Bioaerosol Mass Spectrometry for Rapid Detection of Individual Airborne Mycobacterium tuberculosis H37Ra Particles

Single-particle laser desorption/ionization time-of-flight mass spectrometry, in the form of bioaerosol mass spectrometry (BAMS), was evaluated as a rapid detector for individual airborne, micron-sized, Mycobacterium tuberculosis H37Ra particles, comprised of a single cell or a small number of clumped cells. The BAMS mass spectral signatures for aerosolized M. tuberculosis H37Ra particles were found to be distinct from M. smegmatis, Bacillus atrophaeus, and B. cereus particles, using a distinct biomarker. This is the first time a potentially unique biomarker was measured in M. tuberculosis H37Ra on a single-cell level. In addition, M. tuberculosis H37Ra and M. smegmatis were aerosolized into a bioaerosol chamber and were sampled and analyzed using BAMS, an aerodynamic particle sizer, a viable Anderson six-stage sampler, and filter cassette samplers that permitted direct counts of cells. In a background-free environment, BAMS was able to sample and detect M. tuberculosis H37Ra at airborne concentrations of >1 M. tuberculosis H37Ra-containing particles/liter of air in 20 min as determined by direct counts of filter cassette-sampled particles, and concentrations of >40 M. tuberculosis H37Ra CFU/liter of air in 1 min as determined by using viable Andersen six-stage samplers. This is a first step toward the development of a rapid, stand-alone airborne M. tuberculosis particle detector for the direct detection of M. tuberculosis bioaerosols generated by an infectious patient. Additional instrumental development is currently under way to make BAMS useful in realistic environmental and respiratory particle backgrounds expected in tuberculosis diagnostic scenarios.     

A novel mechanism of soluble guanylate cyclase stimulation: time-dependent activation by bacterial lipopolysaccharide in rat fetal spleen cells

Small amounts of bacterial lipopolysaccharide (LPS) greatly increase cGMP levels in short term cultures of rat fetal liver and spleen cells in a dose and time dependent manner. To determine the role of guanylate cyclase in this response, a series of experiments was undertaken using either intact or broken fetal spleen cells, the most sensitive tissue evaluated to date. The phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, potentiated the LPS-cGMP effect in cultures of these cells even at maximal doses of LPS. Moreover, after incubation of intact cells with LPS for 4 h, soluble guanylate cyclase (EC 4.6.1.2) activity was increased 2-fold, whereas particulate activity was unchanged. This increase in soluble activity was proportional to the dose of LPS, was synchronous with the elevation of cGMP levels, and was not associated with any change in cGMP-phosphodiesterase (EC 3.1.4.17) activity. In contrast to intact cells, neither total nor soluble guanylate cyclase activity was increased by the addition of LPS to spleen cells, neither total cytosol for various times from 10 min to 3.5 h. These results suggest that the LPS-cGMP response is due to a persistent indirect stimulation of soluble guanylate cyclase activity that is both dose and time dependent.     

The role of signaling pathways in osteoblast gravity perception

Bone loss is one of the major problems in long term spaceflight. This physiological consequence of microgravity is the rapid loss of weightbearing bone that is associated with skeletal unloading. Moreover, we have previously noted that sera deprived osteoblasts do not have a normal response to sera in microgravity. Where exercise (mechanical loading) has been shown to increase bone formation and stimulate osteoblastic function, the mechanisms underlying signal transduction of mechano-perception is yet to be fully understood. Osteoblasts have been shown to respond to mechanical stress such as fluid shear, bending, flexing and compression. The type of stress and amount of stress determine the osteoblast response Recently we have discovered that the isolated osteoblast responds to a very short pulse of g-force compression. The possible regulatory sensors include mechano-sensitive calcium channels, autrocrine responses to stress, response to FAK/integrin, alterations in the cytoskeleton as well as other known growth factor and cytokine receptors. The secondary signal may include growth factor related kinases such as ERK, p38 and JNK map kinase (MAPK) pathways. Experimental evidence suggests that normal osteoblast response to stress and sera requires normal earth gravity.     

EVALUATING THE RELATIONSHIP BETWEEN PHOTOPIGMENT SYNTHESIS AND 2-METHYLISOBORNEOL ACCUMULATION IN CYANOBACTERIA

  The relationship between photopigments and the terpene-derived secondary metabolite, 2-methylisoborneol (MIB), was analyzed in photoacclimated cultures of Pseudanabaena articulata Skuja throughout growth, during the diel cycle, and following chemical-induced inhibition of the isoprenoid pathway. Accumulation of MIB coincided with the accumulation of lipophilic and phycobilin pigments during the early to mid-exponential portion of the growth cycle with the greatest accumulation of MIB during the late-exponential phase. Cellular release of MIB occurred as culture populations entered mid- to late-logarithmic phase of growth and was greatest in irradiance-stressed cultures. The greater correspondence of MIB accumulation with photopigments was seen in cultures transferred from a 12:12 h LD photoperiod alone and the consistent relationship between MIB and photopigment accumulation under varying irradiance suggested a photopigment-dependent regulation for MIB synthesis. However, the consistent allocation of carbon into MIB during instances of phytofluene and tetrapyrrole biosynthetic inhibition within P. articulata and Oscillatoria perornata Skuja indicated that MIB accumulation is not limited by isopreniod-carbon availability and does not appear to serve as an "overflow" product. Rather, MIB accumulation simply appears to reflect overall carbon accumulation resulting from increased cell metabolism.