HumanIslets.com: Improving accessibility, integration, and usability of human research islet data.

HumanIslets.com supports diabetes research by offering easy access to islet phenotyping data, analysis tools, and data download. It includes molecular omics, islet and cellular function assays, tissue processing metadata, and phenotypes from 547 donors. As it expands, the resource aims to improve human islet data quality, usability, and accessibility.

Human research islet cell culture outcomes at the Alberta Diabetes Institute IsletCore.

Human islets from deceased organ donors have made important contributions to our understanding of pancreatic endocrine function and continue to be an important resource for research studies aimed at understanding, treating, and preventing diabetes. Understanding the impacts of isolation and culture upon the yield of human islets for research is important for planning research studies and islet distribution to distant laboratories. Here, we examine islet isolation and cell culture outcomes at the Alberta Diabetes Institute (ADI) IsletCore (n = 197). Research-focused isolations typically have a lower yield of islet equivalents (IEQ), with a median of 252,876 IEQ, but a higher purity (median 85%) than clinically focused isolations before culture. The median recovery of IEQs after culture was 75%, suggesting some loss. This was associated with a shift toward smaller islet particles, indicating possible islet fragmentation, and occurred within 24 h with no further loss after longer periods of culture (up to 136 h). No overall change in stimulation index as a measure of islet function was seen with culture time. These findings were replicated in a representative cohort of clinical islet preparations from the Clinical Islet Transplant Program at the University of Alberta. Thus, loss of islets occurs within 24 h of isolation, and there is no further impact of extended culture prior to islet distribution for research.

HumanIslets: An integrated platform for human islet data access and analysis.

Comprehensive molecular and cellular phenotyping of human islets can enable deep mechanistic insights for diabetes research. We established the Human Islet Data Analysis and Sharing (HI-DAS) consortium to advance goals in accessibility, usability, and integration of data from human islets isolated from donors with and without diabetes at the Alberta Diabetes Institute (ADI) IsletCore. Here we introduce HumanIslets.com, an open resource for the research community. This platform, which presently includes data on 547 human islet donors, allows users to access linked datasets describing molecular profiles, islet function and donor phenotypes, and to perform various statistical and functional analyses at the donor, islet and single-cell levels. As an example of the analytic capacity of this resource we show a dissociation between cell culture effects on transcript and protein expression, and an approach to correct for exocrine contamination found in hand-picked islets. Finally, we provide an example workflow and visualization that highlights links between type 2 diabetes status, SERCA3b Ca2+-ATPase levels at the transcript and protein level, insulin secretion and islet cell phenotypes. HumanIslets.com provides a growing and adaptable set of resources and tools to support the metabolism and diabetes research community.

Proteomic predictors of individualized nutrient-specific insulin secretion in health and disease.

Population-level variation and mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized. We defined prototypical insulin secretion responses to three macronutrients in islets from 140 cadaveric donors, including those with type 2 diabetes. The majority of donors' islets exhibited the highest insulin response to glucose, moderate response to amino acid, and minimal response to fatty acid. However, 9% of donors' islets had amino acid responses, and 8% had fatty acid responses that were larger than their glucose-stimulated insulin responses. We leveraged this heterogeneity and used multi-omics to identify molecular correlates of nutrient responsiveness, as well as proteins and mRNAs altered in type 2 diabetes. We also examined nutrient-stimulated insulin release from stem cell-derived islets and observed responsiveness to fat but not carbohydrate or protein-potentially a hallmark of immaturity. Understanding the diversity of insulin responses to carbohydrate, protein, and fat lays the groundwork for personalized nutrition.

Evaluating Islet Cell Isolation and Transplantation From Donors Following Medical Assistance in Dying.

Background: Limited information is available regarding outcomes of islet cell isolation (ICI) and transplantation (ITx) using medical assistance in dying (MAiD) donors. We aimed to assess the feasibility and outcomes of ICI and ITx in MAiD donors. Methods: ICI and ITx from MAiD were compared with donation after circulatory death (DCD) type III between 2016 and 2023. Differences of isolated islet equivalents (IEQs), numeric viability and other quantitative in vitro metabolic measures were assessed. Results: Overall, 81 ICIs were available of whom 34 (42%) and 47 (58%) from MAiD and DCD-III, respectively. There were no differences of pancreas and digested tissue weight and islets viability among the 2 groups; however, cold ischemic time was longer in MAiD (11.5 versus 9.1 h; P = 0.021). The IEQ (P < 0.001) and percent trapped (P < 0.001) were higher in the DCD-III; however, MAiD islets demonstrated a higher purity (P = 0.020). Overall, 15 ITx were performed of whom 3 (8.8%) and 12 (25.5%) from MAiD and DCD-III, respectively (P = 0.056). Patients had a median fasting C-peptide of 0.51 ng/mL (interquartile range, 0.30-0.76 nmol/L), with no differences between groups (MAiD = 0.52 versus DCD-III = 0.51; P = 0.718). The median HbA1c was 6.2% (interquartile range, 5.7%-7%) (MAiD = 6.3% versus DCD-III = 6.1%; P = 0.815) and BETA2 scores (MAiD = 7.4 versus DCD-III = 12.8; P = 0.229) did not differ. Conclusions: ICI from MAiD donor pancreas may be successfully transplanted with comparable outcomes to DCD-III and may be used for research. These results justify additional efforts to consider MAiD as another valuable source of grafts for ITx. Further multicenter studies and larger clinical experience are needed to validate our findings.

Proteomic predictors of individualized nutrient-specific insulin secretion in health and disease.

Population level variation and molecular mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized despite ramifications for personalized nutrition. Here, we define prototypical insulin secretion dynamics in response to the three macronutrients in islets from 140 cadaveric donors, including those diagnosed with type 2 diabetes. While islets from the majority of donors exhibited the expected relative response magnitudes, with glucose being highest, amino acid moderate, and fatty acid small, 9% of islets stimulated with amino acid and 8% of islets stimulated with fatty acids had larger responses compared with high glucose. We leveraged this insulin response heterogeneity and used transcriptomics and proteomics to identify molecular correlates of specific nutrient responsiveness, as well as those proteins and mRNAs altered in type 2 diabetes. We also examine nutrient-responsiveness in stem cell-derived islet clusters and observe that they have dysregulated fuel sensitivity, which is a hallmark of functionally immature cells. Our study now represents the first comparison of dynamic responses to nutrients and multi-omics analysis in human insulin secreting cells. Responses of different people's islets to carbohydrate, protein, and fat lay the groundwork for personalized nutrition. ONE-SENTENCE SUMMARY: Deep phenotyping and multi-omics reveal individualized nutrient-specific insulin secretion propensity.

Evolving Management of Stage IV Melanoma.

Significant advancements have been made in the treatment of advanced melanoma with the use of immune checkpoint inhibitors, novel immunotherapies, and BRAF/MEK-targeted therapies with numerous frontline treatment options. However, there remains suboptimal evidence to guide treatment decisions in many patients. These include patients with newly diagnosed disease, immune checkpoint inhibitor (ICI)-resistant/ICI-refractory disease, CNS metastases, history of autoimmune disease, and/or immune-related adverse events (irAEs). Uveal melanoma (UM) is a rare melanoma associated with a poor prognosis in the metastatic setting. Systemic treatments, including checkpoint inhibitors, failed to demonstrate any survival benefit. Tebentafusp, a bispecific molecule, is the first treatment to improve overall survival (OS) in patients with HLA A*02:01-positive metastatic UM.

Quadriceps Performance and Running Biomechanics Influence Femur BMD Changes after ACL Reconstruction in Collegiate Athletes.

PURPOSE: Reduced bone mineral density of the distal femur (BMD DF ) can persist long term after anterior cruciate ligament reconstruction (ACLR), even in athletes who return to high levels of competition. These deficits may have implications for the onset and progression of knee osteoarthritis. It is unknown if clinically modifiable factors are associated with losses in BMD DF . This study evaluated the potential influence of knee extensor peak torque (PT), rate of torque development (RTD), as well as peak knee flexion (PKF) angle and peak knee extensor moment (PKEM) during running, on longitudinal changes in BMD DF post-ACLR. METHODS: After ACLR, 57 Division I collegiate athletes underwent serial whole-body dual-energy x-ray absorptiometry (DXA) scans between 3 and 24 months post-ACLR. Of these, 43 athletes also had isometric knee extensor testing (21 female, 105 observations), and 54 had running analyses (26 female, 141 observations). Linear mixed-effects models, controlling for sex, assessed the influence of surgical limb quadriceps performance (PT and RTD), running mechanics (PKF and PKEM), and time post-ACLR on BMD DF (5% and 15% of femur length). Simple slope analyses were used to explore interactions. RESULTS: Athletes with RTD less than 7.20 (N·m)·kg -1 ·s -1 (mean) at 9.3 months post-ACLR demonstrated significant decreases in 15% BMD DF over time ( P = 0.03). Athletes with PKEM during running less than 0.92 (N·m)·kg -1 (-1 SD below mean) at 9.8 months post-ACLR demonstrated significant decreases in 15% BMD DF over time ( P = 0.02). Significant slopes were not detected at -1 SD below the mean for PT (1.75 (N·m)·kg -1 , P = 0.07) and PKF (31.3°, P = 0.08). CONCLUSIONS: Worse quadriceps RTD and running PKEM were associated with a greater loss of BMD DF between 3 and 24 months post-ACLR.

Systematic, comprehensive, evidence-based approach to identify neuroprotective interventions for motor neuron disease: using systematic reviews to inform expert consensus.

OBJECTIVES: Motor neuron disease (MND) is an incurable progressive neurodegenerative disease with limited treatment options. There is a pressing need for innovation in identifying therapies to take to clinical trial. Here, we detail a systematic and structured evidence-based approach to inform consensus decision making to select the first two drugs for evaluation in Motor Neuron Disease-Systematic Multi-arm Adaptive Randomised Trial (MND-SMART: NCT04302870), an adaptive platform trial. We aim to identify and prioritise candidate drugs which have the best available evidence for efficacy, acceptable safety profiles and are feasible for evaluation within the trial protocol. METHODS: We conducted a two-stage systematic review to identify potential neuroprotective interventions. First, we reviewed clinical studies in MND, Alzheimer's disease, Huntington's disease, Parkinson's disease and multiple sclerosis, identifying drugs described in at least one MND publication or publications in two or more other diseases. We scored and ranked drugs using a metric evaluating safety, efficacy, study size and study quality. In stage two, we reviewed efficacy of drugs in MND animal models, multicellular eukaryotic models and human induced pluripotent stem cell (iPSC) studies. An expert panel reviewed candidate drugs over two shortlisting rounds and a final selection round, considering the systematic review findings, late breaking evidence, mechanistic plausibility, safety, tolerability and feasibility of evaluation in MND-SMART. RESULTS: From the clinical review, we identified 595 interventions. 66 drugs met our drug/disease logic. Of these, 22 drugs with supportive clinical and preclinical evidence were shortlisted at round 1. Seven drugs proceeded to round 2. The panel reached a consensus to evaluate memantine and trazodone as the first two arms of MND-SMART. DISCUSSION: For future drug selection, we will incorporate automation tools, text-mining and machine learning techniques to the systematic reviews and consider data generated from other domains, including high-throughput phenotypic screening of human iPSCs.

Territory and population attributes affect Florida scrub-jay fecundity in fire-adapted ecosystems.

Fecundity, the number of young produced by a breeding pair during a breeding season, is a primary component in evolutionary and ecological theory and applications. Fecundity can be influenced by many environmental factors and requires long-term study due to the range of variation in ecosystem dynamics. Fecundity data often include a large proportion of zeros when many pairs fail to produce any young during a breeding season due to nest failure or when all young die independently after fledging. We conducted color banding and monthly censuses of Florida scrub-jays (Aphelocoma coerulescens) across 31 years, 15 populations, and 761 territories along central Florida's Atlantic coast. We quantified how fecundity (juveniles/pair-year) was influenced by habitat quality, presence/absence of nonbreeders, population density, breeder experience, and rainfall, with a zero-inflated Bayesian hierarchical model including both a Bernoulli (e.g., brood success) and a Poisson (counts of young) submodel, and random effects for year, population, and territory. The results identified the importance of increasing "strong" quality habitat, which was a mid-successional state related to fire frequency and extent, because strong territories, and the proportion of strong territories in the overall population, influenced fecundity of breeding pairs. Populations subject to supplementary feeding also had greater fecundity. Territory size, population density, breeder experience, and rainfall surprisingly had no or small effects. Different mechanisms appeared to cause annual variation in fecundity, as estimates of random effects were not correlated between the success and count submodels. The increased fecundity for pairs with nonbreeders, compared to pairs without, identified empirical research needed to understand how the proportion of low-quality habitats influences population recovery and sustainability, because dispersal into low-quality habitats can drain nonbreeders from strong territories and decrease overall fecundity. We also describe how long-term study resulted in reversals in our understanding because of complications involving habitat quality, sociobiology, and population density.

A genome-wide CRISPR screen identifies CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk.

Identification of the genes and processes mediating genetic association signals for complex diseases represents a major challenge. As many of the genetic signals for type 2 diabetes (T2D) exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of-function screen in a human pancreatic beta cell line. We assessed the regulation of insulin content as a disease-relevant readout of beta cell function and identified 580 genes influencing this phenotype. Integration with genetic and genomic data provided experimental support for 20 candidate T2D effector transcripts including the autophagy receptor CALCOCO2. Loss of CALCOCO2 was associated with distorted mitochondria, less proinsulin-containing immature granules and accumulation of autophagosomes upon inhibition of late-stage autophagy. Carriers of T2D-associated variants at the CALCOCO2 locus further displayed altered insulin secretion. Our study highlights how cellular screens can augment existing multi-omic efforts to support mechanistic understanding and provide evidence for causal effects at genome-wide association studies loci.

Aging compromises human islet beta cell function and identity by decreasing transcription factor activity and inducing ER stress.

Pancreatic islet beta cells are essential for maintaining glucose homeostasis. To understand the impact of aging on beta cells, we performed meta-analysis of single-cell RNA sequencing datasets, transcription factor (TF) regulon analysis, high-resolution confocal microscopy, and measured insulin secretion from nondiabetic donors spanning most of the human life span. This revealed the range of molecular and functional changes that occur during beta cell aging, including the transcriptional deregulation that associates with cellular immaturity and reorganization of beta cell TF networks, increased gene transcription rates, and reduced glucose-stimulated insulin release. These alterations associate with activation of endoplasmic reticulum (ER) stress and autophagy pathways. We propose that a chronic state of ER stress undermines old beta cell structure function to increase the risk of beta cell failure and type 2 diabetes onset as humans age.

Acquired Hemophilia as a Paraneoplastic Syndrome in a Patient With Small Cell Lung Carcinoma.

This case report describes a 72-year-old female patient diagnosed with small cell lung carcinoma who was found to have elevated partial thromboplastin time (PTT) after reporting diarrhea and melanotic stool. Further investigations revealed the presence of a factor VIII inhibitor, possibly resulting from a side effect of immunotherapy or of possible paraneoplastic origin. The patient's PTT remained elevated following a course of steroid treatment, raising the likelihood of paraneoplastic etiology. This case represents a rare paraneoplastic syndrome, with a previously unreported presentation of melanotic stool as opposed to an acute bleeding episode.

Impacts of the COVID-19 pandemic on a human research islet program.

Designated a pandemic in March 2020, the spread of severe acute respiratory syndrome virus 2 (SARS-CoV2), the virus responsible for coronavirus disease 2019 (COVID-19), led to new guidelines and restrictions being implemented for individuals, businesses, and societies in efforts to limit the impacts of COVID-19 on personal health and healthcare systems. Here we report the impacts of the COVID-19 pandemic on pancreas processing and islet isolation/distribution outcomes at the Alberta Diabetes Institute IsletCore, a facility specializing in the processing and distribution of human pancreatic islets for research. While the number of organs processed was significantly reduced, organ quality and the function of cellular outputs were minimally impacted during the pandemic when compared to an equivalent period immediately prior. Despite the maintained quality of isolated islets, feedback from recipient groups was more negative. Our findings suggest this is likely due to disrupted distribution which led to increased transit times to recipient labs, particularly those overseas. Thus, to improve overall outcomes in a climate of limited research islet supply, prioritization of tissue recipients based on likely tissue transit times may be needed.

Heterogenous impairment of α cell function in type 2 diabetes is linked to cell maturation state.

In diabetes, glucagon secretion from pancreatic α cells is dysregulated. The underlying mechanisms, and whether dysfunction occurs uniformly among cells, remain unclear. We examined α cells from human donors and mice using electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses α cell exocytosis by reducing P/Q-type Ca2+ channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat feeding of mice, α cells shift toward a "ß cell-like" electrophysiological profile in concert with indications of impaired identity. In human α cells we identified links between cell membrane properties and cell surface signaling receptors, mitochondrial respiratory chain complex assembly, and cell maturation. Cell-type classification using machine learning of electrophysiology data demonstrated a heterogenous loss of "electrophysiologic identity" in α cells from donors with type 2 diabetes. Indeed, a subset of α cells with impaired exocytosis is defined by an enrichment in progenitor and lineage markers and upregulation of an immature transcriptomic phenotype, suggesting important links between α cell maturation state and dysfunction.

Cryopreservation and post-thaw characterization of dissociated human islet cells.

The objective of this study is to optimize the cryopreservation of dissociated islet cells and obtain functional cells that can be used in single-cell transcriptome studies on the pathology and treatment of diabetes. Using an iterative graded freezing approach we obtained viable cells after cooling in 10% dimethyl sulfoxide and 6% hydroxyethyl starch at 1°C/min to -40°C, storage in liquid nitrogen, rapid thaw, and removal of cryoprotectants by serial dilution. The expression of epithelial cell adhesion molecule declined immediately after thaw, but recovered after overnight incubation, while that of an endocrine cell marker (HPi2) remained high after cryopreservation. Patch-clamp electrophysiology revealed differences in channel activities and exocytosis of various islet cell types; however, exocytotic responses, and the biophysical properties of voltage-gated Na+ and Ca2+ channels, are sustained after cryopreservation. Single-cell RNA sequencing indicates that overall transcriptome and crucial exocytosis genes are comparable between fresh and cryopreserved dispersed human islet cells. Thus, we report an optimized procedure for cryopreserving dispersed islet cells that maintained their membrane integrity, along with their molecular and functional phenotypes. Our findings will not only provide a ready source of cells for investigating cellular mechanisms in diabetes but also for bio-engineering pseudo-islets and islet sheets for modeling studies and potential transplant applications.

Community suicide rates and related factors within a surveillance platform in Western Kenya.

BACKGROUND: Suicide is an important contributor to the burden of mental health disorders, but community-based suicide data are scarce in many low- and middle-income countries (LMIC) including Kenya. Available data on suicide underestimates the true burden due to underreporting related to stigma and legal restrictions, and under-representation of those not utilizing health facilities. METHODS: We estimated the cumulative incidence of suicide via verbal autopsies from the Health and Demographic Surveillance System (HDSS) in Kisumu County, Kenya. We then used content analysis of open history forms among deaths coded as accidents to identify those who likely died by suicide but were not coded as suicide deaths. We finally conducted a case-control study of suicides (both verbal autopsy confirmed and likely suicides) compared to accident-caused deaths to assess factors associated with suicide in this HDSS. RESULTS: A total of 33 out of 4306 verbal autopsies confirmed suicide as the cause of death. Content analysis of a further 228 deaths originally attributed to accidents identified 39 additional likely suicides. The best estimate of suicide-specific mortality rate was 14.7 per 100,000 population per year (credibility window = 11.3 - 18.0). The most common reported method of death was self-poisoning (54%). From the case-control study interpersonal difficulties and stressful life events were associated with increased odds of suicide in both confirmed suicides and confirmed combined with suspected suicides. Other pertinent factors such as age and being male differed depending upon which outcome was used. CONCLUSION: Suicide is common in this area, and interventions are needed to address drivers. The twofold increase in the suicide-specific mortality rate following incorporation of misattributed suicide deaths exemplify underreporting and misclassification of suicide cases at community level. Further, verbal autopsies may underreport suicide specifically among older and female populations.

Spatial progression and molecular heterogeneity of IDH-mutant glioblastoma determined by DNA methylation-based mapping.

Glioblastoma (GBM) is the most common malignant primary central nervous system (CNS) neoplasm in adults, and has an almost universally poor prognosis. Recently, an emphasis on genetic and epigenetic profiling has revealed a number of molecular features useful in the diagnostic and prognostic classification of GBM, advancing our understanding of the underlying features that make these tumors so aggressive and providing the rationale for the creation of better targeted therapeutics. One such method, DNA methylation profiling, has recently emerged as an important technique for the classification of CNS tumors, with diagnostic accuracy in some cases surpassing traditional methods. However, how DNA methylation profiles change with the course of the disease remains less understood. Here, we present a case of a 30-year-old male with primary IDH-mutant GBM with widespread recurrence and death two years later. Using unsupervised hierarchical clustering of methylation probes, we created a phylogenetic map to trace the tumor path as it spread from the initial biopsy site throughout the right hemisphere, across the corpus callosum to the contralateral hemisphere, and into the brainstem. We identified molecular divergence between the right and left hemisphere GBM samples marked by distinct copy number profile alterations, alterations in specific methylation sites, and regional loss of MGMT promoter methylation, providing a potential mechanism for treatment resistance in this case. In summary, this case both highlights the molecular diversity in GBM, and illustrates a novel use for methylation profiling in establishing a phylogenetic profile to allow for spatial mapping of tumor progression.

Improved glucose tolerance with DPPIV inhibition requires ß-cell SENP1 amplification of glucose-stimulated insulin secretion.

Pancreatic islet insulin secretion is amplified by both metabolic and receptor-mediated signaling pathways. The incretin-mimetic and DPPIV inhibitor anti-diabetic drugs increase insulin secretion, but in humans this can be variable both in vitro and in vivo. We examined the correlation of GLP-1 induced insulin secretion from human islets with key donor characteristics, glucose-responsiveness, and the ability of glucose to augment exocytosis in ß-cells. No clear correlation was observed between several donor or organ processing parameters and the ability of Exendin 4 to enhance insulin secretion. The ability of glucose to facilitate ß-cell exocytosis was, however, significantly correlated with responses to Exendin 4. We therefore studied the effect of impaired glucose-dependent amplification of insulin exocytosis on responses to DPPIV inhibition (MK-0626) in vivo using pancreas and ß-cell specific sentrin-specific protease-1 (SENP1) mice which exhibit impaired metabolic amplification of insulin exocytosis. Glucose tolerance was improved, and plasma insulin was increased, following either acute or 4 week treatment of wild-type (ßSENP1+/+ ) mice with MK-0626. This DPPIV inhibitor was ineffective in ßSENP1+/- or ßSENP1- / - mice. Finally, we confirm impaired exocytotic responses of ß-cells and reduced insulin secretion from islets of ßSENP1- / - mice and show that the ability of Exendin 4 to enhance exocytosis is lost in these cells. Thus, an impaired ability of glucose to amplify insulin exocytosis results in a deficient effect of DPPIV inhibition to improve in vivo insulin responses and glucose tolerance.