Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.     

Determining specific growth rates in different regions of Salmonella typhimurium colonies

Computer-aided image analysis (IA) techniques were used to determine specific growth rates (μ) in different regions of Salmonella typhimurium colonies grown on solid medium at 30°C. Captured transmission images were converted to optical density values, allowing calculation of μ. Data show that growth was exponential throughout the colony during the period studied (between 13 and 21 h after inoculation), but that μ varied from 0·69 to 0·37 h⁻¹ at the periphery and centre, respectively. Results demonstrate regional variation in μ values within colonies, and support conceptual models of colony development. The technique described illustrates that kinetic growth data can be derived within bacterial colonies, and this may lead towards an improved understanding of the growth dynamics of these structures.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

The effects of growth dynamics upon pH gradient formation within and around subsurface colonies of Salmonella typhimurium

pH measurements made in and around submerged colonies of Salmonella typhimurium grown within a model gelatin gel system using pH-sensitive micro- and macroelectrodes indicated some pH heterogeneity occurring in and around the bacterial colony. Inoculation density, initial pH and glucose concentration were all found to influence colony diameter and metabolism of Salmonella colonies. Colony growth in the presence of glucose, at pH 7.0 with an inoculation density of 1 cell ml^-1 led to a pH fall of 1–2 pH units after 2 d. At pH 5.0, with glucose, colony growth rates were much slower than at pH 7.0, and the pH change varied by less than one pH unit often becoming alkaline. In the absence of glucose, only small pH changes were observed within the medium, although growth rates were similar to those in glucose-containing media. At the higher inoculation density ( ca 1000 cells ml^-1), isolated pH changes were not observed. Morphological changes, such as the production of annular rings, were noted in stationary phase colonies as was alkali production in colonies. These results are discussed in relation to observations with surface colonies.     

The effects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria

The growth and morphology of submerged bacterial colonies was investigated. Five separate colonial forms were recognized depending both on species and on agar concentration. These were (i) branched, dendritic structures seen only with Bacillus cereus ; (ii) lenticular colonies for all other species at high agar concentrations; (iii) small lobed to spherical colonies for non-motile organisms at low agar concentrations; (iv) and (v) large diffuse spherical colonies which can be further subdivided into 'snowball' or 'wispy' types for motile bacteria growing at agar concentrations below about 0·65% w/v. Viable count determinations suggested that agar concentration had little effect in the early stages of growth but that motile cells at low agar concentrations achieved higher cell numbers than did those in concentrations greater than 0·65% w/v. Transmission electron microscopy indicated that bacteria in lenticular colonies were tightly packed within lens-shaped splits in the agar whilst at low agar concentrations motile cells were well separated and appeared to move through the agar matrix.     

Immobilization of Burkholderia cepacia in polyurethane-based foams: embedding efficiency and effect on bacterial activity

Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam. All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O₂ consumption and CO₂ evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds. Received 23 December 1996/ Accepted in revised form 13 March 1997     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.      

The effect of Eh on regulatory processes in facultative anaerobes

On increasing aeration rate in chemostat cultures of E. coli the redox potential (E_h) of the culture changes from ?l50 mv to +250 mv. During this period only a slight increase in measured dissolved oxygen concentration is observed. It is suggested that E_h be used as a control variable for aeration in the oxygen limited growth condition, whilst dissolved oxygen concentration be used as control variable in the presence of excess oxygen. Change in cytochrome b₁, three tricarboxylic acid cycle enzymes and hydrogenase can be related to culture E_h. These changes are discussed.     

Measurements of the distribution of adenylate concentrations and adenylate energy charge across Pseudomonas aeruginosa biofilms.

Adenine nucleotide pools and adenylate energy charge distributions were determined by using a laboratory-generated quasi-steady-state Pseudomonas aeruginosa biofilm. The method used involved freezing and sectioning of the intact biofilm, followed by extraction and assay of the adenylates in the sectioned material. Results indicated an increase in adenylate energy charge of about 0.2 units from the bottom to the surface of the biofilm. However, energy charge values were generally low throughout the biofilm, reaching a maximum of only 0.6 units. Of the adenylates measured, AMP was the predominant nucleotide, especially in the deeper parts of the biofilm profile.