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.

Incorporating Latent Variables into Discrete Choice Models - A Simultaneous Estimation Approach Using SEM Software

Integrated choice and latent variable (ICLV) models represent a promising new class of models which merge classic choice models with the structural equation approach (SEM) for latent variables. Despite their conceptual appeal, applications of ICLV models in marketing remain rare. We extend previous ICLV applications by first estimating a multinomial choice model and, second, by estimating hierarchical relations between latent variables. An empirical study on travel mode choice clearly demonstrates the value of ICLV models to enhance the understanding of choice processes. In addition to the usually studied directly observable variables such as travel time, we show how abstract motivations such as power and hedonism as well as attitudes such as a desire for flexibility impact on travel mode choice. Furthermore, we show that it is possible to estimate such a complex ICLV model with the widely available structural equation modeling package Mplus. This finding is likely to encourage more widespread application of this appealing model class in the marketing field.

The Effects of Living in Segregated vs. Mixed Areas in Northern Ireland: A Simultaneous Analysis of Contact and Threat Effects in the Context of Micro-Level Neighbourhoods

This study examines the consequences of living in segregated and mixed neighbourhoods on ingroup bias and offensive action tendencies, taking into consideration the role of intergroup experiences and perceived threat. Using adult data from a cross-sectional survey in Belfast, Northern Ireland, we tested a model that examined the relationship between living in segregated (N = 396) and mixed (N = 562) neighbourhoods and positive contact, exposure to violence, perceived threat and outgroup orientations. Our results show that living in mixed neighbourhoods was associated with lower ingroup bias and reduced offensive action tendencies. These effects were partially mediated by positive contact. However, our analysis also shows that respondents living in mixed neighbourhoods report higher exposure to political violence and higher perceived threat to physical safety. These findings demonstrate the importance of examining both social experience and threat perceptions when testing the relationship between social environment and prejudice.

Exact Solutions to the Symmetric and Asymmetric Vehicle Routing Problem with Simultaneous Delivery and Pick-Up

In reverse logistics networks, products (e.g., bottles or containers) have to be transported from a depot to customer locations and, after use, from customer locations back to the depot. In order to operate economically beneficial, companies prefer a simultaneous delivery and pick-up service. The resulting Vehicle Routing Problem with Simultaneous Delivery and Pick-up (VRPSDP) is an operational problem, which has to be solved daily by many companies. We present two mixed-integer linear model formulations for the VRPSDP, namely a vehicle-flow and a commodity-flow model. In order to strengthen the models, domain-reducing preprocessing techniques, and effective cutting planes are outlined. Symmetric benchmark instances known from the literature as well as new asymmetric instances derived from real-world problems are solved to optimality using CPLEX 12.1.

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.