Tryptophan Fluorescence Quenching in β-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by l,d-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model l,d-transpeptidase (Ldt) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldt. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation

Expanding the scope of the successive ring expansion strategy for macrocycle and medium-sized ring synthesis : unreactive and reactive lactams

New methods are described that expand the scope of the Successive Ring Expansion (SuRE) with respect to synthetically challenging lactams. A protocol has been developed for use with 'unreactive' lactams, enabling SuRE reactions to be performed on subsrates that fail under previously established conditions. Ring expansion is also demonstarted on 'reactive' lactams derived from iminosugars for the first time. The new SuRE methods were used to prepare a diverse array of medium-sized and macrocyclic lactams and lactones, which were evaluted in an anti-bacterial assay against E. coli BW25113WT

Research into the efficacy and cost-effectiveness of brief, free of charge and anonymous sex counselling to improve (mental) health in youth: Design of a randomised controlled trial

<p>Abstract</p> <p>Background</p> <p>The capacity to form romantic relationships and sexual health of adolescents in the Netherlands are compromised by several factors, including young age of first intercourse and adolescent depression. Several thresholds like own expenses, trust and embarrassment prevent adolescents to seek help for their sexual problems. To overcome these thresholds, brief sex counselling has been developed. It has been used since 2006 within the Rotterdam-Rijnmond Public Health Service, but there is lack of information about the (cost-) effectiveness. In the current study we will evaluate the (cost-) effectiveness of brief sex counselling for sexual problems in adolescents and young adults between 18 and 25 years of age.</p> <p>Methods</p> <p>In a randomised controlled trial we will compare (1) brief sex counselling with (2) intensive sexological treatment, and (3) delayed treatment (waiting list). Embedded in this RCT will be a trial-based economic evaluation, looking at the cost-effectiveness and cost-utility of brief sex counselling versus the two other interventions. Four hundred fifty adolescents (aged 18-25) with sexual problems will be recruited among the persons who visit the Public Health Service (PHS) and through various websites. After a screening procedure, eligible participants will be randomly allocated to one of the three intervention groups. Primary outcome measure of the clinical evaluation is the severity of sexual problems. Other outcomes include psychological distress, especially depression. The economic evaluation will be performed from a societal perspective. Costs will be assessed continuously by a retrospective questionnaire covering the last 3 month. All outcome assessments (including those for the economic evaluation) will take place via the internet at baseline, and at 3, 6, 9, and 12 months after baseline.</p> <p>Discussion</p> <p>The proposed research project will be the first study to provide preliminary data about the effect and cost-effectiveness of brief sex counselling in youth in comparison with intensive sexological treatment and delayed treatment. It is anticipated that positive results in (cost-) effectiveness of the proposed intervention will contribute to the improvement of sexual health care for adolescents and young adults.</p> <p>Trial registration</p> <p>The study has been registered at the Netherlands Trial Register, part of the Dutch Cochrane Centre (NTR1952)</p

Discovery of Inhibitors of Leishmania β-1,2-Mannosyltransferases Using a Click-Chemistry-Derived Guanosine Monophosphate Library

Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose–dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-d-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism

The diagnosis of dysplasia of the esophagus with vital staining using Lugol solution

Chez un homme de 70 ans, dysplasie disparaissant sous l'effet du traitement médicamenteux (métoclopramide et antiacides

Selective Anti-Markovnikov Cyclization and Hydrofluorination2 Reaction in Superacid HF/SbF5: A Tool in the Design of Nitrogen-Containing (Fluorinated) Polycyclic Systems

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