Supplementary Material for: Kaempferol ameliorated alcoholic hepatitis through improving intestinal barrier function by targeting miRNA-155 signaling

Introduction: To investigate the effect and mechanism of Kaempferol on alcoholic steatohepatitis. Methods: C57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. Kaempferol was given as the interventional drug to chronic alcohol-fed mice for six weeks to assess its effects. In vitro, intestinal epithelial Caco-2 cells were stimulated by alcohol, and miRNA-155 mimics were used to further study the effect of kaempferol to miRNA-155 signaling in intestinal epithelial cells. HE staining and oil red O staining were used to observe the liver and intestinal tissue damage in each group of mice, and ALT, AST, IL-1B and TNF-a were detected by kits; LPS expression was detected by ELISA kit, and the expression of IL-1B and TNF-a was assessed by qRT-PCR; activated inflammatory response of liver and colon tissue and the related signalling pathway activation. Results: Kaempferol treatment significantly improved pathological changes such as steatosis and vacuolated lesions in liver tissue of the alcohol diet model group, and reduced serum ALT and AST enzyme activities and liver tissue interleukin-1β and tumour necrosis factor-α mRNA expression levels. Kaempferol significantly reduced the expression of miRNA-155 in the intestinal tissue of alcohol-fed mice, significantly increased their SOCS1 protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and ZO-1. What's more, kaempferol significantly reduced serum LPS levels in ASH mice. In vitro experiments showed that compared with the control group, kaempferol significantly inhibited the expression level of miRNA-155 in Caco-2 cells under ethanol exposure, decreased the activation of nuclear factor kappa-B, led to an increase in the expression of SOCS1 protein, and increased the production level of occludin protein in Caco-2 cells under the effect of alcohol. In contrast, overexpression of miRNA-155 significantly decreased occludin and SOCS1 protein production and increased nuclear factor kappa-B activation levels in Caco-2 cells, and the administration of kaempferol significantly inhibited this effect. Conclusion: Kaempferol improved the stability of gut barrier function to ameliorate hepatic injury induced by alcohol intake through enhancing occludin protein expression, by targeting miR-155 to inhibit the excessive inflammatory response in the intestine

Supplementary Material for: Two-Stage Extreme Phenotype Sequencing Design for Discovering and Testing Common and Rare Genetic Variants: Efficiency and Power

Next-generation sequencing technology provides an unprecedented opportunity to identify rare susceptibility variants. It is not yet financially feasible to perform whole-genome sequencing on a large number of subjects, and a two-stage design has been advocated to be a practical option. In stage I, variants are discovered by sequencing the whole genomes of a small number of carefully selected individuals. In stage II, the discovered variants of a large number of individuals are genotyped to assess associations. Individuals with extreme phenotypes are typically selected in stage I. Using simulated data for unrelated individuals, we explore two important aspects of this two-stage design: the efficiency of discovering common and rare single-nucleotide polymorphisms (SNPs) in stage I and the impact of incomplete SNP discovery in stage I on the power of testing associations in stage II. We applied a sum test and a sum of squared score test for gene-based association analyses evaluating the power of the two-stage design. We obtained the following results from extensive simulation studies and analysis of the GAW17 dataset. When individuals with trait values more extreme than the 99.7–99th quantile were included in stage I, the two-stage design could achieve the same power as or even higher than the one-stage design if the rare causal variants had large effect sizes. In such design, fewer than half of the total SNPs including more than half of the causal SNPs were discovered, which included nearly all SNPs with minor allele frequencies (MAFs) ≥5%, more than half of the SNPs with MAFs between 1% and 5%, and fewer than half of the SNPs with MAFs <1%. Although a one-stage design may be preferable to identify multiple rare variants having small to moderate effect sizes, our observations support using the two-stage design as a cost-effective option for next-generation sequencing studies

Supplementary Material for: Protective Actions of Globular and Full-Length Adiponectin on Human Endothelial Cells: Novel Insights into Adiponectin-Induced Angiogenesis

<b><i>Background/Aims:</i></b> Adiponectin levels are decreased in diabetes and atherosclerosis. Coexisting hyperglycaemia and systemic inflammation predisposes to dysregulated angiogenesis and vascular disease. We investigated the effect of globular adiponectin (gAd) and full-length adiponectin (fAd) on angiogenesis and pro-angiogenic molecules, i.e. matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF), in human microvascular endothelial cells (HMEC-1). <b><i>Methods:</i></b> Angiogenesis was assessed by studying capillary tube formation in HMEC-1 on growth factor-reduced Matrigel. Endothelial cell migration assay was performed in a modified Boyden chamber. <b><i>Results:</i></b> Endothelial cell proliferation, in vitro migration and angiogenesis were significantly increased by gAd (mediated by AdipoR1, AMPK-Akt pathways), and gAd significantly increased MMP-2, MMP-9 and VEGF expression levels. The effect of gAd on VEGF appears to be mediated by AdipoR1, whilst the effect of gAd on MMP-2 and MMP-9 appears to be mediated by AdipoR1 and AdipoR2. Only endothelial cell proliferation was significantly increased by fAd in human microvascular endothelial cells and appears to be mediated by AdipoR2. No significant effects on MMP-2, MMP-9 and VEGF were observed. Importantly, gAd decreased glucose and C-reactive protein-induced angiogenesis with a concomitant reduction in MMP-2, MMP-9 and VEGF in HMEC-1 cells. <b><i>Conclusion:</i></b> We report novel insights into the mechanisms of adiponectin on angiogenesis

Supplementary Material for: Constipation in Parkinson’s Disease: A Systematic Review and Meta-Analysis

Introduction: Constipation is a common nonmotor symptom of Parkinson’s disease (PD) and has been reported to increase the risk of developing PD. However, previous studies have yielded conflicting results. Understanding this correlation may promote early diagnosis and treatment of PD, which could help patients improve their quality of life. This study aimed to investigate the association between constipation and PD onset. Methods: The study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Meta-analyses of Observational Studies in Epidemiology (MOOSE) guidelines. We searched the Medline, Embase, Scopus, SINOMED, and Cochrane databases as well as specific journals from inception to September 2021 for observational studies that evaluated the association between constipation and the risk of PD. The Newcastle-Ottawa Scale was used to evaluate the methodological quality of the included studies. Associations were summarized as odds ratios (ORs) using a random-effects model. Subgroup, meta-regression, and sensitivity analyses were performed. Results: Seventeen studies comprising 3,024,193 participants (case-control = 1,636,831; cohort = 1,387,362) were eligible for inclusion. The pooled OR for the association between constipation and PD was 2.36 (95% confidence interval: 1.93–2.88), although strong heterogeneity was observed (I2 = 90%, p p p > 0.05). Conclusion: Constipation has a relatively high incidence in the prodromal phase of PD and is associated with an increased risk of developing PD

Supplementary Material for: Intradialytic Exercise in Hemodialysis Patients: A Systematic Review and Meta-Analysis

<b><i>Background and Objective:</i></b> Hemodialysis (HD) patients are more inactive, leading to poor functional capacity and quality of life; this may be reversed with intradialytic exercise training. To systematically evaluate the efficacy and safety of intradialytic exercise for HD patients, we conducted a meta-analysis of the published randomized controlled trials. <b><i>Data Sources and Methods:</i></b> Medline, Embase, and Cochrane Central Register of Controlled Trials were systematically searched up to February, 2014. The reference lists of eligible studies and relevant reviews were also checked. <b><i>Results:</i></b> 24 studies of 997 patients were included. Compared with control, intradialytic exercise significantly improve Kt/V (SMD = 0.27, 95% CI 0.01-0.53), peak oxygen consumption (VO_2peak) (SMD = 0.53, 95% CI 0.30-0.76), and physical performance of physical function of life (SMD = 0.30, 95% CI 0.04-0.55). However, no significant improvements were found in the mental function of life. There was no significant difference with respect to musculoskeletal and cardiovascular complications between the intradialytic exercise groups and control groups. Further subgroup analysis found that, when the trial duration was more than 6 months, the intervention had significant effects on VO_2peak (SMD = 0.89, 95% CI 0.56-1.22). However, when the trial duration was less than 6 months, the change of VO_2peak was not significant (SMD = 0.19, 95% CI -0.13 to 0.51). <b><i>Conclusion:</i></b> Intradialytic exercise can improve Kt/V, VO_2peak, and the physical quality of life, and intradialytic exercise is safe for HD patients. Therefore, we put forward the suggestion that clinical guideline be updated to inform clinicians on the benefits of intradialytic exercise on HD patients. i 2014 S. Karger AG, Base

Supplementary Material for: Progressive Central Artery Occlusion, Ophthalmic Artery Occlusion, and Hemispheric Intracranial Thrombosis after COVID-19 mRNA Vaccine Application- Case Report

Thromboembolic events as a result of COVID-19 mRNA vaccination are a rare, though life-threatening complication. In this case report, we describe a 40-year-old female patient who developed central retinal artery and ophthalmic artery occlusion progressing to intracranial thrombosis three weeks after vaccination with the Pfizer-BioNTech COVID-19 vaccine. Initially, she presented with progressive acute and painless unilateral vision loss in her left eye. Dilated fundoscopy of left eye showed macular whitening with sparing of the area of cilioretinal artery distribution. Labs revealed a normal erythrocyte sedimentation rate, C-reactive protein, and platelet count. Computerized tomogaphy angiography of the head and neck showed an occlusion of the entire left cervical internal carotid artery and occlusion of the origin of the left external carotid artery. Despite treatment with heparin, her vision declined to no light perception. Ten days later, the patient presented with right peripheral vision loss and was found to have a new left posterior cerebral artery/posterior inferior celebellar artery stroke. Seventeen days later, she presented to the hospital with nausea and vertigo and was found to have a subacute infarction in the left parietal lobe corresponding to left anterior communicating artery/middle celebral artery watershed territory. Hypercoagulable disorders, vasculitis, cardiac arrythmias, and intraventricular thrombi were excluded. Fundus fluorescein angiography confirmed central retinal artery occlusion and ophthalmic artery occlusion with impressive retina and choroid changes in fluorescein angiography patterns. This complication of mRNA COVID-19 vaccination has not been previously described in the literature and should be considered even weeks after initial presentation

Supplementary Material for: Trends in Adherence to Recommended Physical Activity and Its Association with Mortality and Disease Progression among US Adults with Chronic Kidney Disease

Introduction: This study aimed to examine the trends in adherence to the Physical Activity Guidelines (PAG) for aerobic activity and sedentary time and their effects on mortality and disease progression among US adults with chronic kidney disease (CKD). Methods: We studied individuals from the National Health and Nutrition Examination Survey 2007–08 to 2017–18 with a mortality file in 2015. Multivariate regression models were used to evaluate the association between adherence to PAG and sedentary time with mortality, estimated glomerular filtration rate (eGFR), and urine albumin-to-creatinine ratio. Results: For the CKD population, adherence rate increased from 48.2% in 2007–08 to 55.0% in 2017–18, and sedentary time peaked in 2013–14 (7.5 h/day) and then decreased afterward. There was no difference in the trends across the non-CKD and CKD population. For the CKD population, adherence to the PAG was significantly associated with all-cause mortality (HR, 0.49; 95% CI: 0.38–0.63), malignant neoplasm mortality (HR, 0.30; 95% CI: 0.17–0.52), and albumin-creatinine ratio (OR, −0.27; 95% CI: −0.39 to −0.15). Sedentary time was significantly associated with all-cause mortality (HR, 1.12; 95% CI: 1.08–1.15), heart disease mortality (HR, 1.13; 95% CI: 1.08–1.19), and eGFR (OR, −0.49; 95% CI: −0.72 to −0.26). Conclusions: Favorable trends were observed in adherence to the PAG and sedentary time. Adherence to the PAG and reduction in sedentary time reduced all-cause and cause-specific mortality and prevented disease progression differently. Efforts are needed to decrease sedentary time rather than adhering to the PAG for aerobic activity alone

Supplementary Material for: Methods and software to analyze gene-environment interactions under a case-mother - control-mother design with partially missing child genotype

Introduction: The case-mother - control-mother design allows to study fetal and maternal genetic factors together with environmental exposures on early-life outcomes. Mendelian constraints and conditional independence between child genotype and environmental factors enabled semiparametric likelihood methods to estimate logistic models with greater efficiency than standard logistic regression. Difficulties in child genotype collection require methods handling missing child genotype. Methods: We review a stratified retrospective likelihood and two semiparametric likelihood approaches: a prospective one and a modified retrospective one, the latter either modeling the maternal genotype as a function of covariates or leaving their joint distribution unspecified (robust version). We also review software implementing these modelling alternatives, compare their statistical properties in a simulation study, and illustrate their application, focusing on gene-environment interactions and partially missing child genotype. Results; The robust retrospective likelihood provides generally unbiased estimates, with standard errors only slightly larger than when modelling maternal genotype based on exposure. The prospective likelihood encounters maximization problems. In the application to the association of small-for-gestational-age babies with CYP2E1 and drinking water disinfection by-products, the retrospective likelihood allowed a full array of covariates, while the prospective likelihood was limited to few covariates. Conclusion: We recommend the robust version of the modified retrospective likelihood

Supplementary Material for: Methods and software to analyze gene-environment interactions under a case-mother - control-mother design with partially missing child genotype

Introduction: The case-mother - control-mother design allows to study fetal and maternal genetic factors together with environmental exposures on early-life outcomes. Mendelian constraints and conditional independence between child genotype and environmental factors enabled semiparametric likelihood methods to estimate logistic models with greater efficiency than standard logistic regression. Difficulties in child genotype collection require methods handling missing child genotype. Methods: We review a stratified retrospective likelihood and two semiparametric likelihood approaches: a prospective one and a modified retrospective one, the latter either modeling the maternal genotype as a function of covariates or leaving their joint distribution unspecified (robust version). We also review software implementing these modelling alternatives, compare their statistical properties in a simulation study, and illustrate their application, focusing on gene-environment interactions and partially missing child genotype. Results; The robust retrospective likelihood provides generally unbiased estimates, with standard errors only slightly larger than when modelling maternal genotype based on exposure. The prospective likelihood encounters maximization problems. In the application to the association of small-for-gestational-age babies with CYP2E1 and drinking water disinfection by-products, the retrospective likelihood allowed a full array of covariates, while the prospective likelihood was limited to few covariates. Conclusion: We recommend the robust version of the modified retrospective likelihood

Supplementary Material for: Meta-Analysis of Human Leukocyte Antigen-G 3′UTR Polymorphisms Confer Susceptibility to Recurrent Miscarriage

Objectives: The current systematic review and meta-analysis have shown that specific HLA-G 3′ UTR variants are associated with recurrent miscarriage (RM). Our aim was to investigate the relevance of HLA-G 3′ UTR polymorphisms with the risk of RM. Design: A combined meta-analysis was implemented in this study. Participants/Materials, Setting, Methods: Common electronic databases including PubMed, Embase, Web of Science, China National Knowledge Infrastructure, Cochrane Library, and Google Scholar were used to seek eligible articles up to August 2021. Results: Forty-four eligible articles with 4,467 cases and 3,955 controls were finally enrolled. Our meta-results suggested that 14 bp insertion allele was associated with elevated risk of RM (allelic model: OR = 1.18, 95% CI = 1.07–1.31, p = 0.001). Besides, a significant heterogeneity was observed between studies. Further subgroup analyses based on ethnicity revealed similar positive results in both the Caucasian and Asian subgroups but not in the Middle East subgroup. Moreover, rs1063320 G allele conferred elevated susceptibility to RM in Asian group (allelic model: OR = 1.54, 95% CI = 1.17–2.03, p = 0.002). Additionally, pooled results showed a decreased risk of RM in mothers carrying rs1710 G allele (allelic model: OR = 0.68, 95% CI = 0.55–0.85, p p = 0.002). Further stratified analyses by race showed that these positive results were mainly from the associations observed in Asian populations. Limitations: The main limitation is that the enrolled number of individuals was relatively small. Thus, the results should be cautiously considered. Conclusions: Although the current systematic review and meta-analysis have shown that specific HLA-G 3′ UTR variants are associated with RM, a high degree of bias is present and further studies are needed to validate this causative effect