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Defining the neurovascular architecture in the pancreas of individuals with type 1 diabetes (T1D) is crucial to understanding the mechanisms of dysregulated glucagon secretion. In the largest T1D cohort of biobanked tissues analyzed to date, we found that pancreatic capillaries and nerve fibers persist in human T1D despite beta cell loss, suggesting that alpha cell secretory changes may be decoupled from neurovascular components. Because innervation has been studied extensively in rodent T1D models, our studies also provide the first rigorous direct comparisons of neurovascular assembly in mouse and human, indicating dramatic interspecies differences.

Recent studies show that cellular neighborhoods play an important role in evolving biological events such as cancer and diabetes. Therefore, it is critical to accurately and efficiently identify cellular neighborhoods from spatially-resolved single-cell transcriptomic data or single-cell resolution tissue imaging data. In this work, we develop CNTools, a computational toolbox for end-to-end cellular neighborhood analysis on annotated cell images, comprising both the identification and analysis steps. It includes state-of-the-art cellular neighborhood identification methods and post-identification smoothing techniques, with our newly proposed Cellular Neighbor Embedding (CNE) method and Naive Smoothing technique, as well as several established downstream analysis approaches. We applied CNTools on three real-world CODEX datasets and evaluated identification methods with smoothing techniques quantitatively and qualitatively. It shows that CNE with Naive Smoothing overall outperformed other methods and revealed more convincing biological insights. Suggestions on how to choose identification methods and smoothing techniques according to input data are provided at last.

Pancreatic islet cells derived from human pluripotent stem cells hold great promise for modeling and treating diabetes. Differences between stem cell-derived and primary islets remain, but molecular insights to inform improvements are limited. Here, we acquire single-cell transcriptomes and accessible chromatin profiles during in vitro islet differentiation and pancreas from childhood and adult donors for comparison. We delineate major cell types, define their regulomes, and describe spatiotemporal gene regulatory relationships between transcription factors. CDX2 emerged as a regulator of enterochromaffin-like cells, which we show resemble a transient, previously unrecognized, CDX2+ pre-β-cell population in fetal pancreas, arguing against a proposed non-pancreatic origin. Furthermore, we observe insufficient activation of signal-dependent transcriptional programs during in vitro β-cell maturation and identify sex hormones as drivers of β-cell proliferation in childhood. Altogether, our analysis provides a comprehensive understanding of cell fate acquisition in stem cell-derived islets and a framework for manipulating cell identities and maturity.

This protocol describes the tissue preparation processes for the CODEX® (now PhenoCycler™) system by Akoya Biosciences. For the comprehensive multiplexed imaging workflow currently in use at the Vanderbilt Diabetes Research Center, please see CODEX® Multiplexed Imaging | Modality overview.

This protocol describes the workflow for collecting human pancreas samples for multimodal downstream assays, in use by the Vanderbilt University HuBMAP Tissue Mapping Center.

This protocol for H&E staining can be applied to either fixed or unfixed frozen cryosections.

This protocol describes the imaging workflow currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group for CO-Detection by indEXing (CODEX®, now PhenoCycler™; Akoya Biosciences). See also CODEX® Multiplexed Imaging | Modality overview.

This protocol describes the staining and pre-imaging preparation currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group prior to performing multiplexed imaging of the human pancreas using the CO-Detection by indEXing (CODEX®) platform (now PhenoCycler™; Akoya Biosciences). See also CODEX® Multiplexed Imaging | Modality overview.

This protocol describes the antibody conjugation and validation processes for the CODEX® (now PhenoCycler™) system by Akoya Biosciences. For the comprehensive multiplexed imaging workflow currently in use at the Vanderbilt Diabetes Research Center, please see CODEX® Multiplexed Imaging | Modality overview.

This is an overview of all protocols currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group for performing multiplexed imaging of the human pancreas using the CO-Detection by indEXing (CODEX®) platform (now PhenoCycler™; Akoya Biosciences). It includes links to each of the individual protocols that make up the workflow for various applications and projects.

SUMMARYA hallmark of type 2 diabetes (T2D), a major cause of world-wide morbidity and mortality, is dysfunction of insulin-producing pancreatic islet β cells1–3. T2D genome-wide association studies (GWAS) have identified hundreds of signals, mostly in the non-coding genome and overlapping β cell regulatory elements, but translating these into biological mechanisms has been challenging4–6. To identify early disease-driving events, we performed single cell spatial proteomics, sorted cell transcriptomics, and assessed islet physiology on pancreatic tissue from short-duration T2D and control donors. Here, through integrative analyses of these diverse modalities, we show that multiple gene regulatory modules are associated with early-stage T2D β cell-intrinsic defects. One notable example is the transcription factor RFX6, which we show is a highly connected β cell hub gene that is reduced in T2D and governs a gene regulatory network associated with insulin secretion defects and T2D GWAS variants. We validated the critical role of RFX6 in β cells through direct perturbation in primary human islets followed by physiological and single nucleus multiome profiling, which showed reduced dynamic insulin secretion and large-scale changes in the β cell transcriptome and chromatin accessibility landscape. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs, and individuals and thus we anticipate this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits with GWAS data.

Stripe rust disease of wheat, caused by Puccinia striiformis f. sp. tritici, (Pst) is one of the most serious diseases of wheat worldwide. In India, virulent stripe rust races have been constantly evolving in the North-Western Plains Zone leading to the failure of some of the most widely grown resistant varieties in the region. With the goal of studying the recent evolution of virulent races in this region, we conducted whole-genome sequencing of three prevalent Indian Pst pathotypes Pst46S119, Pst78S84 and Pst110S119. We assembled 58.62, 58.33 and 55.78 Mb of Pst110S119, Pst46S119 and Pst78S84 genome, respectively. Pathotypes were found to be highly heterozygous. Comparative phylogenetic analysis indicated the recent evolution of pathotypes Pst110S119 and Pst78S84 from Pst46S119. Pathogenicity-related genes classes (CAZyme, proteases, effectors, and secretome proteins) were identified and found to be under positive selection. Higher rate of gene family expansion was also observed in the three pathotypes. A strong association between the effector genes and transposable elements may be the source of the rapid evolution of these strains. Phylogenetic analysis differentiated the Indian races in this study from other known US, European, African and Asian races. Diagnostic markers developed for the identification of different Pst pathotypes will help tracking of yellow rust at farmers’ field and strategizing resistance gene deployment.

This protocol for H&E staining can be applied to either fixed or unfixed frozen cryosections.

Procuring and preparing human pancreas for paraffin-embedding, fixed cryosections, flash frozen sections, and RNA isolation. This protocol was developed in collaboration with Dr. Rita Bottino of Imagine Pharma and Allegheny Health Network and is based on protocols developed by Dr. Marcela Brissova of Vanderbilt University.

The language in the NHS is said by some to be rather ‘Big Brother’; supervision, governance, targets and plans. I was delighted when I heard, at a conference the other day, about Super Clinical Vision — Clinical Supervision by another name — but so much more positive and much less threatening just by changing a word around.

Let's start this new year, this last year of the millennium with positive regard for the future. It is all too easy to be cynical and negative when, in fact, primary care and nursing are high on the health agenda. The care of patients is at the centre of the new health policies that this government is introducing. The new structures will enable all patients in all areas to receive the best care in illness and the best service in health promotion.

The review of The Regulation of Nurses, Midwives and Health Visitors Act 1997 is well underway. JM Consulting Ltd have been commissioned to undertake this work and have produced a consultation document, and are asking for comments to be sent to them by 27 February. The outcome of this review will have far reaching implications for the future regulation of nursing and it is vital we, the ordinary members of the profession, have our say.