In situ nuclear magnetic resonance microimaging of live biofilms in a microchannel.

Biofilms are comprised of microbial cells and an extracellular polymeric substance (EPS) matrix that supports interactions between community members and with the local environment. The highly hydrated EPS matrix makes the application of many biofilm visualization techniques difficult. Hence, to better visualize how biofilms interact with their environment, there is a need for imaging techniques to monitor hydrated state biofilm dynamics. We employed an in situ dynamic approach to construct label-free images of biofilms. In situ imaging was conducted using a vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface), for biofilm growth; real-time confocal laser scanning microscopy analysis; and nuclear magnetic resonance (NMR) microimaging and spectroscopy. We integrated SALVI microchannel fluids and live biofilms to demonstrate in situ measurement capabilities, including velocity mapping, diffusion coefficient mapping, relaxometry, localized spectroscopy, relaxation times, porosity, and two- and three-dimensional imaging within the microchannel at high spatial resolution. We monitored organic acids adjacent to biofilms, suggesting that kinetic rate and substrate-product yield ratio studies are possible using the SALVI microfluidic reactor for growth characterizations. The integration of NMR microimaging studies into the SALVI platform demonstrates that a multimodal microfluidic platform can serve as an avenue to explore complex biological phenomena, such as biofilm attachment to surfaces, with detailed quantitative physical and chemical mapping. The further incorporation of other SALVI-compatible technologies, such as liquid time-of-flight secondary ion mass spectrometry imaging, with NMR microimaging will produce a powerful correlative approach to monitor in situ biofilm chemistry and dynamics at different spatial scales.

Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.

We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.

Induction and repair of HZE induced cytogenetic damage.

Wistar rats were exposed to high-mass, high energy (HZE) 56Fe particles (1000 GeV/AMU) using the Alternating Gradient Synchrotron (AGS). The animals were sacrificed at 1-5 hours or after a 30-day recovery period. The frequency of micronuclei in the tracheal and the deep lung epithelial cells were evaluated. The relative effectiveness of 56Fe, for the induction of initial chromosome damage in the form of micronuclei, was compared to damage produced in the same biological system exposed to other types of high and low-LET radiation. It was demonstrated that for animals sacrificed at short times after exposure, the tracheal and lung epithelial cells, the 56Fe particles were 3.3 and 1.3 times as effective as 60Co in production of micronuclei, respectively. The effectiveness was also compared to that for exposure to inhaled radon. With this comparison, the 56Fe exposure of the tracheal epithelial cells and the lung epithelial cells were only 0.18 and 0.20 times as effective as radon in the production of the initial cytogenetic damage. It was suggested that the low relative effectiveness was related to potential for 'wasted energy' from the core of the 56Fe particles. When the animals were sacrificed after 30 days, the slopes of the dose-response relationships, which reflect the remaining level of damage, decreased by a factor of 10 for both the tracheal and lung epithelial cells. In both cases, the slope of the dose-response lines were no longer significantly different from zero, and the r2 values were very high. Lung epithelial cells, isolated from the animals sacrificed hours after exposure, were maintained in culture, and the micronuclei frequency evaluated after 4 and 6 subcultures. These cells were harvested at 24 and 36 days after the exposure. There was no dose-response detected in these cultures and no signs of genomic instability at either sample time.

Sonoporation of erythrocytes by lithotripter shockwaves in vitro.

Sonoporation of red blood cells was examined in relation to cavitation-induced hemolysis. FITC-dextran at 580,000 MW was added to suspensions of canine erythrocytes and the mixture was exposed to lithotripter shockwaves. Exposure at 5% or 50% hematocrit in PBS or 50% in plasma yielded not only hemolysis but also FITC-dextran uptake in surviving cells. Hemolysis increased with increasing numbers of shockwaves. The numbers of cells with fluorescent dextran uptake remained roughly constant for 250-1000 shockwaves, but this represented an increasing percentage of the surviving cells. In addition, fluorescent microspheres formed spontaneously in samples with hemolysis. An air bubble was needed in the chamber to obtain substantial effects, implicating the cavitation mechanism. The exposure-response trends could be modeled by simple theory for random interaction of the cells with bubbles.

Induction of micronuclei in respiratory tract following radon inhalation.

Male Wistar rats were exposed to radon and its progeny (0.0, 60, 262 and 564 working level months, WLM), and the frequency of micronuclei was determined in deep lung fibroblasts, and deep lung, trachea and nasal epithelial cells with slopes of 0.28, 0.67, 0.34 and 0.11 micronuclei/1000 binucleated cells/WLM respectively. Micronuclei in deep lung fibroblasts, isolated and cultured using two methods and media, demonstrated no differences in slopes. Biological damage was used as a biodosimeter to calculate the relationship between dosimetric units: alpha particle traversals or 'nuclear hits', dose in mGy and exposure in WLM. The estimated number of nuclear alpha traversals/Gy was 6.3. Radon exposure to 170 WLM resulted in the same frequency of micronuclei in deep lung epithelial cells as produced by one alpha hit/cell nucleus. Absorbed dose/unit of exposure (mGy/WLM) was estimated assuming the damage was related to absorbed dose or to changes in cell sensitivity and ranged from 1.13 to 1.34 for deep lung epithelial cells, 0.47 to 1.09 for deep lung fibroblasts, 0.34 to 0.67 for tracheal epithelial cells and 0.18 to 0.33 for nasal epithelial cells. Biological dosimetry can be used to relate exposure to damage, compare dosimetric units and validate physical dosimetry models. This approach can be applied to any inhaled material capable of producing biological damage.

Comparative clastogenic sensitivity of respiratory tract cells to gamma rays.

To understand the relationships between exposure and damage to different cell populations in the respiratory tract, methods were developed to culture deep-lung fibroblasts and epithelial cells from the nose, trachea and deep lungs. Female F-344 Fischer and male Wistar rats were exposed to 1-5 Gy of 60Co gamma rays at a dose rate of 0.4 Gy/min. Cells were isolated for short-term culture, and the incidences of binucleated cells and micronuclei were determined. The incidences of micronuclei were determined in cytochalasin-B-induced binucleated cells at 72 h for nasal and tracheal tissue and 96 h for deep-lung fibroblasts and epithelial cells. Maximum frequencies of binucleated cells were found in the control nonirradiated cells at these harvest times, and the frequencies were not significantly affected at these harvest times by radiation exposure. No significant differences were found in the frequencies of micronuclei induced in the nasal epithelial cells isolated from female F-344 Fischer or male Wistar rats. Fibroblasts cultured in different media and isolated from either female F-344 Fischer or male Wistar rats also showed a similar frequency of micronuclei. Over the doses tested, the frequency of micronuclei in the respiratory tract cells increased linearly with the dose. The slopes were 92.2 +/- 9.2, 76.2 +/- 7.9, 32.8 +/- 2.4 and 28.7 +/- 3.4 micronuclei/1000 binucleated cells/Gy for deep-lung epithelial cells, deep-lung fibroblasts, tracheal epithelial cells and nasal epithelial cells, respectively. Deep-lung epithelial or fibroblast cells were about two to three times as sensitive for elastogenic damage as nasal and tracheal epithelial cells. The measurement of micronuclei in isolated respiratory tract cells is very useful in assessing cytogenetic damage induced in different cell types by radiation.

Ultrasonically induced hemolysis at high cell and gas body concentrations in a thin-disc exposure chamber.

Ultrasound image contrast may be enhanced by injecting gas bodies into the blood. This in vitro study was undertaken to assess the potential for induction of hemolysis due to ultrasonic activation of the contrast agent gas bodies. Canine whole blood with Albunex (Mallinckrodt Medical, St. Louis, MO, USA) was exposed to near-field ultrasound beams in 1-mm-thick chambers held stationary (i.e., not rotated) in a 37 degrees C water bath. At 2.25 MHz, statistically significant hemolysis occurred in 0.5 hematocrit, 50% Albunex suspensions for 0.28-MPa, 1-s continuous exposure and for 0.58-MPa, 100-s exposures with 10-microsecond pulses and 1.0-ms pulse repetition period. Continuous exposure durations as short as 10 ms produced about 4.5% hemolysis, which only increased slightly to about 5.5% after 100 s. At a constant 1.6 MPa, hemolysis increased with increasing gas body concentration and with decreasing cell concentration. Hemolysis decreased with increasing frequency in a 50/50 mixture of whole blood and Albunex, with thresholds rising from 0.12 MPa continuous (1 s) and 0.47 MPa pulsed (10 microseconds:1.0 ms for 100 s) at 1.06 MHz to 0.47 MPa continuous and 1.9 MPa pulsed at 5.3 MHz.