Single nucleotide polymorphism-based validation of exonic splicing enhancers

Because deleterious alleles arising from mutation are filtered by natural selection, mutations that create such alleles will be underrepresented in the set of common genetic variation existing in a population at any given time. Here, we describe an approach based on this idea called VERIFY (variant elimination reinforces functionality), which can be used to assess the extent of natural selection acting on an oligonucleotide motif or set of motifs predicted to have biological activity. As an application of this approach, we analyzed a set of 238 hexanucleotides previously predicted to have exonic splicing enhancer (ESE) activity in human exons using the relative enhancer and silencer classification by unanimous enrichment (RESCUE)-ESE method. Aligning the single nucleotide polymorphisms (SNPs) from the public human SNP database to the chimpanzee genome allowed inference of the direction of the mutations that created present-day SNPs. Analyzing the set of SNPs that overlap RESCUE-ESE hexamers, we conclude that nearly one-fifth of the mutations that disrupt predicted ESEs have been eliminated by natural selection (odds ratio = 0.82 +/- 0.05). This selection is strongest for the predicted ESEs that are located near splice sites. Our results demonstrate a novel approach for quantifying the extent of natural selection acting on candidate functional motifs and also suggest certain features of mutations/SNPs, such as proximity to the splice site and disruption or alteration of predicted ESEs, that should be useful in identifying variants that might cause a biological phenotype.     

The Effect of Speed of Processing Training on Microsaccade Amplitude

Older adults experience cognitive deficits that can lead to driving errors and a loss of mobility. Fortunately, some of these deficits can be ameliorated with targeted interventions which improve the speed and accuracy of simultaneous attention to a central and a peripheral stimulus called Speed of Processing training. To date, the mechanisms behind this effective training are unknown. We hypothesized that one potential mechanism underlying this training is a change in distribution of eye movements of different amplitudes. Microsaccades are small amplitude eye movements made when fixating on a stimulus, and are thought to counteract the “visual fading” that occurs when static stimuli are presented. Due to retinal anatomy, larger microsaccadic eye movements are needed to move a peripheral stimulus between receptive fields and counteract visual fading. Alternatively, larger microsaccades may decrease performance due to neural suppression. Because larger microsaccades could aid or hinder peripheral vision, we examine the distribution of microsaccades during stimulus presentation. Our results indicate that there is no statistically significant change in the proportion of large amplitude microsaccades during a Useful Field of View-like task after training in a small sample of older adults. Speed of Processing training does not appear to result in changes in microsaccade amplitude, suggesting that the mechanism underlying Speed of Processing training is unlikely to rely on microsaccades.     

Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl(2). Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg(2+), there being at least a 12-fold difference between the activity in the presence of Mg(2+) and of EDTA. There is, however, a difference in the response of the enzymes to Mg(2+) and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl(2) and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl(2) for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.     

Comparative recoveries of airborne fungus spores by viable and non-viable modes of volumetric collection

Summary The suitability of viable and non-viable volumetric collectors as prevalence indicators for potentially allergenic airborne fungi was studied during 124 paired exposures of the Burkard (Hirst) spore trap and a modified, wind-oriented Andersen sampler. Overall, viable recoveries of several Cladosporium form species varied directly with microscopic spore counts (p0.0001). However, as spore levels rose, culture plate data progressively underestimated prevailing concentrations (recoveries falling below 5% at levels above 500 spores/M³). Viable collections yielded low estimates of prevalence (20–40%) even at modest Cladosporium levels (< 100 spores/M³) and substantially understated the abundance and regularity in air of several additional taxa. Spores typical of Penicillium and Aspergillus form species were not sought in spore trap deposits. Careful examination of these failed to reveal typical arthrospores or Fusarium macrospores despite substantial recoveries of corresponding growth in culture. Correlations in the occurrence patterns of arthrospore-forming and non-sporulating colonies with those of Coprinus and other basidiospores (excluding Ganoderma) were noted.This work was supported by Research Grant AI-10181 from the National Institute of Allergy & Infectious Diseases, National Institutes of Health, United States Public Health Service.     

Inactivation by Ionizing Radiation of Salmonella enteritidis Serotype montevideo Grown in Composed Sewage Sludge

S. enteritidis ser. montevideo were grown in composted sewage sludge to levels of approximately 10⁹/g. These bacteria were found to be inactivated by ionizing radiation at approximately the same rate (30 krads/log) as Salmonella species in liquid digested sludge.     

Fungi in libraries: An aerometric survey

The possible role of fungi as allergic contaminants in book collections has been investigated in eleven University of Michigan Libraries. Air in the stacks of each of the eleven libraries was sampled on three occasions (2 or 4–10 minute samples on each occasion) with Andersen Volumetric viable particle samplers. Books were handled during sampling in half the samples each day. In addition on each sampling day a location in the same building away from book storage and an outdoor location were sampled. Library spore levels were generally low. Outdoor levels consistently exceeded indoor levels. Air conditioned (AC) libraries had lower spore levels and indoor/outdoor ratios than conventionally ventilated (CV) libraries. Handling books during sampling increased spore counts in all libraries, but strikingly in CV libraries. Fungus taxa recovered were similar to those encountered in domestic interiors and outside locations in our area. The overall low spore levels and lack of a distinctive library mycoflora suggest that other sources should be sought for librarybased respiratory symptoms.The occurrence of rhinitis or asthma provoked by exposure to libraries and other book collections is well documented (1). While variously ascribed to house dust, arthropods and bindery glues, this symptom pattern generally defies explanation. In this climate of uncertainty, the possible role of fungi as allergenic contaminants of book collections arises naturally for consideration. Among enclosed spaces libraries offer unique substrate characteristics while generally excluding factors (e.g., animal danders and food fragments) present in domestic dusts. Faced with patients describing respiratory symptoms with library use, we have initiated study of the mycoflora of these specialized interiors.Supported by Research Grant A1-10181 from the National Institutes, of Health, USPHS.     

Salmonella regrowth in compost as influenced by substrate (salmonella regrowth in compost)

Composting can eliminate pathogenic organisms, including salmonellae, from sewage sludge. However, if salmonellae are present in the compost at undetectable levels or are inoculated into the compost by infected animals or from other sources, they may regrow presenting a health hazard for certain uses of compost. In this study, we examined dilute mineral-salt extracts of three composts from widely separate composting sites in the United States and found that they supported growth ofSalmonella typhimurium. From kinetic studies of the growth of the organism on these extracts, we concluded that each compost produced on extraction a single water-soluble substrate and that the substrates from the different composts were very similar, if not identical.We thank J. Robert Burge, Statistical Consulting and Analysis, ARS, USDA for invaluable help with the statistical analyses, and Alice V. Gibson of our laboratory for technical help.     

Autophagy,Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence: supporting the next generation of autophagy researchers and fostering international collaborations

Recently, NIH has funded a center for autophagy research named the Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence, located at the University of New Mexico Health Science Center (UNM HSC), with aspirations to promote autophagy research locally, nationally, and internationally. The center has 3 major missions: (i) to support junior faculty in their endeavors to develop investigations in this area and obtain independent funding; (ii) to develop and provide technological platforms to advance autophagy research with emphasis on cellular approaches for high quality reproducible research; and (iii) to foster international collaborations through the formation of an International Council of Affiliate Members and through hosting national and international workshops and symposia. Scientifically, the AIM center is focused on autophagy and its intersections with other processes, with emphasis on both fundamental discoveries and applied translational research.     

A Quantitative Estimation of the Uptake of Two New Electron Stains by the Cytoplasmic Membrane of Ram Sperm

From microdensitometer measurements on electron micrographs of sectioned sperm heads it has been found that the electron stains, triiodobenzoyl chloride, and triiodophenylisocyanate, increase the image contrast of the cell membrane above its immediate background by about 40 per cent and 70 per cent respectively, while the nucleus remains unstained. Assumptions based on current electron scattering theory have been used to deduce the uptake by weight of the stains in terms of the density of the nucleus, which was estimated from complementary measurements made with the interference microscope and electron microscope. The uptake of the stains was found to be about 7 per cent and 12 per cent by weight respectively. It is suggested that the method used in this work could be applied generally for the density measurement of cell structures unresolved by the light microscope.     

Quantification of ergosterol and 3-hydroxy fatty acids in settled house dust by gas chromatography-mass spectrometry: comparison with fungal culture and determination of endotoxin by a Limulus amebocyte lysate assay.

Ergosterol and 3-hydroxy fatty acids, chemical markers for fungal biomass and the endotoxin of gram-negative bacteria, respectively, may be useful in studies of health effects of organic dusts, including domestic house dust. This paper reports a method for the combined determination of ergosterol and 3-hydroxy fatty acids in a single dust sample and a comparison of these chemical biomarkers determined by gas chromatography-mass spectrometry with results from fungal culture and Limulus assay. Analyses of replicate house dust samples resulted in correlations of 0.91 (ergosterol in six replicates; P < 0.01) and 0.94 (3-hydroxy fatty acids in nine replicates; P < 0.001). The amounts of ergosterol (range, 2 to 16.5 ng/mg of dust) correlated with those of total culturable fungi (range, 6 to 1,400 CFU/mg of dust) in 17 samples, (r = 0.65; P < 0.005). The amounts of endotoxin (range, 11 to 243 endotoxin units/mg of dust) measured with a modified chromogenic Limulus assay correlated with those of lipopolysaccharide (LPS) determined from 3-hydroxy fatty acid analysis of 15 samples. The correlation coefficient depended on the chain lengths of 3-hydroxy acids used to compute the LPS content. The correlation was high (r = 0.88 +/- 0.01; P < 0.001) when fatty acid chains of 10 to 14 carbon atoms were included; the correlation was much lower when hydroxy acids of 16- or 18-carbon chains were included. In conclusion, the results of the described extraction and analysis procedure for ergosterol and 3-hydroxy fatty acids are reproducible, and the results can be correlated with fungal culture and endotoxin activity of organic dust samples.