707 Transvenous radiofrequency ablation of epicardial posterior-septal accessory pathways in children with WPW syndrome: can technology and imaging innovations improve the outcome?

Aims The aim of the study was to analyse our recent single-centre experience about epicardial posterior-septal accessory pathways transcatheter ablation in children and young patients using radiofrequency through the coronary sinus, in order to understand which mapping and ablation strategy is associated with higher success rate and safety. Methods and results We reviewed all the cases of ablation of overt accessory pathways (in Wolff–Parkinson–White syndrome) with epicardial posterior-septal localization performed in children or young patients at our institution in the last 5 years. Twenty-two paediatric patients (mean age: 13 ± 3 years) with epicardial posterior-septal accessory pathways (15 in coronary sinus and 7 in the Middle Cardiac Vein) underwent radiofrequency transcatheter ablation with CARTO 3TM. Acute success rate was 77%. No patient was lost to follow-up (mean time 14.4 ± 9 months). The recurrence rate was 18%. Two patients underwent a successful redo-procedure; the overall long-term success rate was 68%. NAVISTAR® catheter presented the highest acute success rate in the coronary sinus. NAVISTAR SMARTTOUCH® was the only catheter that did not present recurrences after the acute success and it was successfully used in two patients previously unsuccessfully treated with a NAVISTAR THERMOCOOL®. Integration with angio-CT of coronary sinus branches obtained with CARTOMERGE was associated with higher success rate in patients with a previous failed ablation attempt. Conclusions Epicardial posterior-septal accessory pathways can be successfully treated with transvenous radiofrequency ablation in more than half of the cases in children/young patients. Acute success rate does not seem to depend on catheters used but contact-force catheter seems to be useful in cases with recurrences. Image integration with cardiac-CT reconstruction of coronary sinus branches anatomy can be useful to better guide ablation in case of previously failed attempts.

A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae

ABSTRACT Control of peptidoglycan assembly is critical to maintain bacterial cell size and morphology. Penicillin-binding proteins (PBPs) are crucial enzymes for the polymerization of the glycan strand and/or their cross-linking via peptide branches. Over the last few years, it has become clear that PBP activity and localization can be regulated by specific cognate regulators. The first regulator of PBP activity in Gram-positive bacteria was discovered in the human pathogen Streptococcus pneumoniae. This regulator, named CozE, controls the activity of the bifunctional PBP1a to promote cell elongation and achieve a proper cell morphology. In this work, we studied a previously undescribed CozE homolog in the pneumococcus, which we named CozEb. This protein displays the same membrane organization as CozE but is much more widely conserved among Streptococcaceae genomes. Interestingly, cozEb deletion results in cells that are smaller than their wild-type counterparts, which is the opposite effect of cozE deletion. Furthermore, double deletion of cozE and cozEb results in poor viability and exacerbated cell shape defects. Coimmunoprecipitation further showed that CozEb is part of the same complex as CozE and PBP1a. However, although we confirmed that CozE is required for septal localization of PBP1a, the absence of CozEb has no effect on PBP1a localization. Nevertheless, we found that the overexpression of CozEb can compensate for the absence of CozE in all our assays. Altogether, our results show that the interplay between PBP1a and the cell size regulators CozE and CozEb is required for the maintenance of pneumococcal cell size and shape. IMPORTANCE Penicillin-binding proteins (PBPs), the proteins catalyzing the last steps of peptidoglycan assembly, are critical for bacteria to maintain cell size, shape, and integrity. PBPs are consequently attractive targets for antibiotics. Resistance to antibiotics in Streptococcus pneumoniae (the pneumococcus) are often associated with mutations in the PBPs. In this work, we describe a new protein, CozEb, controlling the cell size of pneumococcus. CozEb is a highly conserved integral membrane protein that works together with other proteins to regulate PBPs and peptidoglycan synthesis. Deciphering the intricate mechanisms by which the pneumococcus controls peptidoglycan assembly might allow the design of innovative anti-infective strategies, for example, by resensitizing resistant strains to PBP-targeting antibiotics.

FtsW activity and lipid II synthesis are required for recruitment of MurJ to midcell during cell division inEscherichia coli

Peptidoglycan (PG) is the unique cell shape-determining component of the bacterial envelope, and is a key target for antibiotics. PG synthesis requires the transmembrane movement of the precursor lipid II, and MurJ has been shown to provide this activity inE. coli.However, how MurJ functionsin vivohas not been reported. Here we show that MurJ localizes both in the lateral membrane and at midcell, and is recruited to midcell simultaneously with late-localizing divisome proteins and proteins MraY and MurG. MurJ septal localization is dependent on the presence of a complete and active divisome, lipid II synthesis and PBP3/FtsW activities. Inactivation of MurJ, either directly by mutation or through binding with MTSES, did not affect the midcell localization of MurJ. Our study visualizes MurJ localizationin vivoand reveals a possible mechanism of how MurJ functions during cell division, which gives possibilities for future investigations and further antibiotics developments.

Requirements for Septal Localization and Chromosome Segregation Activity of the DNA Translocase SftA from Bacillus subtilis

<i>Bacillus subtilis</i> possesses 2 DNA translocases that affect late stages of chromosome segregation: SftA separates nonsegregated DNA prior to septum closure, while SpoIIIE rescues septum-entrapped DNA. We provide evidence that SftA is associated with the division machinery via a stretch of 47 amino acids within its N-terminus, suggesting that SftA is recruited by protein-protein interactions with a component of the division machinery. SftA was also recruited to mid-cell in the absence of its first 20 amino acids, which are proposed to contain a membrane-binding motif. Cell fractionation experiments showed that SftA can be found in the cytosolic fraction, and to a minor degree in the membrane fraction, showing that it is a soluble protein in vivo. The expression of truncated SftA constructs led to a dominant <i>sftA</i> deletion phenotype, even at very low induction rates of the truncated proteins, indicating that the incorporation of nonfunctional monomers into SftA hexamers abolishes functionality. Mobility shift experiments and surface plasmon binding studies showed that SftA binds to DNA in a cooperative manner, and demonstrated low ATPase activity when binding to short nucleotides rather than to long stretches of DNA.