Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own “affinity matrix,” thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active β-galactosidase was obtained in a single step using the above described method.     

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.     

Evaluating Written Patient Information for Eczema in German: Comparing the Reliability of Two Instruments,DISCERN and EQIP

Patients actively seek information about how to cope with their health problems, but the quality of the information available varies. A number of instruments have been developed to assess the quality of patient information, primarily though in English. Little is known about the reliability of these instruments when applied to patient information in German. The objective of our study was to investigate and compare the reliability of two validated instruments, DISCERN and EQIP, in order to determine which of these instruments is better suited for a further study pertaining to the quality of information available to German patients with eczema. Two independent raters evaluated a random sample of 20 informational brochures in German. All the brochures addressed eczema as a disorder and/or therapy options and care. Intra-rater and inter-rater reliability were assessed by calculating intra-class correlation coefficients, agreement was tested with weighted kappas, and the correlation of the raters’ scores for each instrument was measured with Pearson’s correlation coefficient. DISCERN demonstrated substantial intra- and inter-rater reliability. It also showed slightly better agreement than EQIP. There was a strong correlation of the raters’ scores for both instruments. The findings of this study support the reliability of both DISCERN and EQIP. However, based on the results of the inter-rater reliability, agreement and correlation analyses, we consider DISCERN to be the more precise tool for our project on patient information concerning the treatment and care of eczema.     

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     

Diaphragm thickening during inspiration

Cohn, David, Joshua O. Benditt, Scott Eveloff, and F. DennisMcCool. Diaphragm thickening during inspiration.J. Appl. Physiol. 83(1): 291-296, 1997.Ultrasound has been used to measure diaphragm thickness(T_di) in thearea where the diaphragm abuts the rib cage (zone of apposition).However, the degree of diaphragm thickening during inspiration reportedas obtained by one-dimensional M-mode ultrasound was greater than thatpredicted by using other radiographic techniques. Becausetwo-dimensional (2-D) ultrasound provides greater anatomic definitionof the diaphragm and neighboring structures, we used this technique toreevaluate the relationship between lung volume andT_di. We firstestablished the accuracy and reproducibility of 2-D ultrasound bymeasuring T_diwith a 7.5-MHz transducer in 26 cadavers. We found thatT_di measured byultrasound correlated significantly with that measured by ruler (R² = 0.89), withthe slope of this relationship approximating a line of identity(y = 0.89x + 0.04 mm). The relationship between lung volume andT_di was thenstudied in nine subjects by obtaining diaphragm images at the fivetarget lung volumes [25% increments from residual volume (RV) tototal lung capacity (TLC)]. Plots ofT_di vs. lungvolume demonstrated that the diaphragm thickened as lung volumeincreased, with a more rapid rate of thickening at the higher lungvolumes[T_di = 1.74 vital capacity (VC)² + 0.26 VC + 2.7 mm] (R² = 0.99; P < 0.001) where lung volumeis expressed as a fraction of VC. The mean increase inT_di between RVand TLC for the group was 54% (range 42-78%). We conclude that2-D ultrasound can accurately measureT_di and that theaverage thickening of the diaphragm when a subject is inhaling from RVto TLC using this technique is in the range of what would be predictedfrom a 35% shortening of the diaphragm.

     

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.      

Brahman bull fertility in a north Australian rangeland herd

Low and variable bull fertility was identified as a constraint on reproductive rates in beef cattle grazed in an extensive, multiple sire mating regimen on Mount Bundey station in the Darwin pastoral district of northwestern Australia. Erratic conception patterns were attributed to a high proportion of bulls with low breeding soundness evaluation scores (BSE), a high proportion of aged bulls (40%>8 yr), and to running bulls of mixed age groups. Liveweight, scrotal circumference (SC) and age were positively correlated. An experiment was subsequently designed to investigate the ability of a number of bull measurements to predict fertility in an extensively-managed, multiple-sire mating system. Blood typing was used to match calves to sires. It proved to be an accurate and useful technique which successfully identified the parentage of 94% of calves examined. Single measurements of serum testosterone after administration of gonadotrophin-releasing hormone (GnRH) were not correlated with fertility. However six of the seven most fertile bulls exhibited high peak serum testosterone levels in summer, and lower levels in the winter. In contrast, the less fertile bulls did not exhibit seasonal variation in GnRH-induced serum testosterone levels. Social dominance ratio was weakly ralted to fertility (r=0.51: P<0.05). BSE (r=0.51: P<0.05) and SC (r=0.49: P<0.05) prior to, but not subsequent to, mating were correlated with bull fertility. Under the conditions of this experiment, a bull to cow ratio of 1:20 was excessive for bulls with a satisfactory BSE score.