Identification of Exosite-Targeting Inhibitors of Anthrax Lethal Factor by High-Throughput Screening

SummaryProtease inhibitor discovery has focused almost exclusively on compounds that bind to the active site. Inhibitors targeting protease exosites, regions outside of the active site that influence catalysis, offer potential advantages of increased specificity but are difficult to systematically discover. Here, we describe an assay suitable for detecting exosite-targeting inhibitors of the metalloproteinase anthrax lethal factor (LF) based on cleavage of a full-length mitogen-activated protein kinase kinase (MKK) substrate. We used this assay to screen a small-molecule library and then subjected hits to a secondary screen to exclude compounds that efficiently blocked cleavage of a peptide substrate. We identified a compound that preferentially inhibited cleavage of MKKs compared with peptide substrates and could suppress LF-induced macrophage cytolysis. This approach should be generally applicable to the discovery of exosite-targeting inhibitors of many additional proteases

Conceptual and methodological challenges to measuring political commitment to respond to HIV

Background: Researchers have long recognized the importance of a central government’s political “commitment” in order to mount an effective response to HIV. The concept of political commitment remains ill-defined, however, and little guidance has been given on how to measure this construct and its relationship with HIV-related outcomes. Several countries have experienced declines in HIV infection rates, but conceptual difficulties arise in linking these declines to political commitment as opposed to underlying social and behavioural factors. Methods: This paper first presents a critical review of the literature on existing efforts to conceptualize and measure political commitment to respond to HIV and the linkages between political commitment and HIV-related outcomes. Based on the elements identified in this review, the paper then develops and presents a framework to assist researchers in making choices about how to assess a government's level of political commitment to respond to HIV and how to link political commitment to HIV-related outcomes. Results: The review of existing studies identifies three components of commitment (expressed, institutional and budgetary commitment) as different dimensions along which commitment can be measured. The review also identifies normative and ideological aspects of commitment and a set of variables that mediate and moderate political commitment that need to be accounted for in order to draw valid inferences about the relationship between political commitment and HIV-related outcomes. The framework summarizes a set of steps that researchers can follow in order to assess a government's level of commitment to respond to HIV and suggests ways to apply the framework to country cases. Conclusions: Whereas existing studies have adopted a limited and often ambiguous conception of political commitment, we argue that conceiving of political commitment along a greater number of dimensions will allow researchers to draw a more complete picture of political commitment to respond to HIV that avoids making invalid inferences about the relationship between political commitment and HIV outcomes

Mechanistic basis of an epistatic interaction reducing age at onset in hereditary spastic paraplegia

Many genetic neurological disorders exhibit variable expression within affected families, often exemplified by variations in disease age at onset. Epistatic effects (i.e. effects of modifier genes on the disease gene) may underlie this variation, but the mechanistic basis for such epistatic interactions is rarely understood. Here we report a novel epistatic interaction between SPAST and the contiguous gene DPY30, which modifies age at onset in hereditary spastic paraplegia, a genetic axonopathy. We found that patients with hereditary spastic paraplegia caused by genomic deletions of SPAST that extended into DPY30 had a significantly younger age at onset. We show that, like spastin, the protein encoded by SPAST, the DPY30 protein controls endosomal tubule fission, traffic of mannose 6-phosphate receptors from endosomes to the Golgi, and lysosomal ultrastructural morphology. We propose that additive effects on this pathway explain the reduced age at onset of hereditary spastic paraplegia in patients who are haploinsufficient for both genes.This work was supported by grants to E.R.; Project Grant from United States Spastic Paraplegia Foundation, UK Medical Research Council Project Grant [MR/M00046X/1], Project grant from NIHR Biomedical Research Centre at Addenbrooke’s Hospital, Wellcome Trust Senior Research Fellowship in Clinical Science [082381], Project Grant from Tom Wahlig Stiftung (project 33). J.E. and P.M. are supported by a Wellcome Trust Principal Research Fellowship Grant to Margaret S. Robinson [086598]. T.M.N. was supported by an MRC PhD studentship [G0800117]. B.W. is supported by the Tom Wahlig Advanced Fellowship, the German Federal Ministry of Education and Research (BMBF, 01GQ113), the Bavarian Ministry of Education and Culture, Sciences and Arts in the framework of the Bavarian Molecular Biosystems Research Network and ForIPS, and the Interdisciplinary Centre for Clinical Research (IZKF, University Hospital of Erlangen, N3 and F3). T.R. was supported by research grant DFG GRK2162/1 of the Deutsche Forschungsgemeinschaft. The study was also supported by the European Union within the 7th European Community Framework Program for Research and Technological Development through funding for the NEUROMICS network (F5-2012-305121 to L.S. and A.D.), the E-Rare Network NEUROLIPID (01GM1408B to R.S. and ANR-13-RARE-0003-02 to G.S.), and a Marie Curie International Outgoing Fellowship (grant PIOF-GA-2012-326681 to R.S. and L.S.). This work was further supported by the US National Institutes of Health (NIH) (grant 5R01NS072248 to R.S.), the German HSP-Selbsthilfegruppe e.V. (grant to R.S. and L.S.), and grants to C.B.: Project Grant from Tom Wahlig Stiftung (project 20), grant from the Stiftung für Pathobiochemie und Molekulare Diagnostik. CIMR is supported by a Wellcome Trust Strategic Award [100140] and Equipment Grant [093026]

Measurement of the hadronic photon structure function F_{2}^{γ} at LEP2

The hadronic structure function of the photon F_{2}^{γ} (x, Q²) is measured as a function of Bjorken x and of the photon virtuality Q² using deep-inelastic scattering data taken by the OPAL detector at LEP at e⁺e⁻ centre-of-mass energies from 183 to 209 GeV. Previous OPAL measurements of the x dependence of F_{2}^{γ} are extended to an average Q² of 〈Q²〉=780 GeV² using data in the kinematic range 0.15<x<0.98. The Q² evolution of F_{2}^{γ} is studied for 12.1<〈Q²〉<780 GeV² using three ranges of x. As predicted by QCD, the data show positive scaling violations in F_{2}^{γ} with F_{2}^{γ} (Q²)/α = (0.08±0.02⁺⁰·⁰⁵_₀.₀₃) + (0.13±0.01⁺⁰·⁰¹_₀.₀₁) lnQ², where Q² is in GeV², for the central x region 0.10–0.60. Several parameterisations of F_{2}^{γ} are in qualitative agreement with the measurements whereas the quark-parton model prediction fails to describe the data

Measurement of the charm structure function F_{2,c)^{γ} of the photon at LEP

The production of charm quarks is studied in deep-inelastic electron–photon scattering using data recorded by the OPAL detector at LEP at nominal e⁺e⁻ centre-of-mass energies from 183 to 209 GeV. The charm quarks have been identified by full reconstruction of charged D* mesons using their decays into D⁰π with the D⁰ observed in two decay modes with charged particle final states, Kπ and Kπππ. The cross-section σ^{D*} for production of charged D* in the reaction e⁺e⁻→e⁺e⁻D*Χ is measured in a restricted kinematical region using two bins in Bjorken x, 0.00140.1 the perturbative QCD calculation at next-to-leading order agrees perfectly with the measured cross-section. For x<0.1 the measured cross-section is 43.8±14.3±6.3±2.8 pb with a next-to-leading order prediction of 17.0⁺²·⁹_₂.₃ pb

Measurement of triple gauge boson couplings from W⁺W⁻ production at LEP energies up to 189 GeV

A measurement of triple gauge boson couplings is presented, based on W-pair data recorded by the OPAL detector at LEP during 1998 at a centre-of-mass energy of 189 GeV with an integrated luminosity of 183 pb⁻¹. After combining with our previous measurements at centre-of-mass energies of 161–183 GeV we obtain κ = 0.97_{-0.16}^{+0.20}, g_{1}^{z} = 0.991_{-0.057}^{+0.060} and λ = -0.110_{-0.055}^{+0.058}, where the errors include both statistical and systematic uncertainties and each coupling is determined by setting the other two couplings to their Standard Model values. These results are consistent with the Standard Model expectations

Measurement of the partial widths of the Z into up- and down-type quarks

Using the entire OPAL LEP1 on-peak Z hadronic decay sample, Z -> qbarq gamma decays were selected by tagging hadronic final states with isolated photon candidates in the electromagnetic calorimeter. Combining the measured rates of Z -> qbarq gamma decays with the total rate of hadronic Z decays permits the simultaneous determination of the widths of the Z into up- and down-type quarks. The values obtained, with total errors, were Gamma u = 300 ^{+19}_{-18} MeV and Gamma d = 381 ^{+12}_{-12} MeV. The results are in good agreement with the Standard Model expectation.Comment: 22 pages, 5 figures, Submitted to Phys. Letts.

Search for Yukawa Production of a Light Neutral Higgs Boson at LEP

Within a Two-Higgs-Doublet Model (2HDM) a search for a light Higgs boson in the mass range of 4-12 GeV has been performed in the Yukawa process e+e- -> b bbar A/h -> b bbar tau+tau-, using the data collected by the OPAL detector at LEP between 1992 and 1995 in e+e- collisions at about 91 GeV centre-of-mass energy. A likelihood selection is applied to separate background and signal. The number of observed events is in good agreement with the expected background. Within a CP-conserving 2HDM type II model the cross-section for Yukawa production depends on xiAd = |tan beta| and xihd = |sin alpha/cos beta| for the production of the CP-odd A and the CP-even h, respectively, where tan beta is the ratio of the vacuum expectation values of the Higgs doublets and alpha is the mixing angle between the neutral CP-even Higgs bosons. From our data 95% C.L. upper limits are derived for xiAd within the range of 8.5 to 13.6 and for xihd between 8.2 to 13.7, depending on the mass of the Higgs boson, assuming a branching fraction into tau+tau- of 100%. An interpretation of the limits within a 2HDM type II model with Standard Model particle content is given. These results impose constraints on several models that have been proposed to explain the recent BNL measurement of the muon anomalous magnetic moment.Comment: 24 pages, 9 figures, Submitted to Euro. Phys. J.

Tests of model of color reconnection and a search for glueballs using gluon jets with a rapidity gap

Gluon jets with a mean energy of 22 GeV and purity of 95% are selected from hadronic Z0 decay events produced in e+e- annihilations. A subsample of these jets is identified which exhibits a large gap in the rapidity distribution of particles within the jet. After imposing the requirement of a rapidity gap, the gluon jet purity is 86%. These jets are observed to demonstrate a high degree of sensitivity to the presence of color reconnection, i.e. higher order QCD processes affecting the underlying color structure. We use our data to test three QCD models which include a simulation of color reconnection: one in the Ariadne Monte Carlo, one in the Herwig Monte Carlo, and the other by Rathsman in the Pythia Monte Carlo. We find the Rathsman and Ariadne color reconnection models can describe our gluon jet measurements only if very large values are used for the cutoff parameters which serve to terminate the parton showers, and that the description of inclusive Z0 data is significantly degraded in this case. We conclude that color reconnection as implemented by these two models is disfavored. The signal from the Herwig color reconnection model is less clear and we do not obtain a definite conclusion concerning this model. In a separate study, we follow recent theoretical suggestions and search for glueball-like objects in the leading part of the gluon jets. No clear evidence is observed for these objects.Comment: 42 pages, 18 figure

Search for the Standard Model Higgs Boson with the OPAL Detector at LEP

This paper summarises the search for the Standard Model Higgs boson in e+e- collisions at centre-of-mass energies up to 209 GeV performed by the OPAL Collaboration at LEP. The consistency of the data with the background hypothesis and various Higgs boson mass hypotheses is examined. No indication of a signal is found in the data and a lower bound of 112.7GeV/C^2 is obtained on the mass of the Standard Model Higgs boson at the 95% CL.Comment: 51 pages, 21 figure