Reconstruction of bacterial transcription-coupled repair at single-molecule resolution

International audienc

Stopped in its tracks: The RNA polymerase molecular motor as a robust sensor of DNA damage.

International audience: DNA repair is often a complex, multi-component, multi-step process; this makes detailed kinetic analysis of the different steps of repair a challenging task using standard biochemical methods. At the same time, single-molecule methods are well-suited for extracting kinetic information despite time-averaging due to diffusion of biochemical components and stochasticity of chemical reaction steps. Here we discuss recent experiments using DNA nanomanipulation in a magnetic trap to study the initiation of transcription-coupled repair in a model bacterial system comprising the canonical Escherichia coli RNA polymerase and the Mfd translocase which specifically binds to it. These experiments provide kinetic insight into the reaction process, helping to explain how Mfd discriminates between transcribing RNAP and stalled RNAP. They also identify a reliably long-lived intermediate containing Mfd translocase and, potentially, RNA polymerase. This intermediate presumably serves as a platform for assembly of downstream repair components UvrAB(C)

Enhanced Antibacterial Activity of Substituted Derivatives of NCR169C Peptide

Medicago truncatula in symbiosis with its rhizobial bacterium partner produces more than 700 nodule-specific cysteine-rich (NCR) peptides with diverse physicochemical properties. Most of the cationic NCR peptides have antimicrobial activity and the potential to tackle antimicrobial resistance with their novel modes of action. This work focuses on the antibacterial activity of the NCR169 peptide derivatives as we previously demonstrated that the C-terminal sequence of NCR169 (NCR169C17–38) has antifungal activity, affecting the viability, morphology, and biofilm formation of various Candida species. Here, we show that NCR169C17–38 and its various substituted derivatives are also able to kill ESKAPE pathogens such as Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. The replacement of the two cysteines with serines enhanced the antimicrobial activity against most of the tested bacteria, indicating that the formation of a disulfide bridge is not required. As tryptophan can play role in the interaction with bacterial membranes and thus in antibacterial activity, we replaced the tryptophans in the NCR169C17–38C12,17/S sequence with various modified tryptophans, namely 5-methyl tryptophan, 5-fluoro tryptophan, 6-fluoro tryptophan, 7-aza tryptophan, and 5-methoxy tryptophan, in the synthesis of NCR169C17–38C12,17/S analogs. The results demonstrate that the presence of modified fluorotryptophans can significantly enhance the antimicrobial activity without notable hemolytic effect, and this finding could be beneficial for the further development of new AMPs from the members of the NCR peptide family.</jats:p

Enhanced Antibacterial Activity of Substituted Derivatives of NCR169C Peptide

Medicago truncatula in symbiosis with its rhizobial bacterium partner produces more than 700 nodule-specific cysteine-rich (NCR) peptides with diverse physicochemical properties. Most of the cationic NCR peptides have antimicrobial activity and the potential to tackle antimicrobial resistance with their novel modes of action. This work focuses on the antibacterial activity of the NCR169 peptide derivatives as we previously demonstrated that the C-terminal sequence of NCR169 (NCR169C17&ndash;38) has antifungal activity, affecting the viability, morphology, and biofilm formation of various Candida species. Here, we show that NCR169C17&ndash;38 and its various substituted derivatives are also able to kill ESKAPE pathogens such as Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. The replacement of the two cysteines with serines enhanced the antimicrobial activity against most of the tested bacteria, indicating that the formation of a disulfide bridge is not required. As tryptophan can play role in the interaction with bacterial membranes and thus in antibacterial activity, we replaced the tryptophans in the NCR169C17&ndash;38C12,17/S sequence with various modified tryptophans, namely 5-methyl tryptophan, 5-fluoro tryptophan, 6-fluoro tryptophan, 7-aza tryptophan, and 5-methoxy tryptophan, in the synthesis of NCR169C17&ndash;38C12,17/S analogs. The results demonstrate that the presence of modified fluorotryptophans can significantly enhance the antimicrobial activity without notable hemolytic effect, and this finding could be beneficial for the further development of new AMPs from the members of the NCR peptide family

Interspecific Variation In Juvenile Snapper Otolith Chemical Signatures In the Northern Gulf of Mexico

The objective of this study was to evaluate whether age-0 lane snapper Lutjanus synagris otolith chemical signatures could serve as accurate proxies for those of its congener, red snapper L. campechanus, among northern Gulf of Mexico (GOM) nursery regions. Red (n = 90) and lane (n = 53) snappers were sampled from 3 regions of the northern GOM in fall 2005, and their otolith chemistry was analyzed with sector field-inductively coupled plasma-mass spectrometry (Ba:Ca, Mg:Ca, Mn:Ca, Sr:Ca, Li:Ca) or stable isotope ratio-mass spectrometry (δ13C and δ18O). Chemical signatures were significantly different among regions (MANOVA, p \u3c 0.001) and between species (MANOVA, p = 0.029), with the species effect being driven by significant differences in 4 of the 7 constituents analyzed (ANOVA, p \u3c 0.036). The significant region effect persisted (MANOVA, p \u3c 0.001), but the species effect was non-significant (MANOVA, p = 0.964) when constituent values were normalized to species-specific means. Mean regional classification accuracies from linear discriminant functions computed with otolith constituent data were 84% for lane snapper and 80% for red snapper whether data were normalized or not. Maximum likelihood models parameterized with normalized lane snapper otolith chemistry data estimated red snapper regional composition reasonably well among mixed-region samples (mean error = 9.7% among models). Therefore, it appears age-0 lane snapper otolith chemical signatures can serve as accurate proxies for those of red snapper in the northern GOM. These results have broader implications for deriving natural tags based on otolith chemistry for fishes that may have low abundance in parts of their range

Enantioselective resolution of biologically active dipeptide analogs by high-performance liquid chromatography applying Cinchona alkaloid-based ion-exchanger chiral stationary phases

Sixteen pairs of enantiomeric dipeptides were separated on four chiral ion-exchanger-type stationary phases based on Cinchona alkaloids. Anion-exchangers (QN-AX, QD-AX) and zwitterionic phases [ZWIX(+)(TM) and ZWIX(-)(TM)] were studied in a comparative manner. The effects of the nature and concentrations of the mobile phase solvent components and organic salt additives on analyte retention and enantioseparation were systematically studied in order to get a deeper insight into the enantiorecognition mechanism. Moreover, experiments were performed in the temperature range 10-50 degrees C to calculate thermodynamic parameters like changes in standard enthalpy, Delta(Delta H degrees), entropy, Delta(Delta S degrees), and free energy, Delta(Delta G degrees) on the basis of van't Hoff plots derived from the In alpha vs. 1/T curves. Elution sequences of the dipeptides were determined in all cases and, with a few exceptions, they were found to be opposite on the pseudoenantiomeric stationary phases as of QN-AX/QD-AX and of ZWIX(+) and ZWIX(-). The stere-oselective retention mechanism is based on electrostatically driven intermolecular interactions supported by additional interaction increments mainly determined by the absolute configuration of the chiral C8 and C9 atoms of the quinine and quinidine moieties. (C) 2019 Elsevier B.V. All rights reserved

A Randomized Assessor-Blinded Wait-List-Controlled Trial to Assess the Effectiveness of Acupuncture in the Management of Chemotherapy-Induced Peripheral Neuropathy

Purpose: Chemotherapy-induced peripheral neuropathy is a complex side effect with few available treatment options. The aim of the study was to test the effectiveness of an 8-week course of acupuncture in the management of chemotherapy-induced peripheral neuropathy in cancer patients who were receiving or had received neurotoxic chemotherapy. Methods: Randomized assessor-blinded controlled trial with 2 arms; one arm received acupuncture twice weekly for 8 weeks, while the other arm was a wait-list control group receiving only standard care. Primary outcome was pain intensity and interference over the past week using the Brief Pain Inventory at the end of the intervention. Secondary outcomes included clinical assessment (CTCAE [Common Toxicity Criteria for Adverse Events] grading and Total Neuropathy Score–Clinical Version) and nerve conduction studies; and patient-reported outcome measures (Functional Assessment of Cancer Therapy–Gynecologic Oncology Group–Neurotoxicity Quality of Life scale and Symptom Distress Scale) assessed at baseline, end of treatment (8 weeks), week 14, and week 20 from the beginning of treatment. Results: Eighty-seven patients were randomized to the experimental arm (n = 44) and to the standard care wait-list control arm (n = 43). Significant changes at 8 weeks were detected in relation to primary outcome (pain), the clinical neurological assessment, quality of life domains, and symptom distress (all P &lt; .05). Improvements in pain interference, neurotoxicity-related symptoms, and functional aspects of quality of life were sustained in the 14-week assessment ( P &lt; .05), as were physical and functional well-being at the 20-week assessment ( P &lt; .05). Conclusions: Acupuncture is an effective intervention for treating chemotherapy-induced peripheral neuropathy and improving patients’ quality of life and experience with neurotoxicity-related symptoms with longer term effects evident. </jats:p