Estimation of the viability of Vibrio cholerae 0139 by assessing cell membrane integrity

Changes in the viability of Vibrio cholerae 0139 Bengal, estimated by cellular membrane integrity, in batch culture over 35 days, were investigated. Data indicated an initial period of rapid growth with up to 30% of bacterial mortality, followed by a period of slower growth, lower culturability but higher viability, from day 7 onwards. The size of viable bacteria significantly decreased during the incubation time, whilst the size of dead bacteria showed a less pronounced decrease. V. cholerae 0139 changed from a straight or curved rod shape to a spherical shape. This study shows that BacLight dyes are a fast and useful tool to examine health risk-associated bacteria, providing useful information about their viability and concentration.     

Human immunodeficiency virus tat modulates the Flk-1/KDR receptor, mitogen-activated protein kinases, and components of focal adhesion in Kaposi's sarcoma cells

Kaposi's sarcoma (KS) spindle cell growth and spread have been reported to be modulated by various cytokines as well as the human immunodeficiency virus (HIV) gene product Tat. Recently, HIV-1 Tat has been shown to act like a cytokine and bind to the Flk-1/KDR receptor for the vascular endothelial growth factor A (VEGF-A), which is expressed by KS cells. We have characterized signal transduction pathways stimulated by HIV-1 Tat upon its binding to surface receptors on KS cells. We observed that stimulation in KS 38 spindle cells resulted in tyrosine phosphorylation and activation of the Flk-1/KDR receptor. We also report that HIV-1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions, such as the related adhesion focal tyrosine kinase RAFTK, paxillin, and p130(cas). Further characterization revealed the activation of mitogen-activated protein kinase, c-Jun amino-terminal kinase (JNK), and Src kinase. HIV-1 Tat contains a basic domain which can interact with growth factor tyrosine kinase receptors and a classical RGD sequence which may bind to and activate the surface integrin receptors for fibronectin and vitronectin. We observed that stimulation of KS cells with basic as well as RGD sequence-containing Tat peptides resulted in enhanced phosphorylation of RAFTK and activation of MAP kinase. These studies reveal that Tat stimulation activates a number of signal transduction pathways that are associated with cell growth and migration.     

Flow and pressure field characteristics in the porous block compact modeling of parallel plate heat sinks

Growing complexity has resulted in an increased computational effort in CFD modeling of electronic systems. To reduce the computational effort, one or several heat sinks can be represented by a compact "porous block" model, with an effective thermal conductivity and pressure loss coefficient. In this study of parallel plate heat sinks in laminar forced convection, a methodology is developed to analytically determine the fluid properties of compact heat sink models that provide acceptable levels of approximation. The results of an extensive set of CFD simulations for a three heat sink channel, covering three distinct heat sink geometries, air velocities from 0.25 m/s to 2 m/s and various spacings between the heat sinks, were used to create and evaluate the effectiveness of compact models. Use of a two term, equivalent loss coefficient-reflecting the linear and quadratic components in the pressure drop of a porous block-has led to good agreement between the detailed numerical and compact model predictions, with compact heat sink pressure drops usually slightly higher (<10%) than detailed heat sink pressure drops.     

Thermal compact modeling of parallel plate heat sinks

Growing complexity of electronic systems has resulted in an increased computational effort in CFD modeling of electronic systems. To reduce the computational effort, one or several heat sinks can be represented by a compact "porous block" model, with an effective thermal conductivity and pressure loss coefficient. In this study of parallel plate heat sinks in laminar forced convection, a methodology is developed to rigorously determine the thermal properties of compact heat sink models that provide a high level of accuracy. The results of an extensive set of CFD simulations for a three heat sink channel covering two distinct heat sink geometries, air velocities from 0.25 m/s to 2 m/s and various spacings between the heat sinks, were used to create and evaluate the fidelity of compact models. The results of this study establish the validity and value in using the porous block compact model representation for noncritical heat sinks in an electronic assembly. The results also reveal that a location-independent porous-block representation can yield excellent agreement in the prediction of the thermal characteristics of state-of-the-art heat sinks.     

Reproducibility and simplification of 13C-octanoic acid breath test for gastric emptying of solids

Factor V (FV) activation is the result of cleavages at Arg709, Arg1018 and Arg1545 by thrombin or FXa. The relative importance of these cleavages in tissue factor (TF) induced thrombin generation in plasma and in a purified system was elucidated with recombinant FV in which the three sites had been eliminated one by one or in combinations. The mutants were analyzed with a clotting assay using FV-deficient plasma and in a TF induced thrombin generation system using plasma or purified components. Surprisingly, in the standard FV clotting assay, all mutants gave similar clotting activities and the thrombin generation curves obtained with wild-type and thrombin-resistant FV were similar. Differences in clotting activities and thrombin generation patterns between wild-type and thrombin-resistant FV were only observed when lower TF concentrations were used. The thrombin generation curve obtained in plasma containing wt FV was characterized by a short lag phase and a subsequent phase of rapid thrombin generation (propagation phase). The Arg709 to Gln mutation yielded a slightly prolonged lag phase and the rate of thrombin generation during the propagation phase was approximately 5-fold lower than that observed with wt FV. The Arg1018 to Ile mutation only slightly affected the thrombin generation curve, whereas the Arg1545 to Gln mutation yielded a prolonged lag phase and decreased maximum thrombin activity. Thrombin-resistant FV (mutated at all three sites) yielded a prolonged lag phase and poor thrombin generation during the propagation phase. The purified system further demonstrated the importance of the three cleavage sites for rapid and sustained thrombin generation. The results demonstrate that cleavages at positions 709, 1018 and 1545 are not required for assembly of a FXa-FV complex expressing low but significant prothrombinase activity but that all three sites in different ways are important for the creation of a FVa which maximally supports the FXa-mediated activation of prothrombin.     

The novel heat-stable enterotoxin subtype gene (ystB) of Yersinia enterocolitica: nucleotide sequence and distribution of the yst genes

The gene (ystB) encoding the novel subtype of the heat-stable enterotoxin (Y-STb) was cloned from the chromosome of a clinical isolate of Yersinia enterocolitica 84-50 (serotype O:5, biotype 1A) and the nucleotide sequence was determined. The ystB contained 216 base pairs that encoded a protein of 71 amino acid residues. The C-terminal 30 residues of the precursor protein exactly corresponded to the amino acid sequence of the Y-STb toxin, purified from the culture supernatant of the wild strain. Homology search revealed that there are 76.9% nucleotide sequence similarity between ystB and the Yersinia kristensenii ST gene, and 73.5% with the Y. enterocolitica prototype sequence of yst (ystA). When tested with the PCR generated ystB specific probe, 36 of 304 Y. enterocolitica strains from 18 countries hybridized with the probe. All the ystB probe positive strains belonged to biotype 1A and mostly to the so-called non-pathogenic serotype O:5, O:6, O:7,8 O:7,13 and O:10, while ystA was predominantly found among the pathogenic serotypes (78.5%). Out of 36 ystB gene positive strains, 18 were clinical origin from six countries, which were also positive in the suckling mice assay suggesting that ystB may play an important role in the pathogenesis, and the so-called non-pathogenic serotypes could be virulent for human.     

Hydrogen sulphide production tests and the detection of groundwater faecal contamination by septic seepage.

The 'H2S test' is being advanced for microbiological water quality testing where conventional coliform based methods are impractical or too expensive. It involves ambient temperature incubation of water samples with nutrient formulated to generate hydrogen sulphide when 'faecal' bacteria are present. Recently a WHO review identified several concerns including the limited number of comparative studies, formulation variability, and false positives and negatives. In response we have compared the H2S test's ability to detect and quantify faecal contamination in an aquifer impacted by septic tank leachfields with measurements obtained concurrently using conventional bacterial indicators, coliphages, faecal sterol biomarkers, Cryptosporidium and Giardia. Like these other analytes, H2S testing detected a contamination gradient ranging from high (septic liquid) to moderate (exfiltration zones), to background (e.g. domestic bores), corresponding to indicator removal + dilution by factors > 10(6). Presence/absence tests could not distinguish between heavily and slightly contaminated waters, whereas multi-tube testing (e.g. 10 x 10 mL arrays) did. It was concluded that while the WHO review concerns are justified, the H2S test performance shows promise in sanitary survey work, can be improved by employing an mpn approach and has potential to aid in the protection of source water and identifying contaminated groundwater.