IGFBP7 is upregulated in islets from T2D donors and reduces insulin secretion

\ua9 2024 The Author(s)Intra-islet crosstalk has become a focus area to fully understand the regulation of insulin secretion and impaired β-cell function in type 2 diabetes (T2D). Here, we put forward evidence for insulin-like growth factor binding protein 7 (IGFBP7) as a potential protein involved in autocrine and paracrine β-cell regulation. We showed presence of IGFBP7 in granules of both human α- and β-cells and measured elevated gene expression as well as IGFBP7 protein in T2D. Insulin secretion was reduced in human islets, and the human β-cell line EndoC-βH1, after 72-h incubation with IGFBP7. Mechanistically reduced insulin secretion by IGFBP7 is attributed to reduced p21-activated kinase 1 (PAK1) protein, and decreased oxygen consumption and ATP-production. Knockdown of IGFBP7 in EndoC-βH1 cells verified reduced IGFBP7 levels in the medium, as well as improved insulin secretion. Finally, IGFBP7 knockdown in islets from T2D donors improved insulin secretion, making IGFBP7 a potential drug target in diabetes

High-dimensional tissue profiling of immune cell responses in chronic lung allograft dysfunction

\ua9 2024 The AuthorsPurpose: The immunological drivers of chronic lung allograft dysfunction (CLAD), the major barrier to long-term survival after lung transplantation, are poorly understood at a tissue level. Tissue imaging using mass spectrometry with laser ablation of regions of interest offers single-cell resolution of distinct immune cell populations and their spatial relationships and may improve our understanding of CLAD pathophysiology. Methods: Lung tissue from 23 lung transplant recipients, 20 with and 3 without CLAD, was sectioned and stained with a 40-plex antibody panel before 81 regions of interest from airways, blood vessels and lung parenchyma were laser ablated. Results: 190,851 individual segmented cells across 41 mm2 tissue were captured before 26 distinct immune and structural cell populations were identified and interrogated across CLAD phenotypes. CLAD was associated with expansion of cytotoxic T cells, γδ T cells and plasma cells and M2 macrophage polarization compared with non-CLAD. Within CLAD, bronchiolitis obliterans syndrome was characterized by more γδ T cells and fewer Th1 cells than restrictive allograft syndrome. Both adaptive and innate immune cells were involved in the temporal evolution of fibrotic remodeling. Although fibrosis seemed to be partially associated with different factors in restrictive allograft syndrome (M2 macrophages, Th1 cells) and in bronchiolitis obliterans syndrome (γδ T cells). Conclusion: Imaging mass cytometry enables in-depth analyses of immune cell phenotypes in their local microenvironment. Using this approach, we identified major differences in cell populations in CLAD versus non-CLAD and in BOS versus RAS, with novel insights into the fibrotic progression of CLAD

Single-cell analysis of oxidative phosphorylation protein expression in pancreatic islets in type 2 diabetes

Mitochondrial dysfunction is a key feature of type 2 diabetes and is closely linked to ageing, a major risk factor for the disease. This study investigated islet cell composition and mitochondrial oxidative phosphorylation protein expression in pancreatic tissue from older donors (≥62 years) with and without type 2 diabetes, matched for age, sex, and BMI. Fixed human pancreatic tissue sections were immunolabelled for insulin, glucagon, NDUFB8 (complex I), MTCO1 (complex IV), and VDAC1 (a mitochondrial mass marker) to quantify islet composition and mitochondrial protein levels. A machine learning-based single-cell segmentation pipeline enabled high-resolution profiling of individual cell populations within islets. In type 2 diabetes, islets exhibited an increased alpha:beta cell ratio, altered spatial organisation with fewer beta-beta and more alpha-alpha interactions, and a significantly higher proportion of bi-hormonal cells co-expressing insulin and glucagon. Within beta cells, we observed significant changes in mitochondrial protein expression, including reduced complex I and elevated complex IV levels. Unsupervised clustering of mitochondrial expression patterns identified three distinct beta cell expression clusters. Donors with type 2 diabetes showed a marked shift in the distribution of beta cells across clusters, with increased proportions of beta cells exhibiting low complex I and high complex IV expression. These results highlight significant alterations in islet architecture and mitochondrial protein expression associated with type 2 diabetes, providing new insights into the mechanisms underlying type 2 diabetes

An ultra-conserved poison exon in the <em>Tra2b </em>gene encoding a splicing activator is essential for male fertility and meiotic cell division

\ua9 The Author(s) 2025.The cellular concentrations of splicing factors (SFs) are critical for controlling alternative splicing. Most serine and arginine-enriched (SR) protein SFs regulate their own concentration via a homeostatic feedback mechanism that involves regulation of inclusion of non-coding ‘poison exons’ (PEs) that target transcripts for nonsense-mediated decay. The importance of SR protein PE splicing during animal development is largely unknown despite PE ultra-conservation across animal genomes. To address this, we used mouse genetics to disrupt an ultra-conserved PE in the Tra2b gene encoding the SR protein Tra2β. Focussing on germ cell development, we found that Tra2b PE deletion causes azoospermia due to catastrophic cell death during meiotic prophase. Failure to proceed through meiosis was associated with increased Tra2b expression sufficient to drive aberrant Tra2β protein hyper-responsive splice patterns. Although critical for meiotic prophase, Tra2b PE deletion spared earlier mitotically active germ cells, even though these still required Tra2b gene function. Our data indicate that PE splicing control prevents the accumulation of toxic levels of Tra2β protein that are incompatible with meiotic prophase. This unexpected connection with male fertility helps explain Tra2b PE ultra-conservation and indicates the importance of evaluating PE function in animal models

Distinct lung cell signatures define the temporal evolution of diffuse alveolar damage in fatal COVID-19

\ua9 2023 The Author(s)Background: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with an airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. Methods: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. Findings: Forty patients (32M:8F, age: 22–98), 345 ROIs and &gt;900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. Interpretation: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established. Funding: UK Research and Innovation/ Medical Research Council through the UK Coronavirus Immunology Consortium, Barbour Foundation, General Sir John Monash Foundation, Newcastle University, JGW Patterson Foundation, Wellcome Trust

An Evolutionarily Conserved Laterally Acquired Toolkit Enables Microbiota Targeting by Trichomonas

richomonas species are a diverse group of microbial eukaryotes (also commonly referred to as protists) that are obligate extracellular symbionts associated with or attributed to various inflammatory diseases. They colonize mucosal surfaces across a wide range of hosts, all of which harbor a resident microbiota. Their evolutionary history likely involved multiple host transfers, including zoonotic events from columbiform birds to mammals. Using comparative transcriptomics, this study examines Trichomonas gallinae co-cultured with Escherichia coli, identifying a molecular toolkit that Trichomonas species may use to interact with bacterial members of the microbiota. Integrating transcriptomic data with comparative genomics and phylogenetics revealed a conserved repertoire of protein-coding genes likely acquired through multiple lateral gene transfers (LGTs) in a columbiform-infecting ancestor. These LGT-derived genes encode muramidases, glucosaminidases, and antimicrobial peptides—enzymes and effectors capable of targeting bacterial cell walls, potentially affecting the bacterial-microbiota composition across both avian and mammalian hosts. This molecular toolkit suggests that Trichomonas species can actively compete with and exploit their surrounding microbiota for nutrients, potentially contributing to dysbiosis associated with Trichomonas infections. Their ability to target bacterial populations at mucosal surfaces provides insight into how Trichomonas species may have adapted to diverse hosts and how they could influence inflammatory mucosal diseases in birds and mammals

3D Reconstruction of the Mitochondrial Network within the Neuronal Soma from SBF-SEM Volume Data

\ua9 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature. Neurons contain three compartments, the soma, long axon, and dendrites, which have distinct energetic and biochemical requirements. Mitochondria feature in all compartments and regulate neuronal activity and survival, including energy generation and calcium buffering alongside other roles including proapoptotic signaling and steroid synthesis. Their dynamicity allows them to undergo constant fusion and fission events in response to the changing energy and biochemical requirements. These events, termed mitochondrial dynamics, impact their morphology and a variety of three-dimensional (3D) morphologies exist within the neuronal mitochondrial network. Distortions in the morphological profile alongside mitochondrial dysfunction may begin in the neuronal soma in ageing and common neurodegenerative disorders. However, 3D morphology cannot be comprehensively examined in flat, two-dimensional (2D) images. This highlights a need to segment mitochondria within volume data to provide a representative snapshot of the processes underpinning mitochondrial dynamics and mitophagy within healthy and diseased neurons. The advent of automated high-resolution volumetric imaging methods such as Serial Block Face Scanning Electron Microscopy (SBF-SEM) as well as the range of image software packages allow this to be performed.We describe and evaluate a method for randomly sampling mitochondria and manually segmenting their whole morphologies within randomly generated regions of interest of the neuronal soma from SBF-SEM image stacks. These 3D reconstructions can then be used to generate quantitative data about mitochondrial and cellular morphologies. We further describe the use of a macro that automatically dissects the soma and localizes 3D mitochondria into the subregions created