Risk of endocarditis among patients with coagulase-negative Staphylococcus bacteremia.

Coagulase-negative staphylococci (CoNS) are currently considered typical microorganisms causing infective endocarditis (IE) in patients with prosthetic valves. The objective was to determine variables associated with IE in patients with CoNS bacteremia. We performed an analysis of the clinical characteristics of patients with CoNS bacteremia admitted to a university hospital in Madrid (Spain) from 2021 to December 2022 according to the occurrence of IE. This study is an evaluation of a bacteremia registry. During the study period, 106 patients with CoNS bacteremia were detected. In 85 patients an echocardiogram was performed during hospital admission to rule out IE. Among them, 12 episodes were detected that met IE criteria (14.2%). Of the 6 patients with heart valve prostheses, 5 patients (83.3%) had IE (p < 0.001). Patients with IE more frequently had positive blood cultures more than 12 h after the first draw (58.3% versus 13.4%; p < 0.001). There was a tendency to associate community-acquired bacteremia and to that all blood culture bottles obtained were positive with an increased risk of IE (p = 0.091 and p = 0,057, respectively). Attributable mortality to infection was higher in patients with IE relative to all other patients (16.7% vs. 0%; p = 0.033). The multivariable analysis included having valve prosthesis and persistent bacteremia for more than 12 h. Both were independently associated with IE: valve prosthesis OR 38.6 (95% CI 5.8-258; p < 0.001) and persistent bacteremia OR 2.6 (95% CI 1.1-6.8; p = 0.046). In conclusion, a high percentage of cases of CoNS bacteremia may be due to IE. Some of the variables related to a higher risk of IE, such as having a valvular prosthesis or presenting positive blood cultures for more than 12 h, should lead to rule out or confirm the presence of IE by performing echocardiography.

Intercomparison of Radon Flux Monitors at Low and at High Radium Content Areas under Field Conditions.

Interlaboratory exercises are a good tool to compare the response of different systems to the same quantity and to identify possible inconsistencies between them. One of the main goals of the EMPIR 19ENV01 traceRadon project is to harmonize radon flux measurements based on different systems and methodologies. In the framework of the traceRadon Project, two radon flux intercomparison campaigns were carried out in October 2021 at high and at low radon source areas. Four institutions participated in the field intercomparison exercises with their own systems. Every system was based on a specific radon monitor (diffusion or pump mode) and an accumulation chamber (with manual or automatic opening). Radon fluxes were calculated by each participant using both exponential and linear fittings of the radon activity concentration measured over time within the accumulation chambers. The results of this study show mainly: (i) the exponential approach is not advisable due to the variability of the radon flux and the leakage of the systems during long-time measurements; (ii) the linear approach should be applied to minimize the measurement period in agreement with the time response and sensitivity of the monitors; (iii) radon flux measured at high radon source areas (radium content of about 800 Bq kg-1) risks being underestimated because of the influence of advective effects; (iv) radon flux measured at low radon source areas (radium content of about 30 Bq kg-1) may present large uncertainties if sensitive radon monitors with pump mode are not used.

Intoxicación por monóxido de carbono en una embarazada: reporte de caso / Carbon monoxide poisoning in pregnant women: A case report

Resumen ANTECEDENTES: La intoxicación por monóxido de carbono durante el embarazo es excepcional, aunque puede producir importantes daños al feto. Establecer un diagnóstico de sospecha y aplicar de forma correcta el tratamiento mejora los desenlaces perinatales. CASO CLÍNICO: Paciente de 40 años, en las 32 + 5 semanas del séptimo embarazo, con antecedente de preeclampsia en uno de los embarazos previos. Acudió a Urgencias debido a un cuadro de cefalea intensa, vómitos y tensión arterial elevada luego de un cuadro de convulsiones en una de sus hijas. Al ingreso al hospital la paciente continuó con los síntomas, pero con cifras de tensión arterial normales. En el registro cardiotocográfico se objetivaron desaceleraciones variables. La ecografía de Doppler y los estudios de laboratorio fueron normales, excepto un índice de proteínas-creatinina de 0.41 g/dL. En ese momento el pediatra comunicó que la hija de la paciente cursaba con un cuadro de intoxicación aguda por monóxido de carbono. Con base en esta nueva información se solicitaron estudios de gasometría venosa y cooximetría, con los que se confirmó el diagnóstico de intoxicación por monóxido de carbono. Se le aplicó oxígeno normobárico al 100%. La evolución de la madre y su feto fue favorable, con desaparición de los síntomas de la madre y normalización del registro cardiotocográfico. CONCLUSIONES: Para el diagnóstico de intoxicación por monóxido de carbono es necesario mantener un alto índice de sospecha, sobre todo en las embarazadas en virtud de los daños potencialmente graves que pueden producirse en el feto. Es decisivo el diagnóstico diferencial correcto para no demorar el tratamiento, disminuir la morbilidad y la mortalidad de la madre y el feto.

Abstract BACKGROUND: Carbon monoxide poisoning during pregnancy is exceptional, although it can cause significant damage to the fetus. Establishing a diagnosis of suspicion and applying the correct treatment improves perinatal outcomes. CLINICAL CASE: A 40-year-old woman, at 32 + 5 weeks of her seventh pregnancy, with a history of preeclampsia in one of her previous pregnancies. She came to the emergency department due to severe headache, vomiting and high blood pressure after a seizure in one of her daughters. On admission to the hospital, the patient continued with her symptoms, but with normal blood pressure. Cardiotocographic recording showed variable decelerations. Doppler ultrasound and laboratory studies were normal, except for a protein-creatinine index of 0.41 g/dL. At that time the pediatrician reported that the patient's daughter was suffering from acute carbon monoxide intoxication. Based on this new information, venous blood gas and cooximetry studies were requested, which confirmed the diagnosis of carbon monoxide poisoning. She was given 100% normobaric oxygen. The evolution of the mother and her fetus was favorable, with disappearance of the mother's symptoms and normalization of the cardiotocographic record. CONCLUSIONS: For the diagnosis of carbon monoxide poisoning, it is necessary to maintain a high index of suspicion, especially in pregnant women due to the potentially serious damage that can occur in the fetus. The correct differential diagnosis is decisive in order not to delay treatment and to reduce morbidity and mortality of the mother and fetus.

RESEARCH ON PASSIVE SOIL DEPRESSURIZATION SYSTEMS: STATE OF THE ART AND LAB TEST ON-GOING.

There is strong evidence both internationally and in Ireland that the correct installation of passive prevention systems in new buildings is the most cost-effective way of protecting the population against radon. Previous work considering membranes, granular fill material in the aggregate layer beneath the slab and sump system has been conducted in Ireland to improve the protection of buildings from radon. The implications of research on passive sumps potential to reduce radon concentrations are significant, as if it can be shown that the installation of passive sumps in Irish building is effective; this could constitute a low-cost, passive, sustainable method for minimizing radon levels in buildings. On-going experimental tests investigating the performance of different common cowls used for passive soil depressurization systems are presented, in addition to the impact of different vertical heights and horizontal lengths of pipe with a number of bends investigated.

Phenotypic Spectrum of α-Dystroglycanopathies Associated With the c.919T>a Variant in the FKRP Gene in Humans and Mice.

Mutations in the fukutin-related protein gene, FKRP, are the most frequent single cause of α-dystroglycanopathy. Rare FKRP mutations are clinically not well characterized. Here, we review the phenotype associated with the rare c.919T>A mutation in FKRP in humans and mice. We describe clinical and paraclinical findings in 6 patients, 2 homozygous, and 4-compound heterozygous for c.919T>A, and compare findings with a mouse model we generated, which is homozygous for the same mutation. In patients, the mutation at the homozygous state is associated with a severe congenital muscular dystrophy phenotype invariably characterized by severe multisystem disease and early death. Compound heterozygous patients have a severe limb-girdle muscular dystrophy phenotype, loss of ambulation before age 20 and respiratory insufficiency. In contrast, mice homozygous for the same mutation show no symptoms or signs of muscle disease. Evidence therefore defines the FKRP c.919T>A as a very severe mutation in humans. The huge discrepancy between phenotypes in humans and mice suggests that differences in protein folding/processing exist between human and mouse Fkrp. This emphasizes the need for more detailed structural analyses of FKRP and shows the challenges of developing appropriate animal models of dystroglycanopathies that mimic the disease course in humans.

Review of recent radon research in Ireland, OPTI-SDS project and its impact on the National Radon Control Strategy.

Radon is a radioactive gas originating from uranium, present in all rocks and soils in the Earth's Crust; emanating from the ground, radon can be released into the atmosphere. It is the greatest source of natural radioactivity exposure for the population and, as declared by the World Health Organization (WHO), the leading cause of lung cancer only after smoking. Although radon is a natural gas, its accumulation provoking elevated indoor radon levels is a result from building practices and thus, not natural. In Ireland, exposure to radon is estimated to be responsible for approximately 14% of all lung cancers, which is equivalent to around 300 lung cancers annually. In 2011, an interagency group was established in Ireland to develop a strategy to address indoor radon exposure, considered a significant public health concern. In 2014 a National Radon Control Strategy (NRCS) for Ireland was first published, giving a list of recommendations to be accomplished in a 4-year period Phase 1. A series of research actions to achieve the effective implementation of the strategy were conducted, including the development of a research project (OPTI-SDS) on the optimum specifications for radon mitigation by soil depressurisation systems. An overview of Phase 1 of the NRCS is presented, including outcomes from the research work carried out.

Radon mitigation by soil depressurisation case study: Radon concentration and pressure field extension monitoring in a pilot house in Spain.

A one-year monitoring study was conducted in a pilot house with extremely high radon levels to investigate the ability and efficiency of radon mitigation by soil depressurisation (SD) both active and passive. The study included monitoring of radon concentration, pressure field extension (PFE) under the slab and some atmospheric parameters for different testing phases. Periods in which the house remained closed to foster radon accumulation were alternated with phases of active and passive soil depressurisation under different conditions. The behaviour of the radon concentration in the pilot house was analysed along with the influence of atmospheric variables, significant correlations were found for the radon concentration with atmospheric pressure, outdoor temperature and wind. From the PFE analysis it was proven that the pressure drop with distance from the suction point of the SD system is proportional to the depressurisation generated. A behaviour law was found for the permeability characterisation of the house based on the active SD performance and also, the relationship between wind velocity and extraction airflow during passive SD operation by means of a rotating cowl was obtained. Radon reductions in excess of 85% were achieved for the different testing phases in all cases. Finally, from the results it was postulated that a fan power of 20 W is sufficient to ensure radon reductions over 85% for dwellings with similar aggregate layer and soil permeability.

Investigation of gas flow through soils and granular fill materials for the optimisation of radon soil depressurisation systems.

The purpose of this study is to investigate gas flow through different types of granular fill materials and soil by means of a series of experimental laboratory tests, in relation to soil depressurisation systems for radon reduction under buildings and the soil surrounding the foundation. Gas permeability characterisation of materials used as granular fill material beneath the slab in buildings is a key parameter for the optimum performance of soil depressurisation systems to mitigate radon. A test apparatus was developed, adapted from previous studies, to measure the gas permeability of the samples and Finite Element Method numerical simulations were validated to simulate the flow behaviour through them. Theoretical expressions for permeability were discussed based on the analysis of experimental results and numerical simulations, finding that Darcy-Forchheimer equation provides the best match to the experimental results. Darcy's law also proved to be suitable for low gas velocities, whereas Ergun's equation resulted in a poor fit of the experimental data. Benchmark analysis of the granular fill materials under study and other European standards (Spanish, Irish and British) is also presented.

Performance of radon monitors in a purpose-built radon chamber.

The purpose of this paper is to benchmark several different radon monitors, by quantifying their accuracy and response time. Radon monitors with different characteristics were tested in a purpose-built radon chamber under reference conditions. The radon concentration in the chamber was controlled and maintained at a stable radon concentration of (2648 ± 85) Bq m-3 to evaluate the accuracy and precision of these monitors. The response time of the monitors was analysed for two time intervals. To assess the response time of the monitors, radon concentration was varied from a theoretical value of 0-6441 Bq m-3 and then from 6441 to 2648 Bq m-3. The results from this study show that general purpose radon monitors are less accurate than those used by radon testing service providers and the research community. All monitors tested reported a mean radon concentration within the ±10% of the reference detector value at the radon equilibrium concentration. Different response time analysis methods were proposed and discussed, and for the particular time intervals analysed, response time was found to be slower for those radon monitors intended for general purpose applications.

Investigation of sub-slab pressure field extension in specified granular fill materials incorporating a sump-based soil depressurisation system for radon mitigation.

Design of bearing layers (granular fill material layers) is important for a house with a soil depressurisation (SD) system for indoor radon mitigation. These layers should not only satisfy the bearing capacity and serviceability criteria but should also provide a sufficient degree of the air permeability for the system. Previous studies have shown that a critical parameter for a SD system is the sub-slab pressure field extension in the bearing layers, but this issue has not been systematically investigated. A series of two-dimensional computational fluid dynamic simulations that investigate the behaviour of the sub-slab pressure field extension developed in a SD system is presented in this paper. The SD system considered in this paper consists of a granular fill material layer and a radon sump. The granular fill materials are 'T1 Struc' and 'T2 Perm', which are standard materials for building in the Republic of Ireland. Different conditions, which might be encountered in a practical situation, were examined. The results show that the air permeability and thickness of the granular fill materials are the two key factors which affect the sub slab pressure field extension (SPFE) significantly. Furthermore, the air permeability of native soil is found to be a fundamental factor for the SPFE so that it should be well understood when designing a SD system. Therefore, these factors should be considered sufficiently in each practical situation. Finally, a significant improvement of the pressure field extension can be achieved by ensuring air tightness of the SD system.

Prenatal muscle development in a mouse model for the secondary dystroglycanopathies.

BACKGROUND: The defective glycosylation of α-dystroglycan is associated with a group of muscular dystrophies that are collectively referred to as the secondary dystroglycanopathies. Mutations in the gene encoding fukutin-related protein (FKRP) are one of the most common causes of secondary dystroglycanopathy in the UK and are associated with a wide spectrum of disease. Whilst central nervous system involvement has a prenatal onset, no studies have addressed prenatal muscle development in any of the mouse models for this group of diseases. In view of the pivotal role of α-dystroglycan in early basement membrane formation, we sought to determine if the muscle formation was altered in a mouse model of FKRP-related dystrophy. RESULTS: Mice with a knock-down in FKRP (FKRP(KD)) showed a marked reduction in α-dystroglycan glycosylation and reduction in laminin binding by embryonic day 15.5 (E15.5), relative to wild type controls. In addition, the total number of Pax7(+) progenitor cells in the FKRP(KD) tibialis anterior at E15.5 was significantly reduced, and myotube cluster/myofibre size showed a significant reduction in size. Moreover, myoblasts isolated from the limb muscle of these mice at E15.5 showed a marked reduction in their ability to form myotubes in vitro. CONCLUSIONS: These data identify an early reduction of laminin α2, reduction of myogenicity and depletion of Pax7(+) progenitor cells which would be expected to compromise subsequent postnatal muscle growth and its ability to regenerate postnatally. These findings are of significance to the development of future therapies in this group of devastating conditions.

Síndrome de encefalopatía posterior reversible y puerperio. A propósito de un caso / Reversible posterior encephalopathy syndrome

Se presenta el caso de una mujer puérpera de 31 años de edad, que en el año 2014 padeció un síndrome de encefalopatía posterior reversible durante el puerperio hospitalario. Este síndrome puede presentarse en gestantes o puérperas y en la población general. Se ha asociado a otras patologías, como fallo renal con hipertensión, terapias inmunosupresoras anticancerígenas, enfermedades autoinmunes del tejido conectivo, púrpura trombótica trombocitopénica, infección por el virus de la inmudeficiencia humana, porfiria intermitente, trasplante de órganos e hipercalcemia. Su importancia radica en la dificultad de su diagnóstico, por ser una patología rara, la prontitud del cual se relaciona con la reversibilidad del proceso. Un retraso en el inicio del tratamiento puede derivar en daños neurológicos permanentes

Presenting a case of a 31 years old, who suffered in 2014 a reversible posterior encephalopathy syndrome during the postpartum in hospital. This syndrome can be presented during pregnancy, postpartum and in the rest of the population as well. It has been associated to other pathologies as: renal failure associated with high blood pressure, immunosupressant therapies associated to cancer treatment, connective tissue autoimmune disease, thrombotic thrombocytopenic purpura, HIV infection, intermittent porphyria and hypercalcemia. Diagnosing the syndrome is really difficult, as it is a rare pathology. If diagnosis is done at an early stage it will help the reversibility of the process. If the treatment is delayed it can have consequences such as permanent neurological damage

Detection of Polyclonality among Clinical Isolates from Prosthetic Joint Infections.

Prosthetic joint infection (PJI) is an increasingly important health concern in the Western world due to the rising number of joint arthroplasties. Although most infections are considered to be monomicrobial, the introduction of sonication procedures has led to an increase in the detection of polymicrobial infections. To date, no published studies have investigated the presence of different clones of the same species in the infected patient. The objective of this study was to analyze whether the phenomenon of polyclonality, or the appearance of different clones in the same sample, occurs in PJI. Bacteria isolated by sonication of the retrieved implant from patients with theoretically monomicrobial PJI were included in the study. Two techniques (random amplified polymorphic DNA [RAPD] and matrix-assisted laser desorption ionization-time of flight [MALDI-TOF] mass spectrometry) were used to determine the presence of several clones in the same sample. Results were analyzed to determine bacterial species and infection type (acute versus chronic). RAPD showed a predominance of polyclonal cases (16 of 19). However, when performing the analysis with MALDI-TOF, all cases were shown to be polyclonal. We were unable to establish any relationship between the two methodologies. Polyclonality is a common phenomenon in acute and chronic PJI. Further studies are needed to establish the potential implications of this phenomenon on patient outcomes.

Clinical features, mortality, hospital admission, and length of stay of a cohort of adult patients with diabetic ketoacidosis attending the emergency room of a tertiary hospital in Spain / Características clínicas, mortalidad, hospitalización y duración de la estancia de pacientes adultos con cetoacidosis diabética admitidos en un servicio de urgencias de un hospital terciario en España

AIM: To assess the clinical features, length of stay, incidence rate, mortality, and hospital admissions of patients with episodes of diabetic ketoacidosis (DKA). PATIENTS: It was conducted retrospective, cross-sectional study of 164 consecutive admissions of adult patients (2008-August 2012), with type 1 or type 2 diabetes already known or new onset. RESULTS: Mortality rate was 1.2%. The DKA episodes were mild (18.9%), moderate (31.7%), or severe (49.4%). The cumulative incidence was 2.66 cases/1000 patients with diabetes (DM) in 4.5 years. The most common causes triggering DKA were infection (33.2%) and dietary transgression and/or insulin dose omission (30.7%). A total of 12.8% of patients had new onset DM, 56.7% type 1, and 26.8% type 2 DM. Patients with type 2 DM were older and had at admission higher creatinine, BUN, osmolality, sodium, and anion gap levels. Patients with new-onset of DM had higher levels of glucose and sodium, but lower potassium levels. No differences were found in pH or bicarbonate. Admission to the intensive care unit (ICU) was required in >50% of cases (p < 0.001), and 86.6% of patients were finally admitted to a medical ward (p = 0.005). The length of stay at the ICU (p < 0.001) and hospital (p = 0.013) was significantly different depending on DKA severity. CONCLUSIONS: Most DKA episodes require hospital admission, but mortality is <2%, and length of stay at the ER and medical ward depends on type of DM and initial severity of the episode

OBJETIVO: Se investigaron las características clínicas, la duración, la tasa de incidencia, la mortalidad y los ingresos de episodios de cetoacidosis diabética (CAD). PACIENTES: Se realizó un estudio retrospectivo, transversal, con 164 admisiones consecutivas de adultos (2008-agosto 2012), con diabetes (DM) tipo 1 y 2 ya conocida o debut diabético. RESULTADOS: La tasa de mortalidad fue del 1,2%. Los episodios de CAD fueron leves (18,9%), moderados (31,7%) y graves (49,4%). La incidencia acumulada fue de 2,66 casos/1.000 pacientes con DM en 4,5 años. Las causas desencadenantes más frecuentes fueron las infecciones (33,2%) y la transgresión dietética y/u omisión de la dosis de insulina (30,7%). El 12,8% tuvieron un debut diabético, 56,7% eran DM tipo 1 y el 26,8% tipo 2. Los casos con DM tipo 2 tenían mayor edad y presentaban al ingreso mayores niveles de creatinina, BUN, osmolaridad, sodio y anion GAP. El debut diabético presentaba niveles más elevados de glucosa y sodio, pero valores más bajos de potasio. No se encontró ninguna diferencia en el pH o bicarbonato. La admisión en la unidad de cuidados intensivos (UCI) se requirió en más del 50% de los casos (p < 0,001) y un 86,6% fue finalmente ingresado en una planta de hospitalización médica (p = 0,005). La duración de las estancias en la UCI (p < 0,001) y en el hospital (p = 0,013) fueron significativamente diferentes según la gravedad de la CAD. CONCLUSIONES: La mayoría de las crisis de CAD requieren de ingreso, pero la mortalidad es inferior al 2%, variando la duración de la estancia en urgencias y hospitalización dependiendo del tipo de DM y la gravedad inicial del episodio

Clinical features, mortality, hospital admission, and length of stay of a cohort of adult patients with diabetic ketoacidosis attending the emergency room of a tertiary hospital in Spain.

AIM: To assess the clinical features, length of stay, incidence rate, mortality, and hospital admissions of patients with episodes of diabetic ketoacidosis (DKA). PATIENTS: It was conducted retrospective, cross-sectional study of 164 consecutive admissions of adult patients (2008-August 2012), with type 1 or type 2 diabetes already known or new onset. RESULTS: Mortality rate was 1.2%. The DKA episodes were mild (18.9%), moderate (31.7%), or severe (49.4%). The cumulative incidence was 2.66 cases/1000 patients with diabetes (DM) in 4.5 years. The most common causes triggering DKA were infection (33.2%) and dietary transgression and/or insulin dose omission (30.7%). A total of 12.8% of patients had new onset DM, 56.7% type 1, and 26.8% type 2 DM. Patients with type 2 DM were older and had at admission higher creatinine, BUN, osmolality, sodium, and anion gap levels. Patients with new-onset of DM had higher levels of glucose and sodium, but lower potassium levels. No differences were found in pH or bicarbonate. Admission to the intensive care unit (ICU) was required in >50% of cases (p<0.001), and 86.6% of patients were finally admitted to a medical ward (p=0.005). The length of stay at the ICU (p<0.001) and hospital (p=0.013) was significantly different depending on DKA severity. CONCLUSIONS: Most DKA episodes require hospital admission, but mortality is <2%, and length of stay at the ER and medical ward depends on type of DM and initial severity of the episode.

Regulation of SCN5A by microRNAs: miR-219 modulates SCN5A transcript expression and the effects of flecainide intoxication in mice.

BACKGROUND: The human cardiac action potential in atrial and ventricular cells is initiated by a fast-activating, fast-inactivating sodium current generated by the SCN5A/Nav1.5 channel in association with its ß1/SCN1B subunit. The role of Nav1.5 in the etiology of many cardiac diseases strongly suggests that proper regulation of cell biology and function of the channel is critical for normal cardiac function. Hence, numerous recent studies have focused on the regulatory mechanisms of Nav1.5 biosynthetic and degradation processes as well as its subcellular localization. OBJECTIVE: The purpose of this study was to investigate the role of microRNAs in the Scn5a/Nav1.5 posttranscriptional regulation. METHODS: Quantitative polymerase chain reaction, immunohistochemical and electrophysiological measurements of distinct microRNA gain-of-function experiments in cardiomyocytes for the assessment of Scn5a expression. RESULTS: Functional studies of HL-1 cardiomyocytes and luciferase assays in fibroblasts demonstrate that Scn5a is directly (miR-98, miR-106, miR-200, and miR-219) and indirectly (miR-125 and miR-153) regulated by multiple microRNAs displaying distinct time-dependent profiles. Cotransfection experiments demonstrated that miR-219 and miR-200 have independent opposite effects on Scn5a expression modulation. Of all the microRNAs studied, only miR-219 increases Scn5a expression levels, leading to altered contraction rhythm of HL-1 cardiomyocytes. Electrophysiological analyses in HL-1 cells revealed that miR-219 increases the sodium current. In vivo administration of miR-219 does not alter normal cardiac rhythm, but abolishes some of the effects of flecainide intoxication in mice, particularly QRS prolongation. CONCLUSION: This study demonstrates the involvement of multiple microRNAs in the regulation of Scn5a. Particularly, miR-219 increases Scn5a/Nav1.5 transcript and protein expression. Our data suggest that microRNAs, such as miR-219, constitute a promising therapeutical tool to treat sodium cardiac arrhythmias.

Calcium homeostasis in myogenic differentiation factor 1 (MyoD)-transformed, virally-transduced, skin-derived equine myotubes.

Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human and equine subjects by examining the contractile responses of biopsied muscle strips exposed to caffeine, a potent RYR1 agonist. However, this test is not widely available and, due to its invasive nature, is potentially less suitable for valuable animals in training or in the human paediatric setting. Furthermore, increasingly, RYR1 gene polymorphisms (of unknown pathogenicity and significance) are being identified through next generation sequencing projects. Consequently, we have investigated a less invasive test that can be used to study calcium homeostasis in cultured, skin-derived fibroblasts that are converted to the muscle lineage by viral transduction with a MyoD (myogenic differentiation 1) transgene. Similar models have been utilised to examine calcium homeostasis in human patient cells, however, to date, there has been no detailed assessment of the cells' calcium homeostasis, and in particular, the responses to agonists and antagonists of RYR1. Here we describe experiments conducted to assess calcium handling of the cells and examine responses to treatment with dantrolene, a drug commonly used for prophylaxis of recurrent exertional rhabdomyolysis in horses and malignant hyperthermia in humans.

The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice.

Mutations in fukutin-related protein (FKRP) underlie a group of muscular dystrophies associated with the hypoglycosylation of α-dystroglycan (α-DG), a proportion of which show central nervous system involvement. Our original FKRP knock-down mouse (FKRP(KD)) replicated many of the characteristics seen in patients at the severe end of the dystroglycanopathy spectrum but died perinatally precluding its full phenotyping and use in testing potential therapies. We have now overcome this by crossing FKRP(KD) mice with those expressing Cre recombinase under the Sox1 promoter. Owing to our original targeting strategy, this has resulted in the restoration of Fkrp levels in the central nervous system but not the muscle, thereby generating a new model (FKRP(MD)) which develops a progressive muscular dystrophy resembling what is observed in limb girdle muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) is a bifunctional glycosyltransferase previously shown to hyperglycosylate α-DG. To investigate the therapeutic potential of LARGE up-regulation, we have now crossed the FKRP(MD) line with one overexpressing LARGE and show that, contrary to expectation, this results in a worsening of the muscle pathology implying that any future strategies based upon LARGE up-regulation require careful management.

Adenovirus-mediated expression of myogenic differentiation factor 1 (MyoD) in equine and human dermal fibroblasts enables their conversion to caffeine-sensitive myotubes.

Several human and animal myopathies, such as malignant hyperthermia (MH), central core disease and equine recurrent exertional rhabdomyolysis (RER) are confirmed or thought to be associated with dysfunction of skeletal muscle calcium regulation. For some patients in whom the genetic cause is unknown, or when mutational analysis reveals genetic variants with unclear pathogenicity, defects are further studied through use of muscle histopathology and in vitro contraction tests, the latter in particular, when assessing responses to ryanodine receptor agonists, such as caffeine. However, since muscle biopsy is not always suitable, researchers have used cultured cells to model these diseases, by examining calcium regulation in myotubes derived from skin, following forced expression of muscle-specific transcription factors. Here we describe a novel adenoviral vector that we used to express equine MyoD in dermal fibroblasts. In permissive conditions, transduced equine and human fibroblasts differentiated into multinucleated myotubes. We demonstrate that these cells have a functional excitation-calcium release mechanism and, similarly to primary muscle-derived myotubes, respond in a dose-dependent manner to increasing concentrations of caffeine. MyoD-induced conversion of equine skin-derived fibroblasts offers an attractive method for evaluating calcium homeostasis defects in vitro without the need for invasive muscle biopsy.

Functional characterization of a novel frameshift mutation in the C-terminus of the Nav1.5 channel underlying a Brugada syndrome with variable expression in a Spanish family.

INTRODUCTION: We functionally analyzed a frameshift mutation in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) found in a proband with repeated episodes of ventricular fibrillation who presented bradycardia and paroxysmal atrial fibrillation. Seven relatives also carry the mutation and showed a Brugada syndrome with an incomplete and variable expression. The mutation (p.D1816VfsX7) resulted in a severe truncation (201 residues) of the Nav1.5 C-terminus. METHODS AND RESULTS: Wild-type (WT) and mutated Nav1.5 channels together with hNavß1 were expressed in CHO cells and currents were recorded at room temperature using the whole-cell patch-clamp. Expression of p.D1816VfsX7 alone resulted in a marked reduction (≈90%) in peak Na(+) current density compared with WT channels. Peak current density generated by p.D1816VfsX7+WT was ≈50% of that generated by WT channels. p.D1816VfsX7 positively shifted activation and inactivation curves, leading to a significant reduction of the window current. The mutation accelerated current activation and reactivation kinetics and increased the fraction of channels developing slow inactivation with prolonged depolarizations. However, late INa was not modified by the mutation. p.D1816VfsX7 produced a marked reduction of channel trafficking toward the membrane that was not restored by decreasing incubation temperature during cell culture or by incubation with 300 µM mexiletine and 5 mM 4-phenylbutirate. CONCLUSION: Despite a severe truncation of the C-terminus, the resulting mutated channels generate currents, albeit with reduced amplitude and altered biophysical properties, confirming the key role of the C-terminal domain in the expression and function of the cardiac Na(+) channel.