Search

We evaluated the role of intergenerational paternal exercise on fibrosis, inflammatory profile, and redox status in the adipose tissue of male rat offspring fed with high-fat diet (HFD) and explored to what extent programming affects the systemic metabolic profile.Adult wistar rats were randomly divided into two groups: sedentary fathers and trained fathers (8 weeks of resistance training (RT), three times per week). The offspring were obtained by mating with sedentary females. Upon weaning, male offspring were divided into four groups (7 animals per group): offspring of sedentary fathers exposed to either a control diet (SFO-C) or a high-fat diet (SFO-HF); offspring of trained fathers exposed to a control diet (TFO-C) or a high-fat diet (TFO-HF).Paternal RT was effective in attenuating body weight gain, adipocyte size, collagen deposition, as well as downregulating genes (CTGF, VEGF, C/EBPα SREBP1, MCP-1, and NF-kB), pro-inflammatory cytokine levels (Tumor Necrosis Factor alpha and Interleukin-1-beta), matrix metalloproteinase -2 activity, and ROS production in the epididymal adipose tissue of offspring fed with HFD (TFO-HF vs. SFO-HF; P 0.05). Moreover, paternal RT increased adiponectin and superoxide dismutase (SOD) activity in the tissue. These beneficial effects were accompanied by the increase of antioxidant enzymes (SOD and α-Klotho), while decreasing pro-oxidant agents (F2-isoprostanes, protein carbonyls levels), and metabolic markers (insulin and leptin, HOMA-β, and HOMA-IR) in the offspring blood circulation.Our findings reveal protective effects of intergenerational paternal RT on adipose tissue remodeling and metabolic health of offspring fed with HFD.

Cowpea (Vigna unguiculata L. Walp) is a legume of great economic importance, however it is highly affected by nematodes. The present work aimed to identify proteins and genes involved in nematode resistance by proteomic and transcriptomic analysis. Plants of a genotype resistant (CE31) to root-knot nematode (Meloidogyne spp.) were collected 12 days after inoculation with Meloidogyne incognita and the total proteins and RNA were extracted from the root samples. Shotgun proteomic analysis was performed using an Orbitrap Elite mass spectrometer and the construction and sequencing of cDNA libraries were carried out in a Hi-Seq 2000 sequencing system. The proteomic and transcriptomic analyses revealed key processes involved in cowpea defense and some interesting candidates were further analyzed by RT-qPCR. Proteins and genes involved in essential biological processes were differentially accumulated such as, regulation of transcription, cell wall stiffening and microtubule-based process. However, the main defense strategies of Vigna unguiculata seem to be focused on the interaction of NBS-LRR and WRKY genes for the activation of R genes, production of protease inhibitors and maintenance of actin cytoskeleton. These are key processes that can culminate in the suppression of giant cell formation and consequently in the development of Meloidogyne incognita. SIGNIFICANCE: In this study, we identified proteins and transcripts regulated in cowpea resistant to the nematode Meloidogyne spp. upon inoculation. The results revealed key candidate genes involved in the activation of R genes, the production of protease inhibitors and maintenance of the actin cytoskeleton. These processes might be essential for cowpea resistance, as they can impede nematode nutrition, giant cell formation and consequently the development of Meloidogyne incognita.

A significant number of studies have demonstrated that paternal exercise modulates future generations via effects on the sperm epigenome. However, comprehensive information regarding the effects of exercise performed by the father on different tissues and their clinical relevance has not yet been explored in detail. This narrative review is focused on the effects of paternal exercise training on various physiological systems of offspring. A detailed mechanistic understanding of these effects could provide crucial clues for the exercise physiology field and aid the development of therapeutic approaches to mitigate disorders in future generations. Non-coding RNA and DNA methylation are major routes for transmitting epigenetic information from parents to offspring. Resistance and treadmill exercise are the most frequently used modalities of planned and structured exercise in controlled experiments. Paternal exercise orchestrated protective effects over changes in fetus development and placenta inflammatory status. Moreover paternal exercise promoted modifications in the ncRNA profiles, gene and protein expression in the hippocampus, left ventricle, skeletal muscle, tendon, liver and pancreas in the offspring, while the transgenerational effects are unknown. Paternal exercise demonstrates clinical benefits to the offspring and provides a warning on the harmful effects of a paternal unhealthy lifestyle. Exercise in fathers is presented as one of the most logical and cost-effective ways of restoring health in the offspring and, consequently, modifying the phenotype. It is important to consider that paternal programming might have unique significance in the developmental origins of offspring diseases.

Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.

The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.

Scorpion venoms are formed by toxins harmful to various organisms, including humans. Several techniques have been developed to understand the role of proteins in animal venoms, including proteomics approach. Rhopalurus agamemnon (Koch, 1839) is the largest scorpion in the Buthidae family in the Brazilian Cerrado, measuring up to 110 mm in total length. The accident with R. agamemnon is painful and causes some systemic reactions, but the specie's venom remains uninvestigated. We explore the venom protein composition using a proteomic and a biological-directed approach identifying 230 protein compounds including enzymes like Hyaluronidase, metalloproteinase, L-amino acid oxidase and amylase, the last two are first reported for scorpion venoms. Some of those new reports are important to demonstrate how distant we are from a total comprehension of the diversity about venoms in general, due to their diversity in composition and function. Biological significance In this study, we explored the composition of venom proteins from the scorpion Rhopalurus agamemnon. We identified 230 proteins from the venom including new enzyme reports. These data highlight the unique diversity of the venom proteins from the scorpion R. agamemnon, provide insights into new mechanisms of envenomation and enlarge the protein database of scorpion venoms. The discovery of new proteins provides a new scenario for the development of new drugs and suggests molecular targets to venom components.

Dental pulp is a specialized tissue able to respond to infectious processes. Nevertheless, infection progress and root canal colonization trigger an immune-inflammatory response in tooth-surrounding tissues, leading to apical periodontitis and bone tissue destruction, further contributing to tooth loss. In order to shed some light on the effects of IL-4 on periradicular pathology development modulation, microtomographic, histological and proteomic analyses were performed using 60 mice, 30 wild type and 30 IL-4−/−. For that, 5 animals were used for microtomographic and histological analysis, and another 5 for proteomic analysis for 0, 7 and 21 days with/without pulp exposure. The periapical lesions were established in WT and IL-4−/− mice without statistical differences in their volume, and the value of p Significance: IL-4 might play a protective role in diseases involving bone destruction and its activity may contribute to host protection, mainly due to its antiosteoclastogenic action.

Intestinal ischemia reperfusion injury (iIRI) is a severe clinical condition presenting high morbidity and mortality worldwide. Some of the systemic consequences of IRI can be prevented by applying ischemic preconditioning (IPC), a series of short ischemia/reperfusion events preceding the major ischemia. Although neutrophils are key players in the pathophysiology of ischemic injuries, neither the dysregulation presented by these cells in iIRI nor the protective effect of iIPC have their regulation mechanisms fully understood. Protein phosphorylation, as well as the regulation of the respective phosphatases and kinases are responsible for regulating a large number of cellular functions in the inflammatory response. Moreover, in previous work we found hydrolases and transferases to be modulated in iIR and iIPC, suggesting the possible involvement of phosphatases and kinases in the process. Therefore, in the present study, we analyzed the phosphoproteome of neutrophils from rats submitted to mesenteric ischemia and reperfusion, either submitted or not to IPC, compared to quiescent controls and sham laparotomy. Proteomic analysis was performed by multi-step enrichment of phosphopeptides, isobaric labeling, and LC-MS/MS analysis. Bioinformatics was used to determine phosphosite and phosphopeptide abundance and clustering, as well as kinases and phosphatases sites and domains. We found that most of the phosphorylation-regulated proteins are involved in apoptosis and migration, and most of the regulatory kinases belong to CAMK and CMGC families. An interesting finding revealed groups of proteins that are modulated by iIR, but such modulation can be prevented by iIPC. Among the regulated proteins related to the iIPC protective effect, Vamp8 and Inpp5d/Ship are discussed as possible candidates for control of the iIR damage.

Background Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood. Methods After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes. Results After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication. Conclusions To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation.

Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53 kDa monomer and 124 and 178 kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44 kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60 °C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9 μg/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.

The calcaneal tendon (CT) is the most commonly injured tendon in the human body. Moreover, with advancing age, the amount of damage increases further. Resistance training (RT) could be used to minimize such damages. The aim of the present study was to obtain the identification, detailed protein cataloging and biochemical characterization based on the effects of the aging process and the RT in CT of rats. The analysis by liquid chromatography tandem-mass spectrometry showed 142 distinct proteins, however, only 29 proteins met the inclusion criteria and were analyzed. Aging causes a reduction in the abundance of seven proteins related to extracellular matrix organization, biological regulation and cellular processes. However, RT promoted the positive regulation of proteins important for the maintenance of healthy tendons: seven proteins in young trained and two in older trained group. This study contributes to a better understanding of molecular aspects of the tendon. The down regulation of proteins linked to mechanical function and extracellular matrix remodeling of the tendon during aging can contribute to the increase of injury and weakness in the tendon. Nevertheless, RT proved to be a tool to prevent these adverse effects during aging by increasing proteins involved in the functionality of the tendon.

In recent years, the number of new antimicrobial drugs launched on the market has decreased considerably even though there has been an increase in the number of resistant microbial strains. Thus, antimicrobial resistance has become a serious public health problem. Amphibian skin secretions are a rich source of host defense peptides, which generally are cationic and hydrophobic molecules, with a broad-spectrum of activity. In this study, one novel multifunctional defense peptide was isolated from the skin secretion of the Chaco tree frog, Boana raniceps. Figainin 2 (1FLGAILKIGHALAKTVLPMVTNAFKPKQ28) is cationic and hydrophobic, adopts an α-helical structure in 50% (v/v) trifluoroethanol (TFE), and is thermally stable. This peptide exhibited activity against Gram-negative and Gram-positive pathogenic bacteria arboviruses, T. cruzi epimastigotes; however, it did not show activity against yeasts. Figainin 2 also showed antiproliferative activity on cancer cells, is moderately active on human erythrocytes, and activates the oxidative burst in human neutrophils.

Ancestral obesogenic exposure is able to trigger harmful effects in the offspring left ventricle (LV) which could lead to cardiovascular diseases. However, the impact of the father’s lifestyle on the offspring LV is largely unexplored. The aim of this study was to investigate the effects of 8 weeks of paternal resistance training (RT) on the offspring left ventricle (LV) proteome exposed to control or high-fat (HF) diet. Wistar rats were randomly divided into two groups: sedentary fathers and trained fathers (8 weeks, 3 times per week with weights secured to the animals’ tails). The offspring were obtained by mating with sedentary females. Upon weaning, male offspring were divided into 4 groups (5 animals per group): offspring from sedentary fathers, exposed to control diet (SFO-C); offspring from trained fathers, exposed to control diet (TFO-C); offspring from sedentary fathers, exposed to high-fat diet (SFO-HF); and offspring from trained fathers, exposed to high-fat diet (TFO-HF). The LC-MS/MS analysis revealed 537 regulated proteins among groups. Offspring exposure to HF diet caused reduction in the abundance levels of proteins related to cell component organization, metabolic processes, and transport. Proteins related to antioxidant activity, transport, and transcription regulation were increased in TFO-C and TFO-HF as compared with the SFO-C and SFO-HF groups. Paternal RT demonstrated to be an important intervention capable of inducing significant effects on the LV proteome regardless of offspring diet due to the increase of proteins involved into LV homeostasis maintenance. This study contributes to a better understanding of the molecular aspects involved in transgenerational inheritance.

Depsipeptides are a group of biologically active peptides that have at least one of the amide bonds replaced by an ester bond. These peptides sometimes present additional chemical modifications, including unusual amino acid residues in their structures. Depsipeptides are known to exhibit a large array of bioactivities, such as anticancer, antiproliferative, antimicrobial, antiviral and antiplasmodial properties. They are commonly found in marine organisms: bacteria, tunicates, mollusks, sponges, and others. Herein, we summarize the latest insights about marine depsipeptides, their mechanisms of action and potential as therapeutic agents.

This review discusses the importance and properties of antimicrobial peptides from frogs and their synthetic analogues as potential therapeutic alternatives in fighting not only bacterial infections, but also protozoans involved with the major neglected diseases, which afflict human populations (e.g., Chagas disease, African sleeping sickness, Leishmaniasis and malaria). Here, we emphasize their multifunctional properties such as promising broad-spectrum drugs that target protozoan parasites too.

Apoptosis, a genetically directed process of cell death, has been studied for many years, and the biochemical mechanisms that surround it are well known and described. There are at least three pathways by which apoptosis occurs, and each pathway depends on extra or intracellular processes for activation. Apoptosis is a vital process, but disturbances in proliferation and cell death rates can lead to the development of diseases like cancer. Several compounds, isolated from scorpion venoms, exhibit inhibitory effects on different cancer cells. Indeed, some of these compounds can differentiate between healthy and cancer cells within the same tissue. During the carcinogenic process, morphological, biochemical, and biological changes occur that enable these compounds to modulate cancer but not healthy cells. This review highlights cancer cell features that enable modulation by scorpion neurotoxins. The properties of the isolated scorpion neurotoxins in cancer cells and the potential uses of these compounds as alternative treatments for cancer are discussed.

Fever is a brain-mediated increase in body temperature mainly during inflammatory or infectious challenges. Although there is considerable data regarding the inflammation pathways involved in fever, metabolic alterations necessary to orchestrate the complex inflammatory response are not totally understood. We performed proteomic analysis of rat hypothalamus using label-free LC-MS/MS in a model of fever induced by lipopolysaccharide (LPS) or prostaglandin EThe evolutionary persistence of fever, despite the elevated cost for maintenance of this response, suggests that elevation in core temperature may represent an interesting strategy for survival. Fever response is achieved through the integrated behavioral, physiological, immunological and biochemical processes that determine the balance between heat generation and elimination. The development of such complex response arouses interest in studying how the cell metabolism responds or even contributes to promote fever. Our results offered comprehensive information about fever responses, including metabolic and inflammatory pathways, providing new insights into candidate proteins potentially involved in inflammatory signaling and metabolic changes in the hypothalamus during fever induced by systemic LPS and central PGE

Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules.

WOS: 000449272100017, PubMed: 30397208, Male fertility is the ability of sperm to fertilize the egg and sustain embryo development. Several factors determine the fertilizing capacity of mammalian sperm, including those intrinsic to sperm and components of the seminal plasma. The present study analyzed the seminal fluid proteome of Bos taurus and potential associations between proteins and fertility scores. Mass spectrometry coupled with nano HPLC allowed the identification of 1,159 proteins in the dairy bull seminal plasma. There were 50 and 29 seminal proteins more abundant in high (HF) low fertility (LF) bulls, respectively. Based on multivariate analysis, C-type natriuretic peptide, TIMP-2, BSP5 and sulfhydryl oxidase indicated relationship with HF bulls. Clusterin, tissue factor pathway inhibitor 2, galectin-3-binding protein and 5'-nucleotidase were associated with LF bulls. Abundance of NAD(P)(+)-arginine ADP-ribosyltransferase, prosaposin and transmembrane protein 2 proteins had the highest positive correlations with fertility ranking. Quantities of vitamin D-binding protein, nucleotide exchange factor SIL1 and galectin-3-binding protein showed the highest negative correlations with fertility ranking. A fertility ranking score was calculated and the relationship with these proteins was significant (Spearman's rho = 0.94). The present findings represent a major and novel contribution to the study of bovine seminal proteins. Indicators of fertility can be used to improve reproductive biotechnologies., Alta Genetics, Inc.; Mississippi Agricultural and Forestry Experiment Station, Selcuk University, Federal Research Councils of Brazil (CAPES); Research Foundation of Ceara State (FUNCAP); Financiadora de Estudos e Projetos" (FINEP, Brazil)Ciencia Tecnologia e Inovacao (FINEP) [0439/11, 0694/13]; CAPESCAPES; Mississippi Agricultural and Forestry Experiment Station, Selcuk University, Federal Research Councils of Brazil (CNPQ), The present study was funded in part by Alta Genetics, Inc. and Mississippi Agricultural and Forestry Experiment Station, Selcuk University, Federal Research Councils of Brazil (CAPES, CNPq) and Research Foundation of Ceara State (FUNCAP). Grants 0439/11 and 0694/13 from "Financiadora de Estudos e Projetos" (FINEP, Brazil) were conceded to Sousa, M.V. through PROINFRA program. Scholarships for graduate students (Viana, A.G.A. and Pontes, A.F.) were provided by CAPES.

Inflammatory cascades and mechanisms are ubiquitous during host responses to various types of insult. Biological models and interventional strategies have been devised as an effort to better understand and modulate inflammation-driven injuries. Amongst those the two-hit model stands as a plausible and intuitive framework that explains some of the most frequent clinical outcomes seen in injuries like trauma and sepsis. This model states that a first hit serves as a priming event upon which sequential insults can build on, culminating on maladaptive inflammatory responses. On a different front, ischemic preconditioning (IPC) has risen to light as a readily applicable tool for modulating the inflammatory response to ischemia and reperfusion. The idea is that mild ischemic insults, either remote or local, can cause organs and tissues to be more resilient to further ischemic insults. This seemingly contradictory role that the two models attribute to a first inflammatory hit, as priming in the former and protective in the latter, has set these two theories on opposing corners of the literature. The present review tries to reconcile both models by showing that, rather than debunking each other, each framework offers unique insights in understanding and modulating inflammation-related injuries.

OBJECTIVES: Intestinal ischemia/reperfusion often leads to acute lung injury and multiple organ failure. Ischemic preconditioning is protective in nature and reduces tissue injuries in animal and human models. Although hematimetric parameters are widely used as diagnostic tools, there is no report of the influence of intestinal ischemia/reperfusion and ischemic preconditioning on such parameters. We evaluated the hematological changes during ischemia/reperfusion and preconditioning in rats. METHODS: Forty healthy rats were divided into four groups: control, laparotomy, intestinal ischemia/reperfusion and ischemic preconditioning. The intestinal ischemia/reperfusion group received 45 min of superior mesenteric artery occlusion, while the ischemic preconditioning group received 10 min of short ischemia and reperfusion before 45 min of prolonged occlusion. A cell counter was used to analyze blood obtained from rats before and after the surgical procedures and the hematological results were compared among the groups. RESULTS: The results showed significant differences in hematimetric parameters among the groups. The parameters that showed significant differences included lymphocyte, white blood cells and granulocyte counts; hematocrit; mean corpuscular hemoglobin concentration; red cell deviation width; platelet count; mean platelet volume; plateletcrit and platelet distribution width. CONCLUSION: The most remarkable parameters were those related to leukocytes and platelets. Some of the data, including the lymphocyte and granulocytes counts, suggest that ischemic preconditioning attenuates the effect of intestinal ischemia/reperfusion on circulating blood cells. Our work contributes to a better understanding of the hematological responses after intestinal ischemia/reperfusion and IPC, and the present findings may also be used as predictive values.

In efforts to find new antimicrobial peptides (AMPs), we studied the skin secretion of the endemic Colombian frog Dendropsophus columbianus belonging to a genus that has not been investigated previously. From HPLC-fractionated secretion, we identified one peptide with slightly antibacterial activity. Its peptide sequence showed no sequence similarity to current annotated peptides. We named this novel peptide dendropsophin 1 (Dc1). Afterward, two analogues were designed (Dc1.1 and Dc1.2) to improve the cationic and amphipathic features. Then, their antiproliferative and cytotoxic properties were evaluated against several pathogens including bacteria, fungi, protozoa and also mammalian cells. Dc1 and its two analogues exhibited moderate antibacterial activities and no hemolytic and cytotoxic effects on mammalian cells. Analogue Dc1.2 showed slightly improved antibacterial properties. Their secondary structures were characterised using CD spectroscopy and Dc1.2 displayed a higher α-helix content and thermal stability compared to Dc1 and Dc1.1 in hydrophobic experimental conditions.

Introduction Human amniotic membrane (AM) has been used as a biomaterial for sur-gical wound skin and ocular surface reconstruction for several years. Currently, equineAM has been used for corneal reconstruction in several animal species, and appears tohave the same properties as human AM. Despite the observed positive healing abilitiesof this tissue in horses with ulcerative keratitis the proteins of equine AM have notbeen described.Objective To identify proteins known to be associated with corneal healing fromfrozen equine AM.Procedures Placentas were acquired from healthy live foal births from a local Thor-oughbred breeding farm. The amnion was removed from the chorion by blunt dissec-tion, washed with phosphate-buffered saline (PBS), and treated with 0.05% trypsinand 0.02% ethylene diaminetetraacetic acid in PBS. Amnion was attached to nitrocel-lulose paper (epithelial side up), and cut into 4 9 4 cm pieces. The sheets were frozenat 80 °C. The protein samples were solubilized, and analyzed by 2D gel electropho-resis and shotgun proteomics.Results A reference identification map of the equine AM proteins was produced and149 different proteins were identified. From gel-based proteomics, 49 spots wereexcised and 43 proteins identified by liquid chromatography coupled to tandem massspectrometry (LC-MS/MS). Shotgun proteomics identified 116 proteins with an over-lap of 10 proteins in both analyses.Conclusions We have described a reference map for equine AM proteins that mayprovide a background to explain the positive results found in horses with ulcerativekeratopathies using this biomaterial.Key Words: amniotic membrane, dermatopontin, horse, lumican, protein, proteome

Intestinal ischemia and reperfusion injury are widely used models, which result into tissue injury and multiple organ failure also observed after trauma and surgery. Ischemic preconditioning (IPC) preceding ischemia and reperfusion (IR) was shown to attenuate this injury and has a potential therapeutic application; however the exact underlying mechanism is not clear. Neutrophils play an important role in the mechanism of injuries caused by ischemia and reperfusion while IPC led to a decrease in neutrophil stimulation and activation. The effect of preconditioning on the neutrophil proteome is unclear. Proteomic analysis has been ratified as an appropriate tool for studying complex systems. In order to evaluate the effect of IPC preceding 45 min of ischemia on the proteome of neutrophils we used Wistar rats divided in four experimental groups: Control, sham laparotomy, intestinal ischemia reperfusion and ischemic preconditioning. After neutrophil separation, proteins were extracted, trypsin digested and the resulting peptides were iTRAQ labeled followed by HILIC fractionation and nLC-MS/MS analysis. After database searches, normalization and statistical analysis our proteomic analysis resulted in the identification of 2437 protein groups that were assigned to five different clusters based on the relative abundance profiles among the experimental groups. The clustering followed by statistical analysis led to the identification of significantly up and downregulated proteins in IR and IPC. Cluster based KEGG pathways analysis revealed up- regulation of actin cytoskeleton, metabolism, Fc gamma R mediated phagocytosis, chemokine signaling, focal adhesion and leukocyte transendothelial migration whereas downregulation in ribosome, spliceosome, RNA transport, protein processing in endoplasmic reticulum and proteasome, after intestinal ischemic preconditioning. Furthermore, enzyme prediction analysis revealed the regulation of some important antioxidant enzymes and having their role in reactive oxygen species production. To our knowledge, this work describes the most comprehensive and detailed quantitative proteomic study of the neutrophil showing the beneficial role of ischemic preconditioning and its effects on the neutrophil proteome. This data will be helpful to understand the effect of underlying protective mechanisms modulating the role of PMNs after IPC and provide a trustworthy basis for future studies. Biological significance Preconditioning is a relevant strategy to overcome clinical implications from ischemia and reperfusion. Such implications have the neutrophil as a major player. Although many publications describe specific biochemical and physiological roles of the neutrophil in such conditions, there is no report of a proteomic study providing a broader view of this scenario. Here we describe a group of proteins significantly regulated by ischemia and reperfusion being such regulation prevented by preconditioning. Such finding may provide relevant information for a deeper understanding of the mechanisms involved, as well as serve as basis for future biomarker or drug target assays.

In clinical conditions trauma is associated with high mortality and morbidity. Neutrophils play a key role in the development of multiple organ failure after trauma. To have a detailed understanding of the neutrophil activation at primary stages after trauma, neutrophils were isolated from control and surgical trauma rats in this study. Extracted proteins were analyzed using nano liquid chromatography coupled to tandem mass spectrometry. A total of 2924 rat neutrophil proteins were identified in our analysis, of which 393 were found differentially regulated between control and trauma groups. By using functional pathways analysis of the 190 proteins up-regulated in surgical trauma we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations were then confirmed by in silico protein-protein interaction analysis. Collectively, our results reveal that neutrophils drastically regulate their biochemical pathways after the early stages of surgical trauma, showing lower activity. This implies higher susceptibility of the trauma patients to infection and bystander tissues damage. This article is protected by copyright. All rights reserved.

Despite progress in understanding carcinogenesis, cancer is still a major cause of morbidity and mortality worldwide. Treatment is complicated by the resistance to chemotherapeutics acquired by tumors, ability to repair damaged DNA, alteration of the interaction between drug and its target, and defects in the machinery that leads to apoptosis. The development of novel therapies based on biologically active peptides, especially antimicrobial peptides (AMPs), and proteins has emerged as a new strategy to defeat cancer.

Extracellular vesicles (EVs), including exosomes, play a key role in inter and intracellular communication, promoting the proliferation and invasion of recipient cells to support tumor growth and metastasis. Metastasis comprises multiple steps that first include the detachment of tumor cells through epithelial to mesenchymal transition (EMT), allowing the physical dissemination to distant organs. Thereafter, cancer-derived exosomes are still critical components for preparing the tumor microenvironment by (i) enabling tumor cells to escape from the immunological surveillance and (ii) arranging the pre-metastatic site for the engraftment of detached cancer cells. In this review, we discuss the multifaceted role of EVs in the multiple steps of metastasis. Future research directions draw attention to EVs as biological targets for cancer diagnosis, prognosis and therapy. However, due to their significant role in cell communication, they may become a valuable drug delivery system.

Recently the peptide Hy-a1 (IFGAILPLALGALKNLIK), with antimicrobial activity, was isolated from the skin secretion of the frog Hypsiboas albopunctatus. The aim of the present work was to evaluate four analogues with introduction of acetyl group, Asp or Lys at the N-terminus of antimicrobial peptide Hy-al to supply information about the relationship of structure-biological activity. The antimicrobial activities were assayed by measuring growth inhibition of four species of bacteria and four species of fungus. The hemolytic activity was also tested. The peptide containing Trp instead of Leu in position 6 (for fluorescence studies) presented MIC values comparable to wild type sequence: 32 μmol L(-1) , 32 μmol L(-1) , 8 μmol L(-1) , and 2 μmol L(-1) for E. coli, P. aeruginosa, S. aureus, and B. subtilis, respectively. Two peptides with this modification and containing one acetyl group or Asp residue at the N-terminal region showed activities only against Gram-positive bacteria. Different results were observed when the residue added was Lys. In this case, the activity against the microorganisms was sustained or increased. Conformational properties were investigated by CD techniques in water, TFE, and in zwitterionic micelles (LPC). The CD experiments demonstrated that, in water, the peptides had a random structure, but in TFE and LPC solutions they acquired an ordered structure, composed mainly by α-helix. However, these data have no relationship with activity against Gram-positive bacteria. These results showed that the N-terminal region of the peptide Hy-a1 has key roles in its antibacterial action toward different types of bacteria.

RP-HPLC fractionation of the electrically stimulated skin secretion of the arboreal South American frog Hypsiboas albopunctatus (“spotted treefrog”) led to the isolation of a cytolytic C-terminally amidated peptide. This novel peptide, named hylin a1 (Hy-a1), consists of 18 amino acid residues (IFGAILPLALGALKNLIK-NH 2 ). The α-helical structure of the synthetic hylin a1 peptide was confirmed by CD spectroscopy in the presence of 60% (v/v) TFE. The synthetic peptide displayed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria including Escherichia coli , Staphylococcus aureus , Enterococcus faecalis , Bacillus subtilis and Pseudomonas aeruginosa and also against fungi ( Candida albicans , C. krusei , C. parapsilosis and Cryptococcus neoformans ) . Hylin a1 was also able to disrupt human erytrocytes (HC 50 = 18 μM). Similarity analysis using PSI-BLAST revealed 50–44% of identity to maximins Hv, H16, H15 and H10 from Bombina maxima and also to hylins b1 and b2 (Hy-b1 and Hy-b2) from Hypsiboas lundii (synonym: Hyla biobeba ).

Neobatrachia is the amphibian suborder with the largest number of species and a most important source of bioactive peptides from frog skin secretions. However, 90% of the studies on this subject have been focused on the frog families Hylidae and Ranidae, while very little is known about peptides of other families, like Leptodactylidae. Our work reports for the first time the isolation and characterization of ocellatin 4 (GLLDFVTGVGKDIFAQLIKQI-NH 2 ), a cytolytic peptide from the skin secretion of the South American frog Leptodactylus ocellatus . While most cytolytic amphibian skin peptides are selective for microorganisms and harmless for mammalian cells, the HC 50 of ocellatin 4 against human erythrocytes is 14.3 μM. The interaction between ocellatin 4 and zwitterionic phospholipids in mammalian plasma membranes may be favored by its neutral charge at pH 7.0. Ocellatin 4 also shows some antibacterial activity (MICs E. coli and S. aureus =64 μM) and its sequence shares similarities with the only six leptodactylid peptides previously known and with four peptides from Australian hylid frogs of the genus Litoria .

The Nelore bull (Bos taurus indicus) seminal plasma proteome was analyzed by MALDI-TOF MS and two-dimensional gel electrophoresis. A total of 260 spots were visualized in the 2-DE gel (pI range 3-10) and 13 spots could be identified by peptide mass fingerprinting corresponding to 11 different polypeptides. The results allowed the creation of the first proteomic map of Bos taurus indicus seminal plasma. The roles of the identified proteins in the bull seminal plasma are discussed.

Trypanosoma cruzi, the protozoan that causes Chagas disease, possesses a complex life cycle involving different developmental stages. Experimental conditions for two-dimensional electrophoresis (2-DE) analysis of T. cruzi trypomastigote, amastigote and epimastigote proteomes were optimized. Comparative proteome analysis of the cell-cycle stages were carried out, revealing that few proteins included in the 2-DE maps displayed significant differential expression among the three developmental forms of the parasite. In order to identify landmark proteins, spots from the trypomastigote 2-DE map were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mass fingerprinting, resulting in 26 identifications that corresponded to 19 different proteins. Among the identified polypeptides, there were heat shock proteins (HSP; chaperones, HSP 60, HSP 70 and HSP 90), elongation factors, glycolytic pathway enzymes (enolase, pyruvate kinase and 2,3 bisphosphoglycerate mutase) and structural proteins (KMP 11, tubulin and paraflagellar rod components). The relative expression of the identified proteins in the 2-DE maps of the T. cruzi developmental stages is also presented.

The composition of indolealkylamines of Bufo rubescens cutaneous secretions was compared to those from six other Brazilian bufonids. Skin, parotoid and tibial gland secretions were obtained for analysis by thin-layer chromatography. A triple-quadrupole mass spectrometer was used to confirm the indolealkylamines standards (serotonin, 5-HT; bufotenin, BTN; dehydrobufotenin, DHB and bufotenidin, BTD). We observed clear variation in the composition of indolealkylamines of the cutaneous secretions studied and also between those found in the skin and parotoid gland secretions of the same species. We discuss the utility of indolealkylamines to the phylogeny of this group of toads.

Antimicrobial peptides (AMPs) appear as a promising therapeutic candidate against multiresistant pathogens, because they are able to kill microorganisms and have low toxicity of resistance cells. Hylin a1 (Hy-a1, IFGAILPLALGALKNLIK-NH2) is a peptide extracted from the skin secretion of the frog Hypsiboas albopunctatus , which displays antimicrobial and hemolytic activities. We report here structural studies of Hy-a1 using different techniques such as fluorescence, CD and NMR. Our data showed that Hy-a1 acquires a well defined amphipathic α-helix when interacting with a membrane-like environment. Furthermore, Hy-a1 presented different affinity when compared to membranes of zwitterionic or anionic lipid composition. Finally, we proposed a molecular interaction model of this peptide with micelles.

Cowpea (Vigna unguiculata L. Walp) is an important legume species well adapted to low fertility soils and prolonged drought periods. One of the main problems that cause severe yield losses in cowpea is the root-knot nematode Meloidogyne incognita. The aim of this work was to analyze the differential expression of proteins in the contrasting cultivars of cowpea CE 31 (highly resistant) and CE 109 (slightly resistant) during early stages of M. incognita infection. Cowpea roots were collected at 3, 6, and 9 days after inoculation and used for protein extraction and 2-DE analysis. From a total of 59 differential spots, 37 proteins were identified, mostly involved in plant defense, such as spermidine synthase, patatin, proteasome component, and nitrile-specifier protein. A follow-up study was performed by quantitative RT-PCR analysis of nine selected proteins and the results revealed a very similar upregulation trend between the protein expression profiles and the corresponding transcripts. This study also identified ACT and GAPDH as a good combination of reference genes for quantitative RT-PCR analysis of the pathosystem cowpea/nematode. Additionally, an interactome analysis showed three major pathways affected by nematode infection: proteasome endopeptidase complex, oxidative phosphorylation, and flavonoid biosynthesis. Taken together, the results obtained by proteome, transcriptome, and interactome approaches suggest that oxidative stress, ubiquitination, and glucosinolate degradation may be part of cowpea CE 31 resistance mechanisms in response to nematode infection.

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0-10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0-8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

The honey bee (Apis mellifera) is a social insect that shows complex and integrated behaviors. Its ability to read and respond to several sets of extrinsic and intrinsic signals is fundamental for the modulation of individual activities and social systems. For instance, A. mellifera behavior changes upon the ontogenetic differentiation from nurse to forager worker subcastes. In this work, brain proteomes of nurses and foragers were compared by two-dimensional gel electrophoresis within pH range of 4-7 in order to find proteins related to such an ontogenetic and behavioral development. Twenty differentially expressed proteins were detected by gel image computational analysis, and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Nurse brain showed increased expression of major royal jelly proteins (MRJP1, MRJP2 and MRJP7), which are related to determination of castes during the honey bee larvae differentiation. Immunocytochemistry and electron microscopy showed that MRJP1 was localized in the cytoplasm of brain cells, seemingly along filaments of the cytoskeleton, in the antennal lobe, optical lobe and mushroom body. Also, MRJP1 was deposited on the rhabdom, a structure of the retinular cells, composed of numerous tubules. Such evidence suggests that MRJP1 could be associated to proteins of filamentous structures. MRJP1 was also found in intercellular spaces between cells in mushrooms bodies, indicating that it is a secreted protein. Other proteins implicated in protein synthesis and putative functions in the olfactory system were also up-regulated in the nurse brain. Experienced foragers overexpressed proteins possibly involved in energy production, iron binding, metabolic signaling and neurotransmitter metabolism. Such differential expression of proteins may be related to ontogenetic and behavior changes in A. mellifera.

The salivary complex of leeches contains many components able to modulate physiological mechanisms, such as coagulation and fibrinolysis, and it is composed by the salivary glands and proboscis, encompassing two different proteomes. The bidimensional electrophoretic pattern of the salivary complex from the Haementeria depressa leech revealed a total of 352 spots, 103 in common with the muscular tissue and 249 exclusive from the salivary complex as detected by silver staining; these spots showed isoelectric points from 3.5 to 9.5 and covered an apparent molecular weight range from 10 to 105 kDa. The following isoforms of proteins were identified by mass spectrometry analysis: antiplatelet protein, myohemerythrin and carbonic anhydrase. Since the leeches were not fed for about 2-3 months to stimulate the secretion of proteins that facilitates the blood metabolism, these most abundant proteins in the salivary complex excised from leeches, are expected to play a role during feeding and might have some anti-hemostatic properties. Furthermore, by zymography, a gelatinolytic and a fibrinolytic protein were identified.

Brown spiders of the Loxosceles genus are distributed worldwide. In Brazil, eight species are found in Southern states, where the envenomation by Loxosceles venom (loxoscelism) is a health problem. The mechanism of the dermonecrotic action of Loxosceles venom is not totally understood. Two isoforms of dermonecrotic toxins (loxnecrogins) from L. gaucho venom have been previously purified, and showed sequence similarities to sphingomyelinase. Herein we employed a proteomic approach to obtain a global view of the venom proteome, with a particular interest in the loxnecrogin isoforms' pattern. Proteomic two-dimensional gel electrophoresis maps for L. gaucho, L. intermedia, and L. laeta venoms showed a major protein region (30-35 kDa, pI 3-10), where at least eight loxnecrogin isoforms could be separated and identified. Their characterization used a combined approach composed of Edman chemical sequencing, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and electrospray ionization-quadropole-time of flight tandem mass spectrometry leading to the identification of sphingomyelinases D. The venom was also pre-fractionated by gel filtration on a Superose 12 fast protein liqiud chromatography column, followed by capillary liquid chromatography-mass spectrometry. Eleven possible loxnecrogin isoforms around 30-32 kDa were detected. The identification of dermonecrotic toxin isoforms in L. gaucho venom is an important step towards understanding the physiopathology of the envenomation, leading to improvements in the immunotherapy of loxoscelism.

The emergence, in recent years, of microbial resistance to commonly used antibiotics has aroused a search for new naturally occurring bactericidal and fungicidal agents that may have clinical utility. In the present study, three new antimicrobial peptides were purified from the electrical-stimulated skin secretion of the South American frog Leptodactylus ocellatus by reversed-phase chromatographic procedures. Ocellatin 1 (1GVVDILKGAGKDLLAHLVG ISEKV25-CONH2), ocellatin 2 (1GVVDILKGAGKDLLAHLVGKISEKV25-CONH2) and ocellatin 3 (1GVLDILKNAAKNILAHAAEQI21-CONH2) are structurally related peptides. These peptides present hemolytic activity against human erythrocytes and are also active against Escherichia coli. Ocellatins exhibit significant sequence similarity to other amphibian antimicrobial peptides, mainly to brevinin 2ED from Rana esculenta.

Two hemolytic peptides were isolated from the skin secretion of the Brazilian Hylidae frog Hyla biobeba, using reversed-phase chromatographic procedures. Hylins b1 and b2 exhibit short linear polypeptide chains, a large number of hydrophobic residues, amidated C-termini and hemolytic properties. These two novel peptides are the first examples of bombinins H-like peptides isolated from anurans species not related to Bombina species.

Plants are constantly exposed to a large array of pathogenic organisms and the survival in these conditions demands quick defense responses which include the synthesis of defense peptides and proteins with antimicrobial properties. The main groups of antimicrobial peptides found in plants are thionins, defensins and lipid transfer proteins. They constitute interesting candidates to engineer disease resistance in plants.

Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC−MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC−MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes. Keywords: proteomics • proteome • Trypanosoma cruzi • ICAT reagents • isotope-coded affinity tag

This article is an overview of antimicrobial peptides found in anurans skin secretions. These molecules constitute an initial barrier against microbial infections because of their activity against a large array of microorganisms. These peptides hold remarkable pharmaceutical and technological interest since they selectively kill microorganisms and are unlikely to induce resistance in pathogens. Also, outstanding synergism occurs when these peptides are combined with classic antibiotics and other antimicrobial peptides.

In the present study Tityus fasciolatus crude venom toxicity was evaluated and we also report the purification and characterization of a 6.6 kDa neurotoxin isolated from T. fasciolatus venom. This new toxin, named Tf4, has a molecular mass of 6614 Da and its primary structure is homologous to TbIT-I from T. bahiensis and TsTX-VI and TsNTxP from T. serrulatus. Tf4 delays frog sodium channel inactivation reversibly, but it is non-toxic to mammals or crustaceans. An attempt to identify the residues responsible for the partial loss of toxicity in Tf4 was carried out based on homology modeling and sequence comparison.

The most common manifestation of Loxosceles spider envenoming is a dermonecrotic lesion at the bite site. Dermonecrotic toxins from Loxosceles gaucho venom were purified and characterized by mass spectrometry (capillary liquid chromatography followed by mass spectrometry detection). Two components were purified: a major one of 31,444 Da, called loxnecrogin A, and a minor one of 31,626 Da, called loxnecrogin B, being probably two isoforms of the toxin. The N-terminal sequence of loxnecrogin A showed similarity with N termini of other sphingomyelinolytic dermonecrotic toxins isolated from venoms of different Loxosceles species. The internal sequences did not present any statistically significant hits in sequence databases searches. However, loxnecrogin A partial sequence showed high similarity to regions of L. intermedia LiD1 recombinant protein sequence, recently described in the literature but not yet deposited in databanks.

Antimicrobial peptides (AMPs) represent a large and ubiquitous group of peptides. The current crisis in antibiotic therapy has led to an intensified search for new antimicrobial agents. In this regard, scorpion venom constitutes a rich source of biologically active peptides including AMPs. In the present study, the purification of a novel peptide with antimicrobial activity against the Gram-negative bacteria Klebsiella pneumoniae is described. This antimicrobial peptide, named Cm38, was purified from Centruroides margaritatus scorpion venom using a two-step chromatographic strategy using C8 and C18 columns. This toxin inhibits the proliferation of the Gram-negative bacteria Klebsiella pneumoniae with a Minimal Inhibitory Concentration (MIC) of 64 μM. An analysis of the N-terminal sequence of Cm38 revealed a close structural relationship to Cn11, a Na+-channel modulator toxin previously isolated from Centruroides noxius scorpion venom. Therefore, to test Cm38 for effects on ion channels, we measured its effects on action potential firing in cultured dorsal root ganglion neurons. Cm38 depolarized and increased action potential firing in a subset of neurons tested. The present work reports a new peptide related to Cn11 with antimicrobial properties that is also active in neurons.