Arnold M. Saxton

Diversity and Characterization of Sulfate-Reducing Bacteria in Groundwater at a Uranium Mill Tailings Site

ABSTRACT Microbially mediated reduction and immobilization of U(VI) to U(IV) plays a role in both natural attenuation and accelerated bioremediation of uranium-contaminated sites. To realize bioremediation potential and accurately predict natural attenuation, it is important to first understand the microbial diversity of such sites. In this paper, the distribution of sulfate-reducing bacteria (SRB) in contaminated groundwater associated with a uranium mill tailings disposal site at Shiprock, N.Mex., was investigated. Two culture-independent analyses were employed: sequencing of clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) gene fragments and phospholipid fatty acid (PLFA) biomarker analysis. A remarkable diversity among the DSR sequences was revealed, including sequences from δ-Proteobacteria, gram-positive organisms, and theNitrospira division. PLFA analysis detected at least 52 different mid-chain-branched saturate PLFA and included a high proportion of 10me16:0. Desulfotomaculum andDesulfotomaculum-like sequences were the most dominant DSR genes detected. Those belonging to SRB within δ-Proteobacteria were mainly recovered from low-uranium (≤302 ppb) samples. OneDesulfotomaculum-like sequence cluster overwhelmingly dominated high-U (>1,500 ppb) sites. Logistic regression showed a significant influence of uranium concentration over the dominance of this cluster of sequences (P = 0.0001). This strong association indicates that Desulfotomaculum has remarkable tolerance and adaptation to high levels of uranium and suggests the organisms possible involvement in natural attenuation of uranium. The in situ activity level of Desulfotomaculum in uranium-contaminated environments and its comparison to the activities of other SRB and other functional groups should be an important area for future research.

Seed quality QTL in a prominent soybean population.

Soybean [Glycine max (L.) Merr.] is a versatile crop due to its multitude of uses as a high protein meal and vegetable oil. Soybean seed traits such as seed protein and oil concentration and seed size are important quantitative traits. The objective of this study was to identify representative protein, oil, and seed size quantitative trait loci (QTL) in soybean. A recombinant inbred line (RIL) population consisting of 131 F6-derived lines was created from two prominent ancestors of North American soybeans (‘Essex’ and ‘Williams’) and the RILs were grown in six environments. One hundred simple sequence repeat (SSR) markers spaced throughout the genome were mapped in this population. There were a total of four protein, six oil, and seven seed size QTL found in this population. The QTL found in this study may assist breeders in marker-assisted selection (MAS) to retain current positive QTL in modern soybeans while simultaneously pyramiding additional QTL from new germplasm.

Moisture retention properties of a mycorrhizal soil

The water relations of arbuscular mycorrhizal plants have been compared often, but virtually nothing is known about the comparative water relations of mycorrhizal and nonmycorrhizal soils. Mycorrhizal symbiosis typically affects soil structure, and soil structure affects water retention properties; therefore, it seems likely that mycorrhizal symbiosis may affect soil water relations. We examined the water retention properties of a Sequatchie fine sandy loam subjected to three treatments: seven months of root growth by (1) nonmycorrhizal Vigna unguiculata given low phosphorus fertilization, (2) nonmycorrhizal Vigna unguiculata given high phosphorus fertilization, (3) Vigna unguiculata colonized by Glomus intraradices and given low phosphorus fertilization. Mycorrhization of soil had a slight but significant effect on the soil moisture characteristic curve. Once soil matric potential (Ψm) began to decline, changes in Ψm per unit change in soil water content were smaller in mycorrhizal than in the two nonmycorrhizal soils. Within the range of about −1 to −5 MPa, the mycorrhizal soil had to dry more than the nonmycorrhizal soils to reach the same Ψm. Soil characteristic curves of nonmycorrhizal soils were similar, whether they contained roots of plants fed high or low phosphorus. The mycorrhizal soil had significantly more water stable aggregates and substantially higher extraradical hyphal densities than the nonmycorrhizal soils. Importantly, we were able to factor out the possibly confounding influence of differential root growth among mycorrhizal and nonmycorrhizal soils. Mycorrhizal symbiosis affected the soil moisture characteristic and soil structure, even though root mass, root length, root surface area and root volume densities were similar in mycorrhizal and nonmycorrhizal soils.

Transcriptional responses of Arabidopsis thaliana plants to As (V) stress

BackgroundArsenic is toxic to plants and a common environmental pollutant. There is a strong chemical similarity between arsenate [As (V)] and phosphate (Pi). Whole genome oligonucleotide microarrays were employed to investigate the transcriptional responses of Arabidopsis thaliana plants to As (V) stress.ResultsAntioxidant-related genes (i.e. coding for superoxide dismutases and peroxidases) play prominent roles in response to arsenate. The microarray experiment revealed induction of chloroplast Cu/Zn superoxide dismutase (SOD) (at2g28190), Cu/Zn SOD (at1g08830), as well as an SOD copper chaperone (at1g12520). On the other hand, Fe SODs were strongly repressed in response to As (V) stress. Non-parametric rank product statistics were used to detect differentially expressed genes. Arsenate stress resulted in the repression of numerous genes known to be induced by phosphate starvation. These observations were confirmed with qRT-PCR and SOD activity assays.ConclusionMicroarray data suggest that As (V) induces genes involved in response to oxidative stress and represses transcription of genes induced by phosphate starvation. This study implicates As (V) as a phosphate mimic in the cell by repressing genes normally induced when available phosphate is scarce. Most importantly, these data reveal that arsenate stress affects the expression of several genes with little or unknown biological functions, thereby providing new putative gene targets for future research.

Susceptibility of Bovine Germinal Vesicle-Stage Oocytes from Antral Follicles to Direct Effects of Heat Stress In Vitro

Abstract Delineation of maternal versus direct effects of heat stress in reducing development at the germinal vesicle (GV) stage is challenging, because oocytes spontaneously resume meiosis after removal from antral follicles. The use of S-roscovitine (inhibitor of p34cdc2/cyclin B kinase) to hold bovine oocytes at the GV stage without compromising early embryo development was previously validated in our laboratory. The objective of the present study was to assess the direct effects of an elevated temperature commonly seen in heat-stressed dairy cows on cumulus-oocyte complexes (COCs) held at the GV stage using 50 μM S-roscovitine. During roscovitine culture, GV-stage COCs (antral follicle diameter, 3–8 mm) were cultured at 38.5 or 41°C. Thereafter, oocytes were removed from roscovitine medium and allowed to undergo in vitro maturation, fertilization, and culture. Zona pellucida hardening (solubility to 0.5% pronase), nuclear stage (Hoechst 33342), cortical granule type (lens culinaris agglutinin-fluorescein isothiocyanate [FITC]), and early embryo development were evaluated. Culture of GV-stage COCs at 41°C increased the proportion that had type III cortical granules and reduced the proportion that progressed to metaphase II after in vitro maturation. Effects of 41°C on zona pellucida hardening, fertilization (penetration, sperm per oocyte, pronuclear formation, and monospermic and putative embryos), and cleavage of putative zygotes were not noted. However, culture of GV-stage COCs at 41°C for 6 h decreased the proportion of 8- to 16-cell embryos, whereas 41°C for 12 h reduced blastocyst development. In summary, antral follicle COCs are susceptible to direct effects of elevated body temperature, which may account in part for reduced fertility in heat-stressed cows.

Endocrine profiles of dairy cows following experimentally induced clinical mastitis during early lactation

Concentrations of LH, cortisol, estradiol-17beta (E(2)), prolactin and 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM) were determined in cows with experimentally induced clinical mastitis during early lactation. Cows free of intramammary infection (IMI) and in the luteal phase of the estrous cycle were balanced by lactation number and days in milk and assigned to either control (n=5) or treatment (n=5) groups. Treated cows were infected experimentally (day 0), in two mammary quarters, with Streptococcus uberis and developed clinical mastitis within 60 h after inoculation as evidenced by increased mastitis scores, elevated rectal temperatures, mammary swelling and isolation of S. uberis pathogen. Four days following bacterial challenge, blood samples were collected every 20 min for 8 h for determination of PGFM and LH following administration of oxytocin and GnRH, respectively. Blood samples were also collected on days 0, 4 and 7 of the experiment to determine concentrations of E(2), prolactin and cortisol. Four days after bacterial challenge, concentrations of cortisol were higher (P=0.04) in experimentally infected cows than controls. Experimentally challenged cows had increased (P=0.02) concentrations of cortisol on days 4 and 7 compared with day 0. Control cows had no significant increase in blood cortisol during the experimental period. Baseline concentrations of PGFM did not differ between groups; however, peak concentrations of PGFM following oxytocin challenge were elevated (P=0.006) in cows with clinical mastitis compared with control animals. Prolactin, E(2) and LH did not differ between cows with clinical mastitis or controls. Experimentally induced mastitis during early lactation elevated concentrations of cortisol during the luteal phase of the estrous cycle. Furthermore, mastitic cows demonstrated an increased PGFM response following oxytocin administration. Altered reproductive efficiency in cows with clinical mastitis caused by Gram-positive pathogens may be the result of increased uterine sensitivity to prostaglandin F(2alpha) (PGF(2alpha)).

Extracting gene networks for low-dose radiation using graph theoretical algorithms.

Genes with common functions often exhibit correlated expression levels, which can be used to identify sets of interacting genes from microarray data. Microarrays typically measure expression across genomic space, creating a massive matrix of co-expression that must be mined to extract only the most relevant gene interactions. We describe a graph theoretical approach to extracting co-expressed sets of genes, based on the computation of cliques. Unlike the results of traditional clustering algorithms, cliques are not disjoint and allow genes to be assigned to multiple sets of interacting partners, consistent with biological reality. A graph is created by thresholding the correlation matrix to include only the correlations most likely to signify functional relationships. Cliques computed from the graph correspond to sets of genes for which significant edges are present between all members of the set, representing potential members of common or interacting pathways. Clique membership can be used to infer function about poorly annotated genes, based on the known functions of better-annotated genes with which they share clique membership (i.e., “guilt-by-association”). We illustrate our method by applying it to microarray data collected from the spleens of mice exposed to low-dose ionizing radiation. Differential analysis is used to identify sets of genes whose interactions are impacted by radiation exposure. The correlation graph is also queried independently of clique to extract edges that are impacted by radiation. We present several examples of multiple gene interactions that are altered by radiation exposure and thus represent potential molecular pathways that mediate the radiation response.

Transcriptomic and metabolomic profiling of chicken adipose tissue in response to insulin neutralization and fasting

BackgroundDomestic broiler chickens rapidly accumulate adipose tissue due to intensive genetic selection for rapid growth and are naturally hyperglycemic and insulin resistant, making them an attractive addition to the suite of rodent models used for studies of obesity and type 2 diabetes in humans. Furthermore, chicken adipose tissue is considered as poorly sensitive to insulin and lipolysis is under glucagon control. Excessive fat accumulation is also an economic and environmental concern for the broiler industry due to the loss of feed efficiency and excessive nitrogen wasting, as well as a negative trait for consumers who are increasingly conscious of dietary fat intake. Understanding the control of avian adipose tissue metabolism would both enhance the utility of chicken as a model organism for human obesity and insulin resistance and highlight new approaches to reduce fat deposition in commercial chickens.ResultsWe combined transcriptomics and metabolomics to characterize the response of chicken adipose tissue to two energy manipulations, fasting and insulin deprivation in the fed state. Sixteen to 17 day-old commercial broiler chickens (ISA915) were fed ad libitum, fasted for five hours, or fed but deprived of insulin by injections of anti-insulin serum. Pair-wise contrasts of expression data identified a total of 2016 genes that were differentially expressed after correction for multiple testing, with the vast majority of differences due to fasting (1780 genes). Gene Ontology and KEGG pathway analyses indicated that a short term fast impacted expression of genes in a broad selection of pathways related to metabolism, signaling and adipogenesis. The effects of insulin neutralization largely overlapped with the response to fasting, but with more modest effects on adipose tissue metabolism. Tissue metabolomics indicated unique effects of insulin on amino acid metabolism.ConclusionsCollectively, these data provide a foundation for further study into the molecular basis for adipose expansion in commercial poultry and identify potential pathways through which fat accretion may be attenuated in the future through genetic selection or management practices. They also highlight chicken as a useful model organism in which to study the dynamic relationship between food intake, metabolism, and adipose tissue biology.

Relating foliar dehydration tolerance of mycorrhizal Phaseolus vulgaris to soil and root colonization by hyphae

Mycorrhizal symbiosis can modify plant response to drying soil, but little is known about the relative contribution of soil vs. root hyphal colonization to drought resistance of mycorrhizal plants. Foliar dehydration tolerance, characterized as leaf and soil water potential at the end of a lethal drying episode, was measured in bean plants (Phaseolus vulgaris) colonized by Glomus intraradices or by a mix of arbuscular mycorrhizal fungi collected from a semi-arid grassland. Path analysis modeling was used to evaluate how colonization rates and other variables affected these lethal values. Of several plant and soil characteristics tested, variation in dehydration tolerance was best explained by soil hyphal density. Soil hyphal colonization had larger direct and total effects on both lethal leaf water potential and soil water potential than did root hyphal colonization, root density, soil aggregation, soil glomalin concentration, leaf phosphorus concentration or leaf osmotic potential. Plants colonized by the semi-arid mix of mycorrhizal fungi had lower lethal leaf water potential and soil water potential than plants colonized by G. intraradices. Our findings support the assertion that external, soil hyphae may play an important role in mycorrhizal influence on the water relations of host plants.

Comparison of endoscopic, necropsy and histology scoring of equine gastric ulcers

Equine gastric ulcer syndrome (EGUS) represents a major health problem in performance horses. Much debate exists regarding endoscopic gastric ulcer scoring systems and their ability accurately to predict severity or depth of gastric ulcers. The purpose of this study was to evaluate the ability of an endoscopist to count gastric ulcers and predict gastric ulcer severity or depth using 2 endoscopic scoring systems and compare them to the same gastric ulcers see on necropsy and histopathology. Endoscopic examination of the stomach was performed under general anaesthesia on 23 mixed breed yearling horses, after feed was withheld for 24 h. Gastric ulcers were scored using 2 systems, number/severity-scoring (N/S) and practitioner simplified (PS) systems. After endoscopy, the horses were subjected to euthanasia and the stomach mucosa examined blindly and scored again at necropsy using above scoring systems. Representative gastric ulcers were then placed in 10% formalin and processed routinely for histopathology. The gastric ulcers were scored using a histopathology system (HSS) based on ulcer depth. Number scores in the N/S scoring system and PS on endoscopic and necropsy examinations were compared using Friedman 2 way analysis of variance. Where significant differences between variables were found a post hoc analysis was conducted using a Tukeys Studentised range (HSD) test. Severity scores using the N/S (ENGS) and PS scores recorded for the stomach via endoscopy and scores from HSS were evaluated for significant association using a Mantel-Haenszel Chi-square and Pearson moment correlation coefficient analysis. Significance was P < 0.05. All horses had gastric ulcers in the nonglandular mucosa via endoscopic examination and at necropsy examination. Mean nonglandular ulcer number (ENGN) score was significantly (P = 0.0024) lower on endoscopic examination compared to the score at necropsy (NNGN); whereas PS scores were not significantly different on endoscopy when compared to necropsy examination. A significant but weak association was found between ENGS and HSS (3.89, P = 0.048; r = 0.453, P = 0.045) and no correlation was found between PS and HSS (1.2, P = 0.272; r = 0.117; P = 0.622). Only 1/23 horses had glandular ulcers observed via endoscopic examination whereas, 6/23 horses had glandular ulcers at necropsy and on histopathology. The prevalence of EGUS is high in stalled yearling horses. The endoscopist may underestimate the number of gastric ulcers and may not be able accurately to predict the severity or depth of those ulcers present in the nonglandular equine stomach. Furthermore, the endoscopist may miss glandular gastric ulcers.