Vitamin D Supplementation Reduces Intimal Hyperplasia and Restenosis following Coronary Intervention in Atherosclerotic Swine.

Vitamin D is a fat-soluble steroid hormone that activates vitamin D receptor to regulate multiple downstream signaling pathways and transcription of various target genes. There is an association between vitamin D deficiency and increased risk for cardiovascular disease. However, most of the studies are observational and associative in nature with limited data on clinical application. Thus, there is a need for more prospective randomized controlled studies to determine whether or not vitamin D supplementation provides cardiovascular protection. In this study, we examined the effects of the deficiency and supplementation of vitamin D on coronary restenosis following coronary intervention in atherosclerotic Yucatan microswine. Twelve Yucatan microswine were fed vitamin D-deficient (n = 4) or -sufficient (n = 8) high cholesterol diet for 6-months followed by coronary intervention. Post-intervention, swine in the vitamin D-sufficient high cholesterol diet group received daily oral supplementation of either 1,000 IU (n = 4) or 3,000 IU (n = 4) vitamin D3. Six months later, optical coherence tomography (OCT) was performed to monitor the development of intimal hyperplasia and restenosis. Animals were euthanized to isolate arteries for histomorphometric and immunohistochemical studies. Animals had graded levels of serum 25(OH)D; vitamin D-deficient (15.33 ± 1.45 ng/ml), vitamin D-sufficient + 1,000 IU oral vitamin D post-intervention (32.27 ± 1.20 ng/ml), and vitamin D-sufficient + 3,000 IU oral vitamin D post-intervention (51.00 ± 3.47 ng/ml). Findings from the OCT and histomorphometric studies showed a decrease in intimal hyperplasia and restenosis in vitamin D-supplemented compared to vitamin D-deficient swine. Vitamin D supplementation significantly decreased serum levels of TNF-α and IFN-γ, upregulated serum levels of IL-10, and had no effect on serum IL-6 levels. These findings suggest that vitamin D supplementation limits neointimal formation following coronary intervention in atherosclerotic swine and provide the support for vitamin D supplementation to protect against the development of coronary restenosis.

Progress toward clonable inorganic nanoparticles.

Pseudomonas moraviensis stanleyae was recently isolated from the roots of the selenium (Se) hyperaccumulator plant Stanleya pinnata. This bacterium tolerates normally lethal concentrations of SeO3(2-) in liquid culture, where it also produces Se nanoparticles. Structure and cellular ultrastructure of the Se nanoparticles as determined by cellular electron tomography shows the nanoparticles as intracellular, of narrow dispersity, symmetrically irregular and without any observable membrane or structured protein shell. Protein mass spectrometry of a fractionated soluble cytosolic material with selenite reducing capability identified nitrite reductase and glutathione reductase homologues as NADPH dependent candidate enzymes for the reduction of selenite to zerovalent Se nanoparticles. In vitro experiments with commercially sourced glutathione reductase revealed that the enzyme can reduce SeO3(2-) (selenite) to Se nanoparticles in an NADPH-dependent process. The disappearance of the enzyme as determined by protein assay during nanoparticle formation suggests that glutathione reductase is associated with or possibly entombed in the nanoparticles whose formation it catalyzes. Chemically dissolving the nanoparticles releases the enzyme. The size of the nanoparticles varies with SeO3(2-) concentration, varying in size form 5 nm diameter when formed at 1.0 µM [SeO3(2-)] to 50 nm maximum diameter when formed at 100 µM [SeO3(2-)]. In aggregate, we suggest that glutathione reductase possesses the key attributes of a clonable nanoparticle system: ion reduction, nanoparticle retention and size control of the nanoparticle at the enzyme site.

Pseudomonas seleniipraecipitans proteins potentially involved in selenite reduction.

Pseudomonas seleniipraecipitans grows in the presence of high levels of selenite and selenate and reduces both oxyanions to elemental selenium (Se(0)), a property that may make P. seleniipraecipitans useful as an inoculant for biobarriers designed to remove selenite or selenate from ground or surface waters. An earlier study showed that P. seleniipraecipitans nitrate reductase reduced selenate to Se(0), but failed to identify the protein(s) involved in selenite reduction. This study used ammonium sulfate precipitation, hydrophobic interaction chromatography, and native PAGE to isolate two electrophoretic gel regions, identified as bands A and B that showed selenite-reductase-activity. Proteomics was used to identify the proteins present in those regions. Glutathione reductase (GR) was detected in the A-band; based on this information, Saccharomyces cerevisiae GR, obtained from a commercial source, was evaluated and found to have selenite-reductase-activity, confirming that GR can reduce selenite to Se(0). Proteomics was also used to detect the proteins present in the B-band and thioredoxin reductase (ThxR) was detected as a B-band protein; based on this information, E. coli ThxR, obtained from a commercial source, was evaluated and found to have selenite-reductase-activity, confirming that ThxR can reduce selenite to elemental selenium. Thus, evidence presented in this study shows that S. cerevisiae GR and E. coli ThxR can reduce SeO3 (2-) to Se(0) and strongly suggests that P. seleniipraecipitans GR and ThxR can also reduce SeO3 (2-) to Se(0).

The influence of nitrate on selenium in irrigated agricultural groundwater systems.

Selenium (Se) contamination of groundwater is an environmental concern especially in areas where aquifer systems are underlain by Se-bearing geologic formations such as marine shale. This study examined the influence of nitrate (NO3) on Se species in irrigated soil and groundwater systems and presents results from field and laboratory studies that further clarify this influence. Inhibition of selenate (SeO4) reduction in the presence of NO3 and the oxidation of reduced Se from shale by autotrophic denitrification were investigated. Groundwater sampling from piezometers near an alluvium-shale interface suggests that SeO4 present in the groundwater was due in part to autotrophic denitrification. Laboratory shale oxidation batch studies indicate that autotrophic denitrification is a major driver in the release of SeO4 and sulfate. Similar findings occurred for a shale oxidation flow-through column study, with 70 and 31% more reduced Se and S mass, respectively, removed from the shale material in the presence of NO3 than in its absence. A final laboratory flow-through column test was performed with shallow soil samples to assess the inhibition of SeO4 reduction in the presence of NO3, with results suggesting that a concentration of NO3 of approximately 5 mg L or greater will diminish the reduction of SeO4. The inclusion of the fate and transport of NO3 and dissolved oxygen is imperative when studying or simulating the fate and transport of Se species in soil and groundwater systems.

Vitamin D receptor expression and neoadjuvant therapy in esophageal adenocarcinoma.

Esophageal adenocarcinoma carries a poor prognosis. Tumor response to neoadjuvant therapy is a key prognostic factor in patients with adenocarcinoma of the esophagus, but is inconsistent. Identifying tumor characteristics that portend a favorable response to neoadjuvant therapy would be a valuable clinical tool. The anticancer actions of vitamin D and its receptor may have implications. In this study, 15 biopsy specimens were procured retrospectively from patients being treated for adenocarcinoma of the esophagus. The tissue was immunostained for the vitamin D receptor and compared on the basis of response to neoadjuvant therapy. Tumors that did not respond to neoadjuvant therapy had greater expression of VDR than tumors that responded completely. Expression of VDR declined with tumor de-differentiation. The data suggest that a relationship between vitamin D receptor expression and response to neoadjuvant therapy is plausible.

Reduction of selenite to elemental red selenium by Pseudomonas sp. Strain CA5.

A Pseudomonas sp. that may be useful in bioremediation projects was isolated from soil. The strain is of potential value because it reduces selenite to elemental red selenium and is unusual in that it was resistant to high concentrations of both selenate and selenite. Exposure of the strain to 50, 100, and 150 mM selenite reduced growth by 28, 57, and 66%, respectively, while no change in growth was observed when the strain was exposed to 64 mM selenate, the highest level tested. Cells of the strain removed 1.7 mM selenite from the culture fluid during a 7-day incubation. A selenite reductase with a molecular weight of ~115 kD was detected in cell-free extracts and a protein with a molecular weight of ~700 kD was detected that reduced both selenate and nitrate. The bacterial isolate is a strict aerobe, reducing selenite to elemental red selenium under aerobic conditions only. Pseudomonas sp. strain CA5 might be useful as an inoculum for bioreactors used to harvest selenium from selenite-containing groundwater. 16S rRNA gene sequence alignment and fatty acid analysis were used to identify the bacterium as a novel species of Pseudomonas related to P. argentinensis, P. flavescens, and P. straminea.

Nitrogen limited biobarriers remove atrazine from contaminated water: laboratory studies.

Atrazine is one of the most frequently used herbicides. This usage coupled with its mobility and recalcitrant nature in deeper soils and aquifers makes it a frequently encountered groundwater contaminant. We formed biobarriers in sand filled columns by coating the sand with soybean oil; after which, we inoculated the barriers with a consortium of atrazine-degrading microorganisms and evaluated the ability of the barriers to remove atrazine from a simulated groundwater containing 1 mg L(-1) atrazine. The soybean oil provided a carbon rich and nitrogen poor substrate to the microbial consortium. Under these nitrogen-limiting conditions it was hypothesized that bacteria capable of using atrazine as a source of nitrogen would remove atrazine from the flowing water. Our hypothesis proved correct and the biobarriers were effective at removing atrazine when the nitrogen content of the influent water was low. Levels of atrazine in the biobarrier effluents declined with time and by the 24th week of the study no detectable atrazine was present (limit of detection<0.005 mg L(-1)). Larger amounts of atrazine were also removed by the biobarriers; when biobarriers were fed 16.3 mg L(-1) atrazine 97% was degraded. When nitrate (5 mg L(-1) N), an alternate source of nitrogen, was added to the influent water the atrazine removal efficiency of the barriers was reduced by almost 60%. This result supports the hypothesis that atrazine was degraded as a source of nitrogen. Poisoning of the biobarriers with mercury chloride resulted in an immediate and large increase in the amount of atrazine in the barrier effluents confirming that biological activity and not abiotic factors were responsible for most of the atrazine degradation. The presence of hydroxyatrazine in the barrier effluents indicated that dehalogenation was one of the pathways of atrazine degradation. Permeable barriers might be formed in-situ by the injection of innocuous vegetable oil emulsions into an aquifer or sandy soil and used to remove atrazine from a contaminated groundwater or to protect groundwater from an atrazine spill.

Bio-reduction of selenite to elemental red selenium by Tetrathiobacter kashmirensis.

A bacterium that detoxifies selenite by reduction to insoluble elemental red selenium was isolated from soil. The strain showed an unusually high resistance to the toxic effects of selenite by growing in media containing 64 mM selenite. 16S rRNA gene sequence alignment identified the isolate as Tetrathiobacter kashmirensis. Fatty acid analysis and morphology confirmed the identification. The isolate reduced selenite to elemental selenium under aerobic conditions only. Native gel electrophoresis of cell-free extracts revealed a band, corresponding to a molecular weight of approximately 120 kDa, that reduced selenite. In culture, the strain did not reduce selenate; however, a soluble and inducible enzyme with a molecular weight of approximately 90 kDa that reduced both selenate and nitrate was present in cell-free extracts. This organism might be useful in bioreactors designed to remove selenite from contaminated water.

Carbohydrate and ethane release with Erwinia carotovora subspecies betavasculorum--induced necrosis.

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.

Reduction of selenite to elemental red selenium by Rhizobium sp. strain B1.

A bacterium that reduces the soluble and toxic selenite anion to insoluble elemental red selenium (Se(0)) was isolated from a laboratory bioreactor. Biochemical, morphological, and 16S rRNA gene sequence alignment identified the isolate as a Rhizobium sp. that is related to but is genetically divergent from R. radiobacter (syn. Agrobacterium tumefaciens) or R. rubi (syn. A. rubi). The isolate was capable of denitrification and reduced selenite to Se(0) under aerobic and denitrifying conditions. It did not reduce selenate and did not use selenite or selenate as terminal e(-) donors. Native gel electrophoresis revealed two bands, corresponding to molecular weights of approximately 100 and approximately 45 kDa, that reduced selenite. Tungsten inhibited in vivo selenite reduction, suggesting that a molybdenum-containing protein is involved in selenite reduction. This organism, or its enzymes or DNA, might be useful in bioreactors designed to remove selenite from water.

An Azospira oryzae (syn Dechlorosoma suillum) strain that reduces selenate and selenite to elemental red selenium.

A bacterium that reduces the soluble selenium oxyanions, selenate and selenite, to insoluble elemental red selenium (Se(0)) was isolated from a laboratory reactor developed to remove selenate from groundwater. Gene sequence alignment of the 16S rRNA allowed identification of the isolate as Azospira oryzae. Biochemical and morphologic characterization confirm the identification. The isolate reduces selenate and selenite to Se(0) under microaerophilic and denitrifying conditions but not under aerobic conditions. It does not use selenate or selenite as terminal e(-) donors. Se oxyanion reduction causes the formation of Se nanospheres that are 0.25 +/- 0.04 microm in diameter. Nanospheres may be associated with the cells or free in the medium. The enzymatic activity associated with the reduction of selenate has a molecular mass of approximately 500 kD, and the enzymatic activity associated with the reduction of selenite has a mass of approximately 55 kD. Selenite reduction was inhibited by tungsten. The molecular masses of these activities were different from those associated with the reduction of dimethylsulfoxide, sulfate, and nitrite. This bacterium, or perhaps its enzymes or DNA, might be useful for the remediation of waters contaminated with Se oxyanions.

Removing selenate from groundwater with a vegetable oil-based biobarrier.

Vegetable oil-based permeable reactive biobarriers (PRBs) were evaluated as a method for remediating groundwater containing unacceptable amounts of selenate. PRBs formed by packing laboratory columns with sand coated with soybean oil were used. In an initial 24-week study a simulated groundwater containing 10 mg L(-1) selenate-Se was supplied to three soil columns and the selenate and selenite content of the effluent waters monitored. Two of the soil columns were effective at removing selenate and, during the final 21 weeks of the study, effluents from these columns contained almost no selenate or selenite. Almost all (95%) of the selenate removed was recovered as immobilized selenium sequestered in the solid matrix of the column. For unknown reasons, the third column failed to reduce selenate. A second study looked at the ability of PRBs to remove selenate when nitrate was present. As was done in the first study, three columns were evaluated but this time the water supplied to the columns contained 20 mg L(-1 )nitrate-N and 10 mg L(-1) selenate-Se. Nitrate quickly disappeared from the effluents of these columns and during the final 23 weeks of the study, the nitrate content of the effluent water averaged less than 0.03 microg ml(-1) nitrate-N. Selenate was also removed by these columns but at a slower rate than observed with nitrate. In the final 6 weeks of the study, about 95% of the selenate applied to the columns was removed. In situ PRBs containing soybean oil might be used to remediate groundwater contaminated with both selenate and nitrate.

Identification and characterization of an Aeromonas salmonicida (syn Haemophilus piscium) strain that reduces selenite to elemental red selenium.

A bacterium that reduces toxic and mobile selenite to insoluble elemental selenium (Se0) was isolated from a laboratory scale permeable reactive biobarrier. Biochemical tests and 16S rRNA gene sequence alignment identified the isolate as Aeromonas salmonicida. Two colony types were isolated, one more resistant to selenite than the other. Both grew on agar plates containing 16 mM: selenite, although the colony diameter was reduced to 8% of controls with the small colony type and to 18% with the large colony type. Further study was done with the large colony type. In anaerobic culture, this bacterium was able to use nitrate as a term electron acceptor but not selenate or selenite. In aerobic culture, when no nitrate was present, early log phase cells removed selenite at a rate of 2.6 +/- 0.42 micromol SeO3 (-2)/mg protein/day. Reduction was retarded by 25 mM: nitrate. Mutants with a diminished ability to reduce selenite to Se0 also had a reduced ability to reduce nitrate to nitrous oxide. This bacterium, or perhaps its enzymes or DNA, might be used to remove selenite from contaminated groundwaters.

Injection of innocuous oils to create reactive barriers for bioremediation: laboratory studies.

In situ groundwater remediation may be achieved using stationary permeable barriers created by the injection of a substrate, such as innocuous vegetable oil, into the contaminated aquifer. The oil provides the electron donor stimulating microorganisms to degrade or sequester many contaminants. At present, little is known about the best procedures to use when injecting oil into an aquifer. In this investigation, laboratory column and sand tank studies were used as model systems to explore the effect of different injection parameters on the distribution of oil emulsions into water-saturated sand. The parameters investigated included injection pressures of 70, 1400 and 18,000 KPa; injection times of 15, 30, 60 or 120 s; and the influence of an emulsifier, polyoxyethylenesorbitan monooleate (Tween 80), upon the distribution of the injected oil. The longest injection patterns were achieved at 18,000 KPa. A pattern that was 46+/-1.8 cm long was produced with an 18,000 KPa injection for 60 s. Increasing the injection time to 120 s increased the length of the pattern by only 6.5%. Tween 80 at concentrations of 0.05% increased the width of the injection patterns but did not increase the length of the pattern. A multi-ported injection probe might be used to create in situ permeable barriers approximately 1 m wide.

Removing selenite from groundwater with an in situ biobarrier: laboratory studies.

Laboratory biobarriers were evaluated for their ability to remove selenite from flowing groundwater. Microbial activity in aquifers is usually limited by substrate availability, and biobarriers stimulate microbial activity by providing a substrate; for these studies soybean oil was used. Water containing 10 mg L(-1) selenite-Se was pumped through the biobarriers for 74 days and the amount present in the effluent monitored. The amounts remained high for the first 2 weeks of the study but then declined. From day 28 until the end of the study the amount of selenite-Se in the column effluents averaged 0.20 +/- 0.04 mg L(-1), a decrease of approximately 98%. At the end of the study about half of the selenite-Se applied to the columns was recovered as immobilized selenium trapped by the biobarrier. This study suggests that biobarriers containing vegetable oil might be used as a process for removing selenite from contaminated groundwater.

Bioremediation of chlorate or perchlorate contaminated water using permeable barriers containing vegetable oil.

A scale model of an in situ permeable barrier, formed by injecting vegetable oil onto laboratory soil columns, was used to remove chlorate and perchlorate from flowing groundwater. The hypothesis that trapped oil would serve as a substrate enabling native microorganisms to reduce chlorate or perchlorate to chloride as water flowed through the oil-rich zone had merit. Approximately 96% of the 0.2 mM chlorate and 99% of the 0.2 mM perchlorate present in the water was removed as water was pumped through columns containing vegetable oil barriers. The product formed was chloride. When nitrate at 1.4 mM was added to the water, both nitrate and chlorate were removed. High concentrations of chlorate or perchlorate can be treated; 24 m M chlorate and 6 mM perchlorate were completely reduced to chloride during microcosm incubations. Microorganisms capable of reducing perchlorate are plentiful in the environment.