Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR

All living organisms communicate with the external environment for their survival and existence. In prokaryotes, communication is achieved by two-component systems (TCS) comprising histidine kinases and response regulators. In eukaryotes, signalling is accomplished by serine/threonine and tyrosine kinases. Although TCS and serine/threonine kinases coexist in prokaryotes, direct cross-talk between these families was first described in Group B Streptococcus (GBS). A serine/threonine kinase (Stk1) and a TCS (CovR/CovS) co-regulate toxin expression in GBS. Typically, promoter binding of regulators like CovR is controlled by phosphorylation of the conserved active site aspartate (D53). In this study, we show that Stk1 phosphorylates CovR at threonine 65. The functional consequence of threonine phosphorylation of CovR in GBS was evaluated using phosphomimetic and silencing substitutions. GBS encoding the phosphomimetic T65E allele are deficient for CovR regulation unlike strains encoding the non-phosphorylated T65A allele. Further, compared with wild-type or T65A CovR, the T65E CovR is unable to bind promoter DNA and is decreased for phosphorylation at D53, similar to Stk1-phosphorylated CovR. Collectively, we provide evidence for a novel mechanism of response regulator control that enables GBS (and possibly other prokaryotes) to fine-tune gene expression for environmental adaptation.     

Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation

Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.     

Denial of Risk Behavior Does Not Exclude Asymptomatic Anorectal Sexually Transmitted Infection in HIV-Infected Men

Background

The Centers for Disease Control recommend screening for asymptomatic sexually transmitted infection (STI) among HIV-infected men when there is self-report of unprotected anal-receptive exposure. The study goals were: (1) to estimate the validity and usefulness for screening policies of self-reported unprotected anal-receptive exposure as a risk indicator for asymptomatic anorectal infection with Neisseria gonorrhoeae (GC) and/or Chlamydia trachomatis (CT). (2) to estimate the number of infections that would be missed if anal diagnostic assays were not performed among patients who denied unprotected anorectal exposure in the preceding month.

Methods and Findings

Retrospective analysis in HIV primary care and high resolution anoscopy (HRA) clinics. HIV-infected adult men were screened for self-reported exposure during the previous month at all primary care and HRA appointments. Four sub-cohorts were defined based on microbiology methodology (GC culture and CT direct fluorescent antibody vs. GC/CT nucleic acid amplification test) and clinical setting (primary care vs. HRA). Screening question operating characteristics were estimated using contingency table methods and then pooled across subcohorts. Among 803 patients, the prevalence of anorectal GC/CT varied from 3.5–20.1% in the 4 sub-cohorts. The sensitivity of the screening question for self-reported exposure to predict anorectal STI was higher in the primary care than in the HRA clinic, 86–100% vs. 12–35%, respectively. The negative predictive value of the screening question to predict asymptomatic anorectal STI was ≥90% in all sub-cohorts. In sensitivity analyses, the probability of being an unidentified case among those denying exposure increased from 0.4–8.1% in the primary care setting, and from 0.9–18.8% in the HRA setting as the prevalence varied from 1–20%.

Conclusion

As STI prevalence increases, denial of unprotected anal-receptive exposure leads to an increasingly unacceptable proportion of unidentified asymptomatic anorectal STI if used as a criterion not to obtain microbiologic assays.     

A Forward Chemical Screen in Zebrafish Identifies a Retinoic Acid Derivative with Receptor Specificity

Background

Retinoids regulate key developmental pathways throughout life, and have potential uses for differentiation therapy. It should be possible to identify novel retinoids by coupling new chemical reactions with screens using the zebrafish embryonic model.

Principal Findings

We synthesized novel retinoid analogues and derivatives by amide coupling, obtaining 80–92% yields. A small library of these compounds was screened for bioactivity in living zebrafish embryos. We found that several structurally related compounds significantly affect development. Distinct phenotypes are generated depending on time of exposure, and we characterize one compound (BT10) that produces specific cardiovascular defects when added 1 day post fertilization. When compared to retinoic acid (ATRA), BT10 shows similar but not identical changes in the expression pattern of embryonic genes that are known targets of the retinoid pathway. Reporter assays determined that BT10 interacts with all three RAR receptor sub-types, but has no activity for RXR receptors, at all concentrations tested.

Conclusions

Our screen has identified a novel retinoid with specificity for retinoid receptors. This lead compound may be useful for manipulating components of retinoid signaling networks, and may be further derivatized for enhanced activity.     

The X-Ray Crystal Structure of Escherichia coli Succinic Semialdehyde Dehydrogenase; Structural Insights into NADP+/Enzyme Interactions

Background

In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and γ-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells.

Methodology/Principal Findings

Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP⁺ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.

Conclusions/Significance

Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP⁺, whereas in contrast the human enzyme utilises NAD⁺. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease.     

Gene-specific contributions to mumps virus neurovirulence and neuroattenuation

Mumps virus (MuV) is highly neurotropic and was the leading cause of aseptic meningitis in the Western Hemisphere prior to widespread use of live attenuated MuV vaccines. Due to the absence of markers of virus neuroattenuation and neurovirulence, ensuring mumps vaccine safety has proven problematic, as demonstrated by the occurrence of aseptic meningitis in recipients of certain vaccine strains. Here we examined the genetic basis of MuV neuroattenuation and neurovirulence by generating a series of recombinant viruses consisting of combinations of genes derived from a cDNA clone of the neurovirulent wild-type 88-1961 strain (r88) and from a cDNA clone of the highly attenuated Jeryl Lynn vaccine strain (rJL). Testing of these viruses in rats demonstrated the ability of several individual rJL genes and gene combinations to significantly neuroattenuate r88, with the greatest effect imparted by the rJL nucleoprotein/matrix protein combination. Interestingly, no tested combination of r88 genes, including the nucleoprotein/matrix protein combination, was able to convert rJL into a highly neurovirulent virus, highlighting mechanistic differences between processes involved in neuroattenuation and neurovirulence.     

Live-cell microarray surface coatings supporting reverse transduction by adeno-associated viruses

High-throughput live-cell microarray technologies that facilitate combinatorial screening of genes and RNA interference (RNAi) would be invaluable in the identification of key gene expression profiles involved in complex cellular behaviors. Each spot on such a microarray can comprise a unique combination of genes or RNAi packaged into gene delivery vectors. Live target cells seeded on top of the microarrays would express the combination of genetic factors, potentially leading to phenotypic changes within cells. Here, we investigate the feasibility of using adeno-associated virus (AAV) as a gene delivery agent for such live-cell genetic microarrays. A robotic spotter was used to deposit AAV onto gamma-amino propyl silane, amine silane, or nitrocellulose-coated glass slides. Virus deposition and reverse transduction of target cells were found to be surface coating-dependent with nitrocellulose coating yielding the best AAV deposition, while also producing discrete islands of highly transduced cells. Our results demonstrate the feasibility of using nitrocellulose-coated surfaces for the development of AAV-based genetic microarrays.     

Major phenolic compounds in olive oil: metabolism and health effects

It has been postulated that the components in olive oil in the Mediterranean diet, a diet which is largely vegetarian in nature, can contribute to the lower incidence of coronary heart disease and prostate and colon cancers. The Mediterranean diet includes the consumption of large amounts of olive oil. Olive oil is a source of at least 30 phenolic compounds. The major phenolic compounds in olive oil are oleuropein, hydroxytyrosol and tyrosol. Recently there has been a surge in the number of publications that has investigated their biological properties. The phenolic compounds present in olive oil are strong antioxidants and radical scavengers. Olive "waste water" also possesses compounds which are strong antioxidant and radical scavengers. Typically, hydroxytyrosol is a superior antioxidant and radical scavenger to oleuropein and tyrosol. Hydroxytyrosol and oleuropein have antimicrobial activity against ATTC bacterial strains and clinical bacterial strains. Recent syntheses of labeled and unlabelled hydroxytyrosol coupled with superior analytical techniques have enabled its absorption and metabolism to be studied. It has recently been found that hydroxytyosol is renally excreted unchanged and as the following metabolites as its glucuronide conjugate, sulfate conjugate, homovanillic acid, homovanillic alcohol, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylacetaldehyde. Studies with tyrosol have shown that it is excreted unchanged and as its conjugates. This review summarizes the antioxidant abilities; the scavenging abilities and the biological fates of hydroxytyrosol, oleuropein and tyrosol which have been published in recent years.     

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.