Correlated rates of synonymous site evolution across plant genomes

Synonymous substitution rates have been shown to vary among evolutionary lineages of both nuclear and organellar genes across a broad range of taxonomic groups. In animals, rate heterogeneity does not appear to be correlated across nuclear and mitochondrial genes. In this paper, we contrast substitution rates in two plant groups and show that grasses evolve more rapidly than palms at synonymous sites in a mitochondrial, a nuclear, and a plastid gene. Furthermore, we show that the relative rates of synonymous substitution between grasses and palms are similar at the three loci. The correlation in synonymous substitution rates across genes is particularly striking because the three genes evolve at very different absolute rates. In contrast, relative rates of nonsynonymous substitution are not conserved among the three genes.      

Medical education must make room for student-specific ethical dilemmas

Most contemporary undergraduate courses in medical ethics leave a critical gap unfilled because they fail to address student-specific issues, says third-year student Joye St. Onge. In this article, which won third prize in CMAJs 1996 Dr. William Logie Medical Ethics Essay Contest, St. Onge outlines the importance of discussing student-specific ethical dilemmas and suggests ways to introduce such teaching in medical schools.     

Medium‐Chain Triglycerides Increase Energy Expenditure and Decrease Adiposity in Overweight Men

Objective: The objectives of this study were to compare the effects of diets rich in medium‐chain triglycerides (MCTs) or long‐chain triglycerides (LCTs) on body composition, energy expenditure, substrate oxidation, subjective appetite, and ad libitum energy intake in overweight men. Research Methods and Procedures: Twenty‐four healthy, overweight men with body mass indexes between 25 and 31 kg/m² consumed diets rich in MCT or LCT for 28 days each in a crossover randomized controlled trial. At baseline and after 4 weeks of each dietary intervention, energy expenditure was measured using indirect calorimetry, and body composition was analyzed using magnetic resonance imaging. Results: Upper body adipose tissue (AT) decreased to a greater extent (p < 0.05) with functional oil (FctO) compared with olive oil (OL) consumption (?0.67 ± 0.26 kg and ?0.02 ± 0.19 kg, respectively). There was a trend toward greater loss of whole‐body subcutaneous AT volume (p = 0.087) with FctO compared with OL consumption. Average energy expenditure was 0.04 ± 0.02 kcal/min greater (p < 0.05) on day 2 and 0.03 ± 0.02 kcal/min (not significant) on day 28 with FctO compared with OL consumption. Similarly, average fat oxidation was greater (p = 0.052) with FctO compared with OL intake on day 2 but not day 28. Discussion: Consumption of a diet rich in MCTs results in greater loss of AT compared with LCTs, perhaps due to increased energy expenditure and fat oxidation observed with MCT intake. Thus, MCTs may be considered as agents that aid in the prevention of obesity or potentially stimulate weight loss.     

A method for high‐throughput production of sequence‐verified DNA libraries and strain collections

The low costs of array‐synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost‐effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site‐specific recombination to index library DNA, and next‐generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost‐effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.     

Four‐Compartment Cellular Level Body Composition Model: Comparison of Two Approaches**

Objective: The aim of this study was to develop and compare two DXA‐based four‐compartment [body weight = body cell mass (BCM) + extracellular fluid (ECF) + extracellular solids (ECS) + fat] cellular level models. Research Methods and Procedures: Total body potassium (TBK) model: BCM from TBK by whole‐body counting—ECF_TBK = LST ? [BCM_TBK + 0.73 × osseous mineral (Mo)]. Bromide model: ECF from sodium bromide dilution—BCM_BROMIDE = LST ? (ECF_BROMIDE + 0.73 × Mo); Mo and LST measurements came from DXA. The two approaches were evaluated in 99 healthy men and 118 women. Results: BCM estimates were highly correlated (r = 0.97, p < 0.001), as were ECF estimates (r = 0.87, p < 0.001); a small statistically significant mean difference was present (mean ± SD; BCM_TBK model, 30.4 ± 8.9 kg; BCM_BROMIDE, 31.4 ± 9.3 kg; Δ = 1.0 ± 2.8 kg; p < 0.001; ECF_TBK, 18.5 ± 4.2 kg; ECF_BROMIDE, 17.5 ± 3.6 kg; Δ = 1.0 ± 2.8 kg; p < 0.001). A high correlation (r = 0.97, p < 0.001) and good agreement (38.9 ± 9.5 vs. 38.9 ± 9.5 kg; Δ = 0.0 ± 2.4 kg; p = 0.39) were present between TBW, derived as the sum of intracellular water from TBK and ECW from bromide, and measured TBW by ²H₂O dilution. Discussion: Two developed four‐compartment cellular level DXA models, one of which is appropriate for use in most clinical and research settings, provide comparable results and are applicable for BCM and ECF estimation of subject groups with hydration disturbances.     

Inhibition of JC polyomavirus infectivity by the retrograde transport inhibitor Retro-2.1

JC polyomavirus (JCPyV) is a common human pathogen that results in a chronic asymptomatic infection in healthy adults. Under conditions of immunosuppression, JCPyV spreads to the central nervous system and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), a disease for which there are no vaccines or antiviral therapies. Retro-2 is a previously identified small molecule inhibitor that was originally shown to block retrograde transport of toxins such as ricin toxin from endosomes to the Golgi apparatus and endoplasmic reticulum (ER), and Retro-2.1 is a chemical analog of Retro-2 that has been shown to inhibit ricin intoxication of cells at low nanomolar concentrations. Retro-2 has previously been shown to prevent retrograde transport of JCPyV virions to the ER, but the effect of Retro-2.1 on JCPyV infectivity is unknown. Here it is shown that Retro-2.1 inhibits JCPyV with an EC₅₀ of 3.9 μM. This molecule inhibits JCPyV infection at dosages that are not toxic to human tissue culture cells. Retro-2.1 was also tested against two other polyomaviruses, the human BK polyomavirus and simian virus 40, and was also shown to inhibit infection at similar concentrations. Viral uncoating studies demonstrate that Retro-2.1 inhibits BKPyV infectivity in a manner similar to Retro-2. These studies demonstrate that improved analogs of Retro-2 can inhibit infection at lower dosages than Retro-2 and further optimization of these compounds may lead to effective treatment options for those suffering from JCPyV infection and PML.     

Model based dynamics analysis in live cell microtubule images

Background

The dynamic growing and shortening behaviors of microtubules are central to the fundamental roles played by microtubules in essentially all eukaryotic cells. Traditionally, microtubule behavior is quantified by manually tracking individual microtubules in time-lapse images under various experimental conditions. Manual analysis is laborious, approximate, and often offers limited analytical capability in extracting potentially valuable information from the data.

Results

In this work, we present computer vision and machine-learning based methods for extracting novel dynamics information from time-lapse images. Using actual microtubule data, we estimate statistical models of microtubule behavior that are highly effective in identifying common and distinct characteristics of microtubule dynamic behavior.

Conclusion

Computational methods provide powerful analytical capabilities in addition to traditional analysis methods for studying microtubule dynamic behavior. Novel capabilities, such as building and querying microtubule image databases, are introduced to quantify and analyze microtubule dynamic behavior.

     

Immunophenotyping of human HT29 colon cancer cell primary tumours and their metastases in severe combined immunodeficient mice

The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment of metastases. This revised version was published online in November 2006 with corrections to the Cover Date.     

A likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome

A model of DNA sequence evolution applicable to coding regions is presented. This represents the first evolutionary model that accounts for dependencies among nucleotides within a codon. The model uses the codon, as opposed to the nucleotide, as the unit of evolution, and is parameterized in terms of synonymous and nonsynonymous nucleotide substitution rates. One of the model's advantages over those used in methods for estimating synonymous and nonsynonymous substitution rates is that it completely corrects for multiple hits at a codon, rather than taking a parsimony approach and considering only pathways of minimum change between homologous codons. Likelihood-ratio versions of the relative-rate test are constructed and applied to data from the complete chloroplast DNA sequences of Oryza sativa, Nicotiana tabacum, and Marchantia polymorpha. Results of these tests confirm previous findings that substitution rates in the chloroplast genome are subject to both lineage-specific and locus-specific effects. Additionally, the new tests suggest tha the rate heterogeneity is due primarily to differences in nonsynonymous substitution rates. Simulations help confirm previous suggestions that silent sites are saturated, leaving no evidence of heterogeneity in synonymous substitution rates.