RESUMO
Muscle stem (satellite) cells express Pax7, a key transcription factor essential for satellite cell maintenance and adult muscle regeneration. We identify the corepressor transducin-like enhancer of split-4 (TLE4) as a Pax7 interaction partner expressed in quiescent satellite cells under homeostasis. A subset of satellite cells transiently downregulate TLE4 during early time points following muscle injury. We identify these to be activated satellite cells, and that TLE4 downregulation is required for Myf5 activation and myogenic commitment. Our results indicate that TLE4 represses Pax7-mediated Myf5 transcriptional activation by occupying the -111â kb Myf5 enhancer to maintain quiescence. Loss of TLE4 function causes Myf5 upregulation, an increase in satellite cell numbers and altered differentiation dynamics during regeneration. Thus, we have uncovered a novel mechanism to maintain satellite cell quiescence and regulate muscle differentiation mediated by the corepressor TLE4.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Músculo Esquelético , Proteínas Nucleares , Proteínas Repressoras , Diferenciação Celular/genética , Humanos , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Doenças Musculares/fisiopatologia , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX7/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Satélites de Músculo Esquelético/citologiaRESUMO
Myosin heavy chain-embryonic encoded by the Myh3 gene is a skeletal muscle-specific contractile protein expressed during mammalian development and regeneration, essential for proper myogenic differentiation and function. It is likely that multiple trans-factors are involved in this precise temporal regulation of Myh3 expression. We identify a 4230 bp promoter-enhancer region that drives Myh3 transcription in vitro during C2C12 myogenic differentiation and in vivo during muscle regeneration, including sequences both upstream and downstream of the Myh3 TATA-box that are necessary for complete Myh3 promoter activity. Using C2C12 mouse myogenic cells, we find that Zinc-finger E-box binding homeobox 1 (Zeb1) and Transducin-like Enhancer of Split 3 (Tle3) proteins are crucial trans-factors that interact and differentially regulate Myh3 expression. Loss of Zeb1 function results in earlier expression of myogenic differentiation genes and accelerated differentiation, whereas Tle3 depletion leads to reduced expression of myogenic differentiation genes and impaired differentiation. Tle3 knockdown resulted in downregulation of Zeb1, which could be mediated by increased expression of miR-200c, a microRNA that binds to Zeb1 transcript and degrades it. Tle3 functions upstream of Zeb1 in regulating myogenic differentiation since double knockdown of Zeb1 and Tle3 resulted in effects seen upon Tle3 depletion. We identify a novel E-box in the Myh3 distal promoter-enhancer region, where Zeb1 binds to repress Myh3 expression. In addition to regulation of myogenic differentiation at the transcriptional level, we uncover post-transcriptional regulation by Tle3 to regulate MyoG expression, mediated by the mRNA stabilizing Human antigen R (HuR) protein. Thus, Tle3 and Zeb1 are essential trans-factors that differentially regulate Myh3 expression and C2C12 cell myogenic differentiation in vitro.
Assuntos
Proteínas Correpressoras , Músculo Esquelético , Cadeias Pesadas de Miosina , Fatores de Transcrição , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Animais , Humanos , Camundongos , Diferenciação Celular/genética , Proteínas Correpressoras/genética , Proteínas Contráteis , Proteína Semelhante a ELAV 1 , Músculo Esquelético/embriologia , Cadeias Pesadas de Miosina/genética , Fatores de Transcrição/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Myosin is a major component of the sarcomeres of muscle, but its roles during muscle development are still relatively poorly understood. A new paper in Development investigates the function of a developmentally expressed myosin heavy chain isoform during mice myogenesis. We caught up with the paper's four co-first authors, Megha Agarwal, Akashi Sharma, Pankaj Kumar and Amit Kumar, and their supervisor Sam Mathew (Associate Professor in the Regional Centre for Biotechnology in Faridabad, India) to find out more about the project.
Assuntos
Biologia do Desenvolvimento , Desenvolvimento Muscular/fisiologia , Animais , Pesquisa Biomédica/história , Biologia do Desenvolvimento/história , História do Século XX , História do Século XXI , Índia , Camundongos , Desenvolvimento Muscular/genética , Cadeias Pesadas de Miosina/fisiologiaRESUMO
Myosin heavy chain-embryonic (MyHC-emb) is a skeletal muscle-specific contractile protein expressed during muscle development. Mutations in MYH3, the gene encoding MyHC-emb, lead to Freeman-Sheldon and Sheldon-Hall congenital contracture syndromes. Here, we characterize the role of MyHC-emb during mammalian development using targeted mouse alleles. Germline loss of MyHC-emb leads to neonatal and postnatal alterations in muscle fiber size, fiber number, fiber type and misregulation of genes involved in muscle differentiation. Deletion of Myh3 during embryonic myogenesis leads to the depletion of the myogenic progenitor cell pool and an increase in the myoblast pool, whereas fetal myogenesis-specific deletion of Myh3 causes the depletion of both myogenic progenitor and myoblast pools. We reveal that the non-cell-autonomous effect of MyHC-emb on myogenic progenitors and myoblasts is mediated by the fibroblast growth factor (FGF) signaling pathway, and exogenous FGF rescues the myogenic differentiation defects upon loss of MyHC-emb function in vitro Adult Myh3 null mice exhibit scoliosis, a characteristic phenotype exhibited by individuals with Freeman-Sheldon and Sheldon-Hall congenital contracture syndrome. Thus, we have identified MyHC-emb as a crucial myogenic regulator during development, performing dual cell-autonomous and non-cell-autonomous functions.This article has an associated 'The people behind the papers' interview.
Assuntos
Diferenciação Celular/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/embriologia , Cadeias Pesadas de Miosina/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Mamíferos/embriologia , Mamíferos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genéticaRESUMO
Diabetes mellitus is a global pandemic. India, China and USA will be the countries with major diabetic population in the year 2040. Age of onset is a decade earlier in India compared to other European countries. Relative increase in visceral fat vs. subcutaneous fat in Asians and Asian Indians may explain the greater prevalence of metabolic syndrome in those population than in African American men, in whom Subcutaneous fat predominates. It is possible that visceral fat is a marker for excess postprandial free fatty acids in obesity, which is an early major contributor to the development of insulin resistance. Present study attempts to compare and co-relate the association of visceral fat and abdominal wall fat index to Insulin resistance in patients suffering from T2DM and prediabetes. Material and Objectives: To study the relationship between insulin resistance (HOMA-IR) and abdominal wall fat index (AFI) in Prediabetes and type II Diabetes Mellitus patients. To compare the visceral fat volume (VFV) with abdominal wall fat index in relation to insulin resistance in same subset of patients. METHOD: Cross sectional, observational study in 75 subjects (25 T2DM, 25 Prediabetes, 25 Controls). Detailed history including physical examination was performed. Patients were subjected to these investigations; FBS, HbA1C, S. Fasting Insulin levels, Lipid Profile, USG Abdomen to assess Visceral Fat Volume and Abdominal Wall Fat Index. Data were collected and analysed. OBSERVATION: Mean age of T2DM & prediabetes subjects was a decade higher than controls (T2DM 53 ±11.62 years, Prediabetes 55.76±11.97 years, Controls 45.72±10.42 years). Mean Systolic BP in T2DM subjects was 138.56±14.69, subjects with Prediabetes were 139.2±19.63 which is higher (p 0.02) compared to Controls(128±8.26). Average fasting serum insulin levels (mu/ml) of three groups; for T2DM: 25.41±13.7, for Prediabetes: 8.76 ±2.55, Controls: 6.07±2.55. The highest levels were in patients with T2DM, when compared to Prediabetes and controls. There was significant difference in the value of HOMA-IR, AFI, and the parameters of VFV (length between interior of abdominal muscle and splenic vein, length between interior of abdominal muscle and posterior wall of Aorta, Fat thickness of posterior renal wall) p<0.05. A significant correlation between HOMA-IR levels and VFV was found with a p value of <0.05. CONCLUSION: VFV acted as an independent marker in predicting Insulin resistance in subjects with prediabetes and T2DM. Fasting Insulin levels were highest in T2DM group amongst all three groups reflecting inadequate response of the body to appropriate levels of Insulin.
Assuntos
Parede Abdominal , Diabetes Mellitus Tipo 2 , Resistência à Insulina , Estado Pré-Diabético , Parede Abdominal/diagnóstico por imagem , Biomarcadores , Índice de Massa Corporal , Pré-Escolar , Estudos Transversais , Humanos , Insulina , Gordura Intra-Abdominal/diagnóstico por imagem , Masculino , Estado Pré-Diabético/epidemiologiaRESUMO
Missense mutations in the MYH3 gene encoding myosin heavy chain-embryonic (MyHC-embryonic) have been reported to cause two skeletal muscle contracture syndromes, Freeman Sheldon Syndrome (FSS) and Sheldon Hall Syndrome (SHS). Two residues in MyHC-embryonic that are most frequently mutated, leading to FSS, R672 and T178, are evolutionarily conserved across myosin heavy chains in vertebrates and Drosophila. We generated transgenic Drosophila expressing myosin heavy chain (Mhc) transgenes with the FSS mutations and characterized the effect of their expression on Drosophila muscle structure and function. Our results indicate that expressing these mutant Mhc transgenes lead to structural abnormalities in the muscle, which increase in severity with age and muscle use. We find that flies expressing the FSS mutant Mhc transgenes in the muscle exhibit shortening of the inter-Z disc distance of sarcomeres, reduction in the Z-disc width, aberrant deposition of Z-disc proteins, and muscle fiber splitting. The ATPase activity of the three FSS mutant MHC proteins are reduced compared to wild type MHC, with the most severe reduction observed in the T178I mutation. Structurally, the FSS mutations occur close to the ATP binding pocket, disrupting the ATPase activity of the protein. Functionally, expression of the FSS mutant Mhc transgenes in muscle lead to significantly reduced climbing capability in adult flies. Thus, our findings indicate that the FSS contracture syndrome mutations lead to muscle structural defects and functional deficits in Drosophila, possibly mediated by the reduced ATPase activity of the mutant MHC proteins.
Assuntos
Disostose Craniofacial/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Músculo Esquelético/metabolismo , Mutação , Cadeias Pesadas de Miosina/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Disostose Craniofacial/parasitologia , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Humanos , Contração Muscular/genética , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Cadeias Pesadas de Miosina/metabolismo , Sarcômeros/metabolismo , Homologia de Sequência de AminoácidosRESUMO
OBJECTIVE: To enzymatically synthesize enantiomerically pure ß-amino acids from ß-keto nitriles using nitrilase and ω-transaminase. RESULTS: An enzyme cascade system was designed where in ß-keto nitriles are initially hydrolyzed to ß-keto acids using nitrilase from Bradyrhizobium japonicum and subsequently ß-keto acids were converted to ß-amino acids using ω-transaminases. Five different ω-transaminases were tested for this cascade reaction, To enhance the yields of ß-amino acids, the concentrations of nitrilase and amino donor were optimized. Using this enzymatic reaction, enantiomerically pure (S)-ß-amino acids (ee > 99%) were generated. As nitrilase is the bottleneck in this reaction, molecular docking analysis was carried out to depict the poor affinity of nitrilase towards ß-keto acids. CONCLUSIONS: A novel enzymatic route to generate enantiomerically pure aromatic (S)-ß-amino acids from ß-keto nitriles is demonstrated for the first time.
Assuntos
Aminoácidos/metabolismo , Aminoidrolases/metabolismo , Nitrilas/metabolismo , Transaminases/metabolismo , Proteínas de Bactérias/metabolismo , Biotransformação , Bradyrhizobium/enzimologia , Ensaios Enzimáticos , Escherichia coli , Hidrólise , Simulação de Acoplamento Molecular , EstereoisomerismoRESUMO
Corepressors are proteins that cannot bind DNA directly but repress transcription by interacting with partner proteins. The Groucho/Transducin-Like Enhancer of Split (TLE) are a conserved family of corepressor proteins present in animals ranging from invertebrates such as Drosophila to vertebrates such as mice and humans. Groucho/TLE proteins perform important functions throughout the life span of animals, interacting with several pathways and regulating fundamental processes such as metabolism. However, these proteins have especially crucial functions in animal development, where they are required in multiple tissues in a temporally regulated manner. In this review, we summarize the functions of the Groucho/TLE proteins during animal development, emphasizing on specific tissues where they play essential roles.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Repressoras/metabolismo , Adipogenia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Coração/crescimento & desenvolvimento , Hematopoese , Humanos , Rim/crescimento & desenvolvimento , Família Multigênica , Neurogênese , Osteogênese , Pâncreas/crescimento & desenvolvimento , Proteínas Repressoras/genéticaRESUMO
Muscle regeneration requires the coordinated interaction of multiple cell types. Satellite cells have been implicated as the primary stem cell responsible for regenerating muscle, yet the necessity of these cells for regeneration has not been tested. Connective tissue fibroblasts also are likely to play a role in regeneration, as connective tissue fibrosis is a hallmark of regenerating muscle. However, the lack of molecular markers for these fibroblasts has precluded an investigation of their role. Using Tcf4, a newly identified fibroblast marker, and Pax7, a satellite cell marker, we found that after injury satellite cells and fibroblasts rapidly proliferate in close proximity to one another. To test the role of satellite cells and fibroblasts in muscle regeneration in vivo, we created Pax7(CreERT2) and Tcf4(CreERT2) mice and crossed these to R26R(DTA) mice to genetically ablate satellite cells and fibroblasts. Ablation of satellite cells resulted in a complete loss of regenerated muscle, as well as misregulation of fibroblasts and a dramatic increase in connective tissue. Ablation of fibroblasts altered the dynamics of satellite cells, leading to premature satellite cell differentiation, depletion of the early pool of satellite cells, and smaller regenerated myofibers. Thus, we provide direct, genetic evidence that satellite cells are required for muscle regeneration and also identify resident fibroblasts as a novel and vital component of the niche regulating satellite cell expansion during regeneration. Furthermore, we demonstrate that reciprocal interactions between fibroblasts and satellite cells contribute significantly to efficient, effective muscle regeneration.
Assuntos
Tecido Conjuntivo/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Transgênicos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Reação em Cadeia da Polimerase , Fator de Transcrição 4RESUMO
Muscle and its connective tissue are intimately linked in the embryo and in the adult, suggesting that interactions between these tissues are crucial for their development. However, the study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. Here, we show that the transcription factor Tcf4 (transcription factor 7-like 2; Tcf7l2) is strongly expressed in connective tissue fibroblasts and that Tcf4(GFPCre) mice allow genetic manipulation of these fibroblasts. Using this new reagent, we find that connective tissue fibroblasts critically regulate two aspects of myogenesis: muscle fiber type development and maturation. Fibroblasts promote (via Tcf4-dependent signals) slow myogenesis by stimulating the expression of slow myosin heavy chain. Also, fibroblasts promote the switch from fetal to adult muscle by repressing (via Tcf4-dependent signals) the expression of developmental embryonic myosin and promoting (via a Tcf4-independent mechanism) the formation of large multinucleate myofibers. In addition, our analysis of Tcf4 function unexpectedly reveals a novel mechanism of intrinsic regulation of muscle fiber type development. Unlike other intrinsic regulators of fiber type, low levels of Tcf4 in myogenic cells promote both slow and fast myogenesis, thereby promoting overall maturation of muscle fiber type. Thus, we have identified novel extrinsic and intrinsic mechanisms regulating myogenesis. Most significantly, our data demonstrate for the first time that connective tissue is important not only for adult muscle structure and function, but is a vital component of the niche within which muscle progenitors reside and is a critical regulator of myogenesis.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Tecido Conjuntivo/fisiologia , Desenvolvimento Muscular/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/deficiência , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Feminino , Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Desenvolvimento Muscular/genética , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Gravidez , Transdução de Sinais , Fator de Transcrição 4 , beta Catenina/metabolismoRESUMO
Myosin heavy chain-perinatal (MyHC-perinatal) is one of two development-specific myosin heavy chains expressed exclusively during skeletal muscle development and regeneration. The specific functions of MyHC-perinatal are unclear, although mutations are known to lead to contracture syndromes such as Trismus-pseudocamptodactyly syndrome. Here, we characterize the functions of MyHC-perinatal during skeletal muscle differentiation and regeneration. Loss of MyHC-perinatal function leads to enhanced differentiation characterized by increased expression of myogenic regulatory factors and differentiation index as well as reduced reserve cell numbers in vitro. Proteomic analysis revealed that loss of MyHC-perinatal function results in a switch from oxidative to glycolytic metabolism in myofibers, suggesting a shift from slow type I to fast type IIb fiber type, also supported by reduced mitochondrial numbers. Paracrine signals mediate the effect of loss of MyHC-perinatal function on myogenic differentiation, possibly mediated by non-apoptotic caspase-3 signaling along with enhanced levels of the pro-survival apoptosis regulator Bcl2 and nuclear factor kappa-B (NF-κB). Knockdown of MyHC-perinatal during muscle regeneration in vivo results in increased expression of the differentiation marker myogenin (MyoG) and impaired differentiation, evidenced by smaller myofibers, elevated fibrosis and reduction in the number of satellite cells. Thus, we find that MyHC-perinatal is a crucial regulator of myogenic differentiation, myofiber oxidative phenotype and regeneration.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Músculo Esquelético , Cadeias Pesadas de Miosina , Regeneração , Animais , Diferenciação Celular/genética , Regeneração/genética , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Camundongos , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/citologia , Fenótipo , Caspase 3/metabolismo , Caspase 3/genética , Miogenina/metabolismo , Miogenina/genética , OxirreduçãoRESUMO
Rhabdomyosarcoma tumor cells resemble differentiating skeletal muscle cells, which unlike normal muscle cells, fail to undergo terminal differentiation, underlying their proliferative and metastatic properties. We identify the corepressor TLE3 as a key regulator of rhabdomyosarcoma tumorigenesis by inhibiting the Wnt-pathway. Loss of TLE3 function leads to Wnt-pathway activation, reduced proliferation, decreased migration, and enhanced differentiation in rhabdomyosarcoma cells. Muscle-specific TLE3-knockout results in enhanced expression of terminal myogenic differentiation markers during normal mouse development. TLE3-knockout rhabdomyosarcoma cell xenografts result in significantly smaller tumors characterized by reduced proliferation, increased apoptosis and enhanced differentiation. We demonstrate that TLE3 interacts with and recruits the histone methyltransferase KMT1A, leading to repression of target gene activation and inhibition of differentiation in rhabdomyosarcoma. A combination drug therapy regime to promote Wnt-pathway activation by the small molecule BIO and inhibit KMT1A by the drug chaetocin led to significantly reduced tumor volume, decreased proliferation, increased expression of differentiation markers and increased survival in rhabdomyosarcoma tumor-bearing mice. Thus, TLE3, the Wnt-pathway and KMT1A are excellent drug targets which can be exploited for treating rhabdomyosarcoma tumors.
Assuntos
Rabdomiossarcoma , Humanos , Camundongos , Animais , Proteínas Correpressoras/genética , Histona Metiltransferases , Diferenciação Celular/genética , Rabdomiossarcoma/patologia , Antígenos de Diferenciação , Proliferação de Células/genética , Linhagem Celular TumoralRESUMO
Super-resolution imaging techniques have fundamentally changed our understanding of cellular architecture and dynamics by surpassing the diffraction limit and enabling the visualization of subcellular details. The popular super-resolution method known as stochastic optical reconstruction microscopy (STORM) relies on the exact localization of single fluorescent molecules. The significance of employing Vectashield as a mounting medium for the super-resolution imaging scheme called direct STORM has recently been explored. Alexa Fluor 647 (AF647), one of the most popular dyes, shows significant blinking in Vectashield. However, to observe prominent blinking of the fluorophore for the reconstruction of super-resolved images, the power of the excitation laser needs to be tuned. This work demonstrates the tuning of excitation power density in the sample plane for superior imaging performance using AF647 in Vectashield. Samples comprising MDA-MB-231 breast cancer cell line are used for the experiments. The actin filaments of the cell are stained with phalloidin-conjugated AF647 dye. For the experiment, we employ a low-cost openFrame-based STORM system equipped with a programmable Arduino-regulated laser source emitting at 638 nm. An excitation power density of 0.60 kW/cm2 at 638 nm in the sample plane is observed to maximize the signal-to-noise ratio, the number of switching events, and the number of photons detected per event during image acquisition, thereby leading to the best imaging performance in terms of resolution. The outcome of this work will promote further STORM-based super-resolved imaging applications in cell biology using Alexa Fluor 647 in Vectashield.
RESUMO
Mutations in MYH3, the gene encoding the developmental myosin heavy chain-embryonic (MyHC-embryonic) skeletal muscle-specific contractile protein, cause several congenital contracture syndromes. Among these, recessive loss-of-function MYH3 mutations lead to spondylocarpotarsal synostosis (SCTS), characterized by vertebral fusions and scoliosis. We find that Myh3 germline knockout adult mice display SCTS phenotypes such as scoliosis and vertebral fusion, in addition to reduced body weight, muscle weight, myofiber size, and grip strength. Myh3 knockout mice also exhibit changes in muscle fiber type, altered satellite cell numbers and increased muscle fibrosis. A mass spectrometric analysis of embryonic skeletal muscle from Myh3 knockouts identified integrin signaling and cytoskeletal regulation as the most affected pathways. These pathways are closely connected to the mechanosensing Yes-associated protein (YAP) transcriptional regulator, which we found to be significantly activated in the skeletal muscle of Myh3 knockout mice. To test whether increased YAP signaling might underlie the musculoskeletal defects in Myh3 knockout mice, we treated these mice with CA3, a small molecule inhibitor of YAP signaling. This led to increased muscle fiber size, rescue of most muscle fiber type alterations, normalization of the satellite cell marker Pax7 levels, increased grip strength, reduced fibrosis, and decline in scoliosis in Myh3 knockout mice. Thus, increased YAP activation underlies the musculoskeletal defects seen in Myh3 knockout mice, indicating its significance as a key pathway to target in SCTS and other MYH3-related congenital syndromes.
Assuntos
Cadeias Pesadas de Miosina , Escoliose , Animais , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Escoliose/genética , Escoliose/congênito , Escoliose/metabolismo , Transdução de Sinais , SíndromeRESUMO
The purpose of the study is to develop and assess mucoadhesive in situ nasal gel formulations of loratadine and chlorpheniramine maleate to advance the bioavailability of the drug as compared to its conventional dosage forms. The influence of various permeation enhancers, such as EDTA (0.2% w/v), sodium taurocholate (0.5% w/v), oleic acid (5% w/v), and Pluronic F 127 (10% w/v), on the nasal absorption of loratadine and chlorpheniramine from in situ nasal gels containing different polymeric combinations, such as hydroxypropyl methylcellulose, Carbopol 934, sodium carboxymethylcellulose, and chitosan, is studied. Among these permeation enhancers, sodium taurocholate, Pluronic F127 and oleic acid produced a noticeable increase in the loratadine in situ nasal gel flux compared with in situ nasal gels without permeation enhancer. However, EDTA increased the flux slightly, and in most cases, the increase was insignificant. However, in the case of chlorpheniramine maleate in situ nasal gels, the permeation enhancer oleic acid only showed a noticeable increase in flux. Sodium taurocholate and oleic acid seems to be a better and efficient enhancer, enhancing the flux > 5-fold compared with in situ nasal gels without permeation enhancer in loratadine in situ nasal gels. Pluronic F127 also showed a better permeation, increasing the effect by >2-fold in loratadine in situ nasal gels. In chlorpheniramine maleate in situ nasal gels with EDTA, sodium taurocholate and Pluronic F127 were equally effective, enhancing chlorpheniramine maleate permeation. Oleic acid has a better effect as permeation enhancer in chlorpheniramine maleate in situ nasal gels and showed a maximum permeation enhancement of >2-fold.
RESUMO
Overexpression of HDAC 2 promotes cell proliferation in ovarian cancer. HDAC 2 is involved in chromatin remodeling, transcriptional repression, and the formation of condensed chromatin structures. Targeting HDAC 2 presents a promising therapeutic approach for correcting cancer-associated epigenetic abnormalities. Consequently, HDAC 2 inhibitors have evolved as an attractive class of anti-cancer agents. This work intended to investigate the anti-cancer abilities and underlying molecular mechanisms of Rhamnetin in human epithelial ovarian carcinoma cells (SKOV3), which remain largely unexplored. We employed various in vitro methods, including MTT, apoptosis study, cell cycle analysis, fluorescence microscopy imaging, and in vitro enzymatic HDAC 2 protein inhibition, to examine the chemotherapeutic sensitivity of Rhamnetin in SKOV3 cells. Additionally, we conducted in silico studies using molecular docking, MD simulation, MM-GBSA, DFT, and pharmacokinetic analysis to investigate the binding interaction mechanism within Rhamnetin and HDAC 2, alongside the compound's prospective as a lead candidate. The in vitro assay confirmed the cytotoxic effects of Rhamnetin on SKOV3 cells, through its inhibition of HDAC 2 activity. Rhamnetin, a nutraceutical flavonoid, halted at the G1 phase of the cell cycle and triggered apoptosis in SKOV3 cells. Furthermore, computational studies provided additional evidence of its stable binding to the HDAC 2 protein's binding site cavity. Based on our findings, we conclude that Rhamnetin effectively promotes apoptosis and mitigates the proliferation of SKOV3 cells through HDAC 2 inhibition. These results highlight Rhamnetin as a potential lead compound, opening a new therapeutic strategy for human epithelial ovarian cancer.Communicated by Ramaswamy H. Sarma.
RESUMO
A deracemization method was developed to generate optically pure L-homoalanine from racemic homoalanine using D-amino acid oxidase and ω-transaminase. A whole cell reaction using a biphasic system converted 500 mM racemic homoalanine to 485 mM L-homoalanine (>99% ee).
Assuntos
Aminobutiratos/química , D-Aminoácido Oxidase/metabolismo , Transaminases/metabolismo , Aminobutiratos/metabolismo , Escherichia coli/química , Escherichia coli/enzimologia , Cinética , EstereoisomerismoRESUMO
Cancer is a heterogeneous disease characterized by various genetic and phenotypic aberrations. Cancer cells undergo genetic modifications that promote their proliferation, survival, and dissemination as the disease progresses. The unabated proliferation of cancer cells incurs an enormous energy demand that is supplied by metabolic reprogramming. Cancer cells undergo metabolic alterations to provide for increased energy and metabolite requirement; these alterations also help drive the tumor progression. Dysregulation in glucose uptake and increased lactate production via "aerobic glycolysis" were described more than 100 years ago, and since then, the metabolic signature of various cancers has been extensively studied. However, the extensive research in this field has failed to translate into significant therapeutic intervention, except for treating childhood-ALL with amino acid metabolism inhibitor L-asparaginase. Despite the growing understanding of novel metabolic alterations in tumors, the therapeutic targeting of these tumor-specific dysregulations has largely been ineffective in clinical trials. This chapter discusses the major pathways involved in the metabolism of glucose, amino acids, and lipids and highlights the inter-twined nature of metabolic aberrations that promote tumorigenesis in different types of cancer. Finally, we summarise the therapeutic interventions which can be used as a combinational therapy to target metabolic dysregulations that are unique or common in blood, breast, colorectal, lung, and prostate cancer.
Assuntos
Glicólise , Neoplasias da Próstata , Masculino , Humanos , Criança , Redes e Vias Metabólicas , Transformação Celular Neoplásica/metabolismo , Metabolismo dos CarboidratosRESUMO
During the initial stages of the coronavirus disease 2019 (COVID-19) pandemic, the community spread of the virus had efficiently been prevented in Kerala, India. The present study aimed to assess fear and its predictors among the general public following the unforeseen surge of COVID-19 cases in July, 2020 using a reliable and validated tool, the 'Fear of COVID-19 Scale', administered through social media. Of 1,100 responses, 1,046 responses were included in the analysis. The majority of the respondents expressed mild fear 44.6%; moderate fear was found in 39.4% of the respondents, severe fear in 13.6% and very severe fear in 2.4% of the respondents. The mean fear score was found to be 15.93±5.81. Statistically significant (P≤0.05) associations were found between fear and sociodemographic variables, such as age, sex, education and occupation, along with predictors, such as the district of residence, healthcare stakeholders in the family, and the presence of an infected individual in the family. Women and students were found to be the most affected. On the whole, the present study provides sufficient insight into the fear associated with COVID-19. The findings presented herein may enable authorities to take adequate measures to prevent the aftermath.
RESUMO
Three-dimensional (3D) bioprinting has emerged as a promising method for the engineering of tissues and organs. Still, it faces challenges in its widespread use due to issues with the development of bioink materials and the nutrient diffusion barrier inherent to these scaffold materials. Herein, we introduce a method to promote oxygen diffusion throughout the printed constructs using genetically encoded gas vesicles derived from haloarchaea. These hollow nanostructures are composed of a protein shell that allows gases to permeate freely while excluding the water flow. After printing cells with gas vesicles of various concentrations, the cells were observed to have increased activity and proliferation. These results suggest that air-filled gas vesicles can help overcome the diffusion barrier throughout the 3D bioprinted constructs by increasing oxygen availability to cells within the center of the construct. The biodegradable nature of the gas vesicle proteins combined with our promising results encourage their potential use as oxygen-promoting materials in biological samples.