Dose-response Measurements Research Articles

Comprehensive Drug Testing of Patient-derived Conditionally Reprogrammed Cells from Castration-resistant Prostate Cancer

BackgroundTechnology development to enable the culture of human prostate cancer (PCa) progenitor cells is required for the identification of new, potentially curative therapies for PCa. ObjectiveWe established and characterized patient-derived conditionally reprogrammed cells (CRCs) to assess their biological properties and to apply these to test the efficacies of drugs. Design, setting, and participantsCRCs were established from seven patient samples with disease ranging from primary PCa to advanced castration-resistant PCa (CRPC). The CRCs were characterized by genomic, transcriptomic, protein expression, and drug profiling. Outcome measurements and statistical analysisThe phenotypic quantification of the CRCs was done based on immunostaining followed by image analysis with Advanced Cell Classifier using Random Forest supervised machine learning. Copy number aberrations (CNAs) were called from whole-exome sequencing and transcriptomics using in-house pipelines. Dose-response measurements were used to generate multiparameter drug sensitivity scores using R-statistical language. Results and limitationsWe generated six benign CRC cultures which all had an androgen receptor-negative, basal/transit-amplifying phenotype with few CNAs. In three-dimensional cell culture, these cells could re-express the androgen receptor. The CRCs from a CRPC patient (HUB.5) displayed multiple CNAs, many of which were shared with the parental tumor. We carried out high-throughput drug-response studies with 306 emerging and clinical cancer drugs. Using the benign CRCs as controls, we identified the Bcl-2 family inhibitor navitoclax as the most potent cancer-specific drug for the CRCs from a CRPC patient. Other drug efficacies included taxanes, mepacrine, and retinoids. ConclusionsComprehensive cancer pharmacopeia-wide drug testing of CRCs from a CRPC patient highlighted both known and novel drug sensitivities in PCa, including navitoclax, which is currently being tested in clinical trials of CRPC. Patient summaryWe describe an approach to generate patient-derived cancer cells from advanced prostate cancer and apply such cells to discover drugs that could be applied in clinical trials for castration-resistant prostate cancer.

Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors.

Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors.

Instrumentation to study myofibril mechanics from static to artificial simulations of cardiac cycle

Many causes of heart muscle diseases and skeletal muscle diseases are inherited and caused by mutations in genes of sarcomere proteins which play either a structural or contractile role in the muscle cell. Tissue samples from human hearts with mutations can be obtained but often samples are only a few milligrams and it is necessary to freeze them for storage and transportation. Myofibrils are the fundamental contractile components of the muscle cell and retain all structural elements and contractile proteins performing in contractile event; moreover viable myofibrils can be obtained from frozen tissue.•We are describing a versatile technique for measuring the contractility and its Ca2+ regulation in single myofibrils. The control of myofibril length, incubation medium and data acquisition is carried out using a digital acquisition board via computer software. Using computer control it is possible not only to measure contractile and mechanical parameters but also simulate complex protocols such as a cardiac cycle to vary length and medium independently.•This single myofibril force assay is well suited for physiological measurements. The system can be adapted to measure tension amplitude, rates of contraction and relaxation, Ca2+ dependence of these parameters in dose-response measurements, length-dependent activation, stretch response, myofibril elasticity and response to simulated cardiac cycle length changes. Our approach provides an all-round quantitative way to measure myofibrils performance and to observe the effect of mutations or posttranslational modifications. The technique has been demonstrated by the study of contraction in heart with hypertrophic or dilated cardiomyopathy mutations in sarcomere proteins.

Risk assessment of smokeless tobacco among oral precancer and cancer patients in eastern developmental region of Nepal

Background: Oral potentially malignant disorders (OPMDs) and oral cancer (OC) are preventable oral mucosal diseases prevalent in Asian region. This epidemiological study aims to identify oral potentially malignant disorders (OPMDs) and oral cancer (OC), confirm histopathologically, and treat or refer these cases among the population of Eastern Development Region (EDR) of Nepal. It also attempts to assess the risk factors associated in order to compare dose–response measurements of oral habits in these patients. Methods: Cross-sectional epidemiological study was conducted over a period of 2 years in 16 districts of EDR. A total of 3,200 people were screened. A brief history was taken and visual screening examination was conducted in two phases as described by British Columbia Oral Cancer Prevention Program. Suspicious oral lesions were biopsied either by punch or scalpel after toluidine blue staining. Tissue specimen was transported to the institutional lab for histopathological processing. The reports were sent to the patients through the local leaders or organizations. Results: More than 40% of the study population either chewed areca nut and/or tobacco. Eighteen percent were smokers. OPMDs were prevalent among 468 study population with male-female ratio of 3:1. Tobacco pouch keratosis (50.4%) was the most prevalent OPMD, followed by OSF (29.1%). Fifty-two had squamous cell carcinoma and 8 had verrucous carcinoma. Conclusion: Chewing areca nut, tobacco, commercial areca nut/tobacco preparation and smoking being the major risk factors, there is high prevalence of oral cancer and OPMDs in the EDR of Nepal.

SU‐E‐T‐716: Suitability Study of a Unique 3D Dosimeter for Commissioning Radiation Treatment Machines

Purpose:To determine the suitability of a methacrylic acid‐based MAGAT gel dosimeter to serve as a 3D commissioning toolMethods:Different sized MAGAT gel dosimeters were fabricated in‐house to perform dose response, depth dose and dose rate measurements using an orthovoltage treatment unit. Dose response data were obtained by irradiating dosimeters to six dose levels at three different effective beam energies. Depth dose measurements were taken using one dose at one energy. Dose rate dependence was measured by varying only distances for the gel irradiations while dose level and energy remained unchanged. A Bruker Biospecs 4.7T MRI scanner was used to acquire spin‐spin relaxation rates on axial slices at different depths throughout each dosimeter 24h after irradiation.Results:The dose response curves exhibited a separation when compared for the measured energies. MAGAT depth dose data, normalized to ion chamber data at 2 cm depth, showed an increasing divergence with increasing depth. The dose rate decreased when distances were increased and demonstrated a significant dose rate dependence of the measurements. Consequently, dose rate corrections were applied to all measurements, thus, aligning the dose response curves for all three energies and reducing the divergence between MAGAT and ion chamber depth dose data.Conclusion:The MAGAT dosimeter was intended to serve as a 3D commissioning tool for testing radiation treatment plans of small fields with steep dose gradients and complex treatment volumes. A suitable detector for commissioning needs to be dependent on dose but independent of dose rate. This study, however, showed a dose rate dependence of up to 30% eliminating the current composition of MAGAT as a suitable dosimeter. Further investigations of chemical compositions need to be performed.

Trap spectroscopy and dosimetric aspects of natural kyanite mineral

Thermoluminescence (TL) of X-ray irradiate natural kyanite (Al2SiO5) mineral annealed at 473, 573, 673 and 773 K was studied within 300–520 K. For all types of samples, TL glow curves revealed a stable peak at around 405 K. Analyzing the glow curves by computerized glow curve deconvolution process, one electron trap center had been estimated at depth around 1.01 eV from the conduction band. Investigation of TL emission spectra recorded within 300–520 K showed two intense peaks centered at around 366 and 688 nm, which confirmed the presence of two recombination centers at depth around 3.39 and 1.81 eV from the conduction band. Based on the analysis, a band diagram for kyanite crystal had been proposed showing the possible trap and recombination centers. Dose response, fading analysis and reproducibility measurement were carried out for all type of samples. For the highest annealed sample (773 K), linear dose response was observed within 15–1000 mGy. The rate of fading was very high just after irradiation but slowed after 5 days. The reproducibility of 773 K annealed sample was excellent. After ten recycle, the coefficient of variation was found to be only 0.75 %. These analyses demand the potential use of 773 K annealed kyanite as a natural X-ray dosimeter.

Age‐related Changes in Endothelial Ca2+ Signaling Revealed by Imaging from Inside Arteries at Normal Physiological Pressures

Aging is the summation of many, mostly invisible, but deleterious changes associated with altered structure and function of the cardiovascular system. Age‐induced impairment of endothelial function contributes to and reinforces the changes that occur in vascular disease. Normally, the endothelium transduces multiple chemical and mechanical signals into changes in cytoplasmic calcium (Ca2+) concentration that control vascular function. Thus, understanding age‐related functional changes in the endothelium requires an appreciation of Ca2+ control. We developed a wide‐field, gradient‐index (GRIN) fluorescence imaging system to visualize the endothelium from within the lumen of intact, pressurized arteries. The GRIN system enables rapid imaging of a 500 μm diameter area of the curved endothelial surface (~200 endothelial cells; ECs) with subcellular resolution. We also developed a semi‐automated data extraction procedure to quantify signal dynamics in the individual ECs loaded with fluorescent Ca2+ indicators. In the carotid artery of young rats (3 months), acetylcholine (ACh) evoked endothelial Ca2+ signals that were suppressed as intraluminal pressure increased. In aged rats (18 months) the pressure‐dependent suppression of ACh‐evoked Ca2+ signaling and the frequency of ACh‐evoked intracellular Ca2+ oscillations were reduced, whilst the temporal propagation of Ca2+ signals increased. Significantly, dose response measurements revealed that aging increased EC sensitivity to ACh, yet had no effect on the magnitude of the Ca2+ response in each cell. We propose that such alterations in EC Ca2+ signaling may manifest as a loss of functionality, and explain endothelial dysfunction in aging.

A High-Throughput Phenotypic Screen of Cytotoxic T Lymphocyte Lytic Granule Exocytosis Reveals Candidate Immunosuppressants

We screened the National Institutes of Health’s Molecular Libraries Small Molecule Repository for inhibitors of cytotoxic T lymphocyte (CTL) lytic granule exocytosis by measuring binding of an antibody in the extracellular solution to a lysosomal membrane protein (LAMP-1) that is transferred to the plasma membrane by exocytosis. We used TALL-104 human leukemic CTLs stimulated with soluble chemicals. Using high-throughput cluster cytometry to screen 364,202 compounds in a 1536-well plate format, we identified 2404 initial hits: 161 were confirmed on retesting, and dose–response measurements were performed. Seventy-five of those compounds were obtained, and 48 were confirmed active. Experiments were conducted to determine the molecular mechanism of action (MMOA) of the active compounds. Fifteen blocked increases in intracellular calcium >50%. Seven blocked phosphorylation of extracellular signal-regulated kinase (ERK) by upstream mitogen-activated protein kinase kinases >50%. One completely blocked the activity of the calcium-dependent phosphatase calcineurin. None blocked ERK catalytic activity. Eight blocked more than one pathway. For 8 compounds, we were unable to determine an MMOA. The activity of 1 of these compounds was confirmed from powder resupply. We conclude that a screen based on antibody binding to CTLs is a good means of identifying novel candidate immunosuppressants with either known or unknown MMOAs.

Developmentally distinct activities of the exocyst enable rapid cell elongation and determine meristem size during primary root growth in Arabidopsis.

BackgroundExocytosis is integral to root growth: trafficking components of systems that control growth (e.g., PIN auxin transport proteins) to the plasma membrane, and secreting materials that expand the cell wall to the apoplast. Spatiotemporal regulation of exocytosis in eukaryotes often involves the exocyst, an octameric complex that tethers selected secretory vesicles to specific sites on the plasma membrane and facilitates their exocytosis. We evaluated Arabidopsis lines with mutations in four exocyst components (SEC5, SEC8, EXO70A1 and EXO84B) to explore exocyst function in primary root growth.ResultsThe mutants have root growth rates that are 82% to 11% of wild-type. Even in lines with the most severe defects, the organization of the quiescent center and tissue layers at the root tips appears similar to wild-type, although meristematic, transition, and elongation zones are shorter. Reduced cell production rates in the mutants are due to the shorter meristems, but not to lengthened cell cycles. Additionally, mutants demonstrate reduced anisotropic cell expansion in the elongation zone, but not the meristematic zone, resulting in shorter mature cells that are similar in shape to wild-type. As expected, hypersensitivity to brefeldin A links the mutant root growth defect to altered vesicular trafficking. Several experimental approaches (e.g., dose–response measurements, localization of signaling components) failed to identify aberrant auxin or brassinosteroid signaling as a primary driver for reduced root growth in exocyst mutants.ConclusionsThe exocyst participates in two spatially distinct developmental processes, apparently by mechanisms not directly linked to auxin or brassinosteroid signaling pathways, to help establish root meristem size, and to facilitate rapid cell expansion in the elongation zone.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0386-0) contains supplementary material, which is available to authorized users.

Dosimetry of very high energy electrons (VHEE) for radiotherapy applications: using radiochromic film measurements and Monte Carlo simulations

Very high energy electrons (VHEE) in the range from 100–250 MeV have the potential of becoming an alternative modality in radiotherapy because of their improved dosimetry properties compared with MV photons from contemporary medical linear accelerators. Due to the need for accurate dosimetry of small field size VHEE beams we have performed dose measurements using EBT2 Gafchromic® film. Calibration of the film has been carried out for beams of two different energy ranges: 20 MeV and 165 MeV from conventional radio frequency linear accelerators. In addition, EBT2 film has been used for dose measurements with 135 MeV electron beams produced by a laser-plasma wakefield accelerator. The dose response measurements and percentage depth dose profiles have been compared with calculations carried out using the general-purpose FLUKA Monte Carlo (MC) radiation transport code. The impact of induced radioactivity on film response for VHEEs has been evaluated using the MC simulations. A neutron yield of the order of 10−5 neutrons cm−2 per incident electron has been estimated and induced activity due to radionuclide production is found to have a negligible effect on total dose deposition and film response. Neutron and proton contribution to the equivalent doses are negligible for VHEE. The study demonstrates that EBT2 Gafchromic film is a reliable dosimeter that can be used for dosimetry of VHEE. The results indicate an energy-independent response of the dosimeter for 20 MeV and 165 MeV electron beams and has been found to be suitable for dosimetry of VHEE.

Quantification of biological network perturbations for mechanistic insight and diagnostics using two-layer causal models.

BackgroundHigh-throughput measurement technologies such as microarrays provide complex datasets reflecting mechanisms perturbed in an experiment, typically a treatment vs. control design. Analysis of these information rich data can be guided based on a priori knowledge, such as networks or set of related proteins or genes. Among those, cause-and-effect network models are becoming increasingly popular and more than eighty such models, describing processes involved in cell proliferation, cell fate, cell stress, and inflammation have already been published. A meaningful systems toxicology approach to study the response of a cell system, or organism, exposed to bio-active substances requires a quantitative measure of dose-response at network level, to go beyond the differential expression of single genes.ResultsWe developed a method that quantifies network response in an interpretable manner. It fully exploits the (signed graph) structure of cause-and-effect networks models to integrate and mine transcriptomics measurements. The presented approach also enables the extraction of network-based signatures for predicting a phenotype of interest. The obtained signatures are coherent with the underlying network perturbation and can lead to more robust predictions across independent studies. The value of the various components of our mathematically coherent approach is substantiated using several in vivo and in vitro transcriptomics datasets. As a proof-of-principle, our methodology was applied to unravel mechanisms related to the efficacy of a specific anti-inflammatory drug in patients suffering from ulcerative colitis. A plausible mechanistic explanation of the unequal efficacy of the drug is provided. Moreover, by utilizing the underlying mechanisms, an accurate and robust network-based diagnosis was built to predict the response to the treatment.ConclusionThe presented framework efficiently integrates transcriptomics data and “cause and effect” network models to enable a mathematically coherent framework from quantitative impact assessment and data interpretation to patient stratification for diagnosis purposes.

SU‐F‐BRE‐15: Verification of Energy Dependence of MAGAT Polymer Gel at Orthovoltage Energies

Purpose:Investigation of dose response curves of methacrylic acid‐based “MAGAT” gel at different effective energies to verify an energy dependence of polymer‐gel dosimeters for orthovoltage energy x‐rays.Methods:Six small cylindrical MAGAT gel phantoms were exposed to different dose levels; one phantom was unirradiated for background subtraction. This experiment was repeated for three different effective beam energies.24 h post irradiation the spin‐spin relaxation times (T2) were measured with a 4.7 T Bruker MR scanner at 2 cm depth inside the gel. The T2 values were converted to relaxation rates (R2) and plotted against the respective dose levels corresponding to the different effective energies. The resulting dose response curves were compared for a 250 kVp beam, the 250 kVp beam filtered by 6 cm of water, and a 125 kVp beam.Results:The passage of the 250 kVp beam through water resulted in a half‐value‐layer (HVL) change from 1.05 mm Cu to 1.32 mm Cu at 6 cm depth with a change in effective energy from 81.3 keV to 89.5 keV, respectively. The dose response curves showed a shift to higher relaxation rates for the harder beam. The dose response measurements for the 125 kVp beam (HVL: 3.13 mm Al, effective energy: 33.9 keV) demonstrated even higher relaxation rates than for either of the other beams.Conclusion:The MAGAT dose response curves for three different effective energies demonstrate a complex energy dependence, with an apparent decrease in sensitivity at 89.5 keV effective energy. This energy dependence is consistent with observed discrepancies of depth dose data compared with ion‐chamber data. For future investigations of larger volumes, an energy‐dependent sensitivity function is needed to properly assess 3‐dimensional dose distributions.

Aircraft dose-response research in National Park backcountry areas

The Federal Aviation Administration and National Park Service conducted joint research to better understand visitor response to noise from commercial air tour operations over units of the National Park System. Dose-response research in National Parks was conducted during the 1990’s at heavily visited, frontcountry sites. To expand this work, aircraft dose-response measurements were recently performed at seven backcountry sites in four National Parks. These sites provided both day and overnight hiking and camping opportunities, with visits ranging from one hour to several days. The sampling approach included side-by-side use of three survey instruments, enabling the evaluation of multiple response variables (annoyance, interference with natural quiet, and acceptability) and comparison of audio clip and in situ dose-response, along with the effects of wording and question order. The evaluation of visits longer than previously studied necessitated methodological enhancements including the use of global positioning system based tracking of visitor locations. In total, over 4600 visitor surveys and 50 days of simultaneous acoustic data were collected. This paper, the first of two on this research, describes the research methods and visitor surveys and presents summary results of collected data.

Assessment of the inhibition of Dengue virus infection by carrageenan via real-time monitoring of cellular oxygen consumption rates within a microfluidic device.

A microfluidic device combined with a light modulation system was developed to assess the inhibitory effect of carrageenan on Dengue virus (DENV) infection via real-time monitoring of cellular oxygen consumption rates (OCRs). Measuring cellular OCRs, which can reflect cellular metabolic activity, enabled us to monitor the process of viral infection in real time and to rapidly determine the antiviral activity of potential drugs/chemical compounds. The time variation of the cellular OCR of single cells that were infected in situ by DENV at different multiplicity of infection (m.o.i.) values was first successfully measured within a microfluidic device. The influence of the timing of carrageenan treatment on DENV infection was then examined by real-time monitoring of cellular OCRs in three groups. Cells that were pre-treated with carrageenan and then infected with DENV served as a pre-treatment group, cells to which carrageenan was added simultaneously with DENV served as a virucide group, and cells that were pre-infected with DENV and then treated with carrageenan served as a post-treatment group. By monitoring cellular OCRs, we could rapidly evaluate the inhibitory effect of carrageenan on DENV infection, obtaining a result within 7 h and showing that carrageenan had strong and effective anti-DENV activity in the three groups. In particular, a strong inhibitory effect was observed in the virucide group. Moreover, once the virus enters host cells in the post-treatment group, the immediate treatment with carrageenan for the infected cells has higher efficiency of antiviral activity. Our proposed platform enables to perform time-course or dose-response measurements of changes in cellular metabolic activity caused by diseases, chemical compounds, and drugs via monitoring of the cellular OCR, with rapid and real-time detection. This approach provides the potential to study a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery.

Dose-response measurement in gel dosimeter using various imaging modalities

Measurement methods that accurately measure radiation dose distribution in a three dimensional manner in order to allow comparisons of treatment plans are needed for quality assurance. One such measurement method involves the use of a polymer gel dosimeter to measure the dose distribution in three dimensions. During irradiation, a polymerization reaction makes new chemical bonds and induces changes of the chemical structure of the gel of the gel dosimeter. In the present study, dose-response measurement of an environment-friendly material used in the gel dosimeter was performed by imaging with computed tomography (CT) and R1, R2, and fluid-attenuated inversion-recovery (FLAIR) magnetic resonance imaging (MRI) under various imaging conditions. Dose-response characteristics in the gel dosimeter used in the experiment were observed at doses of 5–20 Gy administered by X-ray CT and MRI. Although the FLAIR signal was a relative value, the dose-response values with FLAIR were excellent compared to those with R1, R2, and CT. Determination of more appropriate imaging conditions could help expand the dose-response parameters of each measurement method.

TU‐G‐108‐08: Variable RBE and Incidence of Radiation Pneumonitis in Lung Patients Treated with Proton Therapy

Purpose: To determine the correlation between the Relative Biological Effectiveness (RBE)‐weighted dose and radiation pneumonitis (RP) incidence in cancer patients treated with proton therapy. Methods: The Monte Carlo proton dose calculation system MC2 based on MCNPX was used to recalculate treatment plans taken from the clinical treatment planning system (TPS) and compute the dose‐averaged LET in each voxel of the CT based patient model. The linear scaling model (LSM) for proton RBE was used with biological coefficients for RP to compute the variable RBE‐weighted dose distributions. These were compared with the constant RBE‐weighted doses computed by the TPS and currently used in our clinical practice. A restaging FDG PET/CT was registered to the treatment planning CT and the standard uptake values (SUV) were used as a measure of radiation dose response. Results: The variable RBE model results in higher biological dose at the distal ends of proton beams and the corresponding lung volumes receiving elevated doses can be significant if the beam is terminating inside the low density lung tissue. Preliminary results for the first two patients in the study show that the regions of elevated variable RBE‐weighted doses inside the lung are well correlated to the high SUV values in the restaging PET. Both patients were diagnosed with grade 3 RP after completing the treatment. Conclusion: The preliminary results indicate that variable RBE‐weighted doses may provide additional insight into toxicity risk for patients treated with proton radiotherapy. The complete study will include the analysis of many more patients diagnosed with grade 3 RP or higher, treated with protons in our institution. NCI grant P01CA021239

Notch Transcriptional Control of Vascular Smooth Muscle Regulatory Gene Expression and Function

Notch receptors and ligands mediate heterotypic cell signaling that is required for normal vascular development. Dysregulation of select Notch receptors in mouse vascular smooth muscle (VSM) and in genetic human syndromes causes functional impairment in some regional circulations, the mechanistic basis of which is undefined. In this study, we used a dominant-negative Mastermind-like (DNMAML1) to block signaling through all Notch receptors specifically in VSM to more broadly test a functional role for this pathway in vivo. Mutant DNMAML1-expressing mice exhibited blunted blood pressure responses to vasoconstrictors, and their aortic, femoral, and mesenteric arteries had reduced contractile responses to agonists and depolarization in vitro. The mutant arteries had significant and specific reduction in the expression and activity of myosin light chain kinase (MLCK), a primary regulator of VSM force production. Conversely, activated Notch signaling in VSM cells induced endogenous MLCK transcript levels. We identified MLCK as a direct target of activated Notch receptor as demonstrated by an evolutionarily conserved Notch-responsive element within the MLCK promoter that binds the Notch receptor complex and is required for transcriptional activity. We conclude that Notch signaling through the transcriptional control of key regulatory proteins is required for contractile responses of mature VSM. Genetic or pharmacological manipulation of Notch signaling is a potential strategy for modulating arterial function in human disease.

Dynamical modelling of prostaglandin signalling in platelets reveals individual receptor contributions and feedback properties

Prostaglandins are the key-players in diminishing platelet function. They exert their effects via a variety of surface receptors that are linked to the cAMP/PKA-signalling cascade. However, less is known about the quantitative impact of the individual receptors on the underlying pathway. We present here a comprehensive ordinary differential equation-based model of the platelet cAMP pathway, including the four prostaglandin receptors IP, DP1, EP3 and EP4, the ADP receptor P2Y12, a detailed PKA-module as well as downstream-targets. Parameter estimation along with a comprehensive combination of time-course and dose-response measurements revealed the individual quantitative role of each receptor in elevating or decreasing pathway activity. A comparison of the two inhibiting receptors EP3 and P2Y12 exhibited a greater signalling strength of the EP3 receptor with implications for antithrombotic treatment. Furthermore, analysis of different model topologies revealed a direct influence of PKA on adenylate cyclase, reducing its maximum catalytic speed. Finally, we show here for the first time the dynamic behaviour of VASP-phosphorylation, which is commonly used as a marker for platelet-inhibition. We validate our model by comparing it to further experimental data.

Cross-Species Transcriptomic Analysis of Mouse and Rat Lung Exposed to Chloroprene

β-Chloroprene (2-chloro-1,3-butadiene), a monomer used in the production of neoprene elastomers, is of regulatory interest due to the production of multiorgan tumors in mouse and rat cancer bioassays. A significant increase in female mouse lung tumors was observed at the lowest exposure concentration of 12.8 ppm, whereas a small, but not statistically significant increase was observed in female rats only at the highest exposure concentration of 80 ppm. The metabolism of chloroprene results in the generation of reactive epoxides, and the rate of overall chloroprene metabolism is highly species dependent. To identify potential key events in the mode of action of chloroprene lung tumorigenesis, dose-response and time-course gene expression microarray measurements were made in the lungs of female mice and female rats. The gene expression changes were analyzed using both a traditional ANOVA approach followed by pathway enrichment analysis and a pathway-based benchmark dose (BMD) analysis approach. Pathways related to glutathione biosynthesis and metabolism were the primary pathways consistent with cross-species differences in tumor incidence. Transcriptional BMD values for the pathway were more similar to differences in tumor response than were estimated target tissue dose surrogates based on the total amount of chloroprene metabolized per unit mass of lung tissue per day. The closer correspondence of the transcriptional changes with the tumor response is likely due to their reflection of the overall balance between metabolic activation and detoxication reactions, whereas the current tissue dose surrogate reflects only oxidative metabolism.

Retrospective dosimetry using OSL of tooth enamel and dental repair materials irradiated under wet and dry conditions

Following a radiological or nuclear emergency event, there is a need for quick and reliable dose estimations of potentially exposed people. In situations where dosimeters are not readily available, the dose estimations must be carried out using alternative methods. In the present study, the optically stimulated luminescence (OSL) properties of tooth enamel and different dental repair materials have been examined. Specimens of the materials were exposed to gamma and beta radiation in different types of liquid environments to mimic the actual irradiation situation in the mouth. Measurements were taken using a Risø TL/OSL reader, and irradiations were made using a (90)Sr/(90)Y source and a linear accelerator (6 MV photons). Results show that the OSL signal from tooth enamel decreases substantially when the enamel is kept in a wet environment. Thus, tooth enamel is not reliable for retrospective dose assessment without further studies of the phenomenon. Dental repair materials, on the other hand, do not exhibit the same effect when exposed to liquids. In addition, dose-response and fading measurements of the dental repair materials show promising results, making these materials highly interesting for retrospective dosimetry. The minimum detectable dose for the dental repair materials has been estimated to be 20-185 mGy.