Regular football training down-regulates miR-1303 muscle expression in veterans.

PURPOSE: Regular exercise affects the expression of several genes, proteins and microRNAs (miRNAs) in time- and intensity-dependent manner promoting longevity. We previously identified from GeneChip Array analysis several differentially expressed genes and miRNAs in muscle from veteran football players (VPG) compared to active untrained elderly subjects (CG); here we focussed on miRNA-1303 (miR-1303). The aims of the present research were: to analyse the effects of football training on the expression of miR-1303 and to identify its putative target involved in the longevity pathways in skeletal muscle from VPG compared to CG. METHODS: RNA samples from 12 VPG and 12 CG muscle biopsies were used to validate miR-1303 expression. Crossing four different bioinformatic algorithms, we identified 16 putative targets of miR-1303; from these, BAG-2, KLHL7 and KBTBD6 were chosen for further validation by Western blot analysis in LHCN-M2 human myoblasts transiently transfected with miR-1303. RESULTS: Football training down-regulates miR-1303 expression in muscle from VPG compared to CG and the expression of BAG-2, a chaperon protein involved in the autophagy pathway, inversely correlated to overexpression of miR-1303 in a time-dependent manner, indicating that it is a miR-1303 potential target. CONCLUSIONS: This is the first report, to our knowledge, describing miR-1303 regulation in skeletal muscle by football training and the identification of a target protein, BAG-2, involved in the autophagy pathway. This result contributes to the enlargement of knowledge on the molecular mechanisms linking football training, autophagy and longevity.

Serum from differently exercised subjects induces myogenic differentiation in LHCN-M2 human myoblasts.

Myogenesis is the formation of muscle tissue from muscle precursor cells. Physical exercise induces satellite cell activation in muscle. Currently, C2C12 murine myoblast cells are used to study myogenic differentiation. Herein, we evaluated whether human LHCN-M2 myoblasts can differentiate into mature myotubes and express early (myotube formation, creatine kinase activity and myogenin) and late (MyHC-ß) muscle-specific markers when cultured in differentiation medium (DM) for 2, 4 and 7 days. We demonstrate that treatment of LHCN-M2 cells with DM supplemented with 0.5% serum from long-term (3 years) differently exercised subjects for 4 days induced myotube formation and significantly increased the early (creatine kinase activity and myogenin) and late (MyHC-ß expression) differentiation markers versus cells treated with serum from untrained subjects. Interestingly, serum from aerobic exercised subjects (swimming) had a greater positive effect on late-differentiation marker (MyHC-ß) expression than serum from anaerobic (body building) or from mixed exercised (soccer and volleyball) subjects. Moreover, p62and anti-apoptotic Bcl-2 protein expression was lower in LHCN-M2 cells cultured with human sera from differently exercised subjectst han in cells cultured with DM. In conclusion, LHCN-M2 human myoblasts represent a species-specific system with which to study human myogenic differentiation induced by serum from differently exercised subjects.

Effect of lifelong football training on the expression of muscle molecular markers involved in healthy longevity.

PURPOSE: We investigated whether lifelong football training affects the expression of healthy longevity-related muscle molecular markers. METHODS: Biopsies were collected from the vastus lateralis muscle of 10 lifelong football-trained men (68.2 ± 3.0 years) and of 10 active untrained healthy men (66.7 ± 1.3 years). Gene and protein expression was measured by RTqPCR on RNA and by western blotting on protein extracts from muscle biopsies, respectively. RESULTS: The expression of AMPKα1/α2, NAMPT, TFAM and PGC1α, which are markers of oxidative metabolism, and MyHC ß isoform expression was higher in the muscle of football-trained men vs untrained men. Also citrate synthase activity was higher in trained than in untrained men (109.3 ± 9.2 vs 75.1 ± 9.2 mU/mg). These findings were associated with a healthier body composition in trained than in untrained men [body weight: 78.2 ± 6.5 vs 91.2 ± 11.2 kg; body mass index BMI: 24.4 ± 1.6 vs 28.8 ± 4.0 kg m-2; fat%: 22.6 ± 8.0 vs 31.4 ± 5.0%)] and with a higher maximal oxygen uptake (VO2max: 34.7 ± 3.8 vs 27.3 ± 4.0 ml/min/kg). Also the expression of proteins involved in DNA repair and in senescence suppression (Erk1/2, Akt and FoxM1) was higher in trained than in untrained men. At BMI- and age-adjusted multiple linear regression analysis, fat percentage was independently associated with Akt protein expression, and VO2max was independently associated with TFAM mRNA and with Erk1/2 protein expression. CONCLUSIONS: Lifelong football training increases the expression of key markers involved in muscle oxidative metabolism, and in the DNA repair and senescence suppression pathways, thus providing the molecular basis for healthy longevity.

Differentially expressed and activated proteins associated with non small cell lung cancer tissues.

BACKGROUND: Lung cancer is a leading cause of mortality. The most common cancer subtype, non small cell lung cancer (NSCLC), accounts for 85-90% all cases and is mainly caused by environmental and genetic factors. Mechanisms involved in lung carcinogenesis include deregulation of several kinases and molecular pathways affecting cell proliferation, apoptosis and differentiation. Despite advances in lung cancer detection, diagnosis and staging, survival rate still remains poor and novel biomarkers for both diagnosis and therapy need to be identified. In the present study, we have explored the potential of novel specific biomarkers in the diagnosis of NSCLC, and the over-expression/activation of several kinases involved in disease development and progression. METHOD: Lung tumor tissue specimens and adjacent cancer-free tissues from 8 NSCLC patients undergoing surgery were collected. The differential activation status of ERK1/2, AKT and IKBα/NF-κß was analyzed. Subsequently, protein expression profile of NSCLC vs normal surrounding tissue was compared by a proteomic approach using LC-MS MS. Subsequently, MS/MS outputs were analyzed by the Protein Discoverer platform for label-free quantitation analysis. Finally, results were confirmed by western blotting analysis. RESULTS: This study confirms the involvement of ERK1/2, AKT, IKBα and NF-κß proteins in NSCLC demonstrating a significant over-activation of all tested proteins. Furthermore, we found significant differential expression of 20 proteins (Rsc ≥ 1.50 or ≤ -1.50) of which 7 are under-expressed and 13 over-expressed in NSCLC lung tissues. Finally, we validated, by western blotting, the two most under-expressed NSCLC tissue proteins, carbonic anhydrase I and II isoforms. CONCLUSION: Our data further support the possibility of developing both diagnostic tests and innovative targeted therapy in NSCLC. In addition to selective inhibitors of ERK1/2, AKT, IKBα and NF-κß, as therapeutic options, our data, for the first time, indicates carbonic anhydrase I and II as attractive targets for development of diagnostic tools enabling selection of patients for a more specific therapy in NSCLC.

DHT and IGF-1 in peripheral blood lymphocytes: new markers for the biological passport of athletes.

We performed a pilot study using human peripheral blood lymphocytes (PBL) as a novel system to identify new biomarkers of dihydrotestosterone (DHT) and insulin-like growth factor-1 (IGF-1) abuse in sport. First, to obtain a gene signature, we treated cultures of lymphocytes from sedentary males with three doses of 0.237 microg/ml DHT, each of which is 80-fold the physiological concentration in young adult male serum, at days 0, 2 and 4, or with a single dose of 1.25 microg/ml IGF-1, which is 5-fold the physiological concentration in young adult male serum. We then used the Human Genome U133 Plus 2.0 microarray to identify a gene signature related to DHT or IGF-1 administration. Gene expression was evaluated after 7 and 21 days of DHT treatment, and after 24 h, 72 h and 7 days of IGF-1 treatment. Microarray analysis yielded a list of genes whose expression was altered after DHT or IGF-1 treatment. Among these we selected the genes that are most representative of the pathways associated with skeletal and muscular disorders using the IPA bioinformatics tool. We identified six (IDO1, CXCL13, CCL1, GZMB, VDR and IL2RA) and two (FN1 and RAB31) genes that were up-regulated in lymphocytes from sedentary subjects after 7 days of DHT and IGF-1 treatment, respectively. The expression of these genes in lymphocytes from differently trained athletes was either down-regulated or similar to that in lymphocytes from sedentary subjects. This finding suggests that up-regulation was due to the drug and not to physical exercise. In conclusion, we demonstrate that PBL can be useful in anti-doping checks, and we describe new biomarkers of DHT and IGF-1 abuse which can be included in the Athlete's Biological Passport.

Indole-3-acetic acid regulates the central metabolic pathways in Escherichia coli.

The physiological changes induced by indoleacetic acid (IAA) treatment were investigated in the totally sequenced Escherichia coli K-12 MG1655. DNA macroarrays were used to measure the mRNA levels for all the 4290 E. coli protein-coding genes; 50 genes (1.1 %) exhibited significantly different expression profiles. In particular, genes involved in the tricarboxylic acid cycle, the glyoxylate shunt and amino acid biosynthesis (leucine, isoleucine, valine and proline) were up-regulated, whereas the fermentative adhE gene was down-regulated. To confirm the indications obtained from the macroarray analysis the activity of 34 enzymes involved in central metabolism was measured; this showed an activation of the tricarboxylic acid cycle and the glyoxylate shunt. The malic enzyme, involved in the production of pyruvate, and pyruvate dehydrogenase, required for the channelling of pyruvate into acetyl-CoA, were also induced in IAA-treated cells. Moreover, it was shown that the enhanced production of acetyl-CoA and the decrease of NADH/NAD+ ratio are connected with the molecular process of the IAA response. The results demonstrate that IAA treatment is a stimulus capable of inducing changes in gene expression, enzyme activity and metabolite level involved in central metabolic pathways in E. coli.

Indole-3-acetic acid improves Escherichia coli's defences to stress.

Indole-3-acetic acid (IAA) is a ubiquitous molecule playing regulatory roles in many living organisms. To elucidate the physiological changes induced by IAA treatment, we used Escherichia coli K-12 as a model system. By microarray analysis we found that 16 genes showed an altered expression level in IAA-treated cells. One-third of these genes encode cell envelope components, or proteins involved in bacterial adaptation to unfavourable environmental conditions. We thus investigated the effect of IAA treatment on some of the structural components of the envelope that may be involved in cellular response to stresses. This showed that IAA-treated cells had increased the production of trehalose, lipopolysaccharide (LPS), exopolysaccharide (EPS) and biofilm. We demonstrated further that IAA triggers an increased tolerance to several stress conditions (heat and cold shock, UV-irradiation, osmotic and acid shock and oxidative stress) and different toxic compounds (antibiotics, detergents and dyes) and this correlates with higher levels of the heat shock protein DnaK. We suggest that IAA triggers an increased level of alert and protection against external adverse conditions by coordinately enhancing different cellular defence systems.