The absorption of bovine milk small extracellular vesicles largely depends on galectin 3 and galactose ligands in human intestinal cells and C57BL/6J mice.

Small extracellular vesicles in milk (sMEVs) have attracted attention in drug delivery and as bioactive food compounds. Previous studies implicate galactose residues on the sMEV surface in sMEV transport across intestinal and endothelial barriers in humans, but details of glycoprotein-dependent transport are unknown. We used a combination of cell biology and genetics protocols to identify glycoproteins on the sMEV surface that facilitate sMEV absorption. We identified 256 proteins on the bovine sMEVs surface by using LC-MS/MS, and bioinformatics analysis suggested that 42, 13, and 13 surface proteins were N-, O-, and 13 C-glycosylated, respectively. Lectin blots confirmed the presence of mannose, galactose, N-acetyl galactose, fucose, and neuraminate. When surface proteins were removed by various treatment with various proteases, sMEV uptake decreased by up to 58% and 67% in FHs-74 Int and Caco-2 cells, respectively, compared with controls (P < 0.05). When glycans were removed by treatment with various glycosidases, sMEV uptake decreased by up to 54% and 74% in FHs-74 Int and Caco-2 cells, respectively (P < 0.05). When galactose and N-acetyl galactosamine residues were blocked with agglutinins, sMEV uptake decreased by more than 50% in FHs-74 Int cells (P < 0.05). When bovine sMEVs were administered to Galectin-3 knockout mice by oral gavage, hepatic sMEV accumulation decreased by 56% compared with wild-type mice (P < 0.05), consistent with a role of ß-galactoside glycan structures in the absorption of sMEVs. We conclude that sMEVs are decorated with glycoproteins, and Galectin-3 and its galactose ligands are particularly important for sMEV absorption.NEW & NOTEWORTHY This is the first paper to assess the role of unique glycans and their Galectin-3 receptor in the transport and distribution of small extracellular vesicles ("exosomes") from milk in mammals. The research assessed milk exosome transport and distribution by using multiple approaches and platforms including cell cultures, various exosome labels, knockout and mutant mice, enzymatic removal of surface proteins and glycans, and lectin blocking of glycans.

Metalloproteomic Investigation of Hg-Binding Proteins in Renal Tissue of Rats Exposed to Mercury Chloride.

Results obtained from rat studies indicate that, even at low concentrations, mercurial species cause harmful effects on the kidneys, by inducing the nephrotic oxidative stress response. In the present work, Hg-associated proteins were identified as possible mercury-exposure biomarkers in rat kidneys exposed to low mercury chloride concentrations for 30 days (Hg-30) and 60 days (Hg-60), using metalloproteomic strategies. The renal proteomic profile was fractioned by two-dimensional electrophoresis and the mercury determinations in kidney samples, protein pellets and protein spots were performed using graphite furnace atomic absorption spectrometry. The characterization of Hg-associated protein spots and the analysis of differentially expressed proteins were performed by liquid chromatography, coupled with tandem mass spectrometry. Eleven Hg-associated protein spots with a concentration range of 79 ± 1 to 750 ± 9 mg kg-1 in the Hg-60 group were identified. The characterization and expression analyses allowed the identification of 53 proteins that were expressed only in the Hg-60 group, 13 "upregulated" proteins (p > 0.95) and 47 "downregulated" proteins (p < 0.05). Actin isoforms and hemoglobin subunits were identified in protein spots of the Hg-60 group, with mercury concentrations in the range of 138 to 750 mg kg-1, which qualifies these proteins as potential mercury-exposure biomarkers.

Mercury metalloproteomic profile in muscle tissue of Arapaima gigas from the Brazilian Amazon.

Metalloproteomics is an innovative methodology for identifying of protein-associated mercury. Thus, we analyzed the muscle proteome of Arapaima gigas (pirarucu), collected in the Madeira River of the Brazilian Amazon, to identify protein-associated mercury, with the aim of identifying possible mercury biomarkers in fish muscle tissue. After obtaining the protein pellet, we conducted two-dimensional electrophoresis (2D PAGE) to fractionate the muscle proteome. Total mercury in muscle tissue and protein pellets and mapping of mercury content in protein spots of the 2D PAGE gels was determined using graphite furnace atomic absorption spectrometry (GFAAS). The protein-associated mercury identification was performed using liquid chromatography coupled with sequence mass spectrometry (LC‒MS/MS). Total mercury determinations by GFAAS indicated concentrations on the order of 153 ± 1.90 mg kg-1 and 142 ± 1.50 mg kg-1 (total precipitation of protein fraction) and 139 ± 1.45 mg kg-1 (fractional precipitation of protein fraction) in muscle tissue and protein pellets, respectively. Mercury concentrations in the range of 48 ± 0.90 to 165 ± 3.00 mg kg-1 were found in twelve protein spots. Among the 2D PAGE protein spots, eleven Hg-binding proteins were identified using LC‒MS/MS, which showed characteristics of mercury exposure biomarkers for important metabolic functions, such as five parvalbumin isoforms, triosephosphate isomerase, cofilin 2 (muscle), and fructose-bisphosphate aldolases.

Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles.

Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, ß-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV.

Metallomics in Fish.

Metallomics allows the integration of traditionally analytical studies with inorganic and biochemical studies. The study of metallomics in living organisms allows us to obtain information about how the metal ion is distributed and coordinated with proteins, the essentiality and/or toxicity, and the individual concentrations of metal species, thus contributing to elucidation of the physiological and functional aspects of these biomolecules. In this context, several lines of research have appeared in the literature with different terms and approaches. For example, metallomic, which deals with the characterization of the total metal/metalloid species present in an organism; metalloprotein, which deals with the characterization of the total elements present in a specific site of an organism (cellular behavior, protein, metalloprotein); and metallomic, which deals with a more in-depth study of metallome. In this area, information is sought on the interactions and functional connections of metal/metalloid species with genes, proteins, metabolites and other biomolecules of the organism and, therefore, the elucidation of the biological role exerted by the metal ions bound to the biomolecules. In this chapter, we will describe techniques used in animal studies.

Energy restriction and impact on indirect calorimetry and oxidative stress in cardiac tissue in rat.

Caloric restriction, defined as a reduction in calorie intake below ad libitum, without malnutrition can have beneficial effects. In this study, we evaluated the impact of caloric restriction of 30 and 60% on calorimetric parameters and oxidative stress in cardiac tissue in rats. Rats were randomly divided into 3 groups (n = 8): G1 = control; G2 = rats exposed to dietary restriction of 30%; and G3 = rats exposed to dietary restriction of 60%. Energy restriction decreased final body weight, oxidation of carbohydrates and lipid, oxygen consumption (VO2), carbon dioxide production (VCO2), resting metabolic rate (RMR), but elevated respiratory quotient (RQ). G3 animals also displayed an imbalance in the oxidant/antioxidant system, as revealed by the decrease in the lipid hydroperoxide (LH) level and GSH-Px activity in heart tissue. In conclusion, dietary restriction decreased oxidative metabolism, as seen by the colorimetric profiles and controlled oxidative stress in cardiac tissue.

Comparative polar and lipid plasma metabolomics differentiate KSHV infection and disease states.

BACKGROUND: Kaposi sarcoma (KS) is a neoplastic disease etiologically associated with infection by the Kaposi sarcoma-associated herpesvirus (KSHV). KS manifests primarily as cutaneous lesions in individuals due to either age (classical KS), HIV infection (epidemic KS), or tissue rejection preventatives in transplantation (iatrogenic KS) but can also occur in individuals, predominantly in sub-Saharan Africa (SSA), lacking any obvious immune suppression (endemic KS). The high endemicity of KSHV and human immunodeficiency virus-1 (HIV) co-infection in Africa results in KS being one of the top 5 cancers there. As with most viral cancers, infection with KSHV alone is insufficient to induce tumorigenesis. Indeed, KSHV infection of primary human endothelial cell cultures, even at high levels, is rarely associated with long-term culture, transformation, or growth deregulation, yet infection in vivo is sustained for life. Investigations of immune mediators that distinguish KSHV infection, KSHV/HIV co-infection, and symptomatic KS disease have yet to reveal consistent correlates of protection against or progression to KS. In addition to viral infection, it is plausible that pathogenesis also requires an immunological and metabolic environment permissive to the abnormal endothelial cell growth evident in KS tumors. In this study, we explored whether plasma metabolomes could differentiate asymptomatic KSHV-infected individuals with or without HIV co-infection and symptomatic KS from each other. METHODS: To investigate how metabolic changes may correlate with co-infections and tumorigenesis, plasma samples derived from KSHV seropositive sub-Saharan African subjects in three groups, (A) asymptomatic (lacking neoplastic disease) with KSHV infection only, (B) asymptomatic co-infected with KSHV and HIV, and (C) symptomatic with clinically diagnosed KS, were subjected to analysis of lipid and polar metabolite profiles RESULTS: Polar and nonpolar plasma metabolic differentials were evident in both comparisons. Integration of the metabolic findings with our previously reported KS transcriptomics data suggests dysregulation of amino acid/urea cycle and purine metabolic pathways, in concert with viral infection in KS disease progression. CONCLUSIONS: This study is, to our knowledge, the first to report human plasma metabolic differentials between in vivo KSHV infection and co-infection with HIV, as well as differentials between co-infection and epidemic KS.

The effects of mercury exposure on Amazonian fishes: An investigation of potential biomarkers.

Exposure to mercury can interfere with the expression of proteins and enzymes, compromise important pathways, such as apoptosis and glucose metabolism, and even induce the expression of metallothioneins. In this study, analytical techniques were used to determine the concentration of total mercury (THg) in muscle and liver tissue, protein pellets, and spots [using graphite furnace atomic absorption spectrometry (GFAAS)], and molecular techniques were used to identify metalloproteins present in mercury-associated protein spots. Thirty individuals from three different fish species, Cichla sp. (n = 10), Brachyplatystoma filamentosum (n = 10), and Semaprochilodus sp. (n = 10) from the Brazilian Amazon were used. Oxidative stress indicators [such as glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), a marker of lipid peroxidation (LPO)] and the possible expression of metallothioneins in muscle and liver tissues were investigated. The two piscivorous species, Cichla sp. and B. filamentosum, presented the highest concentrations of mercury in their hepatic tissue, 1219 ± 15.00 and 1044 ± 13.6 µg kg-1, respectively, and in their muscle tissue, 101 ± 1.30 µg kg-1 and 87.4 ± 0.900 µg kg-1, respectively. The non-carnivorous species Semaprochilodus sp. had comparatively low concentrations of mercury in both its hepatic (852 ± 11.1 µg kg-1) and muscle (71.4 ± 0.930 µg kg-1) tissues. The presence of mercury was identified in 24 protein spots using GFAAS; concentrations ranged from 11.5 to 787 µg kg-1, and mass spectrometry identified 21 metal-binding proteins. The activities of GSH-Px, CAT, and SOD, related to oxidative stress, decreased proportionally as tissue Hg concentrations increased, while the levels of LPO markers increased, indicating the presence of stress. Our study results demonstrate possible mercury interference in oxidative stress markers (GSH-Px, CAT, SOD, and LPO), in addition to the identification of 21 metal-binding proteins as possible biomarkers of mercury exposure in fish.

Metalloproteomic approach to liver tissue of rats exposed to mercury.

The aims of this study were to identify mercury-associated protein spots in the liver tissue of rats exposed to low concentrations of mercury and to elucidate the physiological and functional aspects of the proteins identified in the protein spots. Therefore, proteomic analysis of the liver tissue of Wistar rats exposed to mercury chloride (4.60 µg kg-1 in Hg2+) was performed for thirty days (Hg-30 group) and sixty days (Hg-60 group). The proteomic profile of the liver tissue of the rats was obtained by two-dimensional electrophoresis (2D-PAGE), and the determinations of total mercury in the liver tissue, pellets and protein spots were performed by graphite furnace atomic absorption spectrometry (GFAAS). ImageMaster 2D Platinum 7.0 software was used to identify the differentially expressed mercury-associated protein spots, which were then characterized by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The determinations by GFAAS indicated a total mercury bioaccumulation of 2812% in the Hg-30 group and 3298% in the Hg-60 group and 10 mercury-associated protein spots with a concentration range of 51 ± 1.0 to 412 ± 6.00 mg kg-1 in the 2D PAGE gels from the liver tissue of the Hg-60 group. The LC-MS/MS analyses allowed the identification of 11 metal binding proteins in mercury-associated protein spots that presented fold change with upregulation >1.5, downregulation < -1.7 or that were expressed only in the Hg-60 group. Using the FASTA sequences of the proteins identified in the mercury-associated protein spots, bioinformatics analyses were performed to elucidate the physiological and functional aspects of the metal binding proteins, allowing us to infer that enzymes such as GSTM2 presented greater mercury concentrations and downregulation < -3; Acaa2 and Bhmt, which showed expression only in the Hg-60 group, among others, may act as potential mercury exposure biomarkers.

Investigation of Protein Biomarkers and Oxidative Stress in Pinirampus pirinampu Exposed to Mercury Species from the Madeira River, Amazon-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-1) when compared to muscle tissue (60 ± 2.0 µg kg-1). 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-1). 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.

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

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.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Aberrant energy metabolism and redox balance in seizure onset zones of epileptic patients.

Epilepsy is a disorder that affects around 1% of the population. Approximately one third of patients do not respond to anti-convulsant drugs treatment. To understand the underlying biological processes involved in drug resistant epilepsy (DRE), a combination of proteomics strategies was used to compare molecular differences and enzymatic activities in tissue implicated in seizure onset to tissue with no abnormal activity within patients. Label free quantitation identified 17 proteins with altered abundance in the seizure onset zone as compared to tissue with normal activity. Assessment of oxidative protein damage by protein carbonylation identified additional 11 proteins with potentially altered function in the seizure onset zone. Pathway analysis revealed that most of the affected proteins are involved in energy metabolism and redox balance. Further, enzymatic assays showed significantly decreased activity of transketolase indicating a disruption of the Pentose Phosphate Pathway and diversion of intermediates into purine metabolic pathway, resulting in the generation of the potentially pro-convulsant metabolites. Altogether, these findings suggest that imbalance in energy metabolism and redox balance, pathways critical to proper neuronal function, play important roles in neuronal network hyperexcitability and can be used as a primary target for potential therapeutic strategies to combat DRE. SIGNIFICANCE: Epileptic seizures are some of the most difficult to treat neurological disorders. Up to 40% of patients with epilepsy are resistant to first- and second-line anticonvulsant therapy, a condition that has been classified as refractory epilepsy. One potential therapy for this patient population is the ketogenic diet (KD), which has been proven effective against multiple refractory seizure types However, compliance with the KD is extremely difficult, and carries severe risks, including ketoacidosis, renal failure, and dangerous electrolyte imbalances. Therefore, identification of pathways disruptions or shortages can potentially uncover cellular targets for anticonvulsants, leading to a personalized treatment approach depending on a patient's individual metabolic signature.

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

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.

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

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.

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

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.

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.

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.

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

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.

Influence of oxidative damage to proteins on meat tenderness using a proteomics approach.

The objective of this study was to evaluate the association between oxidative damage to proteins (represented by protein carbonylation) and beef tenderness. Three experimental groups were selected by shear force (SF): tender (38.2 ±â€¯2.9 N), intermediate (51.9 ±â€¯6.8 N), and tough meat (74.5 ±â€¯7.8 N). Two-dimensional electrophoresis with hydrazide fluorophore derivatization was used. The structural proteins actin (ACTA1), myosin (MYL1 and MYL3), desmin (DES) and troponin T (TNNT1 and TNNT3), antioxidant proteins (PRDX1, PRDX2 and PARK7) and heat shock proteins (HSPB1, CRYAB and HSPB6) showed an increase in the oxidative damage in tender meat when compared to the intermediate and tough meat (P < .05). Decrease in oxidative damage of the metabolic enzymes (TPI1, GAPDH and ENO3) were observed in tender meat group (P < .05). The present results suggest that oxidation act on the proteins of different metabolic pathways and consequently affect meat tenderness in Angus crossbred cattle.

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

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 ±â€¯0.003 to 131.00 ±â€¯3 mg kg-1. 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.