Modulation of the Circulating Extracellular Vesicles in Response to Different Exercise Regimens and Study of Their Inflammatory Effects.

Exercise-released extracellular vesicles (EVs) are emerging as a novel class of exerkines that promotes systemic beneficial effects. However, slight differences in the applied exercise protocols in terms of mode, intensity and duration, as well as the need for standardized protocols for EV isolation, make the comparison of the studies in the literature extremely difficult. This work aims to investigate the EV amount and EV-associated miRNAs released in circulation in response to different physical exercise regimens. Healthy individuals were subjected to different exercise protocols: acute aerobic exercise (AAE) and training (AT), acute maximal aerobic exercise (AMAE) and altitude aerobic training (AAT). We found a tendency for total EVs to increase in the sedentary condition compared to trained participants following AAE. Moreover, the cytofluorimetric analysis showed an increase in CD81+/SGCA+/CD45- EVs in response to AAE. Although a single bout of moderate/maximal exercise did not impact the total EV number, EV-miRNA levels were affected as a result. In detail, EV-associated miR-206, miR-133b and miR-146a were upregulated following AAE, and this trend appeared intensity-dependent. Finally, THP-1 macrophage treatment with exercise-derived EVs induced an increase of the mRNAs encoding for IL-1ß, IL-6 and CD163 using baseline and immediately post-exercise EVs. Still, 1 h post-exercise EVs failed to stimulate a pro-inflammatory program. In conclusion, the reported data provide a better understanding of the release of circulating EVs and their role as mediators of the inflammatory processes associated with exercise.

Current Methods for the Isolation of Urinary Extracellular Vesicles.

Extracellular vesicles (EVs) are small membrane-bound particles released into extracellular space by almost all cell types, and found in body fluids like blood, urine, and saliva. Mounting evidence has demonstrated the clinical potential of EVs as diagnostic and therapeutic tools to analyse physiological/pathological processes due to their ability to transport biomolecules secreted from diverse tissues of an individual.For example, the urinary EVs (uEVs), released from all regions of the kidney's nephron and from other cells that line the urinary tract, retain proteomic and transcriptomic markers specific to their cell of origin representing a valuable tool for kidney disease diagnosis.Despite the numerous efforts in developing suitable methods to separate EVs from biofluids, providing material of high purity and low variability poses a limit to clinical translation.This chapter focuses on advantages and disadvantages of several EV isolation methodologies, and provides examples of uEV isolation protocols based on time, cost, and equipment considerations, as well as the sample requirements for any downstream analyses.

A high number of 'natural' mitochondrial DNA polymorphisms in a symptomatic Brugada syndrome type 1 patient.

Brugada syndrome (BrS) is a rare genetic arrhythmic disorder with a complex model of transmission. At least 20 different genes have been identified as BrS-causal or susceptibility genes. Of these, SCN5A is the most frequently mutated. Coregulation of different mutations or genetic variants, including mitochondrial DNA (mtDNA), may contribute to the clinical phenotype of the disease. In thepresent study, we analysed the mitochondrial genome of a symptomatic BrS type 1 patient to investigate a possible mitochondrial involvement recently found in the arrhytmogenic diseases. No pathogenic mutation was identified; however, a high number of singlenucleotide polymorphisms were found (n=21) and some of them were already been reported in molecular autopsy case for sudden death.The results reported here further support our hypothesis on the potential role of mtDNA polymorphisms in mitochondrial dysfunction, which may represent a risk factor for arrhythmogenic disease.

Signal Exchange through Extracellular Vesicles in Neuromuscular Junction Establishment and Maintenance: From Physiology to Pathology.

Neuromuscular junction (NMJ) formation involves morphological changes both in motor terminals and muscle membrane. The molecular mechanisms leading to NMJ formation and maintenance have not yet been fully elucidated. During the last decade, it has become clear that virtually all cells release different types of extracellular vesicles (EVs), which can be taken up by nearby or distant cells modulating their activity. Initially, EVs were associated to a mechanism involved in the elimination of unwanted material; subsequent evidence demonstrated that exosomes, and more in general EVs, play a key role in intercellular communication by transferring proteins, lipids, DNA and RNA to target cells. Recently, EVs have emerged as potent carriers for Wnt, bone morphogenetic protein, miRNA secretion and extracellular traveling. Convincing evidence demonstrates that presynaptic terminals release exosomes that are taken up by muscle cells, and these exosomes can modulate synaptic plasticity in the recipient muscle cell in vivo. Furthermore, recent data highlighted that EVs could also be a potential cause of neurodegenerative disorders. Indeed, mutant SOD1, TDP-43 and FUS/TLS can be secreted by neural cells packaged into EVs and enter in neighboring neural cells, contributing to the onset and severity of the disease.

The Combination of Physical Exercise with Muscle-Directed Antioxidants to Counteract Sarcopenia: A Biomedical Rationale for Pleiotropic Treatment with Creatine and Coenzyme Q10.

Sarcopenia represents an increasing public health risk due to the rapid aging of the world's population. It is characterized by both low muscle mass and function and is associated with mobility disorders, increased risk of falls and fractures, loss of independence, disabilities, and increased risk of death. Despite the urgency of the problem, the development of treatments for sarcopenia has lagged. Increased reactive oxygen species (ROS) production and decreased antioxidant (AO) defences seem to be important factors contributing to muscle impairment. Studies have been conducted to verify whether physical exercise and/or AOs could prevent and/or delay sarcopenia through a normalization of the etiologically relevant ROS imbalance. Despite the strong rationale, the results obtained were contradictory, particularly with regard to the effects of the tested AOs. A possible explanation might be that not all the agents included in the general heading of "AOs" could fulfill the requisites to counteract the complex series of events causing/accelerating sarcopenia: the combination of the muscle-directed antioxidants creatine and coenzyme Q10 with physical exercise as a biomedical rationale for pleiotropic prevention and/or treatment of sarcopenia is discussed.

New insights into the trophic and cytoprotective effects of creatine in in vitro and in vivo models of cell maturation.

A growing body of scientific reports indicates that the role of creatine (Cr) in cellular biochemistry and physiology goes beyond its contribution to cell energy. Indeed Cr has been shown to exert multiple effects promoting a wide range of physiological responses in vitro as well as in vivo. Included in these, Cr promotes in vitro neuron and muscle cell differentiation, viability and survival under normal or adverse conditions; anabolic, protective and pro-differentiative effects have also been observed in vivo. For example Cr has been shown to accelerate in vitro differentiation of cultured C2C12 myoblasts into myotubes, where it also induces a slight but significant hypertrophic effect as compared to unsupplemented cultures; Cr also prevents the anti-differentiation effects caused by oxidative stress in the same cells. In trained adults, Cr increases the mRNA expression of relevant myogemic factors, protein synthesis, muscle strength and size, in cooperation with physical exercise. As to neurons and central nervous system, Cr favors the electrophysiological maturation of chick neuroblasts in vitro and protects them from oxidative stress-caused killing; similarly, Cr promotes the survival and differentiation of GABA-ergic neurons in fetal spinal cord cultures in vitro; in vivo, maternal Cr supplementation promotes the morpho-functional development of hippocampal neurons in rat offsprings. This article, which presents also some new experimental data, focuses on the trophic, pro-survival and pro-differentiation effects of Cr and examines the ensuing preventive and therapeutic potential in pathological muscle and brain conditions.

Mutational analysis of mitochondrial DNA in Brugada syndrome.

BACKGROUND: Brugada syndrome (BrS) is a primary electrical disease associated with an increased risk of sudden cardiac death due to ventricular fibrillation. This pathology has nuclear heterogeneous genetic origins, and at present, molecular diagnostic tests on nuclear DNA cover only 30% of BrS patients. The aim of this study was to assess the possible involvement of mitochondrial (mt) DNA variants in BrS since their etiological role in several cardiomyopathies has already been described. METHODS AND RESULTS: The whole mt genome of BrS patients was sequenced and analyzed. A specific mtDNA mutation responsible for BrS can be excluded, but BrS patient d-loop was found to be more polymorphic than that of control cases (P=0.003). Moreover, there appears to be an association between patients with the highest number of variants (n>20) and four mt Single Nucleotide Polymorphism (SNPs) (T4216C, A11251G, C15452A, T16126C) and the most severe BrS phenotype (P=0.002). CONCLUSIONS: The high substitution rate found in BrS patient mtDNA is unlikely to be the primary cause of the disease, but it could represent an important cofactor in the manifestation of the BrS phenotype. Evidence suggesting that a specific mtDNA allelic combination and a high number of mtDNA SNPs may be associated with more severe cases of BrS represents the starting point for further cohort studies aiming to test whether this mt genetic condition could be a genetic modulator of the BrS clinical phenotype.

The pleiotropic effect of physical exercise on mitochondrial dynamics in aging skeletal muscle.

Decline in human muscle mass and strength (sarcopenia) is one of the principal hallmarks of the aging process. Regular physical exercise and training programs are certain powerful stimuli to attenuate the physiological skeletal muscle alterations occurring during aging and contribute to promote health and well-being. Although the series of events that led to these muscle adaptations are poorly understood, the mechanisms that regulate these processes involve the "quality" of skeletal muscle mitochondria. Aerobic/endurance exercise helps to maintain and improve cardiovascular fitness and respiratory function, whereas strength/resistance-exercise programs increase muscle strength, power development, and function. Due to the different effect of both exercises in improving mitochondrial content and quality, in terms of biogenesis, dynamics, turnover, and genotype, combined physical activity programs should be individually prescribed to maximize the antiaging effects of exercise.

Sugar transporters in the black truffle Tuber melanosporum: from gene prediction to functional characterization.

In a natural forest ecosystem, ectomycorrhiza formation is a way for soil fungi to obtain carbohydrates from their host plants. However, our knowledge of sugar transporters in ectomycorrhizal ascomycetous fungi is limited. To bridge this gap we used data obtained from the sequenced genome of the ectomycorrhizal fungus Tuber melanosporum Vittad. to search for sugar transporters. Twenty-three potential hexose transporters were found, and three of them (Tmelhxt1, Tmel2281 and Tmel131), differentially expressed during the fungus life cycle, were investigated. The heterologous expression of Tmelhxt1 and Tmel2281 in an hxt-null Saccharomyces cerevisiae strain restores the growth in glucose and fructose. The functional characterization and expression profiles of Tmelhxt1 and Tmel2281 in the symbiotic phase suggest that they are high affinity hexose transporters at the plant-fungus interface. On the contrary, Tmel131 is preferentially expressed in the fruiting body and its inability to restore the S. cerevisiae mutant strain growth led us to hypothesize that it could be involved in the transport of alternative carbon sources important for a hypothetical saprophytic strategy for the complete maturation of the carpophore.

Effect of 300 mT static and 50 Hz 0.1 mT extremely low frequency magnetic fields on Tuber borchii mycelium.

The present work aimed to investigate whether exposure to static magnetic field (SMF) and extremely low frequency magnetic field (ELF-MF) can induce biomolecular changes on Tuber borchii hyphal growth. Tuber borchii mycelium was exposed for 1 h for 3 consecutive days to a SMF of 300 mT or an ELF-MF of 0.1 mT 50 Hz. Gene expression and biochemical analyses were performed. In mycelia exposed to ELF-MF, some genes involved in hyphal growth, investigated using quantitative real-time polymerase chain reaction, were upregulated, and the activity of many glycolytic enzymes was increased. On the contrary, no differences were observed in gene expression after exposure to SMF treatment, and only the activities of glucose 6-phosphate dehydrogenase and hexokinase increased. The data herein presented suggest that the electromagnetic field can act as an environmental factor in promoting hyphal growth and can be used for applicative purposes, such as the set up of new in vitro cultivation techniques.

Gene expression profile in cultured human umbilical vein endothelial cells exposed to a 300 mT static magnetic field.

In a previous investigation we reported that exposure to a moderate (300 mT) static magnetic field (SMF) causes transient DNA damage and promotes mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs). To better understand the response of HUVECs to the 300 mT SMF, a high-quality subtracted cDNA library representative of genes induced in cells after 4 h of static magnetic exposure was constructed. The global gene expression profile showed that several genes were induced after the SMF exposure. The characterized clones are involved in cell metabolism, energy, cell growth/division, transcription, protein synthesis, destination and storage, membrane injury, DNA damage/repair, and oxidative stress response. Quantitative real-time polymerase chain reaction (qRT-PCR) experiments were performed at 4 and 24 h on four selected genes. Their expression profiles suggest that HUVEC's response to SMF exposure is transient. Furthermore, compared to control cells, an up-regulation of several genes involved in cell growth and division was observed. This up-regulation is likely to be the cause of the slight, but significant, increase in cell proliferation at 12 h post-treatment. These results provide additional support to the notion that SMFs may be harmless to human health, and could support the rationale for their possible use in medical treatments.

Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation.

This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α, and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

Effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer.

Surgical resection at any location in the body leads to stress response with cellular and subcellular change, leading to tissue damage. The intestine is extremely sensitive to surgical stress with consequent postoperative complications. It has been suggested that the increase of reactive oxygen species as subcellular changes plays an important role in this process. This article focuses on the effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer. Mitochondrial DNA copy number, mitochondrial common deletion and nuclear and mitochondrial 8-oxo-2'-deoxyguanosine content were measured. Both the colon and rectal tissue were significantly damaged either at the nuclear or mitochondrial level. In particular, mitochondrial DNA was more damaged in rectum than in colon. The present investigation found an association between surgical stress and nuclear and mitochondrial DNA damage, suggesting that surgery may generate an increase in free radicals, which trigger a cascade of molecular changes, including alterations in DNA.

Effects of a 300 mT static magnetic field on human umbilical vein endothelial cells.

This study describes the effects of a static magnetic field (SMF) on cell growth and DNA integrity of human umbilical vein endothelial cells (HUVECs). Fast halo assay was used to investigate nuclear damage; quantitative polymerase chain reaction (QPCR), standard PCR, and real-time PCR were used to evaluate mitochondrial DNA integrity, content, and gene expression. HUVECs were continually exposed to a 300 mT SMF for 4, 24, 48, and 72 h. Compared to control samples (unexposed cultures) the SMF-exposed cells did not show a statistically significant change in their viability. Conversely, the static field was shown to be significant after 4 h of exposure, inducing damage on both the nuclear and mitochondrial levels, reducing mitochondrial content and increasing reactive oxygen species. Twenty-four hours of exposure increased mitochondrial DNA content as well as expression of one of the main genes related to mitochondrial biogenesis. No significant differences between exposed and sham cultures were found after 48 and 72 h of exposure. The results suggest that a 300 mT SMF does not cause permanent DNA damage in HUVECs and stimulates a transient mitochondrial biogenesis.

Morphological and molecular modifications induced by different carbohydrate sources in Tuber borchii.

During the life cycle of mycorrhizal fungi, morphological, genetic and metabolic modifications are induced in the fungus and its symbiotic partner. These changes are influenced by environmental factors: light, gravity, oxygen, temperature, soil type, nutrients, root exudates and the presence of particular bacterial and perhaps fungal and viral populations in the mycorrhizosphere. To determine whether different carbohydrates lead to cell-signalling events and morphofunctional changes in cultured Tuber borchii mycelia, the expression level of genes involved in morphological modifications was investigated using a macroarray technique and real-time RT-PCR. The morphological study showed an increased growth of Tuber mycelia in glucose, while the hyphae were thinner and less branched in sucrose and maltose. This was accompanied by an upregulation of the genes involved in the general cell metabolism, detoxification processes, hyphal growth and cytoskeleton organization. Since glucose is also present in root exudates, the increased expression of these genes might support the hypothesis that glucose can act as a signal for the fungus to indicate the presence of the plant, and to trigger the complex symbiotic process. These mechanisms can lead to morphological modifications, including increased branching of the root which is necessary for the fungus to establish the symbiosis.

Molecular and functional characterization of a Rho GDP dissociation inhibitor in the filamentous fungus Tuber borchii.

BACKGROUND: Small GTPases of the Rho family function as tightly regulated molecular switches that govern important cellular functions in eukaryotes. Several families of regulatory proteins control their activation cycle and subcellular localization. Members of the guanine nucleotide dissociation inhibitor (GDI) family sequester Rho GTPases from the plasma membrane and keep them in an inactive form. RESULTS: We report on the characterization the RhoGDI homolog of Tuber borchii Vittad., an ascomycetous ectomycorrhizal fungus. The Tbgdi gene is present in two copies in the T. borchii genome. The predicted amino acid sequence shows high similarity to other known RhoGDIs. Real time PCR analyses revealed an increased expression of Tbgdi during the phase preparative to the symbiosis instauration, in particular after stimulation with root exudates extracts, that correlates with expression of Tbcdc42. In a translocation assay TbRhoGDI was able to solubilize TbCdc42 from membranes. Surprisingly, TbRhoGDI appeared not to interact with S. cerevisiae Cdc42, precluding the use of yeast as a surrogate model for functional studies. To study the role of TbRhoGDI we performed complementation experiments using a RhoGDI null strain of Dictyostelium discoideum, a model organism where the roles of Rho signaling pathways are well established. For comparison, complementation with mammalian RhoGDI1 and LyGDI was also studied in the null strain. Although interacting with Rac1 isoforms, TbRhoGDI was not able to revert the defects of the D. discoideum RhoGDI null strain, but displayed an additional negative effect on the cAMP-stimulated actin polymerization response. CONCLUSION: T. borchii expresses a functional RhoGDI homolog that appears as an important modulator of cytoskeleton reorganization during polarized apical growth that antecedes symbiosis instauration. The specificity of TbRhoGDI actions was underscored by its inability to elicit a growth defect in S. cerevisiae or to compensate the loss of a D. discoideum RhoGDI. Knowledge of the cell signaling at the basis of cytoskeleton reorganization of ectomycorrhizal fungi is essential for improvements in the production of mycorrhized plant seedlings used in timberland extension programs and fruit body production.

Differential effect of creatine on oxidatively-injured mitochondrial and nuclear DNA.

Creatine is a naturally occurring compound obtained in humans from endogenous production and consumption through the diet. It is used as an ergogenic aid to improve exercise performance and increase fat-free mass. Lately, creatine's positive therapeutic benefits in various oxidative stress-associated diseases have been reported in literature and, more recently, creatine has also been shown to exert direct antioxidant effects. Oxidatively-challenged DNA was analysed to show possible protective effects of creatine. Acellular and cellular studies were carried out. Acellular assays, performed using molecular approaches, showed that creatine protects circular and linear DNA from oxidative attacks. Nuclear and mitochondrial DNAs from oxidatively-injured human umbilical vein endothelial cells were analyzed. Creatine supplementation showed significant genoprotective activity on mitochondrial DNA. This evidence suggests that creatine may play an important role in mitochondrial genome stability in that it could normalize mitochondrial mutagenesis and its functional consequences. Thus, creatine supplementation could be used to prevent or ameliorate diseases related to mitochondrial DNA mutations, and possibly to delay aging.

Identification and characterization of the Tuber borchii D-mannitol dehydrogenase which defines a new subfamily within the polyol-specific medium chain dehydrogenases.

A novel NADP(+)-dependent D-mannitol dehydrogenase and the corresponding gene from the plant symbiotic ascomycete fungus Tuber borchii was identified and characterized. The enzyme, called TbMDH, is a homotetramer with two zinc atoms per subunit. It catalyzed both D-fructose reduction and D-mannitol oxidation, although it showed the highest substrate specificity and catalytic efficiency for D-fructose. Co-factor specificity was restricted to NADP(H) and the reaction proceeded via a sequential ordered Bi Bi mechanism. The carbon responsive transcriptional pattern showed that Tbmdh is up-regulated when mycelia are transferred to a culture medium containing D-mannitol or D-fructose. The phylogenetic analysis showed TbMDH to be the first example of a fungal D-mannitol-2-dehydrogenase belonging to the medium-chain dehydrogenase/reductases (MDRs). The enzyme identified a new group of proteins, most of them annotated in databases as hypothetical zinc-dependent dehydrogenases, forming a distinct subfamily among the polyol dehydrogenase family.

Hexose uptake in the plant symbiotic ascomycete Tuber borchii Vittadini: biochemical features and expression pattern of the transporter TBHXT1.

Here, we report the first evidence of a hexose transporter gene, Tbhxt1, in the ectomycorrhizal ascomycete Tuber borchii Vittadini. The protein encoded by Tbhxt1 functionally complements the hxt-null mutant Saccharomyces cerevisiae EBYVW.4000. TBHXT1 has a strong preference for d-glucose (K(m)=38+/-10 microM) over d-fructose (K(m)=16+/-5mM) and uncoupling experiments indicate that TBHXT1 catalyzes the transport via a proton-symport mechanism. The investigations on the substrate specificity reveal that TBHXT1 also imports d-mannose, and the use of deoxyglucose analogues shows that the hydroxyl groups at C1, C3 and C4 are important for substrate recognition. Tbhxt1 is not regulated by fructose, but it reaches its highest level of expression at 3mM glucose and is repressed by very high glucose concentration. Prolonged carbon starvation condition upregulates Tbhxt1, while its expression remains at basal level in the ectomycorrhizal tissue. The mode of regulation of Tbhxt1 is consistent with its role as a high-affinity d-glucose transporter.

Expression and purification of a Tuber borchii fruitbody-specific protein, TBF-1, from Escherichia coli: generation of polyclonal antibodies.

TBF-1 is a fruitbody-specific protein present in the white truffle species Tuber borchii Vittad. A similar protein has been found only in the closely related species Tuber dryophilum (TDF-1), but not in other truffles. The protein from T. borchii was overexpressed as fusion protein in E. coli and was purified to homogeneity by affinity chromatography. Recombinant protein was used for generating polyclonal antibodies. The antiserum strongly reacted with TBF-1, weakly recognized TDF-1, and did not detect correlate band in the other white truffle species. The high level of expression of this protein in the fruitbody and the specificity of the antibody anti-TBF-1 make it possible to set up a diagnostic tool for detecting these species in natural samples and foodstuffs.