Supplementary Material for: Transcriptome, Biochemical and Growth Responses of the Marine Phytoplankter Phaeodactylum Tricornutum Bohlin (Bacillariophyta) to Copepod Grazer Presence

<b><i>Background/Aims:</i></b> As a model organism for a pleiomorphic marine planktonic primary producer, <i>Phaeodactylum tricornutum</i> has been studied on a molecular level under diverse cultural conditions. But little is known about its morphological, nutritional or transcriptomic responses under grazing stress. <b><i>Methods:</i></b> To assess microalgal molecular and cellular responses to grazer presence, we conducted transcriptome profiling in combination with growth rate, biovolume, fatty acid content, carbon and nitrogen content measurements in the model diatom <i>Phaeodactylum tricornutum</i>. RNA-sequencing was used to evaluate the transcriptomic response to grazing stress for <i>P. tricornutum</i> strain <i>CCAP 1055/1</i>. <b><i>Results:</i></b> Among the differentially expressed genes, we found down-regulation of genes involved in pathogen resistance, and in fatty acid biosynthesis pathways, while mitosis-involved genes were up-regulated. Experimentally testing morphological and biochemical responses in five strains of the species, we detected strain-specific significant effects of simulated grazing pressure in altered growth rates, biovolume and nutritional composition. <b><i>Conclusion:</i></b> Our research reveals the associated molecular and cellular responses to grazing effects in <i>P. tricornutum</i> and extends the understanding of co-evolutionary roles in regulating grazing defence between <i>P. tricornutum</i> and its grazer

Supplementary Material for: Piceatannol protects against high glucose-induced injury of renal tubular epithelial cells via regulating carbonic anhydrase 2

Introduction: We here evaluated the efficacy of piceatannol (PIC) in high glucose (HG)-induced injury of renal tubular epithelial cells HK-2. Methods: After the establishment of HG-induced cell injury model and the treatment with PIC at both high and low concentrations and/or Acetazolamide (ACZ, the inhibitor of carbonic anhydrase 2 (CA2)), MTT and flow cytometry assays were carried out to confirm the viability and apoptosis of HK-2 cells. The levels of oxidative stress markers lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS), the ratio of glutathione/oxidized glutathione (GSH/GSSG), and the CA2 activity were determined. Both quantitative reverse-transcription polymerase chain reaction and Western blot were used to calculate the expressions of CA2 (the predicted target gene of PIC via intersecting the data from bioinformatic analyses), and AKT pathway- (Phosphatase and tensin homolog (PTEN), phosphorylated (p)-AKT, AKT) and apoptosis-related proteins (Bcl-2 and cleaved caspase-3). Results: HG suppressed cell viability and the levels of GSH/GSSG ratio, CA2, pThr308-AKT/AKT, pSer473-AKT/AKT, and Bcl-2, while promoting cell apoptosis, the levels of LDH, MDA, and ROS and the expressions of PTEN and cleaved caspase-3. All effects of HG were reversed by PIC at a high concentration. CA2 was predicted and identified as the target of PIC. In HG-treated HK-2 cells, additionally, ACZ reversed the effects of PIC on the viability, apoptosis, and the levels of both oxidative stress markers and AKT pathway- and apoptosis-related factors. Conclusion: PIC protects against HG-induced injury of HK-2 cells via regulating CA2

Supplementary Material for: A common variant of ARRB2 promoter region Associated with the Prognosis of Heart Failure

Introduction The role of ARRB2 in cardiovascular disease has recently gained increasing attention. However, the association between ARRB2 polymorphisms and heart failure (HF) has not yet been investigated. Methods A total of 2386 hospitalized patients with chronic heart failure were enrolled as the first cohort and followed up for a mean period of 20.2 months. Meanwhile, ethnically and geographically matched 3000 individuals without evidence of HF were included as healthy controls. We genotyped the common variant in ARRB2 gene to identify the association between variant and HF. A replicated independent cohort enrolling 837 patients with chronic HF was applied to validate the observed association. A series of function analysis were conducted to illuminate the underlying mechanism. Results We identified a common variant rs75428611 associated with the prognosis of HF in two-stage population: adjusted P = 0.001, HR = 1.31 (1.11-1.54) in additive model and adjusted P = 0.001, HR = 1.39 (1.14-1.69) in dominant model in first-stage population; adjusted P = 0.04, HR = 1.41 (1.02-1.95) in additive model and adjusted P = 0.03, HR = 1.51 (1.03-2.20) in dominant model in replicated stage. However, rs75428611 did not significantly associate with the risk of HF. Functional analysis indicated that rs75428611‐G allele increased the promoter activity and the mRNA expression level of ARRB2 by facilitating transcription factor SRF binding, but not the A allele. Conclusions Our findings demonstrate that rs75428611 in promoter of ARRB2 is associated with the risk of HF mortality. It is a promising potential treatment target for HF

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: Advanced Glycation End Products-induced Activation of Keratinocytes: a Mechanism Underlying Cutaneous Immune Response in Psoriasis

Psoriasis is a common inflammatory skin disease, in which epidermal keratinocytes play a vital role in its pathogenesis via acting both as the responder and as the accelerator to the cutaneous psoriatic immune response. Advanced glycation end products (AGEs) are a class of proinflammatory metabolites that are commonly accumulating in cardiometabolic disorders. Recent studies have also observed increased level of AGEs in the serum and skin of psoriasis patients, but the role of AGEs in psoriatic inflammation has not been well investigated. In the present study, we initially detected abnormal accumulation of AGEs in epidermal keratinocytes of psoriatic lesion collected from psoriasis patients. Furthermore, AGEs promoted the proliferation of keratinocytes via up-regulated Keratin 17 (K17)-mediated p27KIP1 inhibition followed by accelerated cell cycle progression. More importantly, AGEs facilitated the production of interleukin-36 alpha (IL-36α) in keratinocytes, which could enhance T helper 17 (Th17) immune response. In addition, the induction of both K17 and IL-36α by AGEs in keratinocytes was dependent on the activation of signal transducer and activator of transcription 1/3 (STAT1/3) signaling pathways. At last, the effects of AGEs on keratinocytes were mediated by receptor for AGEs (RAGE). Taken together, these findings support that AGEs potentiate the innate immune function of keratinocytes, which contributes to the formation of psoriatic inflammation. Our study implicates AGEs as a potential pathogenic link between psoriasis and cardiometabolic comorbidities

Supplementary Material for: Sodium Citrate Inhibits Endoplasmic Reticulum Stress in Rats with Adenine-Induced Chronic Renal Failure

<b><i>Background/Aims:</i></b> Endoplasmic reticulum stress (ERS) is an important self-protective cellular response to harmful stimuli that contribute to various diseases, including chronic renal failure (CRF). Sodium citrate plays an important role in antioxidant and cellular immunity, but whether it improves ERS in CRF is unclear. <b><i>Methods:</i></b> The rats were randomly divided into five groups: the control group, the sodium citrate control group, the model group, model rats with low dose sodium citrate (216 mg/kg), and model rats with a high dose of sodium citrate (746 mg/kg). The rats were euthanized at 6, 8, 12, and 16 weeks with their blood and renal tissue in detection. <b><i>Results:</i></b> The increased concentrations of blood urea nitrogen and serum creatinine in the model group were significantly decreased by sodium citrate treatment. Hematoxylin-eosin and Masson staining showed that sodium citrate treatment apparently improved renal pathological changes in CRF rats. Western blot analysis showed that sodium citrate treatment decreased the protein levels of transforming growth factor-beta 1 and collagen type IV, which were increased in model rats. Moreover, immunohistochemical staining demonstrated that sodium citrate could effectively reduce the protein expression of glucose-regulated protein 78 kDa and CCAAT/enhancer-binding protein homologous protein in the model rats, which was consistent with western blot results. Additionally, the high dose of sodium citrate had a stronger protective effect in CRF rats than the low dose of sodium citrate. <b><i>Conclusions:</i></b> Sodium citrate has a protective effect on CRF through its effects on ERS

Supplementary Material for: MicroRNA-1185 Induces Endothelial Cell Apoptosis by Targeting UVRAG and KRIT1

<i>Background/Aims:</i> Atherosclerosis is a multifactorial chronic disease and is the main cause of death and impairment in the world. Endothelial injury and apoptosis play a crucial role in the onset and development of atherosclerosis. MicroRNAs (miRNAs) have been proven to be involved in the pathogenesis of atherosclerosis. However, studies of the functional role of apoptosis-related miRNAs in the endothelium during atherogenesis are limited. <i>Methods:</i> Cell injury and apoptosis were measured in five types of cells transfected with miR-1185 or co-transfected with miR-1185 and its inhibitor. Bioinformatics analysis and a luciferase reporter assay were used to confirm the targets of miR-1185. The effects of the targets of miR-1185 on endothelial apoptosis were determined using small-interfering RNA. <i>Results:</i> In this study, we first report that miR-1185 significantly promoted apoptosis in endothelial cells but not in vascular smooth muscle cells and macrophages. A mechanistic analysis showed that ultraviolet irradiation resistance-associated gene (UVRAG) and krev1 interaction trapped gene 1 (KRIT1), targets of miR-1185, mediated miR-1185-induced endothelial cell apoptosis. <i>Conclusion:</i> The results revealed the impact of miR-1185 on endothelial apoptosis, suggesting that miR-1185 may be a potential target for the prevention and treatment of atherosclerosis

Supplementary Material for: Polymorphisms in Sex Hormone Metabolism Genes and Risk of Preeclampsia in Taiyuan, China

<i>Objective:</i> Aberrant synthesis and metabolism of sex hormone are likely to be associated with alterations in vascular function in preeclampsia (PE). The study aims to investigate whether single nucleotide polymorphisms (SNPs) in sex hormone-related genes are associated with PE. <i>Method:</i> We performed a nested case-control study including 436 pregnant women (203 PE and 233 healthy or normal pregnant women) to investigate associations between 96 SNPs in 28 sex hormone-related genes and risk of PE. <i>Results:</i><i>TXNRD2/COMT</i> rs3788314 and <i>SULT1A2/SULT1A1 </i>rs4788073 were associated with an increased risk of PE overall (<i>p</i><i>trend</i> = 0.004 and 0.003, respectively), early-onset PE (<i>p</i><i>trend</i> = 0.007 and 0.009, respectively), and severe PE (<i>p</i><i>trend</i> = 0.002 and 0.005, respectively). Additionally, <i>CYP17A1 </i>rs4919690 and rs4919687 and <i>LHCGR rs10180731</i>were associated with an increased risk of severe PE (<i>p</i><i>trend</i> = 0.005, 0.006, and 0.014, respectively), while <i>GNRHR</i> rs2630488 was associated with a decreased risk of severe PE (<i>p</i><i>trend</i> = 0.014). We also observed that <i>HSD17B3</i> rs8190512 was associated with a decreased risk of early-onset PE (<i>p</i><i>trend</i> = 0.003). We observed strong linkage disequilibrium in SULF1 (rs10106958, rs7813987, and rs6990375). <i>Conclusions:</i> Our study suggested that genetic polymorphisms in <i>TXNRD2/COMT, SULT1A2/SULT1A1, CYP17A1, HSD17B3, GNRHR, LHCGR, </i>and SULF1 might play a role in PE, especially in early-onset PE and severe PE. Future studies are warranted to replicate the observed associations and their functional mechanisms

Supplementary Material for: Cyclic Mechanical Stretch Induced Smooth Muscle Cell Changes in Cerebral Aneurysm Progress by Reducing Collagen Type IV and Collagen Type VI Levels

<b><i>Background/Aims:</i></b> Cerebral aneurysm growth is characterized by continuous structural weakness of local smooth muscle cells, though the mechanism is unclear. In this study, we examine protein changes in cerebral aneurysm and human brain vascular smooth muscle cells after cyclic mechanical stretch. We further explore the relationship between the smooth muscle cell changes and reductions in the levels of collagen types IV and VI. <b><i>Methods:</i></b> Saccular cerebral aneurysms (n=10) were collected, and temporal artery samples were used as controls. Quantitative proteomics were analyzed and histopathological changes were examined. Smooth muscle cells were cultured in a flexible silicone chamber and subjected to 15% cyclic mechanical stretch. The effect of stretch on the cell viability, function, gene and protein expression were further studied for the understanding the molecular mechanism of aneurysm development. <b><i>Results:</i></b> Proteomics analysis revealed 92 proteins with increased expression and 88 proteins with decreased expression compared to the controls (<i>p</i><0.05). KEGG pathway analysis showed that the change in focal adhesion and extracellular matrix-receptor interaction, suggesting the involvement of collagen type IV and VI. The aneurysm tissue exhibited fewer smooth muscle cells and lower levels of collagen type IV and VI. Human brain vascular smooth muscle cell culture showed spindle-like cells and obvious smooth muscle cell layer. Cell proteomics analysis showed that decreased expression of 118 proteins and increased expression of 32 proteins in smooth muscle cells after cyclic mechanical stretch. KEGG pathway analysis indicated that focal adhesion and ECM-receptor interaction were involved. After cyclic mechanical stretch, collagen type IV and IV expression were decreased. Moreover, the stretch induced MMP-1 and MMP-3 expression elevation. <b><i>Conclusion:</i></b> We demonstrated that collagen type IV and VI were decreased in cerebral aneurysms and continuous cyclic mechanical stretch induced smooth muscle cell changes. Smooth muscle cell protection provides an additional therapeutic option to prevent the growth of cerebral aneurysms

Supplementary Material for: The Wound Healing of Autologous Regenerative Factor on Recurrent Benign Airway Stenosis: A Canine Experimental and Pilot Study

Introduction: Benign airway stenosis (BAS) is a severe pathologic condition. Complex stenosis has a high recurrence rate and requires repeated bronchoscopic interventions for achieving optimal control, leading to recurrent BAS (RBAS) due to intraluminal granulation. Methods: This study explored the potential of autologous regenerative factor (ARF) for treating RBAS using a post-intubation tracheal stenosis canine model. Bronchoscopic follow-ups were conducted, and RNA-seq analysis of airway tissue was performed. A clinical study was also initiated involving 17 patients with recurrent airway stenosis. Results: In the animal model, ARF demonstrated significant effectiveness in preventing further collapse of the injured airway, maintaining airway patency and promoting tissue regeneration. RNA-seq results showed differential gene expression, signifying alterations in cellular components and signaling pathways. The clinical study found that ARF treatment was well-tolerated by patients with no severe adverse events requiring hospitalization. ARF treatment yielded a high response rate, especially for post-intubation tracheal stenosis and idiopathic tracheal stenosis patients. Conclusion: The study concludes that ARF presents a promising, effective, and less-invasive method for treating RBAS. ARF has shown potential in prolonging the intermittent period and reducing treatment failure in patients with recurrent tracheal stenosis by facilitating tracheal mucosal wound repair and ameliorating tracheal fibrosis. This novel approach could significantly impact future clinical applications