MethylScreen: DNA methylation density monitoring using quantitative PCR

Aberrant gene silencing of genes through cytosine methylation has been demonstrated during the development of many types of cancers including prostate cancer Several genes including GSTP1 have been shown to be methylated in prostate cancer leading to the suggestion and demonstration that methylation status of such genes could be used as cancer diagnosis markers alone or in support of histology. We developed a bisulfite-free alternative, MethylScreen technology, an assay for DNA methylation detection utilizing combined restriction from both methylation-sensitive restriction enzymes (MSRE) and methylation-dependent restriction enzymes (MDRE). MethylScreen was used to analyze the 5' region of GSTP1 in cell lines, in vitro methylated DNA populations, and flash-frozen tissue samples in an effort to characterize the output and analytical performance characteristics of the assay. The output from the quantitative PCR assay suggested that it could not only detect fully methylated molecules in a mixed population below the 1% level, but it could also quantify the abundance of intermediately methylated molecules. Interestingly, the interpreted output from the four quantitative PCRs closely resembled the molecular population as described by clone-based bisulfite genomic sequencing.     

Fractional clearance of urate: validation of measurement in spot-urine samples in healthy subjects and gouty patients

Introduction

Hyperuricemia is the greatest risk factor for gout and is caused by an overproduction and/or inefficient renal clearance of urate. The fractional renal clearance of urate (FCU, renal clearance of urate/renal clearance of creatinine) has been proposed as a tool to identify subjects who manifest inefficient clearance of urate. The aim of the present studies was to validate the measurement of FCU by using spot-urine samples as a reliable indicator of the efficiency of the kidney to remove urate and to explore its distribution in healthy subjects and gouty patients.

Methods

Timed (spot, 2-hour, 4-hour, 6-hour, 12-hour, and 24-hour) urine collections were used to derive FCU in 12 healthy subjects. FCUs from spot-urine samples were then determined in 13 healthy subjects twice a day, repeated on 3 nonconsecutive days. The effect of allopurinol, probenecid, and the combination on FCU was explored in 11 healthy subjects. FCU was determined in 36 patients with gout being treated with allopurinol. The distribution of FCU was examined in 118 healthy subjects and compared with that from the 36 patients with gout.

Results

No substantive or statistically significant differences were observed between the FCUs derived from spot and 24-hour urine collections. Coefficients of variation (CVs) were both 28%. No significant variation in the spot FCU was obtained either within or between days, with mean intrasubject CV of 16.4%. FCU increased with probenecid (P < 0.05), whereas allopurinol did not change the FCU in healthy or gouty subjects. FCUs of patients with gout were lower than the FCUs of healthy subjects (4.8% versus 6.9%; P < 0.0001).

Conclusions

The present studies indicate that the spot-FCU is a convenient, valid, and reliable indicator of the efficiency of the kidney in removing urate from the blood and thus from tissues. Spot-FCU determinations may provide useful correlates in studies investigating molecular mechanisms underpinning the observed range of efficiencies of the kidneys in clearing urate from the blood.

Trial Registration

ACTRN12611000743965     

Combining Affymetrix microarray results

Background  

As the use of microarray technology becomes more prevalent it is not unusual to find several laboratories employing the same microarray technology to identify genes related to the same condition in the same species. Although the experimental specifics are similar, typically a different list of statistically significant genes result from each data analysis.     

Phylogenetic utility of elongation factor-1 alpha in noctuoidea (Insecta: Lepidoptera): the limits of synonymous substitution

To test its phylogenetic utility, nucleotide sequence variation in a 1,240-bp fragment of the elongation factor-1 alpha (EF-1 alpha) gene was examined in 49 moth species representing the major groups of the superfamily Noctuoidea. Both parsimony and distance analyses supported the monophyly of nearly all groups for which there are clear morphological synapomorphies. Clades of subfamily rank and lower, probably mid-Tertiary and younger, were strongly supported. The third codon position contains 88% of variable sites, and approaches saturation at approximately 20% sequence divergence, possibly due to among-site rate heterogeneity and composition bias; higher divergences occur only in association with shifts in composition. Surprisingly, the few nonsynonymous changes appear no more phylogenetically reliable than synonymous changes. Signal strength for basal divergences is weak and fails to improve with character weighting; thus, dense taxon sampling is probably needed for strong inference from EF-1 alpha regarding deeper splits in Noctuoidea (probably early Tertiary). EF-1 alpha synonymous changes show promise for phylogeny reconstruction within Noctuidae and other groups of Tertiary age.      

Human responses to propionic acid. I. Quantification of within- and between-participant variation in perception by normosmics and anosmics

The objective of this study was to fully characterize normosmic perception of stimuli expected to cause widely varying degrees of olfactory and nasal trigeminal stimulation and to directly evaluate the possible role of olfactory nerve stimulation in nasal irritation sensitivity. During each of four identical test sessions, four anosmic and 31 normosmic participants were presented with a range of concentrations extending from peri-threshold for normosmics to supra- threshold for anosmics. For each session, odor (O) and nasal irritation (NI) sensitivities were summarized in terms of the concentrations required to produce four sensation levels ('iso-response' concentrations). Within-participant variation in these iso-response concentrations was < 10-fold for 95% of normosmics, for both O and NI. For O but not NI, these apparent fluctuations in sensitivity were largely accounted for by the uncertainty surrounding the iso-response concentrations calculated for each session. Anosmics exhibited minimal within- and between-participant variation in NI and required, for all but the highest perceptual level, a higher concentration than almost all normosmics. Between-participant variation, expressed in terms of 90% confidence interval widths, was approximately 0.5 log units for both O and NI for the highest perceptual level, but increased to approximately 0.8 and 1.8 log units, respectively, for the lowest (peri- threshold) level. Our findings suggest that: (i) most apparent variation over time in O sensitivity is actually a reflection of the uncertainty surrounding estimates of sensitivity obtained for each session; (ii) within- and between-participant variation in O sensitivity is far less than is commonly reported; and (iii) low to moderate levels of NI in normosmics are the result of relatively weak trigeminal stimulation combined with much greater olfactory activation.      

Monoclonal antibody 91.9H raised against sulfated mucins is specific for the 3'-sulfated Lewisa tetrasaccharide sequence

The IgG1hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewisa(Lea) and Lextypes, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri- and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3'-sulfated Leatetrasaccharide sequence, which is a potent ligand for the E- and L-selectins. The antibody gives a relatively low signal with the 3'-sulfated non-fucosylated backbone, and has no detectable cross- reaction with the 3'-sulfated Lexisomer, nor with sialyl-Leaand - Lexanalogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.      

Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval

Over the past 13 years, glyoxal has become the leading alternative to formaldehyde as a histological fixative because of its low inhalation risk, faster reaction rate and selective control over crosslinking. The latter attribute is especially important, because most of the difficulties relating to use of formaldehyde-fixed specimens for immunohistochemistry stem from its aggressive crosslinking behavior. With suitable catalysts or other reaction accelerators, glyoxal forms 2-carbon adducts with nearly all end groups in proteins and carbohydrates, leaving most of them unimpaired for subsequent immunohistochemical demonstration. Only arginine is seriously impaired by the formation of imidazoles, which is the basis for the well known arginine blockade method using glyoxal. A special glyoxal-specific antigen retrieval method using high pH and high temperature effectively reverses the blockade and restores immunoreactivity. Other methods for antigen retrieval are rarely beneficial and in most cases damage the specimen. Special stains work well, except silver methods for Helicobacter pylori. Routine hematoxylin and eosin preparations exhibit clarity and cellular detail rarely seen with formaldehyde.     

Consumer evaluation of meat quality from barrows,immunocastrates and boars in six countries

The practice of surgical castration of piglets and its alternatives is still under debate. Production of boars may impair meat quality due to boar taint and reduced tenderness compared to meat from surgically castrated male pigs, while immunocastration reduces boar taint and may improve meat quality but seems to be less accepted by the pig chain. In this study, we aimed to evaluate the consumer’s sensory appreciation of meat from barrows (BAs), immunocastrates (ICs) and boars (BOs) in six European countries, taking into account the selection of tainted carcass and consumers’ appreciation of boar taint. Loin chops of 30 BAs, 30 ICs and 30 BOs were evaluated by 752 consumers in six countries: Belgium, Czech Republic, Poland, Portugal, Romania and Spain. Consumers rated odour, flavour, tenderness, juiciness, overall liking and willingness to buy and sensitivity to and liking of androstenone (AND) and liking of skatole (SKA) was also tested. In each of the six countries, consumers liked the odour of the BO samples less than that of BA, and IC intermediate. For flavour, tenderness, juiciness, overall liking and willingness to buy, liking scores given by the Czech, Polish and Portuguese consumers significantly differed between the BA, BO and IC. Willingness to buy was highest for BA by Czech and Polish consumers and for BA and IC by Portuguese consumers. The frequency of the negative check all terms that apply terms also differed, with a higher frequency of disgusting for BO compared to BA and IC and of off-flavour, irritating, manure, sweat, disappointing compared to BA, and intermediate for IC. 31% of the consumers disliked the odour of AND (NEG_AND), and 36% of them were not sensitive; in contrast, 77% of the consumers disliked SKA (NEG_SKA). The decrease in flavour liking score for BO compared to BA and IC was more outspoken by the NEG_AND consumer, while NEG_SKA consumers gave an overall lower liking score independent of the type of male pig. The results of this study indicate that IC can be a valid alternative for surgical castration.