Comparison of specific methane yield of perennial ryegrass prepared by thermal drying versus non-thermal drying in small-scale batch digestion tests.

Dried milled biomass samples are frequently utilised in small-scale batch digestion tests. However, herbage chemical composition can be altered by thermal drying, and this may affect specific methane (CH4) yields. Thus, the specific CH4 yield of herbage pre- and post-ensiling, prepared by two preparation methods were compared. Perennial ryegrass samples were either non-thermally dried (i.e. subject to cryogenic conditions, -196 °C) or thermally dried (40 °C), prior to milling. Specific CH4 yield was subsequently determined in a small-scale batch digestion test. Herbage pre-ensiling yielded 204 and 243 L CH4 kg(-1)VS(added) and herbage post-ensiling yielded 212 and 188 L CH4 kg(-1)VS(added) with non-thermal dried and thermal dried sample preparation methods, respectively. Due to opposing effects of thermal drying on CH4 yields of herbage either pre- or post-ensiling, it is not recommended to use thermal drying. Instead, it is recommended that non-thermal dried herbage samples are used in small-scale batch digestion tests.

Microbial community structure during fluoranthene degradation in the presence of plants.

AIMS: To investigate bacterial and fungal community structure during degradation of varying concentrations (0-5000 mg kg(-1) ) of the polycyclic aromatic hydrocarbon (PAH) fluoranthene in the presence or absence of tomato plants. METHOD AND RESULTS: Fluoranthene amended or unamended growing media, with or without a plant, were incubated in pots in a glasshouse for 30 days. Fluoranthene concentration was quantified using GC-FID, while bacterial and fungal community structure was investigated using trflp and arisa, respectively. The abundance of two ring hydroxylating dioxygenase genes was measured using qPCR. More fluoranthene was degraded in the presence of tomato plants at 500 and 5000 mg fluoranthene kg(-1) (P < 0·001), and it had a toxic effect on plant growth. Bacterial and fungal community composition in the rhizosphere was significantly different from that in nonrhizospheric and unplanted samples. The influence of fluoranthene on bacterial communities overcame that of the plant root such that community composition in rhizosphere and planted nonrhizospheric samples was broadly similar when fluoranthene was present. PAH-RHDα-GP genes were more abundant than PAH-RHDα-GN genes in all treatments. PAH-RHDα-GN abundance was unaffected by either PAH concentration or growing medium type, while the abundance of PAH-RHDα-GP was greatest in the rhizosphere. CONCLUSIONS: Plants promoted microbial growth and diversity and led to increases in abundance of Gram-positive dioxygenase genes, correlated with increased degradation of fluoranthene. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to knowledge in the broad area of biodegradation and also provides useful information for the design of future bioremediation strategies.

Changes in chemical composition and digestibility of three maize stover components digested by white-rot fungi.

Maize stover (total stem and leaves) is not considered a ruminant feed of high nutritive value. Therefore, an improvement in its digestibility may increase the viability of total forage maize production systems in marginal growth regions. The objective of this study was to describe the changes in chemical composition during the storage of contrasting components of maize stover (leaf, upper stem and lower stem) treated with either of two lignin degrading white-rot fungi (WRF; Pleurotus ostreatus, Trametes versicolor). Three components of maize stover (leaf, upper stem and lower stem), harvested at a conventional maturity for silage production, were digested with either of two WRF for one of four digestion durations (1-4 months). Samples taken prior to fungal inoculation were used to benchmark the changes that occurred. The degradation of acid detergent lignin was observed in all sample types digested with P. ostreatus; however, the loss of digestible substrate in all samples inoculated with P. ostreatus was high, and therefore, P. ostreatus-digested samples had a lower dry matter digestibility than samples prior to inoculation. Similarly, T. veriscolor-digested leaf underwent a non-selective degradation of the rumen-digestible components of fibre. The changes in chemical composition of leaf, upper stem and lower stem digested with either P. ostreatus or T. veriscolor were not beneficial to the feed value of the forage, and incurred high DM losses.

Conservation characteristics of corn ears and stover ensiled with the addition of Lactobacillus plantarum MTD-1, Lactobacillus plantarum 30114, or Lactobacillus buchneri 11A44.

The aim of this study was to investigate the effects of inoculating 3 contrasting lactic acid bacteria on the fermentation profile, estimated nutritive value, and aerobic stability of corn ears and stover produced under marginal growing conditions. Ears and stover were separated from whole-crop corn plants obtained from 3 replicate field blocks. Representative subsamples were precision chopped and allocated to 1 of the following treatments: an uninoculated control, Lactobacillus plantarum MTD-1 (LP1), L. plantarum 30114 (LP2), or Lactobacillus buchneri 11A44 (LB). Each bacterial additive was applied at a rate of 1 × 10(6) cfu/g of fresh herbage. Triplicate samples of each treatment were ensiled in laboratory silos at 15°C for 3, 10, 35, or 130 d. No difference was observed between the dry matter recoveries of uninoculated ear or stover silages and silages made with LP1, and the aerobic stability of uninoculated ear and stover silages did not differ from silages made with LB. Stover silages made with LP2 and ensiled for 35 d had a lower proportion of lactic acid in total fermentation products compared with LP1. The aerobic stability and dry matter recovery of ear and stover silages in this study were not improved when made with LB, LP1, or LP2, due to the indigenous highly heterolactic fermentation that prevailed in the uninoculated ear and stover during 130-d ensilage.

The effect of dietary concentrate and soya oil inclusion on microbial diversity in the rumen of cattle.

AIMS: Methane emissions from ruminants are a significant contributor to global greenhouse gas production. The aim of this study was to examine the effect of diet on microbial communities in the rumen of steers. METHODS AND RESULTS: The effects of dietary alteration (50 : 50 vs 90 : 10 concentrate-forage ratio, and inclusion of soya oil) on methanogenic and bacterial communities in the rumen of steers were examined using molecular fingerprinting techniques (T-RFLP and automated ribosomal intergenic spacer analysis) and real-time PCR. Bacterial diversity was greatly affected by diet, whereas methanogen diversity was not. However, methanogen abundance was significantly reduced (P = 0.009) in high concentrate-forage diets and in the presence of soya oil (6%). In a parallel study, reduced methane emissions were observed with these diets. CONCLUSIONS: The greater effect of dietary alteration on bacterial community in the rumen compared with the methanogen community may reflect the impact of substrate availability on the rumen bacterial community. This resulted in altered rumen volatile fatty acid profiles and had a downstream effect on methanogen abundance, but not diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding how rumen microbial communities contribute to methane production and how these microbes are influenced by diet is essential for the rational design of methane mitigation strategies from livestock.

Assessing the impact of various ensilage factors on the fermentation of grass silage using conventional culture and bacterial community analysis techniques.

AIMS: Grass silage is an important ruminant feedstuff on farms during winter. The ensilage of grass involves a natural lactic acid bacterial fermentation under anaerobic conditions, and numerous factors can influence the outcome of preservation. The aim of this study was to investigate the effect of dry matter concentration, ensiling system, compaction and air infiltration on silage bacterial community composition. METHODS AND RESULTS: The impact of these factors was examined using conventional methods of microbial analysis and culture-independent Terminal Restriction Fragment Length Polymorphism (T-RFLP). Silage fermentation was restricted in herbage with a high dry matter concentration, and this was reflected in a shift in the bacterial population present. In contrast, ensiling system had little effect on bacterial community composition. Air infiltration, in the absence of compaction, altered silage bacterial community composition and silage pH. CONCLUSIONS: Dry matter concentration and the absence of compaction were the main factors affecting silage microbial community composition, and this was reflected in both the conventional culture-based and T-RFLP data. SIGNIFICANCE AND IMPACT OF THE STUDY: T-RFLP proved a useful tool to study the factors affecting ensilage. Apart from monitoring the presence or absence of members of the population, shifts in the relative presence of members could be monitored.

Bacterial community dynamics during the ensilage of wilted grass.

AIMS: Grass silage is the product formed by a natural lactic acid bacterial fermentation when grass is stored under anaerobic conditions, and represents an important ruminant feedstuff on farms during winter. Of the two commonly employed methods of ensiling forage, baled silage composition frequently differs from that of comparable precision-chop silage reflecting a different ensiling environment. The aim of this study was to investigate the dynamics of the silage fermentation in wilted grass and between ensiling systems. METHODS AND RESULTS: Fermentation dynamics were examined using traditional methods of silage analyses, including microbial enumeration and analysis of fermentation products, and culture-independent terminal restriction fragment length polymorphism (T-RFLP). A successful fermentation was achieved in both systems, with the fermentation (increase in lactic acid bacteria and lactic acid concentration, decrease in pH) proceeding rapidly once the herbage was ensiled. CONCLUSIONS: Under controlled conditions, little difference in silage quality and microbial composition were observed between ensiling systems and this was further reflected in the T-RFLP community analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: T-RFLP proved a potentially useful tool to study the ensilage process and could provide valid support to traditional methods, or a viable alternative to these methods, for investigating the dynamics of the bacterial community over the course of the fermentation.

Histological grading of epithelial ovarian carcinomas.

The grade of an ovarian epithelial neoplasm provides useful information. However, different approaches to grading exist and many ovarian cancers are not graded. We examined primary ovarian cancers from patients treated at our hospital and applied the 'universal' grading system. We found a significant association between grade and clinical stage, with a survival difference between grades for low-stage tumours. The application of grade is discussed in the light of developments in the grading of other gynaecological cancers.

Radial scars/complex sclerosing lesions and malignancy in a screening programme: incidence and histological features revisited.

AIMS: Radial scars (RS) are benign entities, frequently identified on screening mammography, which may be associated with malignancy. Much debate has been generated with regard to the optimum management of RS. We present our experience of RS in the first 5 years of a screening programme. The aim was to evaluate (i) the incidence of atypia and malignancy and (ii) the value of the preoperative core biopsy. We also further characterize the histological features. METHODS AND RESULTS: One hundred and twenty-five histologically confirmed cases of RS were reviewed (111 had preoperative biopsies). Thirty-one (24.8%) patients had a final malignant diagnosis (11 with invasive malignancy) and 28 (22.4%) showed atypia (including lobular carcinoma in situ). In those with core biopsies and a final malignant diagnosis, 12 cases were categorized as B5 (41.3%), three as B4 (10.3%), 12 as B3 (41.3%) and two as B2 (7%). Common histological features included obliterated ducts and chronic inflammation with, less frequently, neural hyperplasia (16.8%) and perineural invasion (3.2%). CONCLUSIONS: The high incidence of atypia and malignancy identified in our study justifies our policy of removing all mammographically detected RS. We emphasize the utility of preoperative core biopsy evaluation in permitting one-stage surgical therapy in those with B5 diagnoses.

Microbial community changes during the bioremediation of creosote-contaminated soil.

AIMS: To investigate the effects of aeration on the ex situ biodegradation of polycyclic aromatic hydrocarbons (PAHs) in creosote-contaminated soil and its effect on the microbial community present. METHODS AND RESULTS: Aerated and nonaerated microcosms of soil excavated from a former timber treatment yard were maintained and sampled for PAH concentration and microbial community changes by terminal restriction fragment length polymorphism (TRFLP) analysis. After an experimental period of just 13 days, degradation was observed with all the PAHs monitored. Abiotic controls showed no loss of PAH. Results unexpectedly showed greater loss of the higher molecular weight PAHs in the nonaerated control. This may have been due to the soil excavation causing initial decompaction and aeration and the resulting changes caused in the microbial community composition, indicated by TRFLP analysis showing several ribotypes greatly increasing in relative abundance. Similar changes in both microcosms were observed but with several possible key differences. The species of micro-organisms putatively identified included Bacilli, pseudomonad, aeromonad, Vibrio and Clostridia species. CONCLUSIONS: Excavation of the contaminated soil leads to decompaction, aeration and increased nutrient availability, which in turn allow microbial biodegradation of the PAHs and a change in the microbial community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the changes occurring in the microbial community during biodegradation of all PAHs is essential for the development of improved site remediation protocols. TRFLP allows useful monitoring of the total microbial community.

Marrow aplasia developing 13 years after HLA-identical sibling allogeneic transplantation for chronic myeloid leukaemia: successful treatment with antithymocyte globulin and peripheral blood stem cell infusion from the original donor.

Secondary or late graft failure has been defined as the development of inadequate marrow function after initial engraftment has been achieved. We describe a case of profound marrow aplasia occurring 13 years after sibling allogeneic bone marrow transplantation for chronic myeloid leukaemia (CML) in first chronic phase. Although the patient remained a complete donor chimera, thereby suggesting that an unselected infusion of donor peripheral blood stem cells (PBSC) or bone marrow might be indicated, the newly acquired aplasia was thought to be immune in aetiology and some immunosuppression was therefore considered appropriate. Rapid haematological recovery was achieved after the infusion of unselected PBSC from the original donor following conditioning with anti-thymocyte globulin (ATG).

The role of placental cytogenetic cultures in intrauterine and neonatal deaths.

Knowledge of chromosomal status is useful in helping to explain fetal and neonatal deaths, but maceration has been felt to compromise the success rate. As well as skin fibroblast culture, the placenta can be sampled and this may be useful where permission for autopsy is refused. We examined placental cytogenetic results from 250 cases over a 5-year period. There was a success rate of 84.4%, with neither weight (< or > 500 g) nor maceration status showing a significant impact. Placentas sampled within 1 day were significantly more likely to give a positive culture. The results suggested that placental cytogenetic cultures should be attempted in all perinatal deaths where such information is desired.

Biotransformation of halophenols using crude cell extracts of Pseudomonas putida F6.

Crude cell extracts of Pseudomonas putida F6 transformed 4-substituted fluoro-, chloro-, bromo- and iodo-phenol without the exogenous addition of cofactors. The rate of substrate consumption decreased with increasing substituent size (F>Cl>Br>I). Biotransformations resulted in greater than 95% utilisation of the halogenated substrate. Product accumulation was observed in incubations with 4-chloro, 4-bromo- and 4-iodo-phenol. These products were identified as the corresponding 4-substituted catechols. Transformation of 4-fluorophenol did not result in the accumulation of the corresponding catechol; however, manipulation of the reaction conditions by incorporation of ascorbic acid culminated in the formation of 4-fluorocatechol. Cell extracts of P. putida F6 also showed activity towards a 3-substituted phenol, namely 3-fluorophenol, resulting in the formation of a single product, 4-fluorocatechol.

The quality and value of sudden infant death necropsy reporting in Ireland.

BACKGROUND: Infant necropsies are important for identifying cause of death. Recently issued guidelines have recommended investigations to be performed following sudden unexpected death in infants. AIMS: To evaluate the quality and value of infant postmortem reporting. METHODS: Postmortem reports from 1994-1996 and 1998-2000 in Ireland were evaluated using the National Sudden Infant Death Register. Scoring was by a modification of the Rushton system based on the extent of the postmortem data. The finding of additional pathological information was also assessed. RESULTS: Of the 274 cases registered during the selection period, reports were available for 245. Overall quality of necropsy reporting was below the minimum accepted standard in 55.5%; 47% of the necropsies were performed in regional paediatric pathology centres. The quality of necropsies performed in regional centres was significantly higher than those performed elsewhere. Although 86% of the cases were defined as sudden infant death syndrome (SIDS; no cause of death found), the finding of additional pathological information was significantly related to the extent of the necropsy. There was a significant improvement in the quality of necropsies after the postmortem guidelines were issued. CONCLUSIONS: The overall quality of sudden unexpected infant death necropsies in Ireland is less than adequate. A minimum accepted standard of necropsy is required before a diagnosis of SIDS can be made. Although standards have improved recently, this study highlights the need to adhere to published guidelines and the importance of auditing the effect of introducing practice guidelines on clinical practice to complete the audit loop.

Tamoxifen and estrogen lower circulating lipoprotein(a) concentrations in healthy postmenopausal women.

Data in the literature suggest that circulating levels of lipoprotein(a) [Lp(a)] and insulinlike growth factor I (IGF-I) respond similarly to therapy with growth hormone, estrogen, or tamoxifen. To more clearly document these relations, we designed a randomized, double-blind, placebo-controlled study of the effects of tamoxifen and continuous estrogen on circulating levels of Lp(a), IGF-I, and IGF binding protein 3 (IGFBP-3) in healthy postmenopausal women. Both estrogen and tamoxifen decreased serum levels of IGF-I to 30% below baseline during the 3 months of treatment, while IGFBP-3 levels were unchanged. Plasma Lp(a) levels decreased to 24% below baseline after 1 month of treatment with either estrogen or tamoxifen (P < .05 for estrogen only); after 3 months Lp(a) decreased to 34% below baseline with tamoxifen therapy (P < .05) but returned to only 16% below baseline with estrogen. The correlation between Lp(a) and IGF-I was highly significant (P < .0001). We conclude that (1) tamoxifen lowers plasma Lp(a) levels in healthy postmenopausal women, (2) the suppressive effects of tamoxifen and estrogen on circulating Lp(a) concentration diverge after the first month of therapy, and (3) circulating levels of Lp(a) and IGF-I are strongly correlated with each other, an indication that they may share regulatory influences.

Inhibition of inflammatory cell-mediated myelin oxidation and interleukin-1 beta generation by a 21-aminosteroid, U74500A.

Inflammatory cell-mediated myelin injury may be an important cause of tissue damage in both acute and chronic central nervous system (CNS) disorders. The 21-aminosteroids are novel derivatives of methylprednisolone without obvious glucocorticoid or mineralocorticoid side effects. We evaluated the ability of 21-aminosteroid, U74500A, to inhibit oxidation of rat brain myelin by human polymorphonuclear leukocytes (PMN) and monocytes. Myelin samples, as confirmed by SDS-PAGE, were incubated with PMN or monocytes and 100 microM U74500A or vehicle. Myelin oxidation by both PMN and monocytes was significantly reduced by U74500A. These observations demonstrate that U74500A can inhibit myelin oxidation by inflammatory cells. Additionally, 100 microM U74500A significantly reduced production of interleukin 1-beta by monocytes exposed to myelin. The aminosteroids may be beneficial in CNS disorders where myelin injury by inflammatory cells appears to contribute, such as acute focal ischemia or multiple sclerosis.

The high maltose-producing alpha-amylase of the thermophilic actinomycete, Thermomonospora curvata.

The alpha-amylase of Thermomonospora curvata catalyses the formation of very high levels of maltose from starch (73%, w/w) without the attendant production of glucose. The enzyme was produced extracellularly in high yield during batch fermentation in a 5-1 fermentor. Purification was achieved by ammonium sulphate fractionation, Superose-12 gel filtration and DEAE-Sephacel ion-exchange chromatography. The enzyme exhibited maxima for activity at pH 6.0 and 65 degrees C, had a relative molecular mass of 60,900-62,000 and an isoelectric point at 6.2. The exceptionally high levels of maltose produced and the unique action pattern exhibited on starch and related substrates indicate a very unusual maltogenic system. The predominance of maltose as the final end-product may be explained by the participation of reactions other than simple hydrolysis and the preferential cleavage of maltotriose from higher maltooligosaccharides. The enzyme exhibits very low affinity for maltotriose (Km = 7.7 x 10(-3) M) and its conversion to maltose is achieved by synthetic followed by hydrolytic events, which result in the very high levels of maltose observed and preclude glucose formation. This system is distinguished from other very high maltose-producing amylases by virtue of its high temperature maximum, very low affinity for maltotriose and the absence of glucose in the final saccharide mixture.

A 21-aminosteroid inhibits oxidation of human low density lipoprotein by human monocytes and copper.

Oxidation of low density lipoprotein (LDL) leads to more rapid uptake by arterial wall macrophages and foam cell formation. Inhibiting LDL oxidation may impede these processes and offers a new mechanism to retard atherogenesis. The 21-aminosteroids, derived from methylprednisolone, are potent inhibitors of free radical production by stimulated monocytes and also are scavengers of lipid peroxyl radicals. The 21-aminosteroid, U74500A, was added to a mixture of low density lipoprotein cholesterol and human monocytes to which lipopolysaccharide was add to stimulate the monocytes. At a final concentration of 10 microM, U74500A reduced the production of lipid peroxidation from 6.10 +/- 1.11 to 0.84 +/- 0.16 nmol (mean +/- SEM) MDA equivalent/1 x 10(6) monocytes, as measured by a thiobarbituric acid reacting substance (TBARS) assay. Similarly 10 microns U74500A reduced Cu2+ induced LDL oxidation from 12.28 +/- 0.10 (in vehicle) to 0.49 +/- 0.12. These observations suggest that the 21-aminosteroids should be evaluated in animal models as a potential therapy to retard atherogenesis, especially considering their apparent lack of mineralocorticoid and glucocorticoid side-effects.

A 21-aminosteroid inhibits stimulated monocyte hydrogen peroxide and chemiluminescence measurements from MS patients and controls.

Monocytes are recruited to active sites of multiple sclerosis (MS) demyelination and may promote local tissue injury by generating an inflammatory response, mediated in part by the production of toxic oxygen metabolites. Corticosteroids are frequently and effectively used to ameliorate MS exacerbations, despite inadequate knowledge about the mechanism. We assessed the effects of a 21-aminosteroid, U74500A, a new class of steroid derivatives without glucocorticoid or mineralocorticoid effects, on the production of hydrogen peroxide (H2O2) and chemiluminescence by stimulated monocytes harvested from 8 stable MS patients and age- and sex-matched controls. H2O2 measurements and chemiluminescence were significantly reduced in both groups by U74500A. These results demonstrate that 21-aminosteroids reduce production of toxic oxygen metabolites by monocytes and thus their inflammatory potential, suggesting that these agents may be potentially effective and safe treatment for MS exacerbations.