Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.     

Salt-induced Inhibition of Phosphate Transport and Release of Membrane Proteins from Barley Roots

Osmotic shock with sequential 30-minute treatments in ice-cold saline solutions and H₂O released proteins from excised barley roots and inhibited the subsequent uptake of orthophosphate (Pi). The amount of protein released increased sharply at NaCl concentrations above 0.05 molar, approximately the threshold concentration above which Pi uptake was increasingly suppressed. About 60% of the nearly 100 micrograms of protein per gram fresh weight of roots that was eluted in 0.16 molar NaCl treatments apparently had no function in Pi transport, since it was eluted at NaCl concentrations (≤0.05 molar) that did not affect Pi uptake. Although 0.16 molar NaCl completely inhibited Pi uptake, active uptake resumed at about 60% of control rates within 1 to 2 hours. The presence of either puromycin or cycloheximide greatly reduced the recovery of Pi uptake activity after the NaCl treatment. Mannitol and various monovalent and divalent salts at concentrations isosmotic with NaCl also inhibited Pi uptake, but CaCl₂ was consistently the least inhibitory. The correlation between the concentration of the osmotic treatments and the simultaneous loss of protein and Pi uptake activity, together with the evidence that uptake recovery requires protein synthesis, support the hypothesis that the proteins eluted are required for active Pi transport.     

Optimization of isolation and storage of sperm cells from pollen of perennial ryegrass (Lolium perenne L.)

Summary A procedure for isolating sperm cells of perennial ryegrass (Lolium perenne L.) was developed. The sperm cells were released from the pollen grains by osmotic shock, with the right combination of pH and osmolality being important for optimal release. Various combinations of vitamins E, C and fetal calf serum were tested with the aim of improving yield and long-term viability, and their possible mode of action as important components for improvement of these two parameters is discussed. Under optimized conditions, a yield of 12% was established, and the storage time after which 50% of the sperm cells were still viable was improved to 60 h. Cytological observations demonstrated that sperm cells of perennial ryegrass are true protoplasts, which may allow future fusion experiments to be carried out.     

Outcomes in octogenarians undergoing coronary artery bypass grafting

Background

Although octogenarians are being referred for coronary artery bypass grafting (CABG) with increasing frequency, contemporary outcomes have not been well described. We examined data from 4 Canadian centres to determine outcomes of CABG in this age group.

Methods

Data for the years 1996 to 2001 were examined in a comparison of octogenarians with patients less than 80 years of age. Logistic regression analysis was used to adjust for preoperative factors and to generate adjusted rates of mortality and postoperative stroke.

Results

A total of 15 070 consecutive patients underwent isolated CABG during the study period. Overall, 725 (4.8%) were 80 years of age or older, the proportion increasing from 3.8% in 1996 to 6.2% in 2001 (p for linear trend = 0.03). The crude rate of death was higher among the octogenarians (9.2% v. 3.8%; p < 0.001), as was the rate of stroke (4.7% v. 1.6%, p < 0.001). The octogenarians had a significantly greater burden of comorbid conditions and more urgent presentation at surgery. After adjustment, the octogenarians remained at greater risk for in-hospital death (odds ratio [OR] 2.64, 95% confidence interval [CI] 1.95–3.57) and stroke (OR 3.25, 95% CI 2.15–4.93). Mortality declined over time for both age groups (p for linear trend < 0.001 for both groups), but the incidence of postoperative stroke did not change (p for linear trend = 0.61 [age < 80 years] and 0.08 [age ≥ 80 years]). Octogenarians who underwent elective surgery had crude and adjusted rates of death (OR 1.31, 95% CI 0.60–2.90) and stroke (OR 1.59, 95% CI 0.57–4.44) that were higher than but not significantly different from those for non-octogenarians who underwent elective surgery.

Interpretation

In this study, rates of death and stroke were higher among octogenarians, although the adjusted differences in mortality over time were decreasing. The rate of adverse outcomes in association with elective surgery was similar for older and younger patients.The population is rapidly aging, and an increasing number of octogenarians are being referred for coronary artery bypass grafting (CABG).^1,2 Previous single-centre reports from Canada^3,4,5 and from abroad^1,2,6,7,8have concluded that elderly patients undergoing cardiac surgery have worse outcomes than younger patients. In addition, these studies have reported higher costs and slower recovery for octogenarians undergoing CABG, a finding that has generated debate over the appropriate use of health care resources.^1,5,7,9It has become increasingly clear that the results of CABG among octogenarians, although worse than among younger patients, are better than for percutaneous coronary interventions or medical therapy alone when the extent of the patient''s coronary disease is such that revascularization with CABG is indicated.^10,11 Similarly, the superior results of percutaneous coronary intervention relative to medical therapy in elderly patients with coronary disease will likely continue to increase the total number of octogenarians undergoing coronary angiography, which in turn will probably increase the number of patients being referred for CABG.^10,12 Contemporary outcomes for octogenarians undergoing CABG in Canada have not been well described. If we are to have an informed debate and determine appropriate policy, it is important for these outcomes to be known.We aimed to describe the characteristics and outcomes of patients 80 years of age and older undergoing CABG in Canada and to compare their outcomes with those of younger patients. In addition, we examined changes in results over time.     

Cutting edge: molecular portrait of human autoimmune disease

Autoimmune diseases affect 3-5% of the population, are mediated by the immune response to self-Ags, and are characterized by the site of tissue destruction. We compared expression levels of >4,000 genes in PBMC of control individuals before and after immunization to those of individuals with four distinct autoimmune diseases. The gene expression profile of the normal immune response exhibits coordinate changes in expression of genes with related functions over time. In contrast, each individual from all autoimmune diseases displays a similar gene expression profile unrelated to the pattern of the immunized group. To our surprise, genes with a distinct expression pattern in autoimmunity are not necessarily "immune response" genes, but are genes that encode proteins involved in apoptosis, cell cycle progression, cell differentiation, and cell migration.     

Genetic elimination of behavioral sensitization in mice lacking calmodulin-stimulated adenylyl cyclases

Adenylyl cyclase types 1 (AC1) and 8 (AC8), the two major calmodulin-stimulated adenylyl cyclases in the brain, couple NMDA receptor activation to cAMP signaling pathways. Cyclic AMP signaling pathways are important for many brain functions, such as learning and memory, drug addiction, and development. Here we show that wild-type, AC1, AC8, or AC1&8 double knockout (DKO) mice were indistinguishable in tests of acute pain, whereas behavioral responses to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant, were reduced or abolished in AC1&8 DKO mice. AC1 and AC8 are highly expressed in the anterior cingulate cortex (ACC), and contribute to inflammation-induced activation of CREB. Intra-ACC administration of forskolin rescued behavioral allodynia defective in the AC1&8 DKO mice. Our studies suggest that AC1 and AC8 in the ACC selectively contribute to behavioral allodynia.     

A spatial model of tree α-diversity and tree density for the Amazon

Large-scale patterns of Amazonian biodiversity have until now been obscured by a sparse and scattered inventory record. Here we present the first comprehensive spatial model of tree -diversity and tree density in Amazonian rainforests, based on the largest-yet compilation of forest inventories and bolstered by a spatial interpolation technique that allows us to estimate diversity and density in areas that have never been inventoried. These data were then compared to continent-wide patterns of rainfall seasonality. We find that dry season length, while only weakly correlated with average tree -diversity, is a strong predictor of tree density and of maximum tree -diversity. The most diverse forests for any given DSL are concentrated in a narrow latitudinal band just south of the equator, while the least diverse forests for any given DSL are found in the Guayana Shield and Amazonian Bolivia. Denser forests are more diverse than sparser forests, even when we used a measure of diversity that corrects for sample size. We propose that rainfall seasonality regulates tree -diversity and tree density by affecting shade tolerance and subsequently the number of different functional types of trees that can persist in an area.