Proteomic Study of Broiler Plasma Supplemented with Different Levels of Copper and Manganese from Different Sources.

The aim of the present study was to evaluate the differential expression of plasma proteins in broiler chickens supplemented with different sources (sulfates and hydroxychlorides) and levels of copper (15 and 150 mg kg-1) and manganese (80 and 120 mg kg-1). For this, plasma samples from 40 broiler chickens were used, divided into four experimental groups: S15-80 (15 ppm CuSO4 and 80 ppm MnSO4), S150-120 (150 ppm CuSO4 and 120 ppm MnSO4), H15-80 (15 ppm Cu(OH)Cl and 80 ppm Mn(OH)Cl), and H150-120 (150 ppm Cu(OH)Cl and 120 ppm Mn(OH)Cl). From plasma samples obtained from each bird from the same treatment, four pools were made considering 10 birds per group. Plasma proteome fractionation was performed by 2D-PAGE. Concentrations of the studied minerals were also evaluated in both plasma and protein pellet samples. A higher concentration of Cu and Mn was observed in the plasma and protein pellets of groups that received higher mineral supplementation levels compared to those receiving lower levels. Mn concentrations were higher in plasma and protein pellets of the hydroxychloride-supplemented groups than the sulfate-supplemented groups. Analysis of the gels revealed a total of 40 differentially expressed spots among the four treatments. Supplementation with different sources of minerals, particularly at higher levels, resulted in changes in protein regulation, suggesting a potential imbalance in homeostasis.

Preliminary Insights of Brazilian Snake Venom Metalloproteomics.

Snakebite envenoming is one of the most significantly neglected tropical diseases in the world. The lack of diagnosis/prognosis methods for snakebite is one of our motivations to develop innovative technological solutions for Brazilian health. The objective of this work was to evaluate the protein and metallic ion composition of Crotalus durissus terrificus, Bothrops jararaca, B. alternatus, B. jararacussu, B. moojeni, B. pauloensis, and Lachesis muta muta snake venoms. Brazilian snake venoms were subjected to the shotgun proteomic approach using mass spectrometry, and metal ion analysis was performed by atomic spectrometry. Shotgun proteomics has shown three abundant toxin classes (PLA2, serine proteases, and metalloproteinases) in all snake venoms, and metallic ions analysis has evidenced that the Cu2+ ion is present exclusively in the L. m. muta venom; Ca2+ and Mg2+ ions have shown a statistical difference between the species of Bothrops and Crotalus genus, whereas the Zn2+ ion presented a statistical difference among all species studied in this work. In addition, Mg2+ ions have shown 42 times more in the C. d. terrificus venom when compared to the average concentration in the other genera. Though metal ions are a minor fraction of snake venoms, several venom toxins depend on them. We believe that these non-protein fractions are capable of assisting in the development of unprecedented diagnostic devices for Brazilian snakebites.

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.

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.

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.

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.

Prospecting Biomarkers for Diagnostic and Therapeutic Approaches in Pythiosis.

Pythiosis, whose etiological agent is the oomycete Pythium insidiosum, 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 P. insidiosum 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.

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

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.

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

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-1, 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.

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

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 in silico 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.

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.

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.

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.

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

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-1). 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.

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 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.

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.

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

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 ±â€¯0.02-23.90 ±â€¯0.33 µg g-1. The proteins phosphoglycerate mutase 2 (P. squamosissimus), hemoglobin ß and cytochrome P450scc (C. macropomum), identified by ESI-MS/MS and showing the highest values of mercury concentration, may be considered possible mercury biomarkers.

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

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 ß-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.

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

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-1 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.