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.     

Dryoptkirbioside,A New Fructofuranoside Glycerol,and Other Constituents from Dryopteris kirbi Hook et Grav Rhizomes

A new fructofuranoside glycerol, dryoptkirbioside ( 1 ), along with thirteen known compounds ( 2 - 14 ), was isolated from the MeOH extract of Dryopteris kirbi rhizomes by silica gel column chromatography, Sephadex LH-20 column chromatography, and semipreparative HPLC. The structure of the new compound was determined by analyses of its spectroscopic data including nuclear magnetic resonance (NMR), and high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) and chemical conversions. The hexane-soluble portion and the EAF_A fraction showed strong activities against lung (A549), breast (MCF-7), and cervical (HeLa) human cancer cell lines (IC₅₀ values ranging from 4.0 to 8.8 μg/mL). Aspidinol P ( 5 ) and aspidinol B ( 6 ) exhibited moderate to low cytotoxicity on the three cell lines (IC₅₀ values ranging from 20.4 to 58.7 μM). The MeOH extract and hexane-soluble portion had excellent activities against Staphylococcus aureus and Bacillus subtilis (MICs 11.7 and 23.4 μg/mL), whereas the AcOEt- and BuOH-soluble portions were significantly active on S. aureus (MICs 46.9 and 93.8 μg/mL). The main fractions EAF_B, EAF_C and nBF_B displayed excellent activity against S. aureus (MICs 11.7 and 23.4 μg/mL). Aspidinol B ( 6 ) had significant activity, while aspidinol P ( 5 ) was moderately active against S. aureus and B. subtilis (MICs 42.0 and 89.5 μM).     

THE IMPACT OF STORM-FLOW ON RIVER BIOFILM ARCHITECTURE1

The impact of storm-flow on river biofilm architecture was investigated using transmission (TEM) and scanning (SEM) electron microscopy. TEM resin substrata were colonized under light-grown (LG) or dark-grown (DG) conditions for 33 weeks in the Clywedog River, North Wales, prior to exposure to ambient-flow (approx. 60 cm·s^?1) or storm-flow (approx. 235 cm·s^?1+ river sediment) in a laboratory flume. Line transect methodology was used to quantify information from TEM ultrathin sections of LG material. In the LG ambient-flow biofilm, bacteria were more abundant directly adjacent to the substratum and were noticeably denser directly under the adnate diatom Cocconeis. Higher in the biofilm, the bacteria were loosely dispersed in the matrix between other cells. Cyanobacteria occurred most frequently as single cells but were also found in large “palisade” formations adjacent to the substratum. Significant horizontal and vertical nearest-neighbor associations were noted for both bacteria and cyanobacteria. Cells of Cocconeis were common adjacent to the substratum, providing shelter to, and often elevated upon, an “organic pad” of bacteria, cyanobacteria, and densely staining exopolysaccharide. Cyanobacteria and Cocconeis were resistant to removal by storm-flow, but Cocconeis frustules were sometimes damaged. Bacteria in the LG storm-flow samples were less common adjacent to the substratum and were sometimes more dispersed higher in the biofilm than in ambient-flow samples. We suggest that storm-flow hydrodynamic forces may redistribute bacteria adjacent to the substratum into higher areas of the biofilm. In addition, bacteria and the exopolysaccharide matrix were sometimes removed down to the substratum by storm-flow, unless beneath Cocconeis. The DG biofilm consisted almost entirely of bacteria. Storm-flow only removed surface growth from DG biofilms, and SEM revealed peritrich stalk abrasion and “blow-down.” Pre-disturbance biofilm architecture appears to influence the form of destruction. We suggest that the “microcosms” of Cocconeis and their underlying cells not only serve as an inoculum to recolonize the surface when conditions permit but enhance immigration by interrupting flow patterns across the surface.     

Using Betula pendula and Telephora caryophyllea for Soil Pollution Assessment

The concentrations of available arsenic, copper, lead, and zinc in the soil, and the concentration of these elements in vegetal tissues were measured. The most common species at the sites were studied. All the species that were analyzed took up pollutants and could indicate polluted soil. However, all the studied species did not fit to map pollution. The birch (Betula pendula) and most of the fungi species had still quite low concentrations in their tissues compared with the available concentrations in the soil. No direct correlation between the pollution content in the soil and in the vegetation tissues could be observed. Specimens of Thlaspi caerulescens were accumulating zinc. Of the four fungi species collected, only Thelephora caryophyllea seemed to accumulate actively in the studied pollutants. Moreover, it was possible to use an arsenic test with the fungi, which is also interesting from the perspective of preliminary assessment of the degree of pollution. A qualitative judgement of the soil pollution is possible by examining the plant material. To obtain a more quantitative and complete mapping, the method has to be developed further and completed with other investigations when vegetation is missing.     

Comparison of different extractive procedures for proteins from the edible seaweeds Ulva rigida and Ulva rotundata

Proteins have been extracted from the edible seaweeds Ulva rigida Agardh and Ulva rotundata Bliding using classical or enzymatic procedures. The protocols using NaOH under reductive conditions or a two-phase system (PEG/K₂CO₃) produced the best protein yields. The cleavage or the limitation of the linkages between proteins and polysaccharides caused by these experimental conditions probably explains the efficiency of these protocols. In SDS PAGE, the protein fraction obtained after NaOH extraction from U. rotundata is characterised by the presence of three major bands with apparent molecular weights of 45 600, 31 800 and 18 600. The protein fraction from U. rigida presents two specific bands with apparent molecular weights of about 27 000 and 12 000. These fractions are mainly rich in aspartic and glutamic acids, alanine, glycine and contain few hydroxyproline residues (0.91–2.44% total amino acid content). The use of cellulase does not significantly improve the extraction of algal proteins in comparison with the blank procedure (without enzymes). The weak accessibility of the substrates in the intact cell wall could explain these experimental data. The improvement of protein yield after the use of the polysaccharidase mixture (-glucanase, hemicellulase, cellulase) partially confirms this hypothesis.