Spheniscus humboldti) colonies in Peru has been associated with nesting habitat type, presumably due to differences in environmental exposure and activity patterns that may affect energy demands and metabolism. Gas chromatography and mass spectrometry were used to determine serum concentrations of 19 saccharides from 30 Humboldt penguins nesting at Punta San Juan, Peru in order to evaluate differences in metabolic state between penguins nesting in a sheltered burrow or crevice (n = 17) and those in exposed surface nests (n = 13). Univariate and multivariate statistical analyses identified serum saccharides  that were nest-dimorphic with substantial differences between surface- and sheltered-nesting penguins. Four sugars  had ≥ 2-fold difference in concentration between nest types. Seven saccharides were in the top five subsets generated by discriminant analysis; four of these are simple sugars  and three are derivatives . D-ribose had the highest information values  followed by glucose 6-phosphate, levoglucosenone, and D-galactose. Sex was not a significant predictor of saccharide concentration. Levoglucosenone, which is a metabolite of the environmental contaminant levoglucosan, was significantly higher in surface-nesting penguins, reflecting a higher rate of exposure in non-sheltered penguins. Differences in the saccharide profiles of surface- and sheltered-nesting Humboldt penguins likely reflect increased metabolic requirements of surface-nesters at Punta San Juan. Conservation of appropriate sheltered-nesting habitat for penguins is essential for sustained reproductive success and colony health.Reproductive success of endangered Humboldt penguin ( Spheniscus humboldti) inhabits the southern Pacific coast of South America. Population declines have resulted in the species being classified as “vulnerable” by the International Union for Conservation of Nature [The Humboldt penguin  events caused a concomitant reduction in major seabird populations responsible for coastal guano production. Cumulatively, these practices resulted in a substantial decrease in the overall population size of the species, as well as disruption of nesting habitats [Spheniscus species due to a less stable microclimate and higher predation risks [Reproductive colonies of Humboldt penguins in Peru are closely associated with other seabird rookeries, where penguins dig burrows into surface accumulations of dried guano as their preferred nesting habitat. Large-scale commercial harvesting of guano throughout the 19habitats . The loson risks –11. Burron risks .With the global population of mature Humboldt penguins averaging around 32,000 individuals, the continued protection of breeding colonies and appropriate habitat is paramount. Understanding the impact of nesting habitat on energy demands and reproductive success helps guide sound conservation policy development.Using 12 serum metabolites , most of which are involved in lipid and carbohydrate metabolism, the sex of 29/30 Humboldt penguins was able to be correctly classified [Blood was collected from 30 adult Humboldt penguins in 2009 (May 29 to June 2) during their breeding season as part of a population health assessment project at the Punta San Juan (PSJ), Marine Protected Area, Ica, Peru  using previously described collection methods authorized under Peruvian permit 131-2009-AG-DGFFS-DGEFFS and by the Saint Louis Zoo Institutional Animal Care and Use Committee (Protocol Number 09–02) [2. For metabolic profiling, dried polar extracts were derivatized with 80 μL methoxyamine hydrochloride (20 mg mL-1) for 60 minutes at 50°C, then with MSTFA (80 μL) at 70°C for 120 minutes, followed by a 2-hour incubation at room temperature. Ten microliters (10 μL) of the internal standard  was added to each sample prior to derivatization. Samples were analyzed on a GC/MS system consisting of an Agilent 7890 gas chromatograph, an Agilent 5975 mass selective detector, and an HP 7683B autosampler . Gas chromatography was performed on an HP-5MS (60 m × 0.25 mm I.D. and 0.25 μm film thickness) capillary column .Our analytical methods closely followed Jiye et al.  and are The spectra of all chromatogram peaks were compared with electron impact mass spectrum libraries NIST08 , W8N08 , and a custom-built library. To allow comparison between samples, all data were normalized to the internal standards in each chromatogram and the bird’s weight. The spectra of all chromatogram peaks were evaluated using the HP Chemstation  and AMDIS  programs. Validation of extraction, derivatization, and GC-MS analysis was performed for selected mono- and disaccharides and recovery percentage was calculated . MetabolBurrow, Surface) × sex  interaction without explicitly partitioning the 30 penguins into 4 small subsets , preliminary models included sex as a nominal predictor and nest as the binary response in some analyses. In other analyses, sex was the binary response and nest was the nominal predictor.For statistical analysis and reporting, the unitless relative concentrations were divided by the molecular weight (MW) of the saccharide. All analyses were carried out using SAS 9.4 . To place the subset selection process in perspective, there are 50,389 subsets comprised of 1 to 7 of the 19 saccharides (PROC ALLCOMB). To tie to our previous work , and to preliminary analyses of all 19 saccharides (sugars + metabolites) were carried out to identify a relevant saccharide subset. First, we identified saccharides with a fold-change c), Somers’ D, gamma and tau-a were obtained for the top 222 models from discriminant analyses. When the response is binary, the concordance index c is the area under the receiver operating characteristic (ROC) curve [c varies from 0.5 (no evident accuracy) to 1.0 (complete accuracy).Three pothesis  Eq 1). . prelimiC) curve , 18. Thea + b)/N was calculated for each saccharide (predictor) against nest designation. The same analyses were done for sex .SAS tabulates Ψ as the bin number. The nest designations  were added to the Ψ matrix. The simple matching correlation coefficient, S4 =  ANOVA using Proc GLM to estimate effect-size and 2) discriminant analysis for sensitivity and specificity. We defined K as the number of saccharides in a subset. Nineteen subsets had K = 1 saccharide, 3877 subsets had K = 1–4 saccharides, and 50389 subsets had K = 1–7 saccharides. The first filter for subset retention was 0.9 ≤ specificity/sensitivity (S/S) ≤ 1.1, that is max(specificity) ≈ max(sensitivity). A second filter was min(K). Subsets of > 7 saccharides did little to improve discrimination of saccharide profiles between surface- and burrow-nesters.We concluded from the d [WPooled is the pooled standard deviation of the Winsorized data (SDW), and v is a correction factor. The only published value of v we found was 0.642 for two-sided 20% (γ = 0.2) Winsorization. We estimated v = SDW/SDRandom for two-sided and one-sided trimming of 10% (γ = 0.1) and 20% (γ = 0.2) using a random sample of n = 107 values from a normal distribution with mean = 0 and SD = 1. The number of values trimmed on a side is g = γn rounded down to the nearest integer. The Winsorized sample replaces the upper g trimmed values by the largest value not trimmed and the lower g trimmed values by smallest value not trimmed [We included Cohen’s effect size measure d , 20 and  trimmed .Nineteen saccharides were quantified in 30 penguins . Thirteec, Somers’ D, gamma, and tau-a from logistic regression of 222 models were bimodal. Concordance ranged from 0.88 to 0.93, Somers’ D and gamma from 0.76 to 0.86, tau-a from 0.39 to 0.43, and all RSpearman = 1.0. Thus, these measures did not discriminate among the models.Four saccharides had fold-change (FC) Together, the top 100 subsets from discriminant analysis included all 19 saccharides . The topEngraulis ringens) and other pelagic forage fish. The exact diet composition of the animals in this study is unknown, but is postulated to be consistent amongst the cohort given the temporal clustering of sample collection over 5 days with static ocean conditions. The most recent active foraging time relative to blood collection is also unknown. This lack of data does present a minor confounding variable, but is unavoidable when working with wild penguins in free-ranging ocean environments. Known metabolic pathways for healthy humans, non-human vertebrates, and Escherichia coli may be relevant for the evaluation of penguin results, as are pathways in fasting birds  andrhynchos ) and hibrhynchos –27. The rhynchos –30. PollPygoscelis papua), another monomorphic species with shared parental duties, daily energy expenditures throughout the breeding season is similar in both sexes [Pygoscelis adeliae), both sexes lose mass (depot fat and lean tissue) during the early breeding season, but males have a greater cumulative reproductive effort, which could suggest that differences in energy metabolism may exist in some species. Breeding Adélie penguins feed chicks according to their condition and food availability [Although male Humboldt penguins are larger than females, there is such a considerable degree of overlap in size that discriminant function analysis of measurements is required to determine sex with 95% accuracy , 32. Whith sexes . In Adéllability , and adulability . When foE. coli [What do we already know, and how certain are we in what we know? And building on that and the field’s theory, what magnitudes of differences, odds ratios, or other effect sizes are practically important? These questions would naturally lead a researcher, for example, to use existing evidence from a literature review to identify specifically the findings that would be practically important for the key outcomes under study” [Univariate and multivariate statistical analyses identified serum saccharides  in Humboldt penguins that were nest-dimorphic with substantial differences between surface- and sheltered-nesting penguins. Circulating metabolites come from endogenous processes  and exogenous sources including food  and inteE. coli –38. Withr study” . In the r study” , 41, so Aves). Whole blood samples were chilled upon collection to slow cellular metabolism, and serum was separated within 6 hours. Since nucleated avian erythrocytes do not utilize plasma glucose like mammalian cells, variation in the time between collection and centrifugation was not recorded or analyzed.Of the seven saccharides in the top five predictive modeling combinations, 4 are simple sugars  and three are derivatives . As reported in over 30 papers, glucose metabolism has been studied in several penguin species under various conditions and durations of feeding, breeding, and starvation –51. GlucGlucose regulates lipid metabolism in penguins and affects hormonal status differently in stressed and non-stressed individuals . For exaMaltose is a glucose dimer. Metabolism and transport processes for maltose are conserved from bacteria to humans , and so The PSJ peninsula, where this research was conducted, protects the largest breeding colony of Humboldt penguins in Peru . This colony is 25–50% of the entire Peruvian population and up to 15% of the global population on an annual basis , 1, makiPrevious investigation of environmental contaminants in penguins at PSJ found low contaminant levels and population health concerns , 59; how2 burned [The number of understory fires in the southern Amazonia region that includes southeastern Peru dramatically increased in 2005 and 2007, when 14,000 and 26,000 km2 burned . Also, f2 burned , 67. TheSalmonella enterica serovar typhimurium (strain MS14028s), and was cytotoxic against CHO cells and human hepatocarcinoma lines  [Levoglucosenone in serum was unequivocal evidence of exogenous contamination in the nesting environment of Humboldt penguins at PSJ in 2009 when samples were collected. The presence of levoglucosan in the penguins shows that marine and terrestrial species are exposed to , and ads9.39 μM) . It also9.39 μM) .Pygoscelis penguin species detected many essential genes involved in the major innate immunity pathways, which are critical metabolic pathways for maintaining homeostasis against exogenous infections or toxins [Parus major) had substantial differences in gene expression profiles based on environment, with differentially expressed genes functioning in roles related to immune and inflammatory responses, detoxification, protection against oxidative stress, lipid metabolism, and regulation of gene expression. Many genes linked to stress responses were expressed at higher levels in the urban birds [The health effects of wood smoke in Humboldt penguins have not been reported, but there is a chain of evidence that some effects are not benign, and some may be sex-related. For example, exposure of humans to wood smoke altered inflammatory responses to viral infection in a sex-specific manner . KEGG anr toxins . Furtheran birds . Plasma an birds , and Magan birds . In phasan birds . We projStatistical analysis identified differences in saccharide profiles of surface- and sheltered-nesting Humboldt penguins that likely reflect increased metabolic requirements of surface-nesters. Ramifications of increased metabolic demand warrant further investigation as this requirement is proposed to contribute to decreased reproductive success. Surface-nesting penguins also have higher serum concentrations of levoglucosenone, which is likely indicative of greater exposure to environmental contamination via the burning of land biomass. Conservation strategies that preserve burrowing substrate as essential habitat for nesting penguins are warranted and should be continued.S1 Data(XLSX)Click here for additional data file.


Muehlenbeckia australis from high throughput sequencing data (paired-end Illumina reads) generated from total genomic DNA sequencing libraries. M. australis’ chloroplast genome sequence (GenBank: MG604297) is 163,484 bp in length and composed of long single copy  and short single copy  regions flanked by inverted repeats  typical for angiosperms. The plastome includes 131 genes comprising 83 protein-coding genes, 37 transfer RNA genes, eight ribosomal RNA genes, two possible pseudogenes, psbL and rpl23 with internal stop codons, and truncated repeats of ndhF and rps19 at IR boundaries. We assembled the plastome of the temperate, Southern Hemisphere liana  Fagopyrum Mill.  were transferred to the final circular de novo plastome assembly and corrected manually, checking for start and stop codons, intron junctions, and inverted repeat (IR) boundaries.We extracted DNA from ca. 20 mg of silica gel dried leaf material from a specimen growing in the living collection of the Royal Botanic Gardens Victoria  using the DNeasy Plant Mini Kit . A sequencing library was generated following the protocol of Schuster et al. (in press) and sequenced with an Illumina NextSeq 500 machine at The Walter and Eliza Hall Institute of Medical Research (WEHI) using an Illumina mid-output (2 × 150 Paired End) kit. Sequencing yielded 3,356,344 reads, which were M. australis  lies within the rps19 gene, which is 279 bp long. Therefore, IRA includes 108 bp of a truncated inverted repeat of rps19 at JLA. The JSA junction between SSC and IRA lies in the ndhF gene, which is 2244 bp long, and therefore, IRB includes 62 bp of a truncated inverted repeat of ndhF at JSB. There are two possible pseudogenes, rpl23 and psbL, which have internal stop codons. However, Logacheva et al. (The plastome of a et al.  note thaSequencing of additional woody species of Polygonaceae will facilitate studies of shifts in substitution rates comparing herbaceous and woody species.


B = −6.52, P = 0.03 and B = −2.04, P = 0.03), better oral hygiene practices , higher DMFT , and lesser gingivitis . Internet addiction was associated with negative oral health practices and poor clinical outcomes among young Saudis. Holistic health promotion approaches need to address the negative impact of Internet addiction on health and oral health status for this at-risk age group.The study assessed the relationship between Internet addiction and oral health practices and clinical outcomes and whether this was affected by oral health perception. In 2017, a cross-sectional study included university students in the Eastern Province of Saudi Arabia. Questionnaires assessed demographic background, oral health practices , perceived oral health, and Internet addiction. Caries experience and gingivitis were assessed using the World Health Organization criteria. The multivariate general linear analysis assessed the relationship between dependent variables  and exposure (Internet addiction). Data were available for 919 participants, 75.4% females, mean age = 19.8 years, and 1.6% with significant Internet use problem. The mean percentage of teeth with gingivitis was 8.5% and mean DMFT was 2.9. Among those with good perception of oral health and compared with participants with significant Internet use problem, average Internet users had lower consumption of sugar and tobacco ( The use of the Internet has grown over the past few years especially among young people, and it is used for communication, socializing, searching for health information, gaming, and other purposes . With thThe association between Internet addiction and oral health has been assessed in several studies. Young adults who were Internet addicts reported tooth grinding during sleep . InternePositive perception of health may be associated with better health behaviors leading to better health outcomes although this relationship has not been consistently reported . ExistinIn Saudi Arabia, 88.6% of the population had access to the Internet in 2019 . Most SaThe present cross-sectional study was conducted in Dammam, Saudi Arabia, from February to April 2017 after obtaining the approval of Imam Abdulrahman Bin Faisal University's (IAU) Institutional Review Board (IRB-2015-02-188). The study complied with the ethical guidelines of the Helsinki Declaration. A convenience sample of preparatory year (PY) students at IAU was selected. All students at IAU are admitted to PY program, which serves as a foundation year before students join their actual programs of study. Selected students must be 18 years or older and reported using the Internet. The sample size was estimated based on these assumptions: alpha error = 5%, beta error = 20%, percentage of individuals with unfavorable eating habits and Internet use problem = 21% and those with unfavorable eating habits, and no Internet use problem = 16% . The minDependent variables of the study were oral health practices: consumption of sugars, tobacco use, oral hygiene practices, and clinically assessed oral health: caries experience and gingivitis. The independent variable was Internet use. Several confounders such as age, gender, parental education, study track, and previous dental visits were assessed. Information about oral health practices and Internet use was collected using self-administered, validated questionnaires. The first questionnaire was the World Health Organization (WHO) oral health assessment questionnaire for adults . We usedThe questionnaire assessed confounders including personal background  and type of preparatory year studies track . The questionnaire also assessed self-perceived oral health on a three-point Likert scale, poor, fair, and good, and regular dental checkup visits previous year. The next section assessed oral health practices: (a) frequency of tooth cleaning, (b) frequency of sugar consumption, and (c) frequency of tobacco use. The frequency of tooth cleaning was assessed on a six-point Likert scale ranging from never (0) to two or more times/day (5) for each of the four oral hygiene methods. A five-point Likert scale ranging from rarely (1) to several times/day (6) for six sugary products was used to evaluate the frequency of sugar consumption. The frequency of tobacco use was assessed using a seven-point Likert scale ranging from never (0) to several times/day (6) for five tobacco products. We also used the Internet Addiction Test (IAT) . This quOral health was clinically assessed using the WHO methods and criteria for caries assessment including decayed teeth at the cavitation level (D), missing due to caries (M), and filled (F). Gingival inflammation was based on gingival bleeding after gentle probing defined as applying a force <20 g, which can be established by placing the tip of the probe under thumbnail and pressing till blanching .PY students were approached by the study team during the breaks, and the study purpose was explained to them. Agreeing to join the study was considered as a verbal consent. Participants were asked to fill up paper-based questionnaires, which were collected in the same encounter. Full mouth clinical examination was conducted for all teeth using mobile equipment and disposable instruments under natural daylight . Two calThree scores were developed: (1) oral hygiene practices score, (2) sugar score, and (3) tobacco score. The oral hygiene practices score was developed by multiplying the number of oral hygiene methods used by the participant by cleaning frequency. The score ranged from 0 to 20. The sugar and tobacco scores were the sum of the points of the frequency of all respective products and ranged from 1 to 36 and from 0 to 30, respectively. In all scores, higher values indicated greater frequency. The IAT score was categorized into average use (20–49), frequent use (50–79), and Internet use representing a significant problem (80–100) .t-test and one-way ANOVA test. A multivariate general linear model was used with stratification by perceived oral health to assess effect modification. The five dependent variables were (a) oral hygiene practices score, (b) sugar score, (c) tobacco score, (d) DMFT, and (e) percentage of teeth with gingivitis. The independent variable was the Internet addiction level. Internet addiction is also reported as problematic Internet use in the literature and researchers consider problematic Internet use a more appropriate term [η2) as measures of effect size, and P values. The significance level was set at 5%. IBM SPSS for Windows version 22.0  was used for analysis.Caries experience (DMFT) score was the sum of the number of teeth that were decayed (D), missing due to caries (M), or filled (F) . The perate term . Hence, n = 556, 58.8%) had no gingival bleeding. The mean (SD) percentage of teeth with gingivitis was 8.5 (5.2). The mean (SD) DMFT = 2.9 (2.9), D = 0.9 (1.7), M = 0.1 (0.5), and F = 1.9 (2.5).The questionnaires were distributed to 1900 students. Of those, 1830 answered the IAT and 1799 answered the WHO questionnaire. The response rate was 94.7%. Caries data were available for 1723 and gingivitis was assessed for 949. Therefore, further analysis was restricted to 919 students for whom complete data were available. On average, participants were 19.8 years old and most of them were females (75.4%). There were 23.9% and 21.6% from the health and engineering tracks, respectively. Of all participants, 49.3% had university-educated fathers and 40% had university-educated mothers. Only 10% visited the dentist regularly the previous year, and 15.1% had a poor perception of their oral health. Most participants were average Internet users (51.2%) and only 1.6% had significant Internet use problem . Most paN = 585, 62%) cleaned their teeth two or more times/day, 262 (27.8%) cleaned their teeth once daily, 49 (5.2%) 1-2 times/week, 21 (2.2%) 2-3 times/month, 21 (2.2%) rarely, and 5 (0.5%) never cleaned them. The majority  used toothbrush and toothpaste and brushed twice/day using toothpaste 574 (60.9%). Only 224 (23.7%) used floss, 147 (15.6%) used miswak, and 143 (15.1%) used toothpicks. The mean (SD) oral hygiene practices score was 6.8 (3.7). Most participants (P = 0.004). Similarly, participants with regular dental checkup during the last year had a significantly lower IAT score (mean = 46.69) than those without checkup (mean = 50.02) (P = 0.03).B = −5.90, P=0.003 and B = −5.54, P=0.006, for good perception, B = −6.52, P=0.03 and B = −5.26, P=0.09, B = 4.62, P=0.40 and B = 6.44, P=0.24) than those with problematic Internet use. Average and frequent Internet use were associated with lower tobacco score compared to significantly problematic Internet use . Oral hygiene practices score was nonsignificantly higher in average and frequent users compared to users with a significant problem among participants with fair or good perception (P > 0.05). In all oral health perception levels, DMFT was nonsignificantly higher among average and frequent users compared to users with significant problem (P > 0.05). The percentages of sites with gingivitis were nonsignificantly lower in average and frequent users compared to users with significant problems in all oral health perception levels (P ≥ 0.05). Partial eta squared (η2) values indicate small effect sizes in general ranging from <0.0001 to 0.02.Among participants with fair or good perception of oral health, average and frequent Internet use were associated with lower sugar scores than Internet use with significant problem  and more positive oral health practices  than participants with problematic Internet use. However, in participants with poor oral health perception, average/frequent Internet use was associated with more sugar consumption. Across different oral health perceptions, caries experience was higher in frequent/average Internet users while gingivitis was lower in those with significant Internet use problem. Our findings partially support the study hypothesis among those with good/fair perception of oral health. This has implications for risk assessment of caries and gingivitis and the development of health education programs for Internet users.In the present study, 1.6% of participants had problematic Internet use. This level is lower than that reported among Taiwanese (15.3%) , ChineseIn the present study, among participants with good/fair perception of oral health, those with problematic Internet use had a greater consumption of sugar than those with average/frequent Internet use. Our findings agree with the results of studies of adolescents conducted in Korea , multiplIn addition, our study found that participants with a good perception of oral health who were average or frequent Internet users had lower tobacco scores than those with problematic Internet use. This agrees with studies from Korea , 6 and TOur study showed more frequent oral hygiene practices among participants with fair/good perception of oral health who were average/frequent Internet users than those with problematic Internet use. This is in agreement with two studies of Korean adolescents which reported that problematic Internet use was associated with less frequent tooth brushing , 18. ThiIn the present study, participants with a poor oral health perception and Internet addiction had a lower frequency of sugar consumption than those with average/frequent Internet use. This may be attributed to compensatory health beliefs where individuals compensate for one type of unhealthy behavior by engaging in other healthy behavior/s . Thus, pThe present study showed that the gingival condition was better among those with average/frequent Internet use whereas their caries experience was higher than those with problematic Internet use. The greatest portion of DMFT (2.9) consisted of filled teeth . Therefore, the observed association reflects greater exposure to dental care rather than higher level of untreated decay among participants with average/frequent Internet use. The significant association with some oral health practices compared to the nonsignificant relationship with clinical outcomes suggests that more time is needed for Internet addiction to reflect on clinical outcomes.The present study is limited by its cross-sectional design which cannot support causality and can only suggest associations which can be assessed in longitudinal cohort studies. The IAT does not assess the purpose of Internet use. Hence, this gives equal importance to playing games and using educational resources which may have different impacts on oral health. The strength of the study lies in the clinical assessment of oral health outcomes which fills a knowledge gap regarding the oral health effects of Internet addiction. The study also includes male and female students in a number big enough to allow adjustment for confounders. Future longitudinal studies are needed to assess the long-term impact of Internet addiction on oral health and whether this impact persists into adulthood. In addition, future studies are needed to assess the effect of oral health education provided over the Internet on reducing the adverse impact of Internet addiction on oral health outcomes.The present study showed that Internet addiction was adversely and significantly associated with oral health practices and poorer clinical outcomes. Young Saudi adults with problematic Internet use may be at higher risk of oral diseases. Comprehensive health education interventions should promote healthy practices and moderate Internet use in this high-risk group.


The lipoprotein insulin resistance (LPIR) score was shown to predict insulin resistance (IR) and type 2 diabetes (T2D) in healthy adults. However, the molecular basis underlying the LPIR utility for classification remains unclear.n = 980).To identify small molecule lipids associated with variation in the LPIR score, a weighted index of lipoproteins measured by nuclear magnetic resonance, in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study , in the Heredity and Phenotype Intervention (HAPI) Heart Study (n = 590).Linear mixed effects models were used to test the association between the LPIR score and 413 lipid species and their principal component analysis-derived groups. Significant associations were tested for replication with homeostatic model assessment-IR (HOMA-IR), a phenotype correlated with the LPIR score . Factors 1 (triglycerides and diglycerides/storage lipids) and 3 (mixed lipids) were positively  and factor 2 (phospholipids/non-storage lipids) was inversely  associated with the LPIR score. These findings were replicated for HOMA-IR in the HAPI Heart Study .In GOLDN, 319 lipids were associated with the LPIR score (false discovery rate-adjusted Non-storage lipidomics species show a significant inverse association with the LPIR metabolic dysfunction score and present a promising focus for future therapeutic and prevention studies. Dyslipidemia, one of the major determinants of cardiovascular disease (CVD) , is defiRecently, the lipoprotein insulin resistance (LPIR) score has been shown to significantly improve prediction of incident T2D in the JUPITER trial and the Women’s Health Study, even after adjustment for traditional risk factors such as smoking, physical inactivity and obesity –9. The LAlthough the LPIR score is highly variable , the relTo date, there is little published research on the association between lipids and the LPIR score. As both dyslipidemia and IR are central to T2D pathogenesis, it is sensible to speculate that the differences in T2D risk can be related to the LPIR score-associated lipidomic variability. Thus, this study sought to identify a pattern of small molecule lipids associated with the LPIR score in participants of the Genetics of Lipid Lowering Drugs and Diet Network Study (GOLDN), a cohort characterized by uniquely detailed lipid assessments and a variety of –omics data. To reduce the likelihood of false positive findings inherent in the high-dimensional lipidomic analysis, a replication study was pursued in the well characterized Heredity and Phenotype Intervention (HAPI) Heart Study in which the same lipidomics data was collected.GOLDN, the largest study of postprandial dyslipidemia that offers NMR, clinical lipid, and lipidomic measures, was initiated to assess the interaction of genetic factors with environmental interventions  . Brieflyn = 980) collected at baseline after an 8-h overnight fast was used. Targeted metabolomics approach  was implemented to identify NMR spectroscopy signals produced by the methyl group of lipoprotein subfractions: Large (≥ 8.8–13 nm), medium (8.2–8.8 nm) and small (7.3–8.2 nm) HDL, large (≥65 nm), medium (35–65 nm) and small VLDL (27–35 nm), and large (23–27 nm) and small (19.8–21.2 nm) LDL. LPIR is a combined weighted score of six lipoprotein subclasses or size parameters . It was calculated for each participant using the algorithm described by Shalaurova et al. [In the current study, data from participants n = 90 collecte ≥ 8.8–1 nm, mediLaboratory assays were performed on blood samples that were collected from the study participants after an overnight fast. A hexokinase-mediated reaction on the Hitachi commercial kit (Roche Diagnostics) was used to measure fasting plasma glucose. Plasma insulin was examined using competitive RIA . The intra-assay coefficients of variation for the above measurements were 0.984 and 0.975, respectively. HOMA-IR, used to estimate insulin resistance, was calculated as fasting plasma glucose x fasting plasma insulin/22.5 .tert-butyl ether (MTBE), methanol, and water were used to extract plasma lipids. The quality control (QC) samples were method blanks and pooled human plasma (BioreclamationIVT). The separated non-polar phase was injected into a Waters Acquity UPLC CSH C18  with an additional Waters Acquity VanGuard CSH C18 pre-column  maintained at 65 °C was coupled to an Agilent 1290 Infinity UHPLC (Agilent Technologies) for ESI positive and negative modes. Mobile phase modifiers included ammonium formate and formic acid for positive mode and ammonium acetate (Sigma–Aldrich) for negative mode. The same mobile phase composition of (A) 60:40 v/v acetonitrile:water (LC-MS grade) and (B) 90:10 v/v isopropanol:acetonitrile was used for both positive and negative modes. An Agilent 6550 QTOF with a jet stream electrospray source was employed for acquiring full scan data in the mass range m/z 65–1700 in positive and negative modes with scan rate of 2 spectra/second. Instrument parameters were as follows for the ESI (+) mode – gas temperature 325 °C, gas flow 8 l/min, nebulizer 35 psig, sheath gas temperature 350 °C, sheath gas flow 11, capillary voltage 3500 V, nozzle voltage 1000 V, fragmentor voltage 120 V and skimmer 65 V. In negative ion mode, gas temperature 200 °C, gas flow 14 l/min, fragmentor 175 V, with the other parameters identical to positive ion mode. Data are collected in centroid mode at a rate of 2 scans per second. Injection volume was 1.7 μL for the positive mode and 5 μL for the negative mode. The gradient started at 15% B, ramped to 30% at 2 min, 48% at 2.5 min, 82% at 11 min, 99% at 11.5 min and kept at 99% B until 12 min before ramping down to 15% B at 12.1 min which was kept isocratic until 15 min to equilibrate the column. The total run time was 15 min and the flow rate was 0.6 ml/min. Data were acquired in nine batches and every ten samples, one quality control sample was analyzed. MS1 data were acquired for all samples, and MS/MS data were acquired for a set of pooled samples. Data were processed with the Agilent Quant 7.0 software. Lipids levels were reported as chromatographic peak heights and the data were normalized using the SERRF method  [in-silico MS/MS library LipidBlast.GOLDN lipidomics data includes neutral lipids and phospholipids that were collected using UPLC–QTOFMS at the West Coast Metabolomics Center at University of California Davis, Davis, CA, US. The protocol for this measurement was described in detail elsewhere , 19. BriAfter quality control , 413 lipid compounds were included in the study.The HAPI Heart Study, previously described in detail , was iniFasting glucose was measured by a Beckman glucose analyzer using the glucose oxidase method, fasting insulin was examined by radioimmunoassay , and HOMA-IR, was calculated as fasting plasma glucose x fasting plasma insulin/22.5. The same procedures as used in GOLDN were performed to measure small molecule lipids in the HAPI Heart study at the West Coast Metabolomics Center.lmer function). False discovery rate (FDR)-adjusted p-value <  0.05 was considered to be statistically significant in all analyses.Shapiro-Wilk tests were used to examine normality of the data. Quintiles of the LPIR score were compared on baseline characteristics using Chi-squared and Kruskal-Wallis tests for categorical and continuous variables, respectively. Lipid species were rank-inverse transformed to normalize the data for regression modeling. Partial correlations of LPIR score and each of its six component scores  and lipid species were estimated, adjusted for sex, age, center and body mass index (BMI). To explore associations between the LPIR score and lipid species, linear mixed models were fitted, adjusting for sex, age, study center, and BMI as fixed effects, and family structure as a random effect using the R lme4 package  was performed as a dimension reduction method to identify lipidomic patterns associated with the LPIR score. After lipid species were clustered into unrelated groups (components) using principal component analysis (PCA), three principal components were retained based on factors above the break in the scree plot Fig. , to perfr = 0.48), the association of HOMA-IR with all lipid species was investigated in the replication study. Chi-squared and Kruskal-Wallis tests were used to compare categorical and continuous variables, respectively. Lipid metabolites were rank-inverse transformed since they were not normally distributed. Associations between HOMA-IR and lipid species were estimated using statistical models identical to those in the discovery stage.A direct measure of the LPIR score was not available in HAPI Heart because NMR data were not collected in that cohort. However, because the LPIR score and HOMA-IR were strongly correlated in GOLDN , and an exploratory factor analysis (EFA) using varimax rotation was peformed. Then the scores of each of these three factors were calculated by summing standardized variable values within each factor. Thompson’s scores were created using regression. As a result, factors 1, 2 and 3 in the HAPI Heart study consisted of polar lipids, mixed lipids, and storage lipids (triglycerides and diglycerides) respectively. The number of lipid species with factor loading exceeding 0.5 that overlapped between the GOLDN and HAPI Heart studies in the storage, non-storage and mixed lipid patterns were 40, 43, 38, respectively. The first three factors explained 41% in the metabolites in GOLDN and 55% of the variance in the HAPI Heart cohort. The association between HOMA-IR and each of the three factors were tested in HAPI Heart using models identical to the discovery analyses.A secondary analysis was added to determine if the GOLDN LPIR associated-metabolites were also associated with HOMA-IR in GOLDN and then those lipids were compared with HOMA-IR associated lipids in the HAPI Heart study.www.rproject.org).All data analyses were conducted in the statistical framework R 3.1.0 (Table P <  0.05 after FDR adjustment (n = 363 lipids)) are shown in Supplementary Figure n = 139). Of these LPIR-correlated lipid species, triglycerides (TGs) and diglycerides (DGs) (storage lipids), phosphatidylinositols (PIs), phosphoethanolamines (PEs), and ceramides were positively correlated while cholesteryl esters (CEs) and one single sphingomyelin (SM) were inversely correlated with the LPIR score. Correlations with the PCs were more heterogeneous.Partial correlations between the LPIR score as well as its component scores and LPIR-related lipids with p-value <  0.05) with 319 lipid metabolites, controlling for age, sex, center, BMI and family relationship . Supplementary Figure Findings from the linear mixed models revealed that the LPIR score was associated  except for one ceramide, two PCs, two PEs and one SM (Ceramide (d34:2), PC (37:3), PC (p-38:2) or PC (o-38:3), PE (p-34:1) or PE (o-34:2), PE (p-38:4) or PE (o-38:5), SM (d32:2)). These lipids were directly associated with the LPIR score while they were inversely linked to HOMA-IR. Multivariate-adjusted associations of each of the three factors with HOMA-IR are shown in Table A number of 297 lipid species overlapped between 413 and 383 compounds in the discovery and replication studies, respectively. HOMA-IR was significantly associated with 200 lipids, of which 128 overlapped with LPIR-related lipids  in an independent population characterized using the same lipidomics approach, reducing the chance of false positive findings.Findings of this study are concordant with several previous reports of lipid associations with IR, prediabetes and T2D –23. For The assessment of the fatty acid composition of cholesterol esters provides important information about a potential role in health and disease. In this study, cholesterol esters containing fatty acids with carbon number greater or equal to 18 were inversely associated with the LPIR score. Consistently, other research has shown that in groups with impaired glucose tolerance or diabetes compared to those with normal glucose tolerance, the proportion of palmitic acid (16:0) and palmitoleic acid (16:1) in serum cholesterol esters was higher while the proportion of linoleic acid (18:2) was lower . SimilarOther studies have also reported phospholipids as markers of either diabetes or the complications associated with this metabolic dysfunction , 22. Fin2 (PLA2) [2 isoforms could also play a role. For example, to protect from adipose tissue inflammation during obesity, hyperlipidemic LDL is hydrolyzed by PLA2-V to release unsaturated fatty acids which aid saturated adipocytes-released fatty acids to initiate the polarization of macrophages [Regarding LPCs, which are produced when phospholipids such as PCs or PEs are hydrolyzed by phospholipase A2 (PLA2) , results2 (PLA2) , 33. Howrophages .Ceramides, compounds composing of a sphingosine and a fatty acid, were directly associated with higher LPIR scores. Bergman et al. pointed to C18:0, C20:0, and C24:1 ceramides that were increased in T2D, and C16:0 ceramide that was elevated in patients with IR . ImpaireGiven that many correlated metabolites can reside in overlapping pathways, there is value in investigating patterns captured by particular clusters of metabolites. Of the three factors that were assessed in the current study, the relevance of storage lipids and its components to the score and its role in metabolic disease etiology is of particular interest. Specifically, levels of storage lipids, composed of TGs and DGs (storage lipids), increased with the LPIR score. Similar significant patterns of this association were found for all components of the LPIR score, except for VLDL particle size. A consistent finding, with respect to different types of lipoproteins, has been reported during IR status and diabetes , 37. HowIn contrast to factor 1, factor 2, composed of non-storage lipids and polar lipids, was inversely associated with LPIR and all its subclass scores, except for small LDL. A non-significant association of non-storage lipid pattern with the small LDL score might indicate that it is TGs but not phospholipids component of LDL that play an important role in progression of IR and T2D in healthy individuals. The observed relationship between the LPIR score and lipid pattern could be described in terms of the lipid constituents of this pattern. The storage lipid pattern was mostly composed of TGs which are positively related to increased metabolic dysfunction including diabetes . FurtherThe findings should be considered in the context of several limitations. First, the cross-sectional study cannot establish a temporal or causal relationship between lipid species and the LPIR score. Secondly, participants of the GOLDN study were largely metabolically healthy Americans of European descent, which might limit the generalizability of the findings to other ethnic groups and clinical populations. Finally, even though the findings were reported after controlling for potential confounders including family relationship, residual confounding may not be excluded such as for age and/or gender.Also, while we were able to replicate many of the lipids discovered in GOLDN for LPIR score using HOMA-IR as the outcome in the HAPI Heart study, HOMA-IR was not a perfect proxy. However, secondary analysis of LPIR associated lipids showed the majority were also associated with HOMA-IR in GOLDN lending support to using that phenotype in the external replication effort. Furthermore, these two cohorts were different based on gender. This discrepancy is likely explained by inherent differences in the GOLDN and HAPI Heart populations, especially since the latter is an isolated Amish population. However, Using the HAPI Heart Study was the best opportunity for replication given the Amish cohort is of comparable size and race and had the same lipidomic assays conducted at the same lab (West Coast Metabolomics Center). To address this difference in the analysis, sex was adjusted for in all analytical models and many of the results did replicate.In spite of these limitations, the study possesses some major strengths. First, GOLDN and HAPI Heart were large well characterized studies of Caucasian adults that had available clinical metabolic data and lipidomic data from the same lab enabling external validation of the findings. Secondly, a lipidome-wide approach was employed to comprehensively characterize all associations, compared to previous smaller candidate lipid studies. This study was the first to describe molecular correlates of higher LPIR scores prior to dysglycemia onset, and these findings provide the first evidence of potential lipid targets for interventions , 42.a strong positive association was found between storage lipids including TGs and DGs and a strong inverse association was observed between non-storage lipids with the metabolic dysfunction score in a healthy population.Clinical relevance: Additional research should evaluate whether storage and non-storage lipid patterns, especially among those with optimal clinical profiles but a higher LPIR score, could be useful to inform metabolic disease prevention.Take home message: A pattern of higher storage lipids (e.g. TGs) and lower non-storage lipids (e.g. PCs) is associated with higher LPIR score and insulin resistance in Caucasian adults. More studies are needed to determine if these lipid patterns could offer early targets for prevention of metabolic disease.Additional file 1. Supplementary Table 1. Linear mixed models for the association of the lipoprotein insulin resistance score with plasma lipids in the Lipid Lowering Drugs and Diet Network study. Supplementary Table 2. Factors identified from exploratory factor analysis following principal components analysis (PCA) in the Lipid Lowering Drugs and Diet Network study of lipidomics. Only metabolites with a factor loading ≥ 0.5 were reported as composing a given factor. Supplementary Table 3. Results of associations between homeostatic model assessment-insulin resistance and plasma lipids in the HAPI Heart study. Supplementary Table 4. Characteristics of participants by quintile of the homeostatic model assessment-insulin resistance (n = 590). Supplementary Table 5. Partial correlation between cholesterol esters and lipids and glycemic measurements. Supplementary Figure 1. Heat map showing positive (in red), and negative (in purple) partial correlations  of the lipoprotein insulin resistance (LPIR) score and its component scores with LPIR-correlated metabolites (n = 363); metabolites were characterized according to their molecular structure. Each line belongs to one metabolite. Supplementary Figure 2. Bar plot showing positive (in blue) and negative (in red) effect size derived from linear mixed models of the significant lipoprotein insulin resistance (LPIR)-related metabolites (n = 319) characterized with respect to metabolite composition. Each line belongs to one metabolite. To have a better visualization groups with one metabolite (cholesterol and lactosylceramide (d18:1/24:1(15Z))) were not included in the figure.Additional file 2: Supplementary Table 6. Lipidomcs data for UPLC-QTOF/MS.


Intra-articular glucocorticoid (GC) injections are widely used as a symptomatic treatment for osteoarthritis (OA). However, there are also concerns about their potentially harmful effects, and their detailed effects on chondrocyte phenotype remain poorly understood.We studied the effects of dexamethasone on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were isolated from the cartilage from OA patients undergoing knee replacement surgery and cultured with or without dexamethasone for 24 h. Total RNA was isolated and sequenced, and functional analysis was performed against the Gene Ontology (GO) database. Results for selected genes were confirmed with RT-PCR. We also investigated genes linked to OA in recent genome-wide expression analysis (GWEA) studies.Dexamethasone increased the expression of 480 and reduced that of 755 genes with a fold change (FC) 2.0 or greater. Several genes associated with inflammation and cartilage anabolism/catabolism as well as lipid and carbohydrate metabolism were among the most strongly affected genes. In the GO analysis, genes involved in the extracellular matrix organization, cell proliferation and adhesion, inflammation, and collagen synthesis were enriched among the significantly affected genes. In network analysis, NGF, PI3KR1, and VCAM1 were identified as central genes among those most strongly affected by dexamethasone.This is the first study investigating the genome-wide effects of GCs on the gene expression in OA chondrocytes. In addition to clear anti-inflammatory and anticatabolic effects, GCs affect lipid and glucose metabolism in chondrocytes, an observation that might be particularly important in the metabolic phenotype of OA. Osteoarthritis (OA) is a disease that affects over 15% of the global population aged 60 or more, causing pain, disability, and reduced quality of life, as well as major costs to healthcare systems .Chondrocyte gene expression is markedly altered in osteoarthritis . Some ofAs no proven disease-modifying medications are currently available for OA, all pharmacological treatments are essentially symptomatic . Intra-aGlucocorticoids are steroid hormones that are endogenously produced in the adrenal cortex. In target cells, they form a complex with glucocorticoid receptor (GR), which then dimerizes and migrates into the nucleus. After that, GCs cause their anti-inflammatory effects through two main mechanisms. The GR-steroid complex binds to glucocorticoid response elements (GREs) in the promoter region of target genes, promoting the expression of anti-inflammatory genes as well as a number of genes involved in various other functions. In addition, GCs can inhibit the activity of inflammatory transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1) and sequester their coactivators, leading to suppression of the expression of various inflammatory genes and attenuation of inflammation . In chonIn the present study, we set out to study the effects of the glucocorticoid dexamethasone on gene expression in OA chondrocytes. The aim was to identify significantly modulated pathways and/or functional categories of genes that might be important in the pathogenesis of OA. The results were also compared to those of two previous GWEA studies based on the patient material of the RAAK study , 10 to dn = 10, BMI 27.3 [5.8] kg/m2, age 70.0 [14.8] years, median [IQR]; 4/6 females/males) undergoing total knee replacement surgery in Coxa Hospital for Joint Replacement, Tampere, Finland. All patients fulfilled the American College of Rheumatology classification criteria for knee OA [Leftover cartilage pieces were collected from OA patients  were determined by enzyme-linked immunosorbent assay (ELISA) by using reagents from R&D Systems Europe Ltd., 175 Abingdon, UK . The detection limits were 39 pg/mL, 31.3 pg/mL, and 7.8 pg/mL, respectively.The culture medium was removed at the indicated time point, and total RNA of the chondrocytes was extracted with GenElute™ Mammalian Total RNA Miniprep kit (Sigma). Total RNA was treated with DNAse I . RNA concentration and integrity were confirmed with the 2100 Bioanalyzer (Agilent Technologies). RNA (150 ng/sample) was reverse-transcribed to cDNA for RT-PCR using TaqMan Reverse Transcription reagents and random hexamers .p value < 0.05 were deemed biologically and statistically significant. Functional analysis was performed against the Gene Ontology (GO) database [Sequencing of RNA samples (500 ng) was performed in the Finnish Institute of Molecular Medicine (FIMM) sequencing core, Helsinki, Finland, using the Illumina HiSeq 2500 sequencing platform. Sequencing depth was 15 million paired-end reads 100 bp in length. Read quality was first assessed using FastQC , and thedatabase  using radatabase , and prodatabase . Gene fucDNA obtained from the reverse transcriptase reaction was diluted 1:20 with RNAse-free water and subjected to quantitative RT-PCR using TaqMan Universal PCR Master Mix and the ABI Prism 7500 Sequence detection system (Applied Biosystems).Primers and probes for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclooxygenase-2 (COX-2), MAP kinase phosphatase 1 (MKP-1), matrix metalloproteinases (MMPs) 1 and 13, collagen type II, alpha 1 (COL2A1), and aggrecan (ACAN) were obtained from Metabion International AG . The primer and probe sequences (Table S(−ΔΔCT) method was used. According to the method, the cycle threshold (CT) value for genes of each gene was normalized to the CT value of GAPDH mRNA in the same sample.PCR reaction parameters were as follows: incubation at 50 °C for 2 min, incubation at 95 °C for 10 min, and thereafter 40 cycles of denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 1 min. Each experimental reaction was performed in duplicate. The relative mRNA levels of genes listed in Table St test was used to assess the statistical significance of differential expression, and multiple testing was addressed using Bonferroni correction. Data is presented as average + standard error of the mean (SEM).For NGS data analysis, normalization was performed and differential expression studied using a negative binomial model implemented in DESeq2. In ELISA and PCR experiments, paired Student’s p value < 0.05). Of these, 3612 were up- and 3759 downregulated. Twelve most strongly up- and downregulated genes are listed in Table After normalization and correction for multiple testing, 480 genes were upregulated more than 2.0-fold in dexamethasone-treated cells compared to control cells, and 755 downregulated by the same factor (FC < − 2.0). In total, 7371 genes were found to be differentially expressed in dexamethasone-treated versus control cartilage in a statistically significant manner , a known mediator of OA pain  Table 11.Next, we studied, by ranked list enrichment GO analysis, which functional gene categories were enriched among those genes with the largest fold changes into either direction Table . These iAs low-grade inflammation, oxidative stress, and changes in extracellular matrix production and catabolism are central features in the pathogenesis of OA, we set out to separately study genes linked to these processes Table . SeveralAs glucocorticoids regulate glucose and lipid metabolism, and OA is known to be associated with metabolic syndrome, we separately studied genes for proteins participating in the main pathways of carbohydrate and lipid metabolism  . DexametHowever, dexamethasone affected the expression of several other genes regulating lipid and carbohydrate metabolism . For example, pyruvate dehydrogenase kinase 4 (PDK4) and glycogen phosphorylase L (PYGL), as well as the redox regulator Sestrin 3 (SESN3), were markedly upregulated by dexamethasone , several interactions were identified using the STRING database Fig. . PhosphaThe pathology of OA is characterized by eventual cartilage degradation that is caused by imbalanced gene expression profiles in the cartilage. The effects of GCs used in the treatment of OA on this balance are of interest, and RNA-Seq provides a comprehensive view of gene expression in a tissue. Dexamethasone was found to affect the expression of a large number of genes in OA chondrocytes. Among the most strongly affected genes were several involved in inflammation, extracellular matrix organization, and carbohydrate and lipid metabolism.Constant low-grade joint inflammation along with inflammatory exacerbations is a central feature of OA. Glucocorticoids were found to inhibit inflammation, and this might explain part of their therapeutic effects on OA exacerbations. In our data, dexamethasone reduced the expression of well-known inflammatory factors such as cyclooxygenase-2 (COX-2) , interleOsteoarthritis as well as glucocorticoid treatment has been reported to be associated with increased oxidative stress and chondrocyte apoptosis . Activat2, as well as VEGFA are involved in mediating OA pain [Pain in OA is mediated through various intracellular pathways and soluble factors, of which nerve growth factor (NGF) is thought to be of particular importance . Antibod OA pain . Thus, tDexamethasone markedly affected the expression of several genes involved in glucose and lipid metabolism. An interesting example is pyruvate dehydrogenase kinase 4 (PDK4), which was one of the genes most strongly upregulated by dexamethasone. As this gene inactivates pyruvate dehydrogenase and prevents pyruvate produced in glycolysis from progressing to oxidative phosphorylation , this glOsteoarthritis has a large heritable component, as up to 50% of the incidence of the disease is thought to be explained by genetics . SeveralThe microarray-based genome-wide expression analysis (GWEA) study by Ramos et al.  previousThe time course of the effects of glucocorticoids is an important factor to be taken into account when evaluating their potential effects on cartilage. When used at clinically relevant doses (1–3 mg), dexamethasone injected intra-articularly seems to be mostly absorbed from the joint within 24 h . We thusIn conclusion, dexamethasone was found to cause a major phenotypic switch in OA chondrocytes, while the overall effect on genes linked to OA in GWAS and GWEA studies appeared to be modest. In addition to clear anti-inflammatory, anticatabolic, and extracellular matrix-targeting effects, dexamethasone was found to affect lipid and glucose metabolism-related genes, an observation that might be particularly important in the metabolic phenotype of OA.Additional file 1: Table S1. Primers and probes used for quantitative RT-PCR. Table S3. Expression of cartilage constituents in dexamethasone-treated OA chondrocytes (D) relative to controls (Co). Table S4. Selected genes linked to inflammation, oxidative stress, catabolism and extracellular matrix production in dexamethasone-treated OA chondrocytes (D) relative to controls (Co) as determined by NGS and confirmed with RT-PCR. Table S5. Expression of genes belonging to the major pathways of carbohydrate and lipid metabolism in dexamethasone-treated OA chondrocytes (D) relative to controls (Co) [Table S6. Effects of dexamethasone on genes linked to OA in previous GWAS studies [Table S7. Effects of dexamethasone on genes previously linked to OA in the GWEA study by Ramos et al. [Table S8. Effects of dexamethasone on genes previously linked to OA cartilage in the GWEA study by Almeida et al. [Figure S1. Effects of dexamethasone on the production of catabolic and proinflammatory factors in OA chondrocytes. OA chondrocytes / chondrocytes isolated from OA patients were cultured for 24 h with or without dexamethasone (1 μM). MMP-1 (A), MMP-13 (B) and CCL2 (C) levels in the culture media were determined with ELISA. MMP-1 (D), MMP-13 (E) and CCL2 (F) mRNA expression was studied with quantitative RT-PCR and normalized against GAPDH. The results were compared against control, which was set as 100 %. The results are expressed as mean + SEM, n = 9. *: p < 0.05, **: p < 0.01 and ***: p < 0.001, compared to the untreated control.ols (Co) . Table S studies –23. Tabls et al. . Table Sa et al. . Figure Additional file 2: Table S2. All genes differentially expressed in dexamethasone-treated OA chondrocytes (D) relative to controls (Co).


Breast cancer and its treatment imposes a significant effect in the quality of life (QOL) of women. Being a developing country with contrasting social and cultural norms to the West, Sri Lankan women may have a different experience on QOL following surgical treatment of breast cancer. This study was conducted to evaluate post-treatment QOL in breast cancer patients and to determine its association with the type of surgery.A cross sectional study was carried out. Fifty four women with non-metastatic breast cancer who underwent surgery for breast cancer at the Professorial Surgical Unit, Colombo during 2015–2018 and completed a minimum of one year follow up after surgery were invited to participate. Fifty-four women who responded were assessed using the validated EORTC QLQ-C30 and QLQ-BR23 questionnaires. Non-parametric tests were used for statistical analyses.n = 35) underwent mastectomy and the rest  breast conservation surgery (BCS). The mean QLQ-C30 score was 68.8 (range 8.3–100) and the mean scores for physical function, role function, emotional function, cognitive function, and social function were 71.4, 81.5, 77.0, 80.2, and 86.4, respectively. The mean scores for body image, sexual functioning, sexual enjoyment, future perspective, systemic therapy, breast symptoms, arm symptoms, and hair loss assessed by the QLQ-BR23 were 76.4, 18.3, 33.3, 73.6, 30.5, 16.2, 23.4 and 32.7, respectively. No significant differences (P > 0.05) were noted in global health status, physical function, role function, emotional function, cognitive function and social function between BCS and mastectomy. QLQ-BR23 body image, sexual functioning, sexual enjoyment and future perspective also did not differ significantly (p > 0.05) between the two groups.The mean age was 59 years (range 36–81). A majority (61%, Sexual functioning and enjoyment, breast and arm symptoms and hair loss contributed to poor QOL while the impact on global health status including physical, social and emotional functions were minimal. Type of surgery did not appear to be associated with QOL. Future studies with a larger sample sizes will be helpful to further study these factors.The online version contains supplementary material available at 10.1186/s12885-021-08055-5. Breast cancer is the most commonly diagnosed cancer in women with over two million new patients diagnosed in 2018. Furthermore, it is the commonest cause for cancer-related death in women with over 600,000 deaths the same year .Advancements in diagnosis and treatment have substantially improved the survival rates from breast cancer over last two to three decades with a majority of women achieving a complete cure , 5. AlthIn the South East Asian region, the evaluation of response to therapy is largely limited to the use of traditional markers such as disease free survival and overall survival. However, worldwide, especially in the West there is an increasing focus towards optimising the health-related quality of life among survivors . The conTwo main modes of surgical treatment of breast cancer include breast conservation surgery (BCS) and mastectomy. Evidence has proven BCS followed by whole breast irradiation and total mastectomy to carry nearly equal oncological outcomes in early breast cancer . HoweverSeveral studies over the years have focused on QOL after surgery for breast cancer. Although most have shown BCS to be superior to mastectomy, some had shown no significant difference in QOL –18. SeveA cross sectional study was carried out. Fifty four women with non-metastatic breast cancer who underwent surgery for breast cancer at the Professorial Surgical Unit, Colombo during 2015–2018 and completed a minimum of one year follow up after surgery having an ECOG score of 0–2 were invited via phone calls and mail to participate in the study. The patients were recruited in the immediate postoperative period and followed up. Only the ones with a follow-up duration of minimum one year were included in the study. Fifty-four women who responded were assessed using the validated EORTC QLQ-C30 and QLQ-BR23 questionnaires. Validated Sinhala, Tamil and English versions of the questionnaires were used . Non-parWomen with a previous history of breast or other types of cancer were excluded. Ethical approval for this study was obtained from the Ethical Review Committee, Faculty of Medicine Colombo (EC: 17–126).The QLQ-C30 questionnaire includes 30 items to assess five functional scales , three symptom scales  and a global health and quality of life scale . QLQ-BR2χ2) test, Mann-Whitney U test and Spearman correlation test were used for univariate statistical analyses. QOL scores were compared between women undergoing BCS versus mastectomy. A p value < 0.05 was considered as statistically significant.The data were coded and analysed according to the scoring protocol described in the EORTC QLQ-30 manual by Fayers et al. using SPSS version 24 , 22. Nonn = 33) being less than 60 years. Majority of the women were postmenopausal . A majority  underwent mastectomy as the primary surgery and the rest  underwent BCS. None of the women who had undergone mastectomy and included in the study had breast reconstruction. Axillary node dissection was performed in 59.2% (n = 32) and the rest only a sentinel lymph node biopsy. The patients had a median follow-up duration of > 2 years  from a total of 320 eligible women who had undergone surgery for non-metastatic breast cancer and consented to participate were included in the study. The mean age was 59.0 years (range 36–81) with 61.1% (6.8% fromn = 33) was not any different than the older patients  (Table The mean EORTC QLQ-C30 score was 68.8 (range: 8.3–100) and the mean scores for physical function, role function, emotional function, cognitive function, and social function were 71.4 (range: 33.3–100), 81.5 (range: 0–100), 77 (range: 0–100), 80.2 (range: 33.3–100), 86.4 (range: 0–100), respectively. The EORTC QLQ-C30 scores in the less than 60-year age group , 18.3 (range: 0–100), 33.3 (range: 0–100), 73.6 (range: 0–100), 30.5 (range: 0–90.5), 16.2 (range: 0–66.7), 23.45 (range: 0–77.8), 32.7 (range: 0–100), respectively.p = 0.022). No significant difference was noted in the global health status (p = 0.41), physical function (p = 0.96), role function (p = 0.42), emotional function (p = 0.74), cognitive function (p = 0.30), and social function (p = 0.19) parameters in the two groups who underwent BCS versus mastectomy as reported by the EORTC QLQ-C30. The parameters assessed by the QLQ BR-23 such as body image (p = 0.37), sexual functioning (p = 0.17), sexual enjoyment (p = 0.28), future perspective (p = 0.94), systemic therapy (p = 0.81), breast symptoms (p = 0.46), arm symptoms (p = 0.18), and hair loss (p = 0.859) were also similar in the two groups who underwent BCS compared with mastectomy . In addition, a negative correlation was observed with the overall EORTC QLQ-C30 score and the age (p = 0.022) where younger patients showed a significantly better QOL scores. This finding is different to that of most studies from the west which could be because the value of the intact breast is perceived differently in younger women than older women as younger women are more sexually active than older women unlike in the western world [This cross-sectional study evaluated the post-treatment QOL in Sri Lankan female patients diagnosed with breast cancer using the validated EORTC QLQ-C30 and BR-23 tools have shown substantially poor QOL in sexual functioning and enjoyment, breast and arm symptoms and hair loss domains while the impact on global health status including physical, social and emotional functions were minimal. The mean EORTC QLQ-C30 score of 68.8 indicated that our patients health related quality of life (HRQL) is comparable with average global figures (mean 61.8+/− 24.6) as well as previously published local figures (mean 50 +/− 24.3) , 23. Howrn world .A majority of previous studies investigating QOL with type of breast surgery from Western countries have shown conflicting results although most studies have shown BCS to be superior to mastectomy , 26, 27.Few long-term follow-up studies have shown no differences in body image, sexual function over time among patients who underwent BCS in comparison with mastectomy , 27. A lIn comparison with the global values for QLQ-30 in women after breast cancer treatment, the role function, emotional function, and social function showed substantially better QOL values. However, compared to global parameters, the scores of BR-23 such as body image, sexual functioning and sexual enjoyment appear to be lower [According to our findings, the low sores in breast related symptoms measured by BR-23 seems to be a major contributing factor for the lower QOL in breast cancer patients. Taking this into consideration, it is necessary to take measures to address the burden of breast related symptoms of these patients following surgery as these are easily preventable with adequate care. The HRQL of these patients may be improved by simple measures such as addressing sexual issues by referring them for counselling and prescribing topical applications, offering physiotherapy to alleviate arm symptoms, provision of wigs to combat hair loss following treatment. Furthermore, in the Sri Lankan context patients would be reluctant to bring out these sensitive issues themselves with the doctors. Provision of regular contact with the patients through trained cancer care nurses to recognize these issues and provide advice may help improve QOL in these domains which ultimately will help improve overall QOL. Sri Lanka is a country with a strong preventive health care system. With the increasing cancer burden, the healthcare policy makers should strengthen the public health system by incorporating cancer care nurses into the system and they can address the issues of these patients as they arise.Our study was the first of its kind performed in the Sri Lankan context. The main limitations of this study is the smaller sample size which may not be representative, the inequality of age distribution and the absence of a control group. Majority of the sample being older women could also contribute to the QOL scores mainly due to the sociocultural factors coming into play in the local context. Several other contributors for morbidity following surgery for breast cancer such as sensory complaints of the ipsilateral arm, duration of disease, and a comparison before and after surgery was not considered in the present study which seem necessary in evaluating the QOL. Qualitative methods in future studies may provide further insight. Further prospective studies with larger sample sizes are deemed essential in the local context incorporating these other factors that may affect QOL among breast cancer patients.Sexual functioning and enjoyment, breast and arm symptoms and hair loss contributed to poor QOL in women after treatment for breast cancer while the impact on global health status including physical, social and emotional functions were minimal. Type of surgery did not appear to be associated with QOL. Age was the only statistically significant factor associated with QOL, where younger patients showed a significantly better QOL. Measures should be implemented to help women with breast related symptoms especially sexual, arm and hair loss related symptoms which contribute significantly to poor QOL following surgery. Further studies with larger sample sizes will be helpful to confirm these findings and to identify strategies to improve QOL in these specific domains.Additional file 1. STROBE Statement—Checklist of items that should be included in reports of cross-sectional studies.


Angiosarcomas (AS) have poor prognosis and often metastasize to distant sites. The potential predictors of metastatic angiosarcomas (MAS) have not been extensively investigated. The main objective of this study was to identify survival predictors of MAS.Surveillance, Epidemiology, and End Results (SEER) datasets were used to identify patients with MAS from 2010 to 2016. Risk predictors were determined with the aid of Kaplan-Meier and Cox regression model analyses.A total of 284 MAS patients met the study entry criteria. Among these, 121 patients (42.6%) were diagnosed with metastasis in bone, 26 in brain (9.2%), 86 in liver (30.3%) and 171 in lung (60.2%). Overall, 96 patients (33.8%) had two or more metastatic sites. The 1- and 3-year overall survival (OS) rates were 20.8 and 3.8% while 1- and 3-year cancer-specific survival (CSS) rates were 22.0 and 5.2%, respectively. Cox regression analysis revealed chemotherapy, radiation treatment (RT) and tumor size ≤10 cm as independent favorable predictors of OS. In terms of CSS, tumor grade IV, tumor size > 10 cm and absence of chemotherapy were independent adverse predictors. Surgery did not prolong survival outcomes (both OS and CSS) in the current cohort.MAS is associated with extremely poor survival. Chemotherapy, RT, and tumor size are independent predictors of OS. Chemotherapy and tumor size are independent prognostic factors of CSS. Chemotherapy is therefore recommended as the preferred treatment option for MAS patients. Angiosarcomas (AS) are rare, highly malignant soft-tissue sarcomas of vascular or lymphatic origin, which account for approximately 1–2% of all soft tissue sarcomas . While tPatient data (from 2010 to 2016) were accessed from the Surveillance, Epidemiology, and End Results (SEER)18 registry of the National Cancer Institute, an authoritative cancer research center that uses hospital registry data accredited by the Commission on Cancer. Use of SEER data does not require approval by the Institutional Review Board.SEER*Stat (Version 8.3.6) was applied to identify patients diagnosed with angiosarcoma using the International Classification of Diseases for Oncology 3rd Edition morphological code (9120). All patients met the entry criteria based on positive histological findings. Thirteen patients diagnosed with positive exfoliative cytology, radiography or unknown histology were excluded, along with one patient for whom the mode of therapy and survival months were unknown. Statistical variables included age at diagnosis (≤ and > 60 years), gender , race , year of diagnosis , tumor grade , tumor size , chemotherapy, radiation treatment (RT), surgery treatment (ST) and vital status . Metastatic sites were divided into two categories according to number, specifically, 1 and ≥ 2 sites.P < 0.05 in univariate analyses were included for multivariate analyses. Independent predictors of OS and CSS were determined using the multivariate Cox regression model. We used ‘Enter’ method in Cox regression. Hazard ratios (HR) and 95% confidence intervals (CI) were employed to determine the effects of various factors on OS and CSS. Differences were considered statistically significant at P < 0.05.Statistical analyses were performed using Microsoft Excel 2019  and SPSS statistics . Overall survival (OS) was defined as time from diagnosis to death induced by any cause or last follow-up. Cancer-specific survival (CSS) was regarded as time from diagnosis to death specifically due to AS. Observations were censored if patients were alive at the time of last follow-up. Survival curves were constructed using GraphPad Prism 8 software . The Kaplan–Meier method was applied to calculate survival rates and median survival. We also used Kaplan–Meier method to perform univariate analysis. Statistical significance was calculated with the log-rank test. Variables of From 2010 to 2016, 284 patients with MAS were identified from the SEER database. Descriptive statistics are presented in Table P = 0.003, Table P = 0.036, Table P = 0.024, Table P < 0.001, Table P < 0.001, Table P < 0.001) and CSS (P < 0.001) were observed between chemotherapy-administered and non-administered groups  and CSS (P = 0.251). Regarding survival rates of different primary tumor sites, compared with visceral/deep soft tissue in OS, patients of head and neck tumors had better survival , size (≤10 cm vs. > 10 cm), chemotherapy and RT were included for multivariate analysis of OS. Age at diagnosis(years), grade, size (≤10 cm vs. > 10 cm), chemotherapy were included for multivariate analysis of CSS. The results of multivariate analyses for the entire cohort are shown in Table To date, there is no definitive treatment guideline for MAS. Patients with this cancer have a poor prognosis , 11, 25.Two earlier reports which analyzed AS revealed a negative impact of older age on survival , 10, conOur current findings were consistent with prior studies showing that gender , race, aThe results of the earlier studies regarding the relation between tumor grade and patients’ survival time varied. A number of previous studies have documented no association of tumor grade with OS of AS , 26. ConTwo earlier studies reported that larger tumor size does not impair OS of AS , 20. In Treatment results for AS vary significantly. An earlier retrospective analysis of postoperative AS led to the conclusion that chemotherapy dose not confer an OS benefit . ConsistConic et al. analyzed the outcome of cutaneous AS patients and disclosed no significant impacts of RT on OS , same reA retrospective study disclosed no significant impacts of ST on OS for patients presented with metastatic disease , consistOur study has several limitations that should be considered. First, the available information was incomplete due to the retrospective nature of the investigation. Prospective studies should be conducted to verify our conclusions. Second, the SEER database does not provide other important information, such as detailed radiotherapy and chemotherapy regimens, basic health status of patients, and surgical protocols, which may cause bias of results. Further research should focus on inclusion of these variables to provide supplementary information. Third, imbalance ratios of variables , may contribute to results bias in this study. Despite these limitations, we successfully analyzed the predictors and outcomes of MAS for the first time. In addition, the SEER database collects tumor information based on highly unified standards from multiple centers, providing the largest quantity of tumor data, especially for rare tumors.In this study, we analyzed the survival predictors of MAS, known for its extremely poor survival rates, in 284 patients. Chemotherapy, RT and tumor size ≤10 cm were identified as independent protective predictors of OS. Chemotherapy and tumor size ≤10 cm were independent favorable prognostic factors of CSS. Grade IV was associated with poorer survival of CSS. The number of metastatic sites did not appear to affect OS and CSS. Based on the collective findings, we recommend chemotherapy as the preferred treatment option for MAS patients.Additional file 1: Table S1. Demographics of 284 patients diagnosed with metastatic angiosarcomas identified from SEER database between 2010 and 2016.Additional file 2: Table S2. Median survival data (months) of metastatic angiosarcomas.Additional file 3: Table S3. Univariate analysis of primary tumor sites for OS and CSS in patients of metastatic angiosarcomas.Additional file 4: Table S4. The number of metastatic sites in the 284 patients diagnosed with metastatic angiosarcomas.


Patients with lumbar spinal stenosis (LSS) may be at high risk of falls due to various factors. No effective fall risk assessments or fall prevention measures have been performed for patients with LSS because only a few studies have evaluated falls in these patients. This study aimed to evaluate the incidence and preoperative predictors of falls within 12 months of surgery in patients with LSS.In this prospective study of 82 consecutive preoperative patients with LSS, preoperative demographic data, previous fall history, leg pain, low back pain, Japanese Orthopaedic Association (JOA) score, Hospital Anxiety and Depression Scale (HADS) scores, lower extremity muscle strength, walking speed, grip strength, and muscle mass were assessed at baseline. Falls were assessed at 3, 6, 9, and 12 months after surgery. Participants were categorized as fallers and non-fallers and baseline variables were compared. Binomial logistic regression was used to identify predictors of falls within 12 months of surgery.Seventy-four patients (90.2%) completed the 12-month follow-up after surgery, of whom 24 patients (32.4%) experienced falls. A higher proportion of fallers were female and had a history of falls compared to non-fallers. Fallers had a significantly lower JOA score and a higher HADS-depression score compared to non-fallers. Fallers had significantly lower tibialis anterior muscle strength, gait speed, grip strength, and skeletal muscle mass index. Fallers had a higher prevalence of low muscle mass compared with non-fallers. The presence of low muscle mass was significantly predictive of falls within 12 months of surgery .Patients with LSS have a high incidence of falls after surgery and preoperative low muscle mass may be a predictor of postoperative falls. Falls have been reported to be the main cause of fragility fractures, which increase the burden to society due to increased mortality and economic costs as well as lower quality of life , 2. ManySome previous studies on falls have focused on patients with lumbar spinal stenosis (LSS). One study suggested that patients with LSS are at high risk of falling because they experience motor function decline comparable to that of patients with knee osteoarthritis . AnotherIn this prospective single-center study, consecutive patients with both clinically and radiologically defined LSS underwent surgical treatment at one or two levels  between October 2015 and April 2018. Exclusion criteria consisted of (1) medical conditions such as stroke, neuromuscular disease, cancer, high risk of infection, or cardiovascular disease that researchers have determined to affect surgery or might lead to an unusual postoperative course; (2) dementia or inability to complete the questionnaire independently; (3) previous spine surgery; (4) pacemaker use; (5) non-ambulatory status; and (6) need for long-term care insurance certified by the Ministry of Health, Labour and Welfare. There were 82  patients included in this study , symptom duration, neurological findings such as deep tendon reflexes (patellar tendon reflex and Achilles tendon reflex) and sensory disturbance, history of fragility fracture, and comorbidities such as hypertension, hyperlipidemia, and diabetes were extracted from medical records. For deep tendon reflexes, the results on the symptomatic side were used. Deep tendon reflexes were categorized as 0 for absent, 1 for diminished, 2 for normal, and 3 for increased. A history of previous falls in the 12 months before the baseline assessment was recorded.Leg pain and back pain were evaluated using a numerical rating scale (NRS) .Clinical data were evaluated using the Japanese Orthopaedic Association (JOA) score for low back pain .Anxiety and depression were measured using the 14-item Hospital Anxiety and Depression Scale (HADS) . There aLower extremity muscle strength was assessed using manual muscle testing (MMT) . The tarWalking speed was assessed using 10-m walking test. The time required to walk 10 m at the participant’s normal speed was used to calculate walking speed. Normal walking speed is an evaluation metric that has been shown to be reliable .Handgrip strength was measured using a T.K.K. 5401 dynamometer . The highest score was used in the analysis .2).Muscle mass was measured using bioelectrical impedance analysis (BIA) with an MC-780A body composition analyzer  . AppendiDetails of these evaluations are described in our previous article .2 in men and < 5.7 kg/m2 in women). In this study, we defined low muscle mass as less than 7.0 kg/m2 in men and 5.8 kg/m2 in women, based on a previous report using the BIA method in Japanese participants [The Asian Working Group for Sarcopenia 2019 criteria  were useicipants .A fall was defined as an event in which the participant unintentionally came to rest on a lower level or the floor that was not the result of a major intrinsic event. The follow-up period was 12 months . The incParticipants were classified as either non-fallers or fallers, who could have experienced one or more falls, based on data gathered prospectively during the 12 months after surgery.Data are presented as medians and interquartile ranges. In this study, the following analyses were conducted to evaluate preoperative predictors of postoperative falls. The Mann-Whitney U test was used for continuous variables and ordinal variables, and the chi-square test for nominal variables to compare baseline data between groups. The Wilcoxon signed-rank test was used to compare preoperative and 12-month postoperative outcomes. JOA score, HADS scores, walking speed, handgrip strength, and SMI were treated as continuous variables. NRS and MMT were treated as ordinal variables. Sarcopenia and low muscle mass were treated as nominal variables.P values less than 0.05 were considered statistically significant.Binomial logistic regression was used to investigate preoperative factors associated with falls during the 12 months after surgery. Variables that were significantly different between groups were considered independent variables because preoperative factors that are predictive of postoperative falls in patients with LSS are unknown. The Hosmer-Lemeshow test was used to evaluate model fit. All data were analyzed using SPSS statistical software ; Seventy-four patients (90.2%) completed the 12-month postoperative follow-up. Eight patients were lost to follow-up: five patients did not attend follow-up outpatient visits, two patients developed cancer, and one patient became paralyzed after surgery. Mean procedure time was 140.1 ± 62.3 min, mean length of stay was 18.0 ± 4.7 days, and mean postoperative rehabilitation time was 141.6 ± 59.7 min.Falls occurred in 24 (32.4%) participants: 10 experienced a fall by 3 months after surgery, 9 by 6 months after surgery, and 5 by 12 months after surgery. Of the participants who fell, 8 (33.3%) had one fall and 16 (66.7%) had two or more falls. Table Fallers had a significantly lower JOA score and a higher HADS-depression score compared to non-fallers. Fallers had significantly lower muscle strength in the tibialis anterior, gait speed, grip strength, and SMI. They had a higher prevalence of low muscle mass compared with non-fallers.P = 0.967) and surgical intervertebral level (P = 0.638) among fallers and non-fallers. The rate of fusion surgery  among fallers and non-fallers was 62 and 62.5%, respectively.Table Table P = 0.058.Table This study prospectively investigated the incidence and predictors of falls within 12 months of surgery in patients with LSS. In this study, the incidence of falls within 12 months of surgery in patients with LSS was 32.4%. Multivariate analysis showed that preoperative low muscle mass is a predictor of postoperative falls. A previous study reported that the annual incidence of falls among older individuals in the general population was 10–20% . Other sA univariate analysis showed that compared with non-fallers, fallers included a significantly higher percentage of women and patients with previous falls as well as a higher preoperative HADS-depression score before surgery. In addition, motor function as assessed by JOA score, tibialis anterior muscle strength, gait speed, and grip strength was lower among fallers. A previous study of older individuals in the general population reported that women have a higher risk of falls than men ; the preOne strength of this study is that the multivariate analysis, which adjusted for risk factors of falls, showed that a decline in muscle mass is an independent predictor of falls. Although a previous study had suggested an association between a decline in muscle mass and falls in community-dwelling older people , there aThis study has some limitations. First, the sample size was small; however, the multivariate model had good fit statistics and it was presumed that the small sample size did not significantly affect the results. The power of the post hoc test based on low muscle mass percentage was 83.7%. The second limitation was recall bias. It has been reported that self-administered questionnaire survey responses are affected by the respondent’s age and cognitive function . In thisThis study investigated preoperative predictors of falls within 12 months of surgery in patients with LSS. The results suggest that the incidence of falls after surgery is high and preoperative low muscle mass may be a predictor of postoperative falls in patients with LSS. Therefore, in patients with LSS, it may be important to assess postoperative fall risk based on various perspectives, including preoperative muscle mass.


Citrullus colocynthis L. is one of the worldwide famous traditionally medicinal plants and widely applied in watermelon breeding for its multiple resistances. The complete nucleotide sequence of desert watermelon (Citrullus colocythis L.) chloroplast genome has been determined in this study. The genome was composed of 157,147 bp containing a pair of inverted repeats (IRs) of 26,149 bp, which was separated by a large single-copy region of 86,851 bp and a small single-copy region of 17,998 bp. A total of 123 genes were predicted including 86 protein-coding genes, eight rRNA genes and 29 tRNA genes. Phylogenetic analysis revealed that C. colocynthis were closely related to other two species in the genus Citrullus. The complete chloroplast genome of C. colocynthis would provide some significant information for Cucurbitaceae evolutionary and genomic studies. Citrullus colocynthis L., commonly known as ‘bitter apple’, is one of the worldwide famous traditionally medicinal plants belonging to the family Cucurbitaceae. Its fruit, leaf and root are well-known for containing large amounts of bioactive components with great medicinal value  was stored in the College of Horticulture of Northeast Agricultural University , Harbin, China. Genomic DNA was extracted from fresh leaves and subjected to construct a genomic library and pair-end (2 × 150 bp) sequenced by HiSeq X Ten . Whole genome sequence data of 4 Gb were generates and trimmed, high quality pair-end reads of 0.4 Gb were randomly extracted using Seqtk, and assembled with using the Plasmidspades.py in SPAdes (v3.10.1)  as a reference . The draft sequence was then confirmed and manually corrected by PE read mapping. Finally, the draft sequence was annotated using an integrated web server, CpGAVAS  is double-stranded circular DNA with 157,147 bp in length with 37.14% GC contents. It shows a typical quadripartite structure containing a small single-copy (LSC) region of 17,998 bp, a large single-copy (SSC) region of 86,851 bp, separated by a pair of inverted repeat regions (IRa and IRb) of 26,149 bp. The chloroplast genome contains 123 genes were predicted, including 86 protein-coding genes, 29 tRNA genes and eight rRNA genes, of which 15 genes were duplicated in IR regions.The complete chloroplast genome of C. colocynthis was deduced by its comparison with other 15 chloroplast genomes in Cucurbitaceae or others based on 56 common protein-coding genes. Phylogenetic tree was built by using a maximum likelihood (ML) method of MEGA (v7.0) with 1000 bootstrap replicates (Tamura et al. C. colocynthis was closely related to C. lanatus ssp. vulgaris and C. lanatus ssp. mucosospermus, forming a clade included in Citrullus genus. The Citrullus genus and other species in the family Cucurbitaceae were closely clustered into a clade, and other species in different family belonging to same family were also well clustered into their corresponding clades with high bootstrap value (The phylogenetic relationship of ap value .