RESUMO
CircRNAs are a novel type of closed circular molecules formed through a covalent bond lacking a 5'cap and 3' end tail, which mainly arise from mRNA precursor. They are widely distributed in plants and animals and are characterized by stable structure, high conservativeness in cells or tissues, and showed the expression specificity at different stages of development in different tissues. CircRNAs have been gradually attracted wide attention with the development of RNA sequencing, which become a new research hotspot in the field of RNA. CircRNAs play an important role in gene expression regulation. Presently, the related circRNAs research in the regulation of animal muscle development is still at the initial stage. In this review, the formation, properties, biological functions of circRNAs were summarized. The recent research progresses of circRNAs in skeletal muscle growth and development from economic animals including livestock, poultry and fishes were introduced. Finally, we proposed a prospective for further studies of circRNAs in muscle development, and we hope our research could provide new ideas, some theoretical supports and helps for new molecular genetic markers exploitation and animal genetic breeding in future.
Assuntos
RNA Circular , RNA , Animais , RNA Circular/genética , Estudos Prospectivos , RNA/genética , Marcadores Genéticos , Desenvolvimento Muscular/genéticaRESUMO
BACKGROUND: Normative values for different morphometric parameters of muscle fibres during paediatric development, i.e. from 0 to 18 years, are currently unavailable. They would be of major importance to accurately evaluate pathological changes and could be used as reference biomarkers for evaluating treatment response in clinical trials, or physiological adjustments in sports or ageing. METHODS: Data were derived from 482 images with a total of 33 094 fibres from 10 µm cross-sections of snap-frozen muscle from 83 deltoid muscle biopsies from patients, 0-18 years, without neuromuscular pathology stained with ATPase 9.4. Data was acquired and analysed with patented image analysis algorithms from "CARPACCIO.cloud". Several parameters were extracted or calculated, including cross-sectional area (CSA), fibre type, circularity, as well as the Minimum diameter of Feret (MinFeret). FINDINGS: This study illustrates changes in quantitative parameters for muscle morphology over the course of paediatric development and the pivotal changes occurring around puberty. Only fibre size parameters (MinFeret, CSA) are dependent on gender, and only after puberty. All other parameters vary in a similar manner for females and males. The proportion of type 1 fibres is essentially constant from birth to age 10, decreasing to ≈40% by age 18. Circularity decreases with age, to plateau after age 10 for both fibre types. INTERPRETATION: Normative values and reference charts for muscle fibre types in this age range have been generated to allow comparison of data from patients in pathology laboratories working on neuromuscular diseases. FUNDING: BPI FRANCE, PULSALYS, Association de l'Institut de Myologie, French National Research Agency (ANR), LABEX CORTEX of Université de Lyon.
Assuntos
Desenvolvimento Muscular , Fibras Musculares Esqueléticas , Masculino , Feminino , Humanos , Criança , Adolescente , Estudos Transversais , Biópsia , Envelhecimento , Músculo EsqueléticoRESUMO
Skeletal muscle tissue engineering aims at generating biological substitutes that restore, maintain or improve normal muscle function; however, the quality of cells produced by current protocols remains insufficient. Here, we developed a multifactor-based protocol that combines adenovector (AdV)-mediated MYOD expression, small molecule inhibitor and growth factor treatment, and electrical pulse stimulation (EPS) to efficiently reprogram different types of human-derived multipotent stem cells into physiologically functional skeletal muscle cells (SMCs). The protocol was complemented through a novel in silico workflow that allows for in-depth estimation and potentially optimization of the quality of generated muscle tissue, based on the transcriptomes of transdifferentiated cells. We additionally patch-clamped phenotypic SMCs to associate their bioelectrical characteristics with their transcriptome reprogramming. Overall, we set up a comprehensive and dynamic approach at the nexus of viral vector-based technology, bioinformatics, and electrophysiology that facilitates production of high-quality skeletal muscle cells and can guide iterative cycles to improve myo-differentiation protocols.
Assuntos
Desenvolvimento Muscular , Fibras Musculares Esqueléticas , Diferenciação Celular/fisiologia , Humanos , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Células-Tronco , Fluxo de TrabalhoRESUMO
Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.
Assuntos
Células-Tronco Adultas/citologia , Técnicas de Cultura de Células/métodos , Fibras Musculares Esqueléticas/citologia , Células-Tronco/citologia , Animais , Células Cultivadas , Colagenases/metabolismo , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , RNA Interferente Pequeno/metabolismo , Regeneração , Fixação de Tecidos , TransfecçãoRESUMO
OBJECTIVE: To perform anthropometric and dietary evaluation of patients with glycogenosis type Ia and Ib. METHODS: This cross-sectional study is composed of a sample of 11 patients with glycogenosis divided into two subgroups according to the classification of glycogenosis (type Ia=5 and type Ib=6), aged between 4 and 20 years. The analyzed anthropometric variables were weight, height, body mass index, and measures of lean and fat body mass, which were compared with reference values. For dietary assessment, a food frequency questionnaire was used to calculate energy and macronutrients intake as well as the amount of raw cornstarch consumed. Mann-Whitney U test and Fisher's exact test were performed, considering a significance level of 5%. RESULTS: Patients ingested raw cornstarch in the amount of 0.49 to 1.34 g/kg/dose at a frequency of six times a day, which is lower than recommended (1.75-2.50 g/kg/dose, four times a day). The amount of energy intake was, on average, 50% higher than energy requirements; however, carbohydrate intake was below the adequacy percentage in 5/11 patients. Short stature was found in 4/10 patients; obesity, in 3/11; and muscle mass deficit, in 7/11. There were no statistical differences between the subgroups. CONCLUSIONS: In patients with glycogenosis type I, there was deficit in growth and muscle mass, but no differences were found between the subgroups (Ia and Ib). Although the diet did not exceed the adequacy of carbohydrates, about 1/3 of the patients presented obesity, probably due to higher energy intake.
Assuntos
Antropometria/métodos , Dieta/estatística & dados numéricos , Ingestão de Energia/fisiologia , Doença de Depósito de Glicogênio Tipo I/diagnóstico , Avaliação Nutricional , Adolescente , Animais , Composição Corporal , Estatura/fisiologia , Índice de Massa Corporal , Peso Corporal/fisiologia , Criança , Pré-Escolar , Estudos Transversais , Dieta/tendências , Nanismo/epidemiologia , Corpo Adiposo/fisiologia , Feminino , Doença de Depósito de Glicogênio Tipo I/epidemiologia , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/metabolismo , Humanos , Masculino , Desenvolvimento Muscular/fisiologia , Necessidades Nutricionais , Obesidade/epidemiologia , Inquéritos e Questionários/normas , Magreza , Adulto JovemRESUMO
ABSTRACT Objective: To perform anthropometric and dietary evaluation of patients with glycogenosis type Ia and Ib. Methods: This cross-sectional study is composed of a sample of 11 patients with glycogenosis divided into two subgroups according to the classification of glycogenosis (type Ia=5 and type Ib=6), aged between 4 and 20 years. The analyzed anthropometric variables were weight, height, body mass index, and measures of lean and fat body mass, which were compared with reference values. For dietary assessment, a food frequency questionnaire was used to calculate energy and macronutrients intake as well as the amount of raw cornstarch consumed. Mann-Whitney U test and Fisher's exact test were performed, considering a significance level of 5%. Results: Patients ingested raw cornstarch in the amount of 0.49 to 1.34 g/kg/dose at a frequency of six times a day, which is lower than recommended (1.75-2.50 g/kg/dose, four times a day). The amount of energy intake was, on average, 50% higher than energy requirements; however, carbohydrate intake was below the adequacy percentage in 5/11 patients. Short stature was found in 4/10 patients; obesity, in 3/11; and muscle mass deficit, in 7/11. There were no statistical differences between the subgroups. Conclusions: In patients with glycogenosis type I, there was deficit in growth and muscle mass, but no differences were found between the subgroups (Ia and Ib). Although the diet did not exceed the adequacy of carbohydrates, about 1/3 of the patients presented obesity, probably due to higher energy intake.
RESUMO Objetivo: Realizar avaliação antropométrica e dietética de pacientes com glicogenose tipos Ia e Ib. Métodos: Estudo transversal composto de uma amostra de 11 pacientes com glicogenose divididos em dois subgrupos de acordo com a classificação da glicogenose (tipo Ia=5; tipo Ib=6), com idades entre 4 e 20 anos. As variáveis antropométricas analisadas foram peso, estatura, índice de massa corporal e medidas de massa magra e gorda, que foram comparadas com valores de referência. Para avaliação dietética, foi utilizado um questionário de frequência alimentar para cálculo de ingestão de energia e macronutrientes, além da quantidade de amido cru ingerida. Realizaram-se testes U de Mann-Whitney e exato de Fisher, com nível de significância de 5%. Resultados: Os pacientes ingeriram amido cru na quantidade de 0,49 a 1,34 g/kg/dose na frequência de seis vezes ao dia, inferior à dosagem preconizada (1,75-2,50 g/kg/dose quatro vezes ao dia). A quantidade de energia consumida foi, em média, 50% a mais que as necessidades, contudo o consumo de carboidratos foi abaixo da porcentagem de adequação em 5/11 pacientes. Baixa estatura ocorreu em 4/10 pacientes, obesidade em 3/11 e déficit de massa muscular em 7/11. Não houve diferença estatística entre os subgrupos. Conclusões: Em pacientes com glicogenose tipo I, houve déficit de crescimento e de massa muscular, mas não diferença significante entre os subgrupos (Ia e Ib). Embora a dieta não tenha ultrapassado a adequação de carboidratos, 1/3 dos pacientes apresentou obesidade, provavelmente pela maior ingestão de energia.
Assuntos
Humanos , Animais , Masculino , Feminino , Pré-Escolar , Criança , Adolescente , Adulto Jovem , Ingestão de Energia/fisiologia , Doença de Depósito de Glicogênio Tipo I/diagnóstico , Avaliação Nutricional , Antropometria/métodos , Dieta/estatística & dados numéricos , Magreza , Composição Corporal , Estatura/fisiologia , Peso Corporal/fisiologia , Corpo Adiposo/fisiologia , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/mortalidade , Doença de Depósito de Glicogênio Tipo I/epidemiologia , Índice de Massa Corporal , Estudos Transversais , Inquéritos e Questionários/normas , Desenvolvimento Muscular/fisiologia , Dieta/tendências , Nanismo/epidemiologia , Necessidades Nutricionais , Obesidade/epidemiologiaRESUMO
Movement assisted by muscles forms the basis of various behavioural traits seen in Drosophila. Myogenesis involves developmental processes like cellular specification, differentiation, migration, fusion, adherence to tendons and neuronal innervation in a series of coordinated event well defined in body space and time. Gene regulatory networks are switched on-off, fine tuning at the right developmental stage to assist each cellular event. Drosophila is a holometabolous organism that undergoes myogenesis waves at two developmental stages, and is ideal for comparative analysis of the role of genes and genetic pathways conserved across phyla. In this review we have summarized myogenic events from the embryo to adult focussing on the somatic muscle development during the early embryonic stage and then on indirect flight muscles (IFM) formation required for adult life, emphasizing on recent trends of analysing muscle mutants and advances in Drosophila muscle biology.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila , Desenvolvimento Muscular , Animais , Fenômenos Fisiológicos Celulares , Drosophila/embriologia , Drosophila/crescimento & desenvolvimento , Regulação da Expressão GênicaRESUMO
Ultrasmall superparamagnetic iron oxide nanoparticles with a size <5 nm are emerging nanomaterials for their excellent biocompatibility, chemical stability, and tunable surface modifications. The applications explored include dual-modal or multi-modal imaging, drug delivery, theranostics and, more recently, magnetic resonance angiography. Good biocompatibility and biosafety are regarded as the preliminary requirements for their biomedical applications and further exploration in this field is still required. We previously synthesized and characterized ultrafine (average core size of 3 nm) silica-coated superparamagnetic iron oxide fluorescent nanoparticles, named sub-5 SIO-Fl, uniform in size, shape, chemical properties and composition. The cellular uptake and in vitro biocompatibility of the as-synthesized nanoparticles were demonstrated in a human colon cancer cellular model. Here, we investigated the biocompatibility of sub-5 SIO-Fl nanoparticles in human Amniotic Mesenchymal Stromal/Stem Cells (hAMSCs). Kinetic analysis of cellular uptake showed a quick nanoparticle internalization in the first hour, increasing over time and after long exposure (48 h), the uptake rate gradually slowed down. We demonstrated that after internalization, sub-5 SIO-Fl nanoparticles neither affect hAMSC growth, viability, morphology, cytoskeletal organization, cell cycle progression, immunophenotype, and the expression of pro-angiogenic and immunoregulatory paracrine factors nor the osteogenic and myogenic differentiation markers. Furthermore, sub-5 SIO-Fl nanoparticles were intravenously injected into mice to investigate the in vivo biodistribution and toxicity profile for a time period of 7 weeks. Our findings showed an immediate transient accumulation of nanoparticles in the kidney, followed by the liver and lungs, where iron contents increased over a 7-week period. Histopathology, hematology, serum pro-inflammatory response, body weight and mortality studies demonstrated a short- and long-term biocompatibility and biosafety profile with no apparent acute and chronic toxicity caused by these nanoparticles in mice. Overall, these results suggest the feasibility of using sub-5 SIO-Fl nanoparticles as a promising agent for stem cell magnetic targeting as well as for diagnostic and therapeutic applications in oncology.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis , Nanopartículas de Magnetita/química , Teste de Materiais , Células-Tronco Mesenquimais/metabolismo , Dióxido de Silício , Animais , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Dióxido de Silício/química , Dióxido de Silício/farmacologiaRESUMO
OBJECTIVES: Myoblast transfer therapy (MTT) is a technique to replace muscle satellite cells with genetically repaired or healthy myoblasts, to treat muscular dystrophies. However, clinical trials with human myoblasts were ineffective, showing almost no benefit with MTT. One important obstacle is the rapid senescence of human myoblasts. The main purpose of our study was to compare the various methods for scalable generation of proliferative human myoblasts. METHODS: We compared the immortalization of primary myoblasts with hTERT, cyclin D1 and CDK4R24C , two chemically defined methods for deriving myoblasts from pluripotent human embryonic stem cells (hESCs), and introduction of viral MyoD into hESC-myoblasts. RESULTS: Our results show that, while all the strategies above are suboptimal at generating bona fide human myoblasts that can both proliferate and differentiate robustly, chemically defined hESC-monolayer-myoblasts show the most promise in differentiation potential. CONCLUSIONS: Further efforts to optimize the chemically defined differentiation of hESC-monolayer-myoblasts would be the most promising strategy for the scalable generation of human myoblasts, for applications in MTT and high-throughput drug screening.
Assuntos
Mioblastos/citologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Transformação Celular Viral , Células Cultivadas , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/genética , Marcadores Genéticos , Células-Tronco Embrionárias Humanas/citologia , Humanos , Desenvolvimento Muscular , Proteína MyoD/genética , Mioblastos/fisiologia , Mioblastos/transplante , Regeneração , Células Satélites de Músculo Esquelético/citologia , Telomerase/genéticaRESUMO
The minimal criteria for mesenchymal stem/stromal cell (MSC) identification set by the International Society for Cellular Therapy include plastic adherence, presence and absence of a set of surface antigens and in vitro multilineage differentiation. This differentiation is assessed through stimulation of MSCs with defined combination and concentration of growth factors towards specific lineages and histological confirmation of the presence of differentiated cells. Here we provide protocols for multilineage differentiation, namely, osteogenesis, adipogenesis, chondrogenesis and myogenesis. We also provide their respective histological analyses.
Assuntos
Adipócitos/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Células Musculares/citologia , Osteócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/fisiologia , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Imunofluorescência , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Osteogênese/efeitos dos fármacos , Fluxo de TrabalhoRESUMO
Sixty New Zealand White weaned rabbits were divided into three groups and subjected to different dietary treatments: a standard diet for the control (C), a standard feed supplemented with 10% of plain olive leaves (OL) and a standard feed supplemented with 10% of selenium-fortified olive leaves (100â¯mg/L of foliar spray sodium selenate solution; SeOL). The productive performance was recorded at the time of slaughter (after 35â¯days); the carcass and meat traits were determined and estimated indexes of fatty acid metabolism were calculated. No significant differences were found on the rabbit productive performance and the physical-chemical characteristics of the meat. Both group of rabbits on the enriched diet showed leaner and thinner carcasses and a higher meat concentration of oleic acid. The estimated index of Δ5â¯+â¯Δ6-desaturase, starting from n-6 fatty acids, was lower in both groups supplemented with leaves. The use of selenium-fortified olive leaves, positively affected the lipid oxidative stability of rabbit meat.
Assuntos
Ração Animal , Ácidos Graxos Insaturados/metabolismo , Carne/análise , Desenvolvimento Muscular , Olea/química , Folhas de Planta/química , Ácido Selênico/administração & dosagem , Aerossóis , Agricultura/economia , Algoritmos , Ração Animal/economia , Animais , Biofortificação , Feminino , Qualidade dos Alimentos , Humanos , Resíduos Industriais/análise , Resíduos Industriais/economia , Itália , Masculino , Carne/economia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Valor Nutritivo , Olea/crescimento & desenvolvimento , Olea/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Coelhos , Ácido Selênico/metabolismo , Aumento de PesoRESUMO
Myoblast differentiation is a highly complex process that is regulated by proteins as well as by non-coding RNAs. Circular RNAs have been identified as an emerging new class of non-coding RNA in the modulation of skeletal muscle development, whereas their expression profiles and functional regulation in myoblast differentiation remain unknown. In the present study, we performed deep RNA-sequencing of C2C12 myoblasts during cell differentiation and uncovered 37,751 unique circular RNAs derived from 6943 hosting genes. The ensuing qRT-PCR and RNA ï¬uorescence in situ hybridization verification were carried out to confirm the RNA-sequencing results. An unbiased analysis demonstrated dynamic circular RNA expression changes in the process of myoblast differentiation, and the circular RNA abundances were independent from their cognate linear RNAs. Gene ontology analysis showed that many down-regulated circular RNAs were exclusive to cell division and the cell cycle, whereas up-regulated circular RNAs were related to the cell development process. Furthermore, interaction networks of circular RNA-microRNA were constructed. Several microRNAs well-known for myoblast regulation, such as miR-133, miR-24 and miR-23a, were in this network. In summary, this study showed that circular RNA expression dynamics changed during myoblast differentiation. Circular RNAs play a role in regulating the myoblast cell cycle and development by acting as microRNA binding sites to facilitate their regulation of gene expression during myoblast differentiation. These findings open a new avenue for future investigation of this emerging RNA class in skeletal muscle growth and development.
Assuntos
Diferenciação Celular , MicroRNAs/genética , Desenvolvimento Muscular , Mioblastos/citologia , RNA/genética , Animais , Células Cultivadas , Camundongos , Mioblastos/metabolismo , RNA CircularRESUMO
BACKGROUND: Due to the post-mitotic nature of myonuclei, postnatal myogenesis is essential for skeletal muscle growth, repair, and regeneration. This process is facilitated by satellite cells through proliferation, differentiation, and subsequent fusion with a pre-existing muscle fiber (i.e., myonuclear accretion). Current knowledge of myogenesis is primarily based on the in vitro formation of syncytia from myoblasts, which represents aspects of developmental myogenesis, but may incompletely portray postnatal myogenesis. Therefore, we aimed to develop an in vitro model that better reflects postnatal myogenesis, to study the cell intrinsic and extrinsic processes and signaling involved in the regulation of postnatal myogenesis. METHODS: Proliferating C2C12 myoblasts were trypsinized and co-cultured for 3 days with 5 days differentiated C2C12 myotubes. Postnatal myonuclear accretion was visually assessed by live cell time-lapse imaging and cell tracing by cell labeling with Vybrant® DiD and DiO. Furthermore, a Cre/LoxP-based cell system was developed to semi-quantitatively assess in vitro postnatal myonuclear accretion by the conditional expression of luciferase upon myoblast-myotube fusion. Luciferase activity was assessed luminometrically and corrected for total protein content. RESULTS: Live cell time-lapse imaging, staining-based cell tracing, and recombination-dependent luciferase activity, showed the occurrence of postnatal myonuclear accretion in vitro. Treatment of co-cultures with the myogenic factor IGF-I (p < 0.001) and the cytokines IL-13 (p < 0.05) and IL-4 (p < 0.001) increased postnatal myonuclear accretion, while the myogenic inhibitors cytochalasin D (p < 0.001), myostatin (p < 0.05), and TNFα (p < 0.001) decreased postnatal myonuclear accretion. Furthermore, postnatal myonuclear accretion was increased upon recovery from electrical pulse stimulation-induced fiber damage (p < 0.001) and LY29004-induced atrophy (p < 0.001). Moreover, cell type-specific siRNA-mediated knockdown of myomaker in myoblasts (p < 0.001), but not in myotubes, decreased postnatal myonuclear accretion. CONCLUSIONS: We developed a physiologically relevant, sensitive, high-throughput cell system for semi-quantitative assessment of in vitro postnatal myonuclear accretion, which can be used to mimic physiological myogenesis triggers, and can distinguish the cell type-specific roles of signals and responses in the regulation of postnatal myogenesis. As such, this method is suitable for both basal and translational research on the regulation of postnatal myogenesis, and will improve our understanding of muscle pathologies that result from impaired satellite cell number or function.
Assuntos
Modelos Biológicos , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/citologia , Mioblastos/citologia , Animais , Atrofia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Fusão Celular , Células Cultivadas , Técnicas de Cocultura , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.
Assuntos
Sinalização do Cálcio/fisiologia , Separação Celular/métodos , Músculo Esquelético/citologia , Mioblastos/citologia , Mioblastos/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Cálcio/análise , Cálcio/metabolismo , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Citometria de Fluxo , Imunofluorescência/métodos , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Humanos , Imagem Molecular/métodos , Desenvolvimento Muscular/fisiologia , Fatores de Transcrição/metabolismo , Transfecção/métodosRESUMO
For adolescent athletes (14-18 years), data on sport nutrition knowledge, behaviors and beliefs are limited, especially based on sex, race/ethnicity and socioeconomic status. High school soccer players (n = 535; 55% female; 51% White, 41% Latino; 41% National School Lunch Program (NSLP) participants (80% Latino)) completed two questionnaires (demographic/health history and sport nutrition). The sport nutrition knowledge score was 45.6% with higher scores in NSLP-Whites vs. NSLP-Latinos (p < 0.01). Supplement knowledge differed by sex (16% lower in females; p = 0.047) and race/ethnicity (33% lower in Latinos; p < 0.001). Breakfast consumption was 57%; females ate breakfast less (50%) than males (60%; p < 0.001); NSLP-participants ate breakfast less (47%) than non-NSLP (62%; p < 0.001). Supplement use was 46%, with Latinos using more supplements than Whites do (p = 0.016). Overall, 30% used protein shakes, with females using less than males (p = 0.02), while use was twice as likely in Latino vs. White (p = 0.03). Overall, 45% reported their nutrient requirements were different from non-athlete peers. Latinos were less likely (p = 0.03) to report that their diet met nutritional requirements, but more than twice as likely to report that nutritional supplements were necessary for training (p < 0.001). Adolescent athletes, especially females and Latinos, would benefit from sport nutrition education that enhances food selection skills for health and sport performance.
Assuntos
Comportamento do Adolescente , Atletas , Dieta Saudável , Conhecimentos, Atitudes e Prática em Saúde , Cooperação do Paciente , Ciências da Nutrição e do Esporte/educação , Adolescente , Comportamento do Adolescente/etnologia , Desenvolvimento do Adolescente , Dieta/efeitos adversos , Dieta/etnologia , Dieta Saudável/etnologia , Suplementos Nutricionais/efeitos adversos , Feminino , Conhecimentos, Atitudes e Prática em Saúde/etnologia , Hispânico ou Latino , Humanos , Masculino , Desenvolvimento Muscular , Avaliação das Necessidades , Oregon , Cooperação do Paciente/etnologia , Instituições Acadêmicas , Autorrelato , Futebol , Fatores Socioeconômicos , Fenômenos Fisiológicos da Nutrição Esportiva/etnologia , População BrancaRESUMO
Branched-chain amino acids (BCAA) have been clearly demonstrated to have anabolic effects on muscle protein synthesis. However, little is known about their roles in the regulation of net AA fluxes across skeletal muscle in vivo. This study was aimed to investigate the effect and related mechanisms of dietary supplementation of BCAA on muscle net amino acid (AA) fluxes using the hindlimb flux model. In all fourteen 4-week-old barrows were fed reduced-protein diets with or without supplemental BCAA for 28 d. Pigs were implanted with carotid arterial, femoral arterial and venous catheters, and fed once hourly with intraarterial infusion of p-amino hippurate. Arterial and venous plasma and muscle samples were obtained for the measurement of AA, branched-chain α-keto acids (BCKA) and 3-methylhistidine (3-MH). Metabolomes of venous plasma were determined by HPLC-quadrupole time-of-flight-MS. BCAA-supplemented group showed elevated muscle net fluxes of total essential AA, non-essential AA and AA. As for individual AA, muscle net fluxes of each BCAA and their metabolites (alanine, glutamate and glutamine), along with those of histidine, methionine and several functional non-essential AA (glycine, proline and serine), were increased by BCAA supplementation. The elevated muscle net AA fluxes were associated with the increase in arterial and intramuscular concentrations of BCAA and venous metabolites including BCKA and free fatty acids, and were also related to the decrease in the intramuscular concentration of 3-MH. Correlation analysis indicated that muscle net AA fluxes are highly and positively correlated with arterial BCAA concentrations and muscle net BCKA production. In conclusion, supplementing BCAA to reduced-protein diet increases the arterial concentrations and intramuscular catabolism of BCAA, both of which would contribute to an increase of muscle net AA fluxes in young pigs.
Assuntos
Aminoácidos de Cadeia Ramificada/administração & dosagem , Anabolizantes/administração & dosagem , Dieta com Restrição de Proteínas/veterinária , Desenvolvimento Muscular , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Regulação para Cima , Aminoácidos/sangue , Aminoácidos/metabolismo , Aminoácidos de Cadeia Ramificada/sangue , Aminoácidos de Cadeia Ramificada/metabolismo , Anabolizantes/sangue , Anabolizantes/metabolismo , Animais , China , Cruzamentos Genéticos , Dieta com Restrição de Proteínas/efeitos adversos , Ácidos Graxos não Esterificados/sangue , Ácidos Graxos não Esterificados/metabolismo , Membro Posterior , Técnicas de Diluição do Indicador , Cetoácidos/sangue , Cetoácidos/metabolismo , Masculino , Metabolômica/métodos , Metilistidinas/sangue , Metilistidinas/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/crescimento & desenvolvimento , Orquiectomia/veterinária , Fluxo Sanguíneo Regional , Sus scrofa , Aumento de PesoRESUMO
The problem of modeling the dynamical regulation process within a gene network has been of great interest for a long time. We propose to model this dynamical system with a large number of nonlinear ordinary differential equations (ODEs), in which the regulation function is estimated directly from data without any parametric assumption. Most current research assumes the gene regulation network is static, but in reality, the connection and regulation function of the network may change with time or environment. This change is reflected in our dynamical model by allowing the regulation function varying with the gene expression and forcing this regulation function to be zero if no regulation happens. We introduce a statistical method called functional SCAD to estimate a time-varying sparse and directed gene regulation network, and simultaneously, to provide a smooth estimation of the regulation function and identify the interval in which no regulation effect exists. The finite sample performance of the proposed method is investigated in a Monte Carlo simulation study. Our method is demonstrated by estimating a time-varying directed gene regulation network of 20 genes involved in muscle development during the embryonic stage of Drosophila melanogaster.
Assuntos
Redes Reguladoras de Genes , Método de Monte Carlo , Dinâmica não Linear , Animais , Drosophila melanogaster/embriologia , Desenvolvimento Muscular , Fatores de TempoRESUMO
BACKGROUND: While there is a large body of work supporting the importance of early feeding practices on cognitive, immunity, behavioural and mental outcomes, few longitudinal studies have focused on motor development. The relationship between duration of breast feeding and motor development outcomes at 10, 14, and 17years were examined. METHODS: Data were obtained from the Western Australian Pregnancy (Raine) Study. There were 2868 live births recorded and children were examined for motor proficiency at 10 (M=10.54, SD=2.27), 14 (M=14.02, SD=2.33) and 17 (M=16.99, SD=2.97) years using the McCarron Assessment of Neuromuscular Development (MAND). Using linear mixed models, adjusted for covariates known to affect motor development, the influence of predominant breast feeding for <6months and ⩾6months on motor development outcomes was examined. RESULTS: Breast feeding for ⩾6months was positively associated with improved motor development outcomes at 10, 14 and 17yearsof age (p=0.019, ß 1.38) when adjusted for child's sex, maternal age, alcohol intake, family income, hypertensive status, gestational stress and mode of delivery. CONCLUSION: Early life feeding practices have an influence on motor development outcomes into late childhood and adolescence independent of sociodemographic factors.
Assuntos
Aleitamento Materno , Desenvolvimento Infantil/fisiologia , Destreza Motora/fisiologia , Desenvolvimento Muscular/fisiologia , Nervos Periféricos/fisiologia , Adolescente , Austrália , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Modelos Lineares , Estudos Longitudinais , Masculino , Gravidez , Fatores Socioeconômicos , Austrália OcidentalAssuntos
Artrite Reumatoide/patologia , Artrite Reumatoide/psicologia , Exercício Físico/fisiologia , Comportamento Sedentário , Artrite Reumatoide/complicações , Artrite Reumatoide/fisiopatologia , Aterosclerose/fisiopatologia , Efeitos Psicossociais da Doença , Humanos , Inflamação/patologia , Lipase Lipoproteica/metabolismo , Lipoproteínas HDL/metabolismo , Desenvolvimento Muscular/fisiologia , Qualidade de Vida , Fatores de Risco , Comportamento de Redução do RiscoRESUMO
Stem cell differentiation involves multiple cascades of transcriptional regulation that govern the cell fate. To study the real-time dynamics of this complex process, quantitative and high throughput live cell assays are required. Herein, we developed a lentiviral library of promoters and transcription factor binding sites to quantitatively capture the gene expression dynamics over a period of several days during myogenic differentiation of human mesenchymal stem cells (MSCs) harvested from two different anatomic locations, bone marrow and hair follicle. Our results enabled us to monitor the sequential activation of signaling pathways and myogenic gene promoters at various stages of differentiation. In conjunction with chemical inhibitors, the lentiviral array (LVA) results also revealed the relative contribution of key signaling pathways that regulate the myogenic differentiation. Our study demonstrates the potential of LVA to monitor the dynamics of gene and pathway activation during MSC differentiation as well as serve as a platform for discovery of novel molecules, genes and pathways that promote or inhibit complex biological processes.