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Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403+/-) with Lpar1-ablated mice to create mice carrying both genetic changes (403+/- LPAR1 -/-) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT, 403+/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a. Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.
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Cardiomiopatía Hipertrófica , Receptores del Ácido Lisofosfatídico/genética , Animales , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Proteínas Portadoras , Modelos Animales de Enfermedad , Células Endoteliales/patología , Fibrosis , Hipertrofia/patología , RatonesRESUMEN
Quality Improvement Success Stories are published by the American Diabetes Association in collaboration with the American College of Physicians and the National Diabetes Education Program. This series is intended to highlight best practices and strategies from programs and clinics that have successfully improved the quality of care for people with diabetes or related conditions. Each article in the series is reviewed and follows a standard format developed by the editors of Clinical Diabetes. The following article describes a Brooklyn, NY, hospital's initiative to reduce high A1C rates among its patients.
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The modular cell design principle is formulated to devise modular (chassis) cells. These cells can be assembled with exchangeable production modules in a plug-and-play fashion to build microbial cell factories for efficient combinatorial biosynthesis of novel molecules, requiring minimal iterative strain optimization steps. A modular cell is designed to be auxotrophic, containing core metabolic pathways that are necessary but insufficient to support cell growth and maintenance. To be functional, it must tightly couple with an exchangeable production module containing auxiliary metabolic pathways that not only complement cell growth but also enhance production of targeted molecules. We developed a MODCELL (modular cell) framework based on metabolic pathway analysis to implement the modular cell design principle. MODCELL identifies genetic modifications and requirements to construct modular cell candidates and their associated exchangeable production modules. By defining the degree of similarity and coupling metrics, MODCELL can evaluate which exchangeable production module(s) can be tightly coupled with a modular cell candidate. We first demonstrated how MODCELL works in a step-by-step manner for example metabolic networks, and then applied it to design modular Escherichia coli cells for efficient combinatorial biosynthesis of five alcohols (ethanol, propanol, isopropanol, butanol and isobutanol) and five butyrate esters (ethyl butyrate, propyl butyrate, isopropyl butyrate, butyl butyrate and isobutyl butyrate) from pentose sugars (arabinose and xylose) and hexose sugars (glucose, mannose, and galactose) under anaerobic conditions. We identified three modular cells, MODCELL1, MODCELL2 and MODCELL3, that can couple well with Group 1 of modules (ethanol, isobutanol, butanol, ethyl butyrate, isobutyl butyrate, butyl butyrate), Group 2 (isopropanol, isopropyl butyrate), and Group 3 (propanol, isopropanol), respectively. We validated the design of MODCELL1 for anaerobic production of ethanol, butanol, and ethyl butyrate using experimental data available in literature.
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Células/metabolismo , Ingeniería Metabólica/métodos , Alcoholes/metabolismo , Algoritmos , Anaerobiosis , Metabolismo de los Hidratos de Carbono/genética , Carbohidratos , Ésteres/metabolismo , Redes y Vías Metabólicas , Reproducibilidad de los ResultadosRESUMEN
To trigger an effective immune response, antigen and antigen-presenting cells travel to the lymph nodes via collecting lymphatic vessels. However, our understanding of the regulation of collecting lymphatic vessel function and lymph transport is limited. To dissect the molecular control of lymphatic function, we developed a unique mouse model that allows intravital imaging of autonomous lymphatic vessel contraction. Using this method, we demonstrated that endothelial nitric oxide synthase (eNOS) in lymphatic endothelial cells is required for robust lymphatic contractions under physiological conditions. By contrast, under inflammatory conditions, inducible NOS (iNOS)-expressing CD11b(+)Gr-1(+) cells attenuate lymphatic contraction. This inhibition of lymphatic contraction was associated with a reduction in the response to antigen in a model of immune-induced multiple sclerosis. These results suggest the suppression of lymphatic function by the CD11b(+)Gr-1(+) cells as a potential mechanism of self-protection from autoreactive responses during on-going inflammation. The central role for nitric oxide also suggests that other diseases such as cancer and infection may also mediate lymphatic contraction and thus immune response. Our unique method allows the study of lymphatic function and its molecular regulation during inflammation, lymphedema, and lymphatic metastasis.
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Terapia de Inmunosupresión , Sistema Linfático/fisiología , Vasos Linfáticos/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Antígeno CD11b/biosíntesis , Sistema Inmunológico , Inflamación , Cinética , Metástasis Linfática , Vasos Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Microscopía/métodos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oxazolona/farmacología , Piel/efectos de los fármacosRESUMEN
The regeneration of alveolar epithelial cells is a critical aspect of alveolar reorganization after lung injury. Although alveolar Type II (AT2) cells have been described as progenitor cells for alveolar epithelia, more remains to be understood about how their progenitor cell properties are regulated. A nuclear, chromatin-bound green fluorescence protein reporter (H2B-GFP) was driven from the murine surfactant protein-C (SPC) promoter to generate SPC H2B-GFP transgenic mice. The SPC H2B-GFP allele allowed the FACS-based enrichment and gene expression profiling of AT2 cells. Approximately 97% of AT2 cells were GFP-labeled on Postnatal Day 1, and the percentage of GFP-labeled AT2 cells decreased to approximately 63% at Postnatal Week 8. Isolated young adult SPC H2B-GFP(+) cells displayed proliferation, differentiation, and self-renewal capacity in the presence of lung fibroblasts in a Matrigel-based three-dimensional culture system. Heterogeneity within the GFP(+) population was revealed, because cells with distinct alveolar and bronchiolar gene expression arose in three-dimensional cultures. CD74, a surface marker highly enriched on GFP(+) cells, was identified as a positive selection marker, providing 3-fold enrichment for AT2 cells. In vivo, GFP expression was induced within other epithelial cell types during maturation of the distal lung. The utility of the SPC H2B-GFP murine model for the identification of AT2 cells was greatest in early postnatal lungs and more limited with age, when some discordance between SPC and GFP expression was observed. In adult mice, this allele may allow for the enrichment and future characterization of other SPC-expressing alveolar and bronchiolar cells, including putative stem/progenitor cell populations.
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Cromatina/metabolismo , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Pulmón/metabolismo , Proteína C Asociada a Surfactante Pulmonar/metabolismo , Alelos , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Bronquiolos/citología , Bronquiolos/metabolismo , Diferenciación Celular/genética , Procesos de Crecimiento Celular/genética , Células Cultivadas , Cromatina/genética , Células Epiteliales/citología , Femenino , Fibroblastos/citología , Expresión Génica , Perfilación de la Expresión Génica/métodos , Proteínas Fluorescentes Verdes/genética , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Pulmón/citología , Lesión Pulmonar/genética , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Proteína C Asociada a Surfactante Pulmonar/biosíntesis , Proteína C Asociada a Surfactante Pulmonar/genética , Regeneración/genética , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismoRESUMEN
Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies-an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9-to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.
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Cardiomiopatía Hipertrófica , Edición Génica , Animales , Ratones , Mutación Missense , Miocitos Cardíacos , ARNRESUMEN
Otitis media is an extremely common pediatric inflammation of the middle ear that often causes pain and diminishes hearing. Vulnerability to otitis media is due to eustachian tube dysfunction as well as other poorly understood factors, including genetic susceptibility. As EYA4 mutations cause sensorineural hearing loss in humans, we produced and characterized Eya4-deficient (Eya4(-/-)) mice, which had severe hearing deficits. In addition, all Eya4(-/-) mice developed otitis media with effusion. Anatomic studies revealed abnormal middle ear cavity and eustachian tube dysmorphology; thus, Eya4 regulation is critical for the development and function of these structures. We suggest that some human otitis media susceptibility reflects underlying genetic predisposition in genes like EYA4 that regulate middle ear and eustachian tube anatomy.
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Oído Medio/anomalías , Trompa Auditiva/anomalías , Predisposición Genética a la Enfermedad , Pérdida Auditiva Sensorineural/genética , Otitis Media con Derrame/genética , Transactivadores/genética , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Mutantes , MutaciónRESUMEN
LMNA is one of the leading causative genes of genetically inherited dilated cardiomyopathy (DCM). Unlike most DCM-causative genes, which encode sarcomeric or sarcomere-related proteins, LMNA encodes nuclear envelope proteins, lamin A and C, and does not directly associate with contractile function. However, a mutation in this gene could lead to the development of DCM. The molecular mechanism of how LMNA mutation contributes to DCM development remains largely unclear and yet to be elucidated. The objective of this study was to clarify the mechanism of developing DCM caused by LMNA mutation. Methods and Results: We assessed cardiomyocyte phenotypes and characteristics focusing on cell cycle activity in mice with Lmna mutation. Both cell number and cell size were reduced, cardiomyocytes were immature, and cell cycle activity was retarded in Lmna mutant mice at both 5 weeks and 2 years of age. RNA-sequencing and pathway analysis revealed "proliferation of cells" had the most substantial impact on Lmna mutant mice. Cdkn1a, which encodes the cell cycle regulating protein p21, was strongly upregulated in Lmna mutants, and upregulation of p21 was confirmed by Western blot and immunostaining. DNA damage, which is known to upregulate Cdkn1a, was more abundantly detected in Lmna mutant mice. To assess the proliferative capacity of cardiomyocytes, the apex of the neonate mouse heart was resected, and recovery from the insult was observed. A restricted cardiomyocyte proliferating capacity after resecting the apex of the heart was observed in Lmna mutant mice. Conclusions: Our results strongly suggest that loss of lamin function contributes to impaired cell proliferation through cell cycle defects. The inadequate inborn or responsive cell proliferation capacity plays an essential role in developing DCM with LMNA mutation.
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Background Human mutations in the X-linked lysosome-associated membrane protein-2 (LAMP2) gene can cause a multisystem Danon disease or a primary cardiomyopathy characterized by massive hypertrophy, conduction system abnormalities, and malignant ventricular arrhythmias. We introduced an in-frame LAMP2 gene exon 6 deletion mutation (denoted L2Δ6) causing human cardiomyopathy, into mouse LAMP2 gene, to elucidate its consequences on cardiomyocyte biology. This mutation results in in-frame deletion of 41 amino acids, compatible with presence of some defective LAMP2 protein. Methods and Results Left ventricular tissues from L2Δ6 and wild-type mice had equivalent amounts of LAMP2 RNA, but a significantly lower level of LAMP2 protein. By 20 weeks of age male mutant mice developed left ventricular hypertrophy which was followed by left ventricular dilatation and reduced systolic function. Cardiac electrophysiology and isolated cardiomyocyte studies demonstrated ventricular arrhythmia, conduction disturbances, abnormal calcium transients and increased sensitivity to catecholamines. Myocardial fibrosis was strikingly increased in 40-week-old L2Δ6 mice, recapitulating findings of human LAMP2 cardiomyopathy. Immunofluorescence and transmission electron microscopy identified mislocalization of lysosomes and accumulation of autophagosomes between sarcomeres, causing profound morphological changes disrupting the cellular ultrastructure. Transcription profile and protein expression analyses of L2Δ6 hearts showed significantly increased expression of genes encoding activators and protein components of autophagy, hypertrophy, and apoptosis. Conclusions We suggest that impaired autophagy results in cardiac hypertrophy and profound transcriptional reactions that impacted metabolism, calcium homeostasis, and cell survival. These responses define the molecular pathways that underlie the pathology and aberrant electrophysiology in cardiomyopathy of Danon disease.
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Cardiomiopatías/genética , Proteína 2 de la Membrana Asociada a los Lisosomas , Animales , Arritmias Cardíacas/genética , Autofagia , Calcio , Cardiomegalia , Enfermedad por Depósito de Glucógeno de Tipo IIb/genética , Hipertrofia Ventricular Izquierda , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Masculino , RatonesRESUMEN
Catecholamine-induced polymorphic ventricular tachycardia (CPVT) is a familial disorder caused by cardiac ryanodine receptor type 2 (RyR2) or calsequestrin 2 (CASQ2) gene mutations. To define how CASQ2 mutations cause CPVT, we produced and studied mice carrying a human D307H missense mutation (CASQ(307/307)) or a CASQ2-null mutation (CASQ(DeltaE9/DeltaE9)). Both CASQ2 mutations caused identical consequences. Young mutant mice had structurally normal hearts but stress-induced ventricular arrhythmias; aging produced cardiac hypertrophy and reduced contractile function. Mutant myocytes had reduced CASQ2 and increased calreticulin and RyR2 (with normal phosphorylated proportions) but unchanged calstabin levels, as well as reduced total sarcoplasmic reticulum (SR) Ca(2+), prolonged Ca(2+) release, and delayed Ca(2+) reuptake. Stress further diminished Ca(2+) transients, elevated cytosolic Ca(2+), and triggered frequent, spontaneous SR Ca(2+) release. Treatment with Mg(2+), a RyR2 inhibitor, normalized myocyte Ca(2+) cycling and decreased CPVT in mutant mice, indicating RyR2 dysfunction was critical to mutant CASQ2 pathophysiology. We conclude that CPVT-causing CASQ2 missense mutations function as null alleles. In the absence of CASQ2, calreticulin, a fetal Ca(2+)-binding protein normally downregulated at birth, remains a prominent SR component. Adaptive changes to CASQ2 deficiency (increased posttranscriptional expression of calreticulin and RyR2) maintained electrical-mechanical coupling, but increased RyR2 leakiness, a paradoxical response further exacerbated by stress. The central role of RyR2 dysfunction in CASQ2 deficiency unifies the pathophysiologic mechanism underlying CPVT due to RyR2 or CASQ2 mutations and suggests a therapeutic approach for these inherited cardiac arrhythmias.
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Calreticulina/metabolismo , Calsecuestrina/metabolismo , Catecolaminas/biosíntesis , Regulación de la Expresión Génica , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patología , Animales , Calcio/metabolismo , Calsecuestrina/deficiencia , Calsecuestrina/genética , Electrofisiología , Exones/genética , Eliminación de Gen , Magnesio/metabolismo , Ratones , Ratones Noqueados , Mutación/genética , ARN/genética , Retículo Sarcoplasmático/metabolismo , Taquicardia Ventricular/genéticaRESUMEN
The bromodomain and extraterminal (BET) family comprises epigenetic reader proteins that are important regulators of inflammatory and hypertrophic gene expression in the heart. We previously identified the activation of proinflammatory gene networks as a key early driver of dilated cardiomyopathy (DCM) in transgenic mice expressing a mutant form of phospholamban (PLNR9C) - a genetic cause of DCM in humans. We hypothesized that BETs coactivate this inflammatory process, representing a critical node in the progression of DCM. To test this hypothesis, we treated PLNR9C or age-matched WT mice longitudinally with the small molecule BET bromodomain inhibitor JQ1 or vehicle. BET inhibition abrogated adverse cardiac remodeling, reduced cardiac fibrosis, and prolonged survival in PLNR9C mice by inhibiting expression of proinflammatory gene networks at all stages of disease. Specifically, JQ1 had profound effects on proinflammatory gene network expression in cardiac fibroblasts, while having little effect on gene expression in cardiomyocytes. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Mechanistically, we demonstrated that BRD4 serves as a direct and essential regulator of NF-κB-mediated proinflammatory gene expression in cardiac fibroblasts. Suppressing proinflammatory gene expression via BET bromodomain inhibition could be a novel therapeutic strategy for chronic DCM in humans.
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Azepinas/farmacología , Proteínas de Unión al Calcio/fisiología , Cardiomiopatía Dilatada/prevención & control , Fibrosis/prevención & control , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Animales , Cardiomiopatía Dilatada/etiología , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Fibrosis/etiología , Fibrosis/metabolismo , Fibrosis/patología , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
CLINICAL TRIAL REGISTRATION: clinicaltrials.gov (NCT02709135).
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Enfermedades Cardiovasculares/diagnóstico , Troponina/sangre , Adulto , Anciano , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sistemas de Atención de Punto , Medición de Riesgo , Factores de Riesgo , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2, and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.
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Diafragma/crecimiento & desarrollo , Factor de Transcripción GATA6/genética , Corazón/crecimiento & desarrollo , Células Madre Pluripotentes Inducidas/metabolismo , Páncreas/crecimiento & desarrollo , Diferenciación Celular/genética , Epigénesis Genética/genética , Perfilación de la Expresión Génica , Humanos , Mutación Missense/genética , Miocitos Cardíacos/metabolismoRESUMEN
Mutations in the lamin A/C (LMNA) gene, which encodes nuclear membrane proteins, cause a variety of human conditions including dilated cardiomyopathy (DCM) with associated cardiac conduction system disease. To investigate mechanisms responsible for electrophysiologic and myocardial phenotypes caused by dominant human LMNA mutations, we performed longitudinal evaluations in heterozygous Lmna(+/-) mice. Despite one normal allele, Lmna(+/-) mice had 50% of normal cardiac lamin A/C levels and developed cardiac abnormalities. Conduction system function was normal in neonatal Lmna(+/-) mice but, by 4 weeks of age, atrioventricular (AV) nodal myocytes had abnormally shaped nuclei and active apoptosis. Telemetric and in vivo electrophysiologic studies in 10-week-old Lmna(+/-) mice showed AV conduction defects and both atrial and ventricular arrhythmias, analogous to those observed in humans with heterozygous LMNA mutations. Isolated myocytes from 12-month-old Lmna(+/-) mice exhibited impaired contractility. In vivo cardiac studies of aged Lmna(+/-) mice revealed DCM; in some mice this occurred without overt conduction system disease. However, neither histopathology nor serum CK levels indicated skeletal muscle pathology. These data demonstrate cardiac pathology due to heterozygous Lmna mutations reflecting a 50% reduction in lamin protein levels. Lamin haploinsufficiency caused early-onset programmed cell death of AV nodal myocytes and progressive electrophysiologic disease. While lamin haploinsufficiency was better tolerated by non-conducting myocytes, ultimately, these too succumbed to diminished lamin levels leading to dilated cardiomyopathy, which presumably arose independently from conduction system disease.
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Apoptosis , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Sistema de Conducción Cardíaco/patología , Lamina Tipo A/genética , Edad de Inicio , Animales , Arritmias Cardíacas/diagnóstico por imagen , Arritmias Cardíacas/enzimología , Nodo Atrioventricular/patología , Cardiomiopatía Dilatada/diagnóstico por imagen , Cardiomiopatía Dilatada/enzimología , Núcleo Celular/patología , Separación Celular , Electrofisiología , Sistema de Conducción Cardíaco/enzimología , Heterocigoto , Etiquetado Corte-Fin in Situ , Lamina Tipo A/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Enfermedades Musculares/patología , Miocardio/metabolismo , Miocardio/patología , Telemetría , UltrasonografíaRESUMEN
Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology use homology-directed repair (HDR) to efficiently generate custom hiPSC lines harboring a variety of genomic insertions and deletions. Thus, hiPSCs that encode an endogenous protein fused to a fluorescent reporter protein can be rapidly created by employing CRISPR/Cas9 genome editing, enhancing HDR efficiency and optimizing homology arm length. These fluorescently tagged hiPSCs can be used to visualize protein function and dynamics in real time as cells proliferate and differentiate. Given that nearly any intracellular protein can be fluorescently tagged, this system serves as a powerful tool to facilitate new discoveries across many biological disciplines. In this unit, we present protocols for the design, generation, and monoclonal expansion of genetically customized hiPSCs encoding fluorescently tagged endogenous proteins. © 2018 by John Wiley & Sons, Inc.
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Sistemas CRISPR-Cas/genética , Terapia Genética , Células Madre Pluripotentes Inducidas/citología , Reparación del ADN por Recombinación/genética , Fluorescencia , Edición Génica , Genoma Humano/genética , HumanosRESUMEN
Dilated cardiomyopathy (DCM) is defined by progressive functional and structural changes. We performed RNA-seq at different stages of disease to define molecular signaling in the progression from pre-DCM hearts to DCM and overt heart failure (HF) using a genetic model of DCM (phospholamban missense mutation, PLNR9C/+). Pre-DCM hearts were phenotypically normal yet displayed proliferation of nonmyocytes (59% relative increase vs. WT, P = 8 × 10-4) and activation of proinflammatory signaling with notable cardiomyocyte-specific induction of a subset of profibrotic cytokines including TGFß2 and TGFß3. These changes progressed through DCM and HF, resulting in substantial fibrosis (17.6% of left ventricle [LV] vs. WT, P = 6 × 10-33). Cardiomyocytes displayed a marked shift in metabolic gene transcription: downregulation of aerobic respiration and subsequent upregulation of glucose utilization, changes coincident with attenuated expression of PPARα and PPARγ coactivators -1α (PGC1α) and -1ß, and increased expression of the metabolic regulator T-box transcription factor 15 (Tbx15). Comparing DCM transcriptional profiles with those in hypertrophic cardiomyopathy (HCM) revealed similar and distinct molecular mechanisms. Our data suggest that cardiomyocyte-specific cytokine expression, early fibroblast activation, and the shift in metabolic gene expression are hallmarks of cardiomyopathy progression. Notably, key components of these profibrotic and metabolic networks were disease specific and distinguish DCM from HCM.
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BACKGROUND: The American College of Physicians recommends perioperative use of beta-blockers for certain patients to improve outcomes after surgery. Study of physician behavior with respect to guidelines and recommended practices have shown that beta-blockers have been underutilized after myocardial infarction. We evaluated physician concordance with the perioperative use of beta-blockers along with a specialty-related difference in the frequency of perioperative beta-blocker use. METHODS: To determine perioperative use of beta-blockers, we retrospectively analyzed the medical charts of adult patients who underwent open cholecystectomy at a tertiary care medical center from December 1997 through December 2001. Patients met criteria for perioperative beta-blocker use if they had a history of coronary artery disease or if they had the presence of 2 or more of the following risk factors: 65 years or older; history of hypertension, diabetes mellitus, or hypercholesterolemia; or current smoking. RESULTS: Among the 336 cases of cholecystectomy reviewed, criteria for beta-blocker use were met in 146 patients (43%) who did not have emergency operations and/or contraindications to beta-blocker use. Of these 146 patients, 123 (84%) had a documented preoperative medical evaluation by a physician in the medical chart. There were 44 patients (30%) receiving beta-blockers prior to admission, and 102 patients (70%) were not receiving beta-blockers. Of those 102 patients not receiving beta-blockers at admission but who meet criteria for their use, 94 (92%) were not started on beta-blocker therapy preoperatively. Of the 18 patients evaluated by a cardiologist, 4 (22%) were started on beta-blocker therapy compared with 3 (6%) of 47 patients evaluated by a noncardiologist physician (P =.08). CONCLUSION: Perioperative beta-blocker therapy is underutilized in patients with risk factors for coronary artery disease despite evidence that its use in appropriate individuals may be lifesaving.
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Antagonistas Adrenérgicos beta/uso terapéutico , Enfermedad de la Arteria Coronaria/prevención & control , Adhesión a Directriz , Atención Perioperativa/normas , Pautas de la Práctica en Medicina , Adulto , Anciano , Anciano de 80 o más Años , Colecistectomía , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Enfermedad de la Arteria Coronaria/epidemiología , Utilización de Medicamentos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Estados UnidosRESUMEN
OBJECTIVE: To compare medical care provided by hospitalists and primary care physicians to patients with community-acquired pneumonia in order to identify specific practices that might explain the improved efficiency of care provided by hospitalists. PATIENTS AND METHODS: We retrospectively reviewed the medical charts of 455 patients hospitalized with pneumonia at a community-based tertiary care center between January 1, 1998, and January 1, 1999. Exclusion criteria included human immunodeficiency virus infection, lung cancer, active tuberculosis, hospitalization within 7 days, length of stay (LOS) more than 14 days, and requirement of mechanical ventilation. All patients were cared for by either a full-time hospitalist or a primary care physician. Data collected included patient insurance status, variables to calculate each patient's Pneumonia Severity Index score, initial antibiotic selection, door-to-needle time, time to patient stability for switch to oral antibiotics, time to actual switch, unstable variables at discharge, and subspecialty consultation rate. Each patient's initial chest x-ray film was reviewed and classified as diagnostic of pneumonia, indeterminate, or clear. Outcomes measured via administrative database were mortality, LOS, costs, and readmission rate. RESULTS: Primary care physicians cared for 270 patients, and hospitalists cared for 185. Primary care physician patients were older, and this group had a higher proportion of the highest-risk patients. The mean time to stability was 3.2 days for hospitalists and 3.3 days for primary care physicians, and the mean time from stability to actual switch from intravenous to oral antibiotics was 1.6 days and 23 days, respectively (P=.003). The mean adjusted LOS was 5.6 days for hospitalists and 6.5 days for primary care physicians. Similarly adjusted costs were $594 less per patient treated by hospitalists. A difference in door-to-needle time of 0.9 hour favoring primary care physicians did not contribute to LOS. No significant differences were noted in adjusted inpatient mortality or the appropriateness of initial antibiotics used. Primary care physicians were more likely to prescribe clindamycin and ceftazidime, and they requested infectious disease consultations more often. At discharge, 14% of hospitalist patients and 7% of primary care physician patients had at least 1 unstable variable. Differences in hospital readmission rates at 15 and 30 days were not statistically significant in combined or risk-stratified analyses. CONCLUSIONS: Inpatients with community-acquired pneumonia cared for by hospitalists had a shorter adjusted LOS than those seen by primary care physicians primarily because of earlier recognition of stability and more rapid conversion from intravenous to oral antibiotics. Adjusted costs were likewise reduced. However, patients seen by hospitalists were discharged with an unstable clinical variable more often. Other than earlier switch to oral antibiotics, less use of clindamycin and ceftazidime, and fewer infectious disease consultations, hospitalists' processes of care were similar to those of primary care physicians.
Asunto(s)
Antibacterianos/administración & dosificación , Infecciones Comunitarias Adquiridas/terapia , Médicos Hospitalarios , Evaluación de Procesos y Resultados en Atención de Salud , Médicos de Familia , Neumonía/tratamiento farmacológico , Pautas de la Práctica en Medicina/estadística & datos numéricos , Administración Oral , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Distribución de Chi-Cuadrado , Femenino , Costos de la Atención en Salud , Humanos , Infusiones Intravenosas , Tiempo de Internación/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Análisis de Regresión , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
The purpose of this project was to assess if the presence of a mediastinal mass adversely influences peripherally inserted central catheter (PICC) positioning. A retrospective review of all PICC placements over an 18-month period in a pediatric oncology hospital was conducted in which patients were categorized by the presence or absence of a mediastinal mass. A much higher proportion of patients with a mediastinal mass (8 of 13) had a malpositioned catheter than those without a mass (3 of 38; P = .0002). A significant proportion of the malpositioned catheters in patients with a mediastinal mass terminated in the right heart chambers. These findings show that clinicians must be vigilant about checking for PICC malpositioning in this patient population.
Asunto(s)
Cateterismo Venoso Central/efectos adversos , Cateterismo Periférico/efectos adversos , Catéteres de Permanencia/efectos adversos , Enfermedades del Mediastino/complicaciones , Cateterismo Venoso Central/métodos , Cateterismo Venoso Central/enfermería , Cateterismo Periférico/métodos , Cateterismo Periférico/enfermería , Niño , Falla de Equipo , Femenino , Hospitales Pediátricos , Humanos , Masculino , Enfermedades del Mediastino/terapia , Investigación en Evaluación de Enfermería , Estudios Retrospectivos , TennesseeRESUMEN
INTRODUCTION: Severe sepsis is a leading cause of non-coronary death in hospitals across the United States. Early identification and risk stratification in the emergency department (ED) is difficult because there is limited ability to predict escalation of care. In this study we evaluated if a sustained shock index (SI) elevation in the ED was a predictor of short-term cardiovascular collapse, defined as vasopressor dependence within 72 hours of initial presentation. METHODS: Retrospective dual-centered cross-sectional study using patients identified in the Yale-New Haven Hospital Emergency Medicine sepsis registry. RESULTS: We included 295 patients in the study with 47.5% (n=140) having a sustained SI elevation in the ED. Among patients with a sustained SI elevation, 38.6% (54 of 140) required vasopressors within 72 hours of ED admission contrasted to 11.6% (18 of 155) without a sustained SI elevation (p=0.0001; multivariate modeling OR 4.42 with 95% confidence intervals 2.28-8.55) . In the SI elevation group the mean number of organ failures was 4.0 ± 2.1 contrasted to 3.2 ± 1.6 in the non-SI elevation group (p=0.0001). CONCLUSION: ED patients with severe sepsis and a sustained SI elevation appear to have higher rates of short-term vasopressor use, and a greater number of organ failures contrasted to patients without a sustained SI elevation. An elevated SI may be a useful modality to identify patients with severe sepsis at risk for disease escalation and cardiovascular collapse.