Pancreatic Gene Research Articles

Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications

BackgroundDifferentiation of patient-specific induced pluripotent stem cells (iPS) helps researchers to study the individual sensibility to drugs. However, differentiation protocols are time-consuming, and not all tissues have been studied. Few works are available regarding pancreatic exocrine differentiation of iPS cells, and little is known on culturing and cryopreserving these cells.MethodsWe differentiated the iPS cells of two pediatric Crohn’s disease patients into pancreatic progenitors and exocrine cells, adapting and shortening a protocol for differentiating embryonic stem cells. We analyzed the expression of key genes and proteins of the differentiation process by qPCR and immunofluorescence, respectively. We explored the possibility of keeping differentiated cells in culture and freezing and thawing them to shorten the time needed for the differentiation. We analyzed the cell cycle of undifferentiated and differentiated cells by flow cytometry.ResultsThe analysis of mRNA levels of key pancreatic differentiation genes PDX1 and pancreatic amylase indicate that iPS cells were successfully differentiated into pancreatic exocrine cells with expression of PDX1 (one way ANOVA p < 0.0001), and the two isoforms of amylase (one way ANOVA p < 0.05) significantly higher in exocrine cells in comparison to iPS cells. Differentiation efficiency was also confirmed by immunofluorescence analysis of PDX1 and amylase. We confirmed the possibility of shortening the time necessary for obtaining pancreatic cells without losing differentiation efficiency. Pancreatic progenitors and exocrine cells were maintained in culture and cryopreserved. Interestingly, the stemness marker OCT4 resulted significantly lower after subculturing (OCT4 p < 0.001; one-way ANOVA) and after freezing and thawing procedures (p < 0.05, one-way ANOVA) suggesting a reduction of undifferentiated stem cells leading to a purer population of pancreatic progenitor cells. Also, the stemness marker NANOG resulted lower after passaging, corroborating this result.ConclusionsIn this work, we optimized the generation of patient-specific pancreatic differentiated cells and laid the foundation for creating a bank of patient-specific pancreatic lines exploitable for tailored pharmacological assays.Trial registrationThe study was approved by the Ethical Committee of the Institute of Maternal and Child Health IRCCS Burlo Garofolo, with approval number 1556 (internal ID RC 44/22).

Aberrant expression of collagen type X in solid tumor stroma is associated with EMT, immunosuppressive and pro-metastatic pathways, bone marrow stromal cell signatures, and poor survival prognosis.

Collagen type X (ColXα1, encoded by COL10A1 ) is expressed specifically in the cartilage-to-bone transition, in bone marrow cells, and in osteoarthritic (OA) cartilage. We have previously shown that ColXα1 is expressed in breast tumor stroma, correlates with tumor-infiltrating lymphocytes, and predicts poor adjuvant therapy outcomes in ER + /HER2 + breast cancer. However, the underlying molecular mechanisms for these effects are unknown. In this study, we performed bioinformatic analysis of COL10A1 -associated gene modules in breast and pancreatic cancer as well as in cells from bone marrow and OA cartilage. These findings provide important insights into the mechanisms of transcriptional and extracellular matrix changes which impact the local stromal microenvironment and tumor progression. Immunohistochemistry was performed to examine collagen type X expression in solid tumors. WGCNA was used to generate COL10A1 -associated gene networks in breast and pancreatic tumor cohorts using RNA-Seq data from The Cancer Genome Atlas. Computational analysis was employed to assess the impact of these gene networks on development and progression of cancer and OA. Data processing and statistical analysis was performed using R and various publicly-available computational tools. Expression of COL10A1 and its associated gene networks highlights inflammatory and immunosuppressive microenvironments, which identify aggressive breast and pancreatic tumors and contribute to metastatic potential in a sex-dependent manner. Both cancer types are enriched in stroma, and COL10A1 implicates bone marrow-derived fibroblasts as drivers of the epithelial-to-mesenchymal transition (EMT) in these tumors. Heightened expression of COL10A1 and its associated gene networks is correlated with poorer patient outcomes in both breast and pancreatic cancer. Common transcriptional changes and chondrogenic activity are shared between cancer and OA cartilage, suggesting that similar microenvironmental alterations may underlie both diseases. COL10A1 -associated gene networks may hold substantial value as regulators and biomarkers of aggressive tumor phenotypes with implications for therapy development and clinical outcomes. Identification of tumors which exhibit high expression of COL10A1 and its associated genes may reveal the presence of bone marrow-derived stromal microenvironments with heightened EMT capacity and metastatic potential. Our analysis may enable more effective risk assessment and more precise treatment of patients with breast and pancreatic cancer. ColX highlights features of EMT in breast and pancreatic cancerColX gene modules are immunosuppressive and pro-metastaticColX-associated gene networks contribute to sex differences in pancreatic cancerColX-positive fibroblasts define more aggressive tumors with poorer survivalColX is emerging as a biomarker for bone marrow-derived cells in cancer.

Dexamethasone's Clinical Efficacy in Experimental Autoimmune Pancreatitis Correlates with a Unique Transcriptomic Signature, Whilst Kinase Inhibitors Are Not Effective.

(1) Background: Autoimmune pancreatitis (AIP) is mainly treated with steroids. Using an AIP mouse model, we investigated two potential alternatives, the transforming growth factor-β-activated kinase 1 inhibitor, takinib, and the Janus kinase inhibitor, tofacitinib. (2) Methods: In a multicenter preclinical study, MRL/MpJ mice were injected with polyinosinic/polycytidylic acid (poly I:C) for two weeks to induce AIP. They were then treated for four weeks with either takinib (25, 50, or 75 mg/kg body weight), tofacitinib (5, 10 or 15 mg/kg), dexamethasone (1 mg/kg), or solvent, while the poly I:C injections were continued. The severity of AIP was assessed histopathologically. Flow cytometry was used to examine lymphocyte subtypes in the spleen and mesenteric lymph nodes. The pancreatic gene expression profiles were analyzed by RNA sequencing. (3) Results: Poly I:C-treated mice developed severe AIP with inflammation, destruction of acinar tissue, and fibrosis. Dexamethasone significantly attenuated the disease, while takinib or tofacitinib had no effects. Dexamethasone also antagonized the effects of poly I:C on the relative frequencies of the AIP-associated lymphocyte subtypes CD4/CD69, CD8/CD44high, and CD4/CD25/FoxP3 in the spleen. In the principal component analysis of pancreatic transcriptomics, poly I:C-injected mice treated with tofacitinib, takinib, or solvent clustered together, while untreated and dexamethasone-treated mice formed separate, unique clusters. (4) Conclusions: Dexamethasone effectively reduced AIP severity, while takinib and tofacitinib were ineffective. The unique gene expression profile in dexamethasone-treated mice may provide a basis for identifying new drug targets for AIP treatment.

Single-Cell Meta-Analysis Uncovers the Pancreatic Endothelial Cell Transcriptomic Signature and Reveals a Key Role for NKX2-3 in PLVAP Expression.

The pancreatic vasculature displays tissue-specific physiological and functional adaptations that support rapid insulin response by β-cells. However, the digestive enzymes have made it difficult to characterize pancreatic endothelial cells (ECs), resulting in the poor understanding of pancreatic EC specialization. Available single-nuclei/single-cell RNA-sequencing data sets were mined to identify pancreatic EC-enriched signature genes and to develop an integrated atlas of human pancreatic ECs. We validated the findings using independent single-nuclei/single-cell RNA-sequencing data, bulk RNA-sequencing data of isolated ECs, spatial transcriptomics data, immunofluorescence, and RNAScope of selected markers. The TF (transcription factor) NKX2-3 was expressed in HUVECs via gene transfection, and the expression of pancreatic EC-enriched signature genes was assessed via RT-qPCR. We defined a pancreatic EC-enriched gene signature conserved across species and developmental stages that included genes involved in ECM (extracellular matrix) composition (COL15A1 and COL4A1), permeability and barrier function (PLVAP, EHD4, CAVIN3, HSPG2, ROBO4, HEG1, and CLEC14A), and key signaling pathways (S1P, TGF-β [transforming growth factor-β], RHO-RAC GTPase, PI3k-AKT, and PDGF [platelet-derived growth factor]). The integrated atlas revealed the vascular hierarchy within the pancreas. We identified and validated a specialized islet capillary subpopulation characterized by genes involved in permeability (PLVAP and EHD4), immune-modulation (FABP5, HLA-C, and B2M), ECM composition (SPARC and SPARCL1), IGF (insulin-like growth factor) signaling (IGFBP7), and membrane transport (SLCO2A1, SLC2A3, and CD320). Importantly, we identified NKX2-3 as a key TF enriched in pancreatic ECs. DNA-binding motif analysis found NKX2-3 motifs in ≈40% of the signature genes. Induction of NKX2-3 in HUVECs promoted the expression of the islet capillary EC-enriched genes PLVAP and SPARCL1. We defined a validated transcriptomic signature of pancreatic ECs and uncovered their intratissue transcriptomic heterogeneity. We showed that NKX2-3 acts upstream of PLVAP and provided a single-cell online resource that can be further explored by the community: https://vasconcelos.shinyapps.io/pancreatic_endothelial/.

Enhancing differentiation and functionality of insulin-producing cells derived from iPSCs using esterified collagen hydrogel for cell therapy in diabetes mellitus

BackgroundIslet transplantation is a recommended treatment for type 1 diabetes but is limited by donor organ shortage. This study introduces an innovative approach for improving the differentiation and functionality of insulin-producing cells (IPCs) from iPSCs using 3D spheroid formation and hydrogel matrix as an alternative pancreatic islet source. The extracellular matrix (ECM) is crucial for pancreatic islet functionality, but finding the ideal matrix for β-cell differentiation has been challenging. We aimed to advance IPC differentiation and maturation through an esterified collagen hydrogel, comparing its effectiveness with conventional basement membrane extract (BME) hydrogels.MethodsiPSCs were differentiated into IPCs using a small molecule-based sequential protocol, followed by spheroid formation in concave microwells. Rheological analysis, scanning electron microscopy, and proteomic profiling were used to characterize the chemical and physical properties of each matrix. IPCs, both in single-cell form and as spheroids, were embedded in either ionized collagen or BME hydrogels, which was followed by assessments of morphological changes, pancreatic islet-related gene expression, insulin secretion, and pathway activation using comprehensive analytical techniques.ResultsEsterified collagen hydrogels markedly improved the structural integrity, insulin expression, and cell-cell interactions in IPC spheroids, forming densely packed insulin-expressing clusters, in contrast to the dispersed cells observed in BME cultures. Collagen hydrogel significantly enhanced the mRNA expression of crucial endocrine markers and maturation factors, with IPC spheroids showing accelerated differentiation from day 5, suggesting a faster differentiation compared to single cells in hydrogel encapsulation. Insulin secretion in response to glucose in collagen environments, with a GSIS index of 2.46 ± 0.05, exceeded those in 2D and BME, demonstrating superior pancreatic islet functionality. Pathway analysis highlighted enhanced insulin secretion capabilities, evidenced by the upregulation of genes like Secretogranin III and Chromogranin A in collagen cultures. In vivo transplantation results showed that collagen hydrogel enhanced cluster integrity, tissue integration, and insulin secretion compared to non-embedded IPCs and BME groups.ConclusionEsterified collagen hydrogels demonstrated superior efficacy over 2D and BME in promoting IPC differentiation and maturation, possibly through upregulation of the expression of key secretion pathway genes. Our findings suggest that using collagen hydrogels presents a promising approach to enhance insulin secretion efficiency in differentiating pancreatic β-cells, advancing cell therapy in diabetes cell therapy.

Acquisition of durable insulin-producing cells from human adipose tissue-derived mesenchymal stem cells as a foundation for cell- based therapy of diabetes mellitus

This study aimed to identify the suitable induction protocol to produce highly qualified insulin producing cells (IPCs) from human adipose tissue derived stem cells (ADSCs) and evaluate the efficacy of the most functionally IPCs in management of diabetes mellitus (DM) in rats. The ADSCs were isolated and characterized according to the standard guidelines. ADSCs were further induced to be IPCs in vitro using three different protocols. The success of trans-differentiation was assessed in vitro through analysis of pancreatic endocrine genes expression, and insulin release in response to glucose stimulation. Then, the functionalization of the generated IPCs was evaluated in vivo. The in vitro findings revealed that the laminin-coated plates in combination with insulin-transferrin-selenium, B27, N2, and nicotinamide could efficiently up-regulate the expression of pancreatic endocrine genes. The in vivo study indicated effectual homing of the PKH-26-labelled IPCs in the pancreas of treated animals. Moreover, IPCs infusion in diabetic rats induced significant improvement in the metabolic parameters and prompted considerable up-regulation in the expression of the pancreatic related genes. The regenerative effect of infused IPCs was determined through histological examination of pancreatic tissue. Conclusively, the utilization of laminin–coated plates in concomitant with extrinsic factors promoting proliferation and differentiation of ADSCs could efficiently generate functional IPCs.

Genomic analysis defines distinct pancreatic and neuronal subtypes of lung carcinoid.

Lung carcinoids (L-CDs) are rare, poorly characterised neuroendocrine tumours (NETs). L-CDs are more common in women and are not the consequence of cigarette smoking. They are classified histologically as typical carcinoids (TCs) or atypical carcinoids (ACs). ACs confer a worse survival. Histological classification is imperfect, and there is increasing interest in molecular markers. We therefore investigated global transcriptomic and epigenomic profiles of 15 L-CDs resected with curative intent at Royal Brompton Hospital. We identified underlying mutations and structural abnormalities through whole-exome sequencing (WES) and single nucleotide polymorphism (SNP) genotyping. Transcriptomic clustering algorithms identified two distinct L-CD subtypes. These showed similarities either to pancreatic or neuroendocrine tumours at other sites and so were named respectively L-CD-PanC and L-CD-NeU. L-CD-PanC tumours featured upregulation of pancreatic and metabolic pathway genes matched by promoter hypomethylation of genes for beta cells and insulin secretion (p < 1 × 10-6). These tumours were centrally located and showed mutational signatures of activation-induced deaminase/apolipoprotein B editing complex activity, together with genome-wide DNA methylation loss enriched in repetitive elements (p = 2.2 × 10-16). By contrast, the L-CD-NeU group exhibited upregulation of neuronal markers (adjusted p < 0.01) and was characterised by focal spindle cell morphology (p = 0.04), peripheral location (p = 0.01), high mutational load (p = 2.17 × 10-4), recurrent copy number alterations, and enrichment for ACs. Mutations affected chromatin remodelling and SWI/SNF complex pathways. L-CD-NeU tumours carried a mutational signature attributable to aflatoxin and aristolochic acid (p = 0.05), suggesting a possible environmental exposure in their pathogenesis. Immunologically, myeloid and T-cell markers were enriched in L-CD-PanC and B-cell markers in L-CD-NeU tumours. The substantial epigenetic and non-coding differences between L-CD-PanC and L-CD-NeU open new possibilities for biomarker selection and targeted treatment of L-CD. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Generation of Insulin-Producing Cells from Canine Bone Marrow-Derived Mesenchymal Stem Cells: A Preliminary Study.

Cell-based therapy using insulin-producing cells (IPCs) is anticipated as an alternative treatment option to insulin injection or pancreatic islet transplantation for the treatment of diabetes mellitus in both human and veterinary medicine. Several protocols were reported for the differentiation of mesenchymal stem cells (MSCs) into IPCs; to date, glucose-responsive IPCs have only been obtained from canine adipose tissue-derived MSCs (cAD-MSCs), but not from canine bone marrow-derived MSCs (cBM-MSCs). Therefore, this study aims to generate in vitro glucose-responsive IPCs from cBM-MSCs using two differentiation protocols: a two-step protocol using trichostatin (TSA) and a three-step protocol using mercaptoethanol to induce pancreatic and duodenal homeobox gene 1 (PDX-1) expression. A single experiment was carried out for each protocol. BM-MSCs from one dog were successfully cultured and expanded. Cells exposed to the two-step protocol appeared rarely grouped to form small clusters; gene expression analysis showed a slight increase in PDX-1 and insulin expression, but no insulin protein production nor secretion in the culture medium was detected either under basal conditions or following glucose stimulation. Conversely, cells exposed to the three-step protocol under a 3D culture system formed colony-like structures; insulin gene expression was upregulated compared to undifferentiated control and IPCs colonies secreted insulin in the culture medium, although insulin secretion was not enhanced by high-glucose culture conditions. The single experiment results suggest that the three-step differentiation protocol could generate IPCs from cBM-MSCs; however, further experiments are needed to confirm these data. The ability of IPCs from cBM- MSCs to produce insulin, described here for the first time, is a preliminary interesting result. Nevertheless, the IPCs' unresponsiveness to glucose, if confirmed, would affect its clinical application. Further studies are necessary to establish a differentiation protocol in this perspective.

The guidelines for clinical practice for carriers of germline mutations in hereditary breast, ovarian, prostate, and pancreatic cancer predisposition genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 (4.2024).

The Guidelines for Clinical Practice for carriers of pathogenic variants in clinically relevant cancer predisposition genes define the steps of primary and secondary prevention that should be provided to these individuals at high risk of developing hereditary cancer in the Czech Republic. The drafting of the guidelines was organized by the Oncogenetics Working Group of the Society for Medical Genetics and Genomics of J. E. Purkyně Czech Medical Society (SLG ČLS JEP) in cooperation with the representatives of oncology and oncogynecology. The guidelines are based on the current recommendations of the National Comprehensive Cancer Network (NCCN), European Society of Medical Oncology (ESMO) and take into account the capacity of the Czech healthcare system.

Monocarbonyl analogs of curcumin C66 and B2BrBC modulate oxidative stress, JNK activity, and pancreatic gene expression in rats with streptozotocin-induced diabetes

The pathogenesis of type 1 diabetes mellitus (T1DM) involves oxidative stress and inflammation. Curcumin, a natural polyphenolic compound found in turmeric, known to exhibit antioxidative and anti-inflammatory properties, is characterized by poor chemical stability, low bioavailability, and rapid metabolism. Monocarbonyl analogs of curcumin (MACs) with a structural absence of β-diketone and enhanced stability and bioavailability present a potential solution to the challenges associated with the use of curcumin. This study aimed to evaluate the effect of two MACs, C66 and B2BrBC, on oxidative stress markers, antioxidant enzyme activity, expression of diabetes-associated genes, and signaling pathway proteins in the context of T1DM. Streptozotocin (STZ)-induced male Wistar rats or rat pancreatic RIN-m cells were used for in vivo and in vitro experiments, respectively. C66 or B2BrBC were given either before or after STZ treatment. Oxidative stress markers and antioxidant enzyme activities were determined in various tissues. Expression of diabetes-associated genes was assessed using RT-qPCR, and the activity of signaling pathway proteins in the pancreas was determined through Western blot analysis. Treatment with C66 and B2BrBC significantly reduced oxidative stress markers and positively influenced antioxidant enzyme activities. Moreover, both compounds inhibited JNK activity in the pancreas while enhancing the expression of genes crucial for β-cell survival and glucose and redox homeostasis. The findings highlight the multifaceted potential of C66 and B2BrBC in ameliorating oxidative stress, influencing gene expression patterns linked to diabetes, and modulating key signaling pathways in the pancreas. The findings suggest that these compounds can potentially address diabetes-related pathological processes.

Single cell multiome profiling of pancreatic islets reveals physiological changes in cell type-specific regulation associated with diabetes risk.

Physiological variability in pancreatic cell type gene regulation and the impact on diabetes risk is poorly understood. In this study we mapped gene regulation in pancreatic cell types using single cell multiomic (joint RNA-seq and ATAC-seq) profiling in 28 non-diabetic donors in combination with single cell data from 35 non-diabetic donors in the Human Pancreas Analysis Program. We identified widespread associations with age, sex, BMI, and HbA1c, where gene regulatory responses were highly cell type- and phenotype-specific. In beta cells, donor age associated with hypoxia, apoptosis, unfolded protein response, and external signal-dependent transcriptional regulators, while HbA1c associated with inflammatory responses and gender with chromatin organization. We identified 10.8K loci where genetic variants were QTLs for cis regulatory element (cRE) accessibility, including 20% with lineage- or cell type-specific effects which disrupted distinct transcription factor motifs. Type 2 diabetes and glycemic trait associated variants were enriched in both phenotype- and QTL-associated beta cell cREs, whereas type 1 diabetes showed limited enrichment. Variants at 226 diabetes and glycemic trait loci were QTLs in beta and other cell types, including 40 that were statistically colocalized, and annotating target genes of colocalized QTLs revealed genes with putatively novel roles in disease. Our findings reveal diverse responses of pancreatic cell types to phenotype and genotype in physiology, and identify pathways, networks, and genes through which physiology impacts diabetes risk.

Improving Access to Hereditary Testing in Pancreatic Ductal Carcinoma.

Approximately 5%-10% of patients with pancreatic ductal adenocarcinoma (PDAC) have an inherited basis, yet uptake of genetic testing remains low and subject to disparities. This study compared two genetic testing pathways available to patients referred to a provincial cancer center, BC Cancer: a traditional hereditary cancer clinic-initiated testing (HCT) pathway and a new oncology clinic-initiated testing (OCT) pathway. Study subjects were patients with confirmed PDAC referred for genetic testing through the HCT or OCT pathway between June 1, 2020, and February 1, 2022. Charts were retrospectively reviewed for patient characteristics and testing outcomes. The study population was 397 patients (HCT, n = 279 and OCT, n = 118). OCT patients were more likely to have non-European ethnicity compared with HCT patients (41.9% v 25.6%, P = .004), to have earlier-stage disease (P = .012), and to have better Eastern Cooperative Oncology Group performance status than the HCT group (P = .001). A total of 306 patients completed testing (77%). OCT patients had higher test completion rates than HCT patients (odds ratio, 3.74 [95% CI, 1.66 to 9.62]). Median time for results was shorter in OCT than in HCT (53 days [IQR, 44-76] v 107 days [IQR, 63.8-158.3]). Pancreatic cancer susceptibility pathogenic gene variants were identified in 8.5% (26/306). The real-world observations in our study show that oncology clinic-initiated hereditary testing is more effective and faster than testing through hereditary cancer clinic referrals and reaches a more ethnically diverse population. This has important implications for publicly funded environments with limited resources for genetic counseling.

Modulation of naïve mesenchymal stromal cells by extracellular vesicles derived from insulin-producing cells: an in vitro study

This study was to determine whether extracellular vesicles (EVs) derived from insulin-producing cells (IPCs) can modulate naïve mesenchymal stromal cells (MSCs) to become insulin-secreting. MSCs were isolated from human adipose tissue. The cells were then differentiated to generate IPCs by achemical-based induction protocol. EVs were retrieved from the conditioned media of undifferentiated (naïve) MSCs (uneducated EVs) and from that of MSC-derived IPCs (educated EVs) by sequential ultracentrifugation. The obtained EVs were co-cultured with naïve MSCs.The cocultured cells were evaluated by immunofluorescence, flow cytometry, C-peptide nanogold silver-enhanced immunostaining, relative gene expression and their response to a glucose challenge.Immunostaining for naïve MSCs cocultured with educated EVs was positive for insulin, C-peptide, and GAD65. By flow cytometry, the median percentages of insulin-andC-peptide-positive cells were 16.1% and 14.2% respectively. C-peptide nanogoldimmunostaining providedevidence for the intrinsic synthesis of C-peptide. These cells released increasing amounts of insulin and C-peptide in response to increasing glucose concentrations. Gene expression of relevant pancreatic endocrine genes, except for insulin, was modest. In contrast, the results of naïve MSCs co-cultured with uneducated exosomes were negative for insulin, C-peptide, and GAD65. These findings suggest that this approach may overcome the limitations of cell therapy.

Single-cell transcriptomic profiling of human pancreatic islets reveals genes responsive to glucose exposure over 24 h

Aims/hypothesisDisruption of pancreatic islet function and glucose homeostasis can lead to the development of sustained hyperglycaemia, beta cell glucotoxicity and subsequently type 2 diabetes. In this study, we explored the effects of in vitro hyperglycaemic conditions on human pancreatic islet gene expression across 24 h in six pancreatic cell types: alpha; beta; gamma; delta; ductal; and acinar. We hypothesised that genes associated with hyperglycaemic conditions may be relevant to the onset and progression of diabetes.MethodsWe exposed human pancreatic islets from two donors to low (2.8 mmol/l) and high (15.0 mmol/l) glucose concentrations over 24 h in vitro. To assess the transcriptome, we performed single-cell RNA-seq (scRNA-seq) at seven time points. We modelled time as both a discrete and continuous variable to determine momentary and longitudinal changes in transcription associated with islet time in culture or glucose exposure. Additionally, we integrated genomic features and genetic summary statistics to nominate candidate effector genes. For three of these genes, we functionally characterised the effect on insulin production and secretion using CRISPR interference to knock down gene expression in EndoC-βH1 cells, followed by a glucose-stimulated insulin secretion assay.ResultsIn the discrete time models, we identified 1344 genes associated with time and 668 genes associated with glucose exposure across all cell types and time points. In the continuous time models, we identified 1311 genes associated with time, 345 genes associated with glucose exposure and 418 genes associated with interaction effects between time and glucose across all cell types. By integrating these expression profiles with summary statistics from genetic association studies, we identified 2449 candidate effector genes for type 2 diabetes, HbA1c, random blood glucose and fasting blood glucose. Of these candidate effector genes, we showed that three (ERO1B, HNRNPA2B1 and RHOBTB3) exhibited an effect on glucose-stimulated insulin production and secretion in EndoC-βH1 cells.Conclusions/interpretationThe findings of our study provide an in-depth characterisation of the 24 h transcriptomic response of human pancreatic islets to glucose exposure at a single-cell resolution. By integrating differentially expressed genes with genetic signals for type 2 diabetes and glucose-related traits, we provide insights into the molecular mechanisms underlying glucose homeostasis. Finally, we provide functional evidence to support the role of three candidate effector genes in insulin secretion and production.Data availabilityThe scRNA-seq data from the 24 h glucose exposure experiment performed in this study are available in the database of Genotypes and Phenotypes (dbGap; https://www.ncbi.nlm.nih.gov/gap/) with accession no. phs001188.v3.p1. Study metadata and summary statistics for the differential expression, gene set enrichment and candidate effector gene prediction analyses are available in the Zenodo data repository (https://zenodo.org/) under accession number 11123248. The code used in this study is publicly available at https://github.com/CollinsLabBioComp/publication-islet_glucose_timecourse.Graphical

A prognostic model for anoikis-related genes in pancreatic cancer

Anoikis, a distinct form of programmed cell death, is crucial for both organismal development and maintaining tissue equilibrium. Its role extends to the proliferation and progression of cancer cells. This study aimed to establish an anoikis-related prognostic model to predict the prognosis of pancreatic cancer (PC) patients. Gene expression data and patient clinical profiles were sourced from The Cancer Genome Atlas (TCGA-PAAD: Pancreatic Adenocarcinoma) and the International Cancer Genome Consortium (ICGC-PACA: Pancreatic Ductal Adenocarcinoma). Non-cancerous pancreatic tissue gene expression data were obtained from the Genotype-Tissue Expression (GTEx) project. The R package was used to construct anoikis-related PC prognostic models, which were later validated with the ICGC-PACA database. Survival analyses demonstrated a poorer prognosis for patients in the high-risk group, consistent across both TCGA-PAAD and ICGC-PACA datasets. A nomogram was designed as a predictive tool to estimate patient mortality. The study also analyzed tumor mutations and immune infiltration across various risk groups, uncovering notable differences in tumor mutation patterns and immune landscapes between high- and low-risk groups. In conclusion, this research successfully developed a prognostic model centered on anoikis-related genes, offering a novel tool for predicting the clinical trajectory of PC patients.

In vivo CRISPR screens reveal SCAF1 and USP15 as drivers of pancreatic cancer

Functionally characterizing the genetic alterations that drive pancreatic cancer is a prerequisite for precision medicine. Here, we perform somatic CRISPR/Cas9 mutagenesis screens to assess the transforming potential of 125 recurrently mutated pancreatic cancer genes, which revealed USP15 and SCAF1 as pancreatic tumor suppressors. Mechanistically, we find that USP15 functions in a haploinsufficient manner and that loss of USP15 or SCAF1 leads to reduced inflammatory TNFα, TGF-β and IL6 responses and increased sensitivity to PARP inhibition and Gemcitabine. Furthermore, we find that loss of SCAF1 leads to the formation of a truncated, inactive USP15 isoform at the expense of full-length USP15, functionally coupling SCAF1 and USP15. Notably, USP15 and SCAF1 alterations are observed in 31% of pancreatic cancer patients. Our results highlight the utility of in vivo CRISPR screens to integrate human cancer genomics and mouse modeling for the discovery of cancer driver genes with potential prognostic and therapeutic implications.

Global diversity, recurrent evolution, and recent selection on amylase structural haplotypes in humans.

The adoption of agriculture, first documented ~12,000 years ago in the Fertile Crescent, triggered a rapid shift toward starch-rich diets in human populations. Amylase genes facilitate starch digestion and increased salivary amylase copy number has been observed in some modern human populations with high starch intake, though evidence of recent selection is lacking. Here, using 52 long-read diploid assemblies and short read data from ~5,600 contemporary and ancient humans, we resolve the diversity, evolutionary history, and selective impact of structural variation at the amylase locus. We find that amylase genes have higher copy numbers in populations with agricultural subsistence compared to fishing, hunting, and pastoral groups. We identify 28 distinct amylase structural architectures and demonstrate that nearly identical structures have arisen recurrently on different haplotype backgrounds throughout recent human history. AMY1 and AMY2A genes each exhibit multiple duplications/deletions with mutation rates >10,000-fold the SNP mutation rate, whereas AMY2B gene duplications share a single origin. Using a pangenome graph-based approach to infer structural haplotypes across thousands of humans, we identify extensively duplicated haplotypes present at higher frequencies in modern day populations with traditionally agricultural diets. Leveraging 533 ancient human genomes we find that duplication-containing haplotypes (i.e. haplotypes with more amylase gene copies than the ancestral haplotype) have increased in frequency more than seven-fold over the last 12,000 years providing evidence for recent selection in West Eurasians. Together, our study highlights the potential impacts of the agricultural revolution on human genomes and the importance of long-read sequencing in identifying signatures of selection at structurally complex loci.

Single-dose GLP-1-based Pancreatic Gene Therapy Maintains Weight Loss After Semaglutide Withdrawal in a Murine Model of Obesity

Single-dose GLP-1-based Pancreatic Gene Therapy Maintains Weight Loss After Semaglutide Withdrawal in a Murine Model of Obesity

Rosa canina extract relieves methylation alterations of pancreatic genes in STZ-induced diabetic rats : Gene methylation in diabetic rats treated with an extract.

Diabetes is a chronic metabolic disease that affects many parts of the body. Considering diabetes as a beta cells' defect and loss, the focus is on finding mechanisms and compounds involved in stimulating the function and regeneration of pancreatic β-cells. DNA methylation as an epigenetic mechanism plays a pivotal role in the β-cells' function and development. Considering the regenerative and anti-diabetic effects of Rosa canina extract, this study aimed to assess the methylation levels of Pdx-1, Pax-4, and Ins-1 genes in diabetic rats treated with Rosa Canina extract. Streptozotocin-induced diabetic rats were used to evaluate the frequency of Pdx-1, Pax-4, and Ins-1 gene methylation. Treatment groups were exposed to Rosa canina as spray-dried and decoction extracts. Following blood glucose measurement, pancreatic DNA was extracted and bisulfited. Genes' methylation was measured using MSP-PCR and qRT-PCR techniques. Oral administration of Rosa canina extracts significantly reduced blood sugar levels in diabetic rats compared to the control group. The methylation levels of the Pdx-1, Pax-4, and Ins-1 genes promoter in streptozotocin-induced diabetic rats increased compared to the control rats while, the treatment of diabetic rats with Rosa canina extracts, spray-dried samples especially, led to a decreased methylation in these genes. The results of this study showed that Rosa canina extract as a spray-dried sample could be effective in treating diabetes by regulating the methylation of genes including Pdx-1, Pax-4, and Ins-1 involved in the activity and regeneration of pancreatic islet cells.

RadioGWAS links radiome to genome to discover driver genes with somatic mutations for heterogeneous tumor image phenotype in pancreatic cancer

Addressing the significant level of variability exhibited by pancreatic cancer necessitates the adoption of a systems biology approach that integrates molecular data, biological properties of the tumors, medical images, and clinical features of the patients. In this study, a comprehensive multi-omics methodology was employed to examine a distinctive collection of patient dataset containing rapid autopsy tumor and normal tissue samples as well as longitudinal imaging with a focus on pancreatic cancer. By performing a whole exome sequencing analysis on tumor and normal tissues to identify somatic gene variants and a radiomic feature analysis to tumor CT images, the genome-wide association approach established a connection between pancreatic cancer driver genes and relevant radiomic features, enabling a thorough and quantitative assessment of the heterogeneity of pancreatic tumors. The significant association between sets of genes and radiomic features revealed the involvement of genes in shaping tumor morphological heterogeneity. Some results of the association established a connection between the molecular level mechanism and their outcomes at the level of tumor structural heterogeneity. Because tumor structure and tumor structural heterogeneity are related to the patients’ overall survival, patients who had pancreatic cancer driver gene mutations with an association to a certain radiomic feature have been observed to experience worse survival rates than cases without these somatic mutations. Furthermore, the association analysis has revealed potential gene mutations and radiomic feature candidates that warrant further investigation in future research endeavors.