Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides

Translocation of lipid-linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni-encoded ABC-type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate-linked heptasaccharide intermediate involved in the recently identified bacterial N-linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N-linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O-antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.

Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.

The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat

During O antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenylpyrophosphate (Und-P-P)-bound O antigen subunits occurs before their polymerization and ligation to the rest of the LPS molecule. Despite the general nature of the translocation process, putative O-antigen translocases display a low level of amino acid sequence similarity. In this work, we investigated whether complete O antigen subunits are required for translocation. We demonstrate that a single sugar, GlcNAc, can be incorporated to LPS of Escherichia coli K-12. This incorporation required the functions of two O antigen synthesis genes, wecA (UDP-GlcNAc:Und-P GlcNAc-1-P transferase) and wzx (O-antigen translocase). Complementation experiments with putative O-antigen translocases from E. coli O7 and Salmonella enterica indicated that translocation of O antigen subunits is independent of the chemical structure of the saccharide moiety. Furthermore, complementation with putative translocases involved in synthesis of exopolysaccharides demonstrated that these proteins could not participate in O antigen assembly. Our data indicate that recognition of a complete Und-P-P-bound O antigen subunit is not required for translocation and suggest a model for O antigen synthesis involving recognition of Und-P-P-linked sugars by a putative complex made of Wzx translocase and other proteins involved in the processing of O antigen.

Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1)

In previous studies the authors cloned and characterized the DNA sequence of the regions at both ends of the O7-specific lipopolysaccharide (LPS) biosynthesis cluster of Escherichia coli VW187 (O7:K1), and identified the biosynthetic genes for dTDP-rhamnose and GDP-mannose, as well as one of the candidate glycosyltransferases. In this work the complete DNA sequence of a 6.9 kb intervening region is presented. Seven new ORFs were identified. All the functions required for the synthesis and transfer of the O7 LPS were assigned on the basis of complementation experiments of transposon insertion mutants, and amino acid sequence homology to proteins involved in LPS synthesis of other bacteria. Of the seven ORFs, two encoded membrane proteins that were homologous to the O-antigen translocase (Wzx) and polymerase (Wxy), two were involved in the biosynthesis of dTDP-N-acetylviosamine, and the remaining three showed homologies to sugar transferases. The O antigen chain length regulator gene wzz was also identified in the vicinity of the O7 polysaccharide cluster. O7-specific DNA primers were designed and tested for serotyping of O7 E. coli strains.

Cystic fibrosis research in allied health and nursing professions

This report is the result of the "Allied Health and Nursing Professions Working Group" meeting which took place in Verona, Italy, November 2009, which was organised by the European Cystic Fibrosis Society, and involved 32 experts. The meeting was designed to provide a "roadmap" of high priority research questions that can be addressed by Allied Health Professionals (AHP) and nursing. The other goal was to identify research skills that would be beneficial to AHP and nursing researchers and would ultimately improve the research capacity and capability of these professions. The following tasks were accomplished: 1) a Delphi survey was used to identify high priority research areas and themes, 2) common research designs used in AHP and nursing research were evaluated in terms of their strengths and weaknesses, 3) methods for assessing the clinimetric and psychometric properties, as well as feasibility, of relevant outcome measures were reviewed, and 4) a common skill set for AHPs and nurses undertaking clinical research was agreed on and will guide the planning of future research opportunities. This report has identified important areas and themes for future research which include: adherence; physical activity/exercise; nutritional interventions; interventions for the newborn with CF and evaluation of outcome measures for use in AHP and nursing research. It has highlighted the significant challenges AHPs and nurses experience in conducting clinical research, and proposes strategies to overcome these challenges. It is hoped that this report will encourage research initiatives that assess the efficacy/effectiveness of AHP and nursing interventions in order to improve the evidence base. This should increase the quality of research conducted by these professions, justify services they currently provide, and expand their skills in new areas, with the ultimate goal of improving care for patients with CF.

Randomized controlled trial of atorvastatin in mild to moderate Alzheimer disease:LEADe

There is some evidence that statins may have a protective and symptomatic benefit in Alzheimer disease (AD). The LEADe study is a randomized controlled trial (RCT) evaluating the efficacy and safety of atorvastatin in patients with mild to moderate AD.

The Atorvastatin/Donepezil in Alzheimer's Disease Study (LEADe):design and baseline characteristics

Growing evidence suggests that elevated cholesterol levels in mid-life are associated with increased risk of developing Alzheimer's disease (AD), and that statins might have a protective effect against AD and dementia. The Lipitor's Effect in Alzheimer's Dementia (LEADe) study tests the hypothesis that a statin (atorvastatin 80 mg daily) will provide a benefit on the course of mild to moderate AD in patients receiving background therapy of a cholinesterase inhibitor (donepezil 10 mg daily).

Hypercyclic tuples of operators on $C^n$ and $R^n$

A tuple $(T_1,\dots,T_n)$ of continuous linear operators on a topological vector space $X$ is called hypercyclic if there is $x\in X$ such that the the orbit of $x$ under the action of the semigroup generated by $T_1,\dots,T_n$ is dense in $X$. This concept was introduced by N.~Feldman, who have raised 7 questions on hypercyclic tuples. We answer those 4 of them, which can be dealt with on the level of operators on finite dimensional spaces. In
particular, we prove that the minimal cardinality of a hypercyclic tuple of operators on $\C^n$ (respectively, on $\R^n$) is $n+1$ (respectively, $\frac n2+\frac{5+(-1)^n}{4}$), that there are non-diagonalizable tuples of operators on $\R^2$ which possess an orbit being neither dense nor nowhere dense and construct a hypercyclic 6-tuple of operators on $\C^3$ such that every operator commuting with each member of the tuple is non-cyclic.

The effect of radiation technique and bladder filling on the acute toxicity of pelvic radiotherapy for localized high risk prostate cancer

Purpose: The goal of this project was to see if using IMRT to deliver elective pelvic nodal irradiation (EPNI) for prostate cancer reduced acute treatment toxicity.

Methods: Two hundred and thirty patients were enrolled into prospective trials delivering EPNI with a concomitant hypofractionated IMRT boost to the prostate. During accrual, the method of EPNI delivery changed as new literature emerged. Three methods were used (1) 4FB, (2) IMRT with 2 cm CTV margins around the pelvic vessels as suggested by Shih et al. (2005) [7] (IMRT-Shih), and (3) IMRT with nodal volumes suggested by the RTOG (IMRT-RTOG). Initially patients were treated with an empty bladder, with the remainder treated with bladder full.

Results: Patients in the 4FB group had higher rates of grade 2 acute GI toxicities compared to the IMRT-Shih and IMRT-RTOG groups (31.9% vs 20.8% vs 7.2%, p = 0.0009). Patients in the 4FB group had higher rates of grade 3 urinary frequency compared to the two IMRT groups (8.5% vs 0% vs 0%, p = 0.027). However, multivariate analysis suggested the factor that most influenced toxicity was bladder filling followed by IMRT.

Conclusions: Bladder filling appeared to be the dominant factor which predicted for acute toxicity, followed by the use of IMRT.

Production and evaluation of the utility of novel phage display-derived peptide ligands to Salmonella spp. for magnetic separation

Aims: The objectives of this study were to produce Salmonella-specific peptide ligands by phage display biopanning and evaluate their use for magnetic separation (MS).
Methods and Results: Four phage display biopanning rounds were performed and the peptides expressed by the two most Salmonella-specific (on the basis of phage binding ELISA results) phage clones, MSal020401 and MSal020417, were chemically synthesized and coupled to MyOne™ tosylactivated Dynabeads®. Peptide capture capability for whole Salmonella cells from non-enriched broth cultures was quantified by MS + plate counts and MS + Greenlight™ detection, and compared to capture capability of anti-Salmonella (antibody-coated) Dynabeads®. MS + Greenlight™ gave a more comprehensive picture of capture capability than MS + plate counts and showed that Peptide MSal020417-coated beads exhibited at least similar, if not better, capture capability to anti-Salmonella Dynabeads® (mean capture values of 36.0 ± 18.2 % and 31.2 ± 20.1 %, respectively, over Salmonella spp. concentration range 3 x 101 - 3 x 106 cfu ml-1) with minimal cross-reactivity (= 1.9 %) to three other foodborne bacteria.
Conclusions: One of the phage display-derived peptide ligands was demonstrated by MS + Greenlight™ to be a viable antibody-alternative for MS of Salmonella spp.
Significance and Impact of Study: This study demonstrates an antibody-free approach to Salmonella detection and opens substantial possibilities for more rapid tests for this bacterium.

Phage Display-Derived Binders Able to Distinguish Listeria monocytogenes from Other Listeria Species

The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni). Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries. © 2013 Morton et al.

Development of M13 bacteriophage-based SPR detection using Salmonella detection as a case study

Surface plasmon resonance (SPR)-based biosensor is a popular platform for real-time monitoring and sensitive detection for a myriad of targets. However, only a few studies have reported the use of bacteriophages as specific binders for SPR-based detection. This study aimed to demonstrate how filamentous M13 bacteriophages expressing 12-mer peptides can be employed in an SPR-based assay, using a Salmonella-specific bacteriophage as a model binder to detect the foodborne bacterium Salmonella. Several important factors (immobilization buffers and methods, and interaction buffers) for a successful bacteriophage-based SPR assay were optimized. As a result, a Salmonella-specific bacteriophage-based SPR assay was achieved, with very low cross reactivity with other non-target foodborne pathogens and detection limits of 8.0 × 10⁷ and 1.3 × 10⁷ CFU/mL for one-time and five-time immobilized sensors, respectively. This proof-of-concept study demonstrates the feasibility of using M13 bacteriophages expressing target-specific peptides as a binder in a rapid and label-free SPR assay for pathogen detection.