6) C25—N4—C22—C23 179.9 (4) C20—C21—C22—N4 −0.8 (6) C20—C21—C22—C23 N2—C8—H8A 109.0 H25A—C25—H25B C9—C8—H8A 109.0 C25—C26—H26A N2—C8—H8B 109.0 C25—C26—H26B C9—C8—H8B 109.0 H26A—C26—H26B H8A—C8—H8B 107.8 C25—C26—H26C C8—C9—H9A 109.5 H26A—C26—H26C C8—C9—H9B 109.5 H26B—C26—H26C H9A—C9—H9B 109.5 N4—C27—C28 C8—C9—H9C 109.5 N4—C27—H27A H9A—C9—H9C 109.5 C28—C27—H27A H9B—C9—H9C 109.5 N4—C27—H27B N2—C10—C11 113.0 (4) C28—C27—H27B N2—C10—H10A 109.0 H27A—C27—H27B C11—C10—H10A 109.0 C27—C28—H28A N2—C10—H10B 109.0 C27—C28—H28B C11—C10—H10B 109.0 H28A—C28—H28B H10A—C10—H10B 107.8 C27—C28—H28C C10—C11—H11A 109.5 H28A—C28—H28C C10—C11—H11B 109.5 H28B—C28—H28C H11A—C11—H11B 109.5 C30—C29—C34 C10—C11—H11C 109.5 C30—C29—N3 H11A—C11—H11C 109.5 C34—C29—N3 H11B—C11—H11C 109.5 C29—C30—C31 C13—C12—C17 118.3 (4) C29—C30—H30 C13—C12—N1 123.5 (4) C31—C30—H30 C17—C12—N1 118.1 (4) C32—C31—C30 C12—C13—C14 121.2 (4) C32—C31—H31 C12—C13—H13 119.4 C30—C31—H31 C14—C13—H13 119.4 C33—C32—C31 C15—C14—C13 118.9 (4) C33—C32—Br2 C15—C14—H14 120.5 C31—C32—Br2 C13—C14—H14 120.5 C32—C33—C34 C16—C15—C14 121.1 (4) C32—C33—H33 C16—C15—Br1 119.3 (3) C34—C33—H33 C14—C15—Br1 119.7 (3) C33—C34—C29 C17—C16—C15 119.5 (4) C33—C34—H34 C17—C16—H16 120.2 C29—C34—H34 C12—N1—C1—C2 179.3 (4) C29—N3—C18—C19 N1—C1—C2—C3 −165.6 (4) N3—C18—C19—C24 N1—C1—C2—C7 12.7 (7) N3—C18—C19—C20 C7—C2—C3—C4 −0.3 (6) C24—C19—C20—C21 C1—C2—C3—C4 178.2 (4) C18—C19—C20—C21 C2—C3—C4—C5 1.3 (6) C19—C20—C21—C22 C8—N2—C5—C4 4.7 (6) C27—N4—C22—C21 C10—N2—C5—C4 −168.3 (4) C25—N4—C22—C21 C8—N2—C5—C6 −174.5 (4) C27—N4—C22—C23 C10—N2—C5—C6 12.5 (6) C25—N4—C22—C23 C3—C4—C5—N2 179.9 (4) C20—C21—C22—N4 C3—C4—C5—C6 −0.8 (6) C20—C21—C22—C23 109.0 H25A—C25—H25B 107.7 109.0 C25—C26—H26A 109.5 109.0 C25—C26—H26B 109.5 109.0 H26A—C26—H26B 109.5 107.8 C25—C26—H26C 109.5 109.5 H26A—C26—H26C 109.5 109.5 H26B—C26—H26C 109.5 109.5 N4—C27—C28 113.6 (4) 109.5 N4—C27—H27A 108.8 109.5 C28—C27—H27A 108.8 109.5 N4—C27—H27B 108.8 113.0 (4) C28—C27—H27B 108.8 109.0 H27A—C27—H27B 107.7 109.0 C27—C28—H28A 109.5 109.0 C27—C28—H28B 109.5 109.0 H28A—C28—H28B 109.5 107.8 C27—C28—H28C 109.5 109.5 H28A—C28—H28C 109.5 109.5 H28B—C28—H28C 109.5 109.5 C30—C29—C34 117.8 (4) 109.5 C30—C29—N3 119.7 (4) 109.5 C34—C29—N3 122.3 (4) 109.5 C29—C30—C31 121.7 (4) 118.3 (4) C29—C30—H30 119.2 123.5 (4) C31—C30—H30 119.2 118.1 (4) C32—C31—C30 118.6 (4) 121.2 (4) C32—C31—H31 120.7 119.4 C30—C31—H31 120.7 119.4 C33—C32—C31 120.8 (4) 118.9 (4) C33—C32—Br2 120.8 (3) 120.5 C31—C32—Br2 118.3 (3) 120.5 C32—C33—C34 120.5 (4) 121.1 (4) C32—C33—H33 119.7 119.3 (3) C34—C33—H33 119.7 119.7 (3) C33—C34—C29 120.5 (4) 119.5 (4) C33—C34—H34 119.7 120.2 C29—C34—H34 119.7 179.3 (4) C29—N3—C18—C19 175.5 (4) −165.6 (4) N3—C18—C19—C24 −9.6 (7) 12.7 (7) N3—C18—C19—C20 170.5 (4) −0.3 (6) C24—C19—C20—C21 1.5 (6) 178.2 (4) C18—C19—C20—C21 −178.5 (4) 1.3 (6) C19—C20—C21—C22 −0.1 (7) 4.7 (6) C27—N4—C22—C21 175.4 (4) −168.3 (4) C25—N4—C22—C21 −8.7 (6) −174.5 (4) C27—N4—C22—C23 −2.3 (6) 12.5 (6) C25—N4—C22—C23 173.6 (4) 179.9 (4) C20—C21—C22—N4 −179.5 (4) −0.8 (6) C20—C21—C22—C23 −1.6 (6) —C8—H8A 109.0 H25A—C25—H25B 107.7 —C8—H8A 109.0 C25—C26—H26A 109.5 —C8—H8B 109.0 C25—C26—H26B 109.5 —C8—H8B 109.0 H26A—C26—H26B 109.5 A—C8—H8B 107.8 C25—C26—H26C 109.5 —C9—H9A 109.5 H26A—C26—H26C 109.5 —C9—H9B 109.5 H26B—C26—H26C 109.5 A—C9—H9B 109.5 N4—C27—C28 113.6 (4) —C9—H9C 109.5 N4—C27—H27A 108.8 A—C9—H9C 109.5 C28—C27—H27A 108.8 B—C9—H9C 109.5 N4—C27—H27B 108.8 —C10—C11 113.0 (4) C28—C27—H27B 108.8 —C10—H10A 109.0 H27A—C27—H27B 107.7 1—C10—H10A 109.0 C27—C28—H28A 109.5 —C10—H10B 109.0 C27—C28—H28B 109.5 1—C10—H10B 109.0 H28A—C28—H28B 109.5 0A—C10—H10B 107.8 C27—C28—H28C 109.5 0—C11—H11A 109.5 H28A—C28—H28C 109.5 0—C11—H11B 109.5 H28B—C28—H28C 109.5 1A—C11—H11B 109.5 C30—C29—C34 117.8 (4) 0—C11—H11C 109.5 C30—C29—N3 119.7 (4) 1A—C11—H11C 109.5 C34—C29—N3 122.3 (4) 1B—C11—H11C 109.5 C29—C30—C31 121.7 (4) 3—C12—C17 118.3 (4) C29—C30—H30 119.2 3—C12—N1 123.5 (4) C31—C30—H30 119.2 7—C12—N1 118.1 (4) C32—C31—C30 118.6 (4) 2—C13—C14 121.2 (4) C32—C31—H31 120.7 2—C13—H13 119.4 C30—C31—H31 120.7 4—C13—H13 119.4 C33—C32—C31 120.8 (4) 5—C14—C13 118.9 (4) C33—C32—Br2 120.8 (3) 5—C14—H14 120.5 C31—C32—Br2 118.3 (3) 3—C14—H14 120.5 C32—C33—C34 120.5 (4) 6—C15—C14 121.1 (4) C32—C33—H33 119.7 6—C15—Br1 119.3 (3) C34—C33—H33 119.7 4—C15—Br1 119.7 (3) C33—C34—C29 120.5 (4) 7—C16—C15 119.5 (4) C33—C34—H34 119.7 7—C16—H16 120.2 C29—C34—H34 119.7 2—N1—C1—C2 179.3 (4) C29—N3—C18—C19 175.5 (4) —C1—C2—C3 −165.6 (4) N3—C18—C19—C24 −9.6 (7) —C1—C2—C7 12.7 (7) N3—C18—C19—C20 170.5 (4) —C2—C3—C4 −0.3 (6) C24—C19—C20—C21 1.5 (6) —C2—C3—C4 178.2 (4) C18—C19—C20—C21 −178.5 (4) —C3—C4—C5 1.3 (6) C19—C20—C21—C22 −0.1 (7) —N2—C5—C4 4.7 (6) C27—N4—C22—C21 175.4 (4) 0—N2—C5—C4 −168.3 (4) C25—N4—C22—C21 −8.7 (6) —N2—C5—C6 −174.5 (4) C27—N4—C22—C23 −2.3 (6) 0—N2—C5—C6 12.5 (6) C25—N4—C22—C23 173.6 (4) —C4—C5—N2 179.9 (4) C20—C21—C22—N4 −179.5 (4) —C4—C5—C6 −0.8 (6) C20—C21—C22—C23 −1.6 (6) 109.0 H25A—C25—H25B 1 109.0 C25—C26—H26A 1 109.0 C25—C26—H26B 1 109.0 H26A—C26—H26B 1 107.8 C25—C26—H26C 1 109.5 H26A—C26—H26C 1 109.5 H26B—C26—H26C 1 109.5 N4—C27—C28 1 109.5 N4—C27—H27A 1 109.5 C28—C27—H27A 1 109.5 N4—C27—H27B 1 113.0 (4) C28—C27—H27B 1 109.0 H27A—C27—H27B 1 109.0 C27—C28—H28A 1 109.0 C27—C28—H28B 1 109.0 H28A—C28—H28B 1 107.8 C27—C28—H28C 1 109.5 H28A—C28—H28C 1 109.5 H28B—C28—H28C 1 109.5 C30—C29—C34 1 109.5 C30—C29—N3 1 109.5 C34—C29—N3 1 109.5 C29—C30—C31 1 118.3 (4) C29—C30—H30 1 123.5 (4) C31—C30—H30 1 118.1 (4) C32—C31—C30 1 121.2 (4) C32—C31—H31 1 119.4 C30—C31—H31 1 119.4 C33—C32—C31 1 118.9 (4) C33—C32—Br2 1 120.5 C31—C32—Br2 1 120.5 C32—C33—C34 1 121.1 (4) C32—C33—H33 1 119.3 (3) C34—C33—H33 1 119.7 (3) C33—C34—C29 1 119.5 (4) C33—C34—H34 1 120.2 C29—C34—H34 1 179.3 (4) C29—N3—C18—C19 1 −165.6 (4) N3—C18—C19—C24 − 12.7 (7) N3—C18—C19—C20 1 −0.3 (6) C24—C19—C20—C21 1 178.2 (4) C18—C19—C20—C21 − 1.3 (6) C19—C20—C21—C22 − 4.7 (6) C27—N4—C22—C21 1 −168.3 (4) C25—N4—C22—C21 − −174.5 (4) C27—N4—C22—C23 − 12.5 (6) C25—N4—C22—C23 1 179.9 (4) C20—C21—C22—N4 − −0.8 (6) C20—C21—C22—C23 − sup-7 supplementary materials D—H···A ?—?···? supplementary materials N2—C5—C6—C7 178.5 (4) N4—C22—C23—C24 179.8 (4) C4—C5—C6—C7 −0.7 (6) C21—C22—C23—C24 1.9 (6) C5—C6—C7—C2 1.8 (7) C22—C23—C24—C19 −0.5 (6) C3—C2—C7—C6 −1.3 (6) C20—C19—C24—C23 −1.2 (6) C1—C2—C7—C6 −179.7 (4) C18—C19—C24—C23 178.8 (4) C5—N2—C8—C9 −84.0 (5) C22—N4—C25—C26 −75.2 (5) C10—N2—C8—C9 89.5 (5) C27—N4—C25—C26 101.0 (5) C5—N2—C10—C11 −94.6 (5) C22—N4—C27—C28 89.2 (5) C8—N2—C10—C11 92.0 (5) C25—N4—C27—C28 −86.9 (5) C1—N1—C12—C13 48.8 (6) C18—N3—C29—C30 133.9 (4) C1—N1—C12—C17 −136.0 (4) C18—N3—C29—C34 −51.2 (6) C17—C12—C13—C14 1.5 (7) C34—C29—C30—C31 2.1 (6) N1—C12—C13—C14 176.7 (4) N3—C29—C30—C31 177.3 (4) C12—C13—C14—C15 0.6 (7) C29—C30—C31—C32 −2.1 (7) C13—C14—C15—C16 −1.1 (7) C30—C31—C32—C33 0.9 (7) C13—C14—C15—Br1 179.7 (3) C30—C31—C32—Br2 179.6 (3) C14—C15—C16—C17 −0.5 (7) C31—C32—C33—C34 0.2 (7) Br1—C15—C16—C17 178.7 (3) Br2—C32—C33—C34 −178.4 (3) C15—C16—C17—C12 2.7 (7) C32—C33—C34—C29 −0.2 (7) C13—C12—C17—C16 −3.2 (6) C30—C29—C34—C33 −0.9 (7) N1—C12—C17—C16 −178.6 (4) N3—C29—C34—C33 −176.0 (4) Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A ?—?···? ? ? ? ? sup-8 supplementary materials Fig. 1 sup-9
https://openalex.org/W4387983538	https://dergipark.org.tr/tr/download/article-file/3079955	English	null	Architectural Sustainability with cultural heritage values	Cultural heritage and science	2,023	cc-by-sa	4,052	Abstract Sustainability and cultural heritage are two interconnected and interdependent issues that are essential for achieving sustainable development. Cultural heritage plays a critical role in society as it is an integral part of the social and cultural fabric. This article focuses on the relationship between sustainability and cultural heritage, specifically the integration of cultural heritage into urban and environmentally sustainable development. To achieve this, the study used Tabriz Bazaar as a case study to explore how cultural heritage can be preserved while balancing economic expansion and sustainable development. The research question is how cultural heritage can be integrated into urban and environmentally sustainable development using Tabriz Bazaar as a case study. The hypothesis is that the preservation and protection of cultural heritage values are crucial for achieving sustainable development, and an interdisciplinary approach is necessary to balance economic expansion with cultural heritage preservation. Using a case study approach, the research collected data through a literature review and interviews with experts in the fields of cultural heritage preservation, urban and environmental sustainability, and economics. The research analyzes the challenges of balancing economic development with cultural heritage preservation and proposes an interdisciplinary approach to address these challenges. To make the area more sustainable and preserve its cultural heritage values, specific measures such as promoting cycling in Tabriz Bazaar were recommended. Moreover, education and awareness-raising programs are also necessary to promote the importance of cultural heritage and its contribution to sustainable development. Overall, the research highlights the interdependence between sustainability and cultural heritage and emphasizes the importance of preserving cultural heritage to achieve sustainable development. Policymakers, urban planners, and cultural heritage conservationists can benefit from the findings of this study to develop strategies that promote sustainable development while preserving cultural heritage values. Elyasi, S., & Yamaçlı, R. (2023). Architectural sustainability with cultural heritage values. Cultural Heritage and Science, 4(2), 55-61 Cultural Heritage and Science https://dergipark.org.tr/en/pub/cuhes e-ISSN 2757-9050 Cultural Heritage and Science https://dergipark.org.tr/en/pub/cuhes e-ISSN 2757-9050 Sahar Elyasi 1 , Rusen Yamacli 1 1Eskişehir Technical University, Faculty of Architecture and Design, Department of Architecture, Eskişehir, Türki Keywords Sustainability Sustainable Development Cultural Heritage Cultural Heritage Management Research Article DOI: 10.58598/cuhes.1282179 Received:12.04.2023 Revised: 25.05.2023 Accepted:01.06.2023 Published:27.10.2023 Keywords Sustainability Sustainable Development Cultural Heritage Cultural Heritage Management Research Article DOI: 10.58598/cuhes.1282179 Received:12.04.2023 Revised: 25.05.2023 Accepted:01.06.2023 Published:27.10.2023 Corresponding Author (elyasiiisahar@yahoo.com) ORCID ID 0000-0002-9982-2770 (yamaclir@gmail.com) ORCID ID 0000-0001-9659-9246 Cultural Heritage and Science – 2023, 4(2), 55-61 Cultural Heritage and Science – 2023, 4(2), 55-61 Architectural sustainability with cultural heritage values Sahar Elyasi 1 , Rusen Yamacli 1 1. Introduction tourism, increased real estate values, small business improvement, and so on. Repurposing abandoned or underutilized historic structures is critical to regenerating communities and enhancing quality of life. It is critical to acknowledge cultural heritage as a significant resource and development incentive in order to adopt sustainable development methods and improve quality of life. The paper discusses the impact of immovable cultural heritage on the implementation of sustainable development strategies, the role of cultural heritage in the context of globalization as a fundamental means of avoiding the trend of cultural globalization, the formation of sustainable communities, and the potential impact of cultural heritage resources on economic development and resource productivity. It is important Sustainable development encompasses not just ecological but also economic, social, and cultural dimensions. Many research has shown that preserving cultural heritage enhances ecological, social, cultural, and economic sustainability. Cultural legacy may benefit communities' well-being and quality of life, assist in alleviating the consequences of cultural globalization, and serve as an incentive for long-term economic growth. Cultural heritage preservation is frequently viewed as a barrier to economic development, despite the fact that cultural heritage and its preservation can generate a variety of economic benefits, including income and job creation, professional training and the preservation of craft skills, revitalization of city centers, heritage Cite this article (APA); Cultural Heritage and Science – 2023, 4(2), 55-61 economic and social roles as well, including as a tourist attraction, source of employment, and source of revenue [5]. It is also used to denote a cultural landscape that is particular to a region or area. Cultural heritage and the field of architectural science mutually constitute interrelated components that exhibit a synergistic relationship. A fresh outlook on cultural heritage has materialized, evident in the consequential provisions of the Faro Convention. Notably, the Convention's second clause distinctly delineates cultural heritage as an assemblage of historical resources, which individuals perceive as an unattributed manifestation and mirror of their continuously evolving principles, convictions, erudition, and customs [6]. Cultural heritage is a dynamic and evolving field, with new elements constantly being added. Cultural heritage has a variety of roles and functions that are related to the local community and to the global community. Many of these roles are the same as those of other sectors, such as tourism, arts, education, and health [7]. However, these are sometimes overlooked or undervalued. 2.1. Sustainable development Sustainable development aims to improve living standards, preserve ecosystems, and secure basic needs while creating a safer and happier future. It involves designing economic, financial, trade, energy, agricultural, and industrial policies that promote sustainability. This includes investing in education, health, population, and energy to avoid social debt for future generations. Sustainable development is a comprehensive concept that impacts all aspects of human life, requiring significant changes in national and international policies. Ultimately, it is an approach to economic growth that seeks to meet the needs of the present without compromising the ability of future generations to meet their own needs [2]. The breadth of work in the name of sustainability, the diversity of existing concepts, the importance of ordering between principles, and the vast regional differences are some of the first to compel the designer to find the truth. The common purpose of sustainability studies is to examine from an architectural perspective how natural energy resources can be protected and conserved by assessing sustainability in relation to the natural environment [3]. This includes reducing carbon emissions, protecting natural resources, promoting social equity, and ensuring economic growth that is both environmentally responsible and socially inclusive. 1. Introduction They have a vital role to play in society and are essential to maintaining cultural identities and improving quality of life in the community [8]. Key roles and functions of cultural heritage include education and knowledge creation, economic development, tourism, and aesthetic enjoyment. While these are valuable contributions, they have not always been considered in the same way that other sectors of society have been treated. Some of the key challenges facing the field of cultural heritage include globalization and the loss of identity, changes in behavior and attitudes. for cultural heritage cities and urban heritage cities to recognize heritage preservation as a necessary goal for sustainable planning in urban planning. Our past experience shows to date that monument preservation planning makes a positive contribution to sustainable planning research. This is only possible by protecting and enhancing key elements of the natural and cultural heritage through planning [1]. Cultural heritage connects us to the past, serves as the basis of our identity and is a source of knowledge. 2.3. Cultural heritage and architectural sustainable development Short-term interests promote speculative developments, which pose substantial dangers to cultural assets and the historical landscape. Cultural assets and historic surroundings are important not just to individuals who own or reside in historic buildings. Cultural legacy may also contribute to a community's well-being and quality of life by preventing cultural globalization, preserving cultural variety, and positively impacting economic growth. According to D. Rypkema, in a larger perspective, the importance of heritage in the quest of sustainable development is unmistakable: the protection of cultural heritage offers environmental, cultural, and economic sustainability [9]. Cultural heritage plays a marginal role in the 2030 Agenda for Sustainable Development. It is only explicitly mentioned once in Goal 11, which relates to cities, specifically the need to make cities and human settlements inclusive, safe, resilient and sustainable through inclusive and sustainable urbanization, planning and management (Goal 11.3). and increased efforts to protect and preserve the world's cultural and natural heritage (target 11.4). Sustainable development is a broad concept that recognizes the interconnectedness of economic, social, and environmental factors, and it has direct implications for planning, housing, and urban development policies worldwide. Urban planning is an effective tool for steering sustainable development, but it alone is insufficient to ensure sustainability. The United Nations' Sustainable Development Goals (SDGs), particularly Goal 11, emphasize the need to create inclusive, safe, resilient, and sustainable cities and communities. The New Urban Agenda, adopted at the United Nations Conference on Housing and Sustainable Urban Development, also underscores the importance of participatory urban planning and partnerships among stakeholders to achieve sustainable development. 2.3.1. Cultural heritage values in Tabriz The term cultural heritage is used to refers to the “physical and intangible manifestations of a group’s identity” [4]. This includes such elements as art, architecture, stories, rituals, and so on. It has important According to the trend of comprehensive developments in today's cities of the world, the cities of Iran and Azerbaijan in general and the city of Tabriz in 56 Cultural Heritage and Science – 2023, 4(2), 55-61 careful selection of suitable methodologies for the project, and the seamless transmission of precise information across different stages of scientific research. Additionally, it is imperative to establish a comprehensive management and monitoring program that encompasses the pre-implementation, implementation, and post-implementation phases of conservation efforts [12]. In this regard, it is important to draw up a plan for the protection of cultural heritage values and then monitor its implementation to ensure that it is carried out correctly. In Iran, there is not a proper system for the management and protection of cultural heritage. Thus, the contribution of this project is to develop a comprehensive plan for the conservation of cultural heritage in the country. In order to do this, it is necessary to conduct a comprehensive assessment of cultural values and identify their needs for protection and preservation. This assessment will be based on the available data and information. It will also be carried out with the help of experts and authorities who have relevant experience in the field. As a result, the project will develop a model for the conservation and protection of cultural heritage in Iran that can be used to ensure the preservation of this valuable resource for future generations. The work that is being done under this project is expected to help Iran to make a valuable contribution to the global community by ensuring the conservation and preservation of its unique cultural heritage. This will ultimately enable Iran to contribute to the development of the global economy and enhance its reputation as a developing country in the global community. As a result, the success of the project will help to enhance the country’s economy and improve the overall quality of life for all citizens in Iran. particular have undergone many changes in the architecture with historical value and the way of distributing activities in recent decades. 2.3.1. Cultural heritage values in Tabriz The role of the city administration and planning in preserving historical and unique architectures and creating a healthy environment, economic, social, touristic city, etc. has helped to make the urban system healthy and balanced. The historical city of Tabriz has experienced various administrations and plans over the last few decades that have left their impact on the development of the city and the nature of the texture and environmental issues that have caused the destruction of the old architecture and the unequal distribution of the city services that these Problems in different eras and time management were different. Figure 1. The relationship between culture, society, environment and economy with sustainability. Figure 1. The relationship between culture, society, environment and economy with sustainability. Tabriz, located in northwestern Iran, has a rich cultural heritage that reflects its historical importance as a commercial center and cultural center. Some of the main heritage values in Tabriz are shown in Table 1. The sustainability of historical bazaars is only significant if it accurately reflects the everyday busyness of the community through their physical structures. It is crucial to consider how the renewal process affects the daily life of the community. Unfortunately, urban planners often make spatial changes in an effort to enhance functionality, resulting in changes or elimination of current behavioral regulations. When the workplaces of the sellers are significantly altered or removed, the working methods, physical presence, and sensory experiences they produce within the bazaar also change, all of which are integral to the bazaar environment and contribute to its individual memories and collective identity [13]. Therefore, the sustainability of historical bazaars can only be achieved if it accurately portrays the hustle and bustle of daily life through their physical structures, and if any renewal process considers the impact on the community's daily life and preserves the bazaar's unique character and identity. Cities are caught between the desire to be a member of the global network and the need to maintain their individuality and cultural origins. As new progress symbols have shattered conventional contexts, new meanings of community have developed [10]. The value of cultural heritage has always been an important factor in the development of any country. In fact, it has played an important role in the development of nations. In addition, it has played an important role in the development of the economies of these countries [11]. 2.3.1. Cultural heritage values in Tabriz In this sense, it has become a valuable resource for both individual and social development. Accordingly, it is important to develop a system for the protection and management of these values. Therefore, developing a system for the conservation of cultural heritage values is a priority in Iran. To do this, it is necessary to identify these values and then put measures in place to protect them from harm. The successful execution of a comprehensive conservation program, involving scientific surveys, analyses, and evaluations, entails the diligent coordination of proficient technical experts, Rebuilding Tabriz market with sustainable development in mind would involve considering economic, social, and environmental aspects of the project. Here are some steps that can be taken to achieve this (Table 1). 57 Cultural Heritage and Science – 2023, 4(2), 55-61 Table 1. Heritage values of Tabriz. Items Heritage values y z 1 Historic Architecture Tabriz has numerous historic buildings, such as the Tabriz Historic Bazaar Complex, the Blue Mosque and the Arg of Tabriz, which reflect the city's architectural and cultural heritage. 2 Traditional Crafts Tabriz has a long history of producing high-quality handicrafts such as carpets, copperware and pottery, which are highly valued both locally and internationally. 3 Culinary Traditions Tabriz is known for its unique and delicious cuisine, including dishes such as Kofte Tabrizi, a type of meatball and Ash Reshteh, a type of soup. Table 1. Heritage values of Tabriz. g Items Heritage values y z 1 Historic Architecture Tabriz has numerous historic buildings, such as the Tabriz Historic Bazaar Complex, the Blue Mosque and the Arg of Tabriz, which reflect the city's architectural and cultural heritage. 2 Traditional Crafts Tabriz has a long history of producing high-quality handicrafts such as carpets, copperware and pottery, which are highly valued both locally and internationally. 3 Culinary Traditions Tabriz is known for its unique and delicious cuisine, including dishes such as Kofte Tabrizi, a type of meatball and Ash Reshteh, a type of soup. 4 Festivals and Celebrations Tabriz hosts a number of annual festivals and cultural celebrations including the Tabriz International Carpet Fair, the Tabriz Traditional Music Festival and the Tabriz Pistachio Festival. 5 Cultural Diversity Tabriz has a diverse population that includes different ethnic and religious groups that have contributed to the city's cultural heritage over time. 3. Conclusion In summary, rebuilding Tabriz market with sustainable development in mind requires a comprehensive approach that considers economic, social, and environmental factors. By implementing the steps above, it's possible to create a sustainable market that promotes economic growth, environmental protection, and social well-being. 3. Educate the public: Educating the public about the benefits of cycling and the availability of bike lanes in the bazaar is essential to encourage more people to use them. This can be done through outreach programs, signage, and other forms of communication. Making the Tabriz bazaar more sustainable can have a positive impact on cultural heritage in several ways. Here are some examples: 4. Provide bike parking facilities: Providing secure and accessible bike parking facilities near the bazaar will encourage people to cycle to the bazaar. This could include bike racks, bike lockers, or even bike-sharing programs. Preservation of historical architecture: The Tabriz bazaar is a unique cultural heritage site with a rich history and unique architecture. By promoting sustainable practices in the bazaar, such as using sustainable materials and reducing waste production, the historical architecture of the bazaar can be preserved and maintained for future generations. 5. Work with local businesses: Working with local businesses to promote cycling to the bazaar can be a powerful tool in encouraging people to use bike lanes. For example, businesses could offer discounts or incentives to customers who arrive by bike. Promotion of local culture and heritage: Supporting local businesses and promoting sustainable tourism practices in the Tabriz bazaar can help to promote the local culture and heritage of the region. This can include showcasing traditional crafts and products, preserving local customs and traditions, and celebrating local festivals and events. 6. Collaborate with local government: Collaborating with local government to provide funding and support for bike lanes and other cycling infrastructure is critical to their success. This could include providing funding for bike lane construction and maintenance, as well as other initiatives to encourage cycling, such as public awareness campaigns and bike share programs. Increased awareness and education: By promoting sustainable practices in the Tabriz bazaar, people can become more aware of the importance of preserving cultural heritage and the role that sustainability can play in achieving this. This can lead to increased education and awareness of cultural heritage among visitors and residents of the bazaar. 2.3.1. Cultural heritage values in Tabriz Items Heritage values z Tabriz has numerous historic buildings, such as the Tabriz Historic Bazaar Complex, the Blue Mosque and the Arg of Tabriz, which reflect the city's architectural and cultural heritage. 58 Cultural Heritage and Science – 2023, 4(2), 55-61 Figure 2. Iran‘s Map. Figure 3.Tabriz‘s Map. Figure 4. Tabriz Bazaar Map (cyling road). l b l l h f d h d d Cultural Heritage and Science – 2023, 4(2), 55-61 Figure 3.Tabriz‘s Map. Figure 2. Iran‘s Map. Figure 3.Tabriz‘s Map. Figure 3.Tabriz‘s Map. Figure 2. Iran‘s Map. Figure 4. Tabriz Bazaar Map (cyling road). Figure 4. Tabriz Bazaar Map (cyling road). integrated into the design to reduce energy consumption and promote clean energy use. Develop a sustainability plan: The first step is to create a comprehensive plan that outlines the sustainable goals and objectives of the rebuilding project. This plan should consider factors such as reducing energy consumption, waste management, and promoting sustainable economic growth. Promote local economy: To ensure the long-term sustainability of the market, it's essential to promote the local economy by supporting local businesses and suppliers. This will create jobs and stimulate economic growth in the region. Use sustainable materials: In the rebuilding process, it's essential to use sustainable materials that have minimal environmental impacts. For example, materials that are locally sourced, recycled or have a low carbon footprint. Encourage public transportation: The new design of the market should encourage public transportation to reduce the use of private cars, which contribute to air pollution and traffic congestion. For example, bike lanes, bus stops, or public transportation stations can be incorporated into the design. Incorporate renewable energy sources: Renewable energy sources like solar or wind power can be 59 Cultural Heritage and Science – 2023, 4(2), 55-61 Implement waste management strategies: The market should have a comprehensive waste management plan that includes recycling and composting to minimize waste production and promote sustainability. local culture and heritage, increasing awareness and education, collaborating with local communities, and promoting sustainable tourism practices. To encourage public transportation like bike lanes in the bazaar of Tabriz, the following steps can be taken: 1. Conduct a feasibility study: Before implementing any bike lanes or other infrastructure, it's important to conduct a feasibility study to determine if it's possible and practical to create bike lanes in the bazaar. 2.3.1. Cultural heritage values in Tabriz This study should consider factors like the size of the bazaar, the number of visitors, and the availability of alternative transportation options. Consider social aspects: In addition to economic and environmental factors, social aspects such as inclusivity, accessibility, and safety should be considered in the rebuilding process. For example, the market should be designed to be accessible to people with disabilities and provide safe spaces for everyone. 2. Design safe and accessible bike lanes: The bike lanes should be designed to ensure the safety of cyclists and pedestrians, and to provide easy access to the bazaar. This includes separating the bike lane from vehicle traffic, providing clear signage, and ensuring that the bike lanes are well-maintained. 3. Conclusion By taking these steps, it's possible to encourage more people to use public transportation like bike lanes in the bazaar of Tabriz, reducing traffic congestion, improving air quality, and promoting sustainable transportation options. Collaboration with local communities: Making the Tabriz bazaar more sustainable can involve working closely with local communities to ensure that their cultural heritage is respected and preserved. This can involve collaborating with local artisans and craftspeople, supporting local festivals and events, and providing opportunities for community engagement and participation in sustainable practices. Author contributions Sahar Elyasi: Methodology, Field study, writing original draft preparation Ruşen Yamaçlı: Writing-Reviewing and Editing, validation Sustainable tourism: Sustainable tourism practices can help to promote cultural heritage in the Tabriz bazaar, such as supporting local businesses, providing opportunities for cultural exchange and learning, and minimizing negative impacts on the environment and local communities. Conflicts of interest There is no conflict of interest between the authors. In summary, making the Tabriz bazaar more sustainable can have a positive impact on cultural heritage by preserving historical architecture, promoting 60 Cultural Heritage and Science – 2023, 4(2), 55-61 References 7. Sułkowski, Ł. (2020). Covid-19 Pandemic; Recession, Virtual Revolution Leading to De-globalization? Journal of Intercultural Management, 12(1), 1-11. 1. Nocca, F. (2017). The role of cultural heritage in sustainable development: Multidimensional indicators as decision-making tool. Sustainability, 9(10), 1882. 8. Streimikiene, D., Mikalauskiene, A., & Kiausiene, I. (2019). The impact of value created by culture on approaching the sustainable development goals: Case of the Baltic States. Sustainability, 11(22), 6437. 2. Elyasi, S., & Yamacli, R. (2022). Investigation of Iranian residential buildings with sustainable architecture in hot and dry areas. Journal of Architecture, Engineering & Fine Arts, 4(1), 16-29. 9. Rypkema, D. (2005). Cultural heritage and sustainable economic and social development. Europa Nostra. 10. Banerjee, T., & Southworth, M. (1990). City Sense and City Design. The MIT Press. 3. Çiğan, A., & Yamaçlı, R. (2020). Doğal Enerji, Sürdürülebilir Kalkınma ve Mimarlık Politikaları. Duzce University Journal of Science and Technology, 8, 554_571 11. Seyhan, A. K., & Badem, A. (2018). Erzincan ilindeki hayvansal atıkların biyogaz potansiyelinin araştırılması. Academic Platform-Journal of Engineering and Science, 6(1), 25-35. 4. Bonenberg, W. (2019). The Role of Cultural Heritage in Sustainable Development. Values and Valuation as Key Factors in Spatial Planning of Rural Areas. Advances in Human Factors in Architecture, Sustainable Urban Planning and Infrastructure (s. 124-134). Advances in Intelligent Systems and Computing book series. 12. Naycı, N., Abruzzese, D., Güler, C., Tağa, H., Cammarano, P., Vuth, S., & Türkoğlu, H. (2020). Multidisciplinary Researches in Cultural Heritage Studies: An Approach on Akkale Cistern in Erdemli, Mersin. Cultural Heritage and Science, 1(1), 15-22. 13. Karataş, L. (2022). Conservation status of intangible cultural heritage after restoration: Case study of Mardin Spice Bazaar. Cultural Heritage and Science, 3(2), 30-36. 5. Ashfaq, J., Hassan, H., & Khan, A. (2022). The Impact of Mass Media, Word-Of-Mouth on Travel Intention and Mediating Role of Destination Image and Tourist Attitude. International Journal of Academic Research in Business and Social Sciences, 3224-3239. 6. Tepebaş, U. (2021). The Contribution of Archaeological Surveys on the Perception of Cultural Heritage: Cilicia as a Case Study. Cultural Heritage and Science, 25-30. © Author(s) 2023. This work is distributed under https://creativecommons.org/licenses/by-sa/4.0/ 61
https://openalex.org/W2773085329	https://ecojoin.org/index.php/EJM/article/download/254/243	Indonesian	null	MODEL KEPUASAN PELANGGAN: ANALISIS KUALITAS PRODUK DAN KUALITAS LAYANAN TERHADAP CITRA MEREK PADA GIANT CITRA RAYA JAKARTA	Jurnal Manajemen - Fakultas Ekonomi Universitas Tarumanagara/Jurnal manajemen	2,017	cc-by	8,076	Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Keywords: Product Quality, Service Quality, Brand Image and Customer Satisfaction. Keywords: Product Quality, Service Quality, Brand Image and Customer Satisfaction. Keywords: Product Quality, Service Quality, Brand Image and Customer Satisfaction. Abstrak: Penelitian ini bertujuan untuk menganalisis pengaruh kualitas produk dan kualitas layanan terhadap citra merek dan kepuasan pelanggan baik secara parsial maupun simultan pada Giant Citra Raya Tangerang. Populasi penelitian ini adalah Pelanggan Giant Citra Raya yang rata-rata 2.070.823/12/30 = 5752 perhari. Sampel pada penelitian ini menggunakan rumus Slovin adalah 152 pelangan. Metode analisis kuantitatif dengan menggunakan analisis jalur (Path Analysis), dilanjutkan dengan analisis determinasi (R Square), pengujian hipotesis secara parsial (uji t) dan simultan (uji F) dengan alpha 5 persen. Sebelum dianalisis lebih lanjut terlebih dahulu dilakukan uji instrument dan uji asumsi klasik. Alat bantu analisis menggunakan SPSS versi 21.0 for windows. Hasil penelitian menunjukkan kualitas produk dan kualitas layanan secara parsial dan simultan berpengaruh positif dan signifikan terhadap citra merek dan kepuasan pelanggan, begitu pula citra merek memiliki pengaruh terhadap kepuasan pelanggan pada Giant Citra Raya Tangerang. Keyword: Kualitas Produk, Kualitas layanan, Citra Merek dan Kepuasan Pelanggan. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 MODEL KEPUASAN PELANGGAN: ANALISIS KUALITAS PRODUK DAN KUALITAS LAYANAN TERHADAP CITRA MEREK PADA GIANT CITRA RAYA JAKARTA Agussalim M dan Hapzi Ali Fakuktas Ekonomi Universitas Eka Sakti, Padang Indonesia dan Pascasarjana Universitas Mercu Buana, Jakarta Indonesia Email: hapzi.ali@gmail.com Abstract: This study aims to analyze the effect of product quality and service quality on brand image and customer satisfaction either partially or simultaneously on Giant Citra Raya Tangerang. The population of this study is Giant Citra Raya customers which averages 2,070,823 / 12/30 = 5752 per day. The sample in this research using Slovin formula is 152 customers. Quantitative analysis method using path analysis, followed by determination analysis (R Square), partial hypothesis testing (t test) and simultaneous (F test) with alpha 5 percent. Before further analyzed first tested the instrument and test the classical assumption. Analytical tool using SPSS version 21.0 for windows. The result of the research shows that product quality and service quality are partially and simultaneously have positive and significant effect on brand image and customer satisfaction, and brand image has influence to customer satisfaction at Giant Citra Raya Tangerang. PENDAHULUAN Seiring dengan perkembangan bisnis ritel pada saat ini, bisnis ritel tidak lagi di kelola secara tradisional, melainkan telah dikelola dengan cara modern sehingga menjadi bisnis yang inovatif, dinamis, dan kompetitif. Persaingan yang ketat di bisnis ritel saat ini tidak terlepas dari semakin menjamurnya pusat-pusat perbelanjaan baik yang bersifat Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 317 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... lokal, nasional, maupun internasional dengan berbagai skala dan segmen yang di bidik. Berbagai cara dilakukan oleh perusahaan atau pelaku bisnis untuk menarik konsumen agar mau membeli produk yang ditawarkan, serta mempertahankan konsumen untuk tidak berpindah pada produk yang ditawarkan oleh pesaing. Tak terkecuali pula dengan Giant Tangerang Citra Raya yang merupakan salah satu bisnis ritel cabang dari PT Hero Supermarket Tbk, yang menawarkan konsep Low-price modern market kepada masyarakat. Semakin ketatnya persaingan bisnis ritel di wilayah Citra Raya, Cikupa dan sekitarnya menyebabkan tidak tercapainya target kunjungan pelanggan ke Giant Citra Raya yang malah cenderung mengalami penurunan seperti yang tampak pada Tabel 1 berikut: Tabel 1. Data Jumlah Pengunjung Giant Citra Raya No Tahun Actual Last Year % vs Last Year 1 2011 3.139.818 2 2012 2.186.688 3.139.818 -30% 3 2013 2.108.299 2.186.688 -4% 4 2014 2.073.589 2.108.299 -2% 5 2015 2.070.823 2.073.589 -1% Sumber: Giant Citra Raya Tabel 1. Data Jumlah Pengunjung Giant Citra Raya Selain faktor persaingan, keluhan lainnya adalah kualitas produk yang kurang segar, barang kurang lengkap, terjadi kehabisan barang karena kesalahan estimasi (Out Of Stock), jadi barang yang dibutuhkan pelanggan tidak tersedia. Adapun data keluhan yang di inventarisis adalah sebagai berikut. Tabel 2. Daftar Keluhan Pelanggan Tabel 2. Daftar Keluhan Pelanggan No Bagian Keluhan 1 Kualitas Produk 1. Produk-produk tertentu seringkali kehabisan persediaan sehingga pelanggan yang secara khusus hendak membeli produk tersebut merasa kecewa. 2. Kurangnya variasi jenis produk, sehingga pelanggan kesulitan untuk membandingkan. 3. Produk fresh yang dijual kadang kurang fresh bahkan sudah tidak layak. 2 Kualitas Layanan 1. Kurangnya petugas, sehingga pada saat pelanggan membutuhkan informasi harus mencari petugas terlebih dahulu. 2. Kasir yang tersedia kurang mencukupi, sehingga pelanggan harus harus antre pada saat membayar. 3. Kasir kurang ramah. Sumber : Hasil observasi di Giant Citra Raya Tabel 2. Daftar Keluhan Pelanggan g Sumber : Hasil observasi di Giant Citra Raya Melihat dari beberapa keluhan yang ada sudah semestinya perusahaan melakukan tindakan perbaikan produk dan pelayanan, tujuannya yakni untuk menciptakan citra yang baik dan memberikan kepuasan kepada konsumen. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 PENDAHULUAN Karena kepuasan konsumen bukan saja berharga di masa ekonomi baik, tetapi juga di saat ekonomi buruk (Lestari, 2009). Beberapa penelitianpun telah membuktikan jika kualitas produk dan kualitas layanan memiliki pengaruh terhadap citra merek, diantaranya yaitu penelitian yang Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 318 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... dilakukan oleh Flavian, Torres, dan Guinaliu dalam Hapzi Ali (2012), Terdapat hubungan yang signifikan antara Product Quality dengan Brand Image; Zikmund, William G. dalam Hapzi Ali (2012), bahwa Service Quality berpengaruh positif dan signifikan terhadap Brand Image; Hapzi Ali & Adji (2012), Flavian Torres & Guinaliu (2004), dan Malik dan Ghafoor (2012), bahwa Service Quality berpengaruh postif dan signifikan terhadap Brand Image secara parsial. Selain berpengaruh terhadap citra merek, kualitas produk dan kualitas layanan juga memberikan pengaruh terhadap kepuasan pelanggan, hal ini didukung oleh beberapa penelitian yang telah dilakukan sebelumnya oleh Krisna, gantasala & swapna (2010), dan Benjamin Osayawe Ehigie (2006), mengemukakan kualitas produk memiliki pengaruh terhadap kepuasan pelanggan. Selain itu Sanka (2012) menyatakan ada korelasi positif dan signifikan antara dimensi kualitas pelayanan dengan kepuasan pelanggan. Zafar et al. (2012) juga menyatakan ada hubungan positif dan signifikan antara kualitas pelayanan dengan kepuasan pelanggan. Menurut Bedi dalam Ming et al. (2010) pengiriman layanan yang berkualitas tinggi adalah suatu keharusan untuk mencapai kepuasan pelanggan. Penelitian tersebut sejalan dengan Naeem dan Saif (2009) yang menemukan bahwa kepuasan pelanggan adalah hasil dari kualitas pelayanan. Aldlaigan dan Buttle dalam Ladhari et al. (2011) mengidentifikasi ada korelasi yang positif dan signifikan antara dimensi kualitas pelayanan dan kepuasan secara keseluruhan. Berdasarkan latar belakang dan permasalahan maka tujuan yang ingin dicapai dari penelitian ini adalah: (1) Menganalisis pengaruh kualitas produk terhadap citra merek secara parsial; (2) Menganalisis pengaruh kualitas layanan terhadap citra merek secara parsial; (3) Menganalisis pengaruh kualitas produk dan kualitas layanan terhadap citra merek secara simultan; (4) Menganalisis pengaruh kualitas produk terhadap kepuasan pelanggan secara parsial; (5) Menganalisis pengaruh kualitas layanan terhadap kepuasan pelanggan secara parsial; (6) Menganalisis pengaruh citra merek terhadap kepuasan pelanggan secara parsial; (7) Menganalisis pengaruh kualitas produk, kualitas layanan dan citra merek terhadap kepuasan pelanggan secara simultan. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 KAJIAN TEORI Kualitas Produk. Kualitas produk adalah elemen kunci dalam penawaran pasar (market offering). Perencanaan bauran pemasaran dimulai dengan memformulasikan suatu penawaran untuk memenuhi kebutuhan atau keinginan pelanggan sasaran. Pelanggan akan menilai penawaran tersebut atas keistimewaan produk dan kualitas produk. Philip Kotler (2006) mendefinisikan bahwa kualitas produk adalah segala sesuatu yang dapat ditawarkan ke suatu pasar untuk memenuhi keinginan atau kebutuhan konsumen pada produk yang berkualitas". Tingkat persaingan yang semakin tinggi akan menyebabkan pelanggan menghadapi lebih banyak alternatif produk, harga dan kualitas yang bervariasi, sehingga pelanggan akan selalu mencari nilai yang dianggap paling tinggi dari beberapa produk (Kotler, 2008). Sebab titik berat pandangan konsumen adalah barang yang sesuai dengan keinginannya serta kebutuhannya. Lamb, et.,al. (2007). Untuk itu perusahaan senantiasa melakukan pembaharuan dibidang teknologi yang digunakan agar selalu dapat menyesuaikan dengan kebutuhan pelanggan. Untuk bisnis retail yang perlu dicermati dalam hubungannya dengan teknologi antara lain dalam penyimpanan, dan system informasi. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 319 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Dimensi kualitas produk menurut Mullins, Orville, Larreche, dan Boyd (2005:422) terdiri dari: (1) Performance (kinerja), berhubungan dengan karakteristik operasi dasar dari sebuah produk; (2) Durability (daya tahan), yang berarti berapa lama atau umur produk yang bersangkutan bertahan sebelum produk tersebut harus diganti. Semakin besar frekuensi pemakaian konsumen terhadap produk maka semakin besar pula daya tahan produk. (3) Conformance to specifications (kesesuaian dengan spesifikasi), yaitu sejauh mana karakteristik operasi dasar dari sebuah produk memenuhi spesifikasi tertentu dari konsumen atau tidak ditemukannya cacat pada produk; (4) Features (fitur), adalah karakteristik produk yang dirancang untuk menyempurnakan fungsi produk atau menambah ketertarikan konsumen terhadap produk; (5) Reliabilty (reliabilitas), adalah probabilitas bahwa produk akan bekerja dengan memuaskan atau tidak dalam periode waktu tertentu. Semakin kecil kemungkinan terjadinya kerusakan maka produk tersebut dapat diandalkan; (6) Aesthetics (estetika), berhubungan dengan bagaimana penampilan produk bisa dilihat dari tampak, rasa, bau, dan bentuk dari produk. (7) Perceived quality (kesan kualitas), sering dibilang merupakan hasil dari penggunaan pengukuran yang dilakukan secara tidak langsung karena terdapat kemungkinan bahwa konsumen tidak mengerti atau kekurangan informasi atas produk yang bersangkutan. Jadi, persepsi konsumen terhadap produk didapat dari harga, merek, periklanan, reputasi, negara asal, dalam ritell ketersediaan barang. Kualitas Layanan. Kualitas layanan merupakan sesuatu yang dipersepsikan oleh pelanggan Menurut Tjiptono (2012). Pelanggan akan menilai kualitas sebuah jasa yang dirasakan berdasarkan apa yang mereka deskripsikan dalam benak mereka. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 KAJIAN TEORI Pelanggan akan beralih ke penyedia jasa lain yang lebih mampu memahami kebutuhan spesifik pelanggan dan memberikan layanan yang lebih baik. Kualitas layanan mencerminkan semua dimensi penawaran yang menghasilkan manfaat bagi pelanggan. Dalam konteks kualitas layanan jasa, juga menurut Kotler & Keller (2008), ada beberapa dimensi kualitas yang menjadi acuan, diantaranya adalah: (1) Responsiveness (ketanggapan), yaitu kemampuan untuk menolong pelanggan dan ketersedian untuk melayani pelanggan dengan baik; (2) Reliability (keandalan), yaitu kemampuan untuk melakukan pelayanan sesuai yang dijanjikan dengan segera, akurat dan memuaskan; (3) Empathy (empati), yaitu rasa peduli untuk memberikan perhatian secara individual kepada pelanggan, memahami kebutuhan pelanggan, serta kemudahan untuk dihubungi; (4) Assurance (jaminan), yaitu pengetahuan, kesopanan petugas serta sifatnya yang dapat dipercaya sehingga para pelanggan bebas dari resiko; dan (5) Tangibles (bukti langsung), yaitu meliputi fasilitas fisik, perlengkapan karyawan untuk sarana komunikasi. Citra Merek. Citra merek merupakan serangkaian asosiasi (persepsi) yang ada dalam benak konsumen terhadap suatu merek, biasanya terorganisasi menjadi suatu makna. Hubungan terhadap suatu merek akan semakin kuat jika didasarkan pada pengalaman dan mendapat banyak informasi. Citra atau asosiasi merepresentasikan persepsi yang bisa merefleksikan kenyataan yang objektif ataupun tidak. Menurut Kotler dan Amstrong (2012) brand image merupakan seperangkat keyakinan konsumen mengenai merek tertentu. Kuatnya brand image di benak konsumen maka akan menyebabkan semakin kuat rasa percaya diri yang dirasakan oleh konsumen dalam menggunakan produk yang dibelinya. Rasa percaya diri yang tinggi ini menandakan bahwa konsumen tersebut merasa puas dengan produk yang dibelinya. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 320 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... KAJIAN TEORI Menurut Flavian, Torres, dan Guinaliu dalam Hapzi Ali (2010) adapun pengukuran citra merek dihasilkan empat elemen atau dimensi yang dapat membentuk citra, yaitu access to services, service offered, personal contact, security dan reputation, yang akan digunakan pada penelitian ini: (1) Akses pada pelayanan (access to service) terdiri dari indikator kemudahan dalam menggunakan layanan, ketepatan waktu transaksi, kenyamanan pelayanan, jadwal operasional bank, dan kemungkinan untuk mengajukan keluhan/ protes; (2) Jasa yang ditawarkan (service offered) terdiri dari indikator ketersediaan jumlah produk dan jasa, daya tarik produk dan jasa yang ditawarkan, ketertarikan produk jasa simpanan, ketertarikan pembayaran pinjaman, dan pembayaran biaya pelayanan; (3) Kontak personal (personal contact) terdiri dari indikator kebersahabatan pegawai, perhatian individual, kontak pribadi, bantuan konsultasi keuangan, kemudahan untuk konsultasi; (4) Keamanan (security) terdiri dari indikator keamanan dalam transaksi, keamanan dalam menyimpan uang, keamanan dalam data; (5) Reputasi (reputation) perusahaan terdiri dari indikator kepercayaan janji perbankan, reputasi jasa yang ditawarkan, kepercayaan pada bank yang dipilih melebihi institusi lainnya. Kepuasan Pelanggan. Kepuasan adalah tingkat keadaan perasaan seseorang setelah membandingkan kinerja suatu produk yang ia rasakan dibanding harapannya (Kotler, 2008). Menurut hal yang diungkapkan Zeithaml & Bitner (2008:110) mendefinisikan kepuasan pelanggan sebagai respon pelanggan terhadap evaluasi ketidaksesuaian yang dirasakan antara harapan dan kinerja aktual jasa. Oliver (2010) menyatakan bahwa kunci untuk mempertahankan pelanggan adalah kepuasan pelanggan, dimana dimensi kepuasan pelanggan yaitu: (1) Expectations, pelanggan merasa puas hanya apabila kenyataan produk yang sudah mereka beli sesuai atau melebihi dari harapan/ ekspektasi mereka. Produk yang baik adalah produk yang mampu memenuhi harapan/ ekspektasi pelanggannya, sehingga pelanggan akan memiliki pengalaman positif setelah membeli produk tersebut; (2) Subjective Disconfirmation, pelanggan merasa puas apabila tidak ada hal-hal atau alasan terkait ketidaksesuaian produk yang bersifat subyektif; (3) Performance Outcomes, pelanggan merasa puas dengan keseluruhan kinerja produk yang dia beli serta mendapatkan manfaat yang optimal sesuai dengan yang dia inginkan. Berdasarkan rumusan masalah dan tinjauan pustaka yang telah diuraikan pada bab sebelumnya, kerangka konseptual penelitian mengacu kepada teori-teori dan penelitian terdahulu yang relevan. Dimana dapat di tarik hubungan antar variabel kualitas layanan berpengaruh terhadap citra merek secara parsial berdasarkan penelitian Hapzi Ali & Adji (2012), kualitas produk berpengaruh terhadap citra merek secara parsial berdasarkan penelitian Flavian, Torres, dan Guinaliu dalam Hapzi Ali (2012), kualitas layanan berpengaruh terhadap kepuasan pelanggan secara parsial berdasarkan penelitian Krisna, et,. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 KAJIAN TEORI al (2010), kualitas produk berpengaruh terhadap kepuasan pelanggan secara parsial berdasarkan penelitian Ehigie (2006), dan citra merek berpengaruh terhadap kepuasan pelanggan secara parsial berdasarkan penelitian Tombokan dkk (2015). Sebagai gambaran secara komprehenship terhadap keterkaitan antar variabel yang digunakan dalam model penelitian, dibawah ini disajikan bagan kerangka konseptual penelitian. Dimana kerangka konseptual penelitian tersebut dapat digambarkan dalam (gambar 1) sebagai berikut : Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 321 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Gambar 1. Kerangka Konseptual Gambar 1. Kerangka Konseptual Berdasarkan tujuan penelitian dan tinjauan teori maka hipotesis penelitian adalah: (1) Kualitas produk berpengaruh terhadap citra merek secara parsial; (2) Kualitas layanan berpengaruh terhadap citra merek secara parsial; (3) Kualitas produk dan kualitas layanan berpengaruh terhadap citra merek secara simultan; (4) Kualitas produk berpengaruh terhadap kepuasan pelanggan secara parsial; (5) Kualitas layanan berpengaruh terhadap kepuasan pelanggan secara parsial; (6) Citra merek berpengaruh terhadap kepuasan pelanggan secara parsial; (7) Kualitas produk, kualitas layanan dan citra merek berpengaruh terhadap kepuasan pelanggan secara simultan. METODE Populasi pada penelitian ini adalah Pelanggan Giant Citra Raya yang rata-rata 2.070.823/12/30 = 5752 perhari. Penentuan sampel digunakan suatu metode pengambilan sampel yang tepat agar diperoleh sampel yang representatif dan dapat menggambarkan keadaan populasi secara maksimal. Oleh karena itu teknik pengambilan sampelnya dilakukan dengan accidental sampling untuk menentukan pelanggan yang menjadi anggota sampel. Ukuran atau jumlah sampel dalam penelitian ini ditentukan berdasarkan teori yang dikembangkan oleh Slovin, Umar (2008:141) dengan rumus sukuran sample sebanyak 152 responden. Pendekatan penelitian yang digunakan dalam penelitian ini adalah pendekatan kuantitatif dengan menggunakan analisis jalur (Path Analysis). Analisis ini merupakan pengembangan dari persamaan regresi, yang merupakan salah satu pilihan dalam rangka mempelajari ketergantungan sejumlah variabel didalam model. Analisis ini merupakan metode yang baik untuk menerangkan apabila terdapat seperangkat data yang besar untuk dianalisis dan mencari hubungan kausal. Path analysis merupakan salah satu alat analisis yang dikembangkan oleh (Dillon and Goldstein dalam Hapzi Ali dan Limakrisna, 2013). Wright mengembangkan metode untuk mengetahui pengaruh langsung dan tidak langsung dari suatu variable, dimana terdapat variable yang memberikan pengaruh (eksogenus variables) dan variable yang dipengaruhi (endogenus variables). y g p g Diagram jalur diatas terdiri dari dua sub struktural dengan dua persamaan, dimana X1 dan X2 adalah variabel eksogen (exogenous) yaitu variabel yang tidak ada penyebab 322 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... eksplisit nya, variabel ini berfungsi sebagai variabel bebas/penyebab terhadap variabel urutan sesudahnya yaitu Y serta Z sebagai variabel endogen (endogenous). Sebelum analisis, dilakukan uji instrumen kuesioner dengan uji validitas dan reliabilitas, serta dilakukan uji normalitas untuk menguji apakah dalam model regresi, variabel terikat dan bebas mempunyai distribusi normal atau tidak. Asumsi normalitas merupakan persyaratan yang sangat penting pada pengujian kebermaknaan (signifikansi) koefisien regresi. Model regresi yang baik adalah model regresi yang memiliki distribusi normal atau mendekati normal, sehingga sehingga layak dilakukan pengujian secara statistic. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 323 HASIL DAN PEMBAHASAN Uji Validitas dan realibilitas. Sebelum instrument kuesioner di berikan kepada responden terlebih dahulu di uji validitas dan realibitas. Uji Validitas dengan menganalisi r hitung (koefisien) dan r table. Jika r hitung > dari r table maka kuesioner di simpulkan valid dan dapat terus ke analisis berikutnya. Tabel 3. Uji Validitas Kualitas Produk (X1) Tabel 3. Uji Validitas Kualitas Produk (X1) Persepsi NIlai (X1) KoefisienValiditas rTabel Kesimpulan X1.1 0.831 0.159 Valid X1.2 0.661 0.159 Valid X1.3 0.684 0.159 Valid X1.4 0.639 0.159 Valid X1.5 0.758 0.159 Valid X1.6 0.732 0.159 Valid X1.7 0.840 0.159 Valid Sumber: Hasil Pengolahan data kuesioner Berdasarkan Tabel 3 di atas dapat diketahui bahwa seluruh butir pertanyaan kuesioner untuk variabel kualitas produk seluruhnya mempunyai rhitung > rTabel, (rhitung > 0.159) dengan demikian seluruh butir pertanyaan kuesioner pada variabel persepsi kualitas produk dinyatakan valid. Adapun hasil perhitungan uji validasi untuk variabel kualitas layanan diperoleh hasil sebagai berikut Tabel 4. Uji Validitas Kualitas Layanan (X2) Tabel 4. Uji Validitas Kualitas Layanan (X2) Persepsi Nilai (X2) Koefisien Validitas rTabel Kesimpulan X2.11 0.684 0.159 Valid X2.12 0.472 0.159 Valid X2.13 0.487 0.159 Valid X2.14 0.458 0.159 Valid X2.21 0.454 0.159 Valid X2.22 0.501 0.159 Valid X2.23 0.488 0.159 Valid X2.24 0.459 0.159 Valid X2.25 0.457 0.159 Valid X2.31 0.564 0.159 Valid Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 323 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... X2.32 0.616 0.159 Valid X2.33 0.656 0.159 Valid X2.34 0.702 0.159 Valid X2.35 0.623 0.159 Valid X2.41 0.709 0.159 Valid X2.42 0.631 0.159 Valid X2.43 0.588 0.159 Valid X2.44 0.589 0.159 Valid X2.51 0.518 0.159 Valid X2.52 0.595 0.159 Valid X2.53 0.553 0.159 Valid X2.54 0.431 0.159 Valid Sumber: Hasil Pengolahan data kuesioner Berdasarkan Tabel 4 di atas dapat diketahui bahwa seluruh butir pertanyaan kuesioner untuk variabel kualitas layanan seluruhnya mempunyai rhitung > rTabel, (rhitung > 0.159) dengan demikian seluruh butir pertanyaan kuesioner pada variabel persepsi kualitas layanan dinyatakan valid. Adapun hasil perhitungan uji validasi untuk variabel citra merek diperoleh hasil sebagai berikut: Tabel 5. Uji Validitas Citra Merek (Y) Tabel 5. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 HASIL DAN PEMBAHASAN Uji Validitas Citra Merek (Y) Persepsi Nilai (Y) KoefisienValiditas rTabel Kesimpulan Y.1 0.897 0.159 Valid Y.2 0.741 0.159 Valid Y.3 0.769 0.159 Valid Y.4 0.641 0.159 Valid Y.5 0.659 0.159 Valid Sumber: Hasil Pengolahan data kuesioner Berdasarkan Tabel 5 di atas dapat diketahui bahwa seluruh butir pertanyaan kuesioner untuk variabel citra merek seluruhnya mempunyai rhitung > rTabel, (rhitung > 0.159) dengan demikian seluruh butir pertanyaan kuesioner pada variabel persepsi citra merek dinyatakan valid. Adapun hasil perhitungan uji validasi untuk variabel kepuasan pelanggan diperoleh hasil sebagai berikut Tabel 6. Uji Validitas Kepuasan Pelanggan (Z) Persepsi Nilai (Z) Koefisien Validitas rTabel Kesimpulan Z.1 0.799 0.159 Valid Z.2 0.841 0.159 Valid Z.3 0.754 0.159 Valid Sumber: Hasil Pengolahan data kuesioner Tabel 6. Uji Validitas Kepuasan Pelanggan (Z) Sumber: Hasil Pengolahan data kuesioner Berdasarkan Tabel 6 di atas dapat diketahui bahwa seluruh butir pertanyaan kuesioner untuk variabel persepsi kepuasan pelanggan seluruhnya mempunyai rhitung > rTabel, (rhitung > Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 324 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... 0.159) dengan demikian seluruh butir pertanyaan kuesioner pada variabel persepsi kepuasan pelanggan dinyatakan valid. Tabel 7. Uji Reliabilitas Variabel Cronbach’s Alpha N of Item Kualitas Produk (X1) 0.860 7 Kualitas Layanan (X2) 0.893 22 Citra Merek (Y) 0.713 5 Kepuasan Pelanggan (Z) 0.716 3 Sumber: Hasil Pengolahan data kuesioner Tabel 7. Uji Reliabilitas Sumber: Hasil Pengolahan data kuesioner Berdasarkan Tabel 6 diatas dapat kita lihat bahwa seluruh variabel dependent maupun variabel intervening dan variabel independent memiliki nilai cronbach’s alpha > 0,6 sehingga untuk seluruh variabel tersebut reliabel dan dapat digunakan untuk pengukuran dalam rangka pengumpulan data. Hasil Uji Normalitas. Sebelum melangkah ke pengujian hipotesis, terlebih dahulu dilakukan uji normalitas. Pengujian dilakukan dengan menggunkan bantuan SPSS 21.0 sebagai alat bantu dalam penelitian ini diperoleh hasil bahwasanya data dalam penelitian ini berdistribusi normal, hal ini dibuktikan dari hasil nilai signifikansi (Asymp. Sig. 2- tailed) lebih besar dari 0,05, yaitu (0,427 > 0,05) sehingga dapat disimpulkan bahwa data dalam penelitian ini berdistribusi normal. Path Analysis. Analisis ini merupakan salah satu pilihan dalam rangka mempelajari ketergantungan sejumlah variabel didalam model. Analisis ini merupakan metode yang baik untuk menerangkan apabila terdapat seperangkat data yang besar untuk dianalisis dan mencari hubungan kausal. Untuk menjawab tujuan-tujuan dalam penelitian ini, struktur utama dalam model penelitian di pecah menjadi dua sub struktur. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 325 Secara statistik output tabel coefficients diatas diperoleh persamaan sebagai berikut. Y = Pyx1. X1 + Pyx2. X2 + e1 Y = 0,518.X1 + 0,356.X2 + e1 Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 HASIL DAN PEMBAHASAN Dimana untuk menjawab tujuan 1, 2 dan 3 dengan sub struktur pertama, dan untuk menjawab tujuan 4, 5, 6 dan 7 dengan menggunakan sub struktur kedua. Adapun output persamaan struktur pertama dalam penelitian ini dengan menggunakan software SPSS 21.0 sebagai berikut. Tabel 8. Coefficients regression Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 1.268 .558 2.273 .024 X1_Service_Quality .081 .013 .518 6.162 .000 X2_Product_Quality .134 .032 .356 4.241 .000 a. Dependent Variable: Y_Brand_Image Tabel 8. Coefficients regression ara statistik output tabel coefficients diatas diperoleh persamaan sebagai berikut. Y P X P X Secara statistik output tabel coefficients diatas diperoleh persamaan sebagai berikut. Y = Pyx1. X1 + Pyx2. X2 + e1 Y = 0,518.X1 + 0,356.X2 + e1 Y = Pyx1. X1 + Pyx2. X2 + e1 Y = 0,518.X1 + 0,356.X2 + e1 325 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Hasil persamaan diatas di masukkan ke dalam gambar persamaan struktural seperti dibawah ini: Gambar 2. Hasil Analisis Jalur Sub Struktural I Gambar 2. Hasil Analisis Jalur Sub Struktural I Untuk melihat pengaruh langsung dan tidak langsung, dapat dilihat pada tabel berikut: Untuk melihat pengaruh langsung dan tidak langsung, dapat dilihat pada tabel berikut: Untuk melihat pengaruh langsung dan tidak langsung, dapat dilihat pada tabel berikut: Tabel 9. Rangkuman Koefisien Jalur Kualitas Produk dan Kualitas Layanan terhadap Citra Merek Tabel 9. Rangkuman Koefisien Jalur Kualitas Produk dan Kualitas Layanan terhadap Citra Merek Variabel Langsung Tidak Langsung Sub Total Total X1 X2 X1_Kualitas Produk 26,83% 15,67% 15,67% 42,5% X2_Kualitas Layanan 12,67% 15,67% 15,67% 28,34% Pengaruh X1, X2 70,84% Pengaruh Variabel Lain 29,16% Dari hasil analisis jalur yang dilakukan diperoleh pengaruh langsung kualitas produk terhadap citra merek sebesar 26,83%, kualitas layanan terhadap citra merek sebesar 12,67%, dan total pengaruh kualitas produk dan kualitas layanan terhadap citra merek secara langsung maupun tidak langsung sebesar 70,84. Nilai tersebut menjelaskan nilai koefisien determinasi R2 Square Seperti yang tampak pada tabel berikut. Tabel 10. Model summary Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .841a .708 .704 .798 a. Predictors: (Constant), X2_Product_Quality, X1_Service_Quality Tabel 10. Model summary Nilai R sebesar 0,841 menunjukan korelasi ganda (kualitas produk dan kualitas layanan) dengan citra merek. Dengan mempertimbangkan variasi Nilai R Square sebesar 0,708, memiliki makna besarnya peran atau kontribusi variabel kualitas produk dan kualitas Jurnal Manajemen/Volume XXI, No. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 HASIL DAN PEMBAHASAN 03, Oktober 2017: 317-335 326 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... layanan mampu menjelaskan variabel citra merek sebesar 70,8%. Sedangkan sisanya sebesar 29,2% dipengaruhi oleh variabel lain yang tidak termasuk dalam model ini. Hasil Uji Parsial (Uji t) dan Pengaruh Simultan (Uji F). Pengujian hipotesis bertujuan untuk menjelaskan karakteristik hubungan-hubungan tertentu atau perbedaan-perbedaan antar kelompok atau independensi dari dua faktor atau lebih dalam suatu situasi, (Hapzi Ali dan Limakrisna, N., 2013). Pengujian pengaruh parsial bertujuan untuk menguji apakah masing-masing variabel bebas berpengaruh secara signifikan terhadap variabel terikat secara parsial dengan = 0,05 dan juga penerimaan atau penolakan hipotesis. Uji parsial (Uji t) untuk menjawab hipotesis satu dan dua dari penelitian ini. Tabel 11. t Test Results (Partial) Model t Sig. 1 (Constant) 2.273 .024 X1_Service_Quality 6.162 .000 X2_Product_Quality 4.241 .000 Sumber: Output SPSS 21.0 for windows Tabel 11. t Test Results (Partial) Sumber: Output SPSS 21.0 for windows Pengaruh Kualitas Produk Terhadap Citra Merek. Dari hasil output SPSS seperti yang tampak pada Tabel 11 diperoleh nilai t hitung variabel X1 sebesar 6.162, dengan tingkat signifikansi sebesar 0,000, dikarenakan angka taraf signifikansi lebih < 0.05 (0.000 < 0.05) maka dapat disimpulkan bahwa kualitas produk berpengaruh terhadap citra merek. Berdasarkan dari penjelasan tersebut oleh karena itu H0 ditolak dan H1 diterima, artinya kualitas produk berpengaruh terhadap citra merek. Pengaruh Kualitas Layanan Terhadap Citra Merek. Dari hasil output SPSS seperti yang tampak pada Tabel 11 diperoleh nilai t hitung variabel X2 sebesar 4.162, dengan tingkat signifikansi sebesar 0,000, dikarenakan angka taraf signifikansi lebih < 0.05 (0.000 < 0.05) maka dapat disimpulkan bahwa kualitas layanan berpengaruh terhadap citra merek. Berdasarkan dari penjelasan tersebut oleh karena itu H0 ditolak dan H1 diterima, artinya kualitas layanan berpengaruh terhadap citra merek. Pengaruh Kualitas Produk dan Kualitas Layanan Terhadap Citra Merek Secara Simultan. Untuk menjawab hipotesis ketiga kualitas produk dan kualitas layanan berpengaruh terhadap citra merek secara simultan dapat di lihat dari Tabel 12 berikut. Tabel 12. Hasil Uji F Secara Simultan Tabel 12. Hasil Uji F Secara Simultan ANOVAa Model Sum of Squares df Mean Square F Sig. 1 Regression 230.060 2 115.030 180.642 .000b Residual 94.881 149 .637 Total 324.941 151 a. Dependent Variable: Y_Brand_Image b. Predictors: (Constant), X2_Product_Quality, X1_Service_Quality Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 327 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 HASIL DAN PEMBAHASAN Dari uji Anova atau F test seperti yang tampak pada Tabel 12 dengan diperoleh nilai Fhitung sebesar 180.642 dengan tingkat probabilitas p-value sebesar 0,000, dikarenakan nilai Fhitung > Ftabel (180.642 > 3,10) sehingga dapat disimpulkan bahwa hipotesis nol (H0) ditolak dan hipotesis alternatif (H1) diterima, artinya ada pengaruh secara signifikan antara kualitas produk dan kualitas layanan secara bersama-sama berpengaruh terhadap citra merek. Dengan demikian hipotesis ketiga diterima. Adapun output persamaan struktur kedua dalam penelitian ini dengan menggunakan software SPSS 21.0 sebagai berikut. Tabel 13. Coefficients regression Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) .538 .557 .967 .335 X1_Service_Quality .030 .014 .181 2.066 .041 X2_Product_Quality .152 .033 .385 4.653 .000 Y_Brand_Image .376 .080 .356 4.674 .000 a. Dependent Variable: Z_Customer_Satisfaction Tabel 13. Coefficients regression Tabel 13. Coefficients regression Secara statistik output tabel coefficients diatas diperoleh persamaan sebagai berikut. Z = Pzx1. X1 + Pzx2. X2 + Pzy. Y + e2 Z = 0,181.X1 + 0,385.X2 + 0,356.Y + e2 Secara statistik output tabel coefficients diatas diperoleh persamaan sebagai berikut. Z P X P X P Y Z = Pzx1. X1 + Pzx2. X2 + Pzy. Y + e2 Z = 0,181.X1 + 0,385.X2 + 0,356.Y + e2 Hasil persamaan diatas di masukkan ke dalam gambar persamaan struktural seperti dibawah ini: Hasil persamaan diatas di masukkan ke dalam gambar persamaan struktural seperti dibawah ini: Gambar 3. Hasil Analisis Jalur Sub Struktural II Gambar 3. Hasil Analisis Jalur Sub Struktural II Untuk melihat pengaruh langsung dan tidak langsung, dapat dilihat pada Tabel 14. Dari hasil analisis jalur yang dilakukan diperoleh pengaruh langsung kualitas produk terhadap kepuasan pelanggan sebesar 3,28%, kualitas layanan terhadap kepuasan pelanggan sebesar 14,82%, citra merek terhadap kepuasan pelanggan sebesar 12,67 dan total pengaruh kualitas produk, kualitas layanan dan citra merek terhadap kepuasan kerja secara langsung maupun tidak langsung sebesar 74,99. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 328 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Tabel 14. Rangkuman Koefisien Jalur Kualitas Produk, Kualitas Layanan dan Citra Merek terhadap Kepuasan Pelanggan Variable Langsung Langsung Sub Total Total X1 X2 Y X1_ Kualitas Produk 3,28% 5,92% 5,28% 11,2% 14,48% X2_Kualitas Layanan 14,82% 5,92% 10,91% 16,83% 31,65% Y_Citra Merek 12,67% 5,28% 10,91% 16,19% 28,86% Pengaruh X1, X2, dan Y 74,99% Pengaruh Variabel Lain 25,01% el 14. Pengaruh Kualitas Produk Terhadap Kepuasan Pelanggan. Dari hasil output SPSS seperti yang tampak pada Tabel 16 yang dilakukan diperoleh angka nilai t hitung variabel X1 sebesar 2.066, dengan tingkat signifikansi sebesar 0,041, dikarenakan angka taraf signifikansi lebih < 0.05 (0.051 < 0.05) maka dapat disimpulkan bahwa kualitas produk HASIL DAN PEMBAHASAN Rangkuman Koefisien Jalur Kualitas Produk, Kualitas Layanan dan Citra Merek terhadap Kepuasan Pelanggan Nilai tersebut menjelaskan nilai koefisien determinasi R2 Square Seperti yang tampak pada tabel berikut. Nilai tersebut menjelaskan nilai koefisien determinasi R2 Square Seperti yang tampak pada tabel berikut. Tabel 15. Model summary Tabel 15. Model summary Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .866a .749 .744 .783 a. Predictors: (Constant), Y_Brand_Image, X2_Product_Quality, X1_Service_Quality Tabel 15. Model summary Model Summary Nilai R sebesar 0,866 menunjukan korelasi ganda (kualitas produk, kualitas layanan dan citra merek) dengan kepuasan pelanggan. Dengan mempertimbangkan variasi Nilai R Square sebesar 0,749, memiliki makna besarnya peran atau kontribusi variabel kualitas produk, kualitas layanan dan citra merek mampu menjelaskan variabel kepuasan pelanggan sebesar 74,9%. Sedangkan sisanya sebesar 25,1% dipengaruhi oleh variabel lain yang tidak termasuk dalam model ini. Hasil Uji Parsial (Uji t) dan Pengaruh Simultan (Uji F). Uji parsial (Uji t) untuk struktur dua dilakukan untuk menjawab hipotesis empat, lima dan enam dari penelitian ini. Adapun output uji parsial dapat dilihat pada tabel berikut. Tabel 16. t Test Results (Partial) Model t Sig. 1 (Constant) .967 .335 X1_Service_Quality 2.066 .041 X2_Product_Quality 4.653 .000 Y_Brand_Image 4.674 .000 Sumber: Output SPSS 21.0 for windows Tabel 16. t Test Results (Partial) Pengaruh Kualitas Produk Terhadap Kepuasan Pelanggan. Dari hasil output SPSS seperti yang tampak pada Tabel 16 yang dilakukan diperoleh angka nilai t hitung variabel X1 sebesar 2.066, dengan tingkat signifikansi sebesar 0,041, dikarenakan angka taraf signifikansi lebih < 0.05 (0.051 < 0.05) maka dapat disimpulkan bahwa kualitas produk Pengaruh Kualitas Produk Terhadap Kepuasan Pelanggan. Dari hasil output SPSS seperti yang tampak pada Tabel 16 yang dilakukan diperoleh angka nilai t hitung variabel X1 sebesar 2.066, dengan tingkat signifikansi sebesar 0,041, dikarenakan angka taraf signifikansi lebih < 0.05 (0.051 < 0.05) maka dapat disimpulkan bahwa kualitas produk 329 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... berpengaruh terhadap kepuasan pelanggan. Berdasarkan dari penjelasan tersebut oleh karena itu H0 ditolak dan H1 diterima, artinya kualitas produk berpengaruh terhadap kepuasan pelanggan. berpengaruh terhadap kepuasan pelanggan. Berdasarkan dari penjelasan tersebut oleh karena itu H0 ditolak dan H1 diterima, artinya kualitas produk berpengaruh terhadap kepuasan pelanggan. Pengaruh Kualitas Layanan Terhadap Kepuasan Pelanggan. HASIL DAN PEMBAHASAN Dari hasil output SPSS seperti yang tampak pada Tabel 16 yang dilakukan diperoleh angka nilai t hitung variabel X2 sebesar 4.653, dengan tingkat signifikansi sebesar 0,000, dikarenakan angka taraf signifikansi lebih < 0.05 (0.000 < 0.05) maka dapat disimpulkan bahwa kualitas layanan berpengaruh terhadap kepuasan pelanggan. Berdasarkan dari penjelasan tersebut oleh karena itu H0 ditolak dan H1 diterima, artinya kualitas layanan berpengaruh terhadap kepuasan pelanggan. Pengaruh Citra Merek Terhadap Kepuasan Pelanggan. Dari hasil output SPSS seperti yang tampak pada Tabel 16 yang dilakukan diperoleh angka nilai t hitung variabel Y sebesar 4.674, dengan tingkat signifikansi sebesar 0,000, dikarenakan angka taraf signifikansi lebih < 0.05 (0.000 < 0.05) maka dapat disimpulkan bahwa citra merek berpengaruh terhadap kepuasan kerja. Berdasarkan dari penjelasan tersebut oleh karena itu H0 ditolak dan H1 diterima, artinya citra merek berpengaruh terhadap kepuasan pelanggan. Pengaruh Kualitas Produk, Kualitas Layanan, dan Citra Merek Terhadap Kepuasan Kerja Secara Simultan. Untuk menjawab hipotesis ketujuh bahwa kualitas produk, kualitas layanan dan citra merek berpengaruh terhadap kepuasan pelanggan secara simultan dapat di lihat dari Tabel 17 berikut. Tabel 17. Hasil Uji F Secara Simultan ANOVAa Model Sum of Squares df Mean Square F Sig. 1 Regression 271.024 3 90.341 147.352 .000b Residual 90.739 148 .613 Total 361.763 151 a. Dependent Variable: Z_Customer_Satisfaction b. Predictors: (Constant), Y_Brand_Image, X2_Product_Quality, X1_Service_Quality Tabel 17. Hasil Uji F Secara Simultan p b. Predictors: (Constant), Y_Brand_Image, X2_Product_Quality, X1_Service_Quality Dari uji Anova atau F test seperti yang tampak pada Tabel 17 dengan diperoleh nilai Fhitung sebesar 147.352 dengan tingkat probabilitas p-value sebesar 0,000, dikarenakan nilai Fhitung > Ftabel (147.352 > 3,10) sehingga dapat disimpulkan bahwa hipotesis nol (H0) ditolak dan hipotesis alternatif (H1) diterima, artinya ada pengaruh secara signifikan antara kualitas produk, kualitas layanan dan citra merek secara bersama-sama berpengaruh terhadap kepuasan pelanggan. Dengan demikian hipotesis ketujuh diterima. Dari hasil interpretasi data di atas maka dapat dibahas bahwa pengaruh: 1) Kualitas Produk terhadap Citra Merek. Kualitas produk berpengaruh positif dan signifikan terhadap Brand Image. Dalam ritel kualitas produk dibagi menjadi, kemasan produk, purna jual, dan ketersediaan/variasi produk. Dimensi Product Quality sendiri terdiri dari: performance, durability, conformance to specifications, features (fitur), reliabilty, aesthetics, perceived quality. Citra merek merupakan kunci untuk mempertahankan pelanggan Oliver (2010:129), dimana dimensi citra merek adalah: access to services, service offered, personal contact, security dan reputation. 1) Kualitas Produk terhadap Citra Merek. Kualitas produk berpengaruh positif dan signifikan terhadap Brand Image. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 HASIL DAN PEMBAHASAN Dalam ritel kualitas produk dibagi menjadi, kemasan produk, purna jual, dan ketersediaan/variasi produk. Dimensi Product Quality sendiri terdiri dari: performance, durability, conformance to specifications, features (fitur), reliabilty, aesthetics, perceived quality. Citra merek merupakan kunci untuk mempertahankan pelanggan Oliver (2010:129), dimana dimensi citra merek adalah: access to services, service offered, personal contact, security dan reputation. 330 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Hasil penelitian ini sejalan dengan penelitian Flavian, Torres, dan Guinaliu (2004), dalam Hapzi Ali (2012), bahwa terdapat hubungan yang signifikan antara Service Quality dengan Brand Image. Hasil penelitian ini sejalan dengan penelitian Flavian, Torres, dan Guinaliu (2004), dalam Hapzi Ali (2012), bahwa terdapat hubungan yang signifikan antara Service Quality dengan Brand Image. 2) Kualitas Layanan terhadap Citra Merek. Kualitas layanan berpengaruh positif dan signifikan terhadap citra merek. Kualitas layanan yang baik mencerminkan semua dimensi penawaran yang menghasilkan manfaat bagi pelanggan. Dalam konteks kualitas layanan jasa menurut Kotler & Keller (2008:156), dimensi kualitas layanan adalah responsiveness, reliability, empathy, dan assurance. Kunci untuk mempertahankan pelanggan adalah citra merek, Oliver (2010:129), dimana dimensi Brand Image adalah: access to services, service offered, personal contact, security dan reputation. 2) Kualitas Layanan terhadap Citra Merek. Kualitas layanan berpengaruh positif dan signifikan terhadap citra merek. Kualitas layanan yang baik mencerminkan semua dimensi penawaran yang menghasilkan manfaat bagi pelanggan. Dalam konteks kualitas layanan jasa menurut Kotler & Keller (2008:156), dimensi kualitas layanan adalah responsiveness, reliability, empathy, dan assurance. Kunci untuk mempertahankan pelanggan adalah citra merek, Oliver (2010:129), dimana dimensi Brand Image adalah: access to services, service offered, personal contact, security dan reputation. Apabila perusahaan ingin mempertahankan keunggulan kompetitifnya dalam pasar, perusahaan harus mengerti aspek kualitas layanan yang diharapkan oleh pelangganuntuk membedakan produk yang dijual perusahaan tersebut dengan produk pesaing, Mullins, et. al (2005:422). Hasil penelitian ini di dukung penelitian yang dilakukan Hapzi Ali & Adji (2012), Falvian Torres & Guinaliu (2004), dan Malik dan Ghafoor (2012), bahwa Service Quality berpengaruh postif dan signifikan terhadap Brand Image secara parsial 3) Kualitas Produk dan Kualitas Layanan terhadap Citra Merek. Kualitas produk dan kualitas layanan secara simutan berpengaruh positif dan signifikan terhadap citra merek. Dalam ritel kualitas produk dibagi menjadi kemasan produk, purna jual, dan ketersediaan/variasi produk. Kualitas layanan yang baik mencerminkan semua dimensi penawaran yang menghasilkan manfaat bagi pelanggan. 3) Kualitas Produk dan Kualitas Layanan terhadap Citra Merek. Kualitas produk dan kualitas layanan secara simutan berpengaruh positif dan signifikan terhadap citra merek. HASIL DAN PEMBAHASAN Dalam ritel kualitas produk dibagi menjadi kemasan produk, purna jual, dan ketersediaan/variasi produk. Kualitas layanan yang baik mencerminkan semua dimensi penawaran yang menghasilkan manfaat bagi pelanggan. Dalam konteks kualitas layanan menurut Kotler & Keller (2008:156), dimensi kualitas layanan adalah: responsiveness, reliability, empathy, dan assurance. Kunci untuk mempertahankan pelanggan adalah Brand Image, Oliver (2010:129). Kualitas produk berpengaruh positif dan signifikan terhadap citra merek. Dalam ritel kualitas produk dibagi menjadi kualitas produk, kemasan produk, purna jual, dan ketersediaan/variasi produk. Dimensi kualitas produk sendiri terdiri dari: performance, durability, conformance to specifications, features (fitur), reliabilty, aesthetics, perceived quality. Citra merek merupakan kunci untuk mempertahankan pelanggan Oliver (2010:129), dimana dimensi citra merek adalah: access to services, service offered, personal contact, security dan reputation. ff p y p 4) Kualitas Produk terhadap Kepuasan Pelanggan. Kualitas produk berpengaruh positif dan signifikan terhadap Kepuasan Pelanggan. Dalam ritel kualitas produk dibagi menjadi kualitas produk, kemasan produk, purna jual, dan ketersediaan/variasi produk. Dimensi kualitas produk sendiri terdiri dari: 1) Performance; 2) Durability; 3) Conformance to specifications; 4) Features (fitur); 5) Reliabilty; 6) Aesthetics; 7) Perceived quality. Kepuasan pelanggan merupakan kunci untuk mempertahankan pelanggan Oliver (2010:129), dimana dimensi kepuasan pelanggan adalah: 1) Expectations; 2) Subjective Disconfirmationd; dan 3) Performance Outcomes. Hasil penelitian ini sejalan dengan penelitian Emel (2007), Patricia Huddlestone (2009) dan Bijana (2011), dimana kualitas produk berpengaruhi positif dan signifikan terhadap kepuasan pelanggan. 5) li h d l li l b h i if 4) Kualitas Produk terhadap Kepuasan Pelanggan. Kualitas produk berpengaruh positif dan signifikan terhadap Kepuasan Pelanggan. Dalam ritel kualitas produk dibagi menjadi kualitas produk, kemasan produk, purna jual, dan ketersediaan/variasi produk. Dimensi kualitas produk sendiri terdiri dari: 1) Performance; 2) Durability; 3) Conformance to specifications; 4) Features (fitur); 5) Reliabilty; 6) Aesthetics; 7) Perceived quality. Kepuasan pelanggan merupakan kunci untuk mempertahankan pelanggan Oliver (2010:129), dimana dimensi kepuasan pelanggan adalah: 1) Expectations; 2) Subjective Disconfirmationd; dan 3) Performance Outcomes. Hasil penelitian ini sejalan dengan penelitian Emel (2007), Patricia Huddlestone (2009) dan Bijana (2011), dimana kualitas produk berpengaruhi positif dan signifikan terhadap kepuasan pelanggan. 5) Kualitas Layanan terhadap Kepuasan Pelanggan. Kualitas layanan berpengaruh positif dan signifikan terhadap kepuasan pelanggan. Kualitas layanan yang baik mencerminkan semua dimensi penawaran yang menghasilkan manfaat bagi pelanggan. Dalam konteks kualitas layanan jasa menurut Kotler & Keller (2008;156), dimensi 5) Kualitas Layanan terhadap Kepuasan Pelanggan. Kualitas layanan berpengaruh positif dan signifikan terhadap kepuasan pelanggan. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 HASIL DAN PEMBAHASAN Kualitas layanan yang baik mencerminkan semua dimensi penawaran yang menghasilkan manfaat bagi pelanggan. Dalam konteks kualitas layanan jasa menurut Kotler & Keller (2008;156), dimensi 5) Kualitas Layanan terhadap Kepuasan Pelanggan. Kualitas layanan berpengaruh positif dan signifikan terhadap kepuasan pelanggan. Kualitas layanan yang baik mencerminkan semua dimensi penawaran yang menghasilkan manfaat bagi pelanggan. Dalam konteks kualitas layanan jasa menurut Kotler & Keller (2008;156), dimensi 331 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... kualitas layanan adalah: 1) Responsiveness; 2) Reliability; 3) Empathy; dan 4) Assurance. Kunci untuk mempertahankan pelanggan adalah kepuasan pelanggan, Oliver (2010:129), dimana dimensi kepuasan pelanggan adalah: 1) Expectations; 2) Subjective Disconfirmationd; dan 3) Performance Outcomes. kualitas layanan adalah: 1) Responsiveness; 2) Reliability; 3) Empathy; dan 4) Assurance. Kunci untuk mempertahankan pelanggan adalah kepuasan pelanggan, Oliver (2010:129), dimana dimensi kepuasan pelanggan adalah: 1) Expectations; 2) Subjective Disconfirmationd; dan 3) Performance Outcomes. Apabila perusahaan ingin mempertahankan keunggulan kompetitifnya dalam pasar, perusahaan harus mengerti aspek kualitas layanan yang diharapkan oleh konsumen untuk membedakan produk yang dijual perusahaan tersebut dengan produk pesaing, Mullins, et. al (2005:422). p g Hasil penelitian ini di dukung penelitian yang dilakukan Sanka (2012) menyatakan ada korelasi positif dan signifikan antara dimensi kualitas pelayanan dengan kepuasan pelanggan. Zafar et al (2012) juga menyatakan ada hubungan positif dan signifikan antara kualitas pelayanan dengan kepuasan pelanggan. Disamping itu di perkuat pula oleh hasil penelitian bahwa Kualitas layanan berpengaruh positif dan signifikan terhadap kepuasan pelanggan secara parsial (Hapzi Ali, Nanda Limakrisna & Said Djamalluddin, 2016). 6) Citra Merek terhadap Kepuasan Pelanggan. Citra merek berpengaruh positif dan signifikan terhadap kepuasan pelanggan. Citra merek merupakan seperangkat keyakinan konsumen mengenai merek tertentu (Kotler dan Amstrong, 2012). Kuatnya citra merek di benak konsumen maka akan menyebabkan semakin kuat rasa percaya diri yang dirasakan oleh konsumen dalam menggunakan produk yang dibelinya. Rasa percaya diri yang tinggi ini menandakan bahwa konsumen tersebut merasa puas dengan produk yang dibelinya. Dimensi citra merek sendiri terdiri dari: access to services, service offered, personal contact, security dan reputation. Kepuasan pelanggan merupakan kunci untuk mempertahankan pelanggan Oliver (2010:129), dimana dimensi kepuasan pelanggan adalah:1) Expectations; 2) Subjective Disconfirmationd; dan 3) Performance Outcomes. Dimensi citra merek sendiri terdiri dari: access to services, service offered, personal contact, security dan reputation. Kepuasan pelanggan merupakan kunci untuk mempertahankan pelanggan Oliver (2010:129), dimana dimensi kepuasan pelanggan adalah:1) Expectations; 2) Subjective Disconfirmationd; dan 3) Performance Outcomes. PENUTUP Simpulan. Berdasarkan hasil dan pembahasan maka kesimpulan penelitian ini adalah: (1) Kualitas produk berpengaruh terhadap citra merek Giant. Ini dibenarkan melalui hasil analisis yang mampu memberikan kontribusi yang positif. Hasil lain menunjukkan bahwa kualitas produk secara langsung dan tidak langsung berpengaruh positif terhadap citra merek. (2) Kualitas layanan berpengaruh positif dan signifikan terhadap citra merek Giant. Ini dibenarkan melalui hasil analisis yang mampu memberikan kontribusi yang positif. Begitu juga diketahui bahwa terdapat hasil yang positif untuk pengaruh langsung maupun tidak langsung berpengaruh positif terhadap citra merek. (3) Kualitas produk dan kualitas layanan bersama-sama atau secara simultan berpengaruh terhadap kepuasan pelanggan Giant Citra Raya. Ini dibenarkan melalui hasil analisis yang mampu memberikan kontribusi yang positif dan mempunyai korelasi yang kuat. (4) Kualitas produk berpengaruh terhadap kepuasan pelanggan Giant. Ini dibenarkan melalui hasil analisis yang mampu memberikan kontribusi yang positif. Hasil lain menunjukkan bahwa kualitas produk secara langsung dan tidak langsung berpengaruh positif terhadap kepuasan pelanggan. (5) Kualitas layanan berpengaruh positif dan signifikan terhadap kepuasan pelanggan Giant. Ini dibenarkan melalui hasil analisis yang mampu memberikan kontribusi yang positif. Begitu juga diketahui bahwa terdapat hasil yang positif untuk pengaruh langsung maupun tidak langsung. (6) Citra merek berpengaruh positif dan signifikan terhadap kepuasan pelanggan Giant. Ini dibenarkan melalui hasil analisis yang mampu memberikan kontribusi yang positif. Begitu juga diketahui bahwa terdapat hasil yang positif untuk pengaruh langsung maupun tidak langsung. (7) Kualitas produk, kualitas layanan, dan citra merek bersama- sama atau secara simultan berpengaruh terhadap kepuasan pelanggan Giant Citra Raya. Ini dibenarkan melalui hasil analisis yang mampu memberikan kontribusi yang positif dan mempunyai korelasi yang kuat. Saran. Berdasarkan analisis data, proses perhitungan statistik, pengujian model penelitian empiris dan pembahasan hasil studi yang dilakukan, diajukan beberapa saran sebagai berikut: (1) Untuk meningkatkan kepuasan pelanggan dan citra merek Giant Citra Raya harus memastikan produk yang pelanggan beli adalah benar-benar produk yang bekualitas, serta dapat digunakan secara maksimal dan sesuai dengan harapan, memperhatikan pelayanan yang terbaik sesuai yang dijanjikan. (2) Pada riset-riset selanjutnya, peneliti dapat melakukan riset dengan variable lain yang tidak di teliti seperti kualitas hubungan, kepuasan pelanggan dan lainnya. Disamping itu dapat pula di teliti dengan teknik analisis selain dari path analysis, sehingga dapat diperoleh hasil yang lebih akurat yang dapat menambah atau melengkapi hasil riset yang sudah ada. Akhirnya semoga informasi hasil penelitian ini dapat membantu perusahaan-perusahaan ritel dan sejenisnya dalam meningkatkan penjualannya dalam lingkungan bisnis yang semakin cepat berubah saat ini. HASIL DAN PEMBAHASAN Hasil penelitian ini sejalan dengan penelitian Tombokan dkk (2015), dimana citra merek berpengaruh positif dan signifikan terhadap kepuasan pelanggan. Menurut Nalau, dkk (2012) merek pada hakikatnya merupakan suatu janji penjual kepada konsumen untuk secara konsisten member seperangkat, atribut, manfaat, dan pelayanan. Produk dengan kualitas dan model dari kemasan yang relative sama saat ini dapat memiliki kinerja dan kualitas yang berbeda-beda di pasar karena adanya perbedaan persepsi konsumen terhadap merek produk tersebut. Kuatnya brand image di benak konsumen akan menyebabkan semakin kuat rasa percaya diri yang dirasakan oleh konsumen dalam menggunakan produk yang dibelinya. Rasa percaya diri yang tinggi ini menandakan bahwa konsumen tersebut merasa puas dengan produk yang dibelinya. 7) Kualitas Produk, Kualitas Layanan dan Citra Merek terhadap Kepuasan Pelanggan. Kualitas produk, kualitas layanan dan citra merek secara simutan berpengaruh positif dan signifikan terhadap kepuasan pelanggan. Dalam ritel kualitas produk dibagi menjadi kemasan produk, purna jual, dan ketersediaan/variasi produk. Kualitas layanan yang baik mencerminkan semua dimensi penawaran yang menghasilkan manfaat bagi pelanggan. Sedangkan citra merek yang baik merupakan persepsi konsumen terhadap merek produk tersebut. Kuatnya brand image di benak konsumen akan menyebabkan semakin kuat rasa percaya diri yang dirasakan oleh konsumen dalam menggunakan produk yang dibelinya. Rasa percaya diri yang tinggi ini menandakan bahwa konsumen tersebut merasa puas dengan produk yang dibelinya. 332 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 Emel, Kursunluoglu. (2007) “Customer Service Effects on Customer Satisfaction and Customer Loyalty: A Field Research in Shopping Centers in Izmir City – Turkey”. International Journal of Business and Social Science. 2 (17), 52. DAFTAR RUJUKAN Emel, Kursunluoglu. (2007) “Customer Service Effects on Customer Satisfaction and Customer Loyalty: A Field Research in Shopping Centers in Izmir City – Turkey”. International Journal of Business and Social Science. 2 (17), 52. Emel, Kursunluoglu. (2007) “Customer Service Effects on Customer Satisfaction and Customer Loyalty: A Field Research in Shopping Centers in Izmir City – Turkey”. International Journal of Business and Social Science. 2 (17), 52. Flavian, Torres, dan Guinaliu (2004), dalam Hapzi Ali (2012) “Building corporate image for banking in South Africa”. International Journal of Bank Marketing; 17 (8), MCB University Press, London. Flavian, Torres, dan Guinaliu (2004), dalam Hapzi Ali (2012) “Building corporate image for banking in South Africa”. International Journal of Bank Marketing; 17 (8), MCB University Press, London. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 333 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Hapzi Ali, Adji Djojo (2012) “Information Technology service performance and client’s relationship to increase banking image and its influence on deposits customer banks loyalty (a survey of Banking in Jambi)”. Journal Archives Des Sciences Switzerland (Geneva), ISSN: 1661464x, International Science Index/ISI-Thomson Indexed Journal (2010 Impact Factor : 0.474), 65 (8), 538-551 Hapzi Ali, Nanda Limakrisna (2013) Research Methodology, Practical Guide to Solving Business Problems, Preparation of Theses and Dissertations, ISBN: 978- 602-280-044-6, Deepublish Yogyakarta, Indonesia. Hapzi Ali, Nanda Limakrisna, Said Djamalluddin (2016) “Model of customer satisfaction: the empirical study at BRI in Jambi”. International Journal of Applied Business and Economic Research (IJABER) www.serialsjournal.com,ISSN: 0972- 9380, Terindex Scopus 0.100 (Q4), New Delhi India, 13 (3), 707-719 Kotler, P., & Garry Armstrong, (2012) Prinsip-prinsip Pemasaran. Edisi 12, Jilid 2, Erlangga, Jakarta. Kotler, P., & Keller, K.L., (2008) Marketing Management (13th edition). Prentice- Hall International, Inc., NJ. Krishna, N., Swapna, B.G., dan Gantasala P. (2010) “New York Institute of Technology Sixth circle/Service Quality (Servqual) and its Effect on Customer Satisfaction in Retailing”. European Journal of Social Sciences. 16 (2), 231-243. g Ladhari, R., Ladhari, I., and Morales, M. (2011) “Bank Services Quality: Comparing Canadian and Tunisian Customer Perceptions”. International Journal of Bank Marketing, 29 (33), 224-246. Lamb, Charles. W., et al. (2007) Pemasaran Buku I. Edisi I. Jakarta: Salemba Empat. Malik dan Ghafoor. (2012) “Impact of Brand Image, Service Quality and price on customer satisfaction in Pakistan Telecommunication Setor”. International Journal of Business and Social Science, 3 (23), December 2012. Ming, S.L., Huey, D.H., and Ming, F.Y. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 DAFTAR RUJUKAN (2010) “The Study of The Relationship Among Experiental Marketing, Services Quality, Customer Satisfaction and Customer Loyalty”. International Journal of Organizational Innovation, pp. 352-378. y y Naeem, H., and Saif, I. (2009) “Service Quality and Its Impact on Customer Satisfaction: An Empirical Evidence from the Pakistani Banking Sector”. The International Business and Economics Research Journal, 8 (120, 99-104. Nalau, A., Syarifah, H., dan Wasil, M. (2012) “Brand Image Terhadap Loyalitas Pelanggan J.Co Donuts dan Coffee di Plaza Mulia Samarinda”. Jurnal Ekonomi, Universitas Mulawarman. Oliver, Richard L. (2010) Satisfaction: a Behavioural Perspective on the Consumer. 2nd ed. New York: M.E. Sharpe, Inc. Patricia, Huddleston,. Judith, Rachel Mattick and So Jung Lee. (2009) “Customer Satisfaction in Food Retailing: Comparing Specialty and conventional Grocery Stores”. International Journal of Retail & Distribution Management. 37 (1), 63-80. Sanka, M.S. (2012) “Bank Service Quality, Customer Satisfaction and Loyalty in Ethiopian Banking Sector”. Journal of Business Administration and Management Sciences Research, 1 (1), 001-009. Tjiptono, F. (2012) Perspektif Manajemen dan Pemasaran Kontemporer. Yogyakarta: Andi. Jurnal Manajemen/Volume XXI, No. 03, Oktober 2017: 317-335 334 Agussalim dan Ali: Model Kepuasan Pelanggan: Analisis Kualitas Produk... Tombokan, F., Kawet, L., dan Uhing, Y. (2015) “Pengaruh Kualitas Pelayanan dan Citra Merek Terhadap Kepuasan Konsumen Pengguna Taplus BNI Kantor Cabang Utama Manado”. Jurnal Emba, 3 (3). Umar, H. (2008) Metode riset bisnis: dilengkapi contoh proposal dan riset bidang manajemen dan akuntansi. Jakarta: Gramedia Pustaka Utama. Zafar, M., Zafar, S., dan Asif, A. (2012) “Service Quality, Customer Satisfaction and Loyalty: An Empirical Analysis of Banking Sector in Pakistan”, Information Management and Business Review, 4 (3). Zeithaml, Valerie A & Bitner, Mary Jo. (2008) Service Marketing. McGraw Hill Companies, Inc. Zikmund, William G. 2003, dalam Hapzi Ali. (2012) “Service quality, relationship satisfaction, trust, commitment, image and loyalty”. European Journal of Marketing, Emerald Library. Vol 16. 335
https://openalex.org/W1982926889	https://zenodo.org/records/2095640/files/article.pdf	English	null	CCXXII.—The oxidation of phosphorous acid by iodine	Journal of the Chemical Society. Transactions	1,908	public-domain	5,564	THE OXIDATION OF PHOSPHOROUS ACID BY IODINE. 2203 View Article Online / Journal Homepage / Table of Contents for this issue THE OXIDATION OF PHOSPHOROUS ACID BY IODINE. 2203 View Article Online / Journal Homepage / Table of Contents for this issue STEELE: THE OXIDATION OF Rupp and Finck (Arch. Phurrn., 1902, 240, 663) in developing a method for the estimation of phosphorous and hypophosphorous acids either alone or when mixed. The present communication describes the results that have been obtained during the attempt to elucidate the mechanism of the reaction between iodine and phosphorous acid under different conditions. Federlin (Zeitsch. physikal. Chem., 1902, 41, 565), while in- vestigating the reaction between iodine, phosphorous acid, and potassium persulphate, carried out a few experiments in which he measured the rate of reduction of iodine by a large excess of phosphorous acid, and he concluded that this was a bimolecular reaction. His experiments may, indeed, be interpreted as showing that the reaction is approximately unirnolecular with respect to the iodine, but they give no information as to the number of reacting molecules of phosphorous acid. These experiments were subsidiary, and were carried out by Federlin only to enable him to interpret the results of his other experiments. He does not appear to have noticed the extraordinary effect of reducing the acid concentration. Iodine and phosphorous acid appear to be capable of undergoing two distinct reactions, of which one predominates in acid solution, and the other in the absence of any appreciable concentration of hydrogen ions. The mechanism of the former of these reactions has been worked out with tolerable certainty, and the conclusions which have been arrived at are described in the sequel, but it has not been found possible to suggest a hypothesis which is competent to explain the mechanism of the second reaction. The phosphorous acid for the experiments was prepared by the action of distilled water on phosphorus trichloride which had been previously purified by fractional distillation. The resulting solution containing a mixture of phosphorous and hydrochloric acids was then evaporated in a vacuum, diluted with water, and again evaporated, this process being repeated until the solution was quite free from hydrochloric acid. It was finally diluted so as to obtain a solution containing half a gram-molecule per litre. The reaction was carried out in a thermostat at 35O, at which temperature the iodine completely disappeared in from two to eight hours according to the concentration of the mixture. The reagents were immersed separately in the thermostat, and mixed after they had attained the temperature of the bath. CCXXI1.-The Oxidation of Phosphoyous Acid by Iodine. By BERTRAM DILLON STEELE. IN a former paper (Trans., 1907, 91, 1642), it has been shown that the velocity of the reaction between iodine and hypophosphorous acid in acid solution is independent of the concentration of the former, and proportional to that of the latter, reagent, and that the reaction is catalytically accelerated by hydrogen ions, no appreciable action occurring in their absence. In the same communication, it was pointed out that the oxidation of phosphorous acid by iodine appeared to be retarded by hydrogen ions, and that this remarkable difference in behaviour had been utilised by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. View Article Online View Article Online 2204 STEELE: THE OXIDATION OF STEELE: THE OXIDATION OF Samples of the mixture were then withdrawn from time to time, and the residual iodine was titrated with a fiftieth molar solution of sodium thiosulphate. Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. 2205 View Article Online View Article Online View Article Online y Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. 2205 PHOSPHOROUS ACID BY IODINE. The Injaence of Ph08phorou8 Acid Concentration. The evaluation of the number of reacting molecules of phosphorous acid is complicated by the fact that the reaction is catalytically accelerated by the presence of hydrogen ions, which are not only produced from the phosphorous acid, but are also increased in quantity during the reaction. The reaction, in all probability, undergoes autocatalytic acceleration in the same manner as was found to be the case in the oxidation of hypophosphorous acid by iodine, but partly on account of the extreme slowness of the reaction when small concentra- tions of phosphorous acid were used ; no experiments have been carried out in which the extra quantity of hydrogen ions produced would exert any appreciable effect on the velocity. Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre Published on 01 January 1908. Downloaded by Universite Paris Ou The influence OF‘ the phosphorous acid was investigated by varying its concentration in different experiments. These experiments showed that the reaction is unimolecular with respect to the phosphorous acid, so that the complete equation for the velocity is as follows : dx ~~~- = k(A - x)(B - a+ + k’C(B - x)(B - x)+ dt . . (3). This equation involves the following hypotheses : The velocity is proportional to the concentration of the phosphorous acid and to the square root of the iodine concentration ; it is catalytically accelerated by hydrogen ions, the concentration of which is represented by C. The velocity coefficient, k, of the now catalysed reaction may usually be put equal to zero, since in most cases it is extremely small compared This equation involves the following hypotheses : The velocity is proportional to the concentration of the phosphorous acid and to the square root of the iodine concentration ; it is catalytically accelerated by hydrogen ions the concentration of which is represented by C by hydrogen ions, the concentration of which is represented by C. The velocity coefficient, k, of the now catalysed reaction may usually be put equal to zero, since in most cases it is extremely small compared with k’, the coefficient of the catalysed reaction. I n the present case, this cannot be done, as k and k’ are comparable in magnitude, The hydrogen ion concentration, C, is made up of the sum of H’ produced by the phosphorous, phosphoric, hydriodic, and added acids, and cannot be evaluated with any approach to exactness. PHOSPHOROUS ACID BY IODINE. View Article Online Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. View Article Online 2206 STEELE: TEE OXIDATION OF 08. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:0 Experiments 2 to 18, which are given later and which take into account the variation, not only of the iodine, but also of the phosphorous acid concentration, confirm the assumption that t h e velocity of the reaction is proportional to the square root of the iodine concentration. PHOSPHOROUS ACID BY IODINE. 1908. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17 On account of the practical impossibility of estimating the con- centration of the phosphorous ions during the experiments, the phos- phorous acid was always present in considerable excess. In these circumstances, the degree of ionisation of the acid remained practically constant throughout each experiment, and the concentration of the reacting substance, whether ion or molecule, might without appreciable error be taken as proportional to the total concentration. The InJuence of the Iodine Concentration.--In order to determine the influence of the iodine concentration, a number of experiments was carried out, in which a constant concentration of phosphorous acid was allowed to react with varying concentration of iodine, the acid being always in excess. Published on 01 January 1908. Downloaded by Universite Paris Ouest I n the equation : _.- dx - rE ( A - x)"( j3 - x)n, dt in which A represents the initial concentration of the phosphorous acid and B that of the iodine, it was anticipated that Federlin's con- clusion that n = 1 would be confirmed ; :it was found instead that n. = 0 5 . On the assumption that ( A - x) is constant and that n= 1, integration of the foregoing equation gives the usual equation for a unimolecular reaction : if, however, n = 0.5, we get the equation : if, however, n = 0.5, we get the equation : dn: - dt __ - k(A - z ) y B - 4 0 ' 5 , and on integration : k ( A - x ) " = K = - t JL JKx) . . . . 2( Table I shows a typical example of the manner in which the experi- mental results are described by equations (1) and (2) respectively. TABLE I. t. 23-2. K f o r (.n=l). K for (n=05). 67 0'0154 0'00314 0'00206 113 0'0132 0 00324 0-00203 177 0'0104 000340 0 '00203 232 0-0083 0'00356 000201 266 0.0075 0-00350 OsO0193 - - 0 0.0190 t = time in minutes at which titration was made. ( B - x) =iodine concentration in milligram-mols. per C.C. at the time t. The values of the constants calculated from equations (I) and (2) are given in the third and fourth columns. site Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. The Injaence of Ph08phorou8 Acid Concentration. This is due in the first place to the uncertainty which attaches to the ionisation coefficient of a polybasic acid, and, secondly, to the unknown influence of each acid on the dissociation of the others. Strictly speaking, C = [a(A - x ) + DL’X + a”C + a”’2x1, where A - x, x, 22, and c are the concentrations of the phosphorous, phosphoric, hydriodic, and added acids respectively, and a, a’, a”’, and a” are the corresponding ionisa- tion coefficients of each acid in the presence of all the others. Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. View Article Online View Article Online versite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. 2207 PHOSPHOROUS ACID BY IODINE. 2207 08. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04 On account of the impossibility of accurately evaluating these coefficients, the following simplification has been made : , g p a, a', and a"' are of the same order of magnitude, a''' being greater than a, and a greater than a'. g I n the absence of added acid, the expression for the hydrogen ion concentration becomes aA + (a' + 2a"' - Q)X, which, on account of the small magnitude of ~ t ' compared with A , may without sensible error be put equal to uA. d on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre La Defense on That no appreciable error has been introduced by this assumption will be evident from a consideration of experiments 1 to 9. Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre The accurate evaluation of the hydrogen ion concentration is rendered impossible in the case of the experiments where sulphuric acid has been added by our ignorance of the mutual influence of strong acids on the ionisation coefficients. Published on 01 January 1908. Downloaded by Universite Paris Ou Notwithstanding this, the effect of increasing the hydrogen ion concentration will be clearly seen by reference to the results of experiments 10 to 15. Published on 01 January 1908. Downloaded by Uni p If equation (3) is written in the form dx & =(k+k'C)(A -22) JJB-x, we obtain on integration the equation : g q and the constant obtained by the use of this equation is given in the last column of the following table : No. of expt. STEELE: THE OXIDATION OF smallest A and C values, there is a decrease in k + k'C with increasing iodine concentration. smallest A and C values, there is a decrease in k + k'C with increasing iodine concentration. To ascertain in what direction it would be necessary to modify equation (3) in order that it may more accurately describe the experimental results, k and k' must first be evaluated. This can easily be done, since we have in table I V nine equations giving values of k+k'C, and C is known, at least approximately, for all of them TABLE 111. Expt. A. 1 0.2 2 0.2 3 0 '2 4 0.2 5 0.3 6 0.3 7 0-3 8 0-4 9 0.4 B. 0.01 0.02 0.03 0 '04 0'01 0'02 0.03 0.01 0'02 Espt. 1 5 2 6 9 3 7 a C. 0.087 0.087 0.087 0.087 0'12 0.12 0.12 0.15 0.15 k+k'CxlO3. kx1O8. k'x103. 2-12 0.88 14 -3 2.05 0.90 13'2 1-86 0-71 13.2 1 -69 0.54 13'2 2.68 0 9 2 14-8 2 5 0 0-94 13.0 2-30 0.72 13.2 2.87 0.96 12'7 2 -85 0.94 12-7 TABLE IV. rl: x lo3 calculated from experiments. P- --- . Mean 1and5. l a n d & 5and8. 2and6. 2atnd9. 6and9. 3and7. h!x103. 16.5 12.2 - - - -- - 14'3 16.5 - 13'2 - - - - 14-8 - 12.2 - - - - - 12.7 - - - 13.5 13.0 13.1 - 13'2 - - - 13.5 - 12.5 - 13.0 - - - - 13.0 12.5 - 12.7 - - - - - - 13-2 13'2 - - - - - - 13'2 13'2 13'4 General mean ... ... ... ,.. ... . , . TABLE 111. Expt. A. 1 0.2 2 0.2 3 0 '2 4 0.2 5 0.3 6 0.3 7 0-3 8 0-4 9 0.4 B. 0.01 0.02 0.03 0 '04 0'01 0'02 0.03 0.01 0'02 C. 0.087 0.087 0.087 0.087 0'12 0.12 0.12 0.15 0.15 k+k'CxlO3. kx1O8. k'x103. 2-12 0.88 14 -3 2.05 0.90 13'2 1-86 0-71 13.2 1 -69 0.54 13'2 2.68 0 9 2 14-8 2 5 0 0-94 13.0 2-30 0.72 13.2 2.87 0.96 12'7 2 -85 0.94 12-7 TABLEIV The Injaence of Ph08phorou8 Acid Concentration. A . 1 0.2 2 0.2 3 0.2 4 0.2 5 0-3 6 0.3 7 0.3 8 0'4 9 0'4 B. 0.0093 0'019 0.0285 0-00976 0 -01936 0'0096 0'0192 0-0382 0 e . m C. j 0.0866 0-0866 0-0866 0.0866 0'12 0.12 0'12 0,148 0.148 TABLE 11. Duration of expt., in minutes. 401 266 340 402 167 240 246 111 196 7 Lowest. 0'00193 0~00200 0.00181 0'00152 0'00249 0.00242 000221 0'00263 0 -00260 k -I- k'C. Highest. 0.00225 0 00208 000191 0-00173 0.00275 0.00254 0'00240 0,00304 0.00294 A 3 Mean. 0.00212 0 -00205 0'00186 000169 0.00268 0.00250 000230 0.00287 0.00285 The general correctness of the hypotheses contained in equation (3) is indicated by the very slight variation of the constant in any single experiment. Its insufficiency is shown in table 111 by the variation of k+k'C in experiments 1, 2, 3, and 4, all of which have the same A and C values, and similarly in experiments 5, 6, and 7 and 8 and 9. I n all these cases, but most markedly in those which have the No. of expt. A . 1 0.2 2 0.2 3 0.2 4 0.2 5 0-3 6 0.3 7 0.3 8 0'4 9 0'4 B. 0.0093 0'019 0.0285 0-00976 0 -01936 0'0096 0'0192 0-0382 0 e . m C. j 0.0866 0-0866 0-0866 0.0866 0'12 0.12 0'12 0,148 0.148 TABLE 11. Duration of expt., in minutes. 401 266 340 402 167 240 246 111 196 7 Lowest. 0'00193 0~00200 0.00181 0'00152 0'00249 0.00242 000221 0'00263 0 -00260 k -I- k'C. Highest. 0.00225 0 00208 000191 0-00173 0.00275 0.00254 0'00240 0,00304 0.00294 A 3 Mean. 0.00212 0 -00205 0'00186 000169 0.00268 0.00250 000230 0.00287 0.00285 The general correctness of the hypotheses contained in equation (3) is indicated by the very slight variation of the constant in any single experiment. Its insufficiency is shown in table 111 by the variation of k+k'C in experiments 1, 2, 3, and 4, all of which have the same A and C values, and similarly in experiments 5, 6, and 7 and 8 and 9. I n all these cases, but most markedly in those which have the niversite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. View Article Online 2208 STEELE: THE OXIDATION OF TABLE IV. rl: x lo3 calculated from experiments. Espt. 1 5 2 6 9 3 7 a rl: x lo3 calculated from experiments. P- --- . Mean 1and5. l a n d & 5and8. 2and6. 2atnd9. 6and9. 3and7. h!x103. 16.5 12.2 - - - -- - 14'3 16.5 - 13'2 - - - - 14-8 - 12.2 - - - - - 12.7 - - - 13.5 13.0 13.1 - 13'2 - - - 13.5 - 12.5 - 13.0 - - - - 13.0 12.5 - 12.7 - - - - - - 13-2 13'2 - - - - - - 13'2 13'2 13'4 General mean ... ... ... ,.. ... . , . k and k' were calculated from different pairs of experiments, those experiments being selected which had the same value for the iodine concentration B. The values of k' calculated in this manner from seven pairs of experiments are given in table IV. I n only one calculation, that from experiments 1 and 5, does the value differ to any extent from the general mean of 13.4 x 10-3. This exception appears to be due to an abnormal value for the constant from experiment 5. The values of k which are given in the sixth column of table 111 were obtained by deducting the numbers in column 7 from those in column 5. This method of calculation throws practically all the variation in k + k'C on k, the non-catalytic constant, and this conclusion is confirmed by evaluating k by the method employed for k' and described in table IV; such a calculation yields values for k which, like those in column 6 of table 111, diminish with increasing concentration of iodine, and this suggests that it is the d by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. 2209 View Article Online View Article Online PHOSPHOROUS ACID BY IODINE. 08. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:0 first term in equation (3) which is inaccurate. On the whole, except for this slight variation of k, the experimental results are well expressed by the equation used. The In$uence of Added Acid. I n the series of experiments 10 to 15 (table V), sulphuric acid has been added in varying quantity, the concentration in each experiment being given under c. In all cases an increase in c produces a n increase in the magnitude of the constant k+k'C, but although, as in the former series, the constant is very good in each experiment, no simple relation between its values from different experiments can be detected. This, as already pointed out, is due to the uncertainty as to the total H' concentration. Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterr Published on 01 January 1908. Downloaded by Universite Paris Ou TABLE V. No. of expt. A. 10 0.2 11 0.2 12 0.2 13 0.2 14 0'3 15 0.3 Duration of expt., in B. c. C( = aA + u " ~ ) . minutes. Lowest. 0.019 0.02 0'1146 272 0'00220 0'0187 0-05 0'1146 280 0'00246 0-019 0.10 0.203 311 0.00278 0'0193 0.4 0.447 229 0.00408 0.0194 0.05 0.181 235 0'00284 0'0194 0.10 0'236 227 0'00331 k + k'C. F Highest. Mean. 0'00226 0'00222 0-00265 0'00254 000304 0.00296 0.00468 0.00444 000305 0'00299 0.00369 0'00353 On the assumption that this is given approximately by (aA +a"c), the sum of the concentrations of the hydrogen ions from the phosphorous acid and from the sulphuric acid, each in the absence of the other, values are obtained for k;' which vary between 9.7 and 6.7. The calculation may be reversed, and the total hydrogen ion concentration calculated from the values of k and k' found already from experiments 1 to 9. This has been done, and table VI contains, for experiments 10 to 15, the values of the concentrations of the phosphorous acid ( A ) , iodine (B), and sulphuric acid, c, as well as that of the summed constants (k+k'C), the last two columns showing the difference between (aA +a"c) and C calculated in this manner. The TABLE VI. Expt. A. B. c. k+KC. aA+a"c. C'. The Reaction with Very Low Hydrogen Ion Concentration. The Reaction with Very Low Hydrogen Ion Concentration. It has been already mentioned that if the H' concentration is made very small by the addition of a salt of a very weak acid, such as the sodium salts of acetic, carbonic, or boric acid, the reaction velocity is increased enormously. With moderate concentrations of iodine and excess of phosphorous acid, reaction is complete in from one to seven minutes at 25O. I n order to make any measurements, it is therefore necessary to work at moderately low temperatures, and the following experiments? in which a large excess of phosphorous acid was always used, were carried out in a bath of melting ice. Published on 01 January 1908. Downloaded by Universite Paris Ou Experiment 18, table VII, mill show the nature of the results which have been obtained. TABLE VII.--Experiment 18. TABLE VII.--Experiment 18. A ~0.2. B=0-02. C,H,O,Na=O'4. KI=0.050. t. 0'0 5 5 8-5 13'5 23.5 87.5 6.5 5 99 5 142.5 230-0 B - 2. 0.0189 0.0169 0.0155 0.0144 0-0125 0'0108 0.0084 0'00644 0'0050 0.00328 B B - 2 l/t log-. - 0'0209 0.0238 0'0201 0.0175 0-0156 0'0119 0-0108 0'0093 0-0077 lltB x-. B - x - 1.165 1-37 1 '22 1-15 1-05 0 '98 1 -02 1-04 1 lo Although this and the other experiments of this series yield Although this and the other experiments of this series yield 1 x i l l f h bi l l i Although this and the other experiments of this series yield 1 x approximately constant values for the bimolecular expression - - tB'B - x (column 4, table VII), the conclusion that the reaction is bimolecular with respect to the iodine is not confirmed by the consideration of the value of the constant from different experiments. The relation between the reaction velocity and the concentration of the reagents is, indeed, so complex that the attempt to give quantitative expression to the results has been abandoned. Although this and the other experiments of this series yield 1 x approximately constant values for the bimolecular expression - - tB'B - x (column 4, table VII), the conclusion that the reaction is bimolecular with respect to the iodine is not confirmed by the consideration of the value of the constant from different experiments. The In$uence of Added Acid. 10 0'2 0'02 0 '02 2-22 0'1146 0'1 11 0'2 0'02 0.05 2 -54 0-148 0.123 12 0 '2 0.02 0.10 2-96 0.203 0-157 13 0.2 0.02 0'4 [HCl] 4-44 0.427 0.271 14 0.3 0.02 0 -05 2 -84 0.181 0'16 15 0.3 0-02 0.10 3.53 0.236 0'201 latter is always less than the former, and by an amount which might be expected from the .decrease in ionisation of the phosphorous acid, due to the presence of the sulphuric acid, niversite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. View Article Online Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. View Article Online 2210 STEELE: THE OXIDATION OF STEELE: THE OXIDATION OF 08. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04 This decrease in ionisation would have a dual effect if the reaction were one in which phosphorous ions are oxidised by iodine, since the concentration of the phosphorous ions would also be diminished, and hence also the observed constant. The fact that experiment 13 gives for C a value (0.271) which is less than the H' concentration from the added acid alone, is the only one in the whole of the present investigation which indicates in any way whether the iodine reacts with ion or undissociated molecule. Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre STEELE: THE OXIDATION OF TABLE VII1.-Experiment 16. The Reaction with Very Low Hydrogen Ion Concentration. t .................. 0.0 5.5 11.5 17-0 29'0 48-0 87.0 (B-x) x l o 3... 9'44 6'60 5-66 4'76 3.54 2'36 1.2 Experiment 17 repeats Expeeriment 16. Experiment 18. A = 0.2. B= 0.02. C,H,O,Na = 0.4. KI= 0 050. t .................. 0.0 5-5 8.5 135 23.5 3 7 5 68.5 99.5 142.5 (B-x)x103., 18.9 16'8 15.6 14'4 12.5 10.8 8'4 6.4 5.0 Experiment 1 9. A = 0'2. B= 0-01. C,H,O,Na = 0.6. KI= 0 025. (B-x) x lo3 ... 8-84 6-82 5-54 4.80 2.38 1-86 1-36 t .................. 0.0 2-5 4.5 8.0 22.0 27.0 34.0 Experiment 20. A = 0.2. B = 0'01. C,H30,Na = 0.8. XI= 0.025. t ................... .1 0.0 1.5 4'0 7.5 11.25 18% (B-2)x103 ...... 6.70 5-64 4-12 2-94 2.70 1.50 Experiment 2 1. A = 0.2 B= 0.01. CzHs02Na= 0'4. KI= 0.125. t .................. 0.0 3.0 8.0 14'0 27-0 33.0 62'0 (B-x)x103 ... 9'14 8.20 7.08 6'22 4-82 3-98 2-54 Experiment 22. A=0.2. B=001. C,H,O,Na= 0.4. KI=0225. t .................. 0.0 4-0 12.5 21.0 31.5 49-5 68.5 118.5 ( B - x ) x 10 '.,. 10'06 8.92 7.32 6.16 5.20 4.00 3-06 1'68 The same experiments are summarised in table IX, which gives, in Rxpt. 16 and 17 ... 18 ............ 16 and 17 ... 19 .......... 20 ............ 16 and 17 A. 0 -2 0 2 0'2 0-2 0.2 0 2 TABLE IX. B. C,H,O,Na. 0.01 0.4 0.02 0'4 0.01 0 '4 0.01 0-6 0.01 0'8 0 01 0'4 KI. 0.025 0 -05 0.025 0,025 0.025 0'025 tt. 20 53 20 9.5 7 20 View Article Online 2212 STEELE: THE OXIDATION OF The Reaction with Very Low Hydrogen Ion Concentration. The relation between the reaction velocity and the concentration of the reagents is, indeed, so complex that the attempt to give quantitative expression to the results has been abandoned. View Article Online View Article Online Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23 2211 . Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:2 PHOSPHOROUS ACLD BY IODINE. 2211 PHOSPHOROUS ACLD BY IODINE. The curves of Figs, 1 and 2 are obtained by plotting the experi- This table contains the mental results which are given in table,VIII. FIG. 1. FIG. 1. 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 Time in minutes. FIG. 1. 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 Time in minutes. times of measurement expressed in minutes, and the corresponding values of the iodine concentration for experiments 16 to 22, the con- 0.010 0'009 0~008 0.007 A 0'006 ' 0'005 0'004 0'003 0'002 0.001 2 FIG. 2. 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 l'ime in minutes. centrations of the phosphorous acid (A), iodine (B), sodium acetate, and potasium iodide being given for each experiment, Time in minutes. times of measurement expressed in minutes, and the corresponding values of the iodine concentration for experiments 16 to 22, the con- times of measurement expressed in minutes, and the corresponding values of the iodine concentration for experiments 16 to 22, the con- 0.010 0'009 0~008 0.007 A 0'006 ' 0'005 0'004 0'003 0'002 0.001 2 FIG. 2. 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 l'ime in minutes. centrations of the phosphorous acid (A), iodine (B), sodium acetate, and potasium iodide being given for each experiment, 0.010 0'009 0~008 0.007 A 0'006 ' 0'005 0'004 0'003 0'002 0.001 2 FIG. 2. 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 l'ime in minutes. centrations of the phosphorous acid (A), iodine (B), sodium acetate, and potasium iodide being given for each experiment, Published on 01 January 1908. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. View Article Online View Article Online 2212 STEELE: THE OXIDATION OF TABLE VII1.-Experiment 16. A = 0-2. B = 0.01. C,H,O,Na = 0'4. KI= 0.025. TABLE VII1.-Experiment 16. A = 0-2. B = 0.01. C,H,O,Na = 0'4. KI= 0.025. t .................. 0.0 5.5 11.5 17-0 29'0 48-0 87.0 (B-x) x l o 3... 9'44 6'60 5-66 4'76 3.54 2'36 1.2 A = 0-2. B = 0.01. C,H,O,Na = 0'4. KI= 0.025. t .................. 0.0 5.5 11.5 17-0 29'0 48-0 87.0 (B-x) x l o 3... 9'44 6'60 5-66 4'76 3.54 2'36 1.2 Experiment 17 repeats Expeeriment 16. Experiment 18. A = 0.2. B= 0.02. C,H,O,Na = 0.4. KI= 0 050. t .................. 0.0 5-5 8.5 135 23.5 3 7 5 68.5 99.5 142.5 (B-x)x103., 18.9 16'8 15.6 14'4 12.5 10.8 8'4 6.4 5.0 Experiment 1 9. A = 0'2. B= 0-01. C,H,O,Na = 0.6. KI= 0 025. (B-x) x lo3 ... 8-84 6-82 5-54 4.80 2.38 1-86 1-36 t .................. 0.0 2-5 4.5 8.0 22.0 27.0 34.0 Experiment 20. A = 0.2. B = 0'01. C,H30,Na = 0.8. XI= 0.025. t ................... .1 0.0 1.5 4'0 7.5 11.25 18% (B-2)x103 ...... 6.70 5-64 4-12 2-94 2.70 1.50 Experiment 2 1. A = 0.2 B= 0.01. CzHs02Na= 0'4. KI= 0.125. t .................. 0.0 3.0 8.0 14'0 27-0 33.0 62'0 (B-x)x103 ... 9'14 8.20 7.08 6'22 4-82 3-98 2-54 Experiment 22. A=0.2. B=001. C,H,O,Na= 0.4. KI=0225. t .................. 0.0 4-0 12.5 21.0 31.5 49-5 68.5 118.5 ( B - x ) x 10 '.,. 10'06 8.92 7.32 6.16 5.20 4.00 3-06 1'68 Experiment 17 repeats Expeeriment 16. A = 0.2 B= 0.01. CzHs02Na= 0'4. KI= 0.125. t .................. 0.0 3.0 8.0 14'0 27-0 33.0 62'0 (B-x)x103 ... 9'14 8.20 7.08 6'22 4-82 3-98 2-54 Experiment 22. A=0.2. B=001. C,H,O,Na= 0.4. KI=0225. t .................. 0.0 4-0 12.5 21.0 31.5 49-5 68.5 118.5 ( B - x ) x 10 '.,. 10'06 8.92 7.32 6.16 5.20 4.00 3-06 1'68 The same experiments are summarised in table IX, which gives, in Rxpt. 16 and 17 ... 18 ............ 16 and 17 ... 19 .......... 20 ............ 16 and 17 ... 21 ............ 22 ........... A. 0 -2 0 *2 0'2 0-2 0.2 0-2 0.2 0'2 TABLE IX. B. C,H,O,Na. 0.01 0.4 0.02 0'4 0.01 0 '4 0.01 0-6 0.01 0'8 0.01 0'4 0.01 0.4 0'01 0'4 KI. 0.025 0 -05 0.025 0,025 0.025 0'025 0.125 0.225 tt. 20 53 20 9.5 7 20 29 34 addition to the data contained in table VIII, the time required in the case of each experiment for the reaction to proceed half way to com- y Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04:23. View Article Online View Article Online 2213 PHOSPHOROUS ACID BY IODINE. 908. Downloaded by Universite Paris Ouest Nanterre La Defense on 04/08/2016 17:04 pletion; these figures are given in the sixth column. The great complexity of the reaction and its extreme sensitiveness to concentra- tion changes will be clearly seen from a study of this table and of the figures. g Thus experiments 16, 17, and 18, which are shown in Fig. 1, show the effect of doubling the iodine concentration, the time of half reaction being increased from twenty to fifty-three minutes, an effect which is exactly the opposite to that which should occur in a bimolecular react ion. Experiments 16, 17, 19, and 20 (Fig. 2) show the acceleration of the reaction velocity by an increase in the sodium acetate concentra- tion. This corresponds with a diminution in the concentration of the hydrogen ions, and, if this conditions an increase in the velocity, it is obvious that the reaction in the conditions under discussion cannot be the same as that which takes place in the presence of much free acid. Published on 01 January 1908. Experiment 17 repeats Expeeriment 16. Downloaded by Universite Paris Ouest N Finally, the effect of adding potassium iodide (or iodine ions) is seen from experiments 16, 17, 21, and 22 to be a retardation, the time of half reaction being increased from 20 in experiments 16-17 to 29 and 34 respectively in experiments 21 and 22. Summary. It has been shown that two distinct reactions occur between iodine and phosphorous acid, one of which preponderates in acid solutiop and the other in the absence of any strong acid. The former of these reactions is catalytically accelerated by the presence of hydrogen ions, and the reaction velocity is proportional to the concentrations of the phosphorous acid (or of the phosphorous ion) and to the square root of the concentration of the iodine. The conclusion is drawn that reaction takes place between phosphorous acid molecules (or ions) and iodine atoms, the latter being supplied in extremely small, but sufficient, quantity by the dissociation of the iodine molecules. A slight apparent retardation of the reaction velocity by the iodine is probably due to the incompleteness of the hypothesis which has been auggested to explain the experimental results. The second reaction, which takes place in the presence of salts, such as sodium acetate, bicarbonate, and borate, is extremely complex ; the reaction being accelerated by the presence of sodium acetate, retarded by the presence of iodine, and also retarded by the presence of iodide ions. No hypothesis explanatory of this reaction has been suggested. THE UNIVEKSITY MELBOURNE THE UNIVEKSIT MELBOURN THE UNIVEKSITY MELBOURNE
https://openalex.org/W4224247261	https://hrcak.srce.hr/file/398880	English	null	Energy and Cost Evaluation of Different HVAC Systems in an Office Building	Tehnički vjesnik/Tehnički vjesnik	2,022	cc-by	4,895	1 INTRODUCTION been seen that the air handling unit system and the system with fan-coil units provide energy savings between 1.15% and 12.96% [5]. The climatic conditions of the region where the system will operate affect the energy saving and operating performance of the system. In this context, when VRF and VAV systems are compared in terms of energy saving potential according to the climatic conditions of the USA, it is seen that the VRF system is more advantageous than the VAV system in most of the climates in the USA [6]. In another case study conducted in China, they compared VRF and VAV systems, to operate in office buildings in China, in terms of energy performance and found that the VRF system was more efficient [7]. In another study, energy analysis was performed for VRF, VAV and fan-coil systems under cooling conditions. As a result, it has been seen that the VRF system is the most efficient system among the systems compared in terms of energy consumption [8]. With the developing technology, many different mechanical installation systems can be applied to reach the desired comfort conditions in office buildings. System selection is generally made by considering criteria such as the purpose of use of the building, the climatic conditions of the region where the building is located, the new/existing building condition, mechanical system limitations (material strength classes, device capacities, etc.), architectural structure, operating cost, initial investment cost. However, many different parameters affect the system selection and one of these parameters is the floor height. The researchers experimentally compared the fan-coil system with the underfloor cooling system in terms of thermal comfort at different floor heights. In these experiments, it was seen that the fan-coil system in low ceiling rooms and the floor cooling system in high ceiling rooms are more advantageous [1]. One of the important issues in system selection is the energy source used. The effect of the energy source used on the costs has also been investigated in the literature. Christodoulides et al., in their study, compared the costs of an air source heat pump and a ground source heat pump based on a typical building in Cyprus. It has been seen that the amortization period of the ground source heat pump is twenty years advantageous compared to the air source system [2]. Other important factors in system selection are energy savings and efficiency. Li et al. Energy and Cost Evaluation of Different HVAC Systems in an Office Building Kurtulus YASIN, Deniz YILMAZ* Abstract: Researchers state that people spend about 90 percent of their time indoors, and accordingly, the place of HVAC systems in our lives is increasing day by day. This brings about an increase in energy consumption. Considering the amount of energy consumption for developed and developing countries today, it is seen that the role of HVAC systems is quite high. Therefore, energy consumption costs and investment costs are the main factors to be considered in HVAC system selection. In this study, for a sample office building, six different HVAC systems (water-cooled and air-cooled variable flow cooling systems, air handling unit system, fan coil systems, water source and air source heat pump systems and split air conditioning system) are analyzed in terms of energy consumption, initial investment costs, operating costs and ease of operation. The advantages and disadvantages of the systems were compared with each other, and as a result of the calculations, it was seen that air-cooled systems consume an average of 33% less energy than water-cooled systems and their investment costs are 30% lower. Keywords: HVAC systems; fan-coil; heat pump; VRV systems Keywords: HVAC systems; fan-coil; heat pump; VRV systems ISSN 1330-3651 (Print), ISSN 1848-6339 (Online) ISSN 1330-3651 (Print), ISSN 1848-6339 (Online) https://doi.org/10.17559/TV-20210306185845 Original scientific paper Keywords: HVAC systems; fan-coil; heat pump; VRV systems 2.2 System Design Conditions The building is at 41.0° latitude, ‒28.8° longitude coordinates and is 11 m above sea level. The outdoor design conditions taken into account in the calculations are given in Tab. 1 [13]. 1 INTRODUCTION has discussed the energy savings and economic efficiency of fan-coil and floor cooling systems according to Chinese climates by performing exergy analysis. It has been determined that the underfloor cooling system is approximately 30% more efficient in terms of exergy than the fan-coil system [3]. When the cooling energy performance of the water-cooled VRF system and the AHU system in a commercial building are compared, it has been observed that the VRF system provides 15% energy savings per year in cooling compared to the AHU system [4]. Another important point to be considered in the system to be selected is to save energy in annual electricity consumption. Researchers experimentally and numerically compared the air handling unit system, fan-coil units and heat recovery systems in an office building according to their electricity consumption amounts. As a result, it has One of the factors affecting efficiency in system selection is preferring individual systems instead of compact and complex systems and providing floor-based solutions. When the VRF system is examined centrally and individually for the same office building, it is seen that the individual system provides energy savings compared to the central system [9]. Luo et al. conducted a study on the energy consumption of houses in Beijing. As a result, it has been seen that decentralized systems cause less energy consumption than centralized systems [10]. One of the important factors affecting the efficiency is the use of natural energy resources in the system. The energy obtained from air, water and soil increases efficiency in the long run when used as a source by the system. When the VRF system and the ground source heat pump are compared in terms of energy efficiency, it is seen that the ground source heat pump is 9.4-24.1% more efficient than the VRF system [11]. It should also be taken into account that the soil will remain at a constant temperature in the long term if soil is used as an external source in system selection. Amir et al. compared air source heat pump and ground source heat pump in terms of thermal performance. 2 MATERIALS AND METHODS 2.1 Building and System Description The model office building discussed in this study consists of a basement, ground floor, first floor and attic. It has a total area of 3250 m2, of which the basement floor is 850 m2, the ground floor is 850 m2, the first floor is 800 m2 and the attic is 750 m2. Fig. 1 shows the section view of the model office building. Table 1 Outdoor design conditions Design parameters Temperature Outdoor temperature (winter conditions) ‒3 °C Dry-bulb temperature 33 °C Tday-Tnight 10.5 °C Unconditioned space temperature 30 °C Unheated space temperature 12 °C Ground temperature adjacent to the outer wall 3 °C Figure 1 Cross-section view of the building Figure 1 Cross-section view of the building In the calculations, it is foreseen that the wet areas will not be heated to 20 °C in the winter and not cooled in the summer, while the other spaces will be heated to 20 °C in the winter and cooled to 24 °C in the summer. The internal calculation conditions of the spaces are given in Table 2 [14]. Table 2 Indoor design temperatures Space Temperature (Summer Conditions) Temperature (Winter Conditions) Lounge 24 °C 20 °C WC 30 °C 20 °C Hallway, entree 24 °C 20 °C Office 24 °C 20 °C Meeting room 24 °C 20 °C There are technical offices and a car park in the basement, and office units on the ground floor and the first floor and the attic. Floor heights; 3.75 m in the basement, 3.15 m in the ground and first floors, and 3.45 m in the attic floor. The section view of the model office building is shown in Fig. 2. The thermal conductivity coefficient U (W/m2K) of the building materials was determined by considering the TS 825 (ICS 91.120.10) standards [13]. Calculations were made by accepting the shading coefficient of the glasses as 0.40. Figure 2 Plan of the building 1 INTRODUCTION In this study, in which a house in Canada was examined, they found that the ground source heat pump showed a more stable performance due to the constant temperature Tehnički vjesnik 29, 3(2022), 881-887 881 Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Building of the soil and, accordingly, it was more efficient than the air source heat pump [12]. Heating system, air conditioning system and ventilation system were designed for the model office building. In addition to these, a clean water installation has been designed only in water-cooled systems. The building is located in Istanbul. The calculation and design of the air conditioning and ventilation system has been made in accordance with DIN, BS, ASHRAE, SMACNA standards. Heating and cooling have been made in all parts of the building except for the parking lot and mechanical areas. Alternative systems calculated for the model office building are as follows: Air-cooled Variable Flow Cooling System, Water-cooled Variable Flow Cooling System, Air Handling Unit System, Four-pipe Fan-Coil System, Air- source Heat Pump System (Four-pipes Fan-coil), Water- source Heat Pump System (Four-pipes Fan-Coil). p p In this study, six different mechanical installation systems (Air cooled variable flow cooling systems, Water cooled variable flow cooling systems, Air handling unit system-AHU, Fan coil systems, Air source heat pump systems, Water source heat pump systems) were applied on a model office building. The advantages and disadvantages of the systems against each other, initial investment costs, operating costs, ease of operation and amortization periods are compared, and it is examined which system is the most efficient. For the sample office building, system modeling was done with the Carrier Hap® program and heating/cooling loads were calculated. Devices were selected using the results obtained. According to the devices selected for each system, operating and initial investment costs were calculated and the systems were analyzed using the data obtained. 2.3 Minimum Fresh Air Requirements The number of people in the rooms and the required minimum amount of fresh air must be known for the heat gain calculations of the building. The amount of fresh air and the number of people admitted to the rooms are shown in Tab. 3. The amount of exhausted air and hourly air exchange values from wet areas and storeroom are given in Tab. 4. Figure 2 Plan of the building Technical Gazette 29, 3(2022), 881-887 882 Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Building Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Building Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Building Table 3 Fresh air requirements and occupancy Space Minimum fresh air requirements Number of people Lounge 30 m3/h per person According to furnishing Meeting hall 30 m3/h per person According to furnishing Administration office 36 m3/h per person 1 person in 7 m2 Resting room 36 m3/h per person According to furnishing General areas 30 m3/h per person 1 person in 5 m2 Office 36 m3/h per person 1 person in 5 m2 and the time that people are in the office are shown in Figs. 3, 4 and 5, [14]. Figure 5 Working time rates according to time zones Table 4 Hourly air exhaust amounts Space Hourly air exhaust amounts External air-source WC 8-12 With leakage from neighboring areas Storeroom 2-12 With leakage from neighboring areas Kitchen hood 250-400 m3/h person With leakage from window Table 4 Hourly air exhaust amounts Figure 5 Working time rates according to time zones 2.4 Lighting and Device Loads In this system, the boiler meets the need for heating the domestic water, while the heating and cooling processes of the spaces are provided by the Air-cooled variable flow (VRV) unit, and the ventilation need is provided by the Heat Recovery (HRV) system. The heating and cooling loads needed by the spaces were calculated in the Carrier Hap 4.50 program and device selections were made. The initial investment cost for the air sourced VRV system was calculated by designing in line with the results obtained. Energy analysis was made using the system data created in the program. As a result of the energy analysis, the operating cost was calculated, and system graphics were created on an annual and monthly basis. In the VRV system, there is no annual natural gas fuel consumption since the heating and cooling processes of the spaces are done with VRV devices. One of the factors that cause heat gain is the electrical load of the lamps and devices that provide lighting in the conditioned spaces. The electrical and lighting load values that vary depending on the use of the spaces are given in Tab. 5. Table 5 Lighting and device loads of spaces Space Lighting loads / W/m2 Device loads Office 25 250 W/person Meeting hall 25 500-750 W Administration office 25 1000 W Resting room 25 250 W Figure 3 Operating rates of lighting according to time zones Figure 6 Monthly cost graph of system components Figure 7 Monthly energy cost graph For the air-cooled VRV system, monthly cost graphs and monthly energy cost graphs of system components are shown in Figs. 6 and 7, respectively. While the HVAC system equipment constitutes 25.2% of the annual electricity consumption of the building, the remaining Figure 6 Monthly cost graph of system components Figure 3 Operating rates of lighting according to time zones Figure 3 Operating rates of lighting according to time zones Figure 7 Monthly energy cost graph Figure 4 Operating rates of devices according to time zones Figure 7 Monthly energy cost graph For the air-cooled VRV system, monthly cost graphs and monthly energy cost graphs of system components are shown in Figs. 6 and 7, respectively. While the HVAC system equipment constitutes 25.2% of the annual electricity consumption of the building, the remaining Figure 4 Operating rates of devices according to time zones The building generally consists of offices. 3.2 Water-cooled variable flow cooling system The only difference of this system from the first alternative system is that the VRV outdoor unit, which provides the heating and cooling needs of the spaces, uses water instead of air as an external source. Compared to the air-cooled system, additional devices such as the cooling tower and the pump feeding the cooling tower, the hydrophore, increase the operating and investment costs for this system. Figure 10 Monthly cost graph of system components Figure 11 Monthly energy cost graph Figure 8 Monthly cost graph of system components Figure 9 Monthly energy cost graph Figure 8 Monthly cost graph of system components Figure 11 Monthly energy cost graph Figure 11 Monthly energy cost graph For the air handling unit system, monthly cost graphs and monthly energy cost graphs of system components are shown in Figs. 10 and 11, respectively. While 55.4% of the annual electricity consumption of the building consists of HVAC system equipment, the remaining 44.6% is due to lighting, electrical devices and similar equipment. While 93% of the annual natural gas consumption of the building is due to the need for hot water and the air handling unit coil, 7% is due to the need for the kitchen (cooking). Figure 9 Monthly energy cost graph 3.4 Four-Pipe Fan-Coil System In this system, the heating and cooling needs of the spaces are provided by four-pipe fan coil units. While the fan coil heating need is met by the boiler, the cooling need is met by the chiller. In the four-pipe fan coil system, the cooling and heating needs of the office units are met at the same time. While heating is done in one of the office units, cooling can be done in another unit at the same time. In this system, four pipes feed the fan coil indoor units, namely the heating flow and return, and the cooling flow and return pipes. Figure 9 Monthly energy cost graph In the VRV system, there is no annual natural gas fuel consumption since the heating and cooling processes of the spaces are done with VRV devices. For the water-cooled VRV system, monthly cost graphs and monthly energy cost graphs of system components are shown in Figs. 8 and 9, respectively. While HVAC system equipment constitutes 28.8% of the annual electricity consumption of the building, the remaining 71.2% is due to lighting, electrical devices and similar equipment. While 92% of the annual natural gas consumption of the building is due to the need for hot water and water-cooled VRV, 8% is due to the need for the kitchen (cooking). Figure 12 Monthly cost graph of system components For a four pipe fan coil system monthly cost graphs of Figure 12 Monthly cost graph of system components 2.4 Lighting and Device Loads The amount of lighting in the offices, the working time of the devices Tehnički vjesnik 29, 3(2022), 881-887 883 Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Building Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Building y g Figure 10 Monthly cost graph of system components Figure 11 Monthly energy cost graph Figure 10 Monthly cost graph of system components 74.8% is due to lighting, electrical devices, and similar equipment. While the need for hot water constitutes 81% of the annual natural gas consumption of the building, 19% is due to the need for the kitchen (cooking). 3.6 Water-Source Heat Pump System Figure 13 Monthly energy cost graph 3.5 Air-Source Heat Pump System Figure 13 Monthly energy cost graph In the water source heat pump system, the heating and cooling of the spaces are provided by four-pipe fan coil indoor units, while the alternative of meeting the heating and cooling load with a water source heat pump has been considered. The difference from the air source heat pump is that the heat pump uses water instead of air as an external source. In this alternative system, the heat pump does the work of the boiler, which meets the heating needs and the chiller devices that meet the cooling needs in the four-pipe fan coil system. According to the air-cooled heat pump system, additional devices such as the cooling tower, the pump feeding the cooling tower, and the hydrophore make a difference in the cost of this system. 3.5 Air-Source Heat Pump System Figure 16 Monthly cost graph of system components Figure 16 Monthly cost graph of system components Figure 17 Monthly energy cost graph In the air source heat pump system, the heating and cooling of the spaces are provided by four-pipe fan coil indoor units, while the alternative of meeting the heating and cooling load with an air source heat pump has been considered. In this alternative system, the heat pump does the work of the boiler that meets the heating needs and the chiller devices that meet the cooling needs in the four-pipe fan coil system. In the air source four-pipe heat pump system, heating and cooling can be done simultaneously due to the four-pipe fan coil infrastructure. In the four-pipe heat pump system, in addition to the heating and cooling needs of the fancoil indoor units, the heating need of the domestic hot water tank is also provided by the heat pump. Figure 17 Monthly energy cost graph Figure 14 Monthly cost graph of system components Figure 14 Monthly cost graph of system components Figure 15 Monthly energy cost graph For the air source heat pump system, monthly cost graphs and monthly energy cost graphs of system components are shown in Figs. 14 and 15, respectively. While 30% of the annual electricity consumption of the Figure 17 Monthly energy cost graph Figure 17 Monthly energy cost graph For the water source heat pump system, monthly cost graphs and monthly energy cost graphs of system components are shown in Figs. 3.3 Air Handling Unit System In this system, the boiler meets the domestic water heating requirement and the heating load required for the air handling unit heater coil, while the heating, cooling and ventilation needs of the spaces are provided by the air handling unit. Figure 12 Monthly cost graph of system components For a four-pipe fan coil system, monthly cost graphs of system components and monthly energy cost graphs are Technical Gazette 29, 3(2022), 881-887 884 Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Building building consists of HVAC system equipment, the remaining 70% is due to lighting, electrical devices and similar equipment. All of the annual natural gas consumption of the building is due to the need of the kitchen. shown in Figs. 12 and 13, respectively. While 30.4% of the annual electricity consumption of the building consists of HVAC system equipment, the remaining 69.6% is due to lighting, electrical devices and similar equipment. While 92% of the annual natural gas consumption of the building is due to the need for hot water and fan coil heating, 7% is due to the need of the kitchen (cooking). 3.6 Water-Source Heat Pump System 16 and 17, respectively. While HVAC system equipment constitutes 63.5% of the annual electricity consumption of the building, the remaining 36.5% is due to lighting, electrical devices and similar equipment. While 78% of the building's annual natural gas consumption is due to hot water needs, 22% is due to the need of kitchen (cooking). Figure 15 Monthly energy cost graph Energy analysis was performed for each system and then the annual total energy cost value, annual fuel consumption values and annual fuel cost data per unit area were obtained. These data for each system are shown in Tab. 6. Figure 15 Monthly energy cost graph For the air source heat pump system, monthly cost graphs and monthly energy cost graphs of system components are shown in Figs. 14 and 15, respectively. While 30% of the annual electricity consumption of the For the air source heat pump system, monthly cost graphs and monthly energy cost graphs of system components are shown in Figs. 14 and 15, respectively. While 30% of the annual electricity consumption of the In these installations where different components are used for each system, a quantity study was made to calculate investment costs. In the study, armatures, valves, Tehnički vjesnik 29, 3(2022), 881-887 885 Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Building Tab. 6. As a result of the quantity measurements, the initial investment costs of these systems are shown in Tab. 7. pipes, devices, ducts used for installations, in brief, all parts used in the project are counted separately, and pricing was made for these parts. As a consequence of the pricing study, the systems' investment costs data are shown in Tab. 7. y According to the results obtained from the studies, in general, air source systems can be operated with less energy cost than water source systems. High energy costs arise in all-air systems and water source heat pump systems. Although the operating cost of all-air systems is high, they have a very low cost compared to other systems in terms of investment cost. Since the fresh air, heating and cooling needs of the spaces are met by the supply air in the All-air system solution, it requires more shaft and suspended ceiling space than other systems. 3.6 Water-Source Heat Pump System When criteria such as initial investment costs, operating costs, ease of operation and manufacturing speed are taken into account, air source systems are a more advantageous solution. Table 6 Annual energy analysis of alternative systems System Fuel cost per unit area / $/m² Total energy cost / $ Electricity consumption / kWh Natural gas consumption / m3 Air-cooled variable flow cooling system 31.438 55 101 500 919 0 Water-cooled variable flow cooling system 32.992 57 826 521 069 1 847 Air handling unit system 52.694 92 826 839 605 0 Four-pipe fan-coil system 33.778 59 203 533 952 1 704 Air-source heat pump system 33.579 58 853 531 324 1 517 Water-source heat pump system 64.431 112 928 1 022 722 1 558 5 REFERENCES [1] Cen, C., Jia, Y., Liu, K., & Geng, R. (2018). Experimental comparison of thermal comfort during cooling with a fan coil system and radiant floor system at varying space heights. Building and Environment, 141, 71-79. https://doi.org/10.1016/j.buildenv.2018.05.057 [2] Christodoulides, P., Aresti, L., & Florides, G. (2019). Air- conditioning of a typical house in moderate climates with Ground Source Heat Pumps and cost comparison with Air Source Heat Pumps. Applied Thermal Engineering, 158, 113772. https://doi.org/10.1016/j.applthermaleng.2019.113772 Table 7 Investment cost of alternative systems System Cost of material / $ Workmanship / $ Total cost / $ Air-cooled variable flow cooling system 133 253 15 137 148 390 Water-cooled variable flow cooling system 216 679 17 244 233 923 Air handling unit system 93 918 29 917 123 835 Four-pipe fan- coil system 197 259 20 899 218 158 Air-source heat pump system 182 546 21 015 203 561 Water-source heat pump system 244 793 23 150 267 973 [3] Li, Z., Zhang, D., Chen, X., & Li, C. (2020). A comparative study on energy saving and economic efficiency of different cooling terminals based on exergy analysis. Journal of Building Engineering, 30, 101224. https://doi.org/10.1016/j.jobe.2020.101224 [4] Seo, B., Yoon, Y. B., Yu, B. H., Cho, S., & Lee, K. H. (2020). Comparative analysis of cooling energy performance between water-cooled VRF and conventional AHU systems in a commercial building. Applied Thermal Engineering, 170, 114992. https://doi.org/10.1016/j.applthermaleng.2020.114992 [5] Sarbu, I. & Adam, M. (2014). Experimental and numerical investigations of the energy efficiency of conventional air conditioning systems in cooling mode and comfort assurance in office buildings. Energy and Buildings, 85, 45-58. https://doi.org/10.1016/j.enbuild.2014.09.022 [6] Kim, D., Cox, S. J., Cho, H., & Im, P. (2017). Evaluation of energy savings potential of variable refrigerant flow (VRF) from variable air volume (VAV) in the US climate locations. Energy Reports, 3, 85-93. https://doi.org/10.1016/j.egyr.2017.05.002 4 CONCLUSIONS With the developing technology and increasing world population, energy consumption is increasing rapidly. Meeting this need from exhaustible energy sources has led human beings to use energy efficiently and to produce systems and solutions that will contribute to energy savings. Air conditioning constitutes an important part of energy consumption. Today, studies are carried out to minimize energy consumption in buildings. [7] Yu, X., Yan, D., Sun, K., Hong, T., & Zhu, D. (2016). Comparative study of the cooling energy performance of variable refrigerant flow systems and variable air volume systems in office buildings. Applied Energy, 183, 725-736. https://doi.org/10.1016/j.apenergy.2016.09.033 [8] Zhou, Y. P., Wu, J., Wang, R. Z., & Shiochi, S. (2007). Energy simulation in the variable refrigerant flow air- conditioning system under cooling conditions. Energy and Buildings, 39(2), 212-220. https://doi.org/10.1016/j.enbuild.2006.06.005 With the developing technology, the variety of mechanical air conditioning systems that can be applied in buildings is increasing. Considering the advantages and disadvantages of the systems that can be applied, energy consumption can be minimized by choosing the appropriate and correct system for the building. [9] Liu, C., Zhao, T., Zhang, J., Chen, T., Li, X., Xu, M., & Yang, X. (2015). Operational electricity consumption analyze of VRF air conditioning system and centralized air conditioning system based on building energy monitoring and management system. Procedia Engineering, 121, 1856- 1863. https://doi.org/10.1016/j.proeng.2015.09.167 In this study, operating convenience, operating costs and initial investment costs of six different mechanical air conditioning systems were examined. As a result of the energy analysis, annual fuel consumption and annual energy costs for each alternative system are given in [10] Luo, Q., Yang, L., Liu, N., & Xia, J. (2015). Comparative study on thermal environment and energy consumption of Technical Gazette 29, 3(2022), 881-887 886 Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Building Kurtulus YASIN, Deniz YILMAZ: Energy and Cost Evaluation of Different HVAC Systems in an Office Buildin urban residential houses in Beijing. Procedia Engineering, 121, 2141-2148. https://doi.org/10.1016/j.proeng.2015.09.085 urban residential houses in Beijing. Procedia Engineering, 121, 2141-2148. https://doi.org/10.1016/j.proeng.2015.09.085 [11] Liu, X. & Hong, T. (2010). Comparison of energy efficiency between variable refrigerant flow systems and ground source heat pump systems. Energy and Buildings, 42(5), 584-589. https://doi.org/10.1016/j.enbuild.2009.10.028 [11] Liu, X. & Hong, T. (2010). Comparison of energy efficiency between variable refrigerant flow systems and ground source heat pump systems. Energy and Buildings, 42(5), 584-589. https://doi.org/10.1016/j.enbuild.2009.10.028 [12] Safa, A. 4 CONCLUSIONS A., Fung, A. S., & Kumar, R. (2015). Comparative thermal performances of a ground source heat pump and a variable capacity air source heat pump system for sustainable houses. Applied Thermal Engineering, 81, 279-287. https://doi.org/10.1016/j.applthermaleng.2015.02.039 [12] Safa, A. A., Fung, A. S., & Kumar, R. (2015). Comparative thermal performances of a ground source heat pump and a variable capacity air source heat pump system for sustainable houses. Applied Thermal Engineering, 81, 279-287. https://doi.org/10.1016/j.applthermaleng.2015.02.039 [13] Turkish Standard (2013). TS 825 Thermal insulation requirements for buildings. [13] Turkish Standard (2013). TS 825 Thermal insulation requirements for buildings. [14] ASHRAE (2017). ASHRAE. Pocket Guide for Air Conditioning, Heating, Ventilation, Refrigeration, 219-238. [14] ASHRAE (2017). ASHRAE. Pocket Guide for Air Conditioning, Heating, Ventilation, Refrigeration, 219-238. Contact information: Contact information: Kurtulus YASIM Tanriover Engineering Istanbul, Turkey E-mail: kurtulus@tanriover.com Deniz YILMAZ, Associate Professor, PhD (Corresponding author) Istanbul Arel University, Mechanical Engineering Dept. Istanbul, Turkey E-mail: denizyilmaz@arel.edu.tr Tehnički vjesnik 29, 3(2022), 881-887 887
https://openalex.org/W3213324112	https://www.frontiersin.org/articles/10.3389/fpls.2021.762605/pdf	English	null	Targeted and Untargeted Metabolomics Profiling of Wheat Reveals Amino Acids Increase Resistance to Fusarium Head Blight	Frontiers in plant science	2,021	cc-by	8,838	Edited by: Yiming Wang, Nanjing Agricultural University, China Reviewed by: Haifeng Zhang, Nanjing Agricultural University, China Huiquan Liu, Northwest A and F University, China Correspondence: Qunqing Wang wangqunqing@163.com Qian Xu xuqian@sdau.edu.cn †These authors have contributed equally to this work Edited by: Yiming Wang, Nanjing Agricultural University, China Edited by: Yiming Wang, Nanjing Agricultural University, China Edited by: Yiming Wang, Nanjing Agricultural University, China Reviewed by: Haifeng Zhang, Nanjing Agricultural University, China Huiquan Liu, Northwest A and F University, China Correspondence: Qunqing Wang wangqunqing@163.com Qian Xu xuqian@sdau.edu.cn †These authors have contributed equally to this work Reviewed by: Haifeng Zhang, Nanjing Agricultural University, China Huiquan Liu, Northwest A and F University, China Reviewed by: Haifeng Zhang, Nanjing Agricultural University, China Huiquan Liu, Northwest A and F University, China Correspondence: Qunqing Wang wangqunqing@163.com Qian Xu xuqian@sdau.edu.cn Correspondence: Qunqing Wang wangqunqing@163.com Qian Xu xuqian@sdau.edu.cn †These authors have contributed equally to this work Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 22 August 2021 Accepted: 12 October 2021 Published: 19 November 2021 Keywords: Fusarium head blight, metabolomics, wheat, amino acids, disease resistance Received: 22 August 2021 Accepted: 12 October 2021 Published: 19 November 2021 ORIGINAL RESEARCH published: 19 November 2021 doi: 10.3389/fpls.2021.762605 Targeted and Untargeted Metabolomics Proﬁling of Wheat Reveals Amino Acids Increase Resistance to Fusarium Head Blight Peiying Zhao 1†, Shubo Gu 1†, Chao Han 2, Yaru Lu 1, Chunyang Ma 1, Jichun Tian 1, Jianjie Bi 1, Zhiying Deng 1, Qunqing Wang 2,3* and Qian Xu 1,3* 1 College of Agronomy, Shandong Agricultural University, Tai’an, China, 2 Shandong Province Key Laboratory of Agricultural Microbiology, Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai’an, China, 3 State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, China Peiying Zhao 1†, Shubo Gu 1†, Chao Han 2, Yaru Lu 1, Chunyang Ma 1, Jichun Tian 1, Jianjie Bi 1, Zhiying Deng 1, Qunqing Wang 2,3* and Qian Xu 1,3* Fusarium head blight (FHB), a notorious plant disease caused by Fusarium graminearum (F. graminearum), is severely harmful to wheat production, resulting in a decline in grain quality and yield. In order to develop novel control strategies, metabolomics has been increasingly used to characterize more comprehensive proﬁles of the mechanisms of underlying plant-pathogen interactions. In this research, untargeted and targeted metabolomics were used to analyze the metabolite differences between two wheat varieties, the resistant genotype Sumai 3 and the susceptible genotype Shannong 20, after F. graminearum inoculation. The untargeted metabolomics results showed that differential amino acid metabolic pathways existed in Sumai 3 and Shannong 20 after F. graminearum infection. Additionally, some of the amino acid contents changed greatly in different cultivars when infected with F. graminearum. Exogenous application of amino acids and F. graminearum inoculation assay showed that proline (Pro) and alanine (Ala) increased wheat resistance to FHB, while cysteine (Cys) aggravated the susceptibility. This study provides an initial insight into the metabolite differences of two wheat cultivars under the stress of F. graminearum. Moreover, the method of optimization metabolite extraction presents an effective and feasible strategy to explore the understanding of the mechanisms involved in the FHB resistance. Keywords: Fusarium head blight, metabolomics, wheat, amino acids, disease resistance Citation: Fusarium Head Blight (FHB), which causes a critical yield loss and decline in grain quality, is one of the most destructive wheat diseases in the world (Bai et al., 2018). This serious cereal disease is mainly caused by the pathogenic fungus Fusarium graminearum (Ma et al., 2019). Breeding resistant wheat varieties is an eﬀective method to resist FHB (Buerstmayr et al., 2020). Previous studies employed molecular biology techniques such as QTL (Quantitative Trait Locus) to determine the resistance mechanism of FHB (Liu et al., 2006; Cai, 2016). At present, seven QTL genes (Fhb1–Fhb7) have been identiﬁed in wheat (Liu et al., 2006; Cai, 2016; Wang et al., 2020). Zhao P, Gu S, Han C, Lu Y, Ma C, Tian J, Bi J, Deng Z, Wang Q and Xu Q (2021) Targeted and Untargeted Metabolomics Proﬁling of Wheat Reveals Amino Acids Increase Resistance to Fusarium Head Blight. Front. Plant Sci. 12:762605. doi: 10.3389/fpls.2021.762605 November 2021 \| Volume 12 \| Article 762605 Frontiers in Plant Science \| www.frontiersin.org Amino Acid Increase FHB Resistance Zhao et al. Plant Materials and Inoculation Assay The amino acid levels of wheat are signiﬁcantly increased by deoxynivalenol (DON) treatment, most likely due to an active defense response of the plant triggered by DON alone (Warth et al., 2015). This suggests that pathogenic infection has been found to bring many changes in amino acid metabolism expression (Pratelli and Pilot, 2014). Besides, amino acids play pivotal roles in signaling processes (Fagard et al., 2014). The ﬁnal outcome may be strengthening plant defenses to eﬀectively resist pathogenic attack before infection (Tegeder, 2014). However, metabolites may hold further secrets pursuant to wheat FHB resistance which remain to be investigated. Plant Materials and Inoculation Assay Sumai 3 and Shannong 20 were used as wheat cultivars in this study and provided by Professor Jichun Tian, planted in the greenhouse of Shandong Agricultural University (Taian, China). g g g y F. graminearum strain PH-1 was grown on Potato Dextrose Agar (PDA) medium at 25◦C for 3 days. A total of 14 fungal plugs were transferred into a 400 mL conical ﬂask with 200 mL carboxymethyl cellulose (CMC) liquid medium, and cultured with shaking at 220 rpm at 25◦C for 3 days (Hou et al., 2002). Then conidia were harvested and resuspended with sterile water to the concentration of 1–5×105/mL. Each spike was inoculated with 2 ﬂorets (each ﬂoret was inoculated with 10 µL conidia suspension), as per the protocol described in Feng et al. (2018). In total, 10 biological replicates were performed for each wheat variety. Resistant genotype (Sumai 3) and susceptible genotype (Shannong 20) spike samples were collected at 0, 3, 6, and 9 days post-inoculation (dpi), labeled as R-0, R-3, R-6, and R- 9, and S-0, S-3, S-6, and S-9; each treatment was repeated six times. The spikes were processed with liquid nitrogen freezing and then transferred to a −80◦C refrigerator before detection of the metabolome. The metabolome is the last frontier mediating the communication between the genome and the phenotype of an organism, and now it becomes a potential tool for elucidating host biochemical responses to biotic stress, identifying candidate disease resistance genes, and validating gene function (Kumar et al., 2017; Chen et al., 2019). Most metabolomics studies are based on gas chromatography (GC) or liquid chromatography (LC) coupled with Mass Spectrometry (GC-MS or LC-MS) (Patti et al., 2012; Cajka et al., 2017). The LC-MS system is used to identify the metabolic diﬀerences of barley genotypes resistant to FHB, indicating that phenylpropanes, terpenoids, ﬂavonoids, and fatty acids are the main resistance compounds to barley FHB (Kumaraswamy et al., 2011a,b). Furthermore, the metabolomics approach used to identify hydroxycinnamic acid amides (HCAAs), such as coumaroylagmatine and coumaroylputrescine, were the highest fold change RR metabolites in the rachis of a wheat resistant near-isogenic line (NIL-R) under F. graminearum infection (Kage et al., 2017). In addition to the secondary metabolites, the direct involvement of plants’ primary metabolites such as amino acids not be denied in plant-pathogen interaction (Hildebrandt et al., 2015). MATERIALS AND METHODS Fhb1 was the ﬁrst obtained gene from Sumai 3, which is a strong and stable wheat variety resistant to F. graminearum, and it has been applied to the breeding of wheat varieties resistant to FHB (Liu et al., 2006; Buerstmayr et al., 2009; Rawat et al., 2016). Besides this, the Fhb7 gene has been studied and cloned, which provides a bright prospect for the development of wheat head blight resistance (He et al., 2018; Wang et al., 2020). In the interaction between wheat and F. graminearum (Dweba et al., 2017), plants use complex chemical defense mechanisms involved in metabolic adaptations (Feussner and Polle, 2015; Arbona and Gomez-Cadenas, 2016). Advances in analytical chemistry and high-throughput sequencing were used to acquire extensive metabolic proﬁles during plant-pathogen (Beccari et al., 2019; Chen et al., 2019). A more comprehensive understanding of the mechanisms of underlying plant-pathogen interactions is needed, in order to develop novel, better and safer control strategies. Fhb1 was the ﬁrst obtained gene from Sumai 3, which is a strong and stable wheat variety resistant to F. graminearum, and it has been applied to the breeding of wheat varieties resistant to FHB (Liu et al., 2006; Buerstmayr et al., 2009; Rawat et al., 2016). Besides this, the Fhb7 gene has been studied and cloned, which provides a bright prospect for the development of wheat head blight resistance (He et al., 2018; Wang et al., 2020). In the interaction between wheat and F. graminearum (Dweba et al., 2017), plants use complex chemical defense mechanisms involved in metabolic adaptations (Feussner and Polle, 2015; Arbona and Gomez-Cadenas, 2016). Advances in analytical chemistry and high-throughput sequencing were used to acquire extensive metabolic proﬁles during plant-pathogen (Beccari et al., 2019; Chen et al., 2019). A more comprehensive understanding of the mechanisms of underlying plant-pathogen interactions is needed, in order to develop novel, better and safer control strategies. Frontiers in Plant Science \| www.frontiersin.org Metabolite Extraction All chemicals and solvents were analytical or HPLC grade. Water, methanol, acetonitrile, formic acid, and acetic acid were purchased from CNW Technologies GmbH (Düsseldorf, Germany). The method of metabolite extraction was optimized by referring to previous methods (Guo et al., 2015) for three factors, including extraction solvent, extraction method, and extraction time, with three levels for each factor (Table 1). Due to the multi- factor and multi-level experiment, this experiment adopted L9(34) orthogonal experiments and univariate analysis to assess the optimization of metabolites extraction methods and make variance analysis on the orthogonal test results (Martin et al., 2014). The treatment combination was designed by referring to the L9(34) orthogonal test table (Supplementary Table 1). To obtain suﬃcient compounds, we used a new generation Orbitrap instrument (Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer) with high-performance quadrupole precursor selection and high-resolution accurate-mass Orbitrap detection, combined with UPLC instruments, providing support for obtaining more compound data extraction of metabolites. The metabolite peaks of each combination method were extracted by UPLC-QE-MS, and variance analysis was conducted on the data to screen out the optimal level combination. Each of the frozen wheat spike samples was milled to a ﬁne powder for 1 min using a ball mill (GT200 Grinder, China) with liquid nitrogen pre-cooled 10 mL stainless steel vessels and a 9-mm stainless steel ball. The wheat samples (100 ± 2 mg frozen weight) were extracted with 1 mL extraction solution methanol: acetonitrile: water (2:2:1, v/v/v) in 2 mL Eppendorf tubes. We extracted all homogenates via ultrasonic extraction for 10 min and centrifugation at 13,000 rpm for 5 min. We then transferred 700 µL of each of the supernatants into the Shannong 20 is a new wheat variety cultivated in China with resistance to multiple threats and a high yield, but it shows high susceptibility to FHB (Li et al., 2014). Thus, this study used a new optimized LC-MS method, investigating the metabolites of Shannong 20 and Sumai 3, two wheat genotypes with diﬀerent FHB resistance levels, to clarify the resistance mechanism of wheat to FHB increase. November 2021 \| Volume 12 \| Article 762605 Frontiers in Plant Science \| www.frontiersin.org 2 Amino Acid Increase FHB Resistance Zhao et al. TABLE 1 \| Factors for optimizing extraction method of sample pretreatment. Level Factor Extraction solvent Extraction method Extraction time (min) 1 80%Acetonitrile (0.1%FA) Shock 10 2 80%Acetonitrile (0.1%HAc) Ultrasound 15 3 Methanol:Acetonitrile:H2O(2:2:1) Stand still 30 SIMCA (v. Preprocessing p g A UPLC system (Thermo Fisher Scientiﬁc, Bremen, Germany) was used for the separation of metabolites. To monitor the stability and repeatability of the instrument analysis, the QCs were injected at regular intervals (every 10 samples) throughout the analytical queue (Zhang et al., 2019). The column temperature was 35◦C, and the ﬂow rate was 0.3 mL/min. The eluents were eluent A (0.04% acetic acid in water) and B (0.04% acetic acid in acetonitrile). The solvent gradient was set as follows: 90% A, 0.2 min; 10–0% A, 6 min; 10% A, 8.0 min; 10–90% A, 8.1 min; 90% A, 10 min (Fiehn et al., 2007). The wheat samples (100 ± 2 mg frozen weight), like the untargeted metabolomics samples, were extracted with 1.5 mL sterile water. We extracted all homogenates via ultrasonic extraction at room temperature for 10 min and then centrifuged for 5 min at 13,000 rpm. The supernatant was transferred to a new centrifugal tube, and the amino acids could be detected after ﬁltration by 0.22 µm membrane. A UPLC system (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA) was used for the separation of metabolites. The column temperature was 35◦C, and the ﬂow rate was 0.3 mL/min. The eluents were eluent A (0.1% acetic acid in water) and B (acetonitrile). The solvent gradient was set as follows: 90% A, 0.2 min; 10–90% A, 6 min; 10% A, 8.0 min; 10–90% A, 8.1 min; 90% A, 10 min. Detection of compounds was performed using TSQ Quantive mass spectrometer (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA) operating in the multiple reaction monitoring (MRM) mode, the Quantitive mass spectrometer was operated in positive polarity mode with a spray voltage of 3.8 kV, capillary temperature of 350◦C, sheath gas ﬂow rate of 40 arb and aux gas ﬂow rate of 10 arb, microsweep 1, and AGC target 2e5. Detection of compounds was performed using Q-Exactive quadrupole-Orbitrap mass spectrometer equipped with heated electrospray ionization (ESI) source (Thermo Fisher Scientiﬁc, Bremen, Germany) operating in data-dependent acquisition (DDA) mode. In positive polarity mode, spray voltage was 3.8 kV, capillary temperature 350◦C, sheath gas ﬂow rate 35 arb and aux gas ﬂow rate 10 arb, resolution 17,500, microsweep 1, AGC target 2e5, normalized collision energy was 30. Metabolite Extraction 13.0, 2011, Umetrics, Umea, Sweden) were used for the large-scale identiﬁcation of metabolites and multivariate data analysis from LC-MS/MS data sets. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used to analyze the credibility of the mass spectrum data. The diﬀerences of metabolites were analyzed according to the VIP value of PLS-DA model (VIP > 1), and the P-value of Student’s t-test (P-value < 0.05) of the peak area of metabolites among diﬀerent wheat varieties, and the ploidy changes of the peak area. Preprocessing In negative polarity mode, the voltage was 2.9 kV, capillary temperature 350◦C, sheath gas ﬂow rate 40 arb, aux gas ﬂow rate 10 arb, resolution 17,500, microsweep 1, AGC target 2e5, and the normalized collision energy was 30. Data obtained on the UPLC-QE-MS should be converted into mzML format by ProteoWizard software (Holman et al., 2014). The transformed data were processed by XCMS (https:// xcmsonline.scripps.edu/) (Smith et al., 2006), such as peak extraction, peak alignment, normalization, etc., to obtain the two- dimensional data matrix of mass/charge ratio (m/z), retention time, similarity, and peak area, and to build a multidimensional data set. The missing data were supplemented by the minimum value complement method, and the data results were de-noised by the quad method to ﬁnally determine the positive and negative ion phase variables that met the requirements. Normalization of the peak area was carried out on the data for subsequent analysis. Frontiers in Plant Science \| www.frontiersin.org Absolute Quantitative Analysis of Amino Acids new centrifuge tube and evaporated all ﬂuid with a quick spin concentrator. Dried metabolite pellets were resuspended by 100 µL methanol and ﬁltered through a 0.22 µm ﬁlter membrane, and then could be used in UPLC-QE-MS detection. Quality control (QC) samples were prepared by mixing aliquots of all samples to be a pooled sample. The absolute quantiﬁcation of amino acids (histidine, serine, threonine, arginine, alanine, cysteine, methionine, and proline) was performed by UPLC-QqQ-MS targeted metabolomics. Absolute quantiﬁcation of amino acids was used by the external standard method, the regression equation developed by plotting diﬀerent concentrations of pure amino acids (Sigma-Aldrich R⃝ Inc., MO, USA) (Matuszewski et al., 2003). Student’s t-test was used to analyze the signiﬁcant diﬀerence in amino acid content between the two varieties. Optimization of Metabolites Extraction Methods for Metabolomic Samples As shown in Table 1, the test factors and levels were extraction solvents (A), extraction methods (B), and extraction time (C). Variance analysis on the orthogonal table shows the R-value in level extraction solvent was signiﬁcantly higher in level extraction method and in level extraction time (Supplementary Table 1), suggesting that the factors aﬀecting the number of extraction peaks of plant metabolites are extraction solvents > extraction time > extraction methods. In combination with the results of univariate analysis (Figure 1), the best extraction method of wheat metabolites is conﬁrmed by methanol: acetonitrile: water (2:2:1, v/v/v) and ultrasound for 10 min. Exogenous Application Amino Acid and Inoculation F. graminearum Assay Inoculation and resistance assessment of in wheat leaves in vitro, referring to the method described previously (Chen et al., 2010; Yuan et al., 2011; Kadotani et al., 2016). The experiments were performed in wheat (Shannong 20) at the 3-leaf stage, plants were cultivated in growth chambers under a 16 h light, 28◦C/8 h dark, 25◦C regime. Wheat leaves were cut to 4 cm in length, and a wound gently poked in the middle position of the paraxial surface of the leaves. Two ends of isolated leaves were dipped into one of the 10 mM amino acids (Gly, Met, Arg, Pro, Ala, and Cys) for 24 h. Water was used in mock treatments as a control. Then, we used F. graminearum inoculation on wheat leaves (3 µL), sealed The software MetDNA (Metabolite identiﬁcation and Dysregulated Network Analysis) (http://metdna.zhulab.cn/) and November 2021 \| Volume 12 \| Article 762605 3 Amino Acid Increase FHB Resistance Zhao et al. FIGURE 1 \| Effect of single factor extraction solvent (A), extraction method (B) and extraction time (C) on metabolite extraction. a = 80% Acetonitrile (0.1% Formic acid), b = 80% Acetonitrile (0.1% Acetic acid), c = Methanol: Acetonitrile: H2O (2:2:1, v/v/v). and cultured at 24◦C for 3 days. After 3 days, we observed and recorded the area of disease spots on the leaves, and the statistical data were collected to signiﬁcance analysis by Student’s t-test data we e co ected to s g ca ce a a ys s by Stude ts t test In order to further verify the inﬂuence of exogenous amino acids on FHB resistance, and F. graminearum inoculation of wheat spikes was used for the identiﬁcation. In the wheat ﬂowering stage, spraying 10 mM amino acid on wheat ﬂag leaves, after 24 h, we inoculated 10 µL F. graminearum in the left and right base ﬂorets of the third spikelet. The leaves were then bagged to retain moisture, and the bags discarded after 3 days. After 7 and 14 days, we observed and recorded the number of extended spikelets of FHB, and the statistical data were collected for signiﬁcance analysis by Student’s t-test (Kong et al., 2007). Then, pictures were taken and relative virulence was measured by qRT-PCR. The ratios of F. graminearum DNA (GAPDH, Forward primer: 5′-AAGTTC TACTCTGAGCGTGACCC-3′, reverse primer: 5′-TTGGAGGA AGGACCATCGAC-3′) to wheat DNA (β-actin, Forward primer: 5′-GCAAAGAGATCACGGCCCTT-3′, reverse primer: 5′-GCA ACTTCCACACTTGAGAGG-3′) were quantiﬁed in the infected plants’ tissues. Frontiers in Plant Science \| www.frontiersin.org Exogenous Application Amino Acid and Inoculation F. graminearum Assay All these assays were repeated independently at least three times. RESULTS Optimization of Metabolites Extraction Methods for Metabolomic Samples FHB Resistance Assessment in Sumai 3 and Shannong 20 FIGURE 1 \| Effect of single factor extraction solvent (A), extraction method (B) and extraction time (C) on metabolite extraction. a = 80% Acetonitrile (0.1% Formic acid), b = 80% Acetonitrile (0.1% Acetic acid), c = Methanol: Acetonitrile: H2O (2:2:1, v/v/v). FHB resistance in wheat cultivars Sumai 3 and Shannong 20 was assessed by quantifying the spread of inoculated spikelets to un- inoculated spikelets (Figure 2A). At 9 dpi, the initial symptoms were observed in both Sumai 3 and Shannong 20 inoculated spikelets. Before 9 dpi, there were no signiﬁcant FHB symptoms diﬀerences of Sumai 3 and Shannong 20. Therefore, to explore the potential disease resistance mechanism before FHB symptoms expression, the time before 9 dpi was selected as the emphasis in this study. Wheat Metabolite Proﬁle Under FHB Stress Using UPLC-QE-MS untargeted metabolomics, 5,966, 5,979, 5,987, and 5,997 metabolites were detected in Sumai 3 at 0, 3, 6, and 9 dpi, 5,973, 5,983, 6,014, and 6,033 metabolites were detected in Shannong 20 at 0, 3, 6, and 9 dpi. The disease progress varied drastically between cultivars (Figure 2B). In all 8 bleached spikelets were observed at 14 dpi in Shannong 20 which increased to the entire spike at 21 dpi. Bleached spikelets were observed in Sumai 3 at 14 dpi, and did not spread to other spikelets until 21 dpi. g The stability and repeatability of the UPLC-QE-MS was exhibited in Supplementary Figure 1. Good separation of the wheat metabolites among the R-0 R-3 R-6 R-9, S-0 S-3 S-6 S-9, and QC which were achieved in principle components November 2021 \| Volume 12 \| Article 762605 4 Amino Acid Increase FHB Resistance Zhao et al. FIGURE 2 \| (A) Fusarium head blight (FHB) symptoms at 9, 14, and 21 days post-inoculation (dpi) in wheat cultivars, Sumai 3 and Shannong 20, inoculated with F. graminearum. Shannong 20 is sensitive to FHB and high level of bleaching. (B) Number of diseased spikelets in Sumai 3 and Shannong 20. At 14 and 21 dpi, the number of diseased spikelets in Shannong 20 was signiﬁcantly higher than that in Sumai 3 (P value<0.01). Student t-test P-value: P value<0.01. FIGURE 2 \| (A) Fusarium head blight (FHB) symptoms at 9, 14, and 21 days post-inoculation (dpi) in wheat cultivars, Sumai 3 and Shannong 20, inoculated with F. graminearum. Shannong 20 is sensitive to FHB and high level of bleaching. FHB Resistance Assessment in Sumai 3 and Shannong 20 graminearum infection period were slightly diﬀerent, the common DFMs are shown in Table 2, including L-Serine, betaine, O-Phospho-L-serine, L-Glutamate, L-Histidine, and carnosine. Identiﬁcation results of DFMs between Sumai3 and Shannong20 in 0, 3, 6, and 9 dpi are shown in Supplementary Table 2. biosynthesis, beta-Alanine metabolism, cysteine and methionine metabolism, and arginine and proline metabolism pathways. According to the screening of untargeted metabolites and the analysis of metabolic pathways, it was determined that the metabolites with signiﬁcant diﬀerences were enriched in the amino acid pathway. Detailed data on the results of the other metabolic pathway analysis are provided in Supplementary Table 3. FHB Resistance Assessment in Sumai 3 and Shannong 20 (B) Number of diseased spikelets in Sumai 3 and Shannong 20. At 14 and 21 dpi, the number of diseased spikelets in Shannong 20 was signiﬁcantly higher than that in Sumai 3 (P value<0.01). Student t-test P-value: P value<0.01. analysis (PCA) score plots (Figure 3A). This result indicated that FHB stress and wheat genotypes signiﬁcantly inﬂuenced metabolite variation. As shown in Figure 3B, partial least square discriminant analysis (PLS-DA) was conducted to compare the diﬀerences between groups R-0 vs. S-0, R-3 vs. S-3, R-6 vs. S- 9, and R-9 vs. S-9. The scores plot between PC1 and PC2 in PCA revealed two distinct groups associated with the Sumai 3 and Shannong 20 samples after F. graminearum infection, suggesting the distinction in the metabolite accumulation between two genotypic varieties of wheat. PLS-DA (Figure 3B) and volcano plots (Figure 3C) were used of identifying the important metabolites associated with FHB resistance based on the Variable Importance in the Projection (VIP) score (VIP > 1.0), fold change (FC > 2.0 or FC < 0.5) and P value < 0.05. For Shannong 20 and Sumai 3, the diﬀerential metabolites (DFMs) in F. graminearum infection period were slightly diﬀerent, the common DFMs are shown in Table 2, including L-Serine, betaine, O-Phospho-L-serine, L-Glutamate, L-Histidine, and carnosine. Identiﬁcation results of DFMs between Sumai3 and Shannong20 in 0, 3, 6, and 9 dpi are shown in Supplementary Table 2. analysis (PCA) score plots (Figure 3A). This result indicated that FHB stress and wheat genotypes signiﬁcantly inﬂuenced metabolite variation. As shown in Figure 3B, partial least square discriminant analysis (PLS-DA) was conducted to compare the diﬀerences between groups R-0 vs. S-0, R-3 vs. S-3, R-6 vs. S- 9, and R-9 vs. S-9. The scores plot between PC1 and PC2 in PCA revealed two distinct groups associated with the Sumai 3 and Shannong 20 samples after F. graminearum infection, suggesting the distinction in the metabolite accumulation between two genotypic varieties of wheat. PLS-DA (Figure 3B) and volcano plots (Figure 3C) were used of identifying the important metabolites associated with FHB resistance based on the Variable Importance in the Projection (VIP) score (VIP > 1.0), fold change (FC > 2.0 or FC < 0.5) and P value < 0.05. For Shannong 20 and Sumai 3, the diﬀerential metabolites (DFMs) in F. Frontiers in Plant Science \| www.frontiersin.org Absolute Quantitative Analysis of Amino Acids To validate the content of amino acids, targeted metabolomics analysis was performed on Sumai 3 and Shannong 20 samples. Figure 5 exhibits that the amino acid proﬁles changed a lot between Sumai 3 and Shannong 20 infected with FHB. A total of 9 amino acids accumulated in Sumai 3 and Shannong 20 were subjected to Student’s t-test. Ser, His, and Thr showed no obvious rule in Sumai 3 or Shannong 20 (Figure 5A). Gly, Met, Arg, Pro, Ala, and Cys showed an increasing trend with the extension of inoculation time in Shannong 20 and Sumai 3 (Figures 5B,C). The contents of Arg, Gly, and Cys were higher (P value < 0.01) in Shannong 20 than in Sumai 3 at 3, 6, and 9 dpi (Figure 5B). The contents of Pro, Ala, and Met were higher (P value < 0.05) in Sumai 3 than in Shannong 20 at 6 and 9 dpi (Figure 5C). The results showed that with the days after inoculation of FHB, the The KEGG pathway enrichment analysis from diﬀerential metabolites revealed the top 10 pathways that were aﬀected by each of the two diﬀerent wheat varieties (Figure 4). Among them, most metabolic pathways belong to amino acid metabolism, including histidine metabolism, arginine November 2021 \| Volume 12 \| Article 762605 Frontiers in Plant Science \| www.frontiersin.org 5 Amino Acid Increase FHB Resistance Zhao et al. FIGURE 3 \| F. graminearum inoculation induced a signiﬁcant metabolic response in spikelets of wheat. (A) Principal component analysis (PCA) of metabolic proﬁles in all samples (six biological replicates). The partial least-squares discriminant analysis (PLS-DA) score plots (B) and a volcano plot (C) of the comparison between R-0 and S-0, R-3 and S-3, R-6 and S-6, R-9 and S-9, respectively. QC, Quality Control; R-0/3/6/9, resistant genotype (Sumai 3) at 0/3/6/9 dpi; S-0/3/6/9, susceptible genotype (Shannong 20) at 0/3/6/9 dpi. FIGURE 3 \| F. graminearum inoculation induced a signiﬁcant metabolic response in spikelets of wheat. (A) Principal component analysis (PCA) of metabolic proﬁles in all samples (six biological replicates). The partial least-squares discriminant analysis (PLS-DA) score plots (B) and a volcano plot (C) of the comparison between R-0 and S-0, R-3 and S-3, R-6 and S-6, R-9 and S-9, respectively. QC, Quality Control; R-0/3/6/9, resistant genotype (Sumai 3) at 0/3/6/9 dpi; S-0/3/6/9, susceptible genotype (Shannong 20) at 0/3/6/9 dpi. Exogenous Application Amino Acid Inﬂuence Resistance in Wheat TABLE 2 \| Potential biomarkers of physiological metabolism. TABLE 2 \| Potential biomarkers of physiological metabolism. TABLE 2 \| Potential biomarkers of physiological metabolism. TABLE 2 \| Potential biomarkers of physiological metabolism. Potential biomarkers RT (min) Formula Mass Da Adduct ions KEGG ID L-Serine 1.32 C3H7NO3 [M+H]+ C00065 Betaine 1.34 C5H11NO2 [M+H]+ C00719 O-Phospho-L-serine 6.32 C3H8NO6P [M+H]+ C01005 L-Glutamate 5.22 C5H9NO4 [M+H]+ C00025 L-Histidine 1.02 C6H9N3O2 [M+H]+ C00135 Carnosine 1.41 C9H14N4O3 [M+H]+ C00386 levels of amino acids in the two varieties showed diﬀerent types of changes, and the changes of amino acids might be related to fusarium head blight. To test the eﬀect of diﬀerent amino acids (Pro, Ala, Gly, Met, Arg, and Cys) on disease resistance, Shannong 20 detached leaves were immersed into 10 mM amino acid solutions for 24 h, and F. graminearum was inoculated into to the detached leaves. Then, the symptoms were observed after 3 days. As shown in Figures 6A,B, Pro and Ala treatment reduced necrotic patches on leaves (P value < 0.05), while Gly, Arg, Cys and Met increased the wheat sensitivity of F. graminearum (P value < 0.01). The resistance of diﬀerent amino acids to F. graminearum was also shown in wheat spikelets (Figure 6C). The results showed that Pro and Ala treatments could signiﬁcantly reduce the number of diseased spikelets (P value < 0.01), while Cys levels of amino acids in the two varieties showed diﬀerent types of changes, and the changes of amino acids might be related to fusarium head blight. levels of amino acids in the two varieties showed diﬀerent types of changes, and the changes of amino acids might be related to fusarium head blight. November 2021 \| Volume 12 \| Article 762605 Frontiers in Plant Science \| www.frontiersin.org 6 Amino Acid Increase FHB Resistance Zhao et al. FIGURE 4 \| Top 10 enriched metabolic pathways of the comparison between Sumai 3 and Shannong 20 after F. graminearum infection. The y-axis and x-axis respectively represent the signiﬁcance and importance value of the metabolic pathway, bubble size represents the differential metabolites (DFMs) proportion in the metabolic pathway. Detailed information on metabolic pathways in Supplementary Table 3. inoculation. The result showed that the diﬀerence of metabolic pathways was mainly reﬂected in amino acid metabolic pathways, and selected several amino acids related to FHB resistance (Figures 4, 5). In order to conﬁrm the role of amino acids, exogenous amino acids were applied to the leave and spike of wheat, only Pro and Ala could inhibit the occurrence of F. TABLE 2 \| Potential biomarkers of physiological metabolism. graminearum to a certain extent and signiﬁcantly improve the resistance of F. graminearum in susceptible cultivars, suggesting that Pro and Ala signaling pathways play an important role in wheat resistance to FHB (Figure 6). Amino acid metabolism and biosynthesis pathway take part in resistance to abiotic or biological stress, regulation of plant growth and development, etc. Recently, the role of amino acids in plant disease resistance has been increasingly researched (Kadotani et al., 2016). Amino acids in plants are involved in primary and secondary metabolism and participate in a wide range of cellular enzymatic reactions as constituents of diﬀerent enzymes such as aminotransferases, dehydrogenases, lyases, and decarboxylases (Teixeira et al., 2017). Pro represents a compatible osmolyte, signiﬁcantly increasing during the stress response in several plants to protect subcellular structures and macro-molecules, which acts as a compatible osmolyte during stress, and was adjusted by balancing its synthesis and catabolism both of which were induced both during and after stress treatments (Parre et al., 2007; Häusler et al., 2014; Batista-Silva et al., 2019). In Arabidopsis thaliana, amino acids contents were signiﬁcantly increased after pathogen infection. Overexpressing some genes (Pro deaminase, PRODH, key enzymes that catalyze proline metabolism) in A. thaliana caused an obvious increase in the resistance to pathogen infection (Ward et al., 2010; Cecchini et al., 2011). In addition, Pro provides energy for plant growth, which has distinct protective functions in mitochondria (Welchen et al., 2021). Low doses of exogenous proline could protect plants from salinity, drought, heavy metal, and temperature stress (Teixeira et al., 2017). Similarly, exogenous application Pro to spike and in vitro leaves showed resistance to FHB (Figure 6). Thus, the current study indicated that suitable Pro concentration might contribute to the FHB resistance of wheat. FIGURE 4 \| Top 10 enriched metabolic pathways of the comparison between Sumai 3 and Shannong 20 after F. graminearum infection. The y-axis and x-axis respectively represent the signiﬁcance and importance value of the metabolic pathway, bubble size represents the differential metabolites (DFMs) proportion in the metabolic pathway. Detailed information on metabolic pathways in Supplementary Table 3. treatments could aggravate the course of FHB (P value < 0.05) at 14 dpi (Figure 6D). The biomass quantiﬁcation showed the relative amount of F. graminearum DNA in spikes with Ala and Pro was signiﬁcantly reduced compared to CK, while that with Cys was increased (Figure 6E). TABLE 2 \| Potential biomarkers of physiological metabolism. These results also indicated that Pro and Ala played an important role in wheat FHB resistance. Frontiers in Plant Science \| www.frontiersin.org DISCUSSION One of the most important fungal diseases of wheat and other cereals in the world is Fusarium Head Blight caused mainly by the fungal pathogen Fusarium graminearum. It is a worldwide problem to select wheat varieties resistant to FHB because the currently deployed resistance genes often do not confer adequate protection against the accumulation of damaging mycotoxins in wheat grain. Today, there are major challenges to breeding more disease-resistant wheat cultivars. Ala has previously been identiﬁed as a precursor in mycotoxin formation by Fusarium spp., which was considered as associating with increased susceptibility to disease (Browne, 2007). Nevertheless, in this study, the Ala content in resistant variety Sumai 3 was signiﬁcantly higher than that in susceptible variety Shannong 20 (Figure 5), and the application of Ala can signiﬁcantly improve the resistance of wheat to FHB (Figure 6). Ala is synthesized from Glutamic acid, and its transamination with oxoglutarate produces glutamate and pyruvate, a reversible reaction, granting this amino acid a dual function between carbon and nitrogen metabolism (Kendziorek et al., 2012). Ala plays an important role in storing and transferring amino groups under abiotic stress (Ullah and Lim, 2017). Moreover, Ala serves as a precursor of glucosinolates, and glucosinolates play a role in disease resistance (Halkier and Gershenzon, 2006). Therefore, Ala accumulation might be associated with Plant metabolites continuously responding to biotic and abiotic stress. It is crucial to characterize the metabolome of plants to understand how crops function and respond to stress conditions, produce quality products, learn how to engineer metabolic pathways, and how best to assess food safety. Therefore, in order to increase the understanding of wheat FHB resistance mechanisms, and ﬁnd metabolites related to FHB resistance, this study used metabolomics analysis to dynamic changes of metabolites from two wheat varieties of FHB November 2021 \| Volume 12 \| Article 762605 7 Zhao et al. Amino Acid Increase FHB Resistance FIGURE 5 \| Timeline of amino acids abundances of Sumai 3 and Shannong 20 inoculated F. graminearum. Ser, His, and Thr (A) showed no obvious rule. Arg, Cys, and Gly (B) and Pro, Met, and Ala (C) exhibited increased levels in Sumai 3 and Shannong 20 after F. graminearum treatment. The contents of Arg, Gly, and Cys were higher (P value<0.01) in Shannong 20 than in Sumai 3 at 3, 6, and 9 dpi. Frontiers in Plant Science \| www.frontiersin.org DISCUSSION The contents of Pro, Ala, and Met were higher (P value<0.05) in Sumai 3 than in Shannong 20 at 6 and 9 dpi. Student’s t-test P-value: *P value<0.001, P value<0.01, P value<0.05. FIGURE 5 \| Timeline of amino acids abundances of Sumai 3 and Shannong 20 inoculated F. graminearum. Ser, His, and Thr (A) showed no obvious rule. Arg, Cys, and Gly (B) and Pro, Met, and Ala (C) exhibited increased levels in Sumai 3 and Shannong 20 after F. graminearum treatment. The contents of Arg, Gly, and Cys were higher (P value<0.01) in Shannong 20 than in Sumai 3 at 3, 6, and 9 dpi. The contents of Pro, Ala, and Met were higher (P value<0.05) in Sumai 3 than in Shannong 20 at 6 and 9 dpi. Student’s t-test P-value: P value<0.001, P value<0.01, P value<0.05. synthesis might improve the susceptibility of FHB, which is the idea of cultivating new varieties. precursors storage for protein synthesis to prepare for rapid recovery of plant metabolism following stress (Nostadt et al., 2020). In summary, we selected a few important amino acids for the improvement of wheat resistance to FHB. Pro and Ala can improve wheat resistance to FHB following further validation. This may serve as an osmotic regulator or secondary metabolite precursor metabolite accumulated in plants to reduce damage to biological stress. In addition, since amino acids are metabolites produced by plants, exogenous application does not pollute the environment. It provides a method for preventing FHB safely in wheat production. Cysteine signiﬁcantly increases susceptibility to FHB, it can be utilized through repressing candidate amino acids’ pathway genes by molecular biological methods to enhance resistance FHB. The content of cysteine in the susceptible cultivar Shannong 20 was higher than that in resistant cultivar Sumai 3, and increased with the increase of the infection time (Figure 5). Subsequently, exogenous application of cysteine wheat to demonstrated more susceptibility to FHB (Figure 6). A consistent result was found in previous studies, Cys is reactive and toxic when its accumulation exceeds a certain level (Hildebrandt et al., 2015). DON treatment triggered a strong upregulation of cysteine biosynthesis suggesting that cysteine is used for glutathione formation and conjugation to DON (Gardiner et al., 2010). Therefore, silencing genes related to Cys November 2021 \| Volume 12 \| Article 762605 8 Amino Acid Increase FHB Resistance Zhao et al. DISCUSSION FIGURE 6 \| Exogenous amino acid application inﬂuences FHB resistance in susceptible genotype wheat. (A) The FHB symptoms of detached leaves dipped with an amino acid solution (10mM). Water was used in mock treatments as a control. Pro and Ala treatment can reduce the disease spots on leaves. Cys treatment lead to leaves becoming more seriously diseased. (B) The area of disease spot in Shannong 20 detached leaves. Pro and Ala treatment disease spot areas were smaller than in water treatment (P value<0.05), Gly, Arg, Cys, and Met treatment increased disease spot on leaves compared to water treatment (P value<0.01). (C) The FHB symptoms of spikelets sprayed with amino acid solution (10 mM). At 14 dpi, Pro and Ala treatment can signiﬁcantly reduce the number of diseased spikelets, and lighten the phenomenon of spikelets withered in the upper part of the wheat. (D) The number of spikelets diseased. Pro and Ala treatment signiﬁcantly decreased the treatment at 14 dpi (P value<0.05), (E) The biomass quantiﬁcation of F. graminearum in infected spikelets. Ala and Pro were signiﬁcantly reduced compared to CK (P value<0.05), Cys was increased the F. graminearum relative amount (P value<0.05). Student t-test P value: P value<0.01, P value<0.05. FIGURE 6 \| Exogenous amino acid application inﬂuences FHB resistance in susceptible genotype wheat. (A) The FHB symptoms of detached leaves dipped with an amino acid solution (10mM). Water was used in mock treatments as a control. Pro and Ala treatment can reduce the disease spots on leaves. Cys treatment lead to leaves becoming more seriously diseased. (B) The area of disease spot in Shannong 20 detached leaves. Pro and Ala treatment disease spot areas were smaller than in water treatment (P value<0.05), Gly, Arg, Cys, and Met treatment increased disease spot on leaves compared to water treatment (P value<0.01). (C) The FHB symptoms of spikelets sprayed with amino acid solution (10 mM). At 14 dpi, Pro and Ala treatment can signiﬁcantly reduce the number of diseased spikelets, and lighten the phenomenon of spikelets withered in the upper part of the wheat. (D) The number of spikelets diseased. Pro and Ala treatment signiﬁcantly decreased the treatment at 14 dpi (P value<0.05), (E) The biomass quantiﬁcation of F. graminearum in infected spikelets. Ala and Pro were signiﬁcantly reduced compared to CK (P value<0.05), Cys was increased the F. graminearum relative amount (P value<0.05). Student t-test P value: *P value<0.01, P value<0.05. DATA AVAILABILITY STATEMENT This paper optimized the extraction method of untargeted metabolomics and described the altered metabolic signature of two wheat genotypes (Sumai 3 and Shannong 20) after F. graminearum inoculation. The newly established LC–MS-based untargeted and targeted metabolomics allows the investigation of the wheat metabolism and will be an eﬀective tool for other plant studies. Our study indicated that the main altered metabolic pathways in two wheat genotypes after F. graminearum inoculation were amino acid metabolisms. Signiﬁcantly increased amino acids levels were detected and might be related to wheat FHB resistance. In the subsequent experiment in wheat detached leaves by F. graminearum inoculation, exogenous Pro and Ala addition increased wheat FHB resistance, while Cys addition aggravates wheat FHB susceptibility. Based on the obtained results, we suppose it can be utilized through stacking or repressing of candidate amino acid pathway genes, to enhance resistance in wheat against FHB. The metabolome data were readily acquired by using UPLC-QE-MS, which was potentially used in a complementary way to QTL in marker-assisted plant breeding programs designed to improve FHB resistance in wheat. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors. AUTHOR CONTRIBUTIONS QX and QW designed the experiments and provided the funding for this research. PZ, SG, YL, and CM performed the experiments and analyzed the data. PZ, SG, and YL performed the LC-MS experiments and analyzed the data. QX, QW, and CH wrote the manuscript. ZD, JB, and JT participated in ﬁeld tests or helped with the wheat materials. All authors contributed to the article and approved the submitted version. Frontiers in Plant Science \| www.frontiersin.org REFERENCES Gardiner, S. A., Boddu, J., Berthiller, F., Hametner, C., Stupar, R. M., Adam, G., et al. (2010). Transcriptome analysis of the barley–deoxynivalenol interaction: evidence for a role of glutathione in deoxynivalenol detoxiﬁcation. Mol. Plant- microbe Interact. 23, 962–976. doi: 10.1094/MPMI-23-7-0962 Arbona, V., and Gomez-Cadenas, A. (2016). Metabolomics of disease resistance in crops. Curr. Issues Mol. Biol. 19, 13–30. doi: 10.21775/9781910190357.04 microbe Interact. 23, 962–976. doi: 10.1094/MPMI-23-7-0962 Bai, G., Su, Z., and Cai, J. (2018). Wheat resistance to Fusarium hea Bai, G., Su, Z., and Cai, J. (2018). Wheat resistance to Fusarium head blight. Canad. J. Plant Pathol. 40, 336–346. doi: 10.1080/07060661.2018.1476411 Guo, R., Yang, Z., Li, F., Yan, C., Zhong, X., Liu, Q., et al. (2015). Comparative metabolic responses and adaptive strategies of wheat (Triticum aestivum) to salt and alkali stress. BMC Plant Biol. 15, 1–13. doi: 10.1186/s12870-015-0546-x Batista-Silva, W., Heinemann, B., Rugen, N., Nunes-Nesi, A., Araujo, W. L., Braun, H. P., et al. (2019). The role of amino acid metabolism during abiotic stress release. Plant Cell Environ. 42, 1630–1644. doi: 10.1111/pce.13518 Halkier, B. A., and Gershenzon, J. (2006). Biology and biochemistry of glucosinolates. Ann. Rev. Plant Biol. 57, 303–333. doi: 10.1146/annurev.arplant.57.032905.105228 Beccari, G., Arellano, C., Covarelli, L., Tini, F., Sulyok, M., and Cowger, C. (2019). Eﬀect of wheat infection timing on Fusarium head blight causal agents and secondary metabolites in grain. Int. J. Food Microbiol. 290, 214–225. doi: 10.1016/j.ijfoodmicro.2018.10.014 Häusler, R. E., Ludewig, F., and Krueger, S. (2014). Amino acids– a life between metabolism and signaling. Plant Sci. 229, 225–237. doi: 10.1016/j.plantsci.2014.09.011 Browne, R. (2007). Components of resistance to Fusarium head blight (FHB) in wheat detected in a seed-germination assay with Microdochium majus and the relationship to FHB disease development and mycotoxin accumulation from Fusarium graminearum infection. Plant Pathol. 56, 65–72. doi: 10.1111/j.1365-3059.2006.01509.x He, Y., Zhang, X., Zhang, Y., Ahmad, D., Wu, L., Jiang, P., et al. (2018). Molecular characterization and expression of PFT, an FHB resistance gene at the Fhb1 QTL in wheat. Phytopathology 108, 730–736. doi: 10.1094/PHYTO-11-17-0383-R Hildebrandt, T. M., Nunes Nesi, A., Araujo, W. L., and Braun, H. P. (2015). Amino Acid Catabolism in Plants. Mol. Plant 8, 1563–1579. doi: 10.1016/j.molp.2015.09.005 Buerstmayr, H., Ban, T., and Anderson, J. A. (2009). QTL mapping and marker- assisted selection for Fusarium head blight resistance in wheat: a review. Plant Breed. 128, 1–26. doi: 10.1111/j.1439-0523.2008.01550.x Holman, J. D., Tabb, D. L., and Mallick, P. (2014). REFERENCES Employing proteowizard to convert raw mass spectrometry data. Curr. Protocols Bioinform. 46, 11–13. doi: 10.1002/0471250953.bi1324s46 Buerstmayr, M., Steiner, B., and Buerstmayr, H. (2020). Breeding for Fusarium head blight resistance in wheat-Progress and challenges. Plant Breed. 139, 429–454. doi: 10.1111/pbr.12797 Hou, Z., Xue, C., Peng, Y., Katan, T., Kistler, H. C., and Xu, J.-R. (2002). A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility, heterokaryon formation, and plant infection. Mol. Plant-Microbe Interact. 15, 1119–1127. doi: 10.1094/MPMI.2002.15.11.1119 Cai, J. (2016). Meta-analysis of QTL for Fusarium Head Blight Resistance in Chinese Wheat Landraces Using Genotyping by Sequencing. Kansas: Kansas State University. Cajka, T., Smilowitz, J. T., and Fiehn, O. (2017). Validating quantitative untargeted lipidomics across nine liquid chromatography–high- resolution mass spectrometry platforms. Anal. Chem. 89, 12360–12368. doi: 10.1021/acs.analchem.7b03404 Kadotani, N., Akagi, A., Takatsuji, H., Miwa, T., and Igarashi, D. (2016). Exogenous proteinogenic amino acids induce systemic resistance in rice. BMC Plant Biol. 16:60. doi: 10.1186/s12870-016-0748-x Kage, U., Karre, S., Kushalappa, A. C., and McCartney, C. (2017). Identiﬁcation and characterization of a Fusarium head blight resistance gene TaACTin wheat QTL-2DL. Plant Biotechnol. J. 15, 447–457. doi: 10.1111/pbi. 12641 Cecchini, N. M., Monteoliva, M. I., and Alvarez, M. E. (2011). Proline dehydrogenase contributes to pathogen defense in Arabidopsis. Plant Physiol. 155, 1947–1959. doi: 10.1104/pp.110.167163 Chen, F., Liu, S., Zhao, F., Xu, C., and Xia, G. (2010). Molecular characterisation of the low-molecular weight glutenin subunit genes of tall wheatgrass and functional properties of one clone Ee34. Amino Acids 38, 991–999. doi: 10.1007/s00726-009-0307-y Kendziorek, M., Paszkowski, A., and Zagda´nska, B. (2012). Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially puriﬁed from wheat (Triticum aestivum L.). Phytochemistry 82, 7–14. doi: 10.1016/j.phytochem.2012.07.008 Chen, F., Ma, R., and Chen, X. L. (2019). Advances of metabolomics in fungal pathogen-plant interactions. Metabolites 9:169. doi: 10.3390/metabo9080169 Kong, L., Ohm, H. W., and Anderson, J. M. (2007). Expression analysis of defense- related genes in wheat in response to infection by Fusarium graminearum. Genome 50, 1038–1048. doi: 10.1139/G07-085 Dweba, C., Figlan, S., Shimelis, H., Motaung, T., Sydenham, S., Mwadzingeni, L., et al. (2017). Fusarium head blight of wheat: pathogenesis and control strategies. Crop Prot. 91, 114–122. doi: 10.1016/j.cropro.2016.10.002 Kumar, R., Bohra, A., Pandey, A. K., Pandey, M. K., and Kumar, A. (2017). Metabolomics for Plant Improvement: Status and Prospects. Front. Plant Sci. 8:1302. FUNDING This work was supported by grants from the Major Basic Research Program of Shandong Natural Science Foundation Frontiers in Plant Science \| www.frontiersin.org November 2021 \| Volume 12 \| Article 762605 9 Amino Acid Increase FHB Resistance Zhao et al. Ltd., for providing technical support and suggestions for this research work. (ZR2019ZD15), National Natural Science Foundation of China (31801330), and Key R&D projects in Shandong Province (2019GNC106060 and 2019JZZY020608). ACKNOWLEDGMENTS The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021. 762605/full#supplementary-material We would like to thank the Metabonomics Platform, Agronomy College of SDAU, and Shandong Guocangjian Biotechnology Co., REFERENCES J., Baker, J. M., et al. (2010). The metabolic transition during disease following infection of Arabidopsis thaliana by Pseudomonas syringae pv. tomato. Plant J. 63, 443–457. doi: 10.1111/j.1365-313X.2010.04254.x Martin, A. C., Pawlus, A. D., Jewett, E. M., Wyse, D. L., Angerhofer, C. K., and Hegeman, A. D. (2014). Evaluating solvent extraction systems using metabolomics approaches. RSC Adv. 4, 26325–26334. doi: 10.1039/C4RA02731K Warth, B., Parich, A., Bueschl, C., Schoefbeck, D., Neumann, N. K. N., Kluger, B., et al. (2015). GC–MS based targeted metabolic proﬁling identiﬁes changes in the wheat metabolome following deoxynivalenol treatment. Metabolomics 11, 722–738. doi: 10.1007/s11306-014-0731-1 Matuszewski, B., Constanzer, M., and Chavez-Eng, C. (2003). Strategies for the assessment of matrix eﬀect in quantitative bioanalytical methods based on HPLC–MS/MS. Anal. Chem. 75, 3019–3030. doi: 10.1021/ac020361s Welchen, E., Canal, M. V., Gras, D. E., and Gonzalez, D. H. (2021). Cross-talk between mitochondrial function, growth, and stress signalling pathways in plants. J. Exp. Bot. 72, 4102–4118. doi: 10.1093/jxb/eraa608 Nostadt, R., Hilbert, M., Nizam, S., Rovenich, H., Wawra, S., Martin, J., et al. (2020). A secreted fungal histidine- and alanine-rich protein regulates metal ion homeostasis and oxidative stress. New Phytol. 227, 1174–1188. doi: 10.1111/nph.16606 Yuan, C., Li, C., Yan, L., Jackson, A. O., Liu, Z., Han, C., et al. (2011). A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS ONE 6:e26468. doi: 10.1371/journal.pone.0026468 Parre, E., Ghars, M. A., Leprince, A.-S., Thiery, L., Lefebvre, D., Bordenave, M., et al. (2007). Calcium signaling via phospholipase C is essential for proline accumulation upon ionic but not nonionic hyperosmotic stresses in Arabidopsis. Plant Physiol. 144, 503–512. doi: 10.1104/pp.106.095281 Zhang, W., Tan, B., Ye, G., Wang, J., Dong, X., Yang, Q., et al. (2019). Identiﬁcation of potential biomarkers for soybean meal-induced enteritis in juvenile pearl gentian grouper, Epinephelus lanceolatus♂× Epinephelus fuscoguttatus♀. Aquaculture 512:734337. doi: 10.1016/j.aquaculture.2019.734337 Patti, G. J., Yanes, O., and Siuzdak, G. (2012). Metabolomics: the apogee of the omics trilogy. Nat. Rev. Mol. Cell Biol. 13, 263–269. doi: 10.1038/nr m3314 Conﬂict of Interest: 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. Pratelli, R., and Pilot, G. (2014). Regulation of amino acid metabolic enzymes and transporters in plants. J. Exp. Bot. 65, 5535–5556. doi: 10.1093/jxb/eru320 Rawat, N., Pumphrey, M. O., Liu, S., Zhang, X., Tiwari, V. REFERENCES doi: 10.3389/fpls.2017.01302 Fagard, M., Launay, A., Clément, G., Courtial, J., Dellagi, A., Farjad, M., et al. (2014). Nitrogen metabolism meets phytopathology. J. Exp. Bot. 65, 5643–5656. doi: 10.1093/jxb/eru323 Kumaraswamy, G. K., Bollina, V., Kushalappa, A. C., Choo, T. M., Dion, Y., Rioux, S., et al. (2011a). Metabolomics technology to phenotype resistance in barley against Gibberella zeae. Euro. J. Plant Pathol. 130, 29–43. doi: 10.1007/s10658-010-9729-3 Feng, C., Liu, H., and Tang, Z. (2018). Fusarium graminearum inoculation on wheat head. Bio-protocol 8, e2964–e2964. doi: 10.21769/BioProtoc.2964 Feussner, I., and Polle, A. (2015). What the transcriptome does not tell— proteomics and metabolomics are closer to the plants’ patho-phenotype. Curr. Opin. Plant Biol. 26, 26–31. doi: 10.1016/j.pbi.2015.05.023 Kumaraswamy, K. G., Kushalappa, A. C., Choo, T. M., Dion, Y., and Rioux, S. (2011b). Mass Spectrometry Based Metabolomics to Identify Potential Biomarkers for Resistance in Barley against Fusarium Head Blight (Fusarium graminearum). J. Chem. Ecol. 37, 846–856. doi: 10.1007/s10886-011-9989-1 Fiehn, O., Sumner, L. W., Rhee, S. Y., Ward, J., Dickerson, J., Lange, B. M., et al. (2007). Minimum reporting standards for plant biology context information in metabolomic studies. Metabolomics 3, 195–201. doi: 10.1007/s11306-007-0068-0 Li, J., Deng, Z., Sun, F., Guan, X., Wang, Y., and Tian, J. (2014). Resistance genes of wheat variety Shannong 20 identiﬁed by diagnostic molecular markers. Acta Agronomica Sinica 40, 611–621. doi: 10.3724/SP.J.1006.2014.00611 November 2021 \| Volume 12 \| Article 762605 Frontiers in Plant Science \| www.frontiersin.org 10 Amino Acid Increase FHB Resistance Zhao et al. Ullah, M. S., and Lim, U. T. (2017). Synergism of Beauveria bassiana and Phytoseiulus persimilis in control of Tetranychus urticae on bean plants. Systematic Appl. Acarol. 22, 1924–1935. doi: 10.11158/saa.22.11.11 Liu, S., Zhang, X., Pumphrey, M. O., Stack, R. W., Gill, B. S., and Anderson, J. A. (2006). Complex microcolinearity among wheat, rice, and barley revealed by ﬁne mapping of the genomic region harboring a major QTL for resistance to Fusarium head blight in wheat. Funct. Integrat. Genom. 6, 83–89. doi: 10.1007/s10142-005-0007-y, Wang, H., Sun, S., Ge, W., Zhao, L., Hou, B., Wang, K., et al. (2020). Horizontal gene transfer of Fhb7 from fungus underlies Fusarium head blight resistance in wheat. Science 368:6493. doi: 10.1126/science.aba5435 Ma, R., Zhang, X., Yao, J., and Cheng., S. (2019). Breeding for the resistance to Fusarium head blight of wheat in China. Front. Agricult. Sci. Eng. 6, 251–264. doi: 10.15302/J-FASE-2019262 Ward, J. L., Forcat, S., Beckmann, M., Bennett, M., Miller, S. Frontiers in Plant Science \| www.frontiersin.org REFERENCES K., Ando, K., et al. (2016). Wheat Fhb1 encodes a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain conferring resistance to Fusarium head blight. Nat. Genet. 48, 1576–1580. doi: 10.1038/ng.3706 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Smith, C. A., Want, E. J., O’Maille, G., Abagyan, R., and Siuzdak, G. (2006). XCMS: processing mass spectrometry data for metabolite proﬁling using nonlinear peak alignment, matching, and identiﬁcation. Anal. Chem. 78, 779–787. doi: 10.1021/ac051437y Copyright © 2021 Zhao, Gu, Han, Lu, Ma, Tian, Bi, Deng, Wang and Xu. 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) and the copyright owner(s) 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. Tegeder, M. (2014). Transporters involved in source to sink partitioning of amino acids and ureides: opportunities for crop improvement. J. Exp. Bot. 65, 1865–1878. doi: 10.1093/jxb/eru012 Teixeira, W. F., Fagan, E. B., Soares, L. H., Umburanas, R. C., Reichardt, K., and Neto, D. D. (2017). Foliar and seed application of amino acids aﬀects the antioxidant metabolism of the soybean crop. Front. Plant Sci. 8:327. doi: 10.3389/fpls.2017.00327 November 2021 \| Volume 12 \| Article 762605 Frontiers in Plant Science \| www.frontiersin.org 11

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