Bioquímica nas escolas: uma estratégia educacional para o estudo de Ciência no Ensino Médio

A formação continuada de professores do Ensino Médio, no que tange as ciências da natureza, tem o desafio de incorporar, no conteúdo programático, as pesquisas e tecnologias inovadoras resultantes das novas descobertas no campo da Bioquímica e da Biologia Molecular. O presente trabalho relata a experiência de um projeto de extensão que objetivou a capacitação de professores de Biologia e de Química para o uso de novos métodos de ensino-aprendizagem visando despertar maior interesse dos estudantes pela Ciência. Resultado de parceria entre professores e estudantes universitários com educadores de escolas públicas, o presente projeto possibilitou a capacitação de 34 professores oriundos de 17 diferentes escolas para a utilização de um método de ensino-aprendizagem baseado no emprego de modelos didáticos lúdicos e de problematização da realidade quanto ao consumo de tecnologias e produtos biotecnológicos.Continued education of high school teachers regarding natural sciences has been challenging in terms of incorporating researches and innovative technologies from discoveries in the field of biochemistry and molecular biology in the curriculum. This paper describes the experience of an extension project aimed at training biology and chemistry teachers for the use of new teaching and learning methods to arouse a greater interest of students for science. Resulting from a partnership between professors and undergraduate students with public school teachers, this project enabled the training of 34 teachers from 17 different schools. The teaching-learning method establishes on the use of didactic models as well as the problem-based knowledge of reality regarding the consumption of technology and biotechnology products.La continua formación de los profesores de educación secundaria con respecto a las ciencias naturales, tiene el desafío de ir incorporando en el contenido programático los resultados de las investigaciones y las tecnologías innovadoras descubiertos en el campo de la bioquímica y la biología molecular. El presente trabajo relata la experiencia de un proyecto de extensión cuyo objetivo es la capacitación de profesores de biología y de química para el uso de nuevos métodos de enseñanza y aprendizaje, buscando despertar un mayor interés de los estudiantes por la ciencia. Como resultado de la asociación entre profesores y estudiantes con docentes de colegios públicos, el presente proyecto posibilitó la capacitación de 34 profesores de 17 diferentes colegios en el uso del método de enseñanza-aprendizaje basado en el empleo de modelos ludo-didácticos y de problematización de la realidad en cuanto al consumo de tecnologías y productos biotecnológicos

Chikungunya E2 protein produced in E. coli and HEK293-T cells-comparison of their performances in ELISA

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes a disease characterized by the acute onset of fever accompanied by arthralgia and intense joint pain. Clinical similarities and cocirculation of this and other arboviruses in many tropical countries highlight the necessity for efficient and accessible diagnostic tools. CHIKV envelope proteins are highly conserved among alphaviruses and, particularly, the envelope 2 glycoprotein (CHIKV-E2) appears to be immunodominant and has a considerable serodiagnosis potential. Here, we investigate how glycosylation of CHIKV-E2 affects antigen/antibody interaction and how this affects the performance of CHIKV-E2-based Indirect ELISA tests. We compare two CHIKV-E2 recombinant antigens produced in different expression systems: prokaryotic-versus eukaryotic-made recombinant proteins. CHIKV-E2 antigens are expressed either in E. coli BL21(DE3)-a prokaryotic system unable to produce post-translational modifications-or in HEK-293T mammalian cells-a eukaryotic system able to add post-translational modifications, including glycosylation sites. Both prokaryotic and eukaryotic recombinant CHIKV-E2 react strongly to anti-CHIKV IgG antibodies, showing accuracy levels that are higher than 90%. However, the glycan-added viral antigen presents better sensitivity and specificity (85 and 98%) than the non-glycosylated antigen (81 and 71%, respectively) in anti-CHIKV IgM ELISA assays

Detecting anti–SARS-CoV-2 antibodies in urine samples: a noninvasive and sensitive way to assay COVID-19 immune conversion

Serum-based ELISA (enzyme-linked immunosorbent assay) has been widely used to detect anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. However, to date, no study has investigated patient urine as a biological sample to detect SARS-CoV-2 virus-specific antibodies. An in-house urine-based ELISA was developed using recombinant SARS-CoV-2 nucleocapsid protein. The presence of SARS-CoV-2 antibodies in urine was established, with 94% sensitivity and 100% specificity for the detection of anti–SARS-CoV-2 antibodies with the urine-based ELISA and 88% sensitivity and 100% specificity with a paired serum-based ELISA. The urine-based ELISA that detects anti–SARS-CoV-2 antibodies is a noninvasive method with potential application as a facile COVID-19 immunodiagnostic platform, which can be used to report the extent of exposure at the population level and/or to assess the risk of infection at the individual level.</p

Potential binding sites and probes clustering predicted in the multi-epitope proteins.

Three potential binding sites were predicted by FTSite: #1 (salmon), #2 (lime green), and #3 (blue), all represented by a tridimensional mesh in (A) ZIKV-1 (pale cyan), (B) ZIKV-2 and (C) ZIKV-3. Probes clustering predicted by FTMap for (D) ZIKV-1, (E) ZIKV-2, and (F) ZIKV-3, corroborate FTSite predictions. All images were generated using PyMOL software (v0.99c).</p

Production of ZIKV-1, ZIKV-2, and ZIKV-3 multi-epitope proteins.

(A) SDS-PAGE of cell extracts after induction with 1mM IPTG showing the expression of proteins ZIKV-1 (14 kDa), ZIKV-2 (18 kDa), and ZIKV-3 (20 kDa). (B) Multi-epitope proteins after purification by nickel affinity chromatography using HisTrap columns. (C) Western blot of multi-epitope proteins with pooled sera from ZIKV+ patients. M: molecular weight standards; CN: non-transformed cells; NI: transformed and non-induced cells; I: cells induced with 1M IPTG; ZIKV-E: ZIKV envelope protein (recombinant, positive control).</p

Alignment of African and Asian strains with the epitopes that compose the multiepitope proteins.

(A) Alignment of Epitope 1 with ten African strains and thirty-five Asian strains. (B) Alignment of Epitope 2 with ten African strains and thirty-five Asian strains. (C) Alignment of Epitope 3 with ten African strains and thirty-five Asian strains. (D) Alignment of Epitope 4 with ten African strains and thirty-five Asian strains. (E) Alignment of Epitope 5 with ten African strains and thirty-five Asian strains. (F) Alignment of Epitope 6 with ten African strains and thirty-five Asian strains. (G) Alignment of Epitope 7 with ten African strains and thirty-five Asian strains. (PDF)</p

Indirect anti-ZIKV IgG in-house ELISA.

Multi-epitope proteins were evaluated as antigens in an ELISA, using ZIKV+, DENV+, CHIKV+, RF+ patient samples, and negative samples. (A) ZIKV-1 multi-epitope protein as antigen aside from its corresponding ROC curve. (B) ZIKV-2 multi-epitope protein as antigen and its ROC curve. (C) ZIKV-3 multi-epitope protein as antigen and its ROC curve. Indexes consider the ratio between absorbance readings and cut-off values. Samples dispersions are represented, and significant differences are highlighted with (*** for p>0.001 or **** for p>0.0001). ZIKV-/DENV-: negative samples for Zika and dengue; ZIKV+: positive samples for Zika; DENV+: positive samples for dengue; CHIKV+: positive samples for CHIKV; RF+: samples with increased levels of rheumatoid factor.</p

SASA calculations for multi-epitope proteins.

Zika virus (ZIKV), an arbovirus from the Flaviviridae family, is the causative agent of Zika fever, a mild and frequent oligosymptomatic disease in humans. Nonetheless, on rare occasions, ZIKV infection can be associated with Guillain-Barré Syndrome (GBS), and severe congenital complications, such as microcephaly. The oligosymptomatic disease, however, presents symptoms that are quite similar to those observed in infections caused by other frequent co-circulating arboviruses, including dengue virus (DENV). Moreover, the antigenic similarity between ZIKV and DENV, and even with other members of the Flaviviridae family, complicates serological testing due to the high cross-reactivity of antibodies. Here, we designed, produced in a prokaryotic expression system, and purified three multiepitope proteins (ZIKV-1, ZIKV-2, and ZIKV-3) for differential diagnosis of Zika. The proteins were evaluated as antigens in ELISA tests for the detection of anti-ZIKV IgG using ZIKV- and DENV-positive human sera. The recombinant proteins were able to bind and detect anti-ZIKV antibodies without cross-reactivity with DENV-positive sera and showed no reactivity with Chikungunya virus (CHIKV)- positive sera. ZIKV-1, ZIKV-2, and ZIKV-3 proteins presented 81.6%, 95%, and 66% sensitivity and 97%, 96%, and 84% specificity, respectively. Our results demonstrate the potential of the designed and expressed antigens in the development of specific diagnostic tests for the detection of IgG antibodies against ZIKV, especially in regions with the circulation of multiple arboviruses.</div

Models of the multi-epitope proteins.

Modeled structures obtained with AlphaFold are represented by cartoons with transparent surfaces; (A) ZIKV-1 (pale cyan). (B) ZIKV-2 (aquamarine) presents a sequence addition (gray), as well as (C) ZIKV-3 (light teal), which also presents two sequences intercalation (ruby), evidencing structures’ differences in size. (D) 3D alignment corroborates their structural differences, also showing a similar spatial occupation among the three proteins and their folding variations. Images were generated with PyMOL (v0.99c).</p