Structure and Behavior of Human α-Thrombin upon Ligand Recognition: Thermodynamic and Molecular Dynamics Studies

Thrombin is a serine proteinase that plays a fundamental role in coagulation. In this study, we address the effects of ligand site recognition by alpha-thrombin on conformation and energetics in solution. Active site occupation induces large changes in secondary structure content in thrombin as shown by circular dichroism. Thrombin-D-Phe-Pro-Arg-chloromethyl ketone (PPACK) exhibits enhanced equilibrium and kinetic stability compared to free thrombin, whose difference is rooted in the unfolding step. Small-angle X-ray scattering (SAXS) measurements in solution reveal an overall similarity in the molecular envelope of thrombin and thrombin-PPACK, which differs from the crystal structure of thrombin. Molecular dynamics simulations performed with thrombin lead to different conformations than the one observed in the crystal structure. These data shed light on the diversity of thrombin conformers not previously observed in crystal structures with distinguished catalytic and conformational behaviors, which might have direct implications on novel strategies to design direct thrombin inhibitors

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento CientIfico e Tecnologico (CNPq)Coordenacao para Aperfeicoamento de Pessoal de Ensino Superior (CAPES)Fundo de Defesa da Citricultura (FUNDECITRUS

<i>In vitro</i> antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19

BackgroundCurrent approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity.MethodsSARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19.ResultsDaclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans.ConclusionsDaclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy

Citotoxicidade de tubos de PVC esterilizados em óxido de etileno após exposição à radiação gama

Materiais esterilizados em raios gama, ao serem re-esterilizados em óxido de etileno (EO), formam substâncias tóxicas? Esta questão norteou o objetivo deste estudo, que foi investigar o potencial efeito citotóxico do PVC esterilizado em radiação gama e re-esterilizado em EO pelo método da difusão em ágar em culturas celulares. Nove tubos de PVC foram submetidos à esterilização em radiação gama e re-esterilizados em EO. Os tubos foram divididos em um total de 81 unidades de análise, que foram testadas de forma a representar as superfícies internas, externas e massa de cada tubo. Concluiu-se que os materiais de PVC esterilizados em Radiação Gama e consecutivamente re-esterilizados em EO não são citotóxicos

Vivências maternas na realidade de ter um filho autista: uma compreensão pela enfermagem Experiencias maternas en la realidad de tener un hijo autista: una comprensión para la enfermería Maternal experiences in the reality of having an autistic son: an understanding for nursing

Este estudo objetivou descrever a vivência de ser-mãe de criança autista. Utilizou-se abordagem qualitativa e referencial fenomenológico com conceitos de Martin Heidegger. Foram entrevistadas 14 mães de crianças autistas, com perguntas abertas, gravadas e transcritas na íntegra. O cenário foi a AMA-PI e dados produzidos em maio de 2006. A análise revela que as mães vivenciam a facticidade de ter um filho autista permeada por sentimentos de nulidade, fé e solidão. As mães também deixam de viver o seu cotidiano para viverem o cotidiano do filho. Ao assumirem sua condição existencial -estar-no-mundo e ser mãe de uma criança autista, passam a se compreenderem como ser capaz de lutar pelo bem-estar do filho, sem queixas, demonstrando abnegação, paciência e preocupação.<br>Este estudio que objetivó describir la experiencia de ser-madre del un niño autísta. El abordaje cualitativo fue utilizado según el referencial fenomenológico de Martín Heidegger. Catorce madres de niños autístas fueron entrevistadas con preguntas abiertas, grabadas y transcritas en la integra. El escenario de la investigación fue el AMA-PI y los datos producidos en mayo de 2006. El análisis reveló las madres viven profundamente la facticidad de tener un hijo autísta la cual es permeada por sensaciones negativas, fe y la soledad. Las madres tanbién dejam vivir su vida diaria para vivir la vida diaria de sus hijos. Al asumir la condición existencial - estar -en el mundo y ser madre de un niño autísta, ellas comenzan a comprender cómo son capazes de luchar por lo bien-estar de sus hojos, sin quejas, demuenstrando abnegación, paciencia y preocupación.<br>This study has as objective to describe the experience of being mother of an autistic child. A qualitative approach was accomplished according to the phenomenological concepts of Martin Heidegger. Fourteen mothers of autistic children had been interviewed, with semi-structured questions, tape-recorded and transcribed integrally. The local of data collection was the AMA-PI with data produced in May, 2006. The analysis revealed that mothers who live the facticity of having an autistic child is permeated by negative feelings, faith and solitudeness. Mothers also leave their dalily life to live their children's daily live. When assuming their existential condition - to be -in the world and to be mother of a autistic child, they begin to understand how to be capable to fight for their children's well-being, without complaints, demonstrating self-denial, patience and concern