Clonagem e expressão heteróloga de uma proteína homóloga a alérgeno presente no veneno de aranha-marrom (Loxosceles intermedia)

Orientador : Prof. Dr. Silvio Sanches VeigaCo-orientadora : Profª. Drª. Olga Meiri ChaimDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 26/02/2014Inclui referênciasResumo: O gênero Loxosceles inclui várias espécies popularmente conhecidas como aranhas-marrons. O envenenamento pode causar, entre outras manifestações clínicas, reações inflamatórias que incluem aumento da permeabilidade vascular, infiltrado leucocitário, rash cutâneo e exantema pustuloso. Relatos de acidentes com animais peçonhentos comumente causam reações alérgicas assim como choques anafiláticos. As moléculas descritas no veneno de Loxosceles intermedia incluem fosfolipases-D, hialuronidases, metaloproteases e toxinas não-enzimáticas. Devido ao pequeno volume produzido e a baixa representatividade dessas toxinas no veneno, técnicas de biologia molecular permitem a identificação e expressão de várias dessas toxinas. A partir de uma biblioteca de cDNA de Loxosceles intermedia foi identificado um transcrito que apresentou identidade com alérgenos presentes em uma espécie de vespa e formiga. A sequência nucleotídica dessa proteína homóloga a alérgeno possui 415aa. Por meio de predição da sequência aminoacídica foram encontrados possíveis sítios de Nglicosilação. Segundo o banco de dados ProtParam esta molécula pertence a uma família de proteínas ricas em cisteínas. O objetivo deste trabalho foi a clonagem, expressão heteróloga e avaliação da sua atividade biológica de uma possível toxina alergênica presente no veneno de Loxosceles intermedia, com o intuito de compreender o papel desta no loxoscelismo. De acordo com a nomenclatura da área (WHO/IUIS): a toxina recombinante foi denominada Lox i 1. Foram utilizados modelos heterólogos de expressão procariótico Escherichia coli BL21 (DE3) pLysS e modelo eucariótico Pichia pastoris X-33. A ligação da sequência nucleotídica do alérgeno Lox i 1 foi obtida em vetor de expressão pET-20b e pPICZ_C, assim como, a transformação destas construções em E. coli e P. pastoris, respectivamente. Por meio de testes de indução nas concentrações de 0,1 a 1,0mM de indutor a 37 _ C não houve expressão da proteína recombinante. Entretanto, foi possível obter a toxina recombinante em cultura de leveduras Pichia pastoris, cuja a padronização foi feita em 1% de metanol a 30º C por 5 dias. A purificação foi feita por cromatografia de afinidade utilizando resina Ni-NTA em pH 9,0. Análises espectroscópicas em Dicroísmo Circular foram realizadas para a formação de estruturas secundárias da proteína recombinante, o que indicou o dobramento e solubilidade da toxina recombinante em meio aquoso. Os dados da deconvolução indicaram 16% de ±-hélice e 19% de ±-folha. Os dados obtidos permitiram investir na prospecção da atividade biológica de Lox i 1 por meio de um ensaio de Permeabilidade Vascular em camundongos. O qual foi capaz de aumentar a permeabilidade vascular em camundongos. Portanto, foi pela primeira vez na literatura relatada a expressão e a avaliação da atividade biológica de uma proteína homóloga a alérgeno presente no veneno de L. intermedia. Soma-se a isso o fato de que é crescente a utilização de alérgenos recombinantes em imunoterapia e testes diagnósticos para hipersensibilidade. Futuramente estudos estruturais e imunológicos de Lox i 1 irão contribuir para um maior conhecimento das bases moleculares de alergenicidade no loxoscelismo. Palavras-chave: Reações alérgicas, Loxoscelismo, Alérgeno, Rash cutâneo.Abstract: The Loxosceles genus includes several species known as brown spiders. The envenomation can trigger among other clinical manifestations, inflammatory conditions including increased vascular permeability, cutaneous rash, edema and exanthema pustulosis. Reports of accidents involving venomous animals commonly cause allergic reactions like rash, exanthem pustulosis and anaphylactic shock. The molecules described in the Loxosceles intermedia venom include phospholipase-D, hyaluronidase, metalloproteases and non-enzymatic toxins. Due to the small volume produced and the low representation of these toxins in the venom, molecular biology techniques allow the identification and expression of several of these toxins. By a cDNA library of Loxosceles intermedia a sequence show identity with allergens present in a species of wasp and ant was identified. The nucleotide sequence of this protein homologous to allergen has 415 aminoacids. A putative N-glycosylation site was observed in this sequence. According to the ProtParam database this molecule belongs to a family of cysteine-rich proteins. The objective of this work was the cloning, heterologous expression and evaluation of the biological activity of a putative allergen toxin present in the venom of Loxosceles intermedia, in order to further the knowledge of the loxoscelism. According to the nomenclature of the allergens (WHO / IUIS) the recombinant toxin L. intermedia venom was named Lox i 1. The subcloning of Lox 1 was obtained in vector pET-20b and pPICZ_C to use heterologous expression with a prokaryotic model, E. coli BL21(DE3)pLysS, and eukaryotic model Pichia pastoris X-33. The test of the recombinant protein expression was performed from 0.1 to 1.0 mM IPTG concentrations at 37 _ C. However, it was only possible to obtain the recombinant toxin in cultured yeast Pichia pastoris, whose standardization parameters was 1 % methanol at 30°C for 5 days. Purification was performed by affinity chromatography using Ni-NTA resin with pH 9.0. Analysis by Dichroism Circular were performed for analyses of Lox i 1 secondary structures and solubility The data indicated the deconvolution of 16 % ± - helix of ± 19% sheet. The biological activity of Lox i 1 was tested by vascular permeability assay in mice. The Lox i 1 was able to increase vascular permeability in this model. For the first time is report in the literature the heterologous expression and evaluation of biological activity of a protein homologous to allergens present in the venom of L. intermedia. In addition, future structural and immunological studies of Lox i 1 will contribute to a better understanding of the molecular basis of allergenicity in loxoscelism. Key words: Allergic reactions, Loxoscelism, Allergen, Cutaneous rash

A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. the venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (similar to 45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in similar to 45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a spreading factor. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao Araucaria-PR (FAP)Secretaria de Estado de Ciencia, Tecnologia e Ensino Superior do Parana (SETI)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Fed Parana, Dept Cell Biol, BR-80060000 Curitiba, Parana, BrazilUniv Fed Parana, Clin Hosp, Dept Clin Pathol, BR-80060000 Curitiba, Parana, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Ponta Grossa, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilWeb of Scienc

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Clonagem e expressão heteróloga de uma proteína homóloga a alérgeno presente no veneno de aranha-marrom (Loxosceles intermedia)

Orientador : Prof. Dr. Silvio Sanches VeigaCo-orientadora : Profª. Drª. Olga Meiri ChaimDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 26/02/2014Inclui referênciasResumo: O gênero Loxosceles inclui várias espécies popularmente conhecidas como aranhas-marrons. O envenenamento pode causar, entre outras manifestações clínicas, reações inflamatórias que incluem aumento da permeabilidade vascular, infiltrado leucocitário, rash cutâneo e exantema pustuloso. Relatos de acidentes com animais peçonhentos comumente causam reações alérgicas assim como choques anafiláticos. As moléculas descritas no veneno de Loxosceles intermedia incluem fosfolipases-D, hialuronidases, metaloproteases e toxinas não-enzimáticas. Devido ao pequeno volume produzido e a baixa representatividade dessas toxinas no veneno, técnicas de biologia molecular permitem a identificação e expressão de várias dessas toxinas. A partir de uma biblioteca de cDNA de Loxosceles intermedia foi identificado um transcrito que apresentou identidade com alérgenos presentes em uma espécie de vespa e formiga. A sequência nucleotídica dessa proteína homóloga a alérgeno possui 415aa. Por meio de predição da sequência aminoacídica foram encontrados possíveis sítios de Nglicosilação. Segundo o banco de dados ProtParam esta molécula pertence a uma família de proteínas ricas em cisteínas. O objetivo deste trabalho foi a clonagem, expressão heteróloga e avaliação da sua atividade biológica de uma possível toxina alergênica presente no veneno de Loxosceles intermedia, com o intuito de compreender o papel desta no loxoscelismo. De acordo com a nomenclatura da área (WHO/IUIS): a toxina recombinante foi denominada Lox i 1. Foram utilizados modelos heterólogos de expressão procariótico Escherichia coli BL21 (DE3) pLysS e modelo eucariótico Pichia pastoris X-33. A ligação da sequência nucleotídica do alérgeno Lox i 1 foi obtida em vetor de expressão pET-20b e pPICZ_C, assim como, a transformação destas construções em E. coli e P. pastoris, respectivamente. Por meio de testes de indução nas concentrações de 0,1 a 1,0mM de indutor a 37 _ C não houve expressão da proteína recombinante. Entretanto, foi possível obter a toxina recombinante em cultura de leveduras Pichia pastoris, cuja a padronização foi feita em 1% de metanol a 30º C por 5 dias. A purificação foi feita por cromatografia de afinidade utilizando resina Ni-NTA em pH 9,0. Análises espectroscópicas em Dicroísmo Circular foram realizadas para a formação de estruturas secundárias da proteína recombinante, o que indicou o dobramento e solubilidade da toxina recombinante em meio aquoso. Os dados da deconvolução indicaram 16% de ±-hélice e 19% de ±-folha. Os dados obtidos permitiram investir na prospecção da atividade biológica de Lox i 1 por meio de um ensaio de Permeabilidade Vascular em camundongos. O qual foi capaz de aumentar a permeabilidade vascular em camundongos. Portanto, foi pela primeira vez na literatura relatada a expressão e a avaliação da atividade biológica de uma proteína homóloga a alérgeno presente no veneno de L. intermedia. Soma-se a isso o fato de que é crescente a utilização de alérgenos recombinantes em imunoterapia e testes diagnósticos para hipersensibilidade. Futuramente estudos estruturais e imunológicos de Lox i 1 irão contribuir para um maior conhecimento das bases moleculares de alergenicidade no loxoscelismo. Palavras-chave: Reações alérgicas, Loxoscelismo, Alérgeno, Rash cutâneo.Abstract: The Loxosceles genus includes several species known as brown spiders. The envenomation can trigger among other clinical manifestations, inflammatory conditions including increased vascular permeability, cutaneous rash, edema and exanthema pustulosis. Reports of accidents involving venomous animals commonly cause allergic reactions like rash, exanthem pustulosis and anaphylactic shock. The molecules described in the Loxosceles intermedia venom include phospholipase-D, hyaluronidase, metalloproteases and non-enzymatic toxins. Due to the small volume produced and the low representation of these toxins in the venom, molecular biology techniques allow the identification and expression of several of these toxins. By a cDNA library of Loxosceles intermedia a sequence show identity with allergens present in a species of wasp and ant was identified. The nucleotide sequence of this protein homologous to allergen has 415 aminoacids. A putative N-glycosylation site was observed in this sequence. According to the ProtParam database this molecule belongs to a family of cysteine-rich proteins. The objective of this work was the cloning, heterologous expression and evaluation of the biological activity of a putative allergen toxin present in the venom of Loxosceles intermedia, in order to further the knowledge of the loxoscelism. According to the nomenclature of the allergens (WHO / IUIS) the recombinant toxin L. intermedia venom was named Lox i 1. The subcloning of Lox 1 was obtained in vector pET-20b and pPICZ_C to use heterologous expression with a prokaryotic model, E. coli BL21(DE3)pLysS, and eukaryotic model Pichia pastoris X-33. The test of the recombinant protein expression was performed from 0.1 to 1.0 mM IPTG concentrations at 37 _ C. However, it was only possible to obtain the recombinant toxin in cultured yeast Pichia pastoris, whose standardization parameters was 1 % methanol at 30°C for 5 days. Purification was performed by affinity chromatography using Ni-NTA resin with pH 9.0. Analysis by Dichroism Circular were performed for analyses of Lox i 1 secondary structures and solubility The data indicated the deconvolution of 16 % ± - helix of ± 19% sheet. The biological activity of Lox i 1 was tested by vascular permeability assay in mice. The Lox i 1 was able to increase vascular permeability in this model. For the first time is report in the literature the heterologous expression and evaluation of biological activity of a protein homologous to allergens present in the venom of L. intermedia. In addition, future structural and immunological studies of Lox i 1 will contribute to a better understanding of the molecular basis of allergenicity in loxoscelism. Key words: Allergic reactions, Loxoscelism, Allergen, Cutaneous rash

Aggressive Unicystic Ameloblastoma Affecting The Posterior Mandible: Late Diagnosis During Orthodontic Treatment

Maxillofacial images must be examined to find pathologies not identified during clinical examination. Unicystic ameloblastoma (UA) extending to the mandibular body and ramus was neglected on initial panoramic radiographic examination. After orthodontic therapy, a huge lesion was observed clinically and through imaging exams. After the conservative surgery, no recurrence was observed during five years of follow-up. This case emphasized the need for careful evaluation of patient images focusing on the oral diagnosis before any dental treatment planning, including orthodontic therapy.43211511

Comparison of accuracy between dental and skeletal age in the estimation of chronological age of Down syndrome individuals

This study aimed to evaluate the accuracy of dental age (DA) and skeletal age (SA) methods in order to estimate chronological age (CA) in individuals with Down syndrome (DS), contributing to the Forensic Dentistry and making the identification of these individuals age possible. For this, 278 images of individuals were selected and divided in 2 groups: 216 non-DS patients and 62 with DS. At first, DA was evaluated by Nolla method, on panoramic radiographs, followed by SA, evaluated by Greulich and Pyle method. The linear correlation coefficient of Pearson was used for the analysis of concordance between the methods. Paired t-test with confidence interval was used to evaluate the accuracy and Bland and Altman method was applied to estimate limits of concordance. Complementary to this first analysis, descriptive statistics and ANOVA test were applied for comparison among chronological age (CA), dental age (DA) and skeletal age (SA), with a significance level of 95% (p >= 0.05), ordering to observe the differences among them. DA, estimated by Nolla, is underestimated in both, DS and non-DS individuals, and it is more notable in DS individuals. SA estimated by Greulich and Pyle method is overestimated, except for non-DS males. The range of variance is greater in SA and DS than DA and non-DS individuals, respectively. A greater accordance was found for DA x CA if compared to SA x CA, indicating that DA, estimated by Nolla method, is more accurate than SA, evaluated by Greulich and Pyle method, for estimating CA of both, DS and non-DS individuals. However, neither method seems to be precise and more caution is required for age estimation in DS individuals266578.e1578.e10sem informaçã

Expression of NADPH oxidase in human pancreatic islets

Aims: NADPH oxidase (NOX) is a known source of superoxide anions in phagocytic and non-phagocytic cells. In this study, the presence of this enzyme in human pancreatic islets and the importance of NADPH oxidase in human beta-cell function were investigated. Main methods and key findings: In isolated human pancreatic islets, the expression of NADPH oxidase components was evidenced by real-time PCR (p22(PHOX), p47(PHOX) and p67(PHOX)), Western blotting (p47(PHOX) and p67(PHOX)) and immunohistochemistry (p47(PHOX), p67(PHOX) and gp91(PHOX)). Immunohistochemistry experiments showed co-localization of p47(PHOX), p67(PHOX) and gp91(PHOX) (isoform 2 of NADPH oxidase-NOX2) with insulin secreting cells. Inhibition of NADPH oxidase activity impaired glucose metabolism and glucose-stimulated insulin secretion. Significance: These findings demonstrate the presence of the main intrinsic components of NADPH oxidase comprising the NOX2 isoform in human pancreatic islets, whose activity also contributes to human beta-cell function. (C) 2012 Elsevier Inc. All rights reserved.FAPESPFAPESPCNPqCNPqCAPESCAPESFINEP BrazilFINEP Brazi

Immunological cross-reactivity of whole venom and Dietrich's Hyaluronidase.

<p>Purified recombinant glycosidase (2.0 µg; lanes 1, 2, 5 and 6) or whole venom (40 µg; lanes 3 and 4) were separated by 12.5% SDS-PAGE under reducing conditions, transferred onto nitrocellulose membranes and exposed to antibodies against Dietrich's Hyaluronidase (lanes 2 and 4) or whole venom toxins (lane 6). Lanes 1, 3 and 5 indicate reactions in the presence of pre-immune sera (control for antibody specificity). Molecular mass marker positions are shown in the left of figure.</p