Defensin-like peptides from Capsicum chinense induce increased ROS, loss of mitochondrial functionality, and reduced growth of the fungus Colletotrichum scovillei.

In the present study, we identified and characterized two defensin-like peptides in an antifungal fraction obtained from Capsicum chinense pepper fruits and inhibited the growth of Colletotrichum scovillei, which causes anthracnose. AMPs were extracted from the pericarp of C. chinense peppers and subjected to ion exchange, molecular exclusion, and reversed-phase in a high-performance liquid chromatography system. We investigated the endogenous increase in reactive oxygen species (ROS), the loss of mitochondrial functioning, and the ultrastructure of hyphae. The peptides obtained from the G3 fraction through molecular exclusion chromatography were subsequently fractionated using reverse-phase chromatography, resulting in the isolation of fractions F1, F2, F3, F4, and F5. The F1-Fraction suppressed C. scovillei growth by 90, 70.4, and 44% at 100, 50, and 25 µg mL-1, respectively. At 24 h, the IC50 and minimum inhibitory concentration were 21.5 µg mL-1 and 200 µg mL-1, respectively. We found an increase in ROS, which may have resulted in an oxidative burst, loss of mitochondrial functioning, and cytoplasm retraction, as well as an increase in autophagic vacuoles. MS/MS analysis of the F1-Fraction indicated the presence of two defensin-like proteins, and we were able to identify the expression of three defensin sequences in our C. chinense fruit extract. The F1-Fraction was also found to inhibit the activity of insect α-amylases. In summary, the F1-Fraction of C. chinense exhibits antifungal activity against a major pepper pathogen that causes anthracnose. These defensin-like compounds are promising prospects for further research into antifungal and insecticide biotechnology applications. © 2024 Society of Chemical Industry.

Identification of Immunodominant Proteins of the Leishmania (Viannia) naiffi SubProteome as Pan-Specific Vaccine Targets against Leishmaniasis.

Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. A well-modulated immune response that is established after the long-lasting clinical cure of leishmaniasis can represent a standard requirement for a vaccine. Previous studies demonstrated that Leishmania (Viannia) naiffi causes benign disease and its antigens induce well-modulated immune responses in vitro. In this work we aimed to identify the immunodominant proteins present in the soluble extract of L. naiffi (sLnAg) as candidates for composing a pan-specific anti-leishmaniasis vaccine. After immunoblotting using cured patients of cutaneous leishmaniasis sera and proteomics approaches, we identified a group of antigenic proteins from the sLnAg. In silico analyses allowed us to select mildly similar proteins to the host; in addition, we evaluated the binding potential and degree of promiscuity of the protein epitopes to HLA molecules and to B-cell receptors. We selected 24 immunodominant proteins from a sub-proteome with 328 proteins. Homology analysis allowed the identification of 13 proteins with the most orthologues among seven Leishmania species. This work demonstrated the potential of these proteins as promising vaccine targets capable of inducing humoral and cellular pan-specific immune responses in humans, which may in the future contribute to the control of leishmaniasis.

Structural and Biochemical Characterization of Three Antimicrobial Peptides from Capsicum annuum L. var. annuum Leaves for Anti-Candida Use.

The emergence of resistant microorganisms has reduced the effectiveness of currently available antimicrobials, necessitating the development of new strategies. Plant antimicrobial peptides (AMPs) are promising candidates for novel drug development. In this study, we aimed to isolate, characterize, and evaluate the antimicrobial activities of AMPs isolated from Capsicum annuum. The antifungal potential was tested against Candida species. Three AMPs from C. annuum leaves were isolated and characterized: a protease inhibitor, a defensin-like protein, and a lipid transporter protein, respectively named CaCPin-II, CaCDef-like, and CaCLTP2. All three peptides had a molecular mass between 3.5 and 6.5 kDa and caused morphological and physiological changes in four different species of the genus Candida, such as pseudohyphae formation, cell swelling and agglutination, growth inhibition, reduced cell viability, oxidative stress, membrane permeabilization, and metacaspase activation. Except for CaCPin-II, the peptides showed low or no hemolytic activity at the concentrations used in the yeast assays. CaCPin-II inhibited α-amylase activity. Together, these results suggest that these peptides have the potential as antimicrobial agents against species of the genus Candida and can serve as scaffolds for the development of synthetic peptides for this purpose.

Gallic acid has an inhibitory effect on skin squamous cell carcinoma and acts on the heat shock protein HSP90AB1.

Differences in the features of aggressiveness of non-melanoma skin cancer (NMSC) subtypes, between basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are relevant characteristics. Comparing the characteristics between NMSC subtypes might help identify molecules associated with cancer metastasis and invasion. Considering these facts, the current study aimed to identify a molecular target for inhibiting skin cancer metastasis and invasion. Proteomic analysis suggested that heat shock protein 90 kDa, alpha, class B member 1 (HSP90AB1), pentaxin (PTX3), caspase-14 (CASP14), S100, actin-1, and profilin were the primary targets related to metastasis and invasion. However, after a differential expression comparison between BCC and SCC, HSP90AB1 was identified as the best target to repress metastasis and invasion. Based on molecular docking results, gallic acid (GA) was selected to inhibit HSP90AB1. A specific Hsp90ab1 siRNA targeting was designed and compared to GA. Interestingly, GA was more efficient in silencing HSP90AB1 than siRNAhsp90ab1. Hence, our data suggest that HSP90AB1 is a crucial biomarker for identifying invasion and metastasis and that its inhibition may be a viable strategy for treating skin cancer.

Prepubertal arsenic exposure alters phosphoproteins profile, quality, and fertility of epididymal spermatozoa in sexually mature rats.

Arsenic intoxication affects male reproductive parameters of prepubertal rats. Besides, morphological and functional alterations in their testis and epididymis may remain after withdrawal of arsenic insult, causing potential impairment in male fertility during adulthood. In this study, we aimed at analyzing the effect of prepubertal arsenic exposure on the fecundity of epididymal sperm from sexually mature Wistar rats, assessing fertility indexes, sperm parameters, and sperm phosphoproteins content. Male pups on postnatal day (PND) 21 received filtered water (controls, n = 10) and 10 mg L-1 arsenite (n = 10) daily for 30 days. From PND52 to PND81, rats from both groups received filtered water. During this period, the males mated with non-exposed females between PND72 and PND75. Our results showed that sexually mature rats presented low sperm production, epididymal sperm count, motility, and quality after prepubertal arsenic exposure. These findings possibly contributed to the low fertility potential and high preimplantation loss. Epididymal sperm proteome detected 268 proteins, which 170 were found in animals from both control and arsenic groups, 27 proteins were detected only in control animals and 71 proteins only in arsenic-exposed rats. In these animals, SPATA 18 and other five proteins were upregulated, whereas keratin type II cytoskeletal 1 was downregulated (q < 0.1). The results of KEGG pathway analysis demonstrated an enrichment of pathways related to dopaminergic response, adrenergic signaling, protein degradation, and oocyte meiosis in arsenic-exposed animals. Moreover, 26 proteins were identified by phosphoproteomic with different phosphorylation pattern in animals from both groups, but SPATA18 was phosphorylated only in arsenic-exposed animals. We concluded that prepubertal exposure to arsenic is deleterious to sperm quality and male fertility, altering the sperm phosphoproteins profile.

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus.

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC50 varying from 0.61 to 0.26 mg P mL-1. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.

A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus / Atividade anti-helmíntica in vitro de uma protease cisteínica do látex de Ficus benjamina contra Haemonchus contortus

Abstract Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.

Resumo Haemonchus contortus é um nematoide gastrintestinal, responsável por altas taxas de mortalidade em rebanhos de pequenos ruminantes. A resistência dos nematoides aos anti-helmínticos sintéticos está generalizada e requer uma busca contínua por novos compostos bioativos, como as proteínas. O objetivo deste trabalho foi avaliar o potencial anti-helmíntico da protease purificada do látex de Ficus benjamina contra H. contortus . O látex fresco foi coletado das plantas por pequenas incisões nas hastes verdes e o extrato proteico de látex (EPL) foi obtido. Após o fracionamento do EPL com sulfato de amônio e cromatografia da fração contendo a maior atividade proteolítica da CM-Celulose, uma protease cisteínica (FbP) foi purificada. A FbP tem massa molecular de cerca de 23,97 kDa, a atividade proteolítica foi estável entre pH 6,0 e pH 10 e ao longo de uma ampla faixa de temperatura, com atividade ótima a 60 °C. A FbP inibiu tanto o desenvolvimento quanto o desembainhamento das larvas de H. contortus, com 50% de inibição nas concentrações de 0,26 e 0,79 mg/mL, respectivamente. Concluímos que esta protease cisteínica do látex de F. benjamina, com ação anti-helmíntica contra H. contortus, pode ser uma alternativa promissora para o desenvolvimento de produtos a serem utilizados em programas de controle de parasitos.

A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus.

Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.

Ferritin from the haemolymph of adult ants: an extraction method for characterization and a ferromagnetic study.

Ferritin has been studied in many animals, plants and bacteria. The main functions of ferritin in mammals are iron concentration and stabilization, protection against oxidants and iron storage for later developmental or iron-dependent activities. Although insect ferritin plays a key role in iron transport, only a few studies to date have examined its properties and function. Ferritin isolation from the haemolymph of adult Camponotus sericeiventris ants involved heating at 75 °C, followed by protein fractionation with 3.2 M KBr gradients and ferritin sedimentation with KBr. Protein identification was performed using high-resolution proteomics techniques. SDS-PAGE revealed three subunits with molecular weights (MW) of 26, 28 and 31 kDa. Native PAGE indicated a MW higher than 669 kDa. Proteomic analysis strongly suggested the 26 and 31 kDa bands as F2LCH and F1HCH subunits of ferritin, respectively. Ferromagnetic resonance (FMR) at 100 K showed, at low field, a characteristic broad component of the ferritin iron core, suggesting that its distribution was shifted to values greater than 3000, a higher content than in mammals. The protein yield and MW were comparable to those reported in other studies of insects. To the best of our knowledge, this is the first report on ferritin extracted from adult ants to date. These results are discussed on the basis of the protein structure-function relation of secreted insect and mammal ferritins. This purification method will allow the use of magnetic techniques, which are relevant for understanding the role of ferritin in the biomineralization of magnetic nanoparticles in insects.

Blood coagulation abnormalities in multibacillary leprosy patients.

BACKGROUND: Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae infection. In 2016, more than 200,000 new cases of leprosy were detected around the world, representing the most frequent cause of infectious irreversible deformities and disabilities. PRINCIPAL FINDINGS: In the present work, we demonstrate a consistent procoagulant profile on 40 reactional and non-reactional multibacillary leprosy patients. A retrospective analysis in search of signs of coagulation abnormalities among 638 leprosy patients identified 35 leprosy patients (5.48%) which displayed a characteristic lipid-like clot formed between blood clot and serum during serum harvesting, herein named 'leprosum clot'. Most of these patients (n = 16, 45.7%) belonged to the lepromatous leprosy pole of the disease. In addition, formation of the leprosum clot was directly correlated with increased plasma levels of soluble tissue factor and von Willebrand factor. High performance thin layer chromatography demonstrated a high content of neutral lipids in the leprosum clot, and proteomic analysis demonstrated that the leprosum clot presented in these patients is highly enriched in fibrin. Remarkably, differential 2D-proteomics analysis between leprosum clots and control clots identified two proteins present only in leprosy patients clots: complement component 3 and 4 and inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP). In agreement with those observations we demonstrated that M. leprae induces hepatocytes release of IHRP in vitro. CONCLUSIONS: We demonstrated that leprosy MB patients develop a procoagulant status due to high levels of plasmatic fibrinogen, anti-cardiolipin antibodies, von Willebrand factor and soluble tissue factor. We propose that some of these components, fibrinogen for example, presents potential as predictive biomarkers of leprosy reactions, generating tools for earlier diagnosis and treatment of these events.

Blood coagulation abnormalities in multibacilary leprosy patients

Hemostatic illnesses are frequently associated with acute and chronic infections. In the present work we demonstrated that leprosy patients developed hemostatic abnormalities, like the formation of an atypical lipid clot mass during sera harvesting, a phenomenon previously observed and never unraveled. We characterize the nature of the "leprosum clot", formed during a protrombotic state developed by some patients. During the proteomic analysis of the leprosum clot we discovered a set of potential serum biomarkers to leprosy reactional episodes diagnosis, which at this moment is based only in clinical features. Taking together, our data suggest that leprosy patients are suffering from a procoagulant status, being beneficiated by the introduction of routine coagulation tests during their treatment, which will aloud physicians to prevent some of the acute clinical symptoms related with superficial vein thrombosis such as cyanosis and tissue necrosis observed during severe cases of leprosy reactional episodes. (AU)

In vitro anthelmintic effects of Spigelia anthelmia protein fractions against Haemonchus contortus.

Gastrointestinal nematodes are a significant concern for animal health and well-being, and anthelmintic treatment is mainly performed through the use of chemical products. However, bioactive compounds produced by plants have shown promise for development as novel anthelmintics. The aim of this study is to assess the anthelmintic activity of protein fractions from Spigelia anthelmia on the gastrointestinal nematode Haemonchus contortus. Plant parts were separated into leaves, stems and roots, washed with distilled water, freeze-dried and ground into a fine powder. Protein extraction was performed with sodium phosphate buffer (75 mM, pH 7.0). The extract was fractionated using ammonium sulfate (0-90%) and extensively dialyzed. The resulting fractions were named LPF (leaf protein fraction), SPF (stem protein fraction) and RPF (root protein fraction), and the protein contents and activities of the fractions were analyzed. H. contortus egg hatching (EHA), larval exsheathment inhibition (LEIA) and larval migration inhibition (LMIA) assays were performed. Proteomic analysis was conducted, and high-performance liquid chromatography (HPLC) chromatographic profiles of the fractions were established to identify proteins and possible secondary metabolites. S. anthelmia fractions inhibited H. contortus egg hatching, with LPF having the most potent effects (EC50 0.17 mg mL-1). During LEIA, SPF presented greater efficiency than the other fractions (EC50 0.25 mg mL-1). According to LMIA, the fractions from roots, stems and leaves also reduced the number of larvae, with EC50 values of 0.11, 0.14 and 0.21 mg mL-1, respectively. Protein analysis indicated the presence of plant defense proteins in the S. anthelmia fractions, including protease, protease inhibitor, chitinase and others. Conversely, secondary metabolites were absent in the S. anthemia fractions. These results suggest that S. anthelmia proteins are promising for the control of the gastrointestinal nematode H. contortus.

An in-depth snake venom proteopeptidome characterization: benchmarking Bothrops jararaca

A large-scale proteomic approach was devised to advance the understanding of venom composition. Bothrops jararaca venom was fractionated by OFFGEL followed by chromatography, generating peptidic and proteic fractions. The latter was submitted to trypsin digestion. Both fractions were separately analyzed by reversed-phase nanochromatography coupled to high resolution mass spectrometry. This strategy allowed deeper and joint characterizations of the peptidome and proteome (proteopeptidome) of this venom. Our results lead to the identification of 46 protein classes (with several uniquely assigned proteins per class) comprising eight high abundance bona fide venom components, and 38 additional classes in smaller quantities. This last category included previously described B. jararaca venom proteins, common Elapidae venom constituents (cobra venom factor and three-finger toxin), and proteins typically encountered in lysosomes, cellular membranes and blood plasma. Furthermore, this report is the most complete snake venom peptidome described so far, both in number of peptides and in variety of unique proteins that could have originated them. It is hypothesized that such diversity could enclose cryptides, whose bioactivities would contribute to envenomation in yet undetermined ways. Finally, we propose that the broad range screening of B. jararaca peptidome will facilitate the discovery of bioactive molecules, eventually leading to valuable therapeutical agents. Biological Significance: Our proteopeptidomic strategy yielded unprecedented insights into the remarkable diversity of B. jararaca venom composition, both at the peptide and protein levels. These results bring a substantial contribution to the actual pursuit of large-scale protein-level assignment in snake venomics. The detection of typical elapidic venom components, in a Viperidae venom, reinforces our view that the use of this approach (hand in-hand with transcriptomic and genomic data) for venom proteomic analysis, at the specimen-level, can greatly contribute for venom toxin evolution studies. Furthermore, data were generated in support of a previous hypothesis that venom gland secretory vesicles are specialized forms of lysosomes. Two testable hypotheses also emerge from the results of this work. The first is that a nucleobindin-2-derived protein could lead to prey disorientation during envenomation, aiding in its capture by the snake. The other being that the venom's peptidome might contain a population of cryptides, whose biological activities could lead to the development of new therapeutical agents.

Differential Gel Electrophoresis (DIGE) Evaluation of Naphthoimidazoles Mode of Action: A Study in Trypanosoma cruzi Bloodstream Trypomastigotes.

BACKGROUND: The obligate intracellular protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a neglected illness affecting millions of people in Latin America that recently entered non-endemic countries through immigration, as a consequence of globalization. The chemotherapy for this disease is based mainly on benznidazole and nifurtimox, which are very efficient nitroderivatives against the acute stage but present limited efficacy during the chronic phase. Our group has been studying the trypanocidal effects of naturally occurring quinones and their derivatives, and naphthoimidazoles derived from ß-lapachone N1, N2 and N3 were the most active. To assess the molecular mechanisms of action of these compounds, we applied proteomic techniques to analyze treated bloodstream trypomastigotes, which are the clinically relevant stage of the parasite. METHODOLOGY/PRINCIPAL FINDINGS: The approach consisted of quantification by 2D-DIGE followed by MALDI-TOF/TOF protein identification. A total of 61 differentially abundant protein spots were detected when comparing the control with each N1, N2 or N3 treatment, for 34 identified spots. Among the differentially abundant proteins were activated protein kinase C receptor, tubulin isoforms, asparagine synthetase, arginine kinase, elongation factor 2, enolase, guanine deaminase, heat shock proteins, hypothetical proteins, paraflagellar rod components, RAB GDP dissociation inhibitor, succinyl-CoA ligase, ATP synthase subunit B and methionine sulfoxide reductase. CONCLUSION/SIGNIFICANCE: Our results point to different modes of action for N1, N2 and N3, which indicate a great variety of metabolic pathways involved and allow for novel perspectives on the development of trypanocidal agents.

Cardiac protein changes in rats after soybean oil treatment: a proteomic study.

BACKGROUND: Several studies show that the consumption of vegetable oils, such as soybean oil, rich in polyunsaturated fatty acids (PUFAs) has beneficial health effects by preventing or reducing the risk factors of cardiovascular diseases. While the demonstration of beneficial effects of the consumption of unsaturated fatty acids on the cardiovascular system has been proven in a macroscopic level, the molecular/cellular mechanisms responsible for this phenomenon are poorly understood. METHODS: In this work, a comparative proteomic approach, two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MALDI-TOF/TOF), was applied to investigate proteome differences in the left ventricle (LV) of rats that received 0.1 mL of soybean oil intramuscularly for 15 days (treated group-TR) and rats that had not (control group-CT). RESULTS: Soybean oil treatment improved left ventricular function, TR animals presented lower value of LVEDP and significantly changed LV proteome. The protein profile of VE revealed differences in the expression of 60 protein spots (p<0.05) between the experimental groups (CT and TR), 14 of those were identified by MS and MS/MS, and 12 of the 14 being non-redundant proteins. Robust changes were detected in proteins involved in cellular structure and antioxidant system and muscular contraction. CONCLUSIONS: The TR group presented an increase in the intensity of proteins involved in muscle contraction (myosin light chain-3 (3-MCL), creatine kinase M (CKM)) and thireodoxin, an antioxidant enzyme. Low intensity cytoskeletal protein, desmin, was also detected in TR animals. The results suggest that soybean oil induces changes in the levels of heart proteins which may partially account for the underlying mechanisms involved in the benefits provided by oils rich in polyunsaturated fatty acids.

Proteomic profiling of nipple aspirate fluid (NAF): Exploring the complementarity of different peptide fractionation strategies.

NAF is a breast fluid that is closely related to the tumor microenvironment and a valuable sample for studying breast cancer. To perform an in-depth proteomic analysis of this sample, aliquots of a single NAF digest were analyzed by the following peptide-centric fractionation strategies: a) 30-cm reversed-phase (RP) column on-line with an LTQ-Orbitrap XL; b) off-line strong cation-exchange (SCX) column; and c) pI-based OFFGEL fractionation. All fractions from approaches (b) and (c) were further analyzed on a 10-cm RP column hyphenated to the same mass spectrometer. The RP-30cm, SCX/RP-10cm, and OFFGEL/RP-10cm approaches identified 1676, 2930, and 3240 peptides, which corresponded to 193, 390 and 528 proteins, respectively. In our cumulative dataset, 4466 distinct NAF peptides corresponded to a total of 557 proteins, of which only 34% were identified by all three approaches. No exclusive protein identification was associated to the RP-30cm approach, while SCX/RP-10cm and OFFGEL/RP-10cm contributed to 28 and 166 exclusive identifications, respectively. Each approach provided additional information related to energy metabolism (fermentation process/carbohydrate biosynthesis). In conclusion, the pre-fractionation platforms used were complementary for the comprehensive characterization of NAF and our work provides methodological information for future quantitative cancer-related NAF sample studies. BIOLOGICAL SIGNIFICANCE: High-resolution peptide separation is a sine qua non condition for achieving extensive proteome coverage. Various techniques have been employed to improve peptide fractionation prior to LC-MS/MS, thus allowing a comprehensive characterization of complex biological samples. Although fractionation efficiency is very sample-dependent, this issue is commonly overlooked, and a "cookbook" approach is routinely used during this critical step. The present study provides a systematic comparison of analytical information needed for the successful large-scale differential proteomic analysis of NAF samples from breast cancer patients, a precious and volume-limited biological sample. It reinforces the importance of optimizing sample-specific fractionation protocols for information retrieval from mass spectrometric analysis.

Reevaluating the Trypanosoma cruzi proteomic map: The shotgun description of bloodstream trypomastigotes.

Chagas disease is a neglected disease, caused by the protozoan Trypanosoma cruzi. This kinetoplastid presents a cycle involving different forms and hosts, being trypomastigotes the main infective form. Despite various T. cruzi proteomic studies, the assessment of bloodstream trypomastigote profile remains unexplored. The aim of this work is T. cruzi bloodstream form proteomic description. Employing shotgun approach, 17,394 peptides were identified, corresponding to 7514 proteins of which 5901 belong to T. cruzi. Cytoskeletal proteins, chaperones, bioenergetics-related enzymes, and trans-sialidases are among the top-scoring. GO analysis revealed that all T. cruzi compartments were assessed; and majority of proteins are involved in metabolic processes and/or presented catalytic activity. The comparative analysis between the bloodstream trypomastigotes and cultured-derived or metacyclic trypomastigote proteomic profiles pointed to 2202 proteins exclusively detected in the bloodstream form. These exclusive proteins are related to: (a) surface proteins; (b) non-classical secretion pathway; (c) cytoskeletal dynamics; (d) cell cycle and transcription; (e) proteolysis; (f) redox metabolism; (g) biosynthetic pathways; (h) bioenergetics; (i) protein folding; (j) cell signaling; (k) vesicular traffic; (l) DNA repair; and (m) cell death. This large-scale evaluation of bloodstream trypomastigotes, responsible for the parasite dissemination in the patient, marks a step forward in the comprehension of Chagas disease pathogenesis. BIOLOGICAL SIGNIFICANCE: The hemoflagellate protozoan T. cruzi is the etiological agent of Chagas disease and affects people by the millions in Latin America and other non-endemic countries. The absence of efficient drugs, especially for treatment during the chronic phase of the disease, stimulates the continuous search for novel molecular targets. The identification of essential molecules, particularly those found in clinically relevant forms of the parasite, could be crucial. Inside the vertebrate host, trypomastigotes circulate in the bloodstream before infecting various tissues. The exposure of bloodstream forms of the parasite to the host immune system likely leads to differential protein expression in the parasite. In this context, an extensive characterization of the proteomic profile of bloodstream trypomastigotes could help to find not only promising drug targets but also antigens for vaccines or diagnostics. This work is a large-scale proteomic assessment of bloodstream trypomastigotes that show a considerable number of proteins belonging to different metabolic pathways and functions exclusive to this parasitic form, and provides a valuable dataset for the biological understanding of this clinically relevant form of T. cruzi.

Comparative proteomic analysis of somatic embryo maturation in Carica papaya L.

BACKGROUND: Somatic embryogenesis is a complex process regulated by numerous factors. The identification of proteins that are differentially expressed during plant development could result in the development of molecular markers of plant metabolism and provide information contributing to the monitoring and understanding of different biological responses. In addition, the identification of molecular markers could lead to the optimization of protocols allowing the use of biotechnology for papaya propagation and reproduction. This work aimed to investigate the effects of polyethylene glycol (PEG) on somatic embryo development and the protein expression profile during somatic embryo maturation in papaya (Carica papaya L.). RESULTS: The maturation treatment supplemented with 6% PEG (PEG6) resulted in the greatest number of somatic embryos and induced differential protein expression compared with cultures grown under the control treatment. Among 135 spots selected for MS/MS analysis, 76 spots were successfully identified, 38 of which were common to both treatments, while 14 spots were unique to the control treatment, and 24 spots were unique to the PEG6 treatment. The identified proteins were assigned to seven categories or were unclassified. The most representative class of proteins observed in the control treatment was associated with the stress response (25.8%), while those under PEG6 treatment were carbohydrate and energy metabolism (18.4%) and the stress response (18.4%). CONCLUSIONS: The differential expression of three proteins (enolase, esterase and ADH3) induced by PEG6 treatment could play an important role in maturation, and these proteins could be characterized as candidate biomarkers of somatic embryogenesis in papaya.

Potential correlation between tumor aggressiveness and protein expression patterns of nipple aspirate fluid (NAF) revealed by gel-based proteomic analysis.

Breast cancer is the leading cause of cancer related deaths in women. Most breast cancers stem from mammary ductal cells that secrete nipple aspirate fluid (NAF), a biological sample that contains proteins associated with the tumor microenvironment. In this study, NAF samples from both breasts of 7 Brazilian patients with unilateral breast cancer were analyzed. These samples were systematically compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional fluorescence difference gel electrophoresis (2D-DIGE); substantial qualitative individual differences were observed. In general, when NAF samples were compared from both breasts within the same patient their electrophoretic patterns were very similar, regardless of their cancer status. A comparison of all patients identified 2 main NAF protein profiles. The HomEP, homogeneous expression profile, was characterized by typical SDS-PAGE and 2D-DIGE protein patterns that were observed in patients with a good breast cancer prognosis and were similar to previous Type I NAF classifications that used one-dimensional electrophoresis. The HetEP, heterogeneous expression profile, was characterized by distinct protein patterns that have not been reported in previous studies and have been primarily observed in breast cancer patients with a poor prognosis. The NAF samples were rich in metal-dependent proteolytic enzymes, as visualized by SDS-PAGE zymography. They varied qualitatively with respect to their gelatinolytic band distribution. However, there were no correlations between these characteristics and the pathologic features of these tumors. A comparative analysis of NAF samples taken from each breast in a single patient showed conserved zymographic patterns. In conclusion, the present study highlights important distinctions in the protein content of individual NAF samples and provides insight into the composition of the tumor microenvironment. These data reinforce breast cancer as a heterogeneous disease with a diverse natural history, which is becoming increasingly evident through other recent studies.

Isolation, characterization and antifungal activity of proteinase inhibitors from Capsicum chinense Jacq. Seeds.

Capsicum species belong to the Solanaceae family and have great social, economic and agronomical significance. The present research presents data on the isolation and characterization of Capsicum chinense Jacq. peptides which were scrutinized in relation to their toxicity towards a diverse set of yeast species. The protein extract was separated with C18 reverse-phase chromatography in high performance liquid chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Tricine-SDS-PAGE of the PEF2 revealed peptides with molecular masses of approximately 5.0 and 8.5 kDa. These PEFs also exhibited strong antifungal activity against different yeasts. In the presence of the PEF2, Candida tropicalis exhibited morphological changes, including cellular agglomeration and formation of pseudohyphae. Determined N-terminal sequences of PEF2 and PEF3 were proven to be highly homologous to serine proteinase inhibitors, when analysed by comparative database sequence tools. For this reason were performed protease inhibitory activity assay. The PEFs displayed high inhibitory activity against trypsin and low inhibitory activity against chymotrypsin. PEF2 and PEF3 were considerably unsusceptible to a broad interval of pH and temperatures. Due to the myriad of application of Proteinase inhibitors (PIs) in fields ranging from plant protection against pathogens and pests to medicine such as in cancer and virus replication inhibition, the discovery of new PIs with new properties are of great interest.