Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins

Glucose monitoring in vivo is a crucial issue for gaining new understanding of diabetes. Glucose binding protein (GBP) fused to two fluorescent indicator proteins (FLIP) was used in the present study such as FLIP-glu- 3.2 mM. Recombinant Escherichia coli whole-cells containing genetically encoded nanosensors as well as cell-free extracts were immobilized either on inner epidermis of onion bulb scale or on 96-well microtiter plates in the presence of glutaraldehyde. Glucose monitoring was carried out by Förster Resonance Energy Transfer (FRET) analysis due the cyano and yellow fluorescent proteins (ECFP and EYFP) immobilized in both these supports. The recovery of these immobilized FLIP nanosensors compared with the free whole-cells and cell-free extract was in the range of 50–90%. Moreover, the data revealed that these FLIP nanosensors can be immobilized in such solid supports with retention of their biological activity. Glucose assay was devised by FRET analysis by using these nanosensors in real samples which detected glucose in the linear range of 0–24 mM with a limit of detection of 0.11 mM glucose. On the other hand, storage and operational stability studies revealed that they are very stable and can be re-used several times (i.e. at least 20 times) without any significant loss of FRET signal. To author's knowledge, this is the first report on the use of such immobilization supports for whole-cells and cell-free extract containing FLIP nanosensor for glucose assay. On the other hand, this is a novel and cheap high throughput method for glucose assay.

Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 μmol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 .−) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative ρ0 strain, lacking mitochondrial DNA, was used. The ρ0 strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 .− precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.

Avaliação de massas específicas de cistos de Giardia duodenalis e Entamoeba coli

Cistos de Giardia duodenalis e Entamoeba coli foram observados quanto à flutuação em soluções de sacarose de massas específicas diversas (1.040, 1.050, 1.060, 1.070,1.080,1.090,1.100, 1.150,1.200 e 1.250 kg/m³), contidas em câmaras de contagem de 0,17 mm de altura. Contaram-se os cistos que flutuaram e os que sedimentaram tendo sido calculadas as porcentagens respectivas. As diferenças de flutuabilidade dos cistos de cada uma das espécies não foram consideráveis. Soluções de massa específica igual a 1.200 kg/m³ fizeram flutuar 88,49% dos cistos de G. duodenalis e 95,71% dos de E. coli. Os maiores valores de flutuabilidade estiveram associados à massa específica 1.250 kg/m³ e foram, respectivamente, 89,15% e 98,59% para cistos de G. duodenalis e de E. coli.

Deterministic approach for multiple-source tsunami hazard assessment for Sines, Portugal

In this paper, we present a deterministic approach to tsunami hazard assessment for the city and harbour of Sines, Portugal, one of the test sites of project ASTARTE (Assessment, STrategy And Risk Reduction for Tsunamis in Europe). Sines has one of the most important deep-water ports, which has oil-bearing, petrochemical, liquid-bulk, coal, and container terminals. The port and its industrial infrastructures face the ocean southwest towards the main seismogenic sources. This work considers two different seismic zones: the Southwest Iberian Margin and the Gloria Fault. Within these two regions, we selected a total of six scenarios to assess the tsunami impact at the test site. The tsunami simulations are computed using NSWING, a Non-linear Shallow Water model wIth Nested Grids. In this study, the static effect of tides is analysed for three different tidal stages: MLLW (mean lower low water), MSL (mean sea level), and MHHW (mean higher high water). For each scenario, the tsunami hazard is described by maximum values of wave height, flow depth, drawback, maximum inundation area and run-up. Synthetic waveforms are computed at virtual tide gauges at specific locations outside and inside the harbour. The final results describe the impact at the Sines test site considering the single scenarios at mean sea level, the aggregate scenario, and the influence of the tide on the aggregate scenario. The results confirm the composite source of Horseshoe and Marques de Pombal faults as the worst-case scenario, with wave heights of over 10 m, which reach the coast approximately 22 min after the rupture. It dominates the aggregate scenario by about 60 % of the impact area at the test site, considering maximum wave height and maximum flow depth. The HSMPF scenario inundates a total area of 3.5 km2. © Author(s) 2015.

Prevalence of thermotolerant species of Campylobacter and their biotypes in children and domestic birds and dogs in Southern Chile

The prevalence of thermotolerant Campylobacter in diarrhoeic and healthy children as well as in dogs, hens, ducks and pigeons was determined in Southern Chile. Campylobacter were found in 34.5% of the faecal samples examined. The isolation rate of Campylobacter in diarrhoeic and healthy children was 16.3% and 6.4% respectively. Despite C.jejuni was always more frequent than C. coli, the latter was isolated with a high frequency (29%) from patients with diarrhoea. C.jejuni and C. coli biotypes I and II were found in healthy and diarrhoeic children and were predominant in all the animals species studied. This may point out towards the possible origin of strains infecting children.

Chicken as potential contamination source of Campylocacter lari in Iquitos, Peru

In order to know the importance of chicken as natural reservoir of Campylobacter lari in Iquitos, Peru; samples were obtained by cloacal swabs from 200 chickens and immediately placed into a semisolid enrichment medium; these were streaked on modified Skirrow Agar. The organism was isolated from 21 (10.5%) samples, corresponding 58.8% to biovar I and 41.2% to biovar II (Lior scheme). The results provide evidence that chicken appear to be prominent reservoirs of Campylobacter lari in Iquitos.

Genomic and transcriptomic features of Chlamydia trachomatis. Tracking the basis for the ecological success

Dissertation presented to obtain a Ph.D degree in Biology, speciality in Molecular Genetics, by Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Caracterização preliminar da bioenergética do efluxo pelo sistema AcrAB-TolC em Escherichia coli

A resistência aos antibióticos em bactérias Gram-negativas pode ser aumentada pela extrusão de antibióticos através de sistemas de efluxo. Em Escherichia coli, o principal sistema de efluxo é o AcrAB-TolC o qual tem como principal fonte energética a força proto-motriz. Este trabalho pretendeu estudar alguns aspectos essenciais da bioenergética na actividade de efluxo de E. coli usando três estirpes bem caracterizadas genotipica e fenotipicamente. Foi utilizado um método fluorimétrico semi-automático no qual a fluorescência do fluorocromo brometo de etídeo, substrato de bombas de efluxo foi seguida, permitindo a medição em tempo real da actividade de efluxo e acumulação de fluorocromo (inibição do efluxo). A utilização de brometo de etídeo é particularmente vantajosa pois emite baixa fluorescência no exterior da célula bacteriana tornando-se extremamente fluorescente no seu interior. Este método é uma nova aplicação do termociclador em tempo real RotorGeneTM 3000 que permite o cálculo da cinética de transporte reflectindo o balanço entre acumulação de substrato por difusão passiva através da membrana e a sua extrusão/efluxo, proporcionando uma detecção rápida e económica de inibidores de efluxo. Os resultados obtidos mostram, para todas as estirpes, que a GLU e o pH afectam a acumulação e o efluxo do brometo de etídeo. De todos os inibidores de vias biossintéticas testados, o ortovanadato de sódio, foi o que demonstrou maior actividade inibitória, a qual é revertida na presença de GLU. Em conclusão, este estudo mostra que a actividade de efluxo de E. coli depende não só da fosforilação oxidativa por via da força proto-motriz mas também da energia proveniente da hidrólise de ATP pelas ATPases. O ortovanadato de sódio tem potencial para ser um novo inibidor de bombas de efluxo de largo espectro. A tecnologia utilizada neste trabalho demonstrou ser apropriada para a caracterização bioenergética da actividade de bombas de efluxo e permite a selecção de novos inibidores de bombas de efluxo em bactérias.

Sigla WEBGIS portal: an open source Web-based GIS application for the portuguese bluetongue Entomologiacal surveillance program.

Dissertação apresentada como requisito parcial para obtenção do grau de Mestre em Ciência e Sistemas de Informação Geográfica

Gastrointestinal translocation as a possible source of candidemia in an AIDS patient

Apart from cryptococcosis and histoplasmosis, which are mycoses contained by T cell-mediated mechanisms of host defense, fungemia is rarely found in AIDS patients. The frequency of fungemia due to Candida spp. has been reported to be as low as 1 %. We report a non-neutropenic AIDS patient who presented a candidemia which probably arose from her gastrointestinal tract.

Studies on prevalence of Strongyloides infection in Holambra and Maceió, Brazil, by the agar plate faecal culture method

Prevalence of Strongyloides stercoralis infection in three areas of Brazil was surveyed by a recently developed faecal culture method (an agar plate culture). The Strongyloides infection was confirmed in 11.3% of 432 subjects examined. The diagnostic efficacy of the agar plate culture was as high as 93.9% compared to only 28.5% and 26.5% by the Harada-Mori filter paper culture and faecal concentration methods, when faecal samples were examined simultaneously by these three methods. Among the 49 positive samples, about 60% were confirmed to be positive only by the agar plate culture. These results indicate that the agar plate culture is a sensitive new tool for the correct diagnosis of chronic Strongyloides infection.

Prospective study of enteropathogens in two communities of Misiones, Argentina

Children under five years of age, from two communities of different socio-economic strata (97 from Zaiman and 55 from Las Dolores) were examined epidemiologically during 2 years, by means of quarterly visits of the working team, who carried out the collection of faecal samples. During the study, one or more enteropathogens were identified in 73.9% of samples in children from Zaiman and in 58.3% of the samples from Las Dolores, being associated to diarrhoea in 70.5% and to asymptomatic infections in 65.7%. The number of diarrheic episodes was higher in Zaiman (15.45%) than in Las Dolores (12.35%), being more frequent in the spring-summer seasons. In Zaiman, the bacterial enteropathogen proportion was relevantly higher (p< 0.005) in children with diarrhoea, whereas the presence of parasites was more frequent in asymptomatic children (p< 0.01). Rotavirus had an even distribution within diarrheic and asymptomatic children. In Las Dolores, no relevant differences were found in the detection of enteroparasites between diarrheic and asymptomatic children. Mixed infections were detected; enterotoxigenic Escherichia coli (ETEC)-rotavirus and ETEC-parasites being the most frequent ones. ETEC was involved in 85% of these infections. These data, together with the high enteropathogen carriage, suggest an elevated level of environmental contamination. The latter plays an important role in diarrheic diseases, and added to the most extreme poverty, it affects children's lives.

Purification and parcial characterization of a hemagglutinating factor (HAF): a possible adhesive factor of the diffuse adherent of Escherichia coli (DAEC)

The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains

New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

PCR in the First Oropharynx Aspirate of the Newborn: A Possible Source for Identification of Congenital Infection Agents

We present a case of prenatal diagnosis of congenital rubella. After birth, in addition to traditional serologic and clinical examinations to confirm the infection, we could identify the virus in the "first fluid aspirated from the oropharynx of the newborn", using polimerase chain reaction (PCR). We propose that this first oropharynx fluid (collected routinely immediately after birth) could be used as a source for identification of various congenital infection agents, which may not always be easily identified by current methods