Novel Inhibitors for Isocitrate Lyase as a Potent Antitubercular Agent for Mycobacterium Tuberculosis

Introduction: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB). TB claimed 1.4 million lives in 2019. It is estimated that one-quarter of the world’s population is infected with latent TB. Current first- and second-line drugs used to treat TB require adherence to an extended period of therapy (at least six months) and do not target latent TB. It has been shown that the enzyme isocitrate lyase (ICL) is essential for the survival and persistence of Mtb in latent TB. ICL plays an important role in two metabolic pathways required during TB latency, the glyoxylate and methylcitrate cycle. ICL is absent in humans, and therefore, it is considered a promising drug target. Herein, is reported a novel family of ICL inhibitors that is effective in vitro against both the enzyme and Mtb. Methods: The His-tagged ICL1 enzyme was expressed using E. coli as a host and purified by Ni-NTA chromatography. The recombinant enzyme was used to set up crystallisation trials and in vitro enzymatic assay was performed on ICL1 inhibitors. The synthesis of D/L-threo-2-methylisocitrate and the methylation of five other drug candidates were described. Both co-crystallisation and soaking techniques were performed to obtain an ICL1:CL-54-04 complex crystal, whose structure was solved. The CL-54 drug family was tested against Mtb. A checkerboard assay was utilised to test the combinatory effect of CL-54-04 with rifampicin or bedaquiline in both glycerol and propionate media. To investigate the role of acetate metabolism in drug tolerance, ΔpckA, ICL knock-down and ΔprpD Mtb mutants were assessed. Results: Seven new conditions to crystallise ICL1 were identified, and eleven drugs were tested against the isolated enzyme. A promising new family of drugs have been identified as the most potent ICL1 inhibitors reported to date. One of the analogues, CL-54-04, had a bacteriostatic effect at 10 μM and a bactericidal effect at 100 μM against Mtb in vitro. A drug combination screening of CL-54-04 with rifampicin or bedaquiline led to an additive effect in the checkerboard assay and a significant impact in the colony-forming unit assay. The role of fatty acid metabolism in drug tolerance was investigated by testing three central carbon metabolism mutant strains against isoniazid. Discussion: From the three CL-54 analogues tested, only CL-54-04 caused a bactericidal effect against Mtb. A solved ICL1:CL-54-04 complex crystal structure showed that the catalytic loop had a distinctive move of 13.8 Å, suggesting that the binding of ICL with CL-54-04 leads to a close conformation of the active site. A superimposition of the solved ICL1:CL-54-04 complex and the ICL1 structure alignment with 3-nitropropionate demonstrates that CL-54-04 inhibitor binds and causes the same conformational changes as 3-nitropropionate. Using metabolomics analysis, the combination of rifampicin and CL-54-04 causes an accumulation of the methylcitrate cycle metabolites in propionate media, suggesting that the ICL has been inhibited. Fatty acid as a sole carbon source showed to increase the drug tolerance of all three Mtb mutants against isoniazid. Conclusion: A new drug family has been identified as lead compounds against ICL. The estimated IC50 of CL-54-01 is approximately half of the 3-nitropropionamide, and it causes the exact conformational change to the enzyme as 3-nitropropionate (the analogue of 3-nitropropionamide). These findings suggest that this drug family are the most promising ICL1 inhibitors reported to date

LIMO EEG: A Toolbox for hierarchical LInear MOdeling of ElectroEncephaloGraphic data

Magnetic- and electric-evoked brain responses have traditionally been analyzed by comparing the peaks or mean amplitudes of signals from selected channels and averaged across trials. More recently, tools have been developed to investigate single trial response variability (e.g., EEGLAB) and to test differences between averaged evoked responses over the entire scalp and time dimensions (e.g., SPM, Fieldtrip). LIMO EEG is a Matlab toolbox (EEGLAB compatible) to analyse evoked responses over all space and time dimensions, while accounting for single trial variability using a simple hierarchical linear modelling of the data. In addition, LIMO EEG provides robust parametric tests, therefore providing a new and complementary tool in the analysis of neural evoked responses

The future of spaceborne altimetry. Oceans and climate change: A long-term strategy

The ocean circulation and polar ice sheet volumes provide important memory and control functions in the global climate. Their long term variations are unknown and need to be understood before meaningful appraisals of climate change can be made. Satellite altimetry is the only method for providing global information on the ocean circulation and ice sheet volume. A robust altimeter measurement program is planned which will initiate global observations of the ocean circulation and polar ice sheets. In order to provide useful data about the climate, these measurements must be continued with unbroken coverage into the next century. Herein, past results of the role of the ocean in the climate system is summarized, near term goals are outlined, and requirements and options are presented for future altimeter missions. There are three basic scientific objectives for the program: ocean circulation; polar ice sheets; and mean sea level change. The greatest scientific benefit will be achieved with a series of dedicated high precision altimeter spacecraft, for which the choice of orbit parameters and system accuracy are unencumbered by requirements of companion instruments

Identity ambivalences of the Eurasian Macanese : historical dynamics, political regimes and food practices

This article focuses on the phenomenological experience of identity ambivalence arising from the Eurasian Macanese community through their phenomenological experience of identity ambivalence. Our thematic framework includes the structural impact of colonial and post colonial political regimes in Macao, historical influences on contemporary identity and sociocultural expressions of creolisation. It is argued that the Macanese people illustrate the memory of the ambivalent encounter between the two extremities of the Eurasia (China and Portugal) which started in the 16th-c. and never ceased moving forces to the present day. Furthermore, in the context of fieldwork with the Macanese community in Portugal, an ethnographic approach helps reveal the ambivalent dynamics of similarities and differentiation with respect to food practices and commensality as expressed over dinner by a group of close friends in Lisbon.Cet article présente une étude de cas des macanais euro-asiatiques – ethniquement et culturellement métissés – du point de vue de l’expérience phénoménologique résul- tant de leur identité ambivalente. Les questions traitées ici englobent les forces struc- turelles des régimes politiques coloniaux et post-coloniaux à Macao, les influences historiques sur les identités contemporaines et les expressions socioculturelles de la créolisation. On fait valoir que les macanais représentent la mémoire d’une rencontre ambivalente entre les deux extrémités de l’Eurasie (Chine et Portugal) qui depuis le XVIe s. jusqu’à nos jours n’ont jamais cessé de mesurer leurs forces. Dans le cadre d’une enquête de terrain menée auprès de la communauté macanaise au Portugal, l’approche ethnographique a aussi permis de révéler les dynamiques ambivalentes de similarité et de différence en matière de commensalité et de pratiques alimentaires exprimées par un groupe d’amis proches lors d’un dîner à Lisbonne.Este artigo analisa o estudo de caso dos macaenses euro-asiáticos – indivíduos étnica e culturalmente mestiços – do ponto de vista da experiência fenomenológica resultante da sua identidade ambivalente. As temáticas aqui exploradas abrangem as forças estruturais dos regimes políticos coloniais e pós-coloniais em Macau, as influências históricas sobre as identidades contemporâneas e as expressões socioculturais da crioulização desenvolvidas. É argumentado que os macaenses representam a memória de um encontro ambivalente entre os dois extremos da Eurásia, um processo com início no século XVI e que nunca parou de medir forças até aos dias de hoje. Adotou-se ainda uma perspetiva etnográfica para abordar o conjunto de dinâmicas ambivalentes de semelhança e diferença em relação às práticas alimentares e de comensalidade, expressas por um grupo de amigos íntimos no decorrer de um jantar em Lisboa.info:eu-repo/semantics/publishedVersio

Tools for the automation of large distributed control systems

The new LHC experiments at CERN will have very large numbers of channels to operate. In order to be able to configure and monitor such large systems, a high degree of parallelism is necessary. The control system is built as a hierarchy of sub-systems distributed over several computers. A toolkit - SMI++, combining two approaches: finite state machines and rule-based programming, allows for the description of the various sub-systems as decentralized deciding entities, reacting is real-time to changes in the system, thus providing for the automation of standard procedures and for the automatic recovery from error conditions in a hierarchical fashion. In this paper we will describe the principles and features of SMI++ as well as its integration with an industrial SCADA tool for use by the LHC experiments and we will try to show that such tools, can provide a very convenient mechanism for the automation of large scale, high complexity, applications

Revised Coordinates and Proper Motions of the Stars in the Luyten Half-Second Catalogue

We present refined coordinates and proper motion data for the high proper motion (HPM) stars in the Luyten Half-Second (LHS) catalogue. The positional uncertainty in the original Luyten catalogue is typically >10" and is often >30". We have used the digital scans of the Palomar Observatory Sky Survey (POSS) I and POSS II plates to derive more accurate positions and proper motions of the objects. Out of the 4470 candidates in the LHS catalogue, 4323 objects were manually re-identified in the POSS I and POSS II scans. A small fraction of the stars were not found due to the lack of finder charts and digitized POSS II scans. The uncertainties in the revised positions are typically ~2", but can be as high as ~8" in a few cases; this is a large improvement over the original data. Cross-correlation with the Tycho-2 and Hipparcos catalogues yielded 819 candidates (with m_R < 12). For these brighter sources, the position and proper motion data have been replaced with the more accurate Tycho/Hipparcos data. In total, we have revised proper motion measurements and coordinates for 4040 stars and revised coordinates for 4330 stars, which are presented here.Comment: 108 pages. Accepted for Publication in ApJ Suppl. Some errors caused by the transcription errors in the original LHS catalogue have been corrected in this resubmission. The most current version of the catalogue is also available online at http://www.stsci.edu/~ksahu/lh

Assessment of Lactobacillus crispatus´ role in vaginal infections: friend or foe?

Lactobacillus spp. are the most prevalent microorganisms in the vaginal milleu. Several probiotic mechanisms have been associated with Lactobacillus, but the most relevant one is lactate production – resulting in a low pH value, typical of the healthy vagina. However, its pathogenic state is still unknown. We aim to elucidate the role of a commensal vaginal microorganism, Lactobacillus crispatus, in vaginal infections. Twenty-four vaginal washes have been collected from women attending a gynaecology consultation of a private clinic. The samples were categorized according with clinical diagnosis at the time of sampling. The distribution of bacterial species, and their prevalence was assessed by Next-Generation Sequencing of the 16S rRNA V4 region. In addition, L- and D-lactate concentration, and LDH enzymatic activity was quantified in all washes by a commercial kit. Detection of Candida spp. was performed by PCR. L. crispatus was dominant (>70%) in 11 out of 24 samples – diagnosed for vaginal atrophy (VA, 3), cytolytic vaginosis (CV, 2) and lactobacillosis (LB, 2). Lactate was increased in CV, LB and VA cases only. The remaining samples, diagnosed for vulvovaginal candidosis in its majority, had lower prevalence of L. crispatus; and lower to moderate lactate metabolite. There was not a direct relationship between LDH enzymatic activity and clinical diagnosis. L. crispatus dominance, associated with increased lactate production, was observed in CV, LB and VA cases. These results indicate that this microorganism might have a role in dysbiosis of the vagina associated with these specific pathologies.info:eu-repo/semantics/publishedVersio

Role of Lactobacillus crispatus in vaginal infections: insights from metagenomics and metabolomics studies

Objective: Lactobacillus spp. constitute the most prevalent bacterial microorganism in the vaginal milleu. Several probiotic mechanisms have been associated with Lactobacillus, but the most relevant one is lactate production – resulting in a low pH value, typical of the healthy vagina. We aim to elucidate the role of a native vaginal microorganism, Lactobacillus crispatus, in vaginal infections. Methods: Twenty-four vaginal washes have been collected from women (mean 38±14 years of age) attending a gynecology consultation of a private clinic. The samples were categorized according with clinical diagnosis at the time of sampling. The distribution of bacterial species, and their prevalence was assessed by next-generation sequencing of the 16S V4 region. In addition, lactate was quantified in all washes by a commercial kit. Results: L. crispatus was dominant (>70%) in 11 out of 24 samples – diagnosed for vaginal atrophy (VA, 3), cytolytic vaginosis (CV, 2), lactobacillosis (LB, 2) and vulvovaginal candidosis (VVC, 1). For three samples, diagnostic was inconclusive. Lactate was increased in CV, LB and VA cases only. One sample, diagnosed with VVC had 23% dominance of L. crispatus; and lower lactate metabolite. L. crispatus was not detected in the remaining 12 cases (6 VVC, 4 VA, 1 healthy and 1 inconclusive). Of these, only 5 samples had moderate lactate metabolite. Conclusions: L. crispatus dominance, associated with increased lactate production, was observed in CV, LB and VA cases. These results indicate that this microorganism might have a role in dysbiosis of the vagina associated with these specific pathologies.info:eu-repo/semantics/publishedVersio