Mark R. Riley

Effects of metals Cu, Fe, Ni, V, and Zn on rat lung epithelial cells

Inhalation of combustion-derived particulate matter can have a variety of negative impacts on human health. Metals are known to play a substantial role in these effects, however, the interactions between cellular responses caused by multiple metals is not well understood. The impact of metals (Zn, Cu, Ni, V, and Fe) individually and in combination on a rat lung epithelial cell line (RLE-6TN) was evaluated. Quantifications involved measurement of inhibition of cell culture metabolism (mitochondrial succinate dehydrogenase activity), cell death, mechanisms of cell death, and cytokine secretion. The ranking of metal toxicity based on TC(50) values is V>Zn>Cu>Ni>Fe. Interactions were observed for exposures containing multiple metals: Zn+V, Zn+Cu, Zn+Fe, and Zn+Ni. Zn appears to diminish the negative impact of V and Cu; has an additive effect with Ni, and has no substantial effect on Fe toxicity.

Evaluation of Toxic Agent Effects on Lung Cells by Fiber Evanescent Wave Spectroscopy

Biochemical changes in living cells are detected using a fiber probe system composed of a single chalcogenide fiber acting as both the sensor and transmission line for infrared optical signals. The signal is collected via evanescent wave absorption along the tapered sensing zone of the fiber. We spectroscopically monitored the effects of the surfactant Triton X-100, which serves as a toxic agent simulant on a transformed human lung carcinoma type II epithelial cell line (A549). We observe spectral changes between 2800–3000 cm−1 in four absorptions bands, which are assigned to hydrocarbon vibrations of methylene and methyl groups in membrane lipids. Comparison of fiber and transmission spectra shows that the present technique allows one to locally probe the cell plasma membrane in the lipid spectral region. These optical responses are correlated with cellular metabolic activity measurements and LDH (lactate dehydrogenase) release assays that indicate a loss of cellular function and membrane integrity as would be expected in response to the membrane solubilizing Triton. The spectroscopic technique shows a significantly greater detection resolution in time and concentration.

Utilization of cellulosic waste from tequila bagasse and production of polyhydroxyalkanoate (PHA) bioplastics by Saccharophagus degradans.

Utilization of wastes from agriculture is becoming increasingly important due to concerns of environmental impact. The goals of this work were to evaluate the ability of an unusual organism, Saccharophagus degradans (ATCC 43961), to degrade the major components of plant cell walls and to evaluate the ability of S. degradans to produce polyhydroxyalkanoates (PHAs, also known as bioplastics). S. degradans can readily attach to cellulosic fibers, degrade the cellulose, and utilize this as the primary carbon source. The growth of S. degradans was assessed in minimal media (MM) containing glucose, cellobiose, avicel, and bagasse with all able to support growth. Cells were able to attach to avicel and bagasse fibers; however, growth on these insoluble fibers was much slower and led to a lower maximal biomass production than observed with simple sugars. Lignin in MM alone did not support growth, but did support growth upon addition of glucose, although with an increased adaptation phase. When culture conditions were switched to a nitrogen depleted status, PHA production commences and extends for at least 48 h. At early stationary phase, stained inclusion bodies were visible and two chronologically increasing infrared light absorbance peaks at 1,725 and 1,741 cm−1 confirmed the presence of PHAs. This work demonstrates for what we believe to be the first time, that a single organism can degrade insoluble cellulose and under similar conditions can produce and accumulate PHA. Additional work is necessary to more fully characterize these capabilities and to optimize the PHA production and purification. Biotechnol. Bioeng. 2008;100: 882–888.

Efficient extraction method to collect sugar from sweet sorghum

BackgroundSweet sorghum is a domesticated grass containing a sugar-rich juice that can be readily utilized for ethanol production. Most of the sugar is stored inside the cells of the stalk tissue and can be difficult to release, a necessary step before conventional fermentation. While this crop holds much promise as an arid land sugar source for biofuel production, a number of challenges must be overcome. One lies in the inherent labile nature of the sugars in the stalks leading to a short usable storage time. Also, collection of sugars from the sweet sorghum stalks is usually accomplished by mechanical squeezing, but generally does not collect all of the available sugars.ResultsIn this paper, we present two methods that address these challenges for utilization of sweet sorghum for biofuel production. The first method demonstrates a means to store sweet sorghum stalks in the field under semi-arid conditions. The second provides an efficient water extraction method that can collect as much of the available sugar as feasible. Operating parameters investigated include temperature, stalk size, and solid–liquid ratio that impact both the rate of sugar release and the maximal amount recovered with a goal of low water use. The most desirable conditions include 30°C, 0.6 ratio of solid to liquid (w/w), which collects 90 % of the available sugar. Variations in extraction methods did not alter the efficiency of the eventual ethanol fermentation.ConclusionsThe water extraction method has the potential to be used for sugar extraction from both fresh sweet sorghum stalks and dried ones. When combined with current sugar extraction methods, the overall ethanol production efficiency would increase compared to current field practices.

Phi29 pRNA vector for efficient escort of hammerhead ribozyme targeting survivin in multiple cancer cells

Ribozymes are potential therapeutic agents which suppress specific genes in disease-affected cells. Ribozymes have high substrate cleavage efficiency, yet their medical application has been hindered by RNA degradation, aberrant cell trafficking, or misfolding when fused to a carrier. In this study, we constructed a chimeric ribozyme carried by the motor pRNA of bacteriophage phi29 to achieve proper folding and enhanced stability. A pRNA molecule contains an interlocking loop domain and a 5´/3´ helical domain, which fold independently of one another. When a ribozyme is connected to the helical domain, the chimeric pRNA/ribozyme reorganize into a circularly permuted form, in which the 5´/3´ ends are relocated and buried in the original 71′/75′ positions. Effective silencing of an anti-apoptotic gene survivin by an appropriately designed chimeric ribozyme, as demonstrated at mRNA and protein levels, led to programmed cell death in various human cancer cell lines, including breast, prostate, cervical, nasopharyngeal, and lung, without causing significant non-specific cytotoxicity. Through the interlocking interaction of right and left loops, monomer pRNA/ribozyme chimeras can be incorporated into multi-functional dimer, trimer and hexamer complexes for specific gene delivery. Using the phi29 motor pRNA as an escort may revive the ribozymes strength in medical application again.

Discrimination of bacteria and bacteriophages by raman spectroscopy and surface-enhanced raman spectroscopy

Detection of pathogenic organisms in the environment presents several challenges due to the high cost and long times typically required for identification and quantification. Polymerase chain reaction (PCR) based methods are often hindered by the presence of polymerase inhibiting compounds and so direct methods of quantification that do not require enrichment or amplification are being sought. This work presents an analysis of pathogen detection using Raman spectroscopy to identify and quantify microorganisms without drying. Confocal Raman measurements of the bacterium Escherichia coli and of two bacteriophages, MS2 and PRD1, were analyzed for characteristic peaks and to estimate detection limits using traditional Raman and surface-enhanced Raman spectroscopy (SERS). MS2, PRD1, and E. coli produced differentiable Raman spectra with approximate detection limits for PRD1 and E. coli of 109 pfu/mL and 106 cells/mL, respectively. These high detection concentration limits are partly due to the small sampling volume of the confocal system but translate to quantification of as little as 100 bacteriophages to generate a reliable spectral signal. SERS increased signal intensity 103 fold and presented peaks that were visible using 2-second acquisitions; however, peak locations and intensities were variable, as typical with SERS. These results demonstrate that Raman spectroscopy and SERS have potential as a pathogen monitoring platform.

Simultaneous Measurement of 19 Components in Serum‐Containing Animal Cell Culture Media by Fourier Transform Near‐Infrared Spectroscopy

Animal cell cultures generate maximal amounts of desired products when maintained in a controlled environment with low and constant concentrations of nutrients and wastes. Traditionally this has involved slow addition of glucose and glutamine; however, recent studies have indicated that a number of low concentration amino acids are required to prevent initiation of apoptosis. Therefore, optimal control of animal cell cultures will likely require measurement of a large number of chemical components. We present here the evaluation of a near‐infrared spectroscopic (NIRS) monitoring scheme to quantify 19 cellular nutrients and wastes in culture medium with and without serum. The components include glucose, lactate, ammonia, pyruvate, glutamine, and 14 other amino acids. Spectroscopic calibrations were generated for a synthetic version of a standard culture medium (DMEM) in which the concentrations of 17 DMEM components and ammonia and lactate were varied in a random fashion. This randomization provides a stringent evaluation of the measurement scheme. Reasonably accurate measurements of these 19 components could be accomplished in the absence or presence of 10% horse serum by volume with percent errors ranging from 3% to 37%. Analytes with concentrations as low as 0.3 mM could be reliably quantified. The presence of serum, when properly included in the calibration, has little effect on measurement error. These results provide an important step toward application of NIRS for monitoring the large number of varying components of animal cell cultivations.

Impact of the composition of combustion generated fine particles on epithelial cell toxicity: influences of metals on metabolism☆

Inhaled airborne particulate matter (PM) represents a potentially significant health hazard to humans. Exposure to PM strongly correlates with pulmonary inflammation and incidences of severe respiratory distress, including increased hospital admissions for breathing disorders, asthma, emphysema, and chronic bronchitis. PM generated from the combustion of fuel oils and coals contain a number of water-soluble transition metals including Fe, V, and Zn. We have evaluated the impact of PM types with varying composition collected from the combustion of oils and coals on the health and metabolism of lung cell cultures. Three colorimetric assays (sulforhodamine B (SRB), Janus green, and MTT) have been adapted to quantify the impact of PM on rat lung alveolar type II epithelial cells (RLE-6TN cells). The PM toxicity metrics evaluated were inhibition of cell proliferation (SRB and Janus green) and inhibition of cellular metabolism (MTT). Cell proliferation is inhibited in a consistent dose-dependent manner by PM concentrations from 25 to 250 microg/ml. At a level of 100 microg/ml, oil-derived PM diminishes cell metabolism by as much as 40% relative to controls; the degree of inhibition is strongly dependent on PM particle size and metal content. Conversely, coal-derived PM at the same dosage diminishes cell metabolism by no more than 20% relative to controls. All three assays provide highly repeatable results and consistent toxicity rankings of the PMs evaluated. Overall, metabolic inhibition as measured by the MTT assay was deemed the most appropriate metric for PM toxicity, primarily due to its applicability with in vivo-like confluent cell monolayers.

Rapid calibration of near-infrared spectroscopic measurements of mammalian cell cultivations.

Near‐infrared (NIR) spectroscopy is a flexible method that can be employed to noninvasively monitor the concentrations of multiple nutrients and wastes in mammalian cell bioreactors. Development of suitable calibrations can be a labor‐ and time‐intensive process that must be repeated when process conditions are altered significantly. To address this difficulty, we have produced a new approach for generating NIR spectroscopic calibrations that requires significantly less time compared with standard calibration schemes. This method reduces development time from the present level of several weeks to several hours. A small number of experimentally collected spectra serve as inputs to a computational procedure that yields a large number of simulated spectra, each containing both analyte‐specific and analyte‐independent information. Such simulated spectra may be employed as a calibration set for quantifying analytes in experimentally collected spectra. Spectroscopic measurements of the concentrations of five components (ammonia, glucose, glutamate, glutamine, and lactate) can be accomplished with levels of error similar to those obtained with full experimental calibrations. A key to this process is the utilization of random numbers, which randomizes the influence of natural variations, present in each experimentally collected spectrum, on the resultant composite spectrum. This approach may increase the feasibility of employing NIR spectroscopy to monitor bioreactors and other biological processes subjected to varying operating conditions.

Opto-electrophoretic detection of bio-molecules using conducting chalcogenide glass sensors

Novel telluride glasses with high electrical conductivity, wide infrared transparency and good resistance to crystallization are used to design an opto-electrophoretic sensor for detection and identification of hazardous microorganisms. The sensor is based on an attenuated total reflectance element made of Ge-As-Te glass that serves as both an optical sensing zone and an electrode for driving the migration of bio-molecules within the evanescent wave of the sensor. An electric field is applied between the optical element and a counter electrode in order to induce the migration of bio-molecules carrying surface charges. The effect of concentration and applied voltage is tested and the migration effect is shown to be reversible upon switching the electric field. The collected signal is of high quality and can be used to identify different bacterial genus through statistical spectral analysis. This technique therefore provides the ability to detect hazardous microorganisms with high specificity and high sensitivity in aqueous environments. This has great potential for online monitoring of water quality.