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In this article, we describe the analysis of over 9000 expressed sequence tags (ESTs) from cDNA libraries obtained from various life cycle stages of Globodera pallida. We have identified over 50 G. pallida effectors from this dataset using bioinformatics analysis, by screening clones in order to identify secreted proteins up-regulated after the onset of parasitism and using in situ hybridization to confirm the expression in pharyngeal gland cells. A substantial gene family encoding G. pallida SPRYSEC proteins has been identified. The expression of these genes is restricted to the dorsal pharyngeal gland cell. Different members of the SPRYSEC family of proteins from G. pallida show different subcellular localization patterns in plants, with some localized to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localization may reflect diverse functional roles for each individual protein or, more likely, variety in the compartmentalization of plant proteins targeted by the nematode. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.

Purpose: Telemedicine technology may offer an avenue to implement diabetes self-management education (DSME) for people with diabetes in underserved rural communities. The continuous quality improvement process was used to identify the problem, collect and analyze data, and develop and implement a DSME program via telemedicine (DSME-T) in an underserved rural community., Methods: A pilot study was conducted in 2006, implementing a DSME-T utilizing facilities at the University of Arkansas for Medical Sciences and a rural community hospital in Arkansas (Ozark Health, Inc). A total of 38 people were enrolled to receive DSME-T. Participant knowledge, self-efficacy, and self-care practices were assessed before participants began the education program and after they had completed it. Also, select clinical measures (glycosylated hemoglobin, lipid profile, and urine microalbumin) were collected., Results: A total of 66% of participants (n = 25) completed the DSME-T program. A significantly greater proportion of participants demonstrated improved knowledge (39% vs 83%; P = .012), endorsed greater self-efficacy (54% vs 86%; P = .016), and reported more frequent self-care practices to manage their diabetes at the conclusion of the study period., Conclusions: The results of this pilot study suggest that DSME-T may offer opportunities for DSME among rural residents with diabetes. Plans are in place to explore the possibility of sustaining and expanding the program to other underserved rural communities.

Background: Current management of malignant gastric outlet obstruction (GOO) includes surgical diversion or enteral stent placement for unresectable cancer. We analyzed the long-term results, predictive factors of outcomes, and complications associated with enteral stents with focus on their management., Methods: Between 1997 and 2007, 46 patients with malignant GOO underwent placement of self-expandable metal stents (SEMS) for palliation. Patients were captured prospectively after 2001 and followed until complication or death. Patency, management of complications, and long-term survival were analyzed., Results: Forty-six patients had a mean survival of 152 +/- 235 days and a mean SEMS patency rate of 111 +/- 220 days. SEMS patency rates of 98%, 74%, and 57% at 1, 3, and 6 months were seen. Thirteen patients presented with obstruction and included two SEMS migration, two early occlusion, one fracture, four malignant ingrowth, and four with delayed clinical failure. Interventions included seven endoscopic revisions with three SEMS replacements. Six had percutaneous endoscopic gastrostomy with jejunal arm placed. Two patients eventually underwent surgical bypass. Two patients required surgery for complications including delayed duodenal perforation and aortoenteric fistula., Conclusions: SEMS effectively palliate gastric outlet obstructions that result from upper gastrointestinal malignancies. Their benefits offset potential complications or malfunctions, when a pluridisciplinary approach is adopted.

The Génome Québec and Montreal Heart Institute Pharmacogenomics Centre (Montreal, Canada), created in 2006, is a translational pharmacogenomics platform whose main objectives are to conduct pharmacogenomics research, provide pharmacogenomics services to the academic, biotechnology and pharmaceutical sectors, and integrate pharmacogenomics solutions into the healthcare system. The Centre has brought together a multidisciplinary team of researchers with expertise in genomics, bioinformatics and clinical trial research. All the Centre's clinical research studies are supported by the Centre's unique Good Laboratory Practice facility framework, which has the ability to perform pharmaceutical clinical trials and deliver clinical diagnostics under the highest standards. The Centre has successfully leveraged its experience and expertise in technology development and pharmacogenomics clinical trial work to attract funding and collaborative partnerships in both the public and private sectors.

Initiation of warfarin therapy is associated with bleeding owing to its narrow therapeutic window and unpredictable therapeutic dose. Pharmacogenetic-based dosing algorithms can improve accuracy of initial warfarin dosing but require rapid genotyping for cytochrome P-450 2C9 (CYP2C9) *2 and *3 single nucleotide polymorphisms (SNPs) and a vitamin K epoxide reductase (VKORC1) SNP. We evaluated 4 commercial systems: INFINITI analyzer (AutoGenomics, Carlsbad, CA), Invader assay (Third Wave Technologies, Madison, WI), Tag-It Mutation Detection assay (Luminex Molecular Diagnostics, formerly Tm Bioscience, Toronto, Canada), and Pyrosequencing (Biotage, Uppsala, Sweden). We genotyped 112 DNA samples and resolved any discrepancies with bidirectional sequencing. The INFINITI analyzer was 100% accurate for all SNPs and required 8 hours. Invader and Tag-It were 100% accurate for CYP2C9 SNPs, 99% accurate for VKORC1 -1639/3673 SNP, and required 3 hours and 8 hours, respectively. Pyrosequencing was 99% accurate for CYP2C9 *2, 100% accurate for CYP2C9 *3, and 100% accurate for VKORC1 and required 4 hours. Current commercial platforms provide accurate and rapid genotypes for pharmacogenetic dosing during initiation of warfarin therapy.

Recommendations are provided to assist health care professionals, manufacturers, and consumers in the appropriate handling of tubing with Luer-tip connectors.

This essay considers biotrophic cyst and root-knot nematodes in relation to their biology, host-parasite interactions and molecular genetics. These nematodes have to face the biological consequences of the physical constraints imposed by the soil environment in which they live while their hosts inhabit both above and below ground environments. The two groups of nematodes appear to have adopted radically different solutions to these problems with the result that one group is a host specialist and reproduces sexually while the other has an enormous host range and reproduces by mitotic parthenogenesis. We consider what is known about the modes of parasitism used by these nematodes and how it relates to their host range, including the surprising finding that parasitism genes in both nematode groups have been recruited from bacteria. The nuclear and mitochondrial genomes of these two nematode groups are very different and we consider how these findings relate to the biology of the organisms.

Background: Bilomas have traditionally been treated by either percutaneous drainage or surgery. However, percutaneous drainage is associated with discomfort and infection, whereas surgery, which is usually reserved for refractory cases, has high morbidity and mortality rates. Recently, endoscopic drainage of bilomas adjacent to the GI lumen has been reported in isolated reports., Objective: We analyzed our 4 years' experience with this innovative technique., Design: Patients with symptomatic bilomas were offered EUS-guided drainage and were followed up prospectively for clinical and radiologic responses., Setting: Tertiary care center with long-standing experience in EUS-guided drainage., Patients: A total of 5 patients underwent EUS-guided transenteric drainage of symptomatic bilomas., Intervention: The technique included transenteric EUS-guided puncture, placement of a guidewire into the biloma, and creation of an enteral-biloma fistula with placement of a plastic endoprosthesis after balloon dilation. In 4 cases, the stents were removed at a mean of 6.8 +/- 4.3 weeks. In one patient with malignancy, the stent was left in place for palliation., Main Outcome Measurements: Efficacy and safety of EUS-guided drainage of bilomas., Results: EUS-guided transenteric biloma drainage was successfully performed in 5 patients without any significant morbidity. Biloma resolution was confirmed in all 5 patients, and none of the 4 patients relapsed after stent removal (mean follow-up of 12.8 +/- 6.1 months)., Conclusions: EUS-guided drainage of bilomas is technically feasible, appears safe, and provides an attractive alternative to percutaneous or surgical drainage.

Oncology pharmacogenomics has seen a great deal of progress in the past 10 years. The release of the Human Genome Project data and the availability of fast, affordable genotyping platforms has allowed the field to expand and has provided invaluable data for pharmacogenomics research. The introduction of US FDA-approved targeted therapy (trastuzumab), package insert changes (irinotecan and tamoxifen) and the initiation of a genotype-guided clinical trial for cancer therapy (TYMS TSER in rectal cancer), along with panels of DNA and expression markers (Roche AmpliChip(®) and Oncotype Dx™ panel) are paving the way towards the integration of pharmacogenomics into clinical practice.

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.

With the advent of dense maps of human genetic variation, it is now possible to detect positive natural selection across the human genome. Here we report an analysis of over 3 million polymorphisms from the International HapMap Project Phase 2 (HapMap2). We used 'long-range haplotype' methods, which were developed to identify alleles segregating in a population that have undergone recent selection, and we also developed new methods that are based on cross-population comparisons to discover alleles that have swept to near-fixation within a population. The analysis reveals more than 300 strong candidate regions. Focusing on the strongest 22 regions, we develop a heuristic for scrutinizing these regions to identify candidate targets of selection. In a complementary analysis, we identify 26 non-synonymous, coding, single nucleotide polymorphisms showing regional evidence of positive selection. Examination of these candidates highlights three cases in which two genes in a common biological process have apparently undergone positive selection in the same population:LARGE and DMD, both related to infection by the Lassa virus, in West Africa;SLC24A5 and SLC45A2, both involved in skin pigmentation, in Europe; and EDAR and EDA2R, both involved in development of hair follicles, in Asia.

The discovery that the potato cyst nematode Globodera pallida has a multipartite mitochondrial DNA (mtDNA) composed, at least in part, of six small circular mtDNAs (scmtDNAs) raised a number of questions concerning the population-level processes that might act on such a complex genome. Here we report our observations on the distribution of some scmtDNAs among a sample of European and South American G. pallida populations. The occurrence of sequence variants of scmtDNA IV in population P4A from South America, and that particular sequence variants are common to the individuals within a single cyst, is described. Evidence for recombination of sequence variants of scmtDNA IV in P4A is also reported. The mosaic structure of P4A scmtDNA IV sequences was revealed using several detection methods and recombination breakpoints were independently detected by maximum likelihood and Bayesian MCMC methods.

A 5-year-old African-American girl presented with a CYP2D6*4xN/*10 genotype that was discordant with her poor metabolizer phenotype determined with the probe drug dextromethorphan. Both phenotype and genotype were confirmed in repeat assessments, suggesting that the CYP2D6*10 allele carried a novel debilitating sequence variation(s). The rationale for this study was to resolve the discordance and to describe the novel non-functional allelic variant of CYP2D6 and its frequency in populations of different ethnic backgrounds.

We sequenced four mitochondrial subgenomes from the potato cyst nematode Globodera pallida, previously characterized as one of the few animals to have a multipartite mitochondrial genome. The sequence data indicate that three of these subgenomic mitochondrial circles are mosaics, comprising long, multigenic fragments derived from fragments of the other circles. This pattern is consistent with the operation of intermitochondrial recombination, a process generally considered absent in animal mitochondria. We also report that many of the duplicated genes contain deleterious mutations, ones likely to render the gene nonfunctional; gene conversion does not appear to be homogenizing the different gene copies. The proposed nonfunctional copies are clustered on particular circles, whereas copies that are likely to code functional gene products are clustered on others.

This report describes a novel diagnostic method for virus-vector trichodorid nematodes and associated Tobacco rattle virus (TRV) based on a real-time fluorogenic 5' nuclease PCR assay (TaqMan). Two independent primer/probe sets were designed targeting the 18S gene of the ribosomal cistron for the trichodorid species, Paratrichodorus pachydermus and Trichodorus similis. Assays using purified plasmid DNA containing clones of the 18S region and genomic DNA extracted from individuals from both nematode species displayed high specificity as no cros s-reaction was observed between the species or with two non-target trichodorid species Paratrichodorus anemones and Trichodorus primitivus. Relative quantification of target DNA present in unknown samples was performed by comparison of the fluorescence signals of the samples to those obtained from plasmid standard dilutions. Three primer/probe sets were also used to target TRV; one set for RNA1 and the two other sets for RNA2 of specific isolates (TRV-PpK20 and TRV-TpO1). Detection of both trichodorid species and TRV RNA1 and RNA2 from a single sample was achieved and field samples were used to demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular information in relation to risk assessment in the field.

Background: ABO and D phenotyping of both blood donors and patients receiving transfusions is routinely performed by blood banks to ensure compatibility. These analyses are performed by antibody-based agglutination assays. Blood is not tested for minor blood group antigens on a regular basis, however, because of cost and time constraints. This can result in alloimmunization of the patient against one to several minor antigens and may complicate future transfusions., Study Design and Methods: To address this problem, an assay has been generated on the GenomeLab SNPstream genotyping system to test simultaneously polymorphisms linked to 22 different blood antigens with donor's DNA isolated from minute amounts of white blood cells., Results: The results showed that both the error rate of the assay, as measured by the strand concordance rate, and the no-call rate were very low (0.1%). The concordance rate with the actual red blood cell (RBC) and platelet (PLT) serology data varied from 97 to 100 percent. Experimental or database errors as well as rare polymorphisms contributing to antigen conformation could explain the observed differences. These rates, however, are well above requirements because phenotyping and cross-matching will always be performed before transfusion., Conclusion: Molecular profiling of blood donors for minor RBC and PLT antigens will give blood banks instant access to many different matched donors through the setup of a centralized data storage system.

Purpose: Recommendations of the interdisciplinary Safe Medication Use Expert Committee of the United States Pharmacopeia (USP) to assist health care professionals, manufacturers, and organizations in handling neuromuscular blocking agents (NMBAs) safely and effectively are discussed., Summary: Review and analysis of the USP Medication Errors Reporting Program and MEDMARX program databases showed a continuing risk of patient harm or death due to errors with NMBAs. Medication errors involving wrong concentrations, wrong doses, wrong drugs, look-alike packaging, and sound-alike names, combined with lack of monitoring and communication, have been associated with the use of NMBAs in health care institutions. Serious adverse events occur when NMBAs are used without adequate safeguards. Recommendations for improving safety were developed through review and discussion of root causes and areas of concern with these medications., Conclusion: Medical errors with NMBAs continue to result in patient morbidity and mortality. Increased awareness and action on the part of all parties involved are needed to improve the safety of this class of medications.

The potato cyst nematode Globodera pallida poses a challenge for potato growers. The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida cause damage valued at over pounds 50m per annum in the U.K. and problems in controlling PCN are growing due to the increase in populations and spread of G. pallida, the lack of many commercially attractive cultivars with resistance to this species and the pressure to reduce nematicide use. Over 60% of potato fields in the U.K. are infected with G. pallida (Minnis et al. 2000). The Scottish Agricultural Science Agency (SASA) figures show that the incidence of both species of PCN on Scottish seed potato land, though low, has been increasing. The proportion of potato land in ware production in Scotland is also increasing and now represents 50% of the potato growing area. This situation potentially increases the risk of the spread of PCN unless it is very carefully monitored and managed.

Pollen tubes are an established model system for examining polarized cell growth. The focus here is on pollen tubes of the conifer Norway spruce (Picea abies, Pinaceae); examining the relationship between cytosolic free Ca2+, tip elongation, and intracellular motility. Conifer pollen tubes show important differences from their angiosperm counterparts; they grow more slowly and their organelles move in an unusual fountain pattern, as opposed to reverse fountain, in the tip. Ratiometric ion imaging of growing pollen tubes, microinjected with fura-2-dextran, reveals a tip-focused [Ca2+]i gradient extending from 450 nM at the extreme apex to 225 nM at the base of the tip clear zone. Injection of 5,5' dibromo-BAPTA does not dissipate the apical gradient, but stops cell elongation and uniquely causes rapid, transient increases of apical free Ca2+. The [Ca2+]i gradient is, however, dissipated by reversible perfusion of extracellular caffeine. When the basal cytosolic free Ca2+ concentration falls below 150 nM, again a large increase in apical [Ca2+]i occurs. An external source of calcium is not required for germination but significantly enhances elongation. However, both germination and elongation are significantly inhibited by the inclusion of calcium channels blockers, including lanthanum, gadolinium, or verapamil. Modulation of intracellular calcium also affects organelle position and motility. Extracellular perfusion of lanthanides reversibly depletes the apical [Ca2+]i gradient, altering organelle positioning in the tip. Later, during recovery from lanthanide perfusion, organelle motility switches direction to a reverse fountain. When taken together these data show a unique interplay in Picea abies pollen tubes between intracellular calcium and the motile processes controlling cellular organization.

Objectives: To determine whether hepatitis B virus (HBV) transmission occurred among patients visiting a physician's office and to evaluate potential transmission mechanisms., Design: Serologic survey, retrospective cohort study, and observation of infection control practices., Setting: Private medical office., Patients: Those visiting the office between March 1 and December 26, 2001., Results: We identified 38 patients with acute HBV infection occurring between February 2000 and February 2002. The cohort study, limited to the 10 months before outbreak detection, included 91 patients with serologic test results and available charts representing 18 case-patients and 73 susceptible patients. Overall, 67 patients (74%) received at least one injection during the observation period. Case-patients received a median of 14 injections (range, 2-25) versus 2 injections (range, 0-17) for susceptible patients (P < .001). Acute infections occurred among 18 (27%) of 67 who received at least one injection versus none of 24 who received no injections (RR, 13.6; CI95, 2.4-undefined). Risk of infection increased 5.2-fold (CI95, 0.6-47.3) for those with 3 to 6 injections and 20.0-fold (CI95, 2.8-143.5) for those with more than 6 injections. Typically, injections consisted of doses of atropine, dexamethasone, vitamin B12, or a combination of these mixed in one syringe. HBV DNA genetic sequences of 24 patients with acute infection and 4 patients with chronic infection were identical in the 1,500-bp region examined. Medical staff were seronegative for HBV infection markers. The same surface was used for storing multidose vials, preparing injections, and dismantling used injection equipment., Conclusion: Administration of unnecessary injections combined with failure to separate clean from contaminated areas and follow safe injection practices likely resulted in patient-to-patient HBV transmission in a private physician's office.

Here we report a large, extensively characterized set of single-nucleotide polymorphisms (SNPs) covering the human genome. We determined the allele frequencies of 55,018 SNPs in African Americans, Asians (Japanese-Chinese), and European Americans as part of The SNP Consortium's Allele Frequency Project. A subset of 8333 SNPs was also characterized in Koreans. Because these SNPs were ascertained in the same way, the data set is particularly useful for modeling. Our results document that much genetic variation is shared among populations. For autosomes, some 44% of these SNPs have a minor allele frequency > or =10% in each population, and the average allele frequency differences between populations with different continental origins are less than 19%. However, the several percentage point allele frequency differences among the closely related Korean, Japanese, and Chinese populations suggest caution in using mixtures of well-established populations for case-control genetic studies of complex traits. We estimate that approximately 7% of these SNPs are private SNPs with minor allele frequencies <1%. A useful set of characterized SNPs with large allele frequency differences between populations (>60%) can be used for admixture studies. High-density maps of high-quality, characterized SNPs produced by this project are freely available.

Objective: beta-Blockers require careful initiation and titration when used in patients with heart failure. Some patients tolerate beta-blocker therapy initiation without difficulty, whereas in other patients this period presents clinical challenges. We tested the hypothesis that polymorphisms at codons 389 (Arg389Gly) and 49 (Ser49Gly) of the beta(1)-adrenergic receptor would be associated with differences in initial tolerability of beta-blocker therapy in patients with heart failure. We also tested whether polymorphisms in the beta(2)-adrenergic receptor, G-protein alpha s subunit (G(s)alpha), and cytochrome P450 (CYP) 2D6 genes or S-metoprolol plasma concentrations were associated with beta-blocker tolerability., Methods: Sixty-one beta-blocker-naive patients with systolic heart failure were prospectively enrolled. Patients began taking 12.5 to 25 mg metoprolol controlled release/extended release with titration every 2 weeks (as tolerated) to 200 mg/d or the maximum tolerated dose over a period of 8 to 10 weeks. Decompensation was the composite of death, heart failure hospitalization, increase in other heart failure medications, or need to discontinue metoprolol. End points were assessed during the titration period., Results: The overall rate of decompensation was not different between the codon 49 or 389 genotypes. However, a significantly greater percentage of patients with the Gly389 variant required increases in heart failure medications as compared with Arg389 homozygotes (48% versus 14%, respectively; P = .006). Similarly, patients with the Ser49 homozygous genotype were significantly more likely to require increases in concomitant heart failure therapy as compared with Gly49 carriers (41% versus 11%, respectively; P = .03). Neither CYP2D6 genotypes nor metoprolol pharmacokinetics differed between patients with and those without a decompensation event. There was no association between the beta(2)-adrenergic receptor or G(s)alpha polymorphisms with decompensated heart failure., Conclusions: Patients with the Gly389 variant and Ser49Ser genotype were significantly more likely to require increases in heart failure medications during beta-blocker titration and thus may require more frequent follow-up during titration.

The tomato Hero A gene is the only member of a multigene family that confers a high level (>80%) of resistance to all the economically important pathotypes of potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida. Although the resistance levels of transgenic tomato lines were similar to those of the tomato line LA1792 containing the introgressed Hero multigene family, transgenic potato plants expressing the tomato Hero A gene are not resistant to PCNs. Comparative microscopy studies of in vitro infected roots of PCN-susceptible tomato cv. Money Maker, the resistant breeding line LA1792, and transgenic line L10 with Ro1 pathotype have revealed no statistically significant difference in the number of juveniles invading roots. However, syncytia (specialized feeding cells) induced in LA1792 and L10 roots mostly were found to have degenerated a few days after their induction, and a few surviving syncytia were able to support only the development of males rather than females. Thus, the ratio between males and females was biased towards males on LA1792 and L10 roots. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Although the Hero A gene is expressed in all tissues, including roots, stems, leaves, and flower buds, its expression is upregulated in roots in response to PCN infection. Moreover, the expression profiles of the Hero A correlates with the timing of death of the syncytium.

Objectives: Head and neck squamous cell carcinoma (HNSCC) is notoriously resistant to chemotherapy. The sphingolipid ceramide and its analogs have been demonstrated to exert antitumor activity in many cell types; however, the effectiveness of these analogs has been limited by potency and solubility. This study focuses on the effects of novel highly soluble cationic pyridinium-ceramides, alone and in combination with various chemotherapeutic agents, on cell survival, telomerase activity, and cell cycle arrest in HNSCC cell lines in vitro., Methods: The concentration of pyridinium-ceramides and chemotherapeutic agents that inhibited cell growth by 50% (IC50) was determined by MTT cell survival assays. The cell cycle profiles were determined by flow cytometry. Telomerase activity was determined by telomerase repeat amplification protocol (TRAP) assay., Results: Treatment of the human UM-SCC-22A (SCC of the hypopharynx) cells, as well as various other HNSCC cell lines, with C6-Pyr-Cer resulted in the inhibition of cell survival with an IC50 concentration of approximately 250 to 300 nM at 96 hours, whereas its IC50 was greater than 1000 nM in noncancerous Wi-38 human lung fibroblasts, and adult human epidermal keratinocytes. Moreover, treatment with C6-Pyr-Cer also resulted in cell cycle arrest in G0/G1, which correlated with a significant inhibition of telomerase activity in UM-SCC-22A cells. Additional results demonstrated that the combination of C6-Pyr-Cer with gemcitabine (GMZ) or doxorubicin (DOX), which have the lowest IC50 concentrations among various chemotherapeutic drugs in these cells, enhances the effects of these drugs in the inhibition of telomerase and cell growth., Conclusions: These data suggest that the novel C6-Pyr-Cer with high solubility and bioavailability may lead to the development of new therapeutic strategies that target telomerase for the treatment of HNSCC.

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.

Objective: Beta-Blocker use can be associated with adverse effects that may have an impact on adherence or harm patients. The commonly prescribed beta-blocker metoprolol is metabolized by the polymorphic cytochrome P450 (CYP) 2D6 enzyme, resulting in widely variable drug exposure. We investigated whether metoprolol plasma concentrations, CYP2D6 polymorphisms, or genotype-derived phenotype was associated with adverse effects or efficacy in patients with hypertension., Methods: Fifty hypertensive patients received metoprolol by use of a dose-titration algorithm until target blood pressure was reached, intolerable side effects occurred, or maximal daily dose was achieved. CYP2D6 genotype was determined by methods based on polymerase chain reaction-restriction fragment length polymorphism and included 19 allelic variants. Patients were assigned to standard phenotype groups on the basis of genotype. Patients were also assigned activity scores based on functional activity of the alleles. General and dose-limiting adverse events and blood pressure responses were analyzed in relation to metoprolol steady-state pharmacokinetic profile and CYP2D6 genotype-derived phenotype., Results: Poor metabolizers had a significantly longer elimination half-life, higher S-metoprolol area under the plasma concentration-time curve (AUC), and lower oral clearance (P < or = .007 for all parameters). There was a 29.6-fold variability in AUC among extensive metabolizers, which was largely explained by CYP2D6 activity scores (P = .032 for ordered differences in AUC by activity score among extensive metabolizers). Overall general and dose-limiting adverse event rates were 46% and 14%, respectively. General adverse event rates did not differ by AUC quartile (66.7% [95% confidence interval (CI), 35.4%-88.7%] and 41.7% [95% CI, 16.5%-71.4%] in the lowest and highest quartiles, respectively; P = .09 among all quartiles). Dose-limiting adverse event rates were also not different by AUC quartile (16.7% [95% CI, 2.9%-49.1%] and 8.3% [95% CI, 0.4%-40.2%] in the lowest and highest quartiles; P = .35 among all quartiles). Furthermore, adverse event rates did not differ by activity scores or between extensive, intermediate, or poor metabolizers. Antihypertensive response rate and blood pressure changes also were not influenced by differences in plasma concentrations or CYP2D6 genotypes., Conclusions: As expected, CYP2D6 genotype-phenotype correlates with differences in metoprolol pharmacokinetics. However, there was no association between variable pharmacokinetics or CYP2D6 genotype and beta-blocker-induced adverse effects or efficacy.

Due to their capability of eliciting a form of posttranscriptional gene silencing (termed virus-induced gene silencing or VIGS), plant viruses are increasingly used as reverse-genetics tools for functional characterization of plant genes. RNA viruses have been shown to trigger silencing in a variety of host plants, including members of Solanacae and Arabidopsis (Arabidopsis thaliana). Several factors affect the silencing response, including host range and viral tropism within the plant. The work presented here demonstrates that a modified tobacco rattle virus (TRV) vector retaining the helper protein 2b, required for transmission by a specific vector nematode, not only invades and replicates extensively in whole plants, including meristems, but also triggers a pervasive systemic VIGS response in the roots of Nicotiana benthamiana, Arabidopsis, and tomato (Lycopersicon esculentum). This sustained VIGS response was exemplified by the silencing of genes involved in root development (IRT1, TTG1 [transparent testa glabra], RHL1 [root hairless1], and beta-tubulin), lateral root-meristem function (RML1 [root meristemless1]), and nematode resistance (Mi). Roots of silenced plants exhibit reduced levels of target mRNA and phenocopy previously described mutant alleles. The TRV-2b vector displays increased infectivity and meristem invasion, both key requirements for efficient VIGS-based functional characterization of genes in root tissues. Our data suggest that the TRV helper protein 2b may have an essential role in the host regulatory mechanisms that control TRV invasion.

With the large numbers of single nucleotide polymorphisms (SNPs) available and new technologies that permit high throughput genotyping, we have investigated the possibility of the localization of disease genes with genome-wide panels of SNP markers and taking advantage of the linkage-disequilibrium (LD) between the disease gene and closely linked markers. For this purpose, we selected cases from the Ashkenazi Jewish population, in which the mutant alleles are expected to be identical by descent from a common founder and the regions of LD encompassing these mutant alleles are large. As a validation of this approach for localization, we performed two trials: one in autosomal recessive Bloom syndrome, in which a unique mutation of the BLM gene is present at elevated frequencies in cases, and the other in autosomal dominant hereditary nonpolyposis colorectal cancer (HNPCC), in which a unique mutation of MSH2 is present at elevated frequencies. In the Bloom syndrome trial, we genotyped 3,258 SNPs in 10 Jewish Bloom syndrome cases and 31 non-Bloom syndrome Jewish persons as a comparison group. In the HNPCC trial, we genotyped 8,549 SNPS in 13 Jewish HNPCC cases whose colon cancers exhibited microsatellite instability and in 63 healthy Jews as a comparison group. To identify significant associations, we performed (a) Fisher's exact test comparing genotypes at each locus in cases versus controls and (b) a haplotype analysis by estimating the frequency of haplotypes with the expectation-maximization algorithm and comparing haplotype frequencies in cases versus controls by logistic regression and a maximum likelihood ratio method. In the Bloom syndrome trial, by Fisher's exact test, statistically significant association was detected at a single locus, TSC0754862, which is a locus 1.7 million bp from BLM. Two-locus, three-locus, and four-locus haplotypes that included TSC0754862 and flanked BLM were also statistically more frequent in cases versus controls. In the HNPCC trial, although a significant P value was not obtained by the single SNP genotype analysis, significant associations were detected for several multilocus haplotypes in an 11-million-bp region that contained the MSH2 gene. This work demonstrates the power of the LD mapping approach in an isolated population and its general applicability to the identification of novel cancer-causing genes.

Large-scale association studies hold substantial promise for unraveling the genetic basis of common human diseases. A well-known problem with such studies is the presence of undetected population structure, which can lead to both false positive results and failures to detect genuine associations. Here we examine approximately 15,000 genome-wide single-nucleotide polymorphisms typed in three population groups to assess the consequences of population structure on the coming generation of association studies. The consequences of population structure on association outcomes increase markedly with sample size. For the size of study needed to detect typical genetic effects in common diseases, even the modest levels of population structure within population groups cannot safely be ignored. We also examine one method for correcting for population structure (Genomic Control). Although it often performs well, it may not correct for structure if too few loci are used and may overcorrect in other settings, leading to substantial loss of power. The results of our analysis can guide the design of large-scale association studies.

Genetic studies aimed at understanding the molecular basis of complex human phenotypes require the genotyping of many thousands of single-nucleotide polymorphisms (SNPs) across large numbers of individuals. Public efforts have so far identified over two million common human SNPs; however, the scoring of these SNPs is labor-intensive and requires a substantial amount of automation. Here we describe a simple but effective approach, termed whole-genome sampling analysis (WGSA), for genotyping thousands of SNPs simultaneously in a complex DNA sample without locus-specific primers or automation. Our method amplifies highly reproducible fractions of the genome across multiple DNA samples and calls genotypes at >99% accuracy. We rapidly genotyped 14,548 SNPs in three different human populations and identified a subset of them with significant allele frequency differences between groups. We also determined the ancestral allele for 8,386 SNPs by genotyping chimpanzee and gorilla DNA. WGSA is highly scaleable and enables the creation of ultrahigh density SNP maps for use in genetic studies.

The use of antibiotic resistance analysis (ARA) for microbial source tracking requires the generation of a library of isolates collected from known sources in the watershed. The size and composition of the library are critical in determining if it represents the diversity of patterns found in the watershed. This study was performed to determine the size that an ARA library needs to be to be representative of the watersheds for which it will be used and to determine if libraries from different watersheds can be merged to create multiwatershed libraries. Fecal samples from known human, domesticated, and wild animal sources were collected from six Virginia watersheds. From these samples, enterococci were isolated and tested by ARA. Based on cross-validation discriminant analysis, only the largest of the libraries (2,931 isolates) were found to be able to classify nonlibrary isolates as well as library isolates (i.e., were representative). Small libraries tended to have higher average rates of correct classification, but were much less able to correctly classify nonlibrary isolates. A merged multiwatershed library (6,587 isolates) was created and was found to be large enough to be representative of the isolates from the contributing watersheds. When isolates that were collected from the contributing watersheds approximately 1 year later were analyzed with the multiwatershed library, they were classified as well as the isolates in the library, suggesting that the resistance patterns are temporally stable for at least 1 year. The ability to obtain a representative, temporally stable library demonstrates that ARA can be used to identify sources of fecal pollution in natural waters.

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.

Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved ('haplotype blocks'; refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.

Using an empirical panel of more than 20 000 single base primer extension (SNP-IT) assays we have developed a set of statistical scores for evaluating and rank ordering various parameters of the SNP-IT reaction to facilitate high-throughput assay primer design with improved likelihood of success. Each score predicts either signal magnitude from primer extension or signal noise caused by mispriming of primers and structure of the PCR product. All scores have been shown to correlate with the success/failure rate of the SNP-IT reaction, based on analysis of assay results. A logistic regression analysis was applied to combine all scored parameters into one measure predicting the overall success/failure rate of a given SNP marker. Three training sets for different types of SNP-IT reaction, each containing about 22 000 SNP markers, were used to assign weights to each score and optimize the prediction of the combined measure. c-Statistics of 0.69, 0.77 and 0.72 were achieved for three training sets. This new statistical prediction can be used to improve primer design for the SNP-IT reaction and evaluate the probability of genotyping success for a given SNP based on analysis of the surrounding genomic sequence.

ABSTRACT Polymerase chain reaction amplification of the intergenic spacer region between the 5S and 18S genes from Meloidogyne chitwoodi, M. fallax, and M. hapla enabled these three important temperate species to be differentiated. Length polymorphism was found between M. chitwoodi and M. fallax as a result of differing numbers of short repeats located between the 5S and 18S genes. The presence of the 5S gene within the rDNA cistrons was confirmed in the Meloidogyne spp. included in this study. The region between the 28S and 5S genes for M. chitwoodi and M. fallax was short and lacked variability in repeated sequences compared with the main tropical Meloidogyne spp. and M. hapla. Differences in the number of these repeats resulted in intraspecific length polymorphism for M.hapla.

The Hero gene of tomato is a broad spectrum resistance gene that confers a high level of resistance to all pathotypes of the potato cyst nematodes Globodera rostochiensis and partial resistance to G. pallida. The gene was identified by map-based cloning, sequencing and complementation analysis of two susceptible tomato lines with an array of 13 overlapping cosmids spanning a total distance of 135 kb. Hero encodes a protein with a nucleotide-binding site (NBS) and a leucine-rich-repeat (LRR) domain and is a member of a gene family of 14 highly homologous genes, which are clustered within a continuous 118-kb region. The isolated Hero gene displayed resistance to various G. rostochiensis pathotypes and partial resistance to G. pallida pathotype Pa2/3 in transgenic tomato lines. None of the Hero homologues conferred resistance to G. rostochiensis pathotypes. Hero can be distinguished from its homologues by the length of a compound hexanucleotide microsatellite, which codes for a charged and repetitive amino acid domain within the LRR. We propose that the expansion of this microsatellite may be involved in the evolution of the Hero resistance gene.

As many of the linked chromosome regions that predispose to type 1 diabetes in the NOD mouse have been dissected, it has become apparent that the initially observed effect is in fact attributable to several loci. One such cluster of loci on distal chromosome 3, originally described as Idd10, is now known to comprise three separate loci, Idd10, Idd17, and Idd18. Although these loci have a significant combined effect on diabetes development, their individual effects are barely detectable when diabetes is used as a read-out, which makes fine-mapping them by use of a conventional congenic approach impractical. In this study, we demonstrate that it is possible to map loci, with modest effects, to regions small enough for systematic gene identification by capitalizing on the fact that the combined loci provide more profound, measurable protection. We have mapped the Idd10 and Idd18 loci to 1.3- and 2.0-cM intervals, respectively, by holding the Idd3 allele constant. In addition, we have excluded Csf1 and Nras as candidates for both loci.

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and cause colonization, infection, and pseudo-outbreaks in health care settings. Data suggest that the frequency of nosocomial outbreaks due to NTM may be increasing, and reduced hot water temperatures may be partly responsible for this phenomenon. Attention to adequate high-level disinfection of medical devices and the use of sterile reagents and biologicals will prevent most outbreaks. Because NTM cannot be eliminated from the hospital environment, and because they present an ongoing potential for infection, NTM should be considered in all cases of nosocomial infection, and careful surveillance must be used to identify potential outbreaks. Analysis of the species of NTM and the specimen source may assist in determining the significance of a cluster of isolates. Once an outbreak or pseudo-outbreak is suspected, molecular techniques should be applied promptly to determine the source and identify appropriate control measures.

The cuticle is a major barrier prohibiting the infection of nematodes against micro-organisms. The attachment of bacterial spores of the nematode hyperparasite Pasteuria penetrans (PP1) to field populations of root-knot nematodes (RKN, Meloidogyne spp.) from Burkino Faso, Ecuador, Greece, Malawi, Senegal and Trinidad and Tobago were assayed in standard attachment tests. The attachment of spore population PP1 to different field populations of root-knot nematode showed that the rates of attachment differed between countries. Similar tests were also undertaken on P. penetrans spores from these countries against 2 species of RKN, M. incognita and M. arenaria. The results showed a high degree of variability in spore attachment with no clear distinction between the 2 species of nematode. It has been hypothesized that Pasteuria spore attachment is linked to nematode species designations and this study clearly shows that this is not the case. Further tests showed that variation in spore attachment was not linked to nematode phylogeny. The results therefore beg the question of how do parthenogenetic root-knot nematodes maintain cuticle variability in the face of such an aggressive hyperparasite.

Previous analyses of NOD mice have shown that some genes control the development of both insulitis and diabetes, while other loci influence diabetes without reducing insulitis. Evidence for the existence of a gene only influencing diabetes, Idd9 on mouse chromosome 4, is provided here by the development of a novel congenic mouse strain, NOD.B10 Idd9. NOD.B10 Idd9 mice display profound resistance to diabetes even though nearly all develop insulitis. Subcongenic analysis has demonstrated that alleles of at least three B10 genes, Idd9.1, Idd9.2, and Idd9.3 are required to produce Idd9-mediated diabetes resistance. Candidate genes with amino acid differences between the NOD and B10 strains have been localized to the 5.6 cM Idd9.2 interval (Tnfr2, Cd30) and to the 2.0 cM Idd9.3 interval (Cd137).

Large-scale pharmacogenetics and complex disease association studies will require typing of thousands of single-nucleotide polymorphisms (SNPs) in thousands of individuals. Such projects would benefit from a genotyping system with accuracy >99% and a failure rate <5% on a simple, reliable, and flexible platform. However, such a system is not yet available for routine laboratory use. We have evaluated a modification of the previously reported Invader SNP-typing chemistry for use in a genotyping laboratory and tested its automation. The Invader technology uses a Flap Endonuclease for allele discrimination and a universal fluorescence resonance energy transfer (FRET) reporter system. Three hundred and eighty-four individuals were genotyped across a panel of 36 SNPs and one insertion/deletion polymorphism with Invader assays using PCR product as template, a total of 14,208 genotypes. An average failure rate of 2.3% was recorded, mostly associated with PCR failure, and the typing was 99.2% accurate when compared with genotypes generated with established techniques. An average signal-to-noise ratio (9:1) was obtained. The high degree of discrimination for single base changes, coupled with homogeneous format, has allowed us to deploy liquid handling robots in a 384-well microtitre plate format and an automated end-point capture of fluorescent signal. Simple semiautomated data interpretation allows the generation of approximately 25,000 genotypes per person per week, which is 10-fold greater than gel-based SNP typing and microsatellite typing in our laboratory. Savings on labor costs are considerable. We conclude that Invader chemistry using PCR products as template represents a useful technology for typing large numbers of SNPs rapidly and efficiently.

The mitochondrial genome (mtDNA) of the plant parasitic nematode Globodera pallida exists as a population of small, circular DNAs that, taken individually, are of insufficient length to encode the typical metazoan mitochondrial gene complement. As far as we are aware, this unusual structural organization is unique among higher metazoans, although interesting comparisons can be made with the multipartite mitochondrial genome organizations of plants and fungi. The variation in frequency between populations displayed by some components of the mtDNA is likely to have major implications for the way in which mtDNA can be used in population and evolutionary genetic studies of G. pallida.

ASHP guidelines on the pharmacist's role in clinical drug research and future directions for pharmacists in clinical drug research are described. Health-system pharmacists have a responsibility to the patient and to the institution to ensure that clinical research systems are sound, that patients are protected, and that research is conducted in a safe, effective way. ASHP guidelines list minimum standards that are essential for improving performance. The ASHP Guidelines on Clinical Drug Research, approved in November 1997, update information previously found in the ASHP Guidelines for the Use of Investigational Drugs in Organized Health-Care Settings, approved in 1990, but have an expanded focus. Additions include recognition of relevant business practices, implications of technology, and the expansion of clinical research beyond the academic health center. At a minimum, all pharmacists involved in clinical research should handle the record keeping for drug accountability, provide drug information to patients and to other health care providers, ensure the appropriate care of patients at sites not involved in the study, and provide accountability at nonpharmacy locations. Managing and coordinating clinical drug research is an area of growth that represents a great opportunity for clinical drug research. By providing baseline and higher-level pharmaceutical services for clinical research projects, pharmacists can help to ensure data accuracy and completeness and patient safety.

A gene encoding a novel transmembrane protein was identified by DNA sequence analysis within the insulin-dependent diabetes mellitus (IDDM) locus IDDM4 on chromosome 11q13. Based on its chromosomal position, this gene is a candidate for conferring susceptibility to diabetes. The gene, termed low-density lipoprotein receptor related protein 5 (LRP5), encodes a protein of 1615 amino acids that contains conserved modules which are characteristic of the low-density lipoprotein (LDL) receptor family. These modules include a putative signal peptide for protein export, four epidermal growth factor (EGF) repeats with associated spacer domains, three LDL-receptor (LDLR) repeats, a single transmembrane spanning domain, and a cytoplasmic domain. The encoded protein has a unique organization of EGF and LDLR repeats; therefore, LRP5 likely represents a new category of the LDLR family. Both human and mouse LRP5 cDNAs have been isolated and the encoded mature proteins are 95% identical, indicating a high degree of evolutionary conservation.

The relationships among a number of populations of Globodera pallida from Britian, the Netherlands, Germany, Switzerland, and South America were examined using PCR amplification of the ribosomal cistron between the 18S and 28S genes that include the two intergenic spacer regions (ITS1 and ITS2) and the 5.8S gene. Amplifications produced a similar-sized product of 1150 bp from all populations. Digestion of the amplified fragment with a number of restriction enzymes showed differences among the populations. The restriction enzyme RsaI distinguished the most populations. The RFLP patterns revealed by this enzyme were complex and could have arisen from heterogeneity between individuals within populations and from differences between the repeats of an individual. Sequence analysis from six of the populations, together with RFLP analysis of PCR products, shows that there is intraspecific variation in the rDNA of G. pallida.

The reproduction of single egg-mass isolates of Meloidogyne javanica from Crete that differed in virulence were compared on tomato (Lycopersicon esculentum) genotypes homozygous or heterozygous for the Mi gene. The reproduction of three isolates with partial virulence was much greater on tomato genotypes heterozygous for the Mi gene (cultivars Scala, Bermuda, and 7353) than on two homozygous genotypes (F8 inbred lines derived from Scala). The reproduction of a highly virulent isolate on the homozygous and heterozygous genotypes was similar to that on a susceptible cultivar. These results pose questions regarding the nature of partial virulence and indicate a quantitative effect of the Mi gene in relation to such virulence.