Phenotypic and genotypic characterization of encapsulated Escherichia coli isolated from blooms in two Australian lakes

Escherichia coli has long been used as an indicator organism for water quality assessment. Recently there has been an accumulation of evidence that suggests some strains of this organism are able to proliferate in the environment, a characteristic that would detract from its utility as an indicator of faecal pollution. Phenotypic and genotypic characterization of E. coli isolated from blooms in two Australian lakes, separated by a distance of approximately 200 km, identified that the blooms were dominated by three E. coli strains. A major phenotypic similarity among the three bloom strains was the presence of a group 1 capsule. Genetic characterization of a conserved region of the cps gene cluster, which encodes group 1 capsules, identified a high degree of genetic variation within the bloom isolates. This differs from previously described encapsulated E. coli strains which are highly conserved at the cps locus. The phenotypic or genotypic profiles of the bloom strains were not identified in 435 E. coli strains isolated from vertebrates. The occurrence of these encapsulated strains suggests that some E. coli have evolved a free-living lifestyle and do not require a host in order to proliferate. The presence of the same three strains in bloom events in different geographical regions of a temperate climate, and at different times, indicates that free-living E. coli strains are able to persist in these water reservoirs. This study provides further evidence of circumstances where caution is required in using E. coli as an indicator organism for water quality.     

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

Background  

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform.     

Carbohydrate polymers in amorphous states: an integrated thermodynamic and nanostructural investigation

The effect of water on the structure and physical properties of amorphous polysaccharide matrices is investigated by combining a thermodynamic approach including pressure- and temperature-dependent dilatometry with a nanoscale analysis of the size of intermolecular voids using positron annihilation lifetime spectroscopy. Amorphous polysaccharides are of interest because of a number of unusual properties which are likely to be related to the extensive hydrogen bonding between the carbohydrate chains. Uptake of water by the carbohydrate matrices leads to a strong increase in the size of the holes between the polymer chains in both the glassy and rubbery states while at the same time leading to an increase in matrix free volume. Thermodynamic clustering theory indicates that, in low-moisture carbohydrate matrices, water molecules are closely associated with the carbohydrate chains. Based on these observations, we propose a novel model of plasticization of carbohydrate polymers by water in which the water dynamically disrupts chains the hydrogen bonding between the carbohydrates, leading to an expansion of the matrix originating at the nanolevel and increasing the number of degrees of freedom of the carbohydrate chains. Consequently, even in the glassy state, the uptake of water leads to increased rates of matrix relaxation and mobility of small permeants. In contrast, low-molecular weight sugars plasticize the carbohydrate matrix without appreciably changing the structure and density of the rubbery state, and their role as plasticizer is most likely related to a reduction of the number of molecular entanglements. The improved molecular packing in glassy matrices containing low molecular weight sugars leads to a higher matrix density, explaining, despite the lower glass transition temperature, the reduced mobility of small permeants in such matrices.     

Parallel tandem: a program for parallel processing of tandem mass spectra using PVM or MPI and X!Tandem

A method for the rapid correlation of tandem mass spectra to a list of protein sequences in a database has been developed. The combination of the fast and accurate computational search algorithm, X!Tandem, and a Linux cluster parallel computing environment with PVM or MPI, significantly reduces the time required to perform the correlation of tandem mass spectra to protein sequences in a database. A file of tandem mass spectra is divided into a specified number of files, each containing an equal number of the spectra from the larger file. These files are then searched in parallel against a protein sequence database. The results of each parallel output file are collated into one file for viewing through a web interface. Thousands of spectra can be searched in an accurate, practical, and time effective manner. The source code for running Parallel Tandem utilizing either PVM or MPI on Linux operating system is available from http://www.thegpm.org. This source code is made available under Artistic License from the authors.     

Gene-environment interactions in rare diseases that include common birth defects

Rare syndromes often feature specific types of birth defects that frequently are major diagnostic clues to the presence of a given disorder. Despite this specificity, not everyone with the same syndrome is equally or comparably affected, and not everyone with a specific birth defect manifests the same syndrome or is affected with all the features of a particular syndrome. A symposium sponsored by the National Institutes of Health Office of Rare Diseases, and the National Toxicology Program Center for the Evaluation of Risks to Human Reproduction attempted to explore how much of this variability is due to genetic factors and how much is due to environmental factors. The specific types of birth defects examined included cardiovascular defects, holoprosencephaly, clefts of the lip and/or palate, neural tube defects, and diaphragmatic hernias.     

Genes encoding catalytic subunits of protein kinase A and risk of spina bifida

BACKGROUND: PRKACA and PRKACB are genes encoding the cAMP-dependent protein kinase A (PKA) catalytic subunits alpha and beta, respectively. PKA is known to be involved in embryonic development, as it down-regulates the Hedgehog (Hh) signaling pathway, which is critical to normal pattern formation and morphogenesis. The PKA-deficient mouse model, which has only a single catalytic subunit, provided intriguing evidence demonstrating a relationship between decreased PKA activity and risk for posterior neural tube defects (NTDs) in the thoracic to sacral regions of gene-knockout mice. Unlike most other mutant mouse models of NTDs, the PKA-deficient mice develop spina bifida with 100% penetrance. We hypothesized that sequence variations in human genes encoding the catalytic subunits may alter the PKA activity and similarly increase the risk of spina bifida. METHODS: We sequenced the coding regions and the exon/intron boundaries of PRKACA and PRKACB. We also examined 3 common single-nucleotide polymorphisms (SNPs) of these 2 genes by allele discrimination. RESULTS: Five sequence variants in coding region and 2 intronic sequence variants proximal to exons were detected. None of the 3 SNPs examined in the association study appeared to be associated with substantially increased risk for spina bifida. CONCLUSIONS: Our results did not reveal a strong association between these PKA SNPs and spina bifida risk. Nonetheless, it is important to examine the possible gene-gene interactions between PRKACA and PRKACB when evaluating the risk for NTDs, as well as genes encoding regulatory subunits of PKA. In addition, interactions with other genes such as Sonic Hedgehog (SHH) should also be considered for future investigations.     

Effects of temperature on maximum clinging ability in a diurnal gecko: evidence for a passive clinging mechanism?

The thermal dependence of performance of ectotherms, and particularly locomotor performance in lizards, has received much attention. However, only a single study has examined the effects of temperature on adhesive clinging ability in geckos, despite the importance of adhesion for many pad-bearing lizards and invertebrates. We set out to characterize the thermal response of clinging ability in the diurnal gecko, Phelsuma dubia in the temperature range of 15-35 degrees C. Our findings indicate that there is no significant trend in clinging ability for P. dubia with temperature and that there is high variation about the mean at all temperatures. These findings differ from other whole-organism studies of clinging performance and are suggestive of a passive clinging mechanism that is dominated by intermolecular van der Waals forces. These findings also suggest that clinging ability in this species is not under selective pressures resulting from thermal variation, and that P. dubia does not need to regulate body temperature closely to maximize clinging ability.     

Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy

Urinary proteins may provide clues regarding pathogenesis of kidney disease as well as providing markers of disease activity. We employed two-dimensional differential in-gel electrophoretic analysis (2-D DIGE) to assess multiple urine samples in patients with diabetic nephropathy. Patient samples were collected as timed overnight collections. All the patients had longstanding diabetes, impaired renal function, and overt proteinuria. Control and patient urinary protein were analyzed by 2-D DIGE and DeCyder analysis. Ninety-nine spots were significantly regulated in the urine proteome of the diabetic samples, with 63 up- and 36 down-regulated. One spot corresponding to a pI 5-6 and a molecular weight between 45 and 66 kDa was consistently up-regulated by 19-fold across individuals in the diabetic group. Surface-enhanced laser desorption/ionization-time of flight analysis of in-gel tryptic digest of this spot identified this protein as alpha 1 antitrypsin (AAT). ELISA of urine samples from a separate group of patients and controls confirmed a marked increase of AAT in diabetic patients. Immunostaining of human diabetic kidneys revealed up-regulation of AAT in areas of renal fibrosis. In conclusion, we developed a method to analyze numerous urine samples from patients and allowed for detection and identification of regulated urine protein spots.