HYDROTHERMAL TREATMENT OF EUCALYPTUS GRANDIS WOOD

 Among the technological alternatives to improve the quality and use of eucalyptus wood is heat treatment, as the wood has some features that may limit its use, such as dimensional instability, high anisotropy, and heterogeneous coloring. This study is aimed at evaluating the effect of time of hydrothermal treatment on physical, chemical, and mechanical properties of solid wood of Eucalyptus grandis. We used three trees of E. grandis, selecting only the heartwood, and prepared test specimens with dimensions of 30cmx8cmx3cm (length, width, and thickness). The hydrothermal treatment was performed in a Parr reactor using three reaction times (5, 15, and 25min) at 140°C. Partial removal of extractives occurred, especially in the outer layer of wood. There was an increase of up to 58% of its permeability because of the partial clearing of the vessel elements. There was no degradation of the main constituents of the wood, so loss of mechanical strength was observed. The hydrothermal treatment promoted the partial removal of the hydroxyl groups and/or a structural rearrangement of the hemicelluloses and cellulose, causing a reduction of the hygroscopicity of E. grandis.

Heterologous expression of the nonstructural 1 protein (NS1) of dengue-2 virus in Arabidopsis thaliana

A dengue é a doença mais importante causada por um arbovírus no mundo. O vírus da dengue pertence à família Flaviviridae e possui 4 sorotipos antigenicamente distintos. São vírus de RNA fita simples, polaridade positiva, com o genoma de aproximadamente 11 Kb que é traduzido em uma poliproteína subdividida em três proteínas estruturais e sete proteínas não- estruturais (que estão relacionadas com a replicação viral, expressão das proteínas virais e virulência dos sorotipos). Nos últimos vinte anos têm sido observado um incremento significativo na atividade epidêmica, expansão da distribuição geográfica e transmissão contínua dos diferentes sorotipos em áreas onde a doença não era prevalente. Um dos eventos mais alarmantes tem sido o aumento do número de casos da Febre da Dengue Hemorrágica (FDH) nas Américas. Um ponto ainda limitante dos testes diagnósticos que capturam o anticorpo presente no soro de pacientes infectados consiste na dificuldade de obtenção de baixo custo e produção em grande escala dos antígenos. Diante destas considerações objetivou-se expressar a proteína não-estrutural (NS1) do vírus dengue-2 em plantas transgênicas de Arabidopsis thaliana. O gene da proteína NS1 foi otimizado para a expressão em plantas e clonado no plasmídeo pCAMBIA3301. O vetor de expressão pCAMBIA3301/NS1 foi utilizado para transformação de Arabidopsis thaliana. A terceira geração de plantas transformadas foi selecionada em homozigose dominante. A transformação de Arabidopsis thaliana foi evidenciada pela imunolocalização da proteína NS1 marcada dentro do lúmen do retículo endoplasmático em tecidos foliares. Este sistema será utilizado para obtenção do antígeno NS1 com finalidade de aplicação em testes de diagnóstico da dengue.Dengue fever is the most important disease caused by an arbovirus in the world. The dengue virus belongs to the Flaviviridae family and has four antigenically distinct serotypes. They are single stranded RNA viruses, positive polarity, with a genome of approximately 11 Kb which is translated into a polyprotein subdivided in three structural proteins and seven non-structural proteins (that are related to viral replication, expression of viral proteins and virulence of serotypes). In the last twenty years, it was observed a significant increase in the epidemic activity, expansion of the geographical distribution and streaming of different serotypes in areas where the disease was not prevalent. One of the most alarming events was the increase in the number of cases of dengue hemorrhagic fever (DHF) in the Americas. A further limiting point of the diagnostic tests that capture antibody present in the serum of infected patients is the difficulty of obtaining low-cost and large-scale production of the antigens. Due to this factor, we aimed to build a system in transgenic Arabidopsis thaliana expressing the nonstructural protein (NS1) of the dengue-2 virus. The gene of the NS1 protein was optimized for expression in plants and cloned into the plasmid pCAMBIA3301. The expression vector pCAMBIA3301/NS1 was used for transformation of Arabidopsis thaliana. The third generation of transformed plants were selected by dominant homozygous. The construction of a transgenic Arabidopsis thaliana system was demonstrated by immunolocalization of the NS1 protein into the lumen of the endoplasmic reticulum in the leaf tissues. This system will be used to obtain the NS1 antigen with application purpose of dengue diagnostic tests.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Dengue virus NS1 recombinant protein for diagnosis

A dengue é uma doença que está presente em mais de 120 países, respondendo por 3,9 bilhões de pessoas em risco de infecção em todo o mundo. É uma doença viral transmitida por mosquito do gênero Aedes de grande potencial abrangente, podendo resultar em epidemias que ameaçam a saúde pública global. A transmissão contínua deles está intimamente ligada à emergência do quadro graves da febre hemorrágica e síndrome do choque que causam de altos índices letais da doença. O Dengue virus (DENV) pertence à família Flaviviridae, tem genoma de RNA fita simples, polaridade positiva e possui 4 sorotipos. A proteína não-estrutural 1 (NS1) é a primeira proteína viral presente na circulação sanguínea de paciente infectados, e é utilizada como biomarcador para diagnóstico da doença. Por ser altamente imunogênica, anticorpos circulantes IgM e IgG anti-NS1 são encontrados no soro de pacientes na fase aguda de infecções primárias e secundárias. Com a falta de uma vacina com proteção eficaz contra os 4 sorotipos do vírus, o diagnóstico sorológico é a alternativa mais segura para o tratamento correto da doença. O fator limitante na fabricação de kits diagnósticos de dengue é a produção em larga escala da proteína não-estrutural 1 (NS1) que é utilizada como antígeno na captura de anticorpos do soro de pacientes infectados. No presente trabalho expressamos a proteína NS1 em dois organismos heterólogos diferentes: Arabidopsis thaliana e Pichia pastoris, e avaliamos a atividade antigênica quanto à capacidade de detecção de anticorpos anti-dengue. Os resultados indicam que as proteínas recombinantes são candidatas promissoras para formulação de kit diagnóstico para dengue e testes de detecção rápida, devido ao alto rendimento, integridade antigênica e custo reduzido para produção em escala industrial.Dengue fever is present in more than 120 countries, accounting for 3.9 billion people at risk of infection worldwide. It is a mosquito-borne viral disease that uses Aedes genus as non-human vector and has great epidemics potential which may result in threaten to global public health. The continuous transmission is closely linked to the emergence of severe hemorrhagic fever and shock syndrome causing high rates of lethal disease. Dengue virus (DENV) belongs to the family Flaviviridae, has a positive single strand RNA genome, and 4 serotypes. Nonstructural protein 1 (NS1) is the first viral protein present in the bloodstream of infected patients, and is used as a biomarker for disease diagnostic. Because it is highly immunogenic, circulating anti-NS1 IgM and IgG antibodies are found in the serum of patients in the acute phase of primary and secondary infections. With the lack of effective vaccine against the 4 virus serotypes, the serological diagnosis is the safest alternative for the correct treatment of the disease. Limiting factor in diagnostic kits manufacture for dengue is the large-scale production of nonstructural protein 1 (NS1) which is used as antigen in the capture of serum antibodies from infected patients. In the present work was expressed NS1 protein in two different heterologous organisms: Arabidopsis thaliana and Pichia pastoris. Antigenic activity was assessed for the ability to detect anti-dengue antibodies. Results indicate that recombinant proteins are promising candidates for diagnostic kit and quick tests to dengue fever due to high yield, antigenic integrity and reduced cost for industrial scale production.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Zika Virus Envelope Protein Domain III Produced in <i>K. phaffii</i> Has the Potential for Diagnostic Applications

Zika virus (ZIKV) represents a global human health threat and it is related to severe diseases such as congenital Zika syndrome (CZS) and Guillain-Barré syndrome (GBS). There is no vaccine available nor specific antiviral treatment, so developing sensitive, specific, and low-cost diagnostic tests is necessary. Thus, the objective of this work was to produce the Zika virus envelope protein domain III (ZIKV-EDIII) in Komagataella phaffii KM71H and evaluate its potential for diagnostic applications. After the K. phaffii had been transformed with the pPICZαA-ZIKV-EDIII vector, an SDS-PAGE and Western Blot were performed to characterize the recombinant protein and an ELISA to evaluate the antigenic potential. The results show that ZIKV-EDIII was produced in the expected size, with a good purity grade and yield of 2.58 mg/L. The receiver operating characteristic (ROC) curve showed 90% sensitivity and 87.5% specificity for IgM, and 93.33% sensitivity and 82.76% specificity for IgG. The ZIKV-EDIII protein was efficiently produced in K. phaffi, and it has the potential for diagnostic applications

Genomic and proteomic characterization of vB_SauM-UFV_DC4, a novel Staphylococcus jumbo phage

Staphylococcus aureus is one of the most relevant mastitis pathogens in dairy cattle, and the acquisition of antimicrobial resistance genes presents a significant health issue in both veterinary and human fields. Among the different strategies to tackle S. aureus infection in livestock, bacteriophages have been thoroughly investigated in the last decades; however, few specimens of the so-called jumbo phages capable of infecting S. aureus have been described. Herein, we report the biological, genomic, and structural proteomic features of the jumbo phage vB_SauM-UFV_DC4 (DC4). DC4 exhibited a remarkable killing activity against S. aureus isolated from the veterinary environment and stability at alkaline conditions (pH 4 to 12). The complete genome of DC4 is 263,185 bp (GC content: 25%), encodes 263 predicted CDSs (80% without an assigned function), 1 tRNA (Phe-tRNA), multisubunit RNA polymerase, and an RNA-dependent DNA polymerase. Moreover, comparative analysis revealed that DC4 can be considered a new viral species belonging to a new genus DC4 and showed a similar set of lytic proteins and depolymerase activity with closely related jumbo phages. The characterization of a new S. aureus jumbo phage increases our understanding of the diversity of this group and provides insights into the biotechnological potential of these viruses.This work was financed by Fundo Regional para a Ciência e Tecnologia, project M1.1.a/008/Funcionamento/2020 (ITTAA), Portuguese Foundation for Science and Technology (FCT), under the scope of the project UID/CVT/00153/2019, strategic funding of UIDB/00153/2020 and UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. Sofia PM Silva acknowledges the financial support through grant SFRH/BD/139525/2018 from the Portuguese Foundation for Science and Technology (FCT).info:eu-repo/semantics/publishedVersio