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This work provides a systematic and comprehensive overview of available resources for the molecular-scale modelling of the translation process through agent-based modelling. The case study is the translation in Saccharomyces cerevisiae, one of the most studied yeasts. The data curation workflow encompassed structural information about the yeast (i.e. the simulation environment), and the proteins, ribonucleic acids and other types of molecules involved in the process (i.e. the agents). Moreover, it covers the main process events, such as diffusion (i.e. motion of molecules in the environment) and collision efficiency (i.e. interaction between molecules). Data previously determined by wet-lab techniques were preferred, resorting to computational predictions/extrapolations only when strictly necessary. The computational modelling of the translation processes is of added industrial interest, since it may bring forward knowledge on how to control such phenomena and enhance the production of proteins of interest in a faster and more efficient manner.
Assuntos
Biologia Computacional/métodos , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Análise de Célula Única/métodos , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: We developed machine learning models to understand the predictors of shorter-, intermediate-, and longer-term mortality among hemodialysis (HD) patients affected by COVID-19 in four countries in the Americas. METHODS: We used data from adult HD patients treated at regional institutions of a global provider in Latin America (LatAm) and North America who contracted COVID-19 in 2020 before SARS-CoV-2 vaccines were available. Using 93 commonly captured variables, we developed machine learning models that predicted the likelihood of death overall, as well as during 0-14, 15-30, > 30 days after COVID-19 presentation and identified the importance of predictors. XGBoost models were built in parallel using the same programming with a 60%:20%:20% random split for training, validation, & testing data for the datasets from LatAm (Argentina, Columbia, Ecuador) and North America (United States) countries. RESULTS: Among HD patients with COVID-19, 28.8% (1,001/3,473) died in LatAm and 20.5% (4,426/21,624) died in North America. Mortality occurred earlier in LatAm versus North America; 15.0% and 7.3% of patients died within 0-14 days, 7.9% and 4.6% of patients died within 15-30 days, and 5.9% and 8.6% of patients died > 30 days after COVID-19 presentation, respectively. Area under curve ranged from 0.73 to 0.83 across prediction models in both regions. Top predictors of death after COVID-19 consistently included older age, longer vintage, markers of poor nutrition and more inflammation in both regions at all timepoints. Unique patient attributes (higher BMI, male sex) were top predictors of mortality during 0-14 and 15-30 days after COVID-19, yet not mortality > 30 days after presentation. CONCLUSIONS: Findings showed distinct profiles of mortality in COVID-19 in LatAm and North America throughout 2020. Mortality rate was higher within 0-14 and 15-30 days after COVID-19 in LatAm, while mortality rate was higher in North America > 30 days after presentation. Nonetheless, a remarkable proportion of HD patients died > 30 days after COVID-19 presentation in both regions. We were able to develop a series of suitable prognostic prediction models and establish the top predictors of death in COVID-19 during shorter-, intermediate-, and longer-term follow up periods.
Assuntos
COVID-19 , Adulto , Humanos , Masculino , Vacinas contra COVID-19 , Aprendizado de Máquina , América do Norte/epidemiologia , Diálise Renal , SARS-CoV-2 , FemininoRESUMO
Filamentous fungi are well known for producing secondary metabolites applied in various industrial segments. Among these, lovastatin and itaconic acid, produced by Aspergillus terreus, have applications in the pharmaceutical and chemical industries. Lovastatin is primarily used for the control of hypercholesterolemia, while itaconic acid is a building block for the production of synthetic fibers, coating adhesives, among others. In this study, for the first time, 35 strains of Aspergillus sp. from four Brazilian culture collections were evaluated for lovastatin and itaconic acid production and compared to a reference strain, ATCC 20542. From an initial screening, the strains ATCC 20542, URM 224, URM1876, URM 5061, URM 5254, URM 5256, URM 5650, and URM 5961 were selected for genomic comparison. Among tested strains, the locus corresponding to the lovastatin genomic cluster was assembled, showing that all genes essential for lovastatin biosynthesis were present in producing URM 5961 and URM 5650 strains, with 100% and 98.5% similarity to ATCC 20542, respectively. However, in the no producing URM 1876, URM 224, URM 5254, URM 5061, and URM 5256 strains, this cluster was either fragmented or missing. Among the 35 strains evaluated for itaconic acid production in this study, only three strains had titers above 0.5 g/L, 16 strains had production below 0.5 g/L, and the remaining 18 strains had no production, with the highest production of itaconic acid observed in the URM 5254 strain with 2.2 g/L. The essential genes for itaconic acid production, mttA, cadA msfA were also mapped, where all three genes linked to itaconic acid production were found in a single contig in the assembly of each strain. In contrast to lovastatin loci, there is no correlation between the level of itaconic acid production and genetic polymorphisms in the genes associated with its biosynthesis.
Assuntos
Aspergillus , Lovastatina , Succinatos , Aspergillus/genética , Aspergillus/metabolismo , Biodiversidade , Brasil , Genes Fúngicos , Variação Genética , Genoma Fúngico , Lovastatina/biossíntese , Lovastatina/genética , Filogenia , Succinatos/metabolismoRESUMO
Fluorescence in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of microorganisms. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes (MPs) move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different MPs to diffuse across multiple cellular barriers, as well as the reaction time associated with the formation of the duplex molecular probe-RNA, were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 7.94 × 102 - 4.39 × 103 s), whereas for animal cells, the limiting step should be the diffusion of the probe through the bulk (1.8-5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis for a more rational development of FISH protocols in the future.
Assuntos
Corantes Fluorescentes/química , Hibridização in Situ Fluorescente/métodos , Sondas de Ácido Nucleico/química , Animais , Bactérias , Células Cultivadas , DifusãoRESUMO
The comprehension of how protease networks sculpt proteomes might help to disclose the functional annotation of the peptidome in health and disease. Envisioning to add new insights on the protease networks involved in the regulation of body fluid peptidomes, the authors apply Proteasix software to predict the proteases involved in the generation of the naturally occurring peptides present in six of the most studied human body fluids. Peptidome data is collected from the databases and from experimental studies. The analysis highlights 132 putative proteases from four families with the predominance of serine proteases and metalloproteases. From these, 49 proteases seem to be common to all fluids and are mostly associated to extracellular matrix organization as well as protein/peptide hormone processing. Data analysis also emphasizes: i) the similarity between plasma and CSF protease profiles; ii) that saliva and tears share proteases involved in the generation of peptides with antimicrobial activity; iii) that urine is the body fluid with the highest number of unique putative proteases, precluding an easy tracing of proteolytic events in this case. Taken together, the analysis emphasizes the intricate modus operandi of proteases, challenged by the interconnected pathways and amplification cascades in which they are involved.
Assuntos
Líquidos Corporais/metabolismo , Biologia Computacional/métodos , Fragmentos de Peptídeos/análise , Peptídeo Hidrolases/análise , Proteômica/métodos , HumanosRESUMO
Hyaluronic acid, or HA, is a rigid and linear biopolymer belonging to the class of the glycosaminoglycans, and composed of repeating units of the monosaccharides glucuronic acid and N-acetylglucosamine. HA has multiple important functions in the human body, due to its properties such as bio-compatibility, lubricity and hydrophilicity, it is widely applied in the biomedical, food, health and cosmetic fields. The growing interest in this molecule has motivated the discovery of new ways of obtaining it. Traditionally, HA has been extracted from rooster comb-like animal tissues. However, due to legislation laws HA is now being produced by bacterial fermentation using Streptococcus zooepidemicus, a natural producer of HA, despite it being a pathogenic microorganism. With the expansion of new genetic engineering technologies, the use of organisms that are non-natural producers of HA has also made it possible to obtain such a polymer. Most of the published reviews have focused on HA formulation and its effects on different body tissues, whereas very few of them describe the microbial basis of HA production. Therefore, for the first time this review has compiled the molecular and genetic bases for natural HA production in microorganisms together with the main strategies employed for heterologous production of HA.
Assuntos
Ácido Hialurônico/biossíntese , Streptococcus equi/genética , Streptococcus equi/metabolismo , Fermentação , Engenharia Genética , Humanos , Microbiologia IndustrialRESUMO
BACKGROUND: Crude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous L-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for L-lactic acid production. RESULTS: Batch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g. CONCLUSIONS: We showed that P. pastoris has a great potential as a fermentative organism for producing L-lactic acid using glycerol as the carbon source at limited oxygenation conditions (below 0.05 % DO in the bioreactor). The best strain had both the LDHb and the homologous lactate transporter encoding genes expressed, and reached a titer 1.5 times higher than the strain with the S. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half.
Assuntos
Glicerol/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Pichia/genética , Ácido Acético/metabolismo , Animais , Biocombustíveis , Reatores Biológicos , Bovinos , Fermentação , L-Lactato Desidrogenase/genética , Engenharia Metabólica , Transportadores de Ácidos Monocarboxílicos/isolamento & purificação , Pichia/metabolismoRESUMO
The granular glands of anuran skin secrete an array of bioactive molecules that protect a frog against pathogens and predators. The skin also harbors a microbial community. Although there is evidence to suggest that the microbiota complement the innate immune defense systems against pathogen infection, the effect of the frog bioactive molecules on its resident microbiota has not yet been fully investigated. In the present study, the skin microbiota of Phyllomedusa distincta obtained from two different geographical areas was evaluated with molecular and culture-based approaches. The antagonistic effects exhibited by the host's microbiota and by a novel dermaseptin peptide isolated from P. distincta skin were investigated. Four isolated bacterial colonies displayed antimicrobial activity against known frog pathogens. Our results were consistent with the hypothesis that microbiota from P. distincta may interact with pathogenic microorganisms to protect a frog's health. On the other hand, the novel dermaseptin peptide exhibited an antimicrobial effect on pathogens as well as on some of the bacteria obtained from the skin microbiota. The richness of bacteria on P. distincta skin was further investigated by 16S rRNA gene clone libraries, which revealed that the family Enterobacteriaceae was prevalent, but a high variability at the species level was observed among individual frogs. Differences observed on the microbiota of frogs from contrasting habitats indicated an influence of the environment on the structure of the skin microbiota of P. distincta.
Assuntos
Antibacterianos/farmacologia , Anuros , Microbiota , Pele/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Homologia de Sequência de Aminoácidos , Pele/microbiologiaRESUMO
Cyclotides are a family of plant-derived cyclic peptides comprising six conserved cysteine residues connected by three intermolecular disulfide bonds that form a knotted structure known as a cyclic cystine knot (CCK). This structural motif is responsible for the pronounced stability of cyclotides against chemical, thermal, or proteolytic degradation and has sparked growing interest in this family of peptides. Here, we isolated and characterized a novel cyclotide from Palicourea rigida (Rubiaceae), which was named parigidin-br1. The sequence indicated that this peptide is a member of the bracelet subfamily of cyclotides. Parigidin-br1 showed potent insecticidal activity against neonate larvae of Lepidoptera (Diatraea saccharalis), causing 60% mortality at a concentration of 1 µm but had no detectable antibacterial effects. A decrease in the in vitro viability of the insect cell line from Spodoptera frugiperda (SF-9) was observed in the presence of parigidin-br1, consistent with in vivo insecticidal activity. Transmission electron microscopy and fluorescence microscopy of SF-9 cells after incubation with parigidin-br1 or parigidin-br1-fluorescein isothiocyanate, respectively, revealed extensive cell lysis and swelling of cells, consistent with an insecticidal mechanism involving membrane disruption. This hypothesis was supported by in silico analyses, which suggested that parigidin-br1 is able to complex with cell lipids. Overall, the results suggest promise for the development of parigidin-br1 as a novel biopesticide.
Assuntos
Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Inseticidas/química , Inseticidas/isolamento & purificação , Lepidópteros , Rubiaceae/química , Saccharum , Sequência de Aminoácidos , Animais , Linhagem Celular , Ciclotídeos/genética , Ciclotídeos/metabolismo , Feminino , Fluoresceína-5-Isotiocianato/metabolismo , Regulação da Expressão Gênica de Plantas , Inseticidas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Fosforilcolina/análogos & derivados , Fosforilcolina/metabolismo , Filogenia , Conformação Proteica , Rubiaceae/genética , Estações do Ano , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Antimicrobial peptides (AMPs) are effective antibiotic agents commonly found in plants, animals, and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. It is vital to understand the molecular details that define the mechanism of action of resistance to AMPs for a rational planning of the next antibiotic generation and also to shed some light on the complex AMP mechanism of action. Here, the antibiotic resistance of Escherichia coli ATCC 8739 to magainin I was evaluated in the cytosolic subproteome. Magainin-resistant strains were selected after 10 subsequent spreads at subinhibitory concentrations of magainin I (37.5 mg · liter⻹), and their cytosolic proteomes were further compared to those of magainin-susceptible strains through two-dimensional electrophoresis analysis. As a result, 41 differentially expressed proteins were detected by in silico analysis and further identified by tandem mass spectrometry de novo sequencing. Functional categorization indicated an intense metabolic response mainly in energy and nitrogen uptake, stress response, amino acid conversion, and cell wall thickness. Indeed, data reported here show that resistance to cationic antimicrobial peptides possesses a greater molecular complexity than previously supposed, resulting in cell commitment to several metabolic pathways.
Assuntos
Antibacterianos/farmacologia , Citosol/fisiologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Magaininas/farmacologia , Proteoma/genética , Aminoácidos/metabolismo , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Simulação por Computador , Eletroforese em Gel de Poliacrilamida , Metabolismo Energético/genética , Fermentação , Testes de Sensibilidade Microbiana , Nitrogênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Microcystis aeruginosa is a species of cyanobacteria commonly found in a number of countries and frequently related to animal poisoning episodes due to its capacity to produce the cyanotoxin known as microcystin. Despite vast literature on microcystin structures and their deleterious effects, little is known about its synthesis by cyanobacteria. Therefore, this study used proteomic tools to compare two M. aeruginosa strains, contrasting them for microcystin production. RESULTS: 2-DE gels were performed and 30 differential protein spots were chosen. Among them, 11 protein spots were unique in the toxin producing strain and 8 in the non-toxin producing strain, and 14 protein spots were shown on both 2-DE gels but expressed differently in intensity. Around 57% of the tandem mass spectrometry identified proteins were related to energy metabolism, with these proteins being up-regulated in the toxin producing strain. CONCLUSIONS: These data suggest that the presence of higher quantities of metabolic enzymes could be related to microcystin metabolism in comparison to the non-toxin producing strain. Moreover, it was suggested that the production of microcystin could also be related to other proteins than those directly involved in its production, such as the enzymes involved in the Calvin cycle and glycolysis.
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OBJECTIVE: to construct a prototype of a mobile application on psychological violence against women to facilitate nurses' continuing education. METHOD: an applied methodological research of technological development, based on the Contextualized Instructional Design method. RESULTS: the prototype content was based on the learning objectives developed through a narrative review, which guided an integrative review to compile the information. The prototype is called "EmpodereEnf", and is composed of an initial screen, bringing nurses as a target audience, and, soon after, offering nine general moblets for access to information such as: concepts, causes, manifestations and consequences of psychological violence; means for identification and approach in nursing consultation and health education; compulsory notification and referral; examples of cases of psychological violence and references. FINAL CONSIDERATIONS: the prototype is a possibility for future coping interventions and a work tool for nurses in the face of psychological violence against women.
Assuntos
Enfermeiras e Enfermeiros , Violência , Feminino , Humanos , Educação Continuada , Adaptação Psicológica , AprendizagemRESUMO
It aims to identify how the phenomenon of violence is themed in the curricular components of undergraduate nursing courses of public and private higher education institutions in Northeastern of Brazil.This is a descriptive and exploratory study, with documentary design and qualitative approach, based on the identification of the terms violence/aggression/abuse/aggressive/abusive in discipline menus available online from higher education institutions in northeastern Brazil. The data were organized and processed by the IraMuTeQ software and analyzed in the light of the relevant literature.In total, 412 nursing graduate institutions were found, in which 49 had documents available online, and, of these, 21 had the term violence. The processing of the syllabuses in the program resulted in significant use (79.37%) originating from the Descending Hierarchical Classification, seven classes that reveal the highlight of the approach of violence directed at the female public and in this context, the approach to violence against women was associated with sexual and reproductive aspects, and in a secondary way, it integrated the perspective of female vulnerabilities, power relations, consequences of violence and nursing care to cope with the phenomenon. Violence against children and adolescents and the discussion of violence in the contexts of collective health and health promotion were also approached in a less intense way.There was a predominance of the approach to violence aimed at women's health, under a biological approach, with a deficit in the transversality of the contents. However, it is important to highlight potentialities associated with the approach and teaching of contents that stimulate the critical thinking of the student before female vulnerabilities and power relations between the sexes, which can contribute to the reformulation and reorientation of nursing curricula, restraining deficits, and optimizing the potentialities.
Assuntos
Bacharelado em Enfermagem , Educação em Enfermagem , Estudantes de Enfermagem , Adolescente , Brasil , Criança , Bacharelado em Enfermagem/métodos , Feminino , Humanos , ViolênciaRESUMO
OBJECTIVES: to identify nurses' competences in promoting women's health. METHODS: descriptive study with a qualitative approach that adopted the Galway Consensus as a theoretical-methodological framework. Data collection was performed through semi-structured interviews. RESULTS: most of the Galway Consensus' competence domains were present in the nurses' interventions related to health promotion in women's care. "Assessment of needs" and "Implementation" were the most highlighted domains, followed by "Leadership" and "Impact assessment". The domain "Defending/Advocating Rights" was not identified. FINAL CONSIDERATIONS: within the nurses' work with women, some of the Galway Consensus domains of competencies for health promotion were present. However, there is a need, in the context of continuous health education, to expand strategies and enhance the development and application of these health promotion competences.
Assuntos
Promoção da Saúde , Liderança , Consenso , Feminino , Educação em Saúde , Humanos , Papel do Profissional de Enfermagem , Pesquisa Qualitativa , Saúde da MulherRESUMO
Antimicrobial peptides are widely expressed in organisms and have been linked to innate and acquired immunities in vertebrates. These compounds are constitutively expressed and rapidly induced at different cellular levels to interact directly with infectious agents and/or modulate immunoreactions involved in defense against pathogenic microorganisms. In invertebrates, antimicrobial peptides represent the major humoral defense system against infection, showing a diverse spectrum of action mechanisms, most of them related to plasma membrane disturbance and lethal alteration of microbial integrity. Marine invertebrates are widespread, extremely diverse, and constantly under an enormous microbial challenge from the ocean environment, itself altered by anthropic influences derived from industrialization and transportation. Consequently, this study reexamines the peptides isolated over the past 2 decades from different origins, bringing phyla not previously reviewed up to date. Moreover, a promising novel use of antimicrobial peptides as effective drugs in human and veterinary medicine could be based on their unusual properties and synergic counterparts as immune response humoral effectors, in addition to their direct microbicidal activity. This has been seen in many other marine proteins that are sufficiently immunogenic to humans, not necessarily in terms of antibody generation but as inflammation promoters and recruitment agents or immune enhancers.
Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Invertebrados/metabolismo , Biologia Marinha , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/classificação , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/classificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Humanos , Infecções/tratamento farmacológico , Invertebrados/química , Invertebrados/classificação , Dados de Sequência MolecularRESUMO
Currently, the interactions occurring between oligonucleotides and the cellular envelope of bacteria are not fully resolved at the molecular level. Understanding these interactions is essential to gain insights on how to improve the internalization of the tagged oligonucleotides during fluorescence in situ hybridization (FISH). Agent-based modeling (ABM) is a promising in silico tool to dynamically simulate FISH and bring forward new knowledge on this process. Notably, it is important to simulate the whole bacterial cell, including the different layers of the cell envelope, given that the oligonucleotide must cross the envelope to reach its target in the cytosol. In addition, it is also important to characterize other molecules in the cell to best emulate the cell and represent molecular crowding. Here, we review the main information that should be compiled to construct an ABM on FISH and provide a practical example of an oligonucleotide targeting the 23S rRNA of Escherichia coli .
Assuntos
Hibridização in Situ Fluorescente/métodos , Parede Celular/genética , Citosol/metabolismo , Escherichia coli/genética , Sondas de Oligonucleotídeos/genética , Oligonucleotídeos/genética , RNA Ribossômico 23S/genéticaRESUMO
OBJECTIVE: to describe the health promotion competency domains, performed by nurses, for adolescents, according to the Galway Consensus. METHOD: a qualitative study based on the Galway Consensus theoretical methodological framework. Fifteen nurses from northeastern Brazil participated. Data were collected between April and May 2017 through pre-structured interviews, submitted to the content analysis technique and analyzed according to the Galway Consensus dimensions. RESULTS: the following competency areas were found: catalyzing changes, leadership, needs assessment, planning, implementation, and partnerships. These competencies were contemplated from embracement of adolescents at health unit, guidance, teamwork, educational activities and lectures, as well as active search. CONCLUSION: most health promotion domains were observed; however, it is still suggested that there are challenges to an effective performance of health promotion among adolescents because some competency domains in health promotion have not been evidenced.
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Serviços de Saúde do Adolescente/organização & administração , Promoção da Saúde/métodos , Enfermeiras e Enfermeiros , Enfermagem/normas , Competência Profissional/normas , Adolescente , Brasil , Humanos , Entrevistas como Assunto , Liderança , Pesquisa QualitativaRESUMO
Inflammation is a natural defense mechanism of the immune system; however, when unregulated, it can lead to chronic illness. Glucocorticoids are the most commonly used agents to effectively treat inflammatory conditions, including autoimmune diseases, however these substances can trigger a number of side effects. Thus, viable alternatives to the use of these drugs would be advantageous. In this study, we have analyzed the anti-inflammatory profile of three synthetic peptides first identified in skin secretion of the tree frog Hypsiboas raniceps. Structural characterization was performed using NMR spectroscopy and Mass Spectrometry, and the peptides were tested in vitro in RAW 264.7 cells and in vivo in Balb/c mice for their functional properties. The samples did not show a significant antimicrobial profile. NMR spectroscopy indicated that AC12 (ACFLTRLGTYVC) has a disulfide bond between C2 and C11 and a ß-sheet-turn-ß-sheet conformation in aqueous solution. This peptide showed no cytotoxic effect in mammalian cells and it was the most effective in reducing anti-inflammatory markers, such as NO, TNF-α and IL-12. Peptide DK16 (DKERPICSNTFRGRKC) demonstrated anti-inflammatory properties in vitro, while RC11 (RCFRRRGKLTC) significantly altered the cell viability in RAW 264.7 but was shown to be safe in Balb/c erythrocytes. Our results indicate that, of the three peptides studied, AC12 is the most efficient in reducing anti-inflammatory markers, and it could be a potential agent for the treatment of inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Anuros/metabolismo , Inflamação/tratamento farmacológico , Peptídeos/farmacologia , Pele/metabolismo , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Proteínas de Anfíbios/farmacologia , Animais , Anti-Infecciosos/farmacologia , Biomarcadores , Interleucina-12 , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Células RAW 264.7 , Pele/química , Fator de Necrose Tumoral alfaRESUMO
In recent years, a strong emphasis has been given in deciphering the function of genes unraveled by the completion of several genome sequencing projects. In plants, functional genomics has been massively used in order to search for gene products of agronomic relevance. As far as root-pathogen interactions are concerned, several genes are recognized to provide tolerance/resistance against potential invaders. However, very few proteins have been identified by using current proteomic approaches. One of the major drawbacks for the successful analysis of root proteomes is the inherent characteristics of this tissue, which include low volume content and high concentration of interfering substances such as pigments and phenolic compounds. The proteome analysis of plant-pathogen interactions provides important information about the global proteins expressed in roots in response to biotic stresses. Moreover, several pathogenic proteins superimpose the plant proteome and can be identified and used as targets for the control of viruses, bacteria, fungi and nematode pathogens. The present review focuses on advances in different proteomic strategies dedicated to the challenging analysis of plant defense proteins expressed during bacteria-, fungi- and nematode-root interactions. Recent developments, limitations of the current techniques, and technological perspectives for root proteomics aiming at the identification of resistance-related proteins are discussed.
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Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Proteoma , Proteômica/métodos , Animais , Bactérias/metabolismo , Eletroforese em Gel Bidimensional , Fungos/metabolismo , Espectrometria de Massas , Nematoides/metabolismo , Proteínas de Plantas/isolamento & purificação , Raízes de Plantas/microbiologia , Raízes de Plantas/parasitologia , Plantas/metabolismo , Plantas/microbiologia , Plantas/parasitologiaRESUMO
A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.