Evidence of Infection among Ferrets exposed to high levels of nebulized NC99.

a<p>For exposures to ferrets 2-C and 2-D, the diffusion dryer was inserted into the line exiting the Collison generator, reducing the humidity at the end of the hour nebulization.</p>b<p>Calculation of inhaled virus based on measured viral RNA in aerosol inhaled during 60 min of aerosol exposure.</p>c<p>Nasal wash was collected on day 2 post exposure (pe) in Exp 1, and daily in Exp 2; culture results are reported for the entire collection as FFU/mL; viral RNA is the peak titer on day 3 pe and is reported as GEq/collection.</p

Comparison of impinger and PTFE filter efficiencies for culturable virus and viral RNA.

a<p>Virus NC99 was nebulized in 6 experiments with impinger alone and 9 experiments with impinger and PTFE filter collections in parallel. The mean (range) total dose nebulized was 1.8 (1.1–2.2)×10⁷ FFU, calculated by virus concentration in Collison at beginning of aerosol generation times fluid volume nebulized for each run.</p>b<p>Impinger collection measured for 5.4 (range 5.3–5.5) L/min, reported as total FFU collected during interval of nebulization. N = 5 at each time point as one outlier value was removed from each group.</p>c<p>PTFE filter collection combines two filters in parallel for total flow of 4.0 L/min, reported as total FFU and total genome equivalents (GEq) of RNA measured by T5000 assay.</p>d<p>Ratio of group means of FFU and GEq RNA collected respectively.</p

Characteristics of the aerosol exposure system.

<p><b>a.</b> Photograph of exposure apparatus during nebulization with Collison generator inside the origin chamber shows the visible cloud of airborne particles in both the left (origin) and right (recipient) chambers. Placement of the Collison generator outside of the origin chamber, connected with 20 cm tubing, resulted in no visible suspended airborne particles in the chambers, and all subsequent experiments reported here had this configuration. The Grimm particle spectrometer is placed on top of the left chamber and the sampling port is located on top of the tunnel. <b>b.</b> The number particle size distribution with GRIMM optical counter. The fitted bimodal distribution has the median diameters of 0.44 and 1.70 µm, and geometric standard deviation of 1.25 and 1.46, for the two size modes, respectively. <b>c.</b> Particle number concentrations detected by Grimm laser-based particle counter sampled during four 30–60 sec intervals of the continuous one-hour nebulization.</p

Evidence of viral replication in ferrets exposed to low doses of aerosolized NC99.

<p>a. Actual dose calculated was within 30% of target dose.</p><p>b. Calculations based on viral RNA collected on PTFE filter during 60 min of aerosol exposure.</p

Comparison of sensitivity of the T5000 and RT-PCR Assays.

<p>Comparison of sensitivity of the assays for samples expected to contain relatively higher concentrations (3.0 to 7.0 log₁₀ Geq) of viral RNA (throat swab and nasal wash) and relatively lower concentrations (1.0 to 4.0 log₁₀ Geq) of viral RNA. The Ibis T5000 Influenza assay was not significantly more sensitive for detecting viral RNA in the airway specimens under the conditions of this experimental infection, but was significantly more sensitive in detection viral RNA in filter and impinger aerosol collection devices (Chi-Squared test, p<0.01).</p

Compound 1 inhibited cellular activation of GTPases.

<p>Rab7 (A), Cdc42 (B), and Ras (C) in response to growth factor stimulation. HeLa cells were treated with sequential starvation, compound addition and EGF stimulation. Active GTPases in the cell lysates were quantified using effector linked glutathione beads, probed with fluorescent antibody and analyzed on flow cytometer. Results are given as (sample MCF—negative control MCF)/positive control MCF where the negative control is the MCF reading obtained using lysates from unstimulated cells, and the positive control is the MCF reading obtained from the stimulated cells treated with DMSO.</p

A Pan-GTPase Inhibitor as a Molecular Probe

<div><p>Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.</p></div

Particle Concentration variation during chamber exposure.

<p>Airborne particles were measured continuously by Grimm spectrometer and recorded as particles per liter of air in this typical exposure. Particle numbers per second vary over two orders of magnitude.</p

Lack of covalent bond formation between GTPases and compound 1.

<p>Rab7 (A), Cdc42 (B) or Ras (C) was incubated with or without compound <b>1</b> overnight followed by extensive washing. Binding of BODIPY^-FL GTP to GTPases was indistinguishable whether the enzymes had been treated with compound <b>1</b> or not.</p

Compound 1 inhibited cellular activation of GTPases.

<p>Rab7 (A), Cdc42 (B), and Ras (C) in response to growth factor stimulation. HeLa cells were treated with sequential starvation, compound addition and EGF stimulation. Active GTPases in the cell lysates were quantified using effector linked glutathione beads, probed with fluorescent antibody and analyzed on flow cytometer. Results are given as (sample MCF—negative control MCF)/positive control MCF where the negative control is the MCF reading obtained using lysates from unstimulated cells, and the positive control is the MCF reading obtained from the stimulated cells treated with DMSO.</p