Integrated procedures for accelerating, deepening, and leading genetic inquiry: A first application on human muscle secretome.

PURPOSE: Beyond classical procedures, bioinformatic-assisted approaches and computational biology offer unprecedented opportunities for scholars. However, these amazing possibilities still need epistemological criticism, as well as standardized procedures. Especially those topics with a huge body of data may benefit from data science (DS)-assisted methods. Therefore, the current study dealt with the combined expert-assisted and DS-assisted approaches to address the broad field of muscle secretome. We aimed to apply DS tools to fix the literature research, suggest investigation targets with a data-driven approach, predict possible scenarios, and define a workflow. METHODS: Recognized scholars with expertise on myokines were invited to provide a list of the most important myokines. GeneRecommender, GeneMANIA, HumanNet, and STRING were selected as DS tools. Networks were built on STRING and GeneMANIA. The outcomes of DS tools included the top 5 recommendations. Each expert-led discussion has been then integrated with an DS-led approach to provide further perspectives. RESULTS: Among the results, 11 molecules had already been described as bona-fide myokines in literature, and 11 molecules were putative myokines. Most of the myokines and the putative myokines recommended by the DS tools were described as present in the cargo of extracellular vesicles. CONCLUSIONS: Including both supervised and unsupervised learning methods, as well as encompassing algorithms focused on both protein interaction and gene represent a comprehensive approach to tackle complex biomedical topics. DS-assisted methods for reviewing existent evidence, recommending targets of interest, and predicting original scenarios are worth exploring as in silico recommendations to be integrated with experts' ideas for optimizing molecular studies.

Chronic brain damage in HIV-infected individuals under antiretroviral therapy is associated with viral reservoirs, sulfatide release, and compromised cell-to-cell communication.

HIV infection has become a chronic and manageable disease due to the effective use of antiretroviral therapies (ART); however, several chronic aging-related comorbidities, including cognitive impairment, remain a major public health issue. However, these mechanisms are unknown. Here, we identified that glial and myeloid viral reservoirs are associated with local myelin damage and the release of several myelin components, including the lipid sulfatide. Soluble sulfatide compromised gap junctional communication and calcium wave coordination, essential for proper cognition. We propose that soluble sulfatide could be a potential biomarker and contributor to white matter compromise observed in HIV-infected individuals even in the current ART era.

Identification, Quantification, and Characterization of HIV-1 Reservoirs in the Human Brain.

The major barrier to cure HIV infection is the early generation and extended survival of HIV reservoirs in the circulation and tissues. Currently, the techniques used to detect and quantify HIV reservoirs are mostly based on blood-based assays; however, it has become evident that viral reservoirs remain in tissues. Our study describes a novel multi-component imaging method (HIV DNA, mRNA, and viral proteins in the same assay) to identify, quantify, and characterize viral reservoirs in tissues and blood products obtained from HIV-infected individuals even when systemic replication is undetectable. In the human brains of HIV-infected individuals under ART, we identified that microglia/macrophages and a small population of astrocytes are the main cells with integrated HIV DNA. Only half of the cells with integrated HIV DNA expressed viral mRNA, and one-third expressed viral proteins. Surprisingly, we identified residual HIV-p24, gp120, nef, vpr, and tat protein expression and accumulation in uninfected cells around HIV-infected cells suggesting local synthesis, secretion, and bystander uptake. In conclusion, our data show that ART reduces the size of the brain's HIV reservoirs; however, local/chronic viral protein secretion still occurs, indicating that the brain is still a major anatomical target to cure HIV infection.

Physiactisome: A New Nanovesicle Drug Containing Heat Shock Protein 60 for Treating Muscle Wasting and Cachexia.

Currently, no commercially available drugs have the ability to reverse cachexia or counteract muscle wasting and the loss of lean mass. Here, we report the methodology used to develop Physiactisome-a conditioned medium released by heat shock protein 60 (Hsp60)-overexpressing C2C12 cell lines enriched with small and large extracellular vesicles. We also present evidence supporting its use in the treatment of cachexia. Briefly, we obtain a nanovesicle-based secretion by genetically modifying C2C12 cell lines with an Hsp60-overexpressing plasmid. The secretion is used to treat naïve C2C12 cell lines. Physiactisome activates the expression of PGC-1α isoform 1, which is directly involved in mitochondrial biogenesis and muscle atrophy suppression, in naïve C2C12 cell lines. Proteomic analyses show Hsp60 localisation inside isolated nanovesicles and the localisation of several apocrine and merocrine molecules, with potential benefits for severe forms of muscle atrophy. Considering that Physiactisome can be easily obtained following tissue biopsy and can be applied to autologous muscle stem cells, we propose a potential nanovesicle-based anti-cachexia drug that could mimic the beneficial effects of exercise. Thus, Physiactisome may improve patient survival and quality of life. Furthermore, the method used to add Hsp60 into nanovesicles can be used to deliver other drugs or active proteins to vesicles.

Cancer-Related Cachexia: The Vicious Circle between Inflammatory Cytokines, Skeletal Muscle, Lipid Metabolism and the Possible Role of Physical Training.

Cachexia is a multifactorial and multi-organ syndrome that is a major cause of morbidity and mortality in late-stage chronic diseases. The main clinical features of cancer-related cachexia are chronic inflammation, wasting of skeletal muscle and adipose tissue, insulin resistance, anorexia, and impaired myogenesis. A multimodal treatment has been suggested to approach the multifactorial genesis of cachexia. In this context, physical exercise has been found to have a general effect on maintaining homeostasis in a healthy life, involving multiple organs and their metabolism. The purpose of this review is to present the evidence for the relationship between inflammatory cytokines, skeletal muscle, and fat metabolism and the potential role of exercise training in breaking the vicious circle of this impaired tissue cross-talk. Due to the wide-ranging effects of exercise training, from the body to the behavior and cognition of the individual, it seems to be able to improve the quality of life in this syndrome. Therefore, studying the molecular effects of physical exercise could provide important information about the interactions between organs and the systemic mediators involved in the overall homeostasis of the body.

The role of Pannexin-1 channels and extracellular ATP in the pathogenesis of the human immunodeficiency virus.

Only recently, the role of large ionic channels such as Pannexin-1 channels and Connexin hemichannels has been implicated in several physiological and pathological conditions, including HIV infection and associated comorbidities. These channels are in a closed stage in healthy conditions, but in pathological conditions including HIV, Pannexin-1 channels and Connexin hemichannels become open. Our data demonstrate that acute and chronic HIV infection induces channel opening (Pannexin and Connexin channels), ATP release into the extracellular space, and subsequent activation of purinergic receptors in immune and non-immune cells. We demonstrated that Pannexin and Connexin channels contribute to HIV infection and replication, the long-term survival of viral reservoirs, and comorbidities such as NeuroHIV. Here, we discuss the available data to support the participation of these channels in the HIV life cycle and the potential therapeutic approach to prevent HIV-associated comorbidities.

HIV infection of astrocytes compromises inter-organelle interactions and inositol phosphate metabolism: A potential mechanism of bystander damage and viral reservoir survival.

HIV-associated neurological dysfunction is observed in more than half of the HIV-infected population, even in the current antiretroviral era. The mechanisms by which HIV mediates CNS dysfunction are not well understood but have been associated with the presence of long-lasting HIV reservoirs. In the CNS, macrophage/microglia and a small population of astrocytes harbor the virus. However, the low number of HIV-infected cells does not correlate with the high degree of damage, suggesting that mechanisms of damage amplification may be involved. Here, we demonstrate that the survival mechanism of HIV-infected cells and the apoptosis of surrounding uninfected cells is regulated by inter-organelle interactions among the mitochondria/Golgi/endoplasmic reticulum system and the associated signaling mediated by IP3 and calcium. We identified that latently HIV-infected astrocytes had elevated intracellular levels of IP3, a master regulator second messenger, which diffuses via gap junctions into neighboring uninfected astrocytes resulting in their apoptosis. In addition, using laser capture microdissection, we confirmed that bystander apoptosis of uninfected astrocytes and the survival of HIV-infected astrocytes were dependent on mitochondrial function, intracellular calcium, and IP3 signaling. Blocking gap junction channels did not prevent an increase in IP3 or inter-organelle dysfunction in HIV-infected cells but reduced the amplification of apoptosis into uninfected neighboring cells. Our data provide a mechanistic explanation for bystander damage induced by surviving infected cells that serve as viral reservoirs and provide potential targets for interventions to reduce the devastating consequences of HIV within the brain.

Sex-based differences after a single bout of exercise on PGC1α isoforms in skeletal muscle: A pilot study.

To date, there are limited and incomplete data on possible sex-based differences in fiber-types of skeletal muscle and their response to physical exercise. Adult healthy male and female mice completed a single bout of endurance exercise to examine the sex-based differences of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), heat shock protein 60 (Hsp60), interleukin 6 (IL-6) expression, as well as the Myosin Heavy Chain (MHC) fiber-type distribution in soleus and extensor digitorum longus (EDL) muscles. Our results showed for the first time that in male soleus, a muscle rich of type IIa fibers, endurance exercise activates specifically genes involved in mitochondrial biogenesis such as PGC1 α1 isoform, Hsp60 and IL-6, whereas the expression of PGC1 α2 and α3 was significantly upregulated in EDL muscle, a fast-twitch skeletal muscle, independently from the gender. Moreover, we found that the acute response of different PGC1α isoforms was muscle and gender dependent. These findings add a new piece to the huge puzzle of muscle response to physical exercise. Given the importance of these genes in the physiological response of the muscle to exercise, we strongly believe that our data could support future research studies to personalize a specific and sex-based exercise training protocol.

Function and Fiber-Type Specific Distribution of Hsp60 and αB-Crystallin in Skeletal Muscles: Role of Physical Exercise.

Skeletal muscle is a plastic and complex tissue, rich in proteins that are subject to continuous rearrangements. Skeletal muscle homeostasis can be affected by different types of stresses, including physical activity, a physiological stressor able to stimulate a robust increase in different heat shock proteins (HSPs). The modulation of these proteins appears to be fundamental in facilitating the cellular remodeling processes related to the phenomenon of training adaptations such as hypertrophy, increased oxidative capacity, and mitochondrial activity. Among the HSPs, a special attention needs to be devoted to Hsp60 and αB-crystallin (CRYAB), proteins constitutively expressed in the skeletal muscle, where their specific features could be highly relevant in understanding the impact of different volumes of training regimes on myofiber types and in explaining the complex picture of exercise-induced mechanical strain and damaging conditions on fiber population. This knowledge could lead to a better personalization of training protocols with an optimal non-harmful workload in populations of individuals with different needs and healthy status. Here, we introduce for the first time to the reader these peculiar HSPs from the perspective of exercise response, highlighting the control of their expression, biological function, and specific distribution within skeletal muscle fiber-types.

High-throughput screening against protein:protein interaction interfaces reveals anti-cancer therapeutics as potent modulators of the voltage-gated Na+ channel complex.

Multiple voltage-gated Na+ (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex. Fibroblast growth factor 14 (FGF14) is a functionally relevant component of the Nav1.6 channel complex, a causative link to spinocerebellar ataxia 27 (SCA27) and an emerging risk factor for neuropsychiatric disorders. Yet, how this protein:channel complex is regulated in the cell is still poorly understood. To search for key cellular pathways upstream of the FGF14:Nav1.6 complex, we have developed, miniaturized and optimized an in-cell assay in 384-well plates by stably reconstituting the FGF14:Nav1.6 complex using the split-luciferase complementation assay. We then conducted a high-throughput screening (HTS) of 267 FDA-approved compounds targeting known mediators of cellular signaling. Of the 65 hits initially detected, 24 were excluded based on counter-screening and cellular toxicity. Based on target analysis, potency and dose-response relationships, 5 compounds were subsequently repurchased for validation and confirmed as hits. Among those, the tyrosine kinase inhibitor lestaurtinib was highest ranked, exhibiting submicromolar inhibition of FGF14:Nav1.6 assembly. While providing evidence for a robust in-cell HTS platform that can be adapted to search for any channelopathy-associated regulatory proteins, these results lay the potential groundwork for repurposing cancer drugs for neuropsychopharmacology.

Novel approaches for glioblastoma treatment: Focus on tumor heterogeneity, treatment resistance, and computational tools.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive primary brain tumor. Currently, the suggested line of action is the surgical resection followed by radiotherapy and treatment with the adjuvant temozolomide, a DNA alkylating agent. However, the ability of tumor cells to deeply infiltrate the surrounding tissue makes complete resection quite impossible, and, in consequence, the probability of tumor recurrence is high, and the prognosis is not positive. GBM is highly heterogeneous and adapts to treatment in most individuals. Nevertheless, these mechanisms of adaption are unknown. RECENT FINDINGS: In this review, we will discuss the recent discoveries in molecular and cellular heterogeneity, mechanisms of therapeutic resistance, and new technological approaches to identify new treatments for GBM. The combination of biology and computer resources allow the use of algorithms to apply artificial intelligence and machine learning approaches to identify potential therapeutic pathways and to identify new drug candidates. CONCLUSION: These new approaches will generate a better understanding of GBM pathogenesis and will result in novel treatments to reduce or block the devastating consequences of brain cancers.

Nandrolone decanoate interferes with testosterone biosynthesis altering blood-testis barrier components.

The aim of this study was to investigate whether nandrolone decanoate (ND) use affects testosterone production and testicular morphology in a model of trained and sedentary mice. A group of mice underwent endurance training while another set led a sedentary lifestyle and were freely mobile within cages. All experimental groups were treated with either ND or peanut oil at different doses for 6 weeks. Testosterone serum levels were measured via liquid chromatography-mass spectrometry. Western blot analysis and quantitative real-time PCR were utilized to determine gene and protein expression levels of the primary enzymes implicated in testosterone biosynthesis and gene expression levels of the blood-testis barrier (BTB) components. Immunohistochemistry and immunofluorescence were conducted for testicular morphological evaluation. The study demonstrated that moderate to high doses of ND induced a diminished serum testosterone level and altered the expression level of the key steroidogenic enzymes involved in testosterone biosynthesis. At the morphological level, ND induced degradation of the BTB by targeting the tight junction protein-1 (TJP1). ND stimulation deregulated metalloproteinase-9, metalloproteinase-2 (MMP-2) and the tissue inhibitor of MMP-2. Moreover, ND administration resulted in a mislocalization of mucin-1. In conclusion, ND abuse induces a decline in testosterone production that is unable to regulate the internalization and redistribution of TJP1 and may induce the deregulation of other BTB constituents via the inhibition of MMP-2. ND may well be considered as both a potential inducer of male infertility and a potential risk factor to a low endogenous bioavailable testosterone.

Effects of Conjugated Linoleic Acid Associated With Endurance Exercise on Muscle Fibres and Peroxisome Proliferator-Activated Receptor γ Coactivator 1 α Isoforms.

Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre-type-specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 µl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6-week experimental period, whereas the placebo groups were gavaged with 35 µl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota-Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2, and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre-type-specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre-type-specific hypertrophy induced by endurance exercise (of types I and IIb muscle fibres). J. Cell. Physiol. 232: 1086-1094, 2017. © 2016 Wiley Periodicals, Inc.