Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx(EcO157)) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx(EcO157) consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.

Validation of membrane protein topology models by oxidative labeling and mass spectrometry

Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ·OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ·OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide

WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also determined the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent K(m) and V(max) values for UDP-GlcNAc, Mg(2+), and Mn(2+) suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg(2+), possibly playing a role in orienting the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.

Mixing operators on spaces with weak topology

We prove that a continuous linear operator T on a topological vector space X with weak topology is mixing if and only if the dual operator T' has no finite dimensional invariant subspaces. This result implies the characterization of hypercyclic operators on the space $\omega$ due to Herzog and Lemmert and implies the result of Bayart and Matheron, who proved that for any hypercyclic operator T on $\omega$, $T\oplus T$ is also hypercyclic.

Distinguished algebraic semantics for t-norm based fuzzy logics:Methods and algebraic equivalencies

This paper is a contribution to Mathematical fuzzy logic, in particular to the algebraic study of t-norm based fuzzy logics. In the general framework of propositional core and ?-core fuzzy logics we consider three properties of completeness with respect to any semantics of linearly ordered algebras. Useful algebraic characterizations of these completeness properties are obtained and their relations are studied. Moreover, we concentrate on five kinds of distinguished semantics for these logics-namely the class of algebras defined over the real unit interval, the rational unit interval, the hyperreals (all ultrapowers of the real unit interval), the strict hyperreals (only ultrapowers giving a proper extension of the real unit interval) and finite chains, respectively-and we survey the known completeness methods and results for prominent logics. We also obtain new interesting relations between the real, rational and (strict) hyperreal semantics, and good characterizations for the completeness with respect to the semantics of finite chains. Finally, all completeness properties and distinguished semantics are also considered for the first-order versions of the logics where a number of new results are proved. © 2009 Elsevier B.V. All rights reserved.

PROSTATIC INTRAEPITHELIAL NEOPLASIA (PIN) - PERFORMANCE OF BAYESIAN BELIEF NETWORK FOR DIAGNOSIS AND GRADING

Prostatic intraepithelial neoplasia (PIN) diagnosis and grading are affected by uncertainties which arise from the fact that almost all knowledge of PIN histopathology is expressed in concepts, descriptive linguistic terms, and words. A Bayesian belief network (BBN) was therefore used to reduce the problem of uncertainty in diagnostic clue assessment, while still considering the dependences between elements in the reasoning sequence. A shallow network was used with an open-tree topology, with eight first-level descendant nodes for the diagnostic clues (evidence nodes), each independently linked by a conditional probability matrix to a root node containing the diagnostic alternatives (decision node). One of the evidence nodes was based on the tissue architecture and the others were based on cell features. The system was designed to be interactive, in that the histopathologist entered evidence into the network in the form of likelihood ratios for outcomes at each evidence node. The efficiency of the network was tested on a series of 110 prostate specimens, subdivided as follows: 22 cases of non-neoplastic prostate or benign prostatic tissue (NP), 22 PINs of low grade (PINlow), 22 PINs of high grade (PINhigh), 22 prostatic adenocarcinomas with cribriform pattern (PACcri), and 22 prostatic adenocarcinomas with large acinar pattern (PAClgac). The results obtained in the benign and malignant categories showed that the belief for the diagnostic alternatives is very high, the values being in general more than 0.8 and often close to 1.0. When considering the PIN lesions, the network classified and graded most of the cases with high certainty. However, there were some cases which showed values less than 0.8 (13 cases out of 44), thus indicating that there are situations in which the feature changes are intermediate between contiguous categories or grades. Discrepancy between morphological grading and the BBN results was observed in four out of 44 PIN cases: one PINlow was classified as PINhigh and three PINhigh were classified as PINlow. In conclusion, the network can grade PlN lesions and differentiate them from other prostate lesions with certainty. In particular, it offers a descriptive classifier which is readily implemented and which allows the use of linguistic, fuzzy variables.

Adaptive Measurement-Based Policy-Driven QoS Management with Fuzzy-Rule-based Resource Allocation

Fixed and wireless networks are increasingly converging towards common connectivity with IP-based core networks. Providing effective end-to-end resource and QoS management in such complex heterogeneous converged network scenarios requires unified, adaptive and scalable solutions to integrate and co-ordinate diverse QoS mechanisms of different access technologies with IP-based QoS. Policy-Based Network Management (PBNM) is one approach that could be employed to address this challenge. Hence, a policy-based framework for end-to-end QoS management in converged networks, CNQF (Converged Networks QoS Management Framework) has been proposed within our project. In this paper, the CNQF architecture, a Java implementation of its prototype and experimental validation of key elements are discussed. We then present a fuzzy-based CNQF resource management approach and study the performance of our implementation with real traffic flows on an experimental testbed. The results demonstrate the efficacy of our resource-adaptive approach for practical PBNM systems

Network Topology, Higher Orders of Stability and Efficiency

Stable networks of order r where r is a natural number refer to those networks that are immune to coalitional deviation of size r or less. In this paper, we introduce stability of a finite order and examine its relation with efficient networks under anonymous and component additive value functions and the component-wise egalitarian allocation rule. In particular, we examine shapes of networks or network architectures that would resolve the conflict between stability and efficiency in the sense that if stable networks assume those shapes they would be efficient and if efficient networks assume those shapes, they would be stable with minimal further restrictions on value functions.

Topology synthesis of three-legged spherical parallel manipulators employing Lie group theory

Driven by the requirements of the bionic joint or tracking equipment for the spherical parallel manipulators (SPMs) with three rotational degrees-of-freedom (DoFs), this paper carries out the topology synthesis of a class of three-legged SPMs employing Lie group theory. In order to achieve the intersection of the displacement subgroups, the subgroup characteristics and operation principles are defined in this paper. Mainly drawing on the Lie group theory, the topology synthesis procedure of three-legged SPMs including four stages and two functional blocks is proposed, in which the assembly principles of three legs are defined. By introducing the circular track, a novel class of three-legged SPMs is synthesized, which is the important complement to the existing SPMs. Finally, four typical examples are given to demonstrate the finite displacements of the synthesized three-legged SPMs.

Parental leave regulations and the persistence of the male breadwinner model: Using fuzzy-set ideal type analysis to assess gender equality in an enlarged Europe

his paper uses fuzzy-set ideal type analysis to assess the conformity of European leave regulations to four theoretical ideal typical divisions of labour: male breadwinner, caregiver parity, universal breadwinner and universal caregiver. In contrast to the majority of previous studies, the focus of this analysis is on the extent to which leave regulations promote gender equality in the family and the transformation of traditional gender roles. The results of this analysis demonstrate that European countries cluster into five models that only partly coincide with countries’ geographical proximity. Second, none of the countries considered constitutes a universal caregiver model, while the male breadwinner ideal continues to provide the normative reference point for parental leave regulations in a large number of European states. Finally, we witness a growing emphasis at the national and EU levels concerning the universal breadwinner ideal, which leaves gender inequality in unpaid work unproblematized.

The CACTOS Vision of Context-Aware Cloud Topology Optimization and Simulation

Recent advances in hardware development coupled with the rapid adoption and broad applicability of cloud computing have introduced widespread heterogeneity in data centers, significantly complicating the management of cloud applications and data center resources. This paper presents the CACTOS approach to cloud infrastructure automation and optimization, which addresses heterogeneity through a combination of in-depth analysis of application behavior with insights from commercial cloud providers. The aim of the approach is threefold: to model applications and data center resources, to simulate applications and resources for planning and operation, and to optimize application deployment and resource use in an autonomic manner. The approach is based on case studies from the areas of business analytics, enterprise applications, and scientific computing.