Early versus Later Anticoagulation for Stroke with Atrial Fibrillation.

BACKGROUND: The effect of early as compared with later initiation of direct oral anticoagulants (DOACs) in persons with atrial fibrillation who have had an acute ischemic stroke is unclear. METHODS: We performed an investigator-initiated, open-label trial at 103 sites in 15 countries. Participants were randomly assigned in a 1:1 ratio to early anticoagulation (within 48 hours after a minor or moderate stroke or on day 6 or 7 after a major stroke) or later anticoagulation (day 3 or 4 after a minor stroke, day 6 or 7 after a moderate stroke, or day 12, 13, or 14 after a major stroke). Assessors were unaware of the trial-group assignments. The primary outcome was a composite of recurrent ischemic stroke, systemic embolism, major extracranial bleeding, symptomatic intracranial hemorrhage, or vascular death within 30 days after randomization. Secondary outcomes included the components of the composite primary outcome at 30 and 90 days. RESULTS: Of 2013 participants (37% with minor stroke, 40% with moderate stroke, and 23% with major stroke), 1006 were assigned to early anticoagulation and 1007 to later anticoagulation. A primary-outcome event occurred in 29 participants (2.9%) in the early-treatment group and 41 participants (4.1%) in the later-treatment group (risk difference, -1.18 percentage points; 95% confidence interval [CI], -2.84 to 0.47) by 30 days. Recurrent ischemic stroke occurred in 14 participants (1.4%) in the early-treatment group and 25 participants (2.5%) in the later-treatment group (odds ratio, 0.57; 95% CI, 0.29 to 1.07) by 30 days and in 18 participants (1.9%) and 30 participants (3.1%), respectively, by 90 days (odds ratio, 0.60; 95% CI, 0.33 to 1.06). Symptomatic intracranial hemorrhage occurred in 2 participants (0.2%) in both groups by 30 days. CONCLUSIONS: In this trial, the incidence of recurrent ischemic stroke, systemic embolism, major extracranial bleeding, symptomatic intracranial hemorrhage, or vascular death at 30 days was estimated to range from 2.8 percentage points lower to 0.5 percentage points higher (based on the 95% confidence interval) with early than with later use of DOACs. (Funded by the Swiss National Science Foundation and others; ELAN ClinicalTrials.gov number, NCT03148457.).

Anti-mycobacterial activity evaluation of designed peptides: cryptic and database filtering based approach.

Worldwide, TB is one of the deadly airborne diseases, which accounts for 10.4 million deaths annually. Serious toxicity issue, prolonged treatment regimens of the current drugs, rise in multidrug-resistant strains, and the unique defensive mechanism makes the development of novel therapeutic molecules against Mycobacterium tuberculosis (MT) an urgent need. As MT has a lengthy latent phase and unique cell wall architecture, a reasonable approach is needed to find molecules having a different killing mechanism rather than traditional approaches. Host defence peptides (HDPs) will be the most promising alternative, potential therapeutic candidates as they target the microbial membrane in particular and are an essential part of the innate immunity of humans. This works demonstrates the utility of "Database filtering" and three-dimensional (3D) modelling approach in finding novel AMPs with appreciable activity towards MT. Results of this study indicate that peptides with 70% hydrophobicity, but without hydrophobicity patches (> 4 hydrophobic amino acids in series) and charge of + 4 or + 5 are most likely to be good anti-tubercular candidates.

Down-regulation of PE11, a cell wall associated esterase, enhances the biofilm growth of Mycobacterium tuberculosis and reduces cell wall virulence lipid levels.

PE11 (Rv1169c or LipX) is a cell wall associated esterase/lipase of Mycobacterium tuberculosis (Mtb). Evidences suggest that PE11 is expressed by Mtb both in vitro and in vivo. Previous studies have shown that PE11 leads to modification in cell wall lipid content and enhanced virulence when expressed in the non-pathogenic surrogate Mycobacterium smegmatis. Since cell wall lipids often play different roles in pathogenic and non-pathogenic mycobacteria, we investigated the role of PE11 in its host, Mtb. Mtb with lowered expression of PE11 (PE11 knock-down) displayed significant changes in colony morphology and cell wall lipid profile, confirming the role of PE11 in cell wall architecture. In addition, the levels of phthiocerol dimycocerosates, a cell wall virulence factor, were decreased. Levels of trehalose esters and free mycolic acids were increased. In contrast to M. smegmatis expressing Mtb PE11, a role reversal was observed in Mtb with respect to pellicle/biofilm formation. The PE11 knock-down Mtb strain showed significantly enhanced aggregation and early biofilm growth in detergent-free medium, compared to the wild-type. Knock-down strain also showed nearly 27-fold up-regulation of a fibronectin attachment protein (Rv1759c), linking biofilm growth with over-expression of bacterial proteins that help in aggregation and/or binding to host extracellular matrix. The knock-down also resulted in poor virulence of Mtb in PMA (phorbol 12-myristate 13-acetate) treated and PMA+IFN-γ treated THP-1 macrophages. Therefore, the study not only links PE11 to cell wall virulence lipids but also reveals the involvement of this cell wall associated esterase in down-regulation of biofilm in Mtb.

Mycobacterium tuberculosis can gain access to adipose depots of mice infected via the intra-nasal route and to lungs of mice with an infected subcutaneous fat implant.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis has the remarkable ability to persist as non-replicating forms in the host. These persisters are tolerant to drugs targeting actively replicating bacilli and hence are responsible for the need of an extended duration of anti-tubercular therapy. The anatomical locations and cell types housing Mtb persisters are being investigated in the recent times. Adipose tissue and the adipocytes are proposed niches of Mtb persisters. In the present study, we carried out experiments in the immunocompetent Swiss mice to see the dissemination of Mtb from lungs to adipose tissue and vice versa. Mice infected intra-nasally with ∼ 10(6), 10(4) or 10(2) bacilli harboured Mtb in various adipose depots distal to the lungs such as the visceral, subcutaneous and peri-renal depots. The dissemination was minimal at two weeks post-infection, as evident from culture negative adipose tissue samples. But at seven weeks post-infection, viable Mtb could be detected in 78%, 66% and 66% of the samples from high, moderate and low dose-infection groups respectively. In a separate experiment, Mtb-infected pre-adipocytes were implanted subcutaneously to un-infected mice. At five weeks post-implantation, the intact implants had a mean 7 ± 0.53 log10 CFUs/100 mg tissue, while the lungs had a mean 3.25 ± 0.32 log10 CFUs/100 mg tissue. In conclusion, the study shows that Mtb can disseminate from lungs to distant adipose depots and vice versa.

Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis.

The mycobacterial F0F1-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC50) of these compounds range between 0.36 and 5.45µM. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC50>300µg/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC50=0.51µM, mammalian ATPase IC50>100µM, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12µg/mL. In addition, it also exhibited bactericidal effect (approximately 2.4log10 reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100µg/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2log10 reduction in CFU at 5µg/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague-Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (Vss, 0.41L/kg), moderate clearance (0.06L/h/kg), long half-life (4.2h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12log10 reductions in CFU in both lung and spleen at 173µmol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains.

Predicting outcome after hip fracture: using a frailty index to integrate comprehensive geriatric assessment results.

INTRODUCTION: hip fracture is expensive in terms of mortality, hospital length of stay (LOS) and consequences for independence. Poor outcome reflects the vulnerability of patients who typically sustain this injury, but the impact of different comorbidities and impairments is complex to understand. We consider this in a prospective cohort study designed to examine how a patients' frailty index (FI) predicts outcome. METHODOLOGY: consecutive patients with low trauma hip fracture were assessed, excluding only those unfit for surgery. Comprehensive Geriatric Assessment (CGA) findings were used to derive a FI for each patient, which was examined alongside other assessment and outcome data from our National Hip Fracture Database (NHFD) submission for these individuals. RESULTS: we describe 178 patients; mean age 81 years, 73.5% female. The mean FI was 0.34 (SD = 0.16), and logistic regression identified abbreviated mental test score and FI as the strongest predictors of poor outcome. When patients were stratified by FI, 56 (31.5%) were in the low-frailty group (FI ≤0.25), 58 (32.5%) in intermediate (FI >0.25-0.4), and 64 (36%) in the high-FI group (FI >0.4). All the patients in the low-FI group returned to their original residence within a mean of 21.6 days. The mean LOS for the intermediate group was 36.3 days compared with 67.8 days in the high-FI group (P < 0.01) while 30-day mortality was 3.4% for the intermediate group compared with 17.2% for the high-FI group (P < 0.001). CONCLUSIONS: individual CGA findings proved disappointing as outcome predictors, while FI turned out to be a better predictor of mortality, 30-day residence and length of inpatient stay.

Survival Rate of Immediate Implants in Periodontally Compromised Patients: A Systematic Review.

Background: Immediate implant placement into a fresh extraction socket has been developed as a consistent treatment, allowing for a reduction in the duration of time necessary for prosthetic rehabilitation. The study will evaluate the clinical and radiologic outcomes of implants placed immediately for a 10-year follow-up period. Aim: The aim of this systematic review is to evaluate the durability of the immediate implant in periodontally compromised individual placement. Methods: Studies reporting clinical and radiologic implant outcomes from periodontally compromised individuals who were treated and followed periodontal and implant maintenance for ≥5 years were considered eligible for the review. Screening of the articles, data extraction, and quality assessment were conducted independently and in duplicate. Results: There were 99 papers, and of them, 55 were excluded after title/abstract assessment. The full texts of 28 potentially eligible publications were screened, but only seven studies met the inclusion criteria. Conclusions: The study confirms that immediate implant therapy is safe, effective, and predictable for successful osseointegration and long-term functioning in periodontally compromised individuals, with minimal differences in clinical and radiographic outcomes.

Porphyromonas levii - A Boon for Periodontists?

The majority of species previously categorized as Bacteroides have been reassigned into new genera. Bacteroides levii (Holdeman, Cato, and Mooretaxonomic)'s status has remained uncertain. This species shares a high degree of similarity with members of the genus Porphyromonas based on biochemical, chemical, and comparative 16s rRNA sequence analysis. As a result, Bacteroides levii (Holdeman, Cato, and Moore) was reclassified as Porphyromonas levii comb. now under the genus Porphyromonas.

The use of Mycobacterium tuberculosis H37Ra-infected immunocompetent mice as an in vivo model of persisters.

Persistence of Mycobacterium tuberculosis (Mtb) is one of the challenges to successful treatment of tuberculosis (TB). In vitro models of non-replicating Mtb are used to test the efficacy of new molecules against Mtb persisters. The H37Ra strain is attenuated for growth in macrophages and mice. We validated H37Ra-infected immunocompetent mice for testing anti-TB molecules against slow/non-replicating Mtb in vivo. Swiss mice were infected intravenously with H37Ra and monitored for CFU burden and histopathology for a period of 12 weeks. The bacteria multiplied at a slow pace reaching a maximum load of ∼106 in 8-12 weeks depending on the infection dose, accompanied by time and dose-dependent histopathological changes in the lungs. Surprisingly, four-weeks of treatment with isoniazid-rifampicin-ethambutol-pyrazinamide combination caused only 0.4 log10 and 1 log10 reduction in CFUs in lungs and spleen respectively. The results show that ∼40 % of the H37Ra bacilli in lungs are persisters after 4 weeks of anti-TB therapy. Isoniazid/rifampicin monotherapy also showed similar results. A combination of bedaquiline and isoniazid reduced the CFU counts to <200 (limit of detection), compared to ∼5000 CFUs by isoniazid alone. The study demonstrates an in vivo model of Mtb persisters for testing new leads using a BSL-2 strain.

Mycobacterium tuberculosis Rv2324 is a multifunctional feast/famine regulatory protein involved in growth, DNA replication and damage control.

Feast/famine regulatory proteins (FFRPs) are multifunctional regulators. We show that Mtb Rv2324 is important for growth, survival, and countering DNA damage in Mycobacterium tuberculosis (Mtb). DNA-relaxation activity against linear and supercoiled substrates suggest its involvement in transcription activation, while its high affinity for recombination, replication and repair substrates suggest a role there too. Small-Angle-X-ray scattering supports the adoption of an 'open' quaternary association in response to amino-acid binding. Size-exclusion-chromatography and glutaraldehyde cross-linking identify the adoption of diverse oligomers modulated by amino-acid binding, and DNA interactions. We tested G52A, G101T and D104A mutants which correspond to highly conserved residues, distal to the DNA-binding site, and are important for amino acids binding. G101T exhibits increased DNA affinity, while G52A and D104A exhibit weak DNA-binding thereby suggesting that they mediate effector-binding, and DNA binding activities. Gain and loss-of-function studies show that Rv2324 overexpression promotes growth-rate, while its knock-down leads to retarded growth. Rv2324 down-regulation lowers Mtb survival inside resting and IFN-ϒ-activated macrophages. Rv2324 protects the pathogen from DNA damage, as evidenced by the reduction in the knockdown strain's survival following treatment with H2O2 and UV light. Overall, we show that Rv2324 plays a crucial role in regulating survival and growth of Mtb.

Design, synthesis and biological evaluation of (Quinazoline 4-yloxy)acetamide and (4-oxoquinazoline-3(4H)-yl)acetamide derivatives as inhibitors of Mycobacterium tuberculosis bd oxidase.

New chemical scaffolds with novel mechanism of action are urgently needed for the treatment of drug resistant tuberculosis. The oxidative phosphorylation pathway of Mycobacterium tuberculosis consists of multiple clinically validated drug targets. This pathway can function through any one of the two terminal oxidases-the proton pumping cytochrome bc1-aa3 supercomplex, or the less energy efficient but high affinity cytochrome bd oxidase. Inhibiting the bc1 complex alone has been found bacteriostatic and not bactericidal. On the other hand, inhibition of both these oxidases turns lethal to the pathogen. In the present study, we used a bc1 complex mutant of M. tuberculosis to screen (Quinazoline 4-yloxy)acetamide and (4-oxoquinazoline-3(4H)-yl)acetamide derivatives against the alternate oxidase, i.e., cytochrome bd oxidase. Two molecules, S-021-0601 and S-021-0607 were found to inhibit the mutant with MICs 8 and 16 µM respectively, compared to MICs of 128 and 256 µM against the wild type M. tuberculosis. In the wild type, one of the compounds showed synergism with Q203, an inhibitor of bc1 complex, in inhibiting growth under aerobic conditions. Both compounds showed synergism with Q203 in depleting bacterial ATP and inhibiting oxygen consumption. Both the compounds at 32 µM (one-fourth or one-eighth of their MICs for wild type) were bactericidal to wild type bacteria under hypoxic condition, causing ∼1.9 log10 reduction in viable counts which increased to ∼4-log10 when combined with Q203.

The effects of apixaban on clot characteristics in atrial fibrillation: A novel pharmacodynamic biomarker.

Atrial fibrillation (AF) is a major risk factor for stroke. We aim to characterize AF patients and the effects of apixaban therapy in terms of clot microstructure using gel point analysis, a novel biomarker. Seventy-eight patients were included in the study, 50 Stroke with AF (AF-S), and 28 AF without stroke (AF). Pre- and post-anticoagulation samples were collected: gel point (GP) analysis was performed to obtain (i) TGP (the time taken to reach the GP or the clot formation time) and (ii) df, the fractal dimension of the clot, a quantification of clot fibrin microstructure at the GP. At baseline, the AF-S group had a df  = 1.70 (±0.05) and TGP = 306 (±73 s). The AF group had a df = 1.70 ± 0.05 and TGP = 346 ± 78 s, showing a significantly shortened TGP in the stroke group (p = .008). For both groups, apixaban significantly prolonged TGP, p = .005, but resulted in no change in df. Apixaban prolonged clotting time while having no significant impact on the blood's ability to form stable clots (no change in df ). This indicates that apixaban provides protection from the formation of thrombi by reducing clotting kinetics.

In vitro antimicrobial activities of capuramycin analogues against non-tuberculous mycobacteria.

OBJECTIVES: To determine antibacterial activity of capuramycin analogues SQ997, SQ922, SQ641 and RKS2244 against several non-tuberculous mycobacteria (NTM). METHODS: In vitro antibiotic activities, i.e. MIC, MBC, rate of killing and synergistic interaction with other antibiotics, were evaluated. RESULTS: SQ641 was the most active compound against all the NTM species studied. The MIC of SQ641 was ≤0.06-4 mg/L for Mycobacterium avium complex (MAC; n = 20), 0.125-2 mg/L for M. avium paratuberculosis (MAP; n = 9), 0.125-2 mg/L for Mycobacterium kansasii (MKN;n = 2), 0.25-1 mg/L for Mycobacterium abscessus (MAB; n = 11), 4 mg/L for Mycobacterium smegmatis (MSMG; n = 1), and 1 and 8 mg/L for Mycobacterium ulcerans (MUL; n = 1), by microdilution and agar dilution methods, respectively. SQ641 was bactericidal against NTM, with an MBC/MIC ratio of 1 to 32, and killed all mycobacteria faster than positive control drugs for each strain. In chequerboard titrations, SQ641 was synergistic with ethambutol against both MAC and MSMG, and was synergistic with streptomycin and rifabutin against MAB. CONCLUSIONS: In vitro, SQ641 was the most potent of the capuramycin analogues against all NTM tested, both laboratory and clinical strains.

Rv1717 Is a Cell Wall - Associated ß-Galactosidase of Mycobacterium tuberculosis That Is Involved in Biofilm Dispersion.

Understanding the function of conserved hypothetical protein (CHP)s expressed by a pathogen in the infected host can lead to better understanding of its pathogenesis. The present work describes the functional characterization of a CHP, Rv1717 of Mycobacterium tuberculosis (Mtb). Rv1717 has been previously reported to be upregulated in TB patient lungs. Rv1717 belongs to the cupin superfamily of functionally diverse proteins, several of them being carbohydrate handling proteins. Bioinformatic analysis of the amino acid sequence revealed similarity to glycosyl hydrolases. Enzymatic studies with recombinant Rv1717 purified from Escherichia coli showed that the protein is a ß-D-galactosidase specific for pyranose form rather than the furanose form. We expressed the protein in Mycobacterium smegmatis (Msm), which lacks its ortholog. In Msm Rv1717 , the protein was found to localize to the cell wall (CW) with a preference to the poles. Msm Rv1717 showed significant changes in colony morphology and cell surface properties. Most striking observation was its unusual Congo red colony morphotype, reduced ability to form biofilms, pellicles and autoagglutinate. Exogenous Rv1717 not only prevented biofilm formation in Msm, but also degraded preformed biofilms, suggesting that its substrate likely exists in the exopolysaccharides of the biofilm matrix. Presence of galactose in the extracellular polymeric substance (EPS) has not been reported before and hence we used the galactose-specific Wisteria floribunda lectin (WFL) to test the same. The lectin extensively bound to Msm and Mtb EPS, but not the bacterium per se. Purified Rv1717 also hydrolyzed exopolysaccharides extracted from Msm biofilm. Eventually, to decipher its role in Mtb, we downregulated its expression and demonstrate that the strain is unable to disperse from in vitro biofilms, unlike the wild type. Biofilms exposed to carbon starvation showed a sudden upregulation of Rv1717 transcripts supporting the potential role of Rv1717 in Mtb dispersing from a deteriorating biofilm.

Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11 antimicrobial drugs.

OBJECTIVES: To evaluate the BACTEC(TM) MGIT(TM) 960/MGIT Para TB (MGIT) system for drug susceptibility testing of Mycobacterium avium subsp. paratuberculosis (MAP), a pathogen implicated in some forms of Crohn's disease. METHODS: MICs of 11 drugs for 10 MAP strains were determined using the MGIT system, the BACTEC(TM)460TB system (BACTEC) and conventional agar dilution methods. RESULTS: MICs determined by MGIT methods showed 80%-100% agreement (+/-1 log(2) dilution) with those determined by the BACTEC and agar dilution methods for ciprofloxacin, levofloxacin, azithromycin and clofazimine. The MGIT and BACTEC methods showed 70%, 80% and 90% agreement (+/-1 log(2) dilution) for MICs of ethambutol, rifabutin and rifampicin; agreement for all drugs increased to 100% at 2 log(2) dilution differences. For clarithromycin, the MGIT method had greater agreement with the agar dilution method (70% at the same dilution) than the BACTEC method (60% at +/-1 log(2) dilution); agreement increased to 100% at +/-2 log(2) dilutions in both cases. The MGIT and agar dilution methods agreed 60% and 100% for amikacin MICs at +/-1 log(2) dilution and +/-2 log(2) dilutions, respectively. By all methods MICs were higher than achievable serum concentrations for isoniazid and dapsone. There was 100% agreement between all three methods for azithromycin, clarithromycin and ciprofloxacin, and 80% agreement for rifampicin using published MIC thresholds available for M. avium complex strains. CONCLUSIONS: This study shows that the MGIT system can be used for rapid and reliable drug susceptibility testing of MAP.

Effects of interactions of antibacterial drugs with each other and with 6-mercaptopurine on in vitro growth of Mycobacterium avium subspecies paratuberculosis.

OBJECTIVES: Mycobacterium avium subspecies paratuberculosis (MAP) has been targeted for treatment with clarithromycin and rifamycin derivatives in numerous cases of Crohn's disease (CD). 6-Mercaptopurine and its pro-drug azathioprine are widely used as immunomodulators in the treatment of CD and have recently been shown to have anti-MAP activity in vitro. The objectives of the study were to evaluate the in vitro effects on MAP of (i) 6-mercaptopurine when combined with each of eight conventional antibacterial agents with in vitro anti-MAP activity and (ii) antibacterial combinations consisting of two drugs (clarithromycin combined with amikacin, rifampicin, ciprofloxacin or ethambutol) and three drugs (clarithromycin, rifabutin and clofazimine). METHODS: The drug interaction effects on nine human isolates of MAP were determined by the chequerboard method adapted for the BACTECMGIT960 culture system and by calculation of the fractional inhibitory concentration index (FICI) for drug combinations. RESULTS: Synergism (FICI < or = 0.5) was observed between 6-mercaptopurine and azithromycin (seven isolates), clarithromycin, rifampicin, rifabutin (four isolates each) and ethambutol (two isolates). 6-Mercaptopurine was not antagonistic with any of the antibacterial agents tested. Among the combinations of two and three antibacterials tested, the clarithromycin/rifampicin combination was synergistic against four isolates, while all other combinations showed no interaction. CONCLUSIONS: This in vitro study suggests that 6-mercaptopurine may be synergistic with macrolides and rifamycin derivatives against MAP. The activity of clarithromycin against MAP seems to be enhanced by rifampicin.

Effect of various drugs on differentially detectable persisters of Mycobacterium tuberculosis generated by long-term lipid diet.

Persisters of Mycobacterium tuberculosis (Mtb) that fail to form colonies on agar media when de-stressed are termed as differentially detectable (DD) persisters. Since in the host, Mtb primarily survives by utilizing lipids, we used a long-term lipid diet model to induce DD persisters of M. tuberculosis. Persisters were induced by replacing the dextrose-containing medium with one containing fatty acids instead of dextrose (FAM). After 2, 4 or 6 weeks, CFU and most probable number assays were performed; the difference between the two gave an estimate of DD persisters. Since rifampicin has been shown to induce formation of DD persisters in vitro, one set of FAM cultures were also given short-term rifampicin stress after 2, 4 or 6 weeks. Fraction of DD persisters increased with time and rifampicin treatment enhanced the effect of fatty acids, at 2 and 4 weeks. At six weeks, even in the absence of rifampicin, ∼95% population were DD persisters. The DD persisters were vulnerable to drugs interfering with bacterial respiration such as thioridazine, bedaquiline and clofazimine. The study indicates potential formation of DD persisters of Mtb in a lipid-rich microenvironment in the host even before antibiotic therapy.

Triacylglycerols: Fuelling the Hibernating Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) has the remarkable ability to persist with a modified metabolic status and phenotypic drug tolerance for long periods in the host without producing symptoms of active tuberculosis. These persisters may reactivate to cause active disease when the immune system becomes disrupted or compromised. Thus, the infected hosts with the persisters serve as natural reservoir of the deadly pathogen. Understanding the host and bacterial factors contributing to Mtb persistence is important to devise strategies to tackle the Mtb persisters. Host lipids act as the major source of carbon and energy for Mtb. Fatty acids derived from the host cells are converted to triacylglycerols (triglycerides or TAG) and stored in the bacterial cytoplasm. TAG serves as a dependable, long-term energy source of lesser molecular mass than other storage molecules like glycogen. TAG are found in substantial amounts in the mycobacterial cell wall. This review discusses the production, accumulation and possible roles of TAG in mycobacteria, pointing out the aspects that remain to be explored. Finally, the essentiality of TAG synthesis for Mtb is discussed with implications for identification of intervention strategies.

Challenges in reducing TIA clinic waiting times from 9 to 3 days in an acute Welsh hospital.

A patient impact project which successfully reduced the transient ischaemic attack (TIA) clinic waiting time from 9 to 3 days in an acute Welsh hospital, revealing the challenges faced and how alternative thinking and team work improved care given to our service users. Evaluating current situation, careful planning with multiple brainstorming meetings, 4 N chart and driver diagram with change ideas laid the foundation for this service improvement. Run charts, statistical process control and Pareto charts helped to identify the issues that are hindering the progress, which when rectified, reduced the clinic waiting times. Avoiding clinic cancellations by cross covering TIA clinics with mutual agreement among consultants and redeployment of ward staff to support clinics resulted in a positive impact to the patients. The average waiting time to see a patient in TIA clinic dropped from 9 days to just 3 days as a result of this, reflecting the hard-working and proactive nature of a team following a collaborative leadership journey. The service improvement initiative for 'avoiding clinic cancellations' was implemented in January 2017 and has reduced our waiting times by three times. Repeat analysis by six monthly Plan Do Study Act cycles revealed that this improvement is sustained.

Alternate pathway to ascorbate induced inhibition of Mycobacterium tuberculosis.

Ascorbate has been demonstrated to interfere with the growth of Mycobacterium tuberculosis. It scavenges oxygen in the culture medium to induce dormancy of M. tuberculosis. It kills the mycobacteria by generating reactive oxygen intermediates via iron mediated Fenton reactions. In this study, we observed that ascorbate can inhibit M. tuberculosis isocitrate lyase (MtbICL) with an IC50 of 2.15 mΜ. MtbICL is an essential enzyme for the survival of M. tuberculosis under dormancy. We studied the effect of ascorbate on the growth of M. tuberculosis H37Rv metabolizing through citric acid cycle or glyoxylate cycle with glucose or acetate respectively as the sole carbon source. It was observed that 4 mM ascorbate inhibited ∼89% of the growth in glucose medium, which was confirmed to be mediated by Fenton reaction, as the inhibition was significantly lesser (61%) under low iron condition. On the other hand, in acetate medium, ∼97% of the growth was inhibited and the inhibition was uninfluenced by the iron levels. 3-nitropropionate, a known inhibitor of MtbICL, was seen to cause significantly higher inhibition in the acetate medium than in the glucose medium; however it was indifferent to iron levels in either medium. Molecular docking and dynamic simulation studies confirmed stable binding of ascorbate to MtbICL leading to its inhibition. These observations suggest an additional pathway for ascorbate induced inhibition of M. tuberculosis through inhibition of glyoxylate cycle. Since human immune cells can accumulate ascorbate in millimolar concentrations, the in vitro activity range (1-4 mM) of ascorbate against M. tuberculosis could be extrapolated in vivo. Our result supports the possible benefits of adding high vitamin C diet in TB-treated patients.