Vitamin D receptor gene polymorphisms and prostate cancer.

Prostate cancer (PC) is the most common cancer among men worldwide and its pathogenesis is complex. The development of PC depends on family and environmental factors. Vitamin D can be associated with both of these factors. Its reduced serum concentration has been reported in a number of tumors. However, in the case of PC, the study results are conflicting. Polymorphism of VDR gene may also be involved in the development of this cancer. The aim of the study was to compare the frequency of selected polymorphisms in patients with PC and in men without this disease. Seventy-two Caucasian males aged 35-75 years with histologically proven PC (T1/T2) were enrolled in the study group. Seventy-two random age-matched Caucasian out-patient subjects formed the control group. VDR (FokI, BsmI and TaqI) gene polymorphism (rs2228570, rs1544410, rs731236) was determined by TaqMan® SNP Genotyping. The Hardy-Weinberg Equilibrium (HWE) - p> 0.05 was in all studied polymorphisms. Deviations from the HWE were not found. There were no differences between the study group and the control group. No difference was found when the groups were compared in terms of age or the Gleason score.

Research on anisotropy of fusion-produced protons and neutrons emission from high-current plasma-focus discharges.

The paper concerns fast protons and neutrons from D-D fusion reactions in a Plasma-Focus-1000U facility. Measurements were performed with nuclear-track detectors arranged in "sandwiches" of an Al-foil and two PM-355 detectors separated by a polyethylene-plate. The Al-foil eliminated all primary deuterons, but was penetrable for fast fusion protons. The foil and first PM-355 detector were penetrable for fast neutrons, which were converted into recoil-protons in the polyethylene and recorded in the second PM-355 detector. The "sandwiches" were irradiated by discharges of comparable neutron-yields. Analyses of etched tracks and computer simulations of the fusion-products behavior in the detectors were performed.

Application of quantum dots in clinical and alimentary fields using multicommutated flow injection analysis.

In recent years, the number of scientific papers regarding the use of quantum dots (QDs) has increased almost exponentially, especially emphasizing their use for new applications and describing new approaches. One of the future trends in the development of new methods of analysis is the use of automated methodologies. Among them, Multicommutated Flow Injection Analysis has been here selected in order to show its potentiality in pharmaceutical and food analysis. Using water-soluble CdTe QDs modified by mercaptopropionic acid, a flow system was developed for the determination of ascorbic acid. The system was based on the quenching effect produced by ascorbic acid on the fluorescence of QDs. Under the optimized conditions, the relationship between the fluorescence intensity of the QDs and ascorbic acid concentration was linear in the range of 12-250 µg mL(-1), obtaining a sample throughput of 68 determinations per hour. The proposed method was applied to the determination of ascorbic acid in pharmaceutical formulations, goji capsules and fruit juices. The results obtained were in good agreement with those showed by a reference method, so indicating the utility of the proposed method in the clinical and alimentary fields.

A randomized double-blind crossover study of indoor air filtration and acute changes in cardiorespiratory health in a First Nations community.

UNLABELLED: Few studies have examined indoor air quality in First Nations communities and its impact on cardiorespiratory health. To address this need, we conducted a crossover study on a First Nations reserve in Manitoba, Canada, including 37 residents in 20 homes. Each home received an electrostatic air filter and a placebo filter for 1 week in random order, and lung function, blood pressure, and endothelial function measures were collected at the beginning and end of each week. Indoor air pollutants were monitored throughout the study period. Indoor PM2.5 decreased substantially during air filter weeks relative to placebo (mean difference: 37 µg/m(3) , 95% CI: 10, 64) but remained approximately five times greater than outdoor concentrations owing to a high prevalence of indoor smoking. On average, air filter use was associated with a 217-ml (95% CI: 23, 410) increase in forced expiratory volume in 1 s, a 7.9-mm Hg (95% CI: -17, 0.82) decrease in systolic blood pressure, and a 4.5-mm Hg (95% CI: -11, 2.4) decrease in diastolic blood pressure. Consistent inverse associations were also observed between indoor PM2.5 and lung function. In general, our findings suggest that reducing indoor PM2.5 may contribute to improved lung function in First Nations communities. PRACTICAL IMPLICATIONS: Indoor air quality is known to contribute to adverse cardiorespiratory health, but few studies have examined indoor air quality in First Nations communities. Our findings suggest that indoor PM2.5 may contribute to reduced lung function and that portable air filters may help to alleviate these effects by effectively reducing indoor levels of particulate matter.

Study of Toll-like receptor gene loci in sarcoidosis.

Sarcoidosis is a multi-factorial systemic disease of granulomatous inflammation. Current concepts of the aetiology include interactions of unknown environmental triggers with an inherited susceptibility. Toll-like receptors (TLRs) are main components of innate immunity and therefore TLR genes are candidate susceptibility genes in sarcoidosis. Ten members of the human TLR gene family have been identified and mapped to seven chromosomal segments. The aim of this study was to investigate all known TLR gene loci for genetic linkage with sarcoidosis and to follow positive signals with different methods. We analysed linkage of TLR gene loci to sarcoidosis by use of closely flanking microsatellite markers in 83 families with 180 affected siblings. We found significant linkage between sarcoidosis and markers of the TLR4 gene locus on chromosome 9q (non-parametric linkage score 2.63, P = 0.0043). No linkage was found for the remaining TLR gene loci. We subsequently genotyped 1203 sarcoidosis patients from 997 families, 1084 relatives and 537 control subjects for four single nucleotide polymorphisms of TLR4, including Asp299Gly and Thr399Ile. This genotype data set was studied by case-control comparisons and transmission disequilibrium tests, but showed no significant results. In summary, TLR4 - w ith significant genetic linkage results - appears to be the most promising member of the TLR gene family for further investigation in sarcoidosis. However, our results do not confirm the TLR4 polymorphisms Asp299Gly and Thr399Ile as susceptibility markers. Our results rather point to another as yet unidentified variant within or close to TLR4 that might confer susceptibility to sarcoidosis.

Quantitative analysis of HBV cccDNA from clinical specimens: correlation with clinical and virological response during antiviral therapy.

Attempts to investigate changes in various forms of intrahepatic hepatitis B virus (HBV) DNA during antiviral therapy have been hampered by limitations in technologies and scarcity of adequate tissue for analysis. We used a sensitive, specific assay to detect and quantitate covalently closed circular DNA (cccDNA) from total intrahepatic HBV DNA in clinical liver specimens. Total HBV DNA and cccDNA from 21 needle-biopsy specimens were quantified, with levels ranging from 0.1 to 9.8 copies/cell and 0.3 to 491.0 copies/cell, respectively. Then, we performed the same determinations on baseline and week-52 liver needle-biopsy specimens from eight patients enrolled in a clinical trial and evaluated the association between intrahepatic HBV DNA levels and serological and virological endpoints. In most patients, levels of intrahepatic HBV DNA, including cccDNA, decreased over the 52-week study, regardless of therapy or serological outcome. Higher ratios of cccDNA to total HBV DNA were detected at week 52 than at baseline indicating a shift in predominance of nonreplicating virus in posttreatment specimens. In patients who achieved treatment-related or spontaneous hepatitis B e antigen (HBeAg) responses, including those harbouring tyrosine-methionine-aspartate-aspartate-mutant HBV, levels of intrahepatic and serum HBV DNA suppression were greater than those in patients without HBeAg responses. In conclusion, this pilot study of intrahepatic HBV replicative forms in patients with chronic hepatitis B indicated that total intrahepatic and, specifically, cccDNA levels are not static but change as a reflection of serological and virological events.

Age-dependent prevalence of vascular disease-associated polymorphisms among 2689 volunteer blood donors.

BACKGROUND: The development of vascular disease involves the interaction of genetic and environmental factors. Because vascular disease is a major contributor to mortality in Western societies, we hypothesized that deleterious polymorphisms associated with hemostasis decrease in frequency among a healthy population as a function of age. METHODS: The frequencies of factor V G1691A Leiden (FVL), factor II (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, glycoprotein Ia (GPIa) C807T, glycoprotein IIIa (Pl(A1)/Pl(A2)) T1565C, and angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) alleles were determined among 2689 healthy Caucasian whole-blood donors. For analysis, participants were divided into three age groups: 17-39 years (n = 979; 505 males and 474 females), 40-59 years (n = 900; 526 males and 374 females), and 60-85 years (n = 810; 530 males and 280 females). RESULTS: The Pl(A2) allele frequency decreased from 17.5% to 15.7% and 14.1% in the 17-39 years, 40-59 years, and 60-85 years age groups, respectively (n = 5094 alleles; P = 0.025). Among ACE DD males, the Pl(A2) allele frequency decreased from 20.8% to 16.1% and 9.1% in the same groups, respectively (n = 810 alleles; P = 0.001). No statistically significant decrease in genotype or allele frequency was observed among carriers of FVL, FII 20210A, MTHFR 677T, GPIa 807T, or ACE D. CONCLUSIONS: These data suggest that Pl(A2) carriers, especially those who are ACE DD, are statistically less prevalent among older healthy blood donors compared with their younger counterparts. These observations suggest an important, deleterious, time-dependent impact of the Pl(A2) allele, as well as the ACE DD/Pl(A2) allelic combination, on overall health and longevity.

An invasive cleavage assay for direct quantitation of specific RNAs.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10(7) reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

Large-scale SNP scoring from unamplified genomic DNA.

Discoveries from the Human Genome Project (HGP) continue to spur changes in medical technology that will lead to new diagnostic procedures in the clinical lab. As more single nucleotide polymorphisms (SNPs) are discovered and correlated to human diseases, demands for genetic tests will increase. The enormity of the number of SNPs makes developing inexpensive and reliable high-throughput methods for SNP scoring imperative. High-throughput screening (HTS) means, at a minimum, a production rate of thousands of assays per day. Ideally, the technology will be easy, inexpensive and amenable to automation. The Invader assay offers a simple diagnostic platform to detect single nucleotide changes with high specificity and sensitivity from unamplified, genomic DNA. The Invader assay uses a structure-specific 5' nuclease (or flap endonuclease) to cleave sequence-specific structures in each of two cascading reactions. The cleavage structure forms when two synthetic oligonucleotide probes hybridise in tandem to a target. One of the probes cycles on and off the target and is cut by the nuclease only when the appropriate structure forms. These cleaved probes then participate in a second Invader reaction involving a dye-labelled fluorescence resonance energy transfer (FRET) probe. Cleavage of this FRET probe generates a signal, which can be readily analysed by fluorescence microtitre plate readers. The two cascading reactions amplify the signal significantly; each original target molecule can lead to more than 10(6) cleaved signal probes in one hour. This signal amplification permits identification of single base changes directly from genomic DNA without prior target amplification. The sequences of the oligonucleotide components of the secondary reaction are independent of the target of interest and permit the development of universal secondary reaction components useful to identify any target.

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

Clinical, genetic, and pharmacogenetic applications of the Invader assay.

The Invader technology has been developed for the detection of nucleic acids. It is a signal amplification system able to accurately quantify DNA and RNA targets with high sensitivity. Exquisite specificity is achieved by combining hybridization with enzyme recognition, which provides the ability to discriminate mutant from wild-type at ratios greater than 1/1000 (mutant/wt). The technology is isothermal and flexible and incorporates a homogeneous fluorescence readout. It is therefore readily adaptable for use in clinical reference laboratories, as well as high-throughput applications using 96-, 384-, and 1,536-well microtiter plate formats. The molecular mechanism of the system and specific applications for use in clinical and research laboratories are described. These include direct analysis of unamplified human genomic DNA to detect mutations and single-nucleotide polymorphisms associated with factor V Leiden, factor II, cystic fibrosis, and apolipoprotein E, and gene expression assays that quantify messenger RNA levels in cells using direct lysates.

Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression.

The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nif regulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield with the wild-type strain RMBPC and 17.9% over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that RMBPC -2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.

A closed loop automated seating system.

This technical report presents the recent progress in the design and construction of a closed loop automated seating system. Preliminary test results are reported. The system is designed to measure seating surface forces and control the seating surface geometry of a seated human. It uses force information as feedback to determine custom seating contours which produce desired seating characteristics. Ultimately, the system is intended to be used for research studies with patients. The system consists of an electronically actuated, force-sensing seating surface which is controlled by a computer. Stepper motors are used to move force sensing probes up or down until the desired seating surface characteristic is attained. Preliminary test results are presented and analyzed. A force-equalizing control algorithm has been written and found to produce relatively uniform force distributions for soft, hemispherical loads of various weights.

Statistical needs in national water quality monitoring programs.

The concept that a few well chosen, strategically placed, water quality stations can provide valuable scientific information to water managers is common to many countries. Historically within Canada, water quality regional networks (Great Lakes network, Prairie Provinces Water Board network, Long Range Transport of Airborne Pollutants network, etc.) have been successfully operating for many years. This paper will describe the difficulties associated with developing a national water quality network for a country the size of Canada. In particular, it will describe some of the statistical tools presently being used in regional networks which are suitable for a national network, and discuss the need to develop new statistical tools for environmental monitoring in the 1990's.

Accumulation of creatine kinase mRNA during myogenesis: molecular cloning of a B-creatine kinase cDNA.

Cytosolic creatine kinase isoenzymes MM, MB, and BB are assembled from M or B subunits which occur in different relative amounts in specific tissues. The accumulation of mRNAs encoding the M and B subunits was measured during myogenesis in culture. The relative concentration of the two mRNAs was determined by hybridization with a M-CK cDNA probe isolated previously and a B-CK cDNA probe, the cloning and characterization of which is reported here. The B-CK cDNA hybridizes specifically to a 1.6-kb mRNA found in brain and gizzard but not in adult skeletal muscle tissue. The M-CK cDNA hybridizes to a smaller mRNA 1.4-kb long which is specific to skeletal muscle. In culture, the B-CK mRNA is transiently induced and then declines to a low but detectable level.

Molecular cloning and the complete nucleotide sequence of the creatine kinase-M cDNA from chicken.

The nucleotide sequence of creatine kinase-M (CK-M) cDNA clones has been determined. It includes the entire coding region of 381 amino acids in addition to 5' and 3' untranslated regions. A comparison with a partial sequence from rat CK-M reveals 84% nucleotide sequence homology in the coding region but divergence in the 3' untranslated region. The amino acid sequence is 94% conserved between chicken and rat. Hybridization to RNA immobilized on filters indicates homology between the CK-M 3' untranslated region and additional muscle specific RNA species. The coding region hybridizes only to CK-M RNA.

Molecular cloning of a DNA sequence complementary to creatine kinase M mRNA from chickens.

We have cloned and identified a DNA sequence complementary to the mRNA of creatine kinase (CK) isozyme M, although the mRNA is a minor species of the total mRNA in developing myoblasts. Poly(A)+RNA from breast and thigh muscle of 5-week-old chicks was enriched for CK mRNA by a novel procedure of sucrose gradient centrifugation in the presence of methylmercuric hydroxide. DNA complementary to this mRNA was inserted into pBR322, and colonies containing the recombinant plasmids were screened for the ability of the plasmid DNA to hybridize with and rescue CK mRNA from total muscle mRNA. Three plasmids, pCS195, pCS192, and pM35-4, could specifically rescue CK-M mRNA. CK-M mRNA was detected by in vitro translation and specific immunoprecipitation. The identity of the in vitro translation product was further confirmed by its migration in two-dimensional gels at the isoelectric point and molecular weight of CK-M. The heterogeneity of CK-M observed in vivo also was found upon translation of the CK-M mRNA which hybridizes to the plasmid.