Studies of the transmissibility of the agent of bovine spongiform encephalopathy to the domestic chicken

Background

Escherichia coli O157:H7 is the most common serovar of enterohemorrhagic E. coli associated with serious human disease outbreaks. Cattle are the main reservoir with E. coli O157:H7 inducing hemorrhagic enteritis in persistent shedding beef cattle, however little is known about how this pathogen affects cattle health. Jejunal Hemorrhage Syndrome (JHS) has unclear etiology but the pathology is similar to that described for E. coli O157:H7 challenged beef cattle suggestive that E. coli O157:H7 could be involved. There are no effective treatments for JHS however new approaches to managing pathogen issues in livestock using prebiotics and probiotics are gaining support. The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of dairy cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS and also to identify the presence of potential mycotoxins. The third objective was to determine the impact of a prebiotic, Celmanax?, or probiotic, Dairyman's Choice? paste, on the cytotoxicity associated with feed extracts in vitro. The fourth objective was to determine the impact of a prebiotic or a probiotic on E. coli O157:H7 colonization of mucosal explants and a bovine colonic cell line in vitro. The final objective was to determine if prebiotic and probiotic feed additives could modify the symptoms that preceded JHS losses and the development of new JHS cases.

Findings

Dairy cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium culmorum, F. poae, F. verticillioides, F. sporotrichioides, Aspergillus flavus, Penicillium roqueforti, P. crustosum, P. paneum and P. citrinum. Mixtures of Shiga toxin - producing Escherichia coli (STEC) colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood clot blocking the jejunum. Mycotoxin analysis of the corn crop confirmed the presence of fumonisin, NIV, ZEAR, DON, 15-ADON, 3-ADON, NEO, DAS, HT-2 and T-2. Feed extracts were toxic to enterocytes and 0.1% Celmanax? removed the cytotoxicity in vitro. There was no effect of Dairyman's Choice? paste on feed-extract activity in vitro. Fumonisin, T-2, ZEAR and DON were toxic to bovine cells and 0.1% Celmanax? removed the cytotoxicity in vitro. Celmanax? also directly decreased E. coli O157:H7 colonization of mucosal explants and a colonic cell line in a dose-dependent manner. There was no effect of Dairyman's Choice? paste on E. coli O157:H7 colonization in vitro. The inclusion of the prebiotic and probiotic in the feed was associated with a decline in disease.

Conclusion

The current study confirmed an association between mycotoxigenic fungi in the feed and the development of JHS in cattle. This association was further expanded to include mycotoxins in the feed and mixtures of STECs colonizing the severely hemorrhaged tissues. Future studies should examine the extent of involvement of the different STEC in the infection process. The prebiotic, Celmanax?, acted as an anti-adhesive for STEC colonization and a mycotoxin binder in vitro. Future studies should determine the extent of involvement of the prebiotic in altering disease.     

Tillage and Nitrogen Application Effects on Nitrous and Nitric Oxide Emissions from Irrigated Corn Fields

A 2-year study was conducted to investigate the potential of no-till cropping systems to reduce N₂O and NO emissions under different N application rates in an irrigated corn field in northeastern Colorado. Flux measurements were begun in the spring of 2003, using vented (N₂O) and dynamic (NO) chambers, one to three times per week, year round, within plots that were cropped continuously to corn (Zea mays L.) under conventional-till (CT) and no-till (NT). Plots were fertilized at planting in late April with rates of 0, 134 and 224 kg N ha⁻¹ and corn was harvested in late October or early November each year. N₂O and NO fluxes increased linearly with N application rate in both years. Compared with CT, NT did not significantly affect the emission of N₂O but resulted in much lower emission of NO. In 2003 and 2004 corn growing seasons, the increase in N₂O-N emitted per kg ha⁻¹ of fertilizer N added was 14.5 and 4.1 g ha⁻¹ for CT, and 11.2 and 5.5 g ha⁻¹ for NT, respectively. However, the increase in NO-N emitted per kg ha⁻¹ of fertilizer N added was only 3.6 and 7.4 g ha⁻¹ for CT and 1.6 and 2.0 g ha⁻¹ for NT in 2003 and 2004, respectively. In the fallow season (November 2003 to April 2004), much greater N₂O (2.0–3.1 times) and NO (13.1–16.8 times) were emitted from CT than from NT although previous N application did not show obvious carry-over effect on both gas emissions. Results from this study reveal that NT has potential to reduce NO emission without an obvious change in N₂O emission under continuous irrigated corn cropping compared to CT.     

Differential helical orientations among related G protein-coupled receptors provide a novel mechanism for selectivity. Studies with salvinorin A and the kappa-opioid receptor

Salvinorin A, the active component of the hallucinogenic sage Salvia divinorum, is an apparently selective and highly potent kappa-opioid receptor (KOR) agonist. Salvinorin A is unique among ligands for peptidergic G protein-coupled receptors in being nonnitrogenous and lipid-like in character. To examine the molecular basis for the subtype-selective binding of salvinorin A, we utilized an integrated approach using chimeric opioid receptors, site-directed mutagenesis, the substituted cysteine accessibility method, and molecular modeling and dynamics studies. We discovered that helix 2 is required for salvinorin A binding to KOR and that two residues (Val-108(2.53) and Val-118(2.63)) confer subtype selectivity. Intriguingly, molecular modeling studies predicted that these loci exhibit an indirect effect on salvinorin A binding, presumably through rotation of helix 2. Significantly, and in agreement with our in silico predictions, substituted cysteine accessibility method analysis of helix 2 comparing KOR and the delta-opioid receptor, which has negligible affinity for salvinorin A, revealed that residues known to be important for salvinorin A binding exhibit a differential pattern of water accessibility. These findings imply that differences in the helical orientation of helix 2 are critical for the selectivity of salvinorin A binding to KOR and provide a structurally novel basis for ligand selectivity.     

High anti-TNP plaque-forming cell potential of residual mIg+ cells in a T cell population

In the course of experiments designed to study the immune response of purified populations of B lymphocytes to thymus-independent (TI) antigens, a variety of cell purification procedures were followed. In using anti-immunoglobulin-coated dishes to separate lymphocytes bearing membrane immunoglobulin (mIg) from mIg- lymphocytes, it was found that the nonadherent fraction, which was predominantly mIg-, complement receptor negative, and nonresponsive to the B cell mitogen lipopolysaccharide, gave very substantial anti-TNP plaque-forming cell responses to 2 TI antigens. These responses could be inhibited by incubation of such cells in the presence of anti-mu and thus appeared to be attributable to mIg+ cells. The evidence suggests the existence of a population of B lymphocytes that constitute a minor component of mIg+ cells having a high potential to make in vitro antibody responses. Users of techniques that utilize anti-Ig as a tool for separating B and T lymphocytes should carefully assess the extent to which residual B lymphocytes in the mIg- population contribute to antibody responses being studied.     

Plant Nitrogen Dynamics in Shortgrass Steppe under Elevated Atmospheric Carbon Dioxide

The direct and indirect effects of increasing levels of atmospheric carbon dioxide (CO₂) on plant nitrogen (N) content were studied in a shortgrass steppe ecosystem in northeastern Colorado, USA. Beginning in 1997 nine experimental plots were established: three open-top chambers with ambient CO₂ levels (approximately 365 mol mol^–1), three open-top chambers with twice-ambient CO₂ levels (approximately 720 mol mol^–1), and three unchambered control plots. After 3 years of growing-season CO₂ treatment, the aboveground N concentration of plants grown under elevated atmospheric CO₂ decreased, and the carbon–nitrogen (C:N) ratio increased. At the same time, increased aboveground biomass production under elevated atmospheric CO₂ conditions increased the net transfer of N out of the soil of elevated-CO₂ plots. Aboveground biomass production after simulated herbivory was also greater under elevated CO₂ compared to ambient CO₂. Surprisingly, no significant changes in belowground plant tissue N content were detected in response to elevated CO₂. Measurements of individual species at peak standing phytomass showed significant effects of CO₂ treatment on aboveground plant tissue N concentration and significant differences between species in N concentration, suggesting that changes in species composition under elevated CO₂ will contribute to overall changes in nutrient cycling. Changes in plant N content, driven by changes in aboveground plant N concentration, could have important consequences for biogeochemical cycling rates and the long-term productivity of the shortgrass steppe as atmospheric CO₂ concentrations increase.     

Biomimetic catalysis for hemicellulose hydrolysis in corn stover

Efficient and economical hydrolysis of plant cell wall polysaccharides into monomeric sugars is a significant technical hurdle in biomass processing for renewable fuels and chemicals. One possible approach to overcoming this hurdle is a biomimetic approach with dicarboxylic acid catalyst mimicking the catalytic core microenvironment in natural enzymes. This paper reports developments in the use of a dicarboxylic acid catalyst, maleic acid, for hemicellulose hydrolysis in corn stover. Hemicellulose hydrolysis and xylose degradation kinetics in the presence of maleic acid was compared to sulfuric acid. At optimized reaction conditions for each acid, maleic acid hydrolysis results in minimal xylose degradation, whereas sulfuric acid causes 3-10 times more xylose degradation. These results formed the basis for optimizing the hydrolysis of hemicellulose from corn stover using maleic acid. At 40 g/L dry corn stover solid-loading, both acid catalysts can achieve near-quantitative monomeric xylose yield. At higher solids loadings (150-200 g dry stover per liter), sulfuric acid catalyzed hydrolysis results in more than 30% degradation of the xylose, even under the previously reported optimal condition. However, as a result of minimized xylose degradation, optimized biomimetic hydrolysis of hemicellulose by maleic acid can reach approximately 95% monomeric xylose yields with trace amounts of furfural. Fermentation of the resulting unconditioned hydrolysate by recombinant S. cerevisiae results in 87% of theoretical ethanol yield. Enzyme digestibility experiments on the residual corn stover solids show that >90% yields of glucose can be produced in 160 h from the remaining cellulose with cellulases (15 FPU/g-glucan).     

The effect of estrogen on muscle damage biomarkers following prolonged aerobic exercise in eumenorrheic women

This study assessed the influence of estrogen (E₂) on muscle damage biomarkers [skeletal muscle - creatine kinase (CK); cardiac muscle - CK-MB] responses to prolonged aerobic exercise. Eumenorrheic women (n=10) who were physically active completed two 60-minute treadmill running sessions at ∼60-65% maximal intensity during low E₂ (midfollicular menstrual phase) and high E₂ (midluteal menstrual phase) hormonal conditions. Blood samples were collected prior to exercise (following supine rest), immediately post-, 30 min post-, and 24 hours post-exercise to determine changes in muscle biomarkers. Resting blood samples confirmed appropriate E₂ hormonal levels Total CK concentrations increased following exercise and at 24 hours post-exercise were higher in the midfollicular low E₂ phase (p<0.001). However, CK-MB concentrations were unaffected by E₂ level or exercise (p=0.442) resulting in the ratio of CK-MB to total CK being consistently low in subject responses (i.e., indicative of skeletal muscle damage). Elevated E₂ levels reduce the CK responses of skeletal muscle, but had no effect on CK-MB responses following prolonged aerobic exercise. These findings support earlier work showing elevated E₂ is protective of skeletal muscle from exercise-induced damage associated with prolonged aerobic exercise.     

In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

Background

The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.

Results

A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.

Conclusion

In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users.     

Are biologics more effective than classical disease-modifying antirheumatic drugs?

Major achievements have been reached in the treatment of rheumatoid arthritis during past decades due to the recognition of methotrexate as an anchor drug for treatment of rheumatoid arthritis, due to the notion of a treatment window of opportunity in patients with recent-onset rheumatoid arthritis necessitating early aggressive therapy, due to the development of biologics and due to remission as a treatment target. Most biologics have a much faster onset of action than synthetic disease-modifying anti-rheumatic drugs, but presently there is no convincing evidence that biologic drugs have a superior clinical efficacy in comparison with the synthetic drugs. Biologics are, however, accompanied by less radiological deterioration.