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1.
Nat Commun ; 15(1): 4716, 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38830843

RESUMEN

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.


Asunto(s)
Proteína BRCA2 , Replicación del ADN , ARN Polimerasa II , Ribonucleasa H , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Ribonucleasa H/metabolismo , Ribonucleasa H/genética , ARN Polimerasa II/metabolismo , Transcripción Genética , Terminación de la Transcripción Genética , Daño del ADN , Origen de Réplica , Estructuras R-Loop , Línea Celular Tumoral
2.
Mol Cell ; 84(7): 1224-1242.e13, 2024 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-38458201

RESUMEN

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.


Asunto(s)
Ciclinas , Reparación de la Incompatibilidad de ADN , Animales , Ciclinas/genética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Interfase , Mamíferos/metabolismo
3.
bioRxiv ; 2024 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-38260436

RESUMEN

The large majority of oxidative DNA lesions occurring in the G1 phase of the cell cycle are repaired by base excision repair (BER) rather than mismatch repair (MMR) to avoid long resections that can lead to genomic instability and cell death. However, the molecular mechanisms dictating pathway choice between MMR and BER have remained unknown. Here, we show that, during G1, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins shield p21 from its two ubiquitin ligases CRL1SKP2 and CRL4CDT2 in a CDK4/6-independent manner. In turn, p21 competes through its PCNA-interacting protein degron with MMR components for their binding to PCNA. This inhibits MMR while not affecting BER. At the G1/S transition, the CRL4AMBRA1-dependent degradation of D-type cyclins renders p21 susceptible to proteolysis. These timely degradation events allow the proper binding of MMR proteins to PCNA, enabling the repair of DNA replication errors. Persistent expression of cyclin D1 during S-phase increases the mutational burden and promotes microsatellite instability. Thus, the expression of D-type cyclins inhibits MMR in G1, whereas their degradation is necessary for proper MMR function in S.

4.
Cell ; 186(9): 1985-2001.e19, 2023 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-37075754

RESUMEN

Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer. Here, we describe KaryoCreate (karyotype CRISPR-engineered aneuploidy technology), a system that enables the generation of chromosome-specific aneuploidies by co-expression of an sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to mutant KNL1. We design unique and highly specific sgRNAs for 19 of the 24 chromosomes. Expression of these constructs leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny, with an average efficiency of 8% for gains and 12% for losses (up to 20%) validated across 10 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, frequent in gastrointestinal cancers, promotes resistance to TGF-ß, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe an innovative technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.


Asunto(s)
Centrómero , Técnicas Genéticas , Humanos , Aneuploidia , Centrómero/genética , Deleción Cromosómica , Neoplasias/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
5.
Nat Commun ; 13(1): 1740, 2022 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35365626

RESUMEN

The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by "USP1-trapping". We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.


Asunto(s)
Inestabilidad Genómica , Proteasas Ubiquitina-Específicas , Daño del ADN , Replicación del ADN , Humanos , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismo , Ubiquitinación
6.
Nature ; 592(7856): 789-793, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33854235

RESUMEN

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer1, but the mechanisms that regulate their turnover are still being debated2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4AMBRA1 (also known as CRL4DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , División Celular , Ciclina D1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Sistemas CRISPR-Cas , Ciclina D2/metabolismo , Ciclina D3/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Femenino , Técnicas de Inactivación de Genes , Genes Supresores de Tumor , Células HCT116 , Células HEK293 , Humanos , Masculino , Ratones , Neoplasias/genética , Ubiquitina/metabolismo
7.
Vet Rec ; 187(8): e65, 2020 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-32661182

RESUMEN

BACKGROUND: Canine idiopathic eosinophilic lung disease (ELD) is sparsely documented in the literature. METHODS: Clinical presentation and outcome of dogs diagnosed with ELD (eosinophilic bronchitis or eosinophilic bronchopneumonia) were reviewed. Subgroups were made based on chronicity of clinical signs and findings of thoracic imaging: NCI (no changes in thoracic imaging), BRON (bronchial/peribronchial pattern), INT (bronchointerstitial/interstitial/alveolar). RESULTS: Seventy cases were included. There were more young to adult, crossbreed and female dogs. Compared with the other two groups NCI dogs showed lower bronchoalveolar lavage fluid eosinophilic pleocytosis and absence of circulating eosinophilia, bronchiectasis or death due to respiratory disease. All dogs responded clinically to corticosteroids. Median treatment duration was four months. Remission (no clinical signs after treatment discontinuation for >one month) and long-term remission (>six months) was achieved in 60 per cent, and 51 per cent of patients, respectively. Relapse occurred in 26 per cent of cases after remission but was rare (3 per cent) after long-term remission. The one-year, two-year and four-year survival to death due to respiratory disease was 98 per cent, 97 per cent and 91 per cent, respectively. CONCLUSION: Prognosis and initial clinical response for ELD was generally good although achievement of long-term remission was only seen in 51 per cent of dogs. Different outcomes based on chronicity of signs, corticosteroid dose, thoracic imaging abnormalities and other clinical variables were not appreciated.


Asunto(s)
Corticoesteroides/uso terapéutico , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/tratamiento farmacológico , Eosinofilia Pulmonar/veterinaria , Animales , Perros , Femenino , Estudios de Seguimiento , Masculino , Eosinofilia Pulmonar/diagnóstico , Eosinofilia Pulmonar/tratamiento farmacológico , Estudios Retrospectivos , Resultado del Tratamiento
8.
Nat Struct Mol Biol ; 27(2): 179-191, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32042152

RESUMEN

Long interspersed element-1 (LINE-1, or L1) is the only autonomous retrotransposon that is active in human cells. Different host factors have been shown to influence L1 mobility; however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the double-stranded break (DSB) repair and Fanconi anemia (FA) factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular, BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a 'battleground' at the DNA replication fork between homologous recombination (HR) factors and L1 retrotransposons and reveal a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.


Asunto(s)
Proteína BRCA1/metabolismo , Reparación del ADN , Elementos de Nucleótido Esparcido Largo , Fase S , Proteína BRCA1/genética , Sistemas CRISPR-Cas , Línea Celular , Roturas del ADN de Doble Cadena , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Eliminación de Gen , Células HEK293 , Células HeLa , Recombinación Homóloga , Humanos , Microscopía
9.
Vet Rec ; 186(13): 414, 2020 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-31974267

RESUMEN

BACKGROUND: A previous study showed an association between owner-reported exposure to environmental tobacco smoke (ETS) and lymphoma in cats. This study aimed to investigate the association between ETS exposure and gastrointestinal lymphoma in cats, using hair nicotine concentration (HNC) as a biomarker. METHODS: This was a prospective, multi-centre, case-control study. Gastrointestinal lymphoma was diagnosed on cytology or histopathology. Hair samples were obtained from 35 cats with gastrointestinal lymphoma and 32 controls. Nicotine was extracted from hair by sonification in methanol followed by hydrophilic interaction chromatography with mass spectrometry. Non-parametric tests were used. RESULTS: The median HNC of the gastrointestinal lymphoma and control groups was not significantly different (0.030 ng/mg and 0.029 ng/mg, respectively, p=0.46). When the HNC of all 67 cats was rank ordered and divided into quartiles, there was no significant difference in the proportion of lymphoma cases or controls within these groups (p=0.63). The percentage of cats with an HNC≥0.1 ng/mg was higher for the lymphoma group (22.9%) than the control group (15.6%) but failed to reach significance (p=0.45). CONCLUSION: A significant association was not identified between HNC (a biomarker for ETS) and gastrointestinal lymphoma in cats; however, an association may exist and further studies are therefore required.


Asunto(s)
Enfermedades de los Gatos , Neoplasias Gastrointestinales/veterinaria , Cabello/química , Linfoma/veterinaria , Nicotina/análisis , Animales , Biomarcadores/análisis , Estudios de Casos y Controles , Gatos , Exposición a Riesgos Ambientales/efectos adversos , Estudios Prospectivos , Contaminación por Humo de Tabaco/efectos adversos
11.
Cell ; 179(4): 964-983.e31, 2019 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-31675502

RESUMEN

To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology.


Asunto(s)
Carcinoma de Células Renales/genética , Proteínas de Neoplasias/genética , Proteogenómica , Transcriptoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Supervivencia sin Enfermedad , Exoma/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano/genética , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/inmunología , Fosforilación Oxidativa , Fosforilación/genética , Transducción de Señal/genética , Transcriptoma/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Secuenciación del Exoma
12.
Vet Rec Open ; 6(1): e000315, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30997113

RESUMEN

BACKGROUND: Cytosine arabinoside (CA) and prednisolone are drugs commonly used together in the management of canine non-infectious meningoencephalitis (NIME). The aim of this study was to report the haematological findings before and after CA and prednisolone treatment and identify any adverse haematological events in this clinical setting, following the veterinary cooperative oncology group established common terminology criteria for recording adverse events following administration of chemotherapy or biological antineoplastic therapy. RESULTS: While 48 patients with a presumptive diagnosis of NIME had pretreatment haematology results, only 12 patients met the inclusion criteria of also having post-treatment haematology results available for review after being treated with prednisolone and CA at a standard dose (200 mg/m2) in a single referral hospital in the UK. Forty-nine post-treatment haematology results were available for these 12 patients. CONCLUSIONS: Four adverse haematological events were identified in four patients. None of these events were convincingly attributable to CA administration.

13.
Nat Commun ; 9(1): 3882, 2018 09 24.
Artículo en Inglés | MEDLINE | ID: mdl-30250272

RESUMEN

Homologous recombination (HR) is a crucial pathway for the repair of DNA double-strand breaks. BRCA1/2 breast cancer proteins are key players in HR via their mediation of RAD51 nucleofilament formation and function; however, their individual roles and crosstalk in vivo are unknown. Here we use super-resolution (SR) imaging to map the spatiotemporal kinetics of HR proteins, revealing the interdependent relationships that govern the dynamic interplay and progression of repair events. We show that initial single-stranded DNA/RAD51 nucleofilament formation is mediated by RAD52 or, in the absence of RAD52, by BRCA2. In contrast, only BRCA2 can orchestrate later RAD51 recombinase activity during homology search and resolution. Furthermore, we establish that upstream BRCA1 activity is critical for BRCA2 function. Our analyses reveal the underlying epistatic landscape of RAD51 functional dependence on RAD52, BRCA1, and BRCA2 during HR and explain the phenotypic similarity of diseases associated with mutations in these proteins.


Asunto(s)
Roturas del ADN de Doble Cadena , ADN de Cadena Simple/metabolismo , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Línea Celular Tumoral , ADN de Cadena Simple/genética , Técnicas de Silenciamiento del Gen , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/métodos , ARN Interferente Pequeño/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Imagen Individual de Molécula/métodos
14.
Elife ; 72018 12 31.
Artículo en Inglés | MEDLINE | ID: mdl-30596474

RESUMEN

In response to nutrient deprivation, the cell mobilizes an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and stimulating LC3 lipidation, COPII (Coat Protein Complex II) promotes autophagosome biogenesis. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting SEC23B degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ER-Golgi intermediate compartment, promoting autophagic flux. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.


Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia , Células Epiteliales/fisiología , Proteínas F-Box/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/fisiología , Proteínas de Transporte Vesicular/metabolismo , Línea Celular , Humanos , Fosforilación , Mapas de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteolisis
16.
Proc Natl Acad Sci U S A ; 112(20): E2575-84, 2015 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-25941401

RESUMEN

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/fisiología , ADN Ligasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Western Blotting , Línea Celular Tumoral , ADN Ligasa (ATP) , Transferencia Resonante de Energía de Fluorescencia , Técnica del Anticuerpo Fluorescente , Humanos , Cinética
17.
18.
Cardiovasc Res ; 100(2): 231-40, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-23929525

RESUMEN

AIMS: Cell function requires formation of molecular clusters localized to discrete subdomains. The composition of these interactomes, and their spatial organization, cannot be discerned by conventional microscopy given the resolution constraints imposed by the diffraction limit of light (∼200-300 nm). Our aims were (i) Implement single-molecule imaging and analysis tools to resolve the nano-scale architecture of cardiac myocytes. (ii) Using these tools, to map two molecules classically defined as components 'of the desmosome' and 'of the gap junction', and defined their spatial organization. METHODS AND RESULTS: We built a set-up on a conventional inverted microscope using commercially available optics. Laser illumination, reducing, and oxygen scavenging conditions were used to manipulate the blinking behaviour of individual fluorescent reporters. Movies of blinking fluorophores were reconstructed to generate subdiffraction images at ∼20 nm resolution. With this method, we characterized clusters of connexin43 (Cx43) and of 'the desmosomal protein' plakophilin-2 (PKP2). In about half of Cx43 clusters, we observed overlay of Cx43 and PKP2 at the Cx43 plaque edge. SiRNA-mediated loss of Ankyrin-G expression yielded larger Cx43 clusters, of less regular shape, and larger Cx43-PKP2 subdomains. The Cx43-PKP2 subdomain was validated by a proximity ligation assay (PLA) and by Monte-Carlo simulations indicating an attraction between PKP2 and Cx43. CONCLUSIONS: (i) Super-resolution fluorescence microscopy, complemented with Monte-Carlo simulations and PLAs, allows the study of the nanoscale organization of an interactome in cardiomyocytes. (ii) PKP2 and Cx43 share a common hub that permits direct physical interaction. Its relevance to excitability, electrical coupling, and arrhythmogenic right ventricular cardiomyopathy, is discussed.


Asunto(s)
Conexina 43/análisis , Desmosomas/química , Uniones Comunicantes/química , Microscopía Fluorescente/métodos , Miocitos Cardíacos/ultraestructura , Placofilinas/análisis , Animales , Ancirinas/análisis , Ratones , Método de Montecarlo , Miocitos Cardíacos/química , Ratas
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