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BACKGROUND: The BRCA2 gene is a well-known tumor suppressor gene implicated in breast and ovarian cancers. BRCA1/2 mutations can be sensitive to poly ADP-ribose polymerase (PARP) inhibitors such as olaparib. However, some of these patients develop resistance to this treatment and an essential factor contributing to acquired insensitivity is the occurrence of reversion mutations in the BRCA1/2 genes. CASE PRESENTATION: We report the case of a 65-year-old Brazilian female patient who had previously been diagnosed with metastatic lung carcinoma carrying a BRCA2 mutation that had extended to the central nervous system. Following disease progression, olaparib was administered, resulting in a stabilizing effect on her condition for ~ 30 months. During a routine follow-up, a new triple-negative breast tumor was found. Genetic testing revealed the presence of two distinct BRCA2 gene mutations in the breast tumor. The original mutation (p.Val220Ilefs4) led to a frameshift, culminating in the production of a truncated and non-functional BRCA2 protein; the second mutation, K437fs22, rectified the reading frame of exon 11. Consequently, Rad51 could properly bind to BRCA2-an essential protein crucial for DNA repair. This restoration resulted in a functional BRCA2 protein, effectively elucidating the clinical resistance observed in the new breast tumor in this case. CONCLUSIONS: This case report highlights the clinical significance of comprehensive next-generation sequencing analyses for lung adenocarcinomas, both at diagnosis and upon progression. Such analyses enable informed decisions regarding targeted therapies and facilitate a deeper comprehension of resistance mechanisms.
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Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Neoplasias de Mama Triplo Negativas , Feminino , Humanos , Idoso , Proteína BRCA2/genética , Proteína BRCA1 , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MutaçãoRESUMO
BACKGROUND: Some cationic and amphiphilic α-helical segments of proteins adsorb to prokaryotic membranes when synthesized as individual polypeptide sequences, resulting in broad and potent antimicrobial activity. However, amphiphilicity, a determinant physicochemical property for peptide-membrane interactions, can also be observed in some ß-sheets. METHODS: The software Kamal was used to scan the human reference proteome for short (7-11 amino acid residues) cationic and amphiphilic protein segments with the characteristic periodicity of ß-sheets. Some of the uncovered peptides were chemically synthesized, and antimicrobial assays were conducted. Biophysical techniques were used to probe the molecular interaction of one peptide with phospholipid vesicles, lipopolysaccharides (LPS) and the bacterium Escherichia coli. RESULTS: Thousands of compatible segments were found in human proteins, five were synthesized, and three presented antimicrobial activity in the micromolar range. Hs10, a nonapeptide fragment of the Complement C3 protein, could inhibit only the growth of tested Gram-negative microorganisms, presenting also little cytotoxicity to human fibroblasts. Hs10 interacted with LPS while transitioning from an unstructured segment to a ß-sheet and increased the hydrodynamic radius of LPS particles. This peptide also promoted morphological alterations in E. coli cells. CONCLUSIONS: Data presented herein introduce yet another molecular template to probe proteins in search for encrypted membrane-active segments and demonstrates that, using this approach, short peptides with low cytotoxicity and high selectivity to prokaryotic cells might be obtained. GENERAL SIGNIFICANCE: This work widens the biotechnological potential of the human proteome as a source of antimicrobial peptides with application in human health.
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Anti-Infecciosos , Escherichia coli , Humanos , Escherichia coli/metabolismo , Peptídeos Antimicrobianos , Lipopolissacarídeos/farmacologia , Proteoma , Bactérias Gram-Negativas/metabolismo , Peptídeos/químicaRESUMO
Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.
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Desintegrinas , Peptídeos , Anfíbios/genética , Anfíbios/metabolismo , Animais , DNA Complementar/genética , Desintegrinas/química , Desintegrinas/genética , Desintegrinas/farmacologia , Peptídeos/química , Peptídeos/genética , Peptídeos/farmacologia , Agregação Plaquetária/fisiologiaRESUMO
In order to better understand the relationship between Flagelliform (Flag) spider silk molecular structural organization and the mechanisms of fiber assembly, it was designed and produced the Nephilengys cruentata Flag spidroin analogue rNcFlag2222. The recombinant proteins are composed by the elastic repetitive glycine-rich motifs (GPGGX/GGX) and the spacer region, rich in hydrophilic charged amino acids, present at the native silk spidroin. Using different approaches for nanomolecular protein analysis, the structural data of rNcFlag2222 recombinant proteins were compared in its fibrillar and in its fully solvated states. Based on the results was possible to identify the molecular structural dynamics of NcFlag2222 prior to and after fiber formation. Overal rNcFlag2222 shows a mixture of semiflexible and rigid conformations, characterized mostly by the presence of PPII, ß-turn and ß-sheet. These results agree with previous studies and bring insights about the molecular mechanisms that might driven Flag silk fibers assembly and elastomeric behavior.
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Oesophageal cancer is among the ten most common types of cancer worldwide. More than 80% of the cases and deaths related to the disease occur in developing countries. Local socio-economic, epidemiologic and healthcare particularities led us to create a Brazilian guideline for the management of oesophageal and oesophagogastric junction (OGJ) carcinomas. The Brazilian Group of Gastrointestinal Tumours invited 50 physicians with different backgrounds, including radiology, pathology, endoscopy, nuclear medicine, genetics, oncological surgery, radiotherapy and clinical oncology, to collaborate. This document was prepared based on an extensive review of topics related to heredity, diagnosis, staging, pathology, endoscopy, surgery, radiation, systemic therapy (including checkpoint inhibitors) and follow-up, which was followed by presentation, discussion and voting by the panel members. It provides updated evidence-based recommendations to guide clinical management of oesophageal and OGJ carcinomas in several scenarios and clinical settings.
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This study was designed to evaluate the seasonal expression of seminal plasma proteins from two bovine breeds adapted to a subtropical climate and their associations with post-thawing sperm and environmental characteristics. Semen samples were obtained three times in summer and three times in winter from four Crioulo Lageano and four Angus bulls. Seminal plasma was obtained by centrifugation, and the other portion of the semen was cryopreserved. Seminal plasma proteins were identified by 2D-nanoUPLC-MSE. Post-thawing assessments of sperm kinetics, morphology and membrane integrity were performed. Environmental data such as air temperature, air humidity and black globe temperature (BGT) were recorded, and the temperature-humidity index (THI) was calculated in summer and winter. Results showed that the climate varied significantly between seasons. Although no statistical differences were observed in semen quality between breeds, the protein profiles varied within and between seasons. We suggest that the most critical proteins in summer affecting sperm characteristics were TIMP-2, DNase, Clusterin, CFAH and GPx6. TIMP-2 and DNase showed a higher abundance in Crioulo Lageano in comparison with Angus, while Clusterin, CFAH and GPx6 presented a lower abundance. To the best of our knowledge, this is the first report of a recently evolved type of glutathione peroxidase, GPx6, in seminal plasma of bovines. In winter, five proteins were considered to be more critical: BSP1, BSP3, CCL2, Sulfhydryl oxidase and TIMP-2. BSP1 and TIMP-2 showed a lower abundance while BSP3, CCL2 and Sulfhydryl oxidase presented a higher abundance in this season in Crioulo Lageano in comparison with Angus.
RESUMO: Este estudo foi desenvolvido para avaliar a expressão sazonal de proteínas plasmáticas seminais de duas raças bovinas adaptadas ao clima subtropical e suas associações com espermatozóides pós-descongelamento e características ambientais. Amostras de sêmen foram obtidas três vezes no verão e três no inverno de quatro touros Crioulo Lageano e quatro Angus. O plasma seminal foi obtido por centrifugação e outra porção do sêmen foi criopreservada. As proteínas plasmáticas seminais foram identificadas por 2D-nanoUPLC-MSE. Foram realizadas avaliações pós-descongelamento da cinética espermática, morfologia e integridade da membrana. Dados ambientais como temperatura do ar, umidade do ar e temperatura do globo negro (BGT) foram registrados, e o índice temperatura-umidade (THI) foi calculado no verão e no inverno. Os resultados mostraram que o clima variou significativamente entre as estações. Embora não tenham sido observadas diferenças estatísticas na qualidade do sêmen entre as raças, os perfis proteicos variaram dentro e entre as estações. Sugerimos que as proteínas mais críticas no verão que afetam as características espermáticas foram TIMP-2, DNase, Clusterin, CFAH e GPx6. TIMP-2 e DNase apresentaram maior abundância em Crioulo Lageano em comparação com Angus, enquanto Clusterin, CFAH e GPx6 apresentaram menor abundância. Até onde sabemos, este é o primeiro relato de um tipo recentemente desenvolvido de glutationa peroxidase, GPx6, no plasma seminal de bovinos. No inverno, cinco proteínas foram consideradas mais críticas: BSP1, BSP3, CCL2, sulfidril oxidase e TIMP-2. BSP1 e TIMP-2 apresentaram menor abundância, enquanto BSP3, CCL2 e Sulfidril oxidase apresentaram maior abundância nesta temporada em Crioulo Lageano em comparação com Angus.
Assuntos
Aclimatação , Bovinos/metabolismo , Estações do Ano , Proteínas de Plasma Seminal/metabolismo , Adaptação Fisiológica , Animais , Cruzamento , Criopreservação/veterinária , Umidade , Masculino , Mapas de Interação de Proteínas , Sêmen , Análise do Sêmen/veterinária , Espermatozoides , TemperaturaRESUMO
OBJECTIVES: The present study aimed to identify proteins obtained from pulp tissue and correlate with each clinical diagnosis (healthy pulp, inflamed pulp, and necrotic pulp). MATERIALS AND METHODS: A total of forty-five molars were used. Three biological replicas were evaluated. Lysis and sonication were used for protein extraction. Protein quantification was assessed by using the Bradford technique, and shotgun proteome analysis was performed by nanoUPLC-MSE using a Synapt G2 mass spectrometer. Mass spectra data were processed using the Waters PLGS software, and protein identification was done using the human Uniprot database appended to the PLGS search engine. RESULTS: A total of 123 different proteins were identified in all evaluated pulp conditions. Among these, 66 proteins were observed for healthy pulp, 66 for inflamed pulp, and 91 for necrotic pulp. Most protein identification was related to immune response, multi-organism process, platelet activation, and stress in inflamed pulp samples compared to healthy pulp. Proteins related to cellular component organization or biogenesis, developmental process, growth, immune response, multi-organism process, response to stimulus, signaling, stress, and transport were identified in cases of apical periodontitis compared to inflamed pulp. CONCLUSIONS: The progression of the disease to inflamed pulp promoted a high abundance of proteins related to the immune system and stress. Comparing the necrotic pulp with inflamed pulp conditions, a high abundance of proteins was noticed related to metabolism, transport, and response between organisms. CLINICAL RELEVANCE: This finding may assist in future studies of new markers, understanding of tissue engineering, and development of future products.
Assuntos
Periodontite Periapical , Pulpite , Polpa Dentária , Necrose da Polpa Dentária , Humanos , ProteômicaRESUMO
We present a graphene-based biosensor selective to recombinant cyanovirin-N (rCV-N), an antiviral protein that has proven to be an effective microbicide to inhibit HIV replication. We modified the graphene monolayer devices with 1-pyrenebutanoic acid succinimidyl ester, which interacts with both graphene and the primary and secondary amines of antibodies. By monitoring the change in the electrical resistance of the device, we were able to detect rCV-N in solutions in the range of 0.01 to 10 ng/mL, and found that the detection limit was 0.45 pg/mL, which is much smaller than that obtained with currently available techniques. This is important for applications of this microbicide against HIV, since it may be produced at a large scale from soya bean seeds processed using the available industrial processing technologies. The sensor showed high sensitivity, selectivity, and reproducibility.
Assuntos
Proteínas de Bactérias/análise , Técnicas Biossensoriais , Grafite , Técnicas Eletroquímicas , Humanos , Reprodutibilidade dos Testes , Sementes , Glycine maxRESUMO
Gastric cancer is among the ten most common types of cancer worldwide. Most cases and deaths related to the disease occur in developing countries. Local socio-economic, epidemiologic and healthcare particularities led us to create a Brazilian guideline for the management of gastric carcinomas. The Brazilian Group of Gastrointestinal Tumors (GTG) invited 50 physicians with different backgrounds, including radiology, pathology, endoscopy, nuclear medicine, genetics, oncological surgery, radiotherapy and clinical oncology, to collaborate. This document was prepared based on an extensive review of topics related to heredity, diagnosis, staging, pathology, endoscopy, surgery, radiation, systemic therapy and follow-up, which was followed by presentation, discussion, and voting by the panel members. It provides updated evidence-based recommendations to guide clinical management of gastric carcinomas in several scenarios and clinical settings.
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Recently, new serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. Here, we describe the use of unidirectional genetic switches to evaluate the functionality of six serine integrases in different eukaryotic systems: the HEK 293T cell lineage, bovine fibroblasts and plant protoplasts. Moreover, integrase activity was also tested in human cell types of therapeutic interest: peripheral blood mononuclear cells (PBMCs), neural stem cells (NSCs) and undifferentiated embryonic stem (ES) cells. The switches were composed of plasmids designed to flip two different genetic parts driven by serine integrases. Cell-based assays were evaluated by measurement of EGFP fluorescence and by molecular analysis of attL/attR sites formation after integrase functionality. Our results demonstrate that all the integrases were capable of inverting the targeted DNA sequences, exhibiting distinct performances based on the cell type or the switchable genetic sequence. These results should support the development of tunable genetic circuits to regulate eukaryotic gene expression.
Assuntos
Arabidopsis/enzimologia , Fibroblastos/enzimologia , Integrases/genética , Plasmídeos/genética , Protoplastos/enzimologia , Recombinação Genética , Serina/genética , Animais , Bovinos , Humanos , Integrases/metabolismo , Leucócitos Mononucleares/enzimologia , Regiões Promotoras Genéticas , Serina/metabolismoRESUMO
Arachis stenosperma is a wild peanut relative exclusive to South America that harbors high levels of resistance against several pathogens, including the peanut root-knot nematode (RKN) Meloidogyne arenaria. In this study, a proteomic survey of A. stenosperma-M. arenaria interaction using 2-DE and LC-MS/MS identified approximately 1400 proteins, out of which 222 were differentially abundant (DAPs) when RKN inoculated root samples were compared to the control. Most of these DAPs were assigned to functional categories related to plant responses to pathogens including stress, glycolysis, redox and tricarboxylic acid cycle. The comparison between the transcriptome (RNA-Seq) and proteome expression changes, showed that almost 55% of these DAPs encode genes with a similar expression trend to their protein counterparts. Most of these genes were induced during RKN infection and some were related to plant defense, such as MLP-like protein 34 (MLP34), cinnamoyl-CoA reductase 1 (CCR1), enolase (ENO), alcohol dehydrogenase (ADH) and eukaryotic translation initiation factor 5A (eIF5A). The overexpression of AsMLP34 in Agrobacterium rhizogenes transgenic roots in a susceptible peanut cultivar showed a reduction in the number of M. arenaria galls and egg masses, indicating that AsMLP34 is a promising candidate gene to be exploited in breeding programs for RKN control in peanut. SIGNIFICANCE: The use of an integrated approach to compare plant-nematode transcriptional and translational data enabled the identification of a new gene, AsMLP34, for Meloidogyne resistance.
Assuntos
Tylenchoidea , Agrobacterium , Animais , Arachis/genética , Cromatografia Líquida , Resistência à Doença/genética , Melhoramento Vegetal , Doenças das Plantas/genética , Raízes de Plantas , Proteômica , América do Sul , Espectrometria de Massas em TandemRESUMO
Following the treads of our previous works on the unveiling of bioactive peptides encrypted in plant proteins from diverse species, the present manuscript reports the occurrence of four proof-of-concept intragenic antimicrobial peptides in human proteins, named Hs IAPs. These IAPs were prospected using the software Kamal, synthesized by solid phase chemistry, and had their interactions with model phospholipid vesicles investigated by differential scanning calorimetry and circular dichroism. Their antimicrobial activity against bacteria, yeasts and filamentous fungi was determined, along with their cytotoxicity towards erythrocytes. Our data demonstrates that Hs IAPs are capable to bind model membranes while attaining α-helical structure, and to inhibit the growth of microorganisms at concentrations as low as 1µM. Hs02, a novel sixteen residue long internal peptide (KWAVRIIRKFIKGFIS-NH2) derived from the unconventional myosin 1h protein, was further investigated in its capacity to inhibit lipopolysaccharide-induced release of TNF-α in murine macrophages. Hs02 presented potent anti-inflammatory activity, inhibiting the release of TNF-α in LPS-primed cells at the lowest assayed concentration, 0.1 µM. A three-dimensional solution structure of Hs02 bound to DPC micelles was determined by Nuclear Magnetic Resonance. Our work exemplifies how the human genome can be mined for molecules with biotechnological potential in human health and demonstrates that IAPs are actual alternatives to antimicrobial peptides as pharmaceutical agents or in their many other putative applications.
Assuntos
Anti-Infecciosos/síntese química , Anti-Inflamatórios/síntese química , Peptídeos/farmacologia , Animais , Eritrócitos/efeitos dos fármacos , Humanos , Lipossomos/metabolismo , Macrófagos/metabolismo , Camundongos , Micelas , Peptídeos/análise , Peptídeos/síntese química , Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice , Proteínas/química , Técnicas de Síntese em Fase Sólida , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The present work reports the isolation, characterization and the complete sequence of a phospholipase A2 (PLA2) present in the skin secretion of Pithecopus azureus. Among several peptides and small proteins previously described by our group from some species belonging to this amphibian genus (formerly named Phyllomedusa), a 15â¯kDa N-glycosylated protein showing PLA2 activity was purified, assayed, sequenced and named Pa-PLA2. The Pithecopus azureus skin phospholipase A2 polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA2 enzymatic activity was qualitatively evaluated and compared to classical viperid PLA2 showing that both, native and deglycosylated Pa-PLA2 forms, are catalytically functional. The tridimensional molecular model of Pa-PLA2 indicates that the observed glycan moieties are suggestively placed far from the active site of that enzyme and therefore having little or no significant role on the direct interaction of the Pa-PLA2 catalytic pocket and its substrates.
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Anuros , Fosfolipases A2/química , Sequência de Aminoácidos , Animais , Fracionamento Químico , Cromatografia Líquida , Modelos Moleculares , Fosfolipases A2/isolamento & purificação , Análise de Sequência de Proteína , Espectrometria de Massas em TandemRESUMO
Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra-pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc - Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max-exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT-PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.
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Proteínas de Bactérias/análise , Proteoma/análise , Fatores de Virulência/análise , Xanthomonas campestris/patogenicidade , Proteínas de Bactérias/genética , Meios de Cultura/química , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Proteoma/genética , Virulência/genética , Fatores de Virulência/genética , Xanthomonas campestris/genética , Xanthomonas campestris/isolamento & purificaçãoRESUMO
The pathogenicity of phytonematodes relies on secreted virulence factors to rewire host cellular pathways for the benefits of the nematode. In the root-knot nematode (RKN) Meloidogyne incognita, thousands of predicted secreted proteins have been identified and are expected to interact with host proteins at different developmental stages of the parasite. Identifying the host targets will provide compelling evidence about the biological significance and molecular function of the predicted proteins. Here, we have focused on the hub protein CSN5, the fifth subunit of the pleiotropic and eukaryotic conserved COP9 signalosome (CSN), which is a regulatory component of the ubiquitin/proteasome system. We used affinity purification-mass spectrometry (AP-MS) to generate the interaction network of CSN5 in M. incognita-infected roots. We identified the complete CSN complex and other known CSN5 interaction partners in addition to unknown plant and M. incognita proteins. Among these, we described M. incognita PASSE-MURAILLE (MiPM), a small pioneer protein predicted to contain a secretory peptide that is up-regulated mostly in the J2 parasitic stage. We confirmed the CSN5-MiPM interaction, which occurs in the nucleus, by bimolecular fluorescence complementation (BiFC). Using MiPM as bait, a GST pull-down assay coupled with MS revealed some common protein partners between CSN5 and MiPM. We further showed by in silico and microscopic analyses that the recombinant purified MiPM protein enters the cells of Arabidopsis root tips in a non-infectious context. In further detail, the supercharged N-terminal tail of MiPM (NTT-MiPM) triggers an unknown host endocytosis pathway to penetrate the cell. The functional meaning of the CSN5-MiPM interaction in the M. incognita parasitism is discussed. Moreover, we propose that the cell-penetrating properties of some M. incognita secreted proteins might be a non-negligible mechanism for cell uptake, especially during the steps preceding the sedentary parasitic phase.
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In recent years the antimicrobial peptides (AMPs) have been prospected and designed as new alternatives to conventional antibiotics. Indeed, AMPs have presented great potential toward pathogenic bacterial strains by means of complex mechanisms of action. However, reports have increasingly emerged regarding the mechanisms by which bacteria resist AMP administration. In this context, we performed a comparative proteomic study by using the total bacterial lysate of magainin I-susceptible and -resistant E. coli strains. After nanoUPLC-MSE analyses we identified 742 proteins distributed among the experimental groups, and 25 proteins were differentially expressed in the resistant strains. Among them 10 proteins involved in bacterial resistance, homeostasis, nutrition and protein transport were upregulated, while 15 proteins related to bacterial surface modifications, genetic information and ß-lactams binding-protein were downregulated. Moreover, 60 exclusive proteins were identified in the resistant strains, among which biofilm and cell wall formation and multidrug efflux pump proteins could be observed. Thus, differentially from previous studies that could only associate single proteins to AMP bacterial resistance, data here reported show that several metabolic pathways may be related to E. coli resistance to AMPs, revealing the crucial role of multiple "omics" studies in order to elucidate the global molecular mechanisms involved in this resistance.
Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Magaininas/farmacologia , Espectrometria de Massas , Nanotecnologia , Cromatografia Líquida de Alta Pressão , Proteínas de Escherichia coli/metabolismoRESUMO
Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot, a highly destructive disease that affects all brassicas. This work aimed to study the interaction Xcc-Brassica oleracea using an in vivo system in an attempt to identify proteins involved in pathogenicity. We used label-free shotgun 2D-nanoUPLC/MSE to analyze Xcc proteins in three conditions: in the interaction with susceptible (REK) and resistant (REU) plants and in culture medium (control condition). A model of Xcc-susceptible host interaction is proposed and shows that Xcc increases the abundance of several crucial proteins for infection and cell protection. In this study, we also confirmed the differential expression by qPCR analysis of selected genes. This is the first report showing a large-scale identification of proteins in an in vivo host plant condition. Considering that most studies involving phytopathogens are in vitro (growth in culture medium or in plant extract), this work contributes with relevant information related to the plant-pathogen interaction in planta.
Assuntos
Proteínas de Bactérias/metabolismo , Brassica/metabolismo , Brassica/microbiologia , Fatores de Virulência/metabolismo , Xanthomonas campestris/patogenicidade , Proteínas de Bactérias/genética , Brassica/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteoma/metabolismo , Fatores de Virulência/genéticaRESUMO
The main goal of the present study was a complete proteomic characterization of total proteins eluted from residual substrate-bound proteins (RSBP), and cellulosomes secreted by Clostridium thermocellum B8 during growth in the presence of microcrystalline cellulose as a carbon source. The second goal was to evaluate their potential use as enzymatic blends for hydrolyzing agro-industrial residues to produce fermentable sugars. Protein identification through LC-MS/MS mass spectrometry showed that the RSBP sample, in addition to cellulosomal proteins, contains a wide variety of proteins, including those without a well-characterized role in plant cell wall degradation. The RSBP subsample defined as purified cellulosomes (PC) consists mainly of glycoside hydrolases grouped in families 5, 8, 9, 10 and 48. Dynamic light scattering, DLS, analysis of PC resulted in two protein peaks (pi1 and pi2) presenting molecular masses in agreement with those previously described for cellulosomes and polycellulosomes. These peaks weren't detected after PC treatment with 1.0% Tween. PC and RSBP presented maximal activities at temperatures ranging from 60° to 70°C and at pH 5.0. RSBP retained almost all of its activity after incubation at 50, 60 and 70°C and PC showed remarkable thermostability at 50 and 60°C. RSBP holocellullolytic activities were inhibited by phenolic compounds, while PC showed either increasing activity or a lesser degree of inhibition. RSBP and PC hydrolyze sugar cane straw, cotton waste and microcrystalline cellulose, liberating a diversity of saccharides; however, the highest concentration of released sugar was obtained for assays carried out using PC as an enzymatic blend and after ten days at 50°C.
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Proteínas de Bactérias/metabolismo , Clostridium thermocellum/metabolismo , Lignina/metabolismo , Biocombustíveis , Biomassa , Biotecnologia , Celulossomas/metabolismo , Clostridium thermocellum/enzimologia , Glicosídeo Hidrolases/metabolismo , Hidrólise , Proteoma/metabolismo , Proteômica , Espectrometria de Massas em TandemRESUMO
Tomato chlorotic mottle virus (ToCMoV) is a widespread bipartite Begomovirus species found in tomato fields in Brazil. In this study, plant responses and putative mechanisms associated with the 'Tyking'-derived recessive resistance to ToCMoV were investigated. Changes in the protein profile in the inoculated plants of two near isogenic tomato lines resistant ('LAM 157') and susceptible ('Santa Clara') to ToCMoV were analyzed. Seedlings were biolistically inoculated with an infectious ToCMoV clone. Leaves from infected plants (confirmed by PCR) were sampled at 15days after inoculation. Proteins were extracted using phenol and analyzed by shotgun MS (2D-nanoUPLC/HDMSE). Out of the 534 identified proteins, 82 presented statistically significant differences in abundance, including 35 unique proteins displayed in the resistant tomato inoculated with ToCMoV. Proteins associated to chromatin structure, cytoskeleton structure, cuticle biosynthesis, and ubiquitin pathway were identified and their putative roles during virus infection process were discussed. The protein profile analysis allowed for the development of a hypothetical model showing how the resistant host cell responds to ToCMoV infection. The data obtained provide a better understanding of resistant mechanisms used by the host plant to contain viral infection and could be the basis for further investigation in other plant-begomovirus pathosystems. BIOLOGICAL SIGNIFICANCE: In this study we propose a model of resistance to begomovirus in tomato and highlight host proteins, which could be targets for future investigations in plant-begomovirus pathosystems.
Assuntos
Begomovirus/patogenicidade , Resistência à Doença , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Plantas/análise , Proteômica/métodos , Solanum lycopersicum/virologia , Brasil , Modelos Biológicos , Extratos Vegetais/química , Proteínas de Plantas/fisiologiaRESUMO
Background: Gastric carcinoma (GC) is the third leading cause of death among malignant tumors worldwide, causing approximately 900,000 deaths/year. Changes in oncogenes that encode tyrosine kinase receptors play an important role in the pathogenesis of GC. MET gene is a proto-oncogene that encodes a tyrosine kinase receptor c-MET and it is required for embryonic development and tissue repair. The hepatocyte growth factor (HGF) is the only known ligand for c-Met receptor. The MET oncogene activation suppresses apoptosis and promotes the survival, proliferation, migration, differentiation and angiogenesis of cells. Among the angiogenic factors, VEGF is the main regulator. Its biological function includes the promotion of endothelial cells mitosis to stimulate cells proliferation. These biomarkers expression in GC is relatively recent and population-based studies are required to define the expression pattern. The aim of this study was to determine qPCR technical standardization to evaluate quantitatively, in paraffin tissue samples, the presence of gene 23 expression of the MET, HGF and VEGF in diffuse and intestinal GC types. Methods: Twenty GC patients were studied, 10 patients were intestinal-type GC (average age 72.1 years) and 10 diffuse-type (average age 50.1 years). In all patients, tissue samples were analyzed from the tumor and distant areas of the tumor tissue. The relative expressions of the tumor markers c-Met, HGF and VEGF were performed by qPCR technique by comparing tumor and non-tumoral samples and they were normalized with the GAPDH constitutive gene. Statistical analysis was performed through T-test. Results: For c-Met, 18/20 (90%) patients expressed the marker and 9/20 (45%) overexpressed this gene, in which three were intestinal-type GC and six were diffuse-type GC. For HGF, only 7/20 (35%) patients expressed this gene and it was overexpressed in 4/20 (20%), in which two were intestinal-type GC and two were diffuse-type GC. For VEGF, 20/20 (100%) patients expressed this marker and in 12/20 (60%) were observed overexpression, in which eight patients had diffuse-type GC and four had intestinal-type GC. Conclusions: qPCR technique was standardized and suitable for expression analysis of the three biomarkers using paraffin embedded tissue samples. Further studies should be carried out to characterize the expression pattern of these biomarkers in GC in the Brazilian population (AU)