Primer design and marker clustering for multiplex SNP-IT primer extension genotyping assay using statistical modeling

MOTIVATION: The optimization of the primer design is critical for the development of high-throughput SNP genotyping methods. Recently developed statistical models of the SNP-IT primer extension genotyping reaction allow further improvement of primer quality for the assay. RESULTS: Here we describe how the statistical models can be used to improve primer design for the assay. We also show how to optimize clustering of the SNP markers into multiplex panels using statistical model for multiplex SNP-IT. The primer set failure probability calculated by a model is used as a minimization function for both primer selection and primers clustering. Three clustering algorithms for the multiplex genotyping SNP-IT assay are described and their relative performance is evaluated. We also describe the approaches to improve the speed of primer design and clustering calculations when using the statistical models. Our clustering decreases the average failure probability of the marker set by 7-25%. The experimental marker failure rate in the multiplex reaction was reduced dramatically and success rate can be achieved as high as 96%. AVAILABILITY: The primer design using statistical models is freely available from www.autoprimer.com.     

Coexisting angiomyolipoma and renal cell carcinoma in a kidney of an elderly woman: case report and review of the literature

Angiomyolipoma is a well described but relatively uncommon benign renal neoplasm composed of varying admixtures of mature adipose tissue, smooth muscle, and thick-walled blood vessels. The incidence of angiomyolipoma is about 0.3% overall. It frequently occurs in patients with tuberous sclerosis. Even more uncommon is the simultaneous occurrence of angiomyolipoma and renal cell cancer in the same kidney in a patient without tuberous sclerosis.     

Screening protein refolding using surface plasmon resonance

Surface plasmon resonance (SPR) measurements were used to screen refolding conditions to identify a physicochemical environment which gives an acceptable refolding yield for samples of glutathione-S-transferase (GST) denatured in 6 M guanidine hydrochloride and 32 mM dithiothreitol. The SPR measurements were performed on carboxymethylcellulose coated chips that could accommodate two separate flow paths. One side of the chip was derivatized with immobilized glutathione and the other with goat anti-GST antibody. This created a dual-derivatized chip capable of showing both the presence of GST and providing a measure of enzyme activity. The dual-derivatized chip could be regenerated using a two-step washing procedure and reused to analyze multiple samples from a screening study of protein refolding conditions. SPR measurements have been shown to be suitable for screening protein refolding conditions due to the high sensitivity, ease of chip regeneration and the ability to incorporate a control in the experimental design. The combination of such advantages with the high-throughput automated SPR systems currently available may be a valuable approach to determine conditions suitable for protein refolding following insoluble expression in a bacterial host.     

Auto-validation of fluorescent primer extension genotyping assay using signal clustering and neural networks

Background

SNP genotyping typically incorporates a review step to ensure that the genotype calls for a particular SNP are correct. For high-throughput genotyping, such as that provided by the GenomeLab SNPstream^® instrument from Beckman Coulter, Inc., the manual review used for low-volume genotyping becomes a major bottleneck. The work reported here describes the application of a neural network to automate the review of results.

Results

We describe an approach to reviewing the quality of primer extension 2-color fluorescent reactions by clustering optical signals obtained from multiple samples and a single reaction set-up. The method evaluates the quality of the signal clusters from the genotyping results. We developed 64 scores to measure the geometry and position of the signal clusters. The expected signal distribution was represented by a distribution of a 64-component parametric vector obtained by training the two-layer neural network onto a set of 10,968 manually reviewed 2D plots containing the signal clusters.

Conclusion

The neural network approach described in this paper may be used with results from the GenomeLab SNPstream instrument for high-throughput SNP genotyping. The overall correlation with manual revision was 0.844. The approach can be applied to a quality review of results from other high-throughput fluorescent-based biochemical assays in a high-throughput mode.     

Paraoxonase genotype, LDL-oxidation and carotid atherosclerosis in male life-long smokers

Paraoxonase (PON-1) is a high-density lipoprotein (HDL) associated enzyme that hydrolyzes lipid peroxides in vitro, which may therefore protect against the onset of atherosclerosis. Heavy smokers are more exposed to oxidative stress and hence at high-risk for oxidative modification of LDL. Our hypothesis is that the anti-oxidative properties of PON-1 inhibit LDL oxidation, especially in populations exposed to high oxidative stress. We have studied the effects of PON-1 genotype and smoking to variation in oxidative status parameters and intima-media thickness (IMT), a surrogate marker of atherosclerosis. The contribution of two common polymorphisms in the PON-1 gene (Q192R and L55M) to LDL oxidizability, autoantibodies directed against oxLDL and IMT were studied in 207 male life-long smokers. Smokers were classified into average, heavy and excessive smokers based on pack years of cigarettes smoked. PON-1 genotype was not associated with autoantibodies to oxLDL, LDL oxidizability or IMT. Smoking was associated with IMT in subgroups with the high levels of LDL, but not in the population at large. The lack of association of PON-1 genotype with oxidative status parameters and IMT suggests that PON-1 is not a major inhibitor of LDL oxidation in a population of life-long smokers.     

NO mediates effects of estrogen on central regulation of blood pressure in restrained,ovariectomized rats

We tested the hypotheses that estrogen replacement in ovariectomized (OVX) rats attenuates cardiovascular responses to psychological stress and that nitric oxide (NO) in the brain mediates these effects. Female rats were OVX; one group received 17beta-estradiol (OVX-E) for 11-12 days and the other received vehicle (OVX-V). Seven days after OVX, OVX-E and OVX-V rats were chronically instrumented for arterial pressure measurements and intracerebroventricular injections. Later (4-5 days), OVX-E and OVX-V rats received intracerebroventricular injections of NG-nitro-l-arginine (88 microg/kg), an inhibitor of constitutive NO production, or vehicle. Mean arterial pressure (MAP) and heart rate responses were then measured in conscious rats exposed to two cycles of 1-h restraint/1-h rest. We show that MAP responses in restrained OVX-E rats were attenuated both during restraint and during rest. Although inhibition of NO production in the brain had no effect on MAP responses to restraint in OVX-V rats, it augmented responses in restrained OVX-E rats, especially during periods of rest, so that MAPs in restrained OVX-E and OVX-V rats were indistinguishable. Finally, NO levels in hypothalami and brain stems were elevated in restrained OVX-E, but not OVX-V, rats compared with their respective unrestrained controls. These results show that estrogen replacement in OVX rats reduces arterial pressure responses to psychological stress and that these effects are mediated, at least in part, by NO.     

Exploring the origin of the ion selectivity of the KcsA potassium channel

The availability of structural information about biological ion channels provides an opportunity to gain a detailed understanding of the control of ion selectivity by biological systems. However, accomplishing this task by computer simulation approaches is very challenging. First, although the activation barriers for ion transport can be evaluated by microscopic simulations, it is hard to obtain accurate results by such approaches. Second, the selectivity is related to the actual ion current and not directly to the individual activation barriers. Thus, it is essential to simulate the ion currents and this cannot be accomplished at present by microscopic MD approaches. In order to address this challenge, we developed and refined an approach capable of evaluating ion current while still reflecting the realistic features of the given channel. Our method involves generation of semimacroscopic free energy surfaces for the channel/ions system and Brownian dynamics (BD) simulations of the corresponding ion current. In contrast to most alternative macroscopic models, our approach is able to reproduce the difference between the free energy surfaces of different ions and thus to address the selectivity problem. Our method is used in a study of the selectivity of the KcsA channel toward the K+ and Na+ ions. The BD simulations with the calculated free energy profiles produce an appreciable selectivity. To the best of our knowledge, this is the first time that the trend in the selectivity in the ion current is produced by a computer simulation approach. Nevertheless, the calculated selectivity is still smaller than its experimental estimate. Recognizing that the calculated profiles are not perfect, we examine how changes in these profiles can account for the observed selectivity. It is found that the origin of the selectivity is more complex than generally assumed. The observed selectivity can be reproduced by increasing the barrier at the exit and the entrance of the selectivity filter, but the necessary changes in the barrier approach the limit of the error in the PDLD/S-LRA calculations. Other options that can increase the selectivity are also considered, including the difference between the Na+...Na+ and K+...K+ interaction. However, this interesting effect does not appear to lead to a major difference in selectivity since the Na+ ions at the limit of strong interaction tend to move in a less concerted way than the K+ ions. Changes in the relative binding energies at the different binding sites are also not so effective in changing the selectivity. Finally, it is pointed out that using the calculated profiles as a starting point and forcing the model to satisfy different experimentally based constraints, should eventually provide more detailed understanding of the different complex factors involved in ion selectivity of biological channels.     

CD36 deficiency increases insulin sensitivity in muscle,but induces insulin resistance in the liver in mice

CD36 (fatty acid translocase) is involved in high-affinity peripheral fatty acid uptake. Mice lacking CD36 exhibit increased plasma free fatty acid and triglyceride (TG) levels and decreased glucose levels. Studies in spontaneous hypertensive rats lacking functional CD36 link CD36 to the insulin-resistance syndrome. To clarify the relationship between CD36 and insulin sensitivity in more detail, we determined insulin-mediated whole-body and tissue-specific glucose uptake in CD36-deficient (CD36-/-) mice. Insulin-mediated whole-body and tissue-specific glucose uptake was measured by d-[3H]glucose and 2-deoxy-d-[1-3H]glucose during hyperinsulinemic clamp in CD36-/- and wild-type control littermates (CD36+/+) mice. Whole-body and muscle-specific insulin-mediated glucose uptake was significantly higher in CD36-/- compared with CD36+/+ mice. In contrast, insulin completely failed to suppress endogenous glucose production in CD36-/- mice compared with a 40% reduction in CD36+/+ mice. This insulin-resistant state of the liver was associated with increased hepatic TG content in CD36-/- mice compared with CD36+/+ mice (110.9 +/- 12.0 and 68.9 +/- 13.6 microg TG/mg protein, respectively). Moreover, hepatic activation of protein kinase B by insulin, measured by Western blot, was reduced by 54%. Our results show a dissociation between increased muscle and decreased liver insulin sensitivity in CD36-/- mice.     

UGT73C6 and UGT78D1, glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana

Flavonol glycosides constitute one of the most prominent plant natural product classes that accumulate in the model plant Arabidopsis thaliana. To date there are no reports of functionally characterized flavonoid glycosyltransferases in Arabidopsis, despite intensive research efforts aimed at both flavonoids and Arabidopsis. In this study, flavonol glycosyltransferases were considered in a functional genomics approach aimed at revealing genes involved in determining the flavonol-glycoside profile. Candidate glycosyltransferase-encoding genes were selected based on homology to other known flavonoid glycosyltransferases and two T-DNA knockout lines lacking flavonol-3-O-rhamnoside-7-O-rhamnosides (ugt78D1) and quercetin-3-O-rhamnoside-7-O-glucoside (ugt73C6 and ugt78D1) were identified. To confirm the in planta results, cDNAs encoding both UGT78D1 and UGT73C6 were expressed in vitro and analyzed for their qualitative substrate specificity. UGT78D1 catalyzed the transfer of rhamnose from UDP-rhamnose to the 3-OH position of quercetin and kaempferol, whereas UGT73C6 catalyzed the transfer of glucose from UDP-glucose to the 7-OH position of kaempferol-3-O-rhamnoside and quercetin-3-O-rhamnoside, respectively. The present results suggest that UGT78D1 and UGT73C6 should be classified as UDP-rhamnose:flavonol-3-Orhamnosyltransferase and UDP-glucose:flavonol-3-O-glycoside-7-O-glucosyltransferase, respectively.