Supplementary Material for: VEGF-A Inhibition Ameliorates Podocyte Apoptosis via Repression of Activating Protein 1 in Diabetes

<b><i>Background/Aims:</i></b> Vascular endothelial growth factor-A (VEGF-A) upregulation and podocyte apoptosis have been documented in diabetes. This study was designed to investigate whether inhibiting VEGF-A could ameliorate podocyte apoptosis in diabetes and the underlying mechanisms. <b><i>Methods:</i></b> In vitro, small interfering RNAs (siRNAs) of VEGF-A and activator protein 1 (AP-1, c-fos and c-jun), bevacizumab (VEGF-A inhibitor) and SP600125 (AP-1 inhibitor) were added to high glucose (30 mM) induced podocytes. Luciferase reporter assay was used to investigate whether AP-1 was a direct target of VEGF-A. In vivo, bevacizumab and SP600125 were administered to 12-week-old streptozotocin-induced male Sprague Dawley rats. The level of VEGF-A, c-fos, c-jun and bcl-2 were examined using immunostaining and Western blot analysis. Podocyte apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay, electron microscopy and flow cytometry. <b><i>Results:</i></b> Silencing VEGF-A or AP-1 upregulated bcl-2 and ameliorated podocyte apoptosis. Silencing VEGF-A decreased the level of c-fos and c-jun and bevacizumab and SP600125 treatment attenuated podocyte apoptosis. Luciferase reporter activity of VEGF-A-3′-UTR constructs was significantly provoked when stimulated with TGF-β1. In diabetic rat kidneys, VEGF-A co-localized with bcl-2 in podocytes. With bevacizumab and SP600125 treatment, the level of VEGF-A and AP-1 decreased while bcl-2 increased. Podocyte apoptotic rate was reduced with condensed podocyte nuclei less frequently observed. The urine albumin excretion rate (UAER) and albumin/creatinine were improved. <b><i>Conclusion:</i></b> This study demonstrates VEGF-A inhibition ameliorates podocyte apoptosis by regulating AP-1 and bcl-2 signaling. AP-1 is a direct target of VEGF-A and a novel player in podocyte apoptosis

Supplementary Material for: Tracking Chromosome Evolution in Southern African Gerbils Using Flow-Sorted Chromosome Paints

<i>Desmodillus</i> and <i>Gerbilliscus</i> (formerly <i>Tatera</i>) comprise a monophyletic group of gerbils (subfamily Gerbillinae) which last shared an ancestor approximately 8 million years ago; diploid chromosome number variation among the species ranges from 2n = 36 to 2n = 50. In an attempt to shed more light on chromosome evolution and speciation in these rodents, we compared the karyotypes of 7 species, representing 3 genera, based on homology data revealed by chromosome painting with probes derived from flow-sorted chromosomes of the hairy footed gerbil, <i>Gerbillurus paeba</i> (2n = 36). The fluorescent in situ hybridization data revealed remarkable genome conservation: these species share a high proportion of conserved chromosomes, and differences are due to 10 Robertsonian (Rb) rearrangements (3 autapomorphies, 3 synapomorphies and 4 hemiplasies/homoplasies). Our data suggest that chromosome evolution in <i>Desmodillus </i>occurred at a rate of ∼1.25 rearrangements per million years (Myr), and that the rate among <i>Gerbilliscus</i> over a time period spanning 8 Myr is also ∼1.25 rearrangements/Myr. The recently diverged <i>Gerbillurus (G. tytonis </i>and<i> G. paeba)</i> share an identical karyotype, while <i>Gerbilliscus kempi, G. afra</i> and <i>G. leucogaster</i> differ by 6 Rb rearrangements (a rate of ∼1 rearrangement/Myr). Thus, our data suggests a very slow rate of chromosomal evolution in Southern African gerbils

Supplementary Material for: Familial Coaggregation of Alzheimer's Disease and Parkinson's Disease: Systematic Review and Meta-Analysis

<b><i>Background:</i></b> Familial aggregation has been shown for Alzheimer's disease (AD) and Parkinson's disease (PD) separately, and it has been hypothesized that these diseases also coaggregate in families. <b><i>Methods:</i></b> The authors investigated familial coaggregation of AD and PD by conducting a systematic review and meta-analysis. PubMed was searched for relevant studies published through the end of October 2012. Three independent investigators screened publications and extracted data. Relative risk estimates of AD risk associated with family history of PD or parkinsonism, or PD risk associated with family history of AD or dementia, were summarized into metaestimates using random effects models. Heterogeneity and publication bias were tested using Higgins' and Egger's tests, respectively. <b><i>Results:</i></b> We included 16 studies in the review, with 14 included in any meta-analysis. AD risk associated with family history of PD yielded a summary hazard ratio of 1.18 (95% CI: 1.00-1.39) based on 5 reconstructed cohort studies and a summary odds ratio (OR) of 1.40 (95% CI: 0.92-2.12) based on 7 case-control studies. PD risk associated with family history of AD yielded a summary OR of 0.75 (95% CI: 0.49-1.16) based on 3 studies. There was no significant heterogeneity among studies, nor significant publication bias. <b><i>Conclusions:</i></b> There may be familial coaggregation of AD and PD, although the association was modest and only apparent when studying AD risk associated with family history of PD

Supplementary Material for: Complement Factor I Polymorphism Is Not Associated with Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy in a Chinese Population

<b><i>Purpose:</i></b> To identify the associations of the two complement factor I (CFI) polymorphisms <i>rs10033900</i> and <i>rs2285714</i> with risk of neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in a Chinese case-control study. <b><i>Methods:</i></b> A total of 900 subjects - 300 controls, 300 cases with nAMD and 300 cases with PCV - were included in the present study. Genomic DNA was extracted from venous blood leukocytes. The allelic variants of <i>rs10033900</i> and <i>rs2285714</i> were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The differences in allele distribution between the cases and controls were tested by a χ² test with age and gender adjusted for by logistic regression analysis. We also performed a meta-analysis of the case-control studies of <i>rs10033900</i> and <i>rs2285714</i> based on the currently available evidence from the literature. The meta-analysis was conducted via an inverse-variance, fixed-effects model, as previously described. <b><i>Results:</i></b> No statistically significant association was observed between the two polymorphisms of CFI and AMD risk, including nAMD, PCV and combined AMD (p > 0.05 for all comparisons). By meta-analysis, we detected significant associations between both of the SNPs and late AMD, which is consistent with previous results (odds ratio, OR, <i>rs10033900</i> = 0.814, p <i>rs10033900</i> < 0.001; OR <i>rs2285714</i> = 1.221, p <i>rs2285714</i> < 0.001). For <i>rs2285714</i>, the results of the meta-analysis were less reliable due to its heterogeneity. <b><i>Conclusions:</i></b> In our case-control study, neither of the two SNPs most studied (<i>rs10033900</i> or <i>rs2285714</i>) in the <i>CFI</i> gene was a risk factor for developing nAMD or PCV in a Chinese population. Additional large, comprehensive and well-designed association studies are needed to better understand the role of ethnicity and other gene interactions in the association between the <i>CFI</i> gene and AMD

Supplementary Material for: Using smartphone TechnolOGy to support an EffecTive Home ExeRcise intervention to prevent falls amongst community dwelling older adults The TOGETHER feasibility RCT.

Introduction Falls have major implications for quality-of-life, independence and cost to health services. Strength and balance training has been found to be effective in reducing the rate/risk of falls, as long as there is adequate fidelity to the evidence-based programme. The aims of this study were to: (1) assess the feasibility of using the ‘Motivate Me’ and ‘My Activity Programme’ intervention to support falls rehabilitation when delivered in practice (2) assess study design and trial procedures for the evaluation of the intervention. Methods A two-arm, pragmatic feasibility randomised controlled trial was conducted with five health service providers in the UK. Patients aged 50+ years eligible for a falls rehabilitation exercise programme from community services were recruited and received either: (1) standard service with a smartphone for outcome measurement only or (2) standard service plus the ‘Motivate Me’ and ‘My Activity Programme’ apps. The primary outcome was feasibility of the intervention, study design and procedures (including recruitment rate, adherence and drop-out). Outcome measures include balance, function, falls, strength, fear of falling, health related quality of life, resource use and adherence, measured at baseline, three and six month post-randomisation. Blinded assessors collected the outcome measures. Results 24 patients were randomised to control group, 26 to intervention group, mean age 77.6 (Range 62 to 92) years. We recruited 37.5% of eligible participants across the five clinical sites. 77% in the intervention group completed their full exercise programme (including the use of the app). Response rate for outcome measures at six months were 77%-80% across outcome measures, but this was effected by the COVID19 pandemic. There was a mean 2.6 ± 1.9 point difference between groups in change in BERG balance score from baseline to three months and mean 4.4 ± 2.7 point difference from baseline to six months in favour of the intervention group. Less falls (1.8 ± 2.8 vs 9.1 ± 32.6) and less injurious falls (0.1 ± 0.5 vs 0.4 ± 0.6) in the intervention group and higher adherence scores at three (17.7 ± 6.8 vs 13.1 ± 6.5) and six months (15.3 ± 7.8 vs 14.9 ± 7.8). There were no related adverse events. Health professionals and patients had few technical issues with the apps. Conclusions The motivational apps and trial procedures were feasible for health professionals and patients. There are positive indications from outcome measures in the feasibility trial and key criteria for progression to full trial were met

Supplementary Material for: ABCA1 rs1883025 Polymorphism Shows No Association with Neovascular Age-Related Macular Degeneration or Polypoidal Choroidal Vasculopathy in a Northern Chinese Population

<b><i>Purpose:</i></b> To analyze the association between ABCA1 rs1883025 variants with neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in a northern Chinese population. <b><i>Methods:</i></b> The study enrolled 900 subjects, including 300 controls, 300 cases with nAMD and 300 cases with PCV. Genomic DNA was extracted from venous blood leukocytes. Single-nucleotide polymorphisms in the ABCA1 (rs1883025) gene were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. <b><i>Results:</i></b> The ABCA1 rs1883025 polymorphism was not significantly associated with nAMD (22.5%; p > 0.05) or PCV (20.8%; p > 0.05) in a northern Chinese population. The association remained insignificant after adjustment for age and gender differences (p > 0.05). <b><i>Conclusions:</i></b> This study suggests that ABCA1 rs1883025 variants are not associated with nAMD or PCV in a Chinese population, which is likely due to an ethnic difference

Supplementary Material for: Karyotype Evolution and Phylogenetic Relationships of Cricetulus sokolovi Orlov et Malygin 1988 (Cricetidae, Rodentia) Inferred from Chromosomal Painting and Molecular Data

<p>Sokolov's dwarf hamster (<i>Cricetulus sokolovi</i>) is the least studied representative of the striped hamsters (<i>Cricetulus barabensis</i> species group), the taxonomy of which remains controversial. The species was described based on chromosome morphology, but neither the details of the karyotype nor the phylogenetic relationships with other <i>Cricetulus</i> are known. In the present study, the karyotype of <i>C. sokolovi</i> was examined using cross-species chromosome painting. Molecular and cytogenetic data were employed to determine the phylogenetic position of Sokolov's hamster and to analyze the potential pathways of chromosome evolution in <i>Cricetulus</i>. Both the chromosome and molecular data support the species status of Sokolov's hamster. Phylogenetic analysis of the <i>CYTB</i> data placed <i>C. sokolovi</i> as sister to all other striped hamsters (sequence divergence of 8.1%). FISH data revealed that the karyotype of <i>C. sokolovi</i> is highly rearranged, with the most parsimonious scenario of its origin implying at least 4 robertsonian events and a centromere shift. Comparative cytogenetic data on Cricetinae suggest that their evolutionary history includes both periods of chromosomal conservatism and episodes of rapid chromosomal change.</p

Supplementary Material for: The Protective Effects of Κ-Opioid Receptor Stimulation in Hypoxic Pulmonary Hypertension Involve Inhibition of Autophagy Through the AMPK-MTOR Pathway

<b><i>Background/Aims:</i></b> In a previous study, we showed that κ-opioid receptor stimulation with the selective agonist U50,488H ameliorated hypoxic pulmonary hypertension (HPH). However, the roles that pulmonary arterial smooth muscle cell (PASMC) proliferation, apoptosis, and autophagy play in κ-opioid receptor-mediated protection against HPH are still unknown. The goal of the present study was to investigate the role of autophagy in U50,488H-induced HPH protection and the underlying mechanisms. <b><i>Methods:</i></b> Rats were exposed to 10% oxygen for three weeks to induce HPH. After hypoxia, the mean pulmonary arterial pressure (mPAP) and the right ventricular pressure (RVP) were measured. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by flow cytometry and Western blot. Autophagy was assessed by means of the mRFP-GFP-LC3 adenovirus transfection assay and by Western blot. <b><i>Results:</i></b> Inhibition of autophagy by the administration of chloroquine prevented the development of HPH in the rat model, as evidenced by significantly reduced mPAP and RVP, as well as decreased autophagy. U50,488H mimicked the effects of chloroquine, and the effects of U50,488H were blocked by nor-BNI, a selective κ-opioid receptor antagonist. <i>In vitro</i> experiments showed that the inhibition of autophagy by chloroquine was associated with decreased proliferation and increased apoptosis of PASMCs. Under hypoxia, U50,488H also significantly inhibited autophagy, reduced proliferation and increased apoptosis of PASMCs. These effects of U50,488H were blocked by nor-BNI. Moreover, exposure to hypoxic conditions significantly increased AMPK phosphorylation and reduced mTOR phosphorylation, and these effects were abrogated by U50,488H. The effects of U50,488H on PASMC autophagy were inhibited by AICAR, a selective AMPK agonist, or by rapamycin, a selective mTOR inhibitor. <b><i>Conclusion:</i></b> Our data provide evidence for the first time that κ-opioid receptor stimulation protects against HPH by inhibiting PASMCs autophagy via the AMPK-mTOR pathway

Supplementary Material for: CyclinG1 Amplification Enhances Aurora Kinase Inhibitor-Induced Polyploid Resistance and Inhibition of Bcl-2 Pathway Reverses the Resistance

<i>Background/Aims:</i> CyclinG1 (CycG1) is frequently overexpressed in solid tumors and overexpression of CycG1 promotes cell survival upon paclitaxel exposure by inducing polyploidy. Whether and how CycG1 regulates polyploidization caused by small molecular targeted inhibitors remains unclear. <i>Methods:</i> Immunohistochemistry and immunoblotting were utilized to examine protein expression. Cell proliferation was measured by ATPlite assay, and cell cycle distribution and apoptosis were measured by flow cytometry and/or DNA fragmentation assays. <i>Results:</i> Overexpression of CycG1 in breast cancer cells caused apoptosis-resistant polyploidy upon treatment with Aurora kinase inhibitor, ZM447439 (ZM). Addition of ABT-263, a small-molecule BH3 mimetic, to ZM, produced a synergistic loss of cell viability with greater sustained tumor growth inhibition in breast cancer cell lines. Decrease of Mcl-1 and increase of NOXA caused by ZM treatment, were responsible for the synergy. Furthermore, CycG1 was highly expressed in Triple-Negative-Breast-Cancer patients treated with paclitaxel and was paralleled by decreased cell survival. <i>Conclusion:</i> CycG1 is a crucial factor in ZM-induced polyploidy resistance, and ABT-263/ZM combination hold therapeutic utility in the CycG1-amplified subset of breast cancer and CycG1, thus, is a promising target in breast cancer