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

Iberian cured-ham consumption improves endothelial function in healthy subjects

Objectives: Previous studies have shown that dietary components such as oleic acid or polyphenols exert beneficial effects on endothelium. We aimed to assess the impact of regular consumption of Iberian cured-ham (ICH) on endothelial function. Design: An open-label, randomized controlled parallel study. Setting: Volunteers recruited through advertisements at a hospital in Madrid, Spain. Participants: 102 Caucasian adults (76.8% females) aged 25-55 years, and free from cardiometabolic disease. Intervention: Participants were randomized to an ICH-enriched ad libitum diet or an ad libitum diet without ICH for 6 weeks. Subjects in ICH group were randomly provided with either acorn- or mixed-fed ICH, and followed up for an additional 6-week period under their usual diet. Measurements: Clinical parameters, biomarkers of endothelial function and oxidative stress, microvascular vasodilatory response to hyperemia and arterial stiffness were measured before and after the intervention. Results: After 6 weeks, a larger decrease in PAI-1 was observed in subjects consuming ICH compared to the Control group (-6.2±17.7 vs. 0.3±1.4 ng/ml; p=0.020). Similarly, microvascular vasodilatory response to hyperemia showed a significant increase (112.4±391.7 vs. -56.0±327.9%; p=0.007). However, neither oxidative stress, hemodynamic nor clinical parameters differed significantly over the study. Additionally, after stopping ICH consumption, improvements in PAI-1 remained for 6 additional weeks with respect to baseline (p=0.006). Conclusion: The present study demonstrates, for the first time, that regular consumption of ICH improves endothelial function in healthy adults. Strategies aimed to preserve or improve the endothelial function may have implications in vascular aging beyond the prevention of the atherothrombotic disease

CHK1 expression in gastric cancer is modulated by p53 and RB1/E2F1: Implications in chemo/radiotherapy response

Radiation has a limited but relevant role in the adjuvant therapy of gastric cancer (GC) patients. Since Chk1 plays a critical function in cellular response to genotoxic agents, we aimed to analyze the role of Chk1 in GC as a biomarker for radiotherapy resistance. We analyzed Chk1 expression in AGS and MKN45 human GC cell lines by RT-QPCR and WB and in a small cohort of human patient’s samples. We demonstrated that Chk1 overexpression specifically increases resistance to radiation in GC cells. Accordingly, abrogation of Chk1 activity with UCN-01 and its expression with shChk1 increased sensitivity to bleomycin and radiation. Furthermore, when we assessed Chk1 expression in human samples, we found a correlation between nuclear Chk1 accumulation and a decrease in progression free survival. Moreover, using a luciferase assay we found that Chk1’s expression is controlled by p53 and RB/E2F1 at the transcriptional level. Additionally, we present preliminary data suggesting a posttranscriptional regulation mechanism, involving miR-195 and miR-503, which are inversely correlated with expression of Chk1 in radioresistant cells. In conclusion, Chk1/microRNA axis is involved in resistance to radiation in GC, and suggests Chk1 as a potential tool for optimal stratification of patients susceptible to receive adjuvant radiotherapy after surgeryThis work was supported by Instituto de Salud Carlos III–Fondo de Investigación Sanitaria (PS09/1988 to ISP; PI11-00949, pI014-1495 and Feder Funds to RP); Comunidad Autónoma de Madrid-Universidad Autónoma de Madrid (CCG10-UAM/BIO-5871 to ISP); Fundación Leticia Castillejo Castillo and Ministerio de Ciencia e Innovación (SAF2012-30862 to RSP), Spain

A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient Cavalier King Charles Spaniels is amenable to exon 51 skipping

BACKGROUND Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot". METHODOLOGY/PRINCIPAL FINDINGS Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. CONCLUSIONS/SIGNIFICANCE Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD

Influencia de la temperatura durante el quemado sobre el despellejado en aceitunas variedad Aloreña y Manzanilla Sevillana

Las aceitunas variedad Manzanilla Sevillana y Aloreña se cocieron en recipientes de 8 litros a temperatura controlada a diferentes intervalos. Aloreña y Manzanilla Sevillana se sometieron a 19-20 °C, 22 °C, 23-24 °C, 25-26 °C, 28-29 °C y además solo para Manzanilla Sevillana se agregó el intervalo 32-35 °C. Se utilizó una solución de NaOH al 2,5% p/v en el cocido y en todos los tratamientos, además se emplearon los mismos lavados y la misma salmuera inicial de fermentación con el 10% de concentración de NaCL +0,1% ácido acético glacial. El objetivo de este trabajo es mostrar el efecto de la temperatura en el defecto del despellejado para las diferentes variedades. En todos los tratamientos se evaluó el defecto de despellejado haciendo un corte con cuchillo en toda la circunferencia de cada fruto y se observó si hay o no desprendimiento de la piel adyacente al corte, luego se pasó el dedo en el corte en el mismo sentido para verificar si la piel no se desprendía. La correlación de Pearson observada entre la temperatura de quemado y el porcentaje de despellejado fue de 0,93 (p<0,001) para Aloreña y 0,95 (p<0,001) para Manzanilla indicando una relación directa entre los dos factores. En el intervalo de 19-20 °C se consigue un defecto medio de 1% en Manzanilla y 1,25% en Aloreña.Manzanilla Sevillana and Aloreña variety olives were taken and subject to alkaline treatment in 8 liter containers at a controlled temperature in the following intervals: 19-20 °C, 22 °C, 23-24 °C, 25-26 °C, 28-29 °C, for Aloreña and for Manzanilla Sevillana. In addition to those mentioned, an extra interval of 32-35 °C was performed for Manzanilla Sevillana. A 2.5% w/v NaOH solution was used in the alkaline treatment, in all treatments; the same washes were made and the same initial fermentation brine was used, with a concentration of 10% of NaCl + 0.1% glacial acetic acid. The aim of this work is to show the effect temperature has on the peeling defect these two varieties suffer. The skin detachment or peeling defect was evaluated in every treatment, by making a cut with knife along the entire circumference of each fruit, thus to observe if there was peeling adjacent to the cut, then a finger was passed in the cut in the same direction to check if the skin did not come off. Pearson correlation observed between the lye treatment temperature and the peeling percentage was 0.93 (p<0.001) for Aloreña and y 0.95 (p<0.001) for Manzanilla, indicative of a direct relation between these factors. In the interval 19-20 °C, a mean defect of 1% is obtained in Manzanilla and of 1.25% in Aloreña.EEA ChilecitoFil: Juarez, Julio Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Chilecito. Agencia de Extensión Rural Aimogasta; ArgentinaFil: Fernandez, C. La Rioja (Argentina : provincia) .Departamento Arauco. Dirección de Bromatología; ArgentinaFil: De La Fuente, M. Asociación de productores “Aimoarauco”, Departamento Arauco, La Rioja; Argentina

Allele Copy Number and Underlying Pathology Are Associated with Subclinical Severity in Equine Type 1 Polysaccharide Storage Myopathy (PSSM1)

Equine type 1 polysaccharide storage myopathy (PSSM1), a common glycogenosis associated with an R309H founder mutation in the glycogen synthase 1 gene (GYS1), shares pathological features with several human myopathies. In common with related human disorders, the pathogenesis remains unclear in particular, the marked phenotypic variability between affected animals. Given that affected animals accumulate glycogen and alpha-crystalline polysaccharide within their muscles, it is possible that physical disruption associated with the presence of this material could exacerbate the phenotype. The aim of this study was to compare the histopathological changes in horses with PSSM1, and specifically, to investigate the hypothesis that the severity of underlying pathology, (e.g. vacuolation and inclusion formation) would (1) be higher in homozygotes than heterozygotes and (2) correlate with clinical severity. Resting and post-exercise plasma creatine kinase (CK) and aspartate aminotransferase (AST) enzyme activity measurements and muscle pathology were assessed in matched cohorts of PSSM1 homozygotes, heterozygotes or control horses. Median (interquartile range (IR)) resting CK activities were 364 (332–764) U/L for homozygotes, 301 (222–377) U/L for heterozygotes and 260 (216–320) U/L for controls, and mean (+/− SD) AST activity for homozygotes were 502 (+/116) U/L, for heterozygotes, 357 (+/−92) U/L and for controls, 311 (+/−64) U/L and were significantly different between groups (P = 0.04 and P = 0.01 respectively). Resting plasma AST activity was significantly associated with the severity of subsarcolemmal vacuolation (rho = 0.816; P = 0.01) and cytoplasmic inclusions (rho = 0.766; P = 0.01). There were fewer type 2× and more type 2a muscle fibres in PSSM1-affected horses. Our results indicate that PSSM1 has incomplete dominance. Furthermore, the association between plasma muscle enzyme activity and severity of underlying pathology suggests that physical disruption of myofibres may contribute to the myopathic phenotype. This work provides insight into PSSM1 pathogenesis and has implications for related human glycogenoses

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