Snake venoms are complex mixtures of proteins with toxic activities, with many distinct isoforms, affecting different physiological targets, comprised in a few protein families. It is currently ...accepted that this diversity in venom composition is an adaptive advantage for venom efficacy on a wide range of prey. However, on the other side, variability on isoforms expression has implications in the clinics of human victims of snakebites and in the efficacy of antivenoms. B. atrox snakes are responsible for most of the human accidents in Brazilian Amazon and the type and abundance of protein families on their venoms present individual variability. Thus, in this study we attempted to correlate the individual venom proteome of the snake brought to the hospital by the patient seeking for medical assistance with the clinical signs observed in the same patient. Individual variability was confirmed in venoms of the 14 snakes selected for the study. The abundance of each protein family was quite similar among the venom samples, while the isoforms composition was highly variable. Considering the protein families, the SVMP group presented the best correlation with bleeding disorders and edema. Considering individual isoforms, some isoforms of venom metalloproteinase (SVMP), C-type lectin-like toxins (CTL) and snake venom serine proteinases (SVSP) presented expression levels that with statistically significant positive correlation to signs and symptoms presented by the patients as bleeding disorders, edema, ecchymosis and blister formation. However, some unexpected data were also observed as the correlation between a CTL, CRISP or LAAO isoforms with blister formation, still to be confirmed with a larger number of samples. Although this is still a small number of patient samples, we were able to indicate that venom composition modulates clinical manifestations of snakebites, to confirm at the bedside the prominent role of SVMPs and to include new possible toxin candidates for the development of toxin inhibitors or to improve antivenom selectiveness, important actions for the next generation treatments of snakebites.
Abstract
Breast cancer is one of leading causes of death worldwide in the female population. Deaths from breast cancer could be reduced significantly through earlier and more efficient detection of ...the disease. Saliva, an oral fluid that contains an abundance of protein biomarkers, has been recognized as a promising diagnostic biofluid that is easy to isolate through non-invasive techniques. Assays on saliva can be performed rapidly and are cost-effective. Therefore, our work aimed to identify salivary biomarkers present in the initial stages of breast cancer, where cell alterations are not yet detectable by histopathological analysis. Using state-of-the-art techniques, we employed a transgenic mouse model of mammary cancer to identify molecular changes in precancerous stage breast cancer through protein analysis in saliva. Through corroborative molecular approaches, we established that proteins related to metabolic changes, inflammatory process and cell matrix degradation are detected in saliva at the onset of tumor development. Our work demonstrated that salivary protein profiles can be used to identify cellular changes associated with precancerous stage breast cancer through non-invasive means even prior to biopsy-evident disease.
Infection by the protozoan
causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of ...the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.
Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed ...of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom (“traditional approach”) and immunization with selected key toxins isolated from the snake venom (“toxin oriented” approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.
Trypanosoma cruzi, the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular ...infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi, which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture-derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans-sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin-like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle-free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC-MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain-derived EVs caused higher infection of LLC-MK2 cells than Y strain-derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host-parasite interaction.
Cancer is characterized by the development of abnormal cells that divide in an uncontrolled way and may spread into other tissues where they may infiltrate and destroy normal body tissue. Several ...previous reports have described biochemical anti-tumorigenic properties of crude snake venom or its components, including their capability of inhibiting cell proliferation and promoting cell death. However, to the best of our knowledge, there is no work describing cancer cell proteomic changes following treatment with snake venoms. In this work we describe the quantitative changes in proteomics of MCF7 and MDA-MB-231 breast tumor cell lines following treatment with
snake venom, as well as the functional implications of the proteomic changes. Cell lines were treated with sub-toxic doses at either 0.63 μg/mL (low) or 2.5 μg/mL (high) of
venom for 24 h, conditions that cause no cell death per se. Proteomics analysis was conducted on a nano-scale liquid chromatography coupled on-line with mass spectrometry (nLC-MS/MS). More than 1000 proteins were identified and evaluated from each cell line treated with either the low or high dose of the snake venom. Protein profiling upon venom treatment showed differential expression of several proteins related to cancer cell metabolism, immune response, and inflammation. Among the identified proteins we highlight histone H3, SNX3, HEL-S-156an, MTCH2, RPS, MCC2, IGF2BP1, and GSTM3. These data suggest that sub-toxic doses of
venom have potential to modulate cancer-development related protein targets in cancer cells. This work illustrates a novel biochemical strategy to identify therapeutic targets against cancer cell growth and survival.
Mine tailings are produced by mining activities and contain diverse heavy metal ions, which cause environmental problems and have negative impacts on ecosystems. Different microorganisms, including ...yeasts, play important roles in the absorption and/or adsorption of these heavy metal ions. This work aimed to analyze proteins synthesized by the yeast Yarrowia lipolytica AMJ6 (Yl-AMJ6), isolated from Andean mine tailings in Peru and subjected to stress conditions with common heavy metal ions. Yeast strains were isolated from high Andean water samples impacted by mine tailings from Yanamate (Pasco, Peru). Among all the isolated yeasts, the Yl-AMJ6 strain presented LC50 values of 1.06 mM, 1.42 mM, and 0.49 mM for the Cr+6, Cu+2, and Cd+2 ions, respectively. Proteomic analysis of theYl-AMJ6 strain under heavy metal stress showed that several proteins were up- or downregulated. Biological and functional analysis of these proteins showed that they were involved in the metabolism of proteins, nucleic acids, and carbohydrates; response to oxidative stress and protein folding; ATP synthesis and ion transport; membrane and cell wall; and cell division. The most prominent proteins that presented the greatest changes were related to the oxidative stress response and carbohydrate metabolism, suggesting the existence of a defense mechanism in these yeasts to resist the impact of environmental contamination by heavy metal ions.
The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the
scorpion. Although envenomed patients mostly suffer ...neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in
venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from
venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from
spp. and human testis ACE. These findings advance our understanding of
venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension.
Vogt-Koyanagi-Harada disease (VKH), an autoimmune disease targeted against melanocytes, is associated with HLA-DRB1*0405. This study was undertaken to analyze T-cell recognition and the cytokine ...expression profile induced by melanocyte epitopes in HLA-DRB1*0405-positive and -negative patients with VKH uveitis.
Peripheral blood mononuclear cells (PBMC) proliferation and Th1 (IFN-gamma) and Th2 (IL-4 and IL-5) cytokine production were analyzed in HLA-DRB1*0405-positive (n = 12) and -negative (n = 22) patients with VKH and HLA-DRB1*0405-positive (n = 9) and -negative (n = 8) control subjects in response to human melanoma cell line lysate (HMCLL) and 28 synthetic peptides derived from the human melanocyte differentiation proteins TYR, TRP1, TRP2, and Pmel17. The peptides were selected using the TEPITOPE algorithm, based on their predicted binding to HLA-DRB1*0405 and to the non-disease-related HLA-DRB1*15.
HMCLL was recognized exclusively by the patients' PBMC (44%) but not by those of the control subjects (P < 0.01). PBMC from patients with VKH recognized an increased breadth of melanocyte-derived peptides at lower peptide concentrations than in the control subjects (68% vs. 25%; P < 0.01, at 1 microM) and did not produce the Th2 cytokine IL-4 in response to disease-specific peptides (0% vs. 50%, P < 0.001). Five peptides were exclusively recognized in patients bearing HLA-DRB1*0405. Furthermore, HLA-DRB1*0405-bearing patients, but not those with HLA-DRB1*15, recognized an increased breadth of melanocyte epitopes in comparison to HLA-matched control subjects (60% vs. 28%; P < 0.05).
These data indicate that patients with VKH are sensitized to melanocyte epitopes and display a peptide-specific Th1 cytokine response. In addition, the data indicate that patients bearing HLA-DRB1*0405 recognize a broader melanocyte-derived peptide repertoire, reinforcing the importance of this allele in susceptibility to the development of VKH disease.
The spleen is considered a non-essential organ. However, its importance is increasingly clear, given the serious disorders caused by its absence or dysfunction, e.g., greater susceptibility to ...infections, thromboembolism and cancer. Surgical techniques to preserve the spleen and maintain splenic function have become increasingly common. However, the morbidity and mortality associated with its absence and dysfunction are still high. We used the decellularization technique to obtain a viable splenic scaffold for recellularization
and propose the idea of bioengineered spleen transplantation to the host. We observed the maintenance of important structural components such as white pulp, marginal zone and red pulp, in addition to the network of vascular ducts. The decellularized scaffold presents minimal residual DNA and SDS, which are essential to prevent immunogenic responses and transplantation failure. Also, the main components of the splenic matrix were preserved after decellularization, with retention of approximately 72% in the matrisomal protein content. The scaffold we developed was partially recellularized with stromal cells from the spleen of neonatal rats, demonstrating adhesion, proliferation and viability of cells. Therefore, the splenic scaffold is very promising for use in studies on spleen reconstruction and transplantation, with the aim of complete recovery of splenic function.
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