Exploiting digital droplet PCR and Next Generation Sequencing technologies to determine the relative abundance of individual Eimeria species in a DNA sample.

DNA-based diagnostic assays for detecting infections with Eimeria species have been limited to providing identification and presence/absence data for samples containing oocysts. Modern technologies that generate quantitative data, such as droplet digital PCR (ddPCR) and Next Generation Sequencing (NGS), utilize a relatively short amplicon size containing sufficient species-specific variation for reliable species level identification. Targeting the cytochrome c oxidase subunit III gene in the mitochondrial genome, we established protocols using these technologies to determine the relative abundance of the number of copies/µL of Eimeria species in a sample. Samples from chickens of known and unknown Eimeria species composition were analyzed to determine the suitability of these technologies as diagnostic assays. All technologies demonstrated robust capability of identifying and quantifying the Eimeria species in samples. The new quantitative assays described herein will produce invaluable detail of Eimeria species infections for an array of situations in commercial chicken production systems, enabling further characterization of the disease profile and allowing for the development or enhancement of new intervention strategies.

Restoration of anticoccidial sensitivity to a commercial broiler chicken facility in Canada.

Increasing resistance of Eimeria species to anticoccidial medications is an issue in the broiler chicken industry. Using drug-sensitive strains in live-coccidiosis vaccines has been shown to improve anticoccidial effectiveness in US-based broiler production. In Canada, litter is removed between flocks, which differ from the US industry practice. Thus, we investigated the use of drug-sensitive vaccine strains in a Canadian broiler production facility with suspected anticoccidial resistance. Weekly fecal samples were collected from flocks before, during, and after vaccine seeding to determine oocyst shedding patterns; following the vaccine seeding, OPG counts from similar aged birds were lower than flocks before live-coccidiosis vaccine use. Eimeria species isolates, collected before and after vaccine seeding, were used in 2 anticoccidial sensitivity tests to evaluate their susceptibility to commercially available anticoccidial medications; a low-dose challenge to define parasite replication, and a high-dose challenge to monitor broiler performance. In both experiments, isolates collected after seeding were more susceptible to almost every anticoccidial medication evaluated compared with the isolates collected before seeding. These results demonstrate an improvement in sensitivity to many anticoccidials after the use of live-coccidiosis vaccines at this facility. However, the regulated removal of litter at the end of each flock required under Canadian broiler chicken production management rules could limit the establishment of vaccine-strain Eimeria species in broiler facilities and could shorten the longevity of improved drug sensitivity observed in this study.

Monitoring coccidia in commercial broiler chicken flocks in Ontario: comparing oocyst cycling patterns in flocks using anticoccidial medications or live vaccination.

Coccidiosis, the parasitic disease caused by Eimeria spp., is controlled during broiler chicken production through the inclusion of in-feed anticoccidial medications. Live-coccidiosis vaccination has become an increasingly common alternative to these medications. Monitoring infections with Eimeria spp. in flocks can be accomplished through determining the concentration of oocysts excreted in the fecal material (i.e., oocysts per gram; OPG). The purpose of our study was to sample commercial Ontario broiler chicken flocks at various times of the year to determine weekly OPG counts for flocks that use either an in-feed anticoccidial medication or a live-coccidiosis vaccine. Weekly sampling of 95 flocks from placement to market permitted documentation of oocyst cycling patterns typical of conventional and antibiotic-free flocks, and variation of these patterns in summer and winter. Medicated flocks had higher and later peak oocyst shedding compared with vaccinated flocks. Flocks reared in the summer peaked in oocyst shedding earlier than flocks reared in the winter. Despite what appears to be poorer coccidiosis control in the medicated flocks, the performance data were similar for these flocks compared with vaccinated flocks. This is the first study describing typical patterns of parasite shedding in Ontarian commercial broiler chicken flocks; these data will provide a baseline of expected Eimeria spp. infections in Canadian broiler chicken flocks to ensure optimal coccidiosis prevention.

A goal programming approach for balancing diet costs and feed water use under different environmental conditions.

Water is essential in livestock production systems. In typical dairy production systems, 90% of the total water used by a dairy farm is attributed to feed production. Theoretically, ration manipulation is a method to potentially reduce the irrigation water needed for feed crops without dramatically increasing diet costs. However, published quantitative studies on the relationship between feed production and water use that are integrated with linear programming models are scarce. The overall objective of this study was to develop an optimization framework that could achieve a balance between minimization of dietary costs and dietary irrigation water usage, and that could be used as a framework for future research and models for various livestock production systems. Weighted goal programming models were developed to minimize the dietary costs and irrigation water usage for a hypothetical cow under 8 different environmental scenarios. The environmental conditions used a 2 × 2 × 2 factorial design, including 2 atmospheric CO2 concentrations (400 and 550 ppm), 2 water years (dry and wet), and 2 irrigation methods (furrow and drip). A systematic weighting scheme was used to model the trade-off between minimizing diet cost and minimizing irrigation water use for feedstuffs. Each environmental condition generated a set of distinct diets, which each met the same nutrient requirements of the hypothetical cow but had a different water usage when the weighting scheme was changed from weighting minimum diet costs to minimum irrigation water usage. For water resource planning in areas of dairy production, this set of unique solutions provides the decision maker with different feeding options according to diet cost, water usage, and available feeds. As water was more constrained, dietary dry matter intake increased, concentrations of neutral detergent fiber, ether extract, and energy decreased, and the concentration of lignin increased because less nutritive but more water-saving feedstuffs were included in the diet. Mitigation costs of water usage were calculated from goal programming results and indicated that the potential value of water under water-limited conditions (e.g., in a drought region) was higher than that under water-sufficient conditions. However, a smaller increase in feed costs can initially significantly reduce water usage compared with that of a least-cost diet, which implies that the reduction of water usage through ration manipulation might be possible. This model serves as a framework for the study of irrigation water usage in dairy production and other livestock production systems and for decision-making processes involved in water resources planning in the broader area of animal production.

Responses of broiler chickens to Eimeria challenge when fed a nucleotide-rich yeast extract.

Nucleotide-rich yeast extract (YN) was investigated for effects on growth performance, jejunal physiology, and cecal microbial activity in Eimeria-challenged broiler chickens. A total of 360-day-old male chicks (Ross × Ross 708) were placed on floor pens and provided a corn-soybean meal-based diet without or with YN (500 g/MT; n = 12). On d 10, 6 replicates per diet were orally administered with 1 mL of E. acervulina and E. maxima sporulated oocysts and the rest (non-challenged control) were administered with 1 mL of distilled water. On d 15, 5 birds/pen were then necropsied for intestinal lesion scores, histomorphology and cecal digesta pH, short chain fatty acids (SCFA), and microbial community using Illumina Miseq platform. Supplemental YN improved (P = 0.01) Feed conversion ratio (FCR) during the prechallenge phase (d 0 to 10). In the postchallenge period (d 11 to 15), Eimeria depressed (P < 0.05) Body weight gain (BWG) relative to non-challenged birds, whereas YN-fed birds had a higher (P = 0.05) BWG compared to that of non-YN-fed birds. There was an interaction between YN and Eimeria on jejunal villi height (VH) (P = 0.001) and expression of cationic amino acid transporter 1(CAT1) (P = 0.04). Specifically, in the absence of Eimeria, YN-fed birds had a shorter VH (892 vs. 1,020 µm) relative to that of control but longer VH (533 vs. 447 µm) in the presence of Eimeria. With respect to CAT1, YN-fed birds had a higher (1.65 vs. 0.78) expression when subjected to Eimeria than when not challenged. Independently, Eimeria decreased (P < 0.01) the jejunal expression of maltase, Na glucose transporter 1 and occludin genes, ceca digesta abundance of genus Clostridium cluster XlVa and Oscillibacter but increased (P < 0.01) jejunal proliferating cell nuclear antigen and interleukin 10. Interaction between YN and Eimeria was observed for ceca digesta pH (P = 0.04) and total SCFA (P = 0.01) such that YN increased SCFA in the absence of Eimeria but reduced SCFA and increased pH in the presence of Eimeria. In summary, Eimeria impaired performance and gut function and shifted gut microbiome; YN improved performance independently, attenuated Eimeria damage on indices of gut function, and modulated cecal microbiome.

Weak-link Josephson Junctions Made from Topological Crystalline Insulators.

We report on the fabrication of Josephson junctions using the topological crystalline insulator Pb_{0.5}Sn_{0.5}Te as the weak link. The properties of these junctions are characterized and compared to those fabricated with weak links of PbTe, a similar material yet topologically trivial. Most striking is the difference in the ac Josephson effect: junctions made with Pb_{0.5}Sn_{0.5}Te exhibit a rich subharmonic structure consistent with a skewed current-phase relation. This structure is absent in junctions fabricated from PbTe. A discussion is given on the origin of this effect as an indication of novel behavior arising from the topologically nontrivial surface state.

The utility of routine pre-chemotherapy screening with cardiac gated blood pool scan for patients at low risk of anthracycline toxicity.

Background Anthracycline-based chemotherapy is used in many malignancies. Current recommendations by several groups suggest cardiac monitoring prior to and during anthracycline therapy. We aim to review the usefulness of baseline cardiac screening for left ventricular ejection fraction to assess if it had any impact on chemotherapy decisions in patients to be treated with anthracycline-based regimens or any beneficial effect upon outcomes. Methods We conducted a retrospective three-year audit of cancer patients who underwent GBPS prior to anthracycline (doxorubicin) chemotherapy. Subjects were identified via records from the Department of Nuclear Medicine. Pharmacy dispensing records identified those who received doxorubicin. Patient demographics, cancer type, cardiac risk factors, GBPS ejection fraction (EF), and cumulative anthracycline dose were collected. Results From 1 August 2009 to 31 July 2012, 179 patients underwent GBPS pre-doxorubicin chemotherapy. The mean age was 59 years (range 21-89 years), with 51% being males. Only two patients (1.1%) had an abnormal EF < 50%, while 33 patients (18%) had an EF 51-59% and 144 patients (80%) had EF ≥ 60%. The two patients with reduced baseline EF still received anthracycline-based chemotherapy. All 135 patients without any known cardiovascular risk factors had normal EFs. The total number of patients who received anthracycline chemotherapy during the same period was 207. Thus 28 patients (13%) commenced anthracycline without a prior GBPS. Conclusion Only 1.1% of the screened patients had EF < 50%. These two patients still received doxorubicin chemotherapy despite a compromised EF, as their treating clinicians believed that the benefits of chemotherapy outweighed the risk of potential cardiac toxicity. Our audit questions the practice of routine cardiac evaluation pre-anthracycline screening with GBPS. We propose that routine screening only be requested if cardiac risk factors are present.

More surprises in the global greenhouse: Human health impacts from recent toxic marine aerosol formations, due to centennial alterations of world-wide coastal food webs.

Reductions of zooplankton biomasses and grazing pressures were observed during overfishing-induced trophic cascades and concurrent oil spills at global scales. Recent phytoplankton increments followed, once Fe-, P-, and N-nutrient limitations of commensal diazotrophs and dinoflagellates were also eliminated by respective human desertification, deforestation, and eutrophication during climate changes. Si-limitation of diatoms instead ensued during these last anthropogenic perturbations of agricultural effluents and sewage loadings. Consequently, ~15% of total world-wide annual asthma trigger responses, i.e. amounting to ~45 million adjacent humans during 2004, resulted from brevetoxin and palytoxin poisons in aerosol forms of western boundary current origins. They were denoted by greater global harmful algal bloom [HAB] abundances and breathing attacks among sea-side children during prior decadal surveys of asthma prevalence, compiled here in ten paired shelf ecosystems of western and eutrophied boundary currents. Since 1965, such inferred onshore fluxes of aerosolized DOC poisons of HABs may have served as additional wind-borne organic carriers of toxic marine MeHg, phthalate, and DDT/DDE vectors, traced by radio-iodine isotopes to potentially elicit carcinomas. During these exchanges, as much as 40% of mercury poisonings may instead have been effected by inhalation of collateral HAB-carried marine neurotoxic aerosols of MeHg, not just from eating marine fish. Health impacts in some areas were additional asthma and pneumonia episodes, as well as endocrine disruptions among the same adjacent humans, with known large local rates of thyroid cancers, physician-diagnosed pulmonary problems, and ubiquitous high indices of mercury in hair, pesticides in breast milk, and phthalates in urine.

INO80 is required for oncogenic transcription and tumor growth in non-small cell lung cancer.

Epigenetic regulators are attractive targets for the development of new cancer therapies. Among them, the ATP-dependent chromatin remodeling complexes control the chromatin architecture and have important roles in gene regulation. They are often found to be mutated and de-regulated in cancers, but how they influence the cancer gene expression program during cancer initiation and progression is not fully understood. Here we show that the INO80 chromatin remodeling complex is required for oncogenic transcription and tumor growth in non-small-cell lung cancer (NSCLC). Ino80, the SWI/SNF ATPase in the complex, is highly expressed in NSCLC cells compared with normal lung epithelia cells. Further, its expression, as well as that of another subunit Ino80B, negatively correlates with disease prognosis in lung cancer patients. Functionally, INO80 silencing inhibits NSCLC cell proliferation and anchorage-independent growth in vitro and tumor formation in mouse xenografts. It occupies enhancer regions near lung cancer-associated genes, and its occupancy correlates with increased genome accessibility and enhanced expression of downstream genes. Together, our study defines a critical role of INO80 in promoting oncogenic transcription and NSCLC tumorigenesis, and reveals a potential treatment strategy for inhibiting the cancer transcription network by targeting the INO80 chromatin remodeling complex.

Attenuation of cocaine self-administration by chronic oral phendimetrazine in rhesus monkeys.

Chronic treatment with the monoamine releaser d-amphetamine has been consistently shown to decrease cocaine self-administration in laboratory studies and clinical trials. However, the abuse potential of d-amphetamine is an obstacle to widespread clinical use. Approaches are needed that exploit the efficacy of the agonist approach but avoid the abuse potential associated with dopamine releasers. The present study assessed the effectiveness of chronic oral administration of phendimetrazine (PDM), a pro-drug for the monoamine releaser phenmetrazine (PM), to decrease cocaine self-administration in four rhesus monkeys. Each day, monkeys pressed a lever to receive food pellets under a 50-response fixed-ratio (FR) schedule of reinforcement and self-administered cocaine (0.003-0.56 mg/kg per injection, i.v.) under a progressive-ratio (PR) schedule in the evening. After completing a cocaine self-administration dose-response curve, sessions were suspended and PDM was administered (1.0-9.0 mg/kg, p.o., b.i.d.). Cocaine self-administration was assessed using the PR schedule once every 7 days while food-maintained responding was studied daily. When a persistent decrease in self-administration was observed, the cocaine dose-effect curve was re-determined. Daily PDM treatment decreased cocaine self-administration by 30-90% across monkeys for at least 4 weeks. In two monkeys, effects were completely selective for cocaine. Tolerance developed to initial decreases in food-maintained responding in the third monkey and in the fourth subject, fluctuations were observed that were lower in magnitude than effects on cocaine self-administration. Cocaine dose-effect curves were shifted down and/or rightward in three monkeys. These data provide further support for the use of agonist medications for cocaine abuse, and indicate that the promising effects of d-amphetamine extend to a more clinically viable pharmacotherapy.

Establishment of two quantitative nested qPCR assays targeting the human EPO transgene.

For ethical and safety reasons it is critical to develop easily implemented assays with high sensitivity and specificity for gene doping surveillance. Two nested quantitative real-time PCR (qPCR) assays were developed that target the human EPO (hEPO) cDNA sequence in a circular form, representative of recombinant adeno-associated viral (rAAV) vector genomes found in vivo. Through an interlaboratory evaluation, the assays were validated and utilized in an in vitro blinded study. These assays are specific and extremely sensitive with a limit of detection (LOD) of 1 copy of circular plasmid DNA and a limit of quantification (LOQ) of 10 to 20 copies in the presence of 500 ng of human genomic DNA (hgDNA) extracted from WBCs. Additionally, using the two nested qPCR assays in a non-human primate study, where macaques were injected intramuscularly with a rAAV8 vector harboring a promoterless hEPO cDNA sequence, the viral vector was detected 8 to 14 weeks post-injection in macaque WBCs. The high sensitivity of the nested qPCR approach along with the capability of quantifying target DNA, make this approach a reliable tool for gene doping surveillance and the monitoring of exogenous DNA sequences.

mTORC1 directly phosphorylates and activates ERα upon estrogen stimulation.

Breast cancer is the leading cause of cancer-related deaths among women. Approximately 75% of breast cancers are estrogen receptor-α (ERα) positive, underscoring the dependence of cancer cells on estrogen for growth and survival. Patients treated with endocrine therapy often develop resistance, either de novo or acquired, which in some cases is caused by aberrations within the growth factor signaling pathways. The mechanistic target of rapamycin complex 1 (mTORC1) has emerged as a critical node in estrogenic signaling. We have previously shown that mTORC1 can phosphorylate and activate ERα on S167 via its effector-the 40S ribosomal S6 kinase 1 (S6K1). Presently, we have uncovered a direct link between mTORC1 and ERα. We found that ERα binds to regulatory-associated protein of mTOR (Raptor) and causes it to translocate to the nucleus upon estrogen stimulation. In addition, we identified mTOR as the kinase that phosphorylates ERα on S104/106 and activates transcription of ER target genes. Our findings show a direct link between mTORC1 and ERα, which further implicates mTORC1 signaling in the pathogenesis of ER-positive breast cancer and provides rationale for FDA-approved use of mTORC1 inhibitors in combination with endocrine agents for treatment of this disease.

Critical transport decisions. In time-sensitive emergencies, every moment counts--choose wisely.

Time-sensitive emergencies require early recognition and rapid transport to a facility properly equipped to manage the patient's needs. When managing STEMI, cardiac arrest, suspected stroke, trauma or a severe sepsis patient, use your resources smartly. Manage the patient using all of your capabilities on scene and know the destination best prepared to manage the patient upon ED arrival. When it makes sense to extend transport time to take a patient to a proper facility, it is OK to do so. Considering air medical transport for patients as ground transports exceed 30 minutes is reasonable as long as the flight team provides transport more rapidly or brings additional care that will improve patient outcomes.

Effect of oral nutritional supplementation on wound healing in diabetic foot ulcers: a prospective randomized controlled trial.

AIMS: Among people with diabetes, 10-25% will experience a foot ulcer. Research has shown that supplementation with arginine, glutamine and ß-hydroxy-ß-methylbutyrate may improve wound repair. This study tested whether such supplementation would improve healing of foot ulcers in persons with diabetes. METHODS: Along with standard of care, 270 subjects received, in a double-blinded fashion, (twice per day) either arginine, glutamine and ß-hydroxy-ß-methylbutyrate or a control drink for 16 weeks. The proportion of subjects with total wound closure and time to complete healing was assessed. In a post-hoc analysis, the interaction of serum albumin or limb perfusion, as measured by ankle-brachial index, and supplementation on healing was investigated. RESULTS: Overall, there were no group differences in wound closure or time to wound healing at week 16. However, in subjects with an albumin level of ≤ 40 g/l and/or an ankle-brachial index of < 1.0, a significantly greater proportion of subjects in the arginine, glutamine and ß-hydroxy-ß-methylbutyrate group healed at week 16 compared with control subjects (P = 0.03 and 0.008, respectively). Those with low albumin or decreased limb perfusion in the supplementation group were 1.70 (95% CI 1.04-2.79) and 1.66 (95% CI 1.15-2.38) times more likely to heal. CONCLUSIONS: While no differences in healing were identified with supplementation in non-ischaemic patients or those with normal albumin, addition of arginine, glutamine and ß-hydroxy-ß-methylbutyrate as an adjunct to standard of care may improve healing of diabetic foot ulcers in patients with risk of poor limb perfusion and/or low albumin levels. Further investigation involving arginine, glutamine and ß-hydroxy-ß-methylbutyrate in these high-risk subgroups might prove clinically valuable.

Bisphosphonates as a supplement to exercise to protect bone during long-duration spaceflight.

UNLABELLED: We report the results of alendronate ingestion plus exercise in preventing the declines in bone mass and strength and elevated levels of urinary calcium and bone resorption in astronauts during 5.5 months of spaceflight. INTRODUCTION: This investigation was an international collaboration between NASA and the JAXA space agencies to investigate the potential value of antiresorptive agents to mitigate the well-established bone changes associated with long-duration spaceflight. METHODS: We report the results from seven International Space Station (ISS) astronauts who spent a mean of 5.5 months on the ISS and who took an oral dose of 70 mg of alendronate weekly starting 3 weeks before flight and continuing throughout the mission. All crewmembers had available for exercise a treadmill, cycle ergometer, and a resistance exercise device. Our assessment included densitometry of multiple bone regions using X-ray absorptiometry (DXA) and quantitative computed tomography (QCT) and assays of biomarkers of bone metabolism. RESULTS: In addition to pre- and post-flight measurements, we compared our results to 18 astronauts who flew ISS missions and who exercised using an early model resistance exercise device, called the interim resistance exercise device, and to 11 ISS astronauts who exercised using the newer advanced resistance exercise device (ARED). Our findings indicate that the ARED provided significant attenuation of bone loss compared with the older device although post-flight decreases in the femur neck and hip remained. The combination of the ARED and bisphosphonate attenuated the expected decline in essentially all indices of altered bone physiology during spaceflight including: DXA-determined losses in bone mineral density of the spine, hip, and pelvis, QCT-determined compartmental losses in trabecular and cortical bone mass in the hip, calculated measures of fall and stance computed bone strength of the hip, elevated levels of bone resorption markers, and urinary excretion of calcium. CONCLUSIONS: The combination of exercise plus an antiresoptive drug may be useful for protecting bone health during long-duration spaceflight.

Bacterial mutagenicity screening in the pharmaceutical industry.

Genetic toxicity testing is used as an early surrogate for carcinogenicity testing. Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds. To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition. With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development. Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development. This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs. The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St. Louis, MO in October, 2009. However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices. In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies. The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed.

Survival Benefit of Adjuvant Radiation Therapy for Gastric Cancer following Gastrectomy and Extended Lymphadenectomy.

Purpose. Although randomized trials suggest a survival benefit of adjuvant chemotherapy and radiation therapy (XRT) for gastric adenocarcinoma, its use in patients who undergo an extended lymphadenectomy is less clear. The purpose of this study was to determine if a survival benefit exists in gastric cancer patients who receive adjuvant XRT following resection with extended lymphadenectomy. Methods. The SEER registry was queried for records of patients with resected gastric adenocarcinoma from 1988 to 2007. Multivariable Cox regression models were used to assess independent prognostic factors affecting overall survival (OS) and disease-specific survival (DSS). Results. Of 15,060 patients identified, 3,208 (21%) received adjuvant XRT. Adjuvant XRT was independently associated with improved OS (HR 0.67, CI 0.64-0.71) and DSS (HR 0.69, CI 0.65-0.73) in stages IB through IV (M0). This OS and DSS benefit persisted regardless of the extent of lymphadenectomy. Furthermore, lymphadenectomy with >25 LN resected was associated with improved OS and DSS compared with <15 LN or 15-25 LN. Conclusion. This population-based study shows a survival benefit of adjuvant XRT following gastrectomy that persists in patients who have an extended lymphadenectomy. Furthermore, removal of >25 LNs results in improved OS and DSS compared with patients who have fewer LNs resected.

Accuracy of physician and nurse practitioner colposcopy to effect improved surveillance of cervical cancer.

PURPOSE: To compare physician and nurse practitioner accuracy in recognizing cervical dysplasia during colposcopy. MATERIALS AND METHODS: A retrospective review was performed of cervical excisional biopsies from 2007 to 2009 performed by gynecologists and nurse practitioners in the same patient population. Cervical cone biopsy and loop electrosurgical excision procedure (LEEP) pathology were used as a gold standard compared to the previous colposcopy biopsies. RESULTS: Four hundred fifty-five patients qualified for the study. Patients were stratified according to age: under 30 years, 30-39, and 40 and above. For physicians, 77% of high-grade colposcopy biopsy results agreed with high-grade pathology on cone biopsy or LEEP. This was statistically similar to nurse practitioner results (p = 0.12). Likewise, there was no significant difference between physician and nurse practitioner accuracy within the various patient age strata. CONCLUSION: Colposcopy biopsy results compared to cone biopsy or LEEP results were statistically similar between gynecologists and nurse practitioners.

Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping.

Legitimate uses of gene transfer technology can benefit from sensitive detection methods to determine vector biodistribution in pre-clinical studies and in human clinical trials, and similar methods can detect illegitimate gene transfer to provide sports-governing bodies with the ability to maintain fairness. Real-time PCR assays were developed to detect a performance-enhancing transgene (erythropoietin, EPO) and backbone sequences in the presence of endogenous cellular sequences. In addition to developing real-time PCR assays, the steps involved in DNA extraction, storage and transport were investigated. By real-time PCR, the vector transgene is distinguishable from the genomic DNA sequence because of the absence of introns, and the vector backbone can be identified by heterologous gene expression control elements. After performance of the assays was optimized, cynomolgus macaques received a single dose by intramuscular (IM) injection of plasmid DNA, a recombinant adeno-associated viral vector serotype 1 (rAAV1) or a rAAV8 vector expressing cynomolgus macaque EPO. Macaques received a high plasmid dose intended to achieve a significant, but not life-threatening, increase in hematocrit. rAAV vectors were used at low doses to achieve a small increase in hematocrit and to determine the limit of sensitivity for detecting rAAV sequences by single-step PCR. DNA extracted from white blood cells (WBCs) was tested to determine whether WBCs can be collaterally transfected by plasmid or transduced by rAAV vectors in this context, and can be used as a surrogate marker for gene doping. We demonstrate that IM injection of a conventional plasmid and rAAV vectors results in the presence of DNA that can be detected at high levels in blood before rapid elimination, and that rAAV genomes can persist for several months in WBCs.