Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9.

Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures.

DNA extraction-free quantification of Dehalococcoides spp. in groundwater using a hand-held device.

Nucleic acid amplification of biomarkers is increasingly used to measure microbial activity and predict remedial performance in sites with trichloroethene (TCE) contamination. Field-based genetic quantification of microorganisms associated with bioremediation may help increase accuracy that is diminished through transport and processing of groundwater samples. Sterivex cartridges and a previously undescribed mechanism for eluting biomass was used to concentrate cells. DNA extraction-free loop mediated isothermal amplification (LAMP) was monitored in real-time with a point of use device (termed Gene-Z). A detection limit of 10(5) cells L(­1) was obtained, corresponding to sensitivity between 10 to 100 genomic copies per reaction for assays targeting the Dehalococcoides spp. specific 16S rRNA gene and vcrA gene, respectively. The quantity of Dehalococcoides spp. genomic copies measured from two TCE contaminated groundwater samples with conventional means of quantification including filtration, DNA extraction, purification, and qPCR was comparable to the field ready technique. Overall, this method of measuring Dehalococcoides spp. and vcrA genes in groundwater via direct amplification without intentional DNA extraction and purification is demonstrated, which may provide a more accurate mechanism of predicting remediation rates.

A novel combinatorial biocatalytic approach for producing antibacterial compounds effective against Mycobacterium tuberculosis (TB).

Two bacterial hosts expressing cloned aromatic oxygenases were used to catalyze the oxidation and polymerization of indole and related substrates, creating mixtures of indigoid compounds comprised of novel dimers and trimers. Crude extracts and purified compounds were tested for their ability to inhibit the growth of Gram-positive organisms, in general, and Mycobacterium tuberculosis (TB), in particular. Of the 74 compounds tested against M. tuberculosis, ~66 % had minimum inhibitory concentrations (MIC) of 5 µg/ml or less. The most effective antibiotic found was designated SAB-P1, a heterodimer of indole and anthranil, which had a MIC of 0.16 µg/ml, and did not inhibit kidney cells (IC(50)) at concentrations of >8 µg/ml. Combinatorial biocatalysis was used to create a series of halogenated derivatives of SAB-P1 with a wider therapeutic window. None of the derivatives had MIC values that were superior to SAB-P1, but some had a wider therapeutic window because of decreased kidney cell toxicity. Generally, the indigoid dimers that were effective against TB appeared to be specific for TB. Some of the trimers generated, however, had a broader spectrum of activity inhibiting not only TB (MIC = 1.1 µg/ml) but also the growth of Mycobacterium smegmatis MC2 155, Bacillus cereus, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis 168, and Clostridium acetobutylicum. The structure of two of the novel dimers (SAB-C4 and SAB-P1) and a trimer (SAB-R1) were solved using X-ray crystallography.

3-(3-Aryloxyaryl)imidazo[1,2-a]pyridine sulfones as liver X receptor agonists.

Replacement of a quinoline with an imidazo[1,2-a]pyridine in a series of liver X receptor (LXR) agonists incorporating a [3-(sulfonyl)aryloxyphenyl] side chain provided high affinity LXR ligands 7. In functional assays of LXR activity, good agonist potency and efficacy were found for several analogs.

LXR ligand lowers LDL cholesterol in primates, is lipid neutral in hamster, and reduces atherosclerosis in mouse.

Liver X receptors (LXRs) are ligand-activated transcription factors that coordinate regulation of gene expression involved in several cellular functions but most notably cholesterol homeostasis encompassing cholesterol transport, catabolism, and absorption. WAY-252623 (LXR-623) is a highly selective and orally bioavailable synthetic modulator of LXR, which demonstrated efficacy for reducing lesion progression in the murine LDLR(-/-) atherosclerosis model with no associated increase in hepatic lipogenesis either in this model or Syrian hamsters. In nonhuman primates with normal lipid levels, WAY-252623 significantly reduced total (50-55%) and LDL-cholesterol (LDLc) (70-77%) in a time- and dose-dependent manner as well as increased expression of the target genes ABCA1/G1 in peripheral blood cells. Statistically significant decreases in LDLc were noted as early as day 7, reached a maximum by day 28, and exceeded reductions observed for simvastatin alone (20 mg/kg). Transient increases in circulating triglycerides and liver enzymes reverted to baseline levels over the course of the study. Complementary microarray analysis of duodenum and liver gene expression revealed differential activation of LXR target genes and suggested no direct activation of hepatic lipogenesis. WAY-252623 displays a unique and favorable pharmacological profile suggesting synthetic LXR ligands with these characteristics may be suitable for evaluation in patients with atherosclerotic dyslipidemia.

Transformation of RDX and other energetic compounds by xenobiotic reductases XenA and XenB.

The transformation of explosives, including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), by xenobiotic reductases XenA and XenB (and the bacterial strains harboring these enzymes) under both aerobic and anaerobic conditions was assessed. Under anaerobic conditions, Pseudomonas fluorescens I-C (XenB) degraded RDX faster than Pseudomonas putida II-B (XenA), and transformation occurred when the cells were supplied with sources of both carbon (succinate) and nitrogen (NH4+), but not when only carbon was supplied. Transformation was always faster under anaerobic conditions compared to aerobic conditions, with both enzymes exhibiting a O2 concentration-dependent inhibition of RDX transformation. The primary degradation pathway for RDX was conversion to methylenedinitramine and then to formaldehyde, but a minor pathway that produced 4-nitro-2,4-diazabutanal (NDAB) also appeared to be active during transformation by whole cells of P. putida II-B and purified XenA. Both XenA and XenB also degraded the related nitramine explosives octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine and 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane. Purified XenB was found to have a broader substrate range than XenA, degrading more of the explosive compounds examined in this study. The results show that these two xenobiotic reductases (and their respective bacterial strains) have the capacity to transform RDX as well as a wide variety of explosive compounds, especially under low oxygen concentrations.

Large-scale production of bacterial consortia for remediation of chlorinated solvent-contaminated groundwater.

Chlorinated solvents such as perchloroethylene (PCE) and trichloroethylene (TCE) continue to be significant groundwater contaminants throughout the USA. In many cases efficient bioremediation of aquifers contaminated with these chemicals requires the addition of exogenous microorganisms, specifically members of the genus Dehalococcoides (DHC). This process is referred to as bioaugmentation. In this study a fed-batch fermentation process was developed for producing large volumes (to 3,200 L) of DHC-containing consortia suitable for treating contaminated aquifers. Three consortia enriched from three different sites were grown anaerobically with sodium lactate as an electron donor and PCE or TCE as an electron acceptor. DHC titers in excess of 10(11) DHC/L could be reproducibly obtained at all scales tested and with all three of the enrichment cultures. The mean specific DHC growth rate for culture SDC-9 was 0.036 +/- 0.005 (standard error, SE)/h with a calculated mean doubling time of 19.3 +/- 2.7 (SE) h. Finished cultures could be concentrated approximately tenfold by membrane filtration and stored refrigerated (4 degrees C) for more that 40 days without measurable loss of activity. Dehalogenation of PCE by the fermented cultures was affected by pH with no measurable activity at pH <5.0.

Control of chronic inflammation with pathway selective estrogen receptor ligands.

The discovery of novel intervention points in the inflammatory pathway has been a focus of drug development in recent years. We have identified pathway selective ligands for the estrogen receptor (ER) that inhibit NF-kappaB mediated inflammatory gene expression causing a reduction of cytokines, chemokines, adhesion molecules and inflammatory enzymes. SAR development of a series of 4-(Indazol-3-yl)-phenols has led to the identification of WAY-169916 an orally active non-steroidal ligand with the potential use in the treatment of inflammatory diseases without the classical proliferative effects associated with non-selective estrogens.

The activity of pathway-selective estrogen receptor ligands in experimental septic shock.

Estrogen receptors (ER) are widely expressed in multiple genital and nongenital tissues. Upon engagement of these receptors, multiple genes are affected in target tissues via estrogen response elements. Nonsteroidal pathway-selective ER ligands have recently been identified that inhibit NF-kappaB transcriptional activity and are devoid of conventional estrogenic activities on genital tissues. These pathway-selective ligands are potent anti-inflammatory agents in vivo and may prove to be of therapeutic utility in systemic inflammatory states. These pathway-selective ER ligands were tested in the murine listeriosis model, the neutropenic rat model, and the mouse cecal ligation and puncture model. WAY-204688 did not have any significant activity after systemic infection by Listeria monocytogenes. In the neutropenic rat model, WAY-204688 provided a significant survival benefit against an otherwise lethal challenge of Pseudomonas aeruginosa 12.4.4 compared with the control group (88% versus 25% survival; P < 0.05). Preservation of mucosal weight and prevention of histopathologic changes were observed with the administration of WAY-204688. Similar findings were observed in a cecal ligation and puncture model with WAY-204688 and a related compound WAY-169916. These results indicate that oral administration of these pathway-selective ER ligands preserved gastrointestinal barrier function and improve outcome in experimental models of systemic infection and inflammation. These agents may prove to be useful clinically as a novel treatment strategy for severe sepsis.

Indole trimers with antibacterial activity against Gram-positive organisms produced using combinatorial biocatalysis.

The I100V isoform of toluene-4-monooxygenase was used to catalyze the oxidative polymerization of anthranil and various indoles under mildly acidic conditions, favoring the production of trimers. Compounds produced in sufficient yield were purified and tested for their ability to inhibit the growth of B. anthracis, E. faecalis, L. monocytogenes, S. aureus, and in some cases, F. tularensis. 15 of the compounds displayed promising antibacterial activity (MIC < 5 µg/ml) against one or more of the strains tested, with the best MIC values being <0.8 µg/ml. All of these compounds had good selectivity, showing minimal cytotoxicity towards HepG2 cells. The structure was solved for six of the compounds that could be crystallized, revealing that minimally two classes of indole based trimers were produced. One compound class produced was a group of substituted derivatives of the natural product 2,2-bis(3-indolyl) indoxyl. The other group of compounds identified was classified as tryptanthrin-like compounds, all having multi-ring pendant groups attached at position 11 of tryptanthrin. One compound of particular interest, SAB-J85, had a structure that suggests that any compound, with a ring structure that can be activated by an oxygenase, might serve as a substrate for combinatorial biocatalysis.

Synthesis and activity of substituted 4-(indazol-3-yl)phenols as pathway-selective estrogen receptor ligands useful in the treatment of rheumatoid arthritis.

Pathway-selective ligands for the estrogen receptor (ER) inhibit NF-kappaB-mediated inflammatory gene expression causing a reduction of cytokines, chemokines, adhesion molecules, and inflammatory enzymes. SAR development of a series of 4-(indazol-3-yl)phenols has led to the identification of WAY-169916 an orally active nonsteroidal ligand with the potential use in the treatment of rheumatoid arthritis without the classical proliferative effects associated with estrogens.

Enhancing the attenuation of explosives in surface soils at military facilities: combined sorption and biodegradation.

This research evaluated soil amendments designed to enhance the adsorption and biodegradation of explosives at military training facilities, thus minimizing their potential for transport to subsurface environments. Several carbon cosubstrates were tested in soil slurries for their ability to stimulate the biodegradation of 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (royal demolition exposive [RDX]), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (high-melting explosive [HMX]) by indigenous soil microorganisms. Crude soybean oil and molasses stimulated mineralization of RDX (30-40%) and HMX (approximately 10%). The TNT was not significantly mineralized in any of the treatments, but high-performance liquid chromatography (HPLC) analysis indicated extensive transformation of TNT to amino-containing compounds. The biodegradation of explosives was then examined in unsaturated soil microcosms amended with crude soybean oil and molasses combined with sphagnum peat moss and sawdust. Minimal TNT mineralization was observed, and HMX mineralization was only observed with molasses addition. In contrast, RDX mineralization was extensive in microcosms amended with soybean oil or molasses. The presence of peat moss decreased soybean oil-stimulated RDX mineralization by approximately 5%, but resulted in about 5% greater RDX mineralization compared with molasses only. Sawdust markedly decreased mineralization regardless of cosubstrate type. Mass balance results indicated that the formation of bound residues likely was occurring, especially for TNT. These results indicate that the application of inexpensive adsorbents and cosubstrates to soils may significantly improve the protection of groundwater resources underlying live fire ranges.

Enhancing the attenuation of explosives in surface soils at military facilities: sorption-desorption isotherms.

The primary objective of the present study was to develop inexpensive soil amendments that can be applied to enhance the adsorption of energetic compounds on military training ranges, thus limiting the potential for these compounds to migrate to groundwater. Adsorption and desorption isotherms were determined for 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine with a wide variety of natural and man-made adsorbents, including wheat straw, sawdust, peat moss, ground rubber tires, and clays. Among the various adsorbents tested, peat moss proved to be the most effective sorbent for the three explosives. The adsorption coefficients (Kd(s)) for TNT and RDX with peat (310 and 87 L/kg, respectively) were at least two orders of magnitude higher than that determined for adsorption of these energetics with two surface soils. The adsorption-desorption isotherms for the explosives showed considerable hysteresis (Kd(s) < Kd(d)) with some of the solid adsorbents, suggesting that the sorption process is not readily reversible but, rather, that some fraction of the adsorbed contaminant is either irreversibly bound or present as a slowly desorbed fraction. The data indicate that the application of specific adsorbents to soils at military impact ranges may significantly improve the protection of local groundwater resources.

Detection of Dehalococcoides spp. by peptide nucleic acid fluorescent in situ hybridization.

Chlorinated solvents including tetrachloroethene (perchloroethene and trichloroethene), are widely used industrial solvents. Improper use and disposal of these chemicals has led to a widespread contamination. Anaerobic treatment technologies that utilize Dehalococcoides spp. can be an effective tool to remediate these contaminated sites. Therefore, the aim of this study was to develop, optimize and validate peptide nucleic acid (PNA) probes for the detection of Dehalococcoides spp. in both pure and mixed cultures. PNA probes were designed by adapting previously published DNA probes targeting the region of the point mutations described for discriminating between the Dehalococcoides spp. strain CBDB1 and strain 195 lineages. Different fixation, hybridization and washing procedures were tested. The results indicated that the PNA probes hybridized specifically and with a high sensitivity to their corresponding lineages, and that the PNA probes developed during this work can be used in a duplex assay to distinguish between strain CBDB1 and strain 195 lineages, even in complex mixed cultures. This work demonstrates the effectiveness of using PNA fluorescence in situ hybridization to distinguish between two metabolically and genetically distinct Dehalococcoides strains, and they can have strong implications in the monitoring and differentiation of Dehalococcoides populations in laboratory cultures and at contaminated sites.

Biodegradation of tetrahydrofuran and 1,4-dioxane by soluble diiron monooxygenase in Pseudonocardia sp. strain ENV478.

1,4-Dioxane is an important groundwater contaminant. Pseudonocardia sp. strain ENV478 degrades 1,4-dioxane via cometabolism after the growth on tetrahydrofuran (THF) and other carbon sources. Here, we have identified a THF monooxygenase (thm) in ENV478. The thm genes are transcribed constitutively and are induced to higher levels by THF. Decreased translation of the thmB gene encoding one of the monooxygenase subunits by antisense RNA resulted in the loss of its ability to degrade THF and 1,4-dioxane. This is the first study to link thm genes to THF degradation, as well as the cometabolic oxidation of 1,4-dioxane.

Bioaugmentation for treatment of dense non-aqueous phase liquid in fractured sandstone blocks.

Laboratory experiments were performed in discretely fractured sandstone blocks to evaluate the use of bioaugmentation to treat residual dense non-aqueous phase liquid (DNAPL) tetrachloroethene (PCE). Significant dechlorination of PCE and growth of Dehalococcoides spp. (DHC) occurred within the fractures. DNAPL dissolution was enhanced during bioaugmentation by up to a factor of approximately 3.5, with dissolved PCE concentrations at or near aqueous solubility. The extent of dechlorination and DNAPL dissolution enhancement were dependent upon the fracture characteristics, residence time in the fractures, and dissolved concentration of PCE. No relationship was observed between planktonic DHC concentrations exiting the fracture and the observed extents of PCE dechlorination and DNAPL dissolution. Measured planktonic DHC concentrations exiting the fracture increased with increasing flow rate and bioaugmentation dosage, suggesting that these parameters may be important for distribution of DHC to treat dissolved chlorinated ethenes migrating downgradient of the DNAPL source. Bioaugmentation dosage, for the DHC dosages and conditions studied, did not have a measurable impact on DNAPL dissolution or dechlorination within the fractures themselves. Overall, these results indicate that bioaugmentation may be a viable remedial option for treating DNAPL sources in bedrock.

Aerobic biodegradation of iso-butanol and ethanol and their relative effects on BTEX biodegradation in aquifer materials.

The aerobic biodegradability of iso-butanol, a new biofuel, and its impact on benzene, toluene, ethylbenzene and xylenes (BTEX) degradation was investigated in aerobic microcosms consisting of groundwater and sediment from a California site with a history of gasoline contamination. To the best of our knowledge this is the first study directly examining the effects of iso-butanol on BTEX degradation. Microcosms that received either low (68 µM) or high (3400 µM) concentrations of iso-butanol showed complete biodegradation of iso-butanol within 7 and 23 d, respectively, of incubation at 15°C under aerobic conditions. A maximum utilization rate coefficient of 2.3±0.1×10⁻7 µmol cell⁻¹ h⁻¹ and a half saturation constant of 610±54 µM were regressed from the iso-butanol data. Iso-butanol biodegradation resulted in transient formation of the degradation intermediate products iso-butylaldehyde and iso-butyric acid, and both compounds were subsequently degraded within the timeframe of the experiments. Ethanol was biodegraded more slowly than iso-butanol. Ethanol also exhibited greater adverse impacts on BTEX biodegradation than iso-butanol. Results of the study suggest that iso-butanol added to fuels will be readily biodegraded in the environment under aerobic conditions without the accumulation of major intermediate products (iso-butylaldehyde and iso-butyric acid), and that it will pose less impacts on BTEX biodegradation than ethanol.

Anaerobic biodegradation of iso-butanol and ethanol and their relative effects on BTEX biodegradation in aquifer materials.

Biologically produced iso-butanol is currently being considered as an additive in gasoline blends. To evaluate its potential environmental fate in groundwater aquifers, a laboratory microcosm study was performed to evaluate iso-butanol biodegradation under various anaerobic conditions (nitrate-reducing, sulfate-reducing and methanogenic). The impacts of iso-butanol on benzene, toluene, ethylbenzene, and total xylenes (BTEX) biodegradation were also assessed, and microcosms prepared using ethanol instead of iso-butanol were evaluated to provide a basis for comparison. Iso-butanol was biodegraded under all conditions studied, with an observed apparent first-order rate constant ranging from approximately 0.2 d⁻¹ (nitrate-reducing) to approximately 0.02 d⁻¹ (sulfate-reducing). Iso-butanol typically was degraded in a time frame that was shorter than or similar to BTEX compounds. Iso-butyric acid and trace levels of iso-butylaldehyde were identified as transient intermediates, and both of these compounds were subsequently degraded within the time frame of the experiments. Iso-butanol and ethanol were biodegraded in similar time frames under methanogenic conditions. Under sulfate-reducing conditions, iso-butanol biodegradation initially proceeded more slowly than ethanol, and then increased to a rate greater than that observed for ethanol; this observation likely was due to the growth of iso-butanol degrading bacteria. Iso-butanol generally exhibited less adverse impacts on BTEX biodegradations than ethanol under the anaerobic conditions studied. In some cases, addition of iso-butanol enhanced the rate of TEX biodegradation.

Bioaugmentation for chlorinated ethenes using Dehalococcoides sp.: comparison between batch and column experiments.

Batch and column experiments were performed to evaluate the transport, growth and dechlorination activity of Dehalococcoides sp. (DHC) during bioaugmentation for chlorinated ethenes. Batch experiments showed that the reductive dechlorination of trichloroethene (TCE), cis-1,2-dichloroethene (DCE), and vinyl chloride (VC), as well as growth of the DHC, were well described by the Monod kinetic model. The measured maximum utilization rate coefficients for TCE, DCE, and VC were 1.3x10(-12), 5.2x10(-13), and 1.4x10(-12)mmol Cl(-) (cellh)(-1), respectively. Results of the column experiments showed that dechlorination occurred throughout the length of the column, and that extractable DHC concentrations associated with the soil phase throughout the column were negligible relative to the aqueous phase concentrations. Dechlorination rates relative to aqueous DHC concentrations in the column were approximately 200-times greater than in the batch experiments. Additional batch experiments performed using column effluent water confirmed this result. Incorporation of these enhanced dechlorination kinetics in the transport model provided a reasonable prediction of the column data. Overall results of this study suggest that aqueous phase (as opposed to soil phase) DHC concentrations can be used to estimate dechlorination activity in saturated soils, and DHC dechlorination activity in porous media may be substantially greater than DHC dechlorination activity measured in batch experiments.

Evaluation of a peat moss plus soybean oil (PMSO) technology for reducing explosive residue transport to groundwater at military training ranges under field conditions.

An evaluation of peat moss plus crude soybean oil (PMSO) for mitigation of explosive contamination of soil at military facilities was performed using large soil lysimeters under field conditions. Actual range soils were used, and two PMSO preparations with different ratios of peat moss:soybean oil (1:1, PO1; 1:2, PO2) were compared to a control lysimeter that received no PMSO. PMSO was applied as a 10 cm layer on top of the soil, and Composition B detonation residues from a 55-mm mortar round were applied at the surface of each of the lysimeters. Dissolution of the residues occurred during natural precipitation events over the course of 18 months. Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) emanating from the Composition B residues were significantly reduced by the PO2 PMSO material compared to the untreated control. Soil pore water RDX concentrations and RDX fluxes were reduced over 100-fold compared to the control plots at comparable depths. Residual RDX in the soil profile was also significantly lower in the PMSO treated plots. PO1 PMSO resulted in lower reductions in RDX transport than the PO2 PMSO. The transport of the RDX breakdown product hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) was also greatly reduced by the PMSO materials. Results were in general agreement with a previously developed fate and transport model describing PMSO effectiveness. These results demonstrate the potential effectiveness of the inexpensive and environmentally benign PMSO technology for reducing the subsurface loading of explosives at training ranges and other military facilities.