Interplay of the Wzx translocase and the corresponding polymerase and chain length regulator proteins in the translocation and periplasmic assembly of lipopolysaccharide o antigen

Genetic evidence suggests that a family of bacterial and eukaryotic integral membrane proteins (referred to as Wzx and Rft1, respectively) mediates the transbilayer movement of isoprenoid lipid-linked glycans. Recent work in our laboratory has shown that Wzx proteins involved in O-antigen lipopolysaccharide (LPS) assembly have relaxed specificity for the carbohydrate structure of the O-antigen subunit. Furthermore, the proximal sugar bound to the isoprenoid lipid carrier, undecaprenyl-phosphate (Und-P), is the minimal structure required for translocation. In Escherichia coli K-12, N-acetylglucosamine (GlcNAc) is the proximal sugar of the O16 and enterobacterial common antigen (ECA) subunits. Both O16 and ECA systems have their respective translocases, WzxO16 and WzxE, and also corresponding polymerases (WzyO16 and WzyE) and O-antigen chain-length regulators (WzzO16 and WzzE), respectively. In this study, we show that the E. coli wzxE gene can fully complement a wzxO16 translocase deletion mutant only if the majority of the ECA gene cluster is deleted. In addition, we demonstrate that introduction of plasmids expressing either the WzyE polymerase or the WzzE chain-length regulator proteins drastically reduces the O16 LPS-complementing activity of WzxE. We also show that this property is not unique to WzxE, since WzxO16 and WzxO7 can cross-complement translocase defects in the O16 and O7 antigen clusters only in the absence of their corresponding Wzz and Wzy proteins. These genetic data are consistent with the notion that the translocation of O-antigen and ECA subunits across the plasma membrane and the subsequent assembly of periplasmic O-antigen and ECA Und-PP-linked polymers depend on interactions among Wzx, Wzz, and Wzy, which presumably form a multiprotein complex.

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexaeri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz(pHS2). Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.

Resource utilisation, length of hospital stay, and pattern of investigation during acute medical hospital admission

To describe the patient demographic characteristics and organisational factors that influence length of stay (LOS) among emergency medical admissions. Also, to describe differences in investigation practice among consultant physicians and to examine the impact of these on LOS.

Use of arc-length method for capturing mode jumping in postbuckling aerostructures

The arc-length method has become a widely established solution technique for studying nonlinear structural behavior. By augmenting the set of nonlinear equilibrium equations with a constraint equation, which is a function of both the displacements and load increment, it is capable of traversing limit points. Numerous investigations have shown that highly nonlinear behavior such as sharp "snap-backs" can still lead to numerical difficulties. Two practical examples are presented to assess the effectiveness of this solution technique in capturing secondary instabilities in postbuckling structures, which present themselves as abrupt mode jumps. Although the first example poses no special difficulties, in the second case the nonlinear procedure fails to converge. An improvement to the method's formulation is suggested, which accounts for the residual forces that are usually neglected, when proceeding to the next increment once convergence is reached on the current increment. The choice of a correct load increment at the first iteration, within a predictor-corrector scheme, is central to the method's effectiveness. Current strategies for a choice of this load increment are discussed and are shown to be no longer consistent with the modified formulation; therefore, a new approach is proposed.

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.

Slip distribution model along the anchorage length of prestressing strands

An analytical model to predict strand slips within both transmission and anchorage lengths in pretensioned prestressed concrete members is presented. This model has been derived from an experimental research work by analysing the bond behavior and determining the transmission and anchorage lengths of seven-wire prestressing steel strands in different concrete mixes. A testing technique based on measuring the prestressing strand force in specimens with different embedment lengths has been used. The testing technique allows measurement of free end slip as well as indirect determination of the strand slip at different cross sections of a member without interfering with bond phenomena. The experimental results and the proposed model for strand slip distribution have been compared with theoretical predictions according to different equations in the literature and with experimental results obtained by other researchers. © 2013 Elsevier Ltd.

Electrical conductivity and glass formation in nitrile-functionalized pyrrolidinium bis(trifluoromethylsulfonyl)imide ionic liquids: chain length and odd–even effects of the alkyl spacer between the pyrrolidinium ring and the nitrile group

The electrical conductivity of a series of pyrrolidinium bis(trifluoromethylsulfonyl)imide ionic liquids, functionalized with a nitrile (cyano) group at the end of an alkyl chain attached to the cation, was studied in the temperature range between 173 K and 393 K. The glass formation of the ionic liquids is influenced by the length of the alkyl spacer separating the nitrile function from the pyrrolidinium ring. The electrical conductivity and the viscosity do not show a monotonic dependence on the alkyl spacer length, but rather an odd-even effect. An explanation for this behavior is given, including the potential energy landscape picture for the glass transition.

Influence of embedded length on strength of BFRP rods bonded parallel to the grain in low grade timber by pullout-bending tests

Bonded-in rod connections in timber possess many desirable attributes in terms of efficiency, manufacture, performance, aesthetics and cost. In recent years research has been conducted on such connections using fibre reinforced polymers (FRPs) as an alternative to steel. This research programme investigates the pull-out capacity of Basalt FRP rods bonded-in in low grade Irish Sitka Spruce. Embedded length is thought to be the most influential variable contributing to pull- out capacity of bonded-in rods after rod diameter. Previous work has established an optimum embedded length of 15 times the hole diameter. However, this work only considered the effects of axial stress on the bond using a pull-compression testing system which may have given an artificially high pull out capacity as bending effects were neglected. A hinge system was utilised that allows the effects of bending force to be taken in to consideration along with axial forces in a pull-out test. This paper describes an experimental programme where such pull-bending tests were carried out on samples constructed of 12mm diameter BFRP bars with a 2mm glueline thickness and embedded lengths between 80mm and 280mm bonded-in to low-grade timber with an epoxy resin. Nine repetitions of each were tested. A clear increase in pull-out strength was found with increasing embedded length.