Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence

The genome of the halophilic archaeon Halobacterium sp. NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation: (1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins. This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity. (2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome. (3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain. This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer. (4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes. These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution. (5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome. This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character.     

Clonal origin, restricted natural distribution, and conservation of virulence factors in isolates of enterotoxigenic Escherichia coli serogroup O126.

Enterotoxigenic Escherichia coli serogroup O126 isolates have been isolated in Hong Kong since 1982 from sporadic cases of infantile diarrhea and from one outbreak in a neonatal ward. A 64-megadalton plasmid encoding colonization factor antigen I and heat-stable enterotoxin was identified in all 23 isolates. Enterotoxigenic E. coli strains producing heat-stable enterotoxin from different regions of Southeast Asia were collected and compared by biotyping, antibiotic resistance patterns, and plasmid profiles. Restriction endonuclease digestion of plasmids and subsequent Southern blot analysis with the heat-stable enterotoxin gene probe of representative strains showed a unique plasmid was harbored by all heat-stable enterotoxin-producing O126 strains tested. These results are consistent with conservative inheritance of enterotoxin plasmids within enterotoxigenic E. coli strains over a 2-year period in Hong Kong.     

Prosthetic hip joint infection due to Campylobacter fetus.

A case of postoperative prosthetic hip joint infection due to Campylobacter fetus subsp. fetus is described. Difficulties in isolation and antimicrobial susceptibility testing of this organism are discussed.     

Evaluation and optimization of a latex agglutination assay for detection of cholera toxin and Escherichia coli heat-labile toxin.

The effectiveness of a latex agglutination assay kit for the detection of Escherichia coli heat-labile toxin and cholera toxin was determined for the identification of natural isolates of the corresponding enteric pathogens in Southeast Asia. By selection of the appropriate culture media, the sensitivity of the assay was improved from 90.6% (for the detection of heat-labile toxin) and 75% (for the detection of cholera toxin) to 100%, and the results were confirmed with bioassays and DNA hybridization assays for both clinical and environmental isolates.     

Mesh-independent prediction of skin burns injury

This paper presents a robust finite element model (FEM) with multiple-layers of varying properties for investigation of burn effects on human skin during a burning process resulting from exposure of skin surface to a contact heat source and a hot moving fluid. Henriques' theory of skin burns is used in conjunction with two-dimensional Pennes bioheat transfer equation for determining the spatial and temporal extent of burn injury. The model developed is a two-dimensional axisymmetric model in cylindrical coordinates. The various tissue layers account for changing thermal properties with respect to skin anatomy. A finite element scheme that uses the backward Euler method is used to solve the problem. The injury processes of skin subsequent to the removal of the heat source (post-burn) will also be inspected. The mesh employed in this model consists of a high density of nodes and elements in which a thorough mesh convergence study was done. A comparison of the transient temperature field computed by this model against Diller's results using the FE technique with a comparatively coarse mesh of 125 elements and experimental data by Orgill et al. has been done in the present study. It concluded that improved accurate solutions have been performed using the robust model developed due to the achievement of a mesh-independent solution.     

Cytokine production in lethal and non-lethal murine malaria

Levels of IFN-gamma, IL-2 and IL-4 were measured in vitro during the course of non-lethal Plasmodium chabaudi adami and lethal P. chabaudi strain 1309 infections in BALB/cByJ mice. Spleen cells from mice infected with the non-lethal Plasmodium had a higher initial response to P. chabaudi antigens than mice infected with P. chabaudi strain 1309, as determined by measuring all three lymphokines. We conclude that both Th1 and Th2 subsets of T helper lymphocytes are activated during P. chabaudi adami infection but that these responses are suppressed in mice infected with the more virulent P. chabaudi strain 1309.     

Antigens of virulent and attenuated Rickettsia tsutsugamushi.

We studied the antigens present in L929 mouse fibroblast or rabbit testicular cells, which had been infected or not with a prototype strain of Rickettsia tsutsugamushi, the causal agent of scrub typhus, and its attenuated variant. Immunoblotting revealed four antigens, designated 1, 1a, 2 and 3, which appeared to be specifically associated with infection with this organism. Antigens 1 and 1a had similar mol. wt of about 60 kD and antigen 2 and 3 had mol. wts of 45 kD and 28 kD respectively. Whereas antigen 1a, 2 and 3 were common to infection with either the virulent or the attenuated strains of the organism, antigen 1 was only detected in cells infected with the virulent strain and was reactive only with the antiserum raised against cells infected with that strain. In addition, two antigens were also detected by crossed immunoelectrophoresis, one of which was similarly associated with infection with the virulent strain as antigen 1, while the other was common to infection with either of the strains. It seems that the antigenic cross reaction between the two strains may account, in part at least, for the protection of mice against infection with the virulent strain afforded by the attenuated strain, while the loss or modification of antigen 1 might be associated with attenuation of the organism with respect to its virulence to mice.     

Effect of cyclic adenosine 3'',5''-monophosphate antagonists on endotoxin-induced inhibition of human neutrophil chemotaxis.

We reported previously that Escherichia coli endotoxin inhibited human neutrophil chemotaxis toward C5a. This effect of endotoxin was antagonized by anti-inflammatory steroids. We now report that dibutyryl cyclic adenosine 3',5'-monophosphate, prostaglandin E1, isoproterenol, and cholera toxin also antagonize the suppression of chemotaxis by endotoxin. Each compound inhibited the effect of endotoxin in a dose-dependent fashion. To be effective, each compound except cholera toxin had to be present at the time of endotoxin challenge. Furthermore, propranolol blocked the protective effect of isoproterenol against endotoxin but not the protective effect of dibutyrl cyclic adenosine 3',5'-monophosphate or prostaglandin E1. Dibutyryl cyclic guanosine 3',5'-monophosphate, adenosine 5'-monophosphate, phenylephrine, prostaglandin F2 alpha, and carbachol did not modify the suppression of chemotaxis by endotoxin. Anti-inflammatory steroids and dibutyryl cyclic adenosine 3',5'-monophosphate are thought to stabilize phospholipids in certain cell membranes. This phospholipid-stabilizing action may contribute, at least in part, to the protective effect against endotoxin-mediated suppression of neutrophil chemotaxis.     

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate-methyl methacrylate-methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.     

Toxicity and adaptive immune response to intracellular transgenes delivered by helper-dependent vs. first generation adenoviral vectors

The host immune response to intracellular transgenes delivered by helper-dependent (HDV) vs. first generation (FGV) adenoviral vectors has been relatively unstudied. Previous studies showed short-term correction of bovine and murine argininosuccinate synthetase (ASS) deficiency after first generation adenoviral-mediated liver gene therapy. To determine whether the host adaptive immune response against the intracellular transgene human ASS (hASS) contributed to loss of gene expression in this setting, the same vector (FGV-CAG-hASS) was injected into Rag-/- (immunodeficient) mice. As in wild-type C57BL/6 (B6) mice, Rag-/- mice also showed significant loss of hASS expression and vector by week 4 post-injection, with concomitant elevation of liver enzymes and disruption of liver architecture. Therefore, direct toxicity due to vector rather than adaptive immune response against hASS primarily accounted for loss of expression with FGVs. In contrast to hASS, beta-galactosidase is strongly immunogenic and activates the host adaptive immune response. Loss of transgene expression was observed in B6 mice with either a FGV or a HDV expressing beta-galactosidase. However, the drop in gene expression observed with the HDV was primarily due to the adaptive immune response, since both beta-galactosidase expression and vector genome were sustained in immunodeficient mice treated with HDV. As expected, with weakly immunogenic hASS, vector genome and hASS expression were sustained with a HDV in spite of ubiquitous expression of the transgene. Therefore, viral gene expression is a primary determinant of intermediate and chronic toxicities at day 3 and week 4 post-injection. However, even in the absence of viral gene expression, strongly immunogenic intracellular transgenes can stimulate clearance of transduced hepatocytes.