24036 research outputs found
Sort by
Spatial profiling of human pancreas during type 1 diabetes progression
No full textSpatial transcriptomic profiling on the CosMx platform (NanoString, Seattle, WA). Pancreatic tissue from six nPOD organ donors - three with T1D (6228, 6247, 6456) and three without diabetes (6431, 6339, 6229), matched by age and sex. For each donor, five consecutive FFPE tissue sections from the pancreatic body region were cut at a thickness of 4 microns
RNA-Seq of SMG1 inhibition via SMG1i in human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC)
No full textUPF1 is a multi-domain RNA helicase that constantly monitors the transcriptome by non-specifically binding to mRNAs, dissociating from non-target transcripts, and initiating degradation on selected target RNAs via multiple proposed pathways such as nonsense-mediated decay (NMD). NMD is a translation-coupled mechanism that targets mRNAs harboring a premature stop codon (PTC) for degradation, thereby serving as a quality control and gene regulatory pathway ensuring transcriptome integrity. The execution of NMD requires the phosphorylation of N- and C-terminal tails of the key NMD factor UPF1, which thereby serve as binding platforms for the degradation factors SMG5, SMG6 and SMG7. UPF1 phosphorylation is mediated by the kinase SMG1, which catalytic activity can be inhibited with the SMG1 inhibitor SMG1i, a small molecule that functions as an ATP-competitive inhibitor and binds to the active site of SMG1. We wanted to assess the transcriptome-wide expression changes upon inhibition of SMG1. To this end, we treated human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC) with 1 µM SMG1i inhibitor for 24h. As controls, cells were treated with DMSO for 24h
Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [NuCaptureC]
No full textORGANISM: Homo sapiens.
EXPERIMENT TYPE: Other.
SUMMARY: Transcriptional enhancers are non-coding DNA elements that regulate gene transcription in a temporal and tissue-specific manner. Despite advances in computational and experimental methods, identifying enhancers and their target genes essential for specific biological processes remains challenging. Determining target genes for enhancers is also complex and often relies on indirect, low-resolution, and/or assumptive methodologies. To identify and functionally perturb enhancers at their endogenous sites without altering their sequence, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma development. This screen allowed the identification of CRISPRa- responsive elements (CaREs) that alter cellular growth within the 2 Mb genomic region. To determine CaRE target genes, we developed TESLA-seq (TargEted- SingLe- cell- Activation), which combines CRISPRa screening with targeted single-cell RNA-sequencing, and identified functional CaRE-target gene pairs. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements already active in the system, we found many CaREs and target connections normally active only in other tissue types or with no previous evidence and induced out of context by CRISPRa. This highlights the power of TESLA-seq to reveal gene regulatory networks active outside of a given experimental system.
OVERALL DESIGN: nuCapture C was performed according to (Downes et al., 2022). Single-stranded DNA probes were obtained from IDT as an xGen Lockdown Pool and are listed in the manuscript. Paired-end sequencing (2x150nt) was performed on a NextSeq 500/550 using a HighOutput v2 Kit for 300 cycles (Illumina #FC-420-1004, discontinued)
CNSistent integration and feature extraction from somatic copy number profiles
Get PDFBACKGROUND: Most cancers exhibit somatic copy number alterations (SCNAs)—gains and losses of variable regions of DNA. SCNAs play a key role in cancer adaptation through modulation of gene expression, deletion of tumor suppressor genes, or amplification of oncogenes. Systematic analysis of SCNAs is now a routine task in both the clinic and research and can help identify novel cancer genes, improve our understanding of cancer gene regulation, and enable us to accurately reconstruct cancer phylogenies. However, to conduct such analyses, SCNA profiles have to be integrated between samples, patients, and cohorts—often a nontrivial task, for which dedicated toolkits are lacking. RESULTS: To fill this gap, we developed CNSistent, a Python package for imputation, filtering, consistent segmentation, feature extraction, and visualization of cancer copy number profiles from heterogeneous datasets. We demonstrate the utility of CNSistent by applying it to the following publicly available cohorts: The Cancer Genome Atlas, Pan-Cancer Analysis of Whole Genomes, and TRAcking Cancer Evolution through therapy (Rx). We compare the effect of sample preprocessing and different segmentation and aggregation strategies on cancer type and subtype classification tasks using various classification models. We also evaluate how well a classifier trained on one cohort generalizes to another. Lastly, we introduce 2 segment-based peak and outlier scores to investigate relationships between segments, between samples, and between cancer types. Using these scores, we investigate non–small cell lung cancer samples, highlighting that SOX2 amplification is the dominant copy number alteration in lung squamous cell carcinoma and the main distinction to lung adenocarcinoma. CONCLUSIONS: CNSistent is a general-purpose toolkit for integrated processing of SCNA profiles across many patients and cohorts. It is available at https://bitbucket.org/schwarzlab/cnsistent. The Research Resource Identifier for CNSistent is SCR_027025
Myocardial entropy and risk predictors in hypertrophic cardiomyopathy: an analysis from the NHLBI HCM registry
No full textBACKGROUND: Entropy, a novel measure of myocardial tissue heterogeneity by cardiovascular magnetic resonance imaging, may have clinical value in patients with hypertrophic cardiomyopathy (HCM). We aimed to investigate the associations of entropy with risk predictors in HCM, using the National Heart, Lung, and Blood Institute HCM Registry. METHODS: Entropy values were calculated using the probability distribution of pixel signal intensities of the left ventricular (LV) myocardium on the late gadolinium enhancement (LGE) short-axis stack images. Entropy values were correlated with demographic, genetic, imaging, and serum biomarkers as well as ambulatory Holter recordings and the European Society of Cardiology risk score of sudden cardiac death at 5 years. RESULTS: Among 1736 patients with HCM, LV entropy demonstrated significant associations with sarcomere mutations, history of ventricular tachycardia, atrial fibrillation, and elevation of cTnT (cardiac troponin T) and NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels (P<0.001). Furthermore, LV entropy demonstrated an association with increased maximal LV wall thickness, LGE presence and extent, higher extracellular volume, left atrial area and function, myocardial strain (P<0.001), and was positively correlated with higher values of the European Society of Cardiology risk score (P<0.001). In the subgroup of patients without LGE (n=858), entropy values remained significantly associated with a history of ventricular tachycardia, increased maximal wall thickness, decreased myocardial strain, and the European Society of Cardiology risk score (P<0.05 for all). In both the whole cohort and in patients without LGE, LV entropy was the strongest predictor of ventricular tachycardia on Holter (odds ratio [95% CI] 1.59 [1.33–1.90]; 1.87 [1.28–2.74] respectively, P<0.001 for both). CONCLUSIONS: In patients with HCM, LV entropy demonstrated associations with clinical, imaging, and biological predictors of adverse outcomes independent of LGE presence and was the strongest predictor of ventricular tachycardia on Holter. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01915615
TGFb-induced SMAD1/5 activation drives transient epithelial-to-mesenchymal transition in trophoblasts
No full textSuccessful human placental development requires fetal trophoblasts to undergo epithelial-to-mesenchymal and mesenchymal-to-endothelial transitions, enabling invasion and remodeling of maternal spiral arteries for proper placental perfusion. These trans-differentiation processes are disrupted in preeclampsia, a major cause of maternal and fetal morbidity worldwide. Despite its clinical significance, the molecular mechanisms governing these differentiation trajectories at the fetal-maternal interface remain poorly defined. While Transforming Growth Factor-beta (TGFβ) has been implicated in trophoblast invasion, its role remains controversial. In this study, we report increased SMAD2/3 phosphorylation (pSMAD2/3), but reduced pSMAD1/5 and SNAIL levels in preeclamptic placentas. Mechanistically, we show that TGFβ1 induces transient SMAD1/5 phosphorylation via dual engagement of the TGFβ type I receptor ALK5 and the BMP type I receptor ALK2 in both HTR8/SVneo cells and human trophoblast stem cells. Moreover, single-nucleus transcriptomics of first-trimester placentas revealed co-expression of ACVR1, TGFBR1, SMAD1 and SMAD5 at the transition zone between cytotrophoblasts and extravillous trophoblasts. Our findings suggest that transient TGFβ-induced ALK5/ALK2-SMAD1/5 signaling is critical for EMT initiation, a prerequisite for proper placental differentiation
A novel truncating variant c.1222DupC in RBM20 causes cardiomyopathy through haploinsufficiency
Get PDFRBM20 is a cardiac splicing factor responsible for splicing of several cardiac genes such as TTN, TRDN, RyR2, PDLIM1, and CAMK2D. Mutations in RBM20 are a major cause of familial dilated cardiomyopathy (DCM), and lead to missplicing of RBM20 target genes. Here, we describe a novel heterozygous truncating mutation, RBM20 c.1222DupC, identified in a patient with mitral valve prolapse and late onset familial DCM. This mutation introduces a premature termination codon and generates a truncated protein of ∼55 kDa in vitro. Splicing assays demonstrated complete loss of activity and no dominant-negative effect on wild-type RBM20. Overexpression in NRCMs revealed that the truncated protein localized to both cytoplasm and nucleus, partially co-localizing with wild-type RBM20, despite lacking the RS and RRM domains. To model the patient’s condition, we generated a heterozygous c.1222DupC mutant induced pluripotent stem cell line and differentiated these in cardiomyocytes. Western blot analysis of endogenous RBM20 revealed a strong reduction in RBM20 protein level. RT-PCR revealed splicing defects in canonical RBM20 targets, and RNA-sequencing identified widespread splicing abnormalities, including in established RBM20 targets (TTN, RyR2, CAMK2D, and CACNA1G). Together, these findings establish RBM20 c.1222DupC as a truncating variant that causes DCM primarily through haploinsufficiency
Spatial omics of acute myocardial infarction reveals a novel mode of immune cell infiltration
No full textMyocardial infarction (MI) continues to be a leading cause of death worldwide. Even though it is well-established that the complex interplay between different cell types determines the overall healing response after MI, the precise changes in the tissue architecture are still poorly understood. Here we generated an integrative cellular map of the acute phase after murine MI using a combination of imaging-based transcriptomics (Molecular Cartography) and antibody-based highly multiplexed imaging (Sequential Immunofluorescence), which enabled us to evaluate cell-type compositions and changes at subcellular resolution over time. One striking finding of these analyses was the identification of a novel mode of leukocyte accumulation to the infarcted heart via the endocardium - the inner layer of the heart. To investigate the underlying mechanisms driving this previously unknown infiltration route, we performed unbiased spatial proteomic analysis using Deep Visual Proteomics (DVP). When comparing endocardial cells of homeostatic hearts and infarcted hearts, DVP identified von Willebrand Factor (vWF) as an upregulated mediator of inflammation 24 hours post MI. To further explore the immune mediating capabilities of vWF and its effect on tissue repair, we performed functional blocking of vWF during acute murine MI. This resulted in a reduced amount of infiltration by CCR2+ monocytes and worse cardiac function post-MI. Our study provides the first spatial map of acute murine MI with subcellular resolution and subsequently discovers a novel route of immune infiltration. Furthermore, we identified vWF as a critical immune mediating agent for endocardial immune cell infiltration