Characterisation of the Putative Effector Interaction Site of the Regulatory HbpR Protein from Pseudomonas azelaica by Site-Directed Mutagenesis

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ54-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate PC promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition

Routine Opt-Out HIV Testing Strategies in a Female Jail Setting: A Prospective Controlled Trial

Background: Ten million Americans enter jails annually. The objective was to evaluate new CDC guidelines for routine optout HIV testing and examine the optimal time to implement routine opt-out HIV testing among newly incarcerated jail detainees. Methods: This prospective, controlled trial of routine opt-out HIV testing was conducted among 323 newly incarcerated female inmates in Connecticut’s only women’s jail. 323 sequential entrants to the women’s jail over a five week period in August and September 2007 were assigned to be offered routine opt-out HIV testing at one of three points after incarceration: immediate (same day, n = 108), early (next day, n = 108), or delayed (7 days, n = 107). The primary outcome was the proportion of women in each group consenting to testing. Results: Routine opt-out HIV testing was significantly highest (73%) among the early testing group compared to 55 % for immediate and 50 % for 7 days post-entry groups. Other factors significantly (p = 0.01) associated with being HIV tested were younger age and low likelihood of early release from jail based on bond value or type of charge for which women were arrested. Conclusions: In this correctional facility, routine opt-out HIV testing in a jail setting was feasible, with highest rates of testing if performed the day after incarceration. Lower testing rates were seen with immediate testing, where there is a high prevalence of inability or unwillingness to test, and with delayed testing, where attrition from jail increases with each passing day

Flavonoid-rich orange juice is associated with acute improvements in cognitive function in healthy middle-aged males

Purpose: Epidemiological evidence suggests that chronic consumption of fruit-based flavonoids is associated with cognitive benefits; however, the acute effects of flavonoid-rich (FR) drinks on cognitive function in the immediate postprandial period require examination. The objective was to investigate whether consumption of FR orange juice is associated with acute cognitive benefits over 6 h in healthy middle-aged adults. Methods: Males aged 30–65 consumed a 240-ml FR orange juice (272 mg) and a calorie-matched placebo in a randomized, double-blind, counterbalanced order on 2 days separated by a 2-week washout. Cognitive function and subjective mood were assessed at baseline (prior to drink consumption) and 2 and 6 h post consumption. The cognitive battery included eight individual cognitive tests. A standardized breakfast was consumed prior to the baseline measures, and a standardized lunch was consumed 3 h post-drink consumption. Results: Change from baseline analysis revealed that performance on tests of executive function and psychomotor speed was significantly better following the FR drink compared to the placebo. The effects of objective cognitive function were supported by significant benefits for subjective alertness following the FR drink relative to the placebo. Conclusions: These data demonstrate that consumption of FR orange juice can acutely enhance objective and subjective cognition over the course of 6 h in healthy middle-aged adults

Genetic screening of Fabry patients with EcoTILLING and HRM technology

<p>Abstract</p> <p>Background</p> <p>Anderson-Fabry disease (FD) is caused by a deficit of the α-galactosidase A enzyme which leads to the accumulation of complex sphingolipids, especially globotriaosylceramide (Gb3), in all the cells of the body, causing the onset of a multi-systemic disease with poor prognosis in adulthood. In this article, we describe two alternative methods for screening the <it>GLA </it>gene which codes for the α-galactosidase A enzyme in subjects with probable FD in order to test analysis strategies which include or rely on initial pre-screening.</p> <p>Findings</p> <p>We analyzed 740 samples using EcoTILLING, comparing two mismatch-specific<ul/>endonucleases, CEL I and ENDO-1, while conducting a parallel screening of the same samples using HRM (High Resolution Melting). Afterwards, all samples were subjected to direct sequencing. Overall, we identified 12 different genetic variations: -10C>T, -12G>A, -30G>A, IVS2-76_80del5, D165H, C172Y, IVS4+16A>G, IVS4 +68 A>G, c.718_719delAA, D313Y, IVS6-22C>T, G395A. This was consistent with the high genetic heterogeneity found in FD patients and carriers. All of the mutations were detected by HRM, whereas 17% of the mutations were not found by EcoTILLING. The results obtained by EcoTILLING comparing the CEL I and ENDO-1 endonucleases were perfectly overlapping.</p> <p>Conclusion</p> <p>On the basis of its simplicity, flexibility, repeatability, and sensitivity, we believe that<ul/>HRM analysis of the <it>GLA </it>gene is a reliable presequencing screening tool. This method can be applied to any genomic feature to identify known and unknown genetic alterations, and it is ideal for conducting screening and population studies.</p

Low linkage disequilibrium in wild Anopheles gambiae s.l. populations

<p>Abstract</p> <p>Background</p> <p>In the malaria vector <it>Anopheles gambiae</it>, understanding diversity in natural populations and genetic components of important phenotypes such as resistance to malaria infection is crucial for developing new malaria transmission blocking strategies. The design and interpretation of many studies here depends critically on Linkage disequilibrium (LD). For example in association studies, LD determines the density of Single Nucleotide Polymorphisms (SNPs) to be genotyped to represent the majority of the genomic information. Here, we aim to determine LD in wild <it>An. gambiae s.l</it>. populations in 4 genes potentially involved in mosquito immune responses against pathogens (<it>Gambicin</it>, <it>NOS</it>, <it>REL2 </it>and <it>FBN9</it>) using previously published and newly generated sequences.</p> <p>Results</p> <p>The level of LD between SNP pairs in cloned sequences of each gene was determined for 7 species (or incipient species) of the <it>An. gambiae </it>complex. In all tested genes and species, LD between SNPs was low: even at short distances (< 200 bp), most SNP pairs gave an r²< 0.3. Mean r²ranged from 0.073 to 0.766. In most genes and species LD decayed very rapidly with increasing inter-marker distance.</p> <p>Conclusions</p> <p>These results are of great interest for the development of large scale polymorphism studies, as LD generally falls below any useful limit. It indicates that very fine scale SNP detection will be required to give an overall view of genome-wide polymorphism. Perhaps a more feasible approach to genome wide association studies is to use targeted approaches using candidate gene selection to detect association to phenotypes of interest.</p

Discovery and Genomic Characterization of Noroviruses from a Gastroenteritis Outbreak in Domestic Cats in the US

Norovirus (NoV) RNA was detected in the stools of 6 out 14 (42.8%) 8–12-week-old cats with enteritis from a feline shelter, in New York State. Upon sequence analysis of the complete capsid, the six NoVs were found to be identical, suggesting the spread of a unique NoV strain in the shelter. The full-length genomic sequence (7839 nt) of one feline NoV, CU081210E/2010/US, was determined. In the capsid protein VP1 region, the virus displayed the highest amino acid identity to animal genogroup IV genotype 2 (GIV.2) NoVs: lion/Pistoia-387/06/IT (97.9%) and dog/Bari-170/07/IT (90.4%). These findings document the discovery of a novel feline calicivirus, different from vesiviruses, and extend the spectrum of NoV host range. Epidemiological studies using feline NoV-specific diagnostic tools and experimental infection of cats are required to understand whether NoVs have a pathogenic role in this species

High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene

Background: MUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma. High resolution melting analysis (HRMA) is a mutation scanning method that allows detection of heterozygous sequence changes with high sensitivity, whereas homozygosity for a nucleotide change may not lead to significant curve shape or melting temperature changes compared to homozygous wildtype samples. Therefore, HRMA has been mainly applied to the detection of mutations associated with autosomal dominant or X-linked disorders, while applications to autosomal recessive conditions are less common. Methods: MUTYH coding sequence and UTRs were analyzed by both HRMA and sequencing on 88 leukocyte genomic DNA samples. Twenty-six samples were also examined by SSCP. Experiments were performed both with and without mixing the test samples with wild-type DNA. Results: The results show that all MUTYH sequence variations, including G > C and A > T homozygous changes, can be reliably identified by HRMA when a condition of artificial heterozygosity is created by mixing test and reference DNA. HRMA had a sensitivity comparable to sequencing and higher than SSCP. Conclusions: The availability of a rapid and inexpensive method for the identification of MUTYH sequence variants is relevant for the diagnosis of colorectal cancer susceptibility, since the MAP phenotype is highly variable

Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ70-dependent (p1, pB, and p2) and two σS-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models

A rare exception to Haldane's rule: are X chromosomes key to hybrid incompatibilities?

This work was funded by NERC (NE/G014906/1, NE/L011255/1, NE/I027800/1). Additional funding from the Orthopterists’ Society to PM is also gratefully acknowledged.The prevalence of Haldane’s rule suggests that sex chromosomes commonly have a key role in reproductive barriers and speciation. However, the majority of research on Haldane’s rule has been conducted in species with conventional sex determination systems (XY and ZW) and exceptions to the rule have been understudied. Here we test the role of X-linked incompatibilities in a rare exception to Haldane’s rule for female sterility in field cricket sister species (Teleogryllus oceanicus and T. commodus). Both have an XO sex determination system. Using three generations of crosses, we introgressed X chromosomes from each species onto different, mixed genomic backgrounds to test predictions about the fertility and viability of each cross type. We predicted that females with two different species X chromosomes would suffer reduced fertility and viability compared with females with two parental X chromosomes. However, we found no strong support for such X-linked incompatibilities. Our results preclude X–X incompatibilities and instead support an interchromosomal epistatic basis to hybrid female sterility. We discuss the broader implications of these findings, principally whether deviations from Haldane’s rule might be more prevalent in species without dimorphic sex chromosomes.PostprintPeer reviewe

Understanding Plant-Microbe Interactions for Phytoremediation of Petroleum-Polluted Soil

Plant-microbe interactions are considered to be important processes determining the efficiency of phytoremediation of petroleum pollution, however relatively little is known about how these interactions are influenced by petroleum pollution. In this experimental study using a microcosm approach, we examined how plant ecophysiological traits, soil nutrients and microbial activities were influenced by petroleum pollution in Phragmites australis, a phytoremediating species. Generally, petroleum pollution reduced plant performance, especially at early stages of plant growth. Petroleum had negative effects on the net accumulation of inorganic nitrogen from its organic forms (net nitrogen mineralization (NNM)) most likely by decreasing the inorganic nitrogen available to the plants in petroleum-polluted soils. However, abundant dissolved organic nitrogen (DON) was found in petroleum-polluted soil. In order to overcome initial deficiency of inorganic nitrogen, plants by dint of high colonization of arbuscular mycorrhizal fungi might absorb some DON for their growth in petroleum-polluted soils. In addition, through using a real-time polymerase chain reaction method, we quantified hydrocarbon-degrading bacterial traits based on their catabolic genes (i.e. alkB (alkane monooxygenase), nah (naphthalene dioxygenase) and tol (xylene monooxygenase) genes). This enumeration of target genes suggests that different hydrocarbon-degrading bacteria experienced different dynamic changes during phytoremediation and a greater abundance of alkB was detected during vegetative growth stages. Because phytoremediation of different components of petroleum is performed by different hydrocarbon-degrading bacteria, plants’ ability of phytoremediating different components might therefore vary during the plant life cycle. Phytoremediation might be most effective during the vegetative growth stages as greater abundances of hydrocarbon-degrading bacteria containing alkB and tol genes were observed at these stages. The information provided by this study enhances our understanding of the effects of petroleum pollution on plant-microbe interactions and the roles of these interactions in the phytoremediation of petroleum-polluted soil