Publications by authors named "José Cavalcante Souza Vieira"

26 Publications

  • Page 1 of 1

Feedlot diets containing different starch levels and additives change the cecal proteome involved in cattle's energy metabolism and inflammatory response.

Sci Rep 2022 04 5;12(1):5691. Epub 2022 Apr 5.

Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

Diets for feedlot cattle must be a higher energy density, entailing high fermentable carbohydrate content. Feed additives are needed to reduce possible metabolic disorders. This study aimed to analyze the post-rumen effects of different levels of starch (25%, 35%, and 45%) and additives (monensin or a blend of essential oils and exogenous α-amylase) in diets for Nellore feedlot cattle. The cecum tissue proteome was analyzed via two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and then differentially expressed protein spots were identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The use of blends of essential oils associated with α-amylase as a feed additive promoted the upregulation of enzymes such as triosephosphate isomerase, phosphoglycerate mutase, alpha-enolase, beta-enolase, fructose-bisphosphate aldolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase B, L-lactate dehydrogenase A chain, L-lactate dehydrogenase, and ATP synthase subunit beta, which promote the degradation of carbohydrates in the glycolysis and gluconeogenesis pathways and oxidative phosphorylation, support pyruvate metabolism through the synthesis of lactate from pyruvate, and participate in the electron transport chain, producing ATP from ADP in the presence of a proton gradient across the membrane. The absence of proteins related to inflammation processes (leukocyte elastase inhibitors) in the cecum tissues of animals fed essential oils and amylase may be because feed enzymes can remain active in the intestine and aid in the digestion of nutrients that escape rumen fermentation; conversely, the effect of monensin is more evident in the rumen and less than 10% results in post-ruminal action, corroborating the hypothesis that ionophore antibiotics have a limited effect on the microbiota and intestinal fermentation of ruminants. However, the increase in starch in these diets promoted a downregulation of enzymes linked to carbohydrate degradation, probably caused by damage to the cecum epithelium due to increased responses linked to inflammatory injuries.
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http://dx.doi.org/10.1038/s41598-022-09715-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8983758PMC
April 2022

Investigation of Protein Biomarkers and Oxidative Stress in Pinirampus pirinampu Exposed to Mercury Species from the Madeira River, Amazon-Brazil.

Biol Trace Elem Res 2022 Apr 5;200(4):1872-1882. Epub 2021 Sep 5.

Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

In recent decades, the scientific community has widely debated the contamination of fish in the Amazon region by mercury species. As the diet of riverside populations in the Amazon region is based mainly on fish, these populations are exposed to mercurial species that can cause serious and irreversible damage to their health. The risks of consuming fish exposed to mercurial species in the Amazon region have motivated toxicological investigations. However, the effect of mercurial species on protein and enzyme levels is still controversial. In this work, analytical and bioanalytical techniques Two-dimensional polyacrylamide gel electrophoresis [2D-PAGE] Graphite Furnace Atomic Absorption Spectrometry [GFAAS], and Mass Spectrometry in Sequence with Electrospray Ionization [ESI-MS/MS] were used to identify proteins associated with mercury (metal-binding protein) in muscle and liver tissues of the fish species Pinirampus pirinampu from the Madeira River, in the Brazilian Amazon. Enzymatic and lipid peroxidation analyses were also used to assess changes related to oxidative stress. Determinations of total mercury by GFAAS indicated higher concentrations in liver tissue (555 ± 19.0 µg kg) when compared to muscle tissue (60 ± 2.0 µg kg). The fractionation process of tissue proteomes by 2D-PAGE and subsequent mapping of mercury by GFAAS in the protein spots of the gels identified the presence of mercury in three spots of the liver tissue (concentrations in the range of 0.800 to 1.90 mg kg). The characterization of protein spots associated with mercury by ESI-MS/MS identified the enzymes triosephosphate isomerase A, adenylate kinase 2 mitochondrial, and glyceraldehyde-3-phosphate dehydrogenase as possible candidates for mercury exposure biomarkers. The muscle tissue did not show protein spots associated with mercury. Enzymatic activity decreased proportionally to the increase in mercury concentrations in the tissues.
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http://dx.doi.org/10.1007/s12011-021-02805-zDOI Listing
April 2022

Prospecting Biomarkers for Diagnostic and Therapeutic Approaches in Pythiosis.

J Fungi (Basel) 2021 May 28;7(6). Epub 2021 May 28.

Department of Chemical and Biological Sciences, Institute of Biosciences, São Paulo State University (UNESP), Botucatu 18618-689, Brazil.

Pythiosis, whose etiological agent is the oomycete , is a life-threatening disease that occurs mainly in tropical and subtropical countries, affecting several animal species. It is frequently found in horses in Brazil and humans in Thailand. The disease is difficult to diagnose because the pathogen's hyphae are often misdiagnosed as mucoromycete fungi in histological sections. Additionally, there is no specific antigen to use for rapid diagnosis, the availability of which could improve the prognosis in different animal species. In this scenario, we investigated which antigens are recognized by circulating antibodies in horses and humans with pythiosis from Brazil and Thailand, respectively, using 2D immunoblotting followed by mass spectrometry for the identification of antigens. We identified 23 protein spots, 14 recognized by pooled serum from horses and humans. Seven antigens were commonly recognized by both species, such as the heat-shock cognate 70 KDa protein, the heat-shock 70 KDa protein, glucan 1,3-beta-glucosidase, fructose-bisphosphate aldolase, serine/threonine-protein phosphatase, aconitate hydratase, and 14-3-3 protein epsilon. These results demonstrate that there are common antigens recognized by the immune responses of horses and humans, and these antigens may be studied as biomarkers for improving diagnosis and treatment.
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http://dx.doi.org/10.3390/jof7060423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8229905PMC
May 2021

Supplementation with an Inorganic Zinc Source in the Metalloproteomic Profile of Royal Jelly in Apis mellifera L.

Biol Trace Elem Res 2021 Nov 4;199(11):4308-4318. Epub 2021 Jan 4.

Faculty of Veterinary Medicine and Animal Science, UNESP- São Paulo State University, Botucatu, Brazil.

This study aimed to evaluate the quality of royal jelly produced by honeybees Apis mellifera supplemented with different concentrations of inorganic zinc (zinc sulfate monohydrate-0, 25, 50, and 75 ppm). Two-dimensional electrophoresis for the fractionation of royal jelly proteins was performed, and the zinc level was quantified by the flame atomic absorption spectrometry (FAAS) technique. Proteins were identified by electrospray ionization mass spectrometry (ESI MS MS). Analysis of variance followed by the Tukey test (P < 0.05) was used. Supplementation with the mineral zinc positively affected the quantification of proteins for treatments 50 and 75 ppm. However, all treatments independent of zinc concentrations showed fewer protein spots when compared to the control. All zinc-containing proteins were classified as major royal jelly proteins (MRJPs). The exposure of nursing bees to the mineral zinc in its inorganic form reduced the expression of six different MRJPs involved in larval and glands development of nursing bees (MRJP1, MRJP2, MRJP3, MRJP5, and MRJP7), however promoted an increase in the expression of royal jelly proteins involved in defense systems (MRJP8 and MRJP9). The results demonstrate that vital proteins and metabolic processes are impaired in nursing bees exposed to the mineral zinc in its inorganic form in all doses used affecting nutrition and maintenance of colonies.
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http://dx.doi.org/10.1007/s12011-020-02564-3DOI Listing
November 2021

Identification of potential molecular pathways involved in prostate carcinogenesis in offspring exposed to maternal malnutrition.

Aging (Albany NY) 2020 10 13;12(20):19954-19978. Epub 2020 Oct 13.

Department of Structural and Functional Biology, Institute of Biosciences, Sao Paulo State University (UNESP), Botucatu 18618-689, São Paulo, Brazil.

The developmental origins of health and disease concept links adult diseases with early-life exposure to inappropriate environmental conditions. Intrauterine and postnatal malnutrition may lead to an increased incidence of type 2 diabetes, obesity, and cardiovascular diseases. Maternal malnutrition (MM) has also been associated with prostate carcinogenesis. However, the molecular mechanisms associated with this condition remain poorly understood. Using a proteomic analysis, we demonstrated that MM changed the levels of proteins associated with growth factors, estrogen signaling, detoxification, and energy metabolism in the prostate of both young and old rats. These animals also showed increased levels of molecular markers of endoplasmic reticulum function and histones. We further performed an analysis that identified commonly deregulated proteins in the ventral prostate of old rats submitted to MM with a mouse model and patients with prostate cancer. In conclusion, our results demonstrated that estrogenic signaling pathways, endoplasmic reticulum functions, energy metabolism, and molecular sensors of protein folding and Ca2+ homeostasis, besides histone, and RAS-GTPase family appear to be involved in this process. Knowledge of these factors may raise discussions regarding the role of maternal dietary intervention as a public policy for the lifelong prevention of chronic diseases.
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http://dx.doi.org/10.18632/aging.104093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655221PMC
October 2020

Metalloproteomic Strategies for Identifying Proteins as Biomarkers of Mercury Exposure in Serrasalmus rhombeus from the Amazon Region.

Biol Trace Elem Res 2021 Feb 24;199(2):712-720. Epub 2020 May 24.

School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Rua Prof R. Dr. Valter Maurício Corrêa, s/n, Botucatu, SP, 18.618-681, Brazil.

This manuscript describes the results of a metalloproteomic study of mercury in samples of muscle and liver tissue of the species Serrasalmus rhombeus, popularly known as black piranha and characterised as the most voracious and aggressive predator in the Brazilian Amazon. The metalloproteomic study involved using two-dimensional electrophoresis (2D PAGE) to fractionate the proteome of the muscle and liver tissue samples, along with atomic absorption spectrometry in a graphite furnace (GFAAS) to identify mercury associated with protein SPOTs and mass spectrometry with electrospray ionisation (ESI-MS/MS) to characterise the mercury-binding proteins. The protein SPOTs characterised showed concentrations in the order of 156 mg kg, which ranks as the highest concentrations of mercury determined so far in metalloproteomic studies involving fish species in the Amazon region. Based on FASTA sequences of proteins characterised by ESI-MS/MS, bioinformatics studies were performed that allowed identifying nine proteins with characteristics of biomarkers of mercury exposure. Of those proteins, glutathione peroxidase stands out as an enzyme of great importance in the antioxidant defence of organisms subjected to oxidative stress caused by xenobiotics.
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http://dx.doi.org/10.1007/s12011-020-02178-9DOI Listing
February 2021

Author Correction: Modification of the head proteome of nurse honeybees (Apis mellifera) exposed to field-relevant doses of pesticides.

Sci Rep 2020 Apr 7;10(1):6253. Epub 2020 Apr 7.

Núcleo de Ensino, Ciência e Tecnologia em Apicultura Racional (NECTAR), São Paulo State University (UNESP), School of Veterinary Medicine and Animal Science, Department of Animal Production, Botucatu, SP, Brazil.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-63289-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136240PMC
April 2020

Modification of the head proteome of nurse honeybees (Apis mellifera) exposed to field-relevant doses of pesticides.

Sci Rep 2020 02 10;10(1):2190. Epub 2020 Feb 10.

Núcleo de Ensino, Ciência e Tecnologia em Apicultura Racional (NECTAR), São Paulo State University (UNESP), School of Veterinary Medicine and Animal Science, Department of Animal Production, Botucatu, SP, Brazil.

Understanding the effect of pesticides on the survival of honeybee colonies is important because these pollinators are reportedly declining globally. In the present study, we examined the changes in the head proteome of nurse honeybees exposed to individual and combined pesticides (the fungicide pyraclostrobin and the insecticide fipronil) at field-relevant doses (850 and 2.5 ppb, respectively). The head proteomes of bees exposed to pesticides were compared with those of bees that were not exposed, and proteins with differences in expression were identified by mass spectrometry. The exposure of nurse bees to pesticides reduced the expression of four of the major royal jelly proteins (MRJP1, MRJP2, MRJP4, and MRJP5) and also several proteins associated with carbohydrate metabolism and energy synthesis, the antioxidant system, detoxification, biosynthesis, amino acid metabolism, transcription and translation, protein folding and binding, olfaction, and learning and memory. Overall, when pyraclostrobin and fipronil were combined, the changes in protein expression were exacerbated. Our results demonstrate that vital proteins and metabolic processes are impaired in nurse honeybees exposed to pesticides in doses close to those experienced by these insects in the field, increasing their susceptibility to stressors and affecting the nutrition and maintenance of both managed and natural colonies.
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http://dx.doi.org/10.1038/s41598-020-59070-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010795PMC
February 2020

Parvalbumin and Ubiquitin as Potential Biomarkers of Mercury Contamination of Amazonian Brazilian Fish.

Biol Trace Elem Res 2020 Oct 10;197(2):667-675. Epub 2020 Jan 10.

São Paulo State University (UNESP), Institute of Biosciences, Botucatu, Brazil.

Recent studies have demonstrated the association of mercury (Hg) with some fish proteins, milk, and hair from individuals exposed to the element in the Amazon. However, few studies involve identifying biomarkers of mercury exposure. Therefore, the present study aimed to identify potential biomarkers of Hg exposure in fish. For this, the muscular tissues of two species of fish (Prochilodus lineatus and Mylossoma duriventre) that feed the Amazonian human population were analyzed. Through the analyses obtained by graphite furnace atomic absorption spectrometry (GFAAS), it was possible to identify four protein SPOTS where mercury was present. These SPOTS, identified by mass spectrometry (ESI-MS/MS), included parvalbumin and ubiquitin-40S ribosomal protein S27a, and these being metalloproteins with biomarker characteristics. In addition, the results show the intense Hg/protein ratio observed in the two proteins, which makes metalloproteins strong candidates for biomarkers of mercury exposure. Graphical Abstract.
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http://dx.doi.org/10.1007/s12011-020-02026-wDOI Listing
October 2020

Metalloproteomic approach of mercury-binding proteins in liver and kidney tissues of Plagioscion squamosissimus (corvina) and Colossoma macropomum (tambaqui) from Amazon region: Possible identification of mercury contamination biomarkers.

Sci Total Environ 2020 Apr 22;711:134547. Epub 2019 Nov 22.

São Paulo State University (UNESP), School of Veterinary Medicine and Animal Science, Botucatu, Brazil; São Paulo State University (UNESP), Institute of Biosciences, Botucatu, Brazil. Electronic address:

Fish is an important source of protein, vitamins, and minerals. However, this food is also a major source of human exposure to toxic contaminants such as mercury. Thus, this paper aimed to evaluate mercury-binding proteins for possible application as biomarkers of mercury contamination in hepatic and renal tissues of Plagioscion squamosissimus (carnivorous fish) and Colossoma macropomum (omnivorous fish) from the Amazon region using metalloproteomic approach. The proteome of hepatic and renal tissues of fish species was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the mercury concentrations in protein spots were determined by graphite furnace atomic absorption spectrometry (GFAAS). Finally, the protein spots associated to mercury were characterized by electrospray ionization mass spectrometry (ESI-MS/MS). The activity of antioxidant enzymes (SOD, CAT, GPx, and GST) and lipid peroxidation (LPO) were also determined. The results showed that the highest concentrations of mercury were found in the carnivorous species (P. squamosissimus) and that the accumulation pattern of this metal was higher in hepatic tissues than in renal tissues for both species. A tendency was observed for greater enzymatic activity in the hepatic and renal tissues of P. squamosissimus, the species with the highest concentration of mercury. Only GPx activity in the kidney and GST in the liver were lower for the P. squamosissimus species, and this finding can be explained by the interaction of mercury with these enzymes. The data obtained by ESI-MS/MS allowed for the characterization of the protein spots associated to mercury, revealing proteins involved in energy metabolism, biomolecules transport, protein synthesis and degradation, cell differentiation, gene regulation, and the antioxidant system. The results obtained in the present study can contribute to understanding the physiological processes underlying mercury toxicity and have provided new perspectives on possible candidates for mercury contamination biomarkers in fish.
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http://dx.doi.org/10.1016/j.scitotenv.2019.134547DOI Listing
April 2020

Supplementation with an Inorganic Iron Source Modulates the Metalloproteomic Profile of the Royal Jelly Produced by Apis mellifera L.

Biol Trace Elem Res 2020 Jun 31;195(2):648-657. Epub 2019 Aug 31.

UNESP - São Paulo State University, School of Veterinary Medicine and Animal Science, Botucatu, Brasil.

This study aimed to evaluate the quality of the royal jelly produced by Apis mellifera bees in the presence of different iron concentrations (ferrous sulfate heptahydrate-0, 25, 50, and 100 mg L). Two-dimensional electrophoresis was used for the fractionation of royal jelly proteins, and iron level was quantified using flame atomic absorption spectrometry technique. The proteins were identified using electrospray ionisation mass spectrometry. Analysis of variance followed by the Tukey test (P < 0.05) was utilised. Dietary supplementation with mineral Fe affected the protein content and number of proteins in the experimental period. Further, the diet containing the highest iron concentration showed a greater number of spots containing iron, as well as in the abdomen of the bees. The most protein containing Fe were classified as major royal jelly proteins. These results showed that Fe influenced the quality of royal jelly and can improve its nutritional value.
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http://dx.doi.org/10.1007/s12011-019-01863-8DOI Listing
June 2020

Physiological and functional aspects of metal-binding protein associated with mercury in the liver tissue of pirarucu (Arapaima gigas) from the Brazilian Amazon.

Chemosphere 2019 Dec 9;236:124320. Epub 2019 Jul 9.

São Paulo State University (UNESP), School of Veterinary Medicine and Animal Science, Botucatu, Brazil; São Paulo State University (UNESP), Institute of Biosciences, Botucatu, Brazil. Electronic address:

High concentrations of mercury found in soils, sediments, fish, and humans of the Amazon region have gained prominence in scientific studies during the last decade. However, studies related to the elucidation of mercury toxicity mechanisms in ichthyofauna at the molecular and metallomic levels that seek to elucidate physiological and functional aspects, as well as the search for biomarkers of mercury exposure, are still sparse. In the search for these answers, the present study analyzed the hepatic tissue proteome of the Arapaima gigas (pirarucu) fish species collected in the Jirau hydroelectric power plant reservoir in the state of Rondônia state, Brazil, in order to identify mercury-related metal-binding proteins and to elucidate their physiological and functional aspects. The proteomic profile of the hepatic tissue of Arapaima gigas was obtained by two-dimensional electrophoresis (2D-PAGE) and the presence of mercury was mapped in the protein SPOTS by graphite furnace atomic absorption spectrometry(GFAAS). Mercury was detected in 18 protein SPOTS with concentrations ranging from 0.13 ± 0.003 to 131.00 ± 3 mg kg. The characterization of the protein SPOTS associated with mercury was performed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS), and 10 proteins were identified. Bioinformatics analyses showed that most of the proteins found linked to mercury were involved in cellular component processes and biological processes. For the most part, protein sequences have cellular functions comprising catalytic, binding, sense of localization, and metabolic processes.
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http://dx.doi.org/10.1016/j.chemosphere.2019.07.051DOI Listing
December 2019

Identification of Zinc Absorption Biomarkers in Muscle Tissue of Nile Tilapia Fed with Organic and Inorganic Sources of Zinc Using Metallomics Analysis.

Biol Trace Elem Res 2020 Mar 6;194(1):259-272. Epub 2019 Jun 6.

School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

The development of metallomics techniques has allowed for metallomics analysis of biological systems, enabling a better understanding of the response mechanisms for different stimuli, their relationship to metallic species, and the characterization of biomarkers. In this study, a metallomics analysis of the muscle tissue of Nile tilapia was used to aid the understanding of the molecular mechanisms involved in zinc absorption in this fish species when fed organic and/or inorganic sources of zinc and to identify possible biomarkers for the absorption of this micromineral. To accomplish this, the fish were separated into three groups of 24 g, 74 g, and 85 g initial weights, and each group, respectively, was fed a zinc-free diet (control group, G1), a diet containing zinc found in organic sources (treatment 1, G2), and a diet containing zinc from an inorganic source (treatment 2, G3). Two-dimensional polyacrylamide (2D PAGE) gel electrophoresis was used to separate the proteins of the muscle tissue. Subsequently, the expression profiles of protein spots in the samples where zinc was applied in different concentrations were compared, using the software ImageMaster 2D Platinum version 7.0, to identify proteins that were differentially expressed. The identified proteins were then exposed to atomic absorption spectrometry in a graphite furnace to determine zinc mapping and were subsequently characterized via electrospray ionization tandem mass spectrometry (ESI-MS/MS). The metallomic analysis identified 15 proteins differentially expressed and associated with zinc, leading to the conclusion that three metal-binding proteins presented as possible biomarkers of zinc absorption in fish.
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http://dx.doi.org/10.1007/s12011-019-01765-9DOI Listing
March 2020

Proteomic analysis of the fast-twitch muscle of pacu (Piaractus mesopotamicus) after prolonged fasting and compensatory growth.

Comp Biochem Physiol Part D Genomics Proteomics 2019 06 24;30:321-332. Epub 2019 Apr 24.

Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP), Botucatu, Brazil. Electronic address:

Protocols that improve growth performance in fish while assuring product quality are important for aquaculture. Fasting followed by refeeding may promote compensatory growth, thus optimizing growth performance. During fasting and refeeding, fast-twitch muscle, which comprises most of fish fillet, undergoes intense plasticity. In this work, we studied the proteome of pacu (Piaractus mesopotamicus) fast-twitch muscle after 30 days of fasting (D30), 30 days of refeeding (D60) and 60 days of refeeding (D90) with two-dimensional electrophoresis, mass spectrometry and bioinformatics. Body mass, growth rate and muscle histology were also assessed. At D30, fish presented muscle catabolism and decreased growth. Proteomic analysis showed that metabolism proteins were the most affected, up and downregulated. Cytoskeleton and amino acid biosynthesis proteins were downregulated, while nuclear and regulatory proteins were upregulated. At D60, fish showed accelerated growth, despite the body mass not completely recovering. Metabolism proteins were still the most affected. Amino acid biosynthesis proteins became upregulated, while cytoskeleton proteins remained downregulated. At D90, the fish presented total compensatory growth. Many metabolic proteins were up or downregulated. Few cytoskeleton proteins remained differentially expressed. Amino acid biosynthesis proteins were mostly upregulated, but less than at D60. Prolonged fasting followed by refeeding also led to the regulation of possible meat quality biomarkers, such as antioxidant enzymes. This fact suggests possible consequences of this protocol on fish meat quality. Our work also enriches our knowledge on proteomic changes during muscle plasticity that occur during fasting and refeeding diet protocols.
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http://dx.doi.org/10.1016/j.cbd.2019.04.005DOI Listing
June 2019

Production of milk peptides with antimicrobial and antioxidant properties through fungal proteases.

Food Chem 2019 Apr 30;278:823-831. Epub 2018 Nov 30.

São Paulo State University (UNESP), Institute of Biosciences, Department of Chemistry and Biochemistry, Botucatu, SP, Brazil. Electronic address:

Bioactive peptides can provide health benefits due to different mechanisms. The aims of the present study are to produce bioactive peptides from bovine and goat milk subjected to the proteolytic activity of Aspergillus oryzae and Aspergillus flavipes enzymes, as well as to assess their putative antimicrobial and antioxidant activity. Bioactive peptides were successfully generated from proteases of fungi cultivated in solid-state fermentation. The generated peptides were effective against all tested bacteria and fungi. There was antioxidant activity, up to 92.5% DPPH reduction and ORAC stabilization at 52.5 μmol μL of Trolox Equivalent. The generation of milk-specific sequences peptides in the samples was obtained through 2D-PAGE fractioning followed by mass spectrometry (ESI-MS/MS). Based on results in the present study, milk bioactive peptides presenting broad antimicrobial action and antioxidant activity spectra can be cost-effectively produced through solid-state fermentation. The herein addressed approach can be valuable for the pharmaceutical and food industries.
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http://dx.doi.org/10.1016/j.foodchem.2018.11.119DOI Listing
April 2019

c-Src kinase contributes on endothelial cells mechanotransduction in a heat shock protein 70-dependent turnover manner.

J Cell Physiol 2019 07 22;234(7):11287-11303. Epub 2018 Nov 22.

Department of Chemistry and Biochemistry, São Paulo State University (UNESP), Institute of Biosciences, Botucatu, Brazil.

Shear stress changes are associated with a repertory of signaling cascade modulating vascular phenotype. As shear stress-related tensional forces might be associated with pathophysiological susceptibility, a more comprehensive molecular map needs to be addressed. Thus, we subjected human umbilical vein endothelial cells (HUVECs) to a circuit of different tensional forces in vitro considering the following three groups: (a) physiological blood flow shear stress condition (named Normo), (b) a hypertensive blood flow shear stress (named Hyper), and (c) these hyper-stressed cells were returned to Normo condition (named Return). The samples were properly collected to allow different methodologies analysis. Our data showed a pivotal involvement of c-Src on driving the mechanotransduction cascade by modulating signaling related with adhesion, survival (PI3K/Akt) and proliferative phenotype. Moreover, c-Src seems to develop important role during extracellular matrix remodeling. Additionally, proteomic analysis showed strong involvement of heat shock protein 70 (HSP70) in the hypertensive-stressed cells; it being significantly decreased in return phenotype. This result prompted us to investigate 20S proteasome as an intracellular proteolytic alternative route to promote the turnover of those proteins. Surprisingly, our data reveled significant overexpression of sets of proteasome subunit α-type (PSMA) and β-type (PSMB) genes. In conjunction, our data showed c-Src as a pivotal protein to drive mechanotransduction in endothelial cells in a HSP70-dependent turnover scenario. Because shear patterns is associated with pathophysiological changes, such as atherosclerosis and hypertension, these results paved new road to understand the molecular mechanism on driving mechanotransduction in endothelial cells and, if drugable, these targets must be considered within pharmacological treatment optimization.
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http://dx.doi.org/10.1002/jcp.27787DOI Listing
July 2019

Characterization of molecular biomarkers of mercury exposure to muscle tissue of Plagioscion squamosissimus and Colossoma macropomum from the Amazon region.

Food Chem 2019 Mar 3;276:247-254. Epub 2018 Oct 3.

UNESP - FMVZ - Graduate Program in Animal Sciences, Botucatu, SP, Brazil; UNESP - Institute of Biosciences, Botucatu, SP, Brazil. Electronic address:

Mercury has the ability to bind to a variety of biomolecules, which can compromise its structure and functionality and thus promote its toxic effects. The aim of this study is to identify possible mercury biomarkers in muscle samples of Plagioscion squamosissimus (carnivorous fish) and Colossoma macropomum (omnivorous fish), from the Amazon region. The muscle proteome of fish species was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the total mercury concentrations in protein spots were determined by graphite furnace atomic absorption spectrometry (GFAAS). The protein spots containing mercury were characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The mercury concentrations in the protein spots were in the range of 1.10 ± 0.02-23.90 ± 0.33 μg g. The proteins phosphoglycerate mutase 2 (P. squamosissimus), hemoglobin β and cytochrome P450scc (C. macropomum), identified by ESI-MS/MS and showing the highest values of mercury concentration, may be considered possible mercury biomarkers.
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http://dx.doi.org/10.1016/j.foodchem.2018.10.002DOI Listing
March 2019

Identification of Biomarkers of Mercury Contamination in Brachyplatystoma filamentosum of the Madeira River, Brazil, Using Metalloproteomic Strategies.

Biol Trace Elem Res 2019 Jan 8;187(1):291-300. Epub 2018 May 8.

School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, Brazil.

Predator fish can accumulate high levels of mercury, which qualifies them as potential indicators of this toxic metal. The predatory species Brachyplatystoma filamentosum, popularly known as filhote, is among the most consumed species in the Brazilian Amazon. Continuing the metalloproteomic studies of mercury in Amazonian fishes that have been developed in the last 5 years, the present paper provides the data of protein characterization associated with mercury in muscle and liver samples of filhote (Brachyplatystoma filamentosum) collected in the Madeira River, Brazilian Amazon. The mercury concentration in the muscle and liver samples was determined by graphite furnace atomic absorption spectrometry (GFAAS). The protein fraction was extracted in an aqueous medium, and later, a fractional precipitation procedure was performed to obtain the protein pellets. Then, the proteome of the tissue samples of this fish species was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and a mercury mapping of the protein spots was carried out by GFAAS after acid digestion. Protein spots that had mercury were characterized by mass spectrometry with electrospray ionization in sequence (ESI-MS/MS) after tryptic digestion. It was possible to characterize 11 mercury-associated protein spots that presented biomarker characteristics and could be used to monitor mercury in fish species of the Amazon region. Thus, the metalloproteomic strategies used in the present study allowed us to characterize 11 mercury-associated protein spots. It should be noted that the protein spots identified as GFRP, TMEM186, TMEM57B, and BHMT, which have coordination sites for elements with characteristics of soft acids, such as mercury, can be used as biomarkers of mercury contamination in monitoring studies of this toxic metal in fish species from the Amazon region.
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http://dx.doi.org/10.1007/s12011-018-1363-5DOI Listing
January 2019

Total Mercury Determination in Muscle and Liver Tissue Samples from Brazilian Amazon Fish Using Slurry Sampling.

Biol Trace Elem Res 2018 Aug 2;184(2):517-522. Epub 2017 Dec 2.

School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, Brazil.

This paper presents a slurry sampling method for total mercury determination by graphite furnace atomic absorption spectrometry (GFAAS) in tissue of fish from the Amazon. The tissue samples were lyophilized and macerated, and then the slurry samples were prepared by putting 20 mg of tissue, added to a solution containing Triton X-100, Suprapur HNO, and zirconium nitrate directly in sampling vials of a spectrometer. Mercury standard solutions were prepared under the same conditions as the slurry samples. The slurry samples and the mercury standard solutions were sonicated for 20 s. Twenty microliters of slurry samples were injected into the graphite tube, which contained an internal wall lined with tungsten carbide. Under these conditions, it was possible to thermally stabilize the mercury up to an atomization temperature of 1700 °C. The method was validated by mercury determination in reference materials DORM-4 and DOLT-4. The LOD and LOQ were 0.014 and 0.045 mg kg, respectively, and recovery percentages in relation to the concentration values were certified in the order of 98%.
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http://dx.doi.org/10.1007/s12011-017-1212-yDOI Listing
August 2018

Correction to: Mercury Exposure: Protein Biomarkers of Mercury Exposure in Jaraqui Fish from the Amazon Region.

Biol Trace Elem Res 2018 05;183(1):172

Institute of Bioscience of Botucatu, São Paulo State University (UNESP), Rubião Júnior, Botucatu, São Paulo, 18618-970, Brazil.

In the affiliation section, Luiz Fabricio Zara's affiliation "Pontifical Catholic University of Goiás (PUC), Goiânia, GO, Brazil" was incorrect. The correct affiliation is College of Planaltina, UnB - University of Brasília, Distrito Federal, Brazil.
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http://dx.doi.org/10.1007/s12011-017-1195-8DOI Listing
May 2018

Mercury Exposure: Protein Biomarkers of Mercury Exposure in Jaraqui Fish from the Amazon Region.

Biol Trace Elem Res 2018 May 21;183(1):164-171. Epub 2017 Aug 21.

Institute of Bioscience of Botucatu, São Paulo State University (UNESP), Rubião Júnior, Botucatu, São Paulo, 18618-970, Brazil.

This study presents data on the extraction and characterization of proteins associated with mercury in the muscle and liver tissues of jaraqui (Semaprochilodus spp.) from the Madeira River in the Brazilian Amazon. Protein fractionation was carried out by two-dimensional electrophoresis (2D-PAGE). Mercury determination in tissues, pellets, and protein spots was performed by graphite furnace atomic absorption spectrometry (GFAAS). Proteins in the spots that showed mercury were characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The highest mercury concentrations were found in liver tissues and pellets (426 ± 6 and 277 ± 4 μg kg), followed by muscle tissues and pellets (132 ± 4 and 86 ± 1 μg kg, respectively). Mercury quantification in the protein spots allowed us to propose stoichiometric ratios in the range of 1-4 mercury atoms per molecule of protein in the protein spots. The proteins characterized in the analysis by ESI-MS/MS were keratin, type II cytoskeletal 8, parvalbumin beta, parvalbumin-2, ubiquitin-40S ribosomal S27a, 39S ribosomal protein L36 mitochondrial, hemoglobin subunit beta, and hemoglobin subunit beta-A/B. The results suggest that proteins such as ubiquitin-40S ribosomal protein S27a, which have specific domains, possibly zinc finger, can be used as biomarkers of mercury, whereas mercury and zinc present characteristics of soft acids.
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http://dx.doi.org/10.1007/s12011-017-1129-5DOI Listing
May 2018

Metalloproteomic and differential expression in plasma in a rat model of type 1 diabetes.

Int J Biol Macromol 2017 Nov 8;104(Pt A):414-422. Epub 2017 Jun 8.

Department of Chemistry and Biochemistry, Institute of Bioscience, São Paulo State University (UNESP), Botucatu, SP, Brazil.

Type 1 diabetes is characterized by hyperglycemia, which in the chronic stage is associated with abnormalities in lipids, protein and, carbohydrate metabolism, as well as oxidative stress. New strategies for prevention and treatment are needed, as type 1 diabetes affects life quality and survival, and involves high-cost treatment. Proteomic and metalloproteomic studies can elucidate the functional and physiological aspects of biomolecules. In the present study, differential proteomics was used to identify potential biomarkers of diabetes in rat plasma associated with copper, selenium, zinc, and magnesium fractionation in control and diabetic rats, as well as diabetic rats treated with insulin. 2D-PAGE was used in the plasma protein fractionation; graphite furnace atomic absorption spectrometry (GFAAS) and flame atomic absorption spectrometry (FAAS) were used for quantitative determination of copper, magnesium, selenium, and zinc in the spots that showed different expression; and protein spots were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) after tryptic digestion. ESI-MS/MS analysis characterized 35 different proteins, indicating alpha-1-macroglobulin and haptoglobulin as potential candidates as biomarkers for diabetes treated with insulin; also, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, transmembrane protein 11, serum amyloid P component, vitamin D-binding protein, and biliverdin reductase were identified as potential candidates as biomarkers for uncontrolled diabetes.
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http://dx.doi.org/10.1016/j.ijbiomac.2017.06.032DOI Listing
November 2017

Metalloproteomics Approach to Analyze Mercury in Breast Milk and Hair Samples of Lactating Women in Communities of the Amazon Basin, Brazil.

Biol Trace Elem Res 2018 Feb 25;181(2):216-226. Epub 2017 May 25.

Institute of Biosciences, São Paulo State University (UNESP), Botucatu, Brazil.

Mercury is a potentially toxic element that is present in the environment of the Brazilian Amazon and is responsible for adverse health effects in humans. This study sought to assess possible protein biomarkers of mercury exposure in breast milk samples from lactating women in the Madeira and Negro Rivers in the Brazilian Amazon. The mercury content of hair samples of lactating women was determined, and the proteome of breast milk samples was obtained using two-dimensional electrophoresis after protein precipitation with acetone. Mercury measurements of protein spots obtained via protein fractionation were performed by graphite furnace atomic absorption spectrometry (GFAAS), and it was observed that mercury is linked to proteins with molecular masses in the range of 14-26 kDa. The total mercury concentration was also determined by GFAAS in unprocessed milk, lyophilized milk, and protein pellets, with the purpose of determining the mercury mass balance in relation to the concentration of this element in milk and pellets. Approximately 85 to 97% of mercury present in the lyophilized milk from samples of lactating women of the Madeira River is bound in the protein fraction. From lactating women of the Negro River, approximately 49% of the total mercury is bound in the protein fraction, and a difference of 51% is bound in the lipid fraction.
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http://dx.doi.org/10.1007/s12011-017-1057-4DOI Listing
February 2018

A proteomic approach to identify metalloproteins and metal-binding proteins in liver from diabetic rats.

Int J Biol Macromol 2017 Mar 3;96:817-832. Epub 2017 Jan 3.

Department of Chemistry and Biochemistry, Institute of Bioscience of Botucatu, São Paulo State University, Botucatu, SP, Brazil.

Proteins play crucial roles in biological systems, thus studies comparing the protein pattern present in a healthy sample with an affected sample have been widely used for disease biomarker discovery. Although proteins containing metal ions constitute only a small proportion of the proteome, they are essential in a multitude of structural and functional processes. The correct association between metal ions and proteins is essential because this binding can significantly interfere with normal protein function. Employment of a metalloproteomic study of liver samples from diabetic rats permitted determination of the differential abundance of copper-, selenium-, zinc- and magnesium-associated proteins between diabetic, diabetic treatment with insulin and non-diabetic rats. Proteins were detected by ESI-MS/MS. Seventy-five different proteins were found with alterations in the metal ions of interest. The most prominent pathways affected under the diabetic model included: amino-acid metabolism and its derivates, glycogen storage, metabolism of carbohydrates, redox systems and glucose metabolism. Overall, the current methods employed yielded a greater understanding of metal binding and how type 1 diabetes and insulin treatment can modify some metal bonds in proteins, and therefore affect their mechanism of action and function.
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http://dx.doi.org/10.1016/j.ijbiomac.2016.12.073DOI Listing
March 2017

A Metalloproteomics Study on the Association of Mercury With Breast Milk in Samples From Lactating Women in the Amazon Region of Brazil.

Arch Environ Contam Toxicol 2015 Aug 16;69(2):223-9. Epub 2015 May 16.

Institute of Chemistry, Federal University of Mato Grosso do Sul (INQUI/UFMS), Campo Grande, Mato Grosso do Sul, Brazil.

This study aimed to identify metalloproteins that lose their metal ions in the presence of mercury (Hg) and bind to Hg in breast milk samples collected from the riverine population of the Madeira River, a tributary of the Amazon River. Initially, total Hg was determined from the hair of lactating women to identify individuals who were contaminated followed by a proteomic analysis of breast milk samples through two-dimensional polyacrylamide gel electrophoresis after acetone precipitation. The presence of Hg in the obtained protein spots was determined by cold vapor atomic absorption spectrometry and cold vapor atomic fluorescence spectrometry. These determinations indicated the presence of Hg in one protein spot, which was then characterized through electrospray ionization tandem mass spectrometry. Based on searches in the UniProt database, this protein spot was identified as lysozyme C.
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http://dx.doi.org/10.1007/s00244-015-0161-8DOI Listing
August 2015

Metal ions bound to the human milk immunoglobulin A: metalloproteomic approach.

Food Chem 2015 Jan 17;166:492-497. Epub 2014 Jun 17.

Institute of Biosciences, São Paulo State University-UNESP, Botucatu, SP, Brazil.

The presence of calcium, iron, and zinc bound to human milk secretory IgA (sIgA) was investigated. The sIgA components were first separated by two-dimensional polyacrylamide gel electrophoresis and then identified by electrospray ionization-tandem mass spectrometry (ESI MS MS). The metal ions were detected by flame atomic absorption spectrometry after acid mineralization of the spots. The results showed eight protein spots corresponding to the IgA heavy chain constant region. Another spot was identified as the transmembrane secretory component. Calcium was bound to both the transmembrane component and the heavy chain constant region, while zinc was bound to the heavy chain constant region and iron was not bound with the identified proteins. The association of a metal ion with a protein is important for a number of reasons, and therefore, the findings of the present study may lead to a better understanding of the mechanisms of action and of additional roles that sIgA and its components play in human milk.
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http://dx.doi.org/10.1016/j.foodchem.2014.06.040DOI Listing
January 2015
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