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

Association of VAV2 and VAV3 polymorphisms with cardiovascular risk factors

Hypertension, diabetes and obesity are cardiovascular risk factors closely associated to the development of renal and cardiovascular target organ damage. VAV2 and VAV3, members of the VAV family proto-oncogenes, are guanosine nucleotide exchange factors for the Rho and Rac GTPase family, which is related with cardiovascular homeostasis. We have analyzed the relationship between the presence of VAV2 rs602990 and VAV3 rs7528153 polymorphisms with cardiovascular risk factors and target organ damage (heart, vessels and kidney) in 411 subjects. Our results show that being carrier of the T allele in VAV2 rs602990 polymorphism is associated with an increased risk of obesity, reduced levels of ankle-brachial index and diastolic blood pressure and reduced retinal artery caliber. In addition, being carrier of T allele is associated with increased risk of target organ damage in males. On the other hand, being carrier of the T allele in VAV3 rs7528153 polymorphism is associated with a decreased susceptibility of developing a pathologic state composed by the presence of hypertension, diabetes, obesity or cardiovascular damage, and with an increased risk of developing altered basal glycaemia. This is the first report showing an association between VAV2 and VAV3 polymorphisms with cardiovascular risk factors and target organ damage

A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.European Journal of Human Genetics advance online publication, 4 February 2015; doi:10.1038/ejhg.2014.283

Dense active matter model of motion patterns in confluent cell monolayers

Epithelial cell monolayers show remarkable displacement and velocity correlations over distances of ten or more cell sizes that are reminiscent of supercooled liquids and active nematics. We show that many observed features can be described within the framework of dense active matter, and argue that persistent uncoordinated cell motility coupled to the collective elastic modes of the cell sheet is sufficient to produce swirl-like correlations. We obtain this result using both continuum active linear elasticity and a normal modes formalism, and validate analytical predictions with numerical simulations of two agent-based cell models, soft elastic particles and the self-propelled Voronoi model together with in-vitro experiments of confluent corneal epithelial cell sheets. Simulations and normal mode analysis perfectly match when tissue-level reorganisation occurs on times longer than the persistence time of cell motility. Our analytical model quantitatively matches measured velocity correlation functions over more than a decade with a single fitting parameter.Comment: updated version accepted for publication in Nat. Com

Impact of Liver Inflammation on Bile Acid Side Chain Shortening and Amidation

Bile acid (BA) synthesis from cholesterol by hepatocytes is inhibited by inflammatory cytokines. Whether liver inflammation also affects BA side chain shortening and conjugation was investigated. In human liver cell lines (IHH, HepG2, and HepaRG), agonists of nuclear receptors including the farnesoid X receptor (FXR), liver X receptor (LXR), and peroxisome proliferator-activated receptors (PPARs) did not affect the expression of BA-related peroxisomal enzymes. In contrast, hepatocyte nuclear factor 4? (HNF4?) inhibition down-regulated acyl-CoA oxidase 2 (ACOX2). ACOX2 was repressed by fibroblast growth factor 19 (FGF19), which was prevented by extracellular signal-regulated kinase (ERK) pathway inhibition. These changes were paralleled by altered BA synthesis (HPLC-MS/MS). Cytokines able to down-regulate cholesterol-7?-hydroxylase (CYP7A1) had little effect on peroxisomal enzymes involved in BA synthesis except for ACOX2 and bile acid-CoA:amino acid N-acyltransferase (BAAT), which were down-regulated, mainly by oncostatin M (OSM). This effect was prevented by Janus kinase (JAK) inhibition, which restored BA side chain shortening and conjugation. The binding of OSM to the extracellular matrix accounted for a persistent effect after culture medium replacement. In silico analysis of four databases (n = 201) and a validation cohort (n = 90) revealed an inverse relationship between liver inflammation and ACOX2/BAAT expression which was associated with changes in HNF4? levels. In conclusion, BA side chain shortening and conjugation are inhibited by inflammatory effectors. However, other mechanisms involved in BA homeostasis counterbalance any significant impact on the serum BA profile

Drosophila muscleblind Codes for Proteins with One and Two Tandem Zinc Finger Motifs

Muscleblind-like proteins, Muscleblind (Mbl) in Drosophila and MBNL1-3 in vertebrates, are regulators of alternative splicing. Human MBNL1 is a key factor in the etiology of myotonic dystrophy (DM), a muscle wasting disease caused by the occurrence of toxic RNA molecules containing CUG/CCUG repeats. MBNL1 binds to these RNAs and is sequestered in nuclear foci preventing it from exerting its normal function, which ultimately leads to mis-spliced mRNAs, a major cause of the disease. Muscleblind-proteins bind to RNAs via N-terminal zinc fingers of the Cys3-His type. These zinc fingers are arranged in one (invertebrates) or two (vertebrates) tandem zinc finger (TZF) motifs with both fingers targeting GC steps in the RNA molecule. Here I show that mbl genes in Drosophila and in other insects also encode proteins with two TZF motifs, highly similar to vertebrate MBNL proteins. In Drosophila the different protein isoforms have overlapping but possibly divergent functions in vivo, evident by their unequal capacities to rescue the splicing defects observed in mbl mutant embryos. In addition, using whole transcriptome analysis, I identified several new splicing targets for Mbl in Drosophila embryos. Two of these novel targets, kkv (krotzkopf-verkehrt, coding for Chitin Synthase 1) and cora (coracle, coding for the Drosophila homolog of Protein 4.1), are not muscle-specific but expressed mainly in epidermal cells, indicating a function for mbl not only in muscles and the nervous system

Myofascial trigger points in cluster headache patients: a case series

Active myofascial trigger points (MTrPs) have been found to contribute to chronic tension-type headache and migraine. The purpose of this case series was to examine if active trigger points (TrPs) provoking cluster-type referred pain could be found in cluster headache patients and, if so, to evaluate the effectiveness of active TrPs anaesthetic injections both in the acute and preventive headache's treatment. Twelve patients, 4 experiencing episodic and 8 chronic cluster headache, were studied. TrPs were found in all of them. Abortive infiltrations could be done in 2 episodic and 4 chronic patients, and preemptive infiltrations could be done in 2 episodic and 5 chronic patients, both kind of interventions being successful in 5 (83.3%) and in 6 (85.7%) of the cases respectively. When combined with prophylactic drug therapy, injections were associated with significant improvement in 7 of the 8 chronic cluster patients. Our data suggest that peripheral sensitization may play a role in cluster headache pathophysiology and that first neuron afferent blockade can be useful in cluster headache management