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Pancreatic regeneration after pancreatectomy has been well documented in animal models. However, the phenomenon of pancreatic regeneration in diabetes has not been exploited as yet. We report here the restoration of euglycaemic status in streptozotocin (STZ)-induced diabetic BALB/c mice, after 50% pancreatectomy. We observed that, after pancreatectomy, STZ-diabetic mice showed a rapid improvement in glycaemic status, starting from the 8th postoperative day, and remained normoglycaemic throughout a 90-day follow-up study. STZ-induced diabetic and control non-diabetic BALB/c mice underwent pancreatectomy and were monitored regularly for changes in body weight, plasma glucose and serum insulin concentrations and histological status of the pancreas. All the pancreatectomised animals showed euglycaemic status from about 20 days after operation, whereas a majority (around 70%) of the diabetic, sham-operated animals died of sustained hyperglycaemia by 20-30 days after operation. Examination of the regenerating pancreas indicated nesidioblastotic activity and supported the theory of a ductal origin of islet stem cells. Islets isolated from the regenerating pancreas showed a progressive increase in islet area (1227.9+/-173.2 micrometer(2) on day 5 compared with 2473.8+/-242.0 micrometer(2) by day 20). The increment in insulin concentrations and subsequent decrement in glycaemia of the diabetic pancreatectomised animals indicate islet neogenesis occurring after the operative insult, leading to a normoglycaemic status, probably recapitulating ontogeny. We have shown that induction of a regenerative stimulus (pancreatectomy) in conditions of STZ-induced diabetes may trigger pancreatic regenerative processes, thereby restoring a functional pancreas, in STZ-diabetic mice.

Identifying biomarkers of functional β-cell loss is an important step in the risk stratification of type 1 diabetes. Genetic risk scores (GRS), generated by profiling an array of single nucleotide polymorphisms, are a widely used type 1 diabetes risk-prediction tool. Type 1 diabetes screening studies have relied on a combination of biochemical (autoantibody) and GRS screening methodologies for identifying individuals at high-risk of type 1 diabetes. A limitation of these screening tools is that the presence of autoantibodies marks the initiation of β-cell loss, and is therefore not the best biomarker of progression to early-stage type 1 diabetes. GRS, on the other hand, represents a static biomarker offering a single risk score over an individual's lifetime. In this Personal View, we explore the challenges and opportunities of static and dynamic biomarkers in the prediction of progression to type 1 diabetes. We discuss future directions wherein newer dynamic risk scores could be used to predict type 1 diabetes risk, assess the efficacy of new and emerging drugs to retard, or prevent type 1 diabetes, and possibly replace or further enhance the predictive ability offered by static biomarkers, such as GRS., Competing Interests: Declaration of Interests MVJ declares research funding from JDRF International (Advanced post-doctoral fellowship: 3-APF-2016–178-A-N and JDRF Transition Award: 1-FAC-2021–1063-A-N). FP declares research funding from the Novo Nordisk Foundation (NNF), HORIZON-JU-IHI-2022–03, and the Helmsley Charitable Trust (2202–05774). AAH declares funding from JDRF Australia (1-CDA-2021–1084-M-B), JDRF International (3-SRA-2022–1263-S-B), the Helmsley Charitable Trust (3-SRA-2019–694-M-B and 1-SRA-2021–1067-M-B), the NHMRC Ideas grant (2021/GNT2011557), and Western Sydney University infrastructure support through the Ainsworth Medical Research Funding. SK declares no competing interests., (Copyright © 2024 Published by Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Context: There is substantial evidence that reduced short-chain fatty acids (SCFAs) in the gut are associated with obesity and type 2 diabetes, although findings from clinical interventions that can increase SCFAs are inconsistent., Objective: This systematic review and meta-analysis aimed to assess the effect of SCFA interventions on fasting glucose, fasting insulin, and homeostatic model assessment of insulin resistance (HOMA-IR)., Data Sources: Relevant articles published up to July 28, 2022, were extracted from PubMed and Embase using the MeSH (Medical Subject Headings) terms of the defined keywords [(short-chain fatty acids) AND (obesity OR diabetes OR insulin sensitivity)] and their synonyms. Data analyses were performed independently by two researchers who used the Cochrane meta-analysis checklist and the PRISMA guidelines., Data Extraction: Clinical studies and trials that measured SCFAs and reported glucose homeostasis parameters were included in the analysis. Standardized mean differences (SMDs) with 95%CIs were calculated using a random-effects model in the data extraction tool Review Manager version 5.4 (RevMan 5.4). The risk-of-bias assessment was performed following the Cochrane checklist for randomized and crossover studies., Data Analysis: In total, 6040 nonduplicate studies were identified, 23 of which met the defined criteria, reported fasting insulin, fasting glucose, or HOMA-IR values, and reported change in SCFA concentrations post intervention. Meta-analyses of these studies indicated that fasting insulin concentrations were significantly reduced (overall effect: SMD = -0.15; 95%CI = -0.29 to -0.01, P = 0.04) in treatment groups, relative to placebo groups, at the end of the intervention. Studies with a confirmed increase in SCFAs at the end of intervention also had a significant effect on lowering fasting insulin (P = 0.008). Elevated levels of SCFAs, compared with baseline levels, were associated with beneficial effects on HOMA-IR (P < 0.00001). There was no significant change in fasting glucose concentrations., Conclusion: Increased postintervention levels of SCFAs are associated with lower fasting insulin concentrations, offering a beneficial effect on insulin sensitivity., Systematic Review Registration: PROSPERO registration number CRD42021257248., (© The Author(s) 2023. Published by Oxford University Press on behalf of the International Life Sciences Institute.)

Background: Alcohol-associated liver disease (ALD) is the most common disorder of prolonged drinking. Mechanisms underlying cirrhosis in such patients remain unclear. MicroRNAs play regulatory role in several diseases, are affected by alcohol and may be important players in alcohol use disorders, such as cirrhosis. Methods: We investigated serum samples from heavy chronic alcohol users (80 g/day (male) and 50 g/day (female) for ≥10 years) that were available from our previously reported GenomALC study. A subset of GenomALC drinkers with liver cirrhosis (cases, n = 24) and those without significant liver disease (drinking controls, n = 23) were included. Global microRNA profiling was performed using high-throughput real-time quantitative PCR to identify the microRNA signatures associated with cirrhosis. Ingenuity Pathway Analysis (IPA) software was utilized to identify target mRNAs of significantly altered microRNAs, and molecular pathways were analysed. Identified microRNAs were analysed for correlation with traditional liver disease biomarkers and risk gene variants previously reported from GenomALC genome-wide association study. Results : The expression of 21 microRNAs was significantly downregulated in cases compared to drinking controls ( p < 0.05, ∆∆Ct > 1.5-fold). Seven microRNAs (miR-16, miR-19a, miR-27a, miR-29b, miR-101, miR-130a, and miR-191) had a highly significant correlation ( p < 0.001) with INR, bilirubin and MELD score. Three microRNAs (miR-27a, miR-130a and miR-191) significantly predicted cases with AUC-ROC 0.8, 0.78 and 0.85, respectively ( p < 0.020); however, INR performed best (0.97, p < 0.001). A different set of six microRNAs (miR-19a, miR-26a, miR-101, miR-151-3p, miR-221, and miR-301) showed positive correlation (ranging from 0.32 to 0.51, p < 0.05) with rs10433937: HSD17B13 gene variant, associated with the risk of cirrhosis. IPA analysis revealed mRNA targets of the significantly altered microRNAs associated with cell death/necrosis, fibrosis and increased steatosis, particularly triglyceride metabolism. Conclusions : MicroRNA signatures in drinkers distinguished those with liver cirrhosis from drinkers without liver disease. We identified mRNA targets in liver functions that were enriched for disease pathogenesis pathways.

Purpose: Robust, affordable plasma proteomic biomarker workflows are needed for large-scale clinical studies. We evaluated aspects of sample preparation to allow liquid chromatography-mass spectrometry (LC-MS) analysis of more than 1500 samples from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial of adults with type 2 diabetes., Methods: Using LC-MS with data-independent acquisition we evaluated four variables: plasma protein depletion, EDTA or citrated anti-coagulant blood collection tubes, plasma lipid depletion strategies and plasma freeze-thaw cycles. Optimised methods were applied in a pilot study of FIELD participants., Results: LC-MS of undepleted plasma conducted over a 45 min gradient yielded 172 proteins after excluding immunoglobulin isoforms. Cibachrome-blue-based depletion yielded additional proteins but with cost and time expenses, while immunodepleting albumin and IgG provided few additional identifications. Only minor variations were associated with blood collection tube type, delipidation methods and freeze-thaw cycles. From 65 batches involving over 1500 injections, the median intra-batch quantitative differences in the top 100 proteins of the plasma external standard were less than 2%. Fenofibrate altered seven plasma proteins., Conclusions and Clinical Relevance: A robust plasma handling and LC-MS proteomics workflow for abundant plasma proteins has been developed for large-scale biomarker studies that balance proteomic depth with time and resource costs., (© 2023 The Authors. Proteomics - Clinical Applications published by Wiley-VCH GmbH.)

Background: Type 2 diabetes (T2D) is a critical healthcare challenge and priority in Qatar which is listed amongst the top 10 countries in the world, with its prevalence presently at 17% double the global average. MicroRNAs (miRNAs) are implicated in the pathogenesis of (T2D) and long-term microvascular complications including diabetic retinopathy (DR)., Methods: In this study, a T2D cohort that accurately matches the characteristics of the general population was employed to find microRNA (miRNA) signatures that are correlated with glycemic and β cell function measurements. Targeted miRNA profiling was performed in (471) T2D individuals with or without DR and (491) (non-diabetic) healthy controls from the Qatar Biobank. Discovery analysis identified 20 differentially expressed miRNAs in T2D compared to controls, of which miR-223-3p was significantly upregulated (fold change:5.16, p = 3.6e-02) and positively correlated with glucose and hemoglobin A1c (HbA1c) levels (p-value = 9.88e-04 and 1.64e-05, respectively), but did not show any significant associations with insulin or C-peptide. Accordingly, we performed functional validation using a miR-223-3p mimic (overexpression) under control and hyperglycemia-induced conditions in a zebrafish model., Results: Over-expression of miR-223-3p alone was associated with significantly higher glucose (42.7 mg/dL, n = 75 vs 38.7 mg/dL, n = 75, p = 0.02) and degenerated retinal vasculature, and altered retinal morphology involving changes in the ganglion cell layer and inner and outer nuclear layers. Assessment of retinal angiogenesis revealed significant upregulation in the expression of vascular endothelial growth factor and its receptors, including kinase insert domain receptor. Further, the pancreatic markers, pancreatic and duodenal homeobox 1, and the insulin gene expressions were upregulated in the miR-223-3p group., Conclusion: Our zebrafish model validates a novel correlation between miR-223-3p and DR development. Targeting miR-223-3p in T2D patients may serve as a promising therapeutic strategy to control DR in at-risk individuals., (© 2023. The Author(s).)

Diabetic retinopathy (DR) is a vascular complication of diabetes that can lead to partial or complete loss of vision. Early detection and treatment of DR can prevent blindness. Regular clinical examination is recommended for DR diagnosis; however, it is not always possible or feasible due to limited resources, expertise, time, and infrastructure. Several clinical and molecular biomarkers are proposed for the prediction of DR including microRNAs. MicroRNAs are a class of small non-coding RNAs that are found in biofluids and can be measured using reliable and sensitive methods. The most commonly used biofluid for microRNA profiling is plasma or serum; however, tear fluid (tears) is also demonstrated to contain microRNAs. MicroRNAs isolated from tears present a non-invasive source for DR detection. Different methods of microRNA profiling are available including digital PCR-based methods that can detect up to a single copy of microRNA in the biofluids. Here, we describe microRNA isolation from tears using manual method as well as using a high-throughput automated platform followed by microRNA profiling using digital PCR system., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Leukocyte telomere length (LTL) is suggested to be a biomarker of biological age and reported to be associated with metabolic diseases such as type 2 diabetes. Glucose metabolic traits including glucose and insulin levels have been reported to be associated with LTL in adulthood. However, there is relatively little research focusing on children's LTL and the association with prenatal exposures. This study investigates the relationship between maternal and offspring glucose metabolism with offspring LTL in early life., Methods: This study included 882 mother-child pairs from the HAPO Hong Kong Field Centre, with children evaluated at age 7.0 ± 0.4 (mean ± SD) years. Glucose metabolic traits including maternal post-load glucose during pregnancy, children's glucose and insulin levels, and their derived indices at follow-up were measured or calculated. Offspring LTL was assessed using real-time polymerase chain reaction., Results: Sex- and age-adjusted children's LTL was found to be associated with children's HOMA-IR (β=-0.046 ± 0.016, p=0.005). Interestingly, both children's and maternal post-load glucose levels were positively associated with children's LTL. However, negative associations were observed between children's LTL and children's OGTT insulin levels. In addition, the LTL in females was more strongly associated with pancreatic beta-cell function whilst LTL in males was more strongly associated with OGTT glucose levels., Conclusions: Our findings suggest a close association between maternal and offspring glucose metabolic traits with early life LTL, with the offspring sex as an important modifier of the disparate relationships in insulin production and response., (© 2022. The Author(s).)

With type 2 diabetes presenting at younger ages, there is a growing need to identify biomarkers of future glucose intolerance. A high (20%) prevalence of glucose intolerance at 18 years was seen in women from the Pune Maternal Nutrition Study (PMNS) birth cohort. We investigated the potential of circulating microRNAs in risk stratification for future pre-diabetes in these women. Here, we provide preliminary longitudinal analyses of circulating microRNAs in normal glucose tolerant (NGT@18y, N = 10) and glucose intolerant ( N = 8) women (ADA criteria) at 6, 12 and 17 years of their age using discovery analysis (OpenArray™ platform). Machine-learning workflows involving Lasso with bootstrapping/leave-one-out cross-validation identified microRNAs associated with glucose intolerance at 18 years of age. Several microRNAs, including miR-212-3p, miR-30e-3p and miR-638, stratified glucose-intolerant women from NGT at childhood. Our results suggest that circulating microRNAs, longitudinally assessed over 17 years of life, are dynamic biomarkers associated with and predictive of pre-diabetes at 18 years of age. Validation of these findings in males and remaining participants from the PMNS birth cohort will provide a unique opportunity to study novel epigenetic mechanisms in the life-course progression of glucose intolerance and enhance current clinical risk prediction of pre-diabetes and progression to type 2 diabetes.

Background: The beneficial role of gut microbiota and bacterial metabolites, including short-chain fatty acids (SCFAs), is well recognized, although the available literature around their role in colorectal cancer (CRC) has been inconsistent., Methods: We performed a systematic review and meta-analysis to examine the associations of fecal SCFA concentrations to the incidence and risk of CRC. Data extraction through Medline, Embase, and Web of Science was carried out from database conception to June 29, 2022. Predefined inclusion/exclusion criteria led to the selection of 17 case-control and six cross-sectional studies for quality assessment and analyses. Studies were categorized for CRC risk or incidence, and RevMan 5.4 was used to perform the meta-analyses. Standardized mean differences (SMD) with 95% confidence intervals (CI) were calculated using a random-effects model. Studies lacking quantitation were included in qualitative analyses., Results: Combined analysis of acetic, propionic, and butyric acid revealed significantly lower concentrations of these SCFAs in individuals with a high-risk of CRC (SMD = 2.02, 95% CI 0.31 to 3.74, P = 0.02). Additionally, CRC incidence was higher in individuals with lower levels of SCFAs (SMD = 0.45, 95% CI 0.19 to 0.72, P = 0.0009), compared to healthy individuals. Qualitative analyses identified 70.4% of studies reporting significantly lower concentrations of fecal acetic, propionic, butyric acid, or total SCFAs in those at higher risk of CRC, while 66.7% reported significantly lower concentrations of fecal acetic and butyric acid in CRC patients compared to healthy controls., Conclusions: Overall, lower fecal concentrations of the three major SCFAs are associated with higher risk of CRC and incidence of CRC., (© 2022. The Author(s).)

The ability to detect glucose concentrations in human urine offers a non-invasive approach to monitor changes in blood glucose, kidney health and vascular complications associated with diabetes. We show the potential of employing catalytically active nanoparticles directly grown on textiles to produce a dose-dependent colorimetric sensor for glucose. We use a galvanic replacement (GR) reaction for the synthesis of bimetallic nanoparticles. Here, Cu nanoparticles act as a sacrificial template that undergoes a spontaneous electroless GR reaction when exposed to metal ions of gold, silver, platinum, and palladium to form bimetallic Cu-M nanoparticles (M = Au, Ag, Pt, or Pd). The evaluation of their intrinsic peroxidase-mimicking catalytic activity ("nanozyme") in comparison to that of the Cu nanozyme revealed that the bimetallic systems show a higher catalytic rate with the Cu-Pt nanozyme showing the highest catalytic efficiency. This property of the Cu-Pt nanozyme was then utilized to detect glucose in human urine using the glucose oxidase enzyme as a molecular recognition element. A key outcome of our study is the ability to detect urine glucose without requiring sample dilution which is an advantage over the gold standard GOx-POx method and significantly more reliable performance over commercial urine glucose dipsticks. The difference in the intensity of the colorimetric response between different glucose concentrations further allowed this sensor system to be combined with digital imaging tools for multivariate analysis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Context: Leukocyte telomere length (LTL) is a biomarker of biological aging and is associated with metabolic diseases such as type 2 diabetes. Insufficient maternal vitamin D was associated with increased risk for many diseases and adverse later life outcomes., Objective: This study investigates the relationship between vitamin D levels and offspring LTL at early life., Methods: This observational, longitudinal, hospital-based cohort study included eligible mother-child pairs from the HAPO Hong Kong Field Centre, with 853 offspring at age 6.96 ± 0.44 (mean ± SD) years. LTL was measured using real-time polymerase chain reaction while serum vitamin D metabolites 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3 were measured in maternal blood (at gestation 24-32 weeks) and cord blood by liquid chromatography-mass spectrometry., Results: LTL at follow-up was significantly shorter in boys compared with girls (P < 0.001) at age 7. Childhood LTL was negatively associated with childhood BMI (β ± SE = -0.016 ± 0.007)(P = 0.02) and HOMA-IR (β ± SE = -0.065 ± 0.021)(P = 0.002). Multiple linear regression was used to evaluate the relationship between 25(OH)D and LTL, with covariate adjustments. Childhood LTL was positively correlated with total maternal 25(OH)D (0.048 ± 0.017) (P = 0.004) and maternal 3-epi-25(OH)D3 (0.05 ± 0.017) (P = 0.003), even after adjustment for covariates. A similar association was also noted for cord 3-epi-25(OH)D3 (0.037 ± 0.018) (P = 0.035) after adjustment for offspring sex and age., Conclusion: Our findings suggest 25(OH)D3 and 3-epi-25(OH)D3 in utero may impact on childhood LTLs, highlighting a potential link between maternal vitamin D and biological aging., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The microRNA-29 family members miR-29a-3p, miR-29b-3p, and miR-29c-3p are ubiquitously expressed and consistently increased in various tissues and cell types in conditions of metabolic disease, obesity, insulin resistance, and type 2 diabetes. In pancreatic β cells, miR-29a is required for normal exocytosis, but increased levels are associated with impaired β-cell function. Similarly, in liver, miR-29 species are higher in models of insulin resistance and type 2 diabetes, and either knock-out or depletion using a microRNA inhibitor improves hepatic insulin resistance. In skeletal muscle, miR-29 family upregulation is associated with insulin resistance and altered substrate oxidation, and similarly, in adipocytes, overexpression of miR-29a leads to insulin resistance. Blocking miR-29a using nucleic acid antisense therapeutics show promising results in preclinical animal models of obesity and type 2 diabetes, although the widespread expression pattern of miR-29 family members complicates the exploration of single target tissues. However, in fibrotic diseases, such as in late complications of diabetes and metabolic disease (diabetic kidney disease, nonalcoholic steatohepatitis), miR-29 species expression is suppressed by TGF-β allowing increased extracellular matrix collagen to form. In the clinical setting, circulating levels of miR-29a and miR-29b are consistently increased in type 2 diabetes and in gestational diabetes and are also possible prognostic markers for deterioration of glucose tolerance. In conclusion, miR-29 family miRNAs play an essential role in various organs relevant to intermediary metabolism and its upregulation contributes to impaired glucose metabolism, whereas it suppresses fibrosis development. Thus, a correct balance of levels of miR-29 family miRNA seems important for cellular and organ homeostasis in metabolism.

Machine learning (ML)-workflows enable unprejudiced/robust evaluation of complex datasets. Here, we analyzed over 490,000,000 data points to compare 10 different ML-workflows in a large (N=11,652) training dataset of human pancreatic single-cell (sc-)transcriptomes to identify genes associated with the presence or absence of insulin transcript(s). Prediction accuracy/sensitivity of each ML-workflow was tested in a separate validation dataset (N=2,913). Ensemble ML-workflows, in particular Random Forest ML-algorithm delivered high predictive power (AUC=0.83) and sensitivity (0.98), compared to other algorithms. The transcripts identified through these analyses also demonstrated significant correlation with insulin in bulk RNA-seq data from human islets. The top-10 features, (including IAPP, ADCYAP1, LDHA and SST ) common to the three Ensemble ML-workflows were significantly dysregulated in scRNA-seq datasets from Ire-1α β-/- mice that demonstrate dedifferentiation of pancreatic β-cells in a model of type 1 diabetes (T1D) and in pancreatic single cells from individuals with type 2 Diabetes (T2D). Our findings provide direct comparison of ML-workflows in big data analyses, identify key elements associated with insulin transcription and provide workflows for future analyses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wong, Thorat, Joglekar, Dong, Lee, Chew, Bhave, Hawthorne, Engin, Pant, Dalgaard, Bapat and Hardikar.)

Objective: Several studies support associations between relative leukocyte telomere length (rLTL), a biomarker of biological aging and type 2 diabetes. This study investigates the relationship between rLTL and the risk of glycemic progression in patients with type 2 diabetes., Research Design and Methods: In this cohort study, consecutive Chinese patients with type 2 diabetes (N = 5,506) from the Hong Kong Diabetes Register with stored baseline DNA and available follow-up data were studied. rLTL was measured using quantitative PCR. Glycemic progression was defined as the new need for exogenous insulin., Results: The mean (SD) age of the 5,349 subjects was 57.0 (13.3) years, and mean (SD) follow-up was 8.8 (5.4) years. Baseline rLTL was significantly shorter in the 1,803 subjects who progressed to insulin requirement compared with the remaining subjects (4.43 ± 1.16 vs. 4.69 ± 1.20). Shorter rLTL was associated with a higher risk of glycemic progression (hazard ratio [95% CI] for each unit decrease [to ∼0.2 kilobases]: 1.10 [1.06-1.14]), which remained significant after adjusting for confounders. Baseline rLTL was independently associated with glycemic exposure during follow-up (β = -0.05 [-0.06 to -0.04]). Each 1-kilobase decrease in absolute LTL was on average associated with a 1.69-fold higher risk of diabetes progression (95% CI 1.35-2.11). Two-sample Mendelian randomization analysis showed per 1-unit genetically decreased rLTL was associated with a 1.38-fold higher risk of diabetes progression (95% CI 1.12-1.70)., Conclusions: Shorter rLTL was significantly associated with an increased risk of glycemic progression in individuals with type 2 diabetes, independent of established risk factors. Telomere length may be a useful biomarker for glycemic progression in people with type 2 diabetes., (© 2022 by the American Diabetes Association.)

Introduction: Sports-related concussion (SRC) is a common form of brain injury that lacks reliable methods to guide clinical decisions. MicroRNAs (miRNAs) can influence biological processes involved in SRC, and measurement of miRNAs in biological fluids may provide objective diagnostic and return to play/recovery biomarkers. Therefore, this prospective study investigated the temporal profile of circulating miRNA levels in concussed male and female athletes., Methods: Pre-season baseline blood samples were collected from amateur Australian rules football players (82 males, 45 females). Of these, 20 males and 8 females sustained an SRC during the subsequent season and underwent blood sampling at 2-, 6- and 13-days post-injury. A miRNA discovery Open Array was conducted on plasma to assess the expression of 754 known/validated miRNAs. miRNA target identified were further investigated with quantitative real-time PCR (qRT-PCR) in a validation study. Data pertaining to SRC symptoms, demographics, sporting history, education history and concussion history were also collected., Results: Discovery analysis identified 18 candidate miRNA. The consequent validation study found that plasma miR-221-3p levels were decreased at 6d and 13d, and that miR-27a-3p levels were decreased at 6d, when compared to baseline. Moreover, miR-27a and miR-221-3p levels were inversely correlated with SRC symptom severity., Conclusion: Circulating levels of miR-27a-3p and miR-221-3p were decreased in the sub-acute stages after SRC, and were inversely correlated with SRC symptom severity. Although further studies are required, these analyses have identified miRNA biomarker candidates of SRC severity and recovery that may one day assist in its clinical management., Competing Interests: Declaration Of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Aims/hypothesis: Few large-scale prospective studies have investigated associations between relative leucocyte telomere length (rLTL) and kidney dysfunction in individuals with type 2 diabetes. We examined relationships between rLTL and incident end-stage kidney disease (ESKD) and the slope of eGFR decline in Chinese individuals with type 2 diabetes., Methods: We studied 4085 Chinese individuals with type 2 diabetes observed between 1995 and 2007 in the Hong Kong Diabetes Register with stored baseline DNA and available follow-up data. rLTL was measured using quantitative PCR. ESKD was diagnosed based on the ICD-9 code and eGFR., Results: In this cohort (mean ± SD age 54.3 ± 12.6 years) followed up for 14.1 ± 5.3 years, 564 individuals developed incident ESKD and had shorter rLTL at baseline (4.2 ± 1.2 vs 4.7 ± 1.2, p < 0.001) than the non-progressors (n = 3521). On Cox regression analysis, each ∆∆C t decrease in rLTL was associated with an increased risk of incident ESKD (HR 1.21 [95% CI 1.13, 1.30], p < 0.001); the association remained significant after adjusting for baseline age, sex, HbA 1c , lipids, renal function and other risk factors (HR 1.11 [95% CI 1.03, 1.19], p = 0.007). Shorter rLTL at baseline was associated with rapid decline in eGFR (>4% per year) during follow-up (unadjusted OR 1.22 [95% CI 1.15, 1.30], p < 0.001; adjusted OR 1.09 [95% CI 1.01, 1.17], p = 0.024)., Conclusions/interpretation: rLTL is independently associated with incident ESKD and rapid eGFR loss in individuals with type 2 diabetes. Telomere length may be a useful biomarker for the progression of kidney function and ESKD in type 2 diabetes., (© 2021. The Author(s).)

Background & Aims: Pancreatic islet β-cells are factories for insulin production; however, ectopic expression of insulin also is well recognized. The gallbladder is a next-door neighbor to the developing pancreas. Here, we wanted to understand if gallbladders contain functional insulin-producing cells., Methods: We compared developing and adult mouse as well as human gallbladder epithelial cells and islets using immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assays, RNA sequencing, real-time polymerase chain reaction, chromatin immunoprecipitation, and functional studies., Results: We show that the epithelial lining of developing, as well as adult, mouse and human gallbladders naturally contain interspersed cells that retain the capacity to actively transcribe, translate, package, and release insulin. We show that human gallbladders also contain functional insulin-secreting cells with the potential to naturally respond to glucose in vitro and in situ. Notably, in a non-obese diabetic (NOD) mouse model of type 1 diabetes, we observed that insulin-producing cells in the gallbladder are not targeted by autoimmune cells. Interestingly, in human gallbladders, insulin splice variants are absent, although insulin splice forms are observed in human islets., Conclusions: In summary, our biochemical, transcriptomic, and functional data in mouse and human gallbladder epithelial cells collectively show the evolutionary and developmental similarities between gallbladder and the pancreas that allow gallbladder epithelial cells to continue insulin production in adult life. Understanding the mechanisms regulating insulin transcription and translation in gallbladder epithelial cells would help guide future studies in type 1 diabetes therapy., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are elements of the gene regulatory network and manipulating their abundance is essential toward elucidating their role in patho-physiological conditions. We present a detailed workflow that identifies important miRNAs using a machine learning algorithm. We then provide optimized techniques to validate the identified miRNAs through over-expression/loss-of-function studies. Overall, these protocols apply to any field in biology where high-dimensional data are produced. For complete details on the use and execution of this protocol, please refer to Wong et al. (2021a)., Competing Interests: The authors declare no competing interests., (© 2021.)

To determine whether continuous subcutaneous insulin infusion (CSII) vs. multiple daily injections (MDI) therapy from near-diagnosis of type 1 diabetes is associated with reduced glycaemic variability (GV) and altered microRNA (miRNAs) expression. Adolescents (74% male) within 3-months of diabetes diagnosis (n = 27) were randomized to CSII (n = 12) or MDI. HbA1c, 1-5-Anhydroglucitol (1,5-AG), high sensitivity C-peptide and a custom TaqMan qPCR panel of 52 miRNAs were measured at baseline and follow-up (median (LQ-UQ); 535 (519-563) days). There were no significant differences between groups in baseline or follow-up HbA1c or C-peptide, nor baseline miRNAs. Mean ± SD 1,5-AG improved with CSII vs. MDI (3.1 ± 4.1 vs. - 2.2 ± - 7.0 mg/ml respectively, P = 0.029). On follow-up 11 miRNAs associated with diabetes vascular complications had altered expression in CSII-users. Early CSII vs. MDI use is associated with lower GV and less adverse vascular-related miRNAs. Relationships with future complications are of interest., (© 2021. The Author(s).)

Aims/hypothesis: Type 2 diabetes mellitus is a major cause of morbidity and death worldwide. Women with gestational diabetes mellitus (GDM) have greater than a sevenfold higher risk of developing type 2 diabetes in later life. Accurate methods for postpartum type 2 diabetes risk stratification are lacking. Circulating microRNAs (miRNAs) are well recognised as biomarkers/mediators of metabolic disease. We aimed to determine whether postpartum circulating miRNAs can predict the development of type 2 diabetes in women with previous GDM., Methods: In an observational study, plasma samples were collected at 12 weeks postpartum from 103 women following GDM pregnancy. Utilising a discovery approach, we measured 754 miRNAs in plasma from type 2 diabetes non-progressors (n = 11) and type 2 diabetes progressors (n = 10) using TaqMan-based real-time PCR on an OpenArray platform. Machine learning algorithms involving penalised logistic regression followed by bootstrapping were implemented., Results: Fifteen miRNAs were selected based on their importance in discriminating type 2 diabetes progressors from non-progressors in our discovery cohort. The levels of miRNA miR-369-3p remained significantly different (p < 0.05) between progressors and non-progressors in the validation sample set (n = 82; 71 non-progressors, 11 progressors) after adjusting for age and correcting for multiple comparisons. In a clinical model of prediction of type 2 diabetes that included six traditional risk factors (age, BMI, pregnancy fasting glucose, postpartum fasting glucose, cholesterol and triacylglycerols), the addition of the circulating miR-369-3p measured at 12 weeks postpartum improved the prediction of future type 2 diabetes from traditional AUC 0.83 (95% CI 0.68, 0.97) to an AUC 0.92 (95% CI 0.84, 1.00)., Conclusions: This is the first demonstration of miRNA-based type 2 diabetes prediction in women with previous GDM. Improved prediction will facilitate early lifestyle/drug intervention for type 2 diabetes prevention.

The aim of this cross-sectional study was to compare plasma C-peptide presence and levels in people without diabetes (CON) and with Type 1 diabetes and relate C-peptide status to clinical factors. In a subset we evaluated 50 microRNAs (miRs) previously implicated in beta-cell death and associations with clinical status and C-peptide levels. Diabetes age of onset was stratified as adult (≥ 18 y.o) or childhood (< 18 y.o.), and diabetes duration was stratified as ≤ 10 years, 10-20 years and > 20 years. Plasma C-peptide was measured by ultrasensitive ELISA. Plasma miRs were quantified using TaqMan probe-primer mix on an OpenArray platform. C-peptide was detectable in 55.3% of (n = 349) people with diabetes, including 64.1% of adults and 34.0% of youth with diabetes, p < 0.0001 and in all (n = 253) participants without diabetes (CON). C-peptide levels, when detectable, were lower in the individuals with diabetes than in the CON group [median lower quartile (LQ)-upper quartile (UQ)] 5.0 (2.6-28.7) versus 650.9 (401.2-732.4) pmol/L respectively, p < 0.0001 and lower in childhood versus adult-onset diabetes [median (LQ-UQ) 4.2 (2.6-12.2) pmol/L vs. 8.0 (2.3-80.5) pmol/L, p = 0.02, respectively]. In the childhood-onset group more people with longer diabetes duration (> 20 years) had detectable C-peptide (60%) than in those with shorter diabetes duration (39%, p for trend < 0.05). Nine miRs significantly correlated with detectable C-peptide levels in people with diabetes and 16 miRs correlated with C-peptide levels in CON. Our cross-sectional study results are supportive of (a) greater beta-cell function loss in younger onset Type 1 diabetes; (b) persistent insulin secretion in adult-onset diabetes and possibly regenerative secretion in childhood-onset long diabetes duration; and (c) relationships of C-peptide levels with circulating miRs. Confirmatory clinical studies and related basic science studies are merited.

MicroRNAs in biofluids are potential biomarkers for detecting kidney and other organ injuries. We profiled microRNAs in urine samples from patients with Russell's viper envenoming or acute self-poisoning following paraquat, glyphosate, or oxalic acid [with and without acute kidney injury (AKI)] and on healthy controls. Discovery analysis profiled for 754 microRNAs using TaqMan OpenArray qPCR with three patients per group (12 samples in each toxic agent). From these, 53 microRNAs were selected and validated in a larger cohort of patients (Russell's viper envenoming = 53, paraquat = 51, glyphosate = 51, oxalic acid = 40) and 27 healthy controls. Urinary microRNAs had significantly higher expression in patients poisoned/envenomed by different nephrotoxic agents in both discovery and validation cohorts. Seven microRNAs discriminated severe AKI patients from no AKI for all four nephrotoxic agents. Four microRNAs (miR-30a-3p, miR-30a-5p, miR-92a, and miR-204) had > 17 fold change (p < 0.0001) and receiver operator characteristics area-under-curve (ROC-AUC) > 0.72. Pathway analysis of target mRNAs of these differentially expressed microRNAs showed association with the regulation of different nephrotoxic signaling pathways. In conclusion, human urinary microRNAs could identify toxic AKI early after acute injury. These urinary microRNAs have potential clinical application as early non-invasive diagnostic AKI biomarkers.

Dicer knockout mouse models demonstrated a key role for microRNAs in pancreatic β-cell function. Studies to identify specific microRNA(s) associated with human (pro-)endocrine gene expression are needed. We profiled microRNAs and key pancreatic genes in 353 human tissue samples. Machine learning workflows identified microRNAs associated with (pro-)insulin transcripts in a discovery set of islets (n = 30) and insulin-negative tissues (n = 62). This microRNA signature was validated in remaining 261 tissues that include nine islet samples from individuals with type 2 diabetes. Top eight microRNAs (miR-183-5p, -375-3p, 216b-5p, 183-3p, -7-5p, -217-5p, -7-2-3p, and -429-3p) were confirmed to be associated with and predictive of (pro-)insulin transcript levels. Use of doxycycline-inducible microRNA-overexpressing human pancreatic duct cell lines confirmed the regulatory roles of these microRNAs in (pro-)endocrine gene expression. Knockdown of these microRNAs in human islet cells reduced (pro-)insulin transcript abundance. Our data provide specific microRNAs to further study microRNA-mRNA interactions in regulating insulin transcription., Competing Interests: A patent application (WO2019000017A1) was filed., (© 2021 The Authors.)

Aims: Few studies have investigated the relationship between rLTL and mortality in patients with type 2 diabetes in a large prospective study, particularly in the Asian population. This study investigates the relationship between rLTL and the risk of death in Chinese patients with type 2 diabetes., Methods: Consecutive Chinese patients with type 2 diabetes (N = 5349) from the Hong Kong Diabetes Register with stored baseline DNA and available follow-up data were studied. rLTL was measured using a quantitative polymerase chain reaction. Mortality and clinical outcomes were obtained based on ICD-9 codes., Results: The mean (SD) age of the subjects was 57.5 (13.3) years and mean (SD) follow-up duration was 13.4 (5.5) years. Baseline rLTL was significantly shorter in the 1925 subjects who subsequently died compared with the remaining subjects (4.3 ± 1.2 vs. 4.7 ± 1.2, P < 0.001). Shorter rLTL was associated with a higher risk of mortality (HR: 1.19 (1.14-1.23), P < 0.001), which remained significant after adjusting for traditional risk factors., Conclusions: Shorter rLTL was significantly associated with an increased risk of all-cause and CVD mortality in patients with type 2 diabetes, independent of established risk factors. Telomere length may be a useful biomarker for mortality risk in patients with type 2 diabetes., Competing Interests: Declaration of Competing Interest A.O.L. has served as an advisory committee member for AstraZeneca, Boehringer Ingelheim, Sanofi and Amgen and has received research grants and travel grants from AstraZeneca, Boehringer Ingelheim, MSD, Novartis, Novo Nordisk, Sanofi and Amgen. A.P.K. has received research grants and/or speaker honoraria from Abbott, AstraZeneca, Eli-Lilly, Merck Serono, Nestle, Sanofi and Novo Nordisk. J.C.C. has received research grants and/or honoraria for consultancy or giving lectures, from AstraZeneca, Bayer, Bristol-Myers Squibb, Boehringer Ingelheim, Daiichi-Sankyo, Eli-Lilly, GlaxoSmithKline, Merck Serono, Merck Sharp & Dohme, Novo Nordisk, Pfizer and Sanofi. R.C.M. has received research grants for clinical trials from AstraZeneca, Bayer, MSD, Novo Nordisk, Sanofi, Tricida and honoraria for consultancy or lectures from AstraZeneca, and Boehringer Ingelheim. F.C., C.K.L., L.C., C.H.T., B.F., H.W., A.Y., E.S.L., A.C.N., E.C., H.M.L., A.C.K., M.V.J., W.Y.S., A.J.J., and A.A.H. do not have conflicts of interest to declare., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Telomeres are regions of repetitive nucleotide sequences at the ends of chromosomes. Telomere length is a marker of DNA damage, which is often considered a biomarker for biological ageing, and has also been linked with cardiovascular disease, diabetes, and cancer. Emerging studies have highlighted the role of genetic and environmental factors, and explored the effect of modulating telomere length. We provide an overview of studies to date on diabetes and telomere length, and compare different methods and assays for evaluating telomere length and telomerase activity. We highlight the limitations of current studies and areas that warrant further research to unravel the link between diabetes and telomere length. The value of adding telomere length to clinical risk factors to improve risk prediction of diabetes and related complications also merits further investigation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Type 1 diabetes (T1D) is an autoimmune disease, where insulin-producing β-cells in the pancreas are inappropriately recognized and destroyed by immune cells. Islet transplantation is the most successful cell-based therapy for T1D individuals who experience frequent and severe life-threatening hypoglycemia. However, this therapy is extremely restricted owing to the limited availability of donor pancreas. In recent years, significant progress has been made in generating β-cells from stem/progenitor cells using different approaches of in vitro differentiation. The insulin production from such in vitro generated β-cells is still far less than that observed in islet β-cells. We employed a novel strategy to improve the efficiency of progenitor cell differentiation by performing partial mouse pancreas resection after transplanting in vitro generated insulin-producing cells under the kidney capsule of these mice. Pancreas resection (pancreatectomy) has been shown to induce regenerative pathways, leading to regeneration of almost the entire resected pancreas over 3-5 weeks in mice. We found that in our method, regenerating mouse pancreas promotes better graft differentiation/maturation and insulin production from transplanted cells. In this chapter, we detail the protocols used for transplantation of in vitro differentiated cells in immunocompromised mice, partial pancreatectomy in host (NOD scid) mice, and assessment of graft function. We believe that our protocols provide a solid platform for further studies aimed at understanding growth/differentiation molecules secreted from regenerating pancreas that promote graft maturation.

The colonic mucus layer, comprised of highly O- glycosylated mucins, is vital to mediating host-gut microbiota interactions, yet the impact of dietary changes on colonic mucin O- glycosylation and its associations with the gut microbiota remains unexplored. Here, we used an array of omics techniques including glycomics to examine the effect of dietary fiber consumption on the gut microbiota, colonic mucin O- glycosylation and host physiology of high-fat diet-fed C57BL/6J mice. The high-fat diet group had significantly impaired glucose tolerance and altered liver proteome, gut microbiota composition, and short-chain fatty acid production compared to normal chow diet group. While dietary fiber inclusion did not reverse all high fat-induced modifications, it resulted in specific changes, including an increase in the relative abundance of bacterial families with known fiber digesters and a higher propionate concentration. Conversely, colonic mucin O -glycosylation remained similar between the normal chow and high-fat diet groups, while dietary fiber intervention resulted in major alterations in O -glycosylation. Correlation network analysis revealed previously undescribed associations between specific bacteria and mucin glycan structures. For example, the relative abundance of the bacterium Parabacteroides distasonis positively correlated with glycan structures containing one terminal fucose and correlated negatively with glycans containing two terminal fucose residues or with both an N-acetylneuraminic acid and a sulfate residue. This is the first comprehensive report of the impact of dietary fiber on the colonic mucin O- glycosylation and associations of these mucosal glycans with specific gut bacteria.

MicroRNAs are key regulators of the normal kidney function and development, and altered in acute kidney injury (AKI). However, there is a lack of studies comparing serum and urine miRNA expression in toxic AKI in humans. We aimed to compare the global signature of urinary and serum microRNAs, with and without kidney injury, after human oxalic acid poisoning. We profiled urinary microRNAs in patients who ingested oxalic acid and developed no injury (No AKI n = 3), moderate injury (AKIN2 n = 3) or severe injury (AKIN3 n = 3) and healthy controls (n = 3). We validated a signature of 30 urinary microRNAs identified in the discovery profiling, in a second cohort of individuals exposed to oxalic acid (No AKI n = 15, AKIN2 n=11 & AKIN3 n= 18) and healthy controls (n=-27) and we compared the results with previously published serum data. Global profiling in toxic AKI patients showed a higher expression of urinary microRNAs and lower expression of serum microRNAs. Most urine microRNA in the validation cohort were significantly upregulated (25/30, fold change >2.8 and p < 0.05) in AKIN2/3 patients compared to No AKI. Four urinary microRNAs (miR-191, miR-19b, miR-20a and miR-30b) had good diagnostic performance (AUC greater than 0.8) to predict AKIN2/3 between 4-8 hours post ingestion. Poisoning irrespective of AKI led to significantly lower expression of many microRNAs in serum but relatively few changes in urinary miRNA expression. In conclusion, urinary microRNA signature provides a stronger measure of AKI in oxalic acid poisoning compared to serum microRNA. Kidney injury has the greatest impact on urinary microRNA, while poisoning itself was better reflected in serum miRNA. Plasma and urinary microRNAs signatures provide complementary information in toxic kidney injury., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Several studies support potential links between relative leukocyte telomere length (rLTL), a biomarker of biological aging, and type 2 diabetes. This study investigates relationships between rLTL and incident cardiovascular disease (CVD) in patients with type 2 diabetes., Research Design and Methods: Consecutive Chinese patients with type 2 diabetes ( N = 5,349) from the Hong Kong Diabetes Register for whom DNA obtained at baseline was stored and follow-up data were available were studied. rLTL was measured by using quantitative PCR. CVD was diagnosed on the basis of ICD-9 code., Results: Mean follow-up was 13.4 years (SD 5.5 years). rLTL was correlated inversely with age, diabetes duration, blood pressure, HbA 1c , and urine albumin-to-creatinine ratio (ACR), and positively with estimated glomerular filtration rate (eGFR) (all P < 0.001). Subjects with CVD at baseline had a shorter rLTL (4.3 ± 1.2 ΔΔCt) than did subjects without CVD (4.6 ± 1.2 ΔΔCt) ( P < 0.001). Of the 4,541 CVD-free subjects at baseline, the 1,140 who developed CVD during follow-up had a shorter rLTL (4.3 ± 1.2 ΔΔCt) than those who remained CVD-free after adjusting for age, sex, smoking, and albuminuria status (4.7 ± 1.2 ΔΔCt) ( P < 0.001). In Cox regression models, shorter rLTL was associated with higher risk of incident CVD (for each unit decrease, hazard ratio 1.252 [95% CI 1.195-1.311], P < 0.001), which remained significant after adjusting for age, sex, BMI, systolic blood pressure, LDL cholesterol, HbA 1c , eGFR, and ACR (hazard ratio 1.141 [95% CI 1.084-1.200], P < 0.001)., Conclusions: rLTL is significantly shorter in patients with type 2 diabetes and CVD, is associated with cardiometabolic risk factors, and is independently associated with incident CVD. Telomere length may be a useful biomarker for CVD risk in patients with type 2 diabetes., (© 2020 by the American Diabetes Association.)

Background: Circulating microRNAs (miRNAs) may play a pathogenic role in acute coronary syndromes (ACS). It is not yet known if miRNAs dysregulated in ACS are modulated by colchicine. We profiled miRNAs in plasma samples simultaneously collected from the aorta, coronary sinus, and right atrium in patients with ACS., Methods: A total of 396 of 754 miRNAs were detected by TaqMan real-time polymerase chain reaction from EDTA-plasma in a discovery cohort of 15 patients (n = 3 controls, n = 6 ACS standard therapy, n = 6 ACS standard therapy plus colchicine). Fifty-one significantly different miRNAs were then measured in a verification cohort of 92 patients (n = 13 controls, n = 40 ACS standard therapy, n = 39 ACS standard therapy plus colchicine). Samples were simultaneously obtained from the coronary sinus, aortic root, and right atrium., Results: Circulating levels of 30 of 51 measured miRNAs were higher in ACS standard therapy patients compared to controls. In patients with ACS, levels of 12 miRNAs (miR-17, -106b-3p, -191, -106a, -146a, -130a, -223, -484, -889, -425-3p, -629, -142-5p) were lower with colchicine treatment. Levels of 7 of these 12 miRNA were higher in ACS standard therapy patients compared to controls and returned to levels seen in control individuals after colchicine treatment. Three miRNAs suppressed by colchicine (miR-146a, miR-17, miR-130a) were identified as regulators of inflammatory pathways. MicroRNAs were comparable across sampling sites with select differences in the transcoronary gradient of 4 miRNA., Conclusion: The levels of specific miRNAs elevated in ACS returned to levels similar to control individuals following colchicine. These miRNAs may mediate ACS (via inflammatory pathways) or increase post-ACS risk, and could be potentially used as biomarkers of treatment efficacy.

Our commentary is focused on three studies that used microRNA overexpression methods for directed differentiation of stem cells into insulin-producing cells. Islet transplantation is the only cell-based therapy used to treat type 1 diabetes mellitus. However, due to the scarcity of cadaveric donors and limited availability of good quality and quantity of islets for transplant, alternate sources of insulin-producing cells are being studied and used by researchers. This commentary provides an overview of distinct studies focused on manipulating microRNA expression to optimize differentiation of embryonic stem cells or induced pluripotent stem cells into insulin-producing cells. These studies have used different approaches to overexpress microRNAs that are highly abundant in human islets (such as miR-375 and miR-7) in their differentiation protocol to achieve better differentiation into functional islet beta (β)-cells., Competing Interests: Conflict of interest: Authors state no conflict of interest., (© 2020 Michael D. Williams et al., published by De Gruyter.)

The choice of valve type for aortic valve replacement surgery is sometimes challenging. The main risk for bioprostheses is structural valve degeneration (SVD); however, little is known about what the most important risk factors are. We conducted a systematic review and meta-analysis to identify the risk factors and estimate their pooled effect sizes to aid the prosthesis choice for replacement. We followed PRISMA guidelines and systematically searched three electronic databases (PubMed, Scopus, and Web of Science) using appropriate key terms: 'aortic valve', 'bioprosthesis', 'degeneration', 'durability', 'prosthesis failure', etc. Hazard ratio (HR) and odds ratio (OR) and associated 95% confidence intervals (CI) were extracted. Pooled risk estimates were calculated using a random-effects model. Twenty-nine (29) observational studies were included with a total of 25,490 patients, 981 of whom developed SVD over a mean follow-up time of 18.5 years. Four (4) factors influencing bioprosthetic SVD were identified: increasing age was a protective factor (per 1-yr increase, HR: 0.91 [95% CI 0.89, 0.94], p<0.0001), whereas increased body surface area (HR 1.77 [1.04, 3.01], p=0.034), patient-prosthesis mismatch (HR 1.95 [1.56, 2.43], p<0.001), and smoking (HR 2.28 [1.37, 3.79], p=0.0015) were risk factors for SVD. We found younger age, patient-prosthesis mismatch, body surface area, and smoking, as risk factors for aortic SVD, which should be considered for valve selection. This study generates a further hypothesis that accelerated flow across the valve is a shared key component in the pathophysiology of SVD, thus future research should consider other high cardiac output states., (Copyright © 2019 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.)

Oxalic acid-induced nephrotoxicity and acute kidney injury result from formation of calcium oxalate crystals. Oxalic acid-induced acute kidney injury is a significant problem in many parts of the world. Circulating biomarkers that can accurately and reproducibly detect acute kidney injury are highly desirable. We used a high sensitivity discovery platform to identify signature microRNAs to distinguish healthy individuals never exposed to oxalic acid (n = 4) from those who were exposed to oxalic acid but had no injury (NOAKI; n = 4), moderate injury (AKIN2; n = 4) or severe injury (AKIN3; n = 4). Longitudinal analyses identified 4-8 h post-ingestion as the best time to detect AKIN2/3. We validated a signature of 53 microRNAs identified in the discovery, in a second cohort of individuals exposed to oxalic acid (NOAKI = 11, AKIN2 = 8 and AKIN3 = 18) and healthy controls (n = 19). Thirteen microRNAs were significantly downregulated in acute kidney injury patients compared to NOAKI within 8-h post-ingestion. Five microRNAs (miR-20a, miR-92a, miR-93, miR-195, miR-451) had a highly significant correlation with normalized urinary albumin, serum creatinine at 24 h and creatinine clearance. Logistic regression of these microRNAs had AUC-ROC of 0.85 predicting AKIN2/3 and discriminated patients from healthy controls (AUC-ROC = 0.93). mRNA targets of these microRNAs identified oxidative stress pathways of nephrotoxicity in proximal tubule and glomeruli nephrotoxicity. In conclusion, the downregulation of multiple circulating microRNAs in patients correlated with the severity of oxalic acid-induced acute kidney injury. A set of microRNAs (miR-20a, miR-92a, miR-93, miR-195, miR-451) could be promising biomarkers for early detection of oxalic acid-induced acute kidney injury.

Telomeres represent the nucleotide repeat sequences at the ends of chromosomes and are essential for chromosome stability. They can shorten at each round of DNA replication mainly because of incomplete DNA synthesis of the lagging strand. Reduced relative telomere length is associated with aging and a range of disease states. Different methods such as terminal restriction fragment analysis, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization are available to measure telomere length; however, the qPCR-based method is commonly used for large population-based studies. There are multiple variations across qPCR-based methods, including the choice of the single-copy gene, primer sequences, reagents, and data analysis methods in the different reported studies so far. Here, we provide a detailed step-by-step protocol that we have optimized and successfully tested in the hands of other users. This protocol will help researchers interested in measuring relative telomere lengths in cells or across larger clinical cohort/study samples to determine associations of telomere length with health and disease., Competing Interests: The authors declare no conflict of interest

Our aim was to investigate sebaceous differentiation in thymus tumours and to identify new actionable genomic alterations. To this end we screened 35 normal and 23 hyperplastic thymuses, 127 thymomas and 41 thymic carcinomas for the presence of sebaceous differentiation as defined by morphology and expression of adipophilin and androgen receptor (AR). One primary thymic carcinoma showed morphology of sebaceous carcinomas (keratinizing and foam cells, calcifications, giant cells), a strong expression of adipophilin and AR together with squamous markers. NGS revealed high-level amplification of fibroblast growth factor receptor 2 (FGFR2). In thymuses and thymomas, no cells with sebaceous morphology were present. Occasionally, macrophages or epithelial cells showed adipophilin-positivity, however, without co-expression of AR. Thymic sebaceous carcinoma should be considered if a thymic carcinoma shows clear or foamy features. Testing for FGFR2 amplification might be warranted when searching for actionable genomic alterations in sebaceous carcinomas in the mediastinum and in other locations.

Objective: To determine if continuous subcutaneous insulin infusion (CSII) therapy is associated with lower glycated haemoglobin (HbA1c) variability (long-term glycaemic variability; GV) relative to multiple daily injection (MDI) treatment in adults with type 1 diabetes mellitus (T1DM)., Design: Retrospective audit., Setting and Participants: Clinic records from 506 adults with T1DM from two tertiary Australian hospitals., Outcome Measures: Long-term GV was assessed by HbA1c SD and coefficient of variation (CV) in adults on established MDI or CSII therapy, and in a subset changing from MDI to CSII., Results: Adults (n=506, (164 CSII), 50% women, mean±SD age 38.0±15.3 years, 17.0±13.7 years diabetes, mean HbA1c 7.8%±1.2% (62±13 mmol/mol) on CSII, 8.0%±1.5% (64±16 mmol/mol) on MDI) were followed for 4.1±3.6 years. CSII use was associated with lower GV (HbA1c SD: CSII vs MDI 0.5%±0.41% (6±6 mmol/mol) vs 0.7%±0.7% (9±8 mmol/mol)) and CV: CSII vs MDI 6.7%±4.6% (10±10 mmol/mol) vs 9.3%±7.3% (14±13 mmol/mol), both p<0.001. Fifty-six adults (73% female, age 36±13 years, 16±13 years diabetes, HbA1c 7.8%±0.8% (62±9 mmol/mol)) transitioned from MDI to CSII. Mean HbA1c fell by 0.4%. GV from 1 year post-CSII commencement decreased significantly, HbA1c SD pre-CSII versus post-CSII 0.7%±0.5% (8±5 mmol/mol) vs 0.4%±0.4% (5±4 mmol/mol); p<0.001, and HbA1c CV 9.2%±5.5% (13±8 mmol/mol) vs 6.1%±3.9% (9±5 mmol/mol); p<0.001., Conclusions: In clinical practice with T1DM adults relative to MDI, CSII therapy is associated with lower HbA1c GV., Competing Interests: Competing interests: Funding (scholarships for ES) were provided by NHMRC (Australia), JDRF Australia, the University of Sydney and the Royal Australasian College of Physicians Vincent Fairfax Award. AJJ was supported by a NHMRC Practitioner Fellowship and funding from the NHMRC Clinical Trials Centre and is a Sydney Medical Foundation School Fellow. AAH is supported by a career development award from the JDRF Australia T1D Clinical Research Network., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

: Background: The potential utility of microRNAs (miRNAs) in the diagnosis, prognosis, and treatment of multiple disease states has been an area of great interest since their discovery. In patients with cardiovascular disease, there is a large pool of literature amassed from the last decade assessing their diagnostic and prognostic potential. This systematic review sought to determine whether existing literature supports the use of miRNAs as prognostic markers after an Acute Coronary Syndrome (ACS) presentation. Methods: A systematic review of published articles from 2005-2019 using MEDLINE and EMBASE databases was undertaken independently by two reviewers. Studies addressing prognosis in an ACS population yielded 32 studies and 2 systematic reviews. Results/conclusion: 23 prospective studies reported significant differences in miRNA levels and 16 compared the predictive power of miRNAs. The most common miRNAs assessed included miR-133a, -208b, -21, -1, -34a, -150, and -423, shown to be involved in cell differentiation, apoptosis, and angiogenesis. Barriers to the use of miRNAs as prognostic markers include bias in miRNA selection, small sample size, variable normalization of data, and adjustment for confounders. Therefore, findings from this systematic review do not support the use of miRNAs for prognostication post-ACS beyond traditional cardiovascular risk factors, existing risk scores, and stratifications tools., Competing Interests: The authors declare no conflict of interest.

Polycystic ovary syndrome (PCOS) remains one of the most common endocrine disorder in premenopausal women with an unfavorable metabolic risk profile. Here, we investigate whether biochemical hyperandrogenism, represented by elevated serum free testosterone, resulted in an aberrant circulating microRNA (miRNAs) expression profile and whether miRNAs can identify those PCOS women with metabolic syndrome (MetS). Accordingly, we measured serum levels of miRNAs as well as biochemical markers related to MetS in a case-control study of 42 PCOS patients and 20 Controls. Patients were diagnosed based on the Rotterdam consensus criteria and stratified based on serum free testosterone levels (≥0.034 nmol/l) into either a normoandrogenic ( n = 23) or hyperandrogenic ( n = 19) PCOS group. Overall, hyperandrogenic PCOS women were more insulin resistant compared to normoandrogenic PCOS women and had a higher prevalence of MetS. A total of 750 different miRNAs were analyzed using TaqMan Low-Density Arrays. Altered levels of seven miRNAs (miR-485-3p, -1290, -21-3p, -139-3p, -361-5p, -572, and -143-3p) were observed in PCOS patients when compared with healthy Controls. Stratification of PCOS women revealed that 20 miRNAs were differentially expressed between the three groups. Elevated serum free testosterone levels, adjusted for age and BMI, were significantly associated with five miRNAs (miR-1290, -20a-5p, -139-3p, -433-3p, and -361-5p). Using binary logistic regression and receiver operating characteristic curves (ROC), a combination panel of three miRNAs (miR-361-5p, -1225-3p, and -34-3p) could correctly identify all of the MetS cases within the PCOS group. This study is the first to report comprehensive miRNA profiling in different subgroups of PCOS women with respect to MetS and suggests that circulating miRNAs might be useful as diagnostic biomarkers of MetS for a different subset of PCOS., (Copyright © 2019 Sørensen, Udesen, Maciag, Geiger, Saliani, Januszewski, Jiang, Ma, Hardikar, Wissing, Englund and Dalgaard.)

The loss of functional beta cell mass characterises all forms of diabetes. Beta cells are highly susceptible to stress, including cytokine, endoplasmic reticulum (ER) and oxidative stress. This study examined the role of pleckstrin homology-like, domain family A, member 3 (Phlda3) in beta cell survival under stress conditions and the regulatory basis. We found that the mRNA levels of Phlda3 were markedly upregulated in vivo in the islets of diabetic humans and mice. In vitro, exposure of MIN6 cells or islets to cytokines, palmitate, thapsigargin or ribose upregulated Phlda3 mRNA and protein levels, concurrent with the induction of ER stress (Ddit3 and Trb3) and antioxidant (Hmox1) genes. Furthermore, H 2 O 2 treatment markedly increased PHLDA3 immunostaining in human islets. Phlda3 expression was differentially regulated by adaptive (Xbp1) and apoptotic (Ddit3) unfolded protein response (UPR) mediators. siRNA-mediated knockdown of Xbp1 inhibited the induction of Phlda3 by cytokines and palmitate, whereas knockdown of Ddit3 upregulated Phlda3. Moreover, knockdown of Phlda3 potentiated cytokine-induced apoptosis in association with upregulation of inflammatory genes (iNos, IL1β and IκBα) and NFκB phosphorylation and downregulation of antioxidant (Gpx1 and Srxn1) and adaptive UPR (Xbp1, Hspa5 and Fkbp11) genes. Knockdown of Phlda3 also potentiated apoptosis under oxidative stress conditions induced by ribose treatment. These findings suggest that Phlda3 is crucial for beta cell survival under stress conditions. Phlda3 regulates the cytokine, oxidative and ER stress responses in beta cells via the repression of inflammatory gene expression and the maintenance of antioxidant and adaptive UPR gene expression. Phlda3 may promote beta cell survival in diabetes.

Many acute and chronic lung injuries are incurable and rank as the fourth leading cause of death globally. While stem cell treatment for lung injuries is a promising approach, there is growing evidence that the therapeutic efficacy of stem cells originates from secreted extracellular vesicles (EVs). Consequently, EVs are emerging as next-generation therapeutics. While EVs are extensively researched for diagnostic applications, their therapeutic potential to promote tissue repair is not fully elucidated. By housing and delivering tissue-repairing cargo, EVs refine the cellular microenvironment, modulate inflammation, and ultimately repair injury. Here, the potential use of EVs derived from two placental mesenchymal stem/stromal cell (MSC) lines is presented; a chorionic MSC line (CMSC29) and a decidual MSC cell line (DMSC23) for applications in lung diseases. Functional analyses using in vitro models of injury demonstrate that these EVs have a role in ameliorating injuries caused to lung cells. It is also shown that EVs promote repair of lung epithelial cells. This study is fundamental to advancing the field of EVs and to unlock the full potential of EVs in regenerative medicine., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human islet isolation is a cost-/resource-intensive program generating islets for cell therapy in Type 1 diabetes. However, only a third of cadaveric pancreas get to clinical transplantation due to low quality/number of islets. There is a need to identify biomarker(s) that predict the quality of islets, prior to initiating their isolation. Here, we sequenced transcriptome from 18 human islet preparations stratified into three groups (Gr.1: Best quality/transplantable islets, Gr.2: Intermediary quality, Gr.3: Inferior quality/non-transplantable islets) based on routine measurements including islet purity/viability. Machine-learning algorithms involving penalized regression analyses identified 10 long-non-coding(lnc)RNAs significantly different across all group-wise comparisons (Gr1VsGr2, Gr2vsGr3, Gr1vsGr3). Two variants of Metastasis-Associated Lung Adenocarcinoma Transcript-1(MALAT1) lncRNA were common across all comparisons. We confirmed RNA-seq findings in a "validation set" of 75 human islet preparations. Finally, in 19 pancreas samples, we demonstrate that assessing the levels of MALAT1 variants alone (ROC curve AUC: 0.83) offers highest specificity in predicting post-isolation islet quality and improves the predictive potential for clinical islet transplantation when combined with Edmonton Donor Points/Body Mass Index(BMI)/North American Islet Donor Score(NAIDS). We present this resource of islet-quality-stratified lncRNA transcriptome data and identify MALAT1 as a biomarker that significantly enhances current selection methods for clinical (GMP)-grade islet isolation.

Background: Women with previous gestational diabetes mellitus (GDM) have evidence of postpartum β -cell dysfunction, which increases their risk of developing type 2 diabetes (T2DM) later in life. Elevated levels of circulating cell-free preproinsulin ( INS ) DNA correlate with dying β -cells in both mice and humans. The aim of this study was to determine if cell-free circulating INS DNA levels are higher in women with previous GDM who develop T2DM., Methods: We used droplet digital (dd) PCR to measure the levels of cell-free circulating methylated and unmethylated INS DNA in plasma from 97 women with normal glucose tolerance (NGT), 12 weeks following an index GDM pregnancy. Women were assessed for up to 10 years for the development of T2DM., Results: In the follow-up period, 22% of women developed T2DM. Compared with NGT women, total cell-free INS DNA levels were significantly higher in women who developed T2DM ( P = 0.02). There was no difference in cell-free circulating unmethylated and methylated INS DNA levels between NGT women and women who developed T2DM ( P = 0.09 and P = 0.07, respectively)., Conclusions: In women with a previous index GDM pregnancy, postpartum levels of cell-free circulating INS DNA are significantly higher in those women who later developed T2DM., Competing Interests: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Background: Previous studies indicate that low birth weight and exposure to maternal stress during pregnancy may result in shortened telomeres in infants. Shorter telomere length has in turn been linked with accelerated ageing and with age-related diseases. This study aimed to investigate the association between pregnancy and birth factors and relative telomere length in offspring at 11 years of age., Methods: Participants were aged 11 years enrolled in the Auckland Birthweight Collaborative Study at birth (n = 380). Half of the children were born small for gestational age (SGA = birthweight ≤ 10th percentile) and half were appropriate for gestational age (AGA = birthweight > 10th percentile). Maternal stress during pregnancy was assessed using the Perceived Stress Scale. Relative leukocyte telomere length (RTL) in leukocytes was measured at 11 years of age using quantitative real-time PCR., Results: RTL was normally distributed (mean = 3.78, SD = 1.05). There were no significant associations between RTL at age 11 years and birthweight, sex, maternal smoking, maternal stress during pregnancy or maternal pre-pregnancy body mass index., Conclusion: At age 11 years, RTL did not differ between children by birthweight or pregnancy-related stressors. Further telomere-related studies in newborns, children and adolescents are merited to increase knowledge of potential telomere modulating factors., (Copyright © 2019 Elsevier B.V. All rights reserved.)