Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane of all Gram-negative bacteria, and it is one of the signature molecules recognized by the receptors of the innate immune system. In addition to its lipid A portion (the endotoxin), its O-chain polysaccharide (the O-antigen) plays a critical role in the bacterium-host interplay and, in a number of bacterial pathogens, it is a virulence factor. We present evidence that, in Yersinia enterocolitica serotype O:8, a complex signalling network regulates O-antigen expression in response to temperature. Northern blotting and reporter fusion analyses indicated that temperature regulates the O-antigen expression at the transcriptional level. Promoter cloning showed that the O-antigen gene cluster contains two transcriptional units under the control of promoters P(wb1) and P(wb2). The activity of both promoters is under temperature regulation and is repressed in bacteria grown at 37 degrees C. We demonstrate that the RosA/RosB efflux pump/potassium antiporter system and Wzz, the O-antigen chain length determinant, are indirectly involved in the regulation mainly affecting the activity of promoter P(wb2). The rosAB transcription, under the control of P(ros), is activated at 37 degrees C, and P(wb2) is repressed through the signals generated by the RosAB system activation, i.e. decreased [K+] and increased [H+]. The wzz transcription is under the control of P(wb2), and we show that, at 37 degrees C, overexpression of Wzz downregulates slightly the P(wb1) and P(wb2) activities and more strongly the P(ros) activity, with the net result that more O-antigen is produced. Finally, we demonstrate that overexpression of Wzz causes membrane stress that activates the CpxAR two-component signal transduction system.

Fragmentation of Neutral Amino Acids and Small Peptides by Intense, Femtosecond Laser Pulses

High power femtosecond laser pulses have unique properties that could lead to their application as ionization or activation sources in mass spectrometry. By concentrating many photons into pulse lengths approaching the timescales associated with atomic motion, very strong electric field strengths are generated, which can efficiently ionize and fragment molecules without the need for resonant absorption. However, the complex interaction between these pulses and biomolecular species is not well understood. To address this issue, we have studied the interaction of intense, femtosecond pulses with a number of amino acids and small peptides. Unlike previous studies, we have used neutral forms of these molecular targets, which allowed us to investigate dissociation of radical cations without the spectra being complicated by the action of mobile protons. We found fragmentation was dominated by fast, radical-initiated dissociation close to the charge site generated by the initial ionization or from subsequent ultrafast migration of this charge. Fragments with lower yields, which are useful for structural determinations, were also observed and attributed to radical migration caused by hydrogen atom transfer within the molecule.

Th1 not Th17 cells drive spontaneous MS-like disease despite a functional regulatory T cell response

Multiple sclerosis is considered a disease of complex autoimmune etiology, yet there remains a lack of consensus as to specific immune effector mechanisms. Recent analyses of experimental autoimmune encephalomyelitis, the common mouse model of multiple sclerosis, have investigated the relative contribution of Th1 and Th17 CD4 T cell subsets to initial autoimmune central nervous system (CNS) damage. However, inherent in these studies are biases influenced by the adjuvant and toxin needed to break self-tolerance. We investigated spontaneous CNS disease in a clinically relevant, humanized, T cell receptor transgenic mouse model. Mice develop spontaneous, ascending paralysis, allowing unbiased characterization of T cell immunity in an HLA-DR15-restricted T cell repertoire. Analysis of naturally progressing disease shows that IFN?(+) cells dominate disease initiation with IL-17(+) cells apparent in affected tissue only once disease is established. Tregs accumulate in the CNS but are ultimately ineffective at halting disease progression. However, ablation of Tregs causes profound acceleration of disease, with uncontrolled infiltration of lymphocytes into the CNS. This synchronous, severe disease allows characterization of the responses that are deregulated in exacerbated disease: the correlation is with increased CNS CD4 and CD8 IFN? responses. Recovery of the ablated Treg population halts ongoing disease progression and Tregs extracted from the central nervous system at peak disease are functionally competent to regulate myelin specific T cell responses. Thus, in a clinically relevant mouse model of MS, initial disease is IFN? driven and the enhanced central nervous system responses unleashed through Treg ablation comprise IFN? cytokine production by CD4 and CD8 cells, but not IL-17 responses.

Multiple Regulatory Pathways Associated with High-Level Ciprofloxacin and Multidrug Resistance in Salmonella enterica Serovar Enteritidis: Involvement of ramA and Other Global Regulators

Mechanisms of antibiotic resistance were examined in nalidixic acid-resistant Salmonella enterica serovar Enteritidis field isolates displaying decreased susceptibility to ciprofloxacin and in in vitro-derived ciprofloxacin-resistant mutants (104-cip and 5408-cip). All field isolates harbored a single gyrA mutation (D87Y). Deletion of acrB and complementation with wild-type gyrA increased quinolone susceptibility. Selection for ciprofloxacin resistance was associated with the development of an additional gyrA (S83F) mutation in 104-cip, novel gyrB (E466D) and parE (V461G) mutations in 5408-cip, overexpression of acrB and decreased susceptibility to nonquinolone antibiotics in both mutants, and decreased OmpF production and altered lipopoly- saccharide in 104-cip. Complementation of mutated gyrA and gyrB with wild-type alleles restored susceptibility to quinolones in 104-cip and significantly decreased the ciprofloxacin MIC in 5408-cip. Complementation of parE had no effect on quinolone MICs. Deletion of acrB restored susceptibility to ciprofloxacin and other antibiotics tested. Both soxS and marA were overexpressed in 104-cip, and ramA was overexpressed in 5408-cip. Inactivation of each of these global regulators lowered ciprofloxacin MICs, decreased expression of acrB, and restored susceptibility to other antibiotics. Mutations were found in soxR (R20H) and in soxS (E52K) in 104-cip and in ramR (G25A) in 5408-cip. In conclusion, both efflux activity and a single gyrA mutation contribute to nalidixic acid resistance and reduced ciprofloxacin sensitivity. Ciprofloxacin resistance and decreased susceptibility to multiple antibiotics can result from different genetic events leading to development of target gene mutations, increased efflux activity resulting from differential expression of global regulators associated with mutations in their regulatory genes, and possible altered membrane permeability.

Influence of the experimental design of gene expression studies on the inference of gene regulatory networks: environmental factors

The inference of gene regulatory networks gained within recent years a considerable interest in the biology and biomedical community. The purpose of this paper is to investigate the influence that environmental conditions can exhibit on the inference performance of network inference algorithms. Specifically, we study five network inference methods, Aracne, BC3NET, CLR, C3NET and MRNET, and compare the results for three different conditions: (I) observational gene expression data: normal environmental condition, (II) interventional gene expression data: growth in rich media, (III) interventional gene expression data: normal environmental condition interrupted by a positive spike-in stimulation. Overall, we find that different statistical inference methods lead to comparable, but condition-specific results. Further, our results suggest that non-steady-state data enhance the inferability of regulatory networks.

Attitudes and Ethics: Evaluating Knowledge and Regulatory Constructs in Planning Enforcement Practice

The departure point for the paper is the need to scrutinise previously unconsidered dimensions which are fundamental to understanding the dynamics of the planning enforcement system. Drawing upon emerging themes in regulation theory the paper fuses these with knowledge constructs. The rationale is that regulatory regimes must be informed by knowledge imparted from a range of sources and the resultant quality of decision making is inextricably linked to the robustness and completeness of the evidence base collated.
The theoretical analysis, coupled with proposed radical legislative changes, provides a lens for an empirical investigation which scrutinises tactics, strategies, operational mechanisms, attitudinal dimensions and ethics with a view to identifying key factors impacting upon enforcement efficacy. Prizes and pitfalls are identified in the course of the analysis and evaluation, with evidence-based remedies suggested where appropriate. The paper concludes by reflecting on the importance of theoretical synergy, epistemological advancement, taking cognisance of ethical and attitudinal challenges facing the planning profession; and, stresses the importance of identifying and bringing to book those who flagrantly breach the Code of Professional Conduct.

In pursuit of regulatory compliance: a study of planning enforcement structures in Northern Ireland

As devolution expands across the UK, Northern Ireland (NI) is witnessing the development of new architecture to devolve planning powers. With serious criticism targeted at the legislative provisions for enforcement, this investigation endeavours to assess the robustness of the planning framework through a synergy of theory, law and practice. The paper demonstrates the value of theory in not only supplying a lens that allows both legislative frameworks and praxis to be deconstructed, but also in enabling the identification and scrutiny of underlying problems that pervade the system.

Interferon regulatory factor (IRF) 3 is critical for the development of experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is an animal model of autoimmune inflammatory demyelination that is mediated by Th1 and Th17 cells. The transcription factor interferon regulatory factor 3 (IRF3) is activated by pathogen recognition receptors and induces interferon-beta production.

METHODS: To determine the role of IRF3 in autoimmune inflammation, we immunised wild-type (WT) and irf3-/- mice to induce EAE. Splenocytes from WT and irf3-/- mice were also activated in vitro in Th17-polarising conditions.

RESULTS: Clinical signs of disease were significantly lower in mice lacking IRF3, with reduced Th1 and Th17 cells in the central nervous system. Peripheral T-cell responses were also diminished, including impaired proliferation and Th17 development in irf3-/- mice. Myelin-reactive CD4+ cells lacking IRF3 completely failed to transfer EAE in Th17-polarised models as did WT cells transferred into irf3-/- recipients. Furthermore, IRF3 deficiency in non-CD4+ cells conferred impairment of Th17 development in antigen-activated cultures.

CONCLUSION: These data show that IRF3 plays a crucial role in development of Th17 responses and EAE and warrants investigation in human multiple sclerosis.

Explaining the Stunted Rise of Macroprudential Regulatory Philosophies

In the aftermath of the financial crash of 2008, policy makers operating in international financial regulatory networks discovered macroprudential regulation (MPR), but macroprudential regulation has had a stunted or arrested development that can be explained with reference to five factors that are recounted in this article

The Effect of the Secret Ballot on Party System Fragmentation: A Test of Three Competing Arguments

Few studies have examined the impact that the adoption of the secret ballot had on party system fragmentation outside the United States. This article tests arguments maintaining that the adoption of the secret ballot had a negative, positive or nil effect on party system fragmentation. Using time-series cross-sectional data from
several countries adopting the secret ballot around the turn of the twentieth century, the results demonstrate that the adoption of the secret ballot did not hinder – though did not favour, either – the development of multiparty systems.

Regulating Services Trade Agreements - A Comparative Analysis of Regulatory Disciplines Included in EU and US Free Trade Agreements

Developed countries, led by the EU and the US, have consistently called for ‘deeper integration’ over the course of the past three decades i.e., the convergence of ‘behind-the-border’ or domestic polices and rules such as services, competition, public procurement, intellectual property (“IP”) and so forth. Following the collapse of the Doha Development Round, the EU and the US have pursued this push for deeper integration by entering into deep and comprehensive free trade agreements (“DCFTAs”) that are comprehensive insofar as they are not limited to tariffs but extend to regulatory trade barriers. More recently, the EU and the US launched negotiations on a Transatlantic Trade and Investment Partnership (“TTIP”) and a Trade in Services Agreement (“TISA”), which put tackling barriers resulting from divergences in domestic regulation in the area of services at the very top of the agenda. Should these agreements come to pass, they may well set the template for the rules of international trade and define the core features of domestic services market regulation. This article examines the regulatory disciplines in the area of services included in existing EU and US DCFTAs from a comparative perspective in order to delineate possible similarities and divergences and assess the extent to which these DCFTAs can shed some light into the possible outcome and limitations of future trade negotiations in services. It also discusses the potential impact of such negotiations on developing countries and, more generally, on the multilateral process.

Autonomous collective agreements as a regulatory device in European labour law:How to read Article 139 EC

This article discusses whether European social partners can derive the competence to autonomously devise European collective labour agreements from Article 139 EC (equals Article III-212 Constitution of Europe). Placing the question in the context of discussions of EU governance and private lawmaking in general, the author starts with a comparative overview of legal conceptions for collective labour agreements in Europe, focusing on three Member States' orders where their effects are not or only partly regulated by state legislation. Based on this comparison, she analyses Article 139(2) and offers a new interpretation of its provisions concerning autonomous implementation of European social partner agreements. She concludes that European social partners do have the competence to agree on a basic agreement stating the rules for European collective bargaining autonomously.