RESUMO
RNA purification and cDNA synthesis represents the starting point for molecular analyses of snake venom proteins-enzymes. Usually, the sacrifice of snakes is necessary for venom gland extraction to identify protein-coding transcripts; however, the venom can be used as a source of transcripts. Although there are methods for obtaining RNA from venom, no comparative analysis has been conducted in the Bothrops genus. In the present study, we compared four commercial methods for RNA purification and cDNA synthesis from venom (liquid, lyophilized, or long-term storage) of four clinically relevant species of Peruvian Bothrops. Our results show that the TRIzol method presents the highest yield of RNA purified from venom (59 ± 11 ng/100 µL or 10 mg). The SuperScript First-Strand Synthesis System kit produced high amounts of cDNA (3.2 ± 1.2 ng cDNA/ng RNA), and the highest value was from combination with the Dynabeads mRNA DIRECT kit (4.8 ± 2.0 ng cDNA/ng RNA). The utility of cDNA was demonstrated with the amplification of six relevant toxins: thrombin-like enzymes, P-I and P-III metalloproteinases, acid and basic phospholipases A2, and disintegrins. To our knowledge, this is the first comparative study of RNA purification and cDNA synthesis methodologies from Bothrops genus venom.
Assuntos
Bothrops , Venenos de Crotalídeos , Animais , DNA Complementar/genética , Bothrops/genética , Peru , Relevância Clínica , Venenos de Crotalídeos/genética , Proteínas , RNARESUMO
Due to the high concentration of pollutants, swine wastewater needs to be treated prior to disposal. The combination of anaerobic and aerobic technologies in one hybrid system allows to obtain higher removal efficiencies compared to those achieved via conventional biological treatment, and the performance of a hybrid system depends on the microbial community in the bioreactor. Here, we evaluated the community assembly of an anaerobic-aerobic hybrid reactor for swine wastewater treatment. Sequencing of partial 16S rRNA coding genes was performed using Illumina from DNA and retrotranscribed RNA templates (cDNA) extracted from samples from both sections of the hybrid system and from a UASB bioreactor fed with the same swine wastewater influent. Proteobacteria and Firmicutes were the dominant phyla and play a key role in anaerobic fermentation, followed by Methanosaeta and Methanobacterium. Several differences were found in the relative abundances of some genera between the DNA and cDNA samples, indicating an increase in the diversity of the metabolically active community, highlighting Chlorobaculum, Cladimonas, Turicibacter and Clostridium senso stricto. Nitrifying bacteria were more abundant in the hybrid bioreactor. Beta diversity analysis revealed that the microbial community structure significantly differed among the samples (p < 0.05) and between both anaerobic treatments. The main predicted metabolic pathways were the biosynthesis of amino acids and the formation of antibiotics. Also, the metabolism of C5-branched dibasic acid, Vit B5 and CoA, exhibited an important relationship with the main nitrogen-removing microorganisms. The anaerobic-aerobic hybrid bioreactor showed a higher ammonia removal rate compared to the conventional UASB system. However, further research and adjustments are needed to completely remove nitrogen from wastewater.
Assuntos
Chlorobi , Microbiota , Purificação da Água , Animais , Suínos , Águas Residuárias , Esgotos/química , Eliminação de Resíduos Líquidos , Anaerobiose , Chlorobi/genética , RNA Ribossômico 16S/genética , DNA Complementar , Reatores Biológicos/microbiologiaRESUMO
Amphibians have a great diversity of bioactive peptides in their skin. The cDNA prepro-peptide sequencing allowed the identification of five novel mature peptides expressed in the skin of Boana pulchella, four with similar sequences to hylin peptides having a cationic amphipathic-helical structure. Whole mature peptides and some of their fragments were chemically-synthesized and tested against Gram-positive and Gram-negative bacterial strains. The mature peptide hylin-Pul3 was the most active, with a MIC= 14 µM against Staphylococcus aureus. Circular dichroism assays indicated that peptides are mostly unstructured in buffer solutions. Still, adding large unilamellar vesicles composed of dimyristoyl phosphatidylcholine and dimyristoylphosphatidylglycerol increased the α-helix content of novel hylins. These results demonstrate the strong influence of the environment on peptide conformation and highlight its significance while addressing the pharmacology of peptides and their biological function in frogs.
Assuntos
Anuros , Peptídeos , Animais , Sequência de Aminoácidos , Peptídeos/farmacologia , Peptídeos/química , Lipídeos , Dicroísmo CircularRESUMO
Myrosinases (EC 3.2.1.147) are enzymes known for the generation of hydrolysis products that have a potential beneficial effect on human health. Their reaction mechanisms are widely studied, in order to improve and optimize secondary metabolite production processes. In this work, kinetic and biochemical properties of the broccoli myrosinase enzyme produced from its cDNA cloned in Escherichia coli and Saccharomyces cerevisiae were investigated. The results revealed that the thermal stability of the enzyme produced in S. cerevisiae was slightly higher (30 to 60 °C) than that of myrosinase produced in E. coli (20 to 50 °C). The effect of pH on the enzymatic activity was similar in both enzymes, with pH 3 being the optimum value under the reaction conditions used. The kinetic behavior of both enzymes was adjusted to the Michaelis-Menten model. The catalytic efficiency was up to 4 times higher in myrosinase produced in S. cerevisiae, compared to myrosinase produced in E. coli. The glycosylations present in the enzyme would be related to the formation of a dimeric quaternary structure and would not play an essential role in enzymatic activity, since both enzymes were biologically active. These results will probably allow the development of strategies for the production of bioactive metabolites of medical interest.
Assuntos
Brassica , Brassica/química , Brassica/genética , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Genoma Viral , RNA Viral , Inativação de Vírus , Animais , Células Cultivadas , Chlorocebus aethiops , Efeito Citopatogênico Viral , DNA Complementar/biossíntese , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/fisiologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Vírus da Encefalite Equina do Oeste/genética , Vírus da Encefalite Equina do Oeste/fisiologia , RNA Viral/química , RNA Viral/fisiologia , Ribonucleases/metabolismo , Células VeroRESUMO
The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA "Animal Rule" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other "wild type" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.
Assuntos
Vírus da Encefalite Equina Venezuelana , Vírus da Encefalite Equina do Oeste , Estados Unidos , Humanos , Animais , Cavalos , Camundongos , DNA Complementar/genética , Fenótipo , Células ClonaisRESUMO
The Colombian Creole sheep breed has a high economic and social importance for Colombia. Both males and females of this breed are multipurpose animals, and evaluating the production and meat quality of both sexes is important for small farmers in Colombia. This requires the use of tools that help to evaluate critical production points, such as Real-time quantitative polymerase chain reaction (RT-qPCR), which is a widely used molecular tool for the relative quantification of candidate genes in various tissues. For its correct use, the use of housekeeping genes with stable expression, so-called "reference genes", is required. However, recent studies have shown that the expression of these reference genes can vary among tissues and can be modulated by breed, sex, or external stimuli. Likewise, there is little information regarding the expression of these genes in the Longissimus thoracis et lumborum muscle of male and female Colombian Creole sheep. In this study, the stability in the expression of seven reference genes (ACTB, YWHAZ, SDHA, GAPDH, TUBB2A, B2M, and PGK1) in the Longissimus thoracis et lumborum muscle of male and female Colombian Creole sheep was compared since they are used in RT-qPCR studies to determine the most stable ones for this breed. Twelve animals, six males and six females, with a body weight of 26 ± 4 kg and 12 ± 3 months of age, were used under grazing conditions. Biopsies of the Longissimus thoracis et lumborum muscle were taken, from which RNA was extracted and cDNA was synthesized. Expression was determined using RT-qPCR, and its stability was analyzed by computational algorithms using the geNorm, Normfinder, and BestKeeper software packages, which were integrated using the RefFinder software package. The results indicate that GAPDH, ACTB, and SDHA have the highest stability, whereas the most variable expression was found for B2M. These data provide the basis for more precise results in RT-qPCR studies of gene expression in the muscle of Colombian Creole sheep.(AU)
A raça de ovinos Crioula Colombiana tem grande importância econômica e social para a Colômbia. Avaliar a produção e a qualidade da carne de machos e fêmeas é importante para pequenos produtores do país e, assim, faz necessário o uso ferramentas que ajudam a avaliar os pontos críticos de produção, como a reação em cadeia da polimerase em tempo real (Real-time quantitative polymerase chain reaction [RT-qPCR]). Esta é uma ferramenta molecular amplamente usada para a quantificação relativa de genes candidatos em vários tecidos. Para o seu uso correto, é necessário o uso de genes com expressão estável denominados genes de referência. No entanto, estudos recentes têm mostrado que a expressão desses genes de referência pode variar entre os tecidos e pode ser modulada por raça, sexo ou estímulos externos. Da mesma forma, existem poucas informações sobre a expressão desses genes no músculo Longissimus thoracis et lumborum de ovinos machos e fêmeas da raça Crioula Colombiana. Neste estudo foi comparada a estabilidade na expressão de sete genes de referência (ACTB, YWHAZ, SDHA, GAPDH, TUBB2A, B2M e PGK1) no músculo Longissimus thoracis et lumborum de ovinos Crioulo Colombiano machos e fêmeas, por serem genes utilizados em estudos de RT-qPCR visando determinar os mais estáveis para esta raça. Doze animais com peso corporal de 26 ± 4 kg e 12 ± 3 meses de idade foram utilizados em condições de pastejo. Foram realizadas biópsias do músculo Longissimus thoracis et lumborum, de onde o RNA foi extraído e o cDNA foi sintetizado. A expressão foi determinada usando RT-qPCR e sua estabilidade foi analisada por algoritmos computacionais usando geNorm, Normfinder e BestKeeper pacote de software, os quais foram integrados usando RefFinder pacote de software. Os resultados indicam que GAPDH, ACTB e SDHA apresentam maior estabilidade, enquanto a expressão mais variável foi para B2M. Esses dados fornecem a base para resultados mais precisos em estudos de RT-qPCR de expressão gênica em músculos defeminino ovinos da raça Crioula Colombiana.(AU)
Assuntos
Animais , Feminino , Ovinos , Expressão Gênica , Reação em Cadeia da Polimerase em Tempo Real , GenesRESUMO
INTRODUÇÃO: A Artrite Reumatoide (RA), doença inflamatória crônica autoimune cujos eventos autoimunes primordiais não são totalmente conhecidos, envolve em sua suscetibilidade tanto um elemento genético quanto fatores ambientais. Um deles, a microbiota intestinal, pode modular significativamente o sistema imunológico através de produtos do metabolismo microbiano, muitos dos quais são ligantes do receptor de aril-hidrocarbonetos (AhR). O AhR é ativado por diversos ligantes, exógenos ou endógenos, os quais ativam programas transcricionais específicos, sendo uma ponte entre estímulos ambientais e fisiologia, e situando-se na multifatoriedade da Artrite Reumatoide. A ativação do AhR ocorre mediante interação com o translocador nuclear do AhR (ARNT), e pode ser reprimida através da competição com seu repressor (AhRR). Os genes Arnt e Ahrr estão contidos no intervalo de confiança de dois loci de suscetibilidade à artrite mapeados em camundongos selecionados fenotipicamente para alta (HIII) ou baixa (LIII) produção de anticorpos. Estas linhagens, quando submetidas ao modelo de artrite induzida por pristane (PIA), demonstram suscetibilidade extremamente divergente, possivelmente decorrente de suas genéticas distintas: completamente resistentes e suscetíveis, respectivamente. Elas também albergam microbiotas intestinais distintas, que podem ser parcialmente transferidas entre as linhagens, modulando os fenótipos de suscetibilidade à artrite. O envolvimento dos genes da via do AhR na progressão da artrite reumatoide ainda é pouco explorado, porém é pertinente investigar se os animais HIII e LIII apresentam alelos distintos destes genes, o que poderia sugerir um papel no desenvolvimento da doença, a ser avaliado em estudos posteriores. OBJETIVO: Investigar presença e distribuição de possíveis polimorfismos dos genes Arnt e Ahrr em camundongos HIII e LIII, e suas eventuais relevâncias na suscetibilidade à PIA. METODOLOGIA: RNA de camundongos das linhagens HIII e LIII foi convertido para cDNA por transcrição reversa. O material obtido foi amplificado por PCR com primers específicos para fragmentos dos genes alvo, e então submetido a eletroforese, com posterior purificação do DNA do gel ou do produto de PCR. Os produtos purificados foram então sequenciados. RESULTADOS: Para o Arnt, três de cinco fragmentos foram amplificados. Já para o Ahrr, apenas um de três fragmentos foi amplificado. As reações de sequenciamento realizadas indicaram uma divergência de uma base entre os animais HIII e LIII em um fragmento do Arnt, no entanto em uma sequência de baixa qualidade. DISCUSSÃO: Limitações intrínsecas aos genes, como baixa taxa de expressão e extensas UTRs 3’ de seus mRNAs, além de problemas técnicos que comprometeram o sequenciamento das extremidades dos fragmentos sequenciados, não permitiram concluir que a divergência encontrada seja um polimorfismo, e não um artefato de sequenciamento. CONCLUSÃO: A maior fragmentação da sequência a ser amplificada, ou ainda a clonagem de fragmentos do gene em plasmídeos para posterior sequenciamento poderiam superar os obstáculos encontrados no decorrer deste estudo, e indicam os próximos passos a serem seguidos na investigação do papel que a via do AhR exerce na suscetibilidade à PIA.
RESUMO
In 2016, the world experienced the unprecedented Zika epidemic. The ZIKV emerged as a major human pathogen due to its association with the impairment of perinatal development and Guillain-Barré syndrome. The occurrence of these severe cases of Zika points to the significance of studies for understanding the molecular determinants of flavivirus pathogenesis. Reverse genetics is a powerful method for studying the replication and determinants of pathogenesis, virulence, and viral attenuation of flaviviruses, facilitating the design of vaccines and therapeutics. However, the main hurdle in the development of infectious clones is the instability of full-length cDNA in Escherichia coli. Here, we described the development of a genetically stable and efficient infectious clone based on the ZIKV Rio-U1 isolated in the 2016 epidemic in Brazil. The employed strategy consisted of cloning the viral cDNA genome into two stable plasmid subclones and obtaining a high-quality cDNA template with increment in DNA mass for in vitro transcription by PCR amplification. The strategy for developing a ZIKV infectious cDNA clone designed in this study was successful, yielding a replicative and efficient clone-derived virus with high similarities with its parental virus, Rio-U1, by comparison of the proliferation capacity in mammal and insect cells. The infection of AG129 immunocompromised mice caused identical mortality rates, with similar disease progression and morbidity in the animals infected with the parental and the cDNA-derived virus. Histopathological analyses of mouse brains infected with the parental and the cDNA-derived viruses revealed a similar pathogenesis degree. We observed meningoencephalitis, cellular pyknosis, and neutrophilic invasion adjacent to the choroid plexus and perivascular cuffs with the presence of neutrophils. The developed infectious clone will be a tool for genetic and functional studies in vitro and in vivo to understand viral infection and pathogenesis better.
RESUMO
Together with their undeniable role in the ecology of arid and semiarid ecosystems, Agave species are emerging as a model to dissect the relationships between crassulacean acid metabolism and high efficiency of light and water use, and as an energy crop for bioethanol production. Transcriptome resources from economically valuable Agaves species, such as Agave tequilana and A. salmiana, as well as hybrids for fibers, are now available, and multiple gene expression landscape analyses have been reported. Key components in molecular mechanisms underlying drought tolerance could be uncovered by analyzing gene expression patterns of roots. This study describes an efficient protocol for high-quality total RNA isolation from phenolic compounds-rich Agave roots. Our methodology involves suitable root handling and collecting in the field and using saving-time commercial kits available. RNA isolated from roots free of lignified out-layers and clean cortex showed high values of quality and integrity according to electrophoresis and microfluidics-based platform. Synthesis of long full-length cDNAs and PCR amplification tested the suitability for downstream applications of extracted RNA. The protocol was applied successfully to A. tequilana roots but can be used for other Agave species that also develop lignified epidermis/exodermis in roots.
RESUMO
The isolation of single monoclonal antibodies (mAbs) against a given antigen was only possible with the introduction of the hybridoma technology, which is based on the fusion of specific B lymphocytes with myeloma cells. Since then, several mAbs were described for therapeutic, diagnostic, and research purposes. Despite being an old technique with low complexity, hybridoma-based strategies have limitations that include the low efficiency on B lymphocyte-myeloma cell fusion step, and the need to use experimental animals. In face of that, several methods have been developed to improve mAb generation, ranging from changes in hybridoma technique to the advent of completely new technologies, such as the antibody phage display and the single B cell antibody ones. In this review, we discuss the hybridoma technology along with emerging mAb isolation approaches, taking into account their advantages and limitations. Finally, we explore the usefulness of the hybridoma technology nowadays.
RESUMO
To analyze the impact of Ascaris lumbricoides infection on the pathogenesis and diagnosis of allergic diseases, new allergens should be identified. We report the identification of a new Ascaris lumbricoides allergen, Asc l 5. The aim of this study was to evaluate the physicochemical and immunological features of the Asc l 5 allergen. We constructed an A. lumbricoides cDNA library and Asc l 5 was identified by immunoscreening. After purification, rAsc l 5 was physicochemically characterized. Evaluation of its allergenic activity included determination of Immunoglobulin E (IgE) binding frequency (in two populations: 254 children and 298 all-age subjects), CD203c based-basophil activation tests (BAT) and a passive cutaneous anaphylaxis (PCA) mouse model. We found by amino acid sequence analysis that Asc l 5 belongs to the SXP/RAL-2 protein family of nematodes. rAsc l 5 is a monomeric protein with an alpha-helical folding. IgE sensitization to rAsc l 5 was around 52% in general population; positive BAT rate was 60%. rAsc l 5 induced specific IgE production in mice and a positive PCA reaction. These results show that Asc l 5 has structural and immunological characteristics to be considered as a new allergen from A. lumbricoides.
Assuntos
Alérgenos/imunologia , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Ascaris lumbricoides/imunologia , Asma/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Asma/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Imunoglobulina E/sangue , Lactente , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
BACKGROUND: Coconut tissues consist of a complex network of polysaccharides, proteins, polyphenols, and lipids that can bind to nucleic acids and pose difficulty in isolation. Certainly, a vigorous method is required to isolate high quality and quantity of RNA from such tissues for the purpose of downstream experiments. In this paper, we discuss a newly developed method for the Isolation of RNA from Complex Matrices (IRCM) method from coconut tissues. RESULTS: The method is robust, cheap, and efficient for the extraction of quality RNA in high quantities from the solid endosperm of stored and fresh coconut (150 µg/g FW with A260/280 = 1.89 and 247.5 µg/g FW with A260/280 = 1.91), coconut apple (263.8 µg/g FW with A260/280 = 1.97), and coconut bud (1052.5 µg/g FW with A260/280 = 2.00). The other well established methods, such as Method of RNA Isolation from Palm (MRIP), Cetyl Trimethyl Ammonium Bromide (CTAB), TRIZOL, and RNA plant kit failed to isolate quality RNA in appreciable quantities from the coconut tissues. Furthermore, the resultant RNA performed well in the downstream experiment, that is, RT-PCR for the production and amplification of cDNA. CONCLUSIONS: From the study, we concluded that the present method will play a vital role in the extraction of high quality RNA from complex matrices in a short time.
Assuntos
RNA/isolamento & purificação , Cocos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This work presents a new method and tool to solve a common problem of molecular biologists and geneticists who use molecular markers in their scientific research and developments: curation of sequences. Omic studies conducted by molecular biologists and geneticists usually involve the use of molecular markers. AFLP, cDNA-AFLP, and MSAP are examples of markers that render information at the genomics, transcriptomics, and epigenomics levels, respectively. These three types of molecular markers use adaptors that are the template for PCR amplification. The sequences of the adaptors have to be eliminated for the analysis of the results. Since a large number of sequences are usually obtained in these studies, this clean-up of the data could demand long time and work. To automate this work, an R package, named CleanBSequences, was created that allows the sequences to be curated massively, quickly, without errors and can be used offline. The curating is performed by aligning the forward and/or reverse primers or ends of cloning vectors with the sequences to be removed. After the alignment, new subsequences are generated without biological fragments not desired by the user, i.e., sequences needed by the techniques. In conclusion, the CleanBSequences tool facilitates the work of researchers, reducing time, effort, and working errors. Therefore, the present tool would respond to the problems related to the curation of sequences obtained from the use of some types of molecular markers. In addition to the above, being an open source, CleanBSequences is a flexible tool that has the potential to be used in future improvements to respond to new problems.
Assuntos
Biologia Computacional , Marcadores Genéticos/genética , Biologia Molecular/métodos , Software , Epigenômica/métodos , Genômica/métodos , Anotação de Sequência Molecular/métodos , Alinhamento de Sequência/métodos , Análise de Sequência/métodos , Transcriptoma/genéticaRESUMO
Licensure of a vaccine to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires use of the U.S. Food and Drug Administration (FDA) Animal Rule to assess vaccine efficacy as human studies are not feasible or ethical. An approach to selecting VEEV challenge strains for use under the Animal Rule was developed, taking into account Department of Defense (DOD) vaccine requirements, FDA Animal Rule guidelines, strain availability, and lessons learned from the generation of filovirus challenge agents within the Filovirus Animal Nonclinical Group (FANG). Initial down-selection to VEEV IAB and IC epizootic varieties was based on the DOD objective for vaccine protection in a bioterrorism event. The subsequent down-selection of VEEV IAB and IC isolates was based on isolate availability, origin, virulence, culture and animal passage history, known disease progression in animal models, relevancy to human disease, and ability to generate sufficient challenge material. Methods for the propagation of viral stocks (use of uncloned (wild-type), plaque-cloned, versus cDNA-cloned virus) to minimize variability in the potency of the resulting challenge materials were also reviewed. The presented processes for VEEV strain selection and the propagation of viral stocks may serve as a template for animal model development product testing under the Animal Rule to other viral vaccine programs. This manuscript is based on the culmination of work presented at the "Alphavirus Workshop" organized and hosted by the Joint Vaccine Acquisition Program (JVAP) on 15 December 2014 at Fort Detrick, Maryland, USA.
Assuntos
Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas Virais/uso terapêutico , Animais , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/virologia , Guias como Assunto , Humanos , Programas de Imunização/métodos , Programas de Imunização/normas , Virologia/métodosRESUMO
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.
Assuntos
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
BACKGROUND: Lethal factors are multifunctional oligomeric proteins found in the venomous apparatus of Scorpaeniformes fish. These toxins elicit not only an array of biological responses in vitro but also cardiovascular disorders and strong hemolytic, nociceptive and edematogenic activities in vivo. This work describes the cloning and molecular identification of two toxin subunits, denominated Sp-CTx-α and Sp-CTx-ß, from scorpionfish venom (Scorpaena plumieri). METHODS: The primary structures were deduced after cDNA amplification by PCR with primers from conserved sequences described in Scorpaeniformes toxins. Following DNA sequencing and bioinformatic analysis, the tridimensional structures of both subunits were modeled. RESULTS: The translated sequences (702 amino acids, each subunit) show homology with other lethal factors, while alignment between Sp-CTx-α and Sp-CTx-ß shows 54% identity. The subunits lack N-terminal signal sequences and display masses of approximately 80 kDa each. Both Sp-CTx subunits display a B30.2/SPRY domain at the C-terminal region with typically conserved motifs as described in these toxins. Secondary structure prediction identified six α-helices 18 residues long in both α and ß subunits, some of them amphiphilic with their N-terminal flanked by many basic residues, creating a cationic site associated with the cytolytic activity of these toxins. Antimicrobial potential sites were identified in Sp-CTx and share some features with other peptides presenting variable and broad-spectrum activity. A phylogenetic tree built to represent these toxins supports the proximity between scorpionfish, lionfish and stonefish. CONCLUSION: The study identified a putative toxin protein whose primary structure is similar to other fish toxins and with potential for production of antivenom against scorpionfish envenomation in Brazil. As a prelude to structure-function studies, we propose that the toxin is structurally related to pore-forming marine toxins.
RESUMO
There is few information about troponin gene expression by starvation in insect skeletal muscle and digestive tracts. The objective of this study was to perform molecular cloning of troponin I from the two-spotted cricket, Gryllus bimaculatus (GrybiTnI) and determine its expression patterns in three different skeletal muscles and digestive tracts during starvation. GrybiTnI was translated into a protein encoding 198 amino acids with a theoretical isoelectric point of 9.78 and a molecular weight of 23671.46 Da. The GrybiTnI has both the TnC-binding site and actin/TnC-binding site shown in the typical TnI amino acid sequences. Homology analysis revealed that GrybiTnI exhibited high similarity at the amino acid level to those of other insects already reported; 89~77% identity with those of other insects. Expression of GrybiTnI by starvation did not change in dorsal wing flight muscle and dorsal ventral flight muscle, but showed up-expression in dorsal longitudinal flight muscle. In the digestive tracts, the up-expression of GrybiTnI by starvation was observed only in the hindgut but not in the rest parts including Malpighian tubules. Re-feeding following starvation restored those expressions about the level before starvation in the dorsal longitudinal flight muscle and hindgut. In conclusion, troponin modulates gene expression not only to muscle elements but also to physiological changes such as strains.
Existe pouca informação sobre a expressão gênica da troponina por inanição no músculo esquelético de insetos e no trato digestório. O objetivo deste estudo foi realizar clonagem molecular da troponina I do grilo africano, Gryllus bimaculatus (GrybiTnI) e determinar seus padrões de expressão em três diferentes músculos esqueléticos e tratos digestivos durante a inanição. GrybiTnI foi traduzido em uma proteína codificando 198 aminoácidos com um ponto isoelétrico teórico de 9,78 e um peso molecular de 23671,46 Da. O GrybiTnI tem o local de ligação a TnC e o local de ligação a actina/TnC mostrado nas sequências de aminoácidos TnI típicas. A análise de homologia revelou que GrybiTnI exibiu alta similaridade no nível de aminoácidos em relação àqueles de outros insetos já relatados; 89~77% de identidade com os de outros insetos. A expressão de GrybiTnI pela inanição não se alterou no músculo de vôo da asa dorsal e no músculo de vôo ventral dorsal, mas mostrou expressão positiva no músculo de vôo longitudinal dorsal. Nos tratos digestivos, a expressão positiva de GrybiTnI por inanição foi observada apenas no intestino grosso, mas não nas partes de repouso, incluindo os túbulos de Malpighi. A realimentação após a inanição restaurou as expressões aproximadamente ao nível antes da inanição no músculo de vôo longitudinal dorsal e no intestino grosso. Em conclusão, a troponina modula a expressão gênica não apenas em elementos musculares, mas também em alterações fisiológicas, como as cepas.
Assuntos
Inanição , Troponina , Gryllidae , Clonagem Molecular , Expressão Gênica , Músculo Esquelético , Trato GastrointestinalRESUMO
Human norovirus (HuNoV) is one of the main causes of acute gastroenteritis worldwide and is responsible for at least 20% of all cases. The detailed molecular mechanism of this norovirus remains unknown due to the lack of a suitable in vitro culturing system. An infectious clone of HuNoV would be a useful tool for elucidating the processes of viral infection and the mechanisms of replication. We developed an infectious cDNA clone of HuNoV using the rapid technique of Gibson Assembly. The complete genome of the HuNoV GII.4 Sydney subtype was cloned into a previously modified pcDNA3.1-based plasmid vector downstream from a cytomegaloviral promoter. We monitored the viral infection in vitro by inserting the reporter gene of the green fluorescent protein (GFP) between the NTPase and p22 genes, also by Gibson Assembly, to construct a HuNoV-GFP replicon. Human Caco-2 cells were transfected with the full-length genomic clone and the replicon containing GFP. The gene encoding the VP1/VP2 capsid protein was expressed, which was indirect evidence of the synthesis of subgenomic RNAs and thus the negative strand of the genome. We successfully constructed the infectious clone and its replicon containing GFP for the HuNoV GII.4 Sydney subtype, a valuable tool that will help the study of noroviral infection and replication.
Assuntos
Norovirus/crescimento & desenvolvimento , Norovirus/genética , Replicon , Células CACO-2 , Citomegalovirus/genética , Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Plasmídeos , Regiões Promotoras Genéticas , Genética Reversa , Coloração e Rotulagem , TransfecçãoRESUMO
Background: Cathepsin C (CTSC) (dipeptidyl peptidase I, DPPI), is a member of the papain superfamily of cysteine proteases and involves in a variety of host reactions. However, the information of CTST in Chinese giant salamander (Andrias davidianus), an amphibian species with important evolutionary position and economic values, remained unclear. Results: The full-length salamander CTSC cDNA contained a 96 bp of 5'-UTR, a 1392 bp of ORF encoding 463 amino acids, and a 95 bp of 3'-UTR. The salamander CTSC possessed several sequence features similar to other reported CTSCs such as a signal peptide, a propeptide and a mature peptide. The active site triad of Cys, His and Asn were also found existing in salamander CTSC. Salamander CTSC mRNA was constitutively expressed in all the examined tissues with significantly variant expression level. The highest expression of CTSC was in intestine, followed with stomach, spleen, lung and brain. Following Aeromonas hydrophila infection for 12 h, salamander CTSC was significantly up-regulated in several tissues including lung, spleen, brain, kidney, heart, stomach and skin. Conclusion: CTSC plays roles in the immune response to bacterial infection, which provided valuable information for further studying the functions of CTSC in salamander.