Slow Skeletal Muscle Research Articles

Laing Myopathy: Report of 4 New Families With Novel MYH7 Mutations, Double Mutations, and Severe Phenotype.

Laing distal myopathy (LDM) is an autosomal dominant disorder caused by mutations in the slow skeletal muscle fiber myosin heavy chain (MYH7) gene on chromosome 14q11.2. The classic LDM phenotype-including early-onset, initial involvement of foot dorsiflexors and great toe extensors, followed by weakness of neck flexors and finger extensors-is well documented. Since the original report by Laing et al in 1995, the spectrum of MYH7-related myopathies has expanded to include congenital myopathies, late-onset myopathies, myosin storage myopathy, and scapuloperoneal myopathies. Most patients with LDM harbor mutations in the midrod domain of the MYH7 gene, but rare cases document disease-associated mutations in the globular head region. In this report, we add to the medical literature by describing the clinicopathological findings in 8 affected family members from 4 new LDM families-including 2 with novel MYH7 mutations (Y162D and A1438P), one with dual mutations (V39M and K1617del), and one family (E1508del) with severe early-onset weakness associated with contractures, respiratory insufficiency, and dilated cardiomyopathy. Our families highlight the ever-expanding clinical spectrum and genetic variation of the skeletal myopathies related to MYH7 gene mutations.

Protein Array-Based Approach to Evaluate Biomarkers of Beef Tenderness and Marbling in Cows: Understanding of the Underlying Mechanisms and Prediction.

This study evaluated the potential of a panel of 20 protein biomarkers, quantified by Reverse Phase Protein Array (RPPA), to explain and predict two important meat quality traits, these being beef tenderness assessed by Warner–Bratzler shear force (WBSF) and the intramuscular fat (IMF) content (also termed marbling), in a large database of 188 Protected Designation of Origin (PDO) Maine-Anjou cows. Thus, the main objective was to move forward in the progression of biomarker-discovery for beef qualities by evaluating, at the same time for the two quality traits, a list of candidate proteins so far identified by proteomics and belonging to five interconnected biological pathways: (i) energy metabolic enzymes, (ii) heat shock proteins (HSPs), (iii) oxidative stress, (iv) structural proteins and (v) cell death and protein binding. Therefore, three statistical approaches were applied, these being Pearson correlations, unsupervised learning for the clustering of WBSF and IMF into quality classes, and Partial Least Squares regressions (PLS-R) to relate the phenotypes with the 20 biomarkers. Irrespective of the statistical method and quality trait, seven biomarkers were related with both WBSF and IMF, including three small HSPs (CRYAB, HSP20 and HSP27), two metabolic enzymes from the oxidative pathway (MDH1: Malate dehydrogenase and ALDH1A1: Retinal dehydrogenase 1), the structural protein MYH1 (Myosin heavy chain-IIx) and the multifunctional protein FHL1 (four and a half LIM domains 1). Further, three more proteins were retained for tenderness whatever the statistical method, among which two were structural proteins (MYL1: Myosin light chain 1/3 and TNNT1: Troponin T, slow skeletal muscle) and one was glycolytic enzyme (ENO3: β-enolase 3). For IMF, two proteins were, in this trial, specific for marbling whatever the statistical method: TRIM72 (Tripartite motif protein 72, negative) and PRDX6 (Peroxiredoxin 6, positive). From the 20 proteins, this trial allowed us to qualify 10 and 9 proteins respectively as strongly related with beef tenderness and marbling in PDO Maine-Anjou cows.

Mechanisms of disturbance of the contractile function of slow skeletal muscles induced by myopathic mutations in the tropomyosin TPM3 gene.

Several congenital myopathies of slow skeletal muscles are associated with mutations in the tropomyosin (Tpm) TPM3 gene. Tropomyosin is an actin-binding protein that plays a crucial role in the regulation of muscle contraction. Two Tpm isoforms, γ (Tpm3.12) and β (Tpm2.2) are expressed in human slow skeletal muscles forming γγ-homodimers and γβ-heterodimers of Tpm molecules. We applied various methods to investigate how myopathy-causing mutations M9R, E151A, and K169E in the Tpm γ-chain modify the structure-functional properties of Tpm dimers, and how this affects the muscle functioning. The results show that the features of γγ-Tpm and γβ-Tpm with substitutions in the Tpm γ-chain vary significantly. The characteristics of the γγ-Tpm depend on whether these mutations located in only one or both γ-chains. The mechanism of the development of nemaline myopathy associated with the M9R mutation was revealed. At the molecular level, a cause-and-effect relationship has been established for the development of myopathy by the K169E mutation. Also, we described the structure-functional properties of the Tpm dimers with the E151A mutation, which explain muscle weakness linked to this substitution. The results demonstrate a diversity of the molecular mechanisms of myopathy pathogenesis induced by studied Tpm mutations.

MiR-206 enforces a slow muscle phenotype.

Striated muscle is a highly specialized collection of tissues with contractile properties that vary according to functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-dependent adaptation. Functional adaptation requires molecular adaptation, which is partially provided by miRNA-mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-206 expression increases in both physiological (including female sex and endurance exercise) and pathological conditions (muscular dystrophy and adrenergic agonism) that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-206-knockout mice displays a faster phenotype than wild-type mice. Moreover, left ventricles of male miR-206 knockout mice have a faster myosin profile, accompanied by dilation and systolic dysfunction. Thus, miR-206 appears to be necessary to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms.

Dynamic localization of αB-crystallin at the microtubule cytoskeleton network in beating heart cells.

αB-crystallin is highly expressed in the heart and slow skeletal muscle; however, the roles of αB-crystallin in the muscle are obscure. Previously, we showed that αB-crystallin localizes at the sarcomere Z-bands, corresponding to the focal adhesions of cultured cells. In myoblast cells, αB-crystallin completely colocalizes with microtubules and maintains cell shape and adhesion. In this study, we show that in beating cardiomyocytes α-tubulin and αB-crystallin colocalize at the I- and Z-bands of the myocardium, where it may function as a molecular chaperone for tubulin/microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the striated patterns of GFP-αB-crystallin fluorescence recovered quickly at 37°C. FRAP mobility assay also showed αB-crystallin to be associated with nocodazole-treated free tubulin dimers but not with taxol-treated microtubules. The interaction of αB-crystallin and free tubulin was further confirmed by immunoprecipitation and microtubule sedimentation assay in the presence of 1-100 μM calcium, which destabilizes microtubules. Förster resonance energy transfer analysis showed that αB-crystallin and tubulin were at 1-10 nm apart from each other in the presence of colchicine. These results suggested that αB-crystallin may play an essential role in microtubule dynamics by maintaining free tubulin in striated muscles, such as the soleus or cardiac muscles.

Orthophosphate increases the efficiency of slow muscle-myosin isoform in the presence of omecamtiv mecarbil

Omecamtiv mecarbil (OM) is a putative positive inotropic tool for treatment of systolic heart dysfunction, based on the finding that in vivo it increases the ejection fraction and in vitro it prolongs the actin-bond life time of the cardiac and slow-skeletal muscle isoforms of myosin. OM action in situ, however, is still poorly understood as the enhanced Ca2+-sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca2+. Here we show, by combining fast sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus, that the depressant effect of OM on the force per attached motor is reversed, without effect on the ATPase rate, by physiological concentrations of inorganic phosphate (Pi) (1-10 mM). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients, whenever [Pi] increases with heart-beat frequency.

Comparison of Functional Characteristics of Myosin in Fast and Slow Skeletal Muscles.

Myosins of fast and slow skeletal muscles differ by the isoform composition of the heavy and light chains. We compared functional characteristics of myosin from the fast (m. psoas) and slow (m. soleus) muscles of rabbits. The parameters of single actin-myosin interaction were measured in an optical trap, and the characteristics of the Ca2+ regulation of actin-myosin interaction were studied using an in vitro motility assay. The duration of interaction of myosin from the fast muscle with actin was shorter and the filament sliding velocity over this myosin was higher than the corresponding parameters for myosin from the slow muscle. The dependence pCa-velocity for myosin from the fast muscle was less sensitive to Ca2+ than that of slow muscle myosin. Thus, functional properties of myosin determine not only mechanical and kinetic characteristics of muscle contraction, but also the peculiarities of its Ca2+ regulation.

A long-read RNA-seq approach to identify novel transcripts of very large genes.

RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap: 5 kb), large (Neb: 22 kb), and very large (Ttn: 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type-specific differential expression of these novel transcripts. The improved transcript identification and quantification shown by our approach removes previous impediments to studies aimed at quantitative differential expression of ultralong transcripts.

Effect of eight weeks of resistance training on the expression of klotho protein and insulin-like growth factor 1 genes in slow twitch and fast twitch skeletal muscles of aged Wistar rats

Background and Aims: Klotho protein is a substance effective in increasing life expectancy. Moreover, it prevents muscle atrophy, osteoporosis, and cardiovascular disease. Therefore, the present aimed to assess changes in the expression of klotho protein and insulin-like growth factor 1 (IGF-1) genes in the muscles of aged Wistar rats after eight weeks of resistance training. Materials and Methods: The present experimental study was connected on 16 Wistar male rats, aged 20 months, which were assigned to two groups of resistance training group and control. Resistance training was performed three sessions a week over eight weeks. After dissecting the rats, the soleus and flexor hallucis longus muscles of the rats were extracted to measure the expression of klotho protein and (IGF-1) genes using the real-time polymerase chain reaction technique. Results: After the resistance training program, Klotho protein in the fast-twitch muscle tissue of the trained rats did not show a significant difference, compared to that in the control group. Furthermore, IGF-1 in the fast-twitch and slow-twitch muscles of the training group did not display a marked difference, compared to that in the control group. Conclusion: As evidenced by the obtained results, the values of klotho protein and IGF-1 did not improve after a period of resistance training. It can be argued that resistance training should be conducted in longer periods and with different intensities, which should be scrutinized in future research.

Unique functional properties of slow skeletal muscle tropomyosin

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein playing an essential role in the regulation of muscle contraction. The α- (Tpm 1.1) and γ- (Tpm 3.12) Tpm isoforms are expressed in fast and slow human skeletal muscles, respectively, while β-Tpm (Tpm 2.2) is expressed in both muscle types. This results in the formation of Tpm αα- and γγ-homodimers as well as αβ- and γβ-heterodimers. The properties of αα-homodimer are well studied, whereas very little is known about the functional properties of γγ-homodimer and γβ-heterodimer. We investigated interaction characteristics of Tpm γγ-homodimer and γβ-heterodimer with actin filaments and Ca2+-regulation of actin-myosin interaction on myosin from fast and slow skeletal muscles. The results showed that complexes formed by γγ-Tpm and γβ-Tpm with F-actin are more stable than those with αα-Tpm and αβ-Tpm. The maximum sliding speed of regulated thin filaments with either γγ-Tpm or γβ-Tpm moving over skeletal myosin was significantly less than that of the filaments with αα-Tpm or αβ-Tpm. The results indicate that isoforms of Tpm along with isoforms of myosin determine of functional properties of skeletal muscles and support an idea on the combined expression of myosin and Tpm isoforms.

Characterization and Functional Analysis of Polyadenylation Sites in Fast and Slow Muscles.

Many increasing documents have proved that alternative polyadenylation (APA) events with different polyadenylation sites (PAS) contribute to posttranscriptional regulation. However, little is known about the detailed molecular features of PASs and its role in porcine fast and slow skeletal muscles through microRNAs (miRNAs) and RNA binding proteins (RBPs). In this study, we combined single-molecule real-time sequencing and Illumina RNA-seq datasets to comprehensively analyze polyadenylation in pigs. We identified a total of 10,334 PASs, of which 8734 were characterized by reference genome annotation. 32.86% of PAS-associated genes were determined to have more than one PAS. Further analysis demonstrated that tissue-specific PASs between fast and slow muscles were enriched in skeletal muscle development pathways. In addition, we obtained 1407 target genes regulated by APA events through potential binding 69 miRNAs and 28 RBPs in variable 3′ UTR regions and some are involved in myofiber transformation. Furthermore, the de novo motif search confirmed that the most common usage of canonical motif AAUAAA and three types of PASs may be related to the strength of motifs. In summary, our results provide a useful annotation of PASs for pig transcriptome and suggest that APA may serve as a role in fast and slow muscle development under the regulation of miRNAs and RBPs.

Annals of Neurology: Volume 87, Number 4, April 2020

A fluorescence photomicrograph of skeletal muscle from a zebrafish larva in which the slow skeletal muscle troponin T protein was knocked down resulting in a myopathic phenotype characterized by reduced muscle mass and curvature of the body. A novel mutation in the slow skeletal muscle troponin T (TNNT1) gene causes a type of congenital core‐rod myopathy in humans. Actin is immunostained green and nuclei are stained blue. The muscle fibers in the affected fish model are disrupted and irregular in appearance compared to normal control fish (see Pellerin et al., pp. 568–583). Injecting the wild type human TNNT1 mRNA corrected both the morphological abnormalities and the body curvature.

Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice

Investigating protein abundance profiles is important to understand the differences in the slow and fast skeletal muscle characteristics. The profiles in soleus (Sol) and extensor digitorum longus (EDL) muscles in mice exposed to 1 g or 3 g for 28 d were compared. The biological implications of the profiles revealed that hypergravity exposure activated a larger number of pathways involved in protein synthesis in Sol. In contrast, the inactivation of signalling pathways involved in oxidative phosphorylation were conspicuous in EDL. These results suggested that the reactivity of molecular pathways in Sol and EDL differed. Additionally, the levels of spermidine synthase and spermidine, an important polyamine for cell growth, increased in both muscles following hypergravity exposure, whereas the level of spermine oxidase (SMOX) increased in EDL alone. The SMOX level was negatively correlated with spermine content, which is involved in muscle atrophy, and was higher in EDL than Sol, even in the 1 g group. These results indicated that the contribution of SMOX to the regulation of spermidine and spermine contents in Sol and EDL differed. However, contrary to expectations, the difference in the SMOX level did not have a significant impact on the growth of these muscles following hypergravity exposure. SignificanceThe skeletal muscle-specific protein abundance profiles result in differences in the characteristics of slow and fast skeletal muscles. We investigated differences in the profiles in mouse slow-twitch Sol and fast-twitch EDL muscles following 28-d of 1 g and 3 g exposure by LC-MS/MS analysis and label-free quantitation. A two-step solubilisation of the skeletal muscle proteins increased the coverage of proteins identified by LC-MS/MS analysis. Additionally, this method reduced the complexity of samples more easily than protein or peptide fractionation by SDS-PAGE and offline HPLC while maintaining the high operability of samples and was reproducible. A larger number of hypergravity-responsive proteins as well as a prominent increase in the wet weights was observed in Sol than EDL muscles. The biological implications of the difference in the protein abundance profiles in 1 g and 3 g groups revealed that the reactivity of each molecular pathway in Sol and EDL muscles to hypergravity exposure differed significantly. In addition, we found that the biosynthetic and interconversion pathway of polyamines, essential factors for cell growth and survival in mammals, was responsive to hypergravity exposure; spermidine and spermine contents in Sol and EDL muscles were regulated by different mechanisms even in the 1 g group. However, our results indicated that the difference in the mechanism regulating polyamine contents is unlikely to have a significant effect on the differences in Sol and EDL muscle growth following hypergravity exposure.

Novel Recessive TNNT1 Congenital Core-Rod Myopathy in French Canadians.

Recessive null variants of the slow skeletal muscle troponin T1 (TNNT1) gene are a rare cause of nemaline myopathy that is fatal in infancy due to respiratory insufficiency. Muscle biopsy shows rods and fiber type disproportion. We report on 4 French Canadians with a novel form of recessive congenital TNNT1 core-rod myopathy. Patients underwent full clinical characterization, lower limb magnetic resonance imaging (MRI), muscle biopsy, and genetic testing. A zebrafish loss-of-function model using morpholinos was created to assess the pathogenicity of the identified variant. Wild-type or mutated human TNNT1 mRNAs were coinjected with morpholinos to assess their abilities to rescue the morphant phenotype. Three adults and 1 child shared a novel missense homozygous variant in the TNNT1 gene (NM_003283.6: c.287T > C; p.Leu96Pro). They developed from childhood very slowly progressive limb-girdle weakness with rigid spine and disabling contractures. They suffered from restrictive lung disease requiring noninvasive mechanical ventilation in 3 patients, as well as recurrent episodes of rhabdomyolysis triggered by infections, which were relieved by dantrolene in 1 patient. Older patients remained ambulatory into their 60s. MRI of the leg muscles showed fibrofatty infiltration predominating in the posterior thigh and the deep posterior leg compartments. Muscle biopsies showed multiminicores and lobulated fibers, rods in half the patients, and no fiber type disproportion. Wild-type TNNT1 mRNA rescued the zebrafish morphants, but mutant transcripts failed to do so. This study expands the phenotypic spectrum of TNNT1 myopathy and provides functional evidence for the pathogenicity of the newly identified missense mutation. ANN NEUROL 2020;87:568-583.

Large increases in plasma fast skeletal muscle troponin I after whole-body eccentric exercises

ObjectivesIt has been reported that plasma fast skeletal muscle troponin I (fsTnI) but not slow skeletal muscle troponin I (ssTnI) increases after a bout of eccentric exercise of the elbow flexors. The present study compared the first and second bouts of whole-body eccentric exercises for changes in plasma fsTnI and ssTnI concentrations. DesignObservational study in an experimental group. MethodsFifteen sedentary men (20–25 y) performed nine eccentric exercises targeting arm, leg and trunk muscles, and repeated them two weeks later. Blood samples were taken before and for five days following each bout, and plasma ssTnI and fsTnl concentrations were measured by enzyme-linked immunosorbent assays. Their changes were compared between bouts and their relationships to plasma CK activity and myoglobin concentrations were analysed. ResultsPlasma fsTnI concentration increased after the first bout and peaked at 4 days post-exercise (2152–40,295 ng/mL), but no significant increases were evident after the second bout. Plasma ssTnI concentration did not change significantly from the baseline (<0.08 ng/mL) after either bout. Peak plasma fsTnI concentration was significantly (p < 0.005) correlated with peak plasma CK activity (peak: 23,238–207,304 IU/L, r = 0.727) and myoglobin concentration (1047–3936 μg/L, r = 0.625) after the first bout. ConclusionsThese results suggest that plasma TnI concentrations are more specific biomarker of muscle damage than plasma CK activity and myoglobin concentration. It seems that the whole-body eccentric exercises induced damage preferentially to fast-twitch muscle fibres, and increases in plasma CK activity and myoglobin concentration after eccentric exercise may reflect fast-twitch muscle fibre damage.

Antioxidant Effect of Sechium edule (Chayote) Supplement Intake in Older Adults with Metabolic Syndrome: An Exploratory Study

Chlorpyrifos (CPF) is a toxic organophosphate commonly used worldwide. Its residues are being detected in different environmental matrixes and hence in the food chain. Repeated CPF exposure might pose health risk for the general population on long term. This data article contains the data of contractility impairment further to dietary exposure to CPF on a hind limb skeletal muscle; soleus, a typical slow twitch skeletal muscle. Thirty adult male rats Sprague Dawley are divided into three groups receiving the following daily diet for 6 weeks: Group 1 (vehicle), Group 2: CPF1 (CPF 1mg/kg/day) and Group 3: CPF5 (CPF 5 mg/kg/day). Soleus twitch tension and fatigability index are determined at the end of the treatment. The activity of acteylcholinesterase enzyme is assessed in the tissues homogenate. Additionally, we examined the expression levels of ryanodine type 1 receptor (RyR1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 1 (Atp2a1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2 (Atp2a2) and nicotinic acetylcholine receptor (nAChR) in CPF-exposed skeletal muscle tissue using quantitative real time polymerase chain reaction.CPF exposure at two different doses induced an increase in twitch contraction in soleus muscle along with an increase in fatigability index. These increases are accompanied by low level of acetylcholinesterase enzyme activity as well as modification in genes level expression of nAChR, RyR1, Atp2a1 and Atp2a2 involved in contractility.

Effect of External Calcium and other Divalent Cations on K+ Contractures in Denervated Slow Skeletal Muscle Fibers of the Frog

The influence of Ca2+ and other divalent cations on contractile responses of slow skeletal muscle fibers of the frog (Rana pipiens) under conditions of chronic denervation was investigated.Isometric tension was recorded from slow bundles of normal and denervated cruralis muscle in normal solution and in solutions with free calcium concentration solution or in solutions where other divalent cations (Sr2+, Ni2+, Co2+ or Mn2+) substituted for calcium. In the second week after nerve section, in Ca2+-free solutions, we observed that contractures (evoked from 40 to 80 mM-K+) of non-denervated muscles showed significantly higher tensions (p&lt;0.05), than those from denervated bundles. Likewise, in solutions where calcium was substituted by all divalent cations tested, with exception of Mn2+, the denervated bundles displayed lower tension than non-denervated, also in the second week of denervation. In this case, the Ca2+ substitution by Sr2+ caused the higher decrease in tension, followed by Co2+ and Ni2+, which were different to non-denervated bundles, as the lowest tension was developed by Mn2+, followed by Co2+, and then Ni2+ and Sr2+. After the third week, we observed a recovery in tension. These results suggest that denervation altering the binding capacity to divalent cations of the voltage sensor.

CARDIAC TROPONIN T MEDIATED AUTOIMMUNE RESPONSE AND ITS ROLE IN SKELETAL MUSCLE AGING

Cardiac troponin T (cTnT), a key component of contractile machinery essential for muscle contraction, is also expressed in skeletal muscle under certain conditions (e.g. neuromuscular diseases and aging). We have reported that skeletal muscle cTnT regulates neuromuscular junction denervation preferentially in fast skeletal muscle of old mice. Here, we further report that cTnT is also enriched within some myofibers, and/or along microvascular walls in old mice fast skeletal muscle. Strikingly, immunoglobulin G (IgG), together with markers of complement system activation, cell death (necroptosis or apoptosis), and macrophage infiltration, were all found to be co-localized with cTnT and IgG in those areas. In addition, elevated cTnT and IgG are associated with lower dystrophin expression on muscle fiber membrane, lower muscle capillary density, and reduced muscle performance (wire hanging test). Using purified recombinant TnT proteins, we confirmed that only cTnT, but not slow or fast skeletal muscle TnT1 or TnT3, was detected by immunoblot using sera from old (but not young) mice with pre-determined elevated cTnT and IgG in their skeletal muscle, indicating the existence of anti-cTnT autoantibodies in sera (previously found in human blood) and skeletal muscle of old mice. Immunoblotting further revealed that the age related changes in skeletaI muscle cTnT and IgG are more prominent in fast skeletal muscle than in slow. Importantly, elevated cTnT and IgG were also detected in skeletal muscles from 4 older adults (65-70 yrs, IMFIT). Our finding suggests a novel autoimmune mechanism mediated by cTnT that underlies age related skeletal muscle abnormalities and dysfunction.

Slow skeletal muscle troponin T, titin and myosin light chain 3 are candidate prognostic biomarkers for Ewing's sarcoma.

Ewing's sarcoma (ES) is a common malignant bone tumor in children and adolescents. Although great efforts have been made to understand the pathogenesis and development of ES, the underlying molecular mechanism remains unclear. The present study aimed to identify new key genes as potential biomarkers for the diagnosis, targeted therapy or prognosis of ES. mRNA expression profile chip data sets GSE17674, GSE17679 and GSE45544 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were screened using the R software limma package, and functional and pathway enrichment analyses were performed using the enrichplot package and GSEA software. The NetworkAnalyst online tool, as well as Cytoscape and its plug-ins cytoHubba and NetworkAnalyzer, were used to construct a protein-protein interaction network (PPI) and conduct module analysis to screen key (hub) genes. LABSO COX regression and overall survival (OS) analysis of the Hub genes were performed. A total of 211 DEGs were obtained by integrating and analyzing the three data sets. The functions and pathways of the DEGs were mainly associated with the regulation of small-molecule metabolic processes, cofactor-binding, amino acid, proteasome and ribosome biosynthesis in eukaryotes, as well as the Rac1, cell cycle and P53 signaling pathways. A total of one important module and 20 hub genes were screened from the PPI network using the Maximum Correlation Criteria algorithm of cytoHubba. LASSO COX regression results revealed that titin (TTN), fast skeletal muscle troponin T, skeletal muscle actin α-actin, nebulin, troponin C type 2 (fast), myosin light-chain 3 (MYL3), slow skeletal muscle troponin T (TNNT1), myosin-binding protein C1 slow-type, tropomyosin 3 and myosin heavy-chain 7 were associated with prognosis in patients with ES. The Kaplan-Meier curves demonstrated that high mRNA expression levels of TNNT1 (P<0.001), TTN (P=0.049), titin-cap (P=0.04), tropomodulin 1 (P=0.011), troponin I2 fast skeletal type (P=0.021) and MYL3 (P=0.017) were associated with poor OS in patients with ES. In conclusion, the DEGs identified in the present study may be key genes in the pathogenesis of ES, three of which, namely TNNT1, TTN and MYL3, may be potential prognostic biomarkers for ES.

PP-115 - Extended half-life product (rFVIIIFC) for treatment of children with hemophilia A: preliminary post-switch experience from a tertiary care center

Chlorpyrifos (CPF) is a toxic organophosphate commonly used worldwide. Its residues are being detected in different environmental matrixes and hence in the food chain. Repeated CPF exposure might pose health risk for the general population on long term. This data article contains the data of contractility impairment further to dietary exposure to CPF on a hind limb skeletal muscle; soleus, a typical slow twitch skeletal muscle. Thirty adult male rats Sprague Dawley are divided into three groups receiving the following daily diet for 6 weeks: Group 1 (vehicle), Group 2: CPF1 (CPF 1mg/kg/day) and Group 3: CPF5 (CPF 5 mg/kg/day). Soleus twitch tension and fatigability index are determined at the end of the treatment. The activity of acteylcholinesterase enzyme is assessed in the tissues homogenate. Additionally, we examined the expression levels of ryanodine type 1 receptor (RyR1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 1 (Atp2a1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2 (Atp2a2) and nicotinic acetylcholine receptor (nAChR) in CPF-exposed skeletal muscle tissue using quantitative real time polymerase chain reaction.CPF exposure at two different doses induced an increase in twitch contraction in soleus muscle along with an increase in fatigability index. These increases are accompanied by low level of acetylcholinesterase enzyme activity as well as modification in genes level expression of nAChR, RyR1, Atp2a1 and Atp2a2 involved in contractility.