Effect of Aromatase Inhibition (Exemestane) on Urine Concentration of Osteoprotegerin in Healthy Postmenopausal Women.

This pilot study examined how exemestane (an aromatase inhibitor [AI]) affected osteoprotegerin (OPG) urine concentrations in postmenopausal women. Exemestane (25 mg, single dose) was given to 14 disease-free women past menopause in this nonrandomized, open-label study. Before dosing, urine specimens were gathered. Three days later, these women returned to provide urine specimens for pharmacokinetic (measurement of major parent drug and enzymatic product) and pharmacodynamic (profiling of OPG) analysis. Urine concentrations of the major parent drug (exemestane) and enzymatic product (17-hydroexemestane) were quantified using liquid chromatography-tandem mass spectrometry. An analyst software package was used for data processing. Following the manufacturer's guidelines, OPG urine concentrations were quantified using a human osteoprotegerin TNFRSF11b ELISA kit from Sigma-Aldrich. A microplate reader helped to carry out OPG data analysis and processing. Our results highlight that OPG urine concentrations were decreased 3 days after drug dosage (mean predosage OPG concentration, 61.4 ± 24.1 pg/mL; vs mean postdosage OPG concentration, 45.7 ± 22.1 pg/mL; P = .02, Wilcoxon rank test). Among the 14 volunteers enrolled in the study, 4 subjects had an increase of less than 1-fold, and the rest showed an average of a 2-fold decrease in OPG concentration (range, 1.1-5.4; standard deviation, 1.3) after exemestane administration. There was no association between fold decrease in OPG urine concentration and the pharmacokinetics of the major parent drug (exemestane) and its enzymatic product (17-hydroexemestane). We concluded that one of the off-target pharmacological effects of AIs (eg ,exemestane) may result in the reduction of osteoprotegerin.

Indirect detection of Bacillus anthracis using real-time PCR to detect amplified gamma phage DNA.

Typical real-time PCR methods used to identify Bacillus anthracis do not distinguish between viable and non-viable spores, which would be critical in any first response and remediation scenarios. This study combined both real-time PCR, using primers specifically designed for gamma phage, with the highly specific gamma phage amplification into one simple assay to indirectly detect Bacillus anthracis. Since the amplification of gamma phage only occurs in the presence of a suitable host, the detection of increasing concentrations of progeny gamma phage DNA using real-time PCR implies the presence of viable Bacillus anthracis cells. This method detected a starting Bacillus anthracis concentration of 207 cfu/mL, equivalent to less than one cell in 20 microL, in less than 5 h.

Impact of UGT2B17 Gene Deletion on the Pharmacokinetics of 17-Hydroexemestane in Healthy Volunteers.

Exemestane is an aromatase inhibitor drug used for the treatment of hormone-dependent breast cancer. 17-Hydroexemestane, the major and biologically active metabolite of exemestane in humans, is eliminated via glucuronidation by the polymorphic UGT2B17 phase II drug-metabolizing enzyme. Previous microsomal studies have shown that UGT2B17 gene deletion affects the intrinsic hepatic clearances of 17-hydroexemestane in vitro. In this open-label study we set out to assess the effect of UGT2B17 gene deletion on the pharmacokinetics of 17-hydroexemestane in healthy female volunteers with and without UGT2B17. To achieve this goal, 14 healthy postmenopausal women (8 carriers of the homozygous UGT2B17 wild-type allele and 6 carriers of the homozygous UGT2B17 gene-deletion allele) were enrolled and invited to receive a single 25-mg oral dose of exemestane. Pharmacokinetics was assessed over 72 hours postdosing. Our results showed that there were statistically significant differences in plasma 17-hydroexemestane AUC0-∞ (P = .0007) and urine 17-hydroexemestane C24h (P = .001) between UGT2B17 genotype groups. Our data suggest that UGT2B17 gene deletion influences 17-hydroexemestane pharmacokinetics in humans.

Identification of Aedes aegypti and its respective life stages by real-time polymerase chain reaction.

An Aedes aegypti-specific, fluorogenic probe hydrolysis (Taq-Man), polymerase chain reaction assay was developed for real-time screening using a field-deployable thermocycler. Laboratory-based testing of A. aegypti, A. aegypti (Trinidad strain), Culex pipiens, Culex quinquefasciatus, Anopheles stephensi, and Ochlerotatus taeniorhynchus individual adult mosquitoes and mixed pools (n = 10) demonstrated 100% concordance in both in vitro sensitivity (six of six samples) and specificity (10 of 10 samples). A single adult A. aegypti was identified in a pool of 100 non-A. aegypti mosquitoes. The limit of detection of A. aegypti egg pools was five individual eggs. Field testing was conducted in central Honduras. An A. aegypti and Culex spp. panel of individual and mixed pools (n = 30) of adult mosquitoes, pupae, and larvae demonstrated 100% concordance in sensitivity (22 of 22 samples) and 97% concordance in specificity (29 of 30 samples), with one false-positive result. Field testing of an A. aegypti and Culex spp. blind panel (n = 16) consisting of individual and mixed pools of adult mosquitoes, pupae, and larvae demonstrated 90% concordance in sensitivity (nine of 10 samples) and 88% concordance in specificity (14 of 16 samples).

Rapid identification of dengue virus by reverse transcription-polymerase chain reaction using field-deployable instrumentation.

Dengue virus universal and dengue serotype 1 to 4, fluorogenic probe hydrolysis (TaqMan), reverse transcription-polymerase chain reaction assays were developed for screening and serotype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler. Dengue universal and dengue 1 to 4 serotype assay in vitro sensitivity and specificity results were 100% concordant when tested with total nucleic acid extracts of multiple strains of dengue serotype 1 to 4, yellow fever, Japanese encephalitis, West Nile, and St. Louis encephalitis viruses. The in vitro sensitivity and specificity results for all five assays were concordant when tested with a blind panel of 27 dengue virus-infected mosquitoes, 21 non-dengue (yellow fever, West Nile, or St. Louis encephalitis) flavivirus-infected mosquitoes, and 11 uninfected mosquitoes and with clinical specimens consisting of a human serum panel of eight dengue viremic and 31 non-dengue-infected febrile patient serum samples. No cross-reaction occurred with vector species or human genomic DNA. Sample processing and polymerase chain reaction required <2 hours.

Time-kill Kinetics of a Novel Antimicrobial Silver Wound Gel Against Select Wound Pathogens.

UNLABELLED: New treatments are needed as infection risk associated with diabetic, venous, and pressure ulcers are becoming more prevalent as comorbidities of obesity, aging, and major disease. Postsurgical, burn, and immunocompromised patients are also at an increased risk of wounds and infection. Silver has been utilized in treating various wounds associated with infections and, although highly effective, caution is required for use beyond 2 weeks due to potential silver cytotoxicity. To overcome this obstacle, an antimicrobial wound gel (CelaCare Technologies, Inc, Dallas, TX) was designed to allow low concentrations of a proprietary silver salt combined with acemannan, which has been demonstrated to aid wound healing. MATERIALS AND METHODS: This study's objective was to determine the time-kill kinetics of the antimicrobial wound gel vs 4 commercial topical silver products against 6 common wound pathogens and Bacillus subtilis as a spore-forming bacteria. RESULTS: The antimicrobial wound gel achieved a 2.9 log reduction in growth of Pseudomonas aeruginosa within 30 minutes, a 2.3 log reduction in Streptococcus pyogenes within 8 hours, a 2.1 log reduction in methicillin-resistant Staphylococcus aureus within 48 hours, a 2.3 log reduction in S. aureus within 24 hours, a 4.1 log reduction in Escherichia coli within 30 minutes, a 2.9 log reduction in B. subtilis within 60 minutes, and a 3.4 log reduction in Candida albicans within 90 minutes. Overall, the antimicrobial wound gel demonstrated broad antimicrobial coverage against all wound pathogens evaluated, and it was comparable to, or better than, other tested topical silver products containing substantially higher silver concentrations. CONCLUSION: The broad-spectrum antimicrobial activity of the wound gel indicates it could become a product alternative to current commercial products.

One step purification of alpha(1)-acid glycoprotein from human plasma. Fractionation of its polymorphic allele products.

Alpha(1)-acid glycoprotein is a plasma protein that exhibits both microheterogeneity and polymorphism. Its purification from human plasma is usually performed using a sequence of different fractionation steps. Here we report a one-step isolation technique of this protein based upon pseudo-ligand affinity chromatography on immobilized Cibacron Blue F3GA at acidic pH. In addition, the use of two narrow pH elution buffers allows us to separate the two genetic products of this protein, which differ from each other by 21 amino acid substitutions. This technique will facilitate the study of the structural, biological and pharmacokinetic properties of each individual allele product.

Identification of Francisella tularensis using real-time fluorescence polymerase chain reaction.

A Francisella tularensis-specific, TaqMan probe-based, real-time fluorescence polymerase chain reaction (PCR) assay required approximately 60 minutes and consistently achieved a sensitivity of < or = 10 fg of F. tularensis genomic DNA (five genome equivalents). Specificity testing against a genomic DNA cross-reaction panel comprised of 22 bacterial organisms representing closely related species, diverse genera, and human genomic DNA resulted in no false positives of significance. The assay was conducted on a field-deployable thermocycler, the R.A.P.I.D. ("Ruggedized" Advanced Pathogen Identification Device), a microbial identification system that can provide rapid and accurate identification F. tularensis.

Characterization of the effects of heat stress on the DNA-intercalating dye EvaGreen for potential use with the joint biological agent identification and diagnostic system.

Although advances in real-time polymerase chain reaction (PCR) technology and equipment have facilitated field research, only a limited selection of reagents do not require cold storage. This study explored the temperature stability of the commercially available DNA-intercalating dye EvaGreen after exposure to a spectrum of temperatures for 176 days by analyzing quantification cycle (Cq) and end fluorescence levels during amplification of the invA gene of Salmonella typhimurium. To further characterize potential dye stability, the effects of small differences in dye volume were examined and dye samples were subjected to an Air Force deployment to the Middle East. Significant differences in Cq and end fluorescence were found; however, the magnitude of mean Cq differences was less than one cycle and the magnitude of mean fluorescence differences was less than that attributable to a difference of 0.25 µL of dye per 25 µL reaction. Liquid EvaGreen dye may thus be stable at temperatures as high as 65°C for up to 6 months for use in real-time PCR. These results warrant further investigation by using liquid EvaGreen dye to adapt traditional lab-based real-time PCR assays for Joint Biological Agent Identification and Diagnostic System use and testing the assays in the field.

Comparison of TaqMan and Epoch Dark Quenchers during real-time reverse transcription PCR.

Several biotechnology companies have recently introduced novel quencher fluors for use with dual-labeled fluorogenic hydrolysis probes. The Epoch Dark Quencher trade mark fluorochrome consists of a non-fluorescent moiety capable of absorption at higher wavelengths (400-650 nm). The aim of this study was to: (1) evaluate the feasibility of using Epoch Dark Quencher fluorochromes in real-time PCR pathogen detection assays that were previously optimized with TaqMan (TAMRA) quenching fluors, and (2) compare the sensitivity based on cycle threshold (CT) between probes containing either TaqMan or Epoch Dark Quencher fluors. Our data indicate Epoch Dark Quencher probes can be used in place of TaqMan probes and their performance was not better than traditional TaqMan (TAMRA) quenchers. Marginal differences observed between quenching fluorochromes may arise from concentration differences during probe synthesis.