RESUMEN
OBJECTIVE: Hypercholesterolemia and hypertension are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in hypercholesterolemic/hypertensive mice. APPROACH AND RESULTS: Control, hypertensive, hypercholesterolemic (Apoe(-/-)), and hypercholesterolemic/hypertensive mice were studied. Severe aortic stenosis (echocardiography) occurred only in hypercholesterolemic/hypertensive mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or seam between leaflets) was longer in hypercholesterolemic/hypertensive mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In hypercholesterolemic/hypertensive mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA sequencing was used to identify molecular targets during the developmental phase of stenosis. Genes related to the structure of the valve were identified, which differentially expressed before fibrotic AVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1, were increased greatly in hypercholesterolemic/hypertensive mice. CONCLUSIONS: Hypercholesterolemic/hypertensive mice are the first model of fibrotic AVS. Hypercholesterolemic/hypertensive mice develop severe AVS in the absence of significant calcification, a feature that resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to the development of fibrotic AVS.
Asunto(s)
Estenosis de la Válvula Aórtica/etiología , Válvula Aórtica/patología , Hipercolesterolemia/complicaciones , Hipertensión/complicaciones , Angiotensinógeno/genética , Angiotensinógeno/metabolismo , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/fisiopatología , Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/fisiopatología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Modelos Animales de Enfermedad , Femenino , Fibrosis , Regulación de la Expresión Génica , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipertensión/genética , Hipertensión/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Renina/genética , Renina/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Rigor, reproducibility, and transparency (RR&T) are essential components of all scientific pursuits. Shared research resources, also known as core facilities, are on the frontlines of ensuring robust RR&T practices. The Association of Biomolecular Resource Facilities Committee on Core Rigor and Reproducibility conducted a follow-up survey 4 years after the initial 2017 survey to determine if core facilities have seen a positive impact of new RR&T initiatives (including guidance from the National Institutes of Health, new scientific journal requirements on transparency and data provenance, and educational tools from professional organizations). While there were fewer participants in the most recent survey, the respondents' opinions on the role of core facilities and level of best practices adoption remained the same. Overall, the respondents agreed that procedures should be implemented by core facilities to ensure scientific RR&T. They also indicated that there is a strong correlation between institutions that emphasize RR&T and core customers using this expertise in grant applications and publications. The survey also assessed the impact of the COVID-19 pandemic on core operations and RR&T. The answers to these pandemic-related questions revealed that many of the strategies aimed at increasing efficiencies are also best practices related to RR&T, including the development of standard operating procedures, supply chain management, and cross training. Given the consistent and compelling awareness of the importance of RR&T expressed by core directors in 2017 and 2021 contrasted with the lack of apparent improvements over this time period, the authors recommend an adoption of RR&T statements by all core laboratories. Adhering to the RR&T guidelines will result in more efficient training, better compliance, and improved experimental approaches empowering cores to become "rigor champions."
Asunto(s)
COVID-19 , Pandemias , Humanos , Reproducibilidad de los Resultados , Estudios de Seguimiento , Encuestas y CuestionariosRESUMEN
Prolonged obesity is associated with blunted feeding and thermogenic autonomic responses to leptin, but cardiovascular responses to leptin are maintained. This state of selective leptin resistance is, therefore, proposed to contribute to the pathogenesis and maintenance of obesity-associated hypertension. Cells of the arcuate nucleus of the hypothalamus detect leptin, and although the cellular and molecular mechanisms remain unclear, altered arcuate nucleus biology is hypothesized to contribute to selective leptin resistance. Male C57BL/6J mice were fed a high-fat diet (HFD) or chow from 8 to 18 weeks of age, as this paradigm models selective leptin resistance. Nuclei were then isolated from arcuate nucleus for single-nucleus RNA sequencing. HFD caused expected gains in adiposity and circulating leptin. Twenty-three unique cell-type clusters were identified, and Ingenuity Pathway Analysis was used to explore changes in gene expression patterns due to chronic HFD within each cluster. Notably, gene expression signatures related to leptin signaling exhibited suppression predominantly in neurons identified as the Agouti-related peptide (Agrp) subtype. Ingenuity Pathway Analysis results were also consistent with alterations in CREB (cAMP response element-binding protein) signaling in Agrp neurons after HFD, and reduced phosphorylated CREB was confirmed in arcuate nucleus after prolonged HFD by capillary electrophoresis-based Western blotting. These findings support the concept that prolonged HFD-induced obesity is associated with selective changes in Agrp neuron biology, possibly secondary to altered CREB signaling.
Asunto(s)
Adiposidad/fisiología , Núcleo Arqueado del Hipotálamo/metabolismo , Dieta Alta en Grasa/efectos adversos , Neuronas/metabolismo , Obesidad/metabolismo , Proteína Relacionada con Agouti/genética , Proteína Relacionada con Agouti/metabolismo , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Leptina/sangre , Masculino , Ratones , Obesidad/etiología , Obesidad/genética , Fosforilación , Análisis de Secuencia de ARN , Transducción de Señal/fisiologíaRESUMEN
Shared research resource facilities, also known as core laboratories (Cores), are responsible for generating a significant and growing portion of the research data in academic biomedical research institutions. Cores represent a central repository for institutional knowledge management, with deep expertise in the strengths and limitations of technology and its applications. They inherently support transparency and scientific reproducibility by protecting against cognitive bias in research design and data analysis, and they have institutional responsibility for the conduct of research (research ethics, regulatory compliance, and financial accountability) performed in their Cores. The Association of Biomolecular Resource Facilities (ABRF) is a FASEB-member scientific society whose members are scientists and administrators that manage or support Cores. The ABRF Research Groups (RGs), representing expertise for an array of cutting-edge and established technology platforms, perform multicenter research studies to determine and communicate best practices and community-based standards. This review provides a summary of the contributions of the ABRF RGs to promote scientific rigor and reproducibility in Cores from the published literature, ABRF meetings, and ABRF RGs communications.
Asunto(s)
Investigación Biomédica/normas , Laboratorios/normas , Reproducibilidad de los Resultados , Investigación Biomédica/organización & administración , Biología Computacional/métodos , Biología Computacional/normas , Citometría de Flujo/métodos , Citometría de Flujo/normas , Genómica/métodos , Genómica/normas , Humanos , Laboratorios/organización & administración , Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Metabolómica/métodos , Metabolómica/normas , Microscopía/métodos , Microscopía/normas , Proteómica/métodos , Proteómica/normasRESUMEN
Shared scientific resources, also known as core facilities, support a significant portion of the research conducted at biomolecular research institutions. The Association of Biomolecular Resource Facilities (ABRF) established the Committee on Core Rigor and Reproducibility (CCoRRe) to further its mission of integrating advanced technologies, education, and communication in the operations of shared scientific resources in support of reproducible research. In order to first assess the needs of the scientific shared resource community, the CCoRRe solicited feedback from ABRF members via a survey. The purpose of the survey was to gain information on how U.S. National Institutes of Health (NIH) initiatives on advancing scientific rigor and reproducibility influenced current services and new technology development. In addition, the survey aimed to identify the challenges and opportunities related to implementation of new reporting requirements and to identify new practices and resources needed to ensure rigorous research. The results revealed a surprising unfamiliarity with the NIH guidelines. Many of the perceived challenges to the effective implementation of best practices (i.e., those designed to ensure rigor and reproducibility) were similarly noted as a challenge to effective provision of support services in a core setting. Further, most cores routinely use best practices and offer services that support rigor and reproducibility. These services include access to well-maintained instrumentation and training on experimental design and data analysis as well as data management. Feedback from this survey will enable the ABRF to build better educational resources and share critical best-practice guidelines. These resources will become important tools to the core community and the researchers they serve to impact rigor and transparency across the range of science and technology.
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Investigación Biomédica/normas , Reproducibilidad de los Resultados , Proyectos de Investigación/normas , Investigación Biomédica/legislación & jurisprudencia , Investigación Biomédica/métodos , Costos y Análisis de Costo , Equipos y Suministros/normas , Equipos y Suministros/provisión & distribución , Humanos , National Institutes of Health (U.S.) , Guías de Práctica Clínica como Asunto , Investigadores , Encuestas y Cuestionarios , Factores de Tiempo , Estados UnidosRESUMEN
The DNA Sequencing Research Group (DSRG) of the ABRF conducted a study to assess the ability of DNA sequencing core facilities to successfully sequence a set of well-defined templates containing difficult repeats. The aim of this study was to determine whether repetitive templates could be sequenced accurately by using equipment and chemistries currently utilized in participating sequencing laboratories. The effects of primer and template concentrations, sequencing chemistries, additives, and instrument formats on the ability to successfully sequence repeat elements were examined. The first part of this study was an analysis of the results of 361 chromatograms from participants representing 40 different laboratories who attempted to sequence a panel of difficult-to-sequence templates using their best in-house protocols. The second part of this study was a smaller multi-laboratory evaluation of a single robust protocol with the same panel of templates. This study provides a measure of the potential success of different approaches to sequencing across homopolymer tracts and repetitive elements.
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ADN/química , Proteómica/métodos , Análisis de Secuencia de ADN/métodos , Animales , Cromatografía , Cartilla de ADN/química , Estudios de Evaluación como Asunto , Ratones , Reproducibilidad de los Resultados , Análisis de Secuencia/métodosRESUMEN
Over the past several years, microarray technology has evolved into a critical component of any discovery-based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a profile of microarray service laboratories and, more importantly, an overview of technology development and implementation. Survey questions addressed instrumentation, protocols, staffing, funding, and work flow in a microarray facility. Presented herein are the results of the MARG 2005 survey; where possible, trends in the field are discussed and compared to data collected from previous surveys.
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Biología Computacional/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteómica/métodos , Proteómica/tendencias , Animales , Interpretación Estadística de Datos , Genómica/métodos , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Proteómica/instrumentación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Programas InformáticosRESUMEN
The mouse is useful in studies of vascular biology because of its well-defined genetics and because the mouse genome can be manipulated. However, because only small amounts of mRNA can be extracted from blood vessels, the quantification of gene expression in individual mice is difficult. Endothelial NO synthase (eNOS) plays a major role in the regulation of vascular tone and growth. In addition, there appear to be sex differences in the production of NO under basal conditions in mouse aortas. The goals of this study were to develop a real-time polymerase chain reaction (PCR) method to quantify eNOS mRNA in blood vessels from mice and to examine eNOS mRNA levels in vessels from male and female mice. Blood vessels were isolated from C57BL/6 mice. Total RNA from individual mice was isolated and reverse-transcribed. The number of molecules of eNOS mRNA (after reverse transcription) was determined against cDNA standards, with 18S rRNA used as a control for RNA input and reverse-transcription efficiency. When expressed as copy numbers per nanogram of total RNA or as the ratio of eNOS to 18S rRNA, eNOS mRNA was lower in the aortas of female mice than in those of male mice at 7 to 9 months of age. In contrast, no difference in eNOS mRNA was found in the aortas of 2-month-old mice. In addition, eNOS mRNA levels were similar in the carotid, cerebral, and coronary arteries. These findings provide the first quantitative measurements of eNOS mRNA by using real-time PCR in the vessels of mice and suggest age- and sex-related differences in the basal levels of eNOS mRNA in mice. In addition, the eNOS region that was used for real-time PCR was amplified and sequenced for monkeys and other species. With modifications, this region may be used to design real-time PCR for eNOS in other species.
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Vasos Sanguíneos/enzimología , Óxido Nítrico Sintasa/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , ARN Ribosómico 18S/aislamiento & purificación , Caracteres SexualesRESUMEN
Glaucoma and age-related macular degeneration (AMD) are the two leading causes of visual loss in the United States. We utilized a novel study design to perform a genome-wide association for both primary open angle glaucoma (POAG) and AMD. This study design utilized a two-stage process for hypothesis generation and validation, in which each disease cohort was utilized as a control for the other. A total of 400 POAG patients and 400 AMD patients were ascertained and genotyped at 500,000 loci. This study identified a novel association of complement component 7 (C7) to POAG. Additionally, an association of central corneal thickness, a known risk factor for POAG, was found to be associated with ribophorin II (RPN2). Linked monogenic loci for POAG and AMD were also evaluated for evidence of association, none of which were found to be significantly associated. However, several yielded putative associations requiring validation. Our data suggest that POAG is more genetically complex than AMD, with no common risk alleles of large effect.
Asunto(s)
Estudio de Asociación del Genoma Completo , Glaucoma de Ángulo Abierto/genética , Degeneración Macular/genética , Sitios de Carácter Cuantitativo , Anciano , Alelos , Secuencia de Aminoácidos , Complemento C7/química , Complemento C7/genética , Córnea/patología , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Glaucoma de Ángulo Abierto/patología , Humanos , Degeneración Macular/patología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Carácter Cuantitativo Heredable , Alineación de SecuenciaRESUMEN
We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F(2) rats generated from an SR/JrHsd x SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (alpha = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5' flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor beta2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.
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Oftalmopatías/genética , Ojo/metabolismo , Regulación de la Expresión Génica , Análisis de Varianza , Animales , Mapeo Cromosómico , Ligamiento Genético , Marcadores Genéticos , Humanos , Sitios de Carácter Cuantitativo/genética , Ratas , Ratas Endogámicas , Tamaño de la MuestraRESUMEN
Specific binding of IGF-binding protein (IGFBP)-3 was shown to be present in the isolated, beating rat heart. The uptake of perfused (125)I-labeled IGF-I in the beating heart was decreased to 9% by blocking IGF-I binding sites with the IGF-I analog Long R(3) (LR(3)) IGF-I. When LR(3) was perfused with complexes of (125)I-IGF-I. IGFBP-3, uptake of (125)I-IGF-I was decreased to 41%, which was significantly greater than LR(3) and (125)I-IGF-I (41 vs. 9%). These data suggest that both microvessel IGF-I and IGFBP-3 binding sites contribute to the transport of IGF-I in the perfused rat heart. This also suggests a novel and plausible mechanism whereby circulating IGFs reach sites of IGF bioactivity.