Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Were last glacial climate events simultaneous between Greenland and western Europe?

During the last glacial period, several large abrupt climate fluctuations took place on the Greenland ice cap and elsewhere. Often these Dansgaard/Oeschger events are assumed to have been synchronous, and then used as tie-points to link chronologies between the proxy archives. However, if temporally separate events are lumped into one illusionary event, climatic interpretations of the tuned events will obviously be flawed. Here, we compare Dansgaard/Oeschger-type events in a well-dated record from south-eastern France with those in Greenland ice cores. Instead of assuming simultaneous climate events between both archives, we keep their age models independent. Even these well-dated archives possess large chronological uncertainties, that prevent us from inferring synchronous climate events at decadal to multi-centennial time scales. If possible, tuning of proxy archives should be avoided.

Influence of Particle Size of Deproteinized Bovine Bone Mineral on New Bone Formation and Implant Stability after Simultaneous Sinus Floor Elevation: A Histomorphometric Study in Minipigs

BACKGROUND Deproteinized bovine bone mineral (DBBM) is one of the best-documented bone substitute materials for sinus floor elevation (SFE). PURPOSE DBBM is available in two particle sizes. Large particles are believed to facilitate improved neoangiogenesis compared with small ones. However, their impact on the rate of new bone formation, osteoconduction, and DBBM degradation has never been reported. In addition, the implant stability quotient (ISQ) has never been correlated to bone-to-implant contact (BIC) after SFE with simultaneous implant placement. MATERIALS AND METHODS Bilateral SFE with simultaneous implant placement was performed in 10 Göttingen minipigs. The two sides were randomized to receive large or small particle size DBBM. Two groups of 5 minipigs healed for 6 and 12 weeks, respectively. ISQ was recorded immediately after implant placement and at sacrifice. Qualitative histological differences were described and bone formation, DBBM degradation, BIC and bone-to-DBBM contact (osteoconduction) were quantified histomorphometrically. RESULTS DBBM particle size had no qualitative or quantitative impact on the amount of newly formed bone, DBBM degradation, or BIC for either of the healing periods (p > 0.05). Small-size DBBM showed higher osteoconduction after 6 weeks than large-size DBBM (p < 0.001). After 12 weeks this difference was compensated. There was no significant correlation between BIC and ISQ. CONCLUSION Small and large particle sizes were equally predictable when DBBM was used for SFE with simultaneous implant placement.

Piezoelectric alveolar ridge-splitting technique with simultaneous implant placement: a cohort study with 2-year radiographic results

PURPOSE Extended grafting procedures in atrophic ridges are invasive and time-consuming and increase cost and patient morbidity. Therefore, ridge-splitting techniques have been suggested to enlarge alveolar crests. The aim of this cohort study was to report techniques and radiographic outcomes of implants placed simultaneously with a piezoelectric alveolar ridge-splitting technique (RST). Peri-implant bone-level changes (ΔIBL) of implants placed with (study group, SG) or without RST (control group, CG) were compared. MATERIALS AND METHODS Two cohorts (seven patients in each) were matched regarding implant type, position, and number; superstructure type; age; and gender and received 17 implants each. Crestal implant bone level (IBL) was measured at surgery (T0), loading (T1), and 1 year (T2) and 2 years after loading (T3). For all implants, ΔIBL values were determined from radiographs. Differences in ΔIBL between SG and CG were analyzed statistically (Mann-Whitney U test). Bone width was assessed intraoperatively, and vertical bone mapping was performed at T0, T1, and T3. RESULTS After a mean observation period of 27.4 months after surgery, the implant survival rate was 100%. Mean ΔIBL was -1.68 ± 0.90 mm for SG and -1.04 ± 0.78 mm for CG (P = .022). Increased ΔIBL in SG versus CG occurred mainly until T2. Between T2 and T3, ΔIBL was limited (-0.11 ± 1.20 mm for SG and -0.05 ± 0.16 mm for CG; P = .546). Median bone width increased intraoperatively by 4.7 mm. CONCLUSIONS Within the limitations of this study, it can be suggested that RST is a well-functioning one-stage alternative to extended grafting procedures if the ridge shows adequate height. ΔIBL values indicated that implants with RST may fulfill accepted implant success criteria. However, during healing and the first year of loading, increased IBL alterations must be anticipated.

A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC–MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-13C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3–6.3%) and accurate (3.8–7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, −20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

Campylobacter spp., Yersinia enterocolitica, and Salmonella enterica and their simultaneous occurrence in German fattening pig herds and their environment.

Campylobacter spp., Salmonella enterica, and Yersinia enterocolitica are common causes of foodborne infections in humans with pork as a potential source. Monitoring programs at farm level are, to date, only implemented for S. enterica, while epidemiological knowledge of the other two pathogens is still lacking. This study aimed to assess the pathogen load (in the pigs' environment) in fattening pig herds, their simultaneous occurrence, and the occurrence of Campylobacter spp. and Y. enterocolitica in herds in different Salmonella risk categories. In 50 fattening pig herds in northern Germany, four pooled fecal samples and 10 swab samples from the pigs' direct environment (pen walls, nipple drinkers), indirect environment (hallways, drive boards), and flies and rodent droppings were collected from each herd and submitted for cultural examination. Campylobacter spp. were detected in 38.1% of fecal, 32.7% of direct environment, 5.3% of indirect environment, and 4.6% of flies/pests samples collected, and Y. enterocolitica in 17.1, 8.1, 1.2, and 3.1% and S. enterica in 11.2, 7.7, 4.1, and 1.5%, respectively. For Campylobacter spp., Y. enterocolitica, and S. enterica, 80, 48, and 32% of herds were positive, respectively; 22 herds were positive for both Campylobacter spp. and Y. enterocolitica, 12 for Campylobacter spp. and S. enterica, and 7 for Y. enterocolitica and S. enterica. There was no significant association between the pathogens at herd level. Campylobacter spp. and Y. enterocolitica were found more often in samples from the low Salmonella risk category (odds ratio, 0.51; confidence interval, 0.36 to 0.73, and 0.3, 0.17 to 0.57), and this was also the case for Y. enterocolitica at herd level (odds ratio, 0.08; confidence interval, 0.02 to 0.3). This study provides evidence that the pigs' environment should be accounted for when implementing control measures on farms against Campylobacter spp. and Y. enterocolitica. An extrapolation from the current Salmonella monitoring to the other two pathogens does not seem feasible.