Infecção por SARS-CoV-2 em paciente com agamaglobulinemia ligada ao X tratado com altas doses de imunoglobulina endovenosa e plasma de convalescente / SARS-CoV-2 infection in a patient with X-linked agammaglobulinemia treated with high doses of intravenous immunoglobulin and convalescent plasma

A pandemia pelo vírus SARS-CoV-2 atingiu adultos, crianças e penalizou indivíduos idosos e com comorbidades como diabetes, doença cardíaca, hipertensão e obesidade. A maioria dos infectados são assintomáticos ou têm sintomas leves, entretanto 15% podem apresentar pneumonia e 5% síndrome respiratória aguda grave. Apresentamos um caso de agamaglobulinemia ligada ao X (XLA) em paciente masculino de 27 anos que se infectou com SARS-CoV-2. Os pacientes com XLA não possuem linfócitos B e não produzem anticorpos devido a uma mutação no gene Bruton tirosino-quinase (BTK), responsável pela maturação dos linfócitos B. Ele infectou-se e foi internado em hospital de Ivoti/RS. A evolução da pneumonia foi rápida, necessitando transferência para o Hospital de Clínicas de Porto Alegre (HCPA) no 10° dia de evolução. Iniciou com infusão de imunoglobulinas, tendo utilizado o total de 400 gramas devido ao intenso catabolismo da IgG, mantendo-se sua concentração entre 700-900 mg/dL. Necessitou de ventilação mecânica, oxigenação por membrana extracorpórea (ECMO) e hemodiálise. Foi administrado plasma de convalescente (PC), 300 mL, por duas vezes, com melhora clínico-radiológica e retirada da ventilação mecânica. Piorou e repetiu outras 4 infusões de PC (total de 1717 mL), negativando o vírus na orofaringe (RT-PCR). Em 3 ocasiões teve sepse, debelada rapidamente. Apresentou anemia, com necessidade de transfusão frequente. Identificou-se linfopenia de CD3, CD4, CD8, NK e ausência de linfócitos B. A linfopenia foi revertida com a recuperação clínica e a alta hospitalar aconteceu no 70° dia de internação.

The SARS-CoV-2 pandemic has affected adults and children and penalized older people and those with comorbidities such as diabetes, heart disease, hypertension and obesity. Most of those infected are asymptomatic or have mild symptoms, but 15% may have pneumonia and 5% acute respiratory distress syndrome. We report a case of X-linked agammaglobulinemia (XLA) in a 27-yearold man who was infected with SARS-CoV-2. XLA patients do not have B lymphocytes and do not produce antibodies because of mutations in the Bruton tyrosine-kinase gene, responsible for the maturation of B cells. This patient was infected and then admitted to a hospital in Ivoti, southern Brazil. Pneumonia progressed rapidly, requiring transfer to the Hospital de Clínicas de Porto Alegre on the 10th day. Intravenous immunoglobulin infusions were initiated, using a total of 400 grams because of an intense catabolism of IgG, and the concentration was kept around 700- 900 mg/dL. Mechanical ventilation, extracorporeal membrane oxygenation and hemodialysis were necessary. Convalescent plasma (CP) was administered (2x300 mL) and then followed by clinical and radiological improvement and interruption of mechanical ventilation. Then he got sicker and had to return to invasive support and received 4 extra CP infusions (total of 1717 mL), until a negative reverse-transcriptase polymerase chain reaction (RT-PCR) for SARS-CoV-2 was obtained. On 3 occasions he had sepsis, promptly managed. He had anemia, requiring frequent transfusion, and lymphopenia (CD3, CD4, CD8, NK), with absence of B lymphocytes. Lymphopenia was reverted during recovery, and he was discharged from the hospital on the 70th day.

Nontuberculous mycobacteria in patients with suspected tuberculosis and the genetic diversity of Mycobacterium avium in the extreme south of Brazil / Micobactérias não tuberculosas em pacientes com suspeita de tuberculose e a diversidade genética de Mycobacterium avium no extremo sul do Brasil

ABSTRACT Objective: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. Methods: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. Results: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. Conclusions: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.

RESUMO Objetivo: As micobactérias não tuberculosas (MNT) são um grupo heterogêneo de bactérias amplamente distribuídas na natureza e relacionadas com infecções oportunistas em seres humanos. Os objetivos deste estudo foram identificar MNT em pacientes com suspeita de tuberculose e culturas positivas e avaliar a diversidade genética de cepas identificadas como Mycobacterium avium. Métodos: Foram estudadas amostras pulmonares e extrapulmonares provenientes de 1.248 pacientes. As amostras que apresentaram resultado positivo em cultura e negativo para o complexo M. tuberculosis na identificação molecular foram avaliadas por meio da detecção dos genes hsp65 e rpoB e de sequenciamento de fragmentos conservados desses genes. Todas as cepas identificadas como M. avium foram genotipadas pelo método mycobacterial interspersed repetitive unit-variable-number tandem-repeat com oito loci. Resultados: Das 332 micobactérias isoladas, 25 (7,5%) eram MNT. Dessas 25, 18 (72%) eram M. avium, 5 (20%) eram M. abscessus, 1 (4%) era M. gastri e 1 (4%) era M. kansasii. As 18 cepas de M. avium apresentaram alta diversidade, e apenas duas eram geneticamente relacionadas. Conclusões: Esses resultados mostram a necessidade de considerar a investigação de MNT em pacientes com suspeita de tuberculose ativa e culturas positivas e de avaliar a diversidade genética de cepas de M. avium.

New molecular target for the phylogenetic identification of Leptospira species directly from clinical samples: an alternative gene to 16S rRNA

Abstract INTRODUCTION: Phylogenetic analysis of the 16S ribosomal gene initial region is used to identify Leptospira isolates at the species level from clinical samples. Unfortunately, this method cannot differentiate between some intermediates and saprophytic species. METHODS: We used comparative genomic analysis between 35 Leptospira species to find new molecular targets for Leptospira species identification. RESULTS: We proposed the use of the rpoC gene, encoding the DNA-directed RNA polymerase β-subunit, for identifying 35 Leptospira species. CONCLUSIONS: The rpoC gene can be a molecular target to identify the main species of the Leptospira genus directly from clinical samples.

Alternativas terapéuticas para el manejo de la COVID-19 / Therapeutic alternatives for the management of COVID-19

Introducción: La COVID-19 es una pandemia causada por el Coronavirus 2 del Síndrome Respiratorio Agudo Severo (SARS-CoV-2); no existe hasta el momento tratamiento específico completamente eficaz para esta enfermedad, pero el mundo está trabajando incesantemente para buscar una cura. Objetivo: Describir las alternativas terapéuticas de la COVID-19, según los mecanismos fisiopatológicos descritos hasta el momento. Material y Método: Se realizó una revisión bibliográfica a partir de un total de 31 referencias bibliográficas. Se revisaron artículos, en idioma inglés y español, en revistas nacionales e internacionales en bases de datos como Pubmed/Medline, y Elsevier. Se analizó la calidad, fiabilidad y validez de los artículos seleccionados para realizar una adecuada revisión. Desarrollo: La aparición de la COVID-19 ha causado revuelo internacional por la necesidad de encontrar tratamientos efectivos. Debido a que es una enfermedad frecuentemente autolimitada, se vuelve difícil probar si una estrategia terapéutica es eficaz o la enfermedad ha seguido su curso. Las fases del ciclo de vida viral del SARS-COV proporcionan los objetivos potenciales para la terapia con medicamentos, como son: los inhibidores de la fusión de membrana de la envoltura viral, inhibidores de la proteasa similar a la 3-quimotripsina, inhibidores de la ARN polimerasa dependiente de ARN viral, inhibidores de la entrada y endocitosis y otros medicamentos con alguna función inmunomuduladora. Conclusiones: La pandemia actual representa un desafío para la comunidad médica internacional. Aunque no hay tratamiento específico recomendado, se utilizan diversos medicamentos con cierta efectividad como la hidroxicloroquina, azitromicina, kaletra y el remdesivir con sus respectivas combinaciones(AU)

Introduction: COVID-19 is a pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), so far there is no fully effective specific treatment for this disease, but worldwide effort is incessant in the search for a cure. Objective: To describe the therapeutic alternatives for COVID-19 according to the pathophysiological mechanisms described up until now. Material and Method: A bibliographic review was made from a total of 31 bibliographic references. Articles, in English and Spanish, from national and international journals were searched over on-line databases such as Pubmed/Medline and Elsevier. The quality, reliability and validity of the selected articles were analyzed to carry out an adequate review. Development: The appearance of COVID-19 has caused an international stir due to the need to find effective treatments. Because it is a frequently self-limited disease, it becomes difficult to prove whether a therapeutic strategy is effective or the disease has run its course. The SARS-CoV viral life cycle phases provide potential targets for drug therapy, such as: viral envelope membrane fusion inhibitors, 3-chymotrypsin-like protease inhibitors, virus RNA-dependent RNA polymerase, endocytosis and entry inhibitors, and other medications with some immunomodulatory function. Conclusions: The current pandemic represents a challenge for the international medical community. Although there is no specific recommended treatment, various drugs are used with some effectiveness such as hydroxychloroquine, azithromycin, kaletra and remdesivir with their respective combinations(AU)

Detection of multidrug-resistant Mycobacterium tuberculosis strains isolated in Brazil using a multimarker genetic assay for katG and rpoB genes

Abstract Multidrug-resistant tuberculosis (MDRTB) is a serious world health problem that limits public actions to control tuberculosis, because the most used anti-tuberculosis first-line drugs fail to stop mycobacterium spread. Consequently, a quick detection through molecular diagnosis is essential to reduce morbidity and medical costs. Despite the availability of several molecular-based commercial-kits to diagnose multidrug-resistant tuberculosis, their diagnostic value might diverge worldwide since Mycobacterium tuberculosis genetic variability differs according to geographic location. Here, we studied the predictive value of four common mycobacterial mutations in strains isolated from endemic areas of Brazil. Mutations were found at the frequency of 41.9% for katG, 25.6% for inhA, and 69.8% for rpoB genes in multidrug-resistant strains. Multimarker analysis revealed that combination of only two mutations (“katG/S315T + rpoB/S531L”) was a better surrogate of multidrug-resistant tuberculosis than single-marker analysis (86% sensitivity vs. 62.8%). Prediction of multidrug-resistant tuberculosis was not improved by adding a third or fourth mutation in the model. Therefore, rather than using diagnostic kits detecting several mutations, we propose a simple dual-marker panel to detect multidrug-resistant tuberculosis, with 86% sensitivity and 100% specificity. In conclusion, this approach (previous genetic study + analysis of only prevalent markers) would considerably decrease the processing costs while retaining diagnostic accuracy.

Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data

BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.

Characterization of Francisella species isolated from the cooling water of an air conditioning system

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Occurrence of Lutzomyia longipalpis (Phlebotominae) and canine visceral leishmaniasis in a rural area of Ilha Solteira, SP, Brazil / Ocorrência de Lutzomyia longipalpis (Phlebotominae) e leishmaniose visceral canina em uma área rural de Ilha Solteira, SP, Brasil

This study aimed to investigate the occurrence of Lutzomyia longipalpis and also the canine visceral leishmaniasis (CVL) in a rural area of Ilha Solteira, state of São Paulo. Blood samples were collected from 32 dogs from different rural properties (small farms) and were analyzed by ELISA and the indirect immunofluorescence antibody test (IFAT) in order to diagnose CVL. From these serological tests, 31.25% of the dogs were positive for CVL and these were distributed in 66.7% (8/12) of the rural properties, which were positive for L. longipalpis. CDC (Center for Disease Control and Prevention) light traps were installed in 12 properties (one per property) and insects were caught on three consecutive days per month for one year. L. longipalpis was present on 100% of the rural properties visited, at least once during the twelve-month interval, totaling 64 males and 25 females. The insects were more numerous after the peak of the rain, but the association between prevalence of peridomestic vectors and the climatic data (precipitation, relative air humidity and temperature) and the occurrences of CVL among dogs on each rural property were not statistical significant (p <0.05). However, the occurrence of CVL cases in dogs and the presence of L. longipalpis indicate that more attention is necessairy for the control of this disease in the rural area studied.

O objetivo desse trabalho foi o estudo da prevalência de Lutzomyia longipalpis e da leishmaniose visceral canina (LVC) em uma área rural do município de Ilha Solteira do estado de São Paulo. Amostras de sangue foram coletadas de 32 cães provenientes de pequenas propriedades rurais e analisadas por meio dos métodos sorológicos ELISA (imunoensaio enzimático indireto) e RIFI (reação de imunofluorescência indireta) para o diagnóstico da LVC. Pelos exames sorológicos, dos 32 cães avaliados, 31,25% foram diagnosticados positivos para LVC, os quais estavam diostribuídos em 66,67% (8/12) das propriedades positivas para Lutzomyia longipalpis. Armadilhas luminosas do tipo CDC (Center for Disease Control and Prevention) foram instaladas em 12 propriedades, sendo uma por propriedade, e as coletas dos insetos foram realizadas três dias consecutivos a cada mês, durante um ano. O inseto L. longipalpis foi encontrado em 100% das propriedades visitadas, pelo menos uma vez no ano, totalizando 65 machos e 25 fêmeas. A maior quantidade de insetos foi observada principalmente após a ocorrência dos maiores picos de precipitação pluvial, mas a associação entre a prevalência dos vetores peridomiciliares e os dados climáticos (precipitação, umidade relativa do ar e temperatura) assim como a ocorrência da CVL em cães em cada propriedade não foi estatisticamente significante (p<0.05). No entanto, alerta-se que pela presença dos casos de LVC nos cães amostrados e também de L. longipalpis, maior atenção deve ser dada durante as investigações epidemiológicas para o controle dessa doença nessa área rural estudada.

Clinical Alarms in intensive care: implications of alarm fatigue for the safety of patients / Alarmes clínicos em terapia intensiva: implicações da fadiga de alarmes para a segurança do paciente / Alarmas clínicas en terapia intensiva: implicaciones de la fatiga de alarmas para la seguridad del paciente

OBJECTIVES: to identify the number of electro-medical pieces of equipment in a coronary care unit, characterize their types, and analyze implications for the safety of patients from the perspective of alarm fatigue. METHOD: this quantitative, observational, descriptive, non-participatory study was conducted in a coronary care unit of a cardiology hospital with 170 beds. RESULTS: a total of 426 alarms were recorded in 40 hours of observation: 227 were triggered by multi-parametric monitors and 199 were triggered by other equipment (infusion pumps, dialysis pumps, mechanical ventilators, and intra-aortic balloons); that is an average of 10.6 alarms per hour. CONCLUSION: the results reinforce the importance of properly configuring physiological variables, the volume and parameters of alarms of multi-parametric monitors within the routine of intensive care units. The alarms of equipment intended to protect patients have increased noise within the unit, the level of distraction and interruptions in the workflow, leading to a false sense of security. .

OBJETIVOS: identificar o número de alarmes dos equipamentos eletromédicos numa unidade coronariana, caracterizar o tipo e analisar as implicações para a segurança do paciente na perspectiva da fadiga de alarmes. MÉTODO: trata-se de estudo quantitativo observacional descritivo, não participante, desenvolvido numa unidade coronariana de um hospital de cardiologia, com capacidade para 170 leitos. RESULTADOS: registrou-se o total de 426 sinais de alarmes, sendo 227 disparados por monitores multiparamétricos e 199 alarmes disparados por outros equipamentos (bombas infusoras, hemodiálise, ventiladores mecânicos e balão intra-aórtico), nas 40h, numa média total de 10,6 alarmes/hora. CONCLUSÃO: os resultados encontrados reforçam a importância da configuração de variáveis fisiológicas, do volume e dos parâmetros de alarmes dos monitores multiparamétricos à rotina das unidades de terapia intensiva. Os alarmes dos equipamentos destinados a proteger os pacientes têm conduzido ao aumento do ruído na unidade, à fadiga de alarmes, a distrações e interrupções no fluxo de trabalho e à falsa sensação de segurança. .

OBJETIVOS: identificar el número de alarmas de los equipamientos electromédicos en una unidad coronariana, caracterizar el tipo y analizar las implicaciones para la seguridad del paciente en la perspectiva de fatiga de alarmas. MÉTODO: se trata de un estudio cuantitativo, observacional, descriptivo, no participante, desarrollado en una unidad coronariana de un hospital de cardiología, con capacidad de 170 camas. RESULTADOS: se registró un total de 426 señales de alarmas, siendo 227 disparadas por monitores multiparamétricos y 199 disparadas por otros equipamientos (bombas de infusión, hemodiálisis, ventiladores mecánicos y balón intraaórtico), durante 40h, con un promedio total de 10,6 alarmas/hora. CONCLUSIÓN: los resultados encontrados refuerzan la importancia de la configuración de las variables fisiológicas, del volumen y de los parámetros de alarma de los monitores multiparamétricos, a la rutina de las unidades de terapia intensiva. Las alarmas de los equipamientos destinados a proteger a los pacientes, han llevado al aumento del ruido en la unidad, a la fatiga de alarmas, a las distracciones e interrupciones en el flujo de trabajo y a una falsa sensación de seguridad. .

Molecular characterization of the first leptospires isolated from goats in Brazil

Two Leptospira sp. isolates were obtained by the first time from goats in Brazil and characterized by sequencing rrs, rpoB and secY genes, PFGE and typing with monoclonal antibodies. Both isolates are identical and belong to Leptospira santarosai. Analysis of the rrs and the rpoB genes sequences revealed 100% identity between the goat isolates and the Bananal reference strain. When secY sequences of the two isolates were compared to each other, it was observed that they had identical sequences. However, when compared to that of the Bananal reference strain, there were 15 mismatches along the 549 bp secY sequence. In conclusion, molecular methods are increasingly useful for the characterization of leptospires and allowed to identify those isolates of caprine origin as closely related but not identical to serovar Bananal, and constitute a new type named Carioca.

Determinants of emergency contraception non-use among women in unplanned or ambivalent pregnancies / Determinantes del no uso de la anticoncepción de emergencia entre mujeres con embarazo no planeado u ambivalente / Determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente

Objective To analyze the determinants of emergency contraception non-use among women in unplanned and ambivalent pregnancies. Method Cross-sectional study with a probabilistic sample of 366 pregnant women from 12 primary health care units in the city of São Paulo, Brazil. A multinomial logistic regression was performed, comparing three groups: women who used emergency contraception to prevent ongoing pregnancies (reference); women who made no use of emergency contraception, but used other contraceptive methods; and women who made no use of any contraceptive methods at all. Results Cohabitation with a partner was the common determinant of emergency contraception non-use. No pregnancy risk awareness, ambivalent pregnancies and no previous use of emergency contraception also contributed to emergency contraception non-use. Conclusion Apart from what is pointed out in the literature, knowledge of emergency contraception and the fertile period were not associated to its use. .

Objetivo Analizar los determinantes del no uso de la anticoncepción de emergencia entre las mujeres con embarazo no planeado o ambivalente. Método Estudio transversal en una muestra probabilística de 366 mujeres embarazadas de 12 Unidades Básicas de Salud de São Paulo. Mediante regresión logística multinomial, se comparó tres grupos de mujeres: aquellas que usaron la anticoncepción de emergencia para prevenir el embarazo en curso (referencia), aquellas que usaron algún método anticonceptivo, pero no la anticoncepción de emergência; y aquellas que no usaron ningún método. Resultados Los hallazgos mostraron que vivir com la pareja fue el determinante común del no uso de la anticoncepción de emergencia. No tener conciencia del riesgo de embarazo, estar en un embarazo ambivalente y nunca tener utilizado la anticoncepción de emergencia también fueron associados con su no uso para prevenir el embarazo en curso. Conclusión Contrariamente a lo que reporta la literatura, el conocimiento de la anticoncepción de emergencia y el período fértil no mostró asociación con el no uso. .

Objetivo Analisar os determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente. Método Estudo transversal com amostra probabilística de 366 gestantes de 12 Unidades Básicas de Saúde da cidade de São Paulo. Por meio de regressão logística multinomial, compararam-se três grupos de mulheres: as que usaram anticoncepção de emergência para prevenir a gravidez em curso (referência); as que usaram algum método contraceptivo, mas não anticoncepção de emergência; e as que não usaram nenhum método. Resultados Os achados mostraram que morar com o parceiro foi o determinante comum do não uso da anticoncepção de emergência. Não ter consciência do risco de engravidar, estar em uma gravidez ambivalente e nunca ter usado anticoncepção de emergência também foram associados ao seu não uso para prevenir a gravidez em curso. Conclusão Diferentemente do que relata a literatura, o conhecimento sobre anticoncepção de emergência e sobre o período fértil não mostrou qualquer associação ao não uso. .

Expression of rna polymerase IV and V in oryza sativa

RNA polymerase IV and V are principal players in the RdDM pathway, where their current study has shown interaction of several factors that control DNA silencing of intergenic regions and siRNA production. DNA silencing is an important process during cell differentiation, nuclear structure and viral control. However, RNA pol IV and V are yet to be study in model monocot systems like Oryza sativa that can provide further information on genetic silence mechanism in plats. We show the expression pattern of these polymerases in tissues extracts of Oryza sativa. Detectable amounts of these polymerases are found in specific adult plant tissues and particularly expressed during somatic embryogenesis but not during early stages of normal embryo development. The use of synthetic auxin leads to an induction of both RNA pol IV and V in scutellum tissue where nuclear localization may be required for genome reorganization and gene silencing.

An MLSA-based online scheme for the rapid identification of Stenotrophomonas isolates

An online scheme to assign Stenotrophomonas isolates to genomic groups was developed using the multilocus sequence analysis (MLSA), which is based on the DNA sequencing of selected fragments of the housekeeping genes ATP synthase alpha subunit (atpA), the recombination repair protein (recA), the RNA polymerase alpha subunit (rpoA) and the excision repair beta subunit (uvrB). This MLSA-based scheme was validated using eight of the 10 Stenotrophomonas species that have been previously described. The environmental and nosocomial Stenotrophomonas strains were characterised using MLSA, 16S rRNA sequencing and DNA-DNA hybridisation (DDH) analyses. Strains of the same species were found to have greater than 95 percent concatenated sequence similarity and specific strains formed cohesive readily recognisable phylogenetic groups. Therefore, MLSA appeared to be an effective alternative methodology to amplified fragment length polymorphism fingerprint and DDH techniques. Strains of Stenotrophomonas can be readily assigned through the open database resource that was developed in the current study (www.steno.lncc.br/).

Explorando o transcriptoma de Trypanossoma vivax / Exploring the transcriptome of Trypanosoma vivax

Trypanosoma vivax foi descrito por Zieman, (1905) do sub-gênero Dutonella, família Trypanosomatidae, é um protozoário patogênico causador de tripanossomose na África e na América Latina. Infecta bovinos, ovinos e outros mamíferos silvestres, não infecta o homem e equinos. Os principais sinais clínicos são: anemia, perda de apetite, efeitos na fertilidade, no sistema nervoso central, aborto e até á morte. Transmitido pela mosca tsétsé e tabanídeos, causa grandes perdas econômicas na pecuária. No Brasil, surtos tem sido relatados em Belém- PA, Calçoene-AP, no Pantanal (MS e MT) e Catolé da Rocha-PB. Apesar do impacto econômico relevante e importância médico-veterinária, há pouco conhecimento sobre sua biologia e genoma. Um fator limitante é a dificuldade de cultivo in vivo e in vitro. Com o intuito de obter informações sobre quais genes estão sendo transcritos e realizar uma análise comparativa com a cepa africana Y486, foram sequenciados mais de 4208 clones de cDNAs de um isolado do Pantanal-Brasil VTA-01 de T. vivax e 768 clones genômicos da cepa africana Y486. As sequências foram armazenadas e analisadas no sistema STINGRAY (http://stingray.biowebdb.org/). Após remoção das sequências de baixa qualidade e agrupamento foram analisadas 2557 ESTs e 81 GSSs, com tamanho médio de 497 pb e 500 pb e conteúdo G+C de 43% e 55%, respectivamente. Após pesquisa de similaridade usando diferentes programas como BLAST, RPS-Blast, InterProScan e HMMER, 77% (1746/2638) apresentaram hit. Os principais domínios encontrados foram NADH, RhoA, Rac1 GTPases e Drf_FH1. Usando o programa RepeatMasker foram encontrados as repetições (CA)n, (GA)n e (TA)n e os retrotransposons SINE e LINE. Um total de 217 ESTs espécie-específicas para T. vivax foram identificadas e apresentaram preferencialmente A ou T na terceira posição do codon nas regiões codificantes (CDSs). Destas 88% (191/217) possuem índice RCBS 0,5, com alto nível de expressão e 12% (26/217) RCBS menor 0,5 com baixo nível de expressão. Os genes altamente expressos foram associados com vias de duplicação do DNA, transcrição, tradução, organização celular e formação de actina no citoesqueleto. A localização de domínios G-quadruplex foram encontrados tanto dentro quanto fora da CDS e próximas as extremidades 5’ e 3’ das CDSs e dos ESTs. Nas análises comparativas, a proteína extensina está presente no isolado VTA-01 e cepa Y486 de T. vivax. Aproximadamente 83% das ESTs validaram sequências do projeto genoma de T. vivax. As informações geradas neste projeto irão contribuir para um melhor conhecimento sobre a biologia deste parasita.

The mechanism of cancer involves short interfering (RNA) (siRNA)

The recent discoveries of the RNA-mediated interference system in cells could explain all of the known features of human carcinogenesis. A novel idea, proposed here, is that the cell has the ability to recognize a mutated protein and/or mRNA. Secondly, the cell can generate its own short interfering RNA (siRNA) using an RNA polymerase to destroy mutated mRNA, even when only a single base pair in the gene has mutated. The anti-sense strand of the short RNA molecule (called sicRNA), targets the mutated mRNA of an oncogene or a tumour suppressor. During cell mitosis, the sicRNA complex can move into the nucleus to target the mutated gene. The sicRNA triggers the assembly of protein complexes leading to epigenetic modification of the promoter site of the mutant gene. In some instances, instead of methylation, the homologous DNA is degraded, leading to loss of heterozygosity. The factors controlling these two actions are unknown but the result is gene silencing or physical destruction of the mutant gene. An error in RNAi defence occurs when the sicRNA during methylation of the target gene, inadvertently interferes with the production of a miRNA specific for that tissue. This produces a change in the profile of correct proteins for that tissue. On a rare occasion, a preneoplastic stem cell will survive if miRNA interference switches on/off a gene involved in apoptosis, as well as a gene involved in cell proliferation and DNA damage surveillance. The driving force of carcinogenesis is the sequential loss of specific miRNAs.

Análisis molecular del proceso de transcripción de genes en eucariontes / Molecular analysis of genome transcription in eukaryotes

El proceso de transcripción es altamente regulado en el que intervienen la RNA polimerasa y varios factores adicionales. La enzima RNA polimerasa II contiene entre 8 y 14 subunidades y es la enzima que transcribe los RNA que codifican para proteínas. La subunidad mayor de la RNA polimerasa II contiene en su región carboxilo terminal un dominio denominado CTD que consiste en repeticiones de un heptapépido, el cual es fundamental para la regulación de la transcripción. El proceso de transcripción consiste de tres etapas, iniciación, elongación y terminación. A pesar que la RNA polimerasa II es una enzima multimérica, no puede reconocer los promotores e iniciar la transcripción en forma específica. Para iniciar la transcripción en forma específica requiere de factores adicionales, denominados factores generales de transcripción, los cuales se denominan TFIIA, TFIIB, TFIID, TFIIE, TFIIF y TFIIH. Estos factores y la RNA polimerasa II se ensamblan sobre el promotor en forma secuencial, o preensamblados con la RNA polimerasa II. Los genes son activados en respuesta a señales fisiológicas, llevada a cabo por los activadores de la transcripción, los que se unen a secuencias intensificadoras que están ubicadas río arriba del sitio de iniciación. Para la activación de la transcripción, además de los activadores, se requiere de un complejo MED, el cual puede encontrarse libre o bien unido al CTD de la RNA polimerasa II. El DNA se encuentra compactado dentro del núcleo por historias, las cuales presentan un impedimento físico para que se forme un complejo de iniciación sobre el promotor. Existen enzimas que poseen la capacidad de modificar las historias, que se denominan acetilasas, las cuales acetilan el dominio aminoterminal de las historias, provocando una inestabilidad en el nucleosoma, lo cual permite que se forme un complejo de preiniciación de la transcripción. También existen factores que son capaces de desplazr los nucleosomas para permitir la unión de la RNA polimerasa II y sus factores al promotor

Uso de una proteína recombinante del virus de la fiebre aftosa, la polimerasa 3D, en una prueba de inmunodifusión en gel de agar / Use of a recombinant foot and mouth disease virus 3D polymerase in an agarose gel immunodiffusion test

En este estudio, se examinó la efectividad del uso del polipéptido 3D recombinante, obtenido en su forma nativa en una prueba de IDGA (IDGA-3D), para uso en la detección de anticuerpos específicos de infección con VFA, independientemente de la condición de vacunación. Los resultados indican que en relación a la tradicional prueba de IDGA-VIAA, la IDGA 3D ofrece, particularmente cuando se evalúan sueros de bajo título, un método más consistente, con especificidad comparable, y por lo menos la misma sensibilidad. Ninguno de los antígenos ofreció una ventaja particular con respecto a la definición de las bandas de precipitación. El reemplazo del VIAA por la proteína 3D recombinante tiene considerables atracciones, dado que proporciona un suministro ilimitado de material inocuo, económico, de fácil purificación y consistente, eliminando la presencia potencial de antígenos no específicos de células BHK o componentes de la cápside del VFA(au)

Expresión como proteínas de fusión de las subunidades PB1 y PB2 de la polimerasa del virus de la influenza / Expression how proteins of fusion of the subunits PB1 and PB2 of the influenza viral of polymerase

Los cADNs de los genes que codifican las proteínas PB1 y PB2 de la ARN polimerasa del virus de la influenza, se clonaron en fase en un sitio distal del ATG que codifica la proteína 10 del bacteriógafo T7, en el vector de expresión pAR3040, bajo el control de un promotor de la ARN polimerasa de T7. Los plásmidos recombinantes se introdujeron en la cepa bacteriana. E coli BL21 (DE3)plysS, que contiene en su genoma el gen de la ARN polimerasa del fago T7, bajo el control de un promotor (lac uv5) inducible por IPTG. La inducción de la ARN polimerasa del fago T7 en las células hospederas, resultó en la expresión de las proteínas PB1 y PB2 en forma de proteínas de fusión. Las proteínas expresadas se acumularon en grandes cantidades en el interior de las células bacterianas y fueron recuperadas de los lisados en forma de precipitados insolubles.

In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli

The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10 5. The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other piconaviruses