Reconstitution and substrate specificity for isopentenyl pyrophosphate of the antiviral radical SAM enzyme viperin.

Viperin is a radical SAM enzyme that has been shown to possess antiviral activity against a broad spectrum of viruses; however, its molecular mechanism is unknown. We report here that recombinant fungal and archaeal viperin enzymes catalyze the addition of the 5'-deoxyadenosyl radical (5'-dA•) to the double bond of isopentenyl pyrophosphate (IPP), producing a new compound we named adenylated isopentyl pyrophosphate (AIPP). The reaction is specific for IPP, as other pyrophosphate compounds involved in the mevalonate biosynthetic pathway did not react with 5'-dA• Enzymatic reactions employing IPP derivatives as substrates revealed that any chemical change in IPP diminishes its ability to be an effective substrate of fungal viperin. Mutational studies disclosed that the hydroxyl group on the side chain of Tyr-245 in fungal viperin is the likely source of hydrogen in the last step of the radical addition, providing mechanistic insight into the radical reaction catalyzed by fungal viperin. Structure-based molecular dynamics (MD) simulations of viperin interacting with IPP revealed a good fit of the isopentenyl motif of IPP to the active site cavity of viperin, unraveling the molecular basis of substrate specificity of viperin for IPP. Collectively, our findings indicate that IPP is an effective substrate of fungal and archaeal viperin enzymes and provide critical insights into the reaction mechanism.

Protocol for quantifying SA-ß-gal activity as a measure of senescence in islets of a mouse model of type 1 diabetes.

A subpopulation of pancreatic beta cells becomes senescent during type 1 diabetes (T1D) progression, and removal of these populations protects against T1D in mice. Here, we present a protocol to measure senescence in murine pancreatic islet cells through analysis of senescence-associated ß-galactosidase activity. We describe steps for staining with the fluorogenic substrate C12FDG and analysis by flow cytometry. Increased cell size is another marker of senescence and can also be concurrently measured in the same experiment. For complete details on the use and execution of this protocol, please refer to Lee et al.1 and Helman et al.2.

Ormdl3 regulation of specific ceramides is dispensable for ß-cell function and glucose homeostasis under obesogenic conditions.

Chronic elevation of sphingolipids contributes to ß-cell failure. ORMDL3 has been identified as a key regulator of sphingolipid homeostasis, however, its function in pancreatic ß-cell pathophysiology remains unclear. Here, we generated a mouse model lacking Ormdl3 within pancreatic ß-cells ( Ormdl3 ß-/- ). We show that loss of ß-cell Ormdl3 does not alter glucose tolerance, insulin sensitivity, insulin secretion, islet morphology, or cellular ceramide levels on standard chow diet. When challenged with a high fat diet, while Ormdl3 ß-/- mice did not exhibit any alteration in metabolic parameters or islet architecture, lipidomics analysis revealed significantly higher levels of very long chain ceramides in their islets. Taken together, our results reveal that loss of Ormdl3 alone is not sufficient to impinge upon ß-cell function or whole-body glucose and insulin homeostasis, but loss of Ormdl3 does alter specific sphingolipid levels.

Ormdl3 regulation of specific ceramides is dispensable for mouse ß-cell function and glucose homeostasis under obesogenic conditions.

Chronic elevation of sphingolipids contributes to ß-cell failure. ORMDL3 has been identified as a key regulator of sphingolipid homeostasis, however, its function in pancreatic ß-cell pathophysiology remains unclear. Here, we generated a mouse model lacking Ormdl3 within pancreatic ß-cells (Ormdl3 ß-/-). We show that loss of ß-cell Ormdl3 does not alter glucose tolerance, insulin sensitivity, insulin secretion, islet morphology, or cellular ceramide levels on standard chow diet. When challenged with a high fat diet, while Ormdl3 ß-/- mice did not exhibit any alteration in metabolic parameters or islet architecture, lipidomics analysis revealed significantly higher levels of very long chain ceramides in their islets. Taken together, our results reveal that loss of Ormdl3 alone is not sufficient to impinge upon ß-cell function or whole-body glucose and insulin homeostasis, however, ß-cell-specific loss of Ormdl3 does significantly alter levels of specific sphingolipid species in islets upon high fat feeding.

Bayesian weighting of climate models based on climate sensitivity.

Using climate model ensembles containing members that exhibit very high climate sensitivities to increasing CO2 concentrations can result in biased projections. Various methods have been proposed to ameliorate this 'hot model' problem, such as model emulators or model culling. Here, we utilize Bayesian Model Averaging as a framework to address this problem without resorting to outright rejection of models from the ensemble. Taking advantage of multiple lines of evidence used to construct the best estimate of the earth's climate sensitivity, the Bayesian Model Averaging framework produces an unbiased posterior probability distribution of model weights. The updated multi-model ensemble projects end-of-century global mean surface temperature increases of 2 oC for a low emissions scenario (SSP1-2.6) and 5 oC for a high emissions scenario (SSP5-8.5). These estimates are lower than those produced using a simple multi-model mean for the CMIP6 ensemble. The results are also similar to results from a model culling approach, but retain some weight on low-probability models, allowing for consideration of the possibility that the true value could lie at the extremes of the assessed distribution. Our results showcase Bayesian Model Averaging as a path forward to project future climate change that is commensurate with the available scientific evidence.

Stress-induced ß cell early senescence confers protection against type 1 diabetes.

During the progression of type 1 diabetes (T1D), ß cells are exposed to significant stress and, therefore, require adaptive responses to survive. The adaptive mechanisms that can preserve ß cell function and survival in the face of autoimmunity remain unclear. Here, we show that the deletion of the unfolded protein response (UPR) genes Atf6α or Ire1α in ß cells of non-obese diabetic (NOD) mice prior to insulitis generates a p21-driven early senescence phenotype and alters the ß cell secretome that significantly enhances the leukemia inhibitory factor-mediated recruitment of M2 macrophages to islets. Consequently, M2 macrophages promote anti-inflammatory responses and immune surveillance that cause the resolution of islet inflammation, the removal of terminally senesced ß cells, the reduction of ß cell apoptosis, and protection against T1D. We further demonstrate that the p21-mediated early senescence signature is conserved in the residual ß cells of T1D patients. Our findings reveal a previously unrecognized link between ß cell UPR and senescence that, if leveraged, may represent a novel preventive strategy for T1D.

Analysis of Half a Billion Datapoints Across Ten Machine-Learning Algorithms Identifies Key Elements Associated With Insulin Transcription in Human Pancreatic Islet Cells.

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-1αß-/- mice that demonstrate dedifferentiation of pancreatic ß-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.

Adaptation to chronic ER stress enforces pancreatic ß-cell plasticity.

Pancreatic ß-cells are prone to endoplasmic reticulum (ER) stress due to their role in insulin secretion. They require sustainable and efficient adaptive stress responses to cope with this stress. Whether episodes of chronic stress directly compromise ß-cell identity is unknown. We show here under reversible, chronic stress conditions ß-cells undergo transcriptional and translational reprogramming associated with impaired expression of regulators of ß-cell function and identity. Upon recovery from stress, ß-cells regain their identity and function, indicating a high degree of adaptive plasticity. Remarkably, while ß-cells show resilience to episodic ER stress, when episodes exceed a threshold, ß-cell identity is gradually lost. Single cell RNA-sequencing analysis of islets from type 1 diabetes patients indicates severe deregulation of the chronic stress-adaptation program and reveals novel biomarkers of diabetes progression. Our results suggest ß-cell adaptive exhaustion contributes to diabetes pathogenesis.

An accomplice more than a mere victim: The impact of ß-cell ER stress on type 1 diabetes pathogenesis.

BACKGROUND: Pancreatic ß-cells are the insulin factory of an organism with a mission to regulate glucose homeostasis in the body. Due to their high secretory activity, ß-cells rely on a functional and intact endoplasmic reticulum (ER). Perturbations to ER homeostasis and unmitigated stress lead to ß-cell dysfunction and death. Type 1 diabetes (T1D) is a chronic inflammatory disease caused by the autoimmune-mediated destruction of ß-cells. Although autoimmunity is an essential component of T1D pathogenesis, accumulating evidence suggests an important role of ß-cell ER stress and aberrant unfolded protein response (UPR) in disease initiation and progression. SCOPE OF REVIEW: In this article, we introduce ER stress and the UPR, review ß-cell ER stress in various mouse models, evaluate its involvement in inflammation, and discuss the effects of ER stress on ß-cell plasticity and demise, and islet autoimmunity in T1D. We also highlight the relationship of ER stress with other stress response pathways and provide insight into ongoing clinical studies targeting ER stress and the UPR for the prevention or treatment of T1D. MAJOR CONCLUSIONS: Evidence from ex vivo studies, in vivo mouse models, and tissue samples from patients suggest that ß-cell ER stress and a defective UPR contribute to T1D pathogenesis. Thus, restoration of ß-cell ER homeostasis at various stages of disease presents a plausible therapeutic strategy for T1D. Identifying the specific functions and regulation of each UPR sensor in ß-cells and uncovering the crosstalk between stressed ß-cells and immune cells during T1D progression would provide a better understanding of the molecular mechanisms of disease process, and may reveal novel targets for development of effective therapies for T1D.

Preparing Highly Viable Single-Cell Suspensions from Mouse Pancreatic Islets for Single-Cell RNA Sequencing.

Pancreatic islets consist of several cell types, including alpha, beta, delta, epsilon, and PP cells. Due to cellular heterogeneity, it is challenging to interpret whole-islet transcriptome data. Single-cell transcriptomics offers a powerful method for investigating gene expression at the single-cell level and identifying cellular heterogeneity and subpopulations. Here, we describe a protocol for mouse pancreatic islet isolation, culturing, and dissociation into a single-cell suspension. This protocol yields highly viable cells for successful library preparation and single-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020).

Differential Expression of Ormdl Genes in the Islets of Mice and Humans with Obesity.

The orosomucoid-like (Ormdl) proteins play a critical role in sphingolipid homeostasis, inflammation, and ER stress, all of which are associated with obesity and ßcell dysfunction. However, their roles in ß cells and obesity remain unknown. Here, we show that islets from overweight/obese human donors displayed marginally reduced ORMDL1-2 expression, whereas ORMDL3 expression was significantly downregulated compared with islets from lean donors. In contrast, Ormdl3 was substantially upregulated in the islets of leptin-deficient obese (ob/ob) mice compared with lean mice. Treatment of ob/ob mice and their islets with leptin markedly reduced islet Ormld3 expression. Ormdl3 knockdown in a ß cell line induced expression of pro-apoptotic markers, which was rescued by ceramide synthase inhibitor fumonisin B1. Our results reveal differential expression of Ormdl3 in the islets of a mouse model and humans with obesity, highlight the potential effect of leptin in this differential regulation, and suggest a role for Ormdl3 in ß cell apoptosis.

Beta Cell Dedifferentiation Induced by IRE1α Deletion Prevents Type 1 Diabetes.

Immune-mediated destruction of insulin-producing ß cells causes type 1 diabetes (T1D). However, how ß cells participate in their own destruction during the disease process is poorly understood. Here, we report that modulating the unfolded protein response (UPR) in ß cells of non-obese diabetic (NOD) mice by deleting the UPR sensor IRE1α prior to insulitis induced a transient dedifferentiation of ß cells, resulting in substantially reduced islet immune cell infiltration and ß cell apoptosis. Single-cell and whole-islet transcriptomics analyses of immature ß cells revealed remarkably diminished expression of ß cell autoantigens and MHC class I components, and upregulation of immune inhibitory markers. IRE1α-deficient mice exhibited significantly fewer cytotoxic CD8+ T cells in their pancreata, and adoptive transfer of their total T cells did not induce diabetes in Rag1-/- mice. Our results indicate that inducing ß cell dedifferentiation, prior to insulitis, allows these cells to escape immune-mediated destruction and may be used as a novel preventive strategy for T1D in high-risk individuals.

Changing patterns of injury associated with low-energy falls in the elderly: a 10-year analysis at an Australian Major Trauma Centre.

BACKGROUND: The rate of hospitalization in elderly patients because of falls is increasing. The objective of this study was to investigate long-term trends in injury profiles of low-energy falls and to identify injuries associated with need for in-patient rehabilitation. METHODS: A single-centre retrospective study was performed at an inner city Major Trauma Centre in Sydney. Trauma registry data were obtained from patients who were 65 years of age or over with low-energy falls (trip and fall from height ≤ 1 m, including falls from standing) from the trauma registry between January 2000 and December 2011. Demographic data, time and date of presentation and injury characteristics were collected. Outcomes of interests were proportions of hip fractures, head injuries and discharge to in-patient rehabilitation facilities. RESULTS: A total of 4964 cases were identified. There was a 6.5% per annum decrease in the proportion of elderly patients with low-energy falls who sustained hip fractures compared with a relative increase in severe head injuries, 5.7% per annum. Around 25% of patients were transferred to in-patient rehabilitation. Severe head injuries and lower-limb injuries were the two injuries most associated with transfer to in-patient rehabilitation. CONCLUSION: In elderly patients with low-energy falls, a significant decrease in hip fractures was associated with a rise in severe head injuries over the past decade.