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Metabolic syndrome (MetS), characterized by a set of conditions that include obesity, hypertension, and dyslipidemia, is associated with increased cardiovascular risk. Exercise training (EX) has been reported to improve MetS management, although the underlying metabolic adaptations that drive its benefits remain poorly understood. This work aims to characterize the molecular changes induced by EX in skeletal muscle in MetS, focusing on gastrocnemius metabolic remodelling. 1H NMR metabolomics and molecular assays were employed to assess the metabolic profile of skeletal muscle tissue from lean male ZSF1 rats (CTL), obese sedentary male ZSF1 rats (MetS-SED), and obese male ZF1 rats submitted to 4 weeks of treadmill EX (5 days/week, 60 min/day, 15 m/min) (MetS-EX). EX did not counteract the significant increase of body weight and circulating lipid profile, but had an anti-inflammatory effect and improved exercise capacity. The decreased gastrocnemius mass observed in MetS was paralleled with glycogen degradation into small glucose oligosaccharides, with the release of glucose-1-phosphate, and an increase in glucose-6-phosphate and glucose levels. Moreover, sedentary MetS animals' muscle exhibited lower AMPK expression levels and higher amino acids' metabolism such as glutamine and glutamate, compared to lean animals. In contrast, the EX group showed changes suggesting an increase in fatty acid oxidation and oxidative phosphorylation. Additionally, EX mitigated MetS-induced fiber atrophy and fibrosis in the gastrocnemius muscle. EX had a positive effect on gastrocnemius metabolism by enhancing oxidative metabolism and, consequently, reducing susceptibility to fatigue. These findings reinforce the importance of prescribing EX programs to patients with MetS. • EX had an anti-inflammatory effect in animals with metabolic syndrome (MetS). • EX changed the metabolic profile of gastrocnemius from MetS rats. • EX counteracted the MetS-induced decrease of the amino acids Asp, Glu and Gly. • EX improved fatty acid oxidation and OXPHOS in the muscle of MetS subjects. • Data support the prescription of supervised EX to patients with MetS. [ABSTRACT FROM AUTHOR]

Abstract: Pulmonary arterial hypertension (PAH) is a complex and multifactorial disease characterized by vascular remodeling, vasoconstriction, inflammation and thrombosis. Although the available therapies have resulted in improvements in morbidity and survival, PAH remains a severe and devastating disease with a poor prognosis and a high mortality, justifying the need of novel therapeutic targets. An increasing number of studies have demonstrated that endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts of the pulmonary vessel wall, as well as platelets and inflammatory cells have a role in PAH pathogenesis. This review aims to integrate the interplay among different types of cells, during PAH development and progression, and the impact of current therapies in cellular modulation. The interplay among endothelial cells, smooth muscle cells and fibroblasts present in pulmonary vessels wall, platelets and inflammatory cells is regulated by several mediators produced by these cells, contributing to the pathophysiologic features of PAH. Current therapies are mainly focused in the pulmonary vascular tone and in the endothelial dysfunction. However, once they have not been effective, novel therapies targeting other PAH features, such as inflammation and platelet dysfunction are emerging. Further understanding of the interplay among different vascular cell types involved in PAH development and progression can contribute to find novel therapeutic targets, decreasing PAH mortality and morbidity in the future. [Copyright &y& Elsevier]

Left-right patterning and asymmetric morphogenesis arise from a complex set of molecular and cellular interactions that are particularly dynamic and associated with mechanical forces. How do mechanical forces translate into tissular asymmetries? Are these forces asymmetrical de novo , or do they build up from pre-existing asymmetries? Advances in developmental genetics, live imaging and cell biology have recently shed light on the origins of mechanical forces generated at the cell scale and their implication in asymmetric patterning and morphogenesis is now emerging. Here we ask when and how, molecular asymmetries and mechanical forces contribute to left-right patterning and organ asymmetries. [ABSTRACT FROM AUTHOR]

Summary: Internal organs are asymmetrically positioned inside the body. Embryonic motile cilia play an essential role in this process by generating a directional fluid flow inside the vertebrate left-right organizer. Detailed characterization of how fluid flow dynamics modulates laterality is lacking. We used zebrafish genetics to experimentally generate a range of flow dynamics. By following the development of each embryo, we show that fluid flow in the left-right organizer is asymmetric and provides a good predictor of organ laterality. This was tested in mosaic organizers composed of motile and immotile cilia generated by dnah7 knockdowns. In parallel, we used simulations of fluid dynamics to analyze our experimental data. These revealed that fluid flow generated by 30 or more cilia predicts 90% situs solitus, similar to experimental observations. We conclude that cilia number, dorsal anterior motile cilia clustering, and left flow are critical to situs solitus via robust asymmetric charon expression. [ABSTRACT FROM AUTHOR]

Abstract: Chlamydia trachomatis maintain a conserved plasmid, which is a primary regulator of chromosomal genes, but there is no experimental evidences associating it with the strains’ differential tissue tropism (ocular and genital mucosae, and lymph nodes). We investigated if the number of plasmids per strain correlate with expression profiles of plasmid ORFs and small anti-sense RNAs (sRNAs), and also if these molecular features underlie tropism dissimilarities. We performed absolute and relative qPCR to determine both the plasmid load and expression throughout C. trachomatis development. Our findings suggest that plasmid load (never exceeding 8 copies) is not a function of expression needs and does not reflect tissue tropism. However, for most ORFs, ocular strains presented lower expression than genital or lymphogranuloma venereum (LGV) strains, and ORF6/pgp4 (transcriptional regulator of virulence associated genes) presented the highest mean expression among strains, followed by the virulence factor ORF5/pgp3 (also regulated by ORF6/pgp4). More, the mean expression levels of the sRNA-2 (anti-sense to ORF2/pgp8) were up to 100-fold higher than those of the ORFs, and up to 12-fold higher than that of sRNA-7 (anti-sense to ORF7/pgp5) for the LGV strains. Overall, besides the known regulatory role of C. trachomatis plasmid, its transcriptional dynamics sustains tropism differences. [Copyright &y& Elsevier]

Abstract: In cardiac tissue two mitochondria subpopulations, the subsarcolemmal and the intermyofibrillar mitochondria, present different functional emphasis, although limited information exists about the underlying molecular mechanisms. Our study evidenced higher OXPHOS activity of intermyofibrillar compared to subsarcolemmal mitochondria, paralleled by distinct membrane proteins susceptibility to oxidative damage and not to quantitative differences of OXPHOS composition. Indeed, subsarcolemmal subunits of respiratory chain complexes were more prone to carbonylation while intermyofibrillar mitochondria were more susceptible to nitration. Among membrane protein targets to posttranslational modifications, ATP synthase subunits alpha and beta were notoriously more carbonylated in both subpopulations, although more intensely in subsarcolemmal mitochondria. Our data highlight a localization dependence of cardiac mitochondria OXPHOS activity and susceptibility to posttranslational modifications. [Copyright &y& Elsevier]

RNA Polymerase I (PolI)-mediated transcription of ribosomal RNAs is the limiting factor of ribosomal biogenesis, is often dysregulated in cancer 1 and is a new target for cancer therapy 2,3. Decreased levels of PolI transcription is also associated with cell differentiation 4 suggesting that high levels of PolI transcription are required for stemness. Mammalian cells contain over 200 copies of the ribosomal genes (rDNA) clustered in specific chromosomal regions that aggregate in the nucleolus, where only a proportion acquires an active chromatic configuration allowing PolI transcription to occur. Malignant transformation is associated with an increase in the portion of active rDNA loci in the nucleolus. We showed that during malignant progression of B-cell lymphoma, the rDNA chromatin acquires an open state and rDNA-genome contacts are reorganized, which correlate with the repression of genes involved in B-cell differentiation 5. These data support the intriguing hypothesis that dynamic changes in rDNA chromatin and long-range rDNA-genomic interactions regulate distinct programs of gene expression required for stemness, differentiation and malignant transformation. To address this hypothesis we are; (i) examining the molecular mechanisms by which nucleolar-associated chromatin domains dynamically regulate spatial organisation of the genome; (ii) testing whether the concomitant changes in distinct patterns of gene expression actively drive developmental programs such as differentiation and contribute to malignant transformations; and (iii) examining whether the therapeutic efficacy of Pol I inhibitors is mediated in part through the disruption of long-range rDNA genomic interaction. [ABSTRACT FROM AUTHOR]

Background Skeletal muscle wasting contributes to the poor functional status and quality of life of patients with pulmonary arterial hypertension (PAH). The present study aims to characterize the molecular mechanism underlying skeletal muscle wasting in experimental PAH induced by monocrotaline (MCT). Methods Male Wistar rats were randomly injected with saline solution (CONT; n = 10) or MCT (MCT; 60 mg/kg, s.c.; n = 15). After 4 weeks of MCT or vehicle administration, animals were anesthetized and submitted to right ventricular (RV) hemodynamic evaluation. Blood and gastrocnemius sample s were collected and stored for analysis. Results MCT group developed PAH (70% increase in RV peak systolic pressure) RV dysfunction (increased end-diastolic pressure and Tau), and body and muscle wasting (reduction of 20%, 16% and 30% on body weight, gastrocnemius mass and fiber cross sectional area, respectively). Muscle atrophy was associated with a decrease in type I MHC. Circulating (C reactive protein, myostatin and IL-1beta) and local catabolic markers (MAFbx/atrogin-1, protease activity) were increased in MCT animals, while Akt/mTOR pathway was preserved. Mitochondria isolated from gastrocnemius of MCT animals showed decreased activity of ATP synthase, lower levels of Tfam, accumulation of oxidatively modified proteins together with reduced levels of paraplegin. Conclusions Our data suggests an anabolic/catabolic imbalance in gastrocnemius from MCT-induced PAH rats. Accumulation of dysfunctional mitochondria due to the inefficiency of protein quality control systems to eliminate damaged proteins could also contribute to muscle atrophy in PAH. [ABSTRACT FROM AUTHOR]

[Display omitted] • The MSC-derived secretome from 3D cultures enhances fibroblast and keratinocyte mitogenic and motogenic capacity in vitro , respectively. • The cargo of the 3D MSC-derived exosomes (Exo3D) reveals wound healing-related proteins and promotes wound resolution in a wound healing in vivo model. • Loading MSC-derived exosomes with A151 ODN further reduces the systemic levels of IL-6 and TNF-α pro-inflammatory cytokines at the late stage of wound healing in vivo , crucial for a full regenerated tissue. • A151-loaded Exo3D have a great potential as a noncellular off-the-shelf therapy for non-healing wound treatment. Non-healing wounds remain a major burden due to the lack of effective treatments. Mesenchymal stem cell-derived exosomes (MSC-Exo) have emerged as therapeutic options given their pro-regenerative and immunomodulatory features. Still, little is known on the exact mechanisms mediated by MSC-Exo. Importantly, modulation of their efficacy through 3D-physiologic cultures together with loading strategies continues underexplored. To uncover the MSC-Exo-mediated mechanism via proteomic analyses, and to use 3D-culture and loading technologies to expand MSC-Exo efficacy for cutaneous wound healing. MSC-Exo were produced in either 3D or 2D cultures (Exo3D/Exo2D) and loaded with an exogenous immunosuppressive oligodeoxynucleotide (A151 ODN). Both, loaded and naïve exosomes were characterised regarding size, morphology and the presence of specific protein markers; while IPA analyses enabled to correlate their protein content with the effects observed in vitro and in vivo. The Exo3D/Exo2D regenerative potential was evaluated in vitro by assessing keratinocyte and fibroblast mitogenicity, motogenicity, and cytokine secretion as well as using an in vivo wound splinting model. Accordingly, the modulation of inflammatory and immune responses by A151-loaded Exo3D/Exo2D was also assessed. Exo3D stimulated mitogenically and motogenically keratinocytes and fibroblasts in vitro , with upregulation of IL-1α and VEGF-α or increased secretion of TGF-β, TNF-α and IL-10. In vivo, Exo3D reduced the granulation tissue area and promoted complete re-epithelization of the wound. These observations were sustained by the proteomic profiling of the Exo3D cargo that identified wound healing-related proteins, such as TGF-β, ITGA1-3/5, IL-6, CDC151, S100A10 and Wnt5α. Moreover, when loaded with A151 ODN, Exo3D differentially mediated wound healing-related trophic factors reducing the systemic levels of IL-6 and TNF-α at the late stage of wound healing in vivo. Our results support the potential of A151-loaded Exo3D for the treatment of chronic wounds by promoting skin regeneration, while modulating the systemic levels of the pro-inflammatory cytokines. [ABSTRACT FROM AUTHOR]

Objective: To design a culture method allowing the quantitative and qualitative analysis of terminal erythroid differentiation. Methods: Primary erythroid progenitors derived either from mouse tissues or from human umbilical cord blood were differentiated using hanging drop cultures and compared to methylcellulose cultures. Cultured cells were analyzed by FACS to assess differentiation. Results: We describe a practical culture method by adapting the previously described hanging drop culture system to conditions allowing terminal differentiation of primary erythroid progenitors. Using minimal volumes of media and small numbers of cells, we obtained quantitative terminal erythroid differentiation within two days of culture in the case of murine cells and 4 days in the case of human cells. Conclusions: The established methods for ex vivo culture of primary erythroid progenitors, such as methylcellulose-based burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) assays, allow the detection of committed erythroid progenitors but are of limited value to study terminal erythroid differentiation. We show that the application of hanging drop cultures is a practical alternative that, in combination with clonogenic assays, enables a comprehensive assessment of the behavior of primary erythroid cells ex vivo in the context of genetic and drug-induced perturbations. [Copyright &y& Elsevier]

Abstract Protein nanofibers are gaining much attention due to their unique mechanical strength, thermal stability and functional properties, thus promoting research on new preparation methodologies. Most fibrillation processes developed so far are time consuming, usually taking from 8 h to 15 h, which represents a major drawback in their use. The use of alternative solvents, such as ionic liquids (ILs) and deep eutectic solvents, as fibrillation agents has been recently reported with considerable reduction in the fibrillation time. This fact encouraged us to study the fibrillation of hen egg white lysozyme (HEWL) in the presence of several ILs from two different families, based on i) imidazolium and ii) cholinium cations, combined with different anions derived from organic acids. The properties of the obtained protein nanofibers were studied using UV–vis and fluorescence spectroscopy, electrophoresis, circular dichroism and electron microscopy. All ILs used were shown to fibrillate HEWL within a few hours, generally achieving over 70–80% of conversion ratios. Typically, worm-like nanofibers were obtained, with 0.3–1 μm of length and 15–40 nm of width, depending on the ILs used. Furthermore, the presence of the acetate anion increases the ability of HEWL to form β-sheet structures. These results show that there is a correlation between the IL chemical structure and the structural features HEWL fibrils, which can be advantageously used in multiple applications in the bionanotechnological field. Graphical abstract Unlabelled Image Highlights • Ionic liquids based on imidazolium and cholinium cations fibrillate lysozyme. • Ionic liquids fibrillate lysozyme within a few hours, achieving conversion ratios over 70%. • Worm-like nanofibers with variable dimensions, depending on the ILs used, were obtained. • Ionic liquids accelerate the fibrillations process and tune the nanofiber dimensions. [ABSTRACT FROM AUTHOR]

Infectious diseases caused by multidrug-resistant (MDR) Enterobacteriaceae have exponentially increased in the past decade, and are a major concern in hospitals. In the first part of the work, we compared the proteome profile of MDR and susceptible clinical isolates of Escherichia coli and Klebsiella pneumoniae in order to identify possible biological processes associated with drug resistance and susceptible phenotypes, using a label-free approach. In the second part, we used an immunoproteomics approach to identify immunoreactive proteins in the same isolates. A total of 388 and 377 proteins were identified in MDR and susceptible E. coli , respectively, evidencing that biological processes related to translation are upregulated in E. coli MDR, while there is an upregulation of processes related to catalytic activity in K. pneumoniae MDR. Both MDR strains show downregulation of processes related to amino acid activation and tRNA amino-acylation. Our data also suggest that MDR strains have higher immunoreactivity than the susceptible strains. The application of high-throughput mass spectrometry (MS) and bioinformatics to the study of modulation of biological processes might shed light on the characterization of multidrug resistance in bacteria. [ABSTRACT FROM AUTHOR]

The stem cell leukemia (Scl)/Tal1 gene is essential for normal blood and endothelial development, and is expressed in hematopoietic stem cells (HSCs), progenitors, erythroid, megakaryocytic, and mast cells. The Scl +19 enhancer is active in HSCs and progenitor cells, megakaryocytes, and mast cells, but not mature erythroid cells. Here we demonstrate that in vivo deletion of the Scl +19 enhancer (Scl Δ19/Δ19 ) results in viable mice with normal Scl expression in mature hematopoietic lineages. By contrast, Scl expression is reduced in the stem/progenitor compartment and flow cytometry analysis revealed that the HSC and megakaryocyte-erythroid progenitor populations are enlarged in Scl Δ19/Δ19 mice. The increase in HSC numbers contributed to enhanced expansion in bone marrow transplantation assays, but did not affect multilineage repopulation or stress responses. These results affirm that the Scl +19 enhancer plays a key role in the development of hematopoietic stem/progenitor cells, but is not necessary for mature hematopoietic lineages. Moreover, active histone marks across the Scl locus were significantly reduced in Scl Δ19/Δ19 fetal liver cells without major changes in steady-state messenger RNA levels, suggesting post-transcriptional compensation for loss of a regulatory element, a result that might be widely relevant given the frequent observation of mild phenotypes after deletion of regulatory elements. [ABSTRACT FROM AUTHOR]

Abstract: Hyperglycaemia-related mitochondrial impairment is suggested as a contributor to skeletal muscle dysfunction. Aiming a better understanding of the molecular mechanisms that underlie mitochondrial dysfunction in type 1 diabetic skeletal muscle, the role of the protein quality control system in mitochondria functionality was studied in intermyofibrillar mitochondria that were isolated from gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Hyperglycaemic rats showed more mitochondria but with lower ATP production ability, which was related with increased carbonylated protein levels and lower mitochondrial proteolytic activity assessed by zymography. LC-MS/MS analysis of the zymogram bands with proteolytic activity allowed the identification of an AAA protease, Lon protease; the metalloproteases PreP, LAP-3 and MIP; and cathepsin D. The content and activity of the Lon protease was lower in the STZ animals, as well as the expression of the m-AAA protease paraplegin, evaluated by western blotting. Data indicated that in muscle from diabetic rats the mitochondrial protein quality control system was compromised, which was evidenced by the decreased activity of AAA proteases, and was accompanied by the accumulation of oxidatively modified proteins, thereby causing adverse effects on mitochondrial functionality. [Copyright &y& Elsevier]

Summary: Hematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes. [ABSTRACT FROM AUTHOR]

Abstract: Protective immunity in paracoccidioidomycosis (PCM) is believed to be mediated by cellular immunity, but the role of T cell subsets has never been investigated. The aim of this study was to characterize the function of CD4+ and CD8+ T cells in the immunity developed by susceptible, intermediate and resistant mice after P. brasiliensis infection. In susceptible mice, depletion of CD4+ T cells did not alter disease severity and anergy of cellular immunity but diminished antibody production. Anti-CD8 treatment led to increased fungal loads, but restored DTH reactivity. In resistant mice, both CD4+ and CD8+ T cells control fungal burdens and cytokines although only the former regulate DTH reactions and antibody production. In the intermediate strain, deficiency of whole T and CD8+ T cells but not of CD4+ T or B cells led to increased mortality rates. Thus, in pulmonary PCM: (a) irrespective of the host susceptibility pattern, fungal loads are mainly controlled by CD8+ T cells, whereas antibody production and DTH reactions are regulated by CD4+ T cells; (c) CD4+ T cells play a protective role in the resistant and intermediate mouse strains, whereas in susceptible mice they are deleted or anergic; (d) genetic resistance to PCM is associated with concomitant CD4+ and CD8+ T cell immunity secreting type 1 and type 2 cytokines. [Copyright &y& Elsevier]

Objective: To study the role of mammalian target of rapamycin (mTOR) in the regulation of human Sertoli cell (hSC) metabolism, mitochondrial activity, and oxidative stress.Design: Experimental study.Setting: University research center and private assisted reproductive technology centers.Patient(s): Six men with anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis.Intervention(s): Testicular biopsies were used from patients under treatment for recovery of male gametes. Primary hSCs cultures were established from each biopsy and divided into a control group and one treated with rapamycin, the inhibitor of mTOR, for 24 hours.Main Outcome Measure(s): Cytotoxicity of hSCs to rapamycin was evaluated by sulforhodamine B assay. The glycolytic profile of hSCs was assessed by proton nuclear magnetic resonance and by studying protein expression of key glycolysis-related transporters and enzymes. Expression of mitochondrial complexes and citrate synthase activity were determined. Protein carbonylation, nitration, lipid peroxidation, and sulfhydryl protein group contents were quantified. The mTOR signaling pathway was studied.Result(s): Rapamycin increased glucose consumption by hSCs, maintaining lactate production. Alanine production by rapamycin-exposed hSCs was affected, resulting in an unbalanced intracellular redox state. Rapamycin-exposed hSCs had decreased expression of mitochondrial complex III and increased lipid peroxidation, whereas other oxidative stress markers were unaltered. Treatment of hSCs with rapamycin down-regulated phospho-mTOR (Ser-2448) levels, illustrating an effective partial inhibition of mTORC1. Protein levels of downstream signaling molecule p-4E-BP1 were not altered, suggesting that during treatment it became rephosphorylated.Conclusion(s): We show that mTOR regulates the nutritional support of spermatogenesis by hSCs and redox balance in these cells. [ABSTRACT FROM AUTHOR]