James T. Fleming

Attributing Effects of Aqueous C60 Nano-Aggregates to Tetrahydrofuran Decomposition Products in Larval Zebrafish by Assessment of Gene Expression

Background C60 is a highly insoluble nanoparticle that can form colloidal suspended aggregates in water, which may lead to environmental exposure in aquatic organisms. Previous research has indicated toxicity from C60 aggregate; however, effects could be because of tetrahydrofuran (THF) vehicle used to prepare aggregates. Objective Our goal was to investigate changes in survival and gene expression in larval zebrafish Danio rerio after exposure to aggregates of C60 prepared by two methods: a) stirring and sonication of C60 in water (C60–water); and b) suspension of C60 in THF followed by rotovaping, resuspension in water, and sparging with nitrogen gas (THF–C60). Results Survival of larval zebrafish was reduced in THF–C60 and THF–water but not in C60–water. The greatest differences in gene expression were observed in fish exposed to THF–C60 and most (182) of these genes were similarly expressed in fish exposed to THF–water. Significant up-regulation (3- to 7-fold) of genes involved in controlling oxidative damage was observed after exposure to THF–C60 and THF–water. Analyses of THF–C60 and THF–water by gas chromatography–mass spectrometry did not detect THF but found THF oxidation products γ-butyrolactone and tetrahydro-2-furanol. Toxicity of γ-butyrolactone (72-hr lethal concentration predicted to kill 50% was 47 ppm) indicated effects in THF treatments can result from γ-butyrolactone toxicity. Conclusion This research is the first to link toxic effects directly to a THF degradation product (γ-butyrolactone) rather than to C60 and may explain toxicity attributed to C60 in other investigations. The present work was first presented at the meeting “Overcoming Obstacles to Effective Research Design in Nanotoxicology” held 24–26 April 2006 in Cambridge, Massachusetts, USA.

Whole-cell biocomputing

The ability to manipulate systems on the molecular scale naturally leads to speculation about the rational design of molecular-scale machines. Cells might be the ultimate molecular-scale machines and our ability to engineer them is relatively advanced when compared with our ability to control the synthesis and direct the assembly of man-made materials. Indeed, engineered whole cells deployed in biosensors can be considered one of the practical successes of molecular-scale devices. However, these devices explore only a small portion of cellular functionality. Individual cells or self-organized groups of cells perform extremely complex functions that include sensing, communication, navigation, cooperation and even fabrication of synthetic nanoscopic materials. In natural systems, these capabilities are controlled by complex genetic regulatory circuits, which are only partially understood and not readily accessible for use in engineered systems. Here, we focus on efforts to mimic the functionality of man-made information-processing systems within whole cells.

Nucleic acid analytical approaches in bioremediation: site assessment and characterization

Abstract Bioremediation, the removal of environmental pollutants by living organisms, has become a viable and promising means of restoring contaminated sites. Gene probing techniques have enhanced our ability to assess the efficacy of microbial-based remediation efforts. DNA probes targeting specific genetic sequences, i.e. those genes responsible for the degradative ability of the microorganism, can be used to characterize a contaminated site throughout the bioremediation program to determine overall community structure and catabolic activity. To do so, however, requires efficient techniques for recovering nucleic acids from environmental sites as well as methods for generating probes to the specific genetic sequences desired. This review discusses procedures for isolating DNA, messenger RNA, and ribosomal RNA from environmental samples, the utilization of polymerase chain reactions to construct gene probes, and hybridization methods to genetically match the probe to the environmental sample. The use of these methods and advancement of techniques at several bioremediation sites is also presented along with typical problems and limitations encountered. The first case study involves monitoring the effects of nutrient addition to stimulate microbial degradation of chlorinated solvents at the DOE Westinghouse Savannah River Site. The next case study describes the bioremediation of chlorinated solvents and low levels of BTEX at Dover Air Force Base, Delaware. The final study is a field-scale natural attenuation project currently underway at Columbus Air Force Base, Mississippi.

Molecular diagnostics of polycyclic aromatic hydrocarbon biodegradation in Manufactured Gas Plant soils

Traditional methods for quantifying specific catabolic bacterial populations underestimate the true population count due to the limitations of the necessary laboratory cultivation methods. Likewise,in situ activity is also difficult to assess in the laboratory without altering the sample environment. To circumvent these problems and achieve a truein situ bacterial population count and activity measurement, new methods based on molecular biological analysis of bacterial nucleic acids were applied to soils heavily contaminated with polycyclic aromatic hydrocarbons (PAH). In addition, a naphthalene-lux reporter system was used to determine bioavailability of naphthalene within these soils. DNA extracted from seven PAH-contaminated soils and hybridized with thenahA gene probe indicated that the naphthalene degradative genes were present in all samples in the range of 0.06 to 0.95 ng/100 µl DNA extract which was calculated to represent 3.2×106 to 1.1×1010 cells/g soil (assuming one copy of these genes per cell).14C-naphthalene mineralization was observed in all contaminated soils with14CO2 mineralization rates ranging from 3.2×10−5 to 304,920.0×10−5 µg g soil−1h−1. Phenanthrene, anthracene, and benzo(a)pyrene were mineralized also in several soils. Messenger RNA transcripts ofnahA were isolated and quantified from 4 soils. Only one soil tested, soil B, was inducible with salicylate above thein situ nahA gene transcript level. Two of the soils, C and G, were already fully inducedin situ. The naphthalene mineralization rate correlated positively with the amount ofnahA gene transcripts present (r=0.99). Naphthalene was bioavailable in soils A, D, E, G, and N as determined by a bioluminescent response from the naphthalene-lux reporter system. Taken together, these data provided information on what the naphthalene-degrading bacterial population was experiencingin situ and what approaches would be necessary to increase activity.

In Vitro Immune Toxicity of Depleted Uranium: Effects on Murine Macrophages, CD4 + T Cells, and Gene Expression Profiles

Depleted uranium (DU) is a by-product of the uranium enrichment process and shares chemical properties with natural and enriched uranium. To investigate the toxic effects of environmental DU exposure on the immune system, we examined the influences of DU (in the form of uranyl nitrate) on viability and immune function as well as cytokine gene expression in murine peritoneal macrophages and splenic CD4+ T cells. Macrophages and CD4+ T cells were exposed to various concentrations of DU, and cell death via apoptosis and necrosis was analyzed using annexin-V/propidium iodide assay. DU cytotoxicity in both cell types was concentration dependent, with macrophage apoptosis and necrosis occurring within 24 hr at 100 μM DU exposure, whereas CD4+ T cells underwent cell death at 500 μM DU exposure. Noncytotoxic concentrations for macrophages and CD4+ T cells were determined as 50 and 100 μM, respectively. Lymphoproliferation analysis indicated that macrophage accessory cell function was altered with 200 μM DU after exposure times as short as 2 hr. Microarray and real-time reverse-transcriptase polymerase chain reaction analyses revealed that DU alters gene expression patterns in both cell types. The most differentially expressed genes were related to signal transduction, such as c-jun, NF-κ Bp65, neurotrophic factors (e.g., Mdk), chemokine and chemokine receptors (e.g., TECK/CCL25), and interleukins such as IL-10 and IL-5, indicating a possible involvement of DU in cancer development, autoimmune diseases, and T helper 2 polarization of T cells. The results are a first step in identifying molecular targets for the toxicity of DU and the elucidation of the molecular mechanisms for the immune modulation ability of DU.

Gene expression monitoring in soils by mRNA analysis and gene lux fusions

Two methods recently developed to monitor the gene expression of microbial communities in soil are the extraction and detection of messenger RNA from soil microorganisms and the construction and use of lux-based bioreporter strains. The goal of these approaches is to assess microbial activity in natural and impacted soil environments.

Ferritin: The role of aluminum in ferritin function

We previously showed that human brain ferritin (HBF) binds aluminum (Al) in vivo and in vitro and HBF isolated from Alzheimers brain had more Al bound compared to aged matched controls (7). To further understand the role ferritin may play in Al neurotoxicity, we have studied in vitro the effect of Al on the function of human ferritin isolated from Alzheimers (AD) and normal brain tissue, and compared the results with other mammalian ferritins. Al causes a concentration-dependent decrease in the initial rate of iron loading into apo-horse spleen and human brain ferritin and the rates were similar for ferritin isolated from both AD and normal brains. The rates of iron release of mammalian ferritins from different tissues were determined: horse spleen much greater than human liver greater than rat brain greater than human brain = rat liver ferritin. The rates of iron release of AD and normal human brain ferritin were similar and were unaffected by preloading with Al. Several mammalian ferritins were compared for their total iron uptake: horse spleen = human liver greater than human brain (normal) = human brain (AD) ferritin. In 20 mM HEPES (pH 6.0) buffer holoferritin is more resistant to precipitation by Al than apoferritin suggesting that holoferritin is a better chelator for nonferrous metal ions.

Molecular site assessment and process monitoring in bioremediation and natural attenuation

A variety of modern biotechnical approaches are available to assist in optimizing and controlling bioremediation processes. These approaches are broad-ranging, and may include genetic engineering to improve biodegradative performance, maintenance of the environment, and process monitoring and control. In addition to direct genetic engineering strategies, molecular diagnostic and monitoring technology using DNA gene probing methods and new quantitative mRNA analytical procedures allows direct analysis of degradative capacity, activity, and response underin situ conditions. Applications of these molecular approaches in process developments for trichloroethylene (TCE), polychlorinated biphenyls (PCB), and polynuclear aromatic hydrocarbons (PAH) bio-oxidation in soils, aquifer sediments, and ground-water treatment reactors have been demonstrated. Molecular genetic technologies permit not only the development of new processes for bioremediation, but also new process monitoring, control strategies, and molecular optimization paradigms that take full advantage of vast and diverse abilities of microorganisms to destroy problem chemicals.

Electronic Interfacing with Living Cells

The direct interfacing of living cells with inorganic electronic materials, components or systems has led to the development of two broad categories of devices that can (1) transduce biochemical signals generated by biological components into electrical signals and (2) transduce electronically generated signals into biochemical signals. The first category of devices permits the monitoring of living cells, the second, enables control of cellular processes. This review will survey this exciting area with emphasis on the fundamental issues and obstacles faced by researchers. Devices and applications that use both prokaryotic (microbial) and eukaryotic (mammalian) cells will be covered. Individual devices described include microbial biofuel cells that produce electricity, bioelectrical reactors that enable electronic control of cellular metabolism, living cell biosensors for the detection of chemicals and devices that permit monitoring and control of mammalian physiology.