Quantifying the short-term effects of air pollution on health in the presence of exposure measurement error: A simulation study of multi-pollutant model results

Background: Most epidemiological studies estimate associations without considering exposure measurement error. While some studies have estimated the impact of error in single-exposure models we aimed to quantify the effect of measurement error in multi-exposure models, specifically in time-series analysis of PM2.5, NO2,and mortality using simulations, under various plausible scenarios for exposure errors. Measurement error in multi-exposure models can lead to effect transfer where the effect estimate is overestimated for the pollutant estimated with more error to the one estimated with less error. This complicates interpretation of the independent effects of different pollutants and thus the relative importance of reducing their concentrations in air pollution policy. Methods: Measurement error was defined as the difference between ambient concentrations and personal exposure from outdoor sources. Simulation inputs for error magnitude and variability were informed by the literature. Error-free exposures with their consequent health 16outcome and error-prone exposures of various error types (classical/Berkson) were generated. Bias was quantified as the relative difference in effect estimates of the error-free and error-prone exposures. Results: Mortality effect estimates were generally underestimated with greater bias observed when low ratios of the true exposure variance over the error variance were assumed (27.4% 21underestimation for NO2). Higher ratios resulted in smaller, but still substantial bias (up to 19% for both pollutants).Effect transfer was observed indicating that less precise measurements for one pollutant (NO2) yield more bias, while the co-pollutant(PM2.5) associations were found closer to the true. Interestingly, the sum of single-pollutant model effect estimates was found closer to the summed true associations than those from multi-pollutant models, due to cancelling out of confounding and measurement error bias. Conclusions: Our simulation study indicated an underestimation of true independent health effects of multiple exposures due to measurement error. Using error parameter information in future epidemiological studies should provide more accurate concentration-response functions

Antitubercular specific activity of ibuprofen and the other 2-arylpropanoic acids using the HT-SPOTi whole-cell phenotypic assay

Objectives: Lead antituberculosis (anti-TB) molecules with novel mechanisms of action are urgently required to fuel the anti-TB drug discovery pipeline. The aim of this study was to validate the use of the high-throughput spot culture growth inhibition (HT-SPOTi) assay for screening libraries of compounds against Mycobacterium tuberculosis and to study the inhibitory effect of ibuprofen (IBP) and the other 2-arylpropanoic acids on the growth inhibition of M tuberculosis and other mycobacterial species. Methods: The HT-SPOTi method was validated not only with known drugs but also with a library of 47 confirmed anti-TB active compounds published in the ChEMBL database. Three over-the-counter non-steroidal anti-inflammatory drugs were also included in the screening. The 2-arylpropanoic acids, including IBP, were comprehensively evaluated against phenotypically and physiologically different strains of mycobacteria, and their cytotoxicity was determined against murine RAW264.7 macrophages. Furthermore, a comparative bioinformatic analysis was employed to propose a potential mycobacterial target. Results: IBP showed antitubercular properties while carprofen was the most potent among the 2-arylpropanoic class. A 3,5-dinitro-IBP derivative was found to be more potent than IBP but equally selective. Other synthetic derivatives of IBP were less active, and the free carboxylic acid of IBP seems to be essential for its anti-TB activity. IBP, carprofen and the 3,5-dinitro-IBP derivative exhibited activity against multidrug-resistant isolates and stationary phase bacilli. On the basis of the human targets of the 2-arylpropanoic analgesics, the protein initiation factor infB (Rv2839c) of M tuberculosis was proposed as a potential molecular target. Conclusions: The HT-SPOTi method can be employed reliably and reproducibly to screen the antimicrobial potency of different compounds. IBP demonstrated specific antitubercular activity, while carprofen was the most selective agent among the 2-arylpropanoic class. Activity against stationary phase bacilli and multidrug-resistant isolates permits us to speculate a novel mechanism of antimycobacterial action. Further medicinal chemistry and target elucidation studies could potentially lead to new therapies against TB

Improving the Tuberculosis Drug Development Pipeline

Mycobacterium tuberculosis is considered one of the most successful pathogens and multidrug-resistant tuberculosis, a disease that urgently requires new chemical entities to be developed for treatment. There are currently several new molecules under clinical investigation in the tuberculosis (TB) drug development pipeline. However, the complex lifestyle of M. tuberculosis within the host presents a barrier to the development of new drugs. In this review, we highlight the reasons that make TB drug discovery and development challenging as well as providing solutions, future directions and alternative approaches to new therapeutics for TB

Percutaneous cement augmentation for the treatment of depression fractures of the tibial plateau

The management of insufficiency fractures of the tibial plateau in osteoporotic patients can be very challenging, since it is difficult to achieve a stable fixation, an essential condition for the patients' early mobilization. We present a minimally invasive technique for the treatment of proximal tibial plateau fractures, "tibiaplasty”, using percutaneous polymethylmethacrylate augmentation. Five osteoporotic patients (7 fractures) with a non-traumatic insufficiency tibial plateau fracture were treated with this technique at the authors' institution from 2006 to 2008. The patients' median age was 79 (range 62-88) years. The intervention was performed percutaneously under general or spinal anesthesia; after the intervention, immediate full weight bearing was allowed. The technique was feasible in all patients and no complications related to the intervention were observed. All patients reported a relevant reduction in pain, were able to mobilize with full weight bearing and would undergo the operation again. No secondary loss of reduction or progression of arthrosis was observed in radiological controls; no revision surgery was required. Our initial results indicate that tibiaplasty is a good treatment option for the management of insufficiency in tibial plateau fractures in osteoporotic patients. The technique is minimally invasive, safe and allows immediate mobilization without restrictions. In our group of patients, we found excellent early to mid-term result

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery

Personal exposure to air pollution and respiratory health of COPD patients in London

Previous studies have investigated the effects of air pollution on chronic obstructive pulmonary disease (COPD) patients using either fixed site measurements or a limited number of personal measurements, usually for one pollutant and a short time period. These limitations may introduce bias and distort the epidemiological associations as they do not account for all the potential sources or the temporal variability of pollution.We used detailed information on individuals' exposure to various pollutants measured at fine spatio-temporal scale to obtain more reliable effect estimates. A panel of 115 patients was followed up for an average continuous period of 128 days carrying a personal monitor specifically designed for this project that measured temperature, PM10, PM2.5, NO2, NO, CO and O3 at one-minute time resolution. Each patient recorded daily information on respiratory symptoms and measured peak expiratory flow (PEF). A pulmonologist combined related data to define a binary variable denoting an "exacerbation". The exposure-response associations were assessed with mixed-effects models.We found that gaseous pollutants were associated with a deterioration in patients' health. We observed an increase of 16.4% (95% confidence interval: 8.6-24.6%), 9.4% (5.4-13.6%) and 7.6% (3.0-12.4%) in the odds of exacerbation for an interquartile range increase in NO2, NO and CO respectively. Similar results were obtained for cough and sputum. O3 was found to have adverse associations with PEF and breathlessness. No association was observed between particles and any outcome.Our findings suggest that, when considering total personal exposure to air pollutants, mainly the gaseous pollutants affect COPD patients' health

Culture-Free Enumeration of Mycobacterium tuberculosis in Mouse Tissues Using the Molecular Bacterial Load Assay for Preclinical Drug Development

BACKGROUND: The turnaround times for phenotypic tests used to monitor the bacterial load of Mycobacterium tuberculosis, in both clinical and preclinical studies, are delayed by the organism’s slow growth in culture media. The existence of differentially culturable populations of M.tuberculosis may result in an underestimate of the true number. Moreover, culture methods are susceptible to contamination resulting in loss of critical data points. Objectives: We report the adaptation of our robust, culture-free assay utilising 16S ribosomal RNA, developed for sputum, to enumerate the number of bacteria present in animal tissues as a tool to improve the read-outs in preclinical drug efficacy studies. METHODS: Initial assay adaptation was performed using naïve mouse lungs spiked with known quantities of M. tuberculosis and an internal RNA control. Tissues were homogenised, total RNA extracted, and enumeration performed using RT-qPCR. We then evaluated the utility of the assay, in comparison to bacterial counts estimated using growth assays on solid and liquid media, to accurately inform bacterial load in tissues from M. tuberculosis-infected mice before and during treatment with a panel of drug combinations. RESULTS: When tested on lung tissues derived from infected mice, the MBL assay produced comparable results to the bacterial counts in solid culture (colony forming units: CFU). Notably, under specific drug treatments, the MBL assay was able to detect a significantly higher number of M. tuberculosis compared to CFU, likely indicating the presence of bacteria that were unable to produce colonies in solid-based culture. Additionally, growth recovery in liquid media using the most probable number (MPN) assay was able to account for the discrepancy between the MBL assay and CFU number, suggesting that the MBL assay detects differentially culturable sub-populations of M. tuberculosis. CONCLUSIONS: The MBL assay can enumerate the bacterial load in animal tissues in real time without the need to wait for extended periods for cultures to grow. The readout correlates well with CFUs. Importantly, we have shown that the MBL is able to measure specific populations of bacteria not cultured on solid agar. The adaptation of this assay for preclinical studies has the potential to decrease the readout time of data acquisition from animal experiments and could represent a valuable tool for tuberculosis drug discovery and development

DNA sequence-selective C8-linked pyrrolobenzodiazepine-heterocyclic polyamide conjugates show anti-tubercular-specific activities.

New chemotherapeutic agents with novel mechanisms of action are in urgent need to combat the tuberculosis pandemic. A library of 12 C8-linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD)-heterocyclic polyamide conjugates (1-12) was evaluated for anti-tubercular activity and DNA sequence selectivity. The PBD conjugates were screened against slow-growing Mycobacterium bovis Bacillus Calmette-Guérin and M. tuberculosis H37Rv, and fast-growing Escherichia coli, Pseudomonas putida and Rhodococcus sp. RHA1 bacteria. DNase I footprinting and DNA thermal denaturation experiments were used to determine the molecules' DNA recognition properties. The PBD conjugates were highly selective for the mycobacterial strains and exhibited significant growth inhibitory activity against the pathogenic M. tuberculosis H37Rv, with compound 4 showing MIC values (MIC=0.08 mg l-1) similar to those of rifampin and isoniazid. DNase I footprinting results showed that the PBD conjugates with three heterocyclic moieties had enhanced sequence selectivity and produced larger footprints, with distinct cleavage patterns compared with the two-heterocyclic chain PBD conjugates. DNA melting experiments indicated a covalent binding of the PBD conjugates to two AT-rich DNA-duplexes containing either a central GGATCC or GTATAC sequence, and showed that the polyamide chains affect the interactions of the molecules with DNA. The PBD-C8 conjugates tested in this study have a remarkable anti-mycobacterial activity and can be further developed as DNA-targeted anti-tubercular drugs

Comparative fitness analysis of D-cycloserine resistant mutants reveals both fitness-neutral and high-fitness cost genotypes

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D-cycloserine resistance conferring mutations