Supplementary Material for: Retinal Nerve Fiber Layer Thickness Changes in the Pseudoexfoliation Syndrome: A Meta-Analysis of Case-Control Studies

<strong><em>Purpose:</em></strong> To evaluate and compare changes in retinal nerve fiber layer (RNFL) thickness in patients with the pseudoexfoliation syndrome (PXS) and healthy controls. <b><i>Methods:</i></b> Case-control studies were selected through an electronic search of the Cochrane Controlled Trials Register, PubMed, and Embase. Results were reviewed to ensure that the included studies met prespecified inclusion/exclusion criteria, and the quality of each study was assessed using the Newcastle-Ottawa Scale. All included studies measured average and 4-quadrant (temporal, superior, nasal, and inferior) RNFL thickness using optical coherence tomography (OCT). For the continuous outcomes, we calculated the weighted mean difference (WMD) and 95% confidence intervals (CIs). <b><i>Results:</i></b> Eight case-control studies were included in this meta-analysis involving 225 eyes of PXS patients and 208 eyes of healthy controls in total. Statistical analysis revealed that the average RNFL thickness in PXS patients was significantly reduced compared to healthy controls (WMD = −6.91, 95% CI: −9.99 to −3.82, <i>p</i> < 0.0001). Additionally, differences in RNFL thickness in the superior quadrant (WMD = −10.68, 95% CI: −16.40 to −4.95, <i>p</i> = 0.0003), inferior quadrant (WMD = −8.20, 95% CI: −10.85 to −5.55, <i>p</i> < 0.00001), nasal quadrant (WMD = −3.05, 95% CI: −5.21 to −0.90, <i>p</i> = 0.005), and temporal quadrant (WMD = −6.39, 95% CI: −9.98 to −2.80, <i>p</i> = 0.0005) were all significant between the two groups. <b><i>Conclusions:</i></b> These results suggest that it is important to evaluate RNFL thickness and the optic nerve head through OCT in patients with PXS in order to determine early glaucomatous damage and start timely intervention prior to visual field loss

Supplementary Material for: Analysis of Differentiation Potentials and Gene Expression Profiles of Mesenchymal Stem Cells Derived from Periodontal Ligament and Wharton’s Jelly of the Umbilical Cord

PDLSC-mediated periodontal tissue regeneration is considered a promising method for periodontitis treatment, but the supply of PDLSCs is limited. As a potential alternative, WJCMSCs are readily available; however, there is a lack of evidence proving that WJCMSCs are suitable for periodontal tissue regeneration. In this study, we investigated the characteristics of WJCMSCs and PDLSCs. We found the osteo-/dentinogenic, adipogenic and chondrogenic differentiation potentials of PDLSCs were more powerful than those of WJCMSCs. Microarray analysis discovered that 903 genes were significantly down-regulated and 726 genes up-regulated in WJCMSCs compared with PDLSCs. Based on the microarray data, we found that several genes may be associated with MSCs characteristics. Further bioinformatic analysis identified that TGF-β and WNT signaling pathways, and several genes, including <i>STAT5B </i>and<i> ITGA4</i>, may play key roles in MSCs. Our results indicate that the differentiation potentials of WJCMSCs are far less than those of PDLSCs, and that unmodified WJCMSCs may not be good seeding cells for periodontal tissue regeneration. Our results also help to elucidate the differentiation mechanisms in MSCs and to find the key factors to prompt WJCMSC-mediated periodontal tissue regeneration

Supplementary Material for: Age-period-cohort analysis of long-term trends in ischemic stroke mortality in China caused by specific risk factors from 1990 to 2019

Objective: To study the primary risk factors for the long-term trends of mortality rates in ischemic stroke (IS) in China. Methods: Using the Global Burden of Disease Study 2019 (GBD 2019) database, research was conducted on the 11 primary risk factors for the mortality rates of IS in China from 1990 to 2019. This study employed Joinpoint regression software and the Age-Period-Cohort (APC) method to evaluate the trends of mortality rates divided by age, period, and cohort over time. Results: From 1990 to 2019, the age-standardized mortality rate (ASMR) caused by a diet high in red meat and high body-mass index (BMI) in China showed an upward trend. ASMR increased first and then decreased due to smoking, diet high in sodium, particulate matter pollution, high fasting plasma glucose, and high systolic blood pressure. Low-density lipoprotein cholesterol (LDL-C), kidney dysfunction, low temperature, and lead exposure remained relatively stable during this period. In the 35-45 age group, the mortality rate of IS due to high LDL-C was up to about 60%, and smoking affected men more than women. Overall, high LDL-C, high systolic blood pressure, and particulate matter pollution were the most common risk factors in patients with IS. The risk of death rose with age. The period and cohort relative risks showed that metabolic risk factors had the greatest impact on the mortality of IS. Conclusion: Metabolic risk factors have become the primary risk factors for the ASMR of IS in China. Relevant authorities should pay attention to their long-term effects on IS. Effective public health policies and interventions should be implemented to reduce the burden of IS

Supplementary Material for: Age-period-cohort analysis of long-term trends in ischemic stroke mortality in China caused by specific risk factors from 1990 to 2019

Objective: To study the primary risk factors for the long-term trends of mortality rates in ischemic stroke (IS) in China. Methods: Using the Global Burden of Disease Study 2019 (GBD 2019) database, research was conducted on the 11 primary risk factors for the mortality rates of IS in China from 1990 to 2019. This study employed Joinpoint regression software and the Age-Period-Cohort (APC) method to evaluate the trends of mortality rates divided by age, period, and cohort over time. Results: From 1990 to 2019, the age-standardized mortality rate (ASMR) caused by a diet high in red meat and high body-mass index (BMI) in China showed an upward trend. ASMR increased first and then decreased due to smoking, diet high in sodium, particulate matter pollution, high fasting plasma glucose, and high systolic blood pressure. Low-density lipoprotein cholesterol (LDL-C), kidney dysfunction, low temperature, and lead exposure remained relatively stable during this period. In the 35-45 age group, the mortality rate of IS due to high LDL-C was up to about 60%, and smoking affected men more than women. Overall, high LDL-C, high systolic blood pressure, and particulate matter pollution were the most common risk factors in patients with IS. The risk of death rose with age. The period and cohort relative risks showed that metabolic risk factors had the greatest impact on the mortality of IS. Conclusion: Metabolic risk factors have become the primary risk factors for the ASMR of IS in China. Relevant authorities should pay attention to their long-term effects on IS. Effective public health policies and interventions should be implemented to reduce the burden of IS

Supplementary Material for: Perilipin1 deficiency prompts lipolysis in lipid droplets and aggravates the pathogenesis of persistent immune activation in Drosophila

Lipid droplets (LDs) are highly dynamic intracellular organelles, which are involved in lots of biological processes. However, the dynamic morphogenesis and functions of intracellular LDs during persistent innate immune responses remain obscure. In this study, we induce long-term systemic immune activation in Drosophila through genetic manipulation. Then, the dynamic pattern of LDs is traced in the Drosophila fat body. We find that deficiency of Plin1, a key regulator of LDs’ reconfiguration, blocks LDs minimization at the initial stage of immune hyperactivation but enhances LDs breakdown at the later stage of sustained immune activation via recruiting the lipase Brummer (Bmm, homologous to human ATGL). The high wasting in LDs shortens the lifespan of flies with high-energy-cost immune hyperactivation. Therefore, these results suggest a critical function of LDs during long-term immune activation and provide a potential treatment for the resolution of persistent inflammation

Supplementary Material for: Perilipin1 deficiency prompts lipolysis in lipid droplets and aggravates the pathogenesis of persistent immune activation in Drosophila

Lipid droplets (LDs) are highly dynamic intracellular organelles, which are involved in lots of biological processes. However, the dynamic morphogenesis and functions of intracellular LDs during persistent innate immune responses remain obscure. In this study, we induce long-term systemic immune activation in Drosophila through genetic manipulation. Then, the dynamic pattern of LDs is traced in the Drosophila fat body. We find that deficiency of Plin1, a key regulator of LDs’ reconfiguration, blocks LDs minimization at the initial stage of immune hyperactivation but enhances LDs breakdown at the later stage of sustained immune activation via recruiting the lipase Brummer (Bmm, homologous to human ATGL). The high wasting in LDs shortens the lifespan of flies with high-energy-cost immune hyperactivation. Therefore, these results suggest a critical function of LDs during long-term immune activation and provide a potential treatment for the resolution of persistent inflammation

Supplementary Material for: Enhanced Liver Regeneration After Partial Hepatectomy in Sterol Regulatory Element-Binding Protein (SREBP)-1c-Null Mice is Associated with Increased Hepatocellular Cholesterol Availability

<b><i>Background/Aims:</i></b> Transient lipid accumulation within hepatocytes preceding the peak proliferative process is a characteristic feature of liver regeneration. However, molecular mediators responsible for this lipid accumulation and their functions are not well defined. Sterol regulatory element-binding proteins-1c (SREBP-1c) are critical transcriptional factors that regulate lipid homeostasis in the liver. We hypothesized that SREBP-1c deficiency induced alterations of lipid metabolism may influence hepatocyte proliferation and liver regeneration. <b><i>Methods:</i></b> 2/3 partial hepatectomy (PH) was performed in wild type C57BL/6J (WT) and <i>Srebp-1c-/-</i> mice. The lipid contents in serum and liver were measured by enzymatic colorimetric methods. Hepatic lipid droplets were detected by Oil Red O staining and immunohistological staining. Hepatic expression of genes involved in lipid metabolism and cellular proliferation was determined by real-time PCR and/or immunoblot. Hepatocyte proliferation and liver regeneration were assessed by BrdU staining and the weight of remanent liver lobes in Srebp-1c-/- mice, respectively. <b><i>Results:</i></b> <i>Srebp-1c-/-</i> mice displayed reduced triglyceride and fatty acids but increased cholesterol in the liver before PH. In response to PH, hepatocellular DNA synthesis was elevated and cell cycle progression was prolonged in <i>Srebp-1c-/-</i> mice, which was associated with enhanced liver regeneration. However, <i>Srebp-1c-/-</i> mice had comparable triglyceride and fatty acid contents and expressions of related genes compared with WT mice during the liver regeneration. In contrast, SREBP-1c-deficiency-induced alteration of cholesterol metabolism was retained during the liver regeneration after PH. <i>Srebp-1c-/-</i> mice exhibited higher cholesterol contents and enhanced expression of SREBP-2 and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR) in the liver than WT mice after PH. Moreover, downregulation of genes involved in cholesterol elimination was observed after PH in <i>Srebp-1c-/-</i> mice. <b><i>Conclusion:</i></b> SREBP-1c deficiency in mice did not interfere with triglyceride and fatty acid metabolism but was associated with significant changes in cholesterol profiles during liver regeneration after PH. These results suggest that increased hepatocellular cholesterol storage and cholesterol availability with the enhanced liver regeneration are identified in <i>Srebp-1c-/-</i> mice. This study also shows that providing requisite cholesterol levels to proliferating hepatocytes and keeping appropriate cholesterol metabolism are required for normal liver regeneration

Supplementary Material for: Enhanced Prophylactic and Therapeutic Effects of Polylysine-Modified Ara h 2 DNA Vaccine in a Mouse Model of Peanut Allergy

<p><b><i>Background:</i></b> The prevalence of food allergy has been increasing, but treatment is very limited. DNA vaccination has been recognized as a promising method for the treatment of allergic diseases; however, poor immunogenicity has hindered its application. <b><i>Methods:</i></b> BALB/c mice were intradermally injected with plasmid DNA encoding the peanut protein Ara h 2 (pAra h 2) or pAra h 2 pretreated with poly-L- lysine (PLL) before or after sensitization with Ara h 2 protein. Ara h 2-specific antibodies were measured by ELISA. CD207+ dendritic cells (DCs) and Treg cells in draining lymph nodes were analyzed by flow cytometry after DNA immunization, and cytokine production in splenocytes was also analyzed. <b><i>Results:</i></b> In the prophylactic study, pretreatment with pAra h 2 or PLL-pAra h 2 resulted in lower levels of Ara h 2-specific IgG1, IgG2a, and IgE after sensitization with Ara h 2 protein, and mice in the PLL-pAra h 2 group had a significantly lower level of antibodies than those in the pAra h 2 group. In the treatment study, intradermal injection with pAra h 2 or PLL-pAra h 2 after Ara h 2 protein sensitization significantly decreased the level of Ara h 2-specific antibodies, and PLL- pAra h 2 had stronger effects than pAra h 2. There were increased numbers of CD207+ DCs and Treg cells in the mice receiving intradermal injection with PLL-pAra h 2, and splenocytes from PLL-pAra h 2-treated mice secreted increased levels of IFN-γ and IL-10. <b><i>Conclusions:</i></b> Modification of pAra h 2 with PLL improved its prophylactic and therapeutic effects in peanut-allergic mice.</p

Supplementary Material for: Prevalence of Atopy and Allergic Diseases in Korean Children: Associations with a Farming Environment and Rural Lifestyle

<i>Background:</i> The results of recent studies suggest that factors in rural environments may protect against the development of allergic diseases, but the underlying mechanisms are not well understood. The aim of this study was to investigate the prevalence of allergic diseases, to establish if this prevalence is influenced by migration from rural to urban areas and to identify environmental risk factors associated with these diseases. <i>Methods:</i> A cross-sectional study of children aged 9–12 years from a rural village, a rural town and an urban city in Korea was conducted. Demographic and disease-related information was obtained via a detailed questionnaire, and skin prick tests were performed. <i>Results:</i> There were significant differences in lifestyle and environmental factors between children from the rural village, the rural town and the urban children. The prevalence of allergic diseases and atopy was higher in urban children. A lower prevalence of allergic diseases and atopy was associated with farming parents, contact with farm animals during pregnancy, owning pets or a stable, breast-feeding and having older siblings. A comparison of rural village and rural town children revealed no evidence of an association of allergic diseases and atopy with farming parents, contact with farm animals during pregnancy or owning a stable. On the other hand, having older siblings and antibiotic use during infancy were significantly associated with allergic diseases and atopy in these children. <i>Conclusions:</i> Protective factors associated with a farming environment and/or rural lifestyle may influence the prevalence of allergic diseases and atopy in Korean children

Supplementary Material for: Association between Testosterone and Mortality Risk among U.S. Males Receiving Dialysis

<strong><em>Background:</em></strong> Among the general population, low circulating testosterone levels are associated with higher risk of cardiovascular disease and death. While testosterone deficiency is common in dialysis patients, studies of testosterone and mortality in this population are ambiguous and overlapping. We hypothesized that lower testosterone levels are associated with higher mortality in male dialysis patients. <b><i>Methods:</i></b> We examined a nationally representative cohort of male dialysis patients from a large US dialysis organization who underwent one or more total testosterone measurements from 1/2007 to 12/2011. The association between total testosterone categorized as quartiles and all-cause mortality was studied using Cox models adjusted for expanded case-mix and laboratory covariates. We also examined total testosterone as a continuous predictor of all-cause mortality using restricted cubic splines. <b><i>Results:</i></b> Among 624 male dialysis patients, 51% of patients demonstrated testosterone deficiency (total testosterone <300 ng/dL); median (IQR) total testosterone levels were 297 (190-424) ng/mL. In expanded case-mix + laboratory adjusted Cox analyses, we observed a graded association between lower testosterone levels and higher mortality risk (ref: quartile 3): adjusted hazard ratios (95% CI) 2.32 (1.33-4.06), 1.80 (0.99-3.28), and 0.68 (0.32-1.42) for Quartiles 1, 2, and 4, respectively. In adjusted spline analyses, the lower testosterone-higher mortality risk association declined with higher testosterone levels until the value reached a threshold of 400 ng/dL above which risk plateaued. <b><i>Conclusion:</i></b> Lower testosterone levels were independently associated with higher mortality risk in male dialysis patients. Further studies are needed to determine underlying mechanisms, and whether testosterone replacement ameliorates death risk in this population