RESUMO
Saccharomyces cerevisiae (S. cerevisiae) is the most widely used yeast in biotechnology in the world because its well-known metabolism and physiology as well as its recognized ability to ferment sugars such as hexoses. However, it does not metabolize pentoses such as arabinose and xylose, which are present in lignocellulosic biomass. Lignocellulose is a widely available raw material, with xylose content of approximately 35% of total sugars. This xylose fraction could be used to obtain high added-value chemical products such as xylitol. One of these yeasts isolated from a Colombian locality, designated as 202-3, showed interesting properties. 202-3 was identified through different approaches as a strain of S. cerevisiae, with an interesting consumption of xylose metabolizing into xylitol, in addition with excellent ability as a hexose fermenter with high ethanol yields and shows resistance to inhibitors present in lignocellulosic hydrolysates. The xylose metabolization by the 202-3 strain and their kinetics parameters had not been previously reported for any other natural strain of S. cerevisiae. These results suggest the great potential of natural strains for obtaining high value-added chemical products using sugars available in lignocellulosic biomass. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01054-z.
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Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numerous biotechnological applications, such as the biological control of agricultural pests, the expression of recombinant proteins or the gene delivery of therapeutic sequences in mammals, among others. Their genomes encode between 80 and 200 proteins, of which 38 are shared by all reported species. Thanks to multi-omic studies, there is remarkable information about the baculoviral proteome and the temporality in the virus gene expression. This allows some functional elements of the genome to be very well described, such as promoters and open reading frames. However, less information is available about the transcription termination signals and, consequently, there are still imprecisions about what are the limits of the transcriptional units present in the baculovirus genomes and how is the processing of the 3' end of viral mRNA. Regarding to this, in this review we provide an update about the characteristics of DNA signals involved in this process and we contribute to their correct prediction through an exhaustive analysis that involves bibliography information, data mining, RNA structure and a comprehensive study of the core gene 3' ends from 180 baculovirus genomes.
Assuntos
Baculoviridae/genética , Regulação Viral da Expressão Gênica , Vírus de Insetos/genética , Poliadenilação , RNA Mensageiro/genética , Transcrição Gênica , Regiões 3' não Traduzidas , Animais , Baculoviridae/metabolismo , Sítios de Ligação , Genoma Viral , Genômica/métodos , Ligação Proteica , Processamento Pós-Transcricional do RNA , Sequências Reguladoras de Ácido Ribonucleico , Replicação ViralRESUMO
St. Louis encephalitis virus (SLEV) is a mosquito-borne re-emerging flavivirus in Argentina. It is currently necessary to develop specific serological tests that can efficiently discriminate the flaviviruses that circulate in our country. The immunoassays to diagnose SLEV lack specificity because they are based on the detection of structural viral proteins and the human immunoglobulins produced during infection against these proteins cross-react with other flaviviruses. Here, we describe an enzyme-immunoassay designed to detect human IgG antibodies specific to the viral non-structural protein NS5. The results indicate that NS5 is a promising antigen useful to discriminate SLEV from other circulating flaviviruses.
Assuntos
Vírus da Encefalite de St. Louis/imunologia , Encefalite de St. Louis/diagnóstico , Testes Sorológicos , Proteínas não Estruturais Virais/imunologia , Argentina , Reações Cruzadas , Flavivirus/imunologia , HumanosRESUMO
Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46â¯kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4-8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from SLEV and WNV positive cases showed reactivity to the recombinant NS1 proteins by western blot. The unfolded NS1 proteins were also used as immunogens. The polyclonal antibodies elicited in immunized mice recognized the two recombinant proteins with differential reactivity.
Assuntos
Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Vírus da Encefalite de St. Louis/imunologia , Encefalite de St. Louis/diagnóstico , Proteínas não Estruturais Virais/imunologia , Febre do Nilo Ocidental/diagnóstico , Vírus do Nilo Ocidental/imunologia , Animais , Especificidade de Anticorpos , Antígenos Virais/biossíntese , Antígenos Virais/genética , Argentina , Western Blotting , Cromatografia de Afinidade , Clonagem Molecular , Reações Cruzadas , Diagnóstico Diferencial , Vírus da Encefalite de St. Louis/química , Vírus da Encefalite de St. Louis/genética , Encefalite de St. Louis/imunologia , Encefalite de St. Louis/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Corpos de Inclusão/química , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Solubilidade , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genética , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/química , Vírus do Nilo Ocidental/genéticaRESUMO
Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties.
Assuntos
Anticorpos Neutralizantes/sangue , Vacinas contra Dengue/imunologia , Vírus da Dengue/genética , Portadores de Fármacos , Vírus Junin/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/genética , Camundongos Endogâmicos C57BL , Testes de Neutralização , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Envelope Viral/genética , Ensaio de Placa ViralRESUMO
Floriculture is a vital agro-industrial sector in the Colombian economy; the export of flowers positively impacts employment and the balance of trade. However, this industry could negatively impact the environment if its waste products are not handled properly. These flower residues, rich in lignin, hemicellulose and cellulose, could be a cost-effective raw material to produce enzymes. Here, we evaluate the production of lignocellulolytic enzymes by degradation of Chrysanthemum and Rosa residues using Pleurotus ostreatus, and manganese sulfate and copper sulfate as inductors. From the two residues, we obtained laccase, manganese peroxidase, endoglucanase, exoglucanase, and (3-glucosidase. The use of inductors, favored all enzyme activities except for (3-glucosidase. The enzymes that displayed the highest activity were laccase (4,693.4 U/L and 2,640 U/L from the residues of Chrysanthemum and Rosa, respectively) and (3-glucosidase (9,513 U/L and 6,811.9 U/L). The enzyme that showed the lowest activity was endoglucanase (11.5 U/L and 15.4 U/L). Under the conditions evaluated, the best substrate for enzyme production is Chrysanthemum wastes; the extracts obtained had higher enzymatic activity than the extracts from Rosa residues.
En Colombia la floricultura es un sector agro-industrial importante, con impactos positivos en el empleo y la balanza comercial. Sin embargo, tiene impacto negativo en el medio ambiente porque genera alto volumen de residuos. Estos residuos, ricos en lignina, hemicelulosa y celulosa, podrían ser una materia prima de bajo costo para la producción de enzimas. En este trabajo se estudió en la producción de enzimas lignocelulíticas por la degradación con Pleurotus ostreatus de residuos de Chrysanthemum y Rosa, usando como inductores sulfato de manganeso y de cobre. A partir de ambos residuos se obtuvieron lacasa, manganeso peroxidasa, endoglucanasa, exoglucanasa y p-glucosidasa. Los inductores favorecieron todas las actividades enzimáticas, excepto para p-glucosidasa. Las enzimas que tuvieron mayores actividades fueron lacasa (4,693.4 U/L y 2,640 U/L a partir del residuo de Chrysanthemum y Rosa, respectivamente) y p-glucosidasa (9,513 U/L y 6,811.9 U/L). La enzima que tuvo menor actividad fue endoglucanasa (11.5 U/L y 15.4 U/L). Bajo las condiciones evaluadas, el mejor residuo para producción de enzimas fue Chrysanthemum, porque los extractos tuvieron mayor actividad enzimática que los producidos a partir de Rosa.
Na Colombia, a floricultura è um importante setor da indùstria agrícola, com impactos positivos sobre o sector laboral na balança comercial. Além disto, tem impacto negativo sobre o meio ambiente, pois gera grandes volumes de resíduos. Estes resíduos, com altos conteùdos em lignina, hemicelulose e celulose, podem ser uma matèria-prima de baixo custo para a produçâo de enzimas. Neste traballio foi estudada a produçâo da enzimas lignoceluliticas pela degradaçâo com Pleurotus ostreatus de residuos de Chrysanthemum e Rosa, utilizando como indutores sulfatos de cobre e manganês. A partir destes resíduos foram obtidos lacase, manganês peroxidase, endoglucanase, exoglucanase e β-glicosidase. O uso de indutores favoreceu as atividades enzimáticas, exceto β-glicosidase. As enzimas que apresentaram atividades mais elevadas foram lacase (4,693.4 U/L e 2,640 U/L a partir do Chrysanthemum e Rosa, respetivamente) e β-glicosidase (9,513 U/L e 6,81.9 U/L). A enzima que apresentou menor atividade foi a endoglucanase (11.5 U/L e 15.4 U/L). Nas condiçôes testadas, o melhor resíduo para a produçâo da enzima foi Chrysanthemum, porque os extratos tinham uma atividade enzimática mais elevada que aqueles produzidos a partir de Rosa.
RESUMO
Designing degenerate PCR primers for templates of unknown nucleotide sequence may be a very difficult task. In this paper, we present a new method to design degenerate primers, implemented in family-specific degenerate primer design (FAS-DPD) computer software, for which the starting point is a multiple alignment of related amino acids or nucleotide sequences. To assess their efficiency, four different genome collections were used, covering a wide range of genomic lengths: Arenavirus (10 × 10(4) nucleotides), Baculovirus (0.9 × 10(5) to 1.8 × 10(5) bp), Lactobacillus sp. (1 × 10(6) to 2 × 10(6) bp), and Pseudomonas sp. (4 × 10(6) to 7 × 10(6) bp). In each case, FAS-DPD designed primers were tested computationally to measure specificity. Designed primers for Arenavirus and Baculovirus were tested experimentally. The method presented here is useful for designing degenerate primers on collections of related protein sequences, allowing detection of new family members.
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The Arenaviridae family includes several hemorrhagic fever viruses which are important emerging pathogens. Junín virus, a member of this family, is the etiological agent of Argentine Hemorrhagic Fever (AHF). A collaboration between the Governments of Argentina and the USA rendered the attenuated Junín virus vaccine strain Candid#1. Arenaviruses are enveloped viruses with genomes consisting of two single-stranded RNA species (L and S), each carrying two coding regions separated by a stably structured, non-coding intergenic region. Molecular characterization of the vaccine strain and of its more virulent ancestors, XJ13 (prototype) and XJ#44, allows a systematic approach for the discovery of key elements in virulence attenuation. We show comparisons of sequence information for the S RNA of the strains XJ13, XJ#44 and Candid#1 of Junín virus, along with other strains from the vaccine lineage and a set of Junín virus field strains collected at the AHF endemic area. Comparisons of nucleotide and amino acid sequences revealed different point mutations which might be linked to the attenuated phenotype. The majority of changes are consistent with a progressive attenuation of virulence between XJ13, XJ#44 and Candid#1. We propose that changes found in genomic regions with low natural variation frequencies are more likely to be associated with the virulence attenuation process. We partially sequenced field strains to analyze the genomic variability naturally occurring for Junín virus. This information, together with the sequence analysis of strains with intermediate virulence, will serve as a starting point to study the molecular bases for viral attenuation.
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Ethanol production derived from Saccharomyces cerevisiae fermentation of a hydrolysate from floriculture waste degradation was studied. The hydrolysate was produced from Chrysanthemum (Dendranthema grandiflora) waste degradation by Pleurotus ostreatus and characterized to determine the presence of compounds that may inhibit fermentation. The products of hydrolysis confirmed by HPLC were cellobiose, glucose, xylose and mannose. The hydrolysate was fermented by S. cerevisiae, and concentrations of biomass, ethanol, and glucose were determined as a function of time. Results were compared to YGC modified medium (yeast extract, glucose and chloramphenicol) fermentation. Ethanol yield was 0.45 g g(-1), 88 % of the maximal theoretical value. Crysanthemum waste hydrolysate was suitable for ethanol production, containing glucose and mannose with adequate nutrients for S. cerevisiae fermentation and low fermentation inhibitor levels.
Assuntos
Agaricales/metabolismo , Celulose/metabolismo , Chrysanthemum/metabolismo , Etanol/metabolismo , Microbiologia Industrial/métodos , Lignina/metabolismo , Saccharomyces cerevisiae/metabolismo , Biomassa , Meios de Cultura , Fermentação , Glucose/metabolismo , Hidrólise , Manose/metabolismo , Folhas de Planta/metabolismo , Caules de Planta/metabolismoRESUMO
INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5% polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4% of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.