Publications by authors named "Giuseppe Palmisano"

81 Publications

Protein glycosylation in extracellular vesicles: Structural characterization and biological functions.

Mol Immunol 2021 Apr 29;135:226-246. Epub 2021 Apr 29.

Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil. Electronic address:

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles involved in intercellular communication, delivery of biomolecules from donor to recipient cells, cellular disposal and homeostasis, potential biomarkers and drug carriers. The content of EVs includes DNA, lipids, metabolites, proteins, and microRNA, which have been studied in various diseases, such as cancer, diabetes, pregnancy, neurodegenerative, and cardiovascular disorders. EVs are enriched in glycoconjugates and exhibit specific glycosignatures. Protein glycosylation is a co- and post-translational modification (PTM) that plays an important role in the expression and function of exosomal proteins. N- and O-linked protein glycosylation has been mapped in exosomal proteins. The purpose of this review is to highlight the importance of glycosylation in EVs proteins. Initially, we describe the main PTMs in EVs with a focus on glycosylation. Then, we explore glycan-binding proteins describing the main findings of studies that investigated the glycosylation of EVs in cancer, pregnancy, infectious diseases, diabetes, mental disorders, and animal fluids. We have highlighted studies that have developed innovative methods for studying the content of EVs. In addition, we present works related to lipid glycosylation. We explored the content of studies deposited in public databases, such as Exocarta and Vesiclepedia. Finally, we discuss analytical methods for structural characterization of glycoconjugates and present an overview of the critical points of the study of glycosylation EVs, as well as perspectives in this field.
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http://dx.doi.org/10.1016/j.molimm.2021.04.017DOI Listing
April 2021

Adverse pregnancy outcomes are associated with Plasmodium vivax malaria in a prospective cohort of women from the Brazilian Amazon.

PLoS Negl Trop Dis 2021 Apr 29;15(4):e0009390. Epub 2021 Apr 29.

Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

Background: Malaria in Brazil represents one of the highest percentages of Latin America cases, where approximately 84% of infections are attributed to Plasmodium (P.) vivax. Despite the high incidence, many aspects of gestational malaria resulting from P. vivax infections remain poorly studied. As such, we aimed to evaluate the consequences of P. vivax infections during gestation on the health of mothers and their neonates in an endemic area of the Amazon.

Methods And Findings: We have conducted an observational cohort study in Brazilian Amazon between January 2013 and April 2015. 600 pregnant women were enrolled and followed until delivery. After applying exclusion criteria, 329 mother-child pairs were included in the analysis. Clinical data regarding maternal infection, newborn's anthropometric measures, placental histopathological characteristics, and angiogenic and inflammatory factors were evaluated. The presence of plasma IgG against the P. vivax (Pv) MSP119 protein was used as marker of exposure and possible associations with pregnancy outcomes were analyzed. Multivariate logistic regression analysis revealed that P. vivax infections during the first trimester of pregnancy are associated with adverse gestational outcomes such as premature birth (adjusted odds ratio [aOR] 8.12, 95% confidence interval [95%CI] 2.69-24.54, p < 0.0001) and reduced head circumference (aOR 3.58, 95%CI 1.29-9.97, p = 0.01). Histopathology analysis showed marked differences between placentas from P. vivax-infected and non-infected pregnant women, especially regarding placental monocytes infiltrate. Placental levels of vasomodulatory factors such as angiopoietin-2 (ANG-2) and complement proteins such as C5a were also altered at delivery. Plasma levels of anti-PvMSP119 IgG in infected pregnant women were shown to be a reliable exposure marker; yet, with no association with improved pregnancy outcomes.

Conclusions: This study indicates that P. vivax malaria during the first trimester of pregnancy represents a higher likelihood of subsequent poor pregnancy outcomes associated with marked placental histologic modification and angiogenic/inflammatory imbalance. Additionally, our findings support the idea that antibodies against PvMSP119 are not protective against poor pregnancy outcomes induced by P. vivax infections.
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http://dx.doi.org/10.1371/journal.pntd.0009390DOI Listing
April 2021

PhyloQuant approach provides insights into Trypanosoma cruzi evolution using a systems-wide mass spectrometry-based quantitative protein profile.

Commun Biol 2021 Mar 11;4(1):324. Epub 2021 Mar 11.

Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

The etiological agent of Chagas disease, Trypanosoma cruzi, is a complex of seven genetic subdivisions termed discrete typing units (DTUs), TcI-TcVI and Tcbat. The relevance of T. cruzi genetic diversity to the variable clinical course of the disease, virulence, pathogenicity, drug resistance, transmission cycles and ecological distribution requires understanding the parasite origin and population structure. In this study, we introduce the PhyloQuant approach to infer the evolutionary relationships between organisms based on differential mass spectrometry-based quantitative features. In particular, large scale quantitative bottom-up proteomics features (MS1, iBAQ and LFQ) were analyzed using maximum parsimony, showing a correlation between T. cruzi DTUs and closely related trypanosomes' protein expression and sequence-based clustering. Character mapping enabled the identification of synapomorphies, herein the proteins and their respective expression profiles that differentiate T. cruzi DTUs and trypanosome species. The distance matrices based on phylogenetics and PhyloQuant clustering showed statistically significant correlation highlighting the complementarity between the two strategies. Moreover, PhyloQuant allows the identification of differentially regulated and strain/DTU/species-specific proteins, and has potential application in the identification of specific biomarkers and candidate therapeutic targets.
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http://dx.doi.org/10.1038/s42003-021-01762-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952728PMC
March 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic Onchocerca lupi.

PLoS Negl Trop Dis 2021 Feb 10;15(2):e0009027. Epub 2021 Feb 10.

Department of Veterinary Medicine, University of Bari, Bari, Italy.

Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.
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http://dx.doi.org/10.1371/journal.pntd.0009027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875354PMC
February 2021

Serum Proteomics Reveals Alterations in Protease Activity, Axon Guidance, and Visual Phototransduction Pathways in Infants With Exposure to Zika Virus Without Congenital Zika Syndrome.

Front Cell Infect Microbiol 2020 18;10:577819. Epub 2020 Nov 18.

GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.

In 2015, ZIKV infection attracted international attention during an epidemic in the Americas, when neurological disorders were reported in infants who had their mothers exposed to ZIKV during pregnancy. World Health Organization (WHO) epidemiological data show that 5 to 15% of neonates exposed to ZIKV in the uterus have complications included in abnormalities related to Congenital Zika Syndrome (CZS). The risk of complications after birth is not well documented, however, clinical evidence shows that 6% of infants exposed to ZIKV during pregnancy have complications present at birth, and this rate rises to 14% when medical monitoring is performed in all exposed infants, regardless of birth condition. Thus, the evaluation and monitoring of all exposed infants are of foremost importance as the development of late complications has been increasingly supported by clinical evidence. The identification of changes in protein profile of infants exposed to ZIKV without CZS could provide valuable findings to better understand molecular changes in this cohort. Here, we use a shotgun-proteomics approach to investigate alterations in the serum of infants without CZS symptoms but exposed to intrauterine ZIKV (ZIKV) compared to unexposed controls (CTRL). A complex pattern of differentially expressed proteins was identified, highlighting the dysregulation of proteins involved in axon orientation, visual phototransduction, and global protease activity in children exposed to ZIKV without CZS. These data support the importance of monitoring children exposed to ZIKV during gestation and without early CZS symptoms. Our study is the first to assess molecular evidence of possible late disorders in children victims of the ZIKV outbreak in the Americas. We emphasize the importance of medical monitoring of symptomatic and asymptomatic children, as apparently unexplained late neurological and eye disorders may be due to intrauterine ZIKV exposure.
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http://dx.doi.org/10.3389/fcimb.2020.577819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708324PMC
November 2020

The intracellular bacterium Rickettsia rickettsii exerts an inhibitory effect on the apoptosis of tick cells.

Parasit Vectors 2020 Dec 1;13(1):603. Epub 2020 Dec 1.

Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.

Background: Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector-pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26.

Methods: The proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined.

Results: Tick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth.

Conclusions: Taken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.
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http://dx.doi.org/10.1186/s13071-020-04477-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706286PMC
December 2020

The Complexity and Dynamics of the Tissue Glycoproteome Associated With Prostate Cancer Progression.

Mol Cell Proteomics 2021 Jan 5;20:100026. Epub 2021 Jan 5.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, USP, São Paulo, Brazil. Electronic address:

The complexity and dynamics of the immensely heterogeneous glycoproteome of the prostate cancer (PCa) tumor microenvironment remain incompletely mapped, a knowledge gap that impedes our molecular-level understanding of the disease. To this end, we have used sensitive glycomics and glycoproteomics to map the protein-, cell-, and tumor grade-specific N- and O-glycosylation in surgically removed PCa tissues spanning five histological grades (n = 10/grade) and tissues from patients with benign prostatic hyperplasia (n = 5). Quantitative glycomics revealed PCa grade-specific alterations of the oligomannosidic-, paucimannosidic-, and branched sialylated complex-type N-glycans, and dynamic remodeling of the sialylated core 1- and core 2-type O-glycome. Deep quantitative glycoproteomics identified ∼7400 unique N-glycopeptides from 500 N-glycoproteins and ∼500 unique O-glycopeptides from nearly 200 O-glycoproteins. With reference to a recent Tissue and Blood Atlas, our data indicate that paucimannosidic glycans of the PCa tissues arise mainly from immune cell-derived glycoproteins. Furthermore, the grade-specific PCa glycosylation arises primarily from dynamics in the cellular makeup of the PCa tumor microenvironment across grades involving increased oligomannosylation of prostate-derived glycoproteins and decreased bisecting GlcNAcylation of N-glycans carried by the extracellular matrix proteins. Furthermore, elevated expression of several oligosaccharyltransferase subunits and enhanced N-glycoprotein site occupancy were observed associated with PCa progression. Finally, correlations between the protein-specific glycosylation and PCa progression were observed including increased site-specific core 2-type O-glycosylation of collagen VI. In conclusion, integrated glycomics and glycoproteomics have enabled new insight into the complexity and dynamics of the tissue glycoproteome associated with PCa progression generating an important resource to explore the underpinning disease mechanisms.
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http://dx.doi.org/10.1074/mcp.RA120.002320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010466PMC
January 2021

Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From , Affecting Host-Pathogen Interactions by Hindering Adhesion to Host Cells.

Front Cell Infect Microbiol 2020 23;10:417. Epub 2020 Sep 23.

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Brazil.

is a microaerophilic protozoan that is an important etiologic agent of diarrhea worldwide. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of EV production, revealing its capacity to shed two different enriched EV populations: large (LEV) and small extracellular vesicles (SEV) and identified relevant adhesion functions associated with the larger population. Proteomic analysis revealed differences in proteins relevant for virulence and host-pathogen interactions between the two EV subsets, such as cytoskeletal and anti-oxidative stress response proteins in LEVS. We assessed the effect of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from . The compounds were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of LEVs and reducing parasite attachment to host cells . Our results suggest that LEVs and SEVs have a different role in host-pathogen interaction, and that treatment with EV-inhibitors may be a novel treatment strategy for recurrent giardiasis.
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http://dx.doi.org/10.3389/fcimb.2020.00417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539837PMC
September 2020

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

Biochem Pharmacol 2020 12 23;182:114230. Epub 2020 Sep 23.

Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, São Paulo, Brazil. Electronic address:

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.
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http://dx.doi.org/10.1016/j.bcp.2020.114230DOI Listing
December 2020

Lights and Shadows of TORCH Infection Proteomics.

Genes (Basel) 2020 08 5;11(8). Epub 2020 Aug 5.

Glycoproteomics Laboratory, Department of Parasitology, University of Sao Paulo, Sao Paulo 05508-000, Brazil.

Congenital abnormalities cause serious fetal consequences. The term TORCH is used to designate the most common perinatal infections, where: (T) refers to toxoplasmosis, (O) means "others" and includes syphilis, varicella-zoster, parvovirus B19, zika virus (ZIKV), and malaria among others, (R) refers to rubella, (C) relates to cytomegalovirus infection, and (H) to herpes simplex virus infections. Among the main abnormalities identified in neonates exposed to congenital infections are central nervous system (CNS) damage, microcephaly, hearing loss, and ophthalmological impairment, all requiring regular follow-up to monitor its progression. Protein changes such as mutations, post-translational modifications, abundance, structure, and function may indicate a pathological condition before the onset of the first symptoms, allowing early diagnosis and understanding of a particular disease or infection. The term "proteomics" is defined as the science that studies the proteome, which consists of the total protein content of a cell, tissue or organism in a given space and time, including post-translational modifications (PTMs) and interactions between proteins. Currently, quantitative bottom-up proteomic strategies allow rapid and high throughput characterization of complex biological mixtures. Investigating proteome modulation during host-pathogen interaction helps in elucidating the mechanisms of infection and in predicting disease progression. This "molecular battle" between host and pathogen is a key to identify drug targets and diagnostic markers. Here, we conducted a survey on proteomic techniques applied to congenital diseases classified in the terminology "TORCH", including toxoplasmosis, ZIKV, malaria, syphilis, human immunodeficiency virus (HIV), herpes simplex virus (HSV) and human cytomegalovirus (HCVM). We have highlighted proteins and/or protein complexes actively involved in the infection. Most of the proteomic studies reported have been performed in cell line models, and the evaluation of tissues (brain, muscle, and placenta) and biofluids (plasma, serum and urine) in animal models is still underexplored. Moreover, there are a plethora of studies focusing on the pathogen or the host without considering the triad mother-fetus-pathogen as a dynamic and interconnected system.
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http://dx.doi.org/10.3390/genes11080894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464470PMC
August 2020

Protein glycosylation in spp.

Mol Omics 2020 10;16(5):407-424

GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Avenida Lineu Prestes 1374, Butantã, Sao Paulo - 05508-000, Brazil.

Protein glycosylation is a co- and post-translational modification that, in Leishmania parasites, plays key roles in vector-parasite-vertebrate host interaction. In the mammalian host, Leishmania protein glycosylation is involved in virulence, host cell invasion, and immune evasion and modulation. The Leishmania glycocalyx is composed by a dense array of glycoconjugates including lipophosphoglycan, glycoinositolphospholipids, glycoproteins and proteophosphoglycans which varies in composition between Leishmania species and developmental stages. The current knowledge on Leishmania protein glycosylation is quite limited. The development of novel analytical tools to characterize the Leishmania glycoproteome and the expanding toolbox to modulate the parasite glycocode will help in deciphering the processes involved in Leishmania-host interaction. This review will recapitulate the current knowledge of Leishmania protein glycosylation, and glycan structures reported, and the potential application of mass spectrometry-based analysis for system-wide Leishmania glycoproteome and glycome analysis.
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http://dx.doi.org/10.1039/d0mo00043dDOI Listing
October 2020

Cellular Imprinting Proteomics Assay: A Novel Method for Detection of Neural and Ocular Disorders Applied to Congenital Zika Virus Syndrome.

J Proteome Res 2020 11 4;19(11):4496-4515. Epub 2020 Aug 4.

GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.

Congenital Zika syndrome was first described due to increased incidence of congenital abnormalities associated with Zika virus (ZIKV) infection. Since the eye develops as part of the embryo central nervous system (CNS) structure, it becomes a specialized compartment able to display symptoms of neurodegenerative diseases and has been proposed as a noninvasive approach to the early diagnosis of neurological diseases. Ocular lesions result from defects that occurred during embryogenesis and can become apparent in newborns exposed to ZIKV. Furthermore, the absence of microcephaly cannot exclude the occurrence of ocular lesions and other CNS manifestations. Considering the need for surveillance of newborns and infants with possible congenital exposure, we developed a method termed cellular imprinting proteomic assay (CImPA) to evaluate the ocular surface proteome specific to infants exposed to ZIKV during gestation compared to nonexposure. CImPA combines surface cells and fluid capture using membrane disks and a large-scale quantitative proteomics approach, which allowed the first-time report of molecular alterations such as neutrophil degranulation, cell death signaling, ocular and neurological pathways, which are associated with ZIKV infection with and without the development of congenital Zika syndrome, CZS. Particularly, infants exposed to ZIKV during gestation and without early clinical symptoms could be detected using the CImPA method. Lastly, this methodology has broad applicability as it could be translated in the study of several neurological diseases to identify novel diagnostic biomarkers. Data are available via ProteomeXchange with identifier PXD014038.
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http://dx.doi.org/10.1021/acs.jproteome.0c00320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640952PMC
November 2020

Global changes in nitration levels and DNA binding profile of Trypanosoma cruzi histones induced by incubation with host extracellular matrix.

PLoS Negl Trop Dis 2020 05 29;14(5):e0008262. Epub 2020 May 29.

Departamento de Bioquímica Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil.

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.
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http://dx.doi.org/10.1371/journal.pntd.0008262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286532PMC
May 2020

Phosphoproteomics of Aspergillus fumigatus Exposed to the Antifungal Drug Caspofungin.

mSphere 2020 05 27;5(3). Epub 2020 May 27.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil

is an opportunistic and allergenic pathogenic fungus, responsible for fungal infections in humans. infections are usually treated with polyenes, azoles, or echinocandins. Echinocandins, such as caspofungin, can inhibit the biosynthesis of the β-1,3-glucan polysaccharide, affecting the integrity of the cell wall and leading to fungal death. In some strains, caspofungin treatment at high concentrations induces an increase of fungal growth, a phenomenon called the aspofungin aradoxical ffect (CPE). Here, we analyze the proteome and phosphoproteome of the wild-type strain and of mitogen-activated protein kinase (MAPK) and null mutant strains during CPE (2 μg/ml caspofungin for 1 h). The wild-type proteome showed 75 proteins and 814 phosphopeptides (corresponding to 520 proteins) altered in abundance in response to caspofungin treatment. The Δ (Δ caspofungin/wild-type caspofungin) and Δ (Δ caspofungin/wild-type caspofungin) strains displayed 626 proteins and 1,236 phosphopeptides (corresponding to 703 proteins) and 101 proteins and 1,217 phosphopeptides (corresponding to 645 proteins), respectively, altered in abundance. Functional characterization of the phosphopeptides from the wild-type strain exposed to caspofungin showed enrichment for transcription factors, protein kinases, and cytoskeleton proteins. Proteomic analysis of the Δ and Δ mutants indicated that control of proteins involved in metabolism, such as in production of secondary metabolites, was highly represented in both mutants. Results of functional categorization of phosphopeptides from both mutants were very similar and showed a high number of proteins with decreased phosphorylation of proteins involved in transcriptional control, DNA/RNA binding, cell cycle control, and DNA processing. This report reveals novel transcription factors involved in caspofungin tolerance. is an opportunistic human-pathogenic fungus causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. Caspofungin is an echinocandin that impacts the construction of the fungal cell wall by inhibiting the biosynthesis of the β-1,3-glucan polysaccharide. Caspofungin is a fungistatic drug and is recommended as a second-line therapy for treatment of aspergillosis. Treatment at high concentrations induces an increase of fungal growth, a phenomenon called the aspofungin aradoxical ffect (CPE). Collaboration between the mitogen-activated protein kinases (MAPK) of the cell wall integrity (MapkA) and high-osmolarity glycerol (SakA) pathways is essential for CPE. Here, we investigate the global proteome and phosphoproteome of wild-type, Δ, and Δ strains upon CPE. This study showed intense cross talk between the two MAPKs for the CPE and identified novel protein kinases and transcription factors possibly important for CPE. Increased understanding of how the modulation of protein phosphorylation may affect the fungal growth in the presence of caspofungin represents an important step in the development of new strategies and methods to combat the fungus inside the host.
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http://dx.doi.org/10.1128/mSphere.00365-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253599PMC
May 2020

Inflammasome activation and IL-1 signaling during placental malaria induce poor pregnancy outcomes.

Sci Adv 2020 03 4;6(10):eaax6346. Epub 2020 Mar 4.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil.

Placental malaria (PM) is associated with severe inflammation leading to abortion, preterm delivery, and intrauterine growth restriction. Innate immunity responses play critical roles, but the mechanisms underlying placental immunopathology are still unclear. Here, we investigated the role of inflammasome activation in PM by scrutinizing human placenta samples from an endemic area and ablating inflammasome components in a PM mouse model. The reduction in birth weight in babies from infected mothers is paralleled by increased placental expression of AIM2 and NLRP3 inflammasomes. Using genetic dissection, we reveal that inflammasome activation pathways are involved in the production and detrimental action of interleukin-1β (IL-1β) in the infected placenta. The IL-1R pharmacological antagonist Anakinra improved pregnancy outcomes by restoring fetal growth and reducing resorption in an experimental model. These findings unveil that IL-1β-mediated signaling is a determinant of PM pathogenesis, suggesting that IL-1R antagonists can improve clinical outcomes of malaria infection in pregnancy.
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http://dx.doi.org/10.1126/sciadv.aax6346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056302PMC
March 2020

Lipoatrophy-Associated Insulin Resistance and Hepatic Steatosis are Attenuated by Intake of Diet Rich in Omega 3 Fatty Acids.

Mol Nutr Food Res 2020 04 3;64(7):e1900833. Epub 2020 Feb 3.

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, 05508000, Brazil.

Scope: Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated.

Methods And Results: Severe lipoatrophic mice induced by PPAR-γ deletion exclusively in adipocytes (A-PPARγ KO) and littermate controls (A-PPARγ WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis.

Conclusion: Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.
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http://dx.doi.org/10.1002/mnfr.201900833DOI Listing
April 2020

Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance.

PLoS Genet 2019 12 30;15(12):e1008551. Epub 2019 Dec 30.

Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil.

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.
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http://dx.doi.org/10.1371/journal.pgen.1008551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948819PMC
December 2019

Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies.

Front Mol Neurosci 2019 5;12:279. Epub 2019 Dec 5.

Molecular and Integrative Physiological Sciences Program, Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, United States.

Importance: Non-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined.

Objectives: The aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches.

Design Settings And Participants: Ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies (4 babies with maternal ZIKV exposure during gestation and presence of clinical signs which included ocular abnormalities and microcephaly; 4 babies with maternal ZIKV exposure during gestation but no clinical signs; and 4 unaffected control babies with negative PCR for Zika virus and without clinical signs). Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments.

Results: Our optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations (cell keratinization, pyknosis, karyolysis, anucleation, and vacuolization). Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies.

Conclusions And Relevance: Our method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research.
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http://dx.doi.org/10.3389/fnmol.2019.00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6907025PMC
December 2019

Tissue Proteome Signatures Associated with Five Grades of Prostate Cancer and Benign Prostatic Hyperplasia.

Proteomics 2019 11 20;19(21-22):e1900174. Epub 2019 Oct 20.

Instituto de Ciências Biomédicas, Departamento de Parasitologia, Universidade de São Paulo, USP, São Paulo, CEP: 05508-000, Brazil.

The histology-based Gleason score (GS) of prostate cancer (PCa) tissue biopsy is the most accurate predictor of disease aggressiveness and an important measure to guide treatment strategies and patient management. The variability associated with PCa tumor sampling and the subjective determination of the GS are challenges that limit accurate diagnostication and prognostication. Thus, novel molecular signatures are needed to distinguish between indolent and aggressive forms of PCa for better patient management and outcomes. Herein, label-free LC-MS/MS proteomics is used to profile the proteome of 50 PCa tissues spanning five grade groups (n = 10 per group) relative to tissues from individuals with benign prostatic hyperplasia (BPH). Over 2000 proteins are identified albeit at different levels between and within the patient groups, revealing biological processes associated with specific grades. A panel of 11 prostate-derived proteins including IGKV3D-20, RNASET2, TACC2, ANXA7, LMOD1, PRCP, GYG1, NDUFV1, H1FX, APOBEC3C, and CTSZ display the potential to stratify patients from low and high PCa grade groups. Parallel reaction monitoring of the same sample cohort validate the differential expression of LMOD1, GYG1, IGKV3D-20, and RNASET2. The four proteins associated with low and high PCa grades reported here warrant further exploration as candidate biomarkers for PCa aggressiveness.
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http://dx.doi.org/10.1002/pmic.201900174DOI Listing
November 2019

Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

Proteomics 2019 11 16;19(21-22):e1900010. Epub 2019 Oct 16.

Rudolf-Becker-Laboratory, Institute of Pathology, University Hospital Bonn, Bonn, 53127, Germany.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man GlcNAc Fuc ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man GlcNAc Fuc , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-β-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.
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http://dx.doi.org/10.1002/pmic.201900010DOI Listing
November 2019

Proteomics and Leishmaniasis: Potential Clinical Applications.

Proteomics Clin Appl 2019 11 26;13(6):e1800136. Epub 2019 Aug 26.

Leishmaniasis laboratory, Institute of Biomedical Sciences, Department of Parasitology, University of São Paulo, São Paulo, Brazil.

Leishmaniases are diseases caused by protozoan parasites of the genus Leishmania. They are endemic in 98 countries, affect around 12 million people worldwide and may present several distinct clinical forms. Unfortunately, there are only a few drugs available for treatment of leishmaniasis, which are toxic and not always effective. Different parasite species and different clinical forms require optimization of the treatment or more specific therapies, which are not available. The emergence of resistance is also a matter of concern. Besides, diagnosis can sometimes be complicated due to atypical manifestations and associations with other pathologies. In this review, proteomic data are presented and discussed in terms of their application in important issues in leishmaniasis such as parasite resistance to chemotherapy, diagnosis of active disease in patients and dogs, markers for different clinical forms, identification of virulence factors, and their potential use in vaccination. It is shown that proteomics has contributed to the discovery of potential biomarkers for prognosis, diagnosis, therapeutics, monitoring of disease progression, treatment follow-up and identification of vaccine candidates for specific diseases. However, the authors believe its capabilities have not yet been fully explored for routine clinical analysis for several reasons, which will be presented in this review.
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http://dx.doi.org/10.1002/prca.201800136DOI Listing
November 2019

Detergent-resistant domains in Spodoptera frugiperda midgut microvillar membranes and their relation to microapocrine secretion.

Comp Biochem Physiol B Biochem Mol Biol 2019 Sep 24;235:8-18. Epub 2019 May 24.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Avenida Professor Lineu Prestes, 748, São Paulo 05508-000, Brazil. Electronic address:

The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery.
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http://dx.doi.org/10.1016/j.cbpb.2019.05.008DOI Listing
September 2019

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

Front Cell Neurosci 2019 15;13:64. Epub 2019 Mar 15.

Department of Parasitology, Institute of Biomedical Science, University of São Paulo, São Paulo, Brazil.

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.
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http://dx.doi.org/10.3389/fncel.2019.00064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436085PMC
March 2019

Liver proteomics unravel the metabolic pathways related to Feed Efficiency in beef cattle.

Sci Rep 2019 03 29;9(1):5364. Epub 2019 Mar 29.

Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of São Paulo, Pirassununga, 13635-900, Brazil.

Improving nutrient utilization efficiency is essential for livestock, given the current scenario of increasing demand for animal protein and sustainable resource use. In this context, understanding the biology of feed efficiency (FE) in beef cattle allows the development of markers for identification and selection of best animals for animal production. Thus, 98 young Nellore bulls were evaluated for FE and at the end of the experiment liver samples from six High Feed Efficient (HFE) and six Low Feed Efficient (LFE) animals were collected for protein extraction, digestion and analysis by HPLC-MS/MS. Data were analyzed for differential abundant proteins (DAPs), protein networks, and functional enrichment. Serum endotoxin was also quantified. We found 42 DAPs and 3 protein networks significantly related to FE. The main pathways associated with FE were: microbial metabolism; biosynthesis of fatty acids, amino acids and vitamins; glycolysis/gluconeogenesis; xenobiotic metabolism and; antigen processing and presentation. Serum endotoxins were significantly higher in LFE animals supporting the results. Therefore, the findings presented here confirmed the altered hepatic metabolism and pronounced hepatic inflammation in LFE animals supporting that the increased bacterial load is at least in part responsible for the hepatic lesions and inflammation in LFE animals.
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http://dx.doi.org/10.1038/s41598-019-41813-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441086PMC
March 2019

Novel site-specific PEGylated L-asparaginase.

PLoS One 2019 12;14(2):e0211951. Epub 2019 Feb 12.

Department of Biochemical and Pharmaceutical Technology, University of São Paulo, São Paulo, Brazil.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211951PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372183PMC
November 2019

Heat shock protein B1 is a key mediator of prolactin-induced beta-cell cytoprotection against oxidative stress.

Free Radic Biol Med 2019 04 27;134:394-405. Epub 2019 Jan 27.

Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo (USP), Av. Prof. Lineu Prestes 748, 05508-000 Sao Paulo, Brazil. Electronic address:

Maintaining islet cell viability in vitro, although challenging, appears to be a strategy for improving the outcome of pancreatic islet transplantation. We have shown that prolactin (PRL) leads to beta-cell cytoprotection against apoptosis, an effect mediated by heat shock protein B1 (HSPB1). Since the role of HSPB1 in beta-cells is still unclear and the hormone concentration used is not compatible with clinical applications because of all the side effects displayed by the hormone in other tissues, we explored the molecular mechanisms by which HSPB1 mediates beta-cell cytoprotection. Lysates from PRL- and/or cytokine-treated MIN6 beta-cells were subjected to HSPB1 immunoprecipitation followed by identification through mass spectrometry. PRL-treated cells presented an enrichment of several proteins co-precipitating with HSPB1. Of note were oxidative stress resistance-, protein degradation- and carbohydrate metabolism-related proteins. Wild type, HSPB1 silenced or overexpressing MIN6 cells were exposed to menadione and hydrogen peroxide and analysed for several oxidative stress parameters. HSPB1 knockdown rendered cells more sensitive to oxidative stress and led to a reduced antioxidant capacity, while prolactin induced an HSPB1-mediated cytoprotection against oxidative stress. HSPB1 overexpression, however, led to opposite effects. PRL treatment, HSPB1 silencing or overexpression did not change the expression nor activities of antioxidant enzymes, it also did not lead to a modulation of total glutathione levels nor G6PD expression. However, HSPB1 levels are related to a modulation of GSH/GSSG ratio, G6PD activity and NADPH/NADP  ratio. We have shown that HSPB1 is important for pro-survival effects against oxidative stress-induced beta-cell death. These results are in accordance with PRL-induced enrichment of HSPB1-interacting proteins related to protection against oxidative stress. Finally, our results outline the need of further studies investigating the importance of HSPB1 for beta-cell viability, since this could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.01.023DOI Listing
April 2019

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.

PLoS Negl Trop Dis 2018 10 26;12(10):e0006873. Epub 2018 Oct 26.

Laboratório de Parasitologia, Instituto Butantan, São Paulo, SP, Brasil.

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.
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http://dx.doi.org/10.1371/journal.pntd.0006873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221359PMC
October 2018

Author Correction: Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity.

Sci Rep 2018 Oct 23;8(1):15908. Epub 2018 Oct 23.

GlycoProteomics Laboratory, Department of Parasitology, ICB, University of São Paulo, São Paulo, Brazil.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-33284-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198013PMC
October 2018