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Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries.

Rationale: Obstructive sleep apnea (OSA)-induced endothelial cell (EC) dysfunction contributes to OSA-related cardiovascular sequelae. The mechanistic basis of endothelial impairment by OSA is unclear. Objectives: The goals of this study were to identify the mechanism of OSA-induced EC dysfunction and explore the potential therapies for OSA-accelerated cardiovascular disease. Methods: The experimental methods include data mining, bioinformatics, EC functional analyses, OSA mouse models, and assessment of OSA human subjects. Measurements and Main Results: Using mined microRNA sequencing data, we found that microRNA 210 (miR-210) conferred the greatest induction by intermittent hypoxia in ECs. Consistently, the serum concentration of miR-210 was higher in individuals with OSA from two independent cohorts. Importantly, miR-210 concentration was positively correlated with the apnea-hypopnea index. RNA sequencing data collected from ECs transfected with miR-210 or treated with OSA serum showed a set of genes commonly altered by miR-210 and OSA serum, which are largely involved in mitochondrion-related pathways. ECs transfected with miR-210 or treated with OSA serum showed reduced [Formula: see text]o2 rate, mitochondrial membrane potential, and DNA abundance. Mechanistically, intermittent hypoxia-induced SREBP2 (sterol regulatory element-binding protein 2) bound to the promoter region of miR-210, which in turn inhibited the iron-sulfur cluster assembly enzyme and led to mitochondrial dysfunction. Moreover, the SREBP2 inhibitor betulin alleviated intermittent hypoxia-increased systolic blood pressure in the OSA mouse model. Conclusions: These results identify an axis involving SREBP2, miR-210, and mitochondrial dysfunction, representing a new mechanistic link between OSA and EC dysfunction that may have important implications for treating and preventing OSA-related cardiovascular sequelae.

The homeostasis of vascular endothelium is crucial for cardiovascular health and endothelial cell (EC) aging and dysfunction could negatively impact vascular function. Leveraging transcriptome profiles from ECs subjected to various stimuli, including time-series data obtained from ECs under physiological pulsatile flow vs. pathophysiological oscillatory flow, we performed principal component analysis (PCA) to identify key genes contributing to divergent transcriptional states of ECs. Through bioinformatics analysis, we identified that a long non-coding RNA (lncRNA) RAMP2-AS1 encoded on the antisense of RAMP2, a determinant of endothelial homeostasis and vascular integrity, is a novel regulator essential for EC homeostasis and function. Knockdown of RAMP2-AS1 suppressed RAMP2 expression and caused EC functional changes promoting aging, including impaired angiogenesis and increased senescence. Our study demonstrates an integrative approach to quantifying EC aging based on transcriptome changes, which also identified a number of novel regulators, including protein-coding genes and many lncRNAs involved EC functional modulation, exemplified by RAMP2-AS1.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects cholesterol homeostasis by targeting hepatic LDL receptor (LDLR) for lysosomal degradation. Clinically, PCSK9 inhibitors effectively reduce LDL-cholesterol (LDL-C) levels and the incidence of cardiovascular events. Because microRNAs (miRs) are integral regulators of cholesterol homeostasis, we investigated the involvement of miR-483 in regulating LDL-C metabolism. Using in silico analysis, we predicted that miR-483-5p targets the 3'-UTR of PCSK9 mRNA. In HepG2 cells, miR-483-5p targeted the PCSK9 3'-UTR, leading to decreased PCSK9 protein and mRNA expression, increased LDLR expression, and enhanced LDL-C uptake. In hyperlipidemic mice and humans, serum levels of total cholesterol and LDL-C were inversely correlated with miR-483-5p levels. In mice, hepatic miR-483 overexpression increased LDLR levels by targeting Pcsk9, with a significant reduction in plasma total cholesterol and LDL-C levels. Mechanistically, the cholesterol-lowering effect of miR-483-5p was significant in mice receiving AAV8 PCSK9-3'-UTR but not Ldlr-knockout mice or mice receiving AAV8 PCSK9-3'-UTR (ΔBS) with the miR-483-5p targeting site deleted. Thus, exogenously administered miR-483 or similarly optimized compounds have potential to ameliorate hypercholesterolemia.

Recent evidence suggests that enhanced protease-mediated inflammation may promote insulin resistance and result in diabetes. This study tested the hypothesis that serine protease plays a pivotal role in type 2 diabetes, and inhibition of serine protease activity prevents hyperglycemia in diabetic animals by modulating insulin signaling pathway. We conducted a single-center, cross-sectional study with 30 healthy controls and 57 patients with type 2 diabetes to compare plasma protease activities and inflammation marker between groups. Correlations of plasma total and serine protease activities with variables were calculated. In an in-vivo study, LDLR-/- mice were divided into normal chow diet, high-fat diet (HFD), and HFD with selective serine protease inhibition groups to examine the differences of obesity, blood glucose level, insulin resistance and serine protease activity among groups. Compared with controls, diabetic patients had significantly increased plasma total protease, serine protease activities, and also elevated inflammatory cytokines. Plasma serine protease activity was positively correlated with body mass index, hemoglobin A1c, homeostasis model assessment-insulin resistance index (HOMA-IR), tumor necrosis factor-α, and negatively with adiponectin concentration. In the animal study, administration of HFD progressively increased body weight, fasting glucose level, HOMA-IR, and upregulated serine protease activity. Furthermore, in-vivo serine protease inhibition significantly suppressed systemic inflammation, reduced fasting glucose level, and improved insulin resistance, and these effects probably mediated by modulating insulin receptor and cytokine expression in visceral adipose tissue. Our findings support the serine protease may play an important role in type 2 diabetes and suggest a rationale for a therapeutic strategy targeting serine protease for clinical prevention of type 2 diabetes.

Pulsatile shear (PS) and oscillatory shear (OS) elicit distinct mechanotransduction signals that maintain endothelial homeostasis or induce endothelial dysfunction, respectively. A subset of microRNAs (miRs) in vascular endothelial cells (ECs) are differentially regulated by PS and OS, but the regulation of the miR processing and its implications in EC biology by shear stress are poorly understood. From a systematic in silico analysis for RNA binding proteins that regulate miR processing, we found that nucleolin (NCL) is a major regulator of miR processing in response to OS and essential for the maturation of miR-93 and miR-484 that target mRNAs encoding Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS). Additionally, anti-miR-93 and anti-miR-484 restore KLF2 and eNOS expression and NO bioavailability in ECs under OS. Analysis of posttranslational modifications of NCL identified that serine 328 (S328) phosphorylation by AMP-activated protein kinase (AMPK) was a major PS-activated event. AMPK phosphorylation of NCL sequesters it in the nucleus, thereby inhibiting miR-93 and miR-484 processing and their subsequent targeting of KLF2 and eNOS mRNA. Elevated levels of miR-93 and miR-484 were found in sera collected from individuals afflicted with coronary artery disease in two cohorts. These findings provide translational relevance of the AMPK-NCL-miR-93/miR-484 axis in miRNA processing in EC health and coronary artery disease.

BackgroundDietary sodium and potassium affect the fluctuation in blood pressure (BP) and renal function. Corin, with its enzymatic activity to convert pro-atrial natriuretic peptide (pro-ANP) to biologically active ANP, regulates BP, cardiac, and renal functions. We investigated whether corin expression responds to a high-salt (HS) diet to regulate salt and water balance.MethodsForty-two volunteers followed 3 sequential diets for 7 days each: a low-salt (LS) diet (3.0 g/day NaCl), a HS diet (18.0 g/day NaCl), followed by an HS diet with K+ supplementation (HS + K+) (18.0 g/day NaCl and 4.5 g/day KCl).ResultsCorin level was higher with the HS diet than the LS and HS + K+ diets and was positively correlated with systolic BP (SBP) and 24-hour urinary Na+ and microalbumin (U-mALB) excretion. In rodents, serum and renal levels of corin were transiently increased with the HS diet and were decreased if the HS diet was continued for up to 7 days. HS loading increased SBP, 24-hour urinary Na+, U-mALB excretion, and the expression of proprotein convertase subtilisin/kexin-6 (PCSK6), a corin activator. Knockdown of PCSK6 or corin in high salt-treated M1-cortical collecting duct (M1-CCD) cells increased the expression of aquaporin 2 (AQP2) and β-epithelial Na+ channel (β-ENaC).ConclusionsShort-term HS may induce the PCSK6-corin-ANP-AQP2/β-ENaC pathway in the kidney. Enhanced serum corin level in humans and rodents is positively correlated with HS-induced SBP and 24-hour urinary Na+ and U-mALB excretion, which suggests that corin is involved in the salt-water balance in response to HS intake.Clinical trials registrationPublic Trials Registry Number NCT02915315.

CKD is an independent risk factor for cardiovascular disease (CVD). The accumulation of uremic toxins in CKD induces oxidative stress and endothelial dysfunction. MicroRNA-92a (miR-92a) is induced by oxidative stress in endothelial cells (ECs) and involved in angiogenesis and atherosclerosis. We investigated a role for oxidative stress-responsive miR-92a in CKD. Our study of patients at three clinical sites showed increased serum miR-92a level with decreased kidney function. In cultured ECs, human CKD serum or uremic toxins (such as indoxyl sulfate), compared with non-CKD serum, induced the levels of miR-92a and suppressed the expression of miR-92a targets, including key endothelial-protective molecules. The antioxidant N-acetylcysteine inhibited these vasculopathic properties. In rats, adenine-induced CKD associated with increased levels of miR-92a in aortas, serum, and CD144+ endothelial microparticles. Furthermore, CD144+ microparticles from human uremic serum contained more miR-92a than those from control serum. Additional analysis showed a positive correlation between serum levels of miR-92a and indoxyl sulfate in a cohort of patients with ESRD undergoing hemodialysis. Collectively, our findings suggest that the uremic toxins accumulated in CKD can upregulate miR-92a in ECs, which impairs EC function and predisposes patients to CVD.

Introduction: The DESyne novolimus‐eluting coronary stent (NES) is a new‐generation drug‐eluting stent (DES) that is widely used, but clinical data are rarely reported for this stent. We compared the safety and effectiveness of the DESyne NES and the Orsiro bioresorbable polymer sirolimus‐eluting stent (SES) in patients undergoing percutaneous coronary intervention (PCI). Methods: This was a retrospective, single‐center, observational study. Between July 2017 and December 2022, patients who presented with chronic or acute coronary syndrome undergoing PCI with DESyne NES or Orsiro SES were consecutively enrolled in the present study. The primary endpoint, major adverse cardiovascular event (MACE), was a composite of cardiovascular death, target‐vessel myocardial infarction, or clinically driven target‐lesion revascularization. Results: A total of 776 patients (age 68.8 ± 12.2; 75.9% male) undergoing PCI were included. Overall, 231 patients with 313 lesions received NES and 545 patients with 846 lesions received SES. During a follow‐up duration of 784 ± 522 days, the primary endpoint occurred in 10 patients (4.3%) in the NES group and in 36 patients (6.6%) in the SES group. After multivariate adjustment, the risk of MACE did not significantly differ between groups (NES vs. SES, hazard ratio 0.74, 95% CI, 0.35–1.55, p = 0.425). The event rate of individual components of the primary endpoint was comparable between the two groups. Conclusions: Favorable and similar clinical outcomes were observed in patients undergoing PCI with either NES or SES in a medium‐term follow‐up duration. Future studies with adequately powered clinical endpoints are required for further evaluation. [ABSTRACT FROM AUTHOR]

Objectives: Despite mounting evidence demonstrates circulating endothelial progenitor cells (cEPCs) quantitative changes in depression, no study has investigated cEPC functions in major depressive disorder (MDD). We investigated the role of cEPC adhesive and apoptotic functions in MDD. Methods: We recruited 68 patients with MDD and 56 healthy controls (HCs). The depression symptoms, anxiety, psychosomatic symptoms, subjective cognitive dysfunction, quality of life, and functional disability were evaluated using the Hamilton Depression Rating Scale and Montgomery–Åsberg Depression Rating Scale, Hamilton Anxiety Rating Scale, Depression and Somatic Symptoms Scale (DSSS), Perceived Deficits Questionnaire-Depression, 12-Item Short Form Health Survey (SF-12), and Sheehan Disability Scale (SDS), respectively. Working memory and executive function were assessed using a 2-back task and Wisconsin Card Sorting Test (WCST). Inflammatory marker (soluble interleukin-6 receptor, C-reactive protein, and tumor necrosis factor-α receptor-1), cEPC adhesive, and apoptotic levels were measured using in vitro assays. Results: The MDD patients showed significantly lower cEPC adhesive levels than the HCs, and this difference in adhesive function remained statistically significant even after adjusting for inflammatory marker levels. The cEPC adhesion levels were in inverse correlations with commission and omission errors in 2-back task, the percent perseverative response and percent perseverative errors in WCST, and the DSSS and SDS scores, but in positive correlations with SF-12 physical and mental component scores. cEPC apoptotic levels did not differ significantly between the groups. Conclusion: The findings indicate that cEPC adhesive function is diminished in MDD and impacts various aspects of cognitive and psychosocial functions associated with the disorder. [ABSTRACT FROM AUTHOR]