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Bioelectrochemical systems (BES) are emerging as a suite of versatile sustainable technologies to produce electricity and added-value compounds from renewable and carbon-neutral sources using electroactive organisms. The incomplete knowledge on the molecular processes that allow electroactive organisms to exchange electrons with electrodes has prevented their real-world implementation. In this manuscript we investigate the extracellular electron transfer processes performed by the thermophilic Gram-positive bacteria belonging to the Thermincola genus, which were found to produce higher levels of current and tolerate higher temperatures in BES than mesophilic Gram-negative bacteria. In our study, three multiheme c -type cytochromes, Tfer_0070, Tfer_0075, and Tfer_1887, proposed to be involved in the extracellular electron transfer pathway of T. ferriacetica , were cloned and over-expressed in E. coli . Tfer_0070 (ImdcA) and Tfer_1887 (PdcA) were purified and biochemically characterized. The electrochemical characterization of these proteins supports a pathway of extracellular electron transfer via these two proteins. By contrast, Tfer_0075 (CwcA) could not be stabilized in solution, in agreement with its proposed insertion in the peptidoglycan wall. However, based on the homology with the outer-membrane cytochrome OmcS, a structural model for CwcA was developed, providing a molecular perspective into the mechanisms of electron transfer across the peptidoglycan layer in Thermincola .

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper, we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2-Å resolution, shows that the overall fold and organization of the hemes are not related to other metal reductases and instead are similar to those of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can work as a multipurpose respiratory enzyme allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis. IMPORTANCE Thermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e., exchange catabolic electrons with solid electron acceptors outside the cell). In this paper, we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein can take the role of a respiratory "Swiss Army knife," allowing this organism to grow in environments with soluble and insoluble substrates. Moreover, it is shown that it is unrelated to terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveal that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes., (Copyright © 2019 Costa et al.)

Objective: The origin and mechanisms of focal and macro-re-entrant atrial tachycardia occurring after ablation of paroxysmal or persistent atrial fibrillation are difficult to determine and are often the cause of distress for both patients and electrophysiologists. The purpose of this study was to describe a novel practical approach which would be helpful in planning the treatment of atrial tachycardia in patients who had received prior atrial fibrillation ablation. We used an algorithm which aims to facilitate mapping and ablation strategies, using both conventional electrophysiological tools and a three-dimensional electroanatomic approach., Methods: We investigated a series of 40 patients with atrial tachycardia who had undergone a prior ablation procedure for paroxysmal or persistent atrial fibrillation with a step-wise approach. This approach consisted of four steps: the use of a decapolar catheter to assess the coronary sinus and an entrainment map to evaluate the cavotricuspid isthmus (CTI). If the CTI was not involved, we used a 20-pole dual-loop circular mapping catheter, AFocusII DL, to map the left atrium chamber rapidly during the rhythm of interest. We identified the target entrainment at the putative channels and performed catheter ablation at the critical isthmus of these macrocircuits until the restoration of sinus rhythm., Results: Seventy-five atrial tachycardias were identified in 40 consecutive patients (1.9 ± 1.6 per patient). In 48, the mechanism was macro-re-entry, and the remaining 27 were focal. During 18 months of follow-up, the treated arrhythmia recurred in only three patients, whereas in another four patients, a different atrial tachycardia was observed. The remaining 33 patients maintained sinus rhythm., Conclusion: The increased acquisition speed by AFocusII DL facilitated the reconstruction of the geometric chamber, and the generation of an isochronal activation map reduced processing time and fluoroscopy time per patient, together with the radiation exposure and patient radiation dose. A novel algorithm combining both conventional electrophysiology and a three-dimensional electroanatomic approach for rapid diagnosis, accurate mapping and ablation of iatrogenic atrial tachycardias showed high reproducibility and a satisfactory overall success rate.

Physicochemical factors involved in the development of a topical solution of a novel corticosteroid, tipredane (1), are described. A cosolvent system consisting of polyethylene glycol 400 (PEG 400), propylene glycol, and water was used to dissolve the concentration (0.1% w/w) of 1 required for the formulation. The solvent mixture was also nonirritating to the skin. Buffering agent, antioxidant, and metal-chelating agent were required to stabilize the drug. Solubilities of hydrophilic and lipophilic excipients were ensured by careful adjustment of their concentrations, as well as that of PEG 400. Two formulations, one containing potassium citrate and the other tromethamine as the buffering agents, were identified. Upon storage, sodium metabisulfite, an antioxidant used in the formulation, oxidized to form K2SO4 in the formulation containing potassium citrate. Potassium citrate decreased the solubility and resulted in the precipitation of K2SO4 by exerting a common ion effect. Lowering of the concentrations of potassium citrate, sodium metabisulfite, and PEG 400 ensured the solubility of K2SO4 formed. There was no such precipitation of K2SO4 in the formulation buffered with tromethamine, thus indicating that tromethamine is a good buffering agent in cosolvent systems.