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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Myogenic differentiation is triggered, among other situations, in response to muscle damage for regenerative purposes. It has been shown that during myogenic differentiation, myotubes release ...extracellular vesicles (EVs) which participate in the signalling pattern of the microenvironment. Here we investigated whether EVs released by myotubes exposed or not to mild oxidative stress modulate the behaviour of targeted differentiating myoblasts and macrophages to promote myogenesis. We found that EVs released by oxidatively challenged myotubes (H₂O₂-EVs) are characterized by an increased loading of nucleic acids, mainly DNA. In addition, incubation of myoblasts with H₂O₂-EVs resulted in a significant decrease of myotube diameter, myogenin mRNA levels and myosin heavy chain expression along with an upregulation of proliferating cell nuclear antigen: these effects collectively lead to an increase of recipient myoblast proliferation. Notably, the EVs from untreated myotubes induced an opposite trend in myoblasts, that is, a slight pro-differentiation effect. Finally, H₂O₂-EVs were capable of eliciting an increased interleukin 6 mRNA expression in RAW264.7 macrophages. Notably, this is the first demonstration that myotubes communicate with surrounding macrophages via EV release. Collectively, the data reported herein suggest that myotubes, depending on their conditions, release EVs carrying differential signals which could contribute to finely and coherently orchestrate the muscle regeneration process.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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
,
and
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The effects of different storage treatments on the most common edible truffle species, such as Tuber magnatum and Tuber borchii (white truffles), Tuber melanosporum and Tuber aestivum (black ...truffles), were analysed. Biochemical and microbiological profiles were monitored, in order to evaluate possible alterations during truffle preservation. After harvesting, some fresh samples were kept at 4°C for 30days, other samples were frozen at −20°C for one month, thawed and preserved at 4°C; the remainder were autoclaved.
The biochemical parameters studied were sugar and protein content, the activity of some enzymes involved in the central metabolism of the fungi and the electrophoretic pattern of soluble proteins. Total mesophilic bacteria were also counted. The results obtained showed that the storage at 4°C is the treatment that best preserves the biochemical and microbiological characteristics of fresh truffles. Black truffles were more resistant to biochemical spoilage than the white ones, while T. magnatum was the most resistant to microbial spoilage.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Growing evidence points to the effectiveness of flywheel (FW) based iso-inertial resistance training in improving physical performance capacities. However, molecular adaptations induced by FW ...exercises are largely unknown. Eight resistance-trained men performed 5 sets of 10 maximal squats on a FW device. Muscle biopsies (fine needle aspiration technique) and blood samples were collected before (t0), and 2 h (t1) after FW exercise. Blood samples were additionally drawn after 24 h (t2) and 48 h (t3). Paired samples
-tests revealed significant increases, at t1, of mRNA expression of the genes involved in inflammation, in both muscle (
α
) and peripheral blood mononuclear cells (
α
). Circulating extracellular vesicles (EVs) and EV-encapsulated miRNA levels (miR-206, miR-146a) significantly increased at t1 as well. Conversely, muscle mRNA level of genes associated with muscle growth/remodeling (
) decreased at t1. One-way repeated measure ANOVAs, with Bonferroni corrected
pairwise comparisons, revealed significant increases in plasma concentrations of IL-6 (t1; t2; t3) and muscle creatine kinase (t1; t2), while IGF-1 significantly increased at t2 only. Our findings show that, even in experienced resistance trained individuals, a single FW training session modifies local and systemic markers involved in late structural remodeling and functional adaptation of skeletal muscle.
•Among potential sugar transporters annotated in T. melanosporum genome, three were characterized.•TmelHXT1, Tmel2281 and Tmel131 are properly located in S. cerevisiae plasma membrane.•TmelHXT1 and ...Tmel2281 are abundant in ectomycorrhiza and function as hexose transporters.•Tmel131 is expressed in fruiting body for a probable saprophytic strategy in maturation.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
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
μM) over
d-fructose (
K
m
=
16
±
5
mM) 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 3
mM 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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.
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