Author contributions M.G., K.Y., and C.B-D. conceived the project. F.B. led CRISPR-Cas9 screening, codeveloped Project Score webportal, performed analyses, verified WRN dependency. F.I. led computational analyses and figure preparation, contributed to the Project Score webportal. G.P. performed experiments to verify WRN dependency, carried out analyses, contributed to in vivo studies. E.G. contributed to computational analysis and figures. D.vdM. contributed to developing the Project Score webportal. G.

Mechanistic information and structure-based design methods have been used to design a series of nonpeptide cyclic ureas that are potent inhibitors of human immunodeficiency virus (HIV) protease and HIV replication. A fundamental feature of these inhibitors is the cyclic urea carbonyl oxygen that mimics the hydrogen-bonding features of a key structural water molecule. The success of the design in both displacing and mimicking the structural water molecule was confirmed by x-ray crystallographic studies. Highly selective, preorganized inhibitors with relatively low molecular weight and high oral bioavailability were synthesized.

Atrophy of multifidus has been used as one of the rationales for spine stabilization exercises. The evidence of coexisting atrophy of psoas and multifidus suggests that a future area for study should be selective exercise training of psoas, which is less commonly used in clinical practice.

We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) (4) after lysis of the cells with detergent. A more detailed characterization of this DNA will be published later. Present information indicates that the Xdvgal (Xd-120) DNA is a circular dimer containing tandem duplications of a sequence of several X phage genes (including CI, 0, and P) joined to the entire galactose operon of E. coli (Berg, D., Mertz, J., & Jackson, D., in preparation). DNA concentrations are given as molecular concentrations.Enzymes. The circular SV40 and Xdvgal DNA molecules were cleaved with the bacterial restriction endonuclease RI (Yoshimori and Boyer, unpublished; the enzyme was generously made available to us by these workers). Phage X-exonuclease (given to us by Peter Lobban) was prepared according to Little et al. (5) Centrifugations. Alkaline sucrose gradients were formed by diffusion from equal volumes of 5, 10, 15, and 20% sucrose solutions with 2 mM EDTA containing, respectively, 0.2, 0.4, 0.6, and 0.8 M NaOH, and 0.8, 0.6, 0.4, 0.2 M NaCl. 100-,ul samples were run on 3.8-ml gradients in a Beckman SW56 Ti rotor in a Beckman L2-65B ultracentrifuge at 4°a nd 55,000 rpm for the indicated times. 2-to 10-drop fractions were collected onto 2.5-cm diameter Whatman 3MM discs, dried without washing, and counted in PPO-dimethyl POPOP-toluene scintillator in a Nuclear Chicago Mark II 2904

Objective To evaluate effectiveness of an exercise programme in a community setting for patients with low back pain to encourage a return to normal activities.

High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure−activity relationships of C 2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and “unbound” (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2‘ pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure−activity relationships, and molecular recognition of this series of C 2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2‘ is also discussed.

Capsaicin is a common dietary constituent and a popular homeopathic treatment for chronic pain. Exposure to capsaicin has been shown to cause various dose-dependent acute physiological responses including the sensation of burning and pain, respiratory depression, and death. In this study, the P450-dependent metabolism of capsaicin by recombinant P450 enzymes and hepatic and lung microsomes from various species, including humans, was determined. A combination of LC/MS, LC/MS/MS, and LC/NMR was used to identify several metabolites of capsaicin that were generated by aromatic (M5 and M7) and alkyl hydroxylation (M2 and M3), O-demethylation (M6), N- (M9) and alkyl dehydrogenation (M1 and M4), and an additional ring oxygenation of M9 (M8). Dehydrogenation of capsaicin was a novel metabolic pathway and produced unique macrocyclic, diene, and imide metabolites. Metabolism of capsaicin by microsomes was inhibited by the nonselective P450 inhibitor 1-aminobenzotriazole (1-ABT). Metabolism was catalyzed by CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Addition of GSH (2 mM) to microsomal incubations stimulated the metabolism of capsaicin and trapped several reactive electrophilic intermediates as their GSH adducts. These results suggested that reactive intermediates, which inactivated certain P450 enzymes, were produced during catalytic turnover. Comparison of the rate and types of metabolites produced from capsaicin and its analogue, nonivamide, demonstrated similar pathways in the P450-dependent metabolism of these two capsaicinoids. However, production of the dehydrogenated (M4), macrocyclic (M1), and omega-1-hydroxylated (M3) metabolites was not observed for nonivamide. These differences may be reflective of the mechanism of formation of these metabolites of capsaicin. The role of metabolism in the cytotoxicity of capsaicin and nonivamide was also assessed in cultured lung and liver cells. Lung cells were markedly more sensitive to cytotoxicity by capsaicin and nonivamide. Cytotoxicity was enhanced 5 and 40% for both compounds by 1-ABT in BEAS-2B and HepG2, respectively. These data suggested that metabolism of capsaicinoids by P450 in cells represented a detoxification mechanism (in contrast to bioactivation).

The 2014 Working Group on California Earthquake Probabilities (WGCEP 2014) presents time-dependent earthquake probabilities for the third Uniform California Earthquake Rupture Forecast (UCERF3). Building on the UCERF3 time-independent model published previously, renewal models are utilized to represent elasticrebound-implied probabilities. A new methodology has been developed that solves applicability issues in the previous approach for unsegmented models. The new methodology also supports magnitude-dependent aperiodicity and accounts for the historic open interval on faults that lack a date-of-last-event constraint. Epistemic uncertainties are represented with a logic tree, producing 5760 different forecasts. Results for a variety of evaluation metrics are presented, including logic-tree sensitivity analyses and comparisons to the previous model (UCERF2). For 30 yr M ≥ 6:7 probabilities, the most significant changes from UCERF2 are a threefold increase on the Calaveras fault and a threefold decrease on the San Jacinto fault. Such changes are due mostly to differences in the time-independent models (e.g., fault-slip rates), with relaxation of segmentation and inclusion of multifault ruptures being particularly influential. In fact, some UCERF2 faults were simply too long to produce M 6.7 size events given the segmentation assumptions in that study. Probability model differences are also influential, with the implied gains (relative to a Poisson model) being generally higher in UCERF3. Accounting for the historic open interval is one reason. Another is an effective 27% increase in the total elastic-rebound-model weight. The exact factors influencing differences between UCERF2 and UCERF3, as well as the relative importance of logic-tree branches, vary throughout the region and depend on the evaluation metric of interest. For example, M ≥ 6:7 probabilities may not be a good proxy for other hazard or loss measures. This sensitivity, coupled with the approximate nature of the model and known limitations, means the applicability of UCERF3 should be evaluated on a case-by-case basis.