Search

In Vibrio species, AphB is essential to activate virulence cascades by sensing low-pH and anaerobiosis signals; however, its regulon remains largely unknown. Here, AphB is found to be a key virulence regulator in Vibrio alginolyticus, a pathogen for marine animals and humans. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) enabled the detection of 20 loci in the V. alginolyticus genome that contained AphB-binding peaks. An AphB-specific binding consensus was confirmed by electrophoretic mobility shift assays (EMSAs), and the regulation of genes flanking such binding sites was demonstrated using quantitative real-time PCR analysis. AphB binds directly to its own promoter and positively controls its own expression in later growth stages. AphB also activates the expression of the exotoxin Asp by binding directly to the promoter regions of asp and the master quorum-sensing (QS) regulator luxR. DNase I footprinting analysis uncovered distinct AphB-binding sites (BBS) in these promoters. Furthermore, a BBS in the luxR promoter region overlaps that of LuxR-binding site I, which mediates the positive control of luxR promoter activity by AphB. This study provides new insights into the AphB regulon and reveals the mechanisms underlying AphB regulation of physiological adaptation and QS-controlled virulence in V. alginolyticus. [ABSTRACT FROM AUTHOR]

Many Proteobacteria synthesize acyl-homoserine lactone (AHL) molecules for use as signals in cell density-dependent gene regulation known as quorum sensing (QS) and response. AHL detection protocols are essential to QS researchers and several techniques are available, including a 14 C-AHL radiolabel assay. This assay is based on the uptake of radiolabeled methionine by living cells and conversion of the radiolabel into S-adenosylmethionine (SAM). The radiolabeled SAM is then incorporated into AHL signal by an AHL synthase enzyme. Here we describe a methodology to perform the AHL radiolabel assay, which is unbiased, relatively fast, and very sensitive compared to other AHL detection protocols.

Binary expression systems are flexible and versatile genetic tools in Drosophila. The Q-system is a recently developed repressible binary expression system that offers new possibilities for transgene expression and genetic manipulations. In this review chapter, we focus on current state-of-the-art Q-system tools and reagents. We also discuss in vivo applications of the Q-system, together with GAL4/UAS and LexA/LexAop systems, for simultaneous expression of multiple effectors, intersectional labeling, and clonal analysis.

Objectives: The hybrid approach achieves stage 1 palliation of hypoplastic left heart syndrome with flow and physiologic characteristics that are different from those of the surgical Norwood circulations. In addition to having branch pulmonary arterial banding regulating the balance between pulmonary and systemic blood flows, coronary and cerebral perfusion are dependent on retrograde flow through the native aortic arch when aortic atresia is present. Accordingly, we used computational modeling to assess the effects of pulmonary artery banding diameter and retrograde aortic arch hypoplasia or obstruction on the hybrid stage 1 circulation, including the influence on systemic and cerebral oxygen deliveries., Methods: A computational modeling technique was used to couple a 3-dimensional geometry of the hybrid palliation with a hydraulic network of the entire circulation based on pre-stage 2 hemodynamics. This validated multiscale approach predicts clinically relevant outcomes, such as flow, pressure, ejection fraction, and oxygen delivery. Simulations with pulmonary artery banding varying between 1.5 and 3.5 mm were performed. To examine the effects of retrograde aortic arch hypoplasia and obstruction, models of differing aortic arch diameter (2-5 mm) and isthmus coarctation (2.5-5 mm) were studied., Results: Banding the branch pulmonary arteries to 2 mm led to pulmonary and systemic blood flows closest to 1:1 and produced the highest mixed venous saturation and systemic oxygen delivery. Both cerebral and coronary perfusion decreased markedly when the retrograde aortic arch or the coarctation was less than 3 mm in diameter. Moreover, flow reversal in the carotid arteries was observed during diastole in all models., Conclusions: These computational simulations of the stage 1 hybrid palliation for hypoplastic left heart syndrome with aortic atresia suggest that small differences in the degree of branch pulmonary arterial banding can result in significant changes in the overall performance of the hybrid palliation. Furthermore, retrograde aortic arch hypoplasia or obstruction can lead to suboptimal cerebral and coronary perfusion. Precise pulmonary artery banding may be important to optimize interstage physiology in patients undergoing the hybrid approach, and pre-interventional imaging of the aortic arch and isthmus should be performed to rule out potential for post-procedural suboptimal cerebral and coronary perfusion., (Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Objective: To assess the safety of the suprabrow approach (SBCA) for aneurysm surgery by comparing intraoperative rupture rates with those for the standard pterional approach., Methods: A systematic review of all literature published in or after 1997 was performed using specified search words. All articles described aneurysm surgery by one of two approaches--pterional or suprabrow--and mentioned the rate of intraoperative rupture. A total of 41 articles were found fit for inclusion for the final analysis. Articles that focused on giant, bilateral, posterior fossa, or previously coiled aneurysms were not included. The χ(2) test was used to compare the two cohorts and various subgroup analyses were carried out. A P value of <0.05 was considered significant., Results: The search of literature yielded 9488 aneurysm reports (41 articles), 7535 operated by the pterional approach and 1953 aneurysms by the SBCA. The overall intraoperative rupture (IOR) rate for the entire group was 9.20%. In the pterional craniotomy approach (PtCA) group, the rate of IOR was 10.09% and in the SBCA group, IOR occurred in 5.78%. The IOR rate in the PtCA group was almost double that in the SBCA group and the odds ratio (OR) for this difference was 1.8 (95% confidence interval [CI] 1.49-2.26; P< 0.001). A total of 3039 ruptured aneurysms were analyzed--2848 aneurysms in the PtCA group and 191 in the SBCA group. The rate of IOR was 14.15% for the overall group, 13.8% in the PtCA group, and 19.37% in the SBCA group. The difference in IOR between the PtCA group and the SBCA group for ruptured aneurysms was found to be significant (OR 1.5, 95% CI 1.003-2.119; P< 0.05). The number of unruptured aneurysms in the PtCA group was 862 (39.4%) and in the SBCA group, it was 232 (49.1%). The difference in the number of unruptured aneurysms between the groups was significant (P< 0.001). The rate of IOR was significantly less with the SBCA than with the pterional approach., Conclusions: The rate of intraoperative rupture is significantly higher when ruptured aneurysms are operated with the SBCA (in comparison to the pterional approach). However, the SBCA may be safer for unruptured and middle cerebral artery aneurysms with a lower rate of IOR., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: The management of patients with tetralogy of Fallot (ToF) and pulmonary atresia (PA) with major aortopulmonary collateral arteries (MAPCAs) is challenging. Frequently it is difficult to predict whether complete repair with closure of the ventricular septal defect (VSD) will be tolerated. The aim of this study was to investigate whether measurements of preoperative pulmonary blood flow are associated with early postoperative outcome after VSD closure., Methods: In this retrospective study, the data from 10 patients who had undergone a cardiac magnetic resonance imaging study with flow measurements before attempted surgical complete repair were collected. Systemic blood flow (Qs) was calculated as the sum of descending aortic blood flow distal to the MAPCA origins and superior vena cava flow. Pulmonary blood flow (Qp) was measured either from the sum of the pulmonary flow (n = 7) or calculated as the difference between ascending aortic flow and Qs., Results: Preoperative Qp/Qs averaged 1.71 ± 0.68 and correlated inversely with right ventricular systolic pressure (relative to systolic blood pressure, r = -0.75, P = .03) and positively with the total neopulmonary artery index (TNPAI, r = 0.66, P = .04), as measured by fluoroscopic angiography. Two children did not tolerate VSD closure. This was likely related to bronchial compression in 1 patient while the other had the lowest TNPAI and the lowest Qp/Qs of all patients., Conclusions: CMR provides not only anatomic but also functional information for surgical decision making in patients with ToF and PA with MAPCAs. Preoperative Qp/Qs is associated with postoperative right ventricular pressure and may be a marker of readiness for VSD closure., (Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Avian pathogenic Escherichia coli (APEC) cause avian colibacillosis, a poultry disease characterized by multiple organ infections resulting in significant economic loss in the poultry industry. Several virulence factors are important for disease manifestation in APEC of which, role of quorum sensing has not been investigated. Quorum sensing is a population dependent cell-cell signaling system which modulates numerous physiological processes such as biofilm formation and virulence in multiple species. LuxS, a well-known controller in the QS, plays a role in regulating virulence in various bacterial species. Here we investigated the role of LuxS in regulating virulence in APEC O78:K80:H9. Mutation of luxS resulted in a significant reduction of virulence in APEC O78:K80:H9, evidenced by both in vivo and in vitro assays such as decreased invasion of internal organs in chicken embryo, reduced lethality in chicken embryo lethality assay, and altered lipopolysaccharide (LPS) profile. In addition, the abilities of the knockout strain to survive in chicken macrophage cell lines and to invade in chicken embryo fibroblast cells were significantly diminished. Further, structure and expression level of the LPS profile was significantly altered in the knockout strain, which may be one of the contributing factors for the persistence and virulence of APEC. Complementation of luxS gene in trans restored the virulence of the knockout strain to the level of wild-type bacteria. Taken together, these results show that LuxS contributes to the virulence in APEC O78:K80:H9 strain and partly explain the role played by LuxS in the pathogenesis of APEC strains., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background & Aims: Adefovir (ADV) resistance mutations induce low-level cross-resistance to tenofovir in vitro. Our aim was to compare viral kinetics, nucleos(t)ide analog resistance mutations, and quasispecies (QS) evolution during therapy with tenofovir disoproxil fumarate (TDF) or emtricitabine + TDF (FTC/TDF) in selected patients with incomplete ADV responses., Methods: Patients with chronic hepatitis B and incomplete response to ADV were randomized in a double-blind trial of TDF vs. FTC/TDF. Extensive analysis of QS evolution was performed in 17 patients through 48 weeks of treatment., Results: At week 24, 48% of patients (9/17) achieved HBV DNA undetectability (<69 IU/ml) with no difference between treatment groups. ADV and/or LAM resistance mutations were detected in all 17 patients at baseline and in 5/6 analyzable patients at week 48. A total of 1224 reverse transcriptase clones were analyzed. Clonal analysis revealed no significant difference at baseline in QS complexity or diversity between treatment groups. There was a trend in both treatment groups for an increase in QS complexity at week 12, followed by a decrease in complexity and diversity by week 48. Analysis of individual patients showed no consistent selection/accumulation of specific viral resistance patterns during treatment, but at week 48, mutations at rtA181 persisted in 4 patients., Conclusions: TDF or FTC/TDF demonstrated strong viral suppression in patients with an incomplete response to ADV and no significant selective pressure on pre-existing ADV or LAM resistant strains. TDF monotherapy and FTC/TDF combination therapy had a comparable impact on QS evolution., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Biofouling in membrane bioreactors (MBRs) remains a primary challenge for their wider application, despite the growing acceptance of MBRs worldwide. Research studies on membrane fouling are extensive in the literature, with more than 200 publications on MBR fouling in the last 3 years; yet, improvements in practice on biofouling control and management have been remarkably slow. Commonly applied cleaning methods are only partially effective and membrane replacement often becomes frequent. The reason for the slow advancement in successful control of biofouling is largely attributed to the complex interactions of involved biological compounds and the lack of representative-for-practice experimental approaches to evaluate potential effective control strategies. Biofouling is driven by microorganisms and their associated extra-cellular polymeric substances (EPS) and microbial products. Microorganisms and their products convene together to form matrices that are commonly treated as a black box in conventional control approaches. Biological-based antifouling strategies seem to be a promising constituent of an effective integrated control approach since they target the essence of biofouling problems. However, biological-based strategies are in their developmental phase and several questions should be addressed to set a roadmap for translating existing and new information into sustainable and effective control techniques. This paper investigates membrane biofouling in MBRs from the microbiological perspective to evaluate the potential of biological-based strategies in offering viable control alternatives. Limitations of available control methods highlight the importance of an integrated anti-fouling approach including biological strategies. Successful development of these strategies requires detailed characterization of microorganisms and EPS through the proper selection of analytical tools and assembly of results. Existing microbiological/EPS studies reveal a number of implications as well as knowledge gaps, warranting future targeted research. Systematic and representative microbiological studies, complementary utilization of molecular and biofilm characterization tools, standardized experimental methods and validation of successful biological-based antifouling strategies for MBR applications are needed. Specifically, in addition, linking these studies to relevant operational conditions in MBRs is an essential step to ultimately develop a better understanding and more effective and directed control strategy for biofouling., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Proteins experience a wide variety of conformational dynamics that can be crucial for facilitating their diverse functions. How is the intrinsic flexibility required for these motions encoded in their three-dimensional structures? Here, the overall flexibility of a protein is demonstrated to be tightly coupled to the total amount of surface area buried within its fold. A simple proxy for this, the relative solvent-accessible surface area (Arel), therefore shows excellent agreement with independent measures of global protein flexibility derived from various experimental and computational methods. Application of Arel on a large scale demonstrates its utility by revealing unique sequence and structural properties associated with intrinsic flexibility. In particular, flexibility as measured by Arel shows little correspondence with intrinsic disorder, but instead tends to be associated with multiple domains and increased α-helical structure. Furthermore, the apparent flexibility of monomeric proteins is found to be useful for identifying quaternary-structure errors in published crystal structures. There is also a strong tendency for the crystal structures of more flexible proteins to be solved to lower resolutions. Finally, local solvent accessibility is shown to be a primary determinant of local residue flexibility. Overall, this work provides both fundamental mechanistic insight into the origin of protein flexibility and a simple, practical method for predicting flexibility from protein structures., (© 2013 Elsevier Ltd. All rights reserved.)

Previously, we reported that biofilm formation of Eikenella corrodens is regulated by autoinducer-2 (AI-2), based on observations that biofilm-forming efficiency of ΔluxS mutant was greater than that of the wild type (Azakami et al., J. Biosci. Bioeng., 102, 110-117, 2006). To determine whether the AI-2 molecule affects biofilm formation directly, we added purified AI-2 to luxS mutant and wild-type E. corrodens and compared biofilm formations by using a static assay. Results indicated that biofilm formation in E. corrodens was enhanced by the addition of AI-2. We also compared the biofilms formed by flow cell system for the luxS mutant and the wild type by using scanning electron microscopy and confocal laser scanning microscopy. The number of viable bacteria in the luxS mutant biofilm was dramatically reduced and more sparsely distributed than that of the wild type, which suggested that AI-2 might enhance the mature biofilm. Conversely, further analysis by modified confocal reflection microscopy indicated that the wild-type biofilm was matured earlier than that of the luxS mutant, and became thinner and more sparsely distributed with time. These data suggest that LuxS may facilitate the maturation and detachment of biofilm in E. corrodens., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)