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1.
Proc Natl Acad Sci U S A ; 119(26): e2201267119, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35733248

RESUMO

Delineating gene regulatory networks that orchestrate cell-type specification is a continuing challenge for developmental biologists. Single-cell analyses offer opportunities to address these challenges and accelerate discovery of rare cell lineage relationships and mechanisms underlying hierarchical lineage decisions. Here, we describe the molecular analysis of mouse pancreatic endocrine cell differentiation using single-cell transcriptomics, chromatin accessibility assays coupled to genetic labeling, and cytometry-based cell purification. We uncover transcription factor networks that delineate ß-, α-, and δ-cell lineages. Through genomic footprint analysis, we identify transcription factor-regulatory DNA interactions governing pancreatic cell development at unprecedented resolution. Our analysis suggests that the transcription factor Neurog3 may act as a pioneer transcription factor to specify the pancreatic endocrine lineage. These findings could improve protocols to generate replacement endocrine cells from renewable sources, like stem cells, for diabetes therapy.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cromatina , Ilhotas Pancreáticas , Proteínas do Tecido Nervoso , Transcriptoma , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Célula Única
2.
Am J Physiol Renal Physiol ; 327(3): F489-F503, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38991008

RESUMO

Fate mapping and genetic manipulation of renin cells have relied on either noninducible Cre lines that can introduce the developmental effects of gene deletion or bacterial artificial chromosome transgene-based inducible models that may be prone to spurious and/or ectopic gene expression. To circumvent these problems, we generated an inducible mouse model in which CreERT2 is under the control of the endogenous Akr1b7 gene, an independent marker of renin cells that is expressed in a few extrarenal tissues. We confirmed the proper expression of Cre using Akr1b7CreERT2/+;R26RmTmG/+ mice in which Akr1b7+/renin+ cells become green fluorescent protein (GFP)+ upon tamoxifen administration. In embryos and neonates, GFP was found in juxtaglomerular cells, along the arterioles, and in the mesangium, and in adults, GFP was present mainly in juxtaglomerular cells. In mice treated with captopril and a low-salt diet to induce recruitment of renin cells, GFP extended along the afferent arterioles and in the mesangium. We generated Akr1b7CreERT2/+;Ren1cFl/-;R26RmTmG/+ mice to conditionally delete renin in adult mice and found a marked reduction in kidney renin mRNA and protein and mean arterial pressure in mutant animals. When subjected to a homeostatic threat, mutant mice were unable to recruit renin+ cells. Most importantly, these mice developed concentric vascular hypertrophy ruling out potential developmental effects on the vasculature due to the lack of renin. We conclude that Akr1b7CreERT2 mice constitute an excellent model for the fate mapping of renin cells and for the spatial and temporal control of gene expression in renin cells.NEW & NOTEWORTHY Fate mapping and genetic manipulation are important tools to study the identity of renin cells. Here, we report on a novel Cre mouse model, Akr1b7CreERT2, for the spatial and temporal regulation of gene expression in renin cells. Cre is properly expressed in renin cells during development and in the adult under basal conditions and under physiological stress. Moreover, renin can be efficiently deleted in the adult, leading to the development of concentric vascular hypertrophy.


Assuntos
Camundongos Transgênicos , Renina , Animais , Renina/metabolismo , Renina/genética , Camundongos , Sistema Justaglomerular/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Captopril/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Regulação da Expressão Gênica , Integrases/genética , Integrases/metabolismo
3.
J Am Pharm Assoc (2003) ; : 102080, 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38556245

RESUMO

BACKGROUND: Burnout among clinical pharmacist practitioners has been well established, but not among those who perform academic detailing. OBJECTIVES: To measure burnout among clinical pharmacist practitioners who perform academic detailing (pharmacist-academic detailers) at the United States Veterans Health Administration and compare the findings using 2 validated burnout instruments for healthcare professionals. METHODS: A cross-sectional study design was performed to measure burnout in VHA pharmacist-academic detailers across all VA regions between April 2023 and May 2023. Burnout was measured using the Oldenburg Burnout Inventory (OLBI) and a validated single-item burnout measure (SIMB). OLBI has 2 domains (exhaustion and disengagement) and categorizes burnout into Low, Moderate, and High based on scores above or below 1 standard deviation (SD) of the mean. The validated SIMB categorized burnout as having a score of 3 or greater (range: 1-5). Interrater reliability testing between the OLBI and the SIMB at detecting burnout among pharmacist-academic detailers was performed using the kappa test. Correlation between the 2 burnout instruments was assessed using the Spearman rho test. RESULTS: A total of 50 pharmacist-academic detailers completed the burnout survey. A large proportion of respondents had Moderate levels of burnout for the total (72%) burnout score, disengagement (64%) domain, and exhaustion (74%) domain. In total, 86% of pharmacist-academic detailers reported having Moderate to High levels of burnout on the total OLBI score. On the SIMB, a total of 14 (28%) pharmacist-academic detailers reported having one or more symptoms of burnout. Interrater reliability was considered poor/slight agreement between the OLBI and SIMB. Correlation between the 2 burnout instruments was considered moderately correlated (rho = 0.67, P < 0.001). CONCLUSION: This study provides an empirical analysis of burnout among pharmacist-academic detailers; however, the ability to detect burnout among pharmacist-academic detailers may be impacted by the selection of burnout instrument used.

4.
BMC Genomics ; 23(1): 299, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35413804

RESUMO

BACKGROUND: Epigenome analysis relies on defined sets of genomic regions output by widely used assays such as ChIP-seq and ATAC-seq. Statistical analysis and visualization of genomic region sets is essential to answer biological questions in gene regulation. As the epigenomics community continues generating data, there will be an increasing need for software tools that can efficiently deal with more abundant and larger genomic region sets. Here, we introduce GenomicDistributions, an R package for fast and easy summarization and visualization of genomic region data. RESULTS: GenomicDistributions offers a broad selection of functions to calculate properties of genomic region sets, such as feature distances, genomic partition overlaps, and more. GenomicDistributions functions are meticulously optimized for best-in-class speed and generally outperform comparable functions in existing R packages. GenomicDistributions also offers plotting functions that produce editable ggplot objects. All GenomicDistributions functions follow a uniform naming scheme and can handle either single or multiple region set inputs. CONCLUSIONS: GenomicDistributions offers a fast and scalable tool for exploratory genomic region set analysis and visualization. GenomicDistributions excels in user-friendliness, flexibility of outputs, breadth of functions, and computational performance. GenomicDistributions is available from Bioconductor ( https://bioconductor.org/packages/release/bioc/html/GenomicDistributions.html ).


Assuntos
Genômica , Software , Sequenciamento de Cromatina por Imunoprecipitação , Epigenômica , Genoma
5.
Bioinformatics ; 37(23): 4299-4306, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34156475

RESUMO

MOTIVATION: Genomic region sets summarize functional genomics data and define locations of interest in the genome such as regulatory regions or transcription factor binding sites. The number of publicly available region sets has increased dramatically, leading to challenges in data analysis. RESULTS: We propose a new method to represent genomic region sets as vectors, or embeddings, using an adapted word2vec approach. We compared our approach to two simpler methods based on interval unions or term frequency-inverse document frequency and evaluated the methods in three ways: First, by classifying the cell line, antibody or tissue type of the region set; second, by assessing whether similarity among embeddings can reflect simulated random perturbations of genomic regions; and third, by testing robustness of the proposed representations to different signal thresholds for calling peaks. Our word2vec-based region set embeddings reduce dimensionality from more than a hundred thousand to 100 without significant loss in classification performance. The vector representation could identify cell line, antibody and tissue type with over 90% accuracy. We also found that the vectors could quantitatively summarize simulated random perturbations to region sets and are more robust to subsampling the data derived from different peak calling thresholds. Our evaluations demonstrate that the vectors retain useful biological information in relatively lower-dimensional spaces. We propose that vector representation of region sets is a promising approach for efficient analysis of genomic region data. AVAILABILITY AND IMPLEMENTATION: https://github.com/databio/regionset-embedding. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Genômica , Ligação Proteica
6.
Clin Gerontol ; : 1-12, 2022 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-36200403

RESUMO

OBJECTIVES: The aim is to pilot a low-touch program for reducing benzodiazepine receptor agonist (BZRA; benzodiazepines, z-drugs) prescriptions among older veterans. METHODS: Pilot randomized controlled trial consists of 2,009 veterans aged ≥ 65 years who received BZRA prescriptions from a Veterans Health Administration pharmacy (Colorado or Montana) during the prior 18 months. Active: Arm 1 was a mailed brochure about BZRA risks that also included information about a free, online cognitive behavioral therapy for the insomnia (CBTI) program. Arm 2 was a mailed brochure (same as arm 1) and telephone reinforcement call. Control: Arm 3 was a mailed brochure without insomnia treatment information. Active BZRA prescriptions at follow-up (6 and 12 months) were measured. RESULTS: In logistic regression analyses, the odds of BZRA prescription at 6- and 12-month follow-ups were not significantly different for arm 1 or 2 (active) versus arm 3 (control), including models adjusted for demographics and prescription characteristics (p-values >0.36). CONCLUSIONS: Although we observed no differences in active BZRA prescriptions, this pilot study provides guidance for conducting a future study, indicating a need for a more potent intervention. A full-scale trial testing an optimized program would provide conclusive results. CLINICAL IMPLICATIONS: Mailing information about BZRA risks and CBTI did not affect BZRA prescriptions.

7.
FASEB J ; 34(11): 15314-15326, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32959931

RESUMO

Lipid perturbations contribute to detrimental outcomes in obesity. We previously demonstrated that nervonic acid, a C24:1 ω-9 fatty acid, predominantly acylated to sphingolipids, including ceramides, are selectively reduced in a mouse model of obesity. It is currently unknown if deficiency of nervonic acid-sphingolipid metabolites contribute to complications of obesity. Mice were fed a standard diet, a high fat diet, or these diets supplemented isocalorically with nervonic acid. The primary objective was to determine if dietary nervonic acid content alters the metabolic phenotype in mice fed a high fat diet. Furthermore, we investigated if nervonic acid alters markers of impaired fatty acid oxidation in the liver. We observed that a nervonic acid-enriched isocaloric diet reduced weight gain and adiposity in mice fed a high fat diet. The nervonic acid enrichment led to increased C24:1-ceramides and improved several metabolic parameters including blood glucose levels, and insulin and glucose tolerance. Mechanistically, nervonic acid supplementation increased PPARα and PGC1α expression and improved the acylcarnitine profile in liver. These alterations indicate improved energy metabolism through increased ß-oxidation of fatty acids. Taken together, increasing dietary nervonic acid improves metabolic parameters in mice fed a high fat diet. Strategies that prevent deficiency of, or restore, nervonic acid may represent an effective strategy to treat obesity and obesity-related complications.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Ácidos Graxos Monoinsaturados/farmacologia , Fígado/efeitos dos fármacos , Obesidade/tratamento farmacológico , Aumento de Peso , Animais , Peso Corporal , Ceramidas/metabolismo , Suplementos Nutricionais , Metabolismo Energético , Resistência à Insulina , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/patologia
9.
J Am Pharm Assoc (2003) ; 60(6): e95-e99, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32747164

RESUMO

As the coronavirus disease (COVID-19) pandemic continues its course in 2020, telehealth technology provides opportunities to connect patients and providers. Health policies have been amended to allow easy access to virtual health care, highlighting the field's dynamic ability to adapt to a public health crisis. Academic detailing, a peer-to-peer collaborative outreach designed to improve clinical decision-making, has traditionally relied on in-person encounters for effectiveness. A growth in the adoption of telehealth technology translates to increases in academic detailing reach for providers unable to meet with academic detailers in person. The U.S. Department of Veterans Affairs (VA) has used academic detailing to promote and reinforce evidence-based practices and has encouraged more virtual academic detailing (e-Detailing). Moreover, VA academic detailers are primarily clinical pharmacy specialists who provide clinical services and education and have made meaningful contributions to improving health care at VA. Amid the COVID-19 pandemic and physical isolation orders, VA academic detailers have continued to meet with providers to disseminate critical health care information in a timely fashion by using video-based telehealth. When working through the adoption of virtual technology for the delivery of medical care, providers may need time and nontraditional delivery of "evidence" before eliciting signals for change. Academic detailers are well suited for this role and can develop plans to help address provider discomfort surrounding the use of telehealth technology. By using e-Detailing as a method for both familiarizing and normalizing health professionals with video-based telehealth technology, pharmacists are uniquely poised to deliver consultation and direct-care services. Moreover, academic detailing pharmacists are ambassadors of change, serving an important role navigating the evolution of health care in response to emergent public health crises and helping define the norms of care delivery to follow.


Assuntos
COVID-19/terapia , Assistência Farmacêutica/organização & administração , Padrões de Prática Médica/organização & administração , Telemedicina/organização & administração , Tomada de Decisão Clínica , Instrução por Computador , Medicina Baseada em Evidências , Disseminação de Informação , Relações Interprofissionais , Assistência Farmacêutica/tendências , Padrões de Prática Médica/tendências , SARS-CoV-2 , Telemedicina/normas , Estados Unidos , United States Department of Veterans Affairs
10.
BMC Plant Biol ; 19(1): 535, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31795928

RESUMO

BACKGROUND: Elucidating the effect of source-sink relations on berry composition is of interest for wine grape production as it represents a mechanistic link between yield, photosynthetic capacity and wine quality. However, the specific effects of carbohydrate supply on berry composition are difficult to study in isolation as leaf area or crop adjustments can also change fruit exposure, or lead to compensatory growth or photosynthetic responses. A new experimental system was therefore devised to slow berry sugar accumulation without changing canopy structure or yield. This consisted of six transparent 1.2 m3 chambers to enclose large pot-grown grapevines, and large soda-lime filled scrubbers that reduced carbon dioxide (CO2) concentration of day-time supply air by approximately 200 ppm below ambient. RESULTS: In the first full scale test of the system, the chambers were installed on mature Shiraz grapevines for 14 days from the onset of berry sugar accumulation. Three chambers were run at sub-ambient CO2 for 10 days before returning to ambient. Canopy gas exchange, and juice hexose concentrations were determined. Net CO2 exchange was reduced from 65.2 to 30 g vine- 1 day- 1, or 54%, by the sub-ambient treatment. At the end of the 10 day period, total sugar concentration was reduced from 95 to 77 g L- 1 from an average starting value of 23 g L- 1, representing a 25% reduction. Scaling to a per vine basis, it was estimated that 223 g of berry sugars accumulated under ambient supply compared to 166 g under sub-ambient, an amount equivalent to 50 and 72% of total C assimilated. CONCLUSIONS: Through supply of sub-ambient CO2 using whole canopy gas exchange chambers system, an effective method was developed for reducing photosynthesis and slowing the rate of berry sugar accumulation without modifying yield or leaf area. While in this case developed for further investigations of grape and wine composition, the system has broader applications for the manipulation and of study of grapevine source-sink relations.


Assuntos
Dióxido de Carbono/metabolismo , Produção Agrícola/métodos , Açúcares/metabolismo , Vitis/fisiologia , Frutas/química , Fotossíntese/fisiologia , Folhas de Planta/fisiologia
11.
J Exp Bot ; 70(2): 397-423, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30388247

RESUMO

Climate change scenarios predict an increase in average temperatures and in the frequency, intensity, and length of extreme temperature events in many wine regions around the world. In already warm and hot regions, such changes may compromise grape growing and the production of high quality wine as high temperature has been found to affect berry composition critically. Most recent studies focusing on the sole effect of temperature, separated from light and water, on grape berry composition found that high temperature affects a wide range of metabolites, and in particular flavonoids-key compounds for berry and wine quality. A decrease in total anthocyanins is reported in most cases, and appears to be directly associated with high temperature. Changes in anthocyanin composition, and flavonol and proanthocyanidin responses are however less consistent, and reflect the complexity of the underlying biosynthetic pathways and diversity of experimental treatments that have been used in these studies. This review examines the impact of high temperature on the biosynthesis, accumulation, and degradation of flavonoids, and attempts to reconcile the diversity of responses in relation to the latest understanding of flavonoid chemistry and molecular regulation.


Assuntos
Calor Extremo , Flavonoides/biossíntese , Frutas/metabolismo , Vitis/metabolismo
12.
Molecules ; 24(23)2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31783632

RESUMO

Climate models predict an increase in the frequency and duration of heatwaves with an increase in intensity already strongly evident worldwide. The aim of this work was to evaluate the effect of two heatwave-related parameters (intensity and duration) during berry ripening and identify a threshold for berry survival and flavonoid accumulation. A Doehlert experimental design was used to test three temperature intensities (maxima of 35, 46, and 54 °C) and five durations (3 to 39 h), with treatments applied at the bunch level shortly after véraison. Berry skin and seeds were analysed by liquid chromatography-triple quadrupole-mass spectrometry (LC-QqQ-MS) for flavonoids (flavonols, anthocyanins, free flavan-3-ols, and tannins). Berries exposed to 46 °C showed little difference compared to 35 °C. However, berries reaching temperatures around 54 °C were completely desiccated, and all flavonoids were significantly decreased except for skin flavonols on a per berry basis and seed tannins in most cases. Some compounds, such as dihydroxylated flavonoids and galloylated flavan-3-ols (free and polymerised), were in higher proportion in damaged berries suggesting they were less degraded or more synthesised upon heating. Overall, irreversible berry damages and substantial compositional changes were observed and the berry survival threshold was estimated at around 50-53 °C for mid-ripe Shiraz berries, regardless of the duration of exposure.


Assuntos
Antocianinas/análise , Flavonoides/análise , Flavonóis/análise , Frutas/química , Temperatura Alta/efeitos adversos , Taninos/análise , Vitis/química , Antocianinas/química , Antocianinas/metabolismo , Cromatografia Líquida de Alta Pressão , Flavonoides/metabolismo , Flavonóis/química , Flavonóis/metabolismo , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Espectrometria de Massas , Sementes/química , Taninos/metabolismo , Fatores de Tempo , Vitis/fisiologia
14.
Physiol Plant ; 164(2): 120-133, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29498442

RESUMO

Leaves are an important contributor toward berry sugar and nitrogen (N) accumulation, and leaf area, therefore, affects fruit composition during grapevine (Vitis vinifera) berry ripening. The aim of this study was to investigate the impact of leaf presence on key berry quality attributes in conjunction with the accumulation of primary berry metabolites. Shortly after the start of véraison (berry ripening), potted grapevines were defoliated (total defoliation and 25% of the control), and the accumulation of berry soluble solids, N and anthocyanins were compared to that of a full leaf area control. An untargeted approach was undertaken to measure the content in primary metabolites by gas chromatography/mass spectrometry. Partial and full defoliation resulted in reduced berry sugar and anthocyanin accumulation, while total berry N content was unaffected. The juice yeast assimilable N (YAN), however, increased upon partial and full defoliation. Remobilized carbohydrate reserves allowed accumulation of the major berry sugars during the absence of leaf photoassimilation. Berry anthocyanin biosynthesis was strongly inhibited by defoliation, which could relate to the carbon (C) source limitation and/or increased bunch exposure. Arginine accumulation, likely resulting from reserve translocation, contributed to increased YAN upon defoliation. Furthermore, assessing the implications on various products of the shikimate pathway suggests the C flux through this pathway to be largely affected by leaf source limitation during fruit maturation. This study provides a novel investigation of impacts of leaf C and N source presence during berry maturation, on the development of key berry quality parameters as underlined by alterations in primary metabolism.


Assuntos
Antocianinas/metabolismo , Carbono/metabolismo , Frutas/metabolismo , Nitrogênio/metabolismo , Folhas de Planta/metabolismo , Vitis/metabolismo
15.
Physiol Plant ; 161(4): 434-450, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28692131

RESUMO

Grapevine (Vitis vinifera) roots and leaves represent major carbohydrate and nitrogen (N) sources, either as recent assimilates, or mobilized from labile or storage pools. This study examined the response of root and leaf primary metabolism following defoliation treatments applied to fruiting vines during ripening. The objective was to link alterations in root and leaf metabolism to carbohydrate and N source functioning under conditions of increased fruit sink demand. Potted grapevine leaf area was adjusted near the start of véraison to 25 primary leaves per vine compared to 100 leaves for the control. An additional group of vines were completely defoliated. Fruit sugar and N content development was assessed, and root and leaf starch and N concentrations determined. An untargeted GC/MS approach was undertaken to evaluate root and leaf primary metabolite concentrations. Partial and full defoliation increased root carbohydrate source contribution towards berry sugar accumulation, evident through starch remobilization. Furthermore, root myo-inositol metabolism played a distinct role during carbohydrate remobilization. Full defoliation induced shikimate pathway derived aromatic amino acid accumulation in roots, while arginine accumulated after full and partial defoliation. Likewise, various leaf amino acids accumulated after partial defoliation. These results suggest elevated root and leaf amino N source activity when leaf N availability is restricted during fruit ripening. Overall, this study provides novel information regarding the impact of leaf source restriction, on metabolic compositions of major carbohydrate and N sources during berry maturation. These results enhance the understanding of source organ carbon and N metabolism during fruit maturation.


Assuntos
Frutas/metabolismo , Raízes de Plantas/metabolismo , Vitis/metabolismo , Aminoácidos/metabolismo , Metabolismo dos Carboidratos/genética , Metabolismo dos Carboidratos/fisiologia , Frutas/fisiologia , Cromatografia Gasosa-Espectrometria de Massas , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Raízes de Plantas/fisiologia , Vitis/fisiologia
16.
NAR Genom Bioinform ; 6(3): lqae073, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38974799

RESUMO

Data from the single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) are now widely available. One major computational challenge is dealing with high dimensionality and inherent sparsity, which is typically addressed by producing lower dimensional representations of single cells for downstream clustering tasks. Current approaches produce such individual cell embeddings directly through a one-step learning process. Here, we propose an alternative approach by building embedding models pre-trained on reference data. We argue that this provides a more flexible analysis workflow that also has computational performance advantages through transfer learning. We implemented our approach in scEmbed, an unsupervised machine-learning framework that learns low-dimensional embeddings of genomic regulatory regions to represent and analyze scATAC-seq data. scEmbed performs well in terms of clustering ability and has the key advantage of learning patterns of region co-occurrence that can be transferred to other, unseen datasets. Moreover, models pre-trained on reference data can be exploited to build fast and accurate cell-type annotation systems without the need for other data modalities. scEmbed is implemented in Python and it is available to download from GitHub. We also make our pre-trained models available on huggingface for public use. scEmbed is open source and available at https://github.com/databio/geniml. Pre-trained models from this work can be obtained on huggingface: https://huggingface.co/databio.

17.
Hypertension ; 81(9): 1869-1882, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38989586

RESUMO

BACKGROUND: Renin-expressing cells are myoendocrine cells crucial for the maintenance of homeostasis. Renin is regulated by cAMP, p300 (histone acetyltransferase p300)/CBP (CREB-binding protein), and Brd4 (bromodomain-containing protein 4) proteins and associated pathways. However, the specific regulatory changes that occur following inhibition of these pathways are not clear. METHODS: We treated As4.1 cells (tumoral cells derived from mouse juxtaglomerular cells that constitutively express renin) with 3 inhibitors that target different factors required for renin transcription: H-89-dihydrochloride, PKA (protein kinase A) inhibitor; JQ1, Brd4 bromodomain inhibitor; and A-485, p300/CBP inhibitor. We performed assay for transposase-accessible chromatin with sequencing (ATAC-seq), single-cell RNA sequencing, cleavage under targets and tagmentation (CUT&Tag), and chromatin immunoprecipitation sequencing for H3K27ac (acetylation of lysine 27 of the histone H3 protein) and p300 binding on biological replicates of treated and control As4.1 cells. RESULTS: In response to each inhibitor, Ren1 expression was significantly reduced and reversible upon washout. Chromatin accessibility at the Ren1 locus did not markedly change but was globally reduced at distal elements. Inhibition of PKA led to significant reductions in H3K27ac and p300 binding specifically within the Ren1 super-enhancer region. Further, we identified enriched TF (transcription factor) motifs shared across each inhibitory treatment. Finally, we identified a set of 9 genes with putative roles across each of the 3 renin regulatory pathways and observed that each displayed differentially accessible chromatin, gene expression, H3K27ac, and p300 binding at their respective loci. CONCLUSIONS: Inhibition of renin expression in cells that constitutively synthesize and release renin is regulated by an epigenetic switch from an active to poised state associated with decreased cell-cell communication and an epithelial-mesenchymal transition. This work highlights and helps define the factors necessary for renin cells to alternate between myoendocrine and contractile phenotypes.


Assuntos
Epigênese Genética , Renina , Fatores de Transcrição , Animais , Camundongos , Renina/metabolismo , Renina/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Sistema Justaglomerular/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Fatores de Transcrição de p300-CBP/genética , Proteínas que Contêm Bromodomínio , Proteínas Nucleares
18.
bioRxiv ; 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-38585851

RESUMO

Renin is crucial for blood pressure regulation and electrolyte balance, and its expressing cells arise from Foxd1+ stromal progenitors. However, the factors guiding these progenitors toward the renin-secreting cell fate are not fully understood. Tcf21, a basic helix-loop-helix (bHLH) transcription factor, is essential in kidney development. Utilizing Foxd1 Cre/+ ;Tcf21 f/f and Ren1 dCre/+ ;Tcf21 f/f mouse models, we investigated the role of Tcf21 in the differentiation of Foxd1+ progenitor cells into juxtaglomerular (JG) cells. Immunostaining and in-situ hybridization demonstrated significantly fewer renin-positive areas and altered renal arterial morphology in Foxd1 Cre/+ ;Tcf21 f/f kidneys compared with controls, indicating Tcf21's necessity for renin cell emergence. However, Tcf21 inactivation in renin-expressing cells ( Ren1 dCre/+ ;Tcf21 f/f ) did not recapitulate this phenotype, suggesting Tcf21 is dispensable once renin cell identity is established. Integrated analysis of single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on GFP+ cells (stromal lineage) from E12, E18, P5, and P30 Foxd1 Cre/+ ;Rosa26 mTmG control kidneys revealed that Tcf21 expression peaks at embryonic day 12, crucial for early JG cell specification. Subsequent analyses confirmed Tcf21's critical role in early kidney development, with expression declining as development progresses. Our results highlight the temporal and spatial dynamics of Tcf21, showing its importance in the early specification of Foxd1+ cells into JG cells. These findings provide new insights into the molecular mechanisms governing JG cell differentiation and underscore Tcf21's pivotal role in kidney development. The data suggest that Tcf21 expression in Foxd1+ progenitors is essential for the specification of renin-expressing JG cells, but once renin cell identity is assumed, Tcf21 becomes redundant. NEW & NOTEWORTHY: This manuscript provides novel insights into the role of Tcf21 in the differentiation of Foxd1+ cells into JG cells. Utilizing integrated scRNA-seq and scATAC-seq, the study reveals that Tcf21 expression is crucial during early embryonic stages, with its peak at embryonic day 12. The findings demonstrate that inactivation of Tcf21 leads to fewer renin-positive areas and altered renal arterial morphology, underscoring the importance of Tcf21 in the specification of renin-expressing JG cells and kidney development.

19.
PLoS Genet ; 6(6): e1000991, 2010 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-20577567

RESUMO

Although more than 2,400 genes have been shown to contain variants that cause Mendelian disease, there are still several thousand such diseases yet to be molecularly defined. The ability of new whole-genome sequencing technologies to rapidly indentify most of the genetic variants in any given genome opens an exciting opportunity to identify these disease genes. Here we sequenced the whole genome of a single patient with the dominant Mendelian disease, metachondromatosis (OMIM 156250), and used partial linkage data from her small family to focus our search for the responsible variant. In the proband, we identified an 11 bp deletion in exon four of PTPN11, which alters frame, results in premature translation termination, and co-segregates with the phenotype. In a second metachondromatosis family, we confirmed our result by identifying a nonsense mutation in exon 4 of PTPN11 that also co-segregates with the phenotype. Sequencing PTPN11 exon 4 in 469 controls showed no such protein truncating variants, supporting the pathogenicity of these two mutations. This combination of a new technology and a classical genetic approach provides a powerful strategy to discover the genes responsible for unexplained Mendelian disorders.


Assuntos
Ligação Genética , Predisposição Genética para Doença , Genoma Humano , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Éxons , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Mutação , Linhagem , Análise de Sequência de DNA
20.
PLoS Genet ; 6(9): e1001111, 2010 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-20838461

RESUMO

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten "case" genomes from individuals with severe hemophilia A and ten "control" genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.


Assuntos
Genoma Humano/genética , Análise de Sequência de DNA , Sequência de Bases , Estudos de Casos e Controles , Variações do Número de Cópias de DNA/genética , Bases de Dados Genéticas , Éxons/genética , Fator VIII/genética , Duplicação Gênica/genética , Técnicas de Inativação de Genes , Genética Populacional , Genótipo , Hemofilia A/genética , Humanos , Mutação INDEL/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único/genética
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