Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery

Does inter-vertebral range of motion increase after spinal manipulation? A prospective cohort study.

Background: Spinal manipulation for nonspecific neck pain is thought to work in part by improving inter-vertebral range of motion (IV-RoM), but it is difficult to measure this or determine whether it is related to clinical outcomes. Objectives: This study undertook to determine whether cervical spine flexion and extension IV-RoM increases after a course of spinal manipulation, to explore relationships between any IV-RoM increases and clinical outcomes and to compare palpation with objective measurement in the detection of hypo-mobile segments. Method: Thirty patients with nonspecific neck pain and 30 healthy controls matched for age and gender received quantitative fluoroscopy (QF) screenings to measure flexion and extension IV-RoM (C1-C6) at baseline and 4-week follow-up between September 2012-13. Patients received up to 12 neck manipulations and completed NRS, NDI and Euroqol 5D-5L at baseline, plus PGIC and satisfaction questionnaires at follow-up. IV-RoM accuracy, repeatability and hypo-mobility cut-offs were determined. Minimal detectable changes (MDC) over 4 weeks were calculated from controls. Patients and control IV-RoMs were compared at baseline as well as changes in patients over 4 weeks. Correlations between outcomes and the number of manipulations received and the agreement (Kappa) between palpated and QF-detected of hypo-mobile segments were calculated. Results: QF had high accuracy (worst RMS error 0.5o) and repeatability (highest SEM 1.1o, lowest ICC 0.90) for IV-RoM measurement. Hypo-mobility cut offs ranged from 0.8o to 3.5o. No outcome was significantly correlated with increased IV-RoM above MDC and there was no significant difference between the number of hypo-mobile segments in patients and controls at baseline or significant increases in IV-RoMs in patients. However, there was a modest and significant correlation between the number of manipulations received and the number of levels and directions whose IV-RoM increased beyond MDC (Rho=0.39, p=0.043). There was also no agreement between palpation and QF in identifying hypo-mobile segments (Kappa 0.04-0.06). Conclusions: This study found no differences in cervical sagittal IV-RoM between patients with non-specific neck pain and matched controls. There was a modest dose-response relationship between the number of manipulations given and number of levels increasing IV-RoM - providing evidence that neck manipulation has a mechanical effect at segmental levels. However, patient-reported outcomes were not related to this

Tetrahydroisoquinolines affect the whole-cell phenotype of Mycobacterium tuberculosis by inhibiting the ATP-dependent MurE ligase

Objectives (S)-Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñan, was found to inhibit the growth of Mycobacterium tuberculosis H37Rv. A biomimetic approach for the chemical synthesis of a wide array of 1-substituted tetrahydroisoquinolines was undertaken with the aim of elucidating a common pharmacophore for these compounds with novel mode(s) of anti-TB action. Methods Biomimetic Pictet–Spengler or Bischler–Napieralski synthetic routes were employed followed by an evaluation of the biological activity of the synthesized compounds. Results In this work, the synthesized tetrahydroisoquinolines were found to inhibit the growth of M. tuberculosis H37Rv and affect its whole-cell phenotype as well as the activity of the ATP-dependent MurE ligase, a key enzyme involved in the early stage of cell wall peptidoglycan biosynthesis. Conclusions As the correlation between the MIC and the half-inhibitory enzymatic concentration was not particularly strong, there is a credible possibility that these compounds have pleiotropic mechanism(s) of action in M. tuberculosis

Optimal and Long-Term Dynamic Transport Policy Design: Seeking Maximum Social Welfare through a Pricing Scheme.

This article presents an alternative approach to the decision-making process in transport strategy design. The study explores the possibility of integrating forecasting, assessment and optimization procedures in support of a decision-making process designed to reach the best achievable scenario through mobility policies. Long-term evaluation, as required by a dynamic system such as a city, is provided by a strategic Land-Use and Transport Interaction (LUTI) model. The social welfare achieved by implementing mobility LUTI model policies is measured through a cost-benefit analysis and maximized through an optimization process throughout the evaluation period. The method is tested by optimizing a pricing policy scheme in Madrid on a cordon toll in a context requiring system efficiency, social equity and environmental quality. The optimized scheme yields an appreciable increase in social surplus through a relatively low rate compared to other similar pricing toll schemes. The results highlight the different considerations regarding mobility impacts on the case study area, as well as the major contributors to social welfare surplus. This leads the authors to reconsider the cost-analysis approach, as defined in the study, as the best option for formulating sustainability measures

Observation of the Baryonic Flavor-Changing Neutral Current Decay Lambda_b -> Lambda mu+ mu-

We report the first observation of the baryonic flavor-changing neutral current decay Lambda_b -> Lambda mu+ mu- with 24 signal events and a statistical significance of 5.8 Gaussian standard deviations. This measurement uses ppbar collisions data sample corresponding to 6.8fb-1 at sqrt{s}=1.96TeV collected by the CDF II detector at the Tevatron collider. The total and differential branching ratios for Lambda_b -> Lambda mu+ mu- are measured. We find B(Lambda_b -> Lambda mu+ mu-) = [1.73+-0.42(stat)+-0.55(syst)] x 10^{-6}. We also report the first measurement of the differential branching ratio of B_s -> phi mu+ mu- using 49 signal events. In addition, we report branching ratios for B+ -> K+ mu+ mu-, B0 -> K0 mu+ mu-, and B -> K*(892) mu+ mu- decays.Comment: 8 pages, 2 figures, 4 tables. Submitted to Phys. Rev. Let

Stress-Induced Reinstatement of Drug Seeking: 20 Years of Progress

In human addicts, drug relapse and craving are often provoked by stress. Since 1995, this clinical scenario has been studied using a rat model of stress-induced reinstatement of drug seeking. Here, we first discuss the generality of stress-induced reinstatement to different drugs of abuse, different stressors, and different behavioral procedures. We also discuss neuropharmacological mechanisms, and brain areas and circuits controlling stress-induced reinstatement of drug seeking. We conclude by discussing results from translational human laboratory studies and clinical trials that were inspired by results from rat studies on stress-induced reinstatement. Our main conclusions are (1) The phenomenon of stress-induced reinstatement, first shown with an intermittent footshock stressor in rats trained to self-administer heroin, generalizes to other abused drugs, including cocaine, methamphetamine, nicotine, and alcohol, and is also observed in the conditioned place preference model in rats and mice. This phenomenon, however, is stressor specific and not all stressors induce reinstatement of drug seeking. (2) Neuropharmacological studies indicate the involvement of corticotropin-releasing factor (CRF), noradrenaline, dopamine, glutamate, kappa/dynorphin, and several other peptide and neurotransmitter systems in stress-induced reinstatement. Neuropharmacology and circuitry studies indicate the involvement of CRF and noradrenaline transmission in bed nucleus of stria terminalis and central amygdala, and dopamine, CRF, kappa/dynorphin, and glutamate transmission in other components of the mesocorticolimbic dopamine system (ventral tegmental area, medial prefrontal cortex, orbitofrontal cortex, and nucleus accumbens). (3) Translational human laboratory studies and a recent clinical trial study show the efficacy of alpha-2 adrenoceptor agonists in decreasing stress-induced drug craving and stress-induced initial heroin lapse

PAAR-repeat proteins sharpen and diversify the Type VI secretion system spike

The bacterial type VI secretion system (T6SS) is a large multi-component, dynamic macromolecular machine that plays an important role in the ecology of many Gram negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells1-5. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike6,7. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (Proline-Alanine-Alanine-aRginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins function to sharpen the tip of the VgrG spike. We demonstrate that PAAR proteins are essential for T6SS- mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results suggest a new model of the T6SS organelle in which the VgrG-PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event

Measurement of the W+W−W^+W^- Production Cross Section and Search for Anomalous WWγWW\gamma and WWZWWZ Couplings in ppˉp \bar p Collisions at s=1.96\sqrt{s} = 1.96 TeV

This Letter describes the current most precise measurement of the $W$ boson pair production cross section and most sensitive test of anomalous $WW\gamma$ and $WWZ$ couplings in $p \bar p$ collisions at a center-of-mass energy of 1.96 TeV. The $WW$ candidates are reconstructed from decays containing two charged leptons and two neutrinos, where the charged leptons are either electrons or muons. Using data collected by the CDF II detector from 3.6 fb$^{-1}$ of integrated luminosity, a total of 654 candidate events are observed with an expected background contribution of $320 \pm 47$ events. The measured total cross section is $\sigma (p \bar p \to W^+ W^- + X) = 12.1 \pm 0.9 \textrm{(stat)} ^{+1.6}_{-1.4} \textrm{(syst)}$ pb, which is in good agreement with the standard model prediction. The same data sample is used to place constraints on anomalous $WW\gamma$ and $WWZ$ couplings.Comment: submitted to Phys. Rev. Let

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

BACKGROUND: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals. RESULTS: For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate. CONCLUSION: We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates

Prebiotic synthesis of phosphoenol pyruvate by α-phosphorylation-controlled triose glycolysis

Phosphoenol pyruvate is the highest-energy phosphate found in living organisms and is one of the most versatile molecules in metabolism. Consequently, it is an essential intermediate in a wide variety of biochemical pathways, including carbon fixation, the shikimate pathway, substrate-level phosphorylation, gluconeogenesis and glycolysis. Triose glycolysis (generation of ATP from glyceraldehyde 3-phosphate via phosphoenol pyruvate) is among the most central and highly conserved pathways in metabolism. Here, we demonstrate the efficient and robust synthesis of phosphoenol pyruvate from prebiotic nucleotide precursors, glycolaldehyde and glyceraldehyde. Furthermore, phosphoenol pyruvate is derived within an α-phosphorylation controlled reaction network that gives access to glyceric acid 2-phosphate, glyceric acid 3-phosphate, phosphoserine and pyruvate. Our results demonstrate that the key components of a core metabolic pathway central to energy transduction and amino acid, sugar, nucleotide and lipid biosyntheses can be reconstituted in high yield under mild, prebiotically plausible conditions