P2RX7 signaling drives the differentiation of Th1 cells through metabolic reprogramming for aerobic glycolysis.

Introduction: This study provides evidence of how Th1 cell metabolism is modulated by the purinergic receptor P2X7 (P2RX7), a cation cannel activated by high extracellular concentrations of adenosine triphosphate (ATP). Methods: In vivo analysis was performed in the Plasmodium chabaudi model of malaria in view of the great relevance of this infectious disease for human health, as well as the availability of data concerning Th1/Tfh differentiation. Results: We show that P2RX7 induces T-bet expression and aerobic glycolysis in splenic CD4+ T cells that respond to malaria, at a time prior to Th1/Tfh polarization. Cell-intrinsic P2RX7 signaling sustains the glycolytic pathway and causes bioenergetic mitochondrial stress in activated CD4+ T cells. We also show in vitro the phenotypic similarities of Th1-conditioned CD4+ T cells that do not express P2RX7 and those in which the glycolytic pathway is pharmacologically inhibited. In addition, in vitro ATP synthase blockade and the consequent inhibition of oxidative phosphorylation, which drives cellular metabolism for aerobic glycolysis, is sufficient to promote rapid CD4+ T cell proliferation and polarization to the Th1 profile in the absence of P2RX7. Conclusion: These data demonstrate that P2RX7-mediated metabolic reprograming for aerobic glycolysis is a key event for Th1 differentiation and suggest that ATP synthase inhibition is a downstream effect of P2RX7 signaling that potentiates the Th1 response.

Immunoproteomic and Immunopeptidomic Analyses of Histoplasma capsulatum Reveal Promiscuous and Conserved Epitopes Among Fungi With Vaccine Potential.

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species.

Correction: Novel site-specific PEGylated L-asparaginase.

[This corrects the article DOI: 10.1371/journal.pone.0211951.].

Zika Virus Impairs Neurogenesis and Synaptogenesis Pathways in Human Neural Stem Cells and Neurons.

Growing evidences have associated Zika virus (ZIKV) infection with congenital malformations, including microcephaly. Nonetheless, signaling mechanisms that promote the disease outcome are far from being understood, affecting the development of suitable therapeutics. In this study, we applied shotgun mass spectrometry (MS)-based proteomics combined with cell biology approaches to characterize altered molecular pathways on human neuroprogenitor cells (NPC) and neurons derived from induced pluripotent stem cells infected by ZIKV-BR strain, obtained from the 2015 Brazilian outbreak. Furthermore, ZIKV-BR infected NPCs showed unique alteration of pathways involved in neurological diseases, cell death, survival and embryonic development compared to ZIKV-AF, showing a human adaptation of the Brazilian viral strain. Besides, infected neurons differentiated from NPC presented an impairment of neurogenesis and synaptogenesis processes. Taken together, these data explain that CNS developmental arrest observed in Congenital Zika Syndrome is beyond neuronal cell death.

Novel site-specific PEGylated L-asparaginase.

L-asparaginase (ASNase) from Escherichia coli is currently used in some countries in its PEGylated form (ONCASPAR, pegaspargase) to treat acute lymphoblastic leukemia (ALL). PEGylation refers to the covalent attachment of poly(ethylene) glycol to the protein drug and it not only reduces the immune system activation but also decreases degradation by plasmatic proteases. However, pegaspargase is randomly PEGylated and, consequently, with a high degree of polydispersity in its final formulation. In this work we developed a site-specific N-terminus PEGylation protocol for ASNase. The monoPEG-ASNase was purified by anionic followed by size exclusion chromatography to a final purity of 99%. The highest yield of monoPEG-ASNase of 42% was obtained by the protein reaction with methoxy polyethylene glycol-carboxymethyl N-hydroxysuccinimidyl ester (10kDa) in 100 mM PBS at pH 7.5 and PEG:ASNase ratio of 25:1. The monoPEG-ASNase was found to maintain enzymatic stability for more days than ASNase, also was resistant to the plasma proteases like asparaginyl endopeptidase and cathepsin B. Additionally, monoPEG-ASNase was found to be potent against leukemic cell lines (MOLT-4 and REH) in vitro like polyPEG-ASNase. monoPEG-ASNase demonstrates its potential as a novel option for ALL treatment, being an inventive novelty that maintains the benefits of the current enzyme and solves challenges.

Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

Background The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. Methodology/Principal findings Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. Conclusions/Significance The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents. Author summary

Site-specific characterization of N-linked glycosylation in human urinary glycoproteins and endogenous glycopeptides.

Glycosylation is a very important post-translational modification involved in various cellular processes, such as cell adhesion, signal transduction and immune response. Urine is a rich source of glycoproteins and attractive biological fluid for biomarker discovery, owing to its availability, ease of collection, and correlation with pathophysiology of diseases. Although the urinary proteomics have been explored previously, the urinary glycoproteome characterization remains challenging requiring the development and optimization of analytical and bioinformatics methods for protein glycoprofiling. This study describes the high confident identification of 472 unique N-glycosylation sites covering 256 urinary glycoproteins. Besides, 202 unique N-glycosylation sites were identified in low molecular weight endogenous glycopeptides, which belong to 90 glycoproteins. Global site-specific characterization of the N-linked glycan heterogeneity was achieved by intact glycopeptide analysis, revealing 303 unique glycopeptides most of them displaying complex/hybrid glycans composed by sialic acid and fucose. These datasets consist in a valuable resource of glycoproteins and N-glycosylation sites found in healthy human urine that can be further explored in different disorders, in which the N-linked glycosylation may be aberrant.

Differences and similarities among parotoid macrogland secretions in South American toads: a preliminary biochemical delineation.

Amphibians are known by cutaneous glands, spread over the skin, containing toxins (proteins, peptides, biogenic amines, steroidal bufadienolides, and alkaloids) used as chemical defense against predators and microbial infection. Toads are characterized by the presence of parotoid macroglands. The common toads have lately been divided into two genera: Bufo (Europe, Asia, and Africa) and Rhinella (South America). Basal Rhaebo genus is exclusively of Central America and Amazon region. Although Rhinella and Rhaebo are related, species may share differences due to the diversity of environments that they live in. In this work, we have performed a biochemical characterization of the components of the poison of eight Rhinella species and one Rhaebo by means of RP-HPLC with either UV or MS detection and by SDS-PAGE, in order to verify whether phylogenetic and biological differences, such as habitat, diet, and defensive strategies, between them may also be reflected in poison composition. Although some components were common among the secretions, we were able to identify exclusive molecules to some species. The fact that closely related animals living in different habitats secrete different molecules into the skin is an indication that biological features, and not only evolution, seem to directly influence the skin secretion composition.