The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic b-cell has not been tested. We used an informatics-based approach to develop a transcriptional signature of b-cell GA stress using existing RNA sequencing and microarray data sets generated using human islets from donors with diabetes and islets where type 1 (T1D) and type 2 (T2D) diabetes had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated. In parallel, we generated an RNA-sequencing data set from human islets treated with brefeldin A (BFA), a known GA stress inducer. Overlapping the T1D and T2D groups with the BFA data set, we identified 120 and 204 differentially expressed genes, respectively. In both the T1D and T2D models, pathway analyses revealed that the top pathways were associated with GA integrity, organization, and trafficking. Quantitative RT-PCR was used to validate a common signature of GA stress that included ATF3, ARF4, CREB3, and COG6. Taken together, these data indicate that GA-associated genes are dysregulated in diabetes and identify putative markers of b-cell GA stress.

ObjectiveLoss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined.DesignROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFα stimulation. Clustering of epithelial cells from human UC (inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (ulex europaeus agglutin-1 lectin) and in vivo with DSS injury.ResultsTNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT enhancer-binding protein delta), DNMT1 (DNA methyltransferase) and HIF1A (hypoxia-inducible factor) baseline. Subclustering unexpectedly showed marked TNFα-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H2O2 and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstrated GP2 (glycoprotein-2), basal lymphoplasmacytosis and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation.ConclusionsNOX1 deficiency plus TNFα stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritise ROS modulation for future therapies.

MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in modulating gene expression and are enriched in cell-derived extracellular vesicles (EVs). We investigated whether miRNAs from human islets and islet-derived EVs could provide insight into β cell stress pathways activated during type 1 diabetes (T1D) evolution, therefore serving as potential disease biomarkers. We treated human islets from 10 cadaveric donors with IL-1β and IFN-γ to model T1D ex vivo. MicroRNAs were isolated from islets and islet-derived EVs, and small RNA sequencing was performed. We found 20 and 14 differentially expressed (DE) miRNAs in cytokine- versus control-treated islets and EVs, respectively. Interestingly, the miRNAs found in EVs were mostly different from those found in islets. Only two miRNAs, miR-155-5p and miR-146a-5p, were upregulated in both islets and EVs, suggesting selective sorting of miRNAs into EVs. We used machine learning algorithms to rank DE EV-associated miRNAs, and developed custom label-free Localized Surface Plasmon Resonance-based biosensors to measure top ranked EVs in human plasma. Results from this analysis revealed that miR-155, miR-146, miR-30c, and miR-802 were upregulated and miR-124-3p was downregulated in plasma-derived EVs from children with recent-onset T1D. In addition, miR-146 and miR-30c were upregulated in plasma-derived EVs of autoantibody positive (AAb+) children compared to matched non-diabetic controls, while miR-124 was downregulated in both T1D and AAb+ groups. Furthermore, single-molecule fluorescence in situ hybridization confirmed increased expression of the most highly upregulated islet miRNA, miR-155, in pancreatic sections from organ donors with AAb+ and T1D.

INTRODUCTION For the treatment of refractory Crohn’s disease (CD) autologous stem cell transplant (auto-SCT) is unparalleled in its ability to induce clinical and endoscopic remission.(1, 2) Auto-SCT is unique as a cellular therapy aimed to reset immune pathophysiology to a pre-disease state using hematopoietic stem cells. As such, the study of how the immune system responds to auto-SCT will provide unique insight into CD pathogenesis and treatment. To date, no studies in any cohort have defined the mucosal and peripheral immune response to auto-SCT. Here we report initial studies of high dimensional immune phenotyping of patients with CD during auto-SCT. METHODS Patients with CD were enrolled in a Phase IIa study of auto-SCT (NCT03219359). 14 patients were transplanted (2018-2022). Paired blood and intestinal samples were taken prior to transplant and 6 months post-transplant. Fresh leukocytes were isolated and analyzed by mass cytometry (CyTOF). Supervised clustering of immune cell populations using canonical markers was performed in parallel with unsupervised clustering by FlowSOM.(3) RESULTS After 6 months post-transplant,12/13 patients had an endoscopic response (↓SES-CD by 50%) and 10/13 patients were in endoscopic remission (SES-CD<4). Supervised clustering of major immune cell subsets demonstrated distinct site-specific responses to transplant in myeloid and lymphoid cell populations (Fig 1). Naïve CD4+ and CD8+ T cells universally decrease in number at 6 months post-transplant whereas all other B and T cell populations have discordant changes in the blood and intestine, especially naïve and transitional CD27- B cell populations which significantly increase in blood and decrease in the intestine. CD14+ populations are universally increased in number post-transplant with a specific increase in CD14+ CD206+ macrophages in the intestine. Unsupervised clustering of blood and intestinal immune cells resolve 100 cell clusters that correlate with canonical immune cell markers (Fig 2). Unsupervised analysis further highlights a significant increase in multiple intestinal CD14+CD206+ immune cell populations reflecting newly arrived and/or differentiating and mature monocyte derived macrophages. Principal component and hierarchical clustering analyses of immune cell clusters suggest a reprograming of the intestinal immune compartment post-transplant whereas changes in circulating immune cells populations fail to separate the pre and post-transplant states. CONCLUSION We demonstrate for the first time differences in the intestinal and peripheral immune response to auto-SCT. These studies highlight the changes in intestinal immune cell networks that define the transplant response perhaps through CD14+ cells.

Previous and current research studies have been reporting or refuting susceptibility genes which have any minor or major genetic significance to Alzheimer disease. In our study we have obtained datasets from R. N. A. sequencing analysis performed on post-mortem brain tissues of clinically diagnosed Alzheimer’s male patients above the age of 80. The samples included in our study are grouped into three datasets as frontal brain, temporal brain and whole brain. Our sole purpose of the study was to decipher new S. N. PS if any and report any unreported or untested S. N. Ps related to genes. We have performed mRNA sequencing analysis with the help of Next Generation Sequencing GALAXY tool available online. In addition, we have utilized Integrated Genome Browser (IGB) to visualize our results. P. E. RL Programming algorithm was generated and NCBI’s dbSNP (Database of Single Nucleotide Polymorphisms) and Clin V.A. R (Clinical Variation) databases helped to obtain rsids and related information for galaxy analyses results. Our study is reporting and/or confirming pathogenicity of P. R. N. P, A. P. O. E, CST3, TUBB2B and F. T. L in the AD brain tissues. A growing study of PRNP gene has indicated a strong genetic association with AD yet more research with a bigger sample size in different populations will be of certain significance. Further research is needed for studying the TUBB2B gene due to its uncertain significance in the earlier research in Alzheimer’s. F. T. L is a recent gene which has been linked to A. D and future research in AD should be conducted targeting this specific gene.