In Silico Prediction and Design of Uropathogenic Escherichia coli Alpha-Hemolysin Generate a Soluble and Hemolytic Recombinant Toxin.

The secretion of α-hemolysin by uropathogenic Escherichia coli (UPEC) is commonly associated with the severity of urinary tract infections, which makes it a predictor of poor prognosis among patients. Accordingly, this toxin has become a target for diagnostic tests and therapeutic interventions. However, there are several obstacles associated with the process of α-hemolysin purification, therefore limiting its utilization in scientific investigations. In order to overcome the problems associated with α-hemolysin expression, after in silico prediction, a 20.48 kDa soluble α-hemolysin recombinant denoted rHlyA was constructed. This recombinant is composed by a 182 amino acid sequence localized in the aa542-723 region of the toxin molecule. The antigenic determinants of the rHlyA were estimated by bioinformatics analysis taking into consideration the tertiary form of the toxin, epitope analysis tools, and solubility inference. The results indicated that rHlyA has three antigenic domains localized in the aa555-565, aa600-610, and aa674-717 regions. Functional investigation of rHlyA demonstrated that it has hemolytic activity against sheep red cells, but no cytotoxic effect against epithelial bladder cells. In summary, the results obtained in this study indicate that rHlyA is a soluble recombinant protein that can be used as a tool in studies that aim to understand the mechanisms involved in the hemolytic and cytotoxic activities of α-hemolysin produced by UPEC. In addition, rHlyA can be applied to generate monoclonal and/or polyclonal antibodies that can be utilized in the development of diagnostic tests and therapeutic interventions.

In silico prediction and design of uropathogenic Escherichia coli alpha-hemolysin generate a soluble and hemolytic recombinant toxin

The secretion of α-hemolysin by uropathogenic Escherichia coli (UPEC) is commonly associated with the severity of urinary tract infections, which makes it a predictor of poor prognosis among patients. Accordingly, this toxin has become a target for diagnostic tests and therapeutic interventions. However, there are several obstacles associated with the process of α-hemolysin purification, therefore limiting its utilization in scientific investigations. In order to overcome the problems associated with α-hemolysin expression, after in silico prediction, a 20.48 kDa soluble α-hemolysin recombinant denoted rHlyA was constructed. This recombinant is composed by a 182 amino acid sequence localized in the aa542–723 region of the toxin molecule. The antigenic determinants of the rHlyA were estimated by bioinformatics analysis taking into consideration the tertiary form of the toxin, epitope analysis tools, and solubility inference. The results indicated that rHlyA has three antigenic domains localized in the aa555–565, aa600–610, and aa674–717 regions. Functional investigation of rHlyA demonstrated that it has hemolytic activity against sheep red cells, but no cytotoxic effect against epithelial bladder cells. In summary, the results obtained in this study indicate that rHlyA is a soluble recombinant protein that can be used as a tool in studies that aim to understand the mechanisms involved in the hemolytic and cytotoxic activities of α-hemolysin produced by UPEC. In addition, rHlyA can be applied to generate monoclonal and/or polyclonal antibodies that can be utilized in the development of diagnostic tests and therapeutic interventions.

Secreted autotransporter toxin (Sat) induces cell damage during enteroaggregative Escherichia coli infection

Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77,O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.

Secreted autotransporter toxin (Sat) induces cell damage during enteroaggregative Escherichia coli infection

Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77,O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.

Biological and Chemical Control of Sclerotinia sclerotiorum using Stachybotrys levispora and Its Secondary Metabolite Griseofulvin.

Sclerotinia sclerotiorum is responsible for the white mold of soybeans, and the difficulty to control the disease in Brazil is causing million-dollar damages. Stachybotrys levispora has shown activity against S. sclerotiorum. In our present investigation, we analyzed the chemical basis of this inhibition. Eight compounds were isolated, and using spectroscopic methods, we identified their structures as the known substances 7-dechlorogriseofulvin, 7-dechlorodehydrogriseofulvin, griseofulvin, dehydrogriseofulvin, 3,13-dihydroxy-5,9,11-trimethoxy-1-methylbenzophenone, griseophenone A, 13-hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone, and 12-chloro-13-hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone. Griseofulvin inhibited the mycelial growth of S. sclerotiorum at 2 µg mL-1. Thus, the antagonistic effect of S. levispora to S. sclerotiorum may well be due to the presence of griseofulvins. Our results stimulate new work on the biosynthesis of griseofulvins, to locate genes that encode key enzymes in these routes and use them to increase the production of these compounds and thus potentiate the fungicide effect of this fungus. S. levispora represents an agent for biocontrol, and griseofulvin represents a fungicide to S. sclerotiorum.

Alkaloids and triterpene from Almeidea coerulea (Nees and Mart.) a. St.-Hil. and anti-leishmanial activity

The dichloromethane extract of Almeidea coerulea stems yielded the (11-hydroxyrutaecarpine alkaloid reported for the first time from this species) and the triterpene (28-hydroxy-28, 29-dihydrolupeol). The dictamine, skimianine, sitosterol and stigmasterol were also isolated from methanol extract. Extracellular forms of Leishmania amazonensis (promastigotes) was tested with dichloromethane extract and 28-hydroxy-28, 29-dihydrolupeol with showed anti-leishmanial activity above 0.1 mg/mL and 75µg/mL (inhibited 50 percent promastigote growth), respectively.

Intubação orotraqueal: avaliação do conhecimento médico e das práticas clínicas adotadas em unidades de terapia intensiva / Orotracheal intubation: physicians knowledge assessment and clinical practices in intensive care units

OBJETIVOS: Avaliar o conhecimento médico sobre as técnicas de intubação e identificar as práticas mais realizadas. MÉTODOS: Estudo prospectivo, envolvendo três diferentes unidades de terapia intensiva de um hospital universitário: da anestesiologia (ANEST), da pneumologia (PNEUMO) e do pronto socorro (PS). Todos os médicos que trabalham nessas unidades e que concordaram em participar do estudo, responderam um questionário contendo dados demográficos e questões sobre intubação orotraqueal. RESULTADOS: Foram obtidos 85 questionários (90,42 por cento dos médicos). ANEST teve maior média de idade (p = 0,001), com 43,5 por cento sendo intensivistas. Foi referido uso da associação hipnótico e opióide (97,6 por cento) e pré oxigenação (91,8 por cento), mas apenas 44,6 por cento referiram utilização de coxim suboccipital, sem diferença entre as UTIs. Na ANEST, referiu-se maior uso de bloqueador neuromuscular (p < 0,000) e maior cuidado com estômago cheio (p = 0,002). O conhecimento sobre sequência rápida foi restrito (nota média - 2,20 ± 0,89, com p = 0,6 entre as unidades de terapia intensiva. A manobra de Sellick era conhecida por (97,6 por cento), mas 72 por cento usaram-na inapropriadamente. CONCLUSÕES: O conhecimento médico sobre intubação orotraqueal em terapia intensiva não é satisfatório, mesmo entre profissionais qualificados para tal procedimento. É necessário avaliar se há concordância entre as respostas dos questionários e as práticas clínicas efetivamente adotadas.

OBJECTIVES: To assess the physician’s knowledge on intubation techniques and to identify the common practices. METHODS: This was a prospective study, involving three different intensive care units within a University hospital: Anesthesiology (ANEST), Pulmonology (PULMO) and Emergency Department (ED). All physicians working in these units and consenting to participate in the study completed a questionnaire with their demographic data and questions on orotracheal intubation. RESULTS: 85 completed questionnaires were retrieved (90.42 percent of the physicians). ANEST had the higher mean age (p=0.001), being 43.5 percent of them intensivists. The use of hypnotic and opioid association was reported by 97.6 percent, and pre-oxygenation by 91.8 percent, but only 44.6 percent reported sub-occipital pad use, with no difference between the ICUs. On ANEST an increased neuromuscular blockade use was reported (p<0.000) as well as increased caution with full stomach (p=0.002). The rapid sequence knowledge was restricted (mean 2.20 ± 0.89), p=0.06 between the different units. The Sellick maneuver was known by 97.6 percent, but 72 percent used it inappropriately. CONCLUSIONS: Physicians knowledge on orotracheal intubation in the intensive care unit is unsatisfactory, even among qualified professionals. It is necessary to check if the responses to the questionnaire and actual clinical practices agree.

Orotracheal intubation: physicians knowledge assessment and clinical practices in intensive care units. / Intubação orotraqueal: avaliação do conhecimento médico e das práticas clínicas adotadas em unidades de terapia intensiva.

OBJECTIVES: To assess the physician’s knowledge on intubation techniques and to identify the common practices. METHODS: This was a prospective study, involving three different intensive care units within a University hospital: Anesthesiology (ANEST), Pulmonology (PULMO) and Emergency Department (ED). All physicians working in these units and consenting to participate in the study completed a questionnaire with their demographic data and questions on orotracheal intubation. RESULTS: 85 completed questionnaires were retrieved (90.42% of the physicians). ANEST had the higher mean age (p=0.001), being 43.5% of them intensivists. The use of hypnotic and opioid association was reported by 97.6%, and pre-oxygenation by 91.8%, but only 44.6% reported sub-occipital pad use, with no difference between the ICUs. On ANEST an increased neuromuscular blockade use was reported (p<0.000) as well as increased caution with full stomach (p=0.002). The rapid sequence knowledge was restricted (mean 2.20 ± 0.89), p=0.06 between the different units. The Sellick maneuver was known by 97.6%, but 72% used it inappropriately. CONCLUSIONS: Physicians knowledge on orotracheal intubation in the intensive care unit is unsatisfactory, even among qualified professionals. It is necessary to check if the responses to the questionnaire and actual clinical practices agree.

Brazilian Sepsis Epidemiological Study (BASES study).

INTRODUCTION: Consistent data about the incidence and outcome of sepsis in Latin American intensive care units (ICUs), including Brazil, are lacking. This study was designed to verify the actual incidence density and outcome of sepsis in Brazilian ICUs. We also assessed the association between the Consensus Conference criteria and outcome METHODS: This is a multicenter observational cohort study performed in five private and public, mixed ICUs from two different regions of Brazil. We prospectively followed 1383 adult patients consecutively admitted to those ICUs from May 2001 to January 2002, until their discharge, 28th day of stay, or death. For all patients we collected the following data at ICU admission: age, gender, hospital and ICU admission diagnosis, APACHE II score, and associated underlying diseases. During the following days, we looked for systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, and septic shock criteria, as well as recording the sequential organ failure assessment score. Infection was diagnosed according to CDC criteria for nosocomial infection, and for community-acquired infection, clinical, radiological and microbiological parameters were used. RESULTS: For the whole cohort, median age was 65.2 years (49-76), median length of stay was 2 days (1-6), and the overall 28-day mortality rate was 21.8%. Considering 1383 patients, the incidence density rates for sepsis, severe sepsis and septic shock were 61.4, 35.6 and 30.0 per 1000 patient-days, respectively. The mortality rate of patients with SIRS, sepsis, severe sepsis and septic shock increased progressively from 24.3% to 34.7%, 47.3% and 52.2%, respectively. For patients with SIRS without infection the mortality rate was 11.3%. The main source of infection was lung/respiratory tract. CONCLUSION: Our preliminary data suggest that sepsis is a major public health problem in Brazilian ICUs, with an incidence density about 57 per 1000 patient-days. Moreover, there was a close association between ACCP/SCCM categories and mortality rate.

Brazilian Sepsis Epidemiological Study (BASES study)

(ICUs), including Brazil, are lacking. This study was designed to verify the actual incidence density and outcome of sepsis in Brazilian ICUs. We also assessed the association between the Consensus Conference criteria and outcome Methods This is a multicenter observational cohort study performed in five private and public, mixed ICUs from two different regions of Brazil. We prospectively followed 1383 adult patients consecutively admitted to those ICUs from May 2001 to January 2002, until their discharge, 28th day of stay, or death. For all patients we collected the following data at ICU admission: age, gender, hospital and ICU admission diagnosis, APACHE II score, and associated underlying diseases. During the following days, we looked for systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, and septic shock criteria, as well as recording the sequential organ failure assessment score.Infection was diagnosed according to CDC criteria for nosocomial infection, and for community-acquired infection, clinical, radiological and microbiological parameters were used. Results For the whole cohort, median age was 65.2 years (49–76), median length of stay was 2 days (1–6), and the overall 28-day mortality rate was 21.8%. Considering 1383 patients, the incidence density rates for sepsis, severe sepsis and septic shock were 61.4, 35.6 and 30.0 per 1000 patient-days, respectively. The mortality rate of patients with SIRS, sepsis, severe sepsis and septic shock increased progressively from 24.3% to 34.7%, 47.3% and 52.2%, respectively. For patients with SIRS without infection the mortality rate was 11.3%. The main source of infection was lung/respiratory tract. Conclusion Our preliminary data suggest that sepsis is a major public health problem in Brazilian ICUs, with an incidence density about 57 per 1000 patient-days. Moreover, there was a close association between ACCP/SCCM categories and mortality rate.

Traqueostomia por punçäo percutanea: resultados iniciais / Percutaneous tracheostomy: preliminary results

Em janeiro de 1997 a dezembro de 1998 foram realizadas 39 traqueostomia percutâneas(19 mulheres e 20 homens).A média de idade foi de 56 anos.As traqueostomias foram realizadas com o conjunto CooK (Ciaglia Percutaneous Tracheostomy Introducer Set).O procedimento consistiu na passagem de uma cânula através de um orifício traqueal puncionado e dilatado de forma percutânea.Após a identificação da cartilagem cricóide puncionamos a traquéia no primeiro ou no segundo interespaço,passamos um fio guia e progressivamente dilatamos o orifício de punção até atingirmos o calibre desejado.Introduzimos a cânula traqueal através da cânula orotraqueal.Em nenhum doente ocorreu pneumotórax,pneumomediastino,falso trajeto ou punção de esôfago.Em um doente o orifício lateral da cânula oroteaqueal foi puncionado e em outro a cânula traqueal colocada ,era de calibre menor que o necessário e foi trocada por outra de maior calibre.Não houve nenhum óbito durante os procedimentos.Podemos concluir que a traqueostomia percutânea e um procedimento seguro e pode ser realizado na Unidade de Terapia Intensiva

Incapacidade físicas em hanseníase: estudo multicêntrico da realidade brasileira / Handicapped in hanseniasis: multicenter study of Brazilian relity

Após contextualizar a ralidade das incapacidades físicas em nossos dias, sobretudo nos países subdesenvolvidos, do ponto de vista näo só do individual mas também do coletivo, säo consideradas natureza e magnitude das incapacidades físicas em nosso meio atualmente. Procede-se o estudo das mesmas em 181 hansenianos amostrados em investigaçäo multicêntrica, a nível nacional, envolvendo nove serviços estatais de controle da moléstia. Foi identificada a existência de 950 incapacidades nos 146 doentes (80,66%), que apresentaram pelo menos uma delas, correspondendo a ocorrência média de 6.51 lesöes por pessoa. Sua distribuiçäo por freqüência, segmento corporal e grau de gravidade é apresentada e referenciada a dez casuísticas externas. Constata-se que as variaçöes no entendimento, descriçöes, registro e cocntagem das diferentes incapacidades físicas säo täo inconsistentes que tornar muito arriscadas eventuais comparaçöes de freqüência e características de diferentes casuísticas. Depreende-se, assim, a legitimidade e necessidade de que os estudos de distribuiçöes de freqüências e de identificaçöes das lesöes incapacitantes representativas na hanseníase sejam desenhados e executados segundo procedimentos metodológicos epidemiológicos adequados, para poderem realmente subsidiar formulaçäo e implementaçäo de políticas setoriais para o atendimento e manejo dos segmentos populacionais acometidos