The development of molecular fluorescent switches

Molecular systems in which fluorescence switches between 'on' and 'off' states when driven by chemical stimuli can be designed according to a few principles. The photochemical mechanisms examined are photoinduced electron transfer, internal charge transfer and excimer formation, with emphasis on the first category. These designs open the way to sharp signalling of small chemical species that perform critical biological functions.     

Quantitative assessment of IgG antibodies to Helicobacter pylori and outcome of ischaemic heart disease

Criticisms of serological studies on Helicobacter pylori and ischaemic heart disease (IHD) include: undiagnosed heart disease in live controls; no assessment of severity or outcome of IHD; and qualitative not quantitative measurements of IgG to the bacteria. The aim was to assess quantitatively IgG levels specific for H. pylori (ng ml(-1)) among patients who survived a myocardial infarction (MI) with those who died of IHD. Sera were from four groups: (1) men who survived one MI; (2) men matched for age and socioeconomic background to group 1; (3) individuals who died suddenly of IHD; (4) accidental deaths matched for age and sex to group 3. Levels of IgG to H. pylori increased with age (P<0.005) but were not associated with smoking or socioeconomic groups. There was a correlation between IgG to the bacteria and decreasing socioeconomic levels only among group 1 (P<0.01). IgG levels were higher for subjects who died of heart disease (median=151 ng ml(-1)) compared with survivors (median=88 ng ml(-1)) (P=0.034) and higher for survivors compared with their controls (median=58 ng ml(-1)) (P=0.039). Future serological studies of H. pylori in relation to IHD should be quantitative and severity of disease considered in analyses.     

Effects of autonomic disruption and inactivity on venous vascular function

The effects of autonomic disruption and inactivity were studied on the venous vascular system. Forty-eight subjects, 24 with spinal cord injury (SCI) and 12 sedentary and 12 active able-bodied controls, participated in this study. Peripheral autonomic data were obtained to estimate sympathetic vasomotor control [low-frequency component of systolic blood pressure (LF(SBP))]. Vascular parameters were determined using strain-gauge venous occlusion plethysmography: venous capacitance (VC), venous emptying rate (VER), and total venous outflow (VO(t)). An additional vascular parameter was calculated: venous compliance [(VC/occlusion pressure) x 100]. VC and VO(t) were significantly different (SCI < sedentary < active). VER adjusted for VC was not different for any group comparison, whereas venous compliance was significantly lower in the SCI group than in the able-bodied groups and in the sedentary group compared with the active group. Regression analysis for the total group revealed a significant relationship between LF(SBP) and venous compliance (r = 0.64, P < 0.0001). After controlling for LF(SBP) through analysis of covariance, we found that mean differences for all venous vascular parameters did not change from unadjusted mean values. Our findings suggest that in subjects with SCI, the loss of sympathetic vasomotor tone contributes more than inactivity to reductions in venous vascular function. Heightened VC, VO(t), vasomotor tone, and venous compliance in the active group compared with the sedentary group imply that regular endurance training contributes to optimal venous vascular function and peripheral autonomic integrity.     

Conditional genotypic probabilities for microsatellite loci

Modern forensic DNA profiles are constructed using microsatellites, short tandem repeats of 2-5 bases. In the absence of genetic data on a crime-specific subpopulation, one tool for evaluating profile evidence is the match probability. The match probability is the conditional probability that a random person would have the profile of interest given that the suspect has it and that these people are different members of the same subpopulation. One issue in evaluating the match probability is population differentiation, which can induce coancestry among subpopulation members. Forensic assessments that ignore coancestry typically overstate the strength of evidence against the suspect. Theory has been developed to account for coancestry; assumptions include a steady-state population and a mutation model in which the allelic state after a mutation event is independent of the prior state. Under these assumptions, the joint allelic probabilities within a subpopulation may be approximated by the moments of a Dirichlet distribution. We investigate the adequacy of this approximation for profiled loci that mutate according to a generalized stepwise model. Simulations suggest that the Dirichlet theory can still overstate the evidence against a suspect with a common microsatellite genotype. However, Dirichlet-based estimators were less biased than the product-rule estimator, which ignores coancestry.     

Marker selection for the transmission/disequilibrium test, in recently admixed populations.

Recent admixture between genetically differentiated populations can result in high levels of association between alleles at loci that are <=10 cM apart. The transmission/disequilibrium test (TDT) proposed by Spielman et al. (1993) can be a powerful test of linkage between disease and marker loci in the presence of association and therefore could be a useful test of linkage in admixed populations. The degree of association between alleles at two loci depends on the differences in allele frequencies, at the two loci, in the founding populations; therefore, the choice of marker is important. For a multiallelic marker, one strategy that may improve the power of the TDT is to group marker alleles within a locus, on the basis of information about the founding populations and the admixed population, thereby collapsing the marker into one with fewer alleles. We have examined the consequences of collapsing a microsatellite into a two-allele marker, when two founding populations are assumed for the admixed population, and have found that if there is random mating in the admixed population, then typically there is a collapsing for which the power of the TDT is greater than that for the original microsatellite marker. A method is presented for finding the optimal collapsing that has minimal dependence on the disease and that uses estimates either of marker allele frequencies in the two founding populations or of marker allele frequencies in the current, admixed population and in one of the founding populations. Furthermore, this optimal collapsing is not always the collapsing with the largest difference in allele frequencies in the founding populations. To demonstrate this strategy, we considered a recent data set, published previously, that provides frequency estimates for 30 microsatellites in 13 populations.     

First Approximations of Prescribed Fire Risks Relative to Other Management Techniques Used on Private Lands

Fire is widely recognized as a critical ecological and evolutionary driver that needs to be at the forefront of land management actions if conservation targets are to be met. However, the prevailing view is that prescribed fire is riskier than other land management techniques. Perceived risks associated with the application of fire limits its use and reduces agency support for prescribed burning in the private sector. As a result, considerably less cost-share support is given for prescribed fire compared to mechanical techniques. This study tests the general perception that fire is a riskier technique relative to other land management options. Due to the lack of data available to directly test this notion, we use a combination of approaches including 1) a comparison of fatalities resulting from different occupations that are proxies for techniques employed in land management, 2) a comparison of fatalities resulting from wildland fire versus prescribed fire, and 3) an exploration of causal factors responsible for wildland fire-related fatalities. This approach establishes a first approximation of the relative risk of fatality to private citizens using prescribed fire compared to other management techniques that are readily used in ecosystem management. Our data do not support using risks of landowner fatalities as justification for the use of alternative land management techniques, such as mechanical (machine-related) equipment, over prescribed fire. Vehicles and heavy machinery are consistently leading reasons for fatalities within occupations selected as proxies for management techniques employed by ranchers and agricultural producers, and also constitute a large proportion of fatalities among firefighters. Our study provides the foundation for agencies to establish data-driven decisions regarding the degree of support they provide for prescribed burning on private lands.     

Comparison of NK-1 Receptor Antagonist (Maropitant) to Morphine as a Pre-Anaesthetic Agent for Canine Ovariohysterectomy

Objective

To compare the NK-1 receptor antagonist maropitant to morphine during and after surgery in dogs undergoing ovariohysterectomy (OHE).

Methods

30 healthy female dogs were randomly divided to receive either a pre-anaesthetic dose of morphine (0.5 mg/kg SQ) or maropitant (1 mg/kg, SQ) prior to OHE. Anaesthesia was induced with propofol and maintained with isoflurane. Expired isoflurane concentration, heart rate (HR), systolic arterial pressure (SAP) and respiratory rate were measured. Post-operative pain scores and appetite were evaluated during the recovery period. Rescue analgesia (morphine 0.1 mg/kg IV) was administered as needed post-operatively based on blinded pain score assessments.

Results

Although clinically comparable; during surgical stimulation, the maropitant group had lower HR (108±18 vs 115±24 bpm; p = 0.04), lower SAP (114±23 vs 125±23 mmHg; p = 0.003) and required slightly lower percent of isoflurane anaesthetic (1.35±0.2 vs 1.51±0.4%; p = 0.005), when compared to the morphine group. In the recovery period, the maropitant group had lower pain scores at extubation (1.7±0.7 vs 3.4±2.3; p = 0.0001) and were more likely to eat within 3 hours after extubation (64.7 vs 15.3%). However, post-operative rescue analgesia requirements were similar between groups. All other measured parameters were similar between groups. The overall difference observed between groups was small and all monitored and measured parameters were within the expected range for anesthetized dogs.

Clinical Significance

No major differences in cardiorespiratory parameters or anaesthetic requirements were observed between maropitant and morphine when used as a pre-anesthetic agent for OHE. Further studies are necessary to fully elucidate the benefits of maropitant as a pre-anaesthetic agent for canine OHE.     

Negative Effects of Sample Pooling on PCR-Based Estimates of Soil Microbial Richness and Community Structure

In this study, we examined the effect of various pooling strategies on the characterization of soil microbial community composition and phylotype richness estimates. Automated ribosomal intergenic spacer analysis (ARISA) profiles were determined from soil samples that were (i) unpooled (extracted and amplified individually), (ii) pooled prior to PCR amplification, or (iii) pooled prior to DNA extraction. Regression analyses suggest that the less even the soil microbial community (i.e., low Shannon equitability, E_H), the greater was the impact of either pooling strategy on microbial detection (R² = 0.766). For example, at a tropical rainforest site, which had the most uneven fungal (E_H of 0.597) and bacterial communities (E_H of 0.822), the unpooled procedure detected an additional 67 fungal and 115 bacterial phylotypes relative to either of the pooled procedures. Phylotype rarity, resulting in missed detection upon pooling, differed between the fungal and bacterial communities. Fungi were typified by locally abundant but spatially rare phylotypes, and the bacteria were typified by locally rare but spatially ubiquitous phylotypes. As a result, pooling differentially influenced plot comparisons, leading to an increase in similarity for the bacterial community and a decrease in the fungal community. In conclusion, although pooling reduces sample numbers and variability, it could mask a significant portion of the detectable microbial community, particularly for fungi due to their higher spatial heterogeneity.Microbial communities in soils are extremely complex, with heterogeneity expressed on a wide variety of scales (6-9, 16). Therefore, soil sampling strategies typically combine multiple small samples, obtained from various locations within the area of interest, into a single homogenized sample that is then subsampled for analysis. Previous studies (5, 11, 15) have compared the sizes of the subsamples to best represent the microbial diversity in the pooled samples. Larger sample sizes are typically recommended for community profiling (5, 11, 15) because they can reduce variability in the subsample and appear to adequately capture the dominant members of the community (3, 11). Conversely, multiple small subsamples have been proposed to be better suited for identifying rare community members and estimating species richness (10-11). While previous studies have largely been conducted to determine the variability of the subsample—and, hence, its ability to represent the larger, homogenized sample—the impact of soil sample size and pooling to best represent the site of interest and its influence on plot comparisons has not been adequately explored. For example, “rare” species in the pooled, homogenized sample may arise from two different scenarios: (i) species are found in high abundance at fine scales but are heterogeneously spaced, and (ii) species are found in low abundance but are ubiquitously distributed. Furthermore, detection of rare species could be problematic with molecular approaches that rely on PCR for amplification and detection.Although molecular techniques can detect many microbial species missed by traditional culturing (20), they suffer from several potential biases (14, 17) that may limit successful PCR amplification and detection. For example, because PCR is a competitive process, species with a low relative abundance will be amplified to a lesser degree and may not reach detection threshold levels. This process is routinely utilized in competitive PCR to analyze starting template concentrations in mixed nucleic acid samples (17, 19). Furthermore, this effect would be seen by any process that could dilute rare phylotypes, such as pooling DNA extracts prior to amplification. Therefore, if the microbial community in the starting template is too complex, reducing the soil sample size will increase the likelihood that less abundant species are successfully amplified and detected.In this study, we analyzed the influence of three different sampling strategies on microbial community profiling using automated ribosomal intergenic spacer analysis (ARISA) and the following types of samples: (i) unpooled, (ii) pooled prior to PCR amplification, or (iii) pooled prior to DNA extraction. This sampling scheme was designed to test the effects of different common sampling strategies on microbial community profiles of samples containing equal soil volumes. Studies were conducted at three different field sites with various types of plant overstory complexity: an agricultural corn field, a ponderosa pine forest, and a tropical rainforest.