RESUMO

In this work, we have designed and generated a Fe(III)-binding protein with thiol oxidoreductase activity. The consensus iron-binding motif EExxED from the frataxin protein family was grafted on a model peptide and on the surface of thioredoxin (TRX) from E. coli. We investigated metal interactions with a family of peptides containing the motif EExxED or altered versions obtained by removing negatively charged residues: EExxEx, xExxED, and xExxEx. The interaction of the metal ion with the peptides was studied by circular dichroism, and our results indicated that the motif EExxED retained its functional properties and also that this motif is able to bind Ga(III) and Al(III). The interaction of the grafted TRX with iron(III) was investigated by NMR, showing that the motif was functional in the context of the protein structure, and also the binding of two equivalents of Fe(III) per TRX molecule was stable in a non-chelating neutral buffer. Protein conformation, stability, and enzymatic activity were studied by applying experimental and computational approaches. Interestingly, the thiol oxidoreductase activity was modulated by interaction with Ga(III), a Fe(III) mimetic ion. Furthermore, the design of functional proteins with both functions, oxidoreductase activity and metal-ion binding ability, should consider the reorganisation of the electrostatic network. Similarly, studying the crosstalk and electrostatic balance among different metal-binding sites may be critical.

Assuntos

RESUMO

Fifty ~20-amino acid (aa)-long peptides were selected from functionally relevant SARS-CoV-2 S, M, and E proteins for trial B-21 and another 53 common ones, plus some new ones derived from the virus' main genetic variants for complementary trial C-21. Peptide selection was based on tremendous SARS-CoV-2 genetic variability for analysing them concerning vast human immunogenetic polymorphism for developing the first supramutational, Colombian SARS-protection (SM-COLSARSPROT), peptide mixture. Specific physicochemical rules were followed, i.e., aa predilection for polyproline type II left-handed (PPIIL) formation, replacing ß-branched, aromatic aa, short-chain backbone H-bond-forming residues, π-π interactions (n→π* and π-CH), aa interaction with π systems, and molecular fragments able to interact with them, disrupting PPIIL propensity formation. All these modified structures had PPIIL formation propensity to enable target peptide interaction with human leukocyte antigen-DRß1* (HLA-DRß1*) molecules to mediate antigen presentation and induce an appropriate immune response. Such modified peptides were designed for human use; however, they induced high antibody titres against S, M, and E parental mutant peptides and neutralising antibodies when suitably modified and chemically synthesised for immunising 61 major histocompatibility complex class II (MHCII) DNA genotyped Aotus monkeys (matched with their corresponding HLA-DRß1* molecules), predicted to cover 77.5% to 83.1% of the world's population. Such chemically synthesised peptide mixture represents an extremely pure, stable, reliable, and cheap vaccine for COVID-19 pandemic control, providing a new approach for a logical, rational, and soundly established methodology for other vaccine development.

Assuntos

RESUMO

Congenital long QT syndrome (LQTS) is a cardiac channelopathy characterized by a prolongation of the QT interval and T-wave abnormalities, caused, in most cases, by mutations in KCNQ1, KCNH2, and SCN5A. Although the predominant pattern of LQTS inheritance is autosomal dominant, compound heterozygous mutations in genes encoding potassium channels have been reported, often with early disease onset and more severe phenotypes. Since the molecular mechanisms underlying severe phenotypes in carriers of compound heterozygous mutations are unknown, it is possible that these compound mutations lead to synergistic or additive alterations to channel structure and function. In this study, all-atom molecular dynamic simulations of KCNQ1 and hERG channels were carried out, including wild-type and channels with compound mutations found in two patients with severe LQTS phenotypes and limited family history of the disease. Because channels can likely incorporate different subunit combinations from different alleles, there are multiple possible configurations of ion channels in LQTS patients. This analysis allowed us to establish the structural impact of different configurations of mutant channels in the activated/open state. Our data suggest that channels with these mutations show moderate changes in folding energy (in most cases of stabilizing character) and changes in channel mobility and volume, differentiating them from each other and from WT. This would indicate possible alterations in K+ ion flow. Hetero-tetrameric mutant channels showed intermediate structural and volume alterations vis-à-vis homo-tetrameric channels. These findings support the hypothesis that hetero-tetrameric channels in patients with compound heterozygous mutations do not necessarily lead to synergistic structural alterations.

Assuntos

RESUMO

Resumen Introducción y objetivo: el tratamiento endodóntico se realiza para tratar la enfermedad pulpoperiapical y puede tener un porcentaje de éxito de más del 90% en condiciones ideales para su realización. Dentro de los factores que condicionan la práctica clínica, se encuentran la anatomía interna del diente, las habilidades del operador, el conocimiento de la técnica, los instrumentos empleados y el tiempo operatorio. El éxito lo determina la supervivencia y la ausencia de signos clínicos y radiográficos en el seguimiento. Determinar los factores asociados al resultado del tratamiento de endodoncia, realizado por estudiantes de pregrado en odontología de una universidad colombiana. Materiales y métodos: estudio de cohorte retrospectivo con una cohorte expuesta y una no expuesta a la periodontitis apical. Se evaluaron clínica y radiográficamente todos los pacientes cuyo motivo de consulta fue endodoncia durante los años 2013-2014. Resultados: la media de edad de los pacientes fue 51,23 (DE = 14,23) con un mayor porcentaje de participación de mujeres (71,1%). En el diagnóstico inicial se encontró un 26% de dientes con Periodontitis apical. Se encontró asociación entre una mediana mayor de consultas y no presentar Periodontitis apical al final del tratamiento. En los análisis bivariados y multivariados se encontró asociación de la presencia de accidentes y endodoncia suboturada con la presencia de Periodontitis apical al final del tratamiento. Conclusión: seguir protocolos y guía de atención que permitan evitar los accidentes y conservar la adecuada longitud en la conformación y obturación pueden incrementar la frecuencia de éxito en los tratamientos.

Abstract Introduction and objetive: an endodontic treatment is performed to treat pulp-periapical disorders and may have a success rate of over 90% under ideal conditions. Among the factors that need to be considered, knowledge of the internal anatomy of the root, operator skills, selected technique, instruments involved and surgical time are the most important to determine the success of such treatment. Success implies the survival of the tooth in the oral cavity as well as the absence of clinical and radiographic signs and symptoms. The purpose of this work was to determine the factors associated to the success of an endodontic treatment performed by undergraduate dental students in a Colombian university. Materials and methods: a retrospective study with cohorts exposed and unexposed to apical periodontitis was carried out. Patients who consulted for endodontic treatment in 2013-2014 were invited to participate and were assessed both clinically and radiographically. Results: mean age was 51.23 (SD 14.23) years and higher percentage of female participation (71.1%) was observed. At initial diagnosis, 26% of teeth were diagnosed with apical periodontitis. An association between a high consultation mean and absence of apical periodontitis at the end of treatment was found. Bivariate and multivariate analyses showed an association between the presence of procedure accidents and under-filled root canal obturation with the presence of apical periodontitis at the end of treatment. Conclusion: success rate of endodontic treatments may be increased by carefully following protocols and attention guidelines to reduce the possibility of accidents and to keep an adequate length of the canal filling.

Resumo Introdução e objetivo: um tratamento endodôntico é realizado para tratar distúrbios pulpar-periapicais e pode ter uma taxa de sucesso superior a 90% em condições ideais. Entre os fatores que precisam ser considerados, o conhecimento da anatomia interna da raiz, as habilidades do operador, a técnica selecionada, os instrumentos envolvidos e o tempo cirúrgico são os mais importantes para determinar o sucesso desse tratamento. O sucesso implica a sobrevivência do dente na cavidade oral, bem como a ausência de sinais e sintomas clínicos e radiográficos. O objetivo deste trabalho foi determinar os fatores associados ao sucesso de um tratamento endodôntico realizado por estudantes de graduação em odontologia de uma universidade colombiana. Materiais e métodos: estudo retrospectivo com coortes expostas e não expostas à periodontite apical. Os pacientes que consultaram para tratamento endodôntico em 2013-2014 foram convidados a participar e foram avaliados clinicamente e radiograficamente. Resultados: a média de idade foi de 51,23 (DP 14,23) anos e maior percentual de participação feminina (71,1%). No diagnóstico inicial, 26% dos dentes foram diagnosticados com periodontite apical. Foi encontrada associação entre alta média de consulta e ausência de periodontite apical ao final do tratamento. As análises bivariadas e multivariadas mostraram associação entre a presença de acidentes do procedimento e a obturação do canal radicular com preenchimento insuficiente com a presença de periodontite apical ao final do tratamento. Conclusão: a taxa de sucesso dos tratamentos endodônticos pode ser aumentada seguindo-se cuidadosamente os protocolos e diretrizes de atenção para reduzir a possibilidade de acidentes e manter um comprimento adequado do preenchimento do canal.

RESUMO

Experimentally estimating peptide-major histocompatibility complex (pMHC) binding affinity has been quite challenging due to the many receptors and the many potential ligands implicated in it. We have thus proposed a straightforward computational methodology considering the different mechanisms involved in pMHC binding to facilitate studying such receptor-ligand interactions. We have developed a pipeline using semi-empirical quantum mechanical methods for calculating pMHC class I and II molecules' binding energy (BE). This pipeline can systematize the methodology for calculating pMHC system BE, enabling the rational design of T-cell epitopes to be used as pharmaceuticals and vaccines.

Assuntos

RESUMO

BACKGROUND: Ichthyosis is a heterogeneous group of diseases caused by genetic disorders related to skin formation. They are characterized by generalized dry skin, scaling, hyperkeratosis and frequently associated with erythroderma. Among its different types, harlequin ichthyosis (HI) stands out due to its severity. HI is caused by mutations in the ABCA12 gene, which encodes essential proteins in epidermal lipid transport, and it helps maintain the homeostasis of the stratum corneum of the epidermis. However, due to the wide spectrum of genetic alterations that can cause ichthyosis, holistic medical care, and genetic studies are required to improve the diagnosis and outcomes of these diseases. CASE PRESENTATION: Here, we presented the case of a 19 years old male patient who was a premature infant and exhibited clinical features consistent with HI, including bright yellow hyperkeratotic plates with erythematous fissures that covered his entire body like a collodion baby. Currently, he exhibited erythroderma, photosensitivity, ectropion, auricular pavilion alterations, and musculoskeletal disorders, such as equinovarus feet, fingers, hands, and hypoplastic feet with contractures in flexion and marked difficulty in fine motor skills. In addition, he presented dyschromatopsia, Achilles reflex hyporeflexia, slight speech, dental alteration and deficient cognitive performance. After the genetic sequencing, variants were found in ABCA12 and HRNR which are related to several skin diseases, including ichthyosis. CONCLUSIONS: Although in clinical practice, ichthyosis is a common entity, a severe type of ichthyosis is presented, highlighting the importance of appropriate genetic diagnosis, given the broad spectrum of genetic alterations with similar phenotypic and clinical characteristics. These pathologies must be known to guarantee initial support measures to prevent complications and offer multidisciplinary management to those patients.

Assuntos

RESUMO

Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol and disulfide behaves as a monomer under near physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this process, the FF → LU and LU → FF transitions were studied using conventional molecular dynamics simulations and an enhanced conformational sampling technique for different oxidation and protonation states of the active site cysteine residues CP and CR. Our results suggest that the FF → LU transition has a higher associated energy barrier than the refolding LU → FF process in agreement with the relatively low experimental turnover number of EcTPx. Furthermore, in silico designed single-point mutants of αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionarily optimized to fully speed-up the conformational transition of wild-type EcTPx.

Assuntos

Assuntos

RESUMO

Estimating peptide-major histocompatibility complex (pMHC) binding using structural computational methods has an impact on understanding overall immune function triggering adaptive immune responses in MHC class II molecules. We developed a strategy for optimizing pMHC structure interacting with water molecules and for calculating the binding energy of receptor + ligand systems, such as HLA-DR1 + HA, HLA-DR1 + CLIP, HLA-DR2 + MBP, and HLA-DR3 + CLIP, as well as a monosubstitution panel. Taking pMHC's structural properties, we assumed that ΔH ≫ -TΔS would generate a linear model for estimating relative free energy change, using three semiempirical quantum methods (PM6, PM7, and FMO-SCC-DFTB3) along with the implicit solvent models, and considering proteins in neutral and charged states. Likewise, we confirmed our approach's effectiveness in calculating binding energies having high correlation with experimental data and low root-mean-square error (<2 kcal/mol). All in all, our pipeline differentiates weak from strong peptide binders as a reliable method for studying pMHC interactions.

Assuntos