Approximately 20% of patients on peritoneal dialysis (PD) in Japan are on combination with once-weekly haemodialysis (HD). This study aimed to compare outcomes of combination therapy and PD alone.This longitudinal study on the Japanese Renal Data Registry included patients on PD from 2010 to 2014. Subjects were followed until the end of 2015. Exposure of interest was combination therapy compared with PD alone. Outcomes were complete transition to HD, all-cause mortality, cardiovascular (CV) mortality and congestive heart failure (CHF)-related mortality. Patients who initiated combination therapy were matched with those on PD alone by propensity scores. Data were analysed using Cox regression models.Among the matched cohort, 608 patients were on combination therapy and 869 were on PD alone. Decline in body weight and residual renal function was more prominent in the combination therapy group. During a median follow-up of 2.5 years, 224 deaths occurred. All-cause mortality {hazard ratio (HR) [95% confidence interval (CI)] 0.56 (0.42–0.75)}, CV mortality [HR 0.48 (0.32–0.72)] and CHF-related mortality [HR 0.19 (0.07–0.55)] were significantly lower, but complete transition to HD was significantly earlier [HR 1.72 (1.45–2.03)] in the combination therapy group. Sensitivity analyses considering the effects of dialysis facilities yielded similar results. Assuming causality, numbers needed to treat to prevent one death per year were 34 patients.Combination therapy was associated with lower all-cause mortality, CV mortality and CHF-related mortality, but earlier transition to HD compared with PD alone, which might be due to better fluid removal by HD.  Combination of once-weekly haemodialysis (HD) with peritoneal dialysis (PD) is a unique type of renal replacement therapy available in Japan , 2. In cOnly a few studies have reported about outcomes of PD + HD therapy. In previous studies, after initiation of combination therapy, authors observed reduction in human atrial natriuretic peptide levels , improveWith the above background, this study aimed to compare outcomes of PD + HD therapy and PD alone using the database from the Japanese Renal Data Registry (JRDR), which is a nationwide cohort of dialysis patients in Japan. Our hypothesis was that PD + HD therapy is associated with lower all-cause mortality, CV mortality and congestive heart failure (CHF)-related mortality by better fluid removal among patients on PD + HD therapy compared with those on PD alone.This is a longitudinal study of the database from JRDR from 2010 to 2015 (SAF 2017-004). Details about JRDR were published previously . In brieThe inclusion criterion was undergoing PD from 2010 to 2014. The observation period terminated at the end of 2015. The exclusion criteria were age <18 years, withdrawal from dialysis or renal transplantation during the study period, unknown combination status with HD, combination of HD with peritoneal lavage only or combination of PD with HD twice or more per week. Those who transitioned from HD to PD were also excluded. Thus, included patients were those who initiated renal replacement therapy by PD, and those who switched to combination therapy or those who remained on PD alone.Exposure of interest was PD + HD therapy compared with PD alone. PD + HD therapy was defined as combination of PD and once-weekly HD. Outcome variables were all-cause mortality, CV mortality and CHF-related mortality. Time to transition to HD was also compared.Data were shown as number (%), mean (SD), or median (interquartile range) as appropriate. Patients who initiated PD + HD therapy from 2011 to 2014 were matched with those on PD alone in the same year according to propensity scores (PSs). PS for PD + HD therapy was derived from age, sex, causes of end-stage renal disease (ESRD), PD vintage, serum blood urea nitrogen, serum creatinine, history of myocardial infarction, history of haemorrhagic stroke, history of ischaemic stroke, history of limb amputation and daily urine volume at the end of the preceding year. Patients who initiated PD + HD were matched with those on PD alone on the logit of PS (±0.25 SD). Patients who were on PD alone were matched only once . Those who initiated PD + HD therapy from 2011 to 2014 were not included in the PD alone group. All-cause mortality, CV mortality, CHF-related mortality and transition to HD were compared using Cox regression analyses. Sensitivity analyses were performed using stratified Cox regression in which data were stratified by PS-matched pairs or dialysis facilities. In addition, shared frailty model analyses were performed in which dialysis facilities were treated as a random effect. In the shared frailty models, data were adjusted for quintiles of PS. In the JRDR database, the modality of renal replacement therapy at the end of each year was available, but not the exact date when combination therapy was initiated or the exact date of transition to HD. Those on PD alone at the end of the preceding year and on PD + HD at the end of the given year were considered to initiate PD + HD therapy in the middle of the given year. Similarly, those on PD or PD + HD at the end of the preceding year and on HD at the end of the given year were considered to have transitioned to HD in the middle of the given year. Time courses of body weight and daily urine volume were compared using mixed effects models. For those on PD + HD therapy or those who transitioned to HD, post-dialysis body weight was considered. Statistical analyses were performed using Stata MP version 15.0 .C-statistics for the models used to estimate PS were 0.74, 0.73, 0.76 and 0.76 in 2011, 2012, 2013 and 2014, respectively. Demographics of PS-matched cohort are shown in The flow of patients in this study is shown in With a median follow-up of 2.5 years, there were 61 and 163 deaths, 30 and 93 CV deaths, and 4 and 31 deaths due to CHF among those on PD + HD therapy and those on PD alone, respectively. All-cause mortality [HR 0.56 (0.42–0.75)], CV mortality [HR 0.48 (0.32–0.72)] and CHF-related mortality [HR 0.19 (0.07–0.55)] were significantly lower in the combination therapy group . AssuminThe time to complete transition to HD (thrice-weekly HD without PD) was compared in the matched cohort. Complete transition to HD was significantly earlier for those on PD + HD therapy [HR 1.72 (1.45–2.03)] . The difWe performed additional matching on serum albumin and body weight  to elimin = 74) and their matched pairs (n = 81) showed similar results  and comparable with that in a previous study at HR 0.60 (0.41–0.86) [2–0.75 anIn this study, we used PS matching to eliminate indication bias as much as possible. As the most common reason to initiate PD + HD therapy was insufficient fluid and solute removal, patients were matched with factors affecting these decisions such as blood urea nitrogen, creatinine, PD vintage and daily urine volume. Patients were also matched with factors affecting outcomes such as age and history of CV events. The two groups were well balanced, and discrimination by the models from which PS was derived was reasonable. PD + HD therapy is almost exclusively performed in Japan, and no data have been available based on a large number of patients that could provide guidance on the optimal time to initiate this therapy or which patients most benefit from the therapy. As a result, some nephrologists start PD + HD therapy early after initiation of PD, believing that HD with little ultrafiltration in addition to PD preserves residual renal function and provides better nutritional status . Many neIn Outcomes of PD patients could be influenced by dialysis facilities , 15. SenThis study had several advantages over the previous study showing that PD + HD therapy was associated with lower all-cause mortality . In the We had expected that the benefit of PD + HD therapy would be more prominent among younger patients or diabetic patients who were more at risk for volume overload and among those on PD for a few years with declining daily urine volume. However, the association between PD + HD therapy and all-cause mortality, CV mortality and CHF death were similar across age, PD vintage, diabetic status and baseline daily urine volume. These results suggest that even those who initiated PD with residual renal function might benefit from early use of PD + HD therapy. However, note that we matched patients who actually switched from PD to PD + HD therapy. It is still unclear whether we should start PD + HD therapy from the beginning of dialysis therapy.To our knowledge, the strength of this study is that this is the largest cohort of PD + HD therapy. As PD + HD therapy is almost exclusively performed in Japan and JRDR covers more than 95% of the dialysis population in Japan, our study presented the best data about outcomes of PD + HD therapy. We also performed multiple rigorous sensitivity analyses to eliminate indication biases or centre effects as much as possible. The analyses excluding frail patients who died within 1 year on PD or analyses considering centre effects did not substantially change the results.Kt/V or protein catabolic ratio (PCR). Moreover, for those on PD + HD therapy, no consensus exists on how to evaluate Kt/V or PCR, and it was unclear when these data were obtained in relation to the timing of HD therapy.This study has limitations. As this is an observational study, there are possibilities of residual confounding. As JRDR is a survey performed at the end of each year, data at the end of the preceding year were used for PS matching, and it was unclear exactly when patients initiated PD + HD therapy within the given year. We had no data immediately before patients transitioned from PD alone to PD + HD therapy. There might have been changes in patient’s health status from the time of matching to the time when they actually initiated PD + HD therapy. Better survival in the PD + HD group might be due to better nutritional status and dialysis adequacy at baseline or after initiation of combination therapy. However, due to the large number of missing data, we could not perform matching on In conclusion, this study showed that PD + HD therapy was associated with lower all-cause mortality, CV mortality and CHF-related mortality, but earlier transition to HD compared with PD alone, which might be due to better fluid removal by HD. The use of PD + HD therapy might improve mortality while maintaining a flexible lifestyle with PD.sfaa173_Supplementary_DataClick here for additional data file.


However, the function of thymol has not been comprehensively studied. Here, we applied an inverse molecular docking approach to identify unappreciated functions of thymol. Potential targets of thymol in humans were identified by the server of DRAR‐CPI, and targets of interest were then assessed by GO and KEGG pathway analysis. Subsequently, homologous proteins of these targets in Caenorhabditis elegans were identified by Blast tool, and their three‐dimensional structures were achieved using Swiss‐Model workspace. Interaction between thymol and the targeted proteins in worms was verified using AutoDock 4.0. Analyses of the targets revealed that thymol could be potentially involved in the glycolysis/gluconeogenesis and fatty acid degradation pathways. To verify the activity of thymol on lipid deposition in vivo, the C. elegans model was established. The lipid content of nematodes induced by high‐dose glucose was determined by Oil Red O and Nile Red staining, and gene expression was assessed by qRT‐PCR. The results showed that thymol might lead to the acceleration of β‐oxidation by upregulating cpt‐1, aco, fabp, and tph‐1, causing the descent of lipid content in nematodes. Our findings indicated that thymol could be potentially used for the treatment of chronic metabolic diseases associated with increased fatty acid deposition.As a natural product isolated from thyme oil in  In this study, we found a novel function of thymol on the inhibition of fat deposition induced by high‐dose glucose in Caenorhabditis elegans based on bioinformatics prediction combined with experimental verification in vivo. Our data suggested that only 1 mg/ml thymol significantly alleviated lipid accumulation induced by 1 mM glucose. Further qPCR analysis revealed that thymol might lead to the acceleration of β‐oxidation by upregulating cpt‐1, aco, fabp, and tph‐1, causing the fall of lipid content in nematodes. Additionally, bioinformatics analysis supported a suspicion about acceleration of β‐oxidation. Obesity is considered to be the result of multiple genetic factors and environmental influences. In modern society, foods and drinks rich in high energy including fat and sugar can be seen everywhere. A high‐fat and high‐sugar diet has become the main diet structure at present and is considered as one of the main causes of obesity caused by the environment.However, sugar‐induced obesity is different from fat‐induced obesity. On the one hand, sugar can inhibit fat degradation; on the other hand, it also can be oxidized and decarboxylated to acetyl‐CoA that is the material for the synthesis of fatty acid. As we have known that glycolysis glucose is metabolized to acetyl‐CoA from pyruvate by pyruvate dehydrogenase under aerobic conditions, and acetyl‐CoA is the substrate of lipid biosynthesis. The rising blood glucose level triggers the release of insulin, leading to the dephosphorylation and activation of acetyl‐CoA carboxylase (ACC) by insulin‐dependent protein phosphatase. ACC catalyzes the formation of the first intermediate “malonyl‐CoA” of fatty acid synthesis, which suppresses carnitine acyltransferase I, thereby preventing fatty acid entry into the mitochondrial matrix for degradation through β‐oxidation  have provided advice on daily diets urging a limited consumption of rapidly digestible starches and sugars Brouns, . A low‐eMonarda punctate, thymol (2‐isopropyl‐5‐methylphenol) has antimicrobial . The eggs of worms were incubated on nematode growth medium (NGM) agar plates at 20°C and seeded with a live Escherichia coli OP50 (E. coli OP50) as food resources.The wild‐type 2.2https://zinc.docking.org/substance/1529592).DRAR‐CPI server, a tool of inverse molecular docking, was developed to predict the drug target based on structural characteristics of protein , a web portal for undermining the integrated function of multiple genes    and the addition of hydrogens (X‐ray diffraction technology cannot obtain the coordinate data for H) in GAPDH molecule, and the setting of active keys number in thymol. The second step is the setting of search sites, mainly including the adjustment of grid size in “Grad Box.” The third step is the establishment and operation of Autogrid parameter file with GPF format for thymol, generating GLG file and a series of MAP files of GAPDH that contain information of van der Waals forces, electrostatic forces, and dissolvent forces of different atom probe et al. The fourth step is the construction and running of Autodock parameter file with DPF format for thymol. Finally, we will achieve a DLG file of thymol. Docking files of GAPDH and thymol should be prepared for Pymol analysis, including the selection of thymol with the lowest energy, the sequence matching for GAPDH and thymol in Notepad++. In the end, a PDB file from Notepad++ needs to be opened in Pymol for the visualization and analysis of GAPDH‐thymol docking. We can adjust the appearance of their conformation and search targeting site of thymol by observing its hydrogen bonding site in Pymol.Inverse docking was analyzed by Autodock 4.0, including AutoGrid and AutoDock. AutoGrid calculates related energy in a grid containing Van der Waals force, electrostatic force, and hydrogen bond force, while AutoDock conducts conformational search and evaluation using the Lamarckian Genetic Algorithm. For example, the docking of thymol with GAPDH could be realized by the following steps. The first step is the preparation of the system's coordinate files (PDBQT file) of GAPDH and thymol, including the removal of H2.6Caenorhabditis elegans at L4s were permeabilized through fixation, freezing and thawing, and dehydration process. Permeabilized worms were stained with ORO in the dark overnight at 1.07 g, 28°C. Decolorization of nematodes was then performed with 1, 2‐propylene glycol and washed with PBS buffer. The retention of ORO in nematodes could be observed with a 5× anatomical lens. The quantitative analysis was carried by the method of elution by ethanol. Specifically, quantified nematodes were eluted with absolute ethyl alcohol after discarding PBS buffer by centrifugation, and the collected supernatant was transferred to a 96‐well plate to measure the adsorption of ORO at 510 nm using a microreader.Worms at the characteristic L4 larvae stage were chosen for synchronization. The procedure for glucose induction treatment was carried out as described previously Zhao, . BrieflyE.coli OP50 and allowed to equilibrate for a minimum of 2 hr. Then, L1s obtained from bleaching were transferred to the plates and allowed to grow for the desired length of time. Stained nematodes were imaged under a 10× objective under a dark field and red fluorescence illumination with an inverted fluorescent microscope.Nile Red (Sigma) staining was also performed as described  and purified with DNase I, RNase‐free (Thermo Scientific™). cDNA was obtained using M‐MuLV First Strand cDNA Synthesis Kit (Sangon). Real‐time PCR was performed using SYBR GreenⅠprobes by QuantStudio® 3 System . These analyses revealed GPD‐1, GPD‐2, GPD‐3, GPD‐4, H24K24.3, TPI‐1, PYK‐1, PYK‐2, 1DH‐1, KAT‐1, T02G5.7, and FBP‐1 in nematodes. However, their 3D structures remain unknown except for TPI‐1. Therefore, we constructed the 3D structure of the remaining protein by homology modeling technology. Homology modeling is a technique that can build a 3D structure for proteins based on the 3D structure of a similar protein. A homology model can provide an alternative for subsequent receptor‐ligand analyses such as molecular docking.To confirm the activity of thymol on lipid synthesis and metabolism in vivo, we chosen nematodes as experimental models due to their advantages such as a short cycle, a fast reproduction rate, and high homology with human genes. Homologous genes of thymol's targets in nematodes were identified by Blast analysis  in nematodes is inheritable across several generations Zhao, . So we c3.4cpt‐1), acyl‐CoA oxidase (aco), fatty acid‐binding protein (fabp) in the process of β‐oxidation and tryptophan hydroxylase (tph‐1) in the upstream pathway of β‐oxidation. The result of qRT‐PCR suggested that the decreased level of cpt‐1, aco, fabp, and tph‐1 in nematodes exposed by 1 mM glucose was upregulated after the exposure of thymol  triggered by the rising blood glucose level. Therefore, we suspected that the reduction of fat accumulation by thymol might be achieved by regulating the upstream glycolysis pathway and downstream of fatty acid β oxidation. To confirm this idea, we determined the transcriptional level of key genes correlating with β‐oxidation including carnitine palmitoyltransferase  and obesity. These provide us with proof that this method has reliability for the prediction of thymol.In silico approaches can improve our ability to predict drug targets, thereby streamlining and accelerating the discovery process of a novel function of the drug. In this study, we found that thymol has a potential interaction with proteins associated with glycolysis/gluconeogenesis and fatty acid degradation pathway through reverse molecular docking approaches. These proteins included GAPDH, ADH1A, ADH1B, TPI‐1, PKLR, ADH7, LDHB, ACAT1, and FBP1 in humans. The homologous proteins in nematode are GPD‐1, GPD‐2, GPD‐3, GPD‐4, H24K24.3, TPI‐1, PYK‐1, PYK‐2, 1DH‐1, KAT‐1, T02G5.7, and FBP‐1, respectively. Some of the putative thymol targets have been identified as having an association with fat storage previously. For example, cpt‐1, aco, fabp, and tph‐1. So thymol probably accelerates β‐oxidation according to the determination of qRT‐PCR combined with bioinformatics prediction. Furthermore, we suppose that thymol may accelerate glucose metabolism by regulating the targets of GPD‐1, GPD‐2, GPD‐3, GPD‐4, PYK‐2, 1DH‐1, and 1MO0 in the glycolytic pathway, causing the reduction of blood glucose level and ACC inactivation. ACC inhibition resulted in the reduction of malonyl‐CoA, which releases carnitine palmitoyltransferase (CPT) from inhibition and causes the transport of fatty acids into the mitochondria for β‐oxidation. And the protein targets we predicted including H24K24.3, KAT‐1, and T02G5.7 in the fatty acid degradation pathway may be located downstream of key genes correlating with β‐oxidation ; Formal analysis ; Investigation ; Methodology ; Software ; Validation ; Visualization ; Writing‐original draft . Liangbin Hu: Conceptualization ; Funding acquisition ; Project administration ; Supervision ; Writing‐review & editing . Xiao‐Hui Zhou: Conceptualization ; Writing‐review & editing . Yanyan Zhao: Project administration ; Software ; Supervision . HaiZhen Mo: Project administration ; Writing‐review & editing . Hongbo Li: Project administration ; Supervision ; Writing‐review & editing . Wei Zhou: Conceptualization ; Funding acquisition ; Project administration ; Writing‐review & editing .This study does not involve any human or animal testing.Table S1Click here for additional data file.


Sex‐specific differences affect multiple aspects of HIV infection, yet few studies have quantified HIV levels in tissues from women. Since an HIV functional cure will likely require a major reduction of infected cells from most tissues, we measured total and intact HIV DNA and the HIV transcription profile in blood, gut, genital tract and liver from HIV‐positive antiretroviral therapy (ART) ‐treated women.Peripheral blood mononuclear cells (PBMC) and biopsies from the gastrointestinal  and genital  tracts were collected from 6 ART‐treated (HIV RNA < 200 copies/mL) women. HIV DNA  and levels of read‐through, initiated , 5’elongated, polyadenylated and multiply spliced HIV transcripts were measured by droplet digital PCR. Immunophenotyping of cells was performed using Cytometry by time of flight (CyTOF).We detected total HIV DNA in all tissues and intact HIV DNA in blood, ileum, colon, rectosigmoid and ectocervix. Initiated HIV transcripts per provirus were higher in PBMC and endometrium than in ileum, colon, rectosigmoid, ectocervix or endocervix, and higher in the rectum than either ileum or colon. 5’Elongated HIV transcripts per provirus were comparable in PBMC and endometrium, but higher than in gut or cervical samples. Polyadenylated and multiply spliced HIV transcripts were detected in PBMC , but rarely in the tissues.These results suggest tissue‐specific differences in the mechanisms that govern HIV expression, with lower HIV transcription in most tissues than blood. Therapies aimed at disrupting latency, such as latency‐reversing or latency‐silencing agents, will be required to penetrate into multiple tissues and target different blocks to HIV transcription. Friedman tests were performed using R to determine that there is significant variation in HIV levels across tissues. Post hoc tests for individual tissue comparisons were performed using multilevel mixed‐effects negative binomial regression using Stata, and corrected for multiple comparisons by Benjamini–Hochberg and Bonferroni methods using R. The number of copies of a given HIV transcript (or HIV DNA) was modelled as the outcome variable, tissue type was a covariate and a random offset was assumed for each individual to ensure that the analysis proceeds in a pairwise manner. The exposure variable was defined as the number of tissue cells for Figure 2.7The study sponsors had no role in the study design, in the collection, analysis and interpretation of data, in the writing of the report or in the decision to publish.33.13 and the median CD4/CD8 ratio was 1.0 [0.5 to 2.4].The study participants included six women who had been living with HIV for a median [range] of 15.2 [7.4 to 23.5] years and taking ART for 11.9 [7.3 to 23.0] years  in PBMC, gut , genital tract  and liver. In the women in whom we detected the highest levels of total HIV DNA, we also quantified intact HIV DNA  in PBMC, ileum, colon, rectosigmoid, ectocervix and endometrium Figure . Using aIntact proviruses were detected in PBMC from three out of three women, in ileum and colon from two of three women and in rectosigmoid and ectocervix from one of three women Figure . The nump < 0.05; Figure p < 0.05; Figure To help account for differences between tissues in the percent of all cells that are T cells, we normalized the total and intact HIV DNA levels to TCRα mRNA expression, as previously described , 66. The+ T cells will contribute to the total TCRα mRNA expression but are not usually infected by HIV, we further normalized the HIV DNA levels per TCRα mRNA by the percent of CD4+ T cells. With this normalization, the total HIV DNA tended to be higher in gut tissues compared to PBMC or endometrium , 5’elongated, polyadenylated and multiply spliced HIV transcripts in the PBMC and tissue samples Figure . To accop < 0.05; Figure Likewise, levels of 5’elongated HIV RNA per provirus were higher in PBMC than in all gut sites, higher in endometrium than in ileum or colon, higher in the rectum than in ileum, and higher in the ileum than in endocervix  and the HIV transcription profile among multiple tissues from ART‐treated women.+ T cells is higher in the gut than in the blood, and demonstrate for the first time that the frequency of HIV‐infected T cells may be higher in the ectocervix than endocervix or endometrium.We detected total HIV DNA in all tissues and intact HIV DNA in blood, ileum, colon, rectosigmoid and ectocervix, demonstrating that the gut, genital tract and liver are all sites of HIV persistence in ART‐treated women. Furthermore, when normalized by T‐cell content, the total HIV DNA was higher in the gut and ectocervix than in the blood, endocervix, endometrium or liver. These data suggest that, as previously observed in HIV‐positive ART‐treated men , 72, 73,HIV transcription initiation was higher in blood and endometrium than in gut and cervical tissues. The differences between blood and gut resemble prior findings from HIV‐positive ART‐suppressed men , in whomHIV transcription initiation was higher in endometrium than in other female genital tract tissues or gut, suggesting that there are regional differences within the female genital tract, with the endometrium being a “hotspot” for HIV transcription. Levels per provirus of elongated HIV transcripts were also higher in PBMC and endometrium than in gut and cervical tissues. The increased transcription in the endometrium did not appear to be the result of peripheral blood contamination. In contrast to initiated and elongated transcripts, polyadenylated and multiply spliced HIV transcripts were less frequently detected in all tissues, including the endometrium, compared to blood. If this finding does not just reflect sampling fewer total infected cells from the tissues, it may indicate greater constitutive blocks to HIV transcriptional completion and/or splicing in these tissues, which could further limit virus production in the tissues. Together, these data suggest that the blocks to HIV expression, and likely the mechanisms that govern HIV latency, differ between peripheral blood and tissues from the gut, genital tract and liver.+ T cells, whole tissue biopsies were used to measure HIV DNA and RNA, which would be expected to result in fewer infected cell equivalents per PCR well and less chance of detecting more rare HIV transcripts (such as multiply spliced HIV RNA). However, by avoiding tissue collagenase treatments and cell sorting prior to HIV DNA and RNA quantification, we were able to minimize the effect of sample processing on the HIV transcription profile. Moreover, by analysing whole tissue samples, we were also able to detect HIV DNA and RNA that may exist in cells other than T cells, such as myeloid cells [Limitations of the study should be acknowledged. First, the gut and genital tissues were available from only six participants, whereas liver biopsies were available only from three participants, which limited our ability to draw conclusions about the liver. Second, instead of purifying CD4id cells , 75, 76.5Our findings demonstrate that blood, gut, liver and female genital tract are all sites of HIV persistence in ART‐treated women, and suggest tissue‐specific differences in the mechanisms that govern HIV latency, with less HIV transcription in most tissues than blood. These results have important implications for understanding HIV persistence and developing strategies aimed at HIV cure. Future studies should investigate the cellular and viral factors that regulate HIV latency in different tissue sanctuaries both in HIV‐positive men and women in order to find effective drugs aimed at disrupting HIV latency.The authors have declared no conflict of interest.S.A.Y., N.R.R. and R.G. designed the study and obtained funding. J.P., N.E., R.G. and P.T. recruited participants and provided samples and clinical data. S.M.L. and G.X. performed experiments. P.K. assisted with sample processing. S.M.L., G.X., D.S., S.L., N.R.R. and S.A.Y. analysed data. S.A.Y., J.W. and N.R.R. supervised the work. S.M.L and S.A.Y. wrote the manuscript. All authors edited the manuscript. All authors have read and accepted the final manuscript.Figure S1. CONSORT study flow diagram.Click here for additional data file.Figure S2. Statistical analysis using Wilcoxon signed‐rank test. (A) Quantification of total (LTRs and 5’LTR) and intact (Ψ + Env) HIV DNA per million total tissue cells, (B) per TCRα mRNA expression  and (C) levels of each HIV transcript per provirus (HIV RNA/DNA). Symbol colours denote different tissues; symbols/shapes show different participants; symbols without borders indicate values below the limit of detection; p‐values (Wilcoxon signed‐rank test) are represented in grey for p = 0.06 and black for p = 0.03.Click here for additional data file.Figure S3. Cells analysed per HIV DNA and RNA assay, and total and intact HIV reservoir size per TCRα and per percent of CD4 T cells. (A) Number of analysed cells per HIV DNA ddPCR reaction. (B) Quantification of total (LTRs and 5’LTR) and intact (Ψ + Env) HIV DNA per TCRα mRNA expression  and per percent of all T cells that are CD4+ (measured by CyTOF in 5 out of the 6 participants). (C) Number of analysed cells per HIV RNA RT‐ddPCR reaction. Symbol colours denote different tissues; symbols/shapes show different participants; symbols without borders indicate values below the limit of quantification.Click here for additional data file.Figure S4. Multilevel mixed‐effects negative binomial regression p‐values of the comparison among tissues in HIV DNA. (A) Total HIV DNA (5’LTR and LTR) per cell. (B) Total HIV DNA per TCRα mRNA expression . Significant p values are shown in yellow and green  and non‐significant p values are shown in blue (p > 0.05). Upper limit of colour scheme shows p > 0.10 (dark blue).Click here for additional data file.Figure S5. Multilevel mixed‐effects negative binomial regression p‐values of the comparison among tissues in HIV transcripts per provirus. RTh: read‐through HIV RNA; TAR: initiated HIV RNA; longLTR: 5’elongated HIV RNA; PolyA: polyadenylated HIV RNA; TatRev: multiply spliced HIV RNA; PBMC: peripheral blood mononuclear cells; ILE: ileum; COL: colon; RS: rectosigmoid; CVX: ectocervix; ECC: endocervix; EMBx: endometrium. Significant p values are shown in yellow and green  and non‐significant p values are shown in blue (p > 0.05). Upper limit of colour scheme shows p > 0.10.Click here for additional data file.Figure S6. tSNE depiction of CyTOF dataset of blood and tissues from representative donor showing expression profiles of antigens previously associated with HIV reservoir cells. (A) tSNE depiction of memory CD4+ T cells (CD3+CD19‐CD8‐CD45RO+CD45RA‐) isolated from blood or from biopsies from endometrium, ileum, colon or rectosigmoid, all procured during the same study visit. Expression levels of markers of T follicular helper (Tfh) cells , central memory (Tcm) cells , Th17 cells (CCR6) and tissue‐resident memory (Trm) cells  are shown as heatmaps, with the highest expression depicted in red and lowest in blue. These subsets of memory CD4+ T cells have all previously shown to harbour HIV reservoir cells. Circled are some regions of the tSNE in the blood specimens that harbour high expression levels of these markers. The tSNE for all five specimens were run at the same time. (B) The tissue specimens in panel A were run in a separate tSNE without the blood specimen to better resolve the tissue cells. Note that all tissues expressed very high levels of at least one of the markers of reservoir cells.Click here for additional data file.Table S1. List of CyTOF antibodies used in this studyClick here for additional data file.


Metabolic abnormalities may exacerbate the risk of adverse outcomes in patients with type 2 diabetes mellitus. The present study aims to assess the predictive value of HbA1c and lipid variability on the risks of sudden cardiac death (SCD) and incident atrial fibrillation (AF).The retrospective observational study consists of type 2 diabetic patients prescribed with insulin, who went to publicly funded clinics and hospitals in Hong Kong between January 1, 2009 and December 31, 2009. Variability in total cholesterol, low‐density lipoprotein‐cholesterol (LDL‐C), high‐density lipoprotein‐cholesterol (HDL‐C), triglyceride, and HbA1c were assessed through their SD and coefficient of variation. The primary outcomes were incident (1) ventricular tachycardia/ventricular fibrillation, actual or aborted SCD and (2) AF.p < .05).A total of 23 329 patients  were included. On multivariable analysis, HbA1c, total cholesterol, LDL‐C and triglyceride variability were found to be predictors of SCD (HbA1c and lipid variability were predictive of SCD. Therefore, poor glucose control and variability in lipid parameters in diabetic patients are associated with aborted or actual SCD. These observations suggest the need to re‐evaluate the extent of glycemic control required for outcome optimization. Although the exact mechanisms remain unclear, increased long‐term glycemic and lipid variability is hypothesized to lead to endothelial dysfunction via an increase in oxidative stress.The aim of the present study was to assess the predictive value of HbA1c and lipid variability towards aborted or successful SCD, as well as incident AF in type 2 diabetic patients receiving insulin therapy.2This study is a retrospective observational study approved by The Joint Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee . The study cohort contains type 2 diabetic patients with insulin prescribed from any hospitals and outpatient clinics under the Hong Kong Hospital Authority from January 1, 2009 to December 31, 2009. The study cohort from 10 years ago was selected to ensure there is adequate follow‐up especially for rarer events such as ventricular arrhythmias and SCD. Clinical and biochemical data of eligible patients were obtained through the clinical data analysis and reporting system (CDARS), an electronic healthcare record database that integrates data across all publicly funded hospitals and clinics in Hong Kong. This system has been used by our team and other teams to conduct population‐based studies on different cardiovascular diseases,2.1Clinical and biochemical data of the present cohort were extracted from CDARS. The outcomes of the present study are the occurrence of SCD and AF from January 1, 2009 to December 31, 2019. SCD is defined as episodes of VT, VF, or nonspecific cardiac arrest, which were diagnosed under clinical judgment with electrocardiographic or biochemical findings and subsequently coded into hospital records. Both aborted and actual SCD events were included. VT/VF on electrophysiological study is not included. Hypoglycemia‐induced cases of SCD or AF were defined as cases with dextrose infusion during the admission episode or had blood glucose measured ≤3.9 mg/mmol. Demographic details, including age and sex, were extracted. Patients were categorized into four groups based on their age: below age 55, between ages 55 and 64, between ages 65 and 74, above and include age 75. The number of baseline acute hospitalization episodes between January 1, 2004 and December 31, 2008 was also obtained. Furthermore, the average daily dose of different classes of cardiovascular medications and antidiabetic agents was calculated by averaging the multiple between the daily dose frequency and drug dose by all patients with prescriptions of the specific drug class. Eight classes of antidiabetic agents were examined: Insulin, sulphonylurea, biguanide, alpha‐glucosidase inhibitor, thiazolidinedione, dipeptidyl peptidase‐4 inhibitor (DPP‐4I), glucagon‐like peptide receptor‐1 agonist (GLPA), and meglitinide. Data on five classes of cardiovascular drugs were obtained: angiotensinogen‐converting‐enzyme inhibitor/angiotensin receptor blocker (ACEI/ARB), beta‐adrenergic inhibitor, calcium channel blocker (CCB), diuretics, and lipid‐lowering agentsIn terms of patient comorbidities, the number of nondiabetic comorbidities and diabetes‐related complications between January 1, 1999 and December 31, 2008 were obtained. The specific diabetic‐related complications recorded include: (1) Amyotrophy, (2) Arthropathy, (3) Hyperosmolar hyperglycemic state/diabetic ketoacidosis, (4) Hypoglycemia, (5) Neuropathy, (6) Retinopathy/maculopathy, (7) Peripheral vascular disease/angiopathy, and (8) Nephropathy. Patient's past medical history of the following conditions that initiated between January 1, 1999 and December 31, 2008 were also extracted: (1) Chronic kidney disease (CKD), (2) Chronic obstructive pulmonary disease (COPD), (3) Chronic liver disease (CLD), (4) Heart failure (HF), (5) Ischemic heart disease (IHD), (6) Hypertension, (7) Acute myocardial infarction, and (8) Stroke. International classification of disease, ninth edition (ICD‐9) codes was used to extract the study outcomes and pre‐existing comorbidities  Urine albumin/creatinine ratio, (2) Creatinine clearance, (3) 24‐hours total urine protein and albumin, (4) Spot urine protein, albumin, and glucose. Indices from complete blood count include: (1) The absolute number of hemoglobin, basophil, eosinophil, platelet, red and white blood cell, (2) Mean corpuscular volume, (3) Mean corpuscular hemoglobin concentration, (4) Mean corpuscular hemoglobin (MCH), (5) Hematocrit. The presence of anaemia, defined by sex‐based thresholds of below 13 g/dl for male and 12 g/dl for female, were obtained. Blood test results extracted include: (1) Serum creatinine, (2) Serum sodium and potassium, (3) Serum urea and urate, (4) Total serum protein and albumin, (5) Total serum bilirubin, alanine aminotransferase and alkaline phosphatase, (6) Fasting and random blood glucose. The presence of hypoglycemia at baseline and frequency of hypoglycemic episodes were extracted. Hypoglycemia was defined by fasting or random blood glucose below 3.9 mg/mmol.The following blood results between January 1, 2004 and December 31, 2008 were extracted for the evaluation of their mean, variability and baseline value: (1) Total cholesterol, (2) High‐density lipoprotein cholesterol (HDL‐C), (3) Low‐density lipoprotein cholesterol (LDL‐C), (4) Total triglyceride, and (5) HbA1c. LDL‐C results included findings from both direct and calculated measurements. Variability analysis of a biochemical index was only executed on patients with at least three measurements.2.2p value <.05. Kaplan–Meier survival curve was used to portray the difference in actual or aborted SCD and AF survival between patients of different age groups, with the statistical significance of the intergroup difference evaluated using the log‐rank test. Temporal variability of HbA1c and lipid indices were evaluated using calculated parameters of SD and coefficient of variation (CV). CV was measured by multiplying 100‐fold of the value calculated by the ratio SD and mean. SD and CV were used to measure variability since they were less affected by outliers. Whilst SD is independent of the mean, CV is independent of the scale thus more sensitive to small changes to the mean. To identify predictors for shorter time to aborted or actual SCD and AF occurrence, univariable Cox regression was first applied to clinical and biochemical parameters. Patients with missing data were excluded from the analysis. Furthermore, due to their limited prescription towards the study cohort, GLPA and meglitinide were not included. Subsequently, parameters with p value <0.10 were included in the multivariable Cox regression model. Only patients with no missing data for the selected parameters, and at least three measurements for the selected variability predictors, were included in the multivariate models. No data imputation was performed.Statistical analysis was performed using R Studio, and statistical significance was defined as The inter‐relations between HbA1c and lipid variability with intermittent hypoglycemia were evaluated using logistic and Poisson regression. Logistic regression was also used to examine the relationship between baseline hypoglycemia frequency and (1) occurrence of SCD/AF or (2) aborted or actual SCD/AF episodes that were associated with hypoglycemia. Odds ratios (ORs) were reported from logistic and Poisson regression, whereas hazard ratios were reported from Cox regression, along with the 95% confidence intervals.3n = 5511), 55–64 (n = 5745), 65–74 (n = 6032), >75 (n = 6041), with a significant intergroup difference in survival for both aborted or actual SCD (p < .0001) and incident AF  were on lipid‐lowering agents over the course of follow up.The present study included 23 329 patients . The cohort was divided into four age groups: <55 (p < .0001). Logistic regression revealed that baseline hypoglycemia frequency was a significant predictor of aborted or actual SCD  and incident AF . In total, 2512 and 1846 patients experienced incident SCD and AF respectively throughout follow‐up.The baseline biochemical characteristics of the present cohort are presented in Table 3.1p < .05).In the present cohort, 10.3% of patients suffered from at least one SCD event, and 7.7% suffered from AF. Amongst these cases, 25 and 10 cases of patients experiencing aborted or actual SCD and AF were associated with hypoglycemia on admission, respectively. Supplementary Table p < .0001), male ; (2) Clinical: Frequency of baseline acute admissions , number of concomitant diabetic complications , number of discrete nondiabetic comorbidities , hypoglycemic frequency ; (3) Glycemic and lipid variability: HbA1c SD  and CV , total cholesterol CV , LDL‐C SD , triglyceride mean  and CV ; (4) Antidiabetic agent: Sulphonylurea , biguanide ; (5) Cardiovascular medications: Beta‐blocker , diuretic . CV of HbA1c  and LDL‐C , in addition to SD of total cholesterol , p = .034) and triglyceride  were predictive of SCD on univariable analysis but became protective after multivariable analysis.Table n = 13 267) are presented in Table p < .0001); (2) Clinical: Frequency of baseline acute admissions , hypoglycemic frequency ; (3) Antidiabetic agent: Sulphonylurea , biguanide ; (4) Cardiovascular medication: ACEI/ARB , p = .004), beta‐blocker , CCB , diuretic , lipid‐lowering agents .The findings of multivariable regression analysis for incident AF (4The present study demonstrated the following major findings: (1) Clinical and biochemical indices are predictive of arrhythmic occurrence amongst diabetics; (2) Both the mean and variability of HbA1c and lipid indices can predict VT/VF/SCD in diabetic patients; (3) HbA1c variability is associated with hypoglycemia frequency. To the best of our knowledge, the present study is the first to report an association between increased variability in HbA1c and lipid markers with increased risk for VT/VF/SCD amongst diabetic patients.The prognostic values of HbA1c and lipid variability have been increasingly explored over the past decades. However, prior studies have mostly focused on the prediction of all‐cause mortality or cardiovascular eventsFurthermore, structural remodeling as a result of chronic hyperglycemia is also involved in the pathogenesis.Similar to glycemic variability, the increase in oxidative stress with fluctuations in lipid levels due to atherogenic substance release from unstable plaque is hypothesized to underlie the increased arrhythmic risk.The change in HbA1c and lipid variability from predictive of SCD, in univariable analysis, to being protective under multivariable analysis, can be attributed to several causes. The limitation of cohort size and multivariate analysis may have selected for patients of more advanced disease and undergone aggressive control. Given that a J‐shaped association between adverse outcome and both glycemic and lipid indices have been described, patients with high variability that returned to the optimal glycemic and lipid range would have had a better prognosis.4.1There are four major strengths for the present study: (1) The independent, and interdependent predictive effects of clinical and biochemical indices towards SCD and AF were assessed by univariable and multivariable analysis; (2) The predictive values of both the value and variability of HbA1c and lipid indices were assessed to examine the effect of biochemical fluctuations on arrhythmic risk in diabetics; (3) The inter‐relationship between intermittent hypoglycemia, HbA1c and lipid variability and chronic inflammation was examined to elucidate the underlying pathogenic mechanism; (4) Long follow‐up durations permitted the capture of adverse outcomes over a long period.n = 9) and SGLT‐2 transporter inhibitor (n = 0), were not assessed due to the fact that this retrospective study recruited patients in the year 2009, at which these agents were not yet developed. Finally, data on blood pressure, body mass index, echocardiogram, severity of HF and lifestyle were absent, which can affect the patients' cardiovascular health.However, several limitations should be recognized. Firstly, the cohort was limited to type 2 diabetic patients prescribed with insulin, which can limit the generalizability of the findings. Given that insulin is only prescribed for diabetic patients in later stages, an advanced disease state can be inferred for the selected patients. Secondly, the observational nature of the present study leads to inevitable errors from missing data, coding errors, and under‐coding. A causational relationship cannot be established from the findings of the observational study, which can only demonstrate associations. Furthermore, unfortunately, ICD coding does not reflect the frequency of events, thus the frequency of SCD events was not evaluated. The coding also does not reflect whether the VT is sustained or associated with hemodynamic collapse. It is based on the assumption that coded VT is clinically significant as nonsustained VT would be clinically irrelevant thus not coded into the database. It should be emphasized that a diagnosis of VT is not the same as SCD, hence the effect of long‐term glycemic variability on the risk of SCD should be interpreted with caution. Novel therapies, such as GLPA (5Poor glucose control and variability in lipid parameters in diabetic patients are associated with SCD. These observations suggest the need to re‐evaluate the extent of glycemic control required for outcome optimization. Further studies on the predictive value of variability in other glycemic measures, such as fasting and random blood glucose, in addition to other methods of measuring variability, should be performed to further examine the predictiveness of glycemic variability towards arrhythmias in diabetic patients.No conflicts of interest.Supplementary Table 1 ICD‐9 Codes for Outcomes and Comorbidities.Supplementary Table 2. Univariable Predictors for Sudden Cardiac Deathz.Supplementary Table 3. Univariable Predictors for Atrial Fibrillation.Supplementary Table 4. Univariable Predictors for Ventricular Tachycardia.Click here for additional data file.


This study aimed at evaluating the correlation between seven different performance tests and two neuromuscular control tests in youth football players and to evaluate the influence of sex and age groups on test results.One-hundred and fifteen football players  mean age 14 ± 0.7 (range 13–16) years from youth teams were tested at the start of the second half of the competitive season. A test battery including agility t-test, 505 agility test, single-leg hop for distance test, side-hop test, countermovement jump test, 10-m sprint test, 20-m sprint test, tuck jump assessment (TJA) and drop vertical jump (DVJ) was completed.r =  – 0.323–0.523). Boys performed better than girls in all performance tests (p < 0.001) and in TJA total score (p = 0.002). In boys, older players performed better than younger players in the majority of the tests, while there was no clear age influence among girls.Correlations between the seven different performance tests of agility, jump and sprint ability were generally moderate to strong (r = 0.534–0.971). DVJ did not correlate with the performance tests (rho = 0.004 to  –  0.101) or with TJA total score (rho = 0.127). There were weak to moderate correlations between TJA total score and the performance tests  contains supplementary material, which is available to authorized users. Performance tests are often used to evaluate agility, jump and sprint performance in athletes, which are important physical attributes for successful participation in team sports , 33. PowA battery of field-based tests has been recommended to assess various aspects of performance and neuromuscular control in male youth football players . ScreeniThe study was approved by the Swedish Ethical Review Authority: Dnr 2017/294–31. All players and their guardians signed an informed consent prior to participation. One-hundred and fifteen football (soccer) players  mean age 14 ± 0.7 (range 13–16) years from eight youth teams were tested. All teams had scheduled football training at least two sessions per week. Characteristics of the players are displayed in Table Players were asked to refrain from physically exhausting training on the day before testing. All players were recommended to wear tight shorts, t-shirt, short socks and indoor shoes. Due to their preference, five players performed the tests barefoot. Two sports physiotherapists and two physiotherapy students served as test leaders. They used standardized instructions and demonstrated the performance of each test before the players made the practice trials. Testing was carried out for one team at a time and it took about 2 h to complete the test battery for the whole team. Between trials other players were tested to make sure the players recovered between the trials and were able to perform at their maximum capacity. Before testing all players performed a standardized warm-up for 5 min consisting of running and agility drills. The test battery included nine tests performed in the same order for all players: drop vertical jump (DVJ), agility t-test, single-leg hop for distance test, 505 agility test, side-hop test, 10- and 20-m sprint test, tuck jump assessment (TJA) and countermovement jump test. Agility tests, jump tests and sprint tests were used to assess performance and DVJ and TJA were used to assess neuromuscular control (online appendix).Timing gates with photoelectric cells  were used for the agility and sprint tests and an infrared contact mat  was used during the test for vertical jump height.Two GoPro Hero5 cameras that started and stopped synchronically were used for the neuromuscular control tests and for the side hop test. Cameras were positioned in the frontal plane (TJA and DVJ) and the sagittal plane (TJA) and films were used for post-test assessment.p < 0.05. Pearson’s product-moment correlation coefficient (r) was used to analyze linear correlations between all tests except DVJ where Spearman´s rank correlation coefficient (rho) was used. Strength of correlation coefficients was interpreted as follows: negligible ≤ 0.30, weak 0.31–0.50, moderate 0.51–0.70, or strong > 0.71. Bias corrected and accelerated (BCa) 95% confidence intervals was used. One-way analysis of variance (ANOVA) with additional pairwise contrasts was used to estimate between age group effects, on all seven performance tests and number of jumps during TJA. Effect size measures, partial omega-squared (ωp2) was calculated for all main effects between age groups, and Cohen’s d was calculated for all pairwise contrasts. Kruskal–Wallis H test with additional Mann–Whitney U-tests for pairwise comparisons was used to compare between age group differences in mean rank of TJA. Effect size measures, partial eta-squared (ηp2), and Cohen’s d were calculated as main and pairwise effects between age groups, respectively. Independent samples t-tests were used to estimate between sex effects on all seven performance tests and number of jumps during TJA. Mann–Whitney U-test was used to compare differences in mean rank of TJA between sexes. Cohen’s d was calculated for all between-sex effects. Associations between DVJ, sex and age groups were assessed with Fisher’s exact tests. Calculation of Cramer’s V was used to measure the strength of the associations. The following limits were used for interpretation of effect size measures: ωp2 ≥ 0.01 = small effect, ωp2 ≥ 0.06 = medium effect, ωp2 ≥ 0.14 = large effect; d ≥ 0.2 = small effect, d ≥ 0.5 = medium effect, d ≥ 0.8 = large effect; ηp2 ≥ 0.01 = small effect, ηp2 ≥ 0.06 = medium effect, ηp2 ≥ 0.14 = large effect.Analyses were performed with SPSS , and all analyses were two-sided, with the significance level set at r =  – 0.487). Agility t-test was strongly correlated to 505-agility test (r = 0.775). Correlations between single-leg hop for distance, side hop and CMJ were moderate (r = 0.534–0.694). 10 m sprint test and 20 m sprint test were strongly correlated (r = 0.971). There were weak to moderate correlations between TJA total score and the seven performance tests (r =  – 0.329–0.523). Number of jumps during TJA did not correlate with any variable (r = 0.017–0.153). The DVJ did not correlate with the seven performance tests (rho = 0.012 to  – 0.064) nor with TJA total score (rho = 0.149) (Table Correlations between different performance tests were moderate to strong (r = 0.534–0.971), except between 505-agility test and side hop (p < 0.001) and in TJA total score    , while there was no age influence in the other tests  Table . There w1) Table . In boys6) Table . In girls Tables .Table 3AThe main findings of the present study were that sprint performance was moderately to strongly correlated with agility and jump performance, and there were weak to moderate correlations between performance tests and TJA, while DVJ did not correlate with the other tests. Boys performed better than girls in performance tests and TJA, and in boys there was an age effect where older players performed better. In contrast, no clear age effect was evident in girls.r2) in explosive jump and endurance jump capacity explained by the performance in the agility and sprint tests were 38–49% and 36–60%, respectively. Despite the different characteristics of the tests, almost all performance tests showed moderate to strong correlations. The strongest correlations were seen between sprint and agility tests, which is expected since these tests measure similar physical abilities. Sprint and jump performance was also strongly correlated, which supports previous data on youth male football players [Different performance tests were chosen to measure different aspects of football relevant performance that may be motivating for the players. Jump performance is a functional measure of power in football players  and jump players  and on m players . Acceler players  and no cBoys performed better than girls in performance tests and TJA, which is expected and may be explained by sex differences in physiological characteristics. In boys there was an age effect where older players performed better, while no clear age effect was evident in girls. Age-related differences in performance in male youth football players are probably related to maturation . SimilarThere were contradictory results on the two tests assessing neuromuscular control, where TJA revealed reduced neuromuscular control in girls compared to boys, but there was no sex difference in DVJ. The DVJ and TJA seem to test different aspects of neuromuscular control and may be used complementary. However, the ecological validity of assessing DVJ for football players has been questioned , which iStrengths with the present study design were the utilization of field tests, commonly used in football players, together with standardized instructions and adequate technical equipment. Players performed a structured warm-up before testing and were allowed practice trials before tests and sufficient rest between tests. Subjective assessments of neuromuscular control tests were performed by an experienced sport physiotherapist.Study limitations involve small samples when comparing different age groups split by sex, and there were unequal distributions in age groups in boys and girls; however, differences were not significant. The test battery included nine different tests to assess different aspects of performance and neuromuscular control, but specific tests of muscle strength and balance were not included. Assessment of adolescents entail special challenges since young athletes might display greater heterogeneity in performance of tests due to large variations in physical maturity, fitness and neuromuscular control compared to adults and elite athletes . Some plThe present data can be used by coaches and medical professionals when deciding on which tests to include in a test battery for football players. The performance tests and neuromuscular control tests assessed in the present study may be used to determine individual player strengths and limitations and evaluate responses to a training regimen or to monitor development during maturation. The results of the tests may be compared among players within the team or with normative data. The present cohort had symmetrical limb performance and, therefore, data for both legs were used integrated in the analyses. However, limb asymmetry might be apparent in football players and could be evaluated in for instance the return to play after injury .Sprint performance was moderately to strongly correlated with agility and jump performance, and performance tests were weakly to moderately correlated to TJA, while DVJ did not correlate with the other tests. Boys performed better than girls on all performance tests and in TJA. Age effect on test performance was evident in boys but not in girls.Supplementary material 1 (DOCX 31 kb)Below is the link to the electronic supplementary material.


Halyomorpha halys) and identified a group of secretory SG phospholipase C (PLC) genes with highest sequence similarity to the bacterial homologs. Further analyses demonstrated that they were most closely related to PLCs of Xenorhabdus, a genus of Gammaproteobacteria living in symbiosis with insect‐parasitizing nematodes. These suggested that H. halys might acquire these PLCs from Xenorhabdus through the mechanism of horizontal gene transfer (HGT), likely mediated by a nematode during its parasitizing an insect host. We also showed that the original HGT event was followed by gene duplication and expansion, leading to functional diversification of the bacterial‐origin PLC genes in H. halys. Thus, this study suggested that an herbivore might enhance adaptation through gaining genes from an endosymbiont of its parasite in the tripartite parasitic and symbiotic interactions.Herbivory is a highly sophisticated feeding behavior that requires abilities of plant defense suppression, phytochemical detoxification, and plant macromolecule digestion. For plant‐sucking insects, salivary glands (SGs) play important roles in herbivory by secreting and injecting proteins into plant tissues to facilitate feeding. Little is known on how insects evolved secretory SG proteins for such specialized functions. Here, we investigated the composition and evolution of secretory SG proteins in the brown marmorated stink bug  genes from Xenorhabdus through the mechanism of horizontal gene transfer (HGT), likely mediated by a nematode during its parasitizing an insect host. This study revealed that an herbivore might enhance adaptation through gaining genes from an endosymbiont of its parasite in the tripartite parasitic and symbiotic interactions. In plant pathogens, new effectors can emerge through gene duplication, the acquisition of a secretion function, or the mechanism of horizontal gene transfer (HGT) from a different species , a polyphagous hemipteran species that has dramatically increased its populations globally with high versatility to feed on a wide array of plants  leaves.2.2Halyomorpha halys adults for total RNA extraction. mRNA molecules were enriched using the oligo‐dT beads and libraries were constructed for RNA sequencing on the Illumina HiSeq 2,500 platform to generate 100‐bp paired‐end reads. Raw reads were cleaned by removing the adapters and low‐quality sequences using Seqtk (https://github.com/lh3/seqtk) to generate a de novo RNA‐seq assembly with the Trinity program by its default parameters  with a standalone Perl script  against the NCBI non‐redundant (NR) database (updated until 2019/03/22), and the gene ontology (GO), enzyme code, and InterPro analyses were run on Blast2GO  using InterProScan. The hit proteins were mapped to the genome loci. Only one protein sequence (the longest one) per gene locus was retrieved for phylogenetic analysis.To identify all possible PLC genes encoded by the 2.5Arabidoposis thaliana), mammal (Mus musculus), insects that included Drosophila melanogaster and several orders of species, spider (Parasteatoda tepidariorum), mite (Tetranychus urticae), fungus, and bacterium species that belong to a number of phyla, and two D. melanogaster PLA proteins were used as outgroups , and the poorly aligned regions were trimmed using TRIMAL (v1.4.1) based on a gap threshold of 0.25. The best‐fit models of protein evolution, which were LG+G for the general PLC phylogenetic analysis and WAG+G + F for the HGT‐related phylogenetic analysis, were determined using ProtTest (v3.4.2) according to Bayesian information criteria . First‐strand cDNA was synthesized using HiScript® II Q RT SuperMix with gDNA wipe and the Oligo(dT)23VN primer . Quantitative reverse transcription PCR (qRT‐PCR) was carried out on LightCycler® 480 II  with a reaction volume of 10 μl mixture containing 1 μl of cDNA, 5 μl of 2×QuantiFast® SYBR® Green PCR Master Mix , 0.2 μl of forward primer, 0.2 μl of reverse primer and 3.6 μl of nuclease‐free water. The sequences of PCR primers are listed in Table H. halys 18S rRNA , salivary gland, midgut, and muscle of 33.1H. halys and sequenced the transcriptome using the Illumina HiSeq 2,500 platform. Four replicates of paired‐end RNA‐seq reads were pooled, yielding a total of 190.8 million raw reads, which, after trimming, generated 186.6 million clean reads. Using these trimmed reads, we assembled a de novo transcriptome consisting of 67,108 transcripts, from which the longest transcript of each transcription locus was extracted to generate 53,618 UniGenes with a mean length of 736‐bp. To assess the completeness of this assembly, we performed BUSCO analysis, revealing the presence of 94.9% BUSCO genes, including 91.0% complete genes and 3.9% fragmented genes, with only 5.1% missing in this transcriptome assembly.We collected the salivary gland tissue from adult H. halys gene.To annotate the salivary gland transcriptome, 53,618 UniGenes were translated into 11,026 proteins with minimum length of 100‐aa, out of which 9,871 sequences containing putatively complete N‐termini were selected for the prediction of secretory characteristics. 691 were predicted to be secretory owing to the presence of N‐terminal signal peptide and absence of transmembrane domain. We further identified 523 of their corresponding genome‐derived gene models  of secretory salivary gland proteins had the best hits from insect species as expected, a small number of proteins surprisingly had those of microorganisms, including bacteria, fungi, and archaea, as the best hits , TIM beta/alpha‐barrel domain‐containing protein (IPR017946)  Table . ThroughH. halys PLC genes evolved, we constructed a phylogenetic tree using the PLC proteins sequences derived from a variety of organisms including bacteria, fungi, plant, mammal, insects, and arachnids  and PLCs of mammal, insect, and plant. The second clade, named the Arthropod Clade, comprised seven Hhal‐PLCs (5–11) and PLCs of all arthropod species including various Orders of insect species and arachnids. The third clade, designated as the HGT Clade here, however, only comprised 10 Hhal‐PLCs (12–21) and bacterial and fungal PLCs. Noteworthily, these 10 Hhal‐PLCs were clustered together with Gammaproteobacteria PLCs with strong support (posterior probability = 1), indicating that the genes encoding these 10 Hhal‐PLCs were gained by H. halys from the class of Gammaproteobacteria through the mechanism of HGT  is the mRNA‐lymphokine‐activated killer T‐cell‐originated protein kinase gene (LOC106678610) whose top BLAST hits against NCBI Non‐Redundant database were all from insect species, it is unlikely that they were derived from the contaminant symbionts during the genome sequencing. To further rule out this possibility, we extracted the genomic DNA from the legs of H. halys, the tissue where symbiont contamination is less likely to occur, for gene‐specific amplification of the fragments from four HGT‐related Hhal‐PLC genes  which is 766‐aa Figure . Similar3.4Xenorhabdus, with strong support (posterior probability = 1). Interestingly, the non‐Xenorhabdus PLCs in this clade formed their own subclade, showing closest phylogenetic relationship with those (Xe_vie‐PLC2 and Xe_vie‐PLC3) of Xenorhabdus vietnamensis, suggesting possible horizontal gene transfers occurring between these species, likely starting with a transfer from X. vietnamensis to one of these non‐Xenorhabdus species  included in the insect genome sequence and adjacent to other insect conserved genes; (ii) identified by RNA Sequencing and qRT‐PCR analyses which only selected eukaryotic genes encoding poly(A) tail‐containing mRNAs; and (iii) detected from insect leg DNA with low symbiont contamination likelihood. Expression and sequence analyses revealed that these HGT Clade of genes contained both secretory and non‐secretory types with variable gene expression across tissues, indicating functional diversification of PLC genes in H. halys following HGT. PLC is a class of enzymes that catalyze the hydrolysis of phosphatidylinositol 4,5‐bisphophate (PIP2) into inositol 1,4,5‐trisphosphate (IP3) and diacylglycerol (DAG), both of which serve as important second messengers to regulate a variety of cellular processes   ; Data curation ; Writing‐original draft . Shaoming Miao: Data curation ; Validation . Yanfang Yin: Data curation ; Validation . Yanjuan Zhu: Data curation ; Validation . Paul D. Nabity: Data curation ; Writing‐review & editing . Raman Bansal: Writing‐review & editing . Chenxi Liu: Conceptualization ; Data curation ; Validation ; Writing‐original draft .Figure S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Substantial evidence supports an association between physical activity and cognitive function. However, the role of muscle mass and function in brain structural changes is not well known. This study investigated whether sarcopenia, defined as low muscle mass and strength, accelerates brain volume atrophy.A total of 1284 participants with sarcopenic measurements and baseline and 4-year follow-up brain magnetic resonance images were recruited from the Korean Genome and Epidemiology Study. Muscle mass was represented as appendicular skeletal muscle mass divided by the body mass index. Muscle function was measured by handgrip strength. The low mass and strength groups were defined as being in the lowest quintile of each variable for one’s sex. Sarcopenia was defined as being in the lowest quintile for both muscle mass and handgrip strength.P < 0·001). The significantly greater atrophy in parietal GM was observed in the sarcopenia group compared with the control group. In a joint regression model, low muscle mass, but not muscle strength, was an independent factor associated with a decrease of GM volume.Of the 1284 participants, 12·6%, 10·8%, and 5·4% were classified as the low mass, low strength, and sarcopenia groups, respectively. The adjusted mean changes of gray matter (GM) volume during 4-year follow-up period were − 9·6 mL in the control group, whereas − 11·6 mL in the other three groups (Sarcopenia is associated with parietal GM volume atrophy, in a middle-aged population. Maintaining good levels of muscle mass could be important for brain health in later adulthood.The online version contains supplementary material available at 10.1186/s12877-021-02581-4. It is widely known that aging is accompanied by changes in body composition, primarily a reduction in lean body mass and growth in fat mass. The term strength . It potestrength .Substantial evidence from cross-sectional studies shows an association between muscle function and brain function , 4 or stIn this study, we investigated whether sarcopenia, defined as low muscle mass and strength together, is associated with brain volume atrophy in a middle-aged population, along with the independent effects of low muscle mass and function on brain volume reduction.All study subjects were taken from the Ansan cohort of the Korean Genome Epidemiology Study (KoGES), an ongoing population-based cohort study that began in 2001. KoGES participants have been biennially evaluated for demographics, medical illness, and medications. During the 6th and 7th examination cycles 2011–2014), brain magnetic resonance imaging (MRI) scans and handgrip strength data were acquired. Four years later, follow-up brain MRI scans were conducted during the 8th and 9th exams (2015–2018). Further details are described elsewhere [–2014, brn = 1), 2) any neuropsychiatric disorders, including anxiety, mood, or psychotic disorders (n = 6), 3) stroke (n = 29), 4) any cancer (n = 21), 5) diabetes mellitus (n = 553), 6) missing data (n = 11). After those exclusions, 1284 participants were enrolled in this study. Each participant signed an informed consent form. This study was performed according to the principles of the Declaration of Helsinki of the World Medical Association and was approved by the Institutional Review Board of Korea University Ansan Hospital.In this study, we included 1905 subjects who completed baseline dual-energy X-ray absorptiometry (DEXA) with their handgrip strength tests and had baseline and follow-up brain MRI scans. The DEXA exams were performed from the 5th examination cycle (2009–2010). We excluded 621 subjects with the following conditions: 1) past history of dementia  was calculated as weight in kilograms divided by height in meters squared. Waist circumference was measured to the nearest 0·5 cm in a horizontal plane at the level of the umbilicus at the end of a normal expiration.Blood was drawn for biochemical analysis after an overnight fast. Plasma glucose, serum triglycerides, and high-density lipoprotein cholesterol levels were measured with an autoanalyzer . High-sensitive C-reactive protein (hsCRP) levels were measured by an immunoassay . Insulin was measured with an immunoradiometric assay kit  using a Packard γ counter system.All 3D T1 MRI scans were acquired using a GE Signa HDxt 1·5 T MRI scanner with an 8-channel head coil. The detailed MRI protocols are described in a previous study . Brain MWhole body composition was determined using DEXA. Total body fat mass (g) and total and regional lean mass (g) were measured using a Lunar DPX-MD + densitometer . Appendicular skeletal muscle mass  was defined as the sum of the lean soft tissue mass of the arms and legs , and BMIDiabetes mellitus was diagnosed when the fasting plasma glucose was ≥7·0 mmol/L, 2-h plasma glucose was ≥11·1 mmol/L after a 75 g oral glucose tolerance test, or participants took anti-diabetic medication . InsulinParticipants were classified into four groups according to the presence or absence of low muscle mass and function. Low mass was defined as being in the lowest same-sex quintile of ASM/BMI but not the lowest quintile of handgrip strength. Low strength was defined as the being in the lowest same-sex quintile of handgrip strength but not the lowest quintile of ASM/BMI. Sarcopenia was defined as being in the lowest same-sex quintile of both ASM/BMI and handgrip strength. Controls were defined as those not in the lowest quintile of either ASM/BMI or handgrip strength. The cutoff point for the lowest quintile of ASM/BMI was 0·776 for males and 0·507 for females, and that for handgrip strength was 31·9 kg for males and 18·8 kg for females.P value < 0·05 was considered statistically significant. Statistical analyses were performed using SAS version 9.1 for Windows .Baseline characteristics were compared among four groups classified by muscle mass and strength using one-way analysis of variance (ANOVA) with the Tukey post hoc test for numeric variables. The chi-square test was used to compare categorical variables. Non-normally distributed variables, such as hemoglobin A1c, triglycerides, HOMA-IR, hsCRP, and handgrip strength, are presented as the median with interquartile range for each group, and differences were tested after logarithmic transformation. The brain volume change was calculated by subtracting the baseline brain volume from the follow-up brain volume. We compared adjusted mean changes in total and regional brain volume according to groups classified by baseline muscle mass and strength using analysis of covariance (ANCOVA) with the Tukey post hoc test. For parietal GM volumes significantly ssociated with sarcopenia, adjusted volume changes of subcompartment were compared for each group by ANCOVA. We also investigated associations of z-scores transformed from regional brain volume changes across groups by ANCOVA after adjusting for several relevant factors. ICV, age, sex, smoking, alcohol, exercise, education, HTN, heart disease, baseline brain volume, time interval from DEXA to the first MRI scan, and time interval between the baseline and follow-up MRI scans were included as variables. Multivariate linear regression analyses were conducted to compare the effects of ASM and handgrip strength on brain volume changes. The regression models included GM volume changes as the dependent variable, and baseline ASM, handgrip strength and BMI were included separately and together, along with adjustments for the aforementioned variables. A 2. Of the 1284 subjects, 71·2% were classified as the control group, and 12·6%, 10·8%, and 5·4% were designated as the low mass, low strength, and sarcopenia groups, respectively. The clinical characteristics of the four strata of study subjects are listed in Table 2, P < 0·001), and had higher triglyceride, HOMA-IR, and hsCRP levels. The sarcopenia group reported more HTN and heart disease than the control group. The average period between the baseline and follow-up brain MRI scans was 4·2 ± 0·5 years, with no significant differences among the four groups.The mean age of all participants was 58·0 ± 6·0 years , and the mean BMI was 24·5 ± 2·8 kg/mP < 0·001). The mean adjusted difference in GM volume atrophy between the control and sarcopenia group was − 2·12 mL (95% CI − 4·07 to − 0·17 mL) during 4-year follow-up period. The GM volume decrease was similarly higher in the low mass, low strength, and sarcopenia groups compared with the control, and the difference was statistically significant in the low mass and low strength groups . The reduction in total brain and WM volume did not differ significantly among the four groups.Table P = 0·003), parietal (P = 0·002), and occipital GM (P = 0·016) areas .In the lobar brain volume analyses, the change in GM volume over four years differed significantly across groups in the frontal . Model 2 included ASM, handgrip strength, and BMI together with other covariates and showed that low muscle mass and high BMI, not muscle strength, were significantly associated with faster GM reduction (P = 0·034 and 0·012). The standardized regression coefficient values for the effect of ASM and handgrip strength on GM atrophy were 0·14 and 0·08, respectively  was used as the sarcopenia parameter, based on a recent guideline. These strengths allowed us to broden our understanding of the longitudinal effects of muscle mass and function on brain atrophy more comprehensively and concisely than any previous studies.Sarcopenia is associated with parietal GM volume atrophy in a middle-aged population. Large muscle mass is independently associated with the preservation of GM volume in this large, longitudinal follow-up study.Additional file 1: Supplementary Table. Adjusted means of regional volume changes in the parietal lobe according to groups classified by muscle mass and strength.


M age equivalents: 53–56 months), children with ASD were less accurate at tracking owner-object relationships based on possessive pronouns and were less accurate at identifying the property of third parties. We also found that children with ASD were less likely to defend their own and others’ ownership rights. We hypothesise that these results may be attributed to differences in representing the self and propose that ASD may be characterised by reduced concern for ownership and associated concepts.This study investigated how autism spectrum disorder (ASD) impacts children’s ability to identify ownership from linguistic cues (proper nouns vs. possessive pronouns) and their awareness of ownership rights. In comparison to typically developing (TD) children matched on receptive language ( In order to become effective navigators of the social world, children must learn to identify ownership and adhere to rules that regulate interactions with property Brown . IdentifThe use of language to identify and establish ownership emerges early in typical development. TD children use first-person possessive pronouns (e.g. “mine”) to disambiguate objects in their environment from 12 months  has examined accuracy of ownership identification in this population. In Hartley and Fisher , TD chilIt is plausible that children with ASD may experience difficulty identifying ownership when relationships between people and property are established and probed using pronouns rather than proper nouns. Atypical pronoun reversals  are a well-documented feature of language and communication in children with ASD Kanner . While tUnlike proper nouns, which have a fixed referent, personal pronouns require deictic shifting—the speaker/listener must continuously remap the same pronoun to different people depending on who is speaking . In other trials, ownership relationships were stated and tested using possessive pronouns (e.g. “This pencil case is yours” and “Which pencil case is mine?”). Based on prior evidence that children with ASD can have difficulty comprehending pronouns  and 19 TD children  recruited from specialist schools, mainstream schools, and preschools. Samples were closely matched on receptive vocabulary as measured by the British Picture Vocabulary Scale , t(35) = 0.60, p = .55. All children with ASD were diagnosed by a qualified educational or clinical psychologist using standardised instruments . Children with ASD were significantly older than TD children t(35) = 9.85, p < .001, d = 3.20, and had significantly higher CARS scores, t(35) = 13.78, p < .001. All procedures performed in this research involving human participants were in accordance with the ethical standards of the institutional and national research committees. Informed consent was obtained from parents/caregivers prior to children’s participation.Participants were 18 children with ASD , the following items were common across many children: lunch box, shoe, hat, school bag, book, toy, jumper and drinking bottle.Stimuli for the ‘ownership rights task’ included pens and paper, playdough, and a brightly-coloured box with removable lid.Participants were tested individually in their own educational settings and were accompanied by a familiar adult. Children were reinforced throughout the session for attention and good behaviour, but did not receive feedback concerning their performance in the tasks.There were two within-subjects conditions delivered on different days: Naming and Pronoun. Half of the participants in each sample received the Naming condition in the first session, followed by the Pronoun condition in the second session approximately 1 week later. The other half of the participants in each sample experienced the two conditions in reverse order.your lunch box, it belongs to you. This is his lunch box, it belongs to him. This is my lunch box, it belongs to me.”). The order that object-owner relations were highlighted was randomised across participants. An experimenter then placed the three items in the middle of the table  and asked the participant to identify the owner of each object. These questions varied between conditions. In the Naming condition, the owners for each object were named (e.g. “Which lunch box is John’s? Which lunch box is Nina’s? Which lunch box is [child]’s?”). In the Pronoun condition, the owners were referred to using possessive pronouns (e.g. “Which is yours? Which is hers? Which is mine?”). The order of questions was counterbalanced across participants. Children responded by verbally or gesturally indicating which of the three items belonged to the stated owner. An experimenter then removed the objects from the table and initiated the next trial with different objects.Children sat at a table opposite two adult experimenters . Each session began with the experimenters introducing themselves and inviting the child to play . Children then completed three trials. In each trial, one experimenter presented one of the six pre-identified items belonging to the child (e.g. a blue lunch box), plus two other examples of the same type of item that belonged to the experimenters (e.g. a green lunchbox and a yellow lunchbox). Wherever possible, the experimenters’ items were gender neutral in terms of their colour and/or design, so owner-object matching could not be facilitated by awareness of social stereotypes. Children were allowed to explore the items for a few seconds before an experimenter placed each object in front of its owner and verbally stated the object-owner relations. These verbal statements differed between conditions. In the Naming condition, each owner was named explicitly . In the Pronoun condition, owners were referenced using possessive pronouns . The craft-making materials were located in a box next to the table . Based on Kanngiesser and Hood , this taThe session began with the experimenters introducing themselves and inviting the child to play . Children then completed two warm-up trials to establish whether they would intervene if one of the experimenters behaved strangely. Experimenter 1 presented a small cup and stated their intention to “drink a cup of tea”. Experimenter 1 then pretended to drink from the cup upside down for about 30 s while stating “I’m drinking a cup of tea!”. If the child spontaneously corrected this odd behaviour, Experimenter 1 thanked the child and demonstrated the appropriate action. If the child did not spontaneously intervene, Experimenter 2 verbally encouraged the child to correct the unusual behaviour (e.g. “You should tell Nina if she is doing something wrong!”). Following the child’s intervention or the expiration of approximately 30 s, the second warm-up trial was administered. This followed an identical format, except Experimenter 1 pretended to brush their teeth using the handle of a toothbrush rather than the bristles.Following the warm-up trials, Experimenter 1 placed a brightly-coloured box next to them on the table, stating that the contents of the box belonged to them . Children then completed two trials that involved drawing pictures or creating models from playdough . At the start of each trial, Experimenter 2 presented the craft materials (playdough or paper and colouring pens) and suggested that all parties use them to create something . The child and experimenters then used the craft materials to draw a picture or make a model for approximately 1 min. The experimenters ensured that their creations were visually distinct from the child’s creation and from each other. Experimenter 2 then asked Experimenter 1 “what do you think of these drawings/models?”. Experimenter 1 pointed at one of the three drawings/models and stated “I really like this one! I want to keep it and put it in my box and never give it back.” After waiting for a few seconds, Experimenter 1 repeated their intention (“I love this one! I am going to take it and put it in my box and never give it back!”). Experimenter 1 then picked up the identified model/drawing and slowly moved to place it in their box. Experimenter 1 then repeated this behaviour until they had tried to claim all three of the newly-made objects. If the child protested against Experimenter 1’s attempt to claim an object, the object was left on the table. Verbal and nonverbal behaviours were categorised as a protest. Children could protest physically by retrieving an object from Experimenter 1 and possessing it for at least 10 s. Alternatively, children could protest verbally with or without explicitly referencing ownership . After Experimenter 1 had attempted to claim each object, the three objects were placed in the middle of the table and Experimenter 2 asked the child to indicate the appropriate owner for each object . We coded whether children attributed ownership to the person who made the object. After the participant had responded to these questions, the second trial was administered following the same format using the alternate crafting materials.In both the Naming and Pronoun conditions, children were scored out of three on trials involving identification of their objects, identification of objects belonging to the male experimenter, and identification of objects belonging to the female experimenter see Fig. . These dF = 9.61, MSE = 0.28, p < .001, ηp2 = .22, and Population, F = 9.47, MSE = 1.20, p = .004, ηp2 = .21, were qualified by a significant Population × Owner interaction, F = 6.00, MSE = 0.28, p = .004, ηp2 = .15. Bonferroni-adjusted pairwise comparisons showed that TD children (M = 2.89) and children with ASD (M = 2.78) did not differ in accuracy when identifying objects belonging to them (p = .32). However, children with ASD were significantly less accurate than TD children when identifying items that belonged to the male experimenter  and the female experimenter . A repeated measures ANOVA revealed a significant effect of Owner for children with ASD, F = 8.53, MSE = 0.25, p = .001, ηp2 = .33. Children with ASD identified objects belonging to them with significantly greater accuracy than items belonging to the male experimenter (p = .016) and the female experimenter (p = .008), which did not differ in accuracy (p = .70). There was no effect of Owner for TD children (p = .47), indicating no significant differences in accuracy when identifying items belonging to them, the male experimenter, and female experimenter.Significant main effects of Owner, F = 3.93, MSE = 0.80, p = .055, ηp2 = .10. Given our a priori hypotheses, we proceeded to deconstruct the relationship between Population and Condition, though note that the comparisons should be treated with caution as the interaction was marginally significant. Bonferroni-adjusted pairwise comparisons showed that the accuracy of children with ASD (M = 2.59) and TD children (M = 2.81) did not differ in the Naming condition (p = .24). However, TD children (M = 2.89) responded with significantly greater accuracy than children with ASD (M = 2.20) in the Pronoun condition (p = .002). While TD children did not differ in accuracy between the Naming and Pronoun conditions (p = .56), children with ASD tended to respond with greater accuracy in the Naming condition than the Pronoun condition (p = .058).The omnibus ANOVA also detected a borderline Population × Condition interaction, 2 = 6.17, p = .013. These data suggest that that children with ASD were less likely to correct the experimenter’s erroneous behaviour, but they do not necessarily reflect children’s willingness to defend their own and others’ ownership rights. Indeed, corrective behaviour during the warm-up did not significantly correlate with protest behaviour in the subsequent test trials for TD children  or children with ASD .Eighteen TD children corrected the experimenter’s unusual behaviour on at least one warm-up trial (94.74%) in comparison to 11 children with ASD (61%), a significant difference, χChildren were scored out of two corresponding to the number of trials on which they protested against an experimenter attempting to claim ownership of the child’s objects, their own objects, and objects belonging to the other experimenter see Fig. . These dF = 8.51, MSE = 0.22, p < .001, ηp2 = .20, and Population, F = 4.70, MSE = 0.77, p = .037, ηp2 = .12, were qualified by a significant Population × Owner interaction, F = 4.43, MSE = 0.22, p = .015, ηp2 = .11. When the thief attempted to claim the child’s object, TD children (M = 0.79) were significantly more likely to protest than children with ASD . When the thief attempted to claim the other experimenter’s object, TD children (M = 0.63) tended to protest more often than children with ASD . Protest frequencies did not significantly differ between TD children (M = 0.05) and children with ASD (M = 0.06) when the thief claimed ownership of their own object (p = .97). For children with ASD, protest frequencies did not significantly differ between the three owners. By contrast, TD children were significantly more likely to protest when then the thief attempted to claim ownership of items made by the participant (p = .007) and the experimenter (p = .017) than the thief’s own items.Significant main effects of Owner, Children were also scored out of six corresponding to the number of ownership questions answered correctly  = 6.80, MSE = 0.21, p = .002, ηp2 = .16. Children identified objects belonging to them (M = 1.81) with significantly greater accuracy than objects belonging to the thief  and marginally greater accuracy than objects belonging to the other experimenter . The main effect of Population was also significant, F = 6.22, MSE = 0.78, p = .017, ηp2 = .15, indicating that TD children (M = 1.79) responded to ownership questions with significantly greater accuracy than children with ASD (M = 1.37).The results revealed a significant main effect of Owner, This study investigated how ASD affects children’s ability to identify ownership from linguistic cues and their understanding of ownership rights. The ownership identification task revealed that children with ASD responded less accurately than TD children when ownership relationships were stated and probed using possessive pronouns. Children with ASD were also less accurate at identifying the belongings of third parties. The results of the ownership rights task showed that children with ASD were less likely to protest in defence of their own and others’ ownership rights than TD children. Both populations reliably identified each party as the owner of the items they created, but children with ASD were less accurate at assigning ownership on this basis than TD children. Together, these findings suggest that children with ASD are less accurate at tracking owner-object relationships when ownership status is not explicitly stated with reference to proper nouns and they may have reduced sensitivity to breaches in ownership rights.As predicted, children with ASD were significantly less accurate at identifying owner-object relationships when they were required to comprehend possessive pronouns . This finding suggests that previously documented difficulties using and comprehending personal pronouns  were broadly consistent with those generated by Kanngiesser and Hood’s (M age = 3.50 years) in their similar task. Our TD participants were approximately 10% less likely to defend their own ownership rights (39.5% vs. ~ 50%), but approximately 10% more likely to defend the ownership rights of a third party (31.5% vs. ~ 20%). The absence of protests in the ASD group cannot be attributed to a failure to map owner-object relationships; like the TD group, children with ASD reliably assigned objects to owners based on investment of creative labour at above-chance rates . We acknowledge that including a sample of children with delayed intellectual development matched to children with ASD on non-verbal intelligence and chronological age would have eliminated this issue. Secondly, it is possible that children with ASD did not protest against the thief’s behaviour in the ownership rights task because they were an adult, and thus perceived to be an authority figure. However, this limitation may be mitigated by the fact that most children with ASD were willing to correct their erroneous behaviour in the warm-up game and the protest rates of our TD participants were similar to those reported by Kanngiesser and Hood  who emplIn summary, the present study reports the first evidence that children with ASD are less accurate than TD children when mapping relationships between people and their property. In particular, children with ASD have difficulty identifying ownership based on possessive pronouns and they may struggle to keep track of others’ belongings. We also showed, for the first time, that children with ASD are less likely to defend their own and others’ ownership rights. We hypothesise that these results may be attributed to differences in representing the self and propose that ASD may be characterised by reduced concern for ownership and atypical development of associated concepts, such as ownership rights. Our findings inform broader understanding of social-cognitive differences associated with autism and highlight the possibility that children with ASD could face problems in social situations that require understanding of ownership rules.