Supplementary Material for: ApoE Polymorphisms and the Risk of Different Subtypes of Stroke in the Chinese Population: A Comprehensive Meta-Analysis

<b><i>Background and Purpose:</i></b> Numerous studies have evaluated the association between apolipoprotein E (ApoE) gene polymorphisms and the risk of different subtypes of stroke. However, the results remain uncertain, and few sources of data specific to the Chinese ethnic population contribute to these outstanding questions. Therefore, we performed a meta-analysis to derive a more comprehensive estimate of the association between ApoE polymorphisms and stroke risk in the Chinese population. <b><i>Methods:</i></b> Case-control studies in Chinese and English publications were identified by searching the PubMed, EMBASE, Web of Science, China Nation Knowledge Infrastructure Platform, Wanfang, and VIP databases and by hand-searching relevant journals and the reference lists of the retrieved articles. ORs and 95% CIs were applied to assess the strength of the associations. Subgroup and sensitivity analyses were performed to explore between-study heterogeneity. <b><i>Results:</i></b> Evidence of a significant association was found between the ApoE ε4 allele and different subtypes of stroke (for ischemic stroke (IS): OR 2.19, 95% CI 1.90-2.52, p < 0.001; for intracerebral hemorrhage (ICH): OR 2.08, 95% CI 1.57-2.75, p < 0.001; and for subarachnoid hemorrhage (SAH): OR 2.03, 95% CI 1.28-3.23, p = 0.003) among the Chinese population. In addition, a significant difference in the risk for different subtypes of stroke between ε4 carriers and ε3ε3 genotype carriers was found (for IS: OR 2.41, 95% CI 2.00-2.89, p < 0.001; for ICH: OR 2.41, 95% CI 1.68-3.47, p < 0.001; and for SAH: OR 2.04, 95% CI 1.21-3.45, p = 0.008). <b><i>Conclusion:</i></b> The ApoE ε4 allele may predict an increased risk for different subtypes of stroke, including IS, ICH and SAH, in the Chinese population, and the results of this genotypic analysis may help to identify populations at an increased risk for stroke. Further studies with larger sample sizes are needed to confirm our findings

Supplementary Material for: Exome Sequencing Identifies a Novel DES Mutation (R227C) in a Chinese Dilated Cardiomyopathy Family

<p><b><i>Objectives:</i></b> Dilated cardiomyopathy (DCM) is a common disease in the clinic, and it is the leading cause of heart failure and sudden cardiac death. Previous studies have proven that genetic factors play a crucial role in the occurrence of DCM; more than 50 disease genes including <i>desmin </i>(<i>DES</i>) have been identified to be associated with DCM. At present, most<i> DES </i>mutations are reported in desmin-related myofibrilla myopathy patients, but variants leading to isolated DCM are rarely reported. <b><i>Methods:</i></b> We applied whole-exome sequencing and cardiomyopathy-related gene filtering strategies to discover the genetic factors in a Chinese DCM family. <b><i>Results:</i></b> A novel mutation (c.679 C>T /p.R227C) in exon 3 of<i> DES</i> was identified and cosegregated with the affected members of a Chinese family with isolated DCM phenotypes (left ventricle and left atrial diameters). <b><i>Conclusion:</i></b> This mutation leads to a substitution of arginine by cysteine and it is predicted to be deleterious by bioinformatics programs. Our study not only contributes to the genetic diagnosis and counseling of families with DCM, but it also further proves that <i>DES </i>mutations may lead to isolated DCM and provides a new case for the study of the relationship between <i>DES</i> mutations and DCM.</p

Supplementary Material for: Hypoxia-Ischemia Upregulates TRAIL and TRAIL Receptors in the Immature Rat Brain

The immature brain is susceptible to inflammatory injury induced by hypoxia-ischemia (HI) or infection, which causes serious neurodevelopmental disabilities in the survivors of preterm births. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (death receptor DR4/5 and decoy receptor DcR1/2) were reported to mediate various neuroinflammatory responses. However, little information is available regarding the role of TRAIL and its receptors in the immature brain after HI. The purpose of this study was to evaluate the expression of TRAIL and its receptors in the immature brain after HI and relate this expression to neurological function. We performed right common carotid artery ligation followed by hypoxia (6% O₂, 37°C) for 2.5 h to induce HI in postnatal day 3 rats. The distribution of TRAIL and its receptors, caspase-3 and CD68-labeled microglia/macrophages was evaluated 24 h after HI by immunostaining. The protein and mRNA expression of TRAIL and DR5 was measured by Western blot and real-time PCR, respectively. Delayed neuronal loss was evaluated by NeuN and Nissl staining 7 days after HI. Furthermore, neurological deficits were evaluated by a righting reflex test, time of eye opening and T-maze test. The expression of TRAIL, DR5 and DcR1/2 receptors and caspase-3 was more pronounced in the ipsilateral hemisphere compared with the contralateral part and the control group 24 h after HI. DR5/active caspase-3 double-positive cells were observed at 24 h after HI in the ipsilateral hemisphere but not in the contralateral hemisphere. The TRAIL and CD68 double-labeled cells were more pronounced in the ipsilateral cortical regions compared with the corresponding regions of the contralateral part. HI also resulted in a significant increase in TRAIL and DR5 protein and mRNA expression at 24 h, which corresponded to neuronal cell loss 7 days after HI. Furthermore, the HI group displayed impaired neurobehavioral development compared with the control group (p < 0.05). Altogether our results show that the TNF-α superfamily ligand TRAIL is induced on CD68+ microglia/macrophages after perinatal HI and that one of its receptors, DR5, is induced on neocortical neurons and glial cells. That many DR5+ cells were also caspase-3+ strongly supports the conclusion that these signaling molecules are involved in the delayed loss of neurons in the neocortex and in the neurobehavioral deficits that are often seen after perinatal HI

Supplementary Material for: Molecular Pathways and Functional Analysis of miRNA Expression Associated with Paclitaxel-Induced Apoptosis in Hepatocellular Carcinoma Cells

<b><i>Background:</i></b> We postulated that microRNAs (miRNAs) might be involved in hepatocellular carcinoma (HCC) targeted chemotherapy with paclitaxel. This study sought to generate a list of potential miRNA-based biomarkers and their potential targets to better understand the response to paclitaxel treatment in HCC. <b><i>Methods:</i></b> Cell viability proliferation assays were conducted to test the sensitivity of the HepG2 cells to paclitaxel. The morphological changes of apoptosis were assessed with 4′,6-diamidino-2-phenylindole staining. Differential expression patterns of miRNA in the HepG2 cells either treated or not treated were analyzed using miRNA microarrays. <b><i>Results:</i></b> The array experiments have identified 54 miRNAs whose basal expression levels differed by >2-fold and p < 0.05 between the two phenotypic groups. The data were validated by a quantitative real-time PCR of 8 selected miRNAs (miR-21, miR-1274a, miR-1260, miR-1290, miR-508-5p, miR-877, miR-1246, miR-183*). The PI3K/Akt, mitogen-activated protein kinase (MAPK), TGF-β, ErbB, p53, cell cycle, mammalian target of rapamycin, and Jak-STAT signaling pathways were involved in paclitaxel-induced apoptosis. <b><i>Conclusions:</i></b> The manipulation of one or more of these miRNAs could be an important approach for the improved management of paclitaxel therapy

Erratum: Clinicopathological Features and Therapeutic Responses of Chinese Patients with Advanced Lung Adenocarcinoma Harboring an Anaplastic Lymphoma Kinase Rearrangement

<b><i>Background:</i></b> Presence of anaplastic lymphoma kinase (<i>ALK</i>) rearrangement is an indication for crizotinib in the treatment of patients with advanced or metastatic lung adenocarcinoma. Here, we sought to elucidate the association between clinicopathological features and <i>ALK</i> rearrangement status in Chinese patients with advanced lung adenocarcinoma harboring an <i>ALK</i> rearrangement. <b><i>Patients and Methods:</i></b><i>ALK</i> rearrangement status was determined using immunohistochemistry (IHC) in tumor tissues from 120 patients with advanced lung adenocarcinoma, and further assessed by fluorescence in situ hybridization (FISH) assay. The associations between <i>ALK</i> rearrangement status and clinicopathological features were analyzed. <b><i>Results:</i></b> According to IHC testing, the <i>ALK</i>-positive rate among the advanced lung adenocarcinoma patients was 6.67% (8/120). FISH validation found 5 patients with <i>ALK</i> rearrangement among the 8 IHC-positive cases. No significant difference was observed regarding age, sex, or smoking status between FISH-positive and -negative patients (p > 0.05). None of the 5 FISH-positive patients benefited from first-line chemotherapy. <b><i>Conclusion:</i></b> IHC can be used as a reliable method for <i>ALK</i> rearrangement screening in patients with lung adenocarcinoma, but further FISH validation is imperative. Presence of <i>ALK</i> rearrangement predicts a more aggressive biological behavior of the tumor and might be indicative of poor response to chemotherapy

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: Lysophosphatidic Acid Induces Ligamentum Flavum Hypertrophy Through the LPAR1/Akt Pathway

<b><i>Background/Aims:</i></b> Hypertrophic ligamentum flavum (LF) is a major cause of lumbar spinal stenosis. Our previous work showed that high levels of lysophosphatidic acid (LPA) expression are positively correlated with LF hypertrophy. This study aimed to further unveil how LPA regulates LF hypertrophy <b><i>Methods:</i></b> We studied LPAR1 expression in human LF cells using PCR and western blotting. Cell viability cell cycle, apoptosis rate and molecular mechanisms were assayed in LPAR1 knockdown or overexpression LF cells. LF hypertrophy and the molecular mechanism was confirmed in human samples and in <i>in vivo</i> studies. <b><i>Results:</i></b> The expression of LPA and its receptor LPAR1 is significantly higher in tissues or cells harvested from hypertrophic LF compared to healthy controls. Moreover, LPA promoted LF cell proliferation by interacting with LPAR1. This conclusion is supported by the fact that depletion or overexpression of LPAR1 changed the effect of LPA on LF cell proliferation. LPA also inhibits apoptosis in LF cells through the receptor LPAR1. Importantly, we demonstrated that the LPA-LPAR1 interaction initiated Akt phosphorylation and determined cell proliferation and apoptosis. Our <i>in vitro</i> findings were supported by our <i>in vivo</i> evidence that lyophilized LPA significantly induced LF hypertrophy via the LPAR1-Akt signaling pathway. More importantly, targeted inhibition of LPAR1 by Ki16425 with a gel sponge implant effectively reduced LPA-associated LF hypertrophy. Taken together, these data indicate that LPA binds to the receptor LPAR1 to induce LF cell proliferation and inhibit apoptosis by activating AKT signaling cascades. Targeting this signaling cascade with Ki16425 is a potential therapeutic strategy for preventing LF hypertrophy. <b><i>Conclusion:</i></b> LPA-LPAR1-Akt activation is positively correlated with the proliferation and survival of LF cells. LPAR1 could be a target for new drugs and the development of new therapeutic methods for treating LF hypertrophy

Supplementary Material for: Prenatal Diagnosis of DNA Copy Number Variations by Genomic Single-Nucleotide Polymorphism Array in Fetuses with Congenital Heart Defects

<b><i>Objectives:</i></b> To evaluate the usefulness of single-nucleotide polymorphism (SNP) array for prenatal genetic diagnosis of congenital heart defect (CHD), we used this approach to detect clinically significant copy number variants (CNVs) in fetuses with CHDs. <b><i>Methods:</i></b> A HumanCytoSNP-12 array was used to detect genomic samples obtained from 39 fetuses that exhibited cardiovascular abnormalities on ultrasound and had a normal karyotype. The relationship between CNVs and CHDs was identified by using genotype-phenotype comparisons and searching of chromosomal databases. All clinically significant CNVs were confirmed by real-time PCR. <b><i>Results:</i></b> CNVs were detected in 38/39 (97.4%) fetuses: variants of unknown significance were detected in 2/39 (5.1%), and clinically significant CNVs were identified in 7/39 (17.9%). In 3 of the 7 fetuses with clinically significant CNVs, 3 rare and previously undescribed CNVs were detected, and these CNVs encompassed the CHD candidate genes <i>FLNA</i> (Xq28 dup), <i>BCOR</i> (Xp11.4 dup), and <i>RBL2</i> (16q12.2 del). <b><i>Conclusion:</i></b> Compared with conventional cytogenetic genomics, SNP array analysis provides significantly improved detection of submicroscopic genomic aberrations in pregnancies with CHDs. Based on these results, we propose that genomic SNP array is an effective method which could be used in the prenatal diagnostic test to assist genetic counseling for pregnancies with CHDs

Supplementary Material for: Sex and gender publications in brain health: a mapping review of the Asia-Pacific region

Introduction. Reporting of sex and gender analysis in medical research has been shown to improve quality of the science and ensures findings are applicable to women and men. There is conflicting evidence on whether efforts by funding agencies and medical journals to encourage reporting of sex and gender analysis has resulted in tangible improvements. This study mapped the inclusion of sex and gender analysis in stroke and dementia research conducted in the Asia-Pacific region. Methods. A systematic search for Asia-Pacific stroke and dementia research was conducted in PubMed and papers included from the period 2012 to 2022. Eligible studies were reviewed for inclusion of a primary sex or gender focus and categorized by type of sex and gender analysis. Author gender was determined using an algorithm and its associations with inclusion of sex and gender analysis examined. Results. Total Asia-Pacific publications increased from 109 in 2012 to 313 in 2022, but the rate of studies with a primary sex or gender focus did not increase significantly (R2 = 0.06, F(1,9) = 0.59, p = 0.46). Australia, China, India, Japan and South Korea produced the most publications over the study period and were the only countries with at least 50 publications. The impact of author gender was mixed, with female first authorship associated with inclusion of sex or gender analysis and last female authorship associated with studies having a primary sex or gender focus. Conclusions. In the Asia-Pacific, brain health research is currently centered around high income countries and efforts are needed to ensure research findings are applicable through out the region. While there was a general increase in brain health publications over the last decade, the rate of sex and gender analysis was unchanged. This demonstrates that even with efforts in some countries in place, there is currently a lack of progress in the Asia-Pacific region to produce more research focusing on sex and gender analysis