Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces.

Methods for direct covalent ligation of microorganism surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. Here, we used N-methylluminol (NML), a fully tyrosine-selective protein anchoring group after one-electron oxidation, to label the surface of viruses, living bacteria and cells. The functionalization was performed electrochemically and in situ by applying an electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), Escherichia coli (Gram-) and Staphyloccocus epidermidis (Gram + ) bacterial strains, and HEK293 and HeLa eukaryotic cell lines. Surface electro-conjugation was achieved in minutes to yield functionalized rAAV2 that conserved both structural integrity and infectivity properties, and living bacteria and cell lines that were still alive and able to divide.

Noninvasive Tidal Volume Measurements, Using a Time-of-Flight Camera, Under High-Flow Nasal Cannula-A Physiological Evaluation, in Healthy Volunteers.

OBJECTIVES: The mechanisms of high-flow nasal cannula are still debated but may be mediated by the generation of low positive end-expiratory pressure and a washout of the airway dead space. The aims of this study were to assess the effects of high-flow nasal cannula on tidal volume using a noninvasive method using a time-of-flight camera, under various conditions. DESIGN: A physiologic evaluation in healthy volunteers. SETTING: An university hospital ICU. SUBJECTS: Ten healthy volunteers were included in a physiologic study (CamOpt study, ClinicalTrials.gov identifier: NCT04096183). INTERVENTIONS: All volunteers were submitted to 12 different conditions (i.e., gas flow [baseline = 0; 30-60 L/min]; mouth [open/closed]; respiratory rate [baseline; baseline + 10 breaths/min]). Tidal volume measurements were performed every minute, during a 6-minute recording period. In all combinations, reference respiratory rate was measured by using chronometric evaluation, over a 30-second period (RRREF), and by using the time-of-flight camera (RRTOF). MEASUREMENTS AND MAIN RESULTS: Tidal volume increased while increasing gas flow whatever the respiratory rate and mouth condition (p < 0.001). Similar results were observed whatever the experimental conditions (p < 0.01), except one (baseline respiratory rate + 10 breaths/min and mouth closed). Tidal volume increased while decreasing respiratory rate (p < 0.001) and mouth closing (p < 0.05). Proportion of tidal volume greater than 10, 15, and 20 mL/kg changed while increasing the flow. RRTOF was in agreement with RRREF (intraclass correlation coefficient, 0.96), with a low mean bias (0.55 breaths/min) and acceptable deviation. CONCLUSIONS: Time-of-flight enables to detect tidal volume changes under various conditions of high-flow nasal cannula application. Tidal volume increased significantly while increasing gas flow and mouth closing. Such technique might be useful to monitor the risk of patient self-inflicted lung injury or under assistance.

New Phosphorylation Sites of Rad51 by c-Met Modulates Presynaptic Filament Stability.

Genomic instability through deregulation of DNA repair pathways can initiate cancer and subsequently result in resistance to chemo and radiotherapy. Understanding these biological mechanisms is therefore essential to overcome cancer. RAD51 is the central protein of the Homologous Recombination (HR) DNA repair pathway, which leads to faithful DNA repair of DSBs. The recombinase activity of RAD51 requires nucleofilament formation and is regulated by post-translational modifications such as phosphorylation. In the last decade, studies have suggested the existence of a relationship between receptor tyrosine kinases (RTK) and Homologous Recombination DNA repair. Among these RTK the c-MET receptor is often overexpressed or constitutively activated in many cancer types and its inhibition induces the decrease of HR. In this study, we show for the first time that c-MET is able to phosphorylate the RAD51 protein. We demonstrate in vitro that c-MET phosphorylates four tyrosine residues localized mainly in the subunit-subunit interface of RAD51. Whereas these post-translational modifications do not affect the presynaptic filament formation, they strengthen its stability against the inhibitor effect of the BRC peptide obtained from BRCA2. Taken together, these results confirm the role of these modifications in the regulation of the BRCA2-RAD51 interaction and underline the importance of c-MET in DNA damage response.

Cpe1786/IscR of Clostridium perfringens represses expression of genes involved in Fe-S cluster biogenesis.

Cpe1786 of Clostridium perfringens is an Rrf2-type regulator containing the three-cysteine residues coordinating a Fe-S in IscR, the repressor controlling Fe-S homeostasis in enterobacteria. The cpe1786 gene formed an operon with iscSU involved in Fe-S biogenesis and tmrU. This operon was transcribed from a σA-dependent promoter. We showed that in the heterologous host Bacillus subtilis, Cpe1786, renamed IscRCp, negatively controlled its own transcription. We constructed an iscR mutant in C. perfringens. We then compared the expression profile of strain 13 and of the iscR mutant. IscRCp controlled expression of genes involved in Fe-S biogenesis, in amino acid or sugar metabolisms, in fermentation pathways and in host compound utilization. We then demonstrated, using a ChIP-PCR experiment, that IscRCp interacted with its promoter region in vivo in C. perfringens and with the promoter of cpe2093 encoding an amino acid ABC transporter. We utilized a comparative genomic approach to infer a candidate IscR binding motif and reconstruct IscR regulons in clostridia. We showed that point mutations in the conserved motif of 29 bp identified upstream of iscR decreased the cysteine-dependent repression of iscR mediated by IscRCp.

Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile.

The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (cre(CD) motif), that is, 'RRGAAAANGTTTTCWW'. Binding of purified CcpA protein to 19 target cre(CD) sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators.

Insights into the Rrf2 repressor family--the structure of CymR, the global cysteine regulator of Bacillus subtilis.

The global regulator CymR represses the transcription of a large set of genes involved in cystine uptake and cysteine biosynthesis in Bacillus subtilis and Staphylococcus aureus. This repressor belongs to the widespread and poorly characterized Rrf2 family of regulators. The crystal structure of CymR from B. subtilis reveals a biologically active dimer, where each monomer folds into two tightly packed domains: a DNA-binding domain, which houses a winged helix-turn-helix (wHTH) motif; and a long dimerization domain, which places the wHTH motifs at the extremes. This architecture explains how these small regulators can span 23-27-bp DNA targets. The wHTH motif of CymR resembles those of the GntR superfamily of regulators, such as FadR and HutC. Superimposing the FadR wHTH motifs bound to their DNA fragments onto the wHTH motifs of the CymR dimer structure suggests that the DNA target and/or the protein must undergo some conformational changes upon binding. The CymR structure also hints at a possible location of the Fe-S centre associated with several Rrf2-type regulators.