An automated homogeneous method for quantifying polysorbate using fluorescence polarization

An automated fluorescence polarization (FP) assay has been developed for the quantitation of polysorbate in bioprocess samples. Using the lipophilic probe 5-dodecanoylaminofluorescein (DAF), polysorbate concentrations above the critical micelle concentration can be quantified by the FP increase that results when DAF inserts into the detergent micelles. The specificity, accuracy, and precision of this assay were defined for samples obtained from vaccine purification processes. Spike recoveries were 98-106% for purified products and 110-120% for crude process intermediates. The coefficients of variation for intra- and interassay precision were less than 9 and 14%, respectively. Because of the operational simplicity of the assay, all of the assay steps from sample preparation to data reduction were automated on a Tecan liquid-handling workstation. The combination of a rapid assay and an automated format makes this method well suited to the routine analysis of samples from trial purification processes which are carried out during the development of a vaccine or therapeutic protein. This method should be adaptable for the quantitation of other detergents into which DAF will insert.     

Human Telomere Length Correlates to the Size of the Associated Chromosome Arm

The majority of human telomere length studies have focused on the overall length of telomeres within a cell. In fact, very few studies have examined telomere length for individual chromosome arms. The objective of this study was to examine the relationship between chromosome arm size and the relative length of the associated telomere. Quantitative Fluorescence In Situ Hybridization (Q-FISH) was used to measure the relative telomere length of each chromosome arm in metaphases from cultured lymphocytes of 17 individuals. A statistically significant positive correlation (r = 0.6) was found between telomere length and the size of the associated chromosome arm, which was estimated based on megabase pair measurements from http://www.ncbi.nlm.nih.gov/projects/mapview/.     

Optimal Population-Level Infection Detection Strategies for Malaria Control and Elimination in a Spatial Model of Malaria Transmission

Mass campaigns with antimalarial drugs are potentially a powerful tool for local elimination of malaria, yet current diagnostic technologies are insufficiently sensitive to identify all individuals who harbor infections. At the same time, overtreatment of uninfected individuals increases the risk of accelerating emergence of drug resistance and losing community acceptance. Local heterogeneity in transmission intensity may allow campaign strategies that respond to index cases to successfully target subpatent infections while simultaneously limiting overtreatment. While selective targeting of hotspots of transmission has been proposed as a strategy for malaria control, such targeting has not been tested in the context of malaria elimination. Using household locations, demographics, and prevalence data from a survey of four health facility catchment areas in southern Zambia and an agent-based model of malaria transmission and immunity acquisition, a transmission intensity was fit to each household based on neighborhood age-dependent malaria prevalence. A set of individual infection trajectories was constructed for every household in each catchment area, accounting for heterogeneous exposure and immunity. Various campaign strategies—mass drug administration, mass screen and treat, focal mass drug administration, snowball reactive case detection, pooled sampling, and a hypothetical serological diagnostic—were simulated and evaluated for performance at finding infections, minimizing overtreatment, reducing clinical case counts, and interrupting transmission. For malaria control, presumptive treatment leads to substantial overtreatment without additional morbidity reduction under all but the highest transmission conditions. Compared with untargeted approaches, selective targeting of hotspots with drug campaigns is an ineffective tool for elimination due to limited sensitivity of available field diagnostics. Serological diagnosis is potentially an effective tool for malaria elimination but requires higher coverage to achieve similar results to mass distribution of presumptive treatment.     

Probabilistic accounting of uncertainty in forecasts of species distributions under climate change

Forecasts of species distributions under future climates are inherently uncertain, but there have been few attempts to describe this uncertainty comprehensively in a probabilistic manner. We developed a Monte Carlo approach that accounts for uncertainty within generalized linear regression models (parameter uncertainty and residual error), uncertainty among competing models (model uncertainty), and uncertainty in future climate conditions (climate uncertainty) to produce site‐specific frequency distributions of occurrence probabilities across a species' range. We illustrated the method by forecasting suitable habitat for bull trout (Salvelinus confluentus) in the Interior Columbia River Basin, USA, under recent and projected 2040s and 2080s climate conditions. The 95% interval of total suitable habitat under recent conditions was estimated at 30.1–42.5 thousand km; this was predicted to decline to 0.5–7.9 thousand km by the 2080s. Projections for the 2080s showed that the great majority of stream segments would be unsuitable with high certainty, regardless of the climate data set or bull trout model employed. The largest contributor to uncertainty in total suitable habitat was climate uncertainty, followed by parameter uncertainty and model uncertainty. Our approach makes it possible to calculate a full distribution of possible outcomes for a species, and permits ready graphical display of uncertainty for individual locations and of total habitat.     

Effect of local aromatase inhibition in endometriosis using a new chick embryo chorioallantoic membrane model

Endometriosis is an oestrogen‐dependent, inflammation‐driven gynaecologic disorder causing severe disability. Endometriosis implants are characterized by unbalanced local oestrogen metabolism leading to hyperoestrogenism and aromatase up‐regulation is one of main mechanism involved. Aromatase inhibitors such as letrozole or anastrozole use in young women are associated with severely side effects limiting their long‐term clinical use. An endometriosis‐targeted inhibition of local aromatase could be a viable alternative, although the role of the local inhibition of this enzyme is still unclear. Using a new chick embryo allantoic membrane (CAM) model incorporating xenografted human endometriosis cyst, we showed that topical treatment with anastrozole reduced lesion size, although oestrogens produced by CAM female embryo blunted this effect. Xenografted human endometriosis CAM is a new efficient model for the screening of new drugs targeting endometriosis tissue.     

Specific resistance upon lentiviral TRAIL transfer by intracellular retention of TRAIL receptors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in many transformed cells, suggesting TRAIL as an ideal candidate for cancer gene therapy. A main obstacle in cancer therapy is intrinsic or acquired therapy resistance of malignant cells. To study induction of resistance against TRAIL, we generated lentiviral vectors allowing efficient TRAIL expression and apoptosis induction in a variety of human cancer cell lines. Within days upon TRAIL overexpression, cells became resistant towards TRAIL, but not to CD95 ligation or DNA damage by cisplatin. Cell surface expression of TRAIL receptors 1 and 2 was completely abrogated in resistant cells due to intracellular retention of the receptors by TRAIL. SiRNA directed against TRAIL resensitized the resistant cells by restoring cell surface expression of TRAIL receptors. These findings represent a novel resistance mechanism towards TRAIL, specifically caused by TRAIL overexpression, and question the use of TRAIL expression in tumor-cell targeting gene therapy.     

Effects of aerobic exercise on strength performance following various periods of recovery

The purpose of this study was to determine if the type and intensity of aerobic training affects performance in a subsequent strength-training session after varying periods of recovery. Sixteen male subjects participated in the study and were divided into 2 groups based on aerobic training, high-intensity intervals (MAX n = 8) and continuous submaximal (SUB n = 8). Each subject performed 4 sets of both bench press and leg press at approximately 75% 1 repetition maximum (1RM) following aerobic training with recovery periods of 4, 8, and 24 hours, as well as once in a control condition. Both the 4- and 8-hour conditions resulted in fewer total leg press repetitions than the control and 24-hour conditions. There was no difference between both the control and 24-hour conditions. No main effect was shown with respect to the type of aerobic training. It was concluded that when aerobic training precedes strength training, the volume of work that can be performed is diminished for up to 8 hours. This impairment appears to be localized to the muscle groups involved in the aerobic training.     

Bi-directional cell contact-dependent regulation of gene expression between endothelial cells and osteoblasts in a three-dimensional spheroidal coculture model

Multiple cell-cell interactions control bone morphogenesis and vascularization. We have employed a spheroidal coculture system of endothelial cells (EC) and osteoblasts (OB) to study cell contact-dependent gene regulation between these two cell types that may play a role in regulating OB differentiation and EC angiogenic properties. Coculture spheroids differentiate spontaneously to organize into a core of OB and a surface layer of endothelial cells. Individual spheroid culture of EC or OB leads to significant alterations in gene expression compared to standard monolayer culture (upregulation of Tie-2 in EC; upregulation of angiopoietin-2 in osteoblasts). More importantly, spheroidal coculture of endothelial cells and osteoblasts leads to significant changes of gene expression in both cell populations (upregulation of VEGFR-2 in EC; downregulation of VEGF, and upregulation of alkaline phosphatase in osteoblasts). These changes are dependent on cell-cell contact and are not seen in stimulation experiments with conditioned supernatants. Collectively, the data demonstrate complex bi-directional gene regulation mechanisms between EC and OB that are likely to play a critical role during OB differentiation and in controlling the properties of angiogenic EC.