Eukarion-134 attenuates endoplasmic reticulum stress-induced mitochondrial dysfunction in human skeletal muscle cells

Maladaptive endoplasmic reticulum (ER) stress is associated with modified reactive oxygen species (ROS) generation and mitochondrial abnormalities; and is postulated as a potential mechanism involved in muscle weakness in myositis, an acquired autoimmune neuromuscular disease. This study investigates the impact of ROS generation in an in vitro model of ER stress in skeletal muscle, using the ER stress inducer tunicamycin (24 h) in the presence or absence of a superoxide dismutase/catalase mimetic Eukarion (EUK)-134. Tunicamycin induced maladaptive ER stress, which was mitigated by EUK-134 at the transcriptional level. ER stress promoted mitochondrial dysfunction, described by substantial loss of mitochondrial membrane potential, as well as a reduction in respiratory control ratio, reserve capacity, phosphorylating respiration, and coupling efficiency, which was ameliorated by EUK-134. Tunicamycin induced ROS-mediated biogenesis and fusion of mitochondria, which, however, had high propensity of fragmentation, accompanied by upregulated mRNA levels of fission-related markers. Increased cellular ROS generation was observed under ER stress that was prevented by EUK-134, even though no changes in mitochondrial superoxide were noticeable. These findings suggest that targeting ROS generation using EUK-134 can amend aspects of ER stress-induced changes in mitochondrial dynamics and function, and therefore, in instances of chronic ER stress, such as in myositis, quenching ROS generation may be a promising therapy for muscle weakness and dysfunction

T2DM GWAS in the Lebanese population confirms the role of TCF7L2 and CDKAL1 in disease susceptibility

Genome-wide association studies (GWAS) of multiple populations with distinctive genetic and lifestyle backgrounds are crucial to the understanding of Type 2 Diabetes Mellitus (T2DM) pathophysiology. We report a GWAS on the genetic basis of T2DM in a 3,286 Lebanese participants. More than 5,000,000 SNPs were directly genotyped or imputed using the 1000 Genomes Project reference panels. We identify genome-wide significant variants in two loci CDKAL1 and TCF7L2, independent of sex, age and BMI, with leading variants rs7766070 (OR = 1.39, P = 4.77 × 10(−9)) and rs34872471 (OR = 1.35, P = 1.01 × 10(−8)) respectively. The current study is the first GWAS to find genomic regions implicated in T2DM in the Lebanese population. The results support a central role of CDKAL1 and TCF7L2 in T2DM susceptibility in Southwest Asian populations and provide a plausible component for understanding molecular mechanisms involved in the disease

First evidence for human occupation of a lava tube in Arabia: The archaeology of Umm Jirsan Cave and its surroundings, northern Saudi Arabia

Recent advances in interdisciplinary archaeological research in Arabia have focused on the evolution and historical development of regional human populations as well as the diverse patterns of cultural change, migration, and adaptations to environmental fluctuations. Obtaining a comprehensive understanding of cultural developments such as the emergence and lifeways of Neolithic groups has been hindered by the limited preservation of stratified archaeological assemblages and organic remains, a common challenge in arid environments. Underground settings like caves and lava tubes, which are prevalent in Arabia but which have seen limited scientific exploration, offer promising opportunities for addressing these issues. Here, we report on an archaeological excavation and a related survey at and around Umm Jirsan lava tube in the Harrat Khaybar, north-western Saudi Arabia. Our results reveal repeated phases of human occupation of the site ranging from at least the Neolithic through to the Chalcolithic/Bronze Age. Pastoralist use of the lava tube and surrounding landscape is attested in rock art and faunal records, suggesting that Umm Jirsan was situated along a pastoral route linking key oases. Isotopic data indicates that herbivores primarily grazed on wild grasses and shrubs rather than being provided with fodder, while humans had a diet consistently high in protein but with increasing consumption of C3 plants through-time, perhaps related to the emergence of oasis agriculture. While underground and naturally sheltered localities are globally prominent in archaeology and Quaternary science, our work represents the first such combined records for Saudi Arabia and highlight the potential for interdisciplinary studies in caves and lava tubes

Multivariate epidemiologic analysis of type 2 diabetes mellitus risks in the Lebanese population

Background: The burden of diabetes in Lebanon requires well-targeted interventions for screening type 2 diabetes mellitus (T2DM) and prediabetes and prevention of risk factors. Newly recruited 998 Lebanese individuals, in addition to 7,292 already available, were studied to investigate the prevalence of diabetes, prediabetes and their associated risk factors. Methods: Participants had fasting blood sugar and glycohemoglobin tests in addition to a lipid profile. Clinical and demographic information were obtained from a detailed questionnaire. The relationship between T2DM, its risk factors, and its complications were tested. Comparisons of these risk factors among diabetics, healthy, and coronary artery disease (CAD) patients were performed. Results: The prevalence of T2DM significantly increased with increasing BMI (p < 0.0001). Exercise activity level negatively correlated with the disease (p = 0.002), whereas the prevalence of T2DM (p < 0.0001) and CAD family history (p = 0.006) positively correlated with the affection status. The mean levels of triglycerides and LDL-C were significantly higher in diabetics (1.87; 1.35) compared to individuals with prediabetes (1.63; 1.26) and unaffected controls (1.49; 1.19). People with T2DM showed a significant decrease in HDL-C levels. A strong correlation of overall hyperlipidemia with the diabetes affection status was shown (p < 0.0001). Other comorbid factors such as hypertension (p < 0.0001) and self-reported obesity (p < 0.0001) were highly associated with T2DM and prediabetes. Reproductive health of women showed a strong correlation between giving birth to a baby with a high weight and the occurrence of T2DM and prediabetes later in life (p < 0.0001). Retinopathy and peripheral neuropathy were significantly correlated with diabetes and prediabetes (p < 0.0001). Conclusions: The present study shows an alarming prevalence of diabetes and prediabetes in the studied subgroups representative of the Lebanese population. Electronic supplementary material The online version of this article (doi:10.1186/1758-5996-6-89) contains supplementary material, which is available to authorized users

Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells

Background: Neuromuscular junctions (NMJs) consist of the presynaptic cholinergic motoneuron terminals and the corresponding postsynaptic motor endplates on skeletal muscle fibers. At the NMJ the action potential of the neuron leads, via release of acetylcholine, to muscle membrane depolarization that in turn is translated into muscle contraction and physical movement. Despite the fact that substantial NMJ research has been performed, the potential of in vivo NMJ investigations is inadequate and difficult to employ. A simple and reproducible in vitro NMJ model may provide a robust means to study the impact of neurotrophic factors, growth factors, and hormones on NMJ formation, structure, and function. Methods: This report characterizes a novel in vitro NMJ model utilizing immortalized human skeletal muscle stem cells seeded on 35 mm glass-bottom dishes, cocultured and innervated with spinal cord explants from rat embryos at ED 13.5. The cocultures were fixed and stained on day 14 for analysis and assessment of NMJ formation and development. Results: This unique serum-and trophic factor-free system permits the growth of cholinergic motoneurons, the formation of mature NMJs, and the development of highly differentiated contractile myotubes, which exhibit appropriate configuration of transversal triads, representative of in vivo conditions. Conclusion: This coculture system provides a tool to study vital features of NMJ formation, regulation, maintenance, and repair, as well as a model platform to explore neuromuscular diseases and disorders affecting NMJs

Cross-talk between motor neurons and myotubes via endogenously secreted neural and muscular growth factors

Neuromuscular junction (NMJ) research is vital to advance the understanding of neuromuscular patho-physiology and development of novel therapies for diseases associated with NM dysfunction. In vivo, the micro-environment surrounding the NMJ has a significant impact on NMJ formation and maintenance via neurotrophic and differentiation factors that are secreted as a result of cross-talk between muscle fibers and motor neurons. Recently we showed the formation of functional NMJs in vitro in a co-culture of immortalized human myoblasts and motor neurons from rat-embryo spinal-cord explants, using a culture medium free from serum and neurotrophic or growth factors. The aim of this study was to assess how functional NMJs were established in this co-culture devoid of exogenous neural growth factors. To investigate this, an ELISA-based microarray was used to compare the composition of soluble endogenously secreted growth factors in this co-culture with an a-neural muscle culture. The levels of seven neurotrophic factors brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor-1 (IGF-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and vascular endothelial growth factor (VEGF) were higher (p < 0.05) in the supernatant of NMJ culture compared to those in the supernatant of the a-neural muscle culture. This indicates that the cross-talk between muscle and motor neurons promotes the secretion of soluble growth factors contributing to the local microenvironment thereby providing a favourable regenerative niche for NMJs formation and maturation

Biallelic loss of function variants in PPP1R21 cause a neurodevelopmental syndrome with impaired endocytic function

Next‐generation sequencing (NGS) has been instrumental in solving the genetic basis of rare inherited diseases, especially neurodevelopmental syndromes. However, functional workup is essential for precise phenotype definition and to understand the underlying disease mechanisms. Using whole exome (WES) and whole genome sequencing (WGS) in four independent families with hypotonia, neurodevelopmental delay, facial dysmorphism, loss of white matter, and thinning of the corpus callosum, we identified four previously unreported homozygous truncating PPP1R21 alleles: c.347delT p.(Ile116Lysfs*25), c.2170_2171insGGTA p.(Ile724Argfs*8), c.1607dupT p.(Leu536Phefs*7), c.2063delA p.(Lys688Serfs*26) and found that PPP1R21 was absent in fibroblasts of an affected individual, supporting the allele's loss of function effect. PPP1R21 function had not been studied except that a large scale affinity proteomics approach suggested an interaction with PIBF1 defective in Joubert syndrome. Our co‐immunoprecipitation studies did not confirm this but in contrast defined the localization of PPP1R21 to the early endosome. Consistent with the subcellular expression pattern and the clinical phenotype exhibiting features of storage diseases, we found patient fibroblasts exhibited a delay in clearance of transferrin‐488 while uptake was normal. In summary, we delineate a novel neurodevelopmental syndrome caused by biallelic PPP1R21 loss of function variants, and suggest a role of PPP1R21 within the endosomal sorting process or endosome maturation pathway

A novel bioengineered functional motor unit platform to study neuromuscular interaction

Background: In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. Objective: to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. Methods: an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. Results: blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. Conclusion: a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions

Circulating levels of dickkopf-1, osteoprotegerin and sclerostin are higher in old compared with young men and women and positively associated with whole-body bone mineral density in older adults

Summary: Bone mineral density declines with increasing older age. We examined the levels of circulating factors known to regulate bone metabolism in healthy young and older adults. The circulating levels of dickkopf-1, osteocalcin, osteoprotegerin and sclerostin were positively associated with WBMD in older adults, despite the average WBMD being lower and circulating dickkopf-1, osteoprotegerin and sclerostin being higher in old than young. Purpose: To investigate the relationship between whole-body bone mineral density (WBMD) and levels of circulating factors with known roles in bone remodelling during 'healthy' ageing. Methods: WBMD and fasting plasma concentrations of dickkopf-1, fibroblast growth factor-23, osteocalcin, osteoprotegerin, osteopontin and sclerostin were measured in 272 older subjects (69 to 81 years; 52% female) and 171 younger subjects (18-30 years; 53% female). Results: WBMD was lower in old than young. Circulating osteocalcin was lower in old compared with young, while dickkopf-1, osteoprotegerin and sclerostin were higher in old compared with young. These circulating factors were each positively associated with WBMD in the older adults and the relationships remained after adjustment for covariates (r-values ranging from 0.174 to 0.254, all p<0.01). In multivariate regression, the body mass index, circulating sclerostin and whole-body lean mass together accounted for 13.8% of the variation with WBMD in the older adults. In young adults, dickkopf-1 and body mass index together accounted for 7.7% of variation in WBMD. Conclusion: Circulating levels of dickkopf-1, osteocalcin, osteoprotegerin and sclerostin are positively associated with WBMD in community-dwelling older adults, despite the average WBMD being lower and circulating dickkopf-1, osteoprotegerin and sclerostin being higher in old than young

Can a standard dose of eicosapentaenoic acid (EPA) supplementation reduce the symptoms of delayed onset of muscle soreness?

Unaccustomed exercise can result in delayed onset of muscle soreness (DOMS) which can affect athletic performance. Although DOMS is a useful tool to identify muscle damage and remodelling, prolonged symptoms of DOMS may be associated with the over-training syndrome. In order to reduce the symptoms of DOMS numerous management strategies have been attempted with no significant effect on DOMS-associated cytokines surge. The present study aimed to investigate the acute and chronic effects of a 2x180 mg per day dose of eicosapentaenoic acid (EPA) on interleukin-6 (IL-6) mediated inflammatory response and symptoms associated with DOMS. Methods: Seventeen healthy non-smoking females (age 20.4 +/- 2.1 years, height 161.2 +/- 8.3cm and mass 61.48 +/- 7.4kg) were randomly assigned to either placebo (N = 10) or EPA (N = 7). Serum IL-6, isometric and isokinetic (concentric and eccentric) strength, and rating of perceived exertion (RPE) were recorded on four occasions: i-prior to supplementation, ii-immediately after three weeks of supplementation (basal effects), iii-48 hours following a single bout of resistance exercise (acute training response effects), and iv-48 hours following the last of a series of three bouts of resistance exercise (chronic training response effects). Results: There was only a group difference in the degree of change in circulating IL-6 levels. In fact, relative to the first baseline, by the third bout of eccentric workout, the EPA group had 103 +/- 60% increment in IL-6 levels whereas the placebo group only had 80 +/- 26% incremented IL-6 levels (P = 0.020). We also describe a stable multiple linear regression model which included measures of strength and not IL-6 as predictors of RPE scale. Conclusion: The present study suggests that in doubling the standard recommended dose of EPA, whilst this may still not be beneficial at ameliorating the symptoms of DOMS, it counter intuitively appears to enhance the cytokine response to exercise. In a context where previous in vitro work has shown EPA to decrease the effects of inflammatory cytokines, it may in fact be that the doses required in vivo is much larger than current recommended amounts. An attempt to dampen the exercise-induced cytokine flux in fact results in an over-compensatory response of this system