Publications by authors named "Michel Batista"

21 Publications

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Integrated analysis of label-free quantitative proteomics and bioinformatics reveal insights into signaling pathways in male breast cancer.

Genet Mol Biol 2021 1;44(1):e20190410. Epub 2021 Mar 1.

Universidade Federal do Paraná, Departamento de Genética, Programa de Pós-graduação em Genética, Curitiba, PR, Brazil.

Male breast cancer (MBC) is a rare malignancy that accounts for about 1.8% of all breast cancer cases. In contrast to the high number of the "omics" studies in breast cancer in women, only recently molecular approaches have been performed in MBC research. High-throughput proteomics based methodologies are promisor strategies to characterize the MBC proteomic signatures and their association with clinico-pathological parameters. In this study, the label-free quantification-mass spectrometry and bioinformatics approaches were applied to analyze the proteomic profiling of a MBC case using the primary breast tumor and the corresponding axillary metastatic lymph nodes and adjacent non-tumor breast tissues. The differentially expressed proteins were identified in the signaling pathways of granzyme B, sirtuins, eIF2, actin cytoskeleton, eNOS, acute phase response and calcium and were connected to the upstream regulators MYC, PI3K SMARCA4 and cancer-related chemical drugs. An additional proteomic comparative analysis was performed with a primary breast tumor of a female patient and revealed an interesting set of proteins, which were mainly involved in cancer biology. Together, our data provide a relevant data source for the MBC research that can help the therapeutic strategies for its management.
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http://dx.doi.org/10.1590/1678-4685-GMB-2019-0410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926483PMC
March 2021

Proteomic analysis of exosomes derived from procyclic and metacyclic-like cultured Leishmania infantum chagasi.

J Proteomics 2020 09 14;227:103902. Epub 2020 Jul 14.

Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil. Electronic address:

Leishmania infantum chagasi is the primary etiological agent of visceral leishmaniasis in Latin America, a lethal disease that afflicts hundreds of thousands of people worldwide every year. Previous studies have shown that the parasite releases microvesicles known as exosomes, which prolong and exacerbate infection in the vertebrate vector. However, little is known of their role in the insect vector, the sand fly Lutzomyia longipalpis. Exosomes were isolated from cultured L. i. chagasi in logarithmic (procyclic) (LOG) and stationary phase (metacyclic-like) (STAT) growth stages, which are the parasite stages found in the vector, and submitted to proteomic analysis. Our studies showed that exosomes from LOG and STAT L. i. chagasi display discrete protein profiles. The presence of approximately 50 known virulence factors was detected, including molecules for immunomodulation and evasion (GP63, EF1α, Oligopeptidase), increased pathogenicity (Casein kinase, KMP-11, Cysteine Peptidase and BiP) and parasite protection (Peroxidoxin). Additionally, the majority of ontological terms were associated with both exosome phases, and no substantial ontological enrichment was observed associated with any of the two exosomal stages. We demonstrated that LOG exosomes show a marked increase in protein number and abundance, including many virulence factors, compared to STAT L. i. chagasi exosomes. SIGNIFICANCE: The knowledge of the role of Leishmania exosomes on leishmaniasis opened up a new world of potential and complexity regarding our understanding of the disease. In Brazil the majority of visceral leishmaniasis cases are caused by the parasite Leishmania infantum chagasi and transmitted by the vector Lutzomyia longipalpis. While Leishmania exosomes were found to play an active role in the mammalian host, little is understood about their effects on the sand fly, or how they might impact on the insect infection by the parasite. For this reason, we isolated exosomes from two developmental stages of L. i. chagasi that occur within the insect with a view to identifying and describing the alterations they undergo. We have identified many hundreds of proteins within both exosome phases and have developed a structure by which to examine potential candidates. Our findings regarding the composition of the exosome proteome raise many questions regarding their function and provide compelling evidence that exosomes play an active role in the parasite's development within the sand fly.
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http://dx.doi.org/10.1016/j.jprot.2020.103902DOI Listing
September 2020

Characterising ISWI chromatin remodeler in Trypanosoma cruzi.

Mem Inst Oswaldo Cruz 2020 18;115:e190457. Epub 2020 May 18.

Laboratório de Ciências e Tecnologias Aplicadas em Saúde, Instituto Carlos Chagas, Fundação Oswaldo Cruz-Fiocruz, Curitiba, PR, Brazil.

BACKGROUND Imitation SWItch (ISWI) ATPase is the catalytic subunit in diverse chromatin remodeling complexes. These complexes modify histone-DNA interactions and therefore play a pivotal role in different DNA-dependent processes. In Trypanosoma cruzi, a protozoan that controls gene expression principally post-transcriptionally, the transcriptional regulation mechanisms mediated by chromatin remodeling are poorly understood. OBJECTIVE To characterise the ISWI remodeler in T. cruzi (TcISWI). METHODS A new version of pTcGW vectors was constructed to express green fluorescent protein (GFP)-tagged TcISWI. CRISPR-Cas9 system was used to obtain parasites with inactivated TcISWI gene and we determined TcISWI partners by cryomilling-affinity purification-mass spectrometry (MS) assay as an approximation to start to unravel the function of this protein. FINDINGS Our approach identified known ISWI partners [nucleoplasmin-like protein (NLP), regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP)], previously characterised in T. brucei, and new components in TcISWI complex [DRBD2, DHH1 and proteins containing a domain characteristic of structural maintenance of chromosomes (SMC) proteins]. Data are available via ProteomeXchange with identifier PXD017869. MAIN CONCLUSIONS In addition to its participation in transcriptional silencing, as it was reported in T. brucei, the data generated here provide a framework that suggests a role for TcISWI chromatin remodeler in different nuclear processes in T. cruzi, including mRNA nuclear export control and chromatin compaction. Further work is necessary to clarify the TcISWI functional diversity that arises from this protein interaction study.
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http://dx.doi.org/10.1590/0074-02760190457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7233268PMC
June 2020

Mixed-Data Acquisition: Next-Generation Quantitative Proteomics Data Acquisition.

J Proteomics 2020 06 5;222:103803. Epub 2020 May 5.

Laboratory for Structural and Computational Proteomics, Carlos Chagas Institute, Fiocruz, Curitiba, Paraná, Brazil. Electronic address:

We present the Mixed-Data Acquisition (MDA) strategy for mass spectrometry data acquisition. MDA combines Data-Dependent Acquisition (DDA) and Data-Independent Acquisition (DIA) in the same run, thus doing away with the requirements for separate DDA spectral libraries. MDA is a natural result from advances in mass spectrometry, such as high scan rates and multiple analyzers, and is tailored toward exploiting these features. We demonstrate MDA's effectiveness on a yeast proteome analysis by overcoming a common bottleneck for XIC-based label-free quantitation; namely, the coelution of precursors when m/z values cannot be distinguished. We anticipate that MDA will become the next mainstream data generation approach for proteomics. MDA can also serve as an orthogonal validation approach for DDA experiments. Specialized software for MDA data analysis is made available on the project's website.
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http://dx.doi.org/10.1016/j.jprot.2020.103803DOI Listing
June 2020

Genome-Wide Proteomics and Phosphoproteomics Analysis of Trypanosoma cruzi During Differentiation.

Methods Mol Biol 2020 ;2116:139-159

Carlos Chagas Institute, Fiocruz Parana, Curitiba, Parana, Brazil.

Trypanosoma cruzi is a pathogenic protozoan that still has an impact on public health, despite the decrease in the number of infection cases along the years. T. cruzi possesses an heteroxenic life cycle in which it differentiates in at least four forms. Among the differentiation processes, metacyclogenesis has been exploited in different views by researchers. An intriguing question that rises is how metacyclogenesis is triggered and controlled by cell signaling and which are the differentially expressed proteins and posttranslational modifications involved in this process. An important cell signaling pathway is the protein phosphorylation, and it is reinforced in T. cruzi in which the gene expression control occurs almost exclusively posttranscriptionally. Additionally, the number of protein kinases in T. cruzi is relatively high compared to other organisms. A way to approach these questions is evaluating the cells through phosphoproteomics and proteomics. In this chapter, we will describe the steps from the cell protein extraction, digestion and fractionation, phosphopeptide enrichment, to LC-MS/MS analysis as well as a brief overview on peptide identification. In addition, a published method for in vitro metacyclogenesis will be detailed.
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http://dx.doi.org/10.1007/978-1-0716-0294-2_11DOI Listing
February 2021

Quantitative phosphoproteome and proteome analyses emphasize the influence of phosphorylation events during the nutritional stress of Trypanosoma cruzi: the initial moments of in vitro metacyclogenesis.

Cell Stress Chaperones 2019 09 31;24(5):927-936. Epub 2019 Jul 31.

Laboratory of Applied Science and Technologies in Health, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil.

Phosphorylation is an important event in cell signaling that is modulated by kinases and phosphatases. In Trypanosoma cruzi, the etiological agent of Chagas disease, approximately 2% of the protein-coding genes encode for protein kinases. This parasite has a heteroxenic life cycle with four different development stages. In the midgut of invertebrate vector, epimastigotes differentiate into metacyclic trypomastigotes in a process known as metacyclogenesis. This process can be reproduced in vitro by submitting parasites to nutritional stress (NS). Aiming to contribute to the elucidation of mechanisms that trigger metacyclogenesis, we applied super-SILAC (super-stable isotope labeling by amino acids in cell culture) and LC-MS/MS to analyze different points during NS. This analysis resulted in the identification of 4205 protein groups and 3643 phosphopeptides with the location of 4846 phosphorylation sites. Several phosphosites were considered modulated along NS and are present in proteins associated with various functions, such as fatty acid synthesis and the regulation of protein expression, reinforcing the importance of phosphorylation and signaling events to the parasite. These modulated sites may be triggers of metacyclogenesis.
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http://dx.doi.org/10.1007/s12192-019-01018-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717228PMC
September 2019

High-throughput mass spectrometry and bioinformatics analysis of breast cancer proteomic data.

Data Brief 2019 Aug 10;25:104125. Epub 2019 Jun 10.

Genetics Department, Federal University of Parana, Curitiba, Brazil.

Data present here describe a comparative proteomic analysis among the malignant [primary breast tumor (PT) and axillary metastatic lymph nodes (LN)], and the non-tumor [contralateral (NCT) and adjacent (ANT)] breast tissues. Protein identification and quantification were performed through label-free mass spectrometry using a nano-liquid chromatography coupled to an electrospray ionization-mass spectrometry (nLC-ESI-MS/MS). The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via PRIDE partner repository with the dataset identifier PXD012431. A total of 462 differentially expressed proteins was identified among these tissues and was analyzed in six groups' comparisons (named NCTxANT, PTxNCT, PTxANT, LNxNCT, LNxANT and PTxLN). Proteins at 1.5 log2 fold change were submitted to the Ingenuity Pathway Analysis (IPA) software version 2.3 (QIAGEN Inc.) to identify biological pathways, disease and function annotation, and interaction networks related to cancer biology. The detailed data present here provides information about the proteome alterations and their role on breast tumorigenesis. This information can lead to novel biological insights on cancer research. For further interpretation of these data, please see our research article 'Quantitative label-free mass spectrometry using contralateral and adjacent breast tissues reveal differentially expressed proteins and their predicted impacts on pathways and cellular functions in breast cancer' [2].
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http://dx.doi.org/10.1016/j.dib.2019.104125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6595893PMC
August 2019

Quantitative label-free mass spectrometry using contralateral and adjacent breast tissues reveal differentially expressed proteins and their predicted impacts on pathways and cellular functions in breast cancer.

J Proteomics 2019 05 14;199:1-14. Epub 2019 Feb 14.

Genetics Department, Federal University of Parana, Curitiba, Brazil. Electronic address:

Proteins play an essential role in the biological processes associated with cancer. Their altered expression levels can deregulate critical cellular pathways and interactive networks. In this study, the mass spectrometry-based label-free quantification followed by functional annotation was performed to investigate the most significant deregulated proteins among tissues of primary breast tumor (PT) and axillary metastatic lymph node (LN) and corresponding non-tumor tissues contralateral (NCT) and adjacent (ANT) from patients diagnosed with invasive ductal carcinoma. A total of 462 proteins was observed as differentially expressed (DEPs) among the groups analyzed. A high level of similarity was observed in the proteome profile of both non-tumor breast tissues and DEPs (n = 12) were mainly predicted in the RNA metabolism. The DEPs among the malignant and non-tumor breast tissues [n = 396 (PTxNCT) and n = 410 (LNxNCT)] were related to pathways of the LXR/RXR, NO, eNOS, eIF2 and sirtuins, tumor-related functions, fatty acid metabolism and oxidative stress. Remarkable similarity was observed between both malignant tissues, which the DEPs were related to metastatic capabilities. Altogether, our findings revealed differential proteomic profiles that affected cancer associated and interconnected signaling processes. Validation studies are recommended to demonstrate the potential of individual proteins and/or pathways as biological markers in breast cancer. SIGNIFICANCE: The proteomic analysis of this study revealed high similarity in the proteomic profile of the contralateral and adjacent non-tumor breast tissues. Significant differences were identified among the proteome of the malignant and non-tumor tissue groups of the same patients, providing relevant insights into the hallmarks, signaling pathways, biological functions, and interactive protein networks that act during tumorigenesis and breast cancer progression. These proteins are suggested as targets of relevant interest to be explored as potential biological markers related to tumor development and metastatic progression in the breast cancer disease.
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http://dx.doi.org/10.1016/j.jprot.2019.02.007DOI Listing
May 2019

Identification of Secreted Proteins Involved in Nonspecific dsRNA-Mediated Lutzomyia longipalpis LL5 Cell Antiviral Response.

Viruses 2018 01 18;10(1). Epub 2018 Jan 18.

Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, Rio de Janeiro 21040-360, RJ, Brazil.

Hematophagous insects transmit infectious diseases. Sand flies are vectors of leishmaniasis, but can also transmit viruses. We have been studying immune responses of , the main vector of visceral leishmaniasis in the Americas. We identified a non-specific antiviral response in LL5 embryonic cells when treated with non-specific double-stranded RNAs (dsRNAs). This response is reminiscent of interferon response in mammals. We are investigating putative effectors for this antiviral response. Secreted molecules have been implicated in immune responses, including interferon-related responses. We conducted a mass spectrometry analysis of conditioned medium from LL5 cells 24 and 48 h after dsRNA or mock treatment. We identified 304 proteins. At 24 h, 19 proteins had an abundance equal or greater than 2-fold change, while the levels of 17 proteins were reduced when compared to control cells. At the 48 h time point, these numbers were 33 and 71, respectively. The two most abundant secreted peptides at 24 h in the dsRNA-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (FKBP), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. The transcription profile of most candidates did not follow the pattern of secreted protein abundance.
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http://dx.doi.org/10.3390/v10010043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795456PMC
January 2018

Mitogen-Activated Protein Kinase Kinase 5 Regulates Proliferation and Biosynthetic Processes in Procyclic Forms of Trypanosoma brucei.

J Proteome Res 2018 01 7;17(1):108-118. Epub 2017 Nov 7.

CRUK Beatson Institute, Glasgow G61 1BD, U.K.

The pathogenic protozoan T. brucei alternates into distinct developmental stages in the mammalian and insect hosts. The mitogen-activated protein kinase (MAPK) signaling pathways transduce extracellular stimuli into a range of cellular responses, which ultimately lead to the adaptation to the external environment. Here, we combined a loss of function approach with stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry (MS) to investigate the role of the mitogen-activated protein kinase kinase 5 (MKK5) in T. brucei. The silencing of MKK5 significantly decreased the proliferation of procyclic forms of T. brucei. To shed light on the molecular alterations associated with this phenotype, we measured the total proteome and phosphoproteome of cells silenced for MKK5. In the total proteome, we observed a general decrease in proteins related to ribosome and translation as well as down-regulation of several components of the fatty acids biosynthesis pathway. In addition, we observed alterations in the protein levels and phosphorylation of key metabolic enzymes, which point toward a suppression of the oxidative metabolism. Taken together, our findings show that the silencing of MKK5 alters cell growth, energy metabolism, protein and fatty acids biosynthesis in procyclic T. brucei.
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http://dx.doi.org/10.1021/acs.jproteome.7b00415DOI Listing
January 2018

Quantitative proteome and phosphoproteome analyses highlight the adherent population during Trypanosoma cruzi metacyclogenesis.

Sci Rep 2017 08 29;7(1):9899. Epub 2017 Aug 29.

Functional Genomics Laboratory, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil.

Trypanosoma cruzi metacyclogenesis is a natural process that occurs inside the triatomine vector and corresponds to the differentiation of non-infective epimastigotes into infective metacyclic trypomastigotes. The biochemical alterations necessary for the differentiation process have been widely studied with a focus on adhesion and nutritional stress. Here, using a mass spectrometry approach, a large-scale phospho(proteome) study was performed with the aim of understanding the metacyclogenesis processes in a quantitative manner. The results indicate that major modulations in the phospho(proteome) occur under nutritional stress and after 12 and 24 h of adhesion. Significant changes involve key cellular processes, such as translation, oxidative stress, and the metabolism of macromolecules, including proteins, lipids, and carbohydrates. Analysis of the signalling triggered by kinases and phosphatases from 7,336 identified phosphorylation sites demonstrates that 260 of these sites are modulated throughout the differentiation process, and some of these modulated proteins have previously been identified as drug targets in trypanosomiasis treatment. To the best of our knowledge, this study provides the first quantitative results highlighting the modulation of phosphorylation sites during metacyclogenesis and the greater coverage of the proteome to the parasite during this process. The data are available via ProteomeXchange with identifier number PXD006171.
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http://dx.doi.org/10.1038/s41598-017-10292-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574995PMC
August 2017

Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae.

Insect Biochem Mol Biol 2017 09 3;88:63-70. Epub 2017 Aug 3.

Instituto Aggeu Magalhães-FIOCRUZ, Recife, PE, 50740-465, Brazil. Electronic address:

A binary mosquitocidal toxin composed of a three-domain Cry-like toxin (Cry48Aa) and a binary-like toxin (Cry49Aa) was identified in Lysinibacillus sphaericus. Cry48Aa/Cry49Aa has action on Culex quinquefasciatus larvae, in particular, to those that are resistant to the Bin Binary toxin, which is the major insecticidal factor from L. sphaericus-based biolarvicides, indicating that Cry48Aa/Cry49Aa interacts with distinct target sites in the midgut and can overcome Bin toxin resistance. This study aimed to identify Cry48Aa/Cry49Aa ligands in C. quinquefasciatus midgut through binding assays and mass spectrometry. Several proteins, mostly from 50 to 120 kDa, bound to the Cry48Aa/Cry49Aa toxin were revealed by toxin overlay and pull-down assays. These proteins were identified against the C. quinquefasciatus genome and after analysis a set of 49 proteins were selected which includes midgut bound proteins such as aminopeptidases, amylases, alkaline phosphatases in addition to molecules from other classes that can be potentially involved in this toxin's mode of action. Among these, some proteins are orthologs of Cry receptors previously identified in mosquito larvae, as candidate receptors for Cry48Aa/Cry49Aa toxin. Further investigation is needed to evaluate the specificity of their interactions and their possible role as receptors.
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http://dx.doi.org/10.1016/j.ibmb.2017.08.001DOI Listing
September 2017

Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host.

Mol Microbiol 2017 Jun 9;104(5):712-736. Epub 2017 May 9.

Instituto Carlos Chagas, FIOCRUZ, Curitiba, PR, Brazil.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell-derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long-term cultured epimastigotes: resistance to complement-mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up-regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host-parasite interactions, showing that rdEpi can be infective to the mammalian host.
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http://dx.doi.org/10.1111/mmi.13653DOI Listing
June 2017

The Protein Content of Extracellular Vesicles Derived from Expanded Human Umbilical Cord Blood-Derived CD133 and Human Bone Marrow-Derived Mesenchymal Stem Cells Partially Explains Why both Sources are Advantageous for Regenerative Medicine.

Stem Cell Rev Rep 2017 Apr;13(2):244-257

Instituto Carlos Chagas, Fiocruz-Paraná, Rua Professor Algacyr Munhoz Mader, 3775, Curitiba, PR, 81350-010, Brazil.

Adult stem cells have beneficial effects when exposed to damaged tissue due, at least in part, to their paracrine activity, which includes soluble factors and extracellular vesicles (EVs). Given the multiplicity of signals carried by these vesicles through the horizontal transfer of functional molecules, human mesenchymal stem cell (hMSCs) and CD133 cell-derived EVs have been tested in various disease models and shown to recover damaged tissues. In this study, we profiled the protein content of EVs derived from expanded human CD133 cells and bone marrow-derived hMSCs with the intention of better understanding the functions performed by these vesicles/cells and delineating the most appropriate use of each EV in future therapeutic procedures. Using LC-MS/MS analysis, we identified 623 proteins for expanded CD133-EVs and 797 proteins for hMSCs-EVs. Although the EVs from both origins were qualitatively similar, when protein abundance was considered, hMSCs-EVs and CD133-EVs were different. Gene Ontology (GO) enrichment analysis in CD133-EVs revealed proteins involved in a variety of angiogenesis-related functions as well proteins related to the cytoskeleton and highly implicated in cell motility and cellular activation. In contrast, when overrepresented proteins in hMSCs-EVs were analyzed, a GO cluster of immune response-related genes involved with immune response-regulating factors acting on phagocytosis and innate immunity was identified. Together our data demonstrate that from the point of view of protein content, expanded CD133-EVs and hMSCs-EVs are in part similar but also sufficiently different to reflect the main beneficial paracrine effects widely reported in pre-clinical studies using expanded CD133 cells and/or hBM-MSCs.
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http://dx.doi.org/10.1007/s12015-016-9715-zDOI Listing
April 2017

The MAP kinase MAPKLK1 is essential to Trypanosoma brucei proliferation and regulates proteins involved in mRNA metabolism.

J Proteomics 2017 02 27;154:118-127. Epub 2016 Dec 27.

Functional Genomics Laboratory, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil; Mass Spectrometry Facility RPT02H, Carlos Chagas Institute, Fiocruz, Parana, Brazil. Electronic address:

Protein phosphorylation and dephosphorylation events regulate many cellular processes. The identification of all phosphorylation sites and their association to a respective protein kinase or phosphatase is a challenging and crucial step to have a deeper understanding of the effects of signaling networks on cells. Pathogenic trypanosomatids have a large number of protein kinases and phosphatases in comparison to other organisms, which reinforces the relevance of the phosphorylation process in these early eukaryotes, nevertheless little is known about protein phosphorylation in these protozoa. In this context, the role of a MAP kinase-like kinase (MAPKLK1), observed to be essential to proliferation of procyclic Trypanosoma brucei, was studied. After silencing MAPKLK1 expression by RNAi, the cells were evaluated by SILAC MS-based proteomics and RNA-Seq. We identified 1756 phosphorylation sites of which 384 were not previously described in T. brucei. Despite being essential, few modulations were observed at the phosphorylation patterns and gene expression levels of MAPKLK1 knockdown. These indirect targets and potential substrates of MAPKLK1 are related to key cellular processes enriched to mRNA processing and stability control.

Significance: The field of cell signaling is a promising topic of study for trypanosomatids, since little is known about this topic and the gene expression regulation occurs at post-transcriptional level. In this sense, the present work increases the knowledge on protein phosphorylation process in Trypanosoma brucei. We depleted one MAP kinase (MAPKLK1) of T. brucei and evaluated the effects on the cell. We showed that MAPKLK1 is essential to the cell, while few modulations on phosphoproteome, proteome and transcriptome are observed with its depletion. Although in low number, the changes in phosphoproteome were significant, presenting possible substrate candidates of MAPKLK1 and indirect targets related to mRNA processing and stability control, metabolic pathways, among others. This result provides insights in the phosphorylation network of T. brucei, a model organism that impacts human and animal health.
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http://dx.doi.org/10.1016/j.jprot.2016.12.011DOI Listing
February 2017

Tissue-Derived Signals for Mesenchymal Stem Cell Stimulation: Role of Cardiac and Umbilical Cord Microenvironments.

Cells Tissues Organs 2017 2;203(3):173-182. Epub 2016 Nov 2.

Laboratory of Basic Biology of Stem Cells, Carlos Chagas Institute, Fiocruz-Parana, Brazil.

The tissue microenvironment regulates such stem cell behaviors as self-renewal and differentiation. Attempts to mimic components of these microenvironments could provide new strategies for culturing and directing the behavior of stem cells. The aim of the present study was to mimic cardiac and umbilical cord tissue microenvironments in vitro and compare the resulting bone marrow-derived mesenchymal stem cell (BM-MSC) behaviors. We generated tissue microenvironments using conditioned medium (CM) and extracellular matrix (ECM) samples obtained from human heart and umbilical cord tissue explant cultures and by tissue decellularization. Mass spectrometry and immunostaining were used to characterize and determine the specific protein profiles of the ECMs and CMs. We demonstrated that the ECMs and CMs were not cytotoxic to BM-MSCs and could thus be tested via cell culture. The BM-MSCs showed a higher proliferation rate when cultured with umbilical cord-derived CM compared with the other analyzed conditions. Furthermore, the ECMs increased cell adhesion and migration. However, although the conditions tested in this work were able to maintain the viability and affect the proliferation, adhesion and migration of BM-MSCs in vitro, mimicking tissue microenvironments using ECM and CM was not sufficient to induce the cardiomyogenic differentiation of BM-MSCs. The present study provides a thorough characterization of the biological activity of these ECMs and CMs in human BM-MSC cultures.
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http://dx.doi.org/10.1159/000450600DOI Listing
May 2017

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation.

Mem Inst Oswaldo Cruz 2015 Aug 21;110(5):687-90. Epub 2015 Jul 21.

Laboratório de Genômica Funcional, Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, BR.

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzigenes and is freely available for scientific community.
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http://dx.doi.org/10.1590/0074-02760150074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569835PMC
August 2015

Synthesis and Antibacterial Activity of Alkylated Diamines and Amphiphilic Amides of Quinic Acid Derivatives.

Chem Biol Drug Des 2015 Sep 20;86(3):344-50. Epub 2015 Jan 20.

Departamento de Química, Universidade Federal de Juiz de Fora, 36036-330, Juiz de Fora, MG, Brazil.

Different series of N-alkylated diamines and their derivatives condensed to quinic acid were synthesized and tested for antibacterial properties against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. The lipophilic chain and carbohydrate moiety modulate the antibacterial activity and the compounds showed a structure-activity relationship. Overall, 11 compounds displayed better activity than chloramphenicol against Gram-positive and Gram-negative bacteria. Monoalkylated amines 2a-h displayed an activity similar to that of ethambutol against Mycobacterium tuberculosis.
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http://dx.doi.org/10.1111/cbdd.12498DOI Listing
September 2015

LM14 defined medium enables continuous growth of Trypanosoma cruzi.

BMC Microbiol 2014 Sep 10;14:238. Epub 2014 Sep 10.

Background: Trypanosoma cruzi, the etiologic agent of Chagas disease, alternates between distinct morphological and functional forms during its life cycle. Axenic multiplication and differentiation processes of this protozoan parasite can be reproduced in vitro, enabling the isolation and study of the different evolutionary forms. Although there are several publications attempting the cultivation of T. cruzi under chemically defined conditions, in our experience none of the published media are capable of maintaining T. cruzi in continuous growth.

Results: In this work we modified a known chemically defined medium for Trypanosoma brucei growth. The resulting LM14 and LM14B defined media enabled cultivation of five different strains of T. cruzi for more than forty passages until now. The parasite's biological characteristics such as morphology and differentiation to metacyclic trypomastigotes were maintained when defined media is used.

Conclusions: The establishment of a defined medium for T. cruzi cultivation is an important tool for basic biological research allowing several different approaches, providing new perspectives for further studies related to cell biology of this parasite.
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http://dx.doi.org/10.1186/s12866-014-0238-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172853PMC
September 2014

Towards the phosphoproteome of trypanosomatids.

Subcell Biochem 2014 ;74:351-78

Functional Genomics Laboratory, Fiocruz - Parana, Brazil.

The identification and localization of protein phosphorylation sites provide clues to what proteins or pathways might be activated in a given condition, helping to improve our understanding about signaling networks. Advances in strategies for enrichment of phosphorylated peptides/proteins, mass spectrometry (MS) instrumentation, and specific MS techniques for identification and quantification of post-translational modifications have allowed for large-scale mapping of phosphorylation sites, promoting the field of phosphoproteomics. The great promise of phosphoproteomics is to unravel the dynamics of signaling networks, a layer of the emerging field of systems biology. Until a few years ago only a small number of phosphorylation sites had been described. Following large-scale trends, recent phosphoproteomic studies have reported the mapping of thousands of phosphorylation sites in trypanosomatids. However, quantitative information about the regulation of such sites in different conditions is still lacking. In this chapter, we provide a historical overview of phosphoproteomic studies for trypanosomatids and discuss some challenges and perspectives in the field.
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http://dx.doi.org/10.1007/978-94-007-7305-9_15DOI Listing
June 2014

A high-throughput cloning system for reverse genetics in Trypanosoma cruzi.

BMC Microbiol 2010 Oct 13;10:259. Epub 2010 Oct 13.

Instituto Carlos Chagas, FIOCRUZ, Curitiba, Parana, Brazil.

Background: The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.

Results: We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.

Conclusions: We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.
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http://dx.doi.org/10.1186/1471-2180-10-259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020659PMC
October 2010