Particle production as a function of respiration in a clinical TB patient.

<p>CO₂ concentration (solid line and left ordinate) and particle counts (dots and right ordinate) in the 1–2.5 μm size range for a TB patient.</p

Isolation and visualization of viable mycobacteria in the RASC.

<p>(A) <i>M</i>. <i>smegmatis</i>::<i>gfp</i> growth on solid 7H10 agar plates from the Six-Stage Viable Andersen Cascade Impactor after wet release of 200 μl diluted culture into the RASC (30 000, 3000 and 300 colony forming units—CFU). The columns indicate the particle sizes captured on each plate across the 6 stages of the impactor, and the rows indicate the estimated total number of CFU passing through the impactor. Each release was repeated three times and the mean and SD for each plate are presented below the typical growth pattern distribution seen in the particle release. In all the releases the sampling was run for 5 minutes at 28 l/min resulting in the potential total capture of 3000, 300 and 30 CFU respectively. (B) SEM (left) and fluorescent microscopy (right) of <i>M</i>. <i>smegmatis</i>::<i>gfp</i> isolated on a PM10 impactor following experimental release.</p

The effect of aerosol hydration on particle size distribution.

<p>The graph shows APS measurements of “wet” and “dry” releases of <i>M</i>. <i>smegmatis</i>::<i>gfp</i>. (see text for details).</p

The Respiratory Aerosol Sampling Chamber (RASC).

<p>(A) Photograph of the RASC (with the door open) on site in a community TB clinic (1) aerodynamic particle sizer (2) Filter samplers (3) Andersen impactor (4) Mixing fan (5) CO2, temperature and RH (6) PM10 impactor (7) Chair for participant. (B) Block diagram depicting the fluidic and electronic configuration of the RASC. Thick connecting lines indicate airflow and aerosol paths; thin lines indicate electronic connections. All air leaving the RASC is HEPA filtered.</p

Isolation of <i>Mtb</i> from a TB patient.

<p>SEM image of patient sample impacted on the lower plate of the PM10 impactor. The dimensions and morphology of the rod-shaped structure (denoted by *) are consistent with the presence of <i>Mtb</i> bacilli in the untreated TB patient. There is also evidence of multiple “splats” of unknown identity (one example is denoted by **) which might comprise organic matter derived from patient lung or respiratory tract. Note the “halo” structures (dark shadows) surrounding each particle.</p

Novel Antitubercular 6‑Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure–Activity Relationship and Target Identification Studies

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against <i>Mycobacterium tuberculosis</i> (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome <i>bc</i>₁ complex, disruption of cell–wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylamino­pyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure–activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties