A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi

Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. in these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D H-1 nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INBEQMeDIUniv Fed Uberlandia, Inst Ciencias Biomed, BR-38400 Uberlandia, MG, BrazilUniv São Paulo, Inst Fis Sao Carlos, Sao Carlos, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Vila Mariana, SP, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Biol Geral, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Vila Mariana, SP, BrazilFAPESP: 2010/51867-6FAPESP: 2012/21153-7FAPEMIG: APQ-00621-11FAPEMIG: APQ-00305-12CAPES: 23038.005295/2011-40Web of Scienc

Biophysical studies on protein P21 of Trypanosoma cruzi

Segundo a Organização Mundial da Saúde, em 2018, entre 6 e 7 milhões de pessoas estavam infectadas pelo parasita Trypanosoma cruzi, agente etiológico da Doença de Chagas. Dentre as piores consequências para os chagásicos estão os problemas cardíacos causados pela infecção, os quais afetam cerca de 25% dos pacientes na fase crônica da doença. Na busca por biomoléculas envolvidas no processo de invasão celular pelo parasita, uma proteína produzida por ele, denominada P21, foi encontrada. Ensaios biológicos mostraram que a proteína P21 de T. cruzi interage com o receptor de quimiocinas CXCR4 e desencadeia diversos processos bioquímicos, como: indução de fagocitose por macrófagos, indução da polimerização de actina e inibição de angiogênese. Os dados sugerem que a P21 também pode desempenhar um papel na cardiomiopatia induzida pelo parasita. Visando contribuir com a caracterização biofísica e estrutural da proteína P21, foi desenvolvido um protocolo para sua obtenção a partir de corpos de inclusão e reenovelamento, o qual forneceu quantidades necessárias da proteína para os experimentos. Espectros de ressonância Magnética Nuclear (RMN) permitiram avaliar de forma precisa a qualidade estrutural da amostra reenovelada frente a proteína P21 produzida de forma solúvel. Resultados de RMN mostraram também que a proteína possui cinco hélices-α em sua estrutura secundária, uma grande porção desestruturada, além de duas populações conformacionais em equilíbrio. Ainda, corroborando com os dados biológicos encontrados na literatura, um ensaio de interação in vitro indicou que a P21 interage com o receptor CXCR4, sendo a porção N-terminal deste receptor a região envolvida em tal processo.According to the World Health Organization, in 2018, between 6 and 7 million people were still infected by the parasite Trypanosoma cruzi, the causative agent of Chagas disease. One of the worst aggravations for chagasics is heart disorder caused by the infection, which affects about 25% of patients in the chronic phase of the disease. Searching for biomolecules involved in the parasitic invasion of human cells, a T. cruzi protein called P21 was found. It has a high probability of being secreted or anchored to the membrane. Biological assays have shown that T. cruzi P21 interacts with the CXCR4 chemokine receptor and mediates several biochemical processes, such as: induction of phagocytosis by macrophages, induction of actin polymerization and inhibition of angiogenesis. The data suggests that P21 may also play a role in cardiomyopathy induced by parasite. In order to contribute to the biophysical and structural characterization of P21, a refolding protocol was developed to obtain P21 from inclusion bodies. The protocol provided the necessary amounts of protein required for the experiments. Nuclear Magnetic Resonance (NMR) spectra allowed to accurately evaluate the structural quality of the refolded sample in comparison to the produced soluble P21 protein. NMR spectra also showed five α-helices in the P21 secondary structure, a large unstructured portion and two ensembles of structures in equilibrium. Furthermore, corroborating with the biological data found in the literature, an in vitro interaction assay indicated that P21 interacts with CXCR4 via the N-terminal portion of the receptor

Laser in Pediatric Dentistry

Laser technology has different applications in dentistry, and, particularly, in Paediatric Dentistry. Depending on laser wavelengths and the physical properties of the tissue which is to be targeted; it is possible obtain different results in three main dental fields: Diagnosis, Prevention and Operative Therapy. Conventional treatments can sometimes be replaced by laser treatments and better results may be achieved. Laser treatments offer new treatment opportunities in the dental field that were unknown in the past. This chapter aims to outline the clinical protocols and possible applications of different laser systems in Paediatric Dentistry