An in vitro agent-based modelling approach to optimization of culture medium for generating muscle cells.

Methodologies for culturing muscle tissue are currently lacking in terms of quality and quantity of mature cells produced. We analyse images from in vitro experiments to quantify the effects of culture media composition on mouse-derived myoblast behaviour and myotube quality. Metrics of early indicators of cell quality were defined. Images of muscle cell differentiation reveal that altering culture media significantly affects quality indicators and myoblast migratory behaviours. To study the effects of early-stage cell behaviours on mature cell quality, metrics drawn from experimental images or inferred by approximate Bayesian computation (ABC) were applied as inputs to an agent-based model (ABM) of skeletal muscle cell differentiation with quality indicator metrics as outputs. Computational modelling was used to inform further in vitro experiments to predict the optimum media composition for culturing muscle cells. Our results suggest that myonuclei production in myotubes is inversely related to early-stage nuclei fusion index and that myonuclei density and spatial distribution are correlated with residence time of fusing myoblasts, the age at which myotube-myotube fusion ends and the repulsion force between myonuclei. Culture media with 5% serum was found to produce the optimum cell quality and to make muscle cells cultured in a neuron differentiation medium viable.

Online solid phase extraction high-performance liquid chromatography - Isotope dilution - Tandem mass spectrometry quantification of organophosphate pesticides, synthetic pyrethroids, and selected herbicide metabolites in human urine.

Analytical methods to quantify pesticide biomarkers in human population studies are critical for exposure assessment given the widespread use of pesticides for pest and weed control and their potential for affecting human health. We developed a method to quantify, in 0.2 mL of urine, concentrations of 10 pesticide biomarkers: four organophosphate insecticide metabolites (3,5,6-trichloro-2-pyridinol (TCPy), 2-isopropyl-6-methyl-4-pyrimidinol, para-nitrophenol, malathion dicarboxylic acid); five synthetic pyrethroid insecticide metabolites (4-fluoro-3-phenoxybenzoic acid, 3-phenoxybenzoic acid, cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCCA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid); and the herbicide 2,4-dichlorophenoxyacetic acid. he method is based on enzymatic hydrolysis of conjugated urinary metabolites, extraction and pre-concentration of the deconjugated metabolites using automated online solid-phase extraction, and separation and quantification using liquid chromatography-isotope dilution tandem mass spectrometry. Depending on the analyte, method detection limits were 0.1-0.6 ng/mL; mean accuracy, calculated as spike recoveries, was 91-102%, and total precision, given as percent variation coefficient, was 5.9-11.5%. Percent differences associated with three freeze-thaw cycles, 24-h benchtop storage, and short-term processed sample stability were <14%. Method suitability was assessed by recurring successful participation in external quality assessment schemes and by analyzing samples from subjects with suspected exposure to pesticides (n = 40) or who self-reported consuming an organic diet (n = 50). Interquartile ranges were considerably lower for people consuming an organic diet than for those potentially exposed for cis-DCCA (0.37 ng/mL vs 0.75 ng/mL), trans-DCCA (0.88 ng/mL vs 1.78 ng/mL) and TCPy (1.81 ng/mL vs 2.48 ng/mL). This method requires one-fifth of the sample used in our previous method and is suitable for assessing background exposures to select pesticides in large human populations and for studies with limited sample volumes.

Generating fast-twitch myotubes in vitro with an optogenetic-based, quantitative contractility assay.

The composition of fiber types within skeletal muscle impacts the tissue's physiological characteristics and susceptibility to disease and ageing. In vitro systems should therefore account for fiber-type composition when modelling muscle conditions. To induce fiber specification in vitro, we designed a quantitative contractility assay based on optogenetics and particle image velocimetry. We submitted cultured myotubes to long-term intermittent light-stimulation patterns and characterized their structural and functional adaptations. After several days of in vitro exercise, myotubes contract faster and are more resistant to fatigue. The enhanced contractile functionality was accompanied by advanced maturation such as increased width and up-regulation of neuron receptor genes. We observed an up-regulation in the expression of fast myosin heavy-chain isoforms, which induced a shift towards a fast-twitch phenotype. This long-term in vitro exercise strategy can be used to study fiber specification and refine muscle disease modelling.

Muscle is a stage, and cells and factors are merely players.

The incredible ability of satellite cells to regenerate muscle has captivated much of the research field's attention over the past decades. Versatile, enigmatic, vigorous, and skillful, the satellite cell is the optimal actor to cast in a regenerative epic, grabbing contracts and making headlines. However, the scenarios that play out during normal muscle usage, such as during exercise and aging, diverge from the experimental setup staged to spotlight satellite cells. Recent studies examining myofibers have highlighted novel attributes, including a capacity for self-repair. We discuss here the distinctions between myofiber self-repair and satellite-cell-dependent regeneration and how they may cooperate to repair damage after exercise, in myopathies, and in aging.

Muscle repair after physiological damage relies on nuclear migration for cellular reconstruction.

Regeneration of skeletal muscle is a highly synchronized process that requires muscle stem cells (satellite cells). We found that localized injuries, as experienced through exercise, activate a myofiber self-repair mechanism that is independent of satellite cells in mice and humans. Mouse muscle injury triggers a signaling cascade involving calcium, Cdc42, and phosphokinase C that attracts myonuclei to the damaged site via microtubules and dynein. These nuclear movements accelerate sarcomere repair and locally deliver messenger RNA (mRNA) for cellular reconstruction. Myofiber self-repair is a cell-autonomous protective mechanism and represents an alternative model for understanding the restoration of muscle architecture in health and disease.

mRNA distribution in skeletal muscle is associated with mRNA size.

Skeletal muscle myofibers are large and elongated cells with multiple and evenly distributed nuclei. Nuclear distribution suggests that each nucleus influences a specific compartment within the myofiber and implies a functional role for nuclear positioning. Compartmentalization of specific mRNAs and proteins has been reported at the neuromuscular and myotendinous junctions, but mRNA distribution in non-specialized regions of the myofibers remains largely unexplored. We report that the bulk of mRNAs are enriched around the nucleus of origin and that this perinuclear accumulation depends on recently transcribed mRNAs. Surprisingly, mRNAs encoding large proteins - giant mRNAs - are spread throughout the cell and do not exhibit perinuclear accumulation. Furthermore, by expressing exogenous transcripts with different sizes we found that size contributes to mRNA spreading independently of mRNA sequence. Both these mRNA distribution patterns depend on microtubules and are independent of nuclear dispersion, mRNA expression level and stability, and the characteristics of the encoded protein. Thus, we propose that mRNA distribution in non-specialized regions of skeletal muscle is size selective to ensure cellular compartmentalization and simultaneous long-range distribution of giant mRNAs.

A flagellate-to-amoeboid switch in the closest living relatives of animals.

Amoeboid cell types are fundamental to animal biology and broadly distributed across animal diversity, but their evolutionary origin is unclear. The closest living relatives of animals, the choanoflagellates, display a polarized cell architecture (with an apical flagellum encircled by microvilli) that resembles that of epithelial cells and suggests homology, but this architecture differs strikingly from the deformable phenotype of animal amoeboid cells, which instead evoke more distantly related eukaryotes, such as diverse amoebae. Here, we show that choanoflagellates subjected to confinement become amoeboid by retracting their flagella and activating myosin-based motility. This switch allows escape from confinement and is conserved across choanoflagellate diversity. The conservation of the amoeboid cell phenotype across animals and choanoflagellates, together with the conserved role of myosin, is consistent with homology of amoeboid motility in both lineages. We hypothesize that the differentiation between animal epithelial and crawling cells might have evolved from a stress-induced switch between flagellate and amoeboid forms in their single-celled ancestors.

An In Vitro System to Measure the Positioning, Stiffness, and Rupture of the Nucleus in Skeletal Muscle.

Nuclear positioning plays important roles for certain cellular functions. This is particularly relevant in skeletal muscle cells also known as myofibers in which nuclear positioning defects were shown to hinder muscle function. Myofibers are multinucleated cells with nuclei equally distributed at the periphery of the cell. However, nuclei can be found centrally located during myogenesis before anchoring at the periphery or in certain muscle disorders, either due to regenerating myofibers or defects in nuclear movement. As such, nuclear localization in myofibers (central or peripheral) can be used to assess myofiber maturity, regeneration, or health. To study how nuclei reach the periphery of myofibers during development, we devised a unique protocol to mature myofibers thereby recapitulating later stages of differentiation, including nuclear movement to the periphery. Here we describe how to use this system to study nuclear positioning and other nuclear characteristics such as nuclear stiffness or rupture.

Local Arrangement of Fibronectin by Myofibroblasts Governs Peripheral Nuclear Positioning in Muscle Cells.

Skeletal muscle cells (myofibers) are rod-shaped multinucleated cells surrounded by an extracellular matrix (ECM) basal lamina. In contrast to other cell types, nuclei in myofibers are positioned just below the plasma membrane at the cell periphery. Peripheral nuclear positioning occurs during myogenesis and is driven by myofibril crosslinking and contraction. Here we show that peripheral nuclear positioning is triggered by local accumulation of fibronectin secreted by myofibroblasts. We demonstrate that fibronectin via α5-integrin mediates peripheral nuclear positioning dependent on FAK and Src activation. Finally, we show that Cdc42, downstream of restricted fibronectin activation, is required for myofibril crosslinking but not myofibril contraction. Thus we identify that local activation of integrin by fibronectin secreted by myofibroblasts activates peripheral nuclear positioning in skeletal myofibers.

Nuclear positioning in skeletal muscle.

Skeletal muscle cells possess a unique cellular architecture designed to fulfill their contractile function. Muscle cells (also known as myofibers) result from the fusion of hundreds of myoblasts and grow into a fiber of several centimeters in length. Cellular structures gradually become organized during muscle development to raise a mature contractile cell. A hallmark of this singular cell architecture is the position of nuclei at the periphery of the myofiber, below the plasma membrane. Nuclei in myofibers are evenly distributed except in specialized regions like the neuromuscular or myotendinous junctions. Disruption of nuclear positioning results in hindered muscle contraction and occurs in a multitude of muscle disorders as well as in regenerative myofibers. We will explore in this review the step by step nuclear migrations during myogenesis for nuclei to reach their evenly distributed anchored position at the periphery.

Myofibril contraction and crosslinking drive nuclear movement to the periphery of skeletal muscle.

Nuclear movements are important for multiple cellular functions, and are driven by polarized forces generated by motor proteins and the cytoskeleton. During skeletal myofibre formation or regeneration, nuclei move from the centre to the periphery of the myofibre for proper muscle function. Centrally located nuclei are also found in different muscle disorders. Using theoretical and experimental approaches, we demonstrate that nuclear movement to the periphery of myofibres is mediated by centripetal forces around the nucleus. These forces arise from myofibril contraction and crosslinking that 'zip' around the nucleus in combination with tight regulation of nuclear stiffness by lamin A/C. In addition, an Arp2/3 complex containing Arpc5L together with γ-actin is required to organize desmin to crosslink myofibrils for nuclear movement. Our work reveals that centripetal forces exerted by myofibrils squeeze the nucleus to the periphery of myofibres.

The effect of compression socks worn during a marathon on hemostatic balance.

INTRODUCTION: Marathon running evokes parallel increases in markers of coagulation and fibrinolysis (i.e. hemostatic activation) immediately following strenuous, endurance exercise such that hemostatic balance is maintained. However, other factors incident to marathon running (i.e. dehydration, travel) may disproportionately activate the coagulatory system, increasing blood clot risk after an endurance event in otherwise healthy individuals. We investigated the effect of compression socks on exercise-induced hemostatic activation and balance in endurance athletes running the 2013 Hartford Marathon. METHODS: Adults (n = 20) were divided into compression sock (SOCK; n = 10) and control (CONTROL; n = 10) groups. Age, anthropometrics, vital signs, training mileage and finishing time were collected. Venous blood samples were collected 1 day before, immediately after and 1 day following the marathon for analysis of coagulatory (i.e. thrombin-antithrombin complex [TAT] and D-dimer) and fibrinolytic (i.e. tissue plasminogen activator [t-PA]) factors. RESULTS: Plasma D-dimer, TAT and t-PA did not differ between groups at baseline (p > 0.16). There were no significant group · time interactions (all p ≥ 0.17), however, average t-PA was lower in SOCK (8.9 ± 0.7 ng/mL) than CONTROL (11.2 ± 0.7 ng/mL) (p = 0.04). Average TAT also tended to be lower in SOCK (2.8 ± 0.2 µg/L) than CONTROL (3.4 ± 0.2 µg/L) (p = 0.07). CONCLUSIONS: Our results suggest that overall hemostatic activation (both coagulation and fibrinolysis) following a marathon tended to be lower with compression socks. Thus, compression socks do not adversely influence markers of hemostasis, appear safe for overall use in runners and may reduce exercise-associated hemostatic activation in individuals at risk for deep vein thrombosis.

N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy.

Mutations in amphiphysin-2/BIN1, dynamin 2, and myotubularin are associated with centronuclear myopathy (CNM), a muscle disorder characterized by myofibers with atypical central nuclear positioning and abnormal triads. Mis-splicing of amphiphysin-2/BIN1 is also associated with myotonic dystrophy that shares histopathological hallmarks with CNM. How amphiphysin-2 orchestrates nuclear positioning and triad organization and how CNM-associated mutations lead to muscle dysfunction remains elusive. We find that N-WASP interacts with amphiphysin-2 in myofibers and that this interaction and N-WASP distribution are disrupted by amphiphysin-2 CNM mutations. We establish that N-WASP functions downstream of amphiphysin-2 to drive peripheral nuclear positioning and triad organization during myofiber formation. Peripheral nuclear positioning requires microtubule/Map7/Kif5b-dependent distribution of nuclei along the myofiber and is driven by actin and nesprins. In adult myofibers, N-WASP and amphiphysin-2 are only involved in the maintenance of triad organization but not in the maintenance of peripheral nuclear positioning. Importantly, we confirmed that N-WASP distribution is disrupted in CNM and myotonic dystrophy patients. Our results support a role for N-WASP in amphiphysin-2-dependent nuclear positioning and triad organization and in CNM and myotonic dystrophy pathophysiology.

Translation initiation factor eIF4F modifies the dexamethasone response in multiple myeloma.

Enhanced protein synthesis capacity is associated with increased tumor cell survival, proliferation, and resistance to chemotherapy. Cancers like multiple myeloma (MM), which display elevated activity in key translation regulatory nodes, such as the PI3K/mammalian target of rapamycin and MYC-eukaryotic initiation factor (eIF) 4E pathways, are predicted to be particularly sensitive to therapeutic strategies that target this process. To identify novel vulnerabilities in MM, we undertook a focused RNAi screen in which components of the translation apparatus were targeted. Our screen was designed to identify synthetic lethal relationships between translation factors or regulators and dexamethasone (DEX), a corticosteroid used as frontline therapy in this disease. We find that suppression of all three subunits of the eIF4F cap-binding complex synergizes with DEX in MM to induce cell death. Using a suite of small molecules that target various activities of eIF4F, we observed that cell survival and DEX resistance are attenuated upon eIF4F inhibition in MM cell lines and primary human samples. Levels of MYC and myeloid cell leukemia 1, two known eIF4F-responsive transcripts and key survival factors in MM, were reduced upon eIF4F inhibition, and their independent suppression also synergized with DEX. Inhibition of eIF4F in MM exerts pleotropic effects unraveling a unique therapeutic opportunity.

Changes in muscle strength in patients with statin myalgia.

Statins can produce myalgia or muscle pain, which may affect medication adherence. We measured the effects of statins on muscle strength in patients with previous statin myalgia. Leg isokinetic extension average power at 60° per second (-8.8 ± 10.5N-M, p = 0.02) and average peak torque at 60° per second (-14.0 ± 19.7N-M, p = 0.04) decreased slightly with statin use, but 8 of 10 other variables for leg strength did not change (all p >0.13). Handgrip, muscle pain, respiratory exchange ratio, and daily activity also did not change (all p >0.09). In conclusion, statin myalgia is not associated with reduced muscle strength or muscle performance.

Treating adult attention deficit hyperactivity disorder in hospitalized psychiatric patients.

OBJECTIVES: We intend to review the importance of appropriately recognizing and managing attention deficit/attention deficit hyperactivity disorder (ADD/ADHD) in the acute psychiatric hospital setting. METHODS: We demonstrate the management of three patients with associated ADD/ADHD diagnosis in the hospital setting. This case series is followed by a review of the literature on the treatment of ADD/ADHD with particular focus on inpatient treatment. RESULTS: Given that the core symptoms of ADD/ADHD are inattention, hyperactivity, poor concentration, impulsivity, poor organization and emotional instability, it follows that a comprehensive inpatient treatment plan should address these issues in order to obtain sustained, focused participation on the part of the patient. Suppression of ADD/ADHD symptoms with stimulants greatly enhanced our patients' ability to more productively and actively participate in the treatment of the acute psychiatric problems which led to their admission. CONCLUSIONS: Currently, no published data exist on prevalence of ADD/ADHD in psychiatric hospitals, rates of treatment and outcome of treatment with regard to recovery and quality of aftercare. Nonetheless, the benefits of treating ADD/ADHD among psychiatric inpatients may be seen in case examples and are also apparent in the data concerning treatment of ADD/ADHD in the dually diagnosed.

Web: formación vocacional / Web: for vocacional training

Se realizó un Sofware didáctico educativo, ejecutable con el Sistema Operativo Window, se visualiza en una computadora 486 o superior que soporte el programa Internet Explorar 4.0, con el objetivo de continuar desarrollando el trabajo vocacional de orientación hacia las carreras de la salud mediante una Página Web con la que nos hemos propuesto fortalecer este trabajo brindando orientaciones generales que servirán de guía a los estudiantes que integran los Pre Destacamentos en los diferentes Pre Universitarios de nuestra Provincia, haciéndolo extensivo también a los Cursos de Superación integral para Jóvenes, donde el aspirante puede interactuar de forma rápida y amena para obtener dicha información de modo didáctico a través del uso de la computación en su propio centro de estudios(AU)

Software training and education performed, executes with the Window operating system is displayed on a computer that supports 486 or higher Internet Explorer 4.0 program, aiming to further develop vocational work orientation to careers in health through a Page Web which we have proposed to strengthen this work by providing general guidelines that will guide the students to integrate the different detachments Pre Pre University of our province, extending it also to the Courses for the Comprehensive Youth, where the applicant can interact quickly and pleasant to get that information so learning through the use of computers in their own study center(AU)

Software para el registro y otorgamiento de carreras universitarias en Salud / Software for the record and granting of university careers in health

Se realizó un Software para el registro y distribución de carreras, utilizando plataforma programática en Visual FoxProx 7.0, ejecutable con el Sistema Operativo Window 98 o superior, en tecnología Celerón AP 500 o superior. Con el propósito de registrar los solicitantes a las carreras universitarias y realizar otorgamiento de las mismas, observando las normas de acceso y protección establecidas por seguridad informática. Con la aplicación del mismo se logra proteger de una forma más efectiva la información almacenada impidiendo que pueda ser modificada sin la debida autorización así como controlar y registrar en una bitácora los accesos, protegiendo los registros por encriptación personalizada y salva, emitiendo los informes establecidos y ampliando el nivel de consultas. Al no atornillar las asignaturas y especialidades a ofertar situándolas en ficheros clasificadores, este programa en sus tres niveles (municipal, provincial y nacional) puede ser adecuado a las características específicas de cada territorio(AU)

Intervención promocional sobre sexualidad en individuos que padecieron infecciones de transmisión sexual / Promotional intervention about sexuality in patients that suffered sexual transmission infections

Se realiza un estudio de intervención comunitaria en el área de promoción de salud, para modificar criterios sobre sexualidad en individuos que padecieron infecciones de transmisión sexual, pertenecientes al Consultorio No. 1 de la Policlínica Comunitaria Docente " Omar Ranedo Pubillones" de la Ciudad de Guantánamo, durante el período de enero de 1996-marzo de 1998. Al universo de 53 individuos se les aplicó un cuestionario sobre temas de sexualidad para el diagnóstico educativo. Predominó el grupo de edades de 25-34 años (37,7 por ciento), sexo femenino (63 por ciento), nivel preuniversitario terminado (54,7 por ciento). Se elaboró y ejecutó un programa basado en técnicas participativas grupales que permitió mejorar la comunicación con el médico de familia, estimular la búsqueda de información científica, modificar criterios erróneos sobre la adquisición de enfermedades de transmisión sexual y medidas de protección, el conocimiento de la responsabilidad de conservar la propia salud sexual y la de los demás. Se recomienda perfeccionar el trabajo promocional comunitario para estimular la conducta sexual responsable(AU)