No abstract

Diabetes Mellitus is one of the most common causes of neuropathies, which can be caused by molecular imbalances that impair metabolic pathways. Studies in rats showed the importance of sirtuins (SIRT), deacetylases that use NAD + as a cofactor, which have a widespread function in metabolism, and their relation when food deprived or calorie restricted. Additionally, diabetic neuropathy presents different structural biomarkers that cause morphological alterations in fibers that can be partially treated. SIRT1 is the principal sirtuin, which acts on hypothalamus, liver, kidney, among other organs, up regulating or down regulating the expression of some genes or enzymes crucial in the process of glucose absorption.

The excessive demand for publications results in high plagiarism and duplicate numbers by scientists who take over existing texts into new publications. In addition to serious ethical problems, this practice hinders the generation of original material. In order to reduce the problem, softwares such as eTBLAST are being used to detect plagiarism and repeated papers. Despite the persistence of fraudsters, these tools have helped to reduce these problems; however, the ideal solution would be the basic ethical establishment principles. Therefore, plagiarism has always been a foible that could lead to fraudulent and dishonorable development of science.

Introduction: Antibodies are glycoproteins, which consist of two types of domains light and heavy chains, each of which is composed of variable and constant domains. Rituximab is a monoclonal antibody used in the treatment of lymphomas and its mechanism of action is based on the interaction of CD20 membrane protein, expressed on B lymphocytes, guiding them to depletion. Single-chain fragments variable (scFv) are composed by the variable domains of antibody heavy and light chains, linked by a flexible polypeptide, making them easier to manipulate on protein engineering techniques, such as rational design, where amino acids are chosen by in silico structural and energetic analysis and changed by site-directed mutagenesis in order to set a better or new biological function. Objective: Perform site-directed mutagenesis, based on structural and energetic features, on the variable region of Rituximab, in order to increase its affinity to the CD20 epitope. Methodology: Rituximab scFv mutants were obtained in vitro by site-directed mutagenesis and their sequences confirmed. The bacterial expression of wide-type (wt) scFv and its mutants was standardized to get soluble proteins. The strategy used accomplishes bacterial strain SHUFFLE and pETSUMO vector on overnight expression at 19 o C. These fragments were confirmed by western blot (WB) nitrocellulose membrane, using 5% defatted milk on overnight blockage (4 o C), 1 μg of each protein and 1:5000 ratio to protein L-HRP. CD on "far-UV" was accessed to obtain the structural secondary profile of scFv's (wt and mutants) using 250 μg/mL of proteins in phosphate buffer pH 7.0. A peptide-based ELISA was achieved sensitizing the plate with 1 μg/well CD20-epitope peptide overnight at 4 o C, using 150 μg/mL of proteins and 1:1500 ratio of protein L-HRP at 1 hour each at room temperature. Results: Five Rituximab's scFv variants were constructed and expressed at soluble grade. WB membrane revealed the presence of scFv fragments. CD analysis showed similar profiles among the mutant and wt fragment, characterized by β-sheet secondary structure. Analysis of ELISA confirmed the capacity of binding to immobilized biotinylated peptide of the CD20 epitope from all the fragments compared (wt and mutants scFv). Conclusion: The results showed an efficient method of bacterial expression of soluble antibody fragments, which were confirmed by WB. The secondary structure was confirmed by CD and besides the mutations; they remained capable of binding to the epitope, based on ELISA assay. This work avenues quantification affinity based on microscale thermophoresis of antibody fragments, followed by construction of the scFv-Fc format, in order to study effector functions of those fragments.

ResumoO objetivo do estudo foi realizar revisão de literatura sobre proteases endógenas no processo carioso patológico. A busca foi realizada nas bases de dados Pubmed e Bireme com os descritores "Dental Caries", "Cathepsin" e "Metalloprotease". Foram incluídos artigos publicados entre 2007 e 2017, publicados em inglês e português. Encontraram-se 74 artigos, dos quais 15 se enquadravam ao tema ambiente ácido criado pelos ácidos bacterianos, e ativadas. O colágeno intacto não pode ser degradado por proteases bacterianas, mas de lesões cariosas, dentes e saliva de indivíduos com cárie, podem estar relacionados à maior atividade proteolítica em dentina cariosa. Também foi sugerido um papel "cicatrizador", reforçado por estudos que indicam que MMPs podem ser secretadas em resposta à agressão cariosa, estimulando a migração de fatores de crescimento para formação de tecido duro. O atual conhecimento revela que não é possível determinar exatamente o papel das proteases endógenas no processo carioso, mas participam da formação de dentina terciária e da degradação do colágeno desmineralizado, sendo necessários estudos que mimetizem a progressão lenta e natural de lesões cariosas.Palavras-chave: Dental Caries. Cathepsin. Metalloprotease.