Determination of growth rate of microorganisms in broth from oxygen-sensitive fluorescence plate reader measurements

A novel method utilizing the BD Oxygen Biosensor System has been developed to rapidly, simply, and accurately determine the growth rate of microorganisms in broth, with no needfor plate counts, standardized inocula, or technically difficult manipulations. The BD Oxygen Biosensor System incorporates an oxygen-sensitive material into the wells of standard Falcon microplates. The time response of this sensor monitored in afluorescence plate reader can be used to quantitate microbe growth. The method entails seeding a dilution series of microorganism onto the plate and reading at regular intervals for 3-10 h. As the organisms grow and consume oxygen, thefluorescence intensities increase over time to form a family of sigmoidal growth curves. A simple mathematical analysis of the time intervals between the curves yields the doubling time, which is independent of the initial concentration of organism. The method is ideally suited as a screening tool for assessing the impact of culture conditions, media composition, or added compounds on growth kinetics.     

An estimation of Canadian population exposure to cosmic rays

The worldwide average exposure to cosmic rays contributes to about 16% of the annual effective dose from natural radiation sources. At ground level, doses from cosmic ray exposure depend strongly on altitude, and weakly on geographical location and solar activity. With the analytical model PARMA developed by the Japan Atomic Energy Agency, annual effective doses due to cosmic ray exposure at ground level were calculated for more than 1,500 communities across Canada which cover more than 85% of the Canadian population. The annual effective doses from cosmic ray exposure in the year 2000 during solar maximum ranged from 0.27 to 0.72 mSv with the population-weighted national average of 0.30 mSv. For the year 2006 during solar minimum, the doses varied between 0.30 and 0.84 mSv, and the population-weighted national average was 0.33 mSv. Averaged over solar activity, the Canadian population-weighted average annual effective dose due to cosmic ray exposure at ground level is estimated to be 0.31 mSv.     

An assessment of intra-patient variability on observed relationships between wall shear stress and plaque progression in coronary arteries

Background

Wall shear stress (WSS) has been associated with sites of plaque localization and with changes in plaque composition in human coronary arteries. Different values have been suggested for categorizing WSS as low, physiologic or high; however, uncertainties in flow rates, both across subjects and within a given individual, can affect the classification of WSS and thus influence the observed relationships between local hemodynamics and plaque changes over time. This study examines the effects of uncertainties in flow rate boundary conditions upon WSS values and investigates the influence of this variability on the observed associations of WSS with changes in VH-IVUS derived plaque components.

Methods

Three patients with coronary artery disease underwent baseline and 12 month follow-up angiography and virtual histology-intravascular ultrasound (VH-IVUS) measurements. Coronary artery models were reconstructed from the data and models with and without side-branches were created. Patient-specific Doppler ultrasound (DUS) data were employed as inflow boundary conditions and computational fluid dynamics was used to calculate the WSS in each model. Further, the influence of representative coronary artery flow waveforms upon WSS values was investigated and the concept of treating WSS using relative, rather than actual, values was explored.

Results

Models that included side-branch outflows and subject-specific DUS velocities were considered to be the reference cases. Hemodynamic differences were caused by the exclusion of side-branches and by imposing alternative velocity waveforms. One patient with fewer side-branches and a scaled generic waveform had little deviation from the reference case, while another patient with several side-branches excluded showed much larger departures from the reference situation. Differences between models and the respective reference cases were reduced when data were analyzed using relative, rather than actual, WSS.

Conclusions

When considering individual subjects, large variations in patient-specific flow rates and exclusion of multiple side-branches in computational models can cause significant differences in observed associations between plaque evolution and ranges of computed WSS. These differences may contribute to the large variability typically found among subjects in pooled populations. Relative WSS may be more useful than actual WSS as a correlative variable when there is a large degree of uncertainty in flow rate data.

     

Structure and primase-mediated activation of a bacterial dodecameric replicative helicase

Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.     

Evidence-based de-implementation for contradicted, unproven, and aspiring healthcare practices

Abandoning ineffective medical practices and mitigating the risks of untested practices are important for improving patient health and containing healthcare costs. Historically, this process has relied on the evidence base, societal values, cultural tensions, and political sway, but not necessarily in that order. We propose a conceptual framework to guide and prioritize this process, shifting emphasis toward the principles of evidence-based medicine, acknowledging that evidence may still be misinterpreted or distorted by recalcitrant proponents of entrenched practices and other biases.     

A study of photochemically-generated protein radical spin adducts on bovine serum albumin: the detection of genuine spin-trapping and artefactual,non-radical addition in the same molecule

Summary

Photo-oxidation of bovine serum albumin (BSA) by porphyrins produces protein-centred radicals that can be spin trapped by 3, 5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) and 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO). In the case of DMPO, a thiyl radical from the Cys-34 residue is trapped, whereas with DBNBS signals from both this thiyl and tertiary carbon-centred species are observed. However, specific chemical modification of the Cys-34 residue, in combination with dual-isotope spin-trapping techniques, shows that the signal assigned to the Cys-34 thiyl adduct with DBNBS is a nitroxide artefact resulting from sequential (non-radical) nucleophilic addition and oxidation. In contrast, both the Cys-34 thiyl DMPO adduct and the tertiary carbon-centred DBNBS adducts result from genuine spintrapping. This study shows that such artefacts can be detected—even with anisotropic EPR spectra—through the use of appropriately substituted spin-traps, and that nitroso spin-traps need to be employed with great care in systems containing free thiol groups.     

Associations between COPD related manifestations: a cross-sectional study

Background

Cardiovascular disease, osteoporosis and emphysema are associated with COPD. Associations between these factors and whether they predict all-cause mortality in COPD patients are not well understood. Therefore, we examined associations between markers of cardiovascular disease (coronary artery calcification [CAC], thoracic aortic calcification [TAC] and arterial stiffness), bone density (bone attenuation of the thoracic vertebrae), emphysema (PI-950 and 15th percentile) and all-cause mortality in a COPD cohort.

Methods

We assessed CAC, TAC, bone attenuation of the thoracic vertebrae, PI-950 and 15th percentile on low-dose chest computed tomography in COPD subjects. We measured arterial stiffness as carotid-radial pulse wave velocity (PWV), and identified deaths from the national register.

Results

We studied 119 COPD subjects; aged 67.8 ±7.3, 66% were males and mean FEV₁% predicted was 46.0 ±17.5. Subjects were classified into three pre-specificed groups: CAC = 0 (n = 14), 0 < CAC ≤ 400 (n = 41) and CAC > 400 (n = 64). Subjects with higher CAC were more likely to be older (p < 0.001) and male (p = 0.03), and more likely to have higher systolic blood pressure (p = 0.001) and a history of hypertension (p = 0.002) or ischemic heart disease (p = 0.003). Higher CAC was associated with higher PWV (OR 1.62, p = 0.04) and lower bone attenuation (OR 0.32, p = 0.02), but not with 15th percentile, after adjustment for age, sex and pack-years of smoking. In a Cox proportional hazards model, CAC, TAC and 15th percentile predicted all-cause mortality (HR 2.01, 2.09 and 0.66, respectively).

Conclusions

Increased CAC was associated with increased arterial stiffness and lower bone density in a COPD cohort. In addition, CAC, TAC and extent of emphysema predicted all-cause mortality.

Trial registration

Lothian NHS Board, Lothian Research Ethics Committee, LREC/2003/8/28.     

Morphological and molecular investigations of a new family,genus and species of rodent (Mammalia: Rodentia: Hystricognatha) from Lao PDR

During biodiversity surveys in Khammouan Province, Lao Peoples Democratic Republic, specimens of an unknown species of hystricognathous rodent were discovered in local markets being sold for food; local hunters explaining that these rock rats were trapped in the nearby limestone karst. These specimens are described here on the basis of their unique combination of external and craniodental features as members of a new family, genus and species, using comparative morphological and molecular data. Phylogenetic analyses of morphological data and of 12S rRNA and cytochrome b are presented on selected taxa from all suborders of Rodentia. The results of the molecular and morphological analyses are compared and provide the basis for a discussion of relationships of the new taxon within the Rodentia and Hystricognatha. The disjunct distribution of hystricognaths is recognised as problematic, with most families occurring in the main distributional area of South America, several others in Africa and only one family distributed in Africa and Asia. The presence of an additional hystricognath family in Southeast Asia poses interesting questions and consideration is given to the way in which this new taxon fits into the theories of the biogeographical and evolutionary history of other hystricognaths. Features of possible ecomorphological significance are briefly discussed, such as the apparent adaptations to a rock dwelling existence evident in various features of the external morphology and comparisons are made to the morphology of other known saxicolous rodents.