Article-processing charges as a barrier for science in low-to-medium income regions.

It is widely accepted that science is universal by nature. However, to make science universal, access to research findings is imperative. The open access model of publication of academic articles was established and consolidated during the last two decades. However, most of the open access journals apply article-processing charges (APCs), which can cost more than USD 10,000.00. In regions where support for research is scarce, these funds are usually not available. Similar problems occur in countries with weak economies and, consequently, unfavorable currency conversion rates. This situation reveals a barrier to the alleged universality of science and the access to research findings. In this manuscript, the barriers faced by authors and institutions from low-to-middle income regions to cover APCs and make their science freely available are discussed and illustrated with recent numbers.

Brazilian science: towards extinction?

Brazilian science is under attack. In this manuscript, we will discuss the most recent events that, if not reverted, will make Brazilian science inviable. We urge the scientific community in Brazil and abroad to stand up and resist in defense of more than a century of essential scientific contributions.

Extracellular Vesicles in Fungi: Composition and Functions.

The comprehension of fungal biology is important for several reasons. Besides being used in biotechnological processes and in the food industry, fungi are also important animal and vegetal pathogens. Fungal diseases in humans have a great importance worldwide, and understanding fungal biology is crucial for treatment and prevention of these diseases, especially because of emerging antifungal resistance that poses great epidemiological risks. Communication through extracellular vesicles is a ubiquitous mechanism of molecule transfer between cells and is used to transport proteins, nucleic acids, lipids, and other biologically active molecules. Several pathogens can produce and transfer extracellular vesicles, and the importance of this pathway in fungal communication with hosts and between fungal cells has been described for several species in the last years, as shown for Saccharomyces cereviseae, Cryptococcus neoformans, Candida albicans, Paracoccidioides braziliensis, Sporothrix schenckii, Candida parapsilosis, Malassezia sympodialis, Histoplasma capsulatum, among others. In this chapter, we review the role of extracellular vesicles in fungal communication, interaction with hosts and with the environment, and also highlighting important molecules found in fungal EVs.

Unveiling the partners of the DRBD2-mRNP complex, an RBP in Trypanosoma cruzi and ortholog to the yeast SR-protein Gbp2.

BACKGROUND: RNA-binding proteins (RBPs) are well known as key factors in gene expression regulation in eukaryotes. These proteins associate with mRNAs and other proteins to form mRNP complexes that ultimately determine the fate of target transcripts in the cell. This association is usually mediated by an RNA-recognition motif (RRM). In the case of trypanosomatids, these proteins play a paramount role, as gene expression regulation is mostly posttranscriptional. Despite their relevance in the life cycle of Trypanosoma cruzi, the causative agent of Chagas' disease, to date, few RBPs have been characterized in this parasite. RESULTS: We investigated the role of DRBD2 in T. cruzi, an RBP with two RRM domains that is associated with cytoplasmic translational complexes. We show that DRBD2 is an ortholog of the Gbp2 in yeast, an SR-rich protein involved in mRNA quality control and export. We used an immunoprecipitation assay followed by shotgun proteomics and RNA-seq to assess the interaction partners of the DRBD2-mRNP complex in epimastigotes. The analysis identified mostly proteins involved in RNA metabolism and regulation, such as ALBA1, ALBA3, ALBA4, UBP1, UBP2, DRBD3, and PABP2. The RNA-seq results showed that most of the transcripts regulated by the DRBD2 complex mapped to hypothetical proteins related to multiple processes, such as to biosynthetic process, DNA metabolic process, protein modification, and response to stress. CONCLUSIONS: The identification of regulatory proteins in the DRBD2-mRNP complex corroborates the important role of DRBD2 in gene expression regulation in T. cruzi. We consider these results an important contribution to future studies regarding gene expression regulation in T. cruzi, especially in the field of RNA-binding proteins.

The Nuclear RNA-binding Protein RBSR1 Interactome in Trypanosoma cruzi.

Trypanosoma cruzi, the etiological agent of Chagas disease, has been widely studied, reflecting both its medical importance and the particular features that make this pathogen an attractive model for basic biological studies. The repression of transcripts by messenger ribonucleoprotein (mRNP) complexes is an important pathway of post-transcriptional regulation in eukaryotes, including T. cruzi. RBSR1 is a serine-arginine (SR)-rich RNA-binding protein (RBP) in T. cruzi that contains one RNA-recognition motif (RRM); this protein has a primarily nuclear localization and is developmentally regulated, not being detected in metacyclic trypomastigotes. RBSR1 interacts with other RBPs, such as UBP1 and UBP2, and the nuclear SR-protein TRRM1. Phylogenetic analysis indicated that RBSR1 is orthologous to the human splicing factor SRSF7, what might indicate its possible involvement in pre-RNA processing. Accordingly, ribonomics data showed the enrichment of snoRNAs and snRNAs in the RBSR1 immunoprecipiatation complex, hence reinforcing the supposition that this protein might be involved in RNA processing in the nucleus.

TcNST2 encodes a Golgi-localized UDP-galactose transporter in Trypanosoma cruzi.

Survival and infectivity of trypanosomatids rely on cell-surface and secreted glycoconjugates, many of which contain a variable number of galactose residues. Incorporation of galactose to proteins and lipids occurs along the secretory pathway from UDP-galactose (UDP-Gal). Before being used in glycosylation reactions, however, this activated sugar donor must first be transported across the endoplasmic reticulum and Golgi membranes by a specific nucleotide sugar transporter (NST). In this study, we identified an UDP-Gal transporter (named TcNST2 and encoded by the TcCLB.504085.60 gene) from Trypanosoma cruzi, the etiological agent of Chagas disease. TcNST2 was identified by heterologous expression of selected putative nucleotide sugar transporters in a mutant Chinese Hamster Ovary cell line. TcNST2 mRNA levels were detected in all T. cruzi life-cycle forms, with an increase in expression in axenic amastigotes. Confocal microscope analysis indicated that the transporter is specifically localized to the Golgi apparatus. A three-dimensional model of TcNST2 suggested an overall structural conservation as compared with members of the metabolite transporter superfamily and also suggested specific features that could be related to its activity. The identification of this transporter is an important step toward a better understanding of glycoconjugate biosynthesis and the role NSTs play in this process in trypanosomatids.

Assessing the partners of the RBP9-mRNP complex in Trypanosoma cruzi using shotgun proteomics and RNA-seq.

Gene expression regulation in trypanosomes differs from other eukaryotes due to absence of transcriptional regulation for most of their genes. RNA-binding proteins (RBPs) associate with mRNAs and other regulatory proteins to form ribonucleoprotein complexes (mRNPs), which play a major role in post-transcriptional regulation. Here, we show that RBP9 is a cytoplasmic RBP in Trypanosoma cruzi with one RNA-recognition motif (RRM). The RBP9 sedimentation profile in a sucrose gradient indicated its presence in cytoplasmic translational complexes, suggesting its involvement in translation regulation. Taking this result as a motivation, we used shotgun proteomics and RNA-seq approaches to assess the core of the RBP9-mRNP complex. In epimastigotes in exponential growth, the complex was composed mostly by RBPs involved in RNA metabolism, such as ZC3H39, UBP1/2, NRBD1, and ALBA3/4. When parasites were subjected to nutritional stress, our analysis identified regulatory RBPs and the translation initiation factors eIF4E5, eIF4G5, eIF4G1, and eIF4G4. The RNA-seq results showed that RBP9-mRNP complex regulates transcripts encoding some RBPs - e.g. RBP5, RBP6, and RBP10 -, and proteins involved in metabolic processes. Therefore, we argue that RBP9 is part of cytoplasmic mRNPs complexes associated with mRNA metabolism and translation regulation in T. cruzi.

Trypanosoma cruzi XRNA granules colocalise with distinct mRNP granules at the nuclear periphery.

BACKGROUND Eukaryotic ribonucleoprotein (RNP) granules are important for the regulation of RNA fate. RNP granules exist in trypanosomatids; however, their roles in controlling gene expression are still not understood. XRNA is a component of granules in Trypanosoma brucei but has not been investigated in Trypanosoma cruzi. OBJECTIVES This study aimed to investigate the TcXRNA dynamic assembly and its interaction with RNP components under conditions that affect the mRNA availability. METHODS We used in vitro metacyclogenesis of T. cruzi to observe changes in RNP granules during the differentiation process. TcXRNA expression was analysed by Western blot and immunofluorescence. Colocalisation assays were performed to investigate the interaction of TcXRNA with other RNP components. FINDINGS TcXRNA is constantly present during metacyclogenesis and is localised in cytoplasmic granules. TcXRNA does not colocalise with TcDHH1 and TcCAF1 granules in the cytoplasm. However, TcXRNA granules colocalise with mRNP granules at the nuclear periphery when mRNA processing is inhibited. MAIN CONCLUSIONS TcXRNA plays a role in mRNA metabolism as a component of mRNP granules whose assembly is dependent on mRNA availability. TcXRNA granules colocalise with distinct RNP granules at the nuclear periphery, suggesting that the perinuclear region is a regulatory compartment in T. cruzi mRNA metabolism.

Proteomic profiling of extracellular vesicles secreted from Toxoplasma gondii.

Toxoplasma gondii infects a wide range of hosts worldwide, including humans and domesticated animals causing toxoplasmosis disease. Recently, exosomes, small extracellular vesicles (EV) that contain nucleic acids, proteins, and lipids derived from their original cells were linked with disease protection. The effect of EVs derived from T. gondii on the immune response and its relevance in a physiological context is unknown. Here we disclose the first proteomic profiling of T. gondii EVs compared to EVs isolated from a human foreskin fibroblast infected cell line cultured in a vesicle-free medium. Our results reveal a broad range of canonical exosomes proteins. Data are available via ProteomeXchange with the identifier PXD004895.

Post-translational Modifications of Trypanosoma cruzi Canonical and Variant Histones.

Chagas disease, caused by Trypanosoma cruzi, still affects millions of people around the world. No vaccines nor treatment for chronic Chagas disease are available, and chemotherapy for the acute phase is hindered by limited efficacy and severe side effects. The processes by which the parasite acquires infectivity and survives in different hosts involve tight regulation of gene expression, mainly post-transcriptionally. Nevertheless, chromatin structure/organization of trypanosomatids is similar to other eukaryotes, including histone variants and post-translational modifications. Emerging evidence suggests that epigenetic mechanisms also play an important role in the biology/pathogenesis of these parasites, making epigenetic targets suitable candidates to drug discovery. Here, we present the first comprehensive map of post-translational modifications of T. cruzi canonical and variant histones and show that its histone code can be as sophisticated as that of other eukaryotes. A total of 13 distinct modification types were identified, including rather novel and unusual ones such as alternative lysine acylations, serine/threonine acetylation, and N-terminal methylation. Some histone marks correlate to those described for other organisms, suggesting that similar regulatory mechanisms may be in place. Others, however, are unique to T. cruzi or to trypanosomatids as a group and might represent good candidates for the development of antiparasitic drugs.

RNA-binding proteins and their role in the regulation of gene expression in Trypanosoma cruzi and Saccharomyces cerevisiae.

RNA-binding proteins (RBPs) have important functions in the regulation of gene expression. RBPs play key roles in post-transcriptional processes in all eukaryotes, such as splicing regulation, mRNA transport and modulation of mRNA translation and decay. RBPs assemble into different mRNA-protein complexes, which form messenger ribonucleoprotein complexes (mRNPs). Gene expression regulation in trypanosomatids occurs mainly at the post-transcriptional level and RBPs play a key role in all processes. However, the functional characterization of RBPs in Trypanosoma cruzi has been impaired due to the lack of reliable reverse genetic manipulation tools. The comparison of RBPs from Saccharomyces cerevisiae and T. cruzi might allow inferring on the function of these proteins based on the information available for the orthologous RNA-binding proteins from the S. cerevisiae model organism. In this review, we discuss the role of some RBPs from T. cruzi and their homologues in regulating gene expression in yeast.

Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi.

BACKGROUND: Due to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms. RESULTS: Here we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect's life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage. CONCLUSIONS: A large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.

Eukaryotic translation elongation factor-1 alpha is associated with a specific subset of mRNAs in Trypanosoma cruzi.

BACKGROUND: Regulation of gene expression in trypanosomatids is mainly posttranscriptional. Tight regulation of mRNA stability and access to polysomes allows Trypanosoma cruzi to adapt to different environmental conditions during its life cycle. Posttranscriptional regulation requires association between mRNAs and specific proteins to form mRNP complexes. Proteins that lack a canonical RNA-binding domain, such as eukaryotic elongation factor-1α (EF-1α), may also associate with mRNPs. EF-1α is conserved in many organisms, and it plays roles in many cellular processes other than translation, including RNA transport, the cell cycle, and apoptosis. RESULTS: In a previous study, EF-1α was found associated with mRNP-forming mRNAs in polysome-free fractions both in epimastigotes growing under normal conditions and in nutritionally stressed parasites. This finding suggested the possibility that EF-1α has a non-canonical function. Thus, we investigated the dynamics of EF-1α in association with T. cruzi epimastigote mRNAs under normal and stressed nutritional conditions. EF-1α is expressed throughout the parasite life cycle, but it shows a slight decrease in protein levels in the metacyclic trypomastigote form. The protein is cytoplasmically localized with a granular pattern in all forms analyzed. Following puromycin treatment, EF-1α migrated with the heaviest gradient fractions in a sucrose polysome profile, indicating that its association with large protein complexes was independent of the translation machinery. We next characterized the EF-1α-associated mRNAs in unstressed and stressed epimastigotes. We observed that specific subsets of mRNAs were associated with EF-1α-mRNPs in unstressed or stressed epimastigotes. Some mRNAs were identified in both physiological conditions, whereas others were condition-specific. Gene ontology analysis identified enrichment of gene sets involved in single-organism metabolic processes, amino acid metabolic processes, ATP and metal ion binding, glycolysis, glutamine metabolic processes, and cobalt and iron ion binding. CONCLUSION: These results indicate that in T. cruzi, as in other eukaryotes, EF-1α may play a non-canonical cellular role. We observed the enrichment of functionally related transcripts bound to EF-1α in normal growth conditions as well as in nutritionally stressed cell indicating a potential role of EF-1α mRNP in stress response.

Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi.

BACKGROUND: Nucleotide sugar transporters (NSTs) play an essential role in translocating nucleotide sugars into the lumen of the endoplasmic reticulum and Golgi apparatus to be used as substrates in glycosylation reactions. This intracellular transport is an essential step in the biosynthesis of glycoconjugates. RESULTS: We have identified a family of 11 putative NSTs in Trypanosoma cruzi, the etiological agent of Chagas' disease. A UDP-N-acetylglucosamine transporter, TcNST1, was identified by a yeast complementation approach. Based on a phylogenetic analysis four candidate genes were selected and used for complementation assays in a Kluyveromyces lactis mutant strain. The transporter is likely expressed in all stages of the parasite life cycle and during differentiation of epimastigotes to infective metacyclics. Immunofluorescence analyses of a GFP-TcNST1 fusion protein indicate that the transporter is localized to the Golgi apparatus. As many NSTs are multisubstrate transporters, we also tested the capacity of TcNST1 to transport GDP-Man. CONCLUSIONS: We have identified a UDP-N-acetylglucosamine transporter in T. cruzi, which is specifically localized to the Golgi apparatus and seems to be expressed, at the mRNA level, throughout the parasite life cycle. Functional studies of TcNST1 will be important to unravel the role of NSTs and specific glycoconjugates in T. cruzi survival and infectivity.

Inhibition of cyclooxygenase-1 and cyclooxygenase-2 impairs Trypanosoma cruzi entry into cardiac cells and promotes differential modulation of the inflammatory response.

The intracellular protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion by T. cruzi and its intracellular replication are essential to the parasite's life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the host's cyclooxygenase (COX) enzymes during T. cruzi invasion. Pharmacological antagonists for COX-1 (aspirin) and COX-2 (celecoxib) caused marked inhibition of T. cruzi infection when rat cardiac cells were pretreated with these nonsteroidal anti-inflammatory drugs (NSAIDs) for 60 min at 37°C before inoculation. This inhibition was associated with an increase in the production of NO and interleukin-1ß and decreased production of transforming growth factor ß (TGF-ß) by cells. Taken together, these results indicate that COX-1 more than COX-2 is involved in the regulation of anti-T. cruzi activity in cardiac cells, and they provide a better understanding of the influence of TGF-ß-interfering therapies on the innate inflammatory response to T. cruzi infection and may represent a very pertinent target for new therapeutic treatments of Chagas disease.

The mRNAs associated to a zinc finger protein from Trypanosoma cruzi shift during stress conditions.

Trypanosome gene expression is regulated almost exclusively at the posttranscriptional level, through mRNA stability, storage and degradation. Here, we characterize the ribonucleoprotein complex (mRNPs) corresponding to the zinc finger protein TcZC3H39 from T. cruzi comparing cells growing in normal conditions and under nutritional stress. The nutritional stress is a key step during T. cruzi differentiation from epimastigote form to human infective metacyclic trypomastigote form. The mechanisms by which the stress, altogether with other stimuli, triggers differentiation is not well understood. This work aims to characterize the TcZC3H39 protein during stress response. Using cells cultured in normal and stress conditions, we observed a dynamic change in TcZC3H39 granule distribution, which appeared broader in stressed epimastigotes. The protein core of the TcZC3H39-mRNP is composed of ribosomes, translation factors and RBPs. The TcZC3H39-mRNP could act sequestering highly expressed mRNAs and their associated ribosomes, potentially slowing translation in stress conditions. A shift were observed in the mRNAs associated with TcZC3H39: the number of targets in unstressed epimastigotes was smaller than that in stressed parasites, with no clear functional clustering in normal conditions. By contrast, in stressed parasites, the targets of TcZC3H39 were mRNAs encoding ribosomal proteins and a remarkable enrichment in mRNAs for the cytochrome c complex (COX), highly expressed mRNAs in the replicative form. This identification of a new component of RNA granules in T. cruzi, the TcZC3H39 protein, provides new insight into the mechanisms involved in parasite stress responses and the regulation of gene expression during T. cruzi differentiation.

Characterization of the pattern of ribosomal protein L19 production during the lifecycle of Leishmania spp.

Leishmania is a genus of protozoan parasites causing a wide clinical spectrum of diseases in humans. Analysis of a region of chromosome 6 from Leishmania major (Iribar et al.) showed that the transcript of a putative L19 ribosomal protein (RPL19) was most abundant at the amastigote stage. We therefore decided to characterize L19 protein abundance throughout the lifecycle of Leishmania. Differential expression of the L19 gene during development has been observed for all Leishmania species studied to date (L. major, L. braziliensis, L. donovani, and L. amazonensis). Immunoblotting with polyclonal antibodies against L. major RPL19 revealed that changes to L19 protein abundance follow a similar pattern in various species. The amount of L19 protein was higher in exponentially growing promastigotes than in stationary phase promastigotes. The L19 protein was barely detectable in amastigotes, despite the abundance of L19 transcripts observed in L. major at this stage. Immunofluorescence assays showed a granular, dispersed distribution of RPL19 throughout the cytoplasm. Subcellular fractionation confirmed the presence of the protein in the ribosomal fraction, but not in the cytosol of L. major. We generated a L. major transfectant bearing a plasmid-borne L19 gene. Overproduction of the L19 transcript and protein resulted in impaired growth of the transfectants in association with high polysome peaks. We also showed by metabolic labeling that L19 overexpressing clones display low rates of translation. These data suggest that L19 overexpression affects negatively translation elongation or termination. The lack of correlation between L19 transcript and protein abundances suggest that the translation of L19 is differentially controlled during development in the various species investigated.

TcZC3HTTP, a regulatory element that contributes to Trypanosoma cruzi cell proliferation.

Post-transcriptional regulation of gene expression is a critical process for adapting to and surviving Trypanosoma cruzi, a parasite with a complex life cycle. RNA-binding proteins (RBPs) are key players in this regulation, forming ribonucleoprotein complexes (messenger ribonucleoproteins) and RNA granules that control transcript stability, localization, degradation, and translation modulation. Understanding the specific roles of individual RBPs is crucial for unraveling the details of this regulatory network. In this study, we generated null mutants of the TcZC3HTTP gene, a specific RBP in the Trypanosoma family characterized by a C3H zinc finger and a DNAJ domain associated with RNA and protein binding, respectively. Through cell growth assays, we demonstrated that the absence of TcZC3HTTP or the expression of an additional tagged version impacted epimastigote growth, indicating its contribution to cell proliferation. TcZC3HTTP was found to associate with mRNAs involved in cell cycle and division in epimastigotes, while in nutritionally stressed parasites it exhibited associations with mRNAs coding for other RBPs and rRNA. Furthermore, our analysis identified that TcZC3HTTP protein partners were different during normal growth conditions compared to starvation conditions, with the latter showing enrichment of ribosomal proteins and other RBPs. Therefore, this study provides insights into TcZC3HTTP's role in the post-transcriptional regulation of gene expression during normal growth and nutritional stress in T. cruzi, uncovering its versatile functions in different cellular contexts.IMPORTANCEUnderstanding how Trypanosoma cruzi, the causative agent of Chagas disease, regulates gene expression is crucial for developing targeted interventions. In this study, we investigated the role of TcZC3HTTP, an RNA-binding protein, in post-transcriptional regulation. Our findings demonstrate that TcZC3HTTP is relevant for the growth and proliferation of epimastigotes, a stage of the parasite's life cycle. We identified its associations with specific mRNAs involved in cell cycle and division and its interactions with enzymes and other RNA-binding proteins (RBPs) under normal and starvation conditions. These insights shed light on the regulatory network underlying gene expression in T. cruzi and reveal the multifaceted functions of RBPs in this parasite. Such knowledge enhances our understanding of the parasite's biology and opens avenues for developing novel therapeutic strategies targeting post-transcriptional gene regulation in T. cruzi.