RESUMEN
BACKGROUND: In Benin, a country in West Africa, breast cancer is the leading cancer in women, both in terms of incidence and mortality. However, evidence on the mortality of breast cancer and its associated factors is lacking in this country. Our aim was to describe and analyze the clinical, histopathological, and prognostic aspects of breast cancer in Benin. METHODS: A descriptive and analytical study was carried out at the CNHU-HKM and the CHU-MEL, two major tertiary referral hospitals for breast cancer management located in Cotonou, the capital city of Benin. All breast cancer medical records with histological evidence and immunohistochemistry studies were retrospectively collected between January 1, 2014, and September 30, 2020, in these two tertiary referral hospitals and analyzed in the current study. RESULTS: Finally, 319 medical records were included. The mean age at diagnosis was 48.74 years. The tumors were most frequently classified as T4 (47.6%) with lymph node involvement N2 (34.5%), and metastases were clinically noted in 21.9% of cases. Stage was reported in the medical records of 284 patients. Tumors were diagnosed at very late AJCC stages: stage III (47.5%) and stage IV (24.7%). Grades SBR 2 (49.2%) and SBR 3 (32.6%) were the most frequent grades. Triple-negative breast cancer (31.3%) was the most common molecular type. The overall 5-year survival was 48.49%. In multivariable analysis, the poor prognostic factors were lymph node invasion (HR = 2.63; p = 0.026; CI: [1.12, 6.17]), the presence of metastasis (HR = 3.64; p < 0.001); CI: [2.36, 5.62] and the immunohistochemical profile (HR = 1.29; p < 0.001; CI: [1.13, 1.48]). CONCLUSIONS: Breast cancer in Beninese is predominant in young adults and is often diagnosed at a late stage. The survival of breast cancer patients in Benin can be improved by enhancing early diagnosis and multidisciplinary management.
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Neoplasias de la Mama , Humanos , Benin/epidemiología , Femenino , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Neoplasias de la Mama/diagnóstico , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Adulto , Estadificación de Neoplasias , Anciano , Metástasis Linfática , Centros de Atención Terciaria/estadística & datos numéricosRESUMEN
Muscle atrophy is associated with many diseases including genetic disorders, sarcopenia, or cachexia syndromes. Myostatin (Mstn), a transforming growth factor-beta (TGF-ß) member, plays a key role in skeletal muscle homeostasis as a powerful negative regulator. Over the last decade, about 15 clinical trials aimed at inhibiting the Mstn pathway, failed to produce conclusive results. In this context, we investigated whether growth and differentiation factor-associated serum protein-1 (GASP-1) or GASP-2, two natural inhibitors of Mstn, might represent a potential therapeutic. As we previously reported, mice overexpressing Gasp-1 (Tg(Gasp-1)) present an increase of muscle mass but develop metabolic disorders with aging. Here, we showed that overexpression of Gasp-2 increases the muscular mass without metabolic defects. We also found that Tg(Gasp-2) mice displayed, like Mstn-/- mice, a switch from slow- to fast-twitch myofibers whereas Tg(Gasp-1) mice exhibit a reverse switch. Our studies supported the fact that GASP-2 has less affinity than GASP-1 for Mstn, leading to a constitutive Mstn upregulation only in Tg(Gasp-1) mice, responsible for the observed phenotypic differences. Altogether, our findings highlighted a gene expression regulatory network of TGF-ß members and their inhibitors in muscle.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Miostatina/metabolismo , Animales , Proliferación Celular/genética , Proliferación Celular/fisiología , Células Cultivadas , Variaciones en el Número de Copia de ADN/genética , Variaciones en el Número de Copia de ADN/fisiología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Genotipo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Miostatina/genéticaRESUMEN
BACKGROUND: Linkage disequilibrium (LD) is a key parameter to study the history of populations and to identify and fine map quantitative trait loci (QTL) and it has been studied for many years in animal populations. The advent of new genotyping technologies has allowed whole-genome LD studies in most cattle populations. However, to date, long-range LD (LRLD) between distant variants on the genome has not been investigated in detail in cattle. Here, we present the first comprehensive study of LRLD in French beef cattle by analysing data on 672 Charolais (CHA), 462 Limousine (LIM) and 326 Blonde d'Aquitaine (BLA) individuals that were genotyped on the Illumina BovineHD Beadchip. Furthermore, whole-genome LD and haplotype block structure were analysed in these three breeds. RESULTS: We computed linkage disequilibrium (r2) values for 5.9, 5.6 and 6.0 billion pairs of SNPs on the 29 autosomes of CHA, LIM and BLA, respectively. Mean r2 values drop to less than 0.1 for distances between SNPs greater than 120 kb. However, for the first time, we detected the existence of LRLD in the three main French beef breeds. In total, 598, 266, and 795 LRLD events (r2 ≥ 0.6) were detected in CHA, LIM and BLA, respectively. Each breed had predominantly population-specific LRLD interactions, although shared LRLD events occurred in a number of regions (55 LRLD events were shared between two breeds and nine between the three breeds). Examples of possible functional gene interactions and QTL co-location were observed with some of these LRLD events, which suggests epistatic selection. CONCLUSIONS: We identified long-range linkage disequilibrium for the first time in French beef cattle populations. Epistatic selection may be the main source of the observed LRLD events, but other forces may also be involved. LRLD information should be accounted for in genome-wide association studies.
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Bovinos/genética , Desequilibrio de Ligamiento , Animales , Estudio de Asociación del Genoma Completo/métodos , Técnicas de Genotipaje/métodos , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Carne Roja/normasRESUMEN
DNAJC2 protein, also known as ZRF1 or MPP11, acts both as chaperone and as chromatin regulator. It is involved in stem cell differentiation and its expression is associated with various cancer malignancies. However, the role of Dnajc2 gene during mouse embryogenesis has not been assessed so far. To this aim, we invalidated Dnajc2 gene in FVB/Nj mice using the CrispR/Cas9 approach. We showed that this invalidation leads to the early post-implantation lethality of the nullizygous embryos. Furthermore, using siRNAs against Dnajc2 in mouse 1-cell embryos, we showed that maternal Dnajc2 mRNAs may allow for the early preimplantation development of these embryos. Altogether, these data demonstrate for the first time the requirement of DNAJC2 for early mouse embryogenesis.
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Proteínas de Unión al ADN/genética , Embrión de Mamíferos/embriología , Regulación del Desarrollo de la Expresión Génica , Ratones/embriología , Chaperonas Moleculares/genética , Proteínas de Unión al ARN/genética , Animales , Sistemas CRISPR-Cas , Implantación del Embrión , Pérdida del Embrión/genética , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Femenino , Eliminación de Gen , Ratones/genética , EmbarazoRESUMEN
Myogenesis is a highly regulated multi-step process involving myoblast proliferation and differentiation. Although studies over the last decades have identified several factors governing these distinct major phases, many of them are not yet known. In order to identify novel genes, we took advantage of the C2C12 myoblastic line to establish a functional siRNA screen combined with quantitative-imaging analysis of a large amount of differentiated myoblasts. We knocked down 100 preselected mouse genes without a previously characterized role in muscle. Using image analysis, we tracked gene-silencing phenotypes by quantitative assessment of cellular density, myotube quantity, myotube morphology and fusion index. Our results have revealed six genes involved in both stages of C2C12 myogenesis and 13 genes specific to the differentiation stage. These findings prove that our RNAi-based screen could be an efficient tool to detect clear and subtle phenotypes allowing the identification of new myogenic regulators in mammals.
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Diferenciación Celular/genética , Pruebas Genéticas , Desarrollo de Músculos/genética , Mioblastos/citología , Mioblastos/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Ratones , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Fenotipo , Interferencia de ARNRESUMEN
BACKGROUND/AIMS: Myostatin is known as a powerful negative regulator of muscle growth playing a key role in skeletal muscle homeostasis. Recent studies revealed that myostatin-deficient mice lead to an increase of insulin sensitivity, a decrease of adiposity and a resistance to obesity, showing that myostatin can also impact on metabolism. Thus, myostatin appeared as a potential therapeutic target to treat insulin resistance. METHODS: We generated transgenic mice overexpressing Gasp-1, a myostatin inhibitor. RESULTS: Surprisingly, we found that these mice gained weight with age due to an increase in fat mass associated with ectopic fat accumulation. In addition, these mice developed an adipocyte hypertrophy, hyperglycemia, hyperinsulinemia, muscle and hepatic insulin resistance. Understanding the molecular networks controlling this insulin resistance responsiveness in overexpressing Gasp-1 mice is essential. Molecular analyses revealed a deregulation of adipokines and muscle cytokines expression, but also an increase in plasma myostatin levels. The increase in myostatin bioactivity by a positive feedback mechanism in the Tg(Gasp-1) transgenic mice could lead to this combination of phenotypes. CONCLUSION: Altogether, these data suggested that overexpressing Gasp-1 mice develop most of the symptoms associated with metabolic syndrome and could be a relevant model for the study of obesity or type 2 diabetes.
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Adiposidad/fisiología , Proteínas Portadoras/metabolismo , Glucosa/metabolismo , Adipoquinas/metabolismo , Tejido Adiposo/patología , Animales , Peso Corporal , Proteínas Portadoras/genética , Citocinas/metabolismo , Prueba de Tolerancia a la Glucosa , Hiperglucemia/etiología , Hiperinsulinismo/etiología , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intracelular , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Miostatina/sangre , Fenotipo , Factores de TiempoRESUMEN
Myostatin (Mstn) deficiency leads to skeletal muscle overgrowth and Mstn inhibition is considered as a promising treatment for muscle-wasting disorders. Mstn gene deletion in mice also causes metabolic changes with decreased mitochondria content, disturbance in mitochondrial respiratory function and increased muscle fatigability. However the impact of MSTN deficiency on these metabolic changes is not fully elucidated. Here, we hypothesized that lack of MSTN will alter skeletal muscle membrane lipid composition in relation with pronounced alterations in muscle function and metabolism. Indeed, phospholipids and in particular cardiolipin mostly present in the inner mitochondrial membrane, play a crucial role in mitochondria function and oxidative phosphorylation process. We observed that Mstn KO muscle had reduced fat membrane transporter levels (FAT/CD36, FABP3, FATP1 and FATP4) associated with decreased lipid oxidative pathway (citrate synthase and ß-HAD activities) and impaired lipogenesis (decreased triglyceride and free fatty acid content), indicating a role of mstn in muscle lipid metabolism. We further analyzed phospholipid classes and fatty acid composition by chromatographic methods in muscle and mitochondrial membranes. Mstn KO mice showed increased levels of saturated and polyunsaturated fatty acids at the expense of monounsaturated fatty acids. We also demonstrated, in this phenotype, a reduction in cardiolipin proportion in mitochondrial membrane versus the proportion of others phospholipids, in relation with a decrease in the expression of phosphatidylglycerolphosphate synthase and cardiolipin synthase, enzymes involved in cardiolipin synthesis. These data illustrate the importance of lipids as a link by which MSTN deficiency can impact mitochondrial bioenergetics in skeletal muscle.
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Ácidos Grasos/metabolismo , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Miostatina/deficiencia , 3-Hidroxiacil-CoA Deshidrogenasas/genética , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Ácidos Grasos/genética , Masculino , Ratones , Ratones Noqueados , Mitocondrias Musculares/genética , Mitocondrias Musculares/patología , Músculo Esquelético/patología , Oxidación-ReducciónRESUMEN
BACKGROUND: Copy number variations (CNV) are known to play a major role in genetic variability and disease pathogenesis in several species including cattle. In this study, we report the identification and characterization of CNV in eight French beef and dairy breeds using whole-genome sequence data from 200 animals. Bioinformatics analyses to search for CNV were carried out using four different but complementary tools and we validated a subset of the CNV by both in silico and experimental approaches. RESULTS: We report the identification and localization of 4178 putative deletion-only, duplication-only and CNV regions, which cover 6% of the bovine autosomal genome; they were validated by two in silico approaches and/or experimentally validated using array-based comparative genomic hybridization and single nucleotide polymorphism genotyping arrays. The size of these variants ranged from 334 bp to 7.7 Mb, with an average size of ~ 54 kb. Of these 4178 variants, 3940 were deletions, 67 were duplications and 171 corresponded to both deletions and duplications, which were defined as potential CNV regions. Gene content analysis revealed that, among these variants, 1100 deletions and duplications encompassed 1803 known genes, which affect a wide spectrum of molecular functions, and 1095 overlapped with known QTL regions. CONCLUSIONS: Our study is a large-scale survey of CNV in eight French dairy and beef breeds. These CNV will be useful to study the link between genetic variability and economically important traits, and to improve our knowledge on the genomic architecture of cattle.
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Bovinos/genética , Variaciones en el Número de Copia de ADN , Estudio de Asociación del Genoma Completo/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación Completa del Genoma/métodos , Animales , Productos Lácteos/normas , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Carácter Cuantitativo Heredable , Carne Roja/normasRESUMEN
BACKGROUND: In recent years, several bovine genome sequencing projects were carried out with the aim of developing genomic tools to improve dairy and beef production efficiency and sustainability. RESULTS: In this study, we describe the first French cattle genome variation dataset obtained by sequencing 274 whole genomes representing several major dairy and beef breeds. This dataset contains over 28 million single nucleotide polymorphisms (SNPs) and small insertions and deletions. Comparisons between sequencing results and SNP array genotypes revealed a very high genotype concordance rate, which indicates the good quality of our data. CONCLUSIONS: To our knowledge, this is the first large-scale catalog of small genomic variations in French dairy and beef cattle. This resource will contribute to the study of gene functions and population structure and also help to improve traits through genotype-guided selection.
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Cruzamiento , Variación Genética , Genoma , Polimorfismo de Nucleótido Simple , Animales , Bovinos , Mapeo Cromosómico , Industria Lechera , Femenino , Genotipo , Mutación INDEL , Masculino , Tasa de Mutación , Fenotipo , Carne RojaRESUMEN
BACKGROUND/AIMS: Overexpression of Gasp-1, an inhibitor of myostatin, leads to a hypermuscular phenotype due to hypertrophy rather than hyperplasia in mice. However to date, the cellular and molecular mechanisms underlying this phenotype are not investigated. METHODS: Skeletal muscles of overexpressing Gasp-1 mice, called Tg(Gasp-1) mice, were analyzed by histological methods. Satellite cell-derived myoblasts from these mice were used to investigate the molecular mechanisms. RESULTS: We demonstrated that hypertrophy in Tg(Gasp-1) mice was related to a myonuclear accretion during the first 3 postnatal weeks and an activation of the pro-hypertrophic Akt/mTORC/p70S6K signaling. In accordance with these results, we showed that overexpressing Gasp-1 primary myoblasts proliferated faster and myonuclei average per myotube was increased during differentiation. Molecular analysis revealed that Gasp-1 overexpression resulted in increased myostatin expression related to its auto-regulation. Despite its inhibition, myostatin led to Pax7 deregulation through its non-canonical Erk1/2 signaling pathway. Consistent with this, inhibition of Erk1/2 signaling pathway as well as neutralization of secreted myostatin rescue the Pax7 expression in overexpressing Gasp-1 myoblasts. CONCLUSION: Our study shows that myostatin is able to act independently of its canonical pathway to regulate the Pax7 expression. Altogether, our results indicate that myostatin could regulate muscle development despite its protein inhibition.
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Proteínas Portadoras/genética , Hiperplasia/genética , Miostatina/genética , Regulación hacia Arriba/genética , Animales , Diferenciación Celular/genética , Hiperplasia/embriología , Hipertrofia/genética , Hipertrofia/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas/genética , Ratones , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Factor de Transcripción PAX7/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
The CRISPR/Cas9 technique applied to modify the cattle genome has value in increasing animal health and welfare. Here, we established a simple, fast, and efficient cloning-free CRISPR/Cas9 protocol for large deletions of genomic loci in the frequently used model bovine MDBK cell line. The main advantages of our protocol are as follows: (i) pre-screening of the sgRNA efficiency with a fast and simple cleavage assay, (ii) reliable detection of genomic edits primarily by PCR and confirmed by DNA sequencing, and (iii) single cell sorting with FACS providing specific genetic information from modified cells of interest. Therefore, our method could be successfully applied in different studies, including functional validation of any genetic or regulatory elements.
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Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Bovinos , Animales , Secuencia de Bases , Línea CelularRESUMEN
Using N-ethyl-N-nitrosourea-induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T- and B-cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b(+) Ly-6G(int) neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody-induced arthritis and of immune complex-mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil-mediated inflammatory and anti-infectious responses.
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Proteínas de Unión al ADN/genética , Neutropenia/genética , Mutación Puntual , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Artritis/genética , Artritis/metabolismo , Linfocitos B/metabolismo , Médula Ósea/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Proteínas de Unión al ADN/metabolismo , Etilnitrosourea , Femenino , Inflamación/genética , Inflamación/metabolismo , Linfocitos/metabolismo , Linfopoyesis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Neutropenia/inducido químicamente , Neutrófilos/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: Myostatin, a member of the TGFß superfamily, is well known as a potent and specific negative regulator of muscle growth. Targeting the myostatin signalling pathway may offer promising therapeutic strategies for the treatment of muscle-wasting disorders. In the last decade, various myostatin-binding proteins have been identified to be able to inhibit myostatin activity. One of these is GASP1 (Growth and Differentiation Factor-Associated Serum Protein-1), a protein containing a follistatin domain as well as multiple domains associated with protease inhibitors. Despite in vitro data, remarkably little is known about in vivo functions of Gasp1. To further address the role of GASP1 during mouse development and in adulthood, we generated a gain-of-function transgenic mouse model that overexpresses Gasp1 under transcriptional control of the human cytomegalovirus immediate-early promoter/enhancer. RESULTS: Overexpression of Gasp1 led to an increase in muscle mass observed not before day 15 of postnatal life. The surGasp1 transgenic mice did not display any other gross abnormality. Histological and morphometric analysis of surGasp1 rectus femoris muscles revealed an increase in myofiber size without a corresponding increase in myofiber number. Fiber-type distribution was unaltered. Interestingly, we do not detect a change in total fat mass and lean mass. These results differ from those for myostatin knockout mice, transgenic mice overexpressing the myostatin propeptide or follistatin which exhibit both muscle hypertrophy and hyperplasia, and show minimal fat deposition. CONCLUSIONS: Altogether, our data give new insight into the in vivo functions of Gasp1. As an extracellular regulatory factor in the myostatin signalling pathway, additional studies on GASP1 and its homolog GASP2 are required to elucidate the crosstalk between the different intrinsic inhibitors of the myostatin.
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Proteínas Portadoras/genética , Fibras Musculares Esqueléticas/fisiología , Hipertonía Muscular/genética , Miostatina/metabolismo , Músculo Cuádriceps/fisiología , Animales , Antígenos Virales/genética , Proteínas Portadoras/biosíntesis , Citomegalovirus/genética , Folistatina/genética , Folistatina/metabolismo , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Transgénicos , Hipertonía Muscular/metabolismo , Miostatina/genética , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Transcripción GenéticaRESUMEN
BACKGROUND/AIMS: Growth and differentiation factor-associated serum protein-1 (GASP-1) is a secreted protein known to be capable of binding and inhibiting the activity of several TGF-beta family members, including myostatin. The present study was designed to characterize murine GASP-1 post-translational modifications and to determine their influence on the biological activity of GASP-1. METHODS: We describe herein the site-directed mutagenesis of single N-glycosylation sites and combinations of them in 4 mutants of murine GASP-1. RESULTS: In vitro and in vivo analysis revealed that GASP-1 is a glycoprotein containing 2 N-glycans and several mucin-type O-glycans. Treatments by the recombinant murine GASP-1 protein enhance C2C12 proliferation and differentiation by inhibition of the myostatin pathway. The loss of N-glycans leads to a decrease in protein secretion rate but does not affect its ability to activate myogenesis. CONCLUSION: Analysis of structure-function relationships of murine GASP-1 provides insights into the involvement of the carbohydrate moiety of mGASP-1 on its biological activity.
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Proteínas Portadoras/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Proteínas Portadoras/genética , Diferenciación Celular , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Glicosilación , Péptidos y Proteínas de Señalización Intracelular , Espectrometría de Masas , Ratones , Mutagénesis Sitio-Dirigida , Mioblastos/citología , Mioblastos/metabolismo , Péptidos/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
PhexL222P mouse is a new ENU mouse model for XLH disease due to Leu to Pro amino acid modification at position 222. PhexL222P mouse is characterized by growth retardation, hypophosphatemia, hypocalcemia, reduced body bone length, and increased epiphyseal growth plate thickness and femur diameter despite the increase in PHEXL222P expression. Actually, PhexL222P mice show an increase in Fgf23, Dmp1, and Mepe and Slc34a1 (Na-Pi IIa cotransporter) mRNA expression similar to those observed in Hyp mice. Femoral osteocalcin and sclerostin and Slc34a1 do not show any significant variation in PhexL222P mice. Molecular dynamics simulations support the experimental data. P222 might locally break the E217-Q224 ß-sheet, which in turn might disrupt inter-ß-sheet interactions. We can thus expect local protein misfolding, which might be responsible for the experimentally observed PHEXL222P loss of function. This model could be a valuable addition to the existing XLH model for further comprehension of the disease occurrence and testing of new therapies.
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Factores de Crecimiento de Fibroblastos , Endopeptidasa Neutra Reguladora de Fosfato PHEX/genética , Animales , Huesos/metabolismo , Modelos Animales de Enfermedad , Factores de Crecimiento de Fibroblastos/genética , Ratones , Mutación , Endopeptidasa Neutra Reguladora de Fosfato PHEX/metabolismoRESUMEN
The mutation T3811 â G3811 (TG3811) discovered in the myostatin gene of the Blonde d'Aquitaine breed is suspected of contributing to the outstanding muscularity of this breed. An experiment was designed to estimate the effect of this mutation in an F2 and back-cross Blonde d'Aquitaine × Holstein population. By genotyping all known mutations in the myostatin gene, it was ensured that the TG3811 mutation was indeed the only known mutation segregating in this population. Fifty-six calves (43 F2, 13 back-cross) were intensively fattened and slaughtered at 24.0 ± 1.4 wk of age. The effects of the mutation were estimated by comparing the calves with the [T/T] (n = 18), [T/G] (n = 30), and [G/G] (n = 8) genotypes. Highly significant substitution effects (P < 0.001), above + 1.2 phenotypic SD, were shown on carcass yield and muscularity scores. Birth weight (P < 0.001) was positively affected by the mutation (+0.8 SD) but not growth rate (P = 0.97), while carcass length (P = 0.03), and fatness (P ≤ 0.03) were negatively affected (-0.5 to -0.7 SD). The characteristics of the Triceps brachii muscle were affected by the mutation (P < 0.001), with lower ICDH activity (oxidative) and a higher proportion of myosin type 2X muscle fibers (fast twitch). The effects of the TG3811 mutation were similar to those of other known myostatin mutations, although the Blonde d'Aquitaine animals, which are predominantly [G/G] homozygous, do not exhibit extreme double muscling.
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Miostatina , Carne Roja , Animales , Bovinos/genética , Genotipo , Mutación , Miostatina/genética , FenotipoRESUMEN
Arc/Arg3.1 is robustly induced by plasticity-producing stimulation and specifically targeted to stimulated synaptic areas. To investigate the role of Arc/Arg3.1 in synaptic plasticity and learning and memory, we generated Arc/Arg3.1 knockout mice. These animals fail to form long-lasting memories for implicit and explicit learning tasks, despite intact short-term memory. Moreover, they exhibit a biphasic alteration of hippocampal long-term potentiation in the dentate gyrus and area CA1 with an enhanced early and absent late phase. In addition, long-term depression is significantly impaired. Together, these results demonstrate a critical role for Arc/Arg3.1 in the consolidation of enduring synaptic plasticity and memory storage.
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Proteínas del Citoesqueleto/fisiología , Memoria/fisiología , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Análisis de Varianza , Animales , Reacción de Prevención/fisiología , Conducta Animal , Southern Blotting/métodos , Western Blotting/métodos , Condicionamiento Clásico/fisiología , Proteínas del Citoesqueleto/deficiencia , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/citología , Técnicas In Vitro , Ácido Kaínico , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Plasticidad Neuronal/genética , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Convulsiones/inducido químicamente , Convulsiones/metabolismo , Conducta Espacial/fisiología , Sinapsis/genética , Factores de TiempoRESUMEN
While GASP-1 and GASP-2 proteins are known to regulate myogenesis by inhibiting myostatin, their structural organization suggests a putative role as multivalent protease inhibitors controlling different protease activities. In this study, we show the noncompetitive and competitive antitrypsin activities of the full-length GASP-1 and GASP-2 proteins, respectively, by using a bacterial system production and in vitro enzymatic experiments. The role of the second Kunitz domain in this functional duality is described by assessing the antitrypsin activity of GASP-1/2 chimeric proteins. Molecular dynamics simulations support the experimental data to rationalize differences in binding modes between trypsin and the GASP-1 and GASP-2 second Kunitz domains. A new inhibition mechanism was evidenced for the second Kunitz domain of GASP-2, in which the conventional cationic residue of trypsin inhibitors was substituted by the strongly interacting glutamine residue.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Simulación de Dinámica Molecular , Animales , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intracelular/química , Cinética , Ratones , Mioblastos/citología , Mioblastos/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Knowledge of population structure is essential to improve the management and conservation of farm animal genetic resources. Microsatellites, which have long been popular for this type of analysis, are more and more neglected in favor of whole-genome single nucleotide polymorphism (SNP) chips that are now available for the main farmed animal species. In this study, we compared genetic patterns derived from microsatellites to that inferred by SNPs, considering three pairs of datasets of sheep and cattle. Population genetic differentiation analyses (Fixation index, FST), as well as STRUCTURE analyses showed a very strong consistency between the two types of markers. Microsatellites gave pictures that were largely concordant with SNPs, although less accurate. The best concordance was found in the most complex dataset, which included 17 French sheep breeds (with a Pearson correlation coefficient of 0.95 considering the 136 values of pairwise FST, obtained with both types of markers). The use of microsatellites reduces the cost and the related analyses do not require specific computer equipment (i.e., information technology (IT) infrastructure able to provide adequate computing and storage capacity). Therefore, this tool may still be a very appropriate solution to evaluate, in a first stage, the general state of livestock at national scales. At a time when local breeds are disappearing at an alarming rate, it is urgent to improve our knowledge of them, in particular by promoting tools accessible to the greatest number.
Asunto(s)
Animales Domésticos/genética , Repeticiones de Microsatélite/genética , Polimorfismo de Nucleótido Simple/genética , Crianza de Animales Domésticos/métodos , Animales , Bovinos/genética , Variación Genética/genética , Genoma/genética , Genotipo , Desequilibrio de Ligamiento/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Filogenia , Ovinos/genéticaRESUMEN
Myogenesis is a multistep process taking place during pre- and postnatal stages for muscle formation, growth, and regeneration. It is a highly regulated process involving many molecular factors which act during myoblast proliferation and differentiation. To provide new insights into the molecular mechanisms and interactions behind the regulation of these different steps, RNA interference is an efficient methodology to implement. We developed a high-throughput siRNA screen in C2C12 murine myoblast cells for identification of genes relevant to signaling pathways controlling muscle growth. The proposed protocol is based on (1) the analyses of a maximum number of cells/myotubes to detect and quantify both clear and subtle phenotypes during proliferation/fusion cells and (2) the use of two cellular fluorescent markers, DAPI and myosin, decorating nuclei and myotubes respectively. Four phenotypic criteria were quantitatively assessed: cellular density, myotubes quantity, fusion index, and size and morphology of myotubes.