RESUMEN
Glucose plays a key role in shaping pancreatic ß cell function. Thus, deciphering the mechanisms by which this nutrient stimulates ß cells holds therapeutic promise for combating ß cell failure in type 2 diabetes (T2D). ß Cells respond to hyperglycemia in part by rewiring their mRNA metabolism, yet the mechanisms governing these changes remain poorly understood. Here, we identify a requirement for the RNA-binding protein PCBP2 in maintaining ß cell function basally and during sustained hyperglycemic challenge. PCBP2 was induced in primary mouse islets incubated with elevated glucose and was required to adapt insulin secretion. Transcriptomic analysis of primary Pcbp2-deficient ß cells revealed impacts on basal and glucose-regulated mRNAs encoding core components of the insulin secretory pathway. Accordingly, Pcbp2-deficient ß cells exhibited defects in calcium flux, insulin granule ultrastructure and exocytosis, and the amplification pathway of insulin secretion. Further, PCBP2 was induced by glucose in primary human islets, was downregulated in islets from T2D donors, and impacted genes commonly altered in islets from donors with T2D and linked to single-nucleotide polymorphisms associated with T2D. Thus, these findings establish a paradigm for PCBP2 in governing basal and glucose-adaptive gene programs critical for shaping the functional state of ß cells.
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Diabetes Mellitus Tipo 2 , Glucosa , Células Secretoras de Insulina , Insulina , Proteínas de Unión al ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Animales , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratones , Humanos , Glucosa/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Insulina/metabolismo , Secreción de Insulina , Ratones Noqueados , Masculino , Adaptación FisiológicaRESUMEN
BACKGROUND & AIMS: Endoscopic assessment of ulcerative colitis (UC) typically reports only the maximum severity observed. Computer vision methods may better quantify mucosal injury detail, which varies among patients. METHODS: Endoscopic video from the UNIFI clinical trial (A Study to Evaluate the Safety and Efficacy of Ustekinumab Induction and Maintenance Therapy in Participants With Moderately to Severely Active Ulcerative Colitis) comparing ustekinumab and placebo for UC were processed in a computer vision analysis that spatially mapped Mayo Endoscopic Score (MES) to generate the Cumulative Disease Score (CDS). CDS was compared with the MES for differentiating ustekinumab vs placebo treatment response and agreement with symptomatic remission at week 44. Statistical power, effect, and estimated sample sizes for detecting endoscopic differences between treatments were calculated using both CDS and MES measures. Endoscopic video from a separate phase 2 clinical trial replication cohort was performed for validation of CDS performance. RESULTS: Among 748 induction and 348 maintenance patients, CDS was lower in ustekinumab vs placebo users at week 8 (141.9 vs 184.3; P < .0001) and week 44 (78.2 vs 151.5; P < .0001). CDS was correlated with the MES (P < .0001) and all clinical components of the partial Mayo score (P < .0001). Stratification by pretreatment CDS revealed ustekinumab was more effective than placebo (P < .0001) with increasing effect in severe vs mild disease (-85.0 vs -55.4; P < .0001). Compared with the MES, CDS was more sensitive to change, requiring 50% fewer participants to demonstrate endoscopic differences between ustekinumab and placebo (Hedges' g = 0.743 vs 0.460). CDS performance in the JAK-UC replication cohort was similar to UNIFI. CONCLUSIONS: As an automated and quantitative measure of global endoscopic disease severity, the CDS offers artificial intelligence enhancement of traditional MES capability to better evaluate UC in clinical trials and potentially practice.
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Colitis Ulcerosa , Humanos , Inteligencia Artificial , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/tratamiento farmacológico , Colonoscopía/métodos , Computadores , Inducción de Remisión , Índice de Severidad de la Enfermedad , Ustekinumab/efectos adversosRESUMEN
The prevalence of childhood obesity is increasing worldwide, along with the associated common comorbidities of type 2 diabetes and cardiovascular disease in later life. Motivated by evidence for a strong genetic component, our prior genome-wide association study (GWAS) efforts for childhood obesity revealed 19 independent signals for the trait; however, the mechanism of action of these loci remains to be elucidated. To molecularly characterize these childhood obesity loci we sought to determine the underlying causal variants and the corresponding effector genes within diverse cellular contexts. Integrating childhood obesity GWAS summary statistics with our existing 3D genomic datasets for 57 human cell types, consisting of high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq, and RNA-seq, we applied stratified LD score regression and calculated the proportion of genome-wide SNP heritability attributable to cell type-specific features, revealing pancreatic alpha cell enrichment as the most statistically significant. Subsequent chromatin contact-based fine-mapping was carried out for genome-wide significant childhood obesity loci and their linkage disequilibrium proxies to implicate effector genes, yielded the most abundant number of candidate variants and target genes at the BDNF, ADCY3, TMEM18 and FTO loci in skeletal muscle myotubes and the pancreatic beta-cell line, EndoC-BH1. One novel implicated effector gene, ALKAL2 - an inflammation-responsive gene in nerve nociceptors - was observed at the key TMEM18 locus across multiple immune cell types. Interestingly, this observation was also supported through colocalization analysis using expression quantitative trait loci (eQTL) derived from the Genotype-Tissue Expression (GTEx) dataset, supporting an inflammatory and neurologic component to the pathogenesis of childhood obesity. Our comprehensive appraisal of 3D genomic datasets generated in a myriad of different cell types provides genomic insights into pediatric obesity pathogenesis.
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BACKGROUND AND AIMS: Histologic disease activity in Inflammatory Bowel Disease (IBD) is associated with clinical outcomes and is an important endpoint in drug development. We developed deep learning models for automating histological assessments in IBD. METHODS: Histology images of intestinal mucosa from phase 2 and phase 3 clinical trials in Crohn's disease (CD) and Ulcerative Colitis (UC) were used to train artificial intelligence (AI) models to predict the Global Histology Activity Score (GHAS) for CD and Geboes histopathology score for UC. Three AI methods were compared. AI models were evaluated on held-back testing sets and model predictions were compared against an expert central reader and five independent pathologists. RESULTS: The model based on multiple instance learning and the attention mechanism (SA-AbMILP) demonstrated the best performance among competing models. AI modeled GHAS and Geboes sub-grades matched central readings with moderate to substantial agreement, with accuracies ranging from 65% to 89%. Furthermore, the model was able to distinguish the presence and absence of pathology across four selected histological features with accuracies for colon, in both CD and UC, ranging from 87% to 94% and, for CD ileum, ranging from 76% to 83%. For both CD and UC, and across anatomical compartments (ileum and colon) in CD, comparable accuracies against central readings were found between the model assigned scores and scores by an independent set of pathologists. CONCLUSIONS: Deep learning models based upon GHAS and Geboes scoring systems were effective at distinguishing between the presence and absence of IBD microscopic disease activity.
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The RNA-binding protein Pcbp2 is widely expressed in the innate and adaptive immune systems and is essential for mouse development. To determine whether Pcbp2 is required for CD4+ T cell development and function, we derived mice with conditional Pcbp2 deletion in CD4+ T cells and assessed their overall phenotype and proliferative responses to activating stimuli. We found that Pcbp2 is essential for T conventional cell (Tconv) proliferation, working through regulation of co-stimulatory signaling. Pcbp2 deficiency in the CD4+ lineage did not impact Treg abundance in vivo or function in vitro. In addition, our data demonstrate a clear association between Pcbp2 control of Runx1 exon 6 splicing in CD4+ T cells and a specific role for Pcbp2 in the maintenance of peripheral CD4+ lymphocyte population size. Last, we show that Pcbp2 function is required for optimal in vivo Tconv cell activation in a T cell adoptive transfer colitis model system.
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We performed genome-wide association study meta-analysis to identify genetic determinants of skeletal age (SA) deviating in multiple growth disorders. The joint meta-analysis (N = 4557) in two multiethnic cohorts of school-aged children identified one locus, CYP11B1 (expression confined to the adrenal gland), robustly associated with SA (rs6471570-A; ß = 0.14; P = 6.2 × 10-12). rs6410 (a synonymous variant in the first exon of CYP11B1 in high LD with rs6471570), was prioritized for functional follow-up being second most significant and the one closest to the first intron-exon boundary. In 208 adrenal RNA-seq samples from GTEx, C-allele of rs6410 was associated with intron 3 retention (P = 8.11 × 10-40), exon 4 inclusion (P = 4.29 × 10-34), and decreased exon 3 and 5 splicing (P = 7.85 × 10-43), replicated using RT-PCR in 15 adrenal samples. As CYP11B1 encodes 11-ß-hydroxylase, involved in adrenal glucocorticoid and mineralocorticoid biosynthesis, our findings highlight the role of adrenal steroidogenesis in SA in healthy children, suggesting alternative splicing as a likely underlying mechanism.
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Empalme Alternativo , Desarrollo Óseo/genética , Esteroide 11-beta-Hidroxilasa/genética , Determinación de la Edad por el Esqueleto , Niño , Femenino , Humanos , Masculino , Esteroide 11-beta-Hidroxilasa/metabolismoRESUMEN
We previously demonstrated that the two paralogous RNA-binding proteins PCBP1 and PCBP2 are individually essential for mouse development: Pcbp1-null embryos are peri-implantation lethal, while Pcbp2-null embryos lose viability at midgestation. Midgestation Pcbp2-/- embryos revealed a complex phenotype that included loss of certain hematopoietic determinants. Whether PCBP2 directly contributes to erythropoietic differentiation and whether PCBP1 has a role in this process remained undetermined. Here, we selectively inactivated the genes encoding these two RNA-binding proteins during differentiation of the erythroid lineage in the developing mouse embryo. Individual inactivation of either locus failed to impact viability or blood formation. However, combined inactivation of the two loci resulted in midgestational repression of erythroid/hematopoietic gene expression, loss of blood formation, and fetal demise. Orthogonal ex vivo analyses of primary erythroid progenitors selectively depleted of these two RNA-binding proteins revealed that they mediate a combination of overlapping and isoform-specific impacts on hematopoietic lineage transcriptome, impacting both mRNA representation and exon splicing. These data lead us to conclude that PCBP1 and PCBP2 mediate functions critical to differentiation of the erythroid lineage.
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Proteínas de Unión al ADN/metabolismo , Eritropoyesis , Proteínas de Unión al ARN/metabolismo , Envejecimiento/fisiología , Animales , Linaje de la Célula , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/metabolismo , Células Eritroides/citología , Exones/genética , Sitios Genéticos , Células Madre Hematopoyéticas/metabolismo , Ratones , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Transcriptoma/genéticaRESUMEN
Ribosome-profiling has uncovered pervasive translation in non-canonical open reading frames, however the biological significance of this phenomenon remains unclear. Using genetic variation from 71,702 human genomes, we assess patterns of selection in translated upstream open reading frames (uORFs) in 5'UTRs. We show that uORF variants introducing new stop codons, or strengthening existing stop codons, are under strong negative selection comparable to protein-coding missense variants. Using these variants, we map and validate gene-disease associations in two independent biobanks containing exome sequencing from 10,900 and 32,268 individuals, respectively, and elucidate their impact on protein expression in human cells. Our results suggest translation disrupting mechanisms relating uORF variation to reduced protein expression, and demonstrate that translation at uORFs is genetically constrained in 50% of human genes.
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Enfermedad/genética , Sistemas de Lectura Abierta , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 5' , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Estudios de Asociación Genética , Genoma Humano , Humanos , Fenotipo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas/genética , Receptor EphB2RESUMEN
BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a polygenic disorder characterized principally by dysregulated inflammation impacting the gastrointestinal tract. However, there also is increasing evidence for a clinical association with stress and depression. Given the role of the hypothalamus in stress responses and in the pathogenesis of depression, useful insights could be gleaned from understanding its genetic role in IBD. METHODS: We conducted genetic correlation analyses on publicly available genome-wide association study summary statistics for depression and IBD traits to identify genetic commonalities. We used partitioned linkage disequilibrium score regression, leveraging our ATAC sequencing and promoter-focused Capture C data, to measure enrichment of IBD single-nucleotide polymorphisms within promoter-interacting open chromatin regions of human embryonic stem cell-derived hypothalamic-like neurons (HNs). Using the same data sets, we performed variant-to-gene mapping to implicate putative IBD effector genes in HNs. To contrast these results, we similarly analyzed 3-dimensional genomic data generated in epithelium-derived colonoids from rectal biopsy specimens from donors without pathologic disease noted at the time of colonoscopy. Finally, we conducted enrichment pathway analyses on the implicated genes to identify putative IBD dysfunctional pathways. RESULTS: We found significant genetic correlations (rg) of 0.122 with an adjusted P (Padj) = 1.4 × 10-4 for IBD: rg = 0.122; Padj = 2.5 × 10-3 for ulcerative colitis and genetic correlation (rg) = 0.094; Padj = 2.5 × 10-3 for Crohn's disease, and significant approximately 4-fold (P = .005) and approximately 7-fold (P = .03) enrichment of IBD single-nucleotide polymorphisms in HNs and colonoids, respectively. We implicated 25 associated genes in HNs, among which CREM, CNTF, and RHOA encode key regulators of stress. Seven genes also additionally were implicated in the colonoids. We observed an overall enrichment for immune and hormonal signaling pathways, and a colonoid-specific enrichment for microbiota-relevant terms. CONCLUSIONS: Our results suggest that the hypothalamus warrants further study in the context of IBD pathogenesis.
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Depresión/genética , Predisposición Genética a la Enfermedad , Hipotálamo/fisiopatología , Enfermedades Inflamatorias del Intestino/genética , Estrés Psicológico/genética , Eje Cerebro-Intestino , Estudios de Casos y Controles , Mapeo Cromosómico , Conjuntos de Datos como Asunto , Depresión/fisiopatología , Estudio de Asociación del Genoma Completo , Humanos , Sistema Hipotálamo-Hipofisario/fisiopatología , Hipotálamo/citología , Enfermedades Inflamatorias del Intestino/fisiopatología , Desequilibrio de Ligamiento , Neuronas , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Estrés Psicológico/fisiopatologíaRESUMEN
Immune cell function is influenced by metabolic conditions. Low-glucose, high-lactate environments, such as the placenta, gastrointestinal tract, and the tumor microenvironment, are immunosuppressive, especially for glycolysis-dependent effector T cells. We report that nicotinamide adenine dinucleotide (NAD+), which is reduced to NADH by lactate dehydrogenase in lactate-rich conditions, is a key point of metabolic control in T cells. Reduced NADH is not available for NAD+-dependent enzymatic reactions involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate dehydrogenase (PGDH). We show that increased lactate leads to a block at GAPDH and PGDH, leading to the depletion of post-GAPDH glycolytic intermediates, as well as the 3-phosphoglycerate derivative serine that is known to be important for T cell proliferation. Supplementing serine rescues the ability of T cells to proliferate in the presence of lactate-induced reductive stress. Directly targeting the redox state may be a useful approach for developing novel immunotherapies in cancer and therapeutic immunosuppression.
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Ácido Láctico/metabolismo , NAD/metabolismo , Proliferación Celular , Humanos , Oxidación-ReducciónRESUMEN
G-quadruplex (G4) sequences are abundant in untranslated regions (UTRs) of human messenger RNAs, but their functional importance remains unclear. By integrating multiple sources of genetic and genomic data, we show that putative G-quadruplex forming sequences (pG4) in 5' and 3' UTRs are selectively constrained, and enriched for cis-eQTLs and RNA-binding protein (RBP) interactions. Using over 15,000 whole-genome sequences, we find that negative selection acting on central guanines of UTR pG4s is comparable to that of missense variation in protein-coding sequences. At multiple GWAS-implicated SNPs within pG4 UTR sequences, we find robust allelic imbalance in gene expression across diverse tissue contexts in GTEx, suggesting that variants affecting G-quadruplex formation within UTRs may also contribute to phenotypic variation. Our results establish UTR G4s as important cis-regulatory elements and point to a link between disruption of UTR pG4 and disease.
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G-Cuádruplex , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas , Estudios de Asociación Genética , Variación Genética , Humanos , Motivos de Nucleótidos , Pliegue del ARN , Proteínas de Unión al ARN/fisiologíaRESUMEN
Single-cell gene expression analyses of mammalian tissues have uncovered profound stage-specific molecular regulatory phenomena that have changed the understanding of unique cell types and signaling pathways critical for lineage determination, morphogenesis, and growth. We discuss here the case for a Pediatric Cell Atlas as part of the Human Cell Atlas consortium to provide single-cell profiles and spatial characterization of gene expression across human tissues and organs. Such data will complement adult and developmentally focused HCA projects to provide a rich cytogenomic framework for understanding not only pediatric health and disease but also environmental and genetic impacts across the human lifespan.
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Desarrollo Embrionario/genética , Redes Reguladoras de Genes/genética , Pediatría/tendencias , Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Distribución Tisular/genéticaRESUMEN
Formation of the mammalian hematopoietic system is under a complex set of developmental controls. Here, we report that mouse embryos lacking the KH domain poly(C) binding protein, Pcbp2, are selectively deficient in the definitive erythroid lineage. Compared to wild-type controls, transcript splicing analysis of the Pcbp2-/- embryonic liver reveals accentuated exclusion of an exon (exon 6) that encodes a highly conserved transcriptional control segment of the hematopoietic master regulator, Runx1. Embryos rendered homozygous for a Runx1 locus lacking this cassette exon (Runx1ΔE6) effectively phenocopy the loss of the definitive erythroid lineage in Pcbp2-/- embryos. These data support a model in which enhancement of Runx1 cassette exon 6 inclusion by Pcbp2 serves a critical role in development of hematopoietic progenitors and constitutes a critical step in the developmental pathway of the definitive erythropoietic lineage.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Eritropoyesis/genética , Eritropoyesis/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/deficiencia , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Exones , Regulación del Desarrollo de la Expresión Génica , Globinas/genética , Hematopoyesis/genética , Hematopoyesis/fisiología , Humanos , Células K562 , Hígado/embriología , Hígado/metabolismo , Ratones , Ratones Noqueados , Empalme del ARN , Eliminación de SecuenciaRESUMEN
RNA-binding proteins participate in a complex array of posttranscriptional controls essential to cell type specification and somatic development. Despite their detailed biochemical characterizations, the degree to which each RNA-binding protein impacts mammalian embryonic development remains incompletely defined, and the level of functional redundancy among subsets of these proteins remains open to question. The poly(C) binding proteins, PCBPs (αCPs and hnRNP E proteins), are encoded by a highly conserved and broadly expressed gene family. The two major Pcbp isoforms, Pcbp2 and Pcbp1, are robustly expressed in a wide range of tissues and exert both nuclear and cytoplasmic controls over gene expression. Here, we report that Pcbp1-null embryos are rendered nonviable in the peri-implantation stage. In contrast, Pcbp2-null embryos undergo normal development until midgestation (12.5 to 13.5 days postcoitum), at which time they undergo a dramatic loss in viability associated with combined cardiovascular and hematopoietic abnormalities. Mice heterozygous for either Pcbp1 or Pcbp2 null alleles display a mild and nondisruptive defect in initial postpartum weight gain. These data reveal that Pcbp1 and Pcbp2 are individually essential for mouse embryonic development and have distinct impacts on embryonic viability and that Pcpb2 has a nonredundant in vivo role in hematopoiesis. These data further provide direct evidence that Pcbp1, a retrotransposed derivative of Pcpb2, has evolved an essential function(s) in the mammalian genome.
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Proteínas Portadoras/genética , Ratones/embriología , Proteínas de Unión al ARN/genética , Animales , Secuencia de Bases , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN , Embrión de Mamíferos/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Sitios Genéticos , Hematopoyesis , Ratones/genética , Datos de Secuencia Molecular , Proteínas de Unión al ARN/metabolismo , TranscriptomaRESUMEN
RNA-binding proteins and corresponding post-transcriptional controls play critical roles in gene expression. The poly-(C) binding proteins, PCBPs (αCPs, hnRNPEs), comprise a well-characterized family of abundant RNA-binding proteins that impact on RNA processing in the nucleus as well as mRNA stability and translation in the cytoplasm. Here we demonstrate that PCBP1 and PCBP2 are abundantly expressed in the gastric epithelium with prominent enrichment in specific cell types within the gastric glandular mucosa. The spatial and intracellular patterns of PCBP1 and PCBP2 expression in these regions are highly correlated. Remarkably, we observe that these proteins are present in the nuclear and cytoplasmic compartments of zymogenic chief cells while they are restricted to the nuclear compartment in acid-secreting parietal cells and poorly expressed in pit cells that line the gland exit. This specificity of expression patterns and subcellular localization of PCBP1 and PCBP2, along with their appearance in the precursor tissues of the gastric epithelium during early postnatal development, suggests these RNA-binding proteins play specific roles in cell differentiation and organismal development within the gastric glandular epithelium.