New apparatus for DTA at 2000 bar: thermodynamic studies on Au, Ag, Al and HTSC oxides

A new DTA (Differential Thermal Analysis) device was designed and installed in a Hot Isostatic Pressure (HIP) furnace in order to perform high-pressure thermodynamic investigations up to 2 kbar and 1200C. Thermal analysis can be carried out in inert or oxidising atmosphere up to p(O2) = 400 bar. The calibration of the DTA apparatus under pressure was successfully performed using the melting temperature (Tm) of pure metals (Au, Ag and Al) as standard calibration references. The thermal properties of these metals have been studied under pressure. The values of DV (volume variation between liquid and solid at Tm), ROsm (density of the solid at Tm) and ALPHAm (linear thermal expansion coefficient at Tm) have been extracted. A very good agreement was found with the existing literature and new data were added. This HP-DTA apparatus is very useful for studying the thermodynamics of those systems where one or more volatile elements are present, such as high TC superconducting oxides. DTA measurements have been performed on Bi,Pb(2223) tapes up to 2 kbar under reduced oxygen partial pressure (p(O2) = 0.07 bar). The reaction leading to the formation of the 2223 phase was found to occur at higher temperatures when applying pressure: the reaction DTA peak shifted by 49C at 2 kbar compared to the reaction at 1 bar. This temperature shift is due to the higher stability of the Pb-rich precursor phases under pressure, as the high isostatic pressure prevents Pb from evaporating.Comment: 6 figures, 3 tables, Thermodynamics, Thermal property, Bi-2223, fundamental valu

Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice

Comparison of 8 weeks standard treatment (rifampicin plus clarithromycin) vs. 4 weeks standard plus amoxicillin/clavulanate treatment [RC8 vs. RCA4] to shorten Buruli ulcer disease therapy (the BLMs4BU trial): study protocol for a randomized controlled multi-centre trial in Benin

Background Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans that affects skin, soft tissues, and bones, causing long-term morbidity, stigma, and disability. The recommended treatment for BU requires 8 weeks of daily rifampicin and clarithromycin together with wound care, physiotherapy, and sometimes tissue grafting and surgery. Recovery can take up to 1 year, and it may pose an unbearable financial burden to the household. Recent in vitro studies demonstrated that beta-lactams combined with rifampicin and clarithromycin are synergistic against M. ulcerans. Consequently, inclusion of amoxicillin/clavulanate in a triple oral therapy may potentially improve and shorten the healing process. The BLMs4BU trial aims to assess whether co-administration of amoxicillin/clavulanate with rifampicin and clarithromycin could reduce BU treatment from 8 to 4 weeks. Methods We propose a randomized, controlled, open-label, parallel-group, non-inferiority phase II, multi-centre trial in Benin with participants stratified according to BU category lesions and randomized to two oral regimens: (i) Standard: rifampicin plus clarithromycin therapy for 8 weeks; and (ii) Investigational: standard plus amoxicillin/clavulanate for 4 weeks. The primary efficacy outcome will be lesion healing without recurrence and without excision surgery 12 months after start of treatment (i.e. cure rate). Seventy clinically diagnosed BU patients will be recruited per arm. Patients will be followed up over 12 months and managed according to standard clinical care procedures. Decision for excision surgery will be delayed to 14 weeks after start of treatment. Two sub-studies will also be performed: a pharmacokinetic and a microbiology study. Discussion If successful, this study will create a new paradigm for BU treatment, which could inform World Health Organization policy and practice. A shortened, highly effective, all-oral regimen will improve care of BU patients and will lead to a decrease in hospitalization-related expenses and indirect and social costs and improve treatment adherence. This trial may also provide information on treatment shortening strategies for other mycobacterial infections (tuberculosis, leprosy, or non-tuberculous mycobacteria infections). Trial registration ClinicalTrials.gov NCT05169554. Registered on 27 December 2021

Algorithmic robustness to preferred orientations in single particle analysis by CryoEM

The presence of preferred orientations in single particle analysis (SPA) by cryo-Electron Microscopy (cryoEM) is currently one of the hurdles preventing many structural analyses from yielding high-resolution structures. Although the existence of preferred orientations is mostly related to the grid preparation, in this technical note, we show that some image processing algorithms used for angular assignment and three-dimensional (3D) reconstruction are more robust than others to these detrimental conditions. We exemplify this argument with three different data sets in which the presence of preferred orientations hindered achieving a 3D reconstruction without artifacts or, even worse, a 3D reconstruction could never be achievedWe acknowledge support from “la Caixa” Foundation (Fellowship LCF/BQ/DI18/11660021. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 713673. We also thank the financial support from the Spanish Ministry of Economy and Competitiveness through Grants BIO2016-76400-R(AEI/FEDER, UE) and SEV 2017-0712, the “Comunidad Autónoma de Madrid” through Grant: S2017/BMD-3817, Instituto de Salud Carlos III, PT17/ 0009/0010 (ISCIII-SGEFI/ERDF), European Union (EU) and Horizon 2020 through grants: CORBEL (INFRADEV-1-2014-1, Proposal: 654248), INSTRUCT-ULTRA (INFRADEV-03-2016-2017, Proposal: 731005), EOSC Life (INFRAEOSC-04-2018, Proposal: 824087), High- ResCells (ERC-2018-SyG, Proposal: 810057), IMpaCT (WIDESPREAD-03-2018 – Proposal: 857203), EOSC-Synergy (EINFRA-EOSC-5, Proposal: 857647), and iNEXT-Discovery (Proposal: 871037). The authors acknowledge the support and the use of resources of Instruct, a Landmark ESFRI projec

Cryo-EM and single-particle analysis with Scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed

Dual-Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis

The historical view of β-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a β-lactamase inhibitor. However, most antimycobacterial β-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 β-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3′ that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a β-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A β-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent β-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening

AbstractThe identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.</jats:p